Genetic Linkage Analysis of the Lesser Grain Borer Rhyzopertha Dominica Identifies Two Loci That Confer High-level Resistance to the Fumigant Phosphine

Genetics. Jun, 2002  |  Pubmed ID: 12072472

High levels of inheritable resistance to phosphine in Rhyzopertha dominica have recently been detected in Australia and in an effort to isolate the genes responsible for resistance we have used random amplified DNA fingerprinting (RAF) to produce a genetic linkage map of R. dominica. The map consists of 94 dominant DNA markers with an average distance between markers of 4.6 cM and defines nine linkage groups with a total recombination distance of 390.1 cM. We have identified two loci that are responsible for high-level resistance. One provides approximately 50x resistance to phosphine while the other provides 12.5x resistance and in combination, the two genes act synergistically to provide a resistance level 250x greater than that of fully susceptible beetles. The haploid genome size has been determined to be 4.76 x 10(8) bp, resulting in an average physical distance of 1.2 Mbp per map unit. No recombination has been observed between either of the two resistance loci and their adjacent DNA markers in a population of 44 fully resistant F5 individuals, which indicates that the genes are likely to reside within 0.91 cM (1.1 Mbp) of the DNA markers.

Intestinal Rather Than Hepatic Microsomal Triglyceride Transfer Protein As a Cause of Postprandial Dyslipidemia in Diabetes

Metabolism: Clinical and Experimental. Jul, 2002  |  Pubmed ID: 12077729

Postprandial dyslipidemia may be a major cause of atherosclerosis in diabetes. Microsomal triglyceride transfer protein (MTP) is essential for the synthesis of the chylomicron particle in the intestine and very low-density lipoprotein (VLDL) in the liver. The purpose of the present study was to examine the effect of diabetes on MTP mRNA expression in a rabbit model of diabetes, which develops atherosclerosis. Male New Zealand white rabbits were fed a 0.5% cholesterol diet. Diabetes was induced with alloxan monohydrate. The lymphatic duct was cannulated and lymph collected for isolation of chylomicrons by ultracentrifugation. Apolipoprotein B48 (apo B48) and apo B100 were separated by polyacrylamide gradient gel electrophoresis and quantified by densitometry. MTP mRNA was determined in liver and intestine by RNase protection analysis, and MTP activity was measured. Diabetic animals had significantly increased plasma triglyceride and decreased high-density lipoprotein (HDL) cholesterol (P <.05). They also secreted more lymph chylomicron apo B48 and apo B100 (P <.05) and more lymph chylomicron total and esterified cholesterol/h (P <.05). Lymph chylomicron particles in the diabetic animals contained significantly less lipid/apo B (P <.05). Intestinal MTP activity and mRNA were significantly higher in diabetic compared with control rabbits (0.07 +/- 0.01 v 0.04 +/- 0.015 fluorescent units/microg microsomal protein and 66 +/- 21 v 37 +/- 11 amol MTP mRNA/microg total RNA (P <.005). There was no difference in MTP activity or mRNA expression in the liver. This study suggests that MTP may play an important role in the postprandial dyslipidemia of diabetes.

Genetics of Resistance to Phosphine in Rhyzopertha Dominica (Coleoptera: Bostrichidae)

Journal of Economic Entomology. Aug, 2002  |  Pubmed ID: 12216832

The inheritance of resistance to phosphine was studied in two strains of the lesser grain borer, Rhyzopertha dominica (F.), labeled 'Weak-R' and 'Strong-R'. These strains were purified versions of field-selected populations collected in Queensland, Australia. Weak-R and Strong-R were, respectively, 23.4 times (20-h exposure) and 600 times (48-h exposure) resistant to phosphine compared with a reference susceptible strain (S-strain). Each -R strain was crossed with the S-strain and the response to phosphine was measured in their respective F1, F2, and F1-backcross (F1-BC) progenies. Data from testing of reciprocal F1 progeny indicated that resistance in Weak-R was autosomal and incompletely recessive with a degree of dominance -0.96. Modified chi-square analysis and contingency analysis of the observed response to phosphine of F1-BC and F2 progenies rejected the hypothesis of single gene inheritance of resistance. Analysis of the response of the F1, F2, and F1-BC progeny from the Strong-R x S-strain cross also rejected the null hypothesis for single gene resistance. Resistance in the Strong-R strain was autosomal and incompletely recessive with a degree of dominance of -0.64. The Weak-R and Strong-R strains were then crossed. Analysis ofthe F1 and F2 progenies of this reciprocal cross revealed that the strong resistance phenotype was coded by a combination of the genes already present in the Weak-R genotype plus an extra major, incompletely recessive gene. There was also evidence of a minor dominant gene present in approximately 5% of Strong-R individuals.

Effects of Time and Concentration on Mortality of Phosphine-resistant Sitophilus Oryzae (L) Fumigated with Phosphine

Pest Management Science. Oct, 2002  |  Pubmed ID: 12400440

The effects of exposure period and phosphine concentration on mortality of susceptible and resistant Sitophilus oryzae (L) were investigated. Although S oryzae is one of the world's most serious pests of stored grain there are few data on the practical significance of phosphine resistance in this species. The strains investigated were an Australian susceptible strain, a homozygous resistant strain exhibiting a level of resistance common in Australia and an unselected field strain from China with a much stronger resistance. Fumigations were carried out at 25 degrees C on adults and mixed-age cultures. For adults of all three strains and mixed-age cultures of the susceptible and resistant Australian strains, the relationship between concentration and time could be described by equations of the form Cnt = k. In all cases n < 1, indicating that time was a more important variable than concentration. In all fumigations of adults the resistant strains were harder to kill than the susceptible strain. However, in fumigations of mixed-age cultures, which contained the tolerant pupal stage, the difference between susceptible and resistant strains was more pronounced at lower concentrations than higher concentrations. For example, at 0.02 mg litre-1 the estimated LT99.9 for mixed-age cultures of the Australian resistant strain (27 days) is 3.4 times that of the susceptible strain (8 days), but at 1 mg litre-1 there is no difference between the two strains (4 days). Limited data on the Chinese resistant strain supported this finding. Twenty-three days exposure at 0.02 mg litre-1 had no effect on mixed-age cultures of this strain, but there were no survivors after 5 days exposure to 1 mg litre-1.

Defective Chylomicron Synthesis As a Cause of Delayed Particle Clearance in Diabetes?

International Journal of Experimental Diabetes Research. Jul-Sep, 2002  |  Pubmed ID: 12458658

Chylomicron metabolism is abnormal in diabetes and the chylomicron particle may play a very important role in atherosclerosis. The aim of this study was to examine the effect of diabetes on the metabolism of chylomicrons in cholesterol-fed alloxan diabetic and nondiabetic rabbits. Five diabetic rabbits and 5 control rabbits were given [14C]linoleic acid and [3H]cholesterol by gavage. Lymph was collected following cannulation of the lymph duct and radiolabelled chylomicrons were isolated by ultracentrifugation. The chylomicrons from each animal were injected into paired control and diabetic recipients. Lymph apolipoprotein (apo) B48, apo B100, and apo E were measured using sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. Mean blood sugar of the diabetic donors and diabetic recipients were 19.7 +/- 2.3 and 17.2 +/- 3.2 mmol/L. Diabetic rabbits had significantly raised plasma triglyceride (10.8 +/- 13.9 versus 0.8 +/- 0.5 mmol/L, P < 0.02). There was a large increase in apo B48 in lymph chylomicrons in the diabetic donor animals (0.19 +/- 0.10 versus 0.04 +/- 0.02 mg/h, P < 0.01) and apo B100 (0.22 +/- 0.15 versus 0.07 +/- 0.07 mg/h, P < 0.05) and a reduction in apo E on the lymph chylomicron particle (0.27 +/- 0.01 versus 0.62 +/- 0.07 mg/mg apo B, P < 0.001). Diabetic recipients cleared both control and diabetic chylomicron triglyceride significantly more slowly than control recipients (P < 0.05). Clearance of control chylomicron cholesterol was delayed when injected into diabetic recipients compared to when these chylomicrons were injected into control recipients (P < 0.005). Clearance of diabetic chylomicron cholesterol was significantly slower when injected into control animals compared to control chylomicron injected into control animals (P < 0.02). In this animal model of atherosclerosis, we have demonstrated that diabetes leads to the production of an increased number of lipid and apo E-deficient chylomicron particles. Chylomicron particles from the control animals were cleared more slowly by the diabetic recipient (both triglyceride and cholesterol). The chylomicron particles obtained from the diabetic animals were cleared even more slowly when injected into the diabetic recipient. Although there was an initial delay in clearance of chylomicron triglyceride from the diabetic particle when injected into the control animals, the clearance over the first 15 minutes was not significantly different when compared to the control chylomicron injected into the control animal. On the other hand, the cholesterol clearance was significantly delayed. Thus, diabetes resulted in the production of an increased number of lipid- and apo E-deficient chylomicron particles. These alterations account, in part, for the delay in clearance of these particles.

