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Specific Substrate (specific + substrate)
Selected AbstractsSubstrate incorporation patterns of bacterioplankton populations in stratified and mixed waters of a humic lakeENVIRONMENTAL MICROBIOLOGY, Issue 7 2009Ulrike Buck Summary Bacterial incorporation of glucose, leucine, acetate and 4-hydroxybenzoic acid (HBA) was investigated in an artificially divided humic lake (Grosse Fuchskuhle, Germany). Two basins with contrasting influx of allochthonous organic carbon were sampled during late summer stratification (oxic and anoxic layers) and after autumn mixing. High total and cell-specific incorporation rates were observed for glucose and HBA in stratified and mixed waters respectively, but only a small fraction of bacteria visibly incorporated HBA. The oxic layer of the more humic-rich basin featured a significantly lower fraction of glucose incorporating cells and substantially higher proportions of acetate assimilating bacteria. Niche differentiation was observed in two betaproteobacterial populations: cells affiliated with the Polynucleobacter C subcluster efficiently incorporated acetate but little glucose, whereas the opposite was found for members of the R-BT065 clade. By contrast, leucine incorporation was variable in both taxa. Considering the high concentrations and rapid photochemical generation of organic acids in humic waters our results may help to explain the success of the Polynucleobacter C lineage in such habitats. Specific substrate or habitat preferences were also present in three subgroups of the actinobacterial acI lineage: The numerically dominant clade in oxic waters (acI-840-1) was absent in the anoxic zone and did not incorporate acetate. A second group (acI-840-2) was found both in the epi- and hypolimnion, whereas the third one (acI-840-3) only occurred in anoxic waters. Altogether our results suggest a constitutive preference for some substrates versus an adaptive utilization of others in the studied microbial groups. [source] In vitro effects of tacrolimus on human cytochrome P450FUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 6 2002K. Lecointre Abstract Tacrolimus, a potent immunosuppressive drug, is known to be metabolized predominantly in the liver by cytochrome P450 3A (CYP3A). In order to determine the potential of tacrolimus to inhibit the metabolism of other drugs, we have investigated its inhibitory effects on specific cytochrome reactions. Specific substrates for the seven cytochromes (CYPs) 1A2, 2A6, 2C9, 2C19, 2D6, 2E1 and 3A4/5 were incubated with human hepatic microsome preparations with or without specific inhibitors or tacrolimus and the metabolites were detected by high-pressure liquid chromatography (HPLC) or fluorimetric methods. All the specific inhibitors reduced or abolished the specific CYP activity. Tacrolimus had no effect on any CYP at concentrations below 1 µm, while at higher concentrations it had a mild inhibitory effect on CYP3A4 and 3A5. These observations suggest that tacrolimus is unlikely to potentiate the effect of coadministered drugs through inhibition of their metabolism in the liver. [source] "Reverse degradomics", monitoring of proteolytic trimming by multi-CE and confocal detection of fluorescent substrates and reaction productsELECTROPHORESIS, Issue 13 2009Helene Piccard Abstract A platform for profiling of multiple proteolytic activities acting on one specific substrate, based on the use of a 96-channel capillary DNA sequencer with CE-LIF of labeled substrate peptides and reaction products is introduced. The approach consists of synthesis of a substrate peptide of interest, fluorescent labeling of the substrate, either aminoterminally by chemical coupling, or carboxyterminally by transglutaminase reaction, proteolysis by a biological mixture of proteases in the absence or presence of protease inhibitors, multi-channel analysis of substrate and reaction products, and data collection and processing. Intact substrate and reaction products, even when varying by only one amino acid, can be relatively semi-quantified in a high-throughput manner, yielding information on proteases acting in complex biological mixtures and without prepurification. Monitoring, classification and inhibition of multiple proteolytic activities are demonstrated on a model substrate, the aminoterminus of the mouse granulocyte chemotactic protein-2. In view of extensive processing of chemokines into various natural forms with different specific biological activities, and of the fragmentary knowledge of processing proteases, examples of processing by neutrophil degranulate, tumor cell culture fluids and plasma are provided. An example of selection and comparison of inhibitory mAbs illustrates that the platform is suitable for inhibitor screening. Whereas classical degradomics technologies analyze the substrate repertoire of one specific protease, here the complementary