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Selected AbstractsSubstrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductaseFEBS JOURNAL, Issue 13 2007Ronnie A. Böck Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80,90%, and D31A and D31L by >,90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in Km for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in kcat/Km. Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in Km; 21-fold decrease in kcat/Km). Only minor variations in the Km for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim. [source] Effects of drying regime on microbial colonization and shredder preference in seasonal woodland wetlandsFRESHWATER BIOLOGY, Issue 3 2008MARTYN D. INKLEY Summary 1. Energy budgets of wetlands in temperate deciduous forests are dominated by terrestrially derived leaf litter that decays under different drying conditions depending on autumn precipitation. We compared decay rates and microbial colonization of maple leaves under different inundation schedules in a field experiment, and then conducted a laboratory study on shredder preference. In the field, litter bags either remained submerged (permanent), were moved to a dried part of the basin once and then returned (semi-permanent), or were alternated between wet and dry conditions for 8 weeks (temporary). 2. There was no difference in decay rates among treatments, but leaves incubated under permanent and semi-permanent conditions had higher fungal and bacterial biomass, and lower C : N ratios than those incubated under alternating drying and wetting conditions. 3. To determine the effects of these differences in litter nutritional quality on shredder preference, we conducted a laboratory preference test with larvae of leaf-shredding caddisflies that inhabit the wetland. Caddisflies spent twice as much time foraging on permanent and semi-permanent litter than on litter incubated under temporary conditions. 4. There is considerable variation among previous studies in how basin drying affects litter breakdown in wetlands, and no previous information on shredder preference. We found that frequent drying in a shallow wetland reduces the nutritional quality of leaf litter (lower microbial biomass and nitrogen content), and therefore preference by invertebrate shredders. These results suggest that inter-annual shifts in drying regime should alter detritus processing rates, and hence the mobilization of the energy and nutrients in leaf litter to the wetland food web. [source] A comparative proteome analysis of human metaphase chromosomes isolated from two different cell lines reveals a set of conserved chromosome-associated proteinsGENES TO CELLS, Issue 3 2007Hideaki Takata A comparative proteome analysis of human metaphase chromosomes between a typical epithelial-like cell, HeLa S3, and a lymphoma-type cell, BALL-1, was performed. One-dimensional (1-D) SDS-PAGE and radical-free and highly reducing two-dimensional electrophoresis (RFHR 2-DE) detected more than 200 proteins from chromosomes isolated from HeLa S3 cells, among which 189 proteins were identified by mass spectrometry (MS). Consistent with our recent four-layer structural model of a metaphase chromosome, all the identified proteins were grouped into four distinct levels of abundance. Both HeLa S3 and BALL-1 chromosomes contained specific sets of abundant chromosome structural and peripheral proteins in addition to less abundant chromosome coating proteins (CCPs). Furthermore, titin array analysis and a proteome analysis of the ultra-high molecular mass region indicated an absence of titin with their molecular weight (MW) more than 1000 kDa. Consequently, the present proteome analyses together with previous information on chromosome proteins provide the comprehensive list of proteins essential for the metaphase chromosome architecture. [source] Estimating age by tooth wear of prehistoric human remains in Brazilian archaeological sitesINTERNATIONAL JOURNAL OF OSTEOARCHAEOLOGY, Issue 5 2006R. N. Oliveira Abstract The occlusal surfaces of 298 permanent maxillary and mandibular molar teeth of prehistoric shellfish-gatherer subjects from the Piaçaguera and Tenorio sites (4930 to 1875 BP), near the central-northern coastline of São Paulo, Brazil, were examined for classification of macro-wear stages. Molar tooth wear is an indication of masticatory activity and can be used in the estimation of age at death. The examination of visual and schematic aspects of occlusal macro-wear used a visual chart proposed by Brothwell, which includes the three superior and inferior, left and right, permanent molars. Three examiners performed the macroscopic observations twice under the same conditions. The resulting age estimates were compared with previous information of age estimated by skeletal examination. A reduced intra- and inter-observer variation was observed; all re-examinations indicated discrepancies of less than two years for the upper and lower limit of the age range estimates. The procedure was also considered consistent with the skeletal method used for age estimation of human remains excavated in Brazilian archaeological shell mounds, with a discrepancy of less than 8.22 years between the upper and lower limits of estimates by both methods. Age classification by the occlusal molar wear may be a useful tool for the classification of archaeological findings, mainly when only fragmentary skeletal remains are excavated. The current results indicate that the application of the Brothwell chart for Brazilian archaeological series presented satisfactory results of consistency, and its expanded use may represent a relevant adjunct for research. Copyright © 2006 John Wiley & Sons, Ltd. [source] Structuring Chemical Space: Similarity-Based Characterization of the PubChem DatabaseMOLECULAR INFORMATICS, Issue 1-2 2010Giovanni Cincilla Abstract The ensemble of conceivable molecules is referred to as the Chemical Space. In this article we describe a hierarchical version of the Affinity Propagation (AP) clustering algorithm and apply it to analyze the LINGO-based similarity matrix of a 500 000-molecule subset of the PubChem database, which contains more than 19,million compounds. The combination of two highly efficient methods, namely the AP clustering algorithm and LINGO-based molecular similarity calculations, allows the unbiased analysis of large databases. Hierarchical clustering generates a numerical diagonalization of the similarity matrix. The target-independent, intrinsic structure of the database , derived without any previous information on the physical or biological properties of the compounds, maps together molecules experimentally shown to bind the same biological target or to have similar physical properties. [source] Electrospray mass spectrometric investigation of the binding of cis -parinaric acid to bovine beta-lactoglobulin and study of the ligand-binding site of the protein using limited proteolysisRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 22 2003Tímea Imre The binding property of parinaric acid, a polyunsaturated fatty acid, to bovine , -lactoglobulin, has been studied by electrospray ionization mass spectrometry. Stable complexation was observed under acidic conditions in a molar ratio of 1:1. Competitive complexation experiments were performed using saturated and unsaturated fatty acid standards with different chain lengths and number of double bonds to study the specificity of the interaction. It can be concluded that formation of the parinaric acid,lactoglobulin complex is preferred even if the molar concentration of the other fatty acids is ten times higher. In cases of specific complex formation the protein must have an active site that is a good acceptor for the ligand molecule. Limited trypsinolysis was performed on the lactoglobulin molecule to identify which part is responsible for the complexation. An intermediate tryptic fragment with molecular mass of 5200,Da was found to have the same ability to bind parinaric acid as the intact protein. This disulfide-bonded residue, [41-70]S-S[149-162], might thus be involved in the specific complexation of parinaric acid to , -lactoglobulin. This conclusion is consistent with previous information on this binding site. Copyright © 2003 John Wiley & Sons, Ltd. [source] Bayesian inference for Rayleigh distribution under progressive censored sampleAPPLIED STOCHASTIC MODELS IN BUSINESS AND INDUSTRY, Issue 3 2006Shuo-Jye Wu Abstract It is often the case that some information is available on the parameter of failure time distributions from previous experiments or analyses of failure time data. The Bayesian approach provides the methodology for incorporation of previous information with the current data. In this paper, given a progressively type II censored sample from a Rayleigh distribution, Bayesian estimators and credible intervals are obtained for the parameter and reliability function. We also derive the Bayes predictive estimator and highest posterior density prediction interval for future observations. Two numerical examples are presented for illustration and some simulation study and comparisons are performed. Copyright © 2006 John Wiley & Sons, Ltd. [source] |