Long-term Effectiveness of Grain Protectants and Structural Treatments Against Liposcelis Decolor (Pearman) (Psocoptera: Liposcelididae), a Pest of Stored Products

Pest Management Science. Dec, 2002  |  Pubmed ID: 12476995

Numerous strains of the psocid pest, Liposcelis decolor (Pearman) were collected from farms and central storages and interbred to form three representative strains from three major grain-growing states of Australia: Queensland, South Australia and New South Wales. These were tested against the grain protectants and structural treatments currently registered for use in Australia. Recently, L decolor has become an important pest of stored grain in Australia, particularly in the eastern and southern parts. There is no published information available on management of this pest and the current pest-management strategy, based predominantly on phosphine fumigation, has failed to control infestations of this pest in numerous grain storages in Australia. Alternative methods of control such as use of contact insecticides were explored in the present work to supplement phosphine fumigation to manage this new pest. From eight grain protectants tested as admixtures, only chlorpyrifos-methyl, bioresmethrin plus piperonyl butoxide, and fenitrothion were found to provide long term (3-9 months) protection against all three strains of L decolor. Chlorpyrifos-methyl gave the best protection, providing a minimum of 7.5 to a maximum of 9 months protection, depending on the strains tested. Three structural treatments, azamethiphos, azamethiphos plus carbaryl and permethrin provided long-term control (8-9 months) of all three strains of L decolor on galvanised steel surfaces, with permethrin delivering 9 months protection against all strains. However, all of these treatments failed to provide long-term control of any strains on concrete surfaces. We conclude that chlorpyrifos-methyl as a grain admixture can be incorporated into fumigation strategies to optimise the control of L decolor infestations. Structural treatments, such as permethrin can be used to support a fumigation strategy in storages made of galvanised steel.

Impaired Fasting Glucose Tolerance in First-episode, Drug-naive Patients with Schizophrenia

The American Journal of Psychiatry. Feb, 2003  |  Pubmed ID: 12562574

This study examined the prevalence of impaired fasting glucose tolerance in first-episode, drug-naive patients with schizophrenia.

Caenorhabditis Elegans Mutants Resistant to Phosphine Toxicity Show Increased Longevity and Cross-resistance to the Synergistic Action of Oxygen

Toxicological Sciences : an Official Journal of the Society of Toxicology. May, 2003  |  Pubmed ID: 12700416

Phosphine (hydrogen phosphide, PH3) is the fumigant most widely used to protect stored products from pest infestation. Despite the importance of this chemical, little is known about its mode of action. We have created three phosphine-resistant lines (pre-1, pre-7, pre-33) in the model organism C. elegans, with LC50 values 2, 5, and 9 times greater than the fully susceptible parental strain. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a nonlethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. We take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, all three mutants have an extended average life expectancy of from 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Because the wild-type and mutant nematodes develop at the same rate, the longevity is unlikely to be caused by a clk-type reduction in oxidative metabolism, a potential alternative mechanism of phosphine resistance.

Identification of a D8S1179 Primer Binding Site Mutation and the Validation of a Primer Designed to Recover Null Alleles

Forensic Science International. May, 2003  |  Pubmed ID: 12787655

A population study of Chamorros and Filipinos using short tandem repeat (STR) loci amplified with the AmpFlSTR Profiler Plus PCR amplification kit demonstrated an excess of observed homozygosity at the D8S1179 locus. Use of a different set of D8S1179 primers to type the same samples did not demonstrate an excess of homozygosity and showed discordant genotypes at the D8S1179 locus. A single point mutation, G-to-A transition, 16 nucleotides from the 3' end of the reverse primer, was identified to cause allele dropout when using the AmpFlSTR Profiler Plus primer set. An additional D8S1179 reverse primer specific for the variant was constructed resulting in the recovery of the null allele. The primer was included in the newly developed AmpFlSTR Identifiler PCR amplification kit. No deleterious effects or non-specific peaks were observed in validation experiments evaluating primer concentration, Mg2+ concentration, annealing temperature and population samples.

Lateral Pterygoid Myotomy with Reattachment to the Condylar Neck: an Adjunct to Restore Function After Total Joint Reconstruction

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics. Jun, 2003  |  Pubmed ID: 12789146

The purpose of this study was to investigate the results of performing a lateral pterygoid myotomy with reattachment to the condylar stump to restore more normal function after total joint reconstructive surgery.

Characterisation of the Active Site of a Newly-discovered and Potentially Significant Post-proline Cleaving Endopeptidase Called ZIP Using LC-UV-MS

The Analyst. Jun, 2003  |  Pubmed ID: 12866886

There are enzymes that specifically recognise the amino acid proline within peptides and proteins that are called post-proline cleaving enzymes. Many of them are implicated in neurodegenerative disorders and psychiatric diseases. ZIP is one such newly-discovered peptidase. In this work, it has been purified from bovine serum and subjected to various analytical studies in order to characterise it. A series of reactions between synthesised peptides and ZIP were carried out in order to elucidate the size and specificity of the active site of the enzyme. On-line LC-MS was carried out on samples before and after incubation and the results obtained allowed us to detect if cleavage of the peptides was taking place, and if so, where in the peptide chain. It was found that the enzyme has a preference for another larger, bulky amino acid to follow the proline and that little or no cleavage was observed when polar acidic or other small amino acids were in that location. In terms of the size of the active site, the endopeptidase was found to have optimum activity when there were two more amino acids after the proline, with a fall-off in activity detected for the longer peptides. Data from kinetic studies confirmed the LC-MS results. The methodology described in this paper, which is a combination of LC separation and UV-MS detection, is required for the accurate monitoring of the reactions between the peptidase and its peptide substrates and for analysis of the products of such enzyme-peptide reactions. This work will assist in the design of site-directed inhibitors for new drug therapies.

Inhibition of Egg Development by Phosphine in the Cosmopolitan Pest of Stored Products Liposcelis Bostrychophila (Psocoptera: Liposcelididae)

Pest Management Science. Nov, 2003  |  Pubmed ID: 14620044

Phosphine-induced delay in development of eggs was investigated as a mechanism of resistance to this fumigant in Liposcelis bostrychophila Badonnel. One-day-old eggs of a susceptible and a strongly resistant strain of L bostrychophila were exposed to a range of phosphine concentrations for 6days at 30 (+/- 1) degrees C and 70 (+/- 2)% RH. Delay in mean hatching period occurred in both susceptible and resistant eggs, although it was more pronounced in the latter. A maximum delay of 2.65 days was recorded for eggs of the susceptible strain at 0.01 mg litre(-1) (the highest concentration at which eggs survived) and 13.39 days for the resistant strain at 1 mg litre(-1) (the highest concentration tested). Delay in egg development time was positively correlated with increasing phosphine concentration. Our results reveal that the most successful strategy to control resistant L bostrychophila is to apply relatively low concentrations of phosphine for extended exposure times (eg 0.05 mg litre(-1) for 16 days) that allow all eggs to hatch to the much less tolerant nymph stage.

