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Biochemical Mechanisms (biochemical + mechanism)
Selected AbstractsIncorporating Physiological and Biochemical Mechanisms into Pharmacokinetic,Pharmacodynamic Models: A Conceptual Framework,BASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 1 2010Svein G. Dahl In general, modelling of data has the purpose (1) to describe experimental data, (2a) to reduce the amount of data resulting from an experiment, e.g. a clinical trial and (2b) to obtain the most relevant parameters, (3) to test hypotheses and (4) to make predictions within the boundaries of experimental conditions, e.g. range of doses tested (interpolation) and out of the boundaries of the experimental conditions, e.g. to extrapolate from animal data to the situation in man. Describing the drug/xenobiotic-target interaction and the chain of biological events following the interaction is the first step to build a biologically based model. This is an approach to represent the underlying biological mechanisms in qualitative and also quantitative terms, thus being inherently connected in many aspects to systems biology. As the systems biology models may contain variables in the order of hundreds connected with differential equations, it is obvious that it is in most cases not possible to assign values to the variables resulting from experimental data. Reduction techniques may be used to create a manageable model which, however, captures the biologically meaningful events in qualitative and quantitative terms. Until now, some success has been obtained by applying empirical pharmacokinetic/pharmacodynamic models which describe direct and indirect relationships between the xenobiotic molecule and the effect, including tolerance. Some of the models may have physiological components built in the structure of the model and use parameter estimates from published data. In recent years, some progress toward semi-mechanistic models has been made, examples being chemotherapy-induced myelosuppression and glucose-endogenous insulin-antidiabetic drug interactions. We see a way forward by employing approaches to bridge the gap between systems biology and physiologically based kinetic and dynamic models. To be useful for decision making, the ,bridging' model should have a well founded mechanistic basis, but being reduced to the extent that its parameters can be deduced from experimental data, however capturing the biological/clinical essential details so that meaningful predictions and extrapolations can be made. [source] Biochemical mechanisms of methoxyfenozide resistance in the cotton leafworm Spodoptera littoralisPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 7 2009Hadi Mosallanejad Abstract BACKGROUND: Methoxyfenozide is a lepidopteran-specific insecticide that belongs to a new group of insecticides, the non-steroidal ecdysteroid agonists, also called moulting accelerating compounds (MACs). To investigate the risk of resistance and possible mechanisms conferring resistance to methoxyfenozide, the authors selected in the laboratory for a resistant strain of the cotton leafworm Spodoptera littoralis (Boisd.), which is a representative lepidopteran model and an important pest in cotton and vegetables worldwide, with a high risk for resistance development. RESULTS: After selection with methoxyfenozide during 13 generations, toxicity data showed that the selected strain developed fivefold resistance to methoxyfenozide in comparison with the susceptible strain. Measurement of the detoxification enzymes demonstrated that the monooxygenase (MO) activity was 2.1 times higher in the selected strain, whereas there was no change for esterases and glutathione- S -transferases. When the inhibitors piperonyl butoxide (PBO), S,S,S -tributyl phosphorotrithioate (DEF) and diethyl maleate were tested as synergists, the respective synergistic ratios were 0.97, 0.96 and 1.0 for the susceptible strain, and 2.2, 0.96 and 1.1 for the resistant strain. The significant synergistic effect by PBO concurs with the increased MO activity in the selected strain. CONCLUSION: Taken overall, the present study supports the importance of MO-mediated metabolism in resistance to methoxyfenozide, directing tactics to fight against resistance development for this novel group of insecticides. Copyright © 2009 Society of Chemical Industry [source] Biochemical mechanisms of insecticide resistance in the diamondback moth (DBM), Plutella xylostella L. (Lepidopterata: Yponomeutidae), in the Sydney region, AustraliaAUSTRALIAN JOURNAL OF ENTOMOLOGY, Issue 4 2009Vincent Y Eziah Abstract Following the detection of resistant diamondback moth (DBM) populations to synthetic pyrethroid, organophosphorus and indoxacarb insecticides in the Sydney Basin, a study of the major biochemical mechanisms was conducted to determine the type of resistance in these populations. The activity of cytochrome P450 monooxygenases increased two- to sixfold when compared with the susceptible strain. Up to a 1.9-fold increase in esterase activity in resistant strains compared with the susceptible strain was observed. In vitro inhibition studies showed that profenofos, methamidophos and chlorpyrifos strongly inhibited the esterases while permethrin and esfenvalerate resulted in less than 30% inhibition. Qualitative analysis of the esterases using native polyacrylamide gel electrophoresis showed four bands in both the susceptible and resistant individuals with more intense staining in the resistant individuals. The development of these bands was inhibited by methamidophos and chlorpyrifos pretreatment of the protein extract while permethrin and esfenvalerate did not exhibit this effect. Glutathione S-transferase (GST) activity was significantly higher in two field populations compared with the remaining populations. Overall, the study showed that the mechanisms of insecticide resistance in the DBM populations in the area studied were due to cytochrome P450 monooxygenases, esterase and GSTs, and possibly other non-metabolic mechanisms that were not investigated in the present study. [source] Evidence of oxidative stress in bluegill sunfish (Lepomis macrochirus) liver microsomes simultaneously exposed to solar ultraviolet radiation and anthraceneENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2000Jonghoon Choi Abstract Many polycyclic aromatic hydrocarbons (PAHs) are acutely toxic to fish and other aquatic organisms in the presence of environmentally realistic intensities of solar ultraviolet radiation (SUVR). However, the biochemical mechanism of this toxicity is not well established. In this study, increased levels of both reactive