concept, namely the study of all proteases acting, in a biological context, on one specific substrate, is developed and tuned to identify key proteases and protease inhibitors for the processing of any biological substrate of interest. [source] NF-ATc2 induces apoptosis in Burkitt's lymphoma cells through signaling via the B cell antigen receptorEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2003Eisaku Kondo Abstract Cross-linking of the B cell antigen receptor (BCR) with an anti-IgM antibody has been shown to induce dramatic apoptosis in type I Burkitt's lymphoma (BL) cells. However, the apoptotic mechanism triggered via BCR remains unknown. Here we reports a mechanism of BCR ligation-induced apoptosis involving protein phosphatase calcineurin and its specific substrate, transcriptional factor NF-AT. In response to BCR cross-linking, endogenous calcineurin was rapidly activated, and this facilitated nuclear translocation of NF-ATc2, a subtype of NF-AT members. Interestingly, nuclear-imported NF-ATc2 functioned pro-apoptotically in BL cells. The effect of NF-ATc2 was efficiently blocked with FK506, which prevented its nuclear translocation through inactivation of calcineurin. In addtion, TR3 induction during BCR cross-linking was reduced by FK506 and the VIVIT peptide, which is a highly selective inhibitor for NF-AT. This strongly suggests that activation of NF-ATc2 by calcineurin is essential for TR3 recruitment, and that TR3 can be considered as a candidate for death effector in BCR-mediated apoptosis. Therefore, NF-ATc2 plays a crucial role in BCR-mediated apoptosis in type IBL, providing greater insight into unique BL characteristics through BCR signaling. [source] A novel protein kinase from Brassica juncea stimulated by a protozoan calcium binding proteinFEBS JOURNAL, Issue 11 2000Purification, partial characterization A novel protein kinase (BjCCaBPk) from etiolated Brassica juncea seedlings has been purified and partially characterized. The purified enzyme migrated on SDS/PAGE as a single band with an apparent molecular mass of 43 kDa. The optimum pH for the kinase activity was 8.0. It was stimulated more than sixfold by the protozoa Entamoeba histolytica calcium binding protein EhCaBP (10.5 nm) but not by calmodulin (CaM) when used at equimolar concentration. Moreover the kinase also did not bind CaM,Sepharose. There was neither inhibition of the kinase activity in the presence of W-7 (a CaM antagonist), KN-62 (a specific calcium/CaM kinase inhibitor) and anti-CaM Ig, nor any effect on BjCCaBPk activity of staurosporine (a protein kinase C inhibitor). Furthermore a CaM-kinase specific substrate, syntide-2, proved to be a poor substrate for the BjCCaBPk compared with histone III-S. The phosphorylation of histone III-S involved serine residues. Southern and Northern blot analysis showed the presence of EhCaBP homologues in Brassica. The data suggest that BjCCaBPk may be a novel protein kinase with an affinity towards a calcium binding protein like EhCaBP. [source] Neutrophil elastase in pressure ulcer fluid degrades fibronectin in the exudatesGERIATRICS & GERONTOLOGY INTERNATIONAL, Issue 3 2004Shingo Ai Background: Pressure ulcers are classified as chronic wounds, which do not heal in a timely fashion. Fibronectin is condensed in granulation tissue, and essential glycoprotein of wound healing. It has been proposed that fibronectin degradation may be involved in delaying wound healing. We have investigated whether pressure ulcer fluid (PUF) contains degraded fibronectin. In addition, we tried to identify the proteinase which contributes to fibronectin degradation in PUF. Methods: Fibronectin degradation and the presence of neutrophil elastase (NE) in PUF were determined by immunoblot analysis. Fibronectin degradation activity in PUF was determined in the presence of various proteinase inhibitors. NE activity was assessed using NE specific substrate. Results: Immunoblot analysis revealed that degraded fibronectin was observed in PUF samples but not in acute wound fluid (AWF). The PUF contained a proteinase capable of degrading freshly added fibronectin and its activity in PUF was blocked by a broad-spectrum serine proteinase inhibitor or sivelestat, a specific neutrophil elastase inhibitor, but not by metalloproteinase and cysteine proteinase inhibitors. Immunoblot analysis of PUF using an antineutrophil elastase antibody revealed that neutrophil elastase was detected as three bands at molecular weights of ,30 kDa, ,38 kDa, and ,54 kDa, indicating that neutrophil elastase in the exudates existed not only as free monomers, but also in polymers or complexes with other molecules. Conclusion: These results suggest that PUF contains a high level of neutrophil elastase which may be involved in the delay of the healing of pressure ulcer through the fibronectin degradation. [source] Calpain-mediated degradation of G-substrate plays a critical role