Coordinated regulation of vascular endothelial cell growth and prostacyclin production by epidermal growth factor: Evidence of clonal variations among rat aortic endothelial cells

Summary Rat aortic endothelial cells were found to exhibit clonal variations in response to EGF stimulation in cell growth and prostacyclin synthesis. EGF-induced growth and prostacyclin synthesis appeared to be regulated in a coordinated manner in that a clone with a higher response to EGF growth stimulation also exhibited a higher response to EGF-stimulated prostacyclin synthesis. This observation implys a possible involvement of prostacyclin synthesis in some of the biological effects of EGF on vascular endothelial cells.     

Modification of cell membrane in variants of chinese hamster cells resistant to abrin

Stable and heritable variants of Chinese hamster ovary (CHO) cells which are resistant to different levels (0.1, 1.0 and 10 μg/ml) of the toxin abrin have been isolated and characterized. The frequency of resistant colonies to abrin was increased with the concentration of a chemical mutagen. There was no effect of cell density or cross-feeding on the recovery of variants. In experiments using fluorescein-labeled abrin and ricin which bind to terminal (non-sialylated) galactose residues of cell-surface oligosaccharides, parental cells exhibited strong binding toward both toxins, whereas no fluorescence was observed in the resistant clones. A fluorescein-conjugated lectin, BS II, which is specific for terminal N-acetyl- -glucosaminyl residues, did not interact with the parental cells, but did with the resistant clones. This suggests that on the surface of resistant cells the number of terminal galactosyl residues of oligosaccharide chains in glycoproteins was reduced, exposing the penultimate N-acetyl- -glucosaminyl residues. The number of available endogenous acceptor sites for galactosyl transferase in the abrin-resistant clones was directly proportional to the degree of resistance. In the presence of great excess of exogenous acceptor, the rates of galactosyl transfer were similar in all the abrin-resistant cell types tested, with levels ranging from 1.4 to 1.7 times parental cell values. Studies with tetraploid cell hybrids reveal that resistance was a recessive trait. Fluctuation analysis showed that abrin resistance occurred in CHO cell populations at a rate of 4−7 × 10⁻⁸/cell/generation. The system may serve as a new marker for quantitative mutagenesis studies.     

The acid-catalyzed decompostion of phenacylcobalamin: evidence for the formation of an enol-Co(III) pi-complex intermediate.

Phenacylcobalamin has been synthesized and characterized by thin-layer chromatography and uv-visible spectroscopy, as well as identification of the cobalt-containing and organic products of its cleavage in acid and base and by aerobic photolysis. The major organic product from all three cleavage reactions is acetophenone and the cobalt-containing product is aquacobalamin (or hydroxocobalamin, its conjugate base). In aqueous acidic solution (pH 0 to 7.3, ionic strength 1.0 M, and 25.0 degrees C), the kinetics of the formation of aquacobalamin are biphasic representing the linear sum of two exponential terms. The pH dependence of the first-order rate constant of both phases shows a first-order dependence on proton concentration but with an inflection point ot pH 3.55 for the faster phase and at pH 4.03 for the slower phase. This behavior is interpreted in terms of the specific acid catalyzed formation of an intermediate from both "base on" and "base off" phenacylcobalamin with different second-order rate constants for each form, followed by an intermediate decompotion step with a similar formal mechanism. The nature of the intermediate is discussed and it is concluded to be a pi-complex between cob(III)alamin and the enol of acetophenone.     

Isolation of the S-peptide formed on digestion of fructose 1,6-bisphosphatase with subtilisin and its non-covalent association with the enzyme protein.

Digestion of rabbit liver fructose 1,6-bisphosphatase with subtilisin results in a several-fold increase in catalytic activity measured at pH 9.2. This change is due to cleavage of a peptide bond located 60 amino acid residues from the NH₂-terminus. The S-peptide and the residual subunit appear as separate peptides in sodium dodecyl sulfate polyacrylamide gel electrophoresis and the S-peptide can be isolated by gel filtration in 9% HCOOH. Under nondissociating conditions, however, the S-peptide remains associated with the protein, and the tetrameric structure and original molecular weight are preserved. Thus the nicking of the peptide chain by subtilisin causes a conformation change that alters the catalytic properties of the enzyme.     

Reconstitution of biological molecular generators of electric current. Cytochrome oxidase.