oxygen species production and lipid peroxidation were hypothesized as a toxic mechanism. To test this hypothesis, the production of superoxide anion and of a lipid peroxidation product (malondialdehyde) was measured in bluegill sunfish (Lepomis machrochirus) liver microsomes. These microsomes were exposed to a representative phototoxic PAH (anthracene [ANT]) and to SUVR and normal laboratory fluorescent light (FLU) in four different combinations: FLU + no ANT, FLU + ANT, SUVR + no ANT, and SUVR + ANT. The highest mean levels of both superoxide anion and malondialdehyde production were observed in the SUVR + ANT group, and these levels were significantly different (p < 0.05) from those in all other treatment groups. We conclude that the photoinduced toxicity of ANT, and possibly of other phototoxic PAHs, manifests at least in part through lipid peroxidation after increased production of reactive oxygen species. [source] Could a common biochemical mechanism underlie addictions?JOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 1 2000C. Betz The subject of ,drug addiction' is multifaceted and many aspects of it (even some of the definitions) are controversial. Collateral medical problems include the spread of HIV and hepatitis C virus secondary to i.v. drug abuse and effects on prenatal brain development ( 1). Progress in the understanding of the causes of addictions and its treatment has been impeded by the lack of a unifying biochemical theory. However, recent evidence suggests that some common mechanism might underlie addictions to otherwise apparently unrelated drugs. A major hypothesis has emerged suggesting that the neurotransmitter dopamine (DA) might play a central role in the molecular mechanisms of at least some addictions. If so, it would represent an important target for discovery of effective pharmacotherapy and revolutionize the pharmacist's role in treating addictions. This short overview outlines the status of the theory of a common biochemical mechanism of drug addiction. [source] Lysophosphatidic Acid Inhibits Ca2+ Signaling in Response to Epidermal Growth Factor Receptor Stimulation in Human Astrocytoma Cells by a Mechanism Involving Phospholipase C, and a G,i ProteinJOURNAL OF NEUROCHEMISTRY, Issue 4 2000Marita Hernández Abstract: The effect of the lysophospholipid mediators lysophosphatidic acid (LPA) and sphingosine 1-phosphate and the polypeptide growth factor epidermal growth factor (EGF) on the human astrocytoma cell line 1321N1 was assessed. These agonists produced a rapid and transient increase of the intracellular Ca2+ concentration. When LPA was perfused before addition of EGF, the EGF-dependent Ca2+ transient was abrogated, whereas this was not observed when EGF preceded LPA addition. This inhibitory effect was not found for other EGF-mediated responses, e.g., activation of the mitogen-activated protein kinase cascade and cell proliferation, thus pointing to the existence of cross-talk between LPA and EGF for only a branch of EGF-induced responses. As 1321N1 cells expressed mRNA encoding the LPA receptors endothelial differentiation gene (Edg)-2, Edg-4, and Edg-7 and as sphingosine 1-phosphate did not interfere with LPA signaling, Edg-2, Edg-4, and/or Edg-7 could be considered as the LPA receptors mediating the aforementioned cross-talk. Attempts to address the biochemical mechanism involved in the cross-talk between the receptors were conducted by the immunoprecipitation approach using antibodies reacting with the EGF receptor (EGFR), phosphotyrosine, phospholipase C, (PLC,)-1, and G,i protein. LPA was found to induce coupling of PLC,-1 to the EGFR by a mechanism involving a G,i protein, in the absence of tyrosine phosphorylation of both PLC, and the EGFR. These data show a cross-talk between LPA and EGF limited to a branch of EGFR-mediated signaling, which may be explained by a LPA-induced, G,i -protein-mediated translocation of PLC,-1 to EGFR in the absence of detectable tyrosine phosphorylation of both proteins. [source] Hairpin telomeres and genome plasticity in Borrelia: all mixed up in the endMOLECULAR MICROBIOLOGY, Issue 3 2005George Chaconas Summary Spirochetes of the genus Borrelia have a highly unusual genome structure composed of over 20 replicons. Most of these replicons are linear and terminated by covalently closed hairpin ends or telomeres. Moreover, the linear replicons are affected by extensive DNA rearrangements, including telomere exchanges, DNA duplications, and harbour a large number of pseudogenes. The mechanism for the unusual genome plasticity in the linear replicons has remained elusive. The enzymatic machinery (the telomere resolvase ResT) responsible for generating the hairpin ends from replicative intermediates has recently been shown to also perform a reverse reaction that fuses telomeres on unrelated replicons. Infrequent stabilization of such fusion events over evolutionary time provides the first proposed biochemical mechanism for the DNA rearrangements that are so prominent in the linear replicons of B. burgdorferi. [source] An overview of the antifungal properties of allicin and its breakdown products , the possibility of a safe and effective antifungal prophylacticMYCOSES, Issue 2 2005Stephen R Davis Summary Reports about the safe and successful intravenous (i.v.) use of garlic derivatives in China against invasive fungal infections have been made, but little has been done to seriously investigate the in vivo use of these derivatives in the West. Laboratories have demonstrated impressive in vitro MICs using allitridium, one of these derivatives, against a range of medically important fungi. In addition, it has been demonstrated that allitridium shows in vitro synergy with amphotericin B, one of the main i.v. antifungal agents. Some of the breakdown products of allicin, the main parent antifungal compound in garlic, have been investigated for their general antimicrobial, anticancer and anticholesterol properties, and it appears that there is a common mode of action that underlies these activities. It appears that these small molecules have the ability to cross cell membranes and combine with sulfur-containing molecular groups in amino acids and proteins, thus interfering with cell metabolism. It has been suggested that the reason human cells are not poisoned by allicin derivatives is that they contain glutathione, a sulfur-containing amino acid that combines with the allicin derivative, thus preventing cell damage. In addition to their biochemical mechanism, these derivatives appear to stimulate cellular immunity, an important ability lacking in conventional antifungal chemotherapy. These derivatives appear