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Biochemical Data (biochemical + data)
Selected AbstractsBackbone dynamics of SDF-1, determined by NMR: Interpretation in the presence of monomer,dimer equilibriumPROTEIN SCIENCE, Issue 11 2006Olga K. Baryshnikova Abstract SDF-1, is a member of the chemokine family implicated in various reactions in the immune system. The interaction of SDF-1, with its receptor, CXCR4, is responsible for metastasis of a variety of cancers. SDF-1, is also known to play a role in HIV-1 pathogenesis. The structures of SDF-1, determined by NMR spectroscopy have been shown to be monomeric while X-ray structures are dimeric. Biochemical data and in vivo studies suggest that dimerization is likely to be important for the function of chemokines. We report here the dynamics of SDF-1, determined through measurement of main chain 15N NMR relaxation data. The data were obtained at several concentrations of SDF-1, and used to determine a dimerization constant of ,5 mM for a monomer,dimer equilibrium. The dimerization constant was subsequently used to extrapolate values for the relaxation data corresponding to monomeric SDF-1,. The experimental relaxation data and the extrapolated data for monomeric SDF-1, were analyzed using the model free approach. The model free analysis indicated that SDF-1, is rigid on the nano- to picosecond timescale with flexible termini. Several residues involved in the dimer interface display slow micro- to millisecond timescale motions attributable to chemical exchange such as monomer,dimer equilibrium. NMR relaxation measurements are shown to be applicable for studying oligomerization processes such as the dimerization of SDF-1,. [source] Biological characterization of glutaraldehyde-modified Parietaria judaica pollen extractsCLINICAL & EXPERIMENTAL ALLERGY, Issue 2 2004I. Ibarrola Summary Background Allergoids are widely used in specific immunotherapy (SIT) for the treatment of IgE-mediated allergic diseases, but all techniques for standardization of conventional allergic extracts may not be appropriate for standardization of a glutaraldehyde (GA)-modified extract because of the unique characteristics of these extracts. Objective To assess an accurate methodology for standardization of chemically modified extracts. Methods GA-modified extracts from Parietaria judaica pollen were purified by diafiltration. Biochemical properties were investigated by determination of amino groups, chromatography, and SDS-PAGE. The IgE-binding activity was determined by skin prick test, enzyme allergosorbent test inhibition, basophil activation, and histamine release tests. Peripheral blood mononuclear cells (PBMCs) from P. judaica pollen-allergic subjects were stimulated with either native or allergoid extracts, and proliferation was measured. Results Biochemical data indicated a high degree of allergen polymerization resulting in extract components higher than 100 kDa. IgE-binding activity, both in vivo and in vitro, was reduced by more than 99.8%. Both allergen and allergoid induced PBMC proliferation and synthesis of blocking IgG antibodies at similar rates. Moreover, no evidence of introduction of new determinants by chemical modification was found. Conclusions The preparation of GA-modified extracts by diafiltration is faster and more reliable than previous chromatographic methods. These modified extracts have drastically reduced their allergenicity while maintaining their immunogenicity, and therefore they can be used in safer and shortened schedules of SIT. [source] Rapid and easy semi-quantitative evaluation method for diacylglycerol and inositol-1,4,5-trisphosphate generation in orexin receptor signallingACTA PHYSIOLOGICA, Issue 3 2010M. E. Ekholm Abstract Aim:, Fluorescent protein-based indicators have enabled measurement of intracellular signals previously nearly inaccessible for studies. However, indicators showing intracellular translocation upon response suffer from serious limitations, especially the very time-consuming data collection. We therefore set out in this study to evaluate whether fixing and counting cells showing translocation could mend this issue. Methods:, Altogether three different genetically encoded indicators for diacylglycerol and inositol-1,4,5-trisphosphate were transiently expressed in Chinese hamster ovary cells stably expressing human OX1 orexin receptors. Upon stimulation with orexin-A, the cells were fixed with six different protocols. Results:, Different protocols showed clear differences in their ability to preserve the indicator's localization (i.e. translocation after stimulus) and its fluorescence, and the best results for each indicator were obtained with a different protocol. The concentration,response data obtained with cell counting are mostly comparable to the real-time translocation and biochemical data. Conclusion:, The counting method, as used here, works at single time point and looses the single-cell-quantitative aspect. However, it also has some useful properties. First, it easily allows processing of a 100- to 1000-fold higher cell numbers than real-time imaging producing statistically consistent population-quantitative data much faster. Secondly, it does not require expensive real-time imaging equipment. Fluorescence in fixed cells can also be quantitated, though this analysis would be more time-consuming than cell counting. Thirdly, in addition to the quantitative data collection, the method could be applied for identifying responsive cells. This might be very useful in identification of e.g. orexin-responding neurones in a large population of non-responsive cells in primary cultures. [source] Improvements in insulin sensitivity and ,-cell function (HOMA) with weight loss in the severely obeseDIABETIC MEDICINE, Issue 2 2003J. B. Dixon Abstract Aims To examine the effect of weight loss on insulin sensitivity and ,-cell