Effect of oleic acid on mitochondrial oxidative phosphorylation in rat brain slices

We tested the effect of oleic acid on oxidative phosphorylation and free fatty acid composition in rat brain slices simultaneously to investigate the relationship between the change in respiratory control ratio and the uptake of oleic acid in the brain mitochondria. The uncoupling of mitochondria was observed when the ratio of oleic acid to stearic acid in the free fatty acid fraction was nearly doubled, but was not recovered even by the addition of fatty acid-free bovine serum albumin. The data suggest that the intactness of oxidative phosphorylation of brain mitochondria is maintained by the precise control of the free fatty acid composition in the mitochondrial membranes.     

Effect of oleic acid on mitochondrial cytochrome c oxidase activity

Both Km and Vmax values of cytochrome c oxidase for cytochrome c were elevated in oleic acid-incorporated mitochondria, whereas the amount of oleic acid incorporated into submitochondrial particles was smaller than that into mitochondria and the fatty acid had little effect on the enzyme activity. The degree of change in the bulk membrane fluidity was, however, almost the same in mitochondria and submitochondrial particles. Solubilized cytochrome c oxidase was insensitive to the effect of oleic acid. Oleic acid may act as a modifier of the interaction between cytochrome c oxidase and membrane lipids.     

Uptake of Gibberellin Methyl Esters by Spores and Induction of Dark Germination in Lygodium japonicum

In the fern Lygodium japonicum, the effect of the exogenousapplication of two gibberellin methyl esters, gibberellin A₄methyl ester (GA₄Me) and gibberellin A₂₀ methyl ester (GA₂₀Me)on spore germination in the dark and uptake of GA₄Me and GA₂₀Meby spores was investigated. Tritiated GA₄Me and GA₂₀Me wereprepared and used as radioactive tracers. The activity of GA₄Mewas more than 100-fold that of GA₂₀Me for the induction of sporegermination. When treated for 24 h, the activity for inducingspore germination remained after removal of the gibberellinmethyl esters from the medium. The amount of GA₄Me taken upby spores was more than three times that of GA₂₀Me throughoutthe 24 h time course of treatment. The uptake of both gibberellinmethyl esters was proportional to the external concentrationfor the range of concentrations between 10^–9 M and 10^–6M. When treated with the tritiated gibberellin methyl estersat 10^–6 M and 10^–7 M for 24 h, most of the gibberellinmethyl esters taken up by the spores were not metabolized. Althoughthe uptake of the two gibberellin methyl esters differed by3- to 5- fold, their abilities to induce spore germination differedby more than 100-fold. Therefore, the difference in the activityof the two gibberellin methyl esters regarding the inductionof spore germination could not be explained solely by the differencein their uptake. (Received January 11, 1988; Accepted May 26, 1988)     

The membrane-associated ATPase from Sulfolobus acidocaldarius is distantly related to F1-ATPase as assessed from the primary structure of its alpha-subunit

Isolation of novel membrane-associated ATPases, presumably soluble parts of the H+-ATPases, from archaebacteria has been recently reported, and their properties were found to be significantly different from the usual F1-ATPase. In order to assess the relationship of the archaebacterial ATPases to the F1-ATPases and other known ATPases, the amino acid sequence of the alpha subunit of the ATPase from Sulfolobus acidocaldarius, an acidothermophilic archaebacterium, was compared with the sequences of other ATPases. The gene encoding its alpha subunit was cloned from the genomic library of S. acidocaldarius, and the nucleotide sequence was determined. The 591-amino acid sequence deduced from the nucleotide sequence contains a small number of short stretches that shows sequence similarity to the alpha and beta subunits of F1-ATPase. However, the overall similarity is too weak to consider it to be a typical member of the F1-ATPase family when the highly conserved sequences of the F1-ATPase subunits among various organisms are taken into account. Moreover, most of these stretches overlap the consensus sequences that are commonly found in some nucleotide-binding proteins. There is no significant sequence similarity to the ion-translocating ATPases, which form phosphorylated intermediates, such as animal Na+,K+-ATPases. Thus, the S. acidocaldarius ATPase and probably other archaebacterial ATPases also appear to belong to a new group of ion-translocating ATPases that has only a distant relationship to F1-ATPase.     