in retinal excitotoxicity for amacrine cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2009Toru Nakazawa Abstract The role of neuronal N-methyl-D-aspartate (NMDA) receptor-mediated intracellular signaling has been elucidated in both physiological and pathological conditions. However, the details of relative vulnerability for excitotoxicity remain unknown. Retinal excitotoxicity is involved in various diseases leading to irreversible blindness. Here, we used the visual system and explored the mechanistic details of the NMDA-elicited intracellular events, especially in the amacrine cells, which are the most vulnerable type of neuron in the retina. G-substrate, a specific substrate of cyclic guanosine 3,,5,-monophosphate (cGMP)-dependent protein kinase, is colocalized with amacrine cells and acts as an endogenous inhibitor of protein phosphatase. To elucidate how G-substrate was involved in NMDA-induced amacrine cell death, the immunohistochemical analysis with G-substrate antibody was performed following NMDA injury. In vivo, NMDA immediately decreased G-substrate immunoreactivity, and the suppression of calpain activation using ALLN or calpain III, an inhibitor of calpain, blocked this decrease. In vitro, degraded fragments of G-substrate were detected within 10 min after coincubation of G-substrate and calpain. Moreover, G-substrate knockout (G-substrate,/,) mice were more susceptible to NMDA injury than wild-type mice. ALLN did not have a neuroprotective effect in G-substrate,/, mice. These data strongly suggest that calpain-mediated loss of G-substrate represents an important mechanism contributing to NMDA-induced amacrine cell death. © 2008 Wiley-Liss, Inc. [source] Risk factors for periodontitis in HIV+ patientsJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2004Tamer Alpagot Objective:, The purpose of this study was to identify risk factors for periodontitis associated with human immunodeficiency virus (HIV) infection. Methods:, A total of 152 HIV+ patients were recruited from the CARE clinic at the University of the Pacific School of Dentistry. Clinical measurements (gingival index, plaque index, bleeding index, probing depth, and attachment loss), gingival crevicular fluid (GCF) and subgingival plaque samples were taken from eight sites of each patient at baseline and 6-month visits. GCF neutrophil elastase was determined by measurement of p -nitroanalide resulting from hydrolysis of an elastase-specific peptide. GCF ,-glucuronidase was determined by release of 4-methylumbelliferone from hydrolysis of a specific substrate. A bacterial concentration fluorescence immunoassay was used to detect periodontopathic bacteria in subgingival plaque samples. Results:, Viral load, age, smoking pack-years, Fusobacterium nucleatum, Prevotella intermedia, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, neutrophil elastase, and ,-glucuronidase were significantly correlated with clinical measurements (0.0001 < p < 0.05). Significantly higher levels of elastase, ,-glucuronidase, F. nucleatum, P. intermedia, and A. actinomycetemcomitans were found at progressing sites than in non-progressing sites (0.001 < p < 0.05). Conclusions:, These data indicate that age, smoking pack-years, viral load, F. nucleatum, P. intermedia, A. actinomycetemcomitans, elastase, and ,-glucuronidase are risk factors for periodontitis in HIV+ patients. [source] Granulocyte elastase activity in static and flow gingival crevicular fluidJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2003Lijian Jin Objectives:, This study aimed to evaluate the volume of gingival crevicular fluid (GCF) and granulocyte elastase activity in static GCF (sGCF) and flow GCF (fGCF) from subjects with various periodontal conditions. Methods:, Eleven periodontally healthy, 10 gingivitis and 12 periodontitis subjects were recruited and the sites investigated consisted of healthy sites from healthy subjects (HH); healthy (HG) and gingivitis sites (GG) from gingivitis subjects; and healthy (HP), gingivitis (GP) and periodontitis sites (PP) from periodontitis subjects. fGCF samples were collected either 1 min or 5 min following sGCF collection by paper strip technique. GCF volume was determined by Periotron® 6000 and granulocyte elastase activity was assayed with a specific substrate [l -pyroglutamyl- l -prolyl- l -valine- p -nitroanilide(pGluProVal-pNA)]. Results:, At baseline, no significant differences existed in clinical and GCF parameters between the two matched sites for subsequent collection of fGCF samples either 1 min or 5 min after sGCF sampling in all subjects. The flow exudate in HG and HP sites quickly replenished to sGCF levels, while a delayed replenishment was found in HH sites, despite the similar sGCF volumes of these sites. The GCF volume and elastase levels in the fGCF at 1 min were higher in GP sites than in GG sites (P < 0.05). Overall, depletion of elastase levels in the fGCF at 1 min