function in severely obese subjects of varying glycaemic control. Patients and methods Subjects were 254 (F:M 209:45) patients having adjustable gastric banding for severe obesity, with paired biochemical data from before operation and at 1-year follow up. The homeostatic model assessment method was used to calculate insulin sensitivity (HOMA%S) and ,-cell function (HOMA%B). Subjects were grouped by diabetic status and by pre-weight loss HbA1c. Results Initial mean (sd) weight and body mass index were 128 (26) kg and 46.2 (7.7) kg/m2, respectively, and at 1-year were 101 (22) kg and 36.4 (6.7) kg/m2. The percentage of excess weight lost (%EWL) was 44.3 (14)%. HOMA%S improved from 37.5 (16)% presurgery to 62 (25)% (P < 0.001). %EWL was the only predictor of HOMA%S improvement (r = 0.28, P < 0.001). Subjects with normal fasting glucose, impaired fasting glucose and Type 2 diabetes had a fall, no change and increase in HOMA%B, respectively. The improvement in HOMA%B in subjects with diabetes (n = 39) was inversely related to the time with diabetes (r = ,0.36, P = 0.02). In non-diabetic subjects the HOMA%S,HOMA%B relationship was favourably altered with weight loss, so that for any given HOMA%S there was an increase in HOMA%B (f = 11.8, P = 0.001). This improvement in HOMA%B was positively related to %EWL (r = 0.25, P = 0.019). Discussion There are beneficial changes in both insulin sensitivity and ,-cell function with weight loss. Modern laparoscopic obesity surgery may have an important early role in the management of Type 2 diabetes in obese subjects. [source] The periplasmic peptidyl prolyl cis,trans isomerases PpiD and SurA have partially overlapping substrate specificitiesFEBS JOURNAL, Issue 13 2008Krista H. Stymest One of the rate-limiting steps in protein folding has been shown to be the cis,trans isomerization of proline residues, catalysed by a range of peptidyl prolyl cis,trans isomerases (PPIases). In the periplasmic space of Escherichia coli and other Gram-negative bacteria, two PPIases, SurA and PpiD, have been identified, which show high sequence similarity to the catalytic domain of the small PPIase parvulin. This observation raises a question regarding the biological significance of two apparently similar enzymes present in the same cellular compartment: do they interact with different substrates or do they catalyse different reactions? The substrate-binding motif of PpiD has not been characterized so far, and no biochemical data were available on how this folding catalyst recognizes and interacts with substrates. To characterize the interaction between model peptides and the periplasmic PPIase PpiD from E. coli, we employed a chemical crosslinking strategy that has been used previously to elucidate the interaction of substrates with SurA. We found that PpiD interacted with a range of model peptides independently of whether they contained proline residues or not. We further demonstrate here that PpiD and SurA interact with similar model peptides, and therefore must have partially overlapping substrate specificities. However, the binding motif of PpiD appears to be less specific than that of SurA, indicating that the two PPIases might interact with different substrates. We therefore propose that, although PpiD and SurA have partially overlapping substrate specificities, they fulfil different functions in the cell. [source] Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesisFEBS JOURNAL, Issue 22 2000Nikolas E. Labrou The 2,,3,-dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mm for the fast phase, 0.45 mm for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min,1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme,oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme,oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP-modified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the modeller 4 program. The model confirmed that Lys360 is positioned at the NAD+ -binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360,Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360,Ala enzyme. The data are discussed in terms of engineering coenzyme specificity. [source] p.Gln200Glu, a putative constitutively active mutant of rod ,-transducin (GNAT1) in autosomal dominant congenital stationary night blindness,,HUMAN MUTATION, Issue 7 2007Viktoria Szabo Abstract Congenital stationary night blindness (CSNB) is a non-progressive Mendelian condition resulting from a functional defect in rod photoreceptors. A small number of unique missense mutations in the genes encoding various members of the rod phototransduction cascade, e.g. rhodopsin (RHO), cGMP phosphodiesterase ,-subunit (PDE6B), and transducin ,-subunit (GNAT1) have been reported to cause autosomal dominant (ad) CSNB. While the RHO and PDE6B mutations result in constitutively active proteins, the only known adCSNB-associa-ted GNAT1 change (p.Gly38Asp) produces an ,-transducin that is unable to activate its downstream effector molecule in vitro. In a multigeneration Danish family with adCSNB, we identified a novel heterozygous C to G transversion (c.598C>G) in exon 6 of GNAT1 that should result in a p.Gln200Glu substitution in the evolutionarily highly conserved Switch 2 region of ,-transducin, a domain that has an important role in binding and hydrolyzing GTP. Computer modeling based on the known crystal structure of transducin suggests that the p.Gln200Glu mutant exhibits impaired GTPase activity, and thereby leads to constitutive activation of phototransduction. This assumption is in line with our results of trypsin protection assays as well as previously published biochemical data on mutants of this glutamine in the GTPase active site of ,-transducin following in vitro expression, and observations that inappropriately activating mutants of various members of the rod phototransduction cascade represent one of the major