The Purification and Characterisation of Novel Dipeptidyl Peptidase IV-like Activity from Bovine Serum

The International Journal of Biochemistry & Cell Biology. Jul, 2004  |  Pubmed ID: 15109572

The discovery of a potentially novel proline-specific peptidase from bovine serum is presented which is capable of cleaving the dipeptidyl peptidase IV (DPIV) substrate Gly-Pro-MCA. The enzyme was isolated and purified with the use of Phenyl Sepharose Hydrophobic Interaction, Sephacryl S-300 Gel Filtration, and Q-Sephacryl Anion Exchange, producing an overall purification factor of 257. SDS PAGE resulted in a monomeric molecular mass of 158kDa while size exclusion chromatography generated a native molecular mass of 328kDa. The enzyme remained active over a broad pH range with a distinct preference for a neutral pH range of 7-8.5. Chromatofocusing and isoelectric focusing (IEF) revealed the enzyme's isoelectric point to be 4.74. DPIV-like activity was not inhibited by serine protease inhibitors but was by the metallo-protease inhibitors, the phenanthrolines. The enzyme was also partially inhibited by bestatin. Substrate specificity studies proved that the enzyme is capable of sequential cleavage of bovine beta-Casomorphin and Substance P. The peptidase cleaved the standard DPIV substrate, Gly-Pro-MCA with a K(M) of 38.4 microM, while Lys-Pro-MCA was hydrolysed with a K(M) of 103 microM. The DPIV-like activity was specifically inhibited by both Diprotin A and B, non-competitively, generating a K(i) of 1.4 x 10(-4) M for both inhibitors. Ile-Thiazolidide and Ile-Pyrrolidide both inhibited competitively with an inhibition constant of 3.7 x 10(-7) and 7.5 x 10(-7) M, respectively. It is concluded that bovine serum DPIV-like activity share many biochemical properties with DPIV and DPIV-like enzymes but not exclusively, suggesting that the purified peptidase may play an important novel role in bioactive oligopeptide degradation.

Purification, Identification and Characterisation of Seprase from Bovine Serum

The International Journal of Biochemistry & Cell Biology. Nov, 2004  |  Pubmed ID: 15313476

The study and identification for the first time of a soluble form of a seprase activity from bovine serum is presented. To date, this activity has only been reported to be an integral membrane protease but has been known to shed from its membrane. The activity was purified 30,197-fold to homogeneity, using a combination of column chromatographies, from bovine serum. Inhibition by DFP, resulting in an IC(50) of 100:nM confirms classification as a serine protease. The protease after separation and visualisation by native PAGE was subjected to tryptic digestion and the subsequent peptides sequenced. Each peptide sequenced was found to be present in the primary structure of seprase/fibroblast activation protein (FAP), a serine gelatinase specific for proline-containing peptides and macromolecules. Substrate specificity studies using kinetic, RP-HPLC and LC-MS analysis of synthetic peptides suggest that this peptidase has an extended substrate-binding region in addition to the primary specificity site S(1). This analysis revealed at least five subsites to be involved in enzyme-substrate binding, with the smallest peptide cleaved being a tetrapeptide. A proline residue in position P(1) was absolutely necessary therefore showing high primary substrate specificity for the Pro-X bond, while a preference for a hydrophobic residue at the C-terminal end of the scissile bond (P'(1)) was evident. The enzyme also showed complete insensitivity to the prolyl oligopeptidase specific inhibitors, JTP-4819, Fmoc-Ala-pyrrCN and Z-Phe-Pro-BT. To date, no physiological substrate has clearly been defined for this protease but its ability to effectively degrade gelatin suggests a candidate protein substrate in vivo and a possible role in extracellular matrix protein degradation.

Developmental Validation of a Single-tube Amplification of the 13 CODIS STR Loci, D2S1338, D19S433, and Amelogenin: the AmpFlSTR Identifiler PCR Amplification Kit

Journal of Forensic Sciences. Nov, 2004  |  Pubmed ID: 15568700

Analysis of length polymorphism at short tandem repeat (STR) loci utilizing the polymerase chain reaction (PCR) process has proven to be an ideal assay for human identification purposes. The short length of STR loci coupled with the amplification of target sequence through PCR allows for a robust, sensitive, and specific assay for highly polymorphic markers. A multiplex containing fifteen STR loci plus the gender-determining locus Amelogenin was developed to provide a single amplification/detection of all CODIS (Combined DNA Index System) STR loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and vWA) as well as two internationally-accepted STRs (D2S1338 and D19S433). By incorporating five-dye fragment analysis technology and non-nucleotide linkers, previously optimized AmpFlSTR kit primer sequences have been maintained. This kit has been developed in accordance with the standards of the forensic community as defined by the DNA Advisory Board. Validation studies were performed to include developmental validation, and the results support the use of the AmpFlSTR Identifiler PCR Amplification Kit for human identity and parentage testing.

Identification of a Capsular Polysaccharide from Moraxella Bovis

Carbohydrate Research. Mar, 2005  |  Pubmed ID: 15721351

The bacterium Moraxella bovis is the causative agent of an economically important disease of cattle: Infectious Bovine Keratoconjunctivitis (IBK), otherwise known as pinkeye. Little is known regarding the structure of the carbohydrates produced by M. bovis. The structure of a capsular polysaccharide from M. bovis (strain Mb25) has been determined using NMR and MS analysis. From these data it is concluded that the polysaccharide is composed of the unmodified chondroitin disaccharide repeat unit.

Low Density Lipoprotein Non-esterified Fatty Acids and Lipoprotein Lipase in Diabetes

Atherosclerosis. Jul, 2005  |  Pubmed ID: 15939061

Fatty acid metabolism is disturbed in poorly controlled diabetes. Low density lipoprotein (LDL) oxidation, thought to be an atherogenic modification, is partly dependent on LDL fatty acid content whether it be in the form of cholesteryl ester, phospholipids, triglyceride or non-esterified fatty acid (NEFA). Lipoprotein lipase (LPL) is deficient in diabetic patients. Lipoprotein lipase bound to LDL may facilitate cholesterol accumulation in the artery wall through the attachment of LDL to the proteoglycans expressed on endothelial cells and collagen. The purpose of this study was to examine the degree of binding of fatty acids and lipoprotein lipase to LDL in type 2 diabetic patients and to examine the relationship between non-esterified fatty acids attached to LDL and LDL oxidisability.

Thermal Illuminators for Far-infrared and Submillimeter Astronomical Instruments

Applied Optics. Jun, 2005  |  Pubmed ID: 15943254

Astronomical instruments operating in the infrared-millimeter region often require internal sources for detector monitoring, instrument calibration, and health checking. We describe the design, modeling, and experimental evaluation of thermal emitters with a fast speed of response and low-power dissipation, suitable for the far-infrared-submillimeter region. The development of an internal illuminator for the Herschel Spectral and Photometric Imaging Receiver (SPIRE) satellite instrument is used as an example of the optimization of the design to meet particular requirements. A prototype illuminator design for SPIRE has been developed that, for a power dissipation of 1.5 mW, provides an equivalent blackbody temperature of 45 K with a 90% settling time of 220 ms.

Mini Dental Implants: an Adjunct for Retention, Stability, and Comfort for the Edentulous Patient

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics. Nov, 2005  |  Pubmed ID: 16243233

This article examines the success of mini-dental implants (MDI'S) by assessing four subjective measures of patient satisfaction for MDI's in the edentulous maxilla and mandible: comfort, retention, chewing ability and speaking ability. Success rates, surgical techniques, and financial advantages of the MDI's are reviewed.

Use of a Mixed Tissue RNA Design for Performance Assessments on Multiple Microarray Formats

Nucleic Acids Research. 2005  |  Pubmed ID: 16377776

The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes). The design provides four known ratio measurements of >200 reference probes, which were chosen for their tissue-selectivity, dynamic range coverage and alignment to the same exemplar transcript sequence across all three platforms. The data generated from testing three biological replicates of the reagent at eight laboratories on three array formats provides a benchmark set for both laboratory and data processing performance assessments. Close agreement with target ratios adjusted for sample complexity was achieved on all platforms and low variance was observed among platforms, replicates and sites. The mixed tissue design produces a reagent with known gene expression changes within a complex sample and can serve as a paradigm for performance standards for microarrays that target other species.

Haemostasis, Inflammation and Renal Function Following Exercise in Patients with Intermittent Claudication on Statin and Aspirin Therapy

Thrombosis Journal. 2006  |  Pubmed ID: 16848885

Previous studies have suggested that exercise in patients with intermittent claudication (IC) may induce a systemic thrombo-inflammatory response. The effect of secondary prevention therapy on this response is unknown. This study aimed to investigate the effects of treadmill exercise on markers of coagulation activation, inflammation and renal function in patients with IC, receiving aspirin and statin therapy compared to healthy controls.

