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1.
An assessment was made of two methods for determining the potency of tissue-type plasminogen activator (TPA). A chromogenic microtitre plate assay was established which contained TPA, plasminogen, a synthetic plasmin substrate (H-D-valyl-L-leucyl-L-lysyl-p-nitroaniline dihydrochloride, S2251) and any one of the following stimulators: native fibrinogen, enzymatic and chemical digests of fibrinogen, poly-D-lysine (PDL) and chemical derivatives of the latter. The chromogen assay was compared with an automated clot-lysis (turbidimetric) assay for sensitivity, reproducibility and validity for potency determination. Reference preparations of TPA were titrated in both assays: in the chromogen assay the dose-response curves were non-parallel, whereas parallelism was observed in the clot-lysis assay. Thus, the chromogen assay was restricted in its applicability and disqualified from any routine regulatory use. The potency of individual lots of recombinant (r)TPA could only be estimated in International Units (IU) of TPA activity with the automated clot-lysis assay and the potency values obtained (IU/vial) were in remarkably close agreement with the manufacturers' values. 相似文献
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Joshua R Edwards Evangelos A Diamantakos Jacob D Peuler Peter C Lamar Walter C Prozialeck 《BMC physiology》2007,7(1):1
Background
Ethidium homodimer is a cell-membrane impermeant nuclear fluorochrome that has been widely used to identify necrotic cells in culture. Here, we describe a novel technique for evaluating necrosis of epithelial cells in the proximal tubule that involves perfusing ethidium homodimer through the intact rat kidney. As a positive control for inducing necrosis, rats were treated with 3.5, 1.75, 0.87 and 0.43 mg/kg mercuric chloride (Hg2+, intraperitoneal), treatments which have previously been shown to rapidly cause dose-dependent necrosis of the proximal tubule. Twenty-four h after the administration of Hg2+, ethidium homodimer (5 μM) was perfused through the intact left kidney while the animal was anesthetized. The kidney was then removed, placed in embedding medium, frozen and cryosectioned at a thickness of 5 μm. Sections were permeabilized with -20°C methanol and then stained with 4',6-diamidino-2-phenylindole (DAPI) to label total nuclei. Total cell number was determined from the DAPI staining in random microscopic fields and the number of necrotic cells in the same field was determined by ethidium homodimer labeling. 相似文献3.
Diuron belongs to the family of halogenophenylureas, one of the main groups of herbicides used for more than 40 years. These herbicides absorb sunlight and can be photochemically transformed in the environment (herbicides are transformed on the soil surface exposed to sunlight) or biotransformed by microorganisms present in soil or in water. The metabolites (chlorohydroxyphenylurea, chlorophenylaniline, respectively) are more toxic than the parent compound, as demonstrated by a bioluminescence inhibition assay performed with a marine bacterium (Vibrio fischeri toxicity test). The lipophilicity of these pesticides makes the cell membrane a target for their action, especially the spermatozoa cell membrane. The aim of this study is to use human spermatozoa to evaluate the effect of this urea pesticide and its biotransformed product on the spermatozoa membrane. We investigated the structural and functional effects of these environmental pollutants on spermatozoa. Three million spermatozoa purified on a 95/47.5% Percoll gradient were suspended in 250 μl of modified Earle’s medium (without phenol red) supplemented with 7.5% of human decomplemented serum. Pesticides (Diuron or 3,4-dichloroaniline (3,4-DCA)) were added at a final concentration of 0.1; 1 and 5 mM. Samples were incubated at room temperature for 24 hours. We show that both Diuron and 3,4-DCA decrease motility and vitality of spermatozoa incubated with the highest concentration of pesticides. Our preliminary results show that the effects are more rapid and more intense with the biotransformed product (3,4-DCA) than with Diuron. Addition of herbicide to human spermatozoa increases membrane fluidity, assessed by measuring the fluorescence polarisation anisotropy with a fluorescent probe: 1,6-diphenyl-1,3,5-hexatriene (DPH). Changes in membrane fluidity may be a primary toxic effect of these herbicides. These results suggest that human spermatozoa may constitute a valuable indicator of the toxic effects of pesticides. 相似文献
4.
After 30 years of continuous research into the mechanisms of human hypertension, we summarize the results obtained by the members of the multidisciplinary research group on hypertension of the Clinical Research Institute of Montreal on the disturbances of minerlocorticoid activity in a rigorously selected group of patients with early, mild essential hypertension. We attempt to integrate these findings with those of many other groups working on other aspects of hypertensive cardiovascular diseases. On the assumption that the increased peripheral resistance responsible for hypertension results from an imbalance or a disturbance of the equilibrium between the sympathetic nervous system and norepinephrine on one hand, and the vascular tone, sensitivity and responsiveness of the arterial smooth muscle to norepinephrine and to angiotensin II on the other hand, three models that fit the experimental and clinical facts as known at present are described. 相似文献
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Parathyroid hormone-related protein (PTHrP) can be secreted from cells and interact with its receptor, the Type 1 PTH/PTHrP Receptor (PTHR1) in an autocrine, paracrine or endocrine fashion. PTHrP can also remain inside cells and be transported into the nucleus, where its functions are unclear, although recent experiments suggest that it may broadly regulate cell survival and senescence. Disruption of either the PTHrP or PTHR1 gene results in many abnormalities including a failure of embryonic mammary gland development in mice and in humans. In order to examine the potential functions of nuclear PTHrP in the breast, we examined mammary gland development in PTHrP (1–84) knock-in mice, which express a mutant form of PTHrP that lacks the C-terminus and nuclear localization signals and which can be secreted but cannot enter the nucleus. Interestingly, we found that PTHrP (1–84) knock-in mice had defects in mammary mesenchyme differentiation and mammary duct outgrowth that were nearly identical to those previously described in PTHrP−/− and PTHR1−/− mice. However, the mammary buds in PTHrP (1–84) knock-in mice had severe reductions in mutant PTHrP mRNA levels, suggesting that the developmental defects were due to insufficient production of PTHrP by mammary epithelial cells and not loss of PTHrP nuclear function. Examination of the effects of nuclear PTHrP in the mammary gland in vivo will require the development of alternative animal models. 相似文献
9.
Separase is a protease that promotes chromosome segregation at anaphase by cleaving cohesin. Several non-proteolytic functions of separase have been identified in other organisms. We created a transgenic C. elegans line that expresses protease-dead separase in embryos to further characterize separase function. We find that expression of protease-dead separase is dominant-negative in C. elegans embryos, not previously reported in other systems. The C. elegans embryo is an ideal system to study developmental processes in a genetically tractable system. However, a major limitation is the lack of an inducible gene expression system for the embryo. We have developed two methods that allow for the propagation of lines carrying dominant-negative transgenes and have applied them to characterize expression of protease-dead separase in embryos. Using these methods, we show that protease-dead separase causes embryo lethality, and that protease-dead separase cannot rescue separase mutants. These data suggest that protease-dead separase interferes with endogenous separase function, possibly by binding substrates and protecting them from cleavage. 相似文献
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