to be safe, cheap, wide-spectrum and immunostimulatory, as well as possibly synergistic with conventional antifungal therapy, making them ideal candidates for investigation into their use as prophylactic antifungal agents. [source] Inhibitory effect of bionic fungicide 2-allylphenol on Botrytis cinerea (Pers. ex Fr.) in vitro,PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 12 2009Shuangjun Gong Abstract BACKGROUND: 2-Allylphenol is a registered fungicide in China to control fungal diseases on tomato, strawberry and apple. It is synthetic and structurally resembles the active ingredient ginkgol isolated from Ginkgo biloba L. bark. 2-Allylphenol has been used in China for 10 years. However, its biochemical mode of action remains unclear. An in vitro study was conducted on the biochemical mechanism of 2-allyphenol inhibiting Botrytis cinerea (Pers. ex Fr.). RESULTS: The inhibition was approximately 3 times stronger when the fungus was grown on non-fermentable source, glycerol, than that on a fermentable carbon source, glucose. Inhibition of B. cinerea and Magnaporthe oryzae (Hebert) Barr mycelial growth was markedly potentiated in the presence of salicylhydroxamic acid (SHAM), an inhibitor of mitochondrial alternative oxidase. Furthermore, at 3 h after treatment with 2-allylphenol, oxygen consumption had recovered, but respiration was resistant to potassium cyanide and sensitive to SHAM, indicating that 2-allylphenol had the ability to induce cyanide-resistant respiration. The mycelium inhibited in the presence of 2-allylphenol grew vigorously after being transferred to a fungicide-free medium, indicating that 2-allylphenol is a fungistatic compound. Adenine nucleotide assay showed that 2-allylphenol depleted ATP content and decreased the energy charge values, which confirmed that 2-allylphenol is involved in the impairment of the ATP energy generation system. CONCLUSION: These results suggested that 2-allylphenol induces cyanide-resistant respiration and causes ATP decrease, and inhibits respiration by an unidentified mechanism. Copyright © 2009 Society of Chemical Industry [source] Structural and biochemical characterization of a novel Mn2+ -dependent phosphodiesterase encoded by the yfcE genePROTEIN SCIENCE, Issue 7 2007Darcie J. Miller Abstract Escherichia coli YfcE belongs to a conserved protein family within the calcineurin-like phosphoesterase superfamily (Pfam00149) that is widely distributed in bacteria and archaea. Superfamily members are metallophosphatases that include monoesterases and diesterases involved in a variety of cellular functions. YfcE exhibited catalytic activity against bis- p -nitrophenyl phosphate, a general substrate for phosphodiesterases, and had an absolute requirement for Mn2+. However, no activity was observed with phosphodiesters and over 50 naturally occurring phosphomonoesters. The crystal structure of the YfcE phosphodiesterase has been determined to 2.25 Ĺ resolution. YfcE has a ,-sandwich architecture similar to metallophosphatases of common ancestral origin. Unlike its more complex homologs that have added structural elements for regulation and substrate recognition, the relatively small 184-amino-acid protein has retained its ancestral simplicity. The tetrameric protein carries two zinc ions per active site from the E. coli extract that reflect the conserved di-Mn2+ active site geometry. A cocrystallized sulfate inhibitor mimics the binding of phosphate moeities in known ligand/phosphatase complexes. Thus, YfcE has a similar active site and biochemical mechanism as well-characterized superfamily members, while the YfcE phosphodiester-containing substrate is unique. [source] Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis: discovery of bifunctional O - and C -glucosyltransferasesTHE PLANT JOURNAL, Issue 6 2008Fernando Gandia-Herrero Summary Plants, as predominantly sessile organisms, have evolved complex detoxification pathways to deal with a diverse range of toxic chemicals. The elasticity of this stress response system additionally enables them to tackle relatively recently produced, novel, synthetic pollutants. One such compound is the explosive 2,4,6-trinitrotoluene (TNT). Large areas of soil and groundwater are contaminated with TNT, which is both highly toxic and recalcitrant to degradation, and persists in the environment for decades. Although TNT is phytotoxic, plants are able to tolerate low levels of the compound. To identify the genes involved in this detoxification process, we used microarray analysis and then subsequently characterized seven uridine diphosphate (UDP) glycosyltransferases (UGTs) from Arabidopsis thaliana (Arabidopsis). Six of the recombinantly expressed UGTs conjugated the TNT-transformation products 2- and 4-hydroxylaminodinitrotoulene, exhibiting individual bias for either the 2- or the 4-isomer. For both 2- and 4-hydroxylaminodinitrotoulene substrates, two monoglucose conjugate products, confirmed by HPLC-MS-MS, were observed. Further analysis indicated that these were conjugated by either an O - or C -glucosidic bond. The other major compounds in TNT metabolism, aminodinitrotoluenes, were also conjugated by the UGTs, but to a lesser extent. These conjugates were also identified in extracts and media from Arabidopsis plants grown in liquid culture containing TNT. Overexpression of two of these UGTs, 743B4 and 73C1, in Arabidopsis resulted in increases in conjugate production, and enhanced root growth in 74B4 overexpression seedlings. Our results show that UGTs play an integral role in the biochemical mechanism of TNT detoxification by plants. [source] Impaired Energy Metabolism after Co-Exposure to Leadand EthanolBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 6 2005Suresh Kumar Verma One such factor, ethanol, might affect the neurotoxicity of lead by regulating its absorption and distribution. However, there is little information regarding the possible biochemical mechanism by which ethanol might be affecting the state of neuronal functions in lead-exposed individuals. Therefore, the present investigation involved the effect of alcohol (3 g/kg body weight, intragastrically, for 8 weeks) on lead-induced (50 mg/kg body weight, intragastrically, for 8 weeks) mitochondrial dysfunction in adult rat brain. Ethanol was found to enhance the toxic effects of lead in terms of decreased cellular energy reserves (ATP levels). Co-exposure to lead and ethanol caused marked decline in the rate of mitochondrial respiration as compared to lead alone. Further the activies of various components of the electron transport chain, viz. NADH dehydrogenase, succinate dehydrogenase and cytochrome oxidase