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141.
Cloning, Expression, and Purification of Glutamine Synthetase from Clostridium acetobutylicum 总被引:7,自引:1,他引:6 下载免费PDF全文
A glutamine synthetase (GS) gene, glnA, from the gram-positive obligate anaerobe Clostridium acetobutylicum was cloned on recombinant plasmid pHZ200 and enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. The cloned C. acetobutylicum gene was expressed from a regulatory region contained within the cloned DNA fragment. glnA expression was subject to nitrogen regulation in E. coli. This cloned glnA DNA did not enable an E. coli glnA ntrB ntrC deletion mutant to utilize arginine or low levels of glutamine as sole nitrogen sources, and failed to activate histidase activity in this strain which contained the Klebsiella aerogenes hut operon. The GS produced by pHZ200 was purified and had an apparent subunit molecular weight of approximately 59,000. There was no DNA or protein homology between the cloned C. acetobutylicum glnA gene and GS and the corresponding gene and GS from E. coli. The C. acetobutylicum GS was inhibited by Mg2+ in the γ-glutamyl transferase assay, but there was no evidence that the GS was adenylylated. 相似文献
142.
Tropomyosin from equine platelets was reacted with N-(1-pyrenyl)iodoacetamide, a sulfhydryl-specific fluorescent reagent, to give an average extent of incorporation of 1.12 pyrene (Py) groups per platelet tropomyosin (P-TM) chain. The predominant site of reaction on P-TM was the penultimate COOH-terminal residue, Cys-246. The high proportion of the total emission that is due to pyrene ecximers and the pretransition observed in thermal denaturation of Py-P-TM point to a rather loose structure for the COOH-terminal amino acid residues of P-TM. The label on Cys-246 also reports on end-to-end overlap interactions that occur between two different tropomyosin molecules. Additions to a Py-P-TM solution at low ionic strength of unlabeled P-TM, rabbit cardiac tropomyosin (C-TM), or a carboxypeptidase A treated, nonpolymerizable derivative of C-TM all reduce the extent of excimer fluorescence from the sample. Addition of salt greatly reduces the effects of the unlabeled TM species on the Py-P-TM emission spectrum. Circular dichroism measurements indicate Py-P-TM still to be greater than 95% helical. However, analysis of excimer fluorescence levels in samples that contained a constant protein concentration but different mole ratios of labeled to unlabeled P-TM suggests that the bulky pyrene group may diminish the tendency of Py-P-TM to polymerize in an end-to-end manner. 相似文献
143.
Effects of several population densities ofMeloidogyne incognita on the sweet potato cultivars Centennial (susceptible) and Jasper (moderately resistant) were studied. Field plots were infested with initial levels (Pi) of 0, 10, 100, 1,000, 5,000, and 10,000 eggs and juveniles/500 cm³ soil in 1980 and 0, 100, 1,000, 2,000, 3,000, 4,000, and 5,000 in 1981. M. incognita population development trends were similar on both cultivars; however, at high Pi, more eggs and juveniles were recovered from Centennial than from Jasper. The highest Pi did not result in the highest mid-season (Pm) counts. Pi was negatively correlated with the number of marketable roots and root weight but positively correlated with total cracked roots, percentage of cracked roots, and cracking severity. Jasper tolerated higher Pi with greater yields and better root quality than Centennial. Cracking of fleshy roots occurred with both cultivars at low Pi. 相似文献
144.
John H. Caldwell Jennifer Van Brunt Franklin M. Harold 《The Journal of membrane biology》1986,89(1):85-97
Summary Injection of depolarizing current into vegetative cells of the water moldBlastocladiella emersonii elicits a regenerative response that has the electrical characteristics of an action potential. Once they have been taken past a threshold of about –40 mV, cells abruptly depolarize to +20 mV or above; after an interval ranging from several hundred milliseconds to a few seconds, the cells spontaneously return to their resting potential near –100 mV. When the action potential was analyzed with voltage-clamp recording, it proved to be biphasic. The initial phase reflects an influx of calcium ions through voltage-sensitive channels that also carry Sr2+ ions. The delayed, and more extended, phase of inward current results from the efflux of chloride and other anions. The anion channels are broadly selective, passing chloride, nitrate, phosphate, acetate, succinate and even PIPES. The anion channels open in response to the entry of calcium ions, but do not recognize Sr2+. Calcium channels, anion channels and calcium-specific receptors that link the two channels appear to form an ensemble whose physiological function is not known. Action potentials rarely occur spontaneously but can be elicited by osmotic downshock, suggesting that the ion channels may be involved in the regulation of turgor. 相似文献
145.
