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1.
Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc. 相似文献
2.
This paper defines a collection of Drosophila deletion mutations (deficiencies) that can be systematically screened for embryonic phenotypes, orphan receptor ligands, and genes affecting protein localization. It reports the results of deficiency screens we have conducted that have revealed new axon guidance phenotypes in the central nervous system and neuromuscular system and permitted a quantitative assessment of the number of potential genes involved in regulating guidance of specific motor axon branches. Deficiency “kits” that cover the genome with a minimum number of lines have been established to facilitate gene mapping. These kits cannot be systematically analyzed for phenotypes, however, since embryos homozygous for many deficiencies in these kits fail to develop due to the loss of key gene products encoded within the deficiency. To create new kits that can be screened for phenotype, we have examined the development of the nervous system in embryos homozygous for more than 700 distinct deficiency mutations. A kit of ∼400 deficiency lines for which homozygotes have a recognizable nervous system and intact body walls encompasses >80% of the genome. Here we show examples of screens of this kit for orphan receptor ligands and neuronal antigen expression. It can also be used to find genes involved in expression, patterning, and subcellular localization of any protein that can be visualized by antibody staining. A subset kit of 233 deficiency lines, for which homozygotes develop relatively normally to late stage 16, covers ∼50% of the genome. We have screened it for axon guidance phenotypes, and we present examples of new phenotypes we have identified. The subset kit can be used to screen for phenotypes affecting all embryonic organs. In the future, these deficiency kits will allow Drosophila researchers to rapidly and efficiently execute genome-wide anatomical screens that require examination of individual embryos at high magnification. 相似文献
3.
Detection of asymptomatic herpes simplex virus infections after vaccination. 总被引:4,自引:1,他引:3
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Twenty-two volunteers seronegative for antibodies to herpes simplex virus (HSV) were enrolled in a trial to determine tolerance and immunogenicity of an HSV-2 glycoprotein subunit vaccine. Vaccine was administered at days 0, 28, and 140, and sera were obtained on days 0, 7, 14, 21, 28, 35, 49, 56, 140, 147, and 365 for determination of HSV neutralizing antibody activity and antibody-dependent cell cytotoxicity (ADCC). Sera were also tested by immunoprecipitation of radiolabeled HSV-2-infected cell proteins and polyacrylamide gel electrophoresis to identify the viral proteins which elicited antibody responses in vaccine recipients. After vaccination two male volunteers presented with atypical first-episode genital herpes: patient 1 with a culture-negative genital lesion at day 53 and patient 3 with urethritis at day 68. Seroconversion to wild-type viral proteins not present in the vaccine was detectable by radioimmunoprecipitation-polyacrylamide gel electrophoresis within 10 days in both patients. Two additional volunteers, one a sex contact of patient 1, seroconverted asymptomatically to nonvaccine proteins during the trial. All four vaccine breakthrough patients were indistinguishable from the other volunteers in the time required to develop neutralizing and ADCC antibodies, in the titer of these antibodies, and the time to seroconversion to gB and gD vaccine proteins. However, only one of the four breakthrough patients had antibodies to g80 (a complex of gC-2 and gE) after vaccination as compared with 15 of the other 18 volunteers (P = 0.05). Neither neutralizing antibody nor ADCC titers consistently identified acquisition of wild-type viral infection; therefore, protein-specific serologies were required to detect wild-type antibodies in these four patients. These data underscore the importance of using serologic assays which will distinguish naturally acquired infection from the immune response to vaccination. 相似文献
4.
T. Ashley 《Genetica》1987,72(2):81-84
It has been previously supposed that meiotic synapsis is restricted to homology during early, but not late pachynema. The synaptic begavior of an inverted X chromosome, In(X)1H as reflected in the synaptonemal complexes of the sex chromosomes has been examined in microspread spermatocytes by electron microscopy and evidence of extensive nonhomologus synapsis between the X and Y during early pachynema has been obtained. 相似文献
5.
M Lovett D Goldgaber P Ashley D R Cox D C Gajdusek C J Epstein 《Biochemical and biophysical research communications》1987,144(2):1069-1075
The human amyloid beta protein is the major constituent of the brain amyloid plaques found in Alzheimer disease. The gene that encodes this protein is located on chromosome 21, and individuals with Down syndrome (trisomy 21) also exhibit an early onset form of Alzheimer disease. We have used the cloned human amyloid beta protein gene and a panel of somatic cell hybrids to map the location of the mouse homolog of this gene. We report here that the mouse gene is located on chromosome 16 within the region 16C3----ter, in common with three other genes which map within the Down syndrome region of human chromosome 21. 相似文献
6.
The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with [1-14C]iodoacetamide. The dithiothreitol-reduced E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of 14C. Sequencing of tryptic peptides shows that 2.8 equiv of 14C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of 14C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of 14C. Sequencing of tryptic peptides shows that 1.4 equiv of 14C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I. 相似文献
7.
