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101.
102.
The primitive protozoan pathogen of humans, Leishmania donovani, resides and multiplies in highly restricted micro-environments within their hosts (i.e. as promastigotes in the gut lumen of their sandfly vectors and as amastigotes in the phagolysosomal compartments of infected mammalian macrophages). Like other trypanosomatid parasites, they are purine auxotrophs (i.e. lack the ability to synthesize purines de novo) and therefore are totally dependent upon salvaging these essential nutrients from their hosts. In that context, in this study we identified a unique 35-kDa, dithiothreitol-sensitive nuclease and showed that it was constitutively released/secreted by both promastigote and amastigote developmental forms of this parasite. By using several different molecular approaches, we identified and characterized the structure of LdNuc(s), a gene that encodes this new 35-kDa class I nuclease family member in these organisms. Homologous episomal expression of an epitope-tagged LdNuc(s) chimeric construct was used in conjunction with an anti-LdNuc(s) peptide antibody to delineate the functional and biochemical properties of this unique 35-kDa parasite released/secreted enzyme. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that this "secretory" enzyme could hydrolyze a variety of synthetic polynucleotides as well as several natural nucleic acid substrates, including RNA and single- and double-stranded DNA. Based on these cumulative observations, we hypothesize that within the micro-environments of its host, this leishmanial "secretory" nuclease could function at a distance away from the parasite to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine requirements of these organisms. Thus, this enzyme might play an important role(s) in facilitating the survival, growth, and development of this important human pathogen. 相似文献
103.
Speransky AS Cimaglia F Krinitsina AA Poltronieri P Fasano P Bogacheva AM Valueva TA Halterman D Shevelev AB Santino A 《Biotechnology journal》2007,2(11):1417-1424
Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors. 相似文献
104.
105.
Ryan L. Parr Shawn W. Carlyle Dennis H. O'Rourke 《American journal of physical anthropology》1996,99(4):507-518
Skeletal remains of 47 individuals from the Great Salt Lake Wetlands, affiliated principally with Bear River (A.D. 400—1000) and Levee Phase (A.D. 1000—1350) Fremont cultural elements, were assessed for four mitochondrial DNA (mtDNA) markers that, in particular association, define four haplogroups (A, B, C, and D) widely shared among contemporary Amerindians groups. The most striking result is the absence of haplogroup A in this Fremont series, despite its predominance in contemporary Amerindian groups. Additionally, haplogroup B, defined by the presence of a 9bp deletion in region V, is present at the moderately high frequency of 60%. Haplogroups C and D are present at low frequencies. An additional haplotype, "N, observed in some modern populations and two other prehistoric samples, is also present in this Fremont skeletal collection. © 1996 Wiley-Liss, Inc. 相似文献
106.
Siyu Wang Stephanie M. Reeve Graham T. Holt Adegoke A. Ojewole Marcel S. Frenkel Pablo Gainza Santosh Keshipeddy Vance G. Fowler Dennis L. Wright Bruce R. Donald 《PLoS computational biology》2022,18(2)
Antimicrobial resistance presents a significant health care crisis. The mutation F98Y in Staphylococcus aureus dihydrofolate reductase (SaDHFR) confers resistance to the clinically important antifolate trimethoprim (TMP). Propargyl-linked antifolates (PLAs), next generation DHFR inhibitors, are much more resilient than TMP against this F98Y variant, yet this F98Y substitution still reduces efficacy of these agents. Surprisingly, differences in the enantiomeric configuration at the stereogenic center of PLAs influence the isomeric state of the NADPH cofactor. To understand the molecular basis of F98Y-mediated resistance and how PLAs’ inhibition drives NADPH isomeric states, we used protein design algorithms in the osprey protein design software suite to analyze a comprehensive suite of structural, biophysical, biochemical, and computational data. Here, we present a model showing how F98Y SaDHFR exploits a different anomeric configuration of NADPH to evade certain PLAs’ inhibition, while other PLAs remain unaffected by this resistance mechanism. 相似文献
107.
108.
Icosahedral double-stranded DNA (dsDNA) bacterial viruses are known to package their genomes into preformed procapsids via a unique portal vertex. Bacteriophage PRD1 differs from the more commonly known icosahedral dsDNA phages in that it contains an internal lipid membrane. The packaging of PRD1 is known to proceed via preformed empty capsids. Now, a unique vertex has been shown to exist in PRD1. We show in this study that this unique vertex extends to the virus internal membrane via two integral membrane proteins, P20 and P22. These small membrane proteins are necessary for the binding of the putative packaging ATPase P9, via another capsid protein, P6, to the virus particle. 相似文献
109.
R S Power H E David M H A Z Mutwakil K Fletcher C Daniells M A Nowell J L Dennis A Martinelli R Wiseman E Wharf D I de Pomerai 《Journal of biosciences》1998,23(4):513-526
This paper reviews the current status of nematodes with stress-inducible transgenes as biosensors responsive to a range of
external stressors, e.g., soil or water pollution, microwave radiation or immunological attack. TransgenicCaenorhabditis elegans carrying reporter genes under heat shock promoter control express reporter products only under stressful conditions. Although
relatively insensitive to single metal ions, these worms respond to complex mixtures present in metal-contaminated watercourses
and to laboratory mixtures containing similar constituents, but not to any of their components singly at comparable concentrations.
Responses to metal mixtures are enhanced by a non-ionic surfactant, Pluronic F-127. Metals taken up by food bacteria and insoluble
metal carbonates can also evoke stress responses, both in soil and aqueous media. However, high concentrations of added metals
are needed to induce clear-cut responses in soil, owing to metal sorption onto clays and organic matter. Transgenic worms
are also stressed by exposure to microwave radiation; pulsed signals generate responses that diminish markedly with distance
from the source. Finally, stress responses are inducible by anti-epicuticle antisera and complement, suggesting that immune
attack can also activite the heat shock system. The development of rapid microplate toxicity assays based on transgenic nematodes
is discussed. 相似文献
110.
Hyung J. Kim Mi-Young Jeong Un Na Dennis R. Winge 《The Journal of biological chemistry》2012,287(48):40670-40679
The enzymatic function of succinate dehydrogenase (SDH) is dependent on covalent attachment of FAD on the ∼70-kDa flavoprotein subunit Sdh1. We show presently that flavinylation of the Sdh1 subunit of succinate dehydrogenase is dependent on a set of two spatially close C-terminal arginine residues that are distant from the FAD binding site. Mutation of Arg582 in yeast Sdh1 precludes flavinylation as well as assembly of the tetrameric enzyme complex. Mutation of Arg638 compromises SDH function only when present in combination with a Cys630 substitution. Mutations of either Arg582 or Arg638/Cys630 do not markedly destabilize the Sdh1 polypeptide; however, the steady-state level of Sdh5 is markedly attenuated in the Sdh1 mutant cells. With each mutant Sdh1, second-site Sdh1 suppressor mutations were recovered in Sdh1 permitting flavinylation, stabilization of Sdh5 and SDH tetramer assembly. SDH assembly appears to require FAD binding but not necessarily covalent FAD attachment. The Arg residues may be important not only for Sdh5 association but also in the recruitment and/or guidance of FAD and or succinate to the substrate site for the flavinylation reaction. The impaired assembly of SDH with the C-terminal Sdh1 mutants suggests that FAD binding is important to stabilize the Sdh1 conformation enabling association with Sdh2 and the membrane anchor subunits. 相似文献