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Other Publications (102)

Articles by Denise L. Loranger in JoVE

 JoVE Clinical and Translational Medicine

A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis

1Orvis School of Nursing, University of Nevada, Reno, 2The State University of New York at Buffalo, St. Joseph's Medical Center, 3Strong Memorial Hospital, University of Rochester Medical Center


JoVE 50124

Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.

Other articles by Denise L. Loranger on PubMed

Endemic Typhus in Manila, Philippine Islands; Report of Cases and Identification of the Murine Rickettsial Agent in Domestic Rats by Complement Fixation

[The Nutrition of French-Canadian Workers in an Aluminum Company of Canada Factory]

Not Available

Critical Flicker Frequency and Some Intellectual Functions in Old Age

The Performance of Aged Females on Five Non-language Tests of Intellectual Functions

The Placebo Effect in Psychiatric Drug Research

Cerebral Dysfunction and Intellectual Impairment in Old Age

There is a marked decline in some intellectual abilities in old age. It is frequently hypothesized that impaired cerebral physiology accounts for some of this deficit. In old age the critical flicker frequency, a measure sensitive to cerebral dysfunction, is correlated with aging intellectual abilities. This is interpreted as evidence supporting the above hypothesis.

A Controlled Evaluation of Deanol and Benactyzine-meprobamate

Treatment of Acute Mental Disorders with an Adrenal Steroid

Long-term Care of Schizophrenia

Intellectual Impairment in Parkinson's Syndrome

Levodopa Treatment of Parkinson's Syndrome. Improved Intellectual Functioning

Parkinsonism, L-dopa, and Intelligence

The Determination of Sulfur, Lead, and Silicon in Atmospheric Aerosols: an Application of X-ray Fluorescence and Confined-spot Paper Techniques

Parkinsonism, Levodopa, and Intelligence

X-linkage and Manic-depressive Illness

Thirty years ago it was suggested that the apparently higher incidence of manic-depressive illness in women might be due to X-linked heredity. The hypothesis was undermined by subsequent reports of the frequent occurrence of father to son transmission. Winokur and his associates recently revived it, providing data which indicated that such transmission is absent or rare in the bipolar form of the illness. Additional support has come from linkage studies with known genetic markers located on the X chromosome. The present study, based on the 400 parents of 100 male and 100 female bipolar manic-depressive probands, failed to discover a lack of father-son compared to other affected parent-child pairs. This finding, together with a review of the literature, would indicate that it is premature to invoke X-linked heredity as a general explanation for bipolar manic-depressive illness, though there is mounting evidence that it may account for the illness in some family pedigrees.

Mental Symptoms in Parkinson's Disease During Chronic Treatment with Levodopa

Mental symptoms increased in frequency among 100 patients with parkinsonism treated with levodopa. Dementia was found in about one-third of patients throughout the 6-year treatment period. Thirteen patients became demented during the study, and dementia worsened severely in seven others. Agitated confusion became increasingly frequent and was observed in 60 percent of patients taking levodopa for 6 years. Withdrawal from levodopa decreased agitation, but not dementia. Ten patients received L-tryptophan along with levodopa, but no change in mentation was observed. In view of previous studies of mentation in Parkinson's disease and reports of widespread neuronal changes in the brain of autopsied patients with parkinsonism, our results suggest that the high incidence of dementia in patients with Parkinson's disease who take levodopa reflects prolongation of the course of the illness rather than a direct effect of the medication.

Age at Onset of Bipolar Affective Illness

The age at onset of bipolar affective illness was determined for 100 males and 100 females who met the newly proposed DSM-III criteria for mania. Three different indices of onset were employed: first hospitalization, first treatment, and first apparent symptoms. A table was constructed indicating the cumulative percentage of those who became ill by the time they passed through each age quinquennium. One third were hospitalized before their 25th birthday, and at least 20% had already shown evidence of illness as adolescents. The early 20s was the peak period of onset. No manic episodes were confirmed prior to age 13, and onset after age 60 was rare. Sex, ethnicity, and socioeconomic status did not significantly affect age at onset. Affective illness should be given serious consideration in the differential diagnosis of mental disorders in young people.

[Phalloidin Counteracts the Destruction of F-actin by Osmic Acid. II. Protection by Phalloidin of F-actin Crosslinked with Aldehydes (author's Transl)]

We have studied by viscometry and spectrophotometry the effects of glutaraldehyde, acetaldehyde, acrolein and formaldehyde on F-actin in vitro. Pretreatment with acrolein and, to a lesser extent, with glutaraldehyde, results in increased destruction of F-actin during the subsequent degradation with osmic acid. Formaldehyde alone disintegrates actin filaments. Acetaldehyde gives the best results and does not seem to damage F-actin. Phalloidin protects F-actin against destruction by osmic acid and this protection is also observed in the F-actin which has been pretreated with the four aldehydes mentioned.

Outcome of Psychotherapy

Genetic Independence of Manic-depression and Schizophrenia

Increasingly sophisticated twin and adoption research has demonstrated major genetic contributions to the etiology of manic-depression and schizophrenia. But studies disagree concerning whether the two are genetically related illnesses. This lack of consensus could be due to individual, regional, and temporal differences in the criteria used to diagnose the two conditions. This study is the first to employ the new DSM-III criteria. Schizophrenia was no more common in the 1,098 first-degree relatives of 100 male and 100 female manic-depressives than it is in the population at-large. This would appear to strengthen the view that manic-depression and schizophrenia are genetically unrelated diseases.

Prolipoprotein Signal Peptidase in Escherichia Coli is Distinct from the M13 Procoat Protein Signal Peptidase

We have previously reported a signal peptidase activity in Escherichia coli cell envelope which processes prolipoprotein modified with glyceride (Tokunaga, M., Tokunaga, H., and Wu, H. C. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2255-2259). To ascertain whether the processing enzyme for prolipoprotein is distinct from the signal peptidase for M13 procoat protein purified by Zwizinski and Wickner (Zwizinski, C., and Wickner, W. (1980) J. Biol. Chem. 255, 7973-7977), we have used antibody against purified procoat protein signal peptidase to study the processings of prolipoprotein and M13 procoat protein in vitro. the signal peptidase for modified prolipoprotein remained fully active in solubilized membrane preparations which had been treated with antibody against purified procoat protein signal peptidase whereas the activity towards procoat protein was completely abolished by immunoadsorption. Furthermore, both unmodified and glyceride-modified prolipoprotein were not cleaved by the highly purified signal peptidase preparation provided by Wickner. These data clearly indicate that prolipoprotein signal peptidase is distinct from the M13 procoat protein signal peptidase.

Effect of Silybin on Phalloidin-actin Interactions in Vitro

The possible antagonism between silybin and phalloidin an F-actin has been examined in vitro. Phalloidin protects F-actin against denaturation by potassium iodine, cytochalasin B, DNase and pronase. Silybin does not counteract the effect of phalloidin on F-actin. However, silybin alone slowed the rate of polymerization of actin, but this effect was moderate and obtained with very high concentrations. These results indicate that the site of action in vitro of silybin is not actin.

Familial Transmission of DSM-III Borderline Personality Disorder

A comparison was made of the types of mental disorders occurring in the first-degree relatives of 83 female patients with DSM-III borderline personality disorder, 100 female patients with DSM-III schizophrenia, and 100 female patients with DSM-III bipolar disorder. Diagnosis of the relatives was made independently by two clinicians who were blind to the diagnosis of the probands. The relative of a borderline patient was about ten times more likely to have been treated for a borderline or borderlinelike personality disorder than was the relative of a schizophrenic or bipolar patient. The borderline patients' relatives were also treated for more unipolar depression than the schizophrenics' relatives. However, the relatives of the borderline patients did not have a higher morbid risk for treated mania or schizophrenia than that usually reported for the population at large.

Isolation and Characterization of an Escherichia Coli Clone Overproducing Prolipoprotein Signal Peptidase

Based on the rationale that Escherichia coli cells containing increased levels of prolipoprotein signal peptidase would be highly resistant to globomycin, a specific inhibitor of the prolipoprotein signal peptidase, we have isolated a clone from the Carbon-Clarke collection, plasmid pLC3-13, which is globomycin-resistant and contains an increased level of prolipoprotein signal peptidase activity. The plasmid pMT521, a subclone of pLC3-13 in pBR322, conferred on its host cells approximately 20 times overproduction of prolipoprotein signal peptidase and an extremely high level of resistance against globomycin. The overproduced prolipoprotein signal peptidase was completely inhibited by the presence of globomycin in the in vitro assay, and the overproduced activity was found in the cell envelope fraction. Several lines of biochemical and genetic evidence suggest that the gene contained in pLC3-13 and its derivative clones is most likely the structure gene (lsp) for prolipoprotein signal peptidase.

Prolipoprotein Modification and Processing Enzymes in Escherichia Coli

Prolipoprotein signal peptidase, a unique endopeptidase which recognizes glycyl glyceride cysteine as a cleavage site, was characterized in an in vitro assay system using purified prolipoprotein as the substrate. This enzyme did not require phospholipids for its catalytic activity and was found to be localized in the inner cytoplasmic membrane of the Escherichia coli cell envelope. Globomycin inhibited this enzyme activity in vitro with a half-maximal inhibiting concentration of 0.76 nM. Nonionic detergent, such as Nikkol or Triton X-100, was required for the in vitro activity. The optimum pH and reaction temperature of prolipoprotein signal peptidase were pH 7.9 and 37-45 degrees C, respectively. Phosphatidylglycerol:prolipoprotein glyceryl transferase (glyceryl transferase) activity was measured using [2-3H]glycerol-labeled JE5505 cell envelope and [35S]cysteine-labeled MM18 cell envelope as the donor and acceptor of glyceryl moiety, respectively. 3H and 35S dual-labeled glyceryl cysteine was identified in the product of this enzymatic reaction. The optimal pH and reaction temperature for glyceryl transferase were pH 7.8 and 37 degrees C, respectively.

A Distinct Signal Peptidase for Prolipoprotein in Escherichia Coli

We have previously demonstrated the modification and processing of Escherichia coli prolipoprotein (Braun's) in vitro ( Tokunaga M, Tokunaga H, Wu HC: Proc Natl Acad Sci USA 79:2255, 1982). Using this in vitro assay of prolipoprotein signal peptidase and globomycin selection, we have isolated and partially characterized an E coli mutant which contained a higher level of prolipoprotein signal peptidase activity. In contrast, the procoat protein signal peptidase activity was not increased in this mutant as compared to the wild-type strain. Furthermore, E coli strains containing cloned procoat protein signal peptidase gene were found to contain elevated levels of procoat protein signal peptidase, but normal levels of prolipoprotein signal peptidase. These two signal peptidase activities were also found to exhibit different stabilities during storage at 4 degrees C. Thus biochemical, immunological, and genetic evidence clearly indicate that prolipoprotein signal peptidase is distinct from procoat protein signal peptidase in E coli.

Nucleotide Sequence of the Escherichia Coli Prolipoprotein Signal Peptidase (lsp) Gene

The nucleotide sequence of the prolipoprotein signal peptidase (lsp) gene has been determined. The lsp gene was found to be adjacent to the isoleucyl-tRNA synthetase ( ileS ) gene, such that the termination codon of the ileS gene overlaps with the initiation codon of lsp. These two genes are transcribed in the same direction and the major promotor for the lsp gene appears to be upstream of ileS . Identification of the lsp gene was established by amplification of prolipoprotein signal peptidase activity in strains carrying a subcloned 1.1-kilobase Stu I-Acc I fragment and was further confirmed by introducing mutational alterations in the COOH terminus of the protein that caused a decrease in prolipoprotein signal peptidase activity. The deduced amino acid sequence indicates that prolipoprotein signal peptidase contains 164 residues. Unlike most exported proteins, there is no apparent signal peptide sequence for the lsp protein. Computer-assisted secondary structure analysis of the deduced amino acid sequence identified four hydrophobic regions that share features common to transmembrane segments in integral membrane proteins.

