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351.
An immunoenzymatic solid-phase assay for quantitative determination of HIV-1 protease activity 总被引:1,自引:0,他引:1
Gutiérrez OA Salas E Hernández Y Lissi EA Castrillo G Reyes O Garay H Aguilar A García B Otero A Chavez MA Duarte CA 《Analytical biochemistry》2002,307(1):18-24
A novel immunoenzymatic procedure for the quantitative determination of HIV protease activity is provided. An N-terminal biotinylated peptide (DU1) that comprises an HIV-1 protease (HIV-PR) cleavage sequence was bound to streptavidin-coated microtiter plates. The bound peptide can be quantified by an immunoenzymatic procedure (enzyme-linked immunosorbent assay, ELISA) that includes a monoclonal antibody (Mab 332) against the peptide (DU1) C-terminal. The incubation of the bound peptide with HIV-PR in solution resulted in a signal decrement, as the peptide was hydrolyzed and the released C-terminal segment washed away. An equation that relates the amount of added enzyme to the kinetics of the reaction was written in order to describe this heterogeneous enzyme-quasi-saturable system. This equation allows quantitative determination of protease activity, a feature widely underrated in previous similar assays. The assay also allows evaluation of the inhibitory activity of HIV-PR inhibitors. Due to the intrinsic advantages of the ELISA format, this method could be used in high-throughput screening of HIV protease inhibitors. The assay can be extended to other proteolytic enzymes. 相似文献
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353.
Conditional loss of PTEN leads to testicular teratoma and enhances embryonic germ cell production 总被引:15,自引:0,他引:15
Kimura T Suzuki A Fujita Y Yomogida K Lomeli H Asada N Ikeuchi M Nagy A Mak TW Nakano T 《Development (Cambridge, England)》2003,130(8):1691-1700
The tumor suppressor gene PTEN, which is frequently mutated in human cancers, encodes a lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3] and antagonizes phosphatidylinositol 3 kinase. Primordial germ cells (PGCs), which are the embryonic precursors of gametes, are the source of testicular teratoma. To elucidate the intracellular signaling mechanisms that underlie germ cell differentiation and proliferation, we have generated mice with a PGC-specific deletion of the Pten gene. Male mice that lacked PTEN exhibited bilateral testicular teratoma, which resulted from impaired mitotic arrest and outgrowth of cells with immature characters. Experiments with PTEN-null PGCs in culture revealed that these cells had greater proliferative capacity and enhanced pluripotent embryonic germ (EG) cell colony formation. PTEN appears to be essential for germ cell differentiation and an important factor in testicular germ cell tumor formation. 相似文献
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356.
Mastoparan induces Ca2+-dependent deflagellation of the unicellular green alga Chlamydomonas moewusii Gerloff, as well as the activation of phospholipase C and the production of inositol 1,4,5-trisphosphate (InsP3; T. Munnik et al., 1998, Planta 207: 133–145). Even in the absence of extracellular Ca2+, mastoparan still induces deflagellation (L.M. Quarmby and H.C. Hartzell, 1994, J Cell Biol 124: 807–815; J.A.J. van Himbergen
et al., 1999, J Exp Bot, in press) suggesting that InsP3 mediates Ca2+ release from intracellular stores. To test this hypothesis, cells were pre-loaded with 45Ca2+ and their plasma membranes permeabilized by digitonin. Subsequent treatment of the cells with mastoparan (3.5 μM) induced
release of intracellular 45Ca2+. Mastoparan also activated phospholipase C in permeabilized cells, as demonstrated by the breakdown of 32P-phosphatidylinositol 4,5-bisphosphate and the production of diacylglycerol. The mastoparan analogues mas7 and mas17 were
also effective and their efficacy was correlated with their biological activity. X-ray microanalysis showed that electron-dense
bodies (EDBs) are a major Ca2+ store in C. moewusii. Analysis of digitonin-permeabilized cells showed that EDBs lost calcium at digitonin concentrations that released radioactivity
from 45Ca2+-labelled cells, suggesting that 45Ca2+ monitored the content of EDBs. X-ray microanaysis of living cells treated with mastoparan also revealed that calcium was
released from EDBs.
Received: 30 December 1998 / Accepted: 25 June 1999 相似文献
357.
María Elena Romero-González Ernesto Zambrano Johan Mesa Hilda Ledo de Medina 《Hydrobiologia》2001,450(1-3):47-55
The distribution of phosphate species in the Catatumbo River, Venezuela, was studied using a sequential extraction scheme. Extraction was performed using 1.0 M NH4Cl, 0.1 M Na2S2O4–NaHCO3, 1.0 M NaOH and 0.5 M HCl. Total phosphate was in the range between 121 and 581 g g–1 D.W. About 64% of the total phosphate is inorganic and present the following composition: 1% NH4Cl-P, 10% BD-P, 20% NaOH-P, 33% HCl-P; while org-P is 36%. The main form of P in sediment from the Catatumbo River is calcium bound-P, like apatite. The metal concentrations present in sediment are in the following order: Fe> Al > Ca > Mg > Mn. Relationships between P and Fe, Al and Ca were found. Cluster analysis showed that speciation is not dependent on the inorganic matrix of the sediments. Analysing the influence of NaOH concentration and duration of the extraction invalidated the use of NaOH as extracting agent in P-speciation. 相似文献
358.
