Induction of the blue form of bacteriohodopsin by low concentrations of sodium dodecyl sulfate

The effects produced on bacteriorhodopsin by low concentrations of several detergents have been studied by absorption and fourth-derivative spectrophotometry. Sodium dodecyl sulfate induces the appearance of the blue form of bacteriorhodopsin (λ_max = 600 nm) at pH values up to 7.0 in a reversible manner. The apparent pK of the purple-to-blue transition raised with increasing concentration of SDS. Of the other detergents tested, only sodium dodecyl-N-sarcosinate showed a slight red-shift of the absorption band to 580 nm, whereas sodium taurocholate, Triton X-100 and cetyltrimethylammonium bromide did not favour the appearance of the blue form. The effect of SDS was found to be consistent with a localized conformational change that moves away the counter-ion of the protonated Schiff base.     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Construction and expression of nonsense suppressor tRNAs which function in plant cells

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.     

MHC-like molecules in some nonmammalian vertebrates can be detected by some cross-reactive xenoantisera

Rabbit antisera raised to human and chicken MHC molecules were used to immunoprecipitate cross-reactive molecules from biosynthetically and cell surface-labeled spleen and/or blood cells of representative vertebrate species. Five major points emerged: 1) There were many nonspecific cross-reactions using these techniques, so various criteria were developed to distinguish these from true MHC-like molecules. 2) Only very small subpopulations of immunogen-specific antibodies cross-reacted with MHC-like molecules in other nonmammalian species. These subpopulations were different for each species and even within a species, sometimes being so limited as to behave like alloantisera. This led to a very scattered pattern of true cross-reactions that sometimes failed to reflect the properties of the bulk antibody population. 3) Antisera containing antibodies to class II beta- and class I alpha-chains cross-reacted better and more widely than those to B-G, class II alpha and, in general, beta 2-microglobulin. 4) Some cross-reactive antibodies were clearly directed to epitopes on the surface of the mature heterodimers, but many seemed to recognize nonlinear cryptic determinants, presumably in the contact regions between the chains. These latter antibodies recognized biosynthetic intermediates and also a variety of unusual cell surface MHC-like molecules present in reptile and amphibian, but absent in the mammal and chicken cells tested. These included E homodimers whose relationship to chicken B-G molecules is unknown. 5) MHC-like molecules were identified in a bird, three reptiles, and two amphibians, but no molecules with the expected properties were found with these reagents in any of the fish tested.     

一种“无血清”培养基对肿瘤细胞生长的支持

本文报告一种为适于杂交瘤细胞生长“无血清”培养基－适量的成牛血清低分子成分超滤液、１０ｍｇ／Ｌ转铁蛋白（Ｔ）、１０ｍｇ／Ｌ胰岛素（Ｉ）、２０μｍｏｌ／Ｌ乙醇胺（Ｅ）、４０ｎｍｏｌ／Ｌ硒酸钠（Ｓ）等诸补充成分替代胎牛血清加到基础液中－也完全适用于培养肿瘤细胞,且观察到与之相似的规律性结果,癌细胞在本“无血清”培养若的生长水平达到在含胎牛血清培养基中生长的水平。对于癌细胞的生长,ＬＭＷ－ＣＳ与Ｔ、Ｉ、     

Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: glutamine or arginine at position 191, for the respective A or B allozymes.

The paraoxonase/arylesterase gene is located close to the cystic fibrosis gene on chromosome 7. Human serum contains two paraoxonase/arylesterase allozymes, A and B, which differ in their substrate specificities and kinetic properties. Purified A, AB, and B esterases were digested with trypsin, and the resultant peptides were compared by high-performance liquid chromatography. The elution profiles were very similar for all three samples, except for (1) one peptide (i.e., peptide A) seen only in the A and AB profiles and (2) another peptide (i.e., peptide B) seen only in the B and AB profiles. Sequencing revealed that peptide A had glutamine at amino acid position 191, whereas peptide B was generated by cleavage on the carboxy side of position 191, presumably because there was a basic (trypsin-specific) amino acid at that position. Working independently, our laboratory and one other laboratory have sequenced the coding region for paraoxonase from human liver cDNA libraries and have identified two polymorphic sites: Arg/Gln at position 191 and Leu/Met at position 54. Using PCR amplification and direct sequencing of nucleotides in both polymorphic regions with genomic DNA, we have estimated the allelic frequencies and have determined their concordance with the serum paraoxonase allozyme phenotypes in 27 unrelated adults and in 16 members of a three-generation pedigree. Among unrelated individuals, the Met/Leu polymorphism at position 54 did not correlate with the serum esterase phenotype. In contrast, the particular amino acid at position 191 correlated perfectly with serum phenotypes: A-type individuals had Gln at position 191, and B-type individuals had Arg at position 191; AB-type serum was found only with the heterozygous (Arg/Gln) combination. Pedigree analysis showed both polymorphisms to be inherited in the expected Mendelian manner and confirmed that only the 191 polymorphism showed concordance with the serum paraoxonase/arylesterase phenotypes.     

在中国壮族家系中发现的一例-α~T/-α~Q复合β~0β~0珠蛋白基因型

本文报道在我国广西隆林壮族中发现一个罕見的HbQ复合α,β地中海贫血家系。先证者女,18岁,贫血面容,肝脾肿大。化学结构分析确证本Hb变异体为HbQ Thailand[α74(EF3)Asp→His]。血红蛋白组成以及α和β珠蛋白基因分析结果表明,先证者的珠蛋白基因型为-α~Q/-α~T复合β°/β°(IVSI-1G→T/Codon17A→T);先证者父的基因型为-‘α~Q/-复合β~O/β~A(IVSI-1G→T/β~A);先证母的基因型为-α~T/αα复合β~O/β~A(Codon17A→T/β~A)。     

1α,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1α,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells

1α,25(OH)₂-16-ene-D₃, a synthetic analog of the steroid hormone, 1α,25(OH)₂D₃, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1α,25(OH)₂-16-ene-D₃ expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently. 1α,25(OH)₂-24-oxo-16-ene-D₃, an intermediary metabolite of 1α,25(OH)₂-16-ene-D₃ formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1α,25(OH)₂-24-oxo-D₃, the corresponding intermediary metabolite of 1α,25(OH)₂D₃. In a subsequent in vivo study, we also reported that 1α,25(OH)₂-24-oxo-16-ene-D₃ exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1α,25(OH)₂-24-oxo-16-ene-D₃, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1α,25(OH)₂-24-oxo-16-ene-D₃ in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1α,25(OH)₂D₃. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1α,25(OH)₂D₃ itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1α,25(OH)₂-24-oxo-16-ene-D₃, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1α,25(OH)₂-16-ene-D₃.