贴壁培养细胞台盼蓝拒染试验的方法学探讨

本工作旨在探讨贴壁培养细胞台盼蓝拒染试验的方法,为其合理应用提出建议.用正常培养及NaN3作致死处理的人视网膜色素上皮细胞株-19,进行原位贴壁细胞和培养体系上悬液中细胞的台盼蓝拒染实验,并比较其原位法与计数板法所测活细胞率.与计数板法相似,随染液浓度增高或染色时间延长,原位台盼蓝拒染实验的活细胞率检测值逐渐增加.培养...     

Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources

A recombinant strain of Pichia pastoris with a phenotype of Mut^S was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O₂ concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l^–1 after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.     

Isolation and characterization of phenanthrene-degrading <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> strain ZX4

Phenanthrene-degrading bacterium strain ZX4 was isolated from an oil-contaminated soil, and identified as Sphingomonas paucimobilis based on 16S rDNA sequence, cellular fatty acid composition, mol% G + C and Biolog-GN tests. Besides phenanthrene, strain ZX4 could also utilize naphthalene, fluorene and other aromatic compounds. The growth on salicylic acid and catechol showed that the strain degraded phenanthrene via salicylate pathway, while the assay of catechol 2, 3-dioxygenase revealed catechol could be metabolized through meta-cleavage pathway. Three genes, including two of meta-cleavage operon genes and one of GST encoding gene were obtained. The order of genes arrangement was similar to S-type meta-pathway operons. The phylogenetic trees based on 16S rDNA sequence and meta-pathway gene both revealed that strain ZX4 is clustered with strains from genus Sphingomonas.     

Calcium and lanthanide affinity of the EF-loops from the C-terminal domain of calmodulin

To obtain site-specific information about individual EF-hand motifs, the EF-hand Ca(2+)-binding loops from site III and site IV of calmodulin (CaM) were inserted separately into a non-Ca(2+)-binding cell adhesion protein, domain 1 of CD2 (denoted as CaM-CD2-III-5G-52 and CaM-CD2-IV-5G-52). Structural analyses using various spectroscopic methods have shown that the host protein CD2 retains its native structure after the insertion of the 12-residue loops. The Tb(3+) fluorescence enhancement upon formation of a Tb(3+)-protein complex and the direct competition by La(3+) and Ca(2+) suggest that native Ca(2+)-binding pockets are formed in both engineered proteins. Moreover, as revealed by NMR, both Ca(2+) and La(3+) specifically interact with the residues at the grafted EF-loop. The CaM-CD2-III-5G-52 has stronger affinities to Ca(2+), Tb(3+) and La(3+) than CaM-CD2-IV-5G-52, indicating differential intrinsic metal-binding affinities of the EF-loops.     

3H-azidopine photoaffinity labeling of high molecular weight proteins in chloroquine resistant falciparum malaria

Using 3H-azidopine, we have succeeded in labeling proteins from chloroquine resistant (CR) human falciparum malaria parasites in the molecular weight range of 155-170 kd. Vinblastine does not compete, but azidopine blocks the labeling using 3H-azidopine. Relatively little or no labeling of the 155-170 kd protein is seen in the chloroquine sensitive strain using 3H-azidopine. Further competition can be seen with nicardipine and reserpine (71%) respectively and verapamil (61%), chloroquine (48%), quinacrine (56%), trifluoperazine (32%) and chlorpromazine (33%). We speculate that this may be the glycoprotein responsible for the resistance to chloroquine in falciparum malaria.     

Plant community complexity in the arid valley of Minjiang River, southwestern China

Biocomplexity theory is becoming increasingly important in understanding natural vegetation dynamics and interrelation among all components of the ecosystem. In this study, based on the field investigation of plant species and environmental factors (altitude, microtopography, soil water content, and soil nutrients) in an arid valley of the upper reaches of Minjiang River, Sichuan Province, southwestern China, plant community complexity and its relationship with environmental factors, community diversity, species evenness and richness were studied. Both total and structural complexities of the communities showed a “high- low-high” tendency with the increase in altitude of the area, which meant that the complexity of communities was the highest at the sites of low and high altitude, whereas it was the lowest at the sites of intermediate altitude. It was found that the total community complexity had significant quadratic correlations with soil organic matter (SOM) content, total nitrogen (N), hydrolyzable N, soil water content, and available potassium (K), whereas it had no significant correlations with soil total K, total phosphorus (P), available P, and pH value. The total community complexity positively correlated with community diversity, species evenness and species richness, whereas the structural complexity negatively correlated with the community evenness. Of the two components of the total community complexity, namely, the structural complexity and the structural diversity, the structural complexity was more sensitive than the structural diversity to the changes of species in the community, which was not only related to the community evenness but also to the community richness. The relative contribution of both the structural complexity and the structural diversity to the total complexity would be different for different study areas or ecosystems.