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Articles by Maria Kolmakova in JoVE

 JoVE Clinical and Translational Medicine

Quantitative Analysis of Chromatin Proteomes in Disease

1Department of Anesthesiology, David Geffen School of Medicine at UCLA, 2Department of Medicine, David Geffen School of Medicine at UCLA, 3Department of Physiology, David Geffen School of Medicine at UCLA, 4Department of Internal Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah


JoVE 4294

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

Other articles by Maria Kolmakova on PubMed

[Evaluation of Glaucoma According to Dynamic Changes of the Blind Spot]

[Functional State of the Liver in Opisthorchosis]

[Results of Tick Control in a Focus of Tick-borne Encephalitis]

[Results of Hexachloroethane Therapy of Opisthorchiasis]

[Cutaneous Reaction to Histamine and Morphine in Patients with Rheumatism]

[Examination of the Blood Pressure in the Central Retinal Artery in Patients with Rheumatism]

[On the Use of Zinc-gelatin Caps and Keratolytic Lac After Roentgen Exposure of Trichomycosis Patients]

[THE PROBLEM OF SANITARY TREATMENT OF THE NATURAL FOCUS OF TICK-BORNE ENCEPHALITIS IN INHABITED AREAS]

[CHANGES IN THE INTERSTITIAL SUBSTANCE OF THE EYE IN RHEUMATISM]

[Data on the Treatment of Trichomycosis Patients with Griseofulvin]

[Compression Osteosynthesis in Ununited Fractures of the Mandible]

[Prevention of Ascariasis in the Svedlovsk Region]

[Interrelation of Systolic Phases of the Left Ventricle and Frequency of Cardiac Rhythm in the Transition from Auricular Fibrillation to Sinus Rhythm]

[Results of Many Years of Observations on the Effectiveness of the Emergency Seroprophylaxis of Tick-borne Encephalitis Using Homologous Gamma-globulin]

[Lupus Erythematosus and Porphyria in Patients with Vitiligo]

[Changes in Catecholamine Metabolism in the Brains of Rabbits After Treatment with Anti-brain Antibodies and Lithium Ions]

The authors studied the changes in the activity of tyrosine hydroxylase and dopamine-beta-hydroxylase (the principal enzymes controlling the synthesis of catecholamines) in the whole brain of rabbits on intracisternal injection of immunoglobulin G isolated from the blood of brain-sensitized dogs or from the serum of schizophrenic humans. Also the changes of the content of principal catecholamines (DOPA, noradrenaline, adrenaline) in the animals brain on intravenous injection of lithium chloride after preliminary treatment of the animals with anticerebral antibodies were studied. The changes of the activity of both enzymes in both series of the experiments appeared to be uniformly directed, the fact, that gives one grounds to regard the mechanisms underlying these disturbances as similar. A positive effect of lithium on the catecholamine metabolism in the rabbit CNS was observed. This makes is possible to recommend the model used in this study for examining the efficacy and mode of action of a number of psychotropic drugs.

[Gottron's Reticulosarcomatosis]

[State of the Erythrocyte Membranes in Patients with Chronic Diseases of the Kidneys]

[Determination of Glyoxalase 1 Groups in Liquid and Dried Blood by Polyacrylamide Gel Electrophoresis]

Vasoactive Peptides from the Vespa Orientalis Hornet Venom

[Morphofunctional Comparisons in Chronic Glomerulonephritis]

[The Possibility of Using Equine Serum Albumin in Place of Bovine Serum Albumin and Ovalbumin in Radioimmunological and Immunoenzyme Analyses and in Virological Practice]

Horse serum albumin has been shown to meet the requirements to protein preparations for microanalysis and thus to be suitable for use in kits of reagents for the radioimmunological determination of insulin and myoglobin, for the determination of tick-borne encephalitis virus antigen by the method of the enzyme immunoassay and for the stabilization of proteins in the hemagglutination test and the hemagglutination inhibition test.

[A Case of Diabetic Glomerulosclerosis Associated with Chronic Glomerulonephritis]

[The Determination of the Phenotypes of 3 Enzyme Systems by the Polyacrylamide Gel Electrophoresis Method]

Possibility of combined determination of phenotypes of three enzyme system (glyoxylase-1, phosphoglucomutase and 6-phosphogluconate dehydrogenase) in the apparatus with two vertical blocks of polyacrilamide gel (PAAG) was shown. The method is based on the principle of combination of differently composed blocks of PAAG and single universal electrode buffer. High productivity and economy of the method equally with good quality of resulting phoregrams make this method useful for medicolegal practical work.

[Clinical, Immunological and Morphological Evaluation of Nephrotic Syndrome]

The results are available of combined examination of the kidneys in 84 patients with manifest nephrotic syndrome varying in etiology: 57 had glomerulonephritis, 18 amyloidosis, 9 diabetic nephropathy. The study covered lipid metabolism, immunological status, lifetime morphological investigation of the kidneys. The latter procedure proved advantageous over biochemical and immunological studies in deciding upon nosological diagnosis of nephrotic syndrome and its prognosis. Destruction of podocytic miner processes revealed in all the syndrome cases can serve a uniform morphological substrate underlying massive proteinuria.

[Plasma Lipids in Patients with Chronic Glomerulonephritis]

The author examined 78 patients with chronic glomerulonephritis and verified their diagnosis on the basis of the kidney biopsy data. With regard to the severity of the tubular interstitial component the patients were enrolled in three groups. It was stated that with the increasing changes developed in the tubular interstitial component, the levels of total cholesterol, cholesterol of high density lipoproteins, low density lipoproteins and triglyceride elevated. In the group of the patients with minimal changes in the tubular interstitial component, the number of those in whom plasma lipid levels were below the upper normal limits was higher than in two other groups. A direct proportional relationship between the levels of plasma lipids and severity of the tubular interstitial component indicated the immediate role of the renal tissue in the pathogenesis of lipid turnover disorders in patients with chronic glomerulonephritis.

[Structural-functional Characteristics of Vasoactive Peptides from the Vespa Orientalis Venom]

Two vasoactive peptides are isolated from the Vespa orientalis venom by gel filtration and ion-exchange chromatography. The physicochemical and functional properties of peptides are studied. Vasoactive peptides show the myotropic activity and hypotensive action which is of prolonged character as compared with bradykinin. Their complete amino acidic sequences are determined. One of the peptides is a similar structural analogue of bradykinin.