Induction and characterization of minor histocompatibility antigens. Specific primary cytotoxic T lymphocyte responses in vitro

A definite cytotoxic activity was developed in a BALB/c (H-2d) anti-DBA/2 primary mixed leukocyte culture (MLC), which received interleukin 2 (IL-2) on day 3 of culture. This cytotoxic activity was minor histocompatibility antigens (MIHA)-specific at the stimulator level, and was not developed in a syngeneic (BALB/c anti-BALB/c) MLC. The addition of IL-2 on day 3 of culture was crucial; no or very weak cytotoxic activity was developed in MLC receiving IL-2 on day 0 or on both day 0 and day 3. Only appropriate MIHA-allogeneic tumor cells were lysed as the target of the cytotoxic activity. The cytotoxic activity seemed MIHA-specific also at the target level; it lysed tumor cells of DBA/2 mouse origin but not those of BALB/c (syngeneic) origin. Phenotypes of the cytotoxic effector cell were Thy-1+ Lyt-2+. We concluded from these results that MIHA-specific cytotoxic T lymphocytes (CTL) were generated in the MIHA-allogeneic primary MLC. In this newly developed system, we studied genetic and antigenic requirements for primary anti-MIHA CTL responses in vitro. We demonstrated; among spleen cells (SC) of seven B10 H-2-congenic strains only SC of B10.D2 strain whose major histocompatibility complex (MHC) (H-2d) was compatible with the responder MHC effectively stimulated responder BALB/c (H-2d) SC for an anti-MIHA (DBA-C57BL-common) CTL response. Similarly, only SC of two out of seven C x B recombinant inbred strains (C x B.H and C x B.D), which were compatible at the MHC with responder SC, activated responder BALB/c SC for the response. The possibility that cells responding to H-2 alloantigens suppressed the anti-MIHA response was ruled out. Additional experiments showed that compatibility at the H-2K-end or the H-2D-end of the MHC was sufficient for a definite anti-MIHA response. These provided formal evidence that primary anti-MIHA CTL responses in vitro were MHC-restricted at the stimulator level. We then showed that sonication-disrupted SC or Sephadex G-10 column-passed nonadherent SC failed to stimulate responder SC for a primary anti-MIHA CTL response, whereas G-10-passed nonadherent SC responded well to adherent stimulator cells. Further study demonstrated that Ia+ adherent cells were the most active cell type as stimulator. Finally, we confirmed that the primary anti-MIHA CTL responses to adherent stimulator cells was MHC-restricted.     

Studies on hst, a transforming gene which belongs to a new superfamily of growth factors

The hst gene was originally identified in surgically obtained human gastric mucosae as a transforming gene which could transform NIH3T3 cells morphologically. The hst cDNA clone was synthesized from mRNA of one of the NIH3T3 transformants. A human leukocyte genomic library was screened with this cDNA clone, and an hst genomic fragment was obtained. This genomic fragment itself had transforming activity, and the protein coding sequences were proved to be completely identical to those of the cDNA clone prepared from mRNA of the NIH3T3 transformant. This fact suggests that rearrangement or other structural alterations in the coding sequence are not required for the activation of the hst gene. The predicted hst protein consists of 206 amino acids and has a significant homology (40-50%) to fibroblast growth factors and int-2 protein. They together make up a new superfamily of growth factors and transforming genes.     

Host-mediated antiviral activity of Lactobacillus casei against cytomegalovirus infection in mice

The protective effect of heat-killed Lactobacillus casei (LC) against murine cytomegalovirus (MCMV) infection was examined. ICR mice treated once with LC 1 day or 2 days before challenge survived lethal infection, but untreated or Lactobacillus fermentum (LF)-treated mice did not. The protective effect was evidenced by an increase in plaque-forming units (PFU) per 50% lethal dose (LD50) and a decrease in titers of infectious viruses replicated in the target organs. This was further confirmed by severity of histopathological damage to the target organs, especially the liver. LC neither inactivated MCMV nor inhibited its replication in mouse embryonic fibroblasts (MEF). The spleen cells from LC-treated mice inhibited its replication in MEF on co-cultivation. Augmentation by LC of splenic natural killer (NK) cell activity correlated with survival of mice from otherwise lethal MCMV infection. Cytotoxic activity of peritoneal cells and level of serum interferon (IFN) were elevated after MCMV infection, but they were not associated with survival of mice nor with treatment of LC. The protective effect of LC was not clear in NK-deficient beige mutant (bgJ/bgJ) mice, when compared with that in their littermate (bgJ/+) mice. Poor protection of bgJ/bgJ mice by LC treatment correlated with failure to induce NK cell activity by LC treatment in the mutant mice. Thus, it is likely that LC protects mice from MCMV infection by augmentation of NK cell activity.     