Sex Difference in Age at Onset of Schizophrenia

The age at onset of schizophrenia was determined in 100 male and 100 female patients who unequivocally met DSM-III criteria for the illness. Three different indexes of onset were used: first treatment, first hospitalization, and the immediate family's first awareness of psychotic symptoms and signs. The mean age at onset of the male patients was approximately five years earlier than that of the female patients according to all three criteria. About nine of ten male patients, compared with only two of three female patients, became schizophrenic before the age of 30 years. The onset of psychosis after the age of 35 years occurred in 17% of women and in only 2% of men. About 10% of women gave no evidence of psychosis until after the age of 40 years. The reason for the sex difference is not readily apparent, but it could be a valuable clue to some of the causes of schizophrenia.

Structured Interviews and Borderline Personality Disorder

A study was designed to determine whether the Diagnostic Interview for Borderlines (DIB) might be scored from the Schedule for Affective Disorders and Schizophrenia (SADS), and also whether DIB scores predicted the clinical diagnosis of DSM-III borderline personality disorder. One pair of clinicians interviewed patients with the DIB, and another pair interviewed the same patients with a slightly modified version of the SADS. Both interviews diagnosed virtually the same patients as borderline according to the criteria of Gunderson and Singer. The sensitivity of DIB scores in predicting a DSM-III diagnosis of borderline was 70%, while the specificity was 90%; the intraclass correlation coefficient was .75. Although there is a substantial concordance, the disparity between the DSM-III and DIB systems of diagnosing borderline patients is sufficiently great to preclude the generalization of findings from studies employing one set of criteria to those employing the other.

Identification of Prolipoprotein Signal Peptidase and Genomic Organization of the Lsp Gene in Escherichia Coli

The product of the lsp gene of Escherichia coli, i.e. the prolipoprotein signal peptidase, was identified by both in vivo pulse labeling experiments using a high expression lambda PL promoter vector and by an in vitro transcription/translation coupled system. The molecular weight of prolipoprotein signal peptidase was estimated to be approximately 18,000 by its mobility on polyacrylamide gel electrophoresis and was found to be the same as that of SPase II purified from the wild-type cells. Analysis of SPase II activities in strains containing various subclones, deletion derivatives generated from plasmid pMT521, and analysis of protein products in a strain harboring an ileS-lsp-fused gene indicated that ileS and lsp genes are transcribed on the same mRNA. This was further supported by the observation that Tn5 insertions in the ileS gene resulted in a reduced expression of the lsp gene. In addition to an upstream promoter shared by the ileS and lsp genes, these analyses also revealed the presence of an internal promoter for the lsp gene within the coding region of the ileS gene.

Family History of Alcoholism in Borderline Personality Disorder

The lifetime expectancy (morbid risk) of alcoholism was determined in the parents and siblings of 83 women with DSM-III borderline personality disorder and compared with that in the parents and siblings of 100 women with DSM-III schizophrenia and 100 women with DSM-III bipolar disorder. The relatives of the borderline probands had two to three times more alcoholism than the relatives of the bipolar and schizophrenic probands. The condition was most common in the fathers of the borderline probands, almost one third of whom were either alcoholics or heavy drinkers. When the three groups of probands were subdivided according to whether they, themselves, had occasionally abused alcohol, there were no longer any significant differences in alcoholism among their relatives.

Toxicity of Peptides of Amanita Virosa Mushrooms in Mice

A study was performed on the hepatic reaction of mice to acute intoxication with virotoxins (alaviroidin, viroisin, deoxoviroisin, viroidin, deoxoviroidin) and phalloidin, cyclic peptides isolated from Amanita virosa mushrooms. Purified fractions were administered intraperitoneally at various dosages to determine the LD50 which ranged from 1.0 to 5.3 mg/kg, with viroidin, phalloidin, and viroisin being the most potent. The virotoxins and phalloidin induced hemorrhagic necrosis of the liver. The development of hepatic lesions was followed by enhanced serum alanine aminotransferase (ALT) activity as well as by light and electron microscopic changes. In additional groups, bile ducts were cannulated and bile was collected for 2 hr after injection of the peptides (1 mg/kg) to determine their cholestatic potential. The earliest changes in hepatocytes were plasma membrane invagination and cytoplasmic vacuole formation. At later time periods, erythrocyte accumulation was evident in vacuoles and in the cytoplasm. The severity of hepatic damage, as judged by morphologic analysis, was correlated with serum ALT activity. Two of the peptides tested (viroisin and phalloidin) decreased bile flow by more than 50% over control values. Mild ultrastructural alterations in the bile canalicular pole of hepatocytes were observed during the development of cholestasis. Since virotoxins, like phalloidin, are bound to actin, it is possible that their affinity to cellular actin may be responsible for their hepatotoxicity.

Health Promotion: a Conceptual Integration

DSM-III-R Personality Disorders in Patients with Eating Disorders

The authors conducted a systematic examination of DSM-III-R personality disorders among 35 patients with eating disorders. Fifty-seven percent of the patients met the criteria for at least one axis II diagnosis; borderline, self-defeating, and avoidant were the most frequently assigned personality disorders. Forty percent of the patients were given two or more diagnoses, and 17% of the patients met criteria for five to seven diagnoses. No differences were found between patients with anorexia nervosa, anorexia and bulimia nervosa, and bulimia nervosa in the distribution of diagnoses or the frequency with which individual criteria (traits) were assigned.

Psychosis Proneness and Clinical Psychopathology: Examination of the Correlates of Schizotypy

The present report examined the associations between the Perceptual Aberration Scale (PAS), a prominent psychometric index of hypothetical psychosis proneness, and several measures of clinical psychopathology in a nonpsychotic psychiatric sample (N = 101). Patients were examined by experienced clinicians using structured psychiatric interviews to assess DSM-III-R Axis I and II conditions and rated for anxiety, depression, severity of illness, and current adult social competence. Elevated scores on the PAS were most closely associated with anxiety and depression as well as schizotypal, schizoid, avoidant, and obsessive-compulsive personality disorder symptomatology. Hierarchical regression analysis identified schizotypal symptoms and anxiety as the two underlying psychopathological processes most useful in explaining variance in PAS scores. Results are interpreted as supporting both the clinical relevance and research utility of the PAS and enhancing the construct validity of Meehl's model of schizotypy.

Detection of Familial Schizophrenia Using a Psychometric Measure of Schizotypy

This study examined the lifetime expectancy (morbid risk) of schizophrenia, unipolar depression, and bipolar disorder in the first-degree relatives of 101 nonpsychotic psychiatric patients (probands) who were classified as schizotypy-positive or schizotypy-negative using a psychometric measure of schizotypy, the Perceptual Aberration Scale. The relatives of schizotypy-positive probands were significantly more likely to have been treated for schizophrenia than the relatives of schizotypy-negative probands. Morbid risk for unipolar depression or bipolar disorder among first-degree relatives did not differ between the proband groups. The results support Meehl's theory of the pathogenesis of schizophrenia and enhance the construct validity of the Perceptual Aberration Scale. The heuristic potential of a psychometric high-risk strategy in schizophrenia research is discussed and the need for replication of the present study is emphasized.

Effects of Bile Acids on Actin Polymerization in Vitro

Bile acids are major determinants of canalicular bile secretion, and there are indications that choleretic bile acids increase bile canalicular contractions, in isolated rat hepatocytes. Therefore, we examined the influence of various bile acids on the rate of actin polymerization in vitro. The free forms of cholic acid, ursodeoxycholic acid, and chenodeoxycholic acid, as well as their taurine and glycine conjugates, were incubated with purified muscle actin, at a concentration of 100-300 nmoles/mg actin. The rate of actin polymerization was measured by viscometry and the fluorescence of the pyrene probe, linked to actin. Results showed that all bile acids slow the rate of polymerization, and that the effect was dose-dependent. However, the reduction by chenodeoxycholic acid was greater than that caused by the other bile acids. The results indicate that bile acids, particularly in high concentrations interact with actin, a finding that may be related to the increased bile canalicular contractility, and altered canalicular membrane morphology, induced by choleretic bile acids.

The Impact of DSM-III on Diagnostic Practice in a University Hospital. A Comparison of DSM-II and DSM-III in 10,914 Patients

The DSM-III is the first criteria-based, multiaxial classification system of mental disorders. Since its introduction in 1980, it has received more attention than any previous nosology in the history of psychiatry. The present report attempts to gauge the impact of DSM-III on diagnostic practice at one of the largest university-affiliated psychiatric hospitals in the United States. It compares the diagnoses given to 10,914 hospitalized patients during the last 5 years of the DSM-II era and the first 5 years of the DSM-III era. There were two major consequences of the change from DSM-II to DSM-III: (1) a marked reduction in the diagnosis of schizophrenia and a corresponding increase in the diagnosis of affective disorders, and (2) a marked increase in the diagnosis of personality disorders.

Trait-state Artifacts and the Diagnosis of Personality Disorders

The multiaxial nature of DSM-III has stimulated interest in the personality disorders. There are also indications that it has produced an increase in their diagnosis. However, there is clinical and psychometric evidence that a personality evaluation undertaken while a patient is in a dysphoric mental state may distort or misrepresent traits, the so-called trait-state problem in personality assessment. The present study appears to be the first to investigate this phenomenon with a clinical interview rather than with personality tests. It examined the effect of anxiety, depression, and level of global impairment on the diagnosis of personality disorder and the assessment of the criteria for the individual Axis II disorders. Eighty-four patients, most of whom had current Axis I diagnoses, were evaluated by seven experienced clinicians with a new semistructured interview, the Personality Disorder Examination. The sample evidenced a trend toward acknowledging fewer maladaptive personality traits at follow-up than at entry. There was no evidence, however, that anxiety or depression had affected either the diagnosis of a personality disorder or the criteria associated with most of the individual personality disorders.

Cytokeratin 14 Expression in Rat Liver Cells in Culture and Localization in Vivo

Rat liver epithelial cells (LECs) are non-parenchymal proliferating cells that readily emerge in primary culture and can be established as cell lines, but their in vivo cell(s) of origin is unclear. We reported recently some evidence indicating that the LEC line, T51B, contains two cytokeratins (CKs) equivalent to human CK8 and CK14 respectively. T51B cells also contain vimentin assembled as a network of intermediate filaments distinct from that of the CKs. In the present study, we examined the expression of CK14 gene in various LEC preparations and a Triton-resistant rat skin cytoskeletal fraction, and then assessed its usefulness as an LEC specific marker in the liver. Northern and Western blot analyses with cDNAs and antibodies for CK8, CK14, CK18 and vimentin confirmed that rat hepatocytes express CK8 and CK18 genes only, whereas T51B cells express CK8, CK14 and vimentin genes in the absence of CK18. CK14 was also present in LECs derived as primary from embryonic-day 12 rat liver and secondary cultures from 4-day-old rat liver. Primary cultures of oval cells isolated from 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) treated rat liver (an enriched source of biliary epithelial cells) contained CK14 mRNAs which were slightly shorter than those in LECs. The analyses of CK5 (the usual partner of CK14) gene expression using specific cDNA and antibody clearly demonstrated its absence in LECs. In situ double immunolocalization analyses by laser scanning confocal microscopy showed that CK14 was not present in hepatocytes (HES6+ cells) and was expressed in some biliary epithelial (BDS7+ cells). CK14-positive cells were also found in the Glisson's capsule. However, CK14-positive cells of the portal region were vimentin negative, whereas those of the Glisson's capsule were vimentin positive. Our results suggest that CK14 gene expression is part of the differentiation program of two types of LECs and that this differential CK14 gene expression can be used as a new means to type LECs in culture and in vivo.

