大鼠肝癌中α_2-巨球蛋白等几种分泌性蛋白基因表达的变化

用Northern blot方法对二乙基亚硝胺所诱发的大鼠肝癌中内源性蛋白酶抑制因子α_2-巨球蛋白(α_2-M)、非特异性免疫抑制剂α_1-酸性糖蛋白(α_1-AGP)及雄性激素正调控的α-2u球蛋白(α-2u)三种分泌性蛋白基因表达情况进行了分析。结果表明在大部分(14/16)肝癌样品中α_2-M RNA水平显著降低;而α_1-AGP RNA水平显著高于正常对照水平;α-2u RNA水平明显下降,但在某些雄性大鼠肝癌样品中该基因却有一定程度的表达。这些结果说明,一些肿瘤宿主血浆中α_2-M水平的显著下降及α_1-AGP水平的明显升高分别是由于基因表达活性的下降及升高所致。α-2u基因表达的异常提示,在癌变过程中机体的内分泌功能发生了某些变化。     

Primary structure of the S-peptide formed by digestion of rabbit liver fructose 1,6-biphosphatase with subtilisin.

Digestion of rabbit liver fructose 1,6-bisphosphatase with subtilisin followed by denaturation of the protein yields a peptide containing 60 amino acid residues, including the blocked NH₂-terminus. This peptide has the following sequence: Ac-Ala-Asp-Lys-Ala-Pr o-Phe-Asp-Thr-Asp-Ile-Ser-Thr-Met-Thr-Arg-Phe-Val-Met-Glu-Glu-Gly-Arg-Ly s-Ala-Gly-Gly-Thr-Gly-Glu-Met-Thr-Gln-Leu-Leu-Asn-Ser-Leu-Cys-Thr-Ala-Va l-Lys-Ala-Ile-Ser-Thr-Ala-Val-Arg-Lys-Ala-Gly-Ile-Ala-His-Leu-Tyr-Gly-Ile-Ala.     

Detection of T-2 toxin by an improved radioimmunoassay

T-2 toxin in serum, urine, and saline was analyzed by a modified radioimmunoassay procedure. The specimens were added directly to the assay tubes without extraction steps. The reaction between antibody and ligands was optimal at 1 h. Albumin-coated charcoal was used to separate bound from free radioactivity. Quenching, which occurred with hemolyzed specimens, was corrected by a wet oxidation process with 60% perchloric acid and 30% hydrogen peroxide. The shorter incubation times resulted in an assay that takes less than 6 h to complete. The average affinity constant of the antibody (Km) was 1.75 X 10(10) liters/mol. The sensitivity was 1 ng per assay or 10 ng/ml. Among the other trichothecenes tested, only H-T-2 cross-reacted significantly (10.3%).     

Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA.

To complete assignment of the 19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-substituted Escherichia coli tRNA(Val), resonances from 5-fluorouracil residues involved in tertiary interactions have been identified. Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed. FU54 and FU55 were identified by 19F homonuclear Overhauser experiments and were then assigned by comparison of their 19F NMR spectra with those of 5-fluorouracil-labeled yeast tRNA(Phe) mutants having FU54 replaced by adenine and FU55 replaced by cytosine. FU8 and FU12, were assigned from the 19F NMR spectrum of the tRNA(Val) mutant in which the base triple G9-C23-G12 substituted for the wild-type A9-A23-FU12. Although replacement of the conserved U8 (FU8) with A or C disrupts the tertiary structure of tRNA(Val), it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme. Analysis of the 19F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAVal indicated a strong correlation to tRNA secondary and tertiary structure. 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5-fluorouracil. However, the signal from FU59, in the T-loop of tRNA(Val), was shifted more than 1 p.p.m. downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops. The 19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m. range, when fluorouracil was base-paired with adenine in helical stems. This upfield shift was less pronounced for the fluorine of the FU7.A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix. An unanticipated finding was that the 19F resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m. downfield of those from fluorouracil residues paired with A. In the 19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.     

Differentiation of extracutaneous melanocytes in embryos of the turtle, Trionyx sinensis japonicus.

The present study investigates the mode of differentiation of neural crest-derived melanocytes in the embryos of the soft-shell turtle, Trionyx sinensis japonicus. DOPA reaction-positive melanoblasts were first detected in 10-day-old embryos. Melanocyte differentiation in terms of pigmentation takes place from the day 16 of development. Melanin pigments were found in the dorsal integument as well as in various extracutaneous tissues such as skeletal muscle, dorsal aorta, peritoneum, blood vessels, choroid, lung, bone marrow, fat tissues and in the connective tissue of the nose. These results suggest the presence of a specific environmental regulation of the melanoblast differentiation in the soft-shell turtle.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

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.