molecular causes of adCSNB. © 2007 Wiley-Liss, Inc. [source] Enzymatic and immunochemical evaluation of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in testes and epididymal spermatozoa of rats of different agesINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 2 2002Federica Tramer Selenium (Se) and selenoproteins such as glutathione peroxidases are necessary for the proper development and fertilizing capacity of sperm cells. Phospholipid hydroperoxide glutathione peroxidase (PHGPx, E.C. 1.11.1.12) is a monomeric seleno-enzyme present in different mammalian tissues in soluble and bound form. Its function, like the other glutathione peroxidases, was originally viewed as a protective role against hydroperoxides, but direct and indirect evidence indicates that it has additional regulatory roles. PHGPx is present in testis cells and sperm cells, and its appearance is hormone regulated. We present here biochemical data, which clearly indicate that the enzyme specific activity in rat is age-dependent during the life-span monitored (from 36 to 365 days), with a maximum at 3 months of age in the testis germ cells and at 6 months of age in the isolated epididymal sperm cells. Western blotting and immunocytochemical analysis by means of anti-PHGPx antibodies show the different distribution and the strong binding of PHGPx in the testes and sperm cell subcellular compartments (nucleus, acrosome, mitochondria and residual bodies) of rats of different age. The presence of the protein exhibits in the testis cells a pattern different from that of the catalytic activity, with a maximum at 6 months of age. The subcellular distribution of PHGPx is qualitatively, but not quantitatively, unchanged during ageing. These different behaviours are compared and discussed. [source] Angiopoietin-2 expression in breast cancer correlates with lymph node invasion and short survivalINTERNATIONAL JOURNAL OF CANCER, Issue 4 2003Christian Sfiligoi Abstract Angiogenic factors produced by tumor cells are essential for tumor growth and metastasis. In our study, the expression of Angiopoietin-1 (ANG1) and Angiopoietin-2 (ANG2) mRNA in archival human breast cancer tumor samples and in 6 breast cancer cell lines was investigated. Total RNA from biopsies of 38 breast cancer patients was extracted and ANG1 and ANG2 mRNA expression was measured by means of quantitative real-time RT-PCR (Taqman®). Matching data with available clinicopathologic and biochemical data revealed a significant association between ANG2 expression and axillary lymph node invasion. Univariate and multivariate survival analysis, by means of Kaplan-Meier method and Cox's proportional hazards model, showed significant and independent association between ANG2 mRNA level and both disease-free (p < 0.0001) and overall survival (p < 0.0003). An important fact is that, notwithstanding the small number of cases examined, this association was confirmed also in the group of lymph node-negative patients (DFS, p < 0.003; OS, p < 0.020). Immunohistochemical analysis demonstrated that Ang2 is expressed by both tumor cells and endothelial elements. Expression in tumor cells was confirmed by studying a panel of human breast carcinoma cell lines in culture by RT-PCR. In ZR75.1 and T47D cells, expression of ANG2 mRNA was increased up to 10-fold by treatment with estrogen within 24 hr. Although preliminary, these data suggest a possible role of ANG2 as a prognostic factor for primary breast cancer. © 2002 Wiley-Liss, Inc. [source] Slow-growing craniopharyngioma masquarading as early-onset eating disorder: Two cases,INTERNATIONAL JOURNAL OF EATING DISORDERS, Issue 5 2009Laura Vad Winkler MD Abstract Background: Craniopharyngiomas are slow-growing tumors, which can either be asymptomatic or present themselves with visual, neuropsychiatric or endocrine disturbances. Eating disorders (EDs) are syndromes with unknown etiology, associated with multiple endocrine abnormalities. In pediatric cases the presentation of EDs may differ markedly from those of adults. Objective: We report on two pediatric patients with craniopharyngioma misinterpreted as ED. Method: Available patient records, psychiatric examinations, neuro-radiographic imaging, and biochemical data were evaluated. Discussion: The reported cases illustrate the importance to consider slow-growing craniopharyngioma in ED. Especially in atypical ED, neuro-radiographic, ophthalmologic and endocrine examination should be carried out. Furthermore, structural hypothalamic lesions in these cases mimicking features of ED, suggesting the possibility of an as yet unidentified structural hypothalamic disorder to be implicated in the etiopathogeny of ED. © 2008 by Wiley Periodicals, Inc. Int J Eat Disord, 2009 [source] Safety evaluation of individual non-fried and fried sunflower oil, paraffin oil, jojoba oil and their binary mixtures on rat healthINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 10 2008Radwan S. Farag Summary Sunflower, jojoba, paraffin oils and binary oil mixtures of sunflower, jojoba and sunflower,paraffin oils were continuously heated at 180 °C for 12 h. Aliquots of potato chips were fried in the aforementioned oil samples. Organoleptic tests were performed on fried chips and safety limits of the oil samples were measured by certain biochemical tests. Histopathological examinations of rat liver and kidney tissues were microscopically done. Organoleptic results for fried potato chips indicate that all types of chips obtained from heated oils were categorised good. Histopathological examinations indicate changes in rat tissues of liver and kidney paralleled the biochemical data. In general, the results suggest that paraffin oil alone and in mixtures with sunflower oil have to ban its use in frying processes. [source] A two-dimensional electrophoresis preliminary approach to human hepatocarcinoma differentiation induced by