Modulation of Cytokeratin and Actin Gene Expression and Fibrillar Organization in Cultured Rat Hepatocytes

Intermediate filaments of rat hepatocytes are composed of cytokeratins 8 and 18 (CK8 and CK18, respectively). Recent work from our laboratory has indicated a close relationship between the synthesis of these cytokeratins, their organization into intermediate filaments, and the promotion of growth and differentiation of cultured rat hepatocytes by insulin, epidermal growth factor, and dexamethasone. In the present study, we examined the mRNA expression, level of protein synthesis, and fibrillar distribution of cytokeratins 8 and 18 and actin in hepatocytes, isolated from normal and dexamethasone-injected rats and cultured as monolayers or spheroids in the presence of insulin, or from normal rat hepatocytes, cultured as monolayers in the presence of dexamethasone, insulin, and dimethyl sulfoxide. The CK8 mRNA level was lower in hepatocytes isolated from noninjected rats and cultured as either monolayers or spheroids, than in those from dexamethasone-injected rats. However, the CK18 mRNA level varied in a manner that was different from that of CK8 mRNA, showing that the modes of expression of the two genes were independent. The various changes in hepatocyte culture conditions led to variations in albumin mRNA levels that largely followed those observed in CK8 mRNA levels. In the case of actin, the amount of mRNAs varied from relatively high levels in hepatocyte monolayers to extremely low levels in hepatocyte spheroids, even though in both cases the cells were isolated from dexamethasone-injected rats. These changes in mRNA levels did not necessarily correlate with changes in the synthesis of cytokeratins 8 and 18, and actin. Changes in culture conditions induced a major reorganization in the distribution of cytokeratin intermediate filaments and actin filament between the region near the surface membrane and the cytoplasm. The most divergent patterns in cytokeratin intermediate filaments and actin filament distributions were observed between hepatocytes cultured as spheroidal aggregates and as monolayers in the presence of dimethyl sulfoxide. The former condition resulted in patterns of cytokeratin and actin gene expression and fibrillar organization that best matched those in situ. In the latter condition, inappropriate patterns were obtained, in spite of the fact that dimethyl sulfoxide treated hepatocytes are known to exhibit survival and functional activities equivalent to that of hepatocyte spheroids. These results demonstrate for the first time that the survival and functional activity (i.e., albumin production) of rat hepatocytes in vitro is not necessarily correlated with a particular pattern of cytokeratin and actin gene expression and fibrillar arrangement.

The Clinical Nurse Specialist As a Consultant for Play on the Pediatric Bone Marrow Transplant Unit

A theory of consultation is reviewed and the practical ways in which to implement the consultation role of the pediatric CNS are discussed. A case study is presented to facilitate understanding of the role of the CNS as a consultant in medical play on a pediatric bone marrow transplant unit.

Play Intervention Strategies for the Hispanic Toddler with Separation Anxiety

Constructive, therapeutic play is an effective nursing intervention for helping the toddler deal with separation anxiety, but, the play must transcend cultural barriers. Using Bowlby's theoretical framework to understand the response of separation anxiety in the toddler, culturally sensitive interventions of play that allow the Hispanic toddler to work through fears and express issues of separation are examined.

Computerized Cognitive Training with Learning Disabled Students: a Pilot Study

The effects of practicing computerized exercises in class by 59 learning disabled students who received an 8-hr. training program, 30 min. per week, were evaluated. Six exercises designed to facilitate basic cognitive skills development were used. Twelve subjects were assigned to a control group without any form of intervention. Covariance analysis (pretest scores used as covariates) showed a significant effect of training on mental arithmetic. These results suggest that practicing a computerized exercise of mental arithmetic can facilitate the automatization of basic arithmetic skills (addition, subtraction, and multiplication). The nature, progress, and evaluation of such types of intervention are discussed.

Localization of DNA Topoisomerase II in Chinese Hamster Fibroblasts by Confocal and Electron Microscopy

The localization of the 170- and the 180-kDa isoforms of the enzyme DNA topoisomerase II in growing Chinese hamster fibroblasts has been studied by confocal immunofluorescence microscopy and immunogold electron microscopy after labeling with affinity-purified isoform-specific polyclonal antibodies. Immunofluorescence and immunogold studies, together with quantitative image analysis, show that the two isoforms are present in the nucleoplasm and in the nucleolus. In the nucleoplasm both isoforms are frequently localized at the periphery of heterochromatin regions. In the nucleolus the immunofluorescence and immunogold signals relative to surface area are higher than in the nucleoplasm; both isoforms are localized predominantly in the fibrillar zones. During mitosis both isoforms remain detectable in the cytoplasm. The differential expression of the two isoforms during the cell cycle, observed in other studies, suggests that they have different functions, and their presence in both the nucleoplasm and the nucleolus suggests that these functions are required in both of these nuclear compartments.

[Manganese in Drinking Water and Its Contribution to Human Exposure]

Methylcyclopentadienyl manganese tricarbonyl (MMT) has been used in Canada since 1976 as an additive in unleaded gasoline. The combustion of MMT leads to the emission of Mn oxides to the environment and may represent a potential risk to public health. It therefore seems important to assess the associated Mn exposure. The present study is part of a broader research program on total human exposure to Mn and aims specifically at assessing the level of exposure to Mn and other metals via drinking water. A comparative study was performed between two groups of workers (garage mechanics and blue collar workers of the University of Montreal) differentiated by their exposure to inhaled Mn. For Pb, Cu and Zn in residential tap water, significant differences were observed between the first sample and the one taken after one minute of flow. A significant difference was also found between the two groups of workers (combined flow time) for Mn, Cu and Ca. The Mn contribution from water is estimated to be 1% of the total dose from ingested food. This low exposure may become important (17%) for persons drinking well water, especially if we consider interactions between metals following multimedia exposure.

Occupational and Environmental Exposure of Garage Workers and Taxi Drivers to Airborne Manganese Arising from the Use of Methylcyclopentadienyl Manganese Tricarbonyl in Unleaded Gasoline

Occupational and environmental exposure to airborne manganese has been measured for two groups of workers in Montreal, taxi drivers and garage mechanics. In Canada methylcyclopentadienyl manganese tricarbonyl (MMT) has replaced lead as an anti-knock agent in gasoline and represents a potentially important source of manganese contamination for the population in general and for the two chosen groups of workers in particular. Twenty workers (10 taxi drivers and 10 garage mechanics) wore a personal air sampler for five consecutive working days and two off-work periods. The amount of total Mn on each filter was determined by neutron activation analysis and then converted to atmospheric Mn concentrations. The values obtained varied from 0.004 microgram m-3 to 2.067 micrograms m-3. At work the garage mechanics were exposed to an average of 0.250 microgram m-3 and the taxi drivers to 0.024 microgram m-3. Off-work, the two groups were exposed respectively to an average of 0.007 microgram m-3 and 0.011 microgram m-3. In the garages there was twice as much Mn in the air on days when the doors were closed compared to days when they were left opened (0.314 micrograms m-3/0.152 microgram m-3). The levels found in this study remain well below the established limits for occupational and environmental airborne exposure. These results will lead to further studies to positively identify the source of Mn as MMT and to explore other pathways leading to the contamination of the general population.

The International Personality Disorder Examination. The World Health Organization/Alcohol, Drug Abuse, and Mental Health Administration International Pilot Study of Personality Disorders

One of the aims of the World Health Organization/Alcohol, Drug Abuse, and Mental Health Administration joint program on psychiatric diagnosis and classification is the development and standardization of diagnostic assessment instruments for use in clinical research worldwide. The International Personality Disorder Examination (IPDE) is a semistructured clinical interview compatible with the International Classification of Diseases, Tenth Revision, and the DMS-III-R classification systems. This is the first report of the results of a field trial to investigate the feasibility of using the IPDE to assess personality disorders worldwide.

A Fast Bioassay for Phytotoxicity Measurements Using Immobilized Photosynthetic Membranes

The potential of thylakoid membranes immobilized in an albumin-glutaraldehyde crosslinked matrix in a fast bioassay for phytotoxicity measurements in aqueous samples is studied. Free and immobilized preparations are compared for their electron transport activity measured as the initial rate of oxygen evolution with 2,5-cichlorobenzoquinone as the artificial electron acceptor. Immobilized thylakoids were much stable under storage conditions; in the dark, at 4 degrees C, they were fully stable in terms of photosynthetic activity for a period of 200 h. The immobilized membranes were as sensitive as the free thylakoids for the detection of most of the compounds tested (metal cations, sulfite, nitrite, and herbicides), all known as inhibitors of photosynthetic electron transport. In some instances, the immobilized preparations were even more sensitive than the free counterparts. The sensitivity could be further increased by lowering chlorophyll concentration in the assay. The short incubation period required ( approximately 10 to 15 min) and the small volume of the assay (3 mL) suggest that this type of material should be useful in the detection of locations or effluents with phytotoxic character. (c) 1994 John Wiley & Sons, Inc.

Environmental and Occupational Exposure to Manganese: a Multimedia Assessment

Methylcyclopentadienyl manganese tricarbonyl (MMT) is an organic additive used in Canada since 1976 as an anti-knock agent in unleaded gasoline. Its combustion leads to the emission of Mn oxides, especially Mn3O4. Since no study has assessed the potential risk of chronic exposure to low concentrations resulting from these emissions, the present investigation was undertaken to assess the level of environmental and occupational exposure of the human population. The multimedia exposure of two groups of workers (garage mechanics and blue-collar workers) potentially exposed to different levels of Mn from the combustion of MMT was assessed using personal air samplers, a dietary compilation, water samples at their places of residence, an epidemiological questionnaire and blood and hair samples. Results show that garage mechanics were exposed on average to higher atmospheric Mn at work (0.42 microgram/m3) than the blue-collar workers (0.04 microgram/m3). However, the contribution of atmospheric Mn to the total absorbed dose was less than 1%, and well below the standards established for occupational or environmental exposure; food contributes more than 95% of the multimedia dose. The average whole blood Mn concentrations were similar for the two groups (0.67-0.76 microgram/100 ml) and fall within the normal adult range. The average hair Mn concentrations were significantly higher for the garage mechanics (0.66 microgram/g) than for the blue-collar workers (0.39 microgram/g). The contribution of exogenous Mn versus endogenous Mn is questioned. As judged by the governmental standards or criteria for occupational and non-occupational environments, the current Mn levels in food, water and air may not cause any problems for the workers.

Occupational and Environmental Exposure of Automobile Mechanics and Nonautomotive Workers to Airborne Manganese Arising from the Combustion of Methylcyclopentadienyl Manganese Tricarbonyl (MMT)

Inhalation exposure to manganese (Mn) was measured for a group of garage mechanics and a control group of nonautomotive workers. The airborne Mn exposure of 35 garage mechanics suspected of being relatively highly exposed to Mn from MMT was measured at the workplace over one-week period. It also was measured for 30 nonautomotive workers at the University of Montreal. The environmental exposure also was measured for the two groups, as was the exposure to three other metals, aluminum (Al), iron (Fe), and zinc (Zn). At work the mechanics were exposed to Mn concentrations varying from 0.010 to 6.673 micrograms m-3 with a mean of 0.45 microgram m-5, while the control group was exposed to concentrations varying from 0.011 to 1.862 microgram m-3 with a mean of 0.04 microgram m-3. The mean environmental exposure for the two groups was similar to the Mn concentrations gathered in Montreal in 1992. Workplace concentrations of Al, Fe, and Zn also were higher for the garage mechanics. The results suggest that less than 10% of the Mn exposure of the garage mechanics was due to MMT. The levels of the metals measured were below the established limits for industrial and even environmental exposure.