Magnetic Linear Birefringence in Rare-earth Garnets: Crystal-field Effects and the Judd-Ofelt Approximation

[Characteristics of Prolactin Secretion in Response to Different Degrees of Lesions of the Vestibular Analyzer]

The prolactin concentration in blood of 58 patients was measured during vestibular crisis and compensation of vestibular disorders. The results were compared with the clinical development of the disease and localization of vestibular problems. Patients with acute vestibular lesions at the central level of vestibular disorders showed a significant increase of prolactin concentrations that was higher than the concentrations in patients with the peripheral vestibular syndrome at the decompensation stage. Hyperprolactinemia during central vestibular dysfunction is viewed as a manifestation of dopamine deficiency of the tuberoinfundibular area. It is suggested that inadequacy of dopaminergic structures is one of the factors reducing adaptive and compensatory capabilities of the vestibular apparatus when its central compartments are injured.

[Certain Parameters of Beta-carotene Metabolism in the Rat]

Concentration of retinoids was decreased from 24.5 micrograms/100 ml to 13.5 micrograms/100 ml of blood serum in rats with ageing, during 2-11 months; these rats were maintained on a standard ration. Enrichment of rat ration with beta-carotene caused a statistically significant increase in content of retinoids in blood serum from 24.5 micrograms/100 ml to 48.6 micrograms/100 ml within early periods (1-72 hrs), while within later periods (0.25-2 months) the retinoid content was similar to that of control intact animals. After single and, especially, in long-term treatments with beta-carotene dose-dependent deposition of the carotenoid and increase in content of retinoids were detected in liver tissue. Use of white rats is of importance in principle for evaluation of beta-carotene caused modifications of pathological states developed under influence of unsuitable environmental factors.

[Lipidemia in the Nephrotic Syndrome and the Atherogenicity of the Plasma]

The nephrotic syndrome is characterized by proteinuria, hypoalbuminemia and hypercholesterolemia. Hypercholesterolemia is in some cases a risk factor for atherosclerosis in this group of patients. The lipid plasma spectrum was studied in 45 patients with the nephrotic syndrome. Most pronounced changes of the lipid composition of the plasma were revealed in patients with systemic lupus erythematosus and a special form of mesangio-proliferative glomerulonephritis which is characterized by a torpid course and rapid development of chronic renal failure. Plasma atherogenicity was calculated according to the index of plasma atherogenicity. A high atherogenicity index was revealed in patients with an association of the nephrotic syndrome and arterial hypertension. Plasma atherogenicity is determined mainly by the level of high-density-lipoprotein cholesterol.

[Hormonal Interrelationships in Symptomatic Epilepsy]

The serum and cerebrospinal fluid (CSF) from patients with epilepsy resultant from cerebral leptomeningitis in the fit-free intervals were examined for some hypophyseal and adrenal hormones with reference to the disease course, severity and duration, frequency of the seizures. The levels of hydrocortisone, aldosterone and STH were found stable, while those of prolactin got elevated, especially in males. Hormonal shifts in the serum and CSF appeared significantly different. STH lowered, but prolactin went up only in CSF. The latter increase was related to the seizures frequency.

[Lipid Metabolic Disorders and Lipid Peroxidation in Patients with the Nephrotic Syndrome]

Lipid metabolism was studied in 161 patients with renal diseases, 62 of them had nephrotic syndrome (NS). The measurements were made of cholesterol, triglycerides, dienic conjugates, B2-microglobulins in urine and blood, functional and morphological parameters, red cell resistance to peroxide hemolysis. It was found that NS patients, especially those with additional renal failure, displayed high-intensity lipoperoxidation (LPO). LPO intensity proved unrelated to NS etiology. NS-associated hyperlipidemia gives rise to multifunctional biomembrane instability responsible for antioxidant disorders.

[Causes of Recurrence of Pulmonary Tuberculosis: Short and Long Term Results of Treatment]

The causes of pulmonary tuberculosis recurrences and efficacy of their treatment have been analyzed for 180 patients with pulmonary tuberculosis. The recurrences arise most often secondary to associated diseases (chronic bronchitis, chronic alcoholism, occupational hazards), in subjects who previously have had focal pulmonary tuberculosis (at the sites of posttuberculous changes). Treatment of the recurrences is longer than that of the primary foci, does not prevent residual changes, is not curative in all the cases.

[Immunoenzyme Analysis in the Diagnosis of Opisthorchiasis. I. The Development of an Immunoenzyme Method for Determining IgM Antibodies to the Opisthorchis Antigen]

An assay was developed to determine IgM antibodies to Opisthorchis felineus antigens for the diagnosis of acute infection. The method is based on indirect solid-phase enzyme immunoassay. Optimal conditions were elaborated for analysis and its high specificity and specifications are shown in the paper.

[Comparative Study of Methods for Determining Antibodies to Measles Virus]

The authors have developed enzyme immunoassay (EIA) for detecting antimeasles antibodies in the serum and compared it to routine methods. Preparation and purification of measles virus antigen used in EIA and optimal conditions of the reaction are described. Results of the routine methods and those of EIA correlated. EIA was used to determine titers of antimeasles antibodies in the sera of vaccinated children. 3-4 years after the vaccination the antibodies were undetectable in the sera of 23% of children.

[The Prevalence of ABO Blood Groups Among Persons of Native Nationality in Buryatia]

Analysis of 609 blood specimens from Buryats and 821 from subjects of all nationalities living in the Republic of Buryatia and in the city of Ulan-Ude revealed differences in the incidence of each AB0 blood group in the Buryats in comparison with the population of the Republic at large and with European population (according to published reports).

[Enzyme Immunoassay in Diagnosis of Opisthorchiasis. Communication 2. Enzyme Immunoassay Test System for Diagnosis of Acute Opisthorchiasis]

A standard, highly specific and sensitive enzyme immunoassay test system has been developed for the diagnosis of acute opisthorchiasis, which is based on indirect solid-phase enzyme immunoassay for determination IgM antibodies to the antigen derived from Opisthorchis felineus marita extracts. The sensitivity of the test system is 87%, its specificity is 96.8%.

