aAd-p53基因靶向治疗癌性胸腹水的临床效果分析

目的:探讨aAd-p53注射液靶向灌注治疗癌性胸腹水的临床疗效和安全性。方法:选择2012年5月-2014年2月在我院接受治疗的癌性胸腹水患者80例,根据治疗方法的不同,将患者随机分为研究组和对照组,每组40例。研究组患者采用腔内灌注rAd-p53治疗,对照组患者采用表阿霉素灌注治疗,观察并比较两组患者的治疗总有效率、不良反应的发生率及KPS功能评分的变化情况。结果:所有患者均顺利完成灌注治疗,病情获得好转,生存质量得到改善。研究组和对照组的治疗总有效率分别为70%、67.5%,两组比较无显著性差异(P0.05)。两组治疗后KPS评分均显著高于治疗前,且研究组高于对照组,差异具有统计学意义(P0.05)。研究组和对照组不良反应的发生率分别为20%和25%,研究组低于对照组,但两组差异并无统计学意义(P0.05)。结论:rAd-p53注射液靶向灌注治疗是一种治疗癌性胸腹水安全有效的方法,值得临床推广。     

Spectrochromatography of photosynthetic pigments as a fingerprinting technique for microbial phototrophs

Abstract: The combination of chromatographic separation using reverse-phase high-performance liquid chromatography and continuous monitoring of the column eluate using a diode-array spectrophotometer allowed qualitative and quantitative pigment profiling of extracts of photosynthetic material in a single run. Carotenoids and the spectrally distinct types of chlorophyll and bacteriochlorophyll can be unambiguously identified even when imperfectly separated on the column. The resulting spectrochromatogram is a fingerprint useful for the rapid characterization of pure cultures or mixed populations. We have developed software to allow recording, manipulation, and presentation of the resulting spectrochromatograms and present results from photosynthetic microbes in pure and mixed cultures. We describe a number of approaches to the presentation of the resulting data in a readily comprehensible form.     

Genetic manipulation of carotenoid biosynthesis in the green sulfur bacterium Chlorobium tepidum

The green sulfur bacterium Chlorobium tepidum is a strict anaerobe and an obligate photoautotroph. On the basis of sequence similarity with known enzymes or sequence motifs, nine open reading frames encoding putative enzymes of carotenoid biosynthesis were identified in the genome sequence of C. tepidum, and all nine genes were inactivated. Analysis of the carotenoid composition in the resulting mutants allowed the genes encoding the following six enzymes to be identified: phytoene synthase (crtB/CT1386), phytoene desaturase (crtP/CT0807), zeta-carotene desaturase (crtQ/CT1414), gamma-carotene desaturase (crtU/CT0323), carotenoid 1',2'-hydratase (crtC/CT0301), and carotenoid cis-trans isomerase (crtH/CT0649). Three mutants (CT0180, CT1357, and CT1416 mutants) did not exhibit a discernible phenotype. The carotenoid biosynthetic pathway in C. tepidum is similar to that in cyanobacteria and plants by converting phytoene into lycopene using two plant-like desaturases (CrtP and CrtQ) and a plant-like cis-trans isomerase (CrtH) and thus differs from the pathway known in all other bacteria. In contrast to the situation in cyanobacteria and plants, the construction of a crtB mutant completely lacking carotenoids demonstrates that carotenoids are not essential for photosynthetic growth of green sulfur bacteria. However, the bacteriochlorophyll a contents of mutants lacking colored carotenoids (crtB, crtP, and crtQ mutants) were decreased from that of the wild type, and these mutants exhibited a significant growth rate defect under all light intensities tested. Therefore, colored carotenoids may have both structural and photoprotection roles in green sulfur bacteria. The ability to manipulate the carotenoid composition so dramatically in C. tepidum offers excellent possibilities for studying the roles of carotenoids in the light-harvesting chlorosome antenna and iron-sulfur-type (photosystem I-like) reaction center. The phylogeny of carotenogenic enzymes in green sulfur bacteria and green filamentous bacteria is also discussed.     

