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41.
Photosynthetic Pod Wall of Pea (Pisum sativum L.): Distribution of Carbon Dioxide-fixing Enzymes in Relation to Pod Structure 下载免费PDF全文
The pod wall of pea (Pisum sativum L.) was shown to contain two distinct photosynthetic layers. The outer, comprising chlorenchyma of the mesocarp, captured CO2 from the outside atmosphere; the inner, a chloroplast-containing epidermis lining the pod gas cavity, was involved in photoassimilation of the CO2 released from respiring seeds. 相似文献
42.
We investigated the effects of the non-phorbol tumor promoter okadaic acid on human leukemia K562 cells. It was found that okadaic acid potently and reversibly inhibited cell growth, with a nearly complete inhibition of thymidine uptake seen at about 10 nM. The cytotoxicity of okadaic acid was characterized by a marked mitotic arrest of the cells exhibiting scattered chromosomes and abnormal anaphase-like structures, a phenomenon distinct from the typical metaphase arrest caused by colchicine. Okadaic acid (10-1,000 nM) greatly stimulated phosphorylation of a number of nuclear proteins in K562 cells. Phosphorylation of many of the same proteins was also stimulated by 12-O-tetradecanoylphorbol-13-O-acetate, a protein kinase C activator. The present findings, consistent with recent reports that okadaic acid is a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A) shown to be essential for normal mitosis, provided evidence for the first time that okadaic acid inhibition of PP1/PP2A resulted in enhanced nuclear protein phosphorylation and subsequent mitotic arrest. 相似文献
43.
G A Omburo J M Kuo L S Mullins F M Raushel 《The Journal of biological chemistry》1992,267(19):13278-13283
The bacterial phosphotriesterase has been found to require a divalent cation for enzymatic activity. This enzyme catalyzes the detoxification of organophosphorus insecticides and nerve agents. In an Escherichia coli expression system significantly higher concentrations of active enzyme could be produced when 1.0 mM concentrations of Mn2+, Co2+, Ni2+, and Cd2+ were included in the growth medium. The isolated enzymes contained up to 2 equivalents of these metal ions as determined by atomic absorption spectroscopy. The catalytic activity of the various metal enzyme derivatives was lost upon incubation with EDTA, 1,10-phenanthroline, and 8-hydroxyquinoline-5-sulfonic acid. Protection against inactivation by metal chelation was afforded by the binding of competitive inhibitors, suggesting that at least one metal is at or near the active site. Apoenzyme was prepared by incubation of the phosphotriesterase with beta-mercaptoethanol and EDTA for 2 days. Full recovery of enzymatic activity could be obtained by incubation of the apoenzyme with 2 equivalents of Zn2+, Co2+, Ni2+, Cd2+, or Mn2+. The 113Cd NMR spectrum of enzyme containing 2 equivalents of 113Cd2+ showed two resonances at 120 and 215 ppm downfield from Cd(ClO4)2. The NMR data are consistent with nitrogen (histidine) and oxygen ligands to the metal centers. 相似文献
44.