depicted a significant decrease in the lead and ethanol co-exposed rats as compared to the lead-treated group. The results of the present study reflect that ethanol makes adult rat brain more vulnerable to the neurotoxic effects of lead in terms of altered mitochondrial energy metabolism. [source] Genetic determinants for activated fluoropyrimidine chemotherapyDRUG DEVELOPMENT RESEARCH, Issue 2 2006William H. GmeinerArticle first published online: 5 JUN 200 Abstract Fluoropyrimidines (FPs) remain widely used for the treatment of diverse malignancies more than four decades following the initial report of 5-fluorouracil (5FU), the archetypal FP, as a novel compound with potential anti-neoplastic activity. Subsequent decades of research have enriched our understanding of the biochemical mechanisms that are important for FP activation as well as the genetic determinants that are predictive of the likely success, or failure, of FP chemotherapy for a particular individual. The concept that chemotherapy should be customized to complement the genetic profiles of cancer patients has become increasingly important as genotyping of tumor samples has become possible and as the number of available anticancer drugs has increased. Significant progress has been made in identifying the gene expression profiles for cancer patients who are likely to benefit from treatment with FPs. In this review, we will summarize the results of retrospective clinical studies correlating response to FP chemotherapy with the expression of specific genes, such as TS and DPD. We will also present a summary of FPs in current clinical use, including orally bioavailable FPs such as capecitabine, as well as FPs that are in pre-clinical development, such as FdUMP[10]. Refinement of a target population through pharmacogenetic analysis and development of novel FPs that evoke very high response rates in this target population will likely result in the use of FP regimens in the coming era when cancer becomes a largely manageable disease. Drug Dev. Res. 67:119,129, 2006. © 2006 Wiley-Liss, Inc. [source] Expression of angiogenic factors in chronic myeloid leukaemia: role of the bcr/abl oncogene, biochemical mechanisms, and potential clinical implicationsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2004C. Sillaber Abstract Chronic myeloid leukaemia (CML) is a stem cell disease characterized by an increased production and accumulation of clonal BCR/ABL,positive cells in haematopoietic tissues. The chronic phase of CML is inevitably followed by an accelerated phase of the disease, with consecutive blast crisis. However, depending on genetic stability, epigenetic events, and several other factors, the clinical course and survival appear to vary among patients. Recent data suggest that angiogenic cytokines such as vascular endothelial growth factor (VEGF), are up-regulated in CML, and play a role in the pathogenesis of the disease. These factors appear to be produced and released in leukaemic cells in patients with CML. In line with this notion, increased serum-levels of angiogenic growth factors are measurable in CML patients. In this study we provide an overview of angiogenic growth factors expressed in CML cells, discuss the possible pathogenetic role of these cytokines, the biochemical basis of their production in leukaemic cells, and their potential clinical implications. [source] Migraine and stroke , why do we talk about it?EUROPEAN JOURNAL OF NEUROLOGY, Issue 3 2006C. Lampl Data from observational studies suggest that migraine may be a risk factor for stroke. Furthermore, a significant association between migraine and ischemic stroke (IS) has been demonstrated in population and case,control studies. The risk of IS appears to be higher for migraine with aura than for migraine without aura. The pathogenesis is not known but several studies report some common biochemical mechanisms in the two diseases. Meta-analysis also demonstrates that subjects with migraine are at higher risk of showing white matter abnormalities on magnetic resonance images than are those without migraine. [source] Antipsoriatic drug anthralin induces EGF receptor phosphorylation in keratinocytes: requirement for H2O2 generationEXPERIMENTAL DERMATOLOGY, Issue 2 2004Dominik Peus Abstract: Even though anthralin is a well-established topical therapeutic agent for psoriasis, little is known about its effects and biochemical mechanisms of signal transduction. In contrast to a previous report, we found that anthralin induced time- and concentration-dependent phosphorylation of epidermal growth factor receptor in primary human keratinocytes. Four lines of evidence show that this process is mediated by reactive oxygen species. First, we found that anthralin induces time-dependent generation of H2O2. Second, there is a correlation between a time-dependent increase in anthralin-induced epidermal growth factor receptor phosphorylation and H2O2 generation. Third, the structurally different antioxidants n -propyl gallate and N -acetylcysteine inhibited epidermal growth factor receptor phosphorylation induced by anthralin. Fourth, overexpression of catalase inhibited this process. The epidermal growth factor receptor-specific tyrosine kinase inhibitor PD153035 abrogated anthralin-induced epidermal growth factor receptor phosphorylation and activation of extracellular-regulated kinase 1/2. These findings establish the following sequence of events: (1) H2O2 generation, (2) epidermal growth factor receptor phosphorylation, and (3) extracellular-regulated kinase activation. Our data identify anthralin-induced reactive oxygen species and, more specifically, H2O2 as an important upstream mediator required for ligand-independent epidermal growth factor receptor phosphorylation and downstream signaling. [source] Pigment epithelium-derived factor binds to cell-surface F1 -ATP synthaseFEBS JOURNAL, Issue 9 2010Luigi Notari Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a , 60 kDa PEDF-binding protein in bovine retina with Bos taurus F1 -ATP synthase ,-subunit, and that F1Fo -ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding of PEDF to F1. Size-exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F1. Real-time binding as determined by surface plasmon resonance demonstrated that yeast F1 interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F1 and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF-binding and angiostatin-binding sites on F1. Surfaces of endothelial cells exhibited affinity for PEDF-binding proteins of , 60 kDa. Antibodies to F1,-subunit specifically captured PEDF-binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell-surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell-surface