Structural modifications of oxidized insulin B-chain induced by glycerol addition

H-NMR studies of the bovine insulin S-sulfonatedB-chain are reported in H₂O/D₂O (9/1) and inglycerol-d₅ (5 M) using two-dimensional NMRspectroscopy. The first results show that the oxidizedinsulin B-chain secondary structure differs from thatof native insulin by a loss of the -helixbetween the two disulfide bridges and that theglycerol favours the structuring of the peptide.     

Organogenesis in Taxus brevifolia tissue cultures

Summary A method is described for multiple shoot and plantlet formation from zygotic embryos of Taxus brevifolia. Adventitious bud primordia were best induced by culturing zygotic embryos on 1/2B5 medium supplemented with 10 M BA for 14 days. Further vegetative buds were produced following subculture to half-strength McCown's basal salt medium containing 1.0% activated charcoal. Individual adventitious shoots were excised and approximately 5% of these formed roots. Rooting frequency was increased to 58% by a single treatment with ABT rooting powder. Vigorous growing Taxus brevifolia plants were established after transfer to plant growth medium.     

Interleukin-6 involvement in mesothelioma pathobiology: inhibition by interferon α immunotherapy

A role for interleukin-6 (IL-6) in malignant mesothelioma has been suggested by the clinically presenting symptoms of mesothelioma patients, which include fever, weight loss and thrombocytosis. A murine model of malignant mesothelioma was therefore used to examine the potential role of IL-6 in this cancer type and whether the effect of interferon (IFN) therapy on mesothelioma might be mediated, in part, by regulating IL-6 levels and/or IL-6-induced pathobiology. A panel of human and murine mesothelioma cell lines was assayed for endogenous IL-6 production in a bioassay, and for IL-6-mRNA expression. Four out of 5 human and 5 out of 15 murine mesothelioma cell lines produced moderate to high levels of bioactive IL-6 in vitro. This result was corroborated by mRNA detection. One of the representative murine cell lines, AB22, was chosen for further in vivo studies in the murine mesothelioma model. In AB22-inoculated mice detectable serum IL-6 levels were found to precede macroscopically detectable tumour growth, clinical signs (cachexia, abdominal distension, diarrhoea) and changes in the peripheral lymphoid organs (cell depletion and functional depression). Treatment with anti-IL-6 antibody curtailed the clinical symptoms (P<0.001), as did treatment with recombinant human (rhu) IFN (P<0.001). Neither anti-IL-6 antibody nor rhuIFN had a direct growth-inhibitory effect on the AB22 mesothelioma cell line in vitro, however, in vivo rhuIFN treatment of mice inoculated with AB22 cells attenuated both IL-6 mRNA expression in the tumours and serum IL-6 levels, ameliorated the depression of lymphocyte activities, and enhanced the number of tumour-infiltrating lymphocytes and macrophages. On the basis of these results it is suggested that IL-6 mediates some of these effects, directly or indirectly, and that a combination therapy of rhuIFN and anti-IL-6 antibody may be an improved palliative treatment for patients with malignant mesothelioma.     

Purification and some properties of β-mannanase from Bacillus sp.

-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH₄)₂ SO₄ precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg²⁺. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan     

Electron paramagnetic resonance studies of membrane proteins in hepatic microsomes

Hepatic microsomal membranes, prepared under various conditions that yield either ‘intact’ or ‘disrupted’ microsomal vesicles, have been labeled via the sulfhydryl groups of intrinsic membrane proteins using nitroxide analogs of $\text{N-}\text{ethylmaleimide}$. Electron paramagnetic resonance spectra revealed the presence of two dominant classes of bound label corresponding to differing degrees of immobilization, the ratio of which were quantitated using a parameter designated the ‘$\text{W/S}$’ ratio. For latent microsomes, the value of this parameter was determined to be $\text{0.65 ± 0.02}$ and was influenced by factors such as label/protein ratio, incubation period, nitroxide structure, temperature and pH. The $\text{W/S}$ ratio was also sensitive to the degree of membrane integrity as revealed by the latency of mannose 6-phosphate activity of glucose-6-phosphohydrolase. In addition, membrane disruption resulted in a corresponding decrease in the order parameter for nitroxide-labeled fatty acids intercalated within the lipid bilayer. The $\text{W/S}$ ratio was observed to be dependent upon the method of microsome preparation yielding values of $\text{1.02 ± 0.02}$ for ‘hypertonically disrupted’ vesicles and $\text{1.28 ± 0.02}$ for ‘mechanically disrupted’ vesicles. Microsomal marker enzymes such as cytochrome $\text{P-450}$ and FAD-containing monooxygenase retained significant levels of functionally following nitroxide incorporation.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Electron transfer by b- and c-type cytochromes in relation to the origin of the ‘slow’ electric potential component