Suhail Rasool Hilda Martinez‐Coria Jessica W. Wu Frank LaFerla Charles G. Glabe 《Journal of neurochemistry》2013,126(4):473-482
Alzheimer's disease (AD) is a devastating disorder that is clinically characterized by a comprehensive cognitive decline. Accumulation of the amyloid‐beta (Aβ) peptide plays a pivotal role in the pathogenesis of AD. In AD, the conversion of Aβ from a physiological soluble monomeric form into insoluble fibrillar conformation is an important event. The most toxic form of Aβ is oligomers, which is the intermediate step during the conversion of monomeric form to fibrillar form. There are at least two types of oligomers: oligomers that are immunologically related to fibrils and those that are not. In transgenic AD animal models, both active and passive anti‐Aβ immunotherapies improve cognitive function and clear the parenchymal accumulation of amyloid plaques in the brain. In this report we studied effect of immunotherapy of two sequence‐independent non‐fibrillar oligomer specific monoclonal antibodies on the cognitive function, amyloid load and tau pathology in 3xTg‐AD mice. Anti‐oligomeric monoclonal antibodies significantly reduce the amyloid load and improve the cognition. The clearance of amyloid load was significantly correlated with reduced tau hyperphosphorylation and improvement in cognition. These results demonstrate that systemic immunotherapy using oligomer‐specific monoclonal antibodies effectively attenuates behavioral and pathological impairments in 3xTg‐AD mice. These findings demonstrate the potential of using oligomer specific monoclonal antibodies as a therapeutic approach to prevent and treat Alzheimer's disease. 相似文献
359.
Background
By mechanisms yet to be discerned, the co-expression of high levels of wild-type human superoxide dismutase 1 (hSOD1) with variants of hSOD1 encoding mutations linked familial amyotrophic lateral sclerosis (fALS) hastens the onset of motor neuron degeneration in transgenic mice. Although it is known that spinal cords of paralyzed mice accumulate detergent insoluble forms of WT hSOD1 along with mutant hSOD1, it has been difficult to determine whether there is co-deposition of the proteins in inclusion structures.Methodology/Principal Findings
In the present study, we use cell culture models of mutant SOD1 aggregation, focusing on the A4V, G37R, and G85R variants, to examine interactions between WT-hSOD1 and misfolded mutant SOD1. In these studies, we fuse WT and mutant proteins to either yellow or red fluorescent protein so that the two proteins can be distinguished within inclusions structures.Conclusions/Significance
Although the interpretation of the data is not entirely straightforward because we have strong evidence that the nature of the fused fluorophores affects the organization of the inclusions that form, our data are most consistent with the idea that normal dimeric WT-hSOD1 does not readily interact with misfolded forms of mutant hSOD1. We also demonstrate the monomerization of WT-hSOD1 by experimental mutation does induce the protein to aggregate, although such monomerization may enable interactions with misfolded mutant SOD1. Our data suggest that WT-hSOD1 is not prone to become intimately associated with misfolded mutant hSOD1 within intracellular inclusions that can be generated in cultured cells. 相似文献360.
John G. Nedrud Nayer Bagheri Karin Sch?n Wei Xin Hilda Bergroth Dubravka Grdic Eliasson Nils Y. Lycke 《PloS one》2013,8(12)
A mucosal vaccine against Helicobacter pylori infection could help prevent gastric cancers and peptic ulcers. While previous attempts to develop such a vaccine have largely failed because of the requirement for safe and effective adjuvants or large amounts of well defined antigens, we have taken a unique approach to combining our strong mucosal CTA1-DD adjuvant with selected peptides from urease B (UreB). The protective efficacy of the selected peptides together with cholera toxin (CT) was first confirmed. However, CT is a strong adjuvant that unfortunately is precluded from clinical use because of its toxicity. To circumvent this problem we have developed a derivative of CT, the CTA1-DD adjuvant, that has been found safe in non-human primates and equally effective compared to CT when used intranasally. We genetically fused the selected peptides into the CTA1-DD plasmid and found after intranasal immunizations of Balb/c mice using purified CTA1-DD with 3 copies of an H. pylori urease T cell epitope (CTA1-UreB3T-DD) that significant protection was stimulated against a live challenge infection. Protection was, however, weaker than with the gold standard, bacterial lysate+CT, but considering that we only used a single epitope in nanomolar amounts the results convey optimism. Protection was associated with enhanced Th1 and Th17 immunity, but immunizations in IL-17A-deficient mice revealed that IL-17 may not be essential for protection. Taken together, we have provided evidence for the rational design of an effective mucosal subcomponent vaccine against H. pylori infection based on well selected protective epitopes from relevant antigens incorporated into the CTA1-DD adjuvant platform. 相似文献