was observed in all subjects, whereas elastase levels in the fGCF at 5 min had replenished to sGCF levels in HP, GP, PP sites and GG sites, but had remained at a lower level in HH and HG sites. An overall positive correlation was found between sGCF and fGCF for GCF volume and elastase activity (P < 0.001); however, this correlation varied with GCF parameters and with site conditions of the subjects concerned. Conclusions:, This study shows that patterns of dynamic changes in GCF flow and elastase activity varied under different periodontal conditions. Assessment of both sGCF and fGCF may allow better insight into the dynamic change of the target components in GCF. [source] Evaluation of the Effect of Ethanol's Toxic Metabolite Acetaldehyde on the Gastrointestinal Oligopeptide Transporter, PEPT1: In Vitro and in Vivo StudiesALCOHOLISM, Issue 1 2008Scott J. Fisher Background:, The effects of alcohol consumption and its subsequent metabolism on drug transport, absorption and pharmacokinetics are poorly understood. This study examines the effects of the ethanol metabolite, acetaldehyde, on the clinically relevant drug transporter, PEPT1. The metabolism of ethanol and the following acetaldehyde formation is thought to modulate the uptake capacity of PEPT1 within the gastrointestinal tract for a variety of clinically important peptidomimetic drug compounds. Methods:, Glycylsarcosine ([3H]-GlySar), a nonhydrolysable PEPT1 specific substrate was used in our studies. In vitro uptake studies were performed in the Caco-2 and Chinese hamster ovary (CHO)-hPEPT1 cell models, measuring cellular uptake of labeled compound against increasing levels of unlabeled compound in the presence of acetaldehyde. In vivo absorption of [3H]-GlySar was measured in male Sprague,Dawley rats that were treated with oral dose of ethanol/disulfiram (5 g/kg / 100 mg/kg) for 6 days. These results were compared to control rats treated with saline, ethanol alone or disulfiram alone. Results:, In vitro uptake of [3H]-GlySar in CHO-hPEPT1 cells treated with 1 mM acetaldehyde was significantly decreased (p < 0.05) as compared to untreated controls. The uptake of [3H]-GlySar in Caco-2 cell monolayers treated with 1 mM acetaldehyde was also significantly decreased as compared to the untreated control cells. In vivo absorption of [3H]-GlySar in ethanol treated rats, as measured by AUC0,12 hours were decreased by approximately 50% versus the control rat group. Conclusion:, The effects of acetaldehyde due to consumption of ethanol on the uptake and bioavailability of therapeutic drug compounds transported by the PEPT1 oligopeptide transporter have not been documented. In the present studies, we demonstrate that acetaldehyde significantly modulates PEPT1 function and, thereby, affects drug bioavailability. To our best knowledge, this is the first report on the effects of an ethanol metabolite on substrate absorption in the gastrointestinal tract, rather than interactions in the liver, which is an under-represented area of research in alcohol pathophysiology. [source] Comparative enzymology of native and recombinant house dust mite allergen Der p 1ALLERGY, Issue 3 2009J. Zhang Background:, The cysteine peptidase activity of group 1 house dust mite allergens is important for their allergenicity and may offer new therapeutic targets for allergy treatment. Hitherto, the design of specific inhibitors has been impeded because the availability of pure, fully active allergens has limited the implementation of drug screening campaigns. Similarly, investigation of the mechanisms by which peptidase allergens promote sensitization has also been restricted. Our aim was to compare the enzymology of recombinant and native forms of Der p 1 to establish if an easily expressed recombinant form of Der p 1 could be used as a drug discovery tool. Methods:, Enzymatic activity of natural and recombinant Der p 1 was compared fluorimetrically using a novel specific substrate (ADZ 50,059) and a novel specific active site titrant (ADZ 50,000). The effect of recombinant Der p 1 prodomain on the catalytic activity of both Der p 1 preparations was also examined. Results:, Although differing substantially in molecular weight, the enzymological properties of recombinant and native Der p 1 were indistinguishable. Our data show clearly by experiment that, in contrast to some suggestions, Der p 1 is not an enzyme of bifunctional mechanism. Conclusion:, The catalytic activity of Der p 1 is tolerant of glycosylation differences that occur at N150 when the protein is expressed in Pichia pastoris. This suggests that this recombinant protein may be suitable for drug design studies and in the elucidation of how peptidase activity promotes sensitization to peptidase and nonpeptidase bystander allergens. [source] Role of the sperm proteasome during fertilization and gamete interaction in the mouseMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2005Consuelo