The Structure of H5N1 Avian Influenza Neuraminidase Suggests New Opportunities for Drug Design

Nature. Sep, 2006  |  Pubmed ID: 16915235

The worldwide spread of H5N1 avian influenza has raised concerns that this virus might acquire the ability to pass readily among humans and cause a pandemic. Two anti-influenza drugs currently being used to treat infected patients are oseltamivir (Tamiflu) and zanamivir (Relenza), both of which target the neuraminidase enzyme of the virus. Reports of the emergence of drug resistance make the development of new anti-influenza molecules a priority. Neuraminidases from influenza type A viruses form two genetically distinct groups: group-1 contains the N1 neuraminidase of the H5N1 avian virus and group-2 contains the N2 and N9 enzymes used for the structure-based design of current drugs. Here we show by X-ray crystallography that these two groups are structurally distinct. Group-1 neuraminidases contain a cavity adjacent to their active sites that closes on ligand binding. Our analysis suggests that it may be possible to exploit the size and location of the group-1 cavity to develop new anti-influenza drugs.

Identification of a Novel Glycosyltransferase Involved in LOS Biosynthesis of Moraxella Catarrhalis

Carbohydrate Research. Nov, 2006  |  Pubmed ID: 16934238

Moraxella catarrhalis is an important human mucosal pathogen that contributes to otitis media in infants and exacerbates conditions such as chronic obstructive pulmonary disease in the elderly. This study describes the identification of a novel gene, lgt5 that encodes a glycosyltransferase involved in the LOS biosynthesis of M. catarrhalis. Analysis of NMR data of LOS-derived oligosaccharide from a Serotype A lgt5 mutant strain of M. catarrhalis indicate that lgt5 encodes an alpha-(1-->4)-galactosyltransferase.

The Agilent in Situ-synthesized Microarray Platform

Methods in Enzymology. 2006  |  Pubmed ID: 16938545

Microarray technology has become a standard tool in many laboratories. Agilent Technologies manufactures a variety of catalog and custom long-oligonucleotide (60-mer) microarrays that can be used in multiple two-color microarray applications. Optimized methods and techniques have been developed for two such applications: gene expression profiling and comparative genomic hybridization. Methods for a third technique, location analysis, are evolving rapidly. This chapter outlines current best methods for using Agilent microarrays, provides detailed instructions for the most recently developed techniques, and discusses solutions to common problems encountered with two-color microarrays.

Evaluation of External RNA Controls for the Assessment of Microarray Performance

Nature Biotechnology. Sep, 2006  |  Pubmed ID: 16964227

External RNA controls (ERCs), although important for microarray assay performance assessment, have yet to be fully implemented in the research community. As part of the MicroArray Quality Control (MAQC) study, two types of ERCs were implemented and evaluated; one was added to the total RNA in the samples before amplification and labeling; the other was added to the copyRNAs (cRNAs) before hybridization. ERC concentration-response curves were used across multiple commercial microarray platforms to identify problematic assays and potential sources of variation in the analytical process. In addition, the behavior of different ERC types was investigated, resulting in several important observations, such as the sample-dependent attributes of performance and the potential of using these control RNAs in a combinatorial fashion. This multiplatform investigation of the behavior and utility of ERCs provides a basis for articulating specific recommendations for their future use in evaluating assay performance across multiple platforms.

Performance Comparison of One-color and Two-color Platforms Within the MicroArray Quality Control (MAQC) Project

Nature Biotechnology. Sep, 2006  |  Pubmed ID: 16964228

Microarray-based expression profiling experiments typically use either a one-color or a two-color design to measure mRNA abundance. The validity of each approach has been amply demonstrated. Here we provide a simultaneous comparison of results from one- and two-color labeling designs, using two independent RNA samples from the Microarray Quality Control (MAQC) project, tested on each of three different microarray platforms. The data were evaluated in terms of reproducibility, specificity, sensitivity and accuracy to determine if the two approaches provide comparable results. For each of the three microarray platforms tested, the results show good agreement with high correlation coefficients and high concordance of differentially expressed gene lists within each platform. Cumulatively, these comparisons indicate that data quality is essentially equivalent between the one- and two-color approaches and strongly suggest that this variable need not be a primary factor in decisions regarding experimental microarray design.

The MicroArray Quality Control (MAQC) Project Shows Inter- and Intraplatform Reproducibility of Gene Expression Measurements

Nature Biotechnology. Sep, 2006  |  Pubmed ID: 16964229

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.

Disruption of Iron Homeostasis Increases Phosphine Toxicity in Caenorhabditis Elegans

Toxicological Sciences : an Official Journal of the Society of Toxicology. Mar, 2007  |  Pubmed ID: 17175555

The aim of this study is to identify the biochemical mechanism of phosphine toxicity and resistance, using Caenorhabditis elegans as a model organism. To date, the precise mode of phosphine action is unclear. In this report, we demonstrate the following dose-dependent actions of phosphine, in vitro: (1) reduction of ferric iron (Fe3+) to ferrous iron (Fe2+), (2) release of iron from horse ferritin, (3) and the peroxidation of lipid as a result of iron release from ferritin. Using in situ hybridization, we show that the ferritin genes of C. elegans, both ferritin-1 and ferritin-2, are expressed along the digestive tract with greatest expression at the proximal and distal ends. Basal expression of the ferritin-2 gene, as determined by quantitative PCR, is approximately 80 times that of ferritin-1. However, transcript levels of ferritin-1 are induced at least 20-fold in response to phosphine, whereas there is no change in the level of ferritin-2. This resembles the reported pattern of ferritin gene regulation by iron, suggesting that phosphine toxicity may be related to an increase in the level of free iron. Indeed, iron overload increases phosphine toxicity in C. elegans at least threefold. Moreover, we demonstrate that suppression of ferritin-2 gene expression by RNAi, significantly increases sensitivity to phosphine. This study identifies similarities between phosphine toxicity and iron overload and demonstrates that phosphine can trigger iron release from storage proteins, increasing lipid peroxidation, leading to cell injury and/or cell death.

Detection and Characterisation of Strong Resistance to Phosphine in Brazilian Rhyzopertha Dominica (F.) (Coleoptera: Bostrychidae)

Pest Management Science. Apr, 2007  |  Pubmed ID: 17315137

As failure to control Rhyzopertha dominica (F.) with phosphine is a common problem in the grain-growing regions of Brazil, a study was undertaken to investigate the frequency, distribution and strength of phosphine resistance in R. dominica in Brazil. Nineteen samples of R. dominica were collected between 1991 and 2003 from central storages where phosphine fumigation had failed to control this species. Insects were cultured without selection until testing in 2005. Each sample was tested for resistance to phosphine on the basis of the response of adults to discriminating concentrations of phosphine (20 and 48 h exposures) and full dose-response assays (48 h exposure). Responses of the Brazilian R. dominica samples were compared with reference susceptible, weak-resistance and strong-resistance strains from Australia in parallel assays. All Brazilian population samples showed resistance to phosphine: five were diagnosed with weak resistance and 14 with strong resistance. Five samples showed levels of resistance similar to the reference strong-resistance strain. A representative highly resistant sample was characterised by exposing mixed-age cultures to a range of constant concentrations of phosphine for various exposure periods. Time to population extinction (TPE) and time to 99.9% suppression of population (LT(99.9)) values of this sample were generally similar to those of the reference strong-resistance strain. For example, at 0.1, 0.5 and 1.0 mg L(-1), LT(99.9) values for BR33 and the reference strong-resistance strain were respectively 21, 6.4 and 3.7 days and 17, 6.2 and 3.8 days. With both strains, doubling phosphine concentrations to 2 mg L(-1) resulted in increased LT(99.9) and TPE. High level and frequency of resistance in all population samples, some of which had been cultured without selection for up to 12 years, suggest little or no fitness deficit associated with phosphine resistance. The present research indicates that widespread phosphine resistance may be developing in Brazil. Fumigation practices should be monitored and resistance management plans implemented to alleviate further resistance development.

Slow Diffusion of Lactose out of Galectin-3 Crystals Monitored by X-ray Crystallography: Possible Implications for Ligand-exchange Protocols

Acta Crystallographica. Section D, Biological Crystallography. Mar, 2007  |  Pubmed ID: 17327679

Galectin-3 is a multifunctional carbohydrate-binding protein that has roles in cancer progression. In addition to carbohydrate-dependent extracellular functions, galectin-3 participates in carbohydrate-independent intracellular signalling pathways, including apoptosis, via protein-protein interactions, some of which engage the carbohydrate-binding groove. When ligands bind within this site, conformational rearrangements are induced and information on unliganded galectin-3 is therefore valuable for structure-based drug design. Removal of cocrystallized lactose from the human galectin-3 carbohydrate-recognition domain was achieved via crystal soaking, but took weeks despite low affinity. Pre-soaking to remove lactose enabled the subsequent binding of cryoprotectant glycerol, whereas when the lactose was not removed a priori the glycerol could not displace it in the short cryosoaking time frame. This slow diffusion of lactose out of the crystals contrasts with the entrance of glycerol, which takes place within minutes. The importance of the removal of incumbent ligands prior to attempts to introduce alternative ligands is indicated, even for proteins exhibiting low affinity for ligands, and has significance for ligand exchange in structure-based drug design.