F1, these results show that PEDF is a ligand for endothelial cell-surface F1Fo -ATP synthase. They suggest that PEDF-mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity. Structured digital abstract ,,MINT-7711286: angiostatin (uniprotkb:P00747) physically interacts (MI:0915) with F-ATPase alpha subunit (uniprotkb:P07251), F-ATPase beta subunit (uniprotkb:P00830), F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase delta subunit (uniprotkb:Q12165) and F-ATPase epsilon subunit (uniprotkb:P21306) by competition binding (MI:0405) ,,MINT-7711113: angiostatin (uniprotkb:P00747) physically interacts (MI:0915) with F-ATPase epsilon subunit (uniprotkb:P21306), F-ATPase delta subunit (uniprotkb:Q12165), F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase beta subunit(uniprotkb:P00830) and F-ATPase alpha subunit (uniprotkb:P07251) by surface plasmon resonance (MI:0107) ,,MINT-7711060: F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase beta subunit (uniprotkb:P00830), F-ATPase alpha subunit (uniprotkb:P07251) and PEDF (uniprotkb:P36955) physically interact (MI:0915) by molecular sieving (MI:0071) ,,MINT-7711313: F-ATPase epsilon subunit (uniprotkb:P21306), F-ATPase delta subunit (uniprotkb:Q12165), PEDF (uniprotkb:P36955), F-ATPase alpha subunit (uniprotkb:P07251), F-ATPase beta subunit (uniprotkb:P00830) and F-ATPase gamma subunit(uniprotkb:P38077) physically interact (MI:0915) by molecular sieving (MI:0071) ,,MINT-7711083: PEDF (uniprotkb:P36955) physically interacts (MI:0915) with F-ATPase epsilon subunit (uniprotkb:P21306), F-ATPase delta subunit (uniprotkb:Q12165), F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase beta subunit (uniprotkb:P00830) and F-ATPase alpha subunit (uniprotkb:P07251) by surface plasmon resonance (MI:0107) [source] Isolation and characterisation of a partial peptide synthetase gene from Trichoderma asperellumFEMS MICROBIOLOGY LETTERS, Issue 2 2005Chanikul Chutrakul Abstract Many species of Trichoderma have attracted interest as agents for the biological control of soil borne fungal pathogens of a range of crop plants. Research on the biochemical mechanisms associated with this application has focused on the ability of these fungi to produce enzymes which lyse fungal cell walls, and antifungal antibiotics. An important group of the latter are the non-ribosomal peptides called peptaibols. In this study Trichoderma asperellum, a strain used in biological control in Malaysia, was found to produce the peptaibol, trichotoxin. This type of peptide molecule is synthesised by a peptide synthetase (PES) enzyme template encoded by a peptide synthetase (pes) gene. Using nucleotide sequences amplified from adenylation (A-) domains as probes, to hybridise against a , FIX®II genomic library from T. asperellum, 25 clones were recovered. These were subsequently identified as representative of four groups based on their encoding properties for specific amino acid incorporation modules in a PES. This was based on analysis of their amino acid sequences which showed up to 86% identity to other PESs including TEX 1. [source] Interaction between leucocytes and human spermatozoa influencing reactive oxygen intermediates releaseINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 2 2004Monika Fr Summary The relationship between the presence of white blood cells (WBCs) and the fertilizing potential of human semen is still an open question. It is well known that the presence of leucocytes in human semen can be related to the production of reactive oxygen intermediates (ROI). Semen samples were obtained from 15 normozoospermic men and leucocytes were isolated from heparinized blood drawn from 15 volunteers. Lucigenin and luminol-mediated chemiluminescence assays were used to determine reactive oxygen species (ROS) generation by non-activated or activated leucocytes through 12-myristate-13-acetate or N-formyl-methionyl-leucyl-phenyalanine (FMLP) before the addition of spermatozoa isolated by swim-up or Percoll procedures. All spermatozoal fractions used in this study were characterized by defining their motility, morphology and viability. The levels of ROS formation by non-activated as well as stimulated leucocytes were significantly decreased after addition of swim-up separated spermatozoa (p < 0.01). The ability to inhibit the basal chemiluminescence was of lower degree for spermatozoa isolated from 90% Percoll fractions than for swim-up sperm. However, addition of sperm cells from 47% Percoll fraction was found to increase both lucigenin and luminol signals. Moreover, the determined ROI levels changed depending on the type of inducing factor used for oxidative burst. Then, spermatozoa selected by swim-up procedure although with only slightly higher viability and morphology than sperm obtained from 90% Percoll fraction clearly exhibited much higher capacity to inhibit ROI secretion by receptor-stimulated leucocytes (FMLP-activation) than Percoll fractionated sperm. Such results may indicate that within normal semen may exist sperm subpopulations with different biochemical mechanisms controlling the interaction between spermatozoa and contaminating leucocytes. When ROI levels contained in normozoospermic semen are dependent on the WBCs activation, it seems that spermatozoa with preserved normal functional competence are able to defend themselves against leucocytes-derived ROI. Also for normozoospermic ejaculates, swim-up sperm may improve semen antioxidant characteristics when comparing with Percoll (90%) separated sperm. It may help for optimal sperm preparation when assisting to infertility treatment. [source] Development of a homologous expression system for rubber oxygenase RoxA from Xanthomonas sp.JOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2010N. Hambsch Abstract Aims:, Natural rubber (poly-[cis -1,4-isoprene]) can be cleaved into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al by rubber oxygenase A (RoxA) isolated from Xanthomonas sp. RoxA is a novel type of dihaem dioxygenase with unknown cleavage mechanism of the rubber carbon backbone. Analysis of mutant RoxA after mutagenesis could be a way to investigate the function of selected amino acids of RoxA during catalysis. Unfortunately, expression of functional RoxA in recombinant Escherichia coli or in recombinant ,-Proteobacteria such as Pseudomonas putida was not possible in our hands. Therefore, expression of recombinant RoxA in the homologous host, Xanthomonas, was performed. Methods and Results:, A transformation system via electroporation was established, and a conjugation system was optimized for