Pasten Abstract In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head. Mol. Reprod. Dev. 71: 209,219, 2005. © 2005 Wiley-Liss, Inc. [source] CD13/aminopeptidase N,induced lymphocyte involvement in inflamed joints of patients with rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 9 2002Teruki Shimizu Objective We previously showed that CD13/aminopeptidase N (EC 3.4.11.2) induces chemotactic migration of T lymphocytes by its enzymatic activity. In this study, we examined the role of CD13/aminopeptidase N in lymphocyte involvement in rheumatoid arthritis (RA). Methods Synovial fluids were obtained from 27 RA patients and 6 osteoarthritis (OA) patients. Synovial tissue specimens were obtained from 3 RA patients and 3 OA patients. Protease activity of aminopeptidase in synovial fluids and synovial fibroblasts was assayed fluorometrically using the specific substrate. Expression of CD13/aminopeptidase N in synovial fibroblasts was determined by flow cytometry analyses, Western blotting, and reverse transcriptase,polymerase chain reaction (RT-PCR). Results The mean value of aminopeptidase activity in synovial fluid samples from RA patients was significantly higher than that in samples from OA patients. Increased enzymatic activity of aminopeptidase was detected on synovial fibroblasts from RA patients compared with those from OA patients. Flow cytometry showed that the expression of CD13/aminopeptidase N on synovial fibroblasts from RA patients was higher than the expression on synovial fibroblasts from OA patients, and Western blots and RT-PCR showed that synovial fibroblasts from RA patients contained a greater amount of CD13/aminopeptidase N. The activity of CD13/aminopeptidase N correlated significantly with lymphocyte counts in synovial fluids from RA patients. Synovial fluids from RA patients in which high aminopeptidase activity was detected contained considerable chemotactic activity for lymphocytes, and bestatin, a specific inhibitor of aminopeptidases, partially inhibited the chemotactic activity. Conclusion CD13/aminopeptidase N may participate in the mechanism of lymphocyte involvement in inflamed joints of RA patients as a lymphocyte chemoattractant. [source] Kinetic modeling of cellulosic biomass to ethanol via simultaneous saccharification and fermentation: Part II.BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Experimental validation using waste paper sludge, anticipation of CFD analysis Abstract A kinetic model of cellulosic biomass conversion to ethanol via simultaneous saccharification and fermentation (SSF) developed previously was validated experimentally using paper sludge as the substrate. Adsorption parameters were evaluated based on the data obtained at various values for fractional cellulose conversion. The adsorption model was then combined with batch SSF data to evaluate the cellulose hydrolysis parameters. With the parameters evaluated for the specific substrate, the discrete model was able to predict SSF successfully both with discrete addition of cellulase only and with discrete feeding of substrate, cellulase, and media. The model tested in this study extends the capability of previous SSF models to semi-continuous feeding configurations, and invites a mechanistic interpretation of the recently observed trend of increasing conversion with decreasing feeding frequency [Fan et al. (2007a) Bioprocess Biosyst Eng 30(1):27,34]. Our results also support the feasibility and utility of determining adsorption parameters based on data obtained at several conversions, particularly when the model is to be applied to extended reaction times rather than only initial hydrolysis rates. The revised model is considerably more computationally efficient than earlier models, and appears for many conditions to be within the capability of simulation using computational fluid dynamics. Biotechnol. Bioeng. 2009;102: 66,72. © 2008 Wiley Periodicals, Inc. [source] Inhibitory Effect Of Reactive Oxygen Species On Angiotensin I-Converting Enzyme (Kininase II)CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 3 2001B Michel SUMMARY 1. Somatic angiotensin I-converting enzyme (ACE) is a protein that contains two similar domains (N- and C-terminal), each possessing an active site. We have examined the effects of a generator of hydroxyl radicals (g,OH: 2,2,-azo-bis(2-amidinopropane)) and hydrogen peroxide (H2O2) on ACE using an in vitro approach. 2. The generator of hydroxyl radicals inactivated ACE in a time (2,6 h)- and concentration (0.3,3 mmol/L)-dependent manner at 37°C. When ACE was coincubated for 4 h with g,OH (3 mmol/L), its activity decreased by 70%. Addition of dimethylthiourea or mannitol + methionine, two ,OH scavengers, resulted in a significant protection of ACE activity. Mercaptoethanol and dithiotreitol, two thiol-reducing agents, also efficiently protected ACE activity. 