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81.
82.
A J van Zonneveld H Veerman M E MacDonald J A van Mourik H Pannekoek 《Journal of cellular biochemistry》1986,32(3):169-178
Full-length tissue-type plasminogen activator (t-PA) cDNA served to construct deletion mutants within the N-terminal "heavy" (H)-chain of the t-PA molecule. The H-chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins ("finger," "epidermal growth factor," "kringles"). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon-intron junctions of the chromosomal t-PA gene. Recombinant t-PA deletion mutant proteins were obtained after transient expression in mouse Ltk- cells, transfected with SV40-pBR322-derived t-PA cDNA plasmids. It is demonstrated that the serine protease moiety of t-PA and its substrate specificity for plasminogen is entirely contained within the C-terminal "light" (L)-chain of the protein. The presence of cDNA, encoding the t-PA signal peptide preceding the remaining portion of t-PA, suffices to achieve secretion of (mutant) t-PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t-PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H-chain, kringle K1, and the epidermal growth-factor-like domain, do not contribute to this property of t-PA. These findings correlate well with the fibrin-binding properties of the rt-PA deletion-mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t-PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L-chain of t-PA. Deleting specific segments of t-PA H-chain cDNA and subsequent transient expression in mouse Ltk- cells of t-PA deletion-mutant proteins did not affect the formation of a stable complex between mutant t-PA and PAI. 相似文献
83.
Toxicity and mutual interactions of cadmium and zinc ions in normal and carcinogen-transformed mouse cells 总被引:1,自引:0,他引:1
The toxicity of chloride salts of physiological (zinc, manganese, nickel) and non physiological (cadmium) bivalent metal ions was studied in normal or carcinogen-transformed mouse embryo fibroblast cells. The dose response curves for toxicity to both types of cells exhibited similar shapes. The transformed cells, however, were about twice as sensitive to zinc toxicity as normal cells. When normal and transformed cells were grown together and incubated for several hours with an appropriate concentration of zinc, the malignant cells were selectively killed. Cadmium was much more toxic than the three other metal ions in both types of cells. Its toxic effect was reversed by simultaneous addition of zinc at nontoxic concentrations.Abbrevications CFA
colony forming ability
- MCA
3-methylcholanthrene 相似文献
84.
M Krieger 《Analytical biochemistry》1983,135(2):383-391
Pores formed in the membranes of animal cells by complexes of sterols and the polyene antibiotic amphotericin B can efficiently kill the cells. Thus, in the absence of exogenous sources of cholesterol, inhibitors of enzymes in the cholesterol biosynthetic pathway render cells resistant to amphotericin B. Preincubation of Chinese hamster ovary cells with compactin or 25-hydroxycholesterol, inhibitors of the synthesis of the key intermediate mevalonate, protected cells from amphotericin B killing and this protection was reversed by the addition of exogenous mevalonate. The ability of compactin to confer amphotericin B resistance on normal cells was abolished when cells were provided exogenous cholesterol by the receptor-mediated endocytosis of low density lipoprotein. Low density lipoprotein receptor-defective Chinese hamster ovary cells were not subject to this low density lipoprotein-dependent amphotericin B killing. Exogenous mevalonate did not prevent 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, an inhibitor of mevalonate conversion to sterols, from protecting cells from amphotericin B. A simple two-step protocol in which cells are preincubated (15-24 h) with potential inhibitors and then treated (3-6 h) with amphotericin B was devised to provide a sensitive method for detecting direct (e.g., competitive) and regulatory inhibitors of cholesterol biosynthesis. This protocol may prove useful in detecting potential antihypercholesterolemia drugs and is currently being used to isolate mutants in receptor-mediated endocytosis. 相似文献
85.
Cerebroside and Sulfatide Biosynthesis in the Brain of Snell Dwarf Mouse: Effects of Thyroxine and Growth Hormone in the Early Postnatal Period 总被引:1,自引:1,他引:0
Snell dwarf mice (dw/dw) and normal mice (+/?) were injected with thyroxine (T4) (1 μg/animal, four injections) and growth hormone (GH) (20 μg/animal, four injections) from the 5th to the 15th day of life. In the untreated dw/dw mouse brain, the specific activities of UDP-galactose:ceramide galactosyltransferase (CGalT), PAPS:cerebroside sulfotransferase (CST), and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) were decreased by 28, 25, and 37%, respectively, compared with the control untreated +/? mice. The major effect of T4 was an increase of the brain CNP in the +/? mice (+40%) and dw/dw mice (+111%). The treatment with T4 also brought to normal the level of CGalT in dw/dw brain; a somewhat less marked effect on CST was observed. The treatment with GH had a great stimulatory effect on CNP: the specific activity of this enzyme increased by 40 and 69% in +/? and dw/dw mouse brain, respectively. On the contrary, no effect of GH on the CGalT activity was observe in this study. Our results suggest that T4 and GH may have both independent and complementary actions on the myelin-associated enzymes during the early postnatal period of brain development. 相似文献
86.