Subcellular localization of chlorosome proteins in Chlorobium tepidum and characterization of three new chlorosome proteins: CsmF, CsmH, and CsmX

Chlorosomes are unique light-harvesting structures found in two families of photosynthetic bacteria. In this study, three chlorosome proteins (CsmF, CsmH, and CsmX) of the green sulfur bacterium Chlorobium tepidum were characterized by cloning and sequencing the genes which encode them, by overproducing the respective proteins in Escherichia coli, and by raising polyclonal antisera to the purified proteins. Three other proteins (AtpF, CT1970, and CT2144) which were identified in chlorosome fractions have similarly been characterized. The antisera were used to establish the distribution of each protein in various cellular fractions. Ten chlorosome proteins (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) copurified in a constant proportion together with bacteriochlorophyll c, and none of these 10 proteins was found in substantial amounts in other subcellular fractions. An antiserum to CsmH was highly effective in agglutinating chlorosomes, and antisera to CsmI, CsmJ, CsmX, and CsmA also immunoprecipitated chlorosomes to varying extents. However, an antiserum to CsmF did not agglutinate chlorosomes. The sequences of chlorosome proteins generally are not significantly similar to the sequences of other proteins in the databases. However, the N-terminal domains of three chlorosome proteins, CsmI, CsmJ, and CsmX, are related to adrenodoxin-type ferredoxins that ligate [2Fe-2S] clusters [Vassilieva, E. V., Antonkine, M. L., Zybailov, B. L., Yang, F., Jakobs, C. U., Golbeck, J. H., and Bryant, D. A. (2001) Biochemistry 40, 464-473]. The sequences of the C-terminal domains of these three proteins appear to be distantly related to CsmA and CsmE. The remaining chlorosome proteins can be divided into two additional structural families, CsmB/F and CsmC/D. CsmH is recovered in water-soluble form after overproduction in E. coli. Interestingly, this protein contains an N-terminal domain that is similar to CsmB/D, while its C-terminal domain is related to CsmC/D. The sequence relationships indicate that, although the protein composition of Chlorobium-type chlorosomes is superficially more complex than that of the chlorosomes of Chloroflexus aurantiacus, this heterogeneity is mostly produced by gene duplication and divergence among a small number of protein types.     

Inter‐specific competition and competition‐free space in the tephritid parasitoids Utetes anastrephae and Doryctobracon areolatus (Hymenoptera: Braconidae: Opiinae)

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Identification of the bchP gene, encoding geranylgeranyl reductase in Chlorobaculum tepidum

The Chlorobaculum tepidum genome contains two paralogous genes, CT2256 and CT1232, whose products are members of the FixC dehydrogenase superfamily and have sequence similarity to geranylgeranyl reductases. Each gene was insertionally inactivated, and the resulting mutants were characterized. CT2256 encodes geranylgeranyl reductase (BchP); CT1232 is not involved in bacteriochlorophyll or chlorophyll biosynthesis.     

The bchU gene of Chlorobium tepidum encodes the c-20 methyltransferase in bacteriochlorophyll c biosynthesis

