Partial purification and characterization of taurodeoxycholate 7α-monooxygenase

Taurodeoxycholate 7α-monooxygenase was partially purified from rat liver microsomes. The enzyme was solubilized with cholate, fractionated with polyethylene glycol and chromatographed on a Sepharose 4B column with cholate as ligand. The enzyme activity was eluted from the column into the fraction eluted with 50 mM phosphate buffer containing cholate and KCl, whereas the benzphetamine demethylase activity was eluted in the non-bound fraction. Thus it was established that both enzymes are different entities. The taurodeoxycholate 7α-monooxygenase activity was reconstituted from the partially purified cytochrome P-450, highly purified NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine and NADPH.     

Induction of competence and progression signals in human T lymphocytes by phorbol esters and calcium ionophores

We have investigated the induction of competence (IL-2 responsiveness) and progression in human T lymphocyte proliferation triggered by phorbol ester and calcium ionophore. The degree of proliferation induced with the phorbol ester, phorbol 12,13-dibutyrate (PDB) and the calcium ionophore ionomycin was dependent on the duration of exposure to these agents, with more than 6 h required for obtaining maximum proliferation. Following brief exposure to both agents for 30 min, which did not cause significant proliferation, T cells became competent to proliferate in response to exogenous interleukin 2 (IL-2). These competent T cells also progressed to DNA synthesis following incubation with PDB in the absence of ionomycin. Induction of competence to proliferate in response to either PDB or IL-2 was blocked by EGTA, suggesting that transmembrane Ca2+ flux was obligatory at this stage. Since other phorbol esters and synthetic diacylglycerols also stimulated DNA synthesis in competent cells, it is likely that progression was triggered by activation of protein kinase C. Following a brief exposure to PDB and ionomycin, subsequent incubation with PDB induced gene expression and secretion of IL-2 and augmented the expression of IL-2 receptors in the competent cells. Thus, we have demonstrated that Ca2+ mobilization is required for rendering T cells competent to express functional IL-2 receptors, to produce IL-2 in response to subsequent incubation with PDB, and that sustained activation of protein kinase C seems necessary for IL-2 production and subsequent progression of competent T cells to DNA synthesis.     

Molecular cloning and sequencing of cDNA encoding goat pre alpha-lactalbumin

In poly(A)+RNA extracted from a lactating goat mammary gland, mRNA of about 750 nucleotides was shown to encode pre alpha-lactalbumin by using in vitro translation and immunoprecipitation. From the total poly(A)+RNA, the cDNA library was constructed using the Escherichia coli plasmid pUC18; it was screened with the oligodeoxyribonucleotide probe corresponding to the amino acid sequence of Trp60-Gln65 of goat alpha-lactalbumin. A plasmid containing almost full-length cDNA of goat pre alpha-lactalbumin, pGLA-1, was identified. The cDNA insert of pGLA-1 comprises 727 base pairs and contains the signal peptide and mature protein sequence.     

Expression of goat alpha-lactalbumin in Escherichia coli and its refolding to biologically active protein

A cDNA encoding the mature region of goat alpha-lactalbumin and the 3'-non-coding region was fused to cDNA of the N-terminal half of porcine adenylate kinase which had been placed under the control of the tac promoter in an expression vector in Escherichia coli. In addition, a methionine codon was inserted between the two cDNAs. When the plasmid carried the full-length 3'-non-coding region, little accumulation of the fused protein was observed. However, the deletion of two-thirds of the 3'-non-coding region produced significant expression of the fused protein in E. coli strain JM105. Since goat alpha-lactalbumin contains no methionine residue, the mature goat alpha-lactalbumin was isolated by CNBr digestion of the fused insoluble protein and refolded using thioredoxin. The homogeneous and biologically active goat alpha-lactalbumin was purified by Ca2+ ion-dependent hydrophobic chromatography.     

Determination of base composition of DNA by high performance liquid chromatography of its nuclease P1 hydrolysate

The base composition of DNA was directly determined by high performance liquid chromatography (HPLC) of its nuclease P1 hydrolysate. This method can satisfactorily be employed even if the sample of DNA contains RNA or the amount of the sample is very small.     