PPAR-agonistsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 2 2005Patrizia Bottoni Abstract Adopting biochemical and proteomic approaches, we investigated the effect of some PPAR-agonists, a new class of differentiating agents, on human hepatocellular carcinoma Hep-G2 cell line. Cancer differentiation was assayed by checking albumin, transferrin and ,-fetoprotein synthesis. Cell metabolism was studied by NMR spectroscopy of cell culture supernatants and by evaluation of mitochondrial respiratory chain enzyme activities. The two dimensional electrophoresis approach was employed to analyze modifications in the expression of cellular proteins linked to cell phenotype differentiation in the attempt to identify potential diagnostic and prognostic biomarkers of hepatocellular carcinoma. Results indicate that PPAR-agonists are able to act as differentiating inducers in human hepatocellular carcinoma Hep-G2 cell line as well as to inhibit mitochondrial respiratory chain Complex I, provoking a selective derangement of cellular oxidative metabolism. Lastly, two dimensional electrophoresis showed interesting modifications in the pattern of expression of cellular proteins that confirm biochemical data (increase in albumin and transferrin, decrease of alpha -fetoprotein synthesis) and, moreover, emphasize the meaning of these data by the increase of spots indicatively ascribed to HSP70 and catalase. [source] Localization of synaptic proteins involved in neurosecretion in different membrane microdomainsJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Elena Taverna Abstract A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Cav2.1 (P/Q-type) channels and soluble N -ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Cav2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-,-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery. [source] Expression of the CD44 variant isoform 5 in the human osteoarthritic knee joint: Correlation with radiological, histomorphological, and biochemical parametersJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2004Susanne Fuchs Abstract Purpose: The purpose of this study was to correlate expression of CD44v5 in osteoarthritic synovium, cartilage, and synovial fluid with radiographical, histomorphological, and biochemical data. Methods: Cartilage and synovia specimens of 27 patients with osteoarthritis were histomorphologically assessed according to Mankin and Pelletier, respectively. Extended weight-bearing antero-posterior radiographs were evaluated according to Kellgren and Ahlback. Expression of membrane-bound CD44v5 was analyzed by immunohistochemistry and levels of soluble CD44v5 were determined by ELISA. Results: Expression of CD44v5 in cartilage and synovia was detected in 67% and 59% of the patients, respectively. Immunohistochemical findings in cartilage correlated significantly with structural cartilage changes (p < 0.001), whereas no correlation was found between expression in synovia and inflammatory synovial changes. Additionally, no relationship was evident between CD44v5 expression and radiographical data, but expression in cartilage and synovium was significantly correlated with each other (p < 0.04). Surprisingly, expression of CD44v5 in both cartilage and synovia was negatively correlated with synovial fluid levels of TNF, (p < 0.03 and p < 0.02, respectively), and no association was evident with levels of IL-1,. Conclusions: The data demonstrate expression of CD44v5 in osteoarthritic cartilage and synovia, probably independent of joint inflammation. But more importantly, expression of this receptor variant in cartilage seems to be strongly related to the degree of cartilage destruction. © 2003 Published by Elsevier Ltd. on behalf of Orthopaedic Research Society. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] CpKLP1: A CALMODULIN-BINDING KINESIN-LIKE PROTEIN FROM CYANOPHORA PARADOXA (GLAUCOPHYTA)JOURNAL OF PHYCOLOGY, Issue 4 2000Salah E. Abdel-Ghany KCBP (kinesin-like calmodulin [CaM]-binding proteins), a member of the carboxy-terminal kinesin-like proteins (KLPs), is unique among KLPs in having a CaM-binding domain (CBD). CaM-binding KLPs have been identified from flowering plants and the sea urchin. To determine if CaM-binding KLP is present in phylogenetically divergent protists, we probed Cyanophora paradoxa protein extract with affinity-purified KCBP antibody. The KCBP antibody detected a polypeptide with a molecular mass of about 133 kDa in the crude extract. In a CaM,Sepharose column-purified fraction, the same band was detected with both KCBP antibody and biotinylated CaM. In a PCR reaction using degenerate primers corresponding to two conserved regions in the motor domain of kinesin, a 500-bp fragment (CpKLP1) was amplified from a cDNA library. The predicted amino acid sequence of CpKLP1 showed significant sequence similarity with KCBPs. In phylogenetic analysis, CpKLP1 fell into the KCBP group within the carboxy-terminal subfamily. These biochemical data, sequence, and phylogenetic analysis strongly suggest the presence of a calmodulin-binding KLP in C. paradoxa and that it is related to Ca2+/calmodulin regulated KLPs from plants. This is the first report on identification of any motor protein in C. paradoxa. Furthermore, our data suggest that CaM-binding KLPs may have evolved long before the divergence of plants and animals. [source] Outcomes of critically ill patients with cirrhosis admitted to intensive care: an important perspective from the non-transplant settingALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 2 2010S. J. Thomson Aliment Pharmacol Ther 2010; 32: 233,243 Summary Background, Hospital admissions for cirrhosis have been increasing in the United Kingdom, leading to increased pressure on intensive care (ICU) services. Outcome data for patients admitted to ICU are currently limited to transplant centre reports, with