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31.
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33.
R C Haddock M L Spell C D Baker J R Grammer J M Parks M Speidel F M Booyse 《The Journal of biological chemistry》1991,266(32):21466-21473
Recombinant human single-chain urokinase (rscu-PA), two-chain urokinase (tcu-PA), and diisopropyl-fluorophosphate-treated tcu-PA (DFP-tcu-PA) bound to cultured human and porcine endothelial cells in a rapid, saturable, dose-dependent and reversible manner. Analysis of specific binding results in cultured human umbilical vein endothelial cells (HUVECs) gave the following estimated values for Kd and Bmax: 0.57 +/- 0.08 nM (mean +/- S.E.) and 188,000 +/- 18,000 sites/cell for 125I-labeled rscu-PA; 0.54 +/- 0.10 nM and 132,000 +/- 23,900 sites/cells for 125I-labeled tcu-PA; 0.89 +/- 0.14 nM and 143,000 +/- 30,300 sites/cell for 125I-labeled DFP-tcu-PA, respectively. Values for Kd were similar for primary and subcultured (six passages) HUVECs, but Bmax values were lower in subcultured HUVECs. Similar Kd values were found in cultured porcine endothelial cells; however, Bmax values varied depending on the endothelial cell type. All 125I-labeled urokinase forms yielded similar cross-linked approximately 110-kDa ligand-receptor complexes with cultured HUVECs, and 125I-labeled DFP-tcu-PA bound to a single major approximately 55-kDa protein in whole-cell lysates (ligand blotting/autoradiography), suggesting the presence of a single major approximately 55-kDa urokinase receptor in cultured HUVECs. The approximately 55-kDa urokinase receptor, isolated from several separate batches of cultured HUVECs (3-5 micrograms of protein, approximately 1 x 10(9) cells), by ligand affinity chromatography, exhibited the following properties: retained biologic activity as evidenced by its ability to bind 125I-labeled rscu-PA by ligand blotting/autoradiography and formation of a cross-linked 125I-labeled approximately 110-kDa rscu-PA-receptor complex; single-chain approximately 55-kDa protein, following reduction; complete conversion to and formation of a single major deglycosylated approximately 35-kDa protein, following treatment with N-glycanase. 相似文献
34.
Using yeast strains with null mutations in structural genes which encode delta-aminolevulinic acid synthetase (HEM1), isozymes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG1 and HMG2), squalene epoxidase (ERG1), and fatty acid delta 9-desaturase (OLE1), we were able to determine the effect of hemes, sterols, and unsaturated fatty acids on both sterol production and the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in Saccharomyces cerevisiae. We found that the HMGR isozymes direct essentially equal amounts of carbon to the biosynthesis of sterols under heme-competent conditions, despite a huge disparity (57-fold) in the specific activities of the reductases. Our results demonstrate that palmitoleic acid (16:1) acts as a rate-limiting positive regulator and that ergosterol acts as a potent inhibitor of sterol production in strains which possess only the HMGR1 isozyme (HMG1 hmg2). In strains which contain only the HMGR2 isozyme (hmg1 HMG2), sterol production was inhibited by oleic acid (18:1) and to a lesser degree by ergosterol. The specific activities of the two reductases (HMGR1 and HMGR2) were found to be differentially regulated by hemes but not by ergosterol, palmitoleic acid, or oleic acid. The disparate effects of unsaturated fatty acids and sterols on these strains lead us to consider the possibility of separate, compartmentalized isoprenoid pathways in S. cerevisiae. 相似文献
35.
36.
Effects of cryopreservation procedures on sperm membranes 总被引:10,自引:0,他引:10
Empirical approaches to semen cryopreservation have resulted in the production of young in a broad range of species. However, acceptable levels of fertility in most domestic animal species has not been achieved. In this review, an attempt has been made to describe the complexity of the sperm plasma membrane and the many steps in a cryopreservation procedure where membrane perturbations can occur. Improvement in sperm cryopreservation procedures will require a careful consideration of the complexity of the sperm plasma membrane, the interaction of its components and the influence of cooling, freezing and thawing on these interactions. 相似文献
37.
Effect of sterol side-chain structure on the feed-back control of sterol biosynthesis in yeast 总被引:4,自引:0,他引:4
We measured the incorporation of radiolabeled methionine and acetate into the sterol component of G204, a Saccharomyces cerevisiae mutant strain which is partially heme competent. By comparing the amount of label incorporated into the sterol pool of a control culture, to which no exogenous sterol was added, with a culture which had various sterols added to the growth medium, we were able to determine the specific structural features of ergosterol which facilitate its ability to restrict the sterol biosynthetic pathway. These experiments demonstrate that sterols which contain both a C22 unsaturation and a C24 methyl group are capable of reducing sterol biosynthesis by approx. 50%, regardless of B-ring structure. We examined the regulatory properties of various oxysterols; 24,25-epoxylanosterol reduced endogenous biosynthesis by 49%, whereas all cholesterol derivatives tested, including 25-hydroxycholesterol, had little effect. A new procedure for the synthesis of ergosterol peroxides is also described. 相似文献
38.
Gastrulation in Drosophila: the formation of the ventral furrow and posterior midgut invaginations 总被引:3,自引:0,他引:3
The ventral furrow and posterior midgut invaginations bring mesodermal and endodermal precursor cells into the interior of the Drosophila embryo during gastrulation. Both invaginations proceed through a similar sequence of rapid cell shape changes, which include apical flattening, constriction of the apical diameter, cell elongation and subsequent shortening. Based on the time course of apical constriction in the ventral furrow and posterior midgut, we identify two phases in this process: first, a slow stochastic phase in which some individual cells begin to constrict and, second, a rapid phase in which the remaining unconstricted cells constrict. Mutations in the concertina or folded gastrulation genes appear to block the transition to the second phase in both the ventral furrow and the posterior midgut invaginations. 相似文献
39.
