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91.
Molecular cloning and characterization of an ML-236B (compactin) biosynthetic gene cluster in Penicillium citrinum 总被引:4,自引:0,他引:4
Abe Y Suzuki T Ono C Iwamoto K Hosobuchi M Yoshikawa H 《Molecular genetics and genomics : MGG》2002,267(5):636-646
Cloning of genes encoding polyketide synthases (PKSs) has allowed us to identify a gene cluster for ML-236B biosynthesis in Penicillium citrinum. Like lovastatin, which is produced by Aspergillus terreus, ML-236B (compactin) inhibits the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Genomic sequencing and Northern analysis showed that nine predicted genes for ML-236B biosynthesis were located within a 38-kb region and were transcribed when ML-236B was produced. The predicted amino acid sequences encoded by these nine genes, designated mlcA- mlcH and mlcR, were similar to those encoded by the genes for lovastatin synthesis, and were therefore assumed to be involved either directly or indirectly in ML-236B biosynthesis. Targeted disruption experiments provided evidence that two PKS genes in the cluster, mlcA and mlcB, are required for the biosynthesis of the nonaketide and the diketide moieties, respectively, of ML-236B, suggesting that the gene cluster as a whole is responsible for ML-236B biosynthesis in P. citrinum. Bioconversion of some of the predicted intermediates by an mlcA-disrupted mutant was also investigated in order to analyze the ML-236B biosynthetic pathway. The molecular organization of the gene cluster and proposed functions for the ML-236B biosynthetic genes in P. citrinum are described. 相似文献
92.
Enhanced UVfemale1 tumor growth in CBF1 mice infected with Schistosoma mansoni due to modulation of Th1-like responses 总被引:1,自引:0,他引:1
Yoshida A Maruyama H Kumagai T Amano T Kobayashi F Wang J Kuribayashi K Ohta N 《Parasitology international》2002,51(2):177-186
Effects of Schistosoma mansoni infection on anti-tumor immunity were examined in CBF1 mice with ultraviolet-induced UVfemale1 fibrosarcoma cells. Although many laboratory established tumor cells had rejection mechanisms independent of CD4(+) T cells, we confirmed that CD4(+) cells had significant roles in rejection of UVfemale1 cells in the syngeneic CBF1 mice. When we prepared two CBF1 mouse groups, S. mansoni-infected and schistosome-free, the former group showed up-regulation of Th2-like response to UVfemale1 cells, whereas the latter group mice showed rather type 1-dominant patterns. Cytotoxic activity against UVfemale1 cells tested in vitro, which was attributed to CD8(+) cells, was significantly weaker in S. mansoni-infected mice compared with infection-free mice. In tumor challenge experiments in vivo, we observed that rapid and complete rejection of UVfemale1 cells required the presence of CD8(+) T cells. Under only CD4-depleted situation, survival of tumor cells in schistosome-free mice was prolonged up to 1 month or more. Under the presence of both CD4(+) and CD8(+) cells, S. mansoni infected mice rejected the challenged UVfemale1 cells as was seen in normal mice. However, when CD8(+) cells were depleted from S. mansoni-infected mice, inoculated UVfemale1 cells grew more rapidly than in infection-free mice. Our results suggest that functionally polarized cytokine patterns in schistosome-infected hosts promote rapid tumor growth. 相似文献
93.
Ishijima S Iwamoto T Nozawa S Matsushita K 《Molecular reproduction and development》2002,63(4):459-463
Electron microscopic examination of the spermatozoa from a man suffering from asthenozoospermia (poor or low sperm motility) showed that approximately 92% of the sperm flagella lacked central pair microtubules but possessed dynein arms and radial spokes while a small percentage of the spermatozoa had complete flagella. The characteristics of the motor apparatus of the spermatozoa and the effects of caffeine on the sperm motility were examined, as were the reactivation of demembranated spermatozoa and the sliding of doublet microtubules. Almost all spermatozoa were immotile in a Tyrode solution while only a small percentage of spermatozoa showed slow forward movement or feeble flagellar vibration, whereas addition of caffeine to the sperm suspension induced forward swimming of approximately half of the spermatozoa. The reactivation of demembranated spermatozoa with MgATP(2-) could not succeed because of disintegration of the demembranated flagella. However, when the demembranated spermatozoa were exposed to MgATP(2-) and then treated with elastase, the microtubular doublets of approximately half the number of the flagella slid from the end or middle of the flagella. These results suggest that the motor apparatus in the sperm flagella that lack the central pair microtubules is functionally assembled and intrinsically capable of undergoing flagellar movement but not strong enough to beat normally. 相似文献
94.