mortality rates exceeding 70%. These tertiary reports could fuel a negative bias when patients with cirrhosis are reviewed for ICU admission in secondary care. Aims, To determine whether disease severity and mortality rates in non-transplant general ICU are less severe than those reported by tertiary datasets. Methods, A prospective dual-centre non-transplant ICU study. Admissions were screened for cirrhosis and physiological and biochemical data were collected. Disease-specific and critical illness scoring systems were evaluated. Results, Cirrhosis was present in 137/4198 (3.3%) of ICU admissions. ICU and hospital mortality were 38% and 47%, respectively; median age 50 [43,59] years, 68% men, 72% alcoholic cirrhosis, median Child Pugh Score (CPS) 10 [8,11], Model for End-Stage Liver Disease (MELD) 18 [12,24], Acute Physiology and Chronic Health Evaluation II score (APACHE II) 16 [13,22]. Conclusions, Mortality rates and disease staging were notably lower than in the published literature, suggesting that patients have a more favourable outlook than previously considered. Transplant centre data should therefore be interpreted with caution when evaluating the merits of intensive care admission for patients in general secondary care ICUs. [source] Apoptotic cell death does not parallel other indicators of liver damage in chronic hepatitis C patientsJOURNAL OF VIRAL HEPATITIS, Issue 3 2000Rodrigues The mechanisms of hepatocyte damage and the events that lead to high rates of chronic liver disease in hepatitis C virus (HCV) infection remain unclear. Recent in vitro studies have suggested that the HCV core protein may disrupt specific signalling pathways of apoptosis. This prompted us to study patients with chronic HCV infection to: determine the extent of apoptosis in the liver; evaluate whether clinical and biochemical data are correlated with histological findings; and to investigate if apoptosis is related to the histological activity of the disease. Twelve patients with chronic hepatitis C were included in the study. Liver histology was scored by using the histological activity index (HAI) of Knodell et al. DNA fragmentation was assessed in liver tissue by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) assay. Routine methods were used to determine serum markers of liver disease. Bile acids were measured in serum and liver by gas chromatography. Patients were placed, according to their HAI score, into group A (3.8 ± 0.3) or group B (7.8 ± 0.8) (P < 0.01). Liver enzymes tended to be higher in group B patients than in patients of group A. Levels of toxic bile acids in serum were greater in patients than in controls (P < 0.01). Chenodeoxycholic acid values were slightly higher in serum and liver of patients in group A. Liver biopsies with low HAI scores showed an increased rate of apoptosis (18.0 ± 4.0 apoptotic cells per field) compared to those with higher HAI scores (6.6 ± 2.1, P < 0.05) or to controls (3.5 ± 0.4, P < 0.01). Hence, less severe liver disease, associated with lower histological grades and biochemistries, as well as increased levels of chenodeoxycholic acid, induces an expanded apoptotic response. The lower apoptotic rate in advanced liver disease may be associated with the high incidence of hepatocellular dysplasia/neoplasia. [source] A multi-angular mass spectrometric view at cyclic nucleotide dependent protein kinases: In vivo characterization and structure/function relationshipsMASS SPECTROMETRY REVIEWS, Issue 4 2008Arjen Scholten Abstract Mass spectrometry has evolved in recent years to a well-accepted and increasingly important complementary technique in molecular and structural biology. Here we review the many contributions mass spectrometry based studies have made in recent years in our understanding of the important cyclic nucleotide activated protein kinase A (PKA) and protein kinase G (PKG). We both describe the characterization of kinase isozymes, substrate phosphorylation, binding partners and post-translational modifications by proteomics based methodologies as well as their structural and functional properties as revealed by native mass spectrometry, H/D exchange MS and ion mobility. Combining all these mass spectrometry based data with other biophysical and biochemical data has been of great help to unravel the intricate regulation of kinase function in the cell in all its magnificent complexity. © 2008 Wiley Periodicals, Inc. Mass Spec Rev 27: 331,353, 2008 [source] Effects of simultaneous kidney-pancreaticoduodenal transplantation on diabetes-induced renal insufficiency in ratsMICROSURGERY, Issue 4 2001Jin Han Yoon M.D., Ph.D. An investigation of the functional and histological changes was done after en-bloc kidney-pancreaticoduodenal transplantation (kpdt) in the diabetes-induced, renal insufficient Lewis rats. For donor preparation, an end-to-side portocaval shunt was performed, and the aortic, vena caval segments, and ureter-bladder patch were obtained. They were anastomosed microsurgically to recipient's aorta, vena cava, and bladder in end-to-side fashion. Of 15 diabetes-induced kpdt rats, 14 survived. Two of the 14 surviving rats showed ischemic necrosis. The remaining 12 transplants showed well-preserved glomeruli and Langerhans islets for 5 months postoperatively. Biochemical data comparing diabetic and sham-operated rats (six rats each), six diabetic controls, and 12 kpdt rats showed no significant statistical difference at said observation period. The diabetes-induced kpdt rats showed improvement of following biochemical data: within 1 week postoperatively, the glucose level fell from 300 to 115 mg/dL; BUN level from >20 to <20 mg/dL; the creatinine level from 1.5 to <1.2 mg/dL. The insulin level returned to normal, 1.1 ng/mL, in 2 weeks. The results demonstrate that the kpdt model is an effective and successful operative technique in diabetic rats and may