3. The hydrolysis of two natural and domain-specific substrates was explored. The hydrolysis of angiotensin I, preferentially cleaved by the C-domain, was significantly inhibited (57,58%) after 4 h exposure to g,OH (0.3,1 mmol/L). Under the same conditions of exposure, the hydrolysis of N -acetyl-Ser-Asp-Lys-Pro, a specific substrate for the N-domain, was only slightly inhibited by 1 mmol/L g,OH. 4. Hydrogen peroxide, another source of ,OH, was used. After exposure to H2O2 (3 mmol/L; 4 h), an 89% decrease in ACE activity was observed. Pretreatment with the iron chelator deferoxamine (1 mmol/L) attenuated H2O2 -mediated ACE inactivation, demonstrating that the effect of H2O2 was partly due to its conversion into ,OH (Fenton reaction). 5. In summary, our findings demonstrate that g,OH and H2O2 inhibit ACE activity and suggest a preferential action of g,OH on the C-domain of the enzyme. [source] Phosphorylation of NF-,B proteins by cyclic GMP-dependent kinaseFEBS JOURNAL, Issue 10 2003A noncanonical pathway to NF-, B activation The transcription factor NF-,B is activated in cellular stress responses. This requires rapid regulation of its function, which is accomplished, in part, by various modes of phosphorylation. Even though diverse DNA binding subunits of NF-,B proteins may transactivate from distinct recognition sequences, the differential regulation of transcription from the large number of NF-,B responsive sites in various gene promoters and enhancers has been incompletely understood. The cyclic GMP-dependent kinase (PKG) is an important mediator of signal transduction that may induce gene expression through cAMP response element binding protein (CREB) and through other, yet undefined, mechanisms. We have previously characterized a signal transduction pathway that leads to activation-induced cell death in T-lymphocytes and involves the activation of PKG. Here we demonstrate that the NF-,B proteins p65, p49 (also called p52), and p50 are specific substrates for this kinase. PKG dose-dependently increases the transactivating activity of p65 from the NF-,B consensus sequence. It also mediates dose-dependently an increase in transcriptional activity by p49 or p50 from a unique CCAAT/enhance binding protein (C/EBP)-associated NF-,B site, but not from the consensus site. Phosphorylation of p65, p50, or p49 does not alter their subcellular distribution. Because the release of cytosolic p65/p50 heterodimers into the nucleus is by itself insufficient to differentiate all the numerous NF-,B promoter sequences, phosphorylation of the DNA-binding subunits reveals a form of differential regulation of NF-,B activity and it implies a novel pathway for PKG-induced gene transcription. These observations may bear on mechanisms of programmed cell death in T-lymphocytes. They may also be relevant to ongoing efforts to induce cancer cell apoptosis through activation of PKG. [source] Activity and sequence characterization of two cysteine proteases in the digestive tract of the reduviid bug Triatoma infestansINSECT MOLECULAR BIOLOGY, Issue 6 2004A. H. Kollien Abstract Cathepsin B- and cathepsin L-like activities were identified in gut extracts of the blood-sucking bug Triatoma infestans using specific substrates and inhibitors. Activities decreased during the first 2 days after feeding but increased to a maximum value at 5 and 10 days post feeding. The deduced 332 and 328 amino acid sequences showed high levels of identity (50,60%) to other insect cathepsin B- and L-like proteases, respectively. The three amino acid residues of the catalytic domain, CHN, and the GCNGG motif were conserved in both cathepsins, but the occluding loop, characterizing B-like cathepsins, was present only in one. ERFNIN and GNFD motifs occurred in the other sequence, defining it as cathepsin L-like. The cathepsin B-like gene was expressed at low, constitutive levels in unfed and fed T. infestans. [source] On the reliability of 13C metabolic modeling with two-compartment neuronal-glial modelsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 15 2007Alexander A. Shestov Abstract Metabolic modeling of 13C NMR spectroscopy (13C MRS) data using two-compartment neuronal-glial models enabled non-invasive measurements of the glutamate-glutamine cycle rate (VNT) in the brain in vivo. However, the reliability of such two-compartment metabolic modeling has not been examined thoroughly. This study uses Monte-Carlo simulations to investigate the reliability of metabolic modeling of 13C positional enrichment time courses measured in brain amino acids such as glutamate and glutamine during [1- 13C]- or [1,6- 13C2]glucose infusion. Results show that the determination of VNT is not very precise under experimental conditions typical of in vivo NMR studies, whereas the neuronal TCA cycle rate VTCA(N) is determined with a much higher precision. Consistent with these results, simulated 13C positional enrichment curves for glutamate and glutamine are much more