A universal T cell epitope-containing peptide from hepatitis B surface antigen can enhance antibody specific for HIV gp120. 总被引:4,自引:0,他引:4
J L Greenstein V C Schad W H Goodwin A B Brauer B K Bollinger R D Chin M C Kuo 《Journal of immunology (Baltimore, Md. : 1950)》1992,148(12):3970-3977
Peptide-based vaccines that directly target T cell or B cell epitopes may have significant advantages over conventional vaccines. Further, synthetic chimeric peptides that combine strong T cell epitopes with poorly immunogenic, but immunodominant, B cell epitopes or strain-conserved B cell epitopes may be useful in eliciting antibody to such important regions. Here we characterize a human T cell epitope analyzed in 54 individuals immunized with a hepatitis B virus surface Ag vaccine. Primary cultures from a total of 59 immunized donors were assessed for their ability to respond to hepatitis B virus surface Ag and peptides, and five were non-responders (8.5%). T cell lines were established from the remaining 54 responders. Of the responders, it was found that the peptide representing amino acids 19 through 33 (19-33) elicited significant proliferation in lines derived from 50 donors. This "universal" T cell epitope, which was recognized in donors of many different HLA-DR and -DQ haplotypes, was then used to construct a chimeric peptide containing 19-33 and the third V region loop structure (V3 loop) of HIV-1 envelope gp 120, in an attempt to augment the immune response to the V3 loop peptide. The V3 loop is the region to which significant neutralizing antibody is directed. Thus, a strong immune response to a synthetic peptide that contains the strain-conserved V3 loop region could have significant therapeutic implications. The V3 loop/19-33 peptide was then used to prime mice, to determine whether V3 loop-specific antibody could be induced. The peptide elicited potent 19-33-specific proliferation in T cells isolated from draining lymph nodes, and in six of six mice anti-V3 loop antibody was elicited. Further, V3 loop/19-33-primed animals made significant levels of antibody that bound rgp120. These data suggest that, when a major T cell epitope is synthesized in tandem with the V3 loop, a significant immune response against the loop can be elicited. Thus, given the finding that neutralizing antibody may play a role in the control and/or prevention of HIV infection, an HIV vaccine composed of a T cell epitope-containing peptide may prove effective. In addition, this type of approach can be generalized to the design of peptide-based vaccines. 相似文献
45.
Four naturally occurring compounds, indole-3-carbinol (I3C), apigenin (Api), ellagic acid (EA) and tannic acid (TA), were tested for their inhibitory effects against 1-nitropyrene- (1-NP) or 1,6-dinitropyrene (1,6-DNP)-induced genotoxicity in Salmonella tester strains and Chinese hamster ovary (CHO) cells. Api and TA strongly inhibited the bacterial mutagenesis induced by nitropyrenes, while 13C and EA had little or no effect. For example, in TA98, 0.2 μmole Api resulted in 48% and 56% inhibition of the mutagenicity induced by 4 nmole 1-NP and 0.035 nmole 1,6-DNP, respectively. With an equal dose, expected, a good correlation was observed between the antimutagenicity of nitropyrenes and their inhibitory effect on nitroreductase activity. This indicated that one of the possible antimutagenic mechanisms of Api or TA was to inactivate the metabolism of nitropyrenes. Two biological end-points, cytotoxicity and sister-chromatid exchange (SCEs), were used to screen the antigenotoxic effects of these compounds in CHO cells. At the sub-cytotoxic dose, 13C, Api and TA all protected against the cytotoxicity induced by 1-NP and 1,6-DNP, but only TA and Api gave a significant reduction of the frequency of SCEs. Moreover, this reduction was found to be highly dose-dependent. 相似文献
46.
Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. 总被引:79,自引:0,他引:79
The macrolide rapamycin blocks cell cycle progression in yeast and various animal cells by an unknown mechanism. We demonstrate that rapamycin blocks the phosphorylation and activation of the 70 kd S6 protein kinases (pp70S6K) in a variety of animal cells. The structurally related drug FK506 had no effect on pp70S6K activation but at high concentrations reversed the rapamycin-induced block, confirming the requirement for the rapamycin and FK506 receptor, FKBP. Rapamycin also interfered with signaling by these S6 kinases, blocking serum-stimulated S6 phosphorylation and delaying entry of Swiss 3T3 cells into S phase. Neither rapamycin nor FK506 blocked activation of a distinct family of S6 kinases (RSKs) or the MAP kinases. These studies identify a rapamycin-sensitive signaling pathway, argue for a ubiquitous role for FKBPs in signal transduction, indicate that FK506-FKBP-calcineurin complexes do not interfere with pp70S6K signaling, and show that in fibroblasts pp70S6K, not RSK, is the physiological S6 kinase. 相似文献
47.
48.