[Incidence of GM-system Factors in Indigenous Peoples of Buryatia]

Distribution of blood groups by the serum Gm system was studied in Buryats and in the entire population of Buryatia without consideration for the national appurtenance and with consideration for sex. Differences in the incidence of the factors were detected.

Germination of Cyanobacterial Akinetes from Bottom Deposits in Water from Blooming and Nonblooming Ponds Under Experimental Conditions

[Utilization of Complex Technologies of Molecular Genetics for Forensic Expert Identification of Unidentified Remains of Victims of Terrorist Acts in Moscow in 1999]

This paper presents the first experience gained in Russia in expert identification practice with complex utilization of technologies of molecular genetic individualization of biological objects for solving identification tasks requiring the maximum complete armory of mutually supplementing means. This is particularly important for forensic expert personality identification in cases with many deaths, when indirect identification has to be resorted to, consisting in the use of biological samples from relatives of the victims as the identifying objects. Methodological approaches and concepts of expert studies, used for identification of the remains of people who died in terroristic acts committed in Moscow in September, 1999, are discussed. The results of this study and methodological experience notably extend the potentialities of expert evaluation as regards forensic medical identification of victims of overall disasters, terroristic acts, and war conflicts.

[Who Thinks Clearly--writes in Plain Terms...(about the Problems of the Terminology in Epidemiology)]

The work deals with the tendency of introducing superfluous terms in epidemiology and other medical sciences. Four groups of foreign terminology, occurring in scientific literature on different problems of epidemiology, are presented. Examples of publications containing terms and notions whose interpretation is absent in dictionaries are given.

Apolipoprotein C-I Induces Apoptosis in Human Aortic Smooth Muscle Cells Via Recruiting Neutral Sphingomyelinase

Apolipoprotein C-I (apoC-I) influences lipoprotein metabolism, but little is known about its cellular effects in aortic smooth muscle cells (ASMC).

Transmembrane Reorientation of the Substrate-binding Site in Vesicular Acetylcholine Transporter

Active transport of acetylcholine (ACh) by vesicular ACh transporter (VAChT) is driven by a proton-motive force established by V-ATPase. A published microscopic kinetics model predicts the ACh-binding site is primarily oriented toward the outside for nontransporting VAChT and toward the inside for transporting VAChT. The allosteric ligand [(3)H]vesamicol cannot bind when the ACh-binding site is outwardly oriented and occupied by ACh, but it can bind when the ACh site is inwardly oriented. The kinetics model was tested in the paper reported here using rat VAChT expressed in PC12(A1237) cells. Equilibrium titrations of [(3)H]vesamicol binding and ACh competition show that ATP blocks competition between vesamicol and ACh in over one-half of the VAChT. NaCl did not mimic ACh chloride, and bafilomycin A(1) and FCCP completely blocked the ATP effect, which shows that it is mediated by a proton-motive force. The data are consistent with reorientation of over one-half of the ACh-binding sites from the outside to the inside of vesicles upon activation of transport. The observations support the proposed microscopic kinetics model, and they should be useful in characterizing effects of mutations on the VAChT transport cycle.

Acetylcholine Binding Site in the Vesicular Acetylcholine Transporter

This study sought primarily to locate the acetylcholine (ACh) binding site in the vesicular acetylcholine transporter (VAChT). The design of the study also allowed us to locate residues linked to (a) the binding site for the allosteric inhibitor vesamicol and (b) the rates of the two transmembrane reorientation steps of a transport cycle. In more characterized proteins, ACh is known to be bound in part through cation-pi solvation by tryptophan, tyrosine, and phenylalanine residues. Each of 11 highly conserved W, Y, and F residues in putative transmembrane domains (TMDs) of rat VAChT was mutated to A and a different aromatic residue to test for loss of cation-pi solvation. Mutated VAChTs were expressed in PC12(A123.7) cells and characterized with the goal of determining whether mutations widely perturbed structure. The thermodynamic affinity for ACh was determined by displacement of trace [(3)H]-(-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol) with ACh, and Michaelis-Menten parameters were determined for [(3)H]ACh transport. Expression levels were determined with [(3)H]vesamicol saturation curves and Western blots, and they were used to normalize V(max) values. "Microscopic" parameters for individual binding and rate steps in the transport cycle were calculated on the basis of a published kinetics model. All mutants were expressed adequately, were properly glycosylated, and bound ACh and vesamicol. Subcellular mistargeting was shown not to be responsible for poor transport by some mutants. Mutation of residue W331, which lies in the beginning of TMD VIII proximal to the vesicular lumen, produced 5- and 9-fold decreased ACh affinities and no change in other parameters. This residue is a good candidate for cation-pi solvation of bound ACh. Mutation of four other residues decreased the ACh affinity up to 6-fold and also affected microscopic rate constants. The roles of these residues in ACh binding and transport thus are complex. Nine mutations allowed us to resolve the ACh and vesamicol binding sites from each other. Other mutations affected only the rates of the transmembrane reorientation steps, and four mutations increased the rate of one or the other. Two mutations increased the value of K(M) up to 5-fold as a result of rate effects with no ACh affinity effect. The results demonstrate that analysis of microscopic kinetics is required for the correct interpretation of mutational effects in VAChT. Results also are discussed in terms of recently determined three-dimensional structures for other transporters in the major facilitator superfamily.

Choline is Transported by Vesicular Acetylcholine Transporter

Previously published results appeared to show that vesicular acetylcholine transporter (VAChT) does not transport choline (Ch). Because it is uniquely suited to detect transport of weakly bound substrates, a recently developed assay that detects transmembrane reorientation of the substrate binding site was used to re-examine transport selectivity. Rat VAChT was expressed in PC12(A1237) cells, postnuclear supernatant-containing microvesicles was prepared, and the reorientation assay was conducted with unlabeled Ch and tetramethylammonium (TMA). Also, [(14)C]Ch and [(3)H]acetylcholine (ACh) were used in an optimized accumulation assay. The results demonstrate that Ch is transported at least as well as ACh is, but with sevenfold lower affinity. Even TMA is transported, but with 26-fold lower affinity. Ch transport by VAChT is of interest in view of the possibilities that Ch (i) occurs at higher concentration than ACh does in terminal cytoplasm under some conditions, and (ii) is an agonist for alpha 7 nicotinic receptors.