Xanthomonas sp. Inactivation of the chromosomal roxA gene by insertional mutagenesis resulted in inability of Xanthomonas sp. to produce active RoxA and to utilize rubber as a sole source of carbon and energy. When an intact copy of roxA was cloned under control of a rhamnose-inducible promoter in a broad host range vector and was transferred to Xanthomonas sp., high expression levels of functional RoxA in the presence of rhamnose were obtained. Conclusions and Significance and Impact of the Study:, Purification of recombinantly expressed RoxA was simplified because of drastically shortened fermentation times and because separation of RoxA from remaining rubber latex particles was not necessary with rhamnose-induced cultures. About 6 mg purified RoxA were obtained from 1 l of cell-free culture fluid. Purified recombinant RoxA was highly active and revealed comparable spectral properties as RoxA purified from the wild type. The results of our study are the methodical basis for molecular biological manipulation in Xanthomonas sp. and will simplify investigation into the biochemical mechanisms by which rubber can be biodegraded in the environment by this novel extracellular dihaem dioxygenase RoxA. [source] The effects of osmotic stress on the structure and function of the cell nucleusJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2010John D. Finan Abstract Osmotic stress is a potent regulator of the normal function of cells that are exposed to osmotically active environments under physiologic or pathologic conditions. The ability of cells to alter gene expression and metabolic activity in response to changes in the osmotic environment provides an additional regulatory mechanism for a diverse array of tissues and organs in the human body. In addition to the activation of various osmotically- or volume-activated ion channels, osmotic stress may also act on the genome via a direct biophysical pathway. Changes in extracellular osmolality alter cell volume, and therefore, the concentration of intracellular macromolecules. In turn, intracellular macromolecule concentration is a key physical parameter affecting the spatial organization and pressurization of the nucleus. Hyper-osmotic stress shrinks the nucleus and causes it to assume a convoluted shape, whereas hypo-osmotic stress swells the nucleus to a size that is limited by stretch of the nuclear lamina and induces a smooth, round shape of the nucleus. These behaviors are consistent with a model of the nucleus as a charged core/shell structure pressurized by uneven partition of macromolecules between the nucleoplasm and the cytoplasm. These osmotically-induced alterations in the internal structure and arrangement of chromatin, as well as potential changes in the nuclear membrane and pores are hypothesized to influence gene transcription and/or nucleocytoplasmic transport. A further understanding of the biophysical and biochemical mechanisms involved in these processes would have important ramifications for a range of fields including differentiation, migration, mechanotransduction, DNA repair, and tumorigenesis. J. Cell. Biochem. 109: 460,467, 2010. © 2009 Wiley-Liss, Inc. [source] PTH-dependent adenylyl cyclase activation in SaOS-2 cells: Passage dependent effects on G protein interactionsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2002Hong Gao Parathyroid hormone (PTH) sensitive adenylyl cyclase activity (ACA) in SaOS-2 cells varies as a function of cell passage. In early passage (EP) cells (<,6), ACA in response to PTH and forskolin (FOR) was relatively low and equivalent, whereas in late passage (LP) cells (>,22), PTH exceeded FOR dependent ACA. Potential biochemical mechanisms for this passage dependent change in ACA were considered. In EP, prolonged exposure to pertussis toxin (PT) markedly enhanced ACA activity in response to PTH, Isoproterenol and Gpp(NH)p, whereas ACA in response to FOR was decreased. In contrast, the identical treatment of LP with PT diminished all ACA in response to PTH, Gpp(NH)p, and FOR. The dose dependent effects of PT on subsequent [32P]ADP-ribosylation of its substrates, GTPase activity, as well as FOR-dependent ACA, were equivalent in EP and LP. The relative amounts of G,i and G,s proteins, as determined both by Western blot, PT and cholera toxin (CT) dependent [32P]ADP-ribosylation, were quantitatively similar in EP and LP. Western blot levels of G,s and G,i proteins were not influenced by prior exposure to PT. Both PT and CT dependent [32P]ADP-ribosylation were dose-dependently decreased following exposure to PT. However, the PT-dependent decline in CT-dependent [32P]ADP-ribosylation occurred with enhanced sensitivity in LP. The protein synthesis inhibitor cycloheximide partially reversed the PT associated decrease in FOR dependent ACA in EP. In contrast, cycloheximide completely reversed the PT associated decrease in FOR and as well as PTH dependent ACA in LP. G,s activity, revealed by cyc, reconstitution, was not altered either by cell passage or exposure to PT. The results suggest that the coupling between the components of the complex may be pivotally important in the differential responsiveness of early and late passage SaOS-2 cells to PTH. J. Cell. Physiol. 193: 10,18, 2002. © 2002 Wiley-Liss, Inc. [source] Diabetic neuropathy: therapies on the horizonJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 9 2009Danish Mahmood Abstract Objectives This is a review of emerging interventions from the recent preclinical and clinical literature that demonstrate the potential for effectiveness in the therapy of diabetic neuropathy (DN). DN is the most common complication of diabetes mellitus and up to 50% of patients with type 1 and type 2 forms have some or other form of neuropathy. The pathology of DN is characterized by progressive nerve fibre loss that gives rise to positive and negative clinical signs and symptoms such as pain, paraesthesiae and loss of sensation. Key findings There are very few drugs available to directly treat DN. Those that are clinically indicated provide symptomatic relief but do not repair or reverse underlying nerve damage. However, some agents are in clinical development that may support adult neurons and direct reparative processes after injury stages. Several disease modifying drugs such as aldose reductase inhibitors and protein kinase C inhibitors are in phase III development. Agents on the horizon include neurotrophic factors, growth factors, gene therapy, immunotherapy, poly(ADP-ribose) polymerase inhibitors and non-immunosuppressive immunophilin ligands. Summary Progress has been made toward understanding the biochemical mechanisms leading to