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61.
The effect of Arg306-->Ala and Arg506-->Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S.
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J. O. Egan M. Kalafatis K. G. Mann 《Protein science : a publication of the Protein Society》1997,6(9):2016-2027
Factor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg306, Arg506, and Arg679. Site-directed mutagenesis of human factor V cDNA was used to substitute Arg306-->Ala (rfVa306A) and Arg506-->Gln (rfVa506Q). Both the single and double mutants (rfVa306A/506Q) were constructed. The activation of these procofactors by alpha-thrombin and their inactivation by APC were assessed in coagulation assays using factor V-deficient plasma. All recombinant and wild-type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVaPLASMA) in HBS Ca2+ +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild-type factor Va (rfVaWT) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVaPLASMA. The rfVa306A and rfVa506Q mutants were each inactivated at rates slower than rfVaWT and fVaPLASMA. Following a 90-min incubation with APC, rfVa306A and rfVa506Q retain approximately 30-40% of the initial cofactor activity. The double mutant, rfVa306A/506Q, was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90-min incubation in the presence of APC. Recombinant fVaWT, rfVa306A, rfVa506Q, and rfVa306A/506Q were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVaWT and rfVa306A inactivation in the presence of protein S were unchanged, indicating cleavage at Arg506 is not affected by protein S. The initial rate of rfVa506Q inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg306. Overall, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg306 and Arg506 are required for efficient factor Va inactivation. 相似文献
62.
The RAD51 gene is a eukaryotic counterpart of the Escherichia coli recA gene which is involved in genetic recombination. Two distinct Xenopus laevis RAD51 cDNA clones (XRAD51.1 and XRAD51.2) were isolated from an oocyte cDNA library using the human RAD51 cDNA (HsRAD51) as a probe. Sequence analysis revealed that 98.2% of the amino-acid residues were identical between XRAD51.1 and XRAD51.2, and that both were 95% identical to HsRAD51. Both of the XRAD51 genes were expressed at a higher level in ovary and testis than in other somatic tissues, suggesting their involvement in meiotic recombination. The expression of XRAD51.1 was about eightfold in excess of that of XRAD51.2 in all of the tissues examined. Analysis of the rates of synonymous substitution in the coding sequences of the two XRAD51 suggests that these two genes diverged about 50 million years ago. The structural similarities of the XRAD51 proteins to RecA in E. coli and Rad51 in yeasts or vertebrates are discussed. 相似文献
63.
The thermal behavior of the Soret band relative to the carbonmonoxy derivatives of some β-chain mutant hemoglobins is studied in the temperature range 300–10 K and compared to that of wild-type carbonmonoxy hemoglobin. The band profile at various temperatures is modeled as a Voigt function that accounts for homogeneous broadening and for the coupling with high- and low-frequency vibrational modes, while inhomogeneous broadening is taken into account with a gaussian distribution of purely electronic transition frequencies. The various contributions to the overall bandwidth are singled out With this analysis and their temperature dependence, in turn, gives information on structural and dynamic properties of the system studied. In the wildtype and mutant hemoglobins, the values of homogeneous bandwidth and of the coupling constants to high-frequency vibrational modes are not modified with respect to natural human hemoglobin, thus indicating that the local electronic and vibrational properties of the heme–CO complex are not altered by the recombinant procedures. On the contrary, differences in the protein dynamic behavior are observed. The most relevant are those relative to the “polar isosteric” βVal-67(Ell) →Thr substitution, localized in the heme pocket, which results in decreased coupling with low-frequency modes and increased anharmonic motions. Mutations involving residue βLys-144(HC1) at the C-terminal and residue βCys-112(G14) at the α1β1 interface have a smaller effect consisting in an increased coupling with low-frequency modes. Mutations at the β-N-terminal and at the α1β2 interface have no effect on the dynamic properties of the heme pocket. © 1995 Wiley-Liss, Inc. 相似文献
64.
Hybrid recombinant human leukocyte interferon inhibits differentiation in murine B-16 melanoma cells 总被引:1,自引:0,他引:1
P B Fisher H Hermo D R Prignoli I B Weinstein S Pestka 《Biochemical and biophysical research communications》1984,119(1):108-115
We have investigated the effects of recombinant human leukocyte interferons (IFN-alpha A and IFN-alpha D) and various hybrid recombinant human leukocyte interferons on differentiation in B-16 mouse melanoma cells. Inhibition of both spontaneous and melanocyte hormone stimulated differentiation was observed with one hybrid construct, IFN-alpha A/D (Bgl) consisting of amino acids 1 to 62 from IFN-alpha A and amino acids 64 to 166 from IFN-alpha D. In contrast, the parental human interferons, IFN-alpha A and IFN-alpha D, when used alone or in combination, as well as other hybrid human leukocyte interferons, did not cause significant inhibition of melanogenesis in B-16 mouse cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also inhibited B-16 differentiation and the combination of TPA with IFN-alpha A/D (Bgl) or mouse L-cell interferon was synergistic in delaying melanogenesis. These studies indicate that the IFN-alpha A/D (Bgl) hybrid that exhibits antiviral activity on mouse cells can also inhibit differentiation of murine cells. 相似文献
65.