Mutational and PH Analysis of Ionic Residues in Transmembrane Domains of Vesicular Acetylcholine Transporter

This research investigated the roles of 7 conserved ionic residues in the 12 putative transmembrane domains (TMDs) of vesicular acetylcholine transporter (VAChT). Rat VAChT in wild-type and mutant forms was expressed in PC12(A123.7) cells. Transport and ligand binding were characterized at different pH values using filter assays. The ACh binding site is shown to exhibit high or low affinity (K(d) values are approximately 10 and 200 mM, respectively). Mutation of the lysine and aspartate residues in TMDs II and IV, respectively, can decrease the fraction of sites having high affinity. In three-dimensional structures of related transporters, these TMDs lie next to each other and distantly from TMDs VIII and X, which probably contain the binding sites for ACh and the allosteric inhibitor vesamicol. Importantly, mutation of the aspartate in TMD XI can create extra-high affinities for ACh (K(d) approximately 4 mM) and vesamicol (K(d) approximately 2 nM compared to approximately 20 nM). Effects of different external pH values on transport indicate a site that must be protonated (apparent pK(a) approximately 7.6) likely is the aspartate in TMD XI. The observations suggest a model in which the known ion pair between lysine in TMD II and aspartate in TMD XI controls the conformation or relative position of TMD XI, which in turn controls additional TMDs in the C-terminal half of VAChT. The pH effects also indicate that sites that must be unprotonated for transport (apparent pK(a) approximately 6.4) and vesamicol binding (apparent pK(a) approximately 6.3) remain unidentified.

New Transport Assay Demonstrates Vesicular Acetylcholine Transporter Has Many Alternative Substrates

The acetylcholine-binding site in vesicular acetylcholine transporter faces predominantly toward the outside of the vesicle when resting but predominantly toward the inside when transporting. Transport-related reorientation is detected by an ATP-induced decrease in the ability of saturating substrate to displace allosterically bound [(3)H]vesamicol. The assay was used here to determine whether structurally diverse compounds are transported by rat VAChT expressed in PC12(A123.7) cells. Competition by ethidium, tetraphenylphosphonium and other monovalent organic cations with [(3)H]vesamicol is decreased when ATP is added, and the effect depends on proton-motive force. The results indicate that many organic molecules carrying +1 charge are transported, even though the compounds do not resemble acetylcholine in structural details.

Radiofrequency-enhanced Vascular Gene Transduction and Expression for Intravascular MR Imaging-guided Therapy: Feasibility Study in Pigs

To evaluate the feasibility of radiofrequency (RF)-enhanced vascular gene transduction and expression by using a magnetic resonance (MR) imaging-heating guidewire as an intravascular heating vehicle during MR imaging-guided therapy.

Structural Requirements for Steady-state Localization of the Vesicular Acetylcholine Transporter

The vesicular acetylcholine transporter (VAChT) regulates the amount of acetylcholine stored in synaptic vesicles. However, the mechanisms that control the targeting of VAChT and other synaptic vesicle proteins are still poorly comprehended. These processes are likely to depend, at least partially, on structural determinants present in the primary sequence of the protein. Here, we use site-directed mutagenesis to evaluate the contribution of the C-terminal tail of VAChT to the targeting of this transporter to synaptic-like microvesicles in cholinergic SN56 cells. We found that residues 481-490 contain the trafficking information necessary for VAChT localization and that within this region L485 and L486 are strictly necessary. Deletion and alanine-scanning mutants lacking most of the carboxyl tail of VAChT, but containing residues 481-490, were still targeted to microvesicles. Moreover, we found that clathrin-mediated endocytosis of VAChT is required for targeting to microvesicles in SN56 and PC12 cells. The data provide novel information on the mechanisms and structural determinants necessary for VAChT localization to synaptic vesicles.

Novel Role of Lactosylceramide in Vascular Endothelial Growth Factor-mediated Angiogenesis in Human Endothelial Cells

Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis associated with coronary heart disease, vascular complications in diabetes, inflammatory vascular diseases, and tumor metastasis. The mechanism of VEGF-driven angiogenesis involving glycosphingolipids such as lactosylceramide (LacCer), however, is not known. To demonstrate the involvement of LacCer in VEGF-induced angiogenesis, we used small interfering RNA (siRNA)-mediated silencing of LacCer synthase expression (GalT-V) in human umbilical vein endothelial cells. This gene silencing markedly inhibited VEGF-induced platelet endothelial cell adhesion molecule-1 (PECAM-1) expression and angiogenesis. Second, we used D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of LacCer synthase and glucosylceramide synthase, that significantly mitigated VEGF-induced PECAM-1 expression and angiogenesis. Interestingly, these phenotypic changes were reversed by LacCer but not by structurally related compounds such as glucosylceramide, digalactosylceramide, and ceramide. In a human mesothelioma cell line (REN) that lacks the endogenous expression of PECAM-1, VEGF/LacCer failed to stimulate PECAM-1 expression and tube formation/angiogenesis. In REN cells expressing human PECAM-1 gene/protein, however, both VEGF and LacCer-induced PECAM-1 protein expression and tube formation/angiogenesis. In fact, VEGF-induced but not LacCer-induced angiogenesis was mitigated by SU-1498, a VEGF receptor tyrosine kinase inhibitor. Also, VEGF/LacCer-induced PECAM-1 expression and angiogenesis was mitigated by protein kinase C and phospholipase A2 inhibitors. These results indicate that LacCer generated in VEGF-treated endothelial cells may serve as an important signaling molecule for PECAM-1 expression and in angiogenesis. This finding and the reagents developed in our report may be useful as anti-angiogenic drugs for further studies in vitro and in vivo.