Growth and turnover of microbial biomass during the decomposition of organic matter(Polygonum cuspidatum) in vitro

Air-dried fresh and dead specimens ofPolygonum cuspidatum were incubated for 250 days in the laboratory, and the growth and turnover of microbial biomass-C in the organic matter were studied. The biomass-C in the fresh leaf and fresh stem attained maximum levels on day 14 and day 7, respectively, and then settled down to stable levels. In the dead leaf and dead stem, increase in biomass-C ceased by day 4 and the biomass-C levels did not change thereafter. The turnover time of the biomass-C was estimated from the amount of biomass-C and the release rate of CO₂-C. The turnover was rapid in the early period of incubation. Then the turnover time became longer and after incubation for 70 days the values approached those in natural soils (longer than 16 days). During the incubation period, nitrogen was not mineralized in any organic matter. In the dead leaf and dead stem, asymbiotic nitrogen fixation activity increased after incubation for about 40 days and disappeared by the end of the incubation period, whereas nitrogen fixation was hardly detected in the fresh leaf and fresh stem.     

Secondary release of thromboxane A2 in aerosol leukotriene C4-induced bronchoconstriction in guinea pigs

Effect of a thromboxane synthetase inhibitor (OKY-046) on bronchoconstriction induced by aerosol leukotriene C4 and histamine was studied in anesthetized, artificially ventilated guinea pigs in order to examine whether secondary release of thromboxane A2 is produced by aerosol leukotriene C4 or not. 0.01–1.0μg/ml of leukotriene C4 and 12.5–400μg/ml of histamine inhaled from ultrasonic nebulizer developed for small animals caused dose-dependent increase of pressure at airway opening (Pao) which is considered to be an index representing bronchial response. Pretreatment of the animals with intravenous OKY-046 (100mg/kg) significantly reduced the airway responses produced by inhalation of 0.1, 0.33 and 1.0μg/ml of leukotriene C4, while the pretreatment did not affect the histamine dose-response curve. Based on these findings and previous reports (6, 7), it is suggested that aerosol leukotriene C4 activates arachidonate cyclooxygenase pathway including thromboxane A2 synthesis and the released cyclooxygenase products have bronchodilating effect as a whole     

Distribution and development of the serotonin-and RFamide-like immunoreactive neurons in the arrowworm,Paraspadella gotoi (Chaetognatha)

Summary The distribution and development of serotonin-and RFamide-like immunoreactivities in the nervous system of Chaetognatha, Paraspadella gotoi, were examined in whole-mount preparations. In adults, a single serotonin-like immunoreactive (5HTLI) neuron and numerous RFamide-like immunoreactive (RFaLI) neurons were found in the central nervous system. Based on the structure of the fins, hooks, and eyes, seven postembryonic developmental stages were recognized. The most obvious features of the stages are: stage 1, newly hatched young; stage 2, elongation of a continuous lateral tail fin; stage 3, separation of the lateral and tail fins; stage 4, appearance of hooks; stage 5, pigmentation of eyes, stage 6, attachment by tail adhesive fins; stage 7, prey capture. Stage 1 did not show any immunoreactivity. The 5HTLI neuron first appeared at stage 4 and its axonal pathway became similar to the adult at stage 6. On the other hand, the RFaLI neurons appeared at stage 3 in the ventral ganglion. Some of their somata disappeared at stage 5 and the neuronal architecture resembled the adult at stage 7 although the RFaLI neurons in the cerebral ganglion were complete at the juvenile stage.We are sad to announce that Dr. M. Yoshida died on 29 October 1988