Towards Understanding the Functional Role of the Glycosyltransferases Involved in the Biosynthesis of Moraxella Catarrhalis Lipooligosaccharide

The FEBS Journal. Apr, 2007  |  Pubmed ID: 17388814

The glycosyltransferase enzymes (Lgts) responsible for the biosynthesis of the lipooligosaccharide-derived oligosaccharide structures from Moraxella catarrhalis have been investigated. This upper respiratory tract pathogen is responsible for a spectrum of illnesses, including otitis media (middle ear infection) in children, and contributes to exacerbations of chronic obstructive pulmonary disease in elderly patients. To investigate the function of the glycosyltransferase enzymes involved in the biosynthesis of lipooligosaccharide of M. catarrhalis and to gain some insight into the mechanism of serotype specificity for this microorganism, mutant strains of M. catarrhalis were produced. Examination by NMR and MS of the oligosaccharide structures produced by double-mutant strains (2951lgt1/4Delta and 2951lgt5/4Delta) and a single-mutant strain (2951lgt2Delta) of the bacterium has allowed us to propose a model for the serotype-specific expression of lipooligosaccharide in M. catarrhalis. According to this model, the presence/absence of Lgt4 and the Lgt2 allele determines the lipooligosaccharide structure produced by a strain. Furthermore, it is concluded that Lgt4 functions as an N-acetylglucosylamine transferase responsible for the addition of an alpha-D-GlcNAc (1-->2) glycosidic linkage to the (1-->4) branch, and also that there is competition between the glycosyltransferases Lgt1 and Lgt4. That is, in the presence of an active Lgt4, GlcNAc is preferentially added to the (1-->4) chain of the growing oligosaccharide, instead of Glc. In serotype B strains, which lack Lgt4, Lgt1 adds a Glc at this position. This implies that active Lgt4 has a much higher affinity/specificity for the beta-(1-->4)-linked Glc on the (1-->4) branch than does Lgt1.

Integrated Analysis of Experimental Data Sets Reveals Many Novel Promoters in 1% of the Human Genome

Genome Research. Jun, 2007  |  Pubmed ID: 17567992

The regulation of transcriptional initiation in the human genome is a critical component of global gene regulation, but a complete catalog of human promoters currently does not exist. In order to identify regulatory regions, we developed four computational methods to integrate 129 sets of ENCODE-wide chromatin immunoprecipitation data. They collectively predicted 1393 regions. Roughly 47% of the regions were unique to one method, as each method makes different assumptions about the data. Overall, predicted regions tend to localize to highly conserved, DNase I hypersensitive, and actively transcribed regions in the genome. Interestingly, a significant portion of the regions overlaps with annotated 3'-UTRs, suggesting that some of them might regulate anti-sense transcription. The majority of the predicted regions are >2 kb away from the 5'-ends of previously annotated human cDNAs and hence are novel. These novel regions may regulate unannotated transcripts or may represent new alternative transcription start sites of known genes. We tested 163 such regions for promoter activity in four cell lines using transient transfection assays, and 25% of them showed transcriptional activity above background in at least one cell line. We also performed 5'-RACE experiments on 62 novel regions, and 76% of the regions were associated with the 5'-ends of at least two RACE products. Our results suggest that there are at least 35% more functional promoters in the human genome than currently annotated.

Transcription Factor Binding and Modified Histones in Human Bidirectional Promoters

Genome Research. Jun, 2007  |  Pubmed ID: 17568000

Bidirectional promoters have received considerable attention because of their ability to regulate two downstream genes (divergent genes). They are also highly abundant, directing the transcription of approximately 11% of genes in the human genome. We categorized the presence of DNA sequence motifs, binding of transcription factors, and modified histones as overrepresented, shared, or underrepresented in bidirectional promoters with respect to unidirectional promoters. We found that a small set of motifs, including GABPA, MYC, E2F1, E2F4, NRF-1, CCAAT, YY1, and ACTACAnnTCC are overrepresented in bidirectional promoters, while the majority (73%) of known vertebrate motifs are underrepresented. We performed chromatin-immunoprecipitation (ChIP), followed by quantitative PCR for GABPA, on 118 regions in the human genome and showed that it binds to bidirectional promoters more frequently than unidirectional promoters, and its position-specific scoring matrix is highly predictive of binding. Signatures of active transcription, such as occupancy of RNA polymerase II and the modified histones H3K4me2, H3K4me3, and H3ac, are overrepresented in regions around bidirectional promoters, suggesting that a higher fraction of divergent genes are transcribed in a given cell than the fraction of other genes. Accordingly, analysis of whole-genome microarray data indicates that 68% of divergent genes are transcribed compared with 44% of all human genes. By combining the analysis of publicly available ENCODE data and a detailed study of GABPA, we survey bidirectional promoters with breadth and depth, leading to biological insights concerning their motif composition and bidirectional regulatory mode.

No Association of CETP Genotype with Cognitive Function or Age-related Cognitive Change

Neuroscience Letters. Jun, 2007  |  Pubmed ID: 17531380

A cholesteryl ester transfer protein (CETP) genotype (V/V homozygosity for I405V, NCBI dbSNP rs5882) has been associated with preservation of cognitive function in old age, in addition to its associations with exceptional longevity and cardiovascular disease. We tested the hypotheses that this polymorphism was associated with either level of cognitive function or lifetime cognitive change in 525 participants who took part in the Scottish Mental Survey of 1932. Participants took the same well-validated mental ability test at ages 11 and 79. Scores were also available for several other mental ability tests at age 79, and history of cardiovascular disease was available. Frequency of the V/I genotype was slightly greater than expected in the sample, possibly consistent with some role for the V polymorphism in longevity, and the V/V genotype was associated with a lower rate of cardiovascular disease history. There were, however, no significant associations of CETP genotype with either childhood IQ or current cognitive function in old age, or with lifetime change in cognitive function.

Walker Repair of the Temporomandibular Joint: a Retrospective Evaluation of 117 Patients

Journal of Oral and Maxillofacial Surgery : Official Journal of the American Association of Oral and Maxillofacial Surgeons. Oct, 2007  |  Pubmed ID: 17884522

This study evaluated the outcome of a high condylar shave with meniscal repositioning (Walker repair) in patients with internal derangement of the temporomandibular joint (TMJ). Changes in incisal opening, pain level, chewing ability, and preoperative TMJ symptoms (tinnitus, vertigo, and crepitus) were evaluated.

The Ets-related Transcription Factor GABP Directs Bidirectional Transcription

PLoS Genetics. Nov, 2007  |  Pubmed ID: 18020712

Approximately 10% of genes in the human genome are distributed such that their transcription start sites are located less than 1 kb apart on opposite strands. These divergent gene pairs have a single intergenic segment of DNA, which in some cases appears to share regulatory elements, but it is unclear whether these regions represent functional bidirectional promoters or two overlapping promoters. A recent study showed that divergent promoters are enriched for consensus binding sequences of a small group of transcription factors, including the ubiquitous ets-family transcription factor GA-binding protein (GABP). Here we show that GABP binds to more than 80% of divergent promoters in at least one cell type. Furthermore, we demonstrate that GABP binding is correlated and associated with bidirectional transcriptional activity in a luciferase transfection assay. In addition, we find that the addition of a strict consensus GABP site into a set of promoters that normally function in only one direction significantly increases activity in the opposite direction in 67% of cases. Our findings demonstrate that GABP regulates the majority of divergent promoters and suggest that bidirectional transcriptional activity is mediated through GABP binding and transactivation at both divergent and nondivergent promoters.

Aldosterone-mediated Apical Targeting of ENaC Subunits is Blunted in Rats with Streptozotocin-induced Diabetes Mellitus

Nephrology, Dialysis, Transplantation : Official Publication of the European Dialysis and Transplant Association - European Renal Association. May, 2008  |  Pubmed ID: 18029369

Diabetes mellitus (DM) is associated with a significant polyuria and natriuesis as well as increased plasma aldosterone and anti-diuretic hormone arginine vasopressin (AVP). This study aimed to determine whether diabetic kidneys compensate for the urinary sodium and water losses by increasing apical targeting of epithelial sodium channel (ENaC) subunits and aquaporin-2 (AQP2) in the collecting duct, in addition to the previously observed changes in ENaC subunit protein expression in different kidney zones.