Platelet Derived Growth Factor Recruits Lactosylceramide to Induce Cell Proliferation in UDP Gal:GlcCer: Beta1 --> 4Galactosyltransferase (GalT-V) Mutant Chinese Hamster Ovary Cells

Recent molecular cloning studies have suggested the presence of at least two beta4Gal transferase genes (beta4GalT-V and beta4GalT-VI) that may encode lactosylceramide synthase but whether they are functional in vivo and whether they mediate growth factor induced phenotypic change such as cell proliferation is not known. Our previous studies lead to the suggestion that various risk factors in atherosclerosis such as oxidized LDL, shear stress, nicotine, tumor necrosis factor-alpha converge upon LacCer synthase to induce critical phenotypic changes such as cell proliferation and cell adhesion. However, whether platelet-derived growth factor also recruits LacCer synthase in mediating cell proliferation is not known. Here we have employed a Chinese hamster ovary mutant cell line Pro(-)5Lec20 to determine whether this enzyme physiologically functions to mediate cell proliferation. We show that PDGF stimulates the activity of UDP galactose:glucosylceramide, beta1,4galactosyltransferase. The activity of LacCer synthase increased about 2.5 fold within 2.5-5 min of incubation with PDGF in both wild type and Pro(-)5Lec20 cells. Concomitantly, there was an increase in the generation of superoxide radicals, p44MAPK phosphorylation and cell proliferation in CHO cells. D-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a potent inhibitor of GlcCer synthase/LacCer synthase impaired PDGF mediated induction of LacCer synthase activity, superoxide generation, p44 MAPK activation and cell proliferation in Pro(-)5Lec20 cells. PDGF-induced superoxide generation was also mitigated by the use of diphenylene iodonium; an inhibitor of NADPH oxidase activity that is required for superoxide generation. This inhibition was bypassed by the addition of lactosylceramide. Thus, beta4GalT-V gene produces a bona fide LacCer synthase that can function in vivo to generate LacCer. Moreover, this enzyme alone can mediate PDGF induced activation of a signal transduction cascade involving superoxide generation, p44MAPK activation, phosphorylation of Akt and cell proliferation.

[Changes in Gum in Cases of Arterial Hypertension Combination with Periodontitis]

Microlymphohemocirculatory bed and leucocytogram of gingival tissue were studied by the light microscopy in patients with chronic periodontitis having normal and high arterial blood pressure. In most cases of arterial hypertension the gingival mucous was characterized by widening of lymphatic vessels and interstitial spaces. In cases of arterial hypertension combination with inflammatory reaction the tendency for widening of lymphatic vessels and interstitial spaces persisted compared with cases of normal blood pressure. It testifies to high probability of lymphogenic generalization of inflammation. Besides, in cases of inflammatory gingival pathology in arterial hypertension the absolute neutrophil number was significantly higher showing for more acute inflammatory process and greater volume of tissue involvement.

The Role of the Phospholipid Sphingomyelin in Heart Disease

Sphingomyelin (SM) is an integral component of mammalian cell membranes and nerves. However, the inability to catabolize SM may lead to its accumulation in various tissues and organs, resulting in pathological disorders such as Niemann Pick disease. Elevated levels of SM have also been identified as an independent risk factor for coronary heart disease. During the past two decades, data have emerged that support an important role for metabolites of SM, such as ceramide and sphingosine-1-phosphate, in the regulation of phenotypic changes such as cell proliferation, cell-cycle arrest, apoptosis and angiogenesis. Further studies of the molecular and pathobiological basis of these phospholipids may facilitate advances in the discovery of drugs with which to mitigate diseases that may result from an elevation in SM and its metabolites.

Mutational and Bioinformatics Analysis of Proline- and Glycine-rich Motifs in Vesicular Acetylcholine Transporter

The vesicular acetylcholine transporter (VAChT) contains six conserved sequence motifs that are rich in proline and glycine. Because these residues can have special roles in the conformation of polypeptide backbone, the motifs might have special roles in conformational changes during transport. Using published bioinformatics insights, the amino acid sequences of the 12 putative, helical, transmembrane segments of wild-type and mutant VAChTs were analyzed for propensity to form non-alpha-helical conformations and molecular notches. Many instances were found. In particular, high propensity for kinks and notches are robustly predicted for motifs D2, C and C'. Mutations in these motifs either increase or decrease Vmax for transport, but they rarely affect the equilibrium dissociation constants for ACh and the allosteric inhibitor, vesamicol. The near absence of equilibrium effects implies that the mutations do not alter the backbone conformation. In contrast, the Vmax effects demonstrate that the mutations alter the difficulty of a major conformational change in transport. Interestingly, mutation of an alanine to a glycine residue in motif C significantly increases the rates for reorientation across the membrane. These latter rates are deduced from the kinetics model of the transport cycle. This mutation is also predicted to produce a more flexible kink and tighter tandem notches than are present in wild-type. For the full set of mutations, faster reorientation rates correlate with greater predicted propensity for kinks and notches. The results of the study argue that conserved motifs mediate conformational changes in the VAChT backbone during transport.

Exploring Writhe in Supercoiled Minicircle DNA

Using λ-Int recombination in E. coli, we have generated milligram quantities of supercoiled minicircle DNA. Intramolecular Int recombination was efficient down to lengths ~254 bp. When nicked and religated in the presence of ethidium bromide, 339 bp minicircles adopted at least seven unique topoisomers that presumably correspond to ΔLk ranging from 0 to -6, which we purified individually. We used these minicircles, with unique ΔLk, to address the partition into twist and writhe as a function of ΔLk. Gel electrophoresis and atomic force microscopy revealed progressively higher writhe conformations in the presence of 10 mM CaCl(2) or MgCl(2). From simplistic calculations of the bending and twisting energies, we predict the elastic free energy of supercoiling for these minicircles to be lower than if the supercoiling was partitioned mainly into twist. The predicted writhe corresponds closely with that which we observed experimentally in the presence of divalent metal ions. However, in the absence of divalent metal ions only limited writhe was observed, demonstrating the importance of electrostatic effects on DNA structure, when the screening of charges on the DNA is weak. This study represents a unique insight into the supercoiling of minicircle DNA, with implications for DNA structure in general.