provide effective therapeutic methods for diabetes-induced renal insufficiency. © 2001 Wiley-Liss, Inc. MICROSURGERY 21:173,178 2001 [source] House cleaning, a part of good housekeepingMOLECULAR MICROBIOLOGY, Issue 1 2006Michael Y. Galperin Summary Cellular metabolism constantly generates by-products that are wasteful or even harmful. Such compounds are excreted from the cell or are removed through hydrolysis to normal cellular metabolites by various ,house-cleaning' enzymes. Some of the most important contaminants are non-canonical nucleoside triphosphates (NTPs) whose incorporation into the nascent DNA leads to increased mutagenesis and DNA damage. Enzymes intercepting abnormal NTPs from incorporation by DNA polymerases work in parallel with DNA repair enzymes that remove lesions produced by modified nucleotides. House-cleaning NTP pyrophosphatases targeting non-canonical NTPs belong to at least four structural superfamilies: MutT-related (Nudix) hydrolases, dUTPase, ITPase (Maf/HAM1) and all-, NTP pyrophosphatases (MazG). These enzymes have high affinity (Km's in the micromolar range) for their natural substrates (8-oxo-dGTP, dUTP, dITP, 2-oxo-dATP), which allows them to select these substrates from a mixture containing a ,1000-fold excess of canonical NTPs. To date, many house-cleaning NTPases have been identified only on the basis of their side activity towards canonical NTPs and NDP derivatives. Integration of growing structural and biochemical data on these superfamilies suggests that their new family members cleanse the nucleotide pool of the products of oxidative damage and inappropriate methylation. House-cleaning enzymes, such as 6-phosphogluconolactonase, are also part of normal intermediary metabolism. Genomic data suggest that house-cleaning systems are more abundant than previously thought and include numerous analogous enzymes with overlapping functions. We discuss the structural diversity of these enzymes, their phylogenetic distribution, substrate specificity and the problem of identifying their true substrates. [source] The putative l -lactate dehydrogenase from Methanococcus jannaschii is an NADPH-dependent l -malate dehydrogenaseMOLECULAR MICROBIOLOGY, Issue 6 2000Dominique Madern The enyme encoded by Methanococcus jannaschii open reading frame (ORF) 0490 was purified and characterized. It was shown to be an NADPH-dependent [lactate dehydrogenase (LDH)-like]l -malate dehydrogenase (MalDH) and not an l -lactate dehydrogenase, as had been suggested previously on the basis of amino acid sequence similarity. The results show the importance of biochemical data in the assignment of ORF function in genomic sequences and have implications for the phylogenetic distribution of members of the MalDH/LDH enzyme superfamilies within the prokaryotic kingdom. [source] Comparative Biochemistry of Eumelanogenesis and the Protective Roles of Phenoloxidase and Melanin in InsectsPIGMENT CELL & MELANOMA RESEARCH, Issue 1 2002Manickam Sugumaran The phenolic biopolymer eumelanin is an important skin pigment found throughout the animal kingdom. The enzyme, tyrosinase, initiates melanogenesis in mammals. The biogenesis is assisted by a number of mammalian protein factors including dopachrome tautomerase and 5,6-dihydroxyindole-2-carboxylate oxidase. Invertebrates, such as insects, employ phenoloxidase and dopachrome (decarboxylating) isomerase for melanin biosynthesis. Recently generated molecular biological and biochemical data indicate that tyrosinase and phenoloxidase are distinctly different enzymes in spite of possessing both monophenol monooxygenase activity as well as o -diphenoloxidase activity. Similarly, insect dopachrome isomerase also differs significantly from its mammalian counterpart in several of its properties including the nature of the enzymatic reaction. In addition, there are considerable differences in the eumelanogenic pathways of these two animal groups that include the utility of substrates, use of dihydroxyindoles and the nature of eumelanin pigment. Thus, the biochemistry and molecular biology of melanogenesis in mammals and insects are significantly different. The advantages of generating different eumelanin pigments and intermediates by the insects are discussed. [source] Differential gene expression in senescing leaves of two silver birch genotypes in response to elevated CO2 and tropospheric ozonePLANT CELL & ENVIRONMENT, Issue 6 2010SARI KONTUNEN-SOPPELA ABSTRACT Long-term effects of elevated CO2 and O3 concentrations on gene expression in silver birch (Betula pendula Roth) leaves were studied during the end of the growing season. Two birch genotypes, clones 4 and 80, with different ozone growth responses, were exposed to 2× ambient CO2 and/or O3 in open-top chambers (OTCs). Microarray analyses were performed after 2 years of exposure, and the transcriptional profiles were compared to key physiological characteristics during leaf senescence. There were genotypic differences in the responses to CO2 and O3. Clone 80 exhibited greater transcriptional response and capacity to alter metabolism, resulting in better stress tolerance. The gene expression patterns of birch leaves indicated contrasting responses of senescence-related genes to elevated CO2 and O3. Elevated CO2 delayed leaf senescence and reduced associated transcriptional changes, whereas elevated O3 advanced leaf senescence because of increased oxidative stress. The combined treatment demonstrated that elevated CO2 only temporarily alleviated the negative effects of O3. Gene expression data alone were insufficient to explain the O3 response in birch, and additional physiological and biochemical data were required to understand the true O3 sensitivity of these clones. [source] The MRG domain of human MRG15 uses a shallow hydrophobic pocket to interact with the N-terminal region of PAM14PROTEIN SCIENCE, Issue 10 2006Peng Zhang