diabetic neuropathy, and as a result, new treatment modalities are being explored. The pathogenesis, types and approaches for treating DN together with the newer therapeutic interventions on the horizon are discussed. [source] Toward an "omic" physiopathology of reactive chemicals: Thirty years of mass spectrometric study of the protein adducts with endogenous and xenobiotic compoundsMASS SPECTROMETRY REVIEWS, Issue 5 2009Federico Maria Rubino Abstract Cancer and degenerative diseases are major causes of morbidity and death, derived from the permanent modification of key biopolymers such as DNA and regulatory proteins by usually smaller, reactive molecules, present in the environment or generated from endogenous and xenobiotic components by the body's own biochemical mechanisms (molecular adducts). In particular, protein adducts with organic electrophiles have been studied for more than 30 [see, e.g., Calleman et al., 1978] years essentially for three purposes: (a) as passive monitors of the mean level of individual exposure to specific chemicals, either endogenously present in the human body or to which the subject is exposed through food or environmental contamination; (b) as quantitative indicators of the mean extent of the individual metabolic processing which converts a non-reactive chemical substance into its toxic products able to damage DNA (en route to cancer induction through genotoxic mechanisms) or key proteins (as in the case of several drugs, pesticides or otherwise biologically active substances); (c) to relate the extent of protein modification to that of biological function impairment (such as enzyme inhibition) finally causing the specific health damage. This review describes the role that contemporary mass spectrometry-based approaches employed in the qualitative and quantitative study of protein,electrophile adducts play in the discovery of the (bio)chemical mechanisms of toxic substances and highlights the future directions of research in this field. A particular emphasis is given to the measurement of often high levels of the protein adducts of several industrial and environmental pollutants in unexposed human populations, a phenomenon which highlights the possibility that a number of small organic molecules are generated in the human organism through minor metabolic processes, the imbalance of which may be the cause of "spontaneous" cases of cancer and of other degenerative diseases of still uncharacterized etiology. With all this in mind, it is foreseen that a holistic description of cellular functions will take advantage of new analytical methods based on time-integrated metabolomic measurements of a new biological compartment, the "adductome," aimed at better understanding integrated organism response to environmental and endogenous stressors. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 28:725,784, 2009 [source] The ozone paradox: Ozone is a strong oxidant as well as a medical drugMEDICINAL RESEARCH REVIEWS, Issue 4 2009Velio Bocci Abstract After five decades characterized by empiricism and several pitfalls, some of the basic mechanisms of action of ozone in pulmonary toxicology and in medicine have been clarified. The present knowledge allows to understand the prolonged inhalation of ozone can be very deleterious first for the lungs and successively for the whole organism. On the other hand, a small ozone dose well calibrated against the potent antioxidant capacity of blood can trigger several useful biochemical mechanisms and reactivate the antioxidant system. In detail, firstly ex vivo and second during the infusion of ozonated blood into the donor, the ozone therapy approach involves blood cells and the endothelium, which by transferring the ozone messengers to billions of cells will generate a therapeutic effect. Thus, in spite of a common prejudice, single ozone doses can be therapeutically used in selected human diseases without any toxicity or side effects. Moreover, the versatility and amplitude of beneficial effect of ozone applications have become evident in orthopedics, cutaneous, and mucosal infections as well as in dentistry. © 2009 Wiley Periodicals, Inc. Med Res Rev [source] From fundamental studies of sporulation to applied spore researchMOLECULAR MICROBIOLOGY, Issue 2 2005Imrich Barák Summary Sporulation in the Gram-positive bacterium, Bacillus subtilis, has been used as an excellent model system to study cell differentiation for almost half a century. This research has given us a detailed picture of the genetic, physiological and biochemical mechanisms that allow bacteria to survive harsh environmental conditions by forming highly robust spores. Although many basic aspects of this process are now understood in great detail, including the crystal and NMR structures of some of the key proteins and their complexes, bacterial sporulation still continues to be a highly attractive model for studying various cell processes at a molecular level. There are several reasons for such scientific interest. First, some of the complex steps in sporulation are not fully understood and/or are only described by ,controversial' models. Second, intensive research on unicellular development of a single microorganism, B. subtilis, left us largely unaware of the multitude of diverse sporulation mechanisms in many other Gram-positive endospore and exospore formers. This diversity would likely be increased if we were to include sporulation processes in the Gram-negative spore formers. Spore formers have great potential in applied research. They have been used for many years as biodosimeters and as natural insecticides, exploited in the industrial production of enzymes, antibiotics, used as probiotics and, more, exploited as possible vectors for drug delivery, vaccine antigens and other immunomodulating molecules. This report describes these and other aspects of current fundamental and applied spore research that were presented at European Spores Conference held in Smolenice Castle, Slovakia, June 2004. [source] Resistance to acidic and alkaline environments in the endodontic pathogen Enterococcus faecalisMOLECULAR ORAL MICROBIOLOGY, Issue 5 2006K. Nakajo Background/aims:, This study aimed to investigate the biochemical mechanisms employed by the endodontic pathogen Enterococcus faecalis to confer acid- and alkali-resistance and to compare these with the mechanisms of representative oral streptococci. Methods:,E. faecalis JCM8728, Streptococcus mutans NCTC10449 and Streptococcus sanguinis ATCC10556 were used to assess both acid- and alkali-resistance by examining: (i) growth in complex media; (ii) stability of intracellular pH (pHin); (iii) cell