Specialization Switch in Differentiating Embryonic Rat Liver Progenitor Cells in Response to Sodium Butyrate

Embryonic (E) 12 rat liver epithelial cells constitute a population of bipotential progenitor cells which can differentiate along the hepatocyte (Hep) or biliary epithelial cell (BEC) lineage in primary culture. In the present study, E12 cells were seeded on fibronectin-coated substratum and exposed to sodium butyrate (SB) for various exposure times, and the emergence of the Hep or BEC phenotype was monitored by following the variations in albumin production and assessing the appearance of the two surface-exposed markers HES6 and BDS7. Continuous exposure to SB resulted into a major reduction in albumin production and, at Day 9 postseeding, few cells coexpressed BDS7 and albumin. When cells were exposed to SB for 5 days and then cultured for an additional 5 days without SB, they massively express BDS7, but very little HES6. Moreover, the reverse sequence, i.e., 5 days without SB followed by 5 days with it, led to the appearance of many cells expressing both HES6 and BDS7. These results indicate that progenitors committed preferentially along the Hep lineage still have the option to switch to BECs, at a transitional stage that we refer to as a "differentiation window."

Bile Formation and Hepatic Plasma Membrane Composition in Guinea-pigs and Rats

We compared bile formation, and biliary and liver plasma membrane composition in guinea-pigs and rats in an attempt to explain the observation that the bile flow rate and the bile acid independent fraction of bile flow (BAIF) in guinea-pigs is about five to seven times higher than in rats. Analysis of electrolytes in bile showed that bicarbonate was significantly [acid] higher in guinea-pigs while Cl-, phosphate and Ca2+ were markedly lower than in rats. High bile independent secretion in guinea-pigs was associated with a significantly lower concentration of total bile acid, phospholipid and cholesterol than in rats. Bile acid distribution studies showed that glycine conjugated chenodeoxycholate and ketolithocholate were the main bile acids in guinea-pigs, while taurine conjugated cholate and muricholate were the predominant bile acids in rats. Total fatty acid analysis of bile indicated that in rats the major fatty acids were palmitic acid (C16:0) and linoleic acid (C18:2, n-6). In guinea-pigs, the contribution of these fatty acids was lower than in rats and compensated with a significantly higher percentage of oleic acid (C18:1, n-9). Concentrations of anionic polypeptide fraction (APF), an acidic calcium binding apoprotein closely associated with biliary phospholipid and cholesterol secretion was also significantly lower in guinea-pigs. Canalicular plasma membrane analysis showed that as compared with rats, specific activities of Na+,K+ ATPase, and cholesterol and phospholipid content were markedly lower in guinea-pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokeratin Expression, Fibrillar Organization, and Subtle Function in Liver Cells

Cytokeratins (CKs) constitute a diverse group of intermediate filament (IF) proteins, expressed as pairs in keratinized and nonkeratinizing epithelial cells. Much is known now about the expression, assembly, and function of CKs in keratinized epithelial cells, the main features being the tight coupling between CK pair switch and cell terminal differentiation (protection barrier) and the vital role of CK IFs in cell mechanical integrity. However, the picture about nonkeratinizing epithelia, like the hepatic tissue, remains quite unclear. The liver forms a multicellular system, where parenchymal cells (i.e., hepatocytes) exert diverse metabolic function(s) and nonparenchymal epithelial cells (e.g., biliary epithelial cells) usually serve structural (or accessory) purposes. In terms of differential CK gene expression, the data accumulated so far demonstrated that parenchymal cells can contain as few as one single CK pair, whereas nonparenchymal cells contain more than two CKs, one of them being a representative of those found in epidermis. Moreover, the distribution of the CK IF networks present in the different cell types varies a lot and can often be linked to the cell specialization. However, the function(s) played by these IF proteins in this multicellular tissue remains a major issue. The use of new experimental approaches, largely based on gene transfer technology, indicates that it is quite subtle.

Biliary Secretion and Actin-cytokeratin Filament Distribution in Rat Hepatocytes During Phalloidin-induced Cholestasis

The relationship between bile secretion (bile flow, bile acids, phospholipids, and cholesterol) and distribution of actin microfilaments (MFs) and cytokeratin (CK) intermediate filaments (IFs) was examined in hepatocytes of rats injected with a single low dose of phalloidin. This treatment induced a transient cholestasis characterized by a rapid development period (0-90 min postinjection) and a slow recovery period (24 h and 5 days postinjection). No significant changes were observed in bile acid secretion during the 5-day period. The phospholipid output dropped to less than 25% at 90 min and was back to the normal value at 24 h postinjection. In a parallel way, the cholesterol secretion dropped to 30% but came back to only 60% of the control level. Nile Red staining demonstrated a concomitant accumulation of lipids both in the cytoplasm and at the surface membrane. Immunostaining of the actin MFs and CK IFs showed that, in contrast with controls where both cytoskeletal networks were preferentially and uniformly localized at the surface membrane (i.e., sinusoidal, basolateral, and canalicular regions), the toxin treatment led to a major targeting of actin to the pericanalicular region at 24 h and a massive accumulation of well-preserved CK IFs in the cytoplasm at 5 days. Interestingly, this accumulation of CK IFs was not linked to any significant variations in CK isoforms. Together, these data indicate that a selective binding of the toxin to sinusoidal membrane actin at the time of injection triggers a sequence of events that culminate in delayed accumulation of actin MFs at the canalicular pole and of CK IFs in the cytoplasm. Moreover, the reversible perturbation of the bile secretory activity implies a functional adaptation of the hepatocytes that parallels the phalloidin-induced reorganization of both cytoskeleton networks.

Health Reform 2. Should Congress Look to the States?

Dependent Personality Disorder. Age, Sex, and Axis I Comorbidity

In the present report, we analyzed the age, sex, and axis I comorbidity of a large sample of patients with dependent personality disorder, and compared the findings with those obtained in patients with other personality disorders. The sample consisted of 3640 consecutive hospital admissions with a DSM-III axis II diagnosis. Of the 342 patients with a dependent diagnosis, 51.2% were over 40 years of age, compared with only 25.7% of the remaining patients with a personality disorder. The dependent sample was 69.6% female compared with 58.6% of those with other types of personality disorders. In general, dependent patients were more likely to have major depression and bipolar disorder than those with other personality disorders. Dysthymia and anxiety disorders occurred no more often than they did in the other personality disorders, as a whole. The rate of alcohol use disorders was lower than that observed in any other personality disorder except schizotypal. The rate of drug use disorders was intermediate between the lowest and highest rates in the other personality disorders.

Structural and Functional Alterations of Hepatocytes During Transient Phalloidin-induced Cholestasis in the Rat

To study the relationship between the dynamic actin web and bile secretion, we developed an acute model of cholestasis, using phalloidin, and examined sequential morphologic and biochemical events in rat liver. Biliary function (bite flow, bile, and canalicular membrane components) and cellular integrity (release of hepatic enzymes in serum and bile, canalicular structure, and microfilaments distribution) in rats given a single iv dose of phalloidin (0.8 mg/kg body weight) were assessed at 15, 45, and 90 min, 24 hr, and 5 days postinjection. Bile flow decreased significantly at 45 and 90 min, but cholestasis was transient since bile secretion returned to control levels at 24 hr. The biliary bile acid secretion rate was not modified during the same time period, indicating that cholestasis may have been due to impairment of the bile acid independent component of bile flow. Serum alanine aminotransferase and lactate dehydrogenase as well as biliary alkaline phosphatase and alkaline phosphodiesterase-1 activities were not altered by phalloidin treatment. These data, coupled with morphologic studies, provide no evidence of cell damage. Electron microscopy revealed that the pericanalicular actin web in both centrilobular and periportal hepatocytes was increased at 90 min and further enlarged at 24 hr and 5 days after phalloidin injection. At all time periods, the canalicular structure was well preserved. Na+K+ -ATPase and Mg2+ -ATPase activities in membrane fractions enriched in bile canalicular complexes decreased significantly at 15 min and remained low up to Day 5. Mg2+ -ATPase activity returned to control levels by Day 5. The lipid constituents of liver cell membranes enriched in canalicular complexes showed no significant variations 90 min after toxin treatment but, at 24 hr, phospholipid content rose and membrane fluidity increased. These results clearly indicate that the bile flow variation after a single low dose of phalloidin can be dissociated from specific pericanalicular microfilament distribution, lending further support to the view that normal biliary function is not strictly dependent on the integrity of the actin filament network.

Exposure of Taxi Drivers and Office Workers to Total and Respirable Manganese in an Urban Environment

This research measured the exposure of two groups of workers to respirable and total manganese (Mn) and characterized the Mn particles emitted from an automobile tailpipe. The exposure of 20 office workers and 9 taxi drivers in Toronto to total airborne Mn and respirable Mn was measured over a 7-day period, 24 hours per day. Subjects were asked to wear two pumps (one included a size-selective cyclone that collected the respirable particles), and two battery chargers were supplied to each person so that the pump batteries could be recharged overnight while sampling continued. All filters were analyzed by neutron activation. In addition, Mn particles emitted from a car were collected directly at the exhaust. Particles were observed using secondary electron images in a scanning electron microscope (SEM), and their elemental composition was determined by energy dispersive x-ray spectrometry. The Mn concentrations obtained for the group of office workers ranged from 0.001 to 0.034 microgram/m3 for respirable Mn and from 0.002 to 0.044 microgram/m3 for total Mn. For the taxi drivers the Mn concentrations ranged from 0.007 to 0.032 microgram/m3 for respirable Mn and from 0.008 to 0.073 microgram/m3 for total Mn. There was a significant difference (p < 0.05) between the two groups for both respirable and total Mn. SEM analysis showed that the particles were mostly heterogeneous agglomerates varying from 1 to 100 microns. Even if the specific exposure to Mn from automobiles has not been directly established, these results suggest that the related increase of exposure may be limited.

Manganese and Other Trace Elements in Urban Snow Near an Expressway

The Mn contamination arising from the combustion of MMT (methylcyclopentadienyl manganese tricarbonyl) in unleaded gasoline was assessed using snow collected at different distances 15, 25, 125 and 150 m from an expressway (Montreal, Canada) in February 1993. The snow samples were analyzed by atomic absorption and by neutron activation for total Mn, Mg, Cu, V, Al, Zn, Fe, Na, and Ca concentrations in the soluble (<0.4 microm) and particulate fractions. ANOVA with ranked values was performed to compare element concentrations and soluble/particulate ratios among receptor sites and depths. Principal component analysis was used to describe the spatiotemporal variations of the deposition rates and the influence of meteorological factors. The average concentration of all trace elements, except Mg, Cu, and V, decreased significantly (p<0.05) from receptor sites near the road (15-25 m) to those farther away (125-150 m). The deposition rates of all metals and ions, except Cu, were highly positively correlated (tau = 0.5-0.9) with each other and inversely correlated with snowfalls. Wind frequency showed no correlation with deposition rate. The spatial trend was similar for all these elements making it difficult to distinguish Mn arising from the combustion of MMT from that due to other sources, such as road dust. Only the soluble/particulate ratio calculated for Mn seemed higher than that for the other metals, which might be explained by the particle size of Mn from MMT (0.2-0.4 microm). The present study only indicates a direct contamination of the snow by road activities and substantial deposition of trace elements near the roadway; no clear link can be established between motor vehicle emissions and the concentration of Mn in snow.