sensitive to the value of VTCA(N) than to the value of VNT. We conclude that the determination of the glutamate-glutamine cycle rate VNT using 13C MRS is relatively unreliable when fitting 13C positional enrichment curves obtained during [1- 13C] or [1,6- 13C2]glucose infusion. Further developments are needed to improve the determination of VNT, for example using additional information from 13C- 13C isotopomers and/or using glial specific substrates such as [2- 13C]acetate. © 2007 Wiley-Liss, Inc. [source] Effects of Light and Dark Beer on Hepatic Cytochrome P-450 Expression in Male Rats Receiving Alcoholic Beverages as Part of Total Enteral NutritionALCOHOLISM, Issue 5 2005Mats Hidestrand Background: Alcoholic beverages contain many congeners in addition to ethanol. Therefore, consumption of alcoholic beverages may have considerably different effects on expression of hepatic microsomal monooxygenases than the relatively selective induction of cytochrome P-450 (CYP) 2E1 observed after consumption of pure ethanol. Methods: In the current study, we compared the effects of two beers: lager (a light roasted beer) and stout (a dark roasted beer) with those of an equivalent amount of pure ethanol on hepatic CYP expression. Beer or pure ethanol was part of a complete total enteral nutrition diet that was infused intragastrically into male Sprague Dawley rats for 21 days. At the end of the infusion period, rats were euthanized, and liver and intestinal microsomes were prepared. Expression and activity of CYP1A1/2, CYP2B1, CYP2E1, CYP3A, and CYP4A were assessed by Western immunoblotting and by using CYP enzyme,specific substrates, respectively. Results: mRNA and protein levels of CYP4A1 were elevated only in stout-treated animals. However, lauric acid 12-hydroxylase activity (a CYP4A-specific activity) was reduced (p, 0.05) in microsomes from lager- and stout-fed rats. After oxidation with potassium ferricyanide, this activity was significantly increased in microsomes from stout-fed animals. The relative expression of CYP2E1 and CYP2B1 and the activities toward p -nitrophenol, pentoxyresorufin, or benzyloxyresorufin did not differ between beers or compared with pure ethanol or controls. However, the mean expression of CYP1A2, CYP3A, and CYP4A apoproteins was greater in liver microsomes from stout-infused rats than in those from lager-infused rats, ethanol-infused rats, and diet controls (p, 0.05). In addition, although no significant differences were observed in ethoxyresorufin O-dealkylase (EROD), methoxyresorufin O-dealkylase (MROD), midazolam, or testosterone hydroxylase activities between groups, stout-infused rats had greater hepatic microsomal erythromycin N -demethylase activity than other groups (p, 0.05). Conclusions: Stout contains components other than ethanol that interact in a complex fashion with the monooxygenase system. [source] Flocculation, adhesion and biofilm formation in yeastsMOLECULAR MICROBIOLOGY, Issue 1 2006Kevin J. Verstrepen Summary Yeast cells possess a remarkable capacity to adhere to abiotic surfaces, cells and tissues. These adhesion properties are of medical and industrial relevance. Pathogenic yeasts such as Candida albicans and Candida glabrata adhere to medical devices and form drug-resistant biofilms. In contrast, cell,cell adhesion (flocculation) is a desirable property of industrial Saccharomyces cerevisiae strains that allows the easy separation of cells from the fermentation product. Adhesion is conferred by a class of special cell wall proteins, called adhesins. Cells carry several different adhesins, each allowing adhesion to specific substrates. Several signalling cascades including the Ras/cAMP/PKA and MAP kinase (MAPK)-dependent filamentous growth pathways tightly control synthesis of the different adhesins. Together, these pathways trigger adhesion in response to stress, nutrient limitation or small molecules produced by the host, such as auxin in plants or NAD in mammals. In addition, adhesins are subject to subtelomeric epigenetic switching, resulting in stochastic expression patterns. Internal tandem repeats within adhesin genes trigger recombination events and the formation of novel adhesins, thereby offering fungi an endless reservoir of adhesion properties. These aspects of fungal adhesion exemplify the impressive phenotypic plasticity of yeasts, allowing them to adapt quickly to stressful environments and exploit new opportunities. [source] A versatile strategy to define the phosphorylation preferences of plant protein kinases and screen for putative substratesTHE PLANT JOURNAL, Issue 1 2008Florina Vlad Summary Most signaling networks are regulated by reversible protein phosphorylation. The specificity of this regulation depends in part on the capacity of protein kinases to recognize and efficiently phosphorylate particular sequence motifs in their substrates. Sequenced plant genomes potentially encode over than 1000 