The chemical properties of Amadori compounds in the presence of transition metal ions were studied, using the analogs 1-deoxy-1-n-butylamino-D-fructose (DBF) and N(alpha)-formyl-fructoselysine (fFL). The following characteristics were revealed: (a) DBF combined easily with Cu2+ (but no other transition metal ions) to form a DBF-Cu2+ complex in phosphate buffer, pH 7.4; (b) the complex was unstable, and degraded with the release of Cu+ during incubation at 37 degrees C; (c) degradation of the complex was associated with the production of hydroxyl radicals by the Fenton reaction and alpha-dicarbonyl compounds by non-autoxidative degradation; and (d) properties of DBF were similar to those of fFL. The above properties were additionally observed in glycated poly-Lys (GPL). Our findings indicate a novel mechanism for the generation of hydroxyl radicals and a-dicarbonyl compounds from Amadori adducts in the presence of Cu2+. 相似文献
95.
The entire amino acid sequence of bifunctional alginate lyase from Pseudoalteromonas sp. strain No. 272 were determined by two approaches, Edman degradation of the peptides obtained from protease digestion of the enzyme protein and analysis of PCR products of the structural gene. The former resulted in incomplete amino acid sequence in the entire sequence, due to lacking of the proper peptides from the protease digestion. To compensate for this lack of sequences we applied the method of PCR of the structural gene that was initially elucidated from the primers designed from N- and C-terminal amino acid sequences of the enzyme. The results of the amino acid sequences from these two approaches showed good agreement. The enzyme consisted of 233 amino acid residues with a molecular mass of 25,549.5, including the sole W and cystine residue. The sequence homology search among the other alginate lyases from different origins indicated that they were very weakly homologous, with the exception of the sequence homology (80.3%) of Pseudoalteromonas elyakovii alginate lyase. The consensus sequence, YFKhG + Y-Q (Wong, T. Y., Preston, L. A., and Schiller, N. L. 2000. Annu. Rev. Microbiol.
54: 289–340) in the C-terminal regions was conserved. The kinetic analyses of chemical modification of some amino acid residues of the enzyme showed that W, K, and Y appeared to be important in the enzyme function. 相似文献
96.
Constant variation in structure and function of geometrical isomers of acitretin under natural light
Murayama A Suzuki T Iwamoto M Kunchala SR 《Indian journal of biochemistry & biophysics》2002,39(1):22-27
Acitretin, a beneficial retinoid, was shown to undergo constant structural interconversions among its geometrical isomers (all-trans-acitretin, 9-cis-acitretin, 13-cis-acitretin, 9, 13-di-cis-acitretin, etc.) by photoisomerization under natural light. The photoisomerization was zero order reaction with an apparent velocity of 4x107 M/min under illumination by white fluorescent lamps (1, 200 lx). An equilibrium mixture of the geometrical isomers (all-trans-acitretin 20%, 9-cis-acitretin 15%, 13-cis-acitretin 30%, 9, 13-di-cis-acitretin 15%, and unidentified compounds 20%) was formed at around 30 min. Equilibrium mixtures with similar composition were obtained by photoisomerization reactions starting from other geometrical isomers. Geometrical isomers of acitretin thus formed, showed different effects to induce differentiation of human acute promyelocytic leukemia cells (HL-60 cells): activity of all-trans-acitretin (ED50, 3.2 x 10(-6) M), 9-cis-acitretin (ED50, 2.3 x 10(-5)M), 13-cis-acitretin (ED50, 1.1 x 10(-5)M), 9, 13-di-cis-acitretin (ED50, 2.6 x 10(-6)M). 9-cis-Acitretin acted synergistically with all-trans-acitretin, 13-cis-acitretin and 9, 13-di-cis-acitretin on HL-60 cells. On the other side, all-trans-acitretin, 13-cis-acitretin and 9, 13-di-cis-acitretin acted additively. Geometrical isomers of acitretin showed different effects on differentiation of human epidermal keratinocytes; expression of keratinocyte differentiation markers, keratin 1 and keratin 10, were suppressed more strongly by 9-cis-acitretin and 13-cis-acitretin as compared to all-trans-acitretin or 9, 13-di-cis-acitretin. 相似文献
97.