Histones in Transit: Cytosolic Histone Complexes and Diacetylation of H4 During Nucleosome Assembly in Human Cells

The organization and acetylation of nascent histones prior to their stable incorporation into chromatin were examined. Through sedimentation and immunoprecipitation analyses of HeLa cytosolic extracts, two somatic non-nucleosomal histone complexes were detected: one containing nascent H3 and H4, and a second containing H2A (and probably H2B) in association with the nonhistone protein NAP-1. The H3/H4 complex has a sedimentation coefficient of 5-6S, consistent with the presence of one or more escort proteins. H4 in the cytosolic H3/H4 complex is diacetylated, fully in accord with the acetylation state of newly synthesized H4 in chromatin. The diacetylation of nascent human H4 is therefore completed prior to nucleosome assembly. As part of our studies of the nascent H3/H4 complex, the cytoplasmic histone acetyltransferase most likely responsible for acetylating newly synthesized H4 was also investigated. HeLa histone acetyltransferase B (HAT B) acetylates H4 but not H3 in vitro, and maximally diacetylates H4 even in the presence of sodium butyrate. Human HAT B acetylates H4 exclusively on the lysine residues at positions 5 and 12, in complete agreement with the highly conserved acetylation pattern of nascent nucleosomal H4 (Sobel et al., 1995), and has a native molecular weight of approximately 100 kDa. Based on our findings a model is presented for the involvement of histone acetylation and NAP-1 in H2A/H2B deposition and exchange, during nucleosome assembly and chromatin remodeling in vivo.

Detecting Personality Disorders in a Nonclinical Population. Application of a 2-stage Procedure for Case Identification

There is no epidemiology of personality disorders (PDs) comparable with that currently available for most other mental disorders. One reason for this is that an Axis II diagnosis usually requires considerable clinical sophistication and it is expensive to deploy clinicians rather than trained laypersons to examine large community samples. This study explores the feasibility of using a 2-stage method in which only subjects who were screened as positive for PD would be interviewed by clinicians.

Health Risk Assessment of an Industrial Site Contaminated with Polycyclic Aromatic Hydrocarbons Using CalTOX, an Environmental Fate/exposure Model

This paper presents the results of a risk assessment study made using CalTOX, a multimedia, multiple pathway risk assessment model. The case study is based on the Polycyclic Aromatic Hydrocarbon (PAH) soil contamination resulting from the activities of a natural gas power station over a period of 70 years. It describes model characteristics and input parameters such as physico-chemical properties, landscape description, and human exposure factors. Model simulations and risk estimations corresponding to different remedial scenarios in an industrial zone are also presented. These estimations were based on soil contamination by 16 PAHs in the root-zone and vadose-zone layer. Results show that adult exposure (workers) to contaminated soil will lead to a potential health risk of carcinogenic effects, and to no potential risk of non-carcinogenic effects. On the other hand, the addition of 10 cm of clean soil over the contaminated soil (mitigated scenario) decreases the lifetime cancer risk to an acceptable level. The sensitivity analysis showed that the half-life of benzo[a]pyrene in the root-zone soil is the most sensitive parameter and that it contributes significantly to the variability of the cancer risk estimation. In addition, the cancer risk level of the workers exposed to this substance, as estimated by CalTOX (point estimate) in the mitigated and unmitigated scenario, corresponds approximately to the 95th percentile value obtained by means of Monte-Carlo simulations. Finally, CalTOX has proven to be a valuable tool to predict and elaborate scenarios for the risk management of sites contaminated as a result of industrial activities.

Environmental Contamination and Human Exposure Assessment to Manganese in the St-Lawrence River Ecozone (Quebec, Canada) Using an Environmental Fate/exposure Model: GEOTOX

Methylcyclopentadienyl manganese tricarbonyl (MMT) is an organic derivative of manganese (Mn) used as an additive in unleaded gasoline. The combustion of MMT leads to the formation of oxides of manganese. The objective of the present study is to predict the environmental levels of Mn and the human exposure in the St-Lawrence ecozone (fluvial section, Quebec, Canada) using an environmental fate/exposure model: GEOTOX. The results of our MMT research program on abiotic and biotic components of the ecosystem and on the human exposure were used to validate the model estimations. Air and surface soil were selected as source terms with an annual Mn input rate in each compartment of 0.083-0.113 mol km-2 d-1 and 0.44-0.87 mol km-2 d-1 respectively (Mn3O4 equivalent). The predicted air, soil, plant, surface water and sediment concentrations were similar (+/- 50%) to values measured in the Montreal region. As expected, the ingestion pathway was the main absorption route for adults (> 99%), with vegetables and fruits contributing almost 80% of the dietary intake of Mn. The multimedia exposure doses for adult men predicted by the model ranged between 0.04 and 0.08 mg kg-1 d-1 compared to 0.004 and 0.201 mg kg-1 d-1 (average = 0.05) for workers from the MMT study. Considering the landscape configuration and the source vectors (air and soil) included in the model, GEOTOX estimations were in good agreement with measured values.

Environmental Contamination and Human Exposure to Airborne Total and Respirable Manganese in Montreal

Methylcyclopentadienyl manganese tricarbonyl (MMT) is an organometallic compound used as an octane improver in unleaded gasoline. The combustion of MMT leads to the formation of manganese (Mn) oxides, mainly Mn3O4. The objective of this study is to assess the variations over time and space of respirable (MnR) and total (MnT) Mn in the urban atmosphere and to evaluate human exposure by inhalation. Two sampling sites were selected on the island of Montreal based on their local traffic density (municipal botanical garden, C- = 10,000-15,000 vehicles d-1; Montreal Waterworks, C+ = 100,000-130,000 vehicles d-1). Air samplings were made during the day at stations located 10 m from the road using portable pumps, some of which were equipped with a cyclone. MnR and MnT and other metals were measured on Teflon filters by neutron activation. Mn exposure doses by inhalation were calculated using Monte-Carlo simulations. MnR and MnT average concentrations were significantly higher at site C+ (MnR = 0.024 microgram m-3; MnT = 0.050 microgram m-3) than at site C- (MnR = 0.015 microgram m-3; MnT = 0.027 microgram m-3). Temporal profiles at sites C+ and site C- were similar, with a coefficient of correlation of 0.24 for MnR and 0.26 for MnT. Trend analyses (ARIMA) also showed that the period of the week (work days vs. off days) was significantly related to MnR and MnT variations at both sites. The average exposure dose by inhalation to MnR and MnT ranged from 0.001 to 0.030 microgram kg-1 day-1 and 0.001 to 0.05 microgram kg-1 day-1. MnR and MnT concentrations reflected a positive relationship with traffic density. However, it remains difficult to attribute these results directly to the combustion of MMT in unleaded gasoline. On average, the MnR and MnT inhalation doses were 2 to 15 times lower than the reference dose (RfC) proposed by the U.S. Environmental Protection Agency (EPA) for the general population.

Simple Epithelium Keratins Are Required for Maintenance of Hepatocyte Integrity

Keratin 8 (K8)-deficient adult mice develop a severe disease of the gastrointestinal tract characterized mainly by colorectal hyperplasia and inflammation. Given that hepatocytes contain K8/K18 heteropolymers only, this animal model was used to assess the contribution of these simple epithelium keratins to hepatocyte structural and functional integrity. Homozygous mutant (HMZ), heterozygous, and wild-type (WT) mice were examined for hepatocyte structural and metabolic features and their survival to partial hepatectomy. Except for the presence of few necrotic foci, no other tissular or cellular alterations were observed in nonhepatectomized HMZ mouse livers; glycogen and lipid peroxidation levels were essentially normal, but a small reduction in bile flow was observed. In response to a single pentobarbital injection, HMZ mice had longer sleeping times than heterozygous and WT mice. After a two-thirds partial hepatectomy under pentobarbital anesthesia, all HMZ mice died within a few hours, whereas those anesthetized with ether survived for 1 to 2 days. One hour after hepatectomy after pentobarbital anesthesia, many hepatocytes contained erythrocytes and large vacuoles in the cytoplasm, which suggests damage at the plasma membrane level in response to a sudden increase in portal blood flow. In line with these findings, an uptake of trypan blue by HMZ but not WT mouse hepatocytes was observed during a 10 ml/minute perfusion via the portal vein with a dye-supplemented buffer. Subsequent cellular dispersion led to viable WT mouse hepatocytes but largely nonviable HMZ mouse hepatocytes. Better viability was obtained at lower perfusion rates. Partially hepatectomized heterozygous mice developed liver steatosis, a condition that was not associated with a change in K8 content but perhaps linked to the presence of the neo gene. Transgenic HMZ mouse rescue experiments with a full-length K8 gene confirmed that the phenotypic alterations observed in partially hepatectomized HMZ mice were caused by the disruption of the K8 gene. Taken together, these findings demonstrate that simple epithelium keratins are essential for the maintenance of hepatocyte structural and functional integrity.

Zonal Induction of Mixed Lineage Kinase ZPK/DLK/MUK Gene Expression in Regenerating Mouse Liver

ZPK/DLK/MUK is a serine/theronine kinase believed to be involved in the regulation of cell growth and differentiation. To further explore the suggested participation of ZPK/DLK/MUK in this process, we examined the expression and cellular localization of ZPK/DLK/MUK mRNA in regenerating mouse liver following partial hepatectomy by ribonuclease protection assay and in situ hybridization. The steady-state level of APK/DLKMUK mRNA was very low in normal and sham-operated mouse livers, whereas a marked and transient increase was observed in the regenerating liver. While ZPK/DLK/MUK mRNAs were rarely detected in hepatocytes from all zones of the normal liver, hepatocytes of regenerating liver exhibit a gradient of expression ranging from low in the periportal zone, to intermediate in the mid-zone, to high in the pericentral zone. These findings demonstrate a transient stimulation of ZPK/DLK/MUK gene expression that correlates with the growth response of hepatocyte subpopulations in regenerating liver.

Bioaccumulation of Manganese and Its Toxicity in Feral Pigeons (Columba Livia) Exposed to Manganese Oxide Dust (Mn3O4)

Manganese tetroxide (Mn3O4) is a product from the combustion of methylcyclopentadienyl manganese tricarbonyl. Exposure to high levels of manganese can lead to serious health effects especially to the central nervous and respiratory systems. Very few studies on the effects of long-term low level exposure to Mn3O4 have been reported. The present study was therefore conducted to examine the bioaccumulation and toxicity of manganese in various organs of feral pigeons (Columba livia) when exposed to low levels of Mn3O4 via inhalation and hence to find any possible relationship between these two parameters. A total of 22 pigeons was exposed to 239 micrograms/m3 of manganese for 7 h/day, 5 days/week for 5, 9, and 13 consecutive weeks. Manganese concentrations in various tissues, e.g., brain (mesencephalon), lung, liver, intestine, pancreas, kidney, muscle, bone, and whole blood, were measured by neutron activation analysis. Various biochemical parameters in blood, e.g., hematocrit, total proteins, glucose, uric acid, alanine aminotransferase, total iron, blood urea nitrogen and triglycerides, were also measured. Manganese concentrations in brain, lung, and bone were significantly higher in Mn3O4-exposed pigeons (0.59, 0.58, and 3.02 micrograms wet tissue, respectively) than in the control group (0.46, 0.19, 1.74 micrograms/g wet tissue, respectively). However, except for total proteins such exposure did not produce any changes in various biochemical parameters which were within the normal values. Thus these results have shown that, despite significant bioaccumulation of manganese in some tissues, no significant toxic effects could be seen.

