Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

The fate of spargana inoculated into the cat brain and sequential changes of anti-sparganum IgG antibody levels in the cerebrospinal fluid

To establish an animal model of intracranial sparganosis, the fate and behavior of the experimentally inoculated spargana were observed. A total of 102 scolices of spargana were injected into 22 cat brains, and the cats were sacrificed at 2 weeks, 1 month, 3 months and 6 months after the inoculation. Neurosparganosis was established in 77% of the cats. Of 43 recovered worms, 19 (44%) were located in the subdural or subarachnoid space, 16 (37%) in the brain parenchyme, and 2 (5%) in the lateral ventricle. One was detected at the diploic space of the skull and 5 were outside the cranial cavity. All but one were alive, and had grown tails. They were distributed in the brain parenchyme randomly. There was no place which they could not invade. No adult was found in the intestine. Cerebrospinal fluid (CSF) was collected before inoculation, 1 week, 2 weeks, 1 month, 3 months and 6 months after inoculation. The level of anti-sparganum IgG antibody in CSF measured by ELISA began to increase above the criteria of positivity 1 month after inoculation. Three months after inoculation, the values markedly increased. The present findings reveal that intracranial inoculation of spargana into the brains of cats would be a good animal model of experimental neurosparganosis.     

The changes of histopathology and serum anti-sparganum IgG in experimental sparganosis of mice

The present study is intended to observe the chronologic changes of experimental sparganosis by histopathological observation and detection of circulating anti-sparganum IgG antibody using ELISA. Each of 25 mice was infected with five spargana, and they were examined after 1, 2, 4, 10 weeks or 6 months from infection. The followings are summarized results. 1. The plerocercoids were detected in the subcutaneous tissue of the trunk, neck or axilla, but a few often extended into the skeletal muscle. The recovery rates were 72% at the first week, 80% at the second week, 95% at the fourth week, 92% at the tenth week and 100% at the sixth month. The larvae grew slowly in both length and weight until 6 months. 2. Histopathologically, most of the larvae were observed alive in the soft tissue or skeletal muscle. Numerous eosinophils, neutrophils, lymphocytes and plasma cells were infiltrated focally around the worms by the second week, but they surrounded the worms to form a layer of inflammatory reaction after 4 weeks of infection. Also histiocytes and fibroblasts began to appear around the inflammatory cells at 4 weeks. After 10 weeks, the worms encircled by a thin fibrous layer were found. After 6 months, the worms were surrounded by either fibrous tissue or active inflammatory cells. The inflammation looked more severe in the tracks left by the worms, rather than around the worms. 3. The level of anti-sparganum IgG antibody in the serum showed an increase by the fourth week, and a rapid and continuous increase was observed thereafter by the tenth week after infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of rebamipide on gastric xanthine oxidase activity and type conversion in ethanol-treated rats

Rebamipide, a novel antipeptic ulcer drug, 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinone-4-yl]-propionic acid, was studied for its inhibitory effect on gastric xanthine oxidase activity and type conversion of the enzyme that has a profound role in free radical generation. Intraperitoneal administration of rebamipide at 60 mg/kg body weight reduced gastric mucosal hemorrhagic lesions and lipid peroxidation, which was proportional to the inhibitory effect of rebamipide on alcohol-induced xanthine oxidase-type conversion and enzyme activity. It was also observed that the activity of xanthine oxidase was significantly inhibited by administration of rebamipide at 60 mg/kg body weight, leading to a significant reduction of lipid peroxide content in alcohol-treated rats. The results suggest that alcohol-induced gastric mucosal lesions might be, in part, due to the increased activity of xanthine oxidase and type conversion rate of the enzyme and the protective effect of rebamipide on gastric mucosal lesions would result from its ability to protect against oxidative stress on gastric mucosal lesions of alcohol-treated rats.     