1. Direct measurement of the electric current generation by cytochrome oxidase has been carried out. To this end, two procedures were used. The simpler one consists in formation of planar artificial membrane from the mixture of decane solution of soya bean phospholipids and beef heart cytochrome oxidase. Addition of cytochrome c and ascorbate to one of the two compartments separated by the cytochrome oxidase-containing planar membrane was found to result in a transmembrane electric potential difference being formed (plus on cytochrome c side of the membrane). Maximal values of potential differences obtained by this method were about 40 mV. Much higher potentials were observed when another ("photeoliposome-planar membrane") method was applied. In this case cytochrome oxidase was reconstituted with phospholipid to form proteoliposomes which adhered to planar phospholipid membrane in the presence of Ca2+ ions. Addition of cytochrome c and ascorbate to the proteoliposome-containing compartment gives rise to generation of an electric potential difference across the planar membrane, which reached 100 mV at a current of about 1 X 10(-11) A (minus in the proteoliposome-free compartment). The electromotive force of this generator was estimated as being about 0.2 V. If ascorbate and proteoliposomes were added into different compartments, a penetrating hydrogen atom carrier (phenazine methosulfate, (PMS) or tetramethyl-p-phenylenediamine (TMPD)) was required for a membrane potential to be formed. Generation of an electric potential difference of the opposite direction (plus in the proteoliposome-free compartment) was revealed in experiments with cytochrome oxidase proteoliposome containing cytochrome c in their interior. In this case, addition of PMS or TMPD was necessary. 2. In the suspension of cytochrome oxidase proteoliposome the uptake of a cationic penetrant (tetraphenyl phosphonium cation) was found to be coupled with electron transfer via external cytochrome c. Electron transfer via intraproteoliposomal cytochrome c induced the uptake of anionic penetrants (tetraphenyl borate and phenyldicarbaundecaborane anions). 3. All the above effects were sensitive to cyanide and protonophorous uncouplers. 4. In proteoliposomes containing both cytochrome oxidase and bacteriorhodopsin, the light- and oxidation-dependent generations of membrane potential have been revealed. 5. The data obtained are in agreement with Mitchell's idea of transmembrane electron flow in the cytochrome oxidase segment of the respiratory chain.     

The ultrastructure of the middle cerebral artery and its associated nerve fibers in the squirrel monkey and baboon

This study describes the ultrastructural characteristics of the middle cerebral artery and its related neural elements in the squirrel monkey and baboon. The cytoarchitecture of the M-1 segment as well as that of the smaller extracerebral and intracerebral vessels is comparable in both animals.Smooth muscle elements are occasionally found within the intimai lining. The nerve bundles associated with vessels contain fewer myelinated fibers as the vessel diameter decreases. The cytological relationship between the neural structures and the smooth muscle cells are discussed.     

Breeding of High Daptomycin-Producing Strain by Streptomycin Resistance Superposition

Daptomycin is a cyclolipopeptide antibiotic produced by Streptomyces roseosporus. It is widely used to treat drug-resistant bacterial infections; however, daptomycin yield in wild strains is very low. To improve the daptomycin production by the strain BNCC 342432, a modified method of ribosome engineering with superposition of streptomycin resistance was adopted in this study. The highest-yield mutant strain SR-2620 was obtained by increasing streptomycin resistance of BNCC 342432, and achieved daptomycin production of 38.5 mg/l in shake-flask fermentation, 1.79-fold higher than the parent strain and its heredity stability was stable. The morphological characteristics of the two strains were significantly different, and the 440^th base G of the rpsL gene in the mutant strain was deleted, which resulted in a frameshift mutation. Our results demonstrate that gradually increasing strain resistance to streptomycin was an effective breeding method to improve daptomycin yield in S. roseosporus. Open in a separate window     

新疆棉花黄萎病菌拮抗细菌的分离、筛选与鉴定

【背景】棉花黄萎病是由大丽轮枝菌(Verticillium dahliae Kleb.)引起的一种世界性病害,近年来对该病害的生物防治因具有环境友好和人畜安全的特性而倍受关注。【目的】筛选棉花黄萎病高效拮抗细菌并对其进行鉴定,为棉花黄萎病的生物防治扩充菌种资源。【方法】采用稀释涂布平板法分离细菌,并进行拮抗细菌的初筛和复筛,通过形态特征、生理生化特征和16S rRNA基因序列分析对筛选到的细菌进行鉴定,确定其分类地位。【结果】初筛分离到535株对病原菌具有拮抗作用的细菌,并选取了108株拮抗细菌进行复筛,最终筛选到了4株优势拮抗细菌。通过形态观察、生理生化特征和16SrRNA基因序列分析,将菌株BHZ-29、SHT-15、SHZ-24和SMT-24分别鉴定为贝莱斯芽孢杆菌(Bacillusvelezensis)、枯草芽孢杆菌斯皮兹仁亚种(Bacillus subtilis subsp. spizizenii)、萎缩芽孢杆菌(Bacillus atrophaeus)和香草芽孢杆菌(Bacillus vanillea)。【结论】获得了4株高效拮抗细菌,并且首次报道了香草芽孢杆菌对棉花黄萎病菌具有抑制作用。     

The mushroom ribosome-inactivating protein lyophyllin exerts deleterious effects on mouse embryonic development in vitro

Earlier investigations disclose that some plant ribosome-inactivating proteins (RIPs) adversely affect mouse embryonic development. In the present study, a mushroom RIP, namely lyophyllin from Lyophyllum shimeji, was isolated, partially sequenced, and its translation inhibitory activity determined. Its teratogenicity was studied by using a technique entailing microinjection and postimplantation whole-embryo culture. It was found that embryonic abnormalities during the period of organogenesis from E8.5 to E9.5 were induced by lyophyllin at a concentration as low as 50 μg/ml, and when the lyophyllin concentration was raised, the number of abnormal embryos increased, the final somite number decreased, and the abnormalities increased in severity. The affected embryonic structures included the cranial neural tube, forelimb buds, branchial arches, and body axis, while optic and otic placodes were more resistant. Lyophyllin at a concentration higher than 500 μg/ml also induced forebrain blisters within the cranial mesenchyme. When the abnormal embryos were examined histologically, an increase of cell death was found to be associated with abnormal structures, indicating that cell death may be one of the underlying causes of teratogenicity of the mushroom RIP. This constitutes the first report on the teratogenicity of a mushroom RIP.