Tariq Ahmad Joanne C. Conover Martin M. Quigley Robert L. Collins Anthony J. Thomas Ralph B. L. Gwatkin 《Molecular reproduction and development》1989,22(4):369-373
Failure of epididymal spermatozoa from T/t mutant mice, but not from t/t individuals, to fertilize oocytes in vitro was partially overcome by opening a small aperture in the zona pellucida with acidified Tyrode's solution to permit direct access of the spermatozoon to the vitellus. This study provides a model system to evaluate requirements for successful zona drilling in the treatment of human infertility and further insights into the effects of the t complex on sperm fertility. 相似文献
87.
M. H. Kaufman R. E. Fowler E. Barratt R. D. McDougall 《Molecular reproduction and development》1989,24(1):35-48
The ultrastructural morphology of the mouse zona pellucida was studied in preovulatory follicles from the ovaries of immature mice treated with exogenous gonadotrophins. The ovaries were fixed in the presence of cetylpyridinium chloride, which precipitates carbohydrates, so that their loss during fixation and processing is substantially reduced. The semi-thin araldite sections obtained from osmicated material were viewed by conventional light microscopy, while the ultra-thin sections were examined by transmission electron microscopy. A parallel series of semi-thin sections of non-osmicated ovaries was deresined and subsequently stained with periodic acid Schiff (PAS). The morphological appearance of the zona pellucida in preovulatory oocytes changed during the final stages of oocyte maturation. A close correlation was observed between the ultrastructural appearance of the zona pellucida and that observed following PAS staining during the period studied. Real differences were observed in the location, density, and distribution of glycoconjugates within the zona pellucida during the final stages of oocyte maturation prior to and immediately following germinal vesicle breakdown. Similar changes in the zona were observed in adult females autopsied during proestrus and oestrus. The changes in the density and distribution of glycoconjugates are likely to have important consequences for fertilization by affecting sperm-zona binding and sperm-egg interactions. 相似文献
88.
The influence of the source of pregnant mares' serum gonadotropin (PMSG) on the num ber, quality, and in vitro development of mouse embryos before and after freezing was evaluated among three genotypes: N:NIH(S), C57BL/6N, and C3H/HeN-MTV?. Immature females were given PMSG from one of five commercial sources. Following col lection ( 116 hr later), embryos were evaluated for stage of development, and four-to eight-cell embryos were pooled within genotype and assigned to standardized fresh or freeze-thaw culture trials. Different PMSG sources stimulated the production of different num bers of total embryos (P < 0.05) but not necessarily more embryos suitable for freezing. Differences in embryo production among genotypes indicated that absolute embryo num bers using a single mouse genotype may not accurately reflect the potency of a specific gonadotropin source. The PMSG source also affected the ability of an embryo to survive in culture either immediately after collection or after frozen storage. The effect, however, was genotype specific, with some mouse strains being relatively insensitive to PMSG source, whereas gonadotropin source played a major role in determining in vitro viability in others. Development rates for freshly collected embryos differed, often inconsistently, from those of thawed embryos regardless of the PMSG source used, demonstrating that fresh embryo development cannot be used to estimate expected post-thaw survival. In vitro development of thawed embryos is influenced not only by genotype, but also the source of the gonadotropin used to promote follicular development and oocyte maturation. These findings may explain, in part, the wide variation in embryo viability and culture rates reported among laboratories and intraspecies animal populations. 相似文献
89.
Summary The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos
were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography.
The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of
Days 8, 10, and 11 of gestation were cultured in Tyrode’s solution. Incubation time and concentration of [3H (or14C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without
any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of
several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations
per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured
under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened)
and placenta, 150 μCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of
proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions.
This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic analysis
of cell proteins). 相似文献
90.
Summary We describe the in vitro influence of 3,5,3′-triiodo-l-thyronine (T3),l-thyroxine (T4), a thyroid-stimulating hormone (TSH), and/or estradiol (E2: chosen as the control of the methodology) on the cell kinetics (cell distribution in the S+G2+M phases) of mouse MXT and human MCF-7 mammary cancer cells. Experiments were performed by means of a cell image processor,
analyzing MCF-7 or MXT cells that had been grown on glass cover slips and whose nuclei had been stained by the Feulgen reaction,
which is selective and quantitative (stoichiometric) with respect to DNA. We show that T3, T4, and TSH at 0.01 μM dramatically stimulate the cell kinetics of the MXT mouse and the MCF-7 human mammary cancer cell lines. Indeed, the three
hormones bring about a significant transient increase in the S+G2+M fraction as does E2. Furthermore, our data indicate that E2 and TSH are antagonistic with regards to MXT or MCF-7 cell kinetics.
This work is supported by grants awarded by the IRSIA and the Fonds de la Recherche Scientifique Médicale (FRSM, Belgium). 相似文献