Influence of Concentration, Temperature and Humidity on the Toxicity of Phosphine to the Strongly Phosphine-resistant Psocid Liposcelis Bostrychophila Badonnel (Psocoptera: Liposcelididae)

Pest Management Science. Sep, 2008  |  Pubmed ID: 18416433

The psocid Liposcelis bostrychophila Badonnel, is a widespread, significant pest of stored commodities, has developed strong resistance to phosphine, the major grain disinfestant. The aim was to develop effective fumigation protocols to control this resistant pest.

Integrated Detection and Population-genetic Analysis of SNPs and Copy Number Variation

Nature Genetics. Oct, 2008  |  Pubmed ID: 18776908

Dissecting the genetic basis of disease risk requires measuring all forms of genetic variation, including SNPs and copy number variants (CNVs), and is enabled by accurate maps of their locations, frequencies and population-genetic properties. We designed a hybrid genotyping array (Affymetrix SNP 6.0) to simultaneously measure 906,600 SNPs and copy number at 1.8 million genomic locations. By characterizing 270 HapMap samples, we developed a map of human CNV (at 2-kb breakpoint resolution) informed by integer genotypes for 1,320 copy number polymorphisms (CNPs) that segregate at an allele frequency >1%. More than 80% of the sequence in previously reported CNV regions fell outside our estimated CNV boundaries, indicating that large (>100 kb) CNVs affect much less of the genome than initially reported. Approximately 80% of observed copy number differences between pairs of individuals were due to common CNPs with an allele frequency >5%, and more than 99% derived from inheritance rather than new mutation. Most common, diallelic CNPs were in strong linkage disequilibrium with SNPs, and most low-frequency CNVs segregated on specific SNP haplotypes.

Integrated Genotype Calling and Association Analysis of SNPs, Common Copy Number Polymorphisms and Rare CNVs

Nature Genetics. Oct, 2008  |  Pubmed ID: 18776909

Accurate and complete measurement of single nucleotide (SNP) and copy number (CNV) variants, both common and rare, will be required to understand the role of genetic variation in disease. We present Birdsuite, a four-stage analytical framework instantiated in software for deriving integrated and mutually consistent copy number and SNP genotypes. The method sequentially assigns copy number across regions of common copy number polymorphisms (CNPs), calls genotypes of SNPs, identifies rare CNVs via a hidden Markov model (HMM), and generates an integrated sequence and copy number genotype at every locus (for example, including genotypes such as A-null, AAB and BBB in addition to AA, AB and BB calls). Such genotypes more accurately depict the underlying sequence of each individual, reducing the rate of apparent mendelian inconsistencies. The Birdsuite software is applied here to data from the Affymetrix SNP 6.0 array. Additionally, we describe a method, implemented in PLINK, to utilize these combined SNP and CNV genotypes for association testing with a phenotype.

Crystal Structures of Oseltamivir-resistant Influenza Virus Neuraminidase Mutants

Nature. Jun, 2008  |  Pubmed ID: 18480754

The potential impact of pandemic influenza makes effective measures to limit the spread and morbidity of virus infection a public health priority. Antiviral drugs are seen as essential requirements for control of initial influenza outbreaks caused by a new virus, and in pre-pandemic plans there is a heavy reliance on drug stockpiles. The principal target for these drugs is a virus surface glycoprotein, neuraminidase, which facilitates the release of nascent virus and thus the spread of infection. Oseltamivir (Tamiflu) and zanamivir (Relenza) are two currently used neuraminidase inhibitors that were developed using knowledge of the enzyme structure. It has been proposed that the closer such inhibitors resemble the natural substrate, the less likely they are to select drug-resistant mutant viruses that retain viability. However, there have been reports of drug-resistant mutant selection in vitro and from infected humans. We report here the enzymatic properties and crystal structures of neuraminidase mutants from H5N1-infected patients that explain the molecular basis of resistance. Our results show that these mutants are resistant to oseltamivir but still strongly inhibited by zanamivir owing to an altered hydrophobic pocket in the active site of the enzyme required for oseltamivir binding. Together with recent reports of the viability and pathogenesis of H5N1 (ref. 7) and H1N1 (ref. 8) viruses with neuraminidases carrying these mutations, our results indicate that it would be prudent for pandemic stockpiles of oseltamivir to be augmented by additional antiviral drugs, including zanamivir.

The Balance of Reproducibility, Sensitivity, and Specificity of Lists of Differentially Expressed Genes in Microarray Studies

BMC Bioinformatics. 2008  |  Pubmed ID: 18793455

Reproducibility is a fundamental requirement in scientific experiments. Some recent publications have claimed that microarrays are unreliable because lists of differentially expressed genes (DEGs) are not reproducible in similar experiments. Meanwhile, new statistical methods for identifying DEGs continue to appear in the scientific literature. The resultant variety of existing and emerging methods exacerbates confusion and continuing debate in the microarray community on the appropriate choice of methods for identifying reliable DEG lists.

Mandibular Midline Bicortical Bone Screw: an Adjunct to Genioglossus Insertion Advancement

Journal of Oral and Maxillofacial Surgery : Official Journal of the American Association of Oral and Maxillofacial Surgeons. Nov, 2008  |  Pubmed ID: 18940516

T-bet Dependent Removal of Sin3A-histone Deacetylase Complexes at the Ifng Locus Drives Th1 Differentiation

Journal of Immunology (Baltimore, Md. : 1950). Dec, 2008  |  Pubmed ID: 19050254

Forming and removing epigenetic histone marks at gene loci are central processes in differentiation. Here, we explored mechanisms establishing long-range H4 acetylation marks at the Ifng locus during Th1 lineage commitment. In Th0 cells, histone deacetylase (HDAC)-Sin3A complexes recruited to the Ifng locus actively prevented accumulation of H4 acetylation marks. Th1 differentiation caused loss of HDAC-Sin3A complexes by T-bet-dependent mechanisms and accumulation of H4 acetylation marks. HDAC-Sin3A complexes were absent from the locus in NOD Th0 cells, obviating the need for Th1 differentiation signals to establish histone marks and Th1 differentiation. Thus, Ifng transcription is actively prevented in Th0 cells via epigenetic mechanisms and epigenetic defects allow unregulated Ifng transcription that may contribute to autoimmunity.

Efficient Synthesis of Highly Active Phospha-isosteres of the Influenza Neuraminidase Inhibitor Oseltamivir

ChemMedChem. Mar, 2009  |  Pubmed ID: 19156651

With a Hunsdiecker-Barton iododecarboxylation strategy, we converted the carboxylate group of the oseltamivir precursor into exemplary phosphonate monoesters. In all cases, K(i) values towards influenza virus sialidase remained in the sub-nanomolar range. We have thus made valuable structural space available for the design of novel oseltamivir-based tools for influenza virus research.

Epigenetics and T Helper 1 Differentiation

Immunology. Mar, 2009  |  Pubmed ID: 19178593

Naïve T helper cells differentiate into two subsets, T helper 1 and 2, which either transcribe the Ifng gene and silence the Il4 gene or transcribe the Il4 gene and silence the Ifng gene, respectively. This process is an essential feature of the adaptive immune response to a pathogen and the development of long-lasting immunity. The 'histone code' hypothesis proposes that formation of stable epigenetic histone marks at a gene locus that activate or repress transcription is essential for cell fate determinations, such as T helper 1/T helper 2 cell fate decisions. Activation and silencing of the Ifng gene are achieved through the creation of stable epigenetic histone marks spanning a region of genomic DNA over 20 times greater than the gene itself. Key transcription factors that drive the T helper 1 lineage decision, signal transducer and activator 4 (STAT4) and T-box expressed in T cells (T-bet), play direct roles in the formation of activating histone marks at the Ifng locus. Conversely, STAT6 and GATA binding protein 3, transcription factors essential for the T helper 2 cell lineage decision, establish repressive histone marks at the Ifng locus. Functional studies demonstrate that multiple genomic elements up to 50 kilobases from Ifng play critical roles in its proper transcriptional regulation. Studies of three-dimensional chromatin conformation indicate that these distal regulatory elements may loop towards Ifng to regulate its transcription. We speculate that these complex mechanisms have evolved to tightly control levels of interferon-gamma production, given that too little or too much production would be very deleterious to the host.

