Very-long-chain fatty acid-containing phospholipids accumulate in fatty acid synthase temperature-sensitive mutant strains of the fission yeast Schizosaccharomyces pombe fas2/lsd1

Fission yeast lsd1 strains show aberrant mitosis with a lsd phenotype, large and small daughter nuclei, and a very thick septum, the phenotypic expression being temperature-sensitive. The lsd1(+) gene is the homologue of the budding yeast FAS2 gene encoding the fatty acid synthase alpha-subunit as reported previously (S. Saitoh, K. Takahashi, K. Nabeshima, Y. Yamashita, Y. Nakaseko, A. Hirata, M. Yanagida, J. Cell Biol. 134 (1996) 949--961). In this paper, lsd1 is considered to represent fas2. Here, three fas2 strains were investigated and found to have missense point mutations at different sites in the gene encoding the alpha-subunit of fatty acid synthase. The mutation affected only slightly the enzymatic activities monitored in vitro. Unexpectedly, abnormal phospholipids, phosphatidylcholine and phosphatidylethanolamine, both of which contain a very-long-chain fatty acyl residue (1-melissoyl-2-oleolyl-sn-glycero-3-phosphocholine and 1-melissoyl-2-oleolyl-sn-glycero-3-phosphoethanolamine), accumulated in fas2 strains in a temperature-sensitive manner. Rescue of the fas2 strains by addition of palmitate to the medium at restrictive temperature was accompanied by disappearance of these abnormal phospholipids. Accumulation of these lipids in membranes may cause alteration of various cellular functions.     

Lecithinized Cu, Zn-superoxide dismutase limits the infarct size following ischemia-reperfusion injury in rat hearts in vivo

Ghrelin is a novel gut-brain peptide that binds to the growth hormone secretagogue receptor (GHS-R), thereby functioning in the regulation of growth hormone (GH) release and food intake. Ghrelin-producing cells are most abundant in the oxyntic glands of the stomach. The regulatory mechanism that governs the biosynthesis and secretion of ghrelin has not been clarified. We report that ghrelin mRNA expression in the gastric fundus was increased, but that ghrelin peptide content decreased after a 48-h fast. Both values returned to control levels after refeeding. The ghrelin plasma concentration in the gastric vein and systemic venous blood increased after 24- and 48-h fasts. Furthermore, des-octanoylated ghrelin and n-octanoylated ghrelin were found in rat stomach, with the ratio of des-octanoylated ghrelin to n-octanoylated ghrelin markedly increased after fasting. The ghrelin mRNA level in the stomach also increased after administration of insulin and leptin. Conversely, db/db mice, which are deficient in the leptin receptor, had lower ghrelin mRNA levels than control mice. These findings suggest that this novel gastrointestinal hormone plays a role in the regulation of energy balance.     

CD24 induces apoptosis in human B cells via the glycolipid-enriched membrane domains/rafts-mediated signaling system

The glycosylphosphatidylinositol-anchored CD24 protein is a B cell differentiation Ag that is expressed on mature resting B cells but disappears upon Ag stimulation. We used Burkitt's lymphoma (BL) cells, which are thought to be related to germinal center B cells, to examine the biological effect of Ab-mediated CD24 cross-linking on human B cells and observed 1) induction of apoptosis in BL cells mediated by cross-linking of CD24; and 2) synergism between the cross-linking of CD24 and that of the B cell receptor for Ag in the effect on apoptosis induction. We also observed activation of mitogen-activated protein kinases following CD24 cross-linking, suggesting that CD24 mediates the intracellular signaling that leads to apoptosis in BL cells. Although CD24 has no cytoplasmic portion to transduce signals intracellularly, analysis of biochemically separated glycolipid-enriched membrane (GEM) fractions indicated enhanced association of CD24 and Lyn protein tyrosine kinase in GEM as well as increased Lyn kinase activity after CD24 cross-linking, suggesting that CD24 mediates intracellular signaling via a GEM-dependent mechanism. Specific microscopic cocapping of CD24 and Lyn, but not of other kinases, following CD24 cross-linking supported this idea. We further observed that apoptosis induction by cross-linking is a common feature shared by GEM-associated molecules expressed on BL cells, including GPI-anchored proteins and glycosphingolipids. CD24-mediated apoptosis in BL cells may provide a model for the cell death mechanism initiated by GEM-associated molecules, which is closely related to B cell receptor for Ag-mediated apoptosis.     

Accumulation of oxidative DNA damage, 8-oxo-2'-deoxyguanosine, and change of repair systems during in vitro cellular aging of cultured human skin fibroblasts

Effects of in vitro cellular aging on the content of 8-oxo-2'-deoxyguanosine, a typical oxidation product of DNA bases, were examined in cultured human skin fibroblasts. The 8-oxo-2'-deoxyguanosine content in the DNA of TIG-3S cells established from skin tissues of a fetal donor increased immediately before the cessation of proliferation. TIG-114 and TIG-104 cells established from skin tissues of adult and aged donors, respectively, showed similar changes in 8-oxo-2'-deoxyguanosine content during in vitro cellular aging. The accumulation of 8-oxo-2'-deoxyguanosine in late-passage cells was dependent on the number of cell divisions, and not on the cultivation time. Increases in the activities of superoxide dismutase and glutathione peroxidase were observed prior to the increase in 8-oxo-2'-deoxyguanosine content, while the catalase activity decreased gradually during in vitro cellular aging at late-passage. Furthermore, the activities of 8-oxo-2'-deoxyguanosine endonuclease and DNA polymerases decreased with the progression of proliferation. These results indicate that defense systems against oxidative stress in late-passage cells remain sufficiently active before the cessation of cell division, but that repair systems against oxidative damage decay at late-passage. Oxidative stress beyond the antioxidant capacity and/or repair activity seems to result in an accumulation of 8-oxo-2'-deoxyguanosine in late-passage cells.     

Patterning neuronal and glia cells on light-assisted functionalised photoresists

A common photosensitive polymeric material used in semiconductor microlithography (diazo-naphto-quinone/novolak resist) was pattern-exposed with near-UV light to create carboxylic-rich areas on the polymer surface. The patterned surfaces were further functionalised via: (1) the anchorage of peptides for specific cell-attachment or cell-detachment functions; or (2) the diffusion of silicon rich chemical species to achieve the cell detachment. Pairs of antagonistic surface characteristics controlled the cell attachment: (1) amino-rich or carboxylic-rich surfaces; and (2) hydrophilic or hydrophobic surfaces; in which the former promoted the adhesion. It was found that common microlithographic materials and techniques can be upgraded to allow an effