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41.
Young RJ Alderton W Angell AD Beswick PJ Brown D Chambers CL Crowe MC Dawson J Hamlett CC Hodgson ST Kleanthous S Knowles RG Russell LJ Stocker R Woolven JM 《Bioorganic & medicinal chemistry letters》2011,21(10):3037-3040
Heteroalicyclic carboxamidines were synthesised and evaluated as inhibitors of nitric oxide synthases. (2R)-2-Pyrrolidinecarboxamidine, in particular, was shown to be a highly potent in vitro (IC50 = 0.12 μM) and selective iNOS inhibitor (>100-fold vs both eNOS and nNOS), with probable binding to the key anchoring glutamate residue and co-ordination to the haem iron. 相似文献
42.
R. E. Gibson-D'ambrosio D. L. Crowe C. E. Shuler S. M. D'ambrosio 《Cell biology and toxicology》1993,9(4):385-403
The use of normal adult liver hepatocytes in cell culture for biochemical, toxicological and pharmacological studies has been greatly limited owing to the loss of replicative capacity and differentiated liver function. This is contrary to the ability of the liver to regenerate following injury in vivo. This suggests that liver stem or transitional hepatocytes exist that upon proper stimulus divide and differentiate into mature hepatocytes. In this study we report the establishment and culture of hepatocytes from normal human adult liver, which: (1) possess replicative capacity sufficient to subpassage 12–15 times (27–37 cumulative population doublings); (2) can be cryopreserved for subsequent use without loss of replica five capacity; and (3) upon differentiation in culture synthesize albumin and keratin 18 and metabolize benzo[a]pyrene. The ability of these cells to divide or express differentia tedfunctions appears to be due to a number of cellular, biochemical and physical characteristics that are present during the primary establishment and subsequent growth phases of the cell cultures. Disassociation of cells ffom excess liver tissue was best achieved by combining the mechanical action of the Stomacher@ with very low amounts of proteolytic enzymes and EGTA. The cell lines appeared to grow best when established and subpassaged in an rnALPHA medium supplemented with insulin, hydrocortisone, transferrin, epithelial growth factor and fetal bovine serum ® rescreened for human hepatocyte cell growth). The seeding density and cell-cell contact in culture appeared to be important for both cell division and expression of liver function. When cells were seeded at a low density and subpassaged before confluency, the cells continued to divide. Albumin and keratin 18 synthesis occurred primarily in tightly packed cell clusters. When cells were seeded at a high density, near confluency, albumin and keratin 18 synthesis occurred uniformly in all of the cells of the culture and the culture metabolized benzo[a]pyrene to water-soluble metabolites, which covalently bound to cellular DNA. This appearance of liver functions was consistent with the transition of hepatocytes to a terminally differentiated state. Nonhepatic markers, i.e., -fetoprotein, factor VIII and -glutamyl transpeptidase activity were not expressed in cells cultured at either low or high density. Thus, the data presented here indicate that normal human adult liver hepatocytes, once established in culture, can be subpassaged to a high number of population doublings, cryopreserved for later use, and modulated to express differentiated liver functions.bl]References 相似文献
43.
M G Crowe 《BMJ (Clinical research ed.)》1982,285(6344):783-784
44.
Summary The distribution of substance P (SP)-, somatostatin (SOM)-, vasoactive intestinal polypeptide (VIP)- and met-enkephalin (mENK)-immunoreactive nerve fibres and cell bodies has been studied in the gastrointestinal tract, lateral blood vessel (heart) and segmental ganglia of the leech (Hirudo medicinalis). In the crop and intestine, there was a sparse distribution of VIP-, SP-, SOM- and mENK-immunoreactive nerves, while in the intestine, a dense network of SP-, a moderate network of SOM-, and a sparse distribution of mENK- and VIP-immunoreactive nerve fibres was seen. SP-, SOM- and VIP-immunoreactive nerve cell bodies were found in all the gut regions studied, the greatest number being in the intestine. No mENK-containing cell bodies were seen in any region of the gastrointestinal tract. The heart contained a few SP-, SOM-, and VIP-immunoreactive nerve fibres, but no nerve cell bodies were found. Immunoreactive nerve cell bodies were also present in the segmentai ganglia. A typical midbody ganglion contained up to seven pairs of SP-containing neurones, four pairs of SOM-containing neurones, two pairs of VIP-containing neurones and one to three pairs of mENK-immunoreactive nerve cell bodies. The lateral pair of large SOM-immunoreactive nerve cell bodies is of similar size and correct position to the lateral N cells. One of the pairs of large SP-immunoreactive nerve cell bodies is probably identical to the Leydig cells. A tentative identification of other immunofluorescent nerve cells is attempted. Immunoreactive nerve fibres to all four peptides were distributed throughout the neuropil, those to SP being the most numerous. 相似文献
45.
Jordan R. Willis Bryan S. Briney Samuel L. DeLuca James E. Crowe Jr Jens Meiler 《PLoS computational biology》2013,9(4)
Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding. 相似文献
46.
Wilna J. Moree Florence Jovic Timothy Coon Jinghua Yu Bin-Feng Li Fabio C. Tucci Dragan Marinkovic Raymond S. Gross Siobhan Malany Margaret J. Bradbury Lisa M. Hernandez Zhihong O’Brien Jianyun Wen Hua Wang Samuel R.J. Hoare Robert E. Petroski Aida Sacaan Ajay Madan Paul D. Crowe Graham Beaton 《Bioorganic & medicinal chemistry letters》2010,20(7):2316-2320
SAR of lead benzothiophene H1-antihistamine 2 was explored to identify backup candidates with suitable pharmacokinetic profiles for an insomnia program. Several potent and selective H1-antihistamines with a range of projected half-lives in humans were identified. Compound 16d had a suitable human half-life as demonstrated in a human microdose study, but variability in pharmacokinetic profile, attributed to metabolic clearance, prevented further development of this compound. Compound 28b demonstrated lower predicted clearance in preclinical studies, and may represent a more suitable backup compound. 相似文献
47.