MRI Visualized Neo-intimal Dissection and Co-localization of Novel Apoptotic Markers Apolipoprotein C-1, Ceramide and Caspase-3 in a Watanabe Hyperlipidemic Rabbit Model

Apoptotic arterial wall vascular smooth muscle cell death is known to contribute to plaque vulnerability and rupture. Novel apoptotic markers like apolipoprotein C-I have been implicated in apoptotic human vascular smooth muscle cell death via recruiting a neutral sphingomyelinase (N-SMase)-ceramide pathway. In vivo relevance of these observations in an animal model of plaque rupture has not been shown.

High-resolution Three-dimensional Aortic Magnetic Resonance Angiography and Quantitative Vessel Wall Characterization of Different Atherosclerotic Stages in a Rabbit Model

Atherosclerosis results in a considerable medical and socioeconomic impact on society. We sought to evaluate novel magnetic resonance imaging (MRI) angiography and vessel wall sequences to visualize and quantify different morphologic stages of atherosclerosis in a Watanabe hereditary hyperlipidemic (WHHL) rabbit model.

Differences in the Amino Acid Content of Dominant Phytoplankton Species from a Eutrophic Reservoir

BRD-NUT Oncoproteins: a Family of Closely Related Nuclear Proteins That Block Epithelial Differentiation and Maintain the Growth of Carcinoma Cells

An unusual group of carcinomas, here termed nuclear protein in testis (NUT) midline carcinomas (NMC), are characterized by translocations that involve NUT, a novel gene on chromosome 15. In about 2/3rds of cases, NUT is fused to BRD4 on chromosome 19. Using a candidate gene approach, we identified two NMCs harboring novel rearrangements that result in the fusion of NUT to BRD3 on chromosome 9. The BRD3-NUT fusion gene encodes a protein composed of two tandem chromatin-binding bromodomains, an extra-terminal domain, a bipartite nuclear localization sequence, and almost the entirety of NUT that is highly homologous to BRD4-NUT. The function of NUT is unknown, but here we show that NUT contains nuclear localization and export sequences that promote nuclear-cytoplasmic shuttling via a leptomycin-sensitive pathway. In contrast, BRD3-NUT and BRD4-NUT are strictly nuclear, implying that the BRD moiety retains NUT in the nucleus via interactions with chromatin. Consistent with this idea, FRAP studies show that BRD4, BRD4-NUT and BRD3-NUT have significantly slower rates of lateral nuclear diffusion than that of NUT. To investigate the functional role of BRD-NUT fusion proteins in NMCs, we investigated the effects of siRNA-induced BRD3-NUT and BRD4-NUT withdrawal. Silencing of these proteins in NMC cell lines resulted in squamous differentiation and cell cycle arrest. Together, these data suggest that BRD-NUT fusion proteins contribute to carcinogenesis by associating with chromatin and interfering with epithelial differentiation.

Regulation of Lactosylceramide Synthase (glucosylceramide Beta1-->4 Galactosyltransferase); Implication As a Drug Target

Lactosylceramide is a ubiquitously present glycosphingolipid in mammalian tissues and has been implicated in cell proliferation, adhesion, migration and angiogenesis. This glycosphingolipid is synthesized by Golgi-localized enzyme LacCer synthase. According to recent nomenclature and gene mapping studies, two LacCer synthases beta1,4GalT-V and beta1,4GalT-VI have been identified and characterized. In addition, beta1,4GalT-V has been implicated in the synthesis of N-glycans of cell surface glycoproteins. During the past two decades data have accumulated suggesting that the cellular level of LacCer can be regulated by various growth factors, cytokines, lipids, lipoproteins and hemodynamic factors, such as fluid shear stress, by altering the activity of LacCer synthase. An interesting feature is that a nuclear regulating factor (SP1) plays a critical role in transcriptional regulation of this enzyme in cancer cells. Moreover, in human umbilical vein endothelial cells, NF-kappaB has been also shown to regulate this enzyme which, in turn, regulates the gene/protein expression of platelet endothelial cell adhesion molecule, intercellular cell adhesion molecule and angiogenesis. Since new blood supply via formation of capillaries is critical in tumor growth, metastasis, and atherogenesis, these findings expand the role of enzyme in these pathologies. Additional studies are warranted to understand the molecular and biochemical basis of how LacCer synthases are regulated. These studies will facilitate advances in discovery of drugs which mitigate diseases, such as atherosclerosis and cancer due to an aberrant regulation of these LacCer synthases.

Direct Resequencing of the Complete ERBB2 Coding Sequence Reveals an Absence of Activating Mutations in ERBB2 Amplified Breast Cancer

Gene amplification is among the most common genetic abnormalities that cause cancer. One of the most clinically important gene amplifications in human cancer causes extensive reduplication of ERBB2. A variety of cancers also occasionally harbor somatic mutations in ERBB2. Gene amplification and activating mutations both have predictive value for clinical response to targeted inhibitors. Since the number of gene copies in an amplicon may exceed 100, and since amplicons may encompass multiple genes, high-resolution analysis of gene amplifications poses considerable technical challenges. We have overcome this obstacle by using emulsion-based resequencing to determine the sequence of many independently-amplified individual DNA molecules in parallel. We used this high throughput sequencing technology to analyze ERBB2 mutational status in five ERBB2 amplified cell lines (four breast, one ovarian) and two breast tumors. Genomic DNA was isolated and the 28 exons of ERBB2 were independently amplified. Amplicons were then pooled at equimolar ratios, subjected to emulsion PCR (emPCR) and finally to picotiter plate pyrosequencing. High-quality sequence data were obtained for all amplicons analyzed and no activating mutations within ERBB2 were identified. Although we did not find activating mutations within the multiple copies of ERBB2 in these samples, the results establish the utility of this technology as a feasible and cost-effective approach for high resolution analysis of amplified genes.