Cytochrome redox changes and electric potential generation are kinetically compared during cyclic electron transfer in Photosystem-I-enriched and Photosystem-II-depleted subchloroplast vesicles (i.e., stroma lamellae membrane vesicles) supplemented with ferredoxin using a suitable electron donating system. In response to a single-turnover flash, the sequence of events is: (1) fast reduction of cytochrome b-563 (t_0.5 ≈ 0.5 ms) (2) oxidation of cytochrome c-554 (t_0.5 ≈ 2 ms), (3) slower reduction of cytochrome b-563 (t_0.5 ≈ 4 ms), (4) generation of the ‘slow’ electric potential component (t_0.5 ≈ 15–20 ms), (5) re-reduction of cytochrome c-554 (t_0.5 ≈ 30 ms) and (6) reoxidation of cytochrome b-563t_0.5 ≈ 90 ms). Per flash two cytochrome b-563 species turn over for one cytochrome c-554. These b-563 cytochromes are reduced with different kinetics via different pathways. The fast reductive pathway proceeds probably via ferredoxin, is insensitive to DNP-INT, DBMIB and HQNO and is independent on the dark redox state of the electron transfer chain. In contrast, the slow reductive pathway is sensitive to DNP-INT and DBMIB, is strongly delayed at suboptimal redox poising (i.e., low $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratio) and is possibly coupled to the reduction of cytochrome c-554. Each reductive pathway seems obligatory for the generation of about 50% of the slow electric potential component. Also cytochrome c-559_LP (LP, low potential) is involved in Photosystem-I-associated cyclic electron flow, but its flash-induced turnover is only observed at low preestablished electron pressure on the electron-transfer chain. Data suggest that cyclic electron flow around Photosystem I only proceeds if cytochrome b-559_LP is in the reduced state before the flash, and a tentative model is presented for electron transfer through the cyclic system.     

Pyrophosphate functions as phosphoryl donor with UDP-glucose-treated mammalian phosphofructokinase

CGP 28392, a novel compound structurally related to the dihydropyridine Ca2+-entry blockers, causes a dose-dependent increase in intracellular free Ca2+ in human platelets, as measured with the Quin-2 Ca2+ indicator, with a semimaximal effective concentration of 2.2 X 10(-7) M. This effect occurs in a concentration range in which CGP 28392 competes for specific [3H]nitrendipine binding in guinea pig heart membranes. It can be inhibited by nitrendipine. The data presented furnish direct evidence of the Ca2+-entry-stimulating properties of CGP 28392 and indicate the presence of dihydropyridine-susceptible structures in human platelets.     

Uptake, release and metabolism of docosahexaenoic acid (DHA, c22:6 omega 3) in human platelets and neutrophils

Exogenous DHA is converted by human platelets to 14- and 11- HDHE and by human neutrophils mainly to 7- HDHE . Human platelets prelabeled with 14C-DHA, 14C-EPA and 14C-AA and stimulated with thrombin release and metabolize DHA only in trace amounts as compared to EPA and AA. 14C-DHA is incorporated into the 2-position of platelet phospholipids and occurs predominantly in phosphatidylethanolamine. DHA and EPA were also incorporated by dietary means into phospholipids of platelets and neutrophils. In resting platelets free DHA as well as free AA and EPA are not detectable. In platelets stimulated ex vivo with thrombin DHA is not significantly released which is in contrast to EPA and AA. After stimulation, 14- HDHE is found only in trace amounts as compared to 12-HETE and 12- HEPE . In DHA enriched neutrophils formation of HDHEs cannot be demonstrated after stimulation with ionophore A 23187. We conclude that even after dietary enrichment of DHA in phospholipids of platelets and neutrophils the level of free DHA and/or formation of HDHEs might be too low to substantially affect arachidonic acid metabolism and related functions of these cells.