T. Ashley 《Genetics》1988,118(2):307-317
An examination of synaptic data from a series of X-autosome translocations and crossover data from an extensive series of autosome-autosome translocations and autosomal inversions in mice has lead to the development of a hypothesis which predicts synaptic and recombinational behavior of chromosomal aberrations during meiosis. This hypothesis predicts that in heterozygotes for chromosomal rearrangements that meiotically align G-light chromatin with G-light chromatin lack of homology will be recognized. If homologous synapsis cannot proceed, synaptonemal complex formation will cease and there will be no physical suppression of crossing over in such rearrangements. However, if a chromosomal rearrangement aligns G-light chromatin with G-dark chromatin at the time of synapsis, lack of homology will not be recognized and synaptonemal complex formation will proceed nonhomologously through the G-dark chromatin. Crossing over will be physically suppressed in this region and this suppression of crossing over will be confined to the chromosome in which the G-light chromatin is nonhomologously synapsed with G-dark chromatin. When G-light chromatin is once again aligned with G-light chromatin, lack of homology again will be recognized and either homologous synapsis will be reinitiated (as in an inversion loop), or will cease altogether (as in some translocations). Unlike the previously described "synaptic adjustment", this nonhomologous synapsis of G-light with G-dark chromatin appears to compete with homologous synapsis during early pachynema. 相似文献
8.
The ribonucleoside triphosphate reductase of Lactobacillus leichmannii converts the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP) into a mixture of 2'-deoxyuridine triphosphate (dUTP) and the unstable product 3'-keto-2'-deoxyuridine triphosphate (3'-keto-dUTP). This ketone can be trapped by reduction with NaBH4, producing a 4:1 mixture of xylo-dUTP and dUTP. When [3'-3H]ClUTP is treated with enzyme in the presence of NaBH4, the isomeric deoxyuridines isolated after alkaline phosphatase treatment retained 15% of the 3H in ClUTP. Degradation of these isomeric nucleosides has established the location of the 3H in 3'-keto-dUTP as predominantly 2'(S). The xylo-dU had 98.6% of its label at the 2'(S) position and 1.5% at 2'(R). The isolated dU had 89.6% of its label at 2'(S) and 1.4% at 2'(R), with the remaining 9% label inferred to be at the 3'-carbon, this resulting from the direct enzymic production of dUTP. These results are consistent with enzymic production of a 1:1000 mixture of dUTP and 3'-keto-dUTP, where the 3'-hydrogen of ClUTP is retained at 3' during production of dUTP and is transferred to 2'(S) during production of 3'-keto-dUTP. The implications of these results and the unique role of the cofactor adenosylcobalamin (Ashley et al., 1986) are discussed in terms of reductase being a model for the B12-dependent rearrangement reactions. 相似文献
9.
The ribonucleoside triphosphate reductase (RTPR) of Lactobacillus leichmannii is inactivated by the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP). Inactivation is due to alkylation by 2-methylene-3(2H)-furanone, a decomposition product of the enzymic product 3'-keto-2'-deoxyuridine triphosphate. The former has been unambiguously identified as 2-[(ethylthio)methyl]-3(2H)-furanone, an ethanethiol trapped adduct, which is identical by 1H NMR spectroscopy with material synthesized chemically. Subsequent to rapid inactivation, a slow process occurs that results in formation of a new protein-associated chromophore absorbing maximally near 320 nm. The terminal stages of the inactivation have now been investigated in detail. The alkylation and inactivation stoichiometries were studied as a function of the ratio of ClUTP to enzyme. At high enzyme concentrations (0.1 mM), 1 equiv of [5'-3H]ClUTP resulted in 0.9 equiv of 3H bound to protein and 83% inactivation. The amount of labeling of RTPR increased with increasing ClUTP concentration up to the maximum of approximately 4 labels/RTPR, yet the degree of inactivation did not increase proportionally. This suggests that (1) RTPR may be inactivated by alkylation of a single site and (2) decomposition of 3'-keto-dUTP is not necessarily enzyme catalyzed. The formation of the new protein chromophore was also monitored during inactivation and found to reach its full extent upon the first alkylation. Thus, out of four alkylation sites, only one appears capable of undergoing the subsequent reaction to form the new chromophore. While chromophore formation was prevented by NaBH4 treatment, the chromophore itself is resistant to reduction.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
10.
F Garza D L Thompson D D French J J Wiest R L St George K B Ashley L S Jones P S Mitchell D R McNeill 《Biology of reproduction》1986,35(2):347-352
Five lighthorse mares were actively immunized against gonadotropin releasing hormone (GnRH) to determine the relative importance of this hypothalamic hormone in the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Five mares immunized against the conjugation protein served as controls. Mares were initially immunized in November and received secondary immunizations 4 wk later, and then at 6-wk intervals until ovariectomy in June. All mares immunized against GnRH exhibited an increase (p less than 0.01) in the binding of tritiated GnRH by plasma, an indication that antibodies against this hormone had been elicited. Concentrations of LH, FSH and progesterone in weekly blood samples were lower (p less than 0.05) in GnRH-immunized mares than in controls after approximately 4 mo of immunization. However, the LH concentrations were affected to a greater degree than were FSH concentrations. All five control mares exhibited normal cycles of estrus and diestrus in spring, whereas no GnRH-immunized mare exhibited cyclic displays of estrus up to ovariectomy. All mares were injected intravenously with a GnRH analog (which cross-reacted less than 0.1% with the anti-GnRH antibodies) in May, after all control mares had displayed normal estrous cycles, to characterize the response of LH and FSH in these mares; two days later, the mares were injected with GnRH. The LH response to the analog, which was assessed by net area under the curve, was lower (p less than 0.01) by approximately 99% in mares immunized against GnRH than in control mares. In contrast, the FSH response to the analog was similar for both groups.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献