Abstract MRG15 is a transcription factor expressed in a variety of human tissues, and its orthologs have been found in many other eukaryotes which constitute the MRG protein family. It plays a vital role in embryonic development and cell proliferation, and is involved in cellular senescence. The C-terminal part of MRG15 forms a conserved MRG domain which is involved in interactions with the tumor suppressor protein retinoblastoma and a nucleoprotein PAM14 during transcriptional regulation. We report here the characterization of the interaction between the MRG domain of human MRG15 and PAM14 using both yeast two-hybrid and in vitro binding assays based on the crystal structure of the MRG domain. The MRG domain is predominantly hydrophobic, and consists of mainly ,-helices that are arranged in a three-layer sandwich topology. The hydrophobic core is stabilized by interactions among a number of conserved hydrophobic residues. The molecular surface is largely hydrophobic, but contains a few hydrophilic patches. Structure-based site-directed mutagenesis studies identified key residues involved in the binding of PAM14. Structural and biochemical data together demonstrate that the PAM14 binding site is consisted of residues Ile160, Leu168, Val169, Trp172, Tyr235, Val268, and Arg269 of MRG15, which form a shallow hydrophobic pocket to interact with the N-terminal 50 residues of PAM14 through primarily hydrophobic interactions. These results provide the molecular basis for the interaction between the MRG domain and PAM14, and reveal insights into the potential biological function of MRG15 in transcription regulation and chromatin remodeling. [source] Allosteric transition pathways in the lactose repressor protein core domains: Asymmetric motions in a homodimerPROTEIN SCIENCE, Issue 11 2003Terence C. Flynn Abstract The crystal structures of lactose repressor protein (LacI) provide static endpoint views of the allosteric transition between DNA- and IPTG-bound states. To obtain an atom-by-atom description of the pathway between these two conformations, motions were simulated with targeted molecular dynamics (TMD). Strikingly, this homodimer exhibited asymmetric dynamics. All asymmetries observed in this simulation are reproducible and can begin on either of the two monomers. Asymmetry in the simulation originates around D149 and was traced back to the pre-TMD equilibrations of both conformations. In particular, hydrogen bonds between D149 and S193 adopt a variety of configurations during repetitions of this process. Changes in this region propagate through the structure via noncovalent interactions of three interconnected pathways. The changes of pathway 1 occur first on one monomer. Alterations move from the inducer-binding pocket, through the N-subdomain ,-sheet, to a hydrophobic cluster at the top of this region and then to the same cluster on the second monomer. These motions result in changes at (1) side chains that form an interface with the DNA-binding domains and (2) K84 and K84', which participate in the monomer,monomer interface. Pathway 2 reflects consequent reorganization across this subunit interface, most notably formation of a H74-H74rsquo; ,-stacking intermediate. Pathway 3 extends from the rear of the inducer-binding pocket, across a hydrogen-bond network at the bottom of the pocket, and transverses the monomer,monomer interface via changes in H74 and H74rsquo;. In general, intermediates detected in this study are not apparent in the crystal structures. Observations from the simulations are in good agreement with biochemical data and provide a spatial and sequential framework for interpreting existing genetic data. [source] Gene regulation during late embryogenesis: the RY motif of maturation-specific gene promoters is a direct target of the FUS3 gene productTHE PLANT JOURNAL, Issue 5 2000Wim Reidt Summary The Arabidopsis mutants fus3 and abi3 show pleiotropic effects during embryogenesis including reduced levels of transcripts encoding embryo-specific seed proteins. To investigate the interaction between the B3-domain-containing transcription factors FUS3 and ABI3 with the RY cis -motif, conserved in many seed-specific promoters, a promoter analysis as well as band-shift experiments were performed. The analysis of promoter mutants revealed the structural requirements for the function of the RY cis -element. It is shown that both the nucleotide sequence and the alternation of purin and pyrimidin nucleotides (RY character) are essential for the activity of the motif. Further, it was shown that FUS3 and ABI3 can act independently of each other in controlling promoter activity and that the RY cis -motif is a target for both transcription factors. For FUS3, which is so far the smallest known member of the B3-domain family, a physical interaction with the RY motif was established. The functional and biochemical data demonstrate that the regulators FUS3 and ABI3 are essential components of a regulatory network acting in concert through the RY-promoter element to control gene expression during late embryogenesis and seed development. [source] Variant Creutzfeldt,Jakob disease in France and the United Kingdom: Evidence for the same agent strain,ANNALS OF NEUROLOGY, Issue 3 2009Jean-Philippe Brandel MD Objective Variant Creutzfeldt,Jakob disease (vCJD) was first reported in the United Kingdom in 1996. Since then, the majority of cases have been observed in the United Kingdom where there was a major epidemic of bovine spongiform encephalopathy. France was the second country affected. To address the hypothesis of the involvement of a common strain of agent, we have compared clinical, neuropathological, and biochemical data on vCJD patients from both countries. Methods In France and the United Kingdom, epidemiological and clinical data were obtained from analysis of medical records and direct interview of the family