Bacteriochlorophylls (BChls) c and d, two of the major light-harvesting pigments in photosynthetic green sulfur bacteria, differ only by the presence of a methyl group at the C-20 methine bridge position in BChl c. A gene potentially encoding the C-20 methyltransferase, bchU, was identified by comparative analysis of the Chlorobium tepidum and Chloroflexus aurantiacus genome sequences. Homologs of this gene were amplified and sequenced from Chlorobium phaeobacteroides strain 1549, Chlorobium vibrioforme strain 8327d, and C. vibrioforme strain 8327c, which produce BChls e, d, and c, respectively. A single nucleotide insertion in the bchU gene of C. vibrioforme strain 8327d was found to cause a premature, in-frame stop codon and thus the formation of a truncated, nonfunctional gene product. The spontaneous mutant of this strain that produces BChl c (strain 8327c) has a second frameshift mutation that restores the correct reading frame in bchU. The bchU gene was inactivated in C. tepidum, a BChl c-producing species, and the resulting mutant produced only BChl d. Growth rate measurements showed that BChl c- and d-producing strains of the same organism (C. tepidum or C. vibrioforme) have similar growth rates at high and intermediate light intensities but that strains producing BChl c grow faster than those with BChl d at low light intensities. Thus, the bchU gene encodes the C-20 methyltransferase for BChl c biosynthesis in Chlorobium species, and methylation at the C-20 position to produce BChl c rather than BChl d confers a significant competitive advantage to green sulfur bacteria living at limiting red and near-infrared light intensities.     

Studies of the location and function of isoprenoid quinones in chlorosomes from green sulfur bacteria

The chlorosome antenna of the green sulfur bacterium Chlorobium tepidum essentially consists of aggregated bacteriochlorophyll (BChl) c enveloped in a glycolipid monolayer. Small amounts of protein and the isoprenoid quinones chlorobiumquinone (CK) and menaquinone-7 (MK-7) are also present. Treatment of isolated chlorosomes from Cb. tepidum with sodium dodecyl sulfate (SDS) did not affect the quinones, demonstrating that these are located in a site which is inaccessible to SDS, probably in the interior of the chlorosomes. About half of the quinones were removed by Triton X-100. The non-ionic character of Triton probably allowed it to extract components from within the chlorosomes. MK-10 in chlorosomes from the green filamentous bacterium Chloroflexus aurantiacus was likewise found to be located in the chlorosome interior. The excitation transfer in isolated chlorosomes from Cb. tepidum is redox-regulated. We found a ratio of BChl c fluorescenceintensity under reducing conditions (Fred) to that under oxidizing conditions (Fox) of approximately 40. The chlorosomal BChl a fluorescence was also redox-regulated. When the chlorosomal BChl c–BChl c interactions were disrupted by 1-hexanol, the BChl c Fred/Fox ratiodecreased to approximately 3. When CK and MK-7 were extracted from isolated chlorosomes with hexane, the BChl c Fred/Fox ratio also decreased to approximately 3. A BChl c Fred/Fox ratio of 3–5 was furthermore observed in aggregates of pure BChl c and in chlorosomes from Cfx. aurantiacus which do not contain CK. We therefore suggest that BChl c aggregates inherently exhibit a small redox-dependent fluorescence (Fred/Fox 3) and that the large redox-dependent fluorescence observed in chlorobial chlorosomes (Fred/Fox 40) is CK-dependent.     

One-step plasmid construction for generation of knock-out mutants in cyanobacteria: studies of glycogen metabolism in <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002

Genome sequences of microorganisms typically contain hundreds of genes with vaguely defined functions. Targeted gene inactivation and phenotypic characterization of the resulting mutant strains is a powerful strategy to investigate the function of these genes. We have adapted the recently reported uracil-specific excision reagent (USER) cloning method for targeted gene inactivation in cyanobacteria and used it to inactivate genes in glycogen metabolism in Synechococcus sp. PCC 7002. Knock-out plasmid constructs were made in a single cloning step, where transformation of E. coli yielded about 90% colonies with the correct construct. The two homologous regions were chosen independently of each other and of restriction sites in the target genome. Mutagenesis of Synechococcus sp. PCC 7002 was tested with four antibiotic resistance selection markers (spectinomycin, erythromycin, kanamycin, and gentamicin), and both single-locus and double-loci mutants were prepared. We found that Synechococcus sp. PCC 7002 contains two glycogen phosphorylases (A0481/glgP and A2139/agpA) and that both need to be genetically inactivated to eliminate glycogen phosphorylase activity in the cells.