RGS4 Binds to Membranes Through an Amphipathic Alpha -helix

RGS4, a mammalian GTPase-activating protein for G protein alpha subunits, requires its N-terminal 33 amino acids for plasma membrane localization and biological activity (Srinivasa, S. P., Bernstein, L. S., Blumer, K. J., and Linder, M. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5584-5589). In this study, we tested the hypothesis that the N-terminal domain mediates membrane binding by forming an amphipathic alpha-helix. RGS4 bound to liposomes containing anionic phospholipids in a manner dependent on the first 33 amino acids. Circular dichroism spectroscopy of a peptide corresponding to amino acids 1-31 of RGS4 revealed that the peptide adopted an alpha-helical conformation in the presence of anionic phospholipids. Point mutations that either neutralized positive charges on the hydrophilic face or substituted polar residues on the hydrophobic face of the model helix disrupted plasma membrane targeting and biological activity of RGS4 expressed in yeast. Recombinant mutant proteins were active as GTPase-activating proteins in solution but exhibited diminished binding to anionic liposomes. Peptides corresponding to mutants with the most pronounced phenotypes were also defective in forming an alpha-helix as measured by circular dichroism spectroscopy. These results support a model for direct interaction of RGS4 with membranes through hydrophobic and electrostatic interactions of an N-terminal alpha-helix.

Information-processing Speed and Assessment of Early Response Latency Among Stroke Patients

The development of assessment methods for estimating and predicting amount of functional impairment among stroke patients is important for planning rehabilitation. This study explored the contribution of speed of information processing and response latency in the assessment of 39 stroke patients. Functional impairment was assessed among these patients using the Functional Independence Measure, administered within 72 hours of admission to a rehabilitation center. The correlations between the scores on this measure and on a computerized measure of speed of information processing, Cognitive Performance Test, were examined. The Functional Independence Measure can be used with an acute stroke population. Scores are correlated with cognitive indicators of functional impairment, and scores discriminate between severity of functional impairment. These results are discussed with regard to their implication in monitoring stroke patients throughout rehabilitation.

[Original Humus Forms in a Semi-deciduous Tropical Forest in Guadeloupe]

Humus profiles underneath the canopy of dominant tree species in two secondary semi-evergreen forest sites in Grande-Terre (Guadeloupe) were analysed with a micromorphological method. In the vertisol of a tree plantation, the humus formed was rather similar under all tree species being an eumull and essentially due to the activity of the endoanecic earthworm Polypheretima elongata. In a natural secondary forest located on a steep slope and associated with a rendzina soil (without endoanecic earthworms), the humus forms were described at lower, mid- and upper slope. In this forest, two particular humus forms were observed. At the middle slope, underneath the canopy of Pisonia subcordata L. that produces nitrogen-rich litter, a calcareous amphimull, characterised by an OH horizon made of millipede faecal pellets, was formed. In the upper slope, underneath the canopy of Bursera simaruba (L.) Sarg. that produces a litter rich in resins and aromatic compounds that are poorly consumed by soil animals, a dysmull with a thick root mat (OFRh horizon) developed. Other humus forms were intermediate. The formation of these humus forms is discussed.

Simple Epithelium Keratins 8 and 18 Provide Resistance to Fas-mediated Apoptosis. The Protection Occurs Through a Receptor-targeting Modulation

Keratins 8 and 18 belong to the keratin family of intermediate filament (IF) proteins and constitute a hallmark for all simple epithelia, including the liver. Hepatocyte IFs are made solely of keratins 8 and 18 (K8/K18). In these cells, the loss of one partner via a targeted null mutation in the germline results in hepatocytes lacking K8/K18 IFs, thus providing a model of choice for examining the function(s) of simple epithelium keratins. Here, we report that K8-null mouse hepatocytes in primary culture and in vivo are three- to fourfold more sensitive than wild-type (WT) mouse hepatocytes to Fas-mediated apoptosis after stimulation with Jo2, an agonistic antibody of Fas ligand. This increased sensitivity is associated with a higher and more rapid caspase-3 activation and DNA fragmentation. In contrast, no difference in apoptosis is observed between cultured K8-null and WT hepatocytes after addition of the Fas-related death-factors tumor necrosis factor (TNF) alpha or TNF-related apoptosis-inducing ligand. Analyses of the Fas distribution in K8-null and WT hepatocytes in culture and in situ demonstrate a more prominent targeting of the receptor to the surface membrane of K8-null hepatocytes. Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8-null versus WT hepatocytes. Together, the results strongly suggest that simple epithelium K8/K18 provide resistance to Fas-mediated apoptosis and that this protection occurs through a modulation of Fas targeting to the cell surface.

Keratin-mediated Resistance to Stress and Apoptosis in Simple Epithelial Cells in Relation to Health and Disease

Epithelial cells such as hepatocytes exhibit highly polarized properties as a result of the asymmetric distribution of subsets of receptors at unique portions of the surface membrane. While the proper targeting of these surface receptors and maintenance of the resulting polarity depend on microtubules (MTs), the Golgi sorting compartment, and different actin-filament networks, the contribution of keratin intermediate filaments (IFs) has been unclear. Recent data show that the latter cytoskeletal network plays a predominant role in providing resistance to various forms of stress and to apoptosis targeted to the surface membrane. In this context, we first summarize our knowledge of the domain- or assembly-related features of IF proteins and the dynamic properties of IF networks that may explain how the same keratin pair K8/K18 can exert multiple resistance-related functions in simple epithelial cells. We then examine the contribution of linker protein(s) that integrate interactions of keratin IFs with MTs and the actin-cytoskeleton network, polarity-dependent surface receptors and cytoplasmic organelles. We next address likely molecular mechanisms by which K8/K18 can selectively provide resistance to a mechanical or toxic stress, or to Fas-mediated apoptosis. Finally, these issues on keratin structure-function are examined within a context of pathological anomalies emerging in tissue architecture as a result of natural or targeted mutations, or posttranslational modifications at specific amino acid residues. Clearly. the data accumulated in recent years provide new and significant insights on the role of K8/K18, particularly under conditions where polarized cells resist to stressful or apoptotic insults.

[Psychometric Properties of a Psychological Well-being Test for People with Physical Impairments]

Measuring the psychological well-being of people with physical impairments could provide relevant information to occupational therapists. The aim of this study was to verify psychometric properties of a psychological well-being test called Test de personnalité PER (PER). This test was administered to two samples of people with physical impairments. Two time measurements were collected within a group of 36 individuals and one single measure within another group of 79 individuals. Comparisons between time measurements, between groups and with the normative group of the PER were performed in an attempt to verify the sensitivity, the capacity to discriminate between known groups, and the construct validity of the PER. One section of the Sickness Impact Profile measuring psychological well-being was administered to the same groups to verify the concurrent criterion validity of the PER. The results indicate that the PER has sufficient psychometric qualities.

Voriconazole and Fluconazole Susceptibility of Candida Isolates

An adapted NCCLS M27-A method was used to evaluate the activity of voriconazole (VRC) and fluconazole (FLC) against 295 Candida isolates collected from 189 patients (including isolates from deep sites). Isolates included 186 C. albicans, 54 C. glabrata, 27 C. tropicalis, 14 C. parapsilosis, 6 C. krusei, 6 C. lusitaniae, 1 C. lypolytica and 1 C. sake. Forty-two isolates had reduced susceptibility to FLC (MIC >8 mg/L); 83.3% of these had VRC MICs < or =2 mg/L (9 of 11 C. albicans, 18 of 19 C glabrata, 6 of 6 C. krusei, 2 of 2 C. lusitaniae and 0 of 4 C. tropicalis), including 60% of isolates collected from deep-seated infections. These results suggested that in the era of azole resistance, VRC has a promising antifungal activity for serious infections with Candida spp., including most species with low susceptibility to FLC and uncommonly isolated species.

SNAP-25 Traffics to the Plasma Membrane by a Syntaxin-independent Mechanism

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are essential for vesicle docking and fusion. SNAP-25, syntaxin 1A, and synaptobrevin/vesicle-associated membrane protein (VAMP) are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. It has been proposed that interactions of SNAP-25 with syntaxin 1A are required for initial membrane attachment of SNAP-25 (Vogel, K., Cabaniols, J.-P., and Roche, P. (2000) J. Biol. Chem. 275, 2959-2965). However, we have shown previously that residues 85-120 of the SNAP-25 interhelical domain, which do not interact with syntaxin, are necessary and sufficient for palmitoylation and plasma membrane localization of a green fluorescent protein reporter molecule (Gonzalo, S., Greentree, W. K., and Linder, M. E. (1999) J. Biol. Chem. 274, 21313-21318). To clarify the role of syntaxin in membrane targeting of SNAP-25, we studied a SNAP-25 point mutant (G43D) that does not interact with syntaxin. SNAP-25 G43D/green fluorescent protein was palmitoylated and localized at the plasma membrane. Newly synthesized SNAP-25 G43D had the same kinetics of membrane association as the wild-type protein. Furthermore, expression of a cytosolic mutant syntaxin 1A did not interfere with SNAP-25 membrane interactions or palmitoylation in the neuronal cell line NG108-15. Exogenously expressed SNAP-25 targets efficiently to the plasma membrane in cells of neuronal origin but only partially in HeLa cells, a neurosecretion-incompetent line. This phenotype was not rescued when syntaxin 1A was co-expressed with SNAP-25. Our data support a syntaxin-independent mechanism of membrane targeting for SNAP-25.

Physiological and Environmental Aspects of Ascospore Discharge in Gibberella Zeae (anamorph Fusarium Graminearum)

We investigated ascospore discharge in the perithecial fungus, Gibberella zeae. In a wind tunnel study that simulated constant rain and varying day and night lengths, the rate of ascospore release was approximately 8-30% greater under light than in complete darkness. Under constant light, ascospore discharge occurred at maximal rates at relative humidity levels greater than 92%. When perithecia were placed under conditions of high external osmolarity, ascospore discharge was significantly reduced. Ascospores were discharged from asci along with droplets of fluid, the epiplasm, from within the ascus. Analysis of discharged epiplasmic fluid by GC-MASS Spectrometry revealed that mannitol was the major simple sugar component of the fluid. Activity of mannitol dehydrogenase, which catalyzes the conversion of fructose to mannitol, was higher in protein extracts from mature perithecia than in extracts from vegetative tissue. Several inhibitors of K(+) and Ca(++) ion channels inhibited ascospore discharge, which suggested that ascospore discharge resulted from the buildup of turgor pressure generated by ion fluxes and mannitol accumulation.

Distinct Chaperone Mechanisms Can Delay the Formation of Aggresomes by the Myopathy-causing R120G AlphaB-crystallin Mutant

A familial form of desmin-related myopathy (DRM) is associated with a missense mutation (R120G) in alphaB-crystallin (alphaB) and is characterized by intracellular desmin aggregation. Because alphaB is a molecular chaperone that participates in the assembly of desmin filaments, it has been suggested that the desmin aggregation might be due to the loss of alphaB function. We report here that alphaBR120G has indeed impaired in vivo function and structure as reflected by a highly reduced capacity to protect cells against heat shock and by an abnormal supramolecular organization even in cells not expressing desmin. In many cells, alphaBR120G accumulated in inclusion bodies that had characteristics of aggresomes concentrating around the centrosome following a microtubule-facilitated process. Three distinct chaperone mechanisms could reduce or even prevent the formation of the alphaBR120G aggresomes. Wild-type alphaB and Hsp27 prevented aggresome formation by co-oligomerizing with alphaBR120G. Hsp70 with its co-chaperone Hdj-1 or Chip-1 but not a mutant of Chip-1 lacking ubiquitin ligase activity, reduced the frequency of aggresome formation likely by targeting alphaBR120G for degradation. Finally, HspB8 interacted only transiently with alphaB but nonetheless rescued the alphaBR120G oligomeric organization, suggesting that it acted as a true chaperone assisting in the folding of the mutant protein. Hence, the formation of inclusion bodies in alphaBR120G-mediated DRM is probably due to the misfolding of alphaBR120G per se and can be delayed or prevented by expression of the wild type alphaB allele or other molecular chaperones, thereby explaining the adult onset of the disease.