P F Agris R Parks L Bowman R H Guenther S A Kovacs S Pelsue 《Experimental cell research》1990,189(2):276-279
Type I topoisomerases (EC 5.99.1.2) are those enzymes capable of relaxing negatively supercoiled DNA without the need for ATP. The central role played by these enzymes in cell function suggests that the structure of type I topoisomerases may be highly conserved in eukaryotic cells. However, the extent of the conservation among eukaryotes is unknown. Human DNA topoisomerase I is an autoimmune antigen (Scl-70) of scleroderma patients. We have found that the autoimmune antibodies in human Scl-70 sera recognize protein from various plants, and these proteins display DNA relaxation function. In addition, Scl-70 antibodies were able to inhibit enzymatic activity of plant topoisomerase I. Therefore, the immunological cross-reactivity of the plant topoisomerase with human antibodies demonstrates that, despite divergence of eukaryotic organisms, these plant and animal enzymes retain structurally similar enzymatic features. 相似文献
40.
Dissolved and suspended mercury. species in the Wabigoon River (Ontario,Canada): seasonal and regional variations 总被引:1,自引:1,他引:0
Jackson T. A. Parks J. W. Jones P. D. Woychuk R. N. Sutton J. A. Hollinger J. D. 《Hydrobiologia》1982,91(1):473-487
Seasonal and regional variations in the speciation, sediment-water partitioning, and dynamics of mercury (Hg) were studied
at selected sites along the Hg-polluted Wabigoon River, and at unpolluted headwater and tributary sites, during April–September,
1979. ‘Dissolved’ and ‘particulate’ forms of Hg in the water were separated by continuous-flow centrifugation in the field.
The Hg and other pollutants such as wood chips and salt had been discharged from a chlor-alkali plant and paper mill at Dryden,
Ontario.
Concentrations and loadings of particulate methyl mercury (CH3Hg+) and total particulate Hg (and loadings of total ‘dissolved’ Hg) were greatest during the spring flood (April-May) owing
to accelerated resuspension and transport of sediments. Concentrations of ‘dissolved’ CH3Hg+, however, were highest in the summer (July–September), probably reflecting stimulation of microbial methylating activity
by elevated temperatures, together with factors such as reduced levels of metal-scavenging particulates and minimal dilution
by runoff. Total dissolved Hg concentrations were relatively high in September at polluted sites only, possibly because of
desorption from sediments due to elevated concentrations of Cl− ions. Loadings of dissolved CH3Hg+ tended to be high in the summer but were generally depressed (suggesting sorption by suspended particles) during the major
spring-flood episode in May.
During July–August dissolved CH3Hg+ was a function of total dissolved Hg, suggesting rapid biomethylation of desorbed inorganic Hg; but in general dissolved
and suspended CH3Hg+ levels depended on environmental variables and were unrelated to total Hg concentrations. In the summer only, total dissolved
Hg was a function of dissolved Cl−. Hg species in particulates were associated with sulfides, hydrated Fe and Mn oxides, organic matter (notably high molecular
weight humic and humic-Fe components), and selenium (Se); but CH3Hg+ and total Hg differed in their specific preferences for binding agents, implying that binding sites discriminate between
CH3Hg+ and Hg2+ ions. CH3Hg+ was associated with sulfide and (in the spring only) with Fe oxides, whereas total Hg was associated with organic matter
and Se and with DTPA- and NaOH-extractable Fe in the spring but with Mn oxide and NaOH-extractable organics in the summer.
Sulfides were most abundant in May, indicating that they were eroded from bottom sediments, but Fe and Mn oxides were most
abundant in the summer, probably owing to activities of filamentous iron bacteria and other micro-organisms.
Particulate Hg was 98–100% nonextractable by mild solvents such as Ca acetate, CaCl2, dilute acetic acid, and (at polluted sites only) DTPA solutions, suggesting that the particulate Hg mobilized in the spring
may not be readily available to organisms; association with Se and high molecular weight humic matter also supports this hypothesis.
Hg probably becomes more bio-available in the summer, as suggested by the upsurge in dissolved CH3Hg+ and total dissolved Hg levels, and by increases in the solubility of particulate Hg in acetic acid, DTPA, H2O2, and NaOH solutions, as well as an increase in the relative importance of lower molecular weight fractions of NaOH-extractable
Hg (in September).
Regional variations in Hg speciation and partitioning reflected a gradient in sediment composition from wood chips near Dryden
to silt-clay mud further downstream. Hg in silt-clay mud relatively far (> 35 km) downstream from the source of pollution
or in unpolluted areas appeared to be more readily solubilized by Cl− ions or chelators such as DTPA, more readily methylated (as indicated by downstream increases in dissolved CH3Hg+ levels and CH3Hg+/total Hg ratios), and was to a greater degree organically bound (H2O2-extractable), and thus was probably more bio-available, than Hg in wood-chip deposits. Possible explanations include weaker
binding of Hg by the mud, the more finely divided state of the mud, and improved microbial growth at lower concentrations
of toxic pollutants. Owing to enrichment in sulfides and Fe oxides, resuspended wood-chip sediments were especially efficient
scavengers of CH3Hg+.
The results indicate that in any pollution abatement plan aimed at lowering the Hg levels in the biota of lakes fed by the
Wabigoon River, immobilization, removal, or detoxification of dissolved as well as particulate forms of Hg in the river would
probably have to be considered. Possibly, Hg species could be ‘scrubbed’ from the river water by increasing the suspended
load and by sedimentation and treatment with Hg-binding agents in special receiving basins. 相似文献