of the patients using the same standardized questionnaire in both countries. When brain material was available, we performed with similar methods a comparative study of brain lesions and PrPres glycoform ratios in both vCJD populations. Results Clinical data, genetic background, neuropathological finding, and biochemical findings in the 185 patients observed in France (n = 23) and the United Kingdom (n = 162) were similar except for age at death. Currently, blood transfusion is a risk factor identified only in the United Kingdom. Interpretation The close similarity between the cases of vCJD in France and the United Kingdom supports the hypothesis that a common strain of infectious agent is involved in both countries. The 5-year delay in the peak that we observed in France compared with the United Kingdom fits well with the increase in the importation of beef products to France from the United Kingdom between 1985 and 1995. Ann Neurol 2009;65:249,256 [source] Prosthesis related sepsis following laparoscopic adjustable gastric bandingANZ JOURNAL OF SURGERY, Issue 7-8 2010Michael Facek Abstract Background:, Laparoscopic adjustable gastric banding (LAGB) is well-recognized as a superior method to achieving durable weight loss in the medium term when compared with non-surgical methods of weight loss. In this paper, we described the clinical presentation and outcomes of patients presenting with band or band-adjustment reservoir sepsis from our series from a single institution. Methods:, We conducted a retrospective review of prospectively collected clinical, anthropometric and biochemical data from patients who underwent LAGB placement over a five-year period at a metropolitan teaching hospital. Those patients requiring surgical intervention for prosthesis-related sepsis were included in the review. Results:, Of the 445 patients in this series, 10 (2.2%) developed prosthesis sepsis and required operative intervention. Three (0.7%) presented with reservoir sepsis requiring removal of the reservoir. One had band erosion identified and the entire prosthesis removed. In seven (1.5%) of the patients, infections occurred at the gastric band. Two patients presented with purulent peritonitis and underwent immediate band removal. The remainder presented with band abscesses and either had their band removed (three patients) or left in position and the sepsis treated with drainage and antibiotics (two patients). Conclusions:, In our current series, a small proportion of LAGB patients developed prosthesis-related infection that typically required port or band removal and usually occurred early in the post-operative course. We have modified our prophylactic antibiotic regime and surgical technique as a result of this review. In selected cases of band infection, bands were salvaged with subsequent acceptable weight loss, suggesting that LAGB salvage in the presence of sepsis may be achievable in some patients. [source] Effects of sublethal levels of tributyltin chloride on a new toxicity test organism, Liza saliens (osteichthyes, mugilidae): a histological studyAPPLIED ORGANOMETALLIC CHEMISTRY, Issue 6 2006P. D'Agati Abstract The histopathological effects of 10,7 and 10,9M tributyltin(IV)chloride,TBTCl, solutions on different Liza saliens organs have been studied by light microscope. The fish were sacrificed after 3,4 h incubation in 10,7M TBTCl solution or after 15 days incubation in 10,9M solution. The observed histopathological changes were dose- and time-dependent. The 10,7M TBTCl concentration resulted in major damage to the gill epithelium, indicating that TBTCl primarily interfered with the respiration, osmoregulation, acid balance and nitrogenous waste excretion processes. After incubation in 10,9M TBTCl solution the fish lived 20 or more days, but many of the organs were altered. Thymus atrophy, reduced spleen and altered head kidney were observed. These histological results indicated that TBTCl interfered with organ immunodefense and altered main metabolic pathways in Liza saliens. The presence of melano-macrophage centers, only in TBT-treated liver and spleen, can be considered a tool to facilitate, with other biomarkers, the detection of alterations by toxicants. Regarding the pancreas activity in 10,7M solutions, it has been noted that, in the exocrine cells, very few zymogen granules were still present and the Langerhans islets were more altered. In 10,9M solution the exocrine pancreatic cells had no granules and the islet cells presented degenerative alterations. In addition, TBTCl, which altered the pancreas and gonad morphology, could again be considered an endocrine disrupter even if biochemical data are still necessary. Finally, the Liza saliens juveniles could be considered an interesting biological model for experiments with contaminants, due to their ease of adaptation to experimental conditions and food chain position. Copyright © 2005 John Wiley & Sons, Ltd. [source] New kinase regulation mechanism found in HipBA: a bacterial persistence switchACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Artem Evdokimov Bacterial persistence is the ability of individual cells to randomly enter a period of dormancy during which the cells are protected against antibiotics. In Escherichia coli, persistence is regulated by the activity of a protein kinase HipA and its DNA-binding partner HipB, which is a strong inhibitor of both HipA activity and hip operon transcription. The crystal structure of the HipBA complex was solved by application of the SAD technique to a mercury derivative. In this article, the fortuitous and interesting effect of mercury soaks on the native HipBA crystals is discussed as well as the intriguing tryptophan-binding pocket found on the HipA surface. A HipA-regulation model is also proposed that is consistent with the available structural and biochemical data. [source] | ||||||||||||||||