The Digital Human: Towards a Unified Ontology

Keratins Modulate C-Flip/extracellular Signal-regulated Kinase 1 and 2 Antiapoptotic Signaling in Simple Epithelial Cells

Among the large family of intermediate filament proteins, the keratin 8 and 18 (K8/K18) pair constitutes a hallmark for all simple epithelial cells, such as hepatocytes and mammary cells. Functional studies with different cell models have suggested that K8/K18 are involved in simple epithelial cell resistance to several forms of stress that may lead to cell death. We have reported recently that K8/K18-deprived hepatocytes from K8-null mice are more sensitive to Fas-mediated apoptosis. Here we show that upon Fas, tumor necrosis factor alpha receptor, or tumor necrosis factor alpha-related apoptosis-inducing ligand receptor stimulation, an inhibition of extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation sensitizes wild-type but not K8-null mouse hepatocytes to apoptosis and that a much weaker ERK1/2 activation occurs in K8-null hepatocytes. In turn, this impaired ERK1/2 activation in K8-null hepatocytes is associated with a drastic reduction in c-Flip protein, an event that also holds in a K8-null mouse mammary cell line. c-Flip, along with Raf-1, is part of a K8/K18-immunoisolated complex from wild-type hepatocytes, and Fas stimulation leads to further c-Flip and Raf-1 recruitment in the complex. This points to a new regulatory role of simple epithelium keratins in the c-Flip/ERK1/2 antiapoptotic signaling pathway.

Uncovering the Roles of Intermediate Filaments in Apoptosis

Keratin 8 Modulation of Desmoplakin Deposition at Desmosomes in Hepatocytes

Keratins, the intermediate filament proteins of epithelial cells, connect to desmosomes, the cell-cell adhesion structures at the surface membrane. The building elements of desmosomes include desmoglein and desmocollin, which provide the actual cell adhesive properties, and desmoplakins, which anchor the keratin intermediate filaments to desmosomes. In the work reported here, we address the role of keratin 8 in modulating desmoplakin deposition at surface membrane in mouse hepatocytes. The experimental approach is based on the use of keratin 8- and keratin 18-null mouse hepatocytes as cell models. In wild-type mouse hepatocytes, desmoplakin is aligned with desmoglein and keratin 8 at the surface membrane. In keratin 8-null hepatocytes, the intermediate filament loss leads to alterations in desmoplakin distribution at the surface membrane, but not of desmoglein. Intriguingly, a significant proportion of keratin 18-null hepatocytes express keratin 8 at the surface membrane, associated with a proper desmoplakin alignment with desmoglein at desmosomes. A Triton treatment of the monolayer reveals that most of the desmoplakin present in either wild-type, keratin 8- or keratin 18-null hepatocytes is insoluble. Deletion analysis of keratin 8 further suggests that the recovery of desmoplakin alignment requires the keratin 8 rod domain. In addition, similarly to other works revealing a key role of desmoplakin phosphorylation on its interaction with intermediate filaments, we find that the phosphorylation status of the keratin 8 head domain affects desmoplakin distribution at desmosomes. Together, the data indicate that a proper alignment/deposition of desmoplakin with keratins and desmoglein in hepatocytes requires keratin 8, through a reciprocal phosphoserine-dependent process.

Keratins Modulate Hepatic Cell Adhesion, Size and G1/S Transition

Keratins (Ks) are the intermediate filament (IF) proteins of epithelial cells. Hepatocyte IFs are made solely of keratins 8 and 18 (K8/K18), the hallmark of all simple epithelia. While K8/K18 are essential for maintaining structural integrity, there is accumulating evidence indicating that they also exert non-mechanical functions. We have reported recently that K8/K18-free hepatocytes from K8-null mice are more sensitive to Fas-mediated apoptosis, in line with an increased Fas density at the cell surface and an altered c-Flip regulation of the anti-apoptotic ERK1/2 signaling pathway. In the present study, we show that K8-null hepatocytes attach more rapidly but spread more slowly on a fibronectin substratum and undergo a more efficient G1/S transition than wild-type hepatocytes. Moreover, plectin, an IF associated protein, receptor for activated C kinase 1 (RACK1), a plectin partner, and vinculin, a key component of focal adhesions, distribute differently in spreading K8-null hepatocytes. Cell seeding leads to no differential activation of ERK1/2 in WT versus K8-null hepatocytes, whereas a stronger Akt activation is detected in K8-null hepatocytes. Insulin stimulation also leads to a differential Akt activation, implying altered Akt signaling capacity as a result of the K8/K18 loss. In addition, a delayed autophosphorylation of FAK, a target for integrin beta1 signaling, was obtained in seeding K8-null hepatocytes. These alterations in cell cycle-related events in hepatocytes in primary culture are also found in a K8-knockdown H4-II-E-C3 rat hepatoma cell line. Besides, K8/K18-free cells are smaller and exhibit a reduced rate of protein synthesis. In addition, a distinctive cyclin interplay is observed in these K8/K18-free hepatic cells, namely a more efficient cyclin A-dependent G1/S phase transition. Furthermore, K8 re-expression in these cells, following transfer of a human K8 cDNA, restores proper cell size, spreading and growth. Together, these results suggest new interrelated signaling roles of K8/18 with plectin/RACK1 in the modulation of cell attachment/spreading, size/protein synthesis and G1/S transition.

DSM-IV Personality Disorders in the National Comorbidity Survey Replication

The population prevalence of DSM-IV personality disorders (PDs) remains largely unknown. Data are reported here on the prevalence and correlates of clinician-diagnosed Clusters A, B, and C DSM-IV PDs in the general population of the United States.

Dual Roles of Intermediate Filaments in Apoptosis

New roles have emerged recently for intermediate filaments (IFs), namely in modulating cell adhesion and growth, and providing resistance to various forms of stress and to apoptosis. In this context, we first summarize findings on the IF association with the cell response to mechanical stress and growth stimulation, in light of growth-related signaling events that are relevant to death-receptor engagement. We then address the molecular mechanisms by which IFs can provide cell resistance to apoptosis initiated by death-receptor stimulation and to necrosis triggered by excessive oxidative stress. In the same way, we examine IF involvement, along with cytolinker participation, in sequential caspase-mediated protein cleavages that are part of the overall cell death execution, particularly those that generate new functional IF protein fragments and uncover neoantigen markers. Finally, we report on the usefulness of these markers as diagnostic tools for disease-related aspects of apoptosis in humans. Clearly, the data accumulated in recent years provide new and significant insights into the multiple functions of IFs, particularly their dual roles in cell response to apoptotic insults.

An Evaluation of a Numbered Surgical Sponge Product

Surgical sponge counting is an essential patient safety measure in the OR in which all members of the surgical team must participate. The RN acting as circulator is responsible for accurately documenting sponge counts during the surgical procedure. A sequentially numbered sponge product was evaluated in a survey of OR personnel to determine ease of use and whether the product affected the flow of the surgical procedure. Survey respondents reported that the numbered sponge product was easy to use and did not lengthen or affect the flow of the surgical procedure. Respondents also indicated that the product may contribute to patient safety.

Open Tools for Storage and Management of Quantitative Image Data

The explosion in quantitative imaging has driven the need to develop tools for storing, managing, analyzing, and viewing large sets of data. In this chapter, we discuss tools we have built for storing large data sets for the lifetime of a typical research project. As part of the Open Microscopy Environment (OME) Consortium, we have built a series of open-source tools that support the manipulation and visualization of large sets of complex image data. Images from a number of proprietary file formats can be imported and then accessed from a single server running in a laboratory or imaging facility. We discuss the capabilities of the OME Server, a Perl-based data management system that is designed for large-scale analysis of image data using a web browser-based user interface. In addition, we have recently released a lighter weight Java-based OME Remote Objects Server that supports remote applications for managing and viewing image data. Together these systems provide a suite of tools for large-scale quantitative imaging that is now commonly used throughout cell and developmental biology.

Keratin-protein Kinase C Interaction in Reactive Oxygen Species-induced Hepatic Cell Death Through Mitochondrial Signaling

Keratins (Ks), the intermediate filament (IF) proteins of epithelia, constitute at least 20 cytoskeletal proteins subdivided into type I (K9-20) and type II (K1-K8) and expressed as type I/type II pairs in a cell differentiation manner. Hepatocyte IFs are made only of K8/K18, the hallmark of simple epithelial cells. We have shown previously that a K8/K18 loss leads to a modulation of apoptosis in Fas-stimulated mouse hepatocytes. Here we report that K8-knockout mouse hepatocytes and K8-knockdown H4-II-E-C3 (shK8b1) rat hepatoma cells were much more resistant than their K8/K18-containing counterparts, wild-type hepatocytes, and H4ev hepatoma cells, in response to excess H2O2 or tert-butyl hydroperoxide, a ROS generator. While excess H2O2 altered glutathione (GSH) and ROS levels in H4ev versus shK8b1 cells, the differential death response was largely GSH level independent. Assessment of key cell death features revealed that hepatic cells exposed to H2O2 die through a mitochondrial involvement. Similarly, administration of the GSH depletor L-buthionine-sulfoximine to generate mitochondrial ROS-sensitized H4-II-E-C3 cells but not shK8b1 cells to death. Treatment with protein kinase C (PKC) inhibitors yielded a resistance of H2O2-treated H4-II-E-C3 cells comparable to that of nontreated shK8b1 cells, which in turn were not affected by the treatment. In addition, this differential death response was associated with altered PKCdelta activation and surface-membrane/mitochondria distribution in H2O2-treated shK8b1 cells. Together, these results point to a key regulatory function for K8/K18 in ROS-induced mitochondria-mediated death through PKCdelta involvement in hepatic cells.

Switch in Fas-activated Death Signaling Pathway As Result of Keratin 8/18-intermediate Filament Loss

Fas-induced apoptosis is initiated through the recruitment of FADD and procaspase 8 to form the death-inducing signaling complex (DISC). In some cells (type I cells) the initiator caspase 8 directly activates effector caspases such as procaspase 3, whereas in others (type II cells) the death signal is amplified through mitochondria. In epithelial cells, Fas-induced hierarchic caspase activation is also linked with DEDD, a member of the DED family that binds to keratin (K) intermediate filaments (IFs). Hepatocytes are type II cells and their IFs are made exclusively of K8/K18. We have shown previously that K8-null mouse hepatocytes, lacking K8/K18 IFs, are more sensitive than their wild-type counterparts to Fas-induced apoptosis. Here, by examining the cell-death kinetics and death-signaling ordering, we found that K8-null hepatocytes exhibited prominent DISC formation, higher procaspase 8 activation and direct procaspase 3 activation as reported for type I cells; however they experienced a reduced Bid cleavage and a stronger procaspase 9 activation. In addition, the K8/K18 loss altered the DEDD ubiquitination status and nuclear/cytoplasmic distribution. Together, the results suggest that the K8/K18 loss induces a switch in Fas-induced death signaling, likely through a DEDD involvement.

Feeding Jejunostomy for the Treatment of Severe Hyperemesis Gravidarum: a Case Series

Hyperemesis gravidarum is severe nausea and vomiting during pregnancy leading to dehydration, nutrition deficiency, and fetal morbidity and mortality. Treatment must maintain fluid and electrolyte balance and caloric intake. Parenteral nutrition is often attempted; however, complication rates are high. Nutrition via nasoenteric and percutaneous endoscopic gastrostomy tubes is limited by poor patient tolerance, tube dislodgement, and altered anatomy in pregnancy.