durability to leakage of preloaded BCECF (2,,7,-bis-(2-carboxyethyl)-5,6-carboxy-fluorescein); and (iv) cell permeability to SYTOX-Green. Results:, Growth was initiated by E. faecalis at pH 4.0,11.0, by S. mutans at pH 4.0,9.0 and by S. sanguinis at pH 5.0,9.0. The pHin was similar to the extracellular pH in S. mutans and S. sanguinis at pH 5,10, while the pHin of E. faecalis was maintained at approximately 7.5,8.5 when extracellular pH was 7.5,10 and was maintained at levels equivalent to the extracellular pH when pH < 7.5. Cell membranes of E. faecalis were resistant to BCECF leakage when extracellular pH was 2.5,12 and to SYTOX-Green permeability at pH 4,10. The cell membrane durability to extracellular pH in E. faecalis was higher than that observed in the Streptococcus strains. Conclusion:, Compared to S. mutans, E. faecalis was found to be equally resistant to acid and more resistant to alkalis. The results suggest that pH-resistance in E. faecalis is attributed to membrane durability against acid and alkali, in addition to cell membrane-bound proton-transport systems. These characteristics may account for why E. faecalis is frequently isolated from acidic caries lesions and from persistently infected root canals where calcium hydroxide medication is ineffective. [source] Protein misfolding in neurodegenerative diseasesNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 3 2004E. I. Agorogiannis A common pathogenic mechanism shared by diverse neurodegenerative disorders, like Alzheimer's disease, Parkinson's disease, Huntington's disease and transmissible spongiform encephalopathies, may be altered protein homeostasis leading to protein misfolding and aggregation of a wide variety of different proteins in the form of insoluble fibrils. Mutations in the genes encoding protein constituents of these aggregates have been linked to the corresponding diseases, thus a reasonable scenario of pathogenesis was based on misfolding of a neurone-specific protein that forms insoluble fibrils that subsequently kill neuronal cells. However, during the past 5 years accumulating evidence has revealed the neurotoxic role of prefibrillar intermediate forms (soluble oligomers and protofibrils) produced during fibril formation. Many think these may be the predominant neurotoxic species, whereas microscopically visible fibrillar aggregates may not be toxic. Large protein aggregates may rather be simply inactive, or even represent a protective state that sequesters and inactivates toxic oligomers and protofibrils. Further understanding of the biochemical mechanisms involved in protein misfolding and fibrillization may optimize the planning of common therapeutic approaches for neurodegenerative diseases, directed towards reversal of protein misfolding, blockade of protein oligomerization and interference with the action of toxic proteins. [source] Basic science and translational research in female pelvic floor disorders: Proceedings of an NIH-sponsored meetingNEUROUROLOGY AND URODYNAMICS, Issue 4 2004Anne M. Weber Abstract Aims To report the findings of a multidisciplinary group of scientists focusing on issues in basic science and translational research related to female pelvic floor disorders, and to produce recommendations for a research agenda for investigators studying female pelvic floor disorders. Methods A National Institutes of Health (NIH)-sponsored meeting was held on November 14,15, 2002, bringing together scientists in diverse fields including obstetrics, gynecology, urogynecology, urology, gastroenterology, biomechanical engineering, neuroscience, endocrinology, and molecular biology. Recent and ongoing studies were presented and discussed, key gaps in knowledge were identified, and recommendations were made for research that would have the highest impact in making advances in the field of female pelvic floor disorders. Results The meeting included presentations and discussion on the use of animal models to better understand physiology and pathophysiology; neuromuscular injury (such as at childbirth) as a possible pathogenetic factor and mechanisms for recovery of function after injury; the use of biomechanical concepts and imaging to better understand the relationship between structure and function; and molecular and biochemical mechanisms that may underlie the development of female pelvic floor disorders. Conclusions While the findings of current research will help elucidate the pathophysiologic pathways leading to the development of female pelvic floor disorders, much more research is needed for full understanding that will result in better care for patients through specific rather than empiric therapy, and lead to the potential for prevention on primary and secondary levels. © 2004 Wiley-Liss, Inc. [source] Cross-resistance study and biochemical mechanisms of thiamethoxam resistance in B-biotype Bemisia tabaci (Hemiptera: Aleyrodidae)PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 3 2010Yuntao Feng Abstract BACKGROUND: B-biotype Bemisia tabaci (Gennadius) has invaded China over the past two decades. To understand the risks and to determine possible mechanisms of resistance to thiamethoxam in B. tabaci, a resistant strain was selected in the laboratory. Cross-resistance and the biochemical mechanisms of thiamethoxam resistance were investigated in the present study. RESULTS: A 66.3-fold thiamethoxam-resistant B. tabaci strain (TH-R) was established after selection for 36 generations. Compared with the susceptible strain (TH-S), the selected TH-R strain showed obvious cross-resistance to imidacloprid (47.3-fold), acetamiprid (35.8-fold), nitenpyram (9.99-fold), abamectin (5.33-fold) and carbosulfan (4.43-fold). No cross-resistance to fipronil, chlorpyrifos or deltamethrin was seen. Piperonyl butoxide (PBO) and triphenyl phosphate (TPP) exhibited significant synergism on thiamethoxam effects in the TH-R strain (3.14- and 2.37-fold respectively). However, diethyl maleate (DEM) did not act synergistically with thiamethoxam. Biochemical assays showed that cytochrome P450 monooxygenase activities increased 1.21- and 1.68-fold respectively, and carboxylesterase activity increased 2.96-fold in the TH-R strain. However, no difference was observed for glutathione S -transferase between the two strains. CONCLUSION: B-biotype B. tabaci develops resistance to thiamethoxam. Cytochrome P450 monooxygenase and carboxylesterase appear to be responsible for the resistance. Reasonable resistance management that avoids the use of cross-resistance insecticides may delay the development of resistance to thiamethoxam in this species. 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