Partial purification from human mononuclear cells and placental plasma membranes of an insulin mediator which stimulates pyruvate dehydrogenase and suppresses glucose-6-phosphatase

A substance capable of stimulating pyruvate dehydrogenase (PDH) and suppressing glucose-6-phosphatase (G-6-Pase) in a cell-free system was prepared from insulin-treated human placental plasma membranes and peripheral blood mononuclear cells by formic acid extraction. This material was partially purified by molecular-exclusion chromatography, ion-exchange chromatography, and hydroxylapatite chromatography. This was found to stimulate pyruvate dehydrogenase and inhibit glucose-6-phosphatase in a dose-dependent manner. The amount or ability of this substance to stimulate pyruvate dehydrogenase was increased in the proportion to the concentration of insulin. The stimulation of pyruvate dehydrogenase by the factor was eliminated when sodium fluoride was presented in the assay of the activation. This result implied that the activation of pyruvate dehydrogenase was mediated by the stimulation of the phosphatase of pyruvate dehydrogenase complex. Each material isolated from insulin-treated human placental plasma membranes and mononuclear cells shared a number of important characteristics of putative second messengers of insulin action as follows: (i) heat and acid stability; (ii) a similar molecular weight; (iii) increased activity of pyruvate dehydrogenase in a insulin-dependent manner; and (iv) stimulated pyruvate dehydrogenase by the sodium fluoride-sensitive mechanism. This human putative second messenger of insulin action was eluted from the anion-exchange resin AG1-X8 at an ionic strength of 3–4 m, as well as from the hydroxylapatite column at a phosphate concentration of 2–3 m.     

Effects of deep seawater on the growth of several species of marine micro-algae

Deep seawater (DSW; seawater under the euphotic layer), obtained from Deep Seawater Laboratory in Muroto, Japan, was applied to the culture ofDunaliella tertiolecta, D. salina, Nannochloropsis oculata, N. salina, Porphyridium cruentum, Tetraselmis tetrahele, andChaetoceros ceratosporum. DSW supported the exponential growth of every species. The growth yields were at 14.7 (±2.3 SD) mg dry weight per liter, and could be heightened by the addition of nitrate to DSW.Author for correspondence     

Extracellular proteolytic activity ofCryptococcus neoformans

Eight strains ofCryptococcus neoformans var.neoformans isolated from AIDS patients in the Infectious Disease Institute, University of Turin, Italy, were examined for growth and extracellular proteolytic activity in culture with solid and liquid media. All of the strains grew well on Yeast Carbon Base (YCB) agar medium supplemented with both 0.1% (w/v) bovine serum albumin (BSA) and 0.01% (w/v) polypeptone (Pp), and produced a clear proteolytic zone around their colonies, whereas they exhibited less growth and proteolytic activity on YCB medium supplemented with BSA alone. Strain #8 with a strong proteolytic activity was cultured in three different liquid media. Its growth was limited in YCB medium supplemented with 0.1% BSA, but was moderate in that with 0.01% Pp. Enhanced growth was supported by the addition of both BSA and Pp to the YCB medium. The relative value of the final cellular yields obtained with the above YCB-0.1% BSA, YCB-0.01% Pp and YCB-0.1% BSA-0.01% Pp media was approximately 1:10:20. In the culture with YCB medium containing both BSA and Pp, a rapid decrease in the amount of BSA was demonstrated by a spectrophotometric assay and gel electrophoresis of the culture supernatant after the log-to-stationary phase. The proteolytic activity in the culture supernatant became detectable after the log phase when tested with skim milk agarose plates. These results allowed us to conclude thatCr. neoformans var.neoformans is able to secrete protease and to utilize protein as a source of nitrogen.     

Lysis of endothelial cells by autologous lymphokine-activated killer cells

The mechanisms of lysis of endothelial cells derived from human umbilical vein (HUVEC) by autologous lymphokine-activated killer (LAK) cells, generated from cord blood lymphocytes of the same donor, were investigated. Freshly isolated HUVEC as well as HUVEC cultured for several passages were efficiently lysed by autologous LAK cells, and their susceptibility to the LAK cells was almost the some as that of allogenic HUVEC. Complement-depletion experiments revealed that the lysis was mainly dependent on CD16-natural killer (NK) LAK cells. Pretreatment of HUVEC with recombinant interferon (rIFN) for 24 h made them resistant to lysis by autologous LAK cells, while pretreatment with either rIL-1. rTNF, or acidic or basic fibroblast growth factor did not alter the lytic sensitivity of HUVEC. The resistance of rIFN-treated HUVEC was specific to lysis by CD16⁺ NK LAK cells, and their lysis by CD3⁺ T-LAK cells was not significantly altered. Moreover, in comparison with control HUVEC or rIL-1-treated HUVEC, rIFN-treated HUVEC had a significantly less potent inhibitory effect on the lysis of untreated HUVEC, when used as an unlabeled target. This suggests that rIFN treatment may down-regulate the recognition of some molecules on HUVEC by rIL-2-activated NK cells. These data suggest that damage of the endothelium during LAK therapy is mainly dependent on LAK cells with a NK phenotype that can specifically recognize a certain molecule on autologous endothelial cells.     

Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells

Endothelial cells (ECs) release ATP in response to shear stress, a mechanical force generated by blood flow, and the ATP released modulates EC functions through activation of purinoceptors. The molecular mechanism of the shear stress-induced ATP release, however, has not been fully elucidated. In this study, we have demonstrated that cell surface ATP synthase is involved in shear stress-induced ATP release. Immunofluorescence staining of human pulmonary arterial ECs (HPAECs) showed that cell surface ATP synthase is distributed in lipid rafts and co-localized with caveolin-1, a marker protein of caveolae. Immunoprecipitation indicated that cell surface ATP synthase and caveolin-1 are physically associated. Measurement of the extracellular metabolism of [(3)H]ADP confirmed that cell surface ATP synthase is active in ATP generation. When exposed to shear stress, HPAECs released ATP in a dose-dependent manner, and the ATP release was markedly suppressed by the membrane-impermeable ATP synthase inhibitors angiostatin and piceatannol and by an anti-ATP synthase antibody. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) disrupted lipid rafts and abolished co-localization of ATP synthase with caveolin-1, which resulted in a marked reduction in shear stress-induced ATP release. Pretreatment of the cells with cholesterol prevented these effects of MbetaCD. Downregulation of caveolin-1 expression by transfection of caveolin-1 siRNA also markedly suppressed ATP-releasing responses to shear stress. Neither MbetaCD, MbetaCD plus cholesterol, nor caveolin-1 siRNA had any effect on the amount of cell surface ATP synthase. These results suggest that the localization and targeting of ATP synthase to caveolae/lipid rafts is critical for shear stress-induced ATP release by HPAECs.     

Anaerobic biodegradation of polychlorinated biphenyls by a microbial consortium originated from uncontaminated paddy soil

Polychlorinated biphenyls (PCBs) in Kanechlor-300 and -400 mixtures dissipated significantly compared with a sterilized control under anaerobic conditions in three Japanese paddy soils with no history of PCB contamination, demonstrating the anaerobic microbial degradation of PCBs. The PCB-degrading activity was maintained successfully in a static flooded soil medium for more than 3 years by serial transfer at intervals of 56 days (13 transfers). Ortho-, meta-, and para-substituted PCBs, 15.2 ± 9.9 mol% in total, were significantly degraded after 56 days of incubation. Analysis of menaquinones-6 and -7 and cloning of 16S rRNA gene fragments from a polymerase chain reaction denaturing gradient gel electrophoresis (DGGE) profile indicated the predominance of Firmicutes in the consortium. A PCR-based identification of the gene fragments showed the frequent presence of Desulfitobacterium sp., but not Dehalobacter sp. or Dehalococcoides sp., in the consortium. It is proposed that Japanese paddy soils with no history of PCB contamination contain an anaerobic microbial consortium consisting predominantly of Firmicutes that have the potential for anaerobic degradation of PCB.     

Asymmetrical Polyhedral Configuration of Giant Vesicles Induced by Orderly Array of Encapsulated Colloidal Particles

Giant vesicles (GVs) encapsulating colloidal particles by a specific volume fraction show a characteristic configuration under a hypertonic condition. Several flat faces were formed in GV membrane with orderly array of inner particles. GV shape changed from the spherical to the asymmetrical polyhedral configuration. This shape deformation was derived by entropic interaction between inner particles and GV membrane. Because a part of inner particles became to form an ordered phase in the region neighboring the GV membrane, free volume for the other part of particles increased. Giant vesicles encapsulating colloidal particles were useful for the model of “crowding effect” which is the entropic interaction in the cell.     

Studies on the Utilization of Hydrocarbons by Microorganisms

The production of microbial cell substances from hydrocarbons has become the object of commercial attention. We have tested hydrocarbon-utilizing bacterial strains for cell production, and found out one strain of high cell yield, which is very similar to Pseudomonas aeruginosa. The effect of medium composition on cell yield and the utilization of individual hydrocarbons by this strain were investigated. Sixty percent of the added kerosene was converted into cell materials in the following medium of optimum composition: kerosene 2.5%, urea 0.13%, dipotassium phosphate 0.25%, magnesium sulfate 7 aq. 0.1% and tap water. Aliphatic series lower than C₁₀, aromatic and naphthenic hydrocarbons were not or very slightly assimilated. However, aliphatic members higher than C₁₂ were utilized with increasing case. Especially, n-docosane and octadecene-1 were utilized very effectively, and 70% of them were converted into cell substances.     

Eremophilanolides and other constituents from the Argentine liverwort Frullania brasiliensis.

Two eremophilanolides, 5-epidilatanolides A and B, as well as a new natural bibenzyl were isolated from an Argentine collection of the liverwort Frullania brasiliensis, along with the known eudesmane-type sesquiterpene lactones nepalensolide A, nepalensolide B, (+)-frullanolide, and (+)-dihydrofrullanolide, the hopanoid zeorin, the four sterols stigmasta-4,22-dien-3,6-dione, stigmasta-4,22-dien-3-one, stigmasterol, and sitosterol, and a trace amount of atraric acid. The structure and stereochemistry of the eremophilanolides and the bibenzyl were established by a combination of extensive NMR spectroscopy experiments and X-ray crystallographic analysis. Absolute configurations of the new compounds were derived on the basis of CD spectra.