The effect of Type I interferon on bone resorption was studied by measuring its effect on parathormone-stimulated calcium release from neonatal murine calvaria in vitro. A pure human recombinant leukocyte interferon hybrid of the A and D subtypes was used, which has high antiviral activity on mouse cells. Calcium release was inhibited in a dose dependent fashion with 50% inhibition at about 10(-10) M or 600 U/ml, and the inhibition was reversible. The presence of interferon was required before or during the activation phase of the resorptive response, when the formation of osteoclasts from precursor cells would occur. When added to actively resorbing bone it had no effect. The data suggest that Type I interferon can inhibit the parathormone-regulated development of active osteoclasts, possibly by inhibiting osteoclast precursor differentiation. 相似文献
66.
Isolation of restriction enzyme EcoVIII, an isoschizomer of HindIII, produced by Escherichia coli E1585-68 总被引:1,自引:0,他引:1
A restriction endonuclease designated EcoVIII, an isoschizomer of HindIII, was isolated from Escherichia coli E1585-68 and purified by dextran-polyethylene glycol (DPG) phase partition, ammonium sulfate precipitation, phospho- and DEAE-cellulose chromatography, and hydroxylapatite chromatography. The purified EcoVIII was stable during the purification procedure and its high specific activity required 10 mM Mg2+. Unlike HindIII, Eco VIII exhibited a high specific activity even at low pH (pH 6.3) and showed the highest activity at 48° C. Transformation of purified plasmid DNA from E. coli E1585-68 into K-12 indicated that the EcoVIII gene was carried on a multicopy 4.4-kb miniplasmid. EcoVIII seems to be preferable to HindIII for its production and use because of easier handling of producer cells and a wider range of activity. 相似文献
67.
68.
R. H. Goodwin G. J. Tompkins P. McCawley 《In vitro cellular & developmental biology. Plant》1978,14(6):485-494
Summary A series of cell lines unique in insect virus susceptibility pattern have been isolated from the ovaries of the gypsy moth
(Lymantria dispar: Lepidoptera: Lymantriidae) on a synthetic medium with mammalian and avian serum supplementation. Growth curves showed the
poorest growth occurring on peptone-based media with somewhat better growth on amino-acid-based media. The best growth was
obtained with combined media. Serological study distinguished the present cell lines from one another and from cell lines
derived from other insect species grown routinely in the same laboratory. Baculovirus susceptibility among the new lines varied
from no response to a specific complete replication response upon challenge by the homologous (gypsy moth) nuclear polyhedrosis
virus.
This research was funded in part through a reimbursable agreement with the U.S. Forest Service. 相似文献
69.
70.
We examined the survival of a host Escherichia coli K-12 bacterium containing two transferable plasmids (pLM2, pSL222-4) and one poorly mobilizable plasmid (pBR322), and the transfer of these three plasmids to endogenous bacteria in the human intestinal tract. The survival of this plasmid-carrying host organism in four human volunteers was 3.5 to 6 days at recovery rates of 10?1 to 10?4. This finding was similar to our previous survival data on the same organism bearing a single plasmid. The K-12 strain appeared to be under a strong selective disadvantage in the human gut, since, even when bearing a tetracycline-resistant plasmid, its titer did not increase despite the administration of tetracycline. Studies of transferability showed that, while the transfer-depressed incFII plasmid pSL222-4 transferred at a frequency of 10?1 in culture, its transfer in the human gut was much less frequent. The number of new recipients per donor cell ingested was about 10?5, which included new recipients arising by multiplication. The recovery of pSL222-4 transcipients was enhanced by the administration of tetracycline on day 6. Neither the transfer-repressed, broad host range incP plasmid pLM2, nor the plasmid pBR322, could be detected in any endogenous host bacteria. Using the transfer and mobilization frequencies obtained in culture and the number of new recipients of pSL222-4 in the intestinal tract, we estimated that any in vivo mobilization of pBR322 to a new recipient could not occur at a frequency higher than 10?12. 相似文献