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A Chinese hamster ovary auxotroph requiring glycine + adenosine + thymidine (CHO AUXB1) was shown by us previously to lack several folylpolyglutamate synthetase (FPGS) type activities. Two revertants of AUXB1 (one spontaneous and one Pt(S04)2 induced) have been isolated and found to contain altered forms of this enzyme. The revertant enzymes are more sensitive to heat inactivation (37 °C, pH 7.4 or 9.0) than the parent CHO enzyme. Increased sensitivity of revertant FPGS is observed irrespective of whether one assays the specific catalysis of radioactive tetrahydropteroyldi- or tetraglutamate synthesis. ATP and MgCl2 protect both revertant and parent CHO FPGS against rapid heat denaturation at pH 9.0, but not at pH 7.4. A genetically related auxotroph (CHO AUXB3) contains one-fifth the parent amount of FPGS. AUXB3 FPGS shows a normal sensitivity to 37 °C heat inactivation, but it has an altered substrate saturation and specificity pattern when assayed for tetrahydropteroyldi[U-14C]glutamate synthesis. Also, unlike the FPGS from parent CHO and a genetically unrelated mutant requiring only glycine (CHO AUXB2), the AUXB3 enzyme specifically lacks tetrahydropteroyltetra[U-14C]glutamate synthetase activity. These findings and polyethylene glycol fusion data with AUXB2 indicate that AUXB1 and AUXB3 each carry a mutation in the structural gene for a CHO FPGS that catalyzes tetrahydropteroyldi- as well as tetraglutamate formation. The altered form of FPGS in AUXB3 is responsible for its glycine + adenosine auxotrophy under standard culture conditions. 相似文献
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Quantum yields (φ) for the aerobic photolysis of 5′-deoxyadenosylcobalamin (dAB12), methylcobalamin (MeB12), propylcobalamin (PrB12), and ethylcobalamin (EtB12) were determined as a function of the irradiation wavelength. φ Determinations were made for both the base-on and base-off forms of each compound (except base-off dAB12) at incident wavelengths from 250 nm to 570 nm. As a rule, the φs were high (0.1–0.5) and they varied significantly with respect to the irradiation wavelength. In general, each alkylcobalamin at pH 7.0 displayed a quantum yield spectrum distinct from its base-off form at pH 1.0. Across most of the spectrum, the φs of the base-off form were appreciably smaller than the base-on φs of the same compound. An exception to this generality was MeB12 for which the φs at pH 1.0 were about the same as, or slightly greater above 450 nm than those at pH 7.0. At pH 7.0 and in the visible region the trend of the φs was dAB12 < MeB12 < PrB12 < EtB12. Under neutral conditions each compound showed a broad quantum yield peak in the 450–470 nm region.From the quantum yield and absorption spectra, photolysis spectra were calculated for 5.0 × 10?5m solutions of each compound. The light-action spectra accurately give the relative rates/μ Einstein that these solutions photolyze at each wavelength. Thus, for example, MeB12 photolyzed faster at pH 7.0 versus pH 1.0 in 510 nm light, but it photolyzed slower at pH 7.0 versus pH 1.0 in 450 nm light. Solutions of each compound photolyzed faster in the ultraviolet region as opposed to the visible (e.g., 310 nm versus 510 nm).Our findings show that the previously reported photolysis rates estimated by others with tungsten lamps provide no valid information about the intrinsic photolability of various alkyl-cobalt bonds. This also applies to the relative white-light photolysis rates reported for the base-on versus the base-off form of MeB12. All such relative rates are artifacts which represent only the extent of overlap between the true action spectrum and the light emission spectrum of an incandescent lamp. 相似文献
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G Prefontaine P Fast P C Lau M A Hefford Z Hanna R Brousseau 《Applied and environmental microbiology》1987,53(12):2808-2814
Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion. 相似文献
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The synthesis of 5'-O-beta-D-glucopyranosyl and 5'-O-beta-D-galactopyranosyl derivatives (13 and 15, respectively) of the antiviral agent ribavirin are described. Direct glycosylation of 2',3'-O-isopropylideneribavirin with either tetra-O-acetyl-alpha-D-glucopyranosyl bromide (4) or tetra-O-acetyl-alpha-D-galactopyranosyl bromide (8) under Koenigs-Knorr conditions (i.e., silver carbonate, silver perchlorate, and Drierite in dichloromethane) followed by O-deacetylation of the reaction product gave the corresponding ortho esters. However, treatment of 2',3'-di-O-acetyl-5'-O-tritylribavirin (11) with 4 under the Bredereck modification of the Koenigs-Knorr reaction (i.e., silver perchlorate and Drierite in nitromethane) and subsequent deacetylation furnished the desired 1-(5-O-beta-D-glucopyranosyl-beta-D-ribofuranosyl)-1,2,4-triazole-3-carb oxamide (13). Similarly, reaction of 11 with 8 in the presence of AgClO4, and deprotection of the condensation product, gave 5'-O-beta-D-galactopyranosylribavirin (15). The beta-anomeric configuration of the D-glucosyl and D-galactosyl groups of 13 and 15 was assigned by 1H-n.m.r. studies. 相似文献
9.
A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized. 相似文献
10.
Divalent cation involvement in the action of Clostridium perfringens type A enterotoxin. Early events in enterotoxin action are divalent cation-independent 总被引:15,自引:0,他引:15
B A McClane A P Wnek K I Hulkower P C Hanna 《The Journal of biological chemistry》1988,263(5):2423-2435
Clostridium perfringens type A enterotoxin (CPE) is a membrane-active cytotoxin. There are a number of recognized early steps in CPE cytotoxicity including binding of CPE to a protein receptor, insertion of CPE into membranes, and CPE-mediated induction of changes in membrane permeability for small molecules such as ions and amino acids. Further support for the existence of these early steps and further characterization of these events are presented in this report. We now report that these early steps in CPE action are largely independent of extracellular divalent cations. It is also shown that 3H-nucleotide release, known to be a later CPE effect, is Ca2+-dependent. A model for CPE cytotoxicity is suggested involving CPE action as a two-step process with Ca2+-independent early steps and Ca2+-dependent late steps. 相似文献