Dickinson DA Iles KE Watanabe N Iwamoto T Zhang H Krzywanski DM Forman HJ 《Free radical biology & medicine》2002,33(7):974
Glutathione is the most abundant non-protein thiol in the cell, with roles in cell cycle regulation, detoxification of xenobiotics, and maintaining the redox tone of the cell. The glutathione content is controlled at several levels, the most important being the rate of de novo synthesis, which is mediated by two enzymes, glutamate cysteine ligase (GCL), and glutathione synthetase (GS), with GCL being rate-limiting generally. The GCL holoenzyme consists of a catalytic (GCLC) and a modulatory (GCLM) subunit, which are encoded by separate genes. In the present study, the signaling mechanisms leading to de novo synthesis of GSH in response to physiologically relevant concentrations of 4-hydroxy-2-nonenal (4HNE), an endproduct of lipid peroxidation, were investigated. We demonstrated that exposure to 4HNE resulted in increased content of both Gcl mRNAs, both GCL subunits, phosphorylated JNK1 and c-Jun proteins, as well as Gcl TRE sequence-specific AP-1 binding activity. These increases were attenuated by pretreating the cells with a novel membrane-permeable JNK pathway inhibitor, while chemical inhibitors of the p38 or ERK pathways were ineffective. These data reveal that de novo GSH biosynthesis in response to 4HNE signals through the JNK pathway and suggests a major role for AP-1 driven expression of both Gcl genes in HBE1 cells. 相似文献
98.
Tyr-199 and charged residues of pharaonis Phoborhodopsin are important for the interaction with its transducer 下载免费PDF全文
pharaonis Phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a retinal protein in Natronobacterium pharaonis and is a receptor of negative phototaxis. It forms a complex with its transducer, pHtrII, in membranes and transmits light signals by protein-protein interaction. Tyr-199 is conserved completely in phoborhodopsins among a variety of archaea, but it is replaced by Val (for bacteriorhodopsin) and Phe (for sensory rhodopsin I). Previously, we (Sudo, Y., M. Iwamoto, K. Shimono, and N. Kamo, submitted for publication) showed that analysis of flash-photolysis data of a complex between D75N and the truncated pHtrII (t-Htr) give a good estimate of the dissociation constant K(D) in the dark. To investigate the importance of Tyr-199, K(D) of double mutants of D75N/Y199F or D75N/Y199V with t-Htr was estimated by flash-photolysis and was approximately 10-fold larger than that of D75N, showing the significant contribution of Tyr-199 to binding. The K(D) of the D75N/t-Htr complex increased with decreasing pH, and the data fitted well with the Henderson-Hasselbach equation with a single pK(a) of 3.86 +/- 0.02. This suggests that certain deprotonated carboxyls at the surface of the transducer (possibly Asp-102, Asp-104, and Asp-106) are needed for the binding. 相似文献
99.
Yuasa T Kataoka H Kinto N Iwamoto M Enomoto-Iwamoto M Iemura S Ueno N Shibata Y Kurosawa H Yamaguchi A 《Journal of cellular physiology》2002,193(2):225-232
The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein-2 (Bmp-2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino-terminal portion of SHH (rSHH-N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3-E1 cells. rSHH-N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH-N-induced ALP activity in C3H10T1/2 and MC3T3-E1 cells, indicating that BMPs are involved in SHH-induced osteoblast differentiation. Simultaneous supplement with rSHH-N and BMP-2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH-N for 6 h enhanced the response to BMP-2 by increasing ALP activity in C3H10T1/2 and MC3T3-E1 cells. Stimulatory effects of rSHH-N and additive effects with rSHH-N and BMP-2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP-2 (1 microg) into muscle of mice induced formation of ectopic bone, whereas transplantation of r-SHH-N (1-5 microg) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP. 相似文献