Homocysteine Increases the Risk Associated with Hyperlipidaemia

European Journal of Cardiovascular Prevention and Rehabilitation : Official Journal of the European Society of Cardiology, Working Groups on Epidemiology & Prevention and Cardiac Rehabilitation and Exercise Physiology. Apr, 2009  |  Pubmed ID: 19276985

The European Concerted Action Project 'Homocysteine and Vascular Disease' showed that an elevated homocysteine is associated with a substantially increased risk of cardiovascular disease, and particularly when combined with other factors such as smoking, hypertension and hypercholesterolaemia. The purpose of this study was to examine the potential interactions between homocysteine and individual lipid subfractions. In addition, it was hypothesized that HDL cholesterol may protect against hyperhomocysteinaemia because HDL cholesterol is associated with the enzyme paroxonase, which reduces oxidization of homocysteine to the harmful metabolite, homocysteine thiolactonase.

Galactose-conjugates of the Oseltamivir Pharmacophore--new Tools for the Characterization of Influenza Virus Neuraminidases

Organic & Biomolecular Chemistry. Jun, 2009  |  Pubmed ID: 19503932

We describe the synthesis of mimetics of the alpha2-3 and alpha2-6 sialogalactoside substrates of influenza neuraminidase which include the oseltamivir pharmacophore, and report the sub-nanomolar affinities for these novel neuraminidase inhibitors. The challenge of synthesizing a Phospha-Oseltamivir/Tamiphosphor monoester involving the secondary 3-hydroxy group of galactose required to mimic the alpha2-3 sialogalactoside has been overcome by palladium-promoted coupling of the oseltamivir-derived vinyl iodide with a protected galactose-3-phosphonate. The difference in binding of these two inhibitors to a given influenza neuraminidase should be a function of its alpha2-3/alpha2-6-selectivity, an important, but not yet fully understood factor in the adaptation of highly pathogenic avian influenza viruses to human hosts.

Biochemical Analysis of Lgt3, a Glycosyltransferase of the Bacterium Moraxella Catarrhalis

Biochemical and Biophysical Research Communications. Mar, 2010  |  Pubmed ID: 20153730

The lipooligosaccharide (LOS) of Moraxella catarrhalis is unusual in that it lacks heptose. The sugar linking oligosaccharide to Lipid A is a trisubstituted glucose. A single enzyme, Lgt3, is suggested to trisubstitute this core sugar. The lgt3 gene encodes two distinct domains with high similarity to glucosyltransferases of the GT-A superfamily, thus encoding a bidomain, multifunctional glucosyltransferase. To characterise Lgt3, the gene was amplified from M. catarrhalis, expressed in Escherichia coli, and purified. Analysis of its glycosyltransferase catalytic activity ascertained the pH and temperature optima for Lgt3. The donor specificity and acceptor specificity were examined. This study confirms that Lgt3 is a glucosyltransferase which catalyses addition of glucose to its cognate receptor, a terminal glucose presented as the core region of LOS.

Cervical Support Collar: a Substitute to the Jaw Thrust/chin Lift Methods of Airway Management During Oral Maxillofacial Surgeries

Journal of Oral and Maxillofacial Surgery : Official Journal of the American Association of Oral and Maxillofacial Surgeons. Mar, 2010  |  Pubmed ID: 20171488

Circulation of Human Influenza Viruses and Emergence of Oseltamivir-resistant A(H1N1) Viruses in Cameroon, Central Africa

BMC Infectious Diseases. 2010  |  Pubmed ID: 20205961

While influenza surveillance has increased in most developing countries in the last few years, little influenza surveillance has been carried out in sub-Saharan Africa and no information is available in Central Africa. The objective of this study was to assess the prevalence of influenza viruses circulating in Yaounde, Cameroon and determine their antigenic and genetic characteristics.

Neuraminidase Receptor Binding Variants of Human Influenza A(H3N2) Viruses Resulting from Substitution of Aspartic Acid 151 in the Catalytic Site: a Role in Virus Attachment?

Journal of Virology. Jul, 2010  |  Pubmed ID: 20410266

Changes in the receptor binding characteristics of human H3N2 viruses have been evident from changes in the agglutination of different red blood cells (RBCs) and the reduced growth capacity of recently isolated viruses, particularly in embryonated eggs. An additional peculiarity of viruses circulating in 2005 to 2009 has been the poor inhibition of hemagglutination by postinfection ferret antisera for many viruses isolated in MDCK cells, including homologous reference viruses. This was shown not to be due to an antigenic change in hemagglutinin (HA) but was shown to be the result of a mutation in aspartic acid 151 of neuraminidase (NA) to glycine, asparagine, or alanine, which caused an oseltamivir-sensitive agglutination of RBCs. The D151G substitution was shown to cause a change in the specificity of NA such that it acquired the capacity to bind receptors, which were refractory to enzymatic cleavage, without altering its ability to remove receptors for HA. Thus, the inhibition of NA-dependent agglutination by the inclusion of oseltamivir carboxylate in the assay was effective in restoring the anti-HA specificity of the hemagglutination inhibition (HI) assay for monitoring antigenic changes in HA. Since the NA-dependent binding activity did not affect virus neutralization, and virus populations in clinical specimens possessed, at most, low levels of the "151 mutant," the biological significance of this feature of NA in, for example, immune evasion is unclear. It is apparent, however, that an important role of aspartic acid 151 in the activity of NA may be to restrict the specificity of the NA interaction and its receptor-destroying activity to complement that of HA receptor binding.

Keeping One's Option Open

Immunity. May, 2010  |  Pubmed ID: 20510865

In the periphery, T helper cell differentiation is a key event orchestrating the adaptive immune response yet recent studies demonstrate considerable plasticity in these cell fate decisions. In this issue of Immunity, Mukasa et al. (2010) describe the epigenetic basis underlying this plasticity.

Distal Regions of the Human IFNG Locus Direct Cell Type-specific Expression

Journal of Immunology (Baltimore, Md. : 1950). Aug, 2010  |  Pubmed ID: 20574006

Genes, such as IFNG, which are expressed in multiple cell lineages of the immune system, may employ a common set of regulatory elements to direct transcription in multiple cell types or individual regulatory elements to direct expression in individual cell lineages. By employing a bacterial artificial chromosome transgenic system, we demonstrate that IFNG employs unique regulatory elements to achieve lineage-specific transcriptional control. Specifically, a one 1-kb element 30 kb upstream of IFNG activates transcription in T cells and NKT cells but not in NK cells. This distal regulatory element is a Runx3 binding site in Th1 cells and is needed for RNA polymerase II recruitment to IFNG, but it is not absolutely required for histone acetylation of the IFNG locus. These results support a model whereby IFNG uses cis-regulatory elements with cell type-restricted function.

Identification of a G2-like Porcine Rotavirus Bearing a Novel VP4 Type, P[32]

Veterinary Research. Sep-Oct, 2010  |  Pubmed ID: 20663474

A porcine group A rotavirus (GARV) strain, 61/07/Ire, was isolated from a 4-5 week asymptomatic piglet, during an epidemiological survey of porcine herds in Southern Ireland, in 2007. The nucleotide (nt) and amino acid (aa) sequence of the full-length VP4 protein of the PoRV strain 61/07/Ire was determined. Based on the entire VP4 open reading frame (nt), strain 61/07/Ire displayed ≤76.5% identity to representatives of the established 31 P-types, a value far lower than the percentage identity cutoff value (80%) established by the Rotavirus Classification Working Group (RCWG) to define a novel P genotype. Strain 61/07/Ire revealed low aa identity, ranging from 57.1% to 83.6%, to the cognate sequences of representatives of the various P genotypes. The aa identity was lower in the VP8* trypsin-cleavage fragment of the VP4, which encompasses the VP4 hypervariable region, ranging from 36.9% to 75.3%. Sequence analyses of the VP7, VP6, and NSP4 genes revealed that the GARV strain 61/07/Ire possessed a G2-like VP7, an E9 NSP4 genotype and an I5 VP6 genotype. Altogether, these results indicate that the GARV strain 61/07/Ire should be considered as a prototype of a new VP4 genotype, P[32], and provide further evidence for the vast heterogeneity of group A rotaviruses.