Production of poly-β-hydroxybutyrate by Alcaligenes eutrophus transformants harbouring cloned phbCAB genes

Summary Three transformants of Alcaligenes eutrophus harbouring the recombinant plasmids containing phbCAB, phbAB, and phbC genes, were cultivated to investigate the effect of cloned genes on cell growth and poly--hydroxybutyrate accumulation. Both in the nutrient-rich and minimal media, the increased PHB accumulation in the transformants was observed compared to the parent strain, and this was the result of the increased enzyme activities in the transformants. Low carbon concentration and high C/N molar ratio favored higher PHB accumulations in the transformants. The transformant harbouring the phbC gene showed the highest PHB accumulation, which indicated that PHB synthase was the most critical enzyme for PHB biosynthesis in the transformant.     

Ginkgolide B production in cultured cells derived from Ginkgo biloba L. leaves

Callus cultures and cell suspension cultures derived from Ginkgo biloba L. leaves produced ginkgolidc B. In cell suspension cultures, the production reached a maximum by the 13th day of subculture and followed by a sharp decrease. The medium of Murashige and Skoog induced the highest ginkgolide B content in cultures while the medium of Schenk and Hildebrandt promoted cell growth. For the maximal production of ginkgolide B, cells were cultured in Murashige and Skoog medium modified to contain 1.0 mg/l of -naphthaleneacetic acid, 0.1 mg/1 of kinetin, 30 g/1 sucrose and 1.25 mM potassium phosphate with a molar ratio of ammonium to nitrate ions of 1 3.Abbreviations B5 Gamborg et al (1968) medium - GKB Ginkgolide B - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic aicd - SH Schenk and Hildebrandt (1972) medium     

High frequency transformation of Alcaligenes eutrophus producing poly-β-hydroxybutyric acid by electroporation

Summary A strain of Alcaligenes eutrophus producing poly--hydroxybutyric acid was successfully transformed by the electroporation. The plasmid used was a broad host range plasmid pKT230 conferring kanamycin resistance. The optimum yield of transformant was 0.8×10²/g DNA when 50 l competent cells at 10¹⁰/ml were pulsed by 11.5 kV/cm for 5 ms with 1 g DNA. Plasmid DNA in the A. eutrophus transformant was stably maintained as a monomeric structure.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

A metal ion-binding site in the kringle region of bovine prothrombin fragment 1.

45Ca(II) binding studies (equilibrium dialysis) on the kringle domain of bovine prothrombin fragment 1 were conducted using a mixture of peptides (residues 43-156 and 46-156) resulting from limited alpha-chymotryptic hydrolysis of fragment 1. Analysis of the Scatchard plot of these data indicates a single, low affinity Ca(II)-binding site to be present. Similar results were obtained from studies on the decarboxylated fragment 1 derivative, 10-gamma-MGlu-fragment 1. Acetylation of bovine fragment 1 in the absence of Ca(II) or Mg(II) ions results in the loss of the metal ion-promoted quenching of the intrinsic Trp fluorescence of the protein and the Ca(II)-mediated binding to phosphatidylserine/phosphatidylcholine (PS/PC) vesicles. The acetylation of the NH2 alpha-group of Ala-1 has been shown (Welsch, D. J., and Nelsestuen, G. L. (1988) Biochemistry 27, 4946-4952) to abolish the PS/PC binding property of fragment 1. The present study demonstrates that acetylation of a second site possibly Ser-79 or Thr-81 using the conditions described in the preceding paper results in loss of both the fluorescence transition and the Ca(II)-mediated PS/PC binding of the resulting protein derivative. Removal of the O-acetyl group at the Ser-79/Thr-81 site is accomplished by aminolysis with 0.2 M hydroxylamine, pH 10, 50 degrees C; the fluorescence transition is partially restored. PS/PC binding is partially restored if the NH2 alpha-group of Ala-1 is trinitrophenylated but is not restored if the NH2 alpha-group of Ala-1 is acetylated. We conclude that the Ser-79/Thr-81 site may represent a portion of the metal ion-binding site within the kringle domain of fragment 1. Occupancy of this site by a Ca(II) ion appears to be important in the binding of the protein to PS/PC vesicles.