Use of a Novel Anti-proliferative Compound Coated on a Biopolymer to Mitigate Platelet-derived Growth Factor-induced Proliferation in Human Aortic Smooth Muscle Cells: Comparison with Sirolimus

Drug eluting stents (DES) have become a common mode of treatment for stenosis in coronary arteries. However, currently, the use of sirolimus/paclitaxel-coated DES has come under scrutiny, because of their pro-thrombotic effects leading to potential adverse outcomes in the long run. We have previously documented that D: -threo-1-phenyl-2-decanoylamino-3-morholino propanol (D-PDMP); an inhibitor of glucosylceramide synthase and lactosylceramide (LacCer) synthase markedly inhibited platelet-derived growth factor (PDGF)-induced cell proliferation. We have fabricated DES wherein, D-PDMP or sirolimus was coated on to a double layer of poly (lactic-co-glycolic acid) on a bare metal stent. The in vitro release of D-PDMP from biopolymer and its consequent effect on PDGF induced proliferation and apoptosis was assessed in human aortic smooth muscle cells (ASMC). D-PDMP was released from biopolymers in a dose-dependent fashion and was accompanied with a decrease in PDGF-induced cell proliferation, but not apoptosis. In contrast, sirolimus markedly increased apoptosis in these cells in addition to inhibiting proliferation. Our mechanistic studies revealed that D-PDMP, but not sirolimus decreased the cellular level of glucosyl and lactosylceramide that accompanied inhibition of PDGF-induced cell proliferation. Our short-term (14 days) in vivo studies in rabbits also attested to the safety and biocompatibility of the D-PDMP coated stents. Our data reveal the superiority of D-PDMP coated biopolymers over sirolimus coated biopolymers in mitigating ASMC proliferation. Such D-PDMP coated stents may be useful for localized delivery of drug to mitigate neo-vascular hyperplasia and other proliferative disorders.

Scavenger Receptor Class B Type I Protein As an Independent Predictor of High-density Lipoprotein Cholesterol Levels in Subjects with Hyperalphalipoproteinemia

In mice, scavenger receptor class B, type I (SR-BI) receptor protein deficiency is associated with elevated high-density lipoprotein (HDL)-cholesterol (HDL-C) levels.

VEGF Recruits Lactosylceramide to Induce Endothelial Cell Adhesion Molecule Expression and Angiogenesis in Vitro and in Vivo

Angiogenesis is largely driven by vascular endothelial growth factor (VEGF). However, the role of lipid second messengers such as lactosylceramide (LacCer) and LacCer synthase in angiogenesis is not well understood. We have determined the distribution of various LacCer synthase mRNA transcripts using sequential analysis of gene expression (SAGE). Endothelial cells from colon cancer tissues had a 4.5-fold increase in a LacCer synthase transcript (beta1,4GalT-V) as compared to normal colon tissue endothelial cells. Consequently, our focus turned to understanding the role of this enzyme in regulating VEGF-induced angiogenesis in vitro and in vivo. Herein, we show that in human endothelial cells, VEGF-induced angiogenesis is mitigated by dimethylsphingosine and suramin; inhibitors of sphingosine kinase 1(SphK-1) and sphingosine1-phosphate receptor 1(S1P (1)), respectively, and this were bypassed by LacCer but not by S1P. VEGF and basic fibroblast growth factor-induced angiogenesis was mitigated by PDMP; an inhibitor of glucosylceramide synthase and LacCer synthase in human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC). Likewise, GalT-V gene ablation using corresponding siRNA also mitigated VEGF-induced angiogenesis. In Matrigel plug angiogenesis assay in nude mice, angiogenesis was markedly inhibited by D-PDMP with concordantly diminished LacCer synthase activity. Mechanistic studies revealed that the use of LY294002, a PI3 kinase inhibitor, mitigated VEGF-induced expression of platelet-endothelial cell adhesion molecule (PECAM-1/CD31); the trans-endothelial migration of a monocyte cell line (U-937) and angiogenesis in HAEC cells. Since this enzyme is a target for VEGF action and LacCer serves as a lipid second messenger in inducing angiogenesis in vitro and in vivo, novel therapeutic approaches may be developed using our findings to mitigate colon cancer.

Association of Constitutively Activated Hepatocyte Growth Factor Receptor (Met) with Resistance to a Dual EGFR/Her2 Inhibitor in Non-small-cell Lung Cancer Cells

There is a pressing need to identify new drug targets and novel approaches for treatment of non-small-cell lung carcinoma (NSCLC). Members of the epidermal growth factor receptor (EGFR) and Met receptor families have been identified as important molecular targets for NSCLC. Two EGFR tyrosine kinase inhibitors (TKIs; erlotinib and gefitinib) are in current clinical use, but a majority of patients do not respond to these targeted therapies. We used receptor TK (RTK) capture arrays to identify receptors active in NSCLC cell lines. As Met and ErbBs were active, we explored the potential therapeutic advantage of combined targeting of Met with ErbB receptor family inhibitors for treatment of NSCLC. We found that Met physically interacts with both EGFR and Her2 in a NSCLC cell line with overexpression/overactivation of Met. Combined use of a dual EGFR/Her2 inhibitor with a Met inhibitor yields maximal growth inhibition compared with the use of EGFR and/or Met inhibitors. This suggests that simultaneous inhibition of multiple RTKs may be needed to effectively abrogate tumour cell growth. Phosphoproteomic analysis by RTK capture arrays may be a valuable tool for identifying the subset of tumours with functional receptor activation, regardless of mechanism.

[Formation of Plasma Cell Infiltrates in Gingiva of Patients with Chronic Apical Periodontitis]

The structure of leukocyte infiltrates in gingiva of 80 patients of various ages and gender with chronic apical periodontitis was studied using light microscopy with the application of monoclonal antibodies detecting CD38 antigen. Gingival tissues of practically of all the patients with periodontitis contained 2 types of leukocyte infiltrates: infiltrates with the low plasma cell content and high numbers of neutrophils and structures with high number of plasmocytes and low concentration of neutrophilic granulocytes. Within the gingival epithelium in patients with chronic apical periodontitis CD38 + cells were absent. The histogenesis of gingival leukocyte infiltrates in chronic apical periodontitis is discussed with the special emphasis on the role of plasma cells in the development of the pathological process.

Efficiency of Transfer of Essential Polyunsaturated Fatty Acids Along Trophic Chains in Aquatic Ecosystems

[B-cellular Lymphoid Follicles of Gingival Mucous Membrane in Cases of Chronic Apical Periodontitis]

The structure of gingival mucous membrane leukocytal infiltration of 80 patients with chronic apical periodontitis of different age groups was studied by the light microscopy with the use of monoclonal antibodies to CD38. It was disclosed that in gingival tissues of practically all patients 2 types of leukocytal infiltration were present with low number of plasmatic cells and high number of neutrophils (true leukocytal infiltration) and structures with high number of plasmatic cells and low number of neutrophils - most likely lymphoid follicle resultant in mucous membranes of different organs in cases of chronic inflammation. In epithelial gingival paving in cases of chronic apical periodontitis CD38(+)-cells were absent.