Follicular B Cell Trafficking Within the Spleen Actively Restricts Humoral Immune Responses

Immunity. Aug, 2010  |  Pubmed ID: 20691614

Follicular (FO) and marginal zone (MZ) B cells are maintained in distinct locations within the spleen, but the genetic basis for this separation is still enigmatic. We now report that B cell sequestration requires lineage-specific regulation of migratory receptors by the transcription factor Klf2. Moreover, using gene-targeted mice we show that altered splenic B cell migration confers a significant in vivo gain-of-function phenotype to FO B cells, including the ability to quickly respond to MZ-associated antigens and pathogens in a T cell-dependent manner. This work demonstrates that in wild-type animals, naive FO B cells are actively removed from the MZ, thus restricting their capacity to respond to blood-borne pathogens.

Galectin Inhibitory Disaccharides Promote Tumour Immunity in a Breast Cancer Model

Cancer Letters. Dec, 2010  |  Pubmed ID: 20826047

High level galectin-1 expression results in cancer cell evasion of the immune response, increased tumour survival and aggressive metastases. Using a galectin-1 polyclonal antibody, high levels of galectin-1 protein were shown to be expressed by breast cancer cells established from FVB/N MMTV-c-neu mice as well as by the B16F10 melanoma cell line. In mixed lymphocyte cultures using tumour cells as antigenic stimulators, addition of recombinant galectin-1 dose-dependently inhibited lymphocyte production. Disaccharides were identified that inhibited galectin-1 function and increased growth and activation of CD8(+) CTL's killing cancer cells. X-ray crystallographic structures of human galectin-1 in complex with inhibitory disaccharides revealed their mode of binding. Combining galectin-blocking carbohydrates as adjuvants with vaccine immunotherapy in vivo to promote immune responses significantly decreased tumour progression and improved the outcomes for tumour challenged mice. This is the first report showing that suitably selected galectin-1 blocking disaccharides will act as adjuvants promoting vaccine stimulated immune responses against tumours in vivo.

[Impact of Avian Influenza Virus H5N1 Neuraminidase Mutations on the Activity of Neuraminidase and the Sensibility to Neuraminidase Inhibitors]

Zhonghua Yi Xue Za Zhi. Jul, 2010  |  Pubmed ID: 20979914

To study the impact of avian influenza virus H5N1 neuraminidase mutations I117V, I314V and I117V + I314V on the sensibility of neuraminidase inhibitors (NAIs) and the activity of neuraminidase (NA).

Integrin α(IIb)β₃ Exists in an Activated State in Subjects with Elevated Plasma Homocysteine Levels

Platelets. 2011  |  Pubmed ID: 21133649

Elevated levels of plasma homocysteine (Hcy) are an independent risk factor for cardiovascular disease and thrombosis. The molecular basis for this phenomenon is not known but may relate to modification of cell surface thiols. The platelet specific integrin α(IIb)β₃ is a cysteine-rich cell adhesion molecule that plays a critical role in platelet aggregation and adhesion in haemostasis and thrombosis. In this study, we looked for evidence of a homocysteine-induced modification of α(IIb)β₃ using a fluorescently labeled PAC-1 antibody that recognizes the activated conformation of the integrin on the platelet surface. We show that exogenous Hcy (10-100 µM) and homocysteine thiolactone (HcyTL) (10-100 µM) increased PAC-1 binding to platelets in a concentration dependent manner in vitro. In parallel, we show subjects with clinical hyperhomocysteinemia exhibit a greater degree of activation of α(IIb)β₃ compared to age-matched controls. These findings demonstrate that circulating Hcy can modulate the activation state of the platelet integrin α(IIb)β₃, a key player in platelet aggregation and thrombosis.

Two-tiered Approach Identifies a Network of Cancer and Liver Disease-related Genes Regulated by MiR-122

The Journal of Biological Chemistry. May, 2011  |  Pubmed ID: 21402708

MicroRNAs function as important regulators of gene expression and are commonly linked to development, differentiation, and diseases such as cancer. To better understand their roles in various biological processes, identification of genes targeted by microRNAs is necessary. Although prediction tools have significantly helped with this task, experimental approaches are ultimately required for extensive target search and validation. We employed two independent yet complementary high throughput approaches to map a large set of mRNAs regulated by miR-122, a liver-specific microRNA implicated in regulation of fatty acid and cholesterol metabolism, hepatitis C infection, and hepatocellular carcinoma. The combination of luciferase reporter-based screening and shotgun proteomics resulted in the identification of 260 proteins significantly down-regulated in response to miR-122 in at least one method, 113 of which contain predicted miR-122 target sites. These proteins are enriched for functions associated with the cell cycle, differentiation, proliferation, and apoptosis. Among these miR-122-sensitive proteins, we identified a large group with strong connections to liver metabolism, diseases, and hepatocellular carcinoma. Additional analyses, including examination of consensus binding motifs for both miR-122 and target sequences, provide further insight into miR-122 function.

Increased Sensitivity to Apoptosis Induced by Methotrexate is Mediated by JNK

Arthritis and Rheumatism. Sep, 2011  |  Pubmed ID: 21618198

Low-dose methotrexate (MTX) is an effective therapy for rheumatoid arthritis (RA), yet its mechanism of action is incompletely understood. The aim of this study was to explore the induction of apoptosis by MTX.

A Neutralizing Antibody Selected from Plasma Cells That Binds to Group 1 and Group 2 Influenza A Hemagglutinins

Science (New York, N.Y.). Aug, 2011  |  Pubmed ID: 21798894

The isolation of broadly neutralizing antibodies against influenza A viruses has been a long-sought goal for therapeutic approaches and vaccine design. Using a single-cell culture method for screening large numbers of human plasma cells, we isolated a neutralizing monoclonal antibody that recognized the hemagglutinin (HA) glycoprotein of all 16 subtypes and neutralized both group 1 and group 2 influenza A viruses. Passive transfer of this antibody conferred protection to mice and ferrets. Complexes with HAs from the group 1 H1 and the group 2 H3 subtypes analyzed by x-ray crystallography showed that the antibody bound to a conserved epitope in the F subdomain. This antibody may be used for passive protection and to inform vaccine design because of its broad specificity and neutralization potency.

Taloside Inhibitors of Galectin-1 and Galectin-3

Chemical Biology & Drug Design. Mar, 2012  |  Pubmed ID: 22136701

Galectin-1 and galectin-3 have roles in cancer and inflammation. Galectin-1 has recently emerged as a significant protein produced by tumour cells to promote tumour development, angiogenesis and metastasis and consequently represents an important target to inhibit. The design of inhibitors targeting the carbohydrate recognition domain that is known to recognize galactose is an important approach in the fight against cancer. Based on the analysis of crystal structures, we pursued the concept that if the galactose was replaced with talose (the C2 epimer of galactose) as a scaffold, then O2 substituents would be directed closer to the protein surface and provide opportunity to design inhibitors that are more specific towards particular galectins. Our elucidation of X-ray crystal structures of two of our synthesized talosides in complex with galectin-1 and galectin-3 provides the first atomic information on the interactions of galectins, and indeed any protein, with talosides. These results have enabled a structure-based rationale for the specificity differences shown by galectin-1 and galectin-3 towards these talosides and demonstrate new opportunities for further exploitation as specific inhibitors of galectins.

Diverse Functions of Distal Regulatory Elements at the IFNG Locus

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2012  |  Pubmed ID: 22246629

Previous studies have identified multiple conserved noncoding sequences (CNS) at the mouse Ifng locus sufficient for enhancer activity in cell-based assays. These studies do not directly address biology of the human IFNG locus in a genomic setting. IFNG enhancers may be functionally redundant or each may be functionally unique. We test the hypothesis that each IFNG enhancer has a unique necessary function using a bacterial artificial chromosome transgenic model. We find that CNS-30, CNS-4, and CNS+20 are required at distinct stages of Th1 differentiation, whereas CNS-16 has a repressive role in Th1 and Th2 cells. CNS+20 is required for IFN-γ expression by memory Th1 cells and NKT cells. CNS-4 is required for IFN-γ expression by effector Th1 cells. In contrast, CNS-16, CNS-4, and CNS+20 are each partially required for human IFN-γ expression by NK cells. Thus, IFNG CNS enhancers have redundant necessary functions in NK cells but unique necessary functions in Th cells. These results also demonstrate that distinct CNSs are required to transcribe IFNG at each stage of the Th1 differentiation pathway.