Three-dimensional structure of endothelial cells in hepatic sinusoids of the rat as revealed by the Golgi method

Summary The three-dimensional structure of endothelial cells in the hepatic sinusoids of the rat was studied by application of light- and electron microscopy on Golgi-impregnated specimens. A number of endothelial cells could thus be individually delineated throughout the hepatic lobules. The cytoplasm, showing heavy silver deposits, consists of two distinct areas, a thick and thin portion. The thick portion, issuing from the region of the perikaryon, branches and tapers toward the cell periphery. The thin portion, occupying the remainder of the cytoplasm, consists largely of highly fenestrated sieve plates. Some intralobular variation can be noted; the thick portion of the endothelial cells is well developed in the periportal zone, while the cells in the centrilobular zone are relatively rich in thin portions. In addition, the area of distribution of an individual endothelial cell is larger in the centrilobular sinusoids than in the periportal zone. Some endothelial cells also possess unique cytoplasmic processes projecting into the intercellular space between hepatocytes and connecting the sinusoidal walls of neighboring sinusoids. These processes may anchor the endothelial cells to the hepatic plates.     

Prerequisite for the induction of lymphokine-activated killer cells from T lymphocytes

Human blood mononuclear cells were separated into Leu-11+7-NK, Leu-11-7+, and Leu-11-7-T cells by means of a combination of the Percoll gradient method and C-mediated cytolysis using mAb. When purified Leu-11+7-NK, Leu-11-7+, and Leu-11-7-T cells were cultured with rIL 2 (500 U/ml) for 6 days in a medium supplemented with 10% FCS, Leu-11+7-NK cells responded at the maximum level and Leu-11-7+ cells responded moderately as shown by both cell-proliferation response and cytotoxic activity generated. On the other hand, Leu-11-7-T cells did not respond at all to rIL-2. However, when Leu-11-7-T cells were cultured with rIL-2 in a medium supplemented with 10% autologous serum, they showed considerable responsiveness to rIL-2. In addition, much greater response to Leu-11-7-T cells were produced by the addition of monocytes. Monocyte cytokines, neither IL 1, IFN-gamma, TNF, nor their combination were able to substitute for monocytes in the induction culture. In contrast, the response level of Leu-11+7- NK cells remained unchanged irrespective of supplementation with autologous serum to medium or the addition of monocytes to the culture. These results indicated that culture conditions in the experiments significantly affected the results as to determination of lymphokine-activated killer cell precursors, especially the result pertaining to the conversion of T lymphocytes to lymphokine-activated killer cells. Under appropriate conditions, not only NK cells but also T cells are important precursors of lymphokine-activated killer cells.     

Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae.

The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene.     

Flowering responses to light-breaks in photomorphogenic mutants of Arabidopsis thaliana, a long-day plant

Flowering response and plant form of photomorphogenic mutants (hy1, hy2, hy3, hy4 and hy5) of Arabidopsis thaliana (L.), a long-day plant, were examined in long and short days. There were only slight differences among genotypes including Landsberg wild type with respect to the flowering time under long days. The effect of 1 h light-(night)-breaks of far-red, red, blue and white light given in the middle of the dark period of plants grown under short days, was studied. Effects of far-red light applied at the end or the beginning of the main photoperiod on flowering and plant form were also examined. The light-breaks with all the above mentioned light qualities promoted floral initiation of all the genotypes including the wild type in terms of both the flowering time and the number of rosette leaves. In general, far-red light was most effective. It is possible to classify the hy-mutants into 3 groups by their responses to light-breaks under short day conditions: (a) Mutants hy2 and hy3, which have a reduced number of rosette leaves, and flower early. Red light is as effective as far-red light. The wavelength of light-breaks is relatively unimportant for flowering response. (b) Mutants hy4, hy5 and Landsberg wild type, which have a greater number of rosette leaves, and flower relatively late. The effectiveness of light-breaks is in the following order, far-red, blue, and red light, which is in reverse order to the transformation of phytochrome to the P_fr form. (c) Mutant hy1, which behaves anomalously with respect to relations between flowering time and number of rosette leaves; late flowering with reduced number of rosette leaves. Red, blue and far-red light are effective, but white light is ineffective for reducing the number of rosette leaves. When far-red light was given in the middle of the night or at the end of the main photoperiod, it markedly reduced the number of rosette leaves compared to those grown under short days for all the genotypes, while when applied at the beginning of the main photoperiod far-red light did not affect the number of rosette leaves. Different effects on the plant form dependent on the time of treatment with far-red light-breaks are also discussed.