Isolation of novel bacteria, including a candidate division, from geothermal soils in New Zealand 总被引:4,自引:0,他引:4
Stott MB Crowe MA Mountain BW Smirnova AV Hou S Alam M Dunfield PF 《Environmental microbiology》2008,10(8):2030-2041
We examined bacterial diversity of three geothermal soils in the Taupo Volcanic Zone of New Zealand. Phylogenetic analysis of 16S rRNA genes recovered directly from soils indicated that the bacterial communities differed in composition and richness, and were dominated by previously uncultured species of the phyla Actinobacteria , Acidobacteria , Chloroflexi , Proteobacteria and candidate division OP10. Aerobic, thermophilic, organotrophic bacteria were isolated using cultivation protocols that involved extended incubation times, low-pH media and gellan as a replacement gelling agent to agar. Isolates represented previously uncultured species, genera, classes, and even a new phylum of bacteria. They included members of the commonly cultivated phyla Proteobacteria , Firmicutes , Thermus/Deinococcus , Actinobacteria and Bacteroidetes , as well as more-difficult-to-cultivate groups. Isolates possessing < 85% 16S rRNA gene sequence identity to any cultivated species were obtained from the phyla Acidobacteria , Chloroflexi and the previously uncultured candidate division OP10. Several isolates were prevalent in 16S rRNA gene clone libraries constructed directly from the soils. A key factor facilitating isolation was the use of gellan-solidified plates, where the gellan itself served as an energy source for certain bacteria. The results indicate that geothermal soils are a rich potential source of novel bacteria, and that relatively simple cultivation techniques are practical for isolating bacteria from these habitats. 相似文献
48.
BACKGROUND: Helicobacter pylori infection leads to gastritis, peptic ulcer, and gastric cancer, in part due to epithelial damage following bacteria binding to the epithelium. Infection with cag pathogenicity island (PAI) bearing strains of H. pylori is associated with increased gastric inflammation and a higher incidence of gastroduodenal diseases. It is now known that various effector molecules are injected into host epithelial cells via a type IV secretion apparatus, resulting in cytoskeletal changes and chemokine secretion. Whether binding of bacteria and subsequent apoptosis of gastric epithelial cells are altered by cag PAI status was examined in this study. METHODS: AGS, Kato III, and N87 human gastric epithelial cell lines were incubated with cag PAI-positive or cag PAI-negative strains of H. pylori in the presence or absence of clarithromycin. Binding was evaluated by flow cytometry and scanning electron microscopy. Apoptosis was assessed by detection of DNA degradation and ELISA detection of exposed histone residues. RESULTS: cag PAI-negative strains bound to gastric epithelial cells to the same extent as cag PAI-positive strains. Both cag PAI-positive and cag PAI-negative strains induced apoptosis. However, cag PAI-positive strains induced higher levels of DNA degradation. Incubation with clarithromycin inactivated H. pylori but did not affect binding. However, pretreatment with clarithromycin decreased infection-induced apoptosis. CONCLUSIONS: cag PAI status did not affect binding of bacteria to gastric epithelial cells but cag PAI-positive H. pylori induced apoptosis more rapidly than cag PAI-negative mutant strains, suggesting that H. pylori binding and subsequent apoptosis are differentially regulated with regard to bacterial properties. 相似文献
49.
Crowe JH Tablin F Wolkers WF Gousset K Tsvetkova NM Ricker J 《Chemistry and physics of lipids》2003,122(1-2):41-52
Human blood platelets are normally stored in blood banks for 3-5 days, after which they are discarded. We have launched an effort at developing means for preserving the platelets for long term storage. In previous studies we have shown that trehalose can be used to preserve biological membranes and proteins during drying and have provided evidence concerning the mechanism. A myth has grown up about special properties of trehalose, which we discuss here and clarify some of what is fact and what is misconception. We have found a simple way of introducing this sugar into the cytoplasm of platelets and have successfully freeze-dried the trehalose-loaded platelets, with very promising results. We present evidence that membrane microdomains are maintained intact in the platelets freeze-dried with trehalose. Finally, we propose a possible mechanism by which the microdomains are preserved. 相似文献
50.
Hunted and unhunted populations of greywing francolin Francolinus africanus have been studied in the eastern Cape Province of South Africa in order to understand the effects of hunting on the demography and genetic structure of these populations. Greywing population density cycled annually for both hunted and unhunted populations. However, there was an apparent pulse of immigration of sub-dominant birds, and earlier reproduction, in the hunted populations immediately after the winter hunting season. Average levels of allozyme heterozygosity (H) for hunted and unhunted populations were both 0.076, and although the proportion of polymorphic loci per sample and the mean number of alleles per locus for each sample were lower for the hunted populations than for the unhunted populations, these differences were not significant. However, the hunted populations displayed higher levels of outbreeding (lower F
IS and F
IT values) than those for unhunted populations. Therefore, it is concluded that although greywing francolin populations contain relatively high levels of genetic heterogeneity, it is probably the increased levels of local immigration following hunting which reduces the effects of any reduction in genetic variation due to a decrease in local population size from hunting. 相似文献