Replacement of a labile aspartyl residue increases the stability of human epidermal growth factor 总被引:5,自引:0,他引:5
C George-Nascimento J Lowenson M Borissenko M Calderón A Medina-Selby J Kuo S Clarke A Randolph 《Biochemistry》1990,29(41):9584-9591
Long-term storage of recombinant human epidermal growth factor (EGF), an important promoter of cell division, results in its conversion to a new species that elutes later than native EGF on a reverse-phase column. This new species, called EGF-X, has only 20% of the biological activity of native EGF. Peptide mapping indicated that the primary structure of EGF-X differs from that of native EGF solely within the first 13 residues. N-Terminal sequencing of EGF-X revealed that about 30% of the polypeptides have been cleaved at the Asp-3/Ser-4 bond. In addition, the yields after the His residue at position 10 were extremely low, indicating that a chemical modification occurs at residue 11 that is incompatible with Edman degradation. We hypothesized that aspartic acid 11 had been converted to an isoaspartyl residue, and this was confirmed with L-isoaspartyl/D-aspartyl methyltransferase, an enzyme that methylates the side-chain carboxyl group of L-isoaspartyl residues but does not recognize normal L-aspartyl residues. EGF-X, but not EGF, was found to be a substrate of this enzyme, and proteolytic digestion of EGF-X with thermolysin localized the site of methylation to a nine-residue peptide containing position 11. We did not observe formation of the isoaspartyl derivative in EGF that had been denatured by reduction of its disulfide bonds. In addition, replacement of the aspartyl residue at position 11 with glutamic acid resulted in a fully active EGF derivative that does not form detectable amounts of EGF-X. We propose that conversion of this aspartyl residue to isoaspartate is a significant nonenzymatic degradation reaction affecting this growth factor.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
49.
Binding of carbamoyl phosphate to Escherichia coli ornithine transcarbamoylase and its relation to turnover have been examined as a function of pH under steady-state conditions. The pH profile of the dissociation constant of carbamoyl phosphate (Kiacp) shows that the affinity of the substrate increases as pH decreases. Two ionizing groups are involved in carbamoyl phosphate binding. Protonation of an enzymic group with pKa 9.6 results in productive binding of the substrate with a moderate affinity of Kiacp approximately 30 microM. Protonation of a second group further enhances binding by roughly another order of magnitude. This ionization occurs with a pKa that shifts from less than 6 in the free enzyme to 7.3 in the binary complex. However, tighter binding of carbamoyl phosphate due to this ionization does not contribute to catalysis. The turnover rate (kcat) of the enzyme diminishes in the acidic pH range and is governed by an ionization with a pKa of 7.2. Both the catalytic pKa of 7.2 and the productive binding pKa of 9.6 appear in the pH profile of kcat/KMcp. Together with earlier kinetic results (Kuo, L. C., Herzberg, W., and Lipscomb, W. N. (1985) Biochemistry 24, 4754-4761), these data suggest that the step which modulates kcat may occur prior to the binding of the second substrate L-ornithine. 相似文献
50.
Differential nuclease sensitivity of the ovalbumin and beta-globin chromatin regions in erythrocytes and oviduct cells of laying hen. 总被引:32,自引:19,他引:13 下载免费PDF全文
We have monitored the differential nuclease sensitivity of defined regions of the chicken genome in different cells using a method which combines restriction enzyme digestion and blotting to diazobenzyloxymethyl (DBM)-paper (see Ref. 11). By using different specific probes and by scanning the bands on the autoradiograms, it is possible to compare on the same blot the digestion patterns of similar-sized fragments from different regions of the genome corresponding to "active" and reference "inactive" genes. We have demonstrated the preferential sensitivity to DNaseI and micrococcal nuclease digestion of the ovalbumin gene region in hen oviduct chromatin. The beta-globin gene region (containing both an adult and an embryonic gene) is also preferentially digested by DNaseI in hen mature erythrocyte nuclei, but at a lower rate than the ovalbumin gene region in oviduct. These observations raise the possibility that there may be several types of preferential nuclease sensitivities, all characterized by increased rates of digestion but to different levels, the highest corresponding to the very actively transcribing genes. 相似文献