[Treatment of the Patient with Anthrax Complicated with Gastric Bleeding]

Deficiency of Scavenger Receptor Class B Type I Negatively Affects Progesterone Secretion in Human Granulosa Cells

Our goal was to examine the effect of deficiency of the lipoprotein receptor, scavenger receptor class B type I (SR-BI), on progesterone secretion in human granulosa cells (HGL5). Scrambled or SR-BI small interfering RNA [knockdown (KD)] cells were exposed to dimethylsulfoxide [DMSO, vehicle for forskolin (Fo)], Fo, serum, high-density lipoprotein, low-density lipoprotein (LDL), or Fo plus lipoproteins or serum for 24 h. Progesterone secretion was lower in all of the SR-BI KD cells regardless of treatment. We examined progesterone secretion in SR-BI KD, LDL receptor KD, and double KD cells incubated with DMSO, Fo, LDL, or Fo + LDL for 6-24 h. As compared with scrambled cells, progesterone secretion was lower in SR-BI and double KD cells regardless of treatment; whereas progesterone secretion was only lower in LDL receptor KD cells incubated with LDL and Fo + LDL. We measured phosphorylation of hormone-sensitive lipase (pHSL) expression, intracellular total cholesterol (TC) mass, and progesterone secretion in scrambled and SR-BI KD cells incubated with DMSO or Fo for 2-24 h. The expression of pHSL was similar between the cells and conditions. The mean change in TC mass and progesterone secretion was lower in SR-BI KD cells exposed to DMSO and Fo. Incubating SR-BI KD cells with 22-hydroxy cholesterol did not overcome the reduction in progesterone secretion. At different time points, RNA expression of steroidogenic acute regulatory protein, side-chain cleavage, and 3β-hydroxysteroid dehydrogenase was significantly lower in SR-BI KD cells incubated with Fo. In conclusion, SR-BI protein deficiency, in part, might explain progesterone deficiency in some infertile women.

[Biochemical Characteristics of Compensation of Posthemorrhagic Anemia in Patients Presenting with Nasal Bleeding]

This work was designed to study the development of compensatory processes during posthemorrhagic anemia in 82 patients presenting with nasal bleeding (NB). The patients were allocated to three groups. Group 1 included patients with isolated episodes of NB, group 2 was comprised of patients in a moderately severe condition with recurring NB, group 3 was composed of patients in a severe condition with recurring NB. The general medical examination was supplemented by the evaluation of factors maintaining the oxygen-transporting function of the blood (hemoglobin affinity for oxygen, erythrocyte content of 2.3-diphosphoglyceric (2.3-DPG) acid as the principal modulator of hemoglobin affinity for oxygen) and indicators of energy (carbohydrate) metabolism in plasma and erythrocytes (glucose-6-phosphate dehydrogenase (G-6-PDH) activity, pyruvic acid (PA), lactate and lactate dehydrogenase (LDH) levels). Changes of biochemical parameters in patients presenting with incidental episodes of NB (group 1) suggested a compensatory increase in functional potential of the blood oxygen-transporting system. Patients of group 2 showed evidence of development of the modulation-type adaptive and compensatory mechanisms. Those of group 3 experienced a decrease of the 2.3-DPH level in erythrocytes and enhancement of hemoglobin affinity for oxygen which slowed down its uptake by the tissues. Tissue hypoxia and accompanying acidosis aggravated the impairment of gas-transporting function of the blood. In is concluded that patients of group 3 are at risk of uncompensated hypoxic hypoxia associated with the unfavourable changes in the oxygen-transporting function and the impairment of the functional potential of erythrocytes. Taken together, these untoward factors may be responsible for the severe clinical conditions of these patients.

Efficiency of Transfer of Essential Polyunsaturated Fatty Acids Versus Organic Carbon from Producers to Consumers in a Eutrophic Reservoir

One of the central paradigms of ecology is that only about 10% of organic carbon production of one trophic level is incorporated into new biomass of organisms of the next trophic level. Many of energy-yielding compounds of carbon are designated as 'essential', because they cannot be synthesized de novo by consumers and must be obtained with food, while they play important structural and regulatory functions. The question arises: are the essential compounds transferred through trophic chains with the same efficiency as bulk carbon? To answer this question, we measured gross primary production of phytoplankton and secondary production of zooplankton and content of organic carbon and essential polyunsaturated fatty acids of ω-3 family with 18-22 carbon atoms (PUFA) in the biomass of phytoplankton and zooplankton in a small eutrophic reservoir during two summers. Transfer efficiency between the two trophic levels, phytoplankton (producers) and zooplankton (consumers), was calculated as ratio of the primary production versus the secondary (zooplankton) production for both carbon and PUFA. We found that the essential PUFA were transferred from the producers to the primary consumers with about twice higher efficiency than bulk carbon. In contrast, polyunsaturated fatty acids with 16 carbon atoms, which are synthesized exclusively by phytoplankton, but are not essential for animals, had significantly lower transfer efficiency than both bulk carbon, and essential PUFA. Thus, the trophic pyramid concept, which implicitly implies that all the energy-yielding compounds of carbon are transferred from one trophic level to the next with the same efficiency of about on average 10%, should be specified for different carbon compounds.

Endogenous Cardiotonic Steroids in Chronic Renal Failure

Previous reports demonstrated that digitalis-like cardiotonic steroids (CTS) contribute to the pathogenesis of end-stage renal disease. The goal of the present study was to define the nature of CTS in patients with chronic kidney disease (CKD) and in partially nephrectomized (PNx) rats.

Clinical Impact of Scavenger Receptor Class B Type I Gene Polymorphisms on Human Female Fertility

The goal of this study was to evaluate the association of SCARB1 single nucleotide polymorphisms (SNPs) and fertility outcomes in women undergoing IVF.

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