Reassortant rotaviruses containing structural proteins vp3 and vp7 from different parents induce antibodies protective against each parental serotype. 总被引:46,自引:20,他引:26 下载免费PDF全文
Genetic studies of reassortant rotaviruses have demonstrated that gene segments 4 and 9 each segregate with the serotype-specific neutralization phenotype in vitro. Reassortant rotaviruses derived by coinfection of MA-104 cells with the simian strain SA11 and the antigenically distinct bovine strain NCDV were used to determine which viral genes coded for proteins which induced a protective immune response in vivo. In addition, reassortant rotaviruses containing only the gene segment 4 or 9 protein products (vp3 and vp7, respectively) from SA11 or NCDV were used to determine the serotypic specificities of both vp3 and vp7 in several mammalian rotavirus strains. vp3 and vp7 from the murine strain Eb were shown to be indistinguishable from the corresponding proteins from strain SA11. Adult mice orally inoculated with strain Eb developed neutralizing antibodies to both vp3 and vp7. The two naturally occurring bovine rotavirus strains NCDV and UK were shown to contain antigenically similar vp7 but distinct vp3 proteins. Mouse dams orally immunized with a reassortant virus containing only gene 9 from NCDV passively protected their progeny against UK challenge, whereas mouse dams orally immunized with a reassortant virus containing only gene 4 from NCDV did not. Finally, we constructed reassortant viruses that immunized against rotaviruses of two distinct serotypes. SA11 X NCDV reassortants that contained vp3 and vp7 from different parents induced a protective immune response against both parental serotypes. vp3 and vp7 were independently capable of inducing a protective immune response after oral immunization. An understanding of the serotypic specificities of both vp3 and vp7 of human rotavirus isolates will be necessary for the development of successful strategies to protect infants against severe rotavirus infections. 相似文献
146.
Vascular smooth muscle caldesmon 总被引:10,自引:0,他引:10
T Clark P K Ngai C Sutherland U Gr?schel-Stewart M P Walsh 《The Journal of biological chemistry》1986,261(17):8028-8035
Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard. 相似文献
147.
The interactions of Mn2+ and Co2+ with glucose isomerases from three microbial sources have been studied using various direct physical methods. Co2+ was found to activate each enzyme, although the degree of activation varied significantly for enzymes from different organisms. EPR spectroscopy measurements revealed that dissimilarities in the coordination sphere of enzyme-bound Mn2+ accompanied the differences in enzyme activity. Variations in the EPR spectra of a nitroxide spin label coupled to two of the three isomerases, possibly near their active sites, were also observed. In no case was the EPR spectrum influenced by Co2+ addition, a result discordant with the hypothesis that Co2+ activates the enzyme by inducing a conformational change. The proximal biochemical environment of enzymebound Co2+ was also examined using EXAFS spectroscopy. This method showed that glucose causes notable changes in the ligand environment of the enzyme-bound metal, suggesting the formation of an enzyme-metal-substrate bridge complex. The significance of these results relative to possible reaction mechanisms is discussed. 相似文献
148.
149.
We explored the pathologic changes in the skin of mice undergoing a chronic graft-versus-host (GVH) reaction. In rodents and in man, chronic GVH includes the deposition of excess collagen in the skin-a reaction which resembles idiopathic scleroderma. GVH disease across minor histocompatibility barriers was produced by injecting B10.D2 cells into irradiated BALB/c mice. These strains are identical at the H-2 and Mls loci but differ in minor histocompatibility antigens. Control BALB/c mice received irradiation and BALB/c cells. Serial skin biopsies were taken and studied for histological changes characteristic of chronic GVHD, for mast cell density, and for the deposition of immunoreactants. GVHD was produced in B10.D2----BALB/c mice as measured by body weight loss and the production of skin changes including dermal fibrosis, loss of fat and appendages, and a mononuclear cell infiltrate. Dermal mast cells, assessed by toluidine blue staining, were normal at Day 11, but had disappeared by Days 21-63 and returned to normal by Day 104. Immunoglobulins IgG, IgA, and IgM appeared at the dermo-epidermal junction and along the basement membrane zone of hair follicles. This deposition was maximal at Day 42 and waned thereafter. Thus the appearance of immunoglobulins in the skin was maximal when mast cell staining was minimal. The changes in this GVHD model leading to a scleroderma-like picture in the skin are compatible with an immune etiology for the fibrosis. Vasodilation following liberation of mast cell mediators would facilitate the deposition of immunoglobulins. The disappearance of mast cell staining may be caused by extensive degranulation. We postulate an interaction between GVHD-activated T cells, mast cell stimulation, fibroblast activation, and fibrosis. 相似文献
150.
Evolution of karyotypic abnormalities and C-MYC oncogene amplification in human colonic carcinoma cell lines 总被引:7,自引:0,他引:7
C. C. Lin Kari Alitalo Manfred Schwab Donna George Harold E. Varmus J. Michael Bishop 《Chromosoma》1985,92(1):11-15
Cell lines (COLO 320 DM and COLO 320 HSR), established from a human neuroendocrine tumor, contain an amplified cellular oncogene (c-myc). We have previously shown that the homogeneously staining regions (HSRs) of a marker chromosome in the COLO 320 HSR cells that evolved in culture from COLO 320 DM cells contain amplified c-myc. Molecular hybridization in situ has now been used to demonstrate that the HSRs are on both arms of what was once an X chromosome. We also show that amplified c-myc copies are present in the isolated double minute chromosomes (DMs) of the COLO 320 DM cells that were characteristic of the tumor cells initially established from the patient. The results suggest that the amplified c-myc appeared first as DMs and was subsequently transposed to engender HSRs on an X chromosome. The initial COLO 320 tumor cell may have acquired two early replicating (i.e., active) X chromosomes and lost the late replicating (i.e., inactive) X. 相似文献