Hydrogen evolution by co-immobilized Chlorella vulgaris and Clostridium butyricum cells

Whole cells of Chlorella vulgaris and Clostridium butyricum were co-immobilized in 2% agar gel. NADP was suitable as an electron carrier. The rate of hydrogen evolution increased with increasing NADP concentration. The optimum conditions for hydrogen evolution were pH 7.0 and 37°C. The immobilized C. vulgaris-NADP-immobilized Cl. butyricum system continuously evolved hydrogen at a rate of 0.29–1.34 μmol/h per mg Chl for 6 days. On the other hand, the system without NADP evolved only a trace amount of hydrogen.     

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.     

Chimeric peptides as a vehicle for peptide pharmaceutical delivery through the blood-brain barrier

A new strategy for peptide delivery through the brain capillary wall, i.e., the blood-brain barrier (BBB), is the synthesis of chimeric peptides which are formed by the covalent coupling of a non-transportable peptide (e.g., beta-endorphin) to a transportable peptide that undergoes receptor- or absorptive-mediated transcytosis at the BBB. beta-endorphin was covalently coupled via disulfide linkage to cationized albumin (pI greater than or equal to 9) which, owing to it's highly basic charge, undergoes rapid absorptive-mediated transport into brain from blood. The [3H]labeled beta-endorphin-cationized albumin chimera was rapidly taken up by isolated brain capillaries in vitro and by rat brain in vivo; conversely, the BBB uptake of native [3H]beta-endorphin was negligible. The synthesis of chimeric peptides is a new strategy for solving the problem of peptide delivery through the BBB.     

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.     

Cellular and Vacuolar Fusion of Protoplasts Electrofused Using Platinum Microelectrodes

Protoplasts isolated from cultured cells of Coptis japonicaand Euphorbia millii were electrically fused using platinummicroelectrodes. The process involved two stages, cellular andvacuolar fusion, which are characterized respectively by transientwrinkling of the membrane and the formation of a dark-red precipitate. (Received June 12, 1987; Accepted October 13, 1987)     

Effects of Physical Parameters upon Protoplast Electrofusion

The effects of three physical parameters upon protoplast electrofusionwere studied using protoplasts from cultured cells of Coptisjaponica and Euphorbia millii. The osmotic potential of themedium did not appreciably affect the AC-field-induced protoplast-pairformation, but significantly influenced the fusion process ofthe paired protoplasts in response to DC pulses. The optimumosmotic potential was 0.55 to 0.60 Osm/kg H₂O in our system.The density of the medium markedly influenced both pair formationand fusion process. The optimum density was 1.13 to 1.14 g/cm₃,and at this density the yield of the fused protoplasts increasedto more than twice that of the control (1.10 g/cm₃). Hydrophiliccoating of the bottom surface of the chamber with Gellan gumor polyacrylamide gel was also effective for both pair formationand the fusion process, while coating with hydrophobic siliconewas entirely inhibitory. Possible interpretations of the effectsof these physical parameters upon protoplast electrofusion arepresented. ¹Permanent address: Biochemical Research Laboratories, KanegafuchiChemical Industry Co., Ltd., Takasago, Hyogo 676, Japan. (Received December 21, 1987; Accepted March 18, 1988)     

Levels of IAA, Cytokinins, ABA and Ethylene in Rice Plants as Affected by a Gibberellin Biosynthesis Inhibitor, Uniconazole-P.

Effects of uniconazole-P, a triazole-type growth retardant,on endogenous levels of IAA, cytokinins, ABA and ethylene inrice seedlings were investigated. Endogenous levels of IAA andABA were similar between control and uniconazole-P-treated riceshoots. Evolution of ethylene was promoted slightly, being 1.8times greater under 0.3 ppm uniconazole-P treatment than thatof control. The most obvious effect was the increase of trans-Zand trans-RZ in shoots. Shoots treated with uniconazole-P (10mg/m² nursery box) contained 3.4 times and 3 times more trans-Zand trans-RZ than control, respectively. No significant differencesof cytokinin levels were recognized in roots except for cis-RZ.The increase of ethylene and active forms of cytokinins, andthe decrease of gibberellin in the shoots may be the basis forphysiological phenomena caused by uniconazole-P, namely thepromotion of flowering in woody plants and the enhancement offemaleness in cucumber. (Received September 9, 1987; Accepted October 20, 1987)