protein kinases, representing 4% of the proteins, twice the proportion found in humans. This plethora of plant kinases requires the development of high-throughput strategies to identify their substrates. In this study, we have implemented a semi-degenerate peptide array screen to define the phosphorylation preferences of four kinases from Arabidopsis thaliana that are representative of the plant calcium-dependent protein kinase and Snf1-related kinase superfamily. We converted these quantitative data into position-specific scoring matrices to identify putative substrates of these kinases in silico in protein sequence databases. Our data show that these kinases display related but nevertheless distinct phosphorylation motif preferences, suggesting that they might share common targets but are likely to have specific substrates. Our analysis also reveals that a conserved motif found in the stress-related dehydrin protein family may be targeted by the SnRK2-10 kinase. Our results indicate that semi-degenerate peptide array screening is a versatile strategy that can be used on numerous plant kinases to facilitate identification of their substrates, and therefore represents a valuable tool to decipher phosphorylation-regulated signaling networks in plants. [source] Tree age is a key factor for the conservation of epiphytic lichens and bryophytes in beech forestsAPPLIED VEGETATION SCIENCE, Issue 1 2009Örjan Fritz Abstract Questions: What factors limit the distribution of epiphytic lichens and bryophytes at plot and tree level in beech forests? At what ages do epiphytic species, and species of conservation concern in particular, occur along a chronosequence of beech? Location: South-west Sweden. Method: Five hundred and seventy-one age-determined trees from 37 plots distributed among 29 beech-dominated stands were surveyed along with a number of environmental (16) and substrate (seven) variables in a landscape of ca. 550 ha. Non-metric multidimensional scaling (NMS) and indicator species analysis (ISA) were used for data analysis. Results: Plots containing old trees, confined to the base of slopes and with low impacts of recent forestry (thinning), generally had a high richness of species of conservation concern. Richness of common species and red-listed bryophytes were mostly related to the surveyed bark area. At tree level, primary factors explaining both species richness and composition were age, diameter at breast height and moss cover. There was a gradual replacement of tree age ranges for 58 lichens and 37 bryophytes along the chronosequence of beech. Red-listed lichens favoured damaged beech trees (,180 years), whereas red-listed bryophytes were found on old and young stems in dense stands. Conclusions: Tree age exerts a profound influence on epiphytic lichens and bryophytes growing on beech. Many of the habitat specialists were found mainly on old beech because they inhabit specific substrates that occur on older trees. The association to high tree age commonly excludes red-listed lichens from conventionally managed beech forests with a 100- to 140-year rotation period. [source] pH-Controllable Supramolecular SystemsCHEMISTRY - AN ASIAN JOURNAL, Issue 3 2009Ken Cham-Fai Leung Prof. Abstract Proton, all that matters! This Focus Review surveys representative examples of pH-controllable supramolecular systems with interesting features and state-of-the-art applications, which can lead to the construction of meaningful molecular machines for electronic and biological applications that can be controlled by simple perturbation with acid and base. This Focus Review surveys representative examples of pH-controllable supramolecular systems with interesting features and state-of-the-art applications such as 1),conformational changes within individual molecules; 2),folding/unfolding of polymers; 3),simultaneous binding of cations and anions; 4),logic function; 5),ON,OFF switchable colorimetric sensing; 6),translocation of macrocycle-in-rotaxane molecules; 7),large-scale movement within molecules; and 8),regulation of the substrate flow in nanocontainers. In particular, systems will be discussed that involve: pH-induced conformational changes of a resorcinarene cavitand and a bis(iron porphyrin) complex; pH control in assembly and disassembly of supramolecular systems stabilized with different major noncovalent interactions; pH-driven movements of interlocked molecules involving rotaxanes, molecular elevators, and molecular muscles; and, finally, multicomponent supramolecular systems immobilized on solid supports as pH-responsive nanovalves for the controlled release of specific substrates. Recent advances in the understanding of pH-controllable supramolecular systems have led to the construction of meaningful molecular machines for electronic and biological applications that are amenable to control by simple perturbation with acids and bases. [source] | ||||||||||||||||