The Mechanism Whereby Heat Shock Induces Apoptosis Depends on the Innate Sensitivity of Cells to Stress

The cellular response to heat shock (HS) is a paradigm for many human diseases collectively known as "protein conformation diseases" in which the accumulation of misfolded proteins induces cell death. Here, we analyzed how cells having a different apoptotic threshold die subsequent to a treatment with HS. Cells with a low apoptotic threshold mainly induced apoptosis through activation of conventional stress kinase signaling pathways. By contrast, cells with a high apoptotic threshold also died by apoptosis but likely after the accumulation of heat-aggregated proteins as revealed by the formation of aggresomes in these cells, which were associated with the generation of atypical nuclear deformations. Inhibition of the proteasome or expression of an aggregation prone protein produced similar nuclear alterations. Furthermore, elevated levels of chaperones markedly suppressed both HS-induced nuclear deformations and apoptosis induced upon protein aggregation whereas they had little effect on stress kinase-mediated apoptosis. We conclude that the relative contribution of stress signaling pathways and the accumulation of protein aggregates to cell death by apoptosis is related to the innate sensitivity of cells to deadly insults.

Keratin 8/18 Modulation of Protein Kinase C-mediated Integrin-dependent Adhesion and Migration of Liver Epithelial Cells

Keratins are intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatocyte and hepatoma cell IFs are made solely of keratins 8/18 (K8/K18), the hallmark of all simple epithelia. Cell attachment/spreading (adhesion) and migration involve the formation of focal adhesions at sites of integrin interactions with extracellular matrix, actin adaptors such as talin and vinculin, and signaling molecules such as focal adhesion kinase (FAK) and member(s) of the protein kinase C (PKC) family. Here, we identify the novel PKCdelta as mediator of the K8/K18 modulation of hepatoma cell adhesion and migration. We also demonstrate a K8/K18-dependent relationship between PKCdelta and FAK activation through an integrin/FAK-positive feedback loop, in correlation with a reduced FAK time residency at focal adhesions. Notably, a K8/K18 loss results to a time course modulation of the receptor of activated C-kinase-1, beta1-integrin, plectin, PKC, and c-Src complex formation. Although the K8/K18 modulation of hepatocyte adhesion also occurs through a PKC mediation, these differentiated epithelial cells exhibit minimal migrating ability, in link with marked differences in protein partner content and distribution. Together, these results uncover a key regulatory function for K8/K18 IFs in the PKC-mediated integrin/FAK-dependent adhesion and migration of simple epithelial cells.

Metadata Matters: Access to Image Data in the Real World

Data sharing is important in the biological sciences to prevent duplication of effort, to promote scientific integrity, and to facilitate and disseminate scientific discovery. Sharing requires centralized repositories, and submission to and utility of these resources require common data formats. This is particularly challenging for multidimensional microscopy image data, which are acquired from a variety of platforms with a myriad of proprietary file formats (PFFs). In this paper, we describe an open standard format that we have developed for microscopy image data. We call on the community to use open image data standards and to insist that all imaging platforms support these file formats. This will build the foundation for an open image data repository.

Outpatient Psychotherapy Practice with Adolescents Following Psychiatric Hospitalization for Suicide Ideation or a Suicide Attempt

Outpatient treatment is standard care for adolescents discharged following a psychiatric hospitalization. There is little research, however, on the amount and types of psychotherapy these clients receive in the community. We examined therapy attendance and therapist report of outpatient therapy practice with adolescents discharged from psychiatric hospitalization following either a suicide attempt or severe suicidal ideation in the Northeastern USA. Therapists (n = 84) completed a packet of self-report questionnaires regarding treatment of these adolescents in the first six months after discharge from the hospital. Information on number of sessions attended, primary presenting problem, therapist orientation, therapy techniques, and therapeutic relationship was collected. The findings indicated that therapists met their clients in both private and community outpatient settings. The most common modality of treatment was individual therapy, but almost all types of therapeutic techniques were endorsed. Adolescents attended an average of 8.1 therapy sessions (SD = 4.7), with 18% terminating treatment against therapist advice within the first three months. Psychologists, psychiatrists, and social workers used cognitive-behavioral, psychodynamic, and family system techniques about equally. Social workers used humanistic techniques more than their counterparts. The variability in number of therapy sessions attended suggests that many adolescents discharged after a psychiatric hospitalization will not receive adequate care. Short-term therapy protocols designed for community practice emphasizing cognitive techniques may be useful to test in future community-based research trials based on the high percentage of adolescents attending relatively few sessions.

Characterizing the Structure of Pharmaceutical Granules Obtained by Wet Granulation with Varying Amounts of Water Via Raman Chemical Imaging

A pharmaceutical formulation containing metformin hydrochloride (MET), hydroxypropyl cellulose (HPC), and microcrystalline cellulose (MCC) was wet granulated with varying amounts of water and the structure of the obtained granules was characterized by Raman chemical mapping. Univariate Raman mapping was found to be satisfactory for producing the images of the two components of interest (HPC and MCC). In addition to the images, the average Raman spectra from the maps as well as the micro-Raman spectra from the hot pixels were analyzed. HPC is found to strongly respond to the addition of water, with its domain dissipating and Raman bands becoming weaker as the water addition increases. MCC is also responsive to water, reacting similarly to HPC but to a much smaller extent and only for the largest amounts of water. Granules made with increasing water content also have improved tabletting properties and flow.

OMERO: Flexible, Model-driven Data Management for Experimental Biology

Data-intensive research depends on tools that manage multidimensional, heterogeneous datasets. We built OME Remote Objects (OMERO), a software platform that enables access to and use of a wide range of biological data. OMERO uses a server-based middleware application to provide a unified interface for images, matrices and tables. OMERO's design and flexibility have enabled its use for light-microscopy, high-content-screening, electron-microscopy and even non-image-genotype data. OMERO is open-source software, available at http://openmicroscopy.org/.

Sequencing Chromosomal Abnormalities Reveals Neurodevelopmental Loci That Confer Risk Across Diagnostic Boundaries

Balanced chromosomal abnormalities (BCAs) represent a relatively untapped reservoir of single-gene disruptions in neurodevelopmental disorders (NDDs). We sequenced BCAs in patients with autism or related NDDs, revealing disruption of 33 loci in four general categories: (1) genes previously associated with abnormal neurodevelopment (e.g., AUTS2, FOXP1, and CDKL5), (2) single-gene contributors to microdeletion syndromes (MBD5, SATB2, EHMT1, and SNURF-SNRPN), (3) novel risk loci (e.g., CHD8, KIRREL3, and ZNF507), and (4) genes associated with later-onset psychiatric disorders (e.g., TCF4, ZNF804A, PDE10A, GRIN2B, and ANK3). We also discovered among neurodevelopmental cases a profoundly increased burden of copy-number variants from these 33 loci and a significant enrichment of polygenic risk alleles from genome-wide association studies of autism and schizophrenia. Our findings suggest a polygenic risk model of autism and reveal that some neurodevelopmental genes are sensitive to perturbation by multiple mutational mechanisms, leading to variable phenotypic outcomes that manifest at different life stages.

Cytoskeleton Keratin Regulation of FasR Signaling Through Modulation of Actin/ezrin Interplay at Lipid Rafts in Hepatocytes

FasR stimulation by Fas ligand leads to rapid formation of FasR microaggregates, which become signaling protein oligomerization transduction structures (SPOTS), through interactions with actin and ezrin, a structural step that triggers death-inducing signaling complex formation, in association with procaspase-8 activation. In some cells, designated as type I, caspase 8 directly activates effector caspases, whereas in others, known as type II, the caspase-mediated death signaling is amplified through mitochondria. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatocyte IFs are made solely of keratins 8/18 (K8/K18), the hallmark of all simple epithelia. We have shown recently that in comparison to type II wild-type (WT) mouse hepatocytes, the absence of K8/K18 IFs in K8-null hepatocytes leads to more efficient FasR-mediated apoptosis, in link with a type II/type I-like switch in FasR-death signaling. Here, we demonstrate that the apoptotic process occurring in type I-like K8-null hepatocytes is associated with accelerated SPOTS elaboration at surface membrane, along with manifestation of FasR cap formation and internalization. In addition, the lipid raft organization is altered in K8-null hepatocytes. While lipid raft inhibition impairs SPOTS formation in both WT and K8-null hepatocytes, the absence of K8/K18 IFs in the latter sensitizes SPOTS to actin de-polymerization, and perturbs ezrin compartmentalization. Overall, the results indicate that the K8/K18 IF loss in hepatocytes alters the initial FasR activation steps through perturbation of ezrin/actin interplay and lipid raft organization, which leads to a type II/type I switch in FasR-death signaling.

Demonstration of an Ultra-high Frequency Picosecond Pulse Generator Using an SBS Frequency Comb and Self Phase-locking

We propose a method to generate phase-locked pulses in the picosecond regime by using Stimulated Brillouin Scattering (SBS). The phase-locked comb is generated using only long length of fiber and a single frequency CW pump laser. We show that there is a phase relationship between multiple Stokes peaks in a cavity, which directly leads to pulsing without the need to add a mode-locking component. This generates highly coherent pulses in the order of ~10 ps. The repetition frequency, which is very stable is in the order of tens of GHz, is based on the SBS frequency shift and has a linear dependence with temperature (1 MHz/°C). Such a laser could therefore be used in high-speed optical clocks and optical communication system. This system allows the pulses to be generated at any wavelength by simply changing the pump wavelength.

Keratin 8/18 Regulation of Glucose Metabolism in Normal Versus Cancerous Hepatic Cells Through Differential Modulation of Hexokinase Status and Insulin Signaling

As differentiated cells, hepatocytes primarily metabolize glucose for ATP production through oxidative phosphorylation of glycolytic pyruvate, whereas proliferative hepatocellular carcinoma (HCC) cells undergo a metabolic shift to aerobic glycolysis despite oxygen availability. Keratins, the intermediate filament (IF) proteins of epithelial cells, are expressed as pairs in a lineage/differentiation manner. Hepatocyte and HCC (hepatoma) cell IFs are made solely of keratins 8/18 (K8/K18), thus providing models of choice to address K8/K18 IF functions in normal and cancerous epithelial cells. Here, we demonstrate distinctive increases in glucose uptake, glucose-6-phosphate formation, lactate release, and glycogen formation in K8/K18 IF-lacking hepatocytes and/or hepatoma cells versus their respective IF-containing counterparts. We also show that the K8/K18-dependent glucose uptake/G6P formation is linked to alterations in hexokinase I/II/IV content and localization at mitochondria, with little effect on GLUT1 status. In addition, we find that the insulin-stimulated glycogen formation in normal hepatocytes involves the main PI-3 kinase-dependent signaling pathway and that the K8/K18 IF loss makes them more efficient glycogen producers. In comparison, the higher insulin-dependent glycogen formation in K8/K18 IF-lacking hepatoma cells is associated with a signaling occurring through a mTOR-dependent pathway, along with an augmentation in cell proliferative activity. Together, the results uncover a key K8/K18 regulation of glucose metabolism in normal and cancerous hepatic cells through differential modulations of mitochondrial HK status and insulin-mediated signaling.

The Use of Weissler Method for Scale-up a Kraft Pulp Oxidation by TEMPO-mediated System from a Batch Mode to a Continuous Flow-through Sonoreactor

The efficiency of cellulose oxidation mediated by the 4-acetamido-TEMPO radical under ultrasonic cavitation was investigated using two ultrasonic systems: a batch lab scale ultrasonic bath with a glass reactor and a semi-continuous flow-through sonoreactor. The main objective was to explore the possibility of scaling up the production of oxidized cellulose under ultrasound, from a lab scale process to a pilot plant process, which served as a precursor for producing nanofibrils cellulose. It was found that under acoustic cavitation, the efficiency of TEMPO-mediation oxidation of native cellulose was significantly improved, particularly in the flow-through sonoreactor. In comparison with the glass reactor, the flow-through sonoreactor reduce the applied energy by 88% while increasing 7.8 times the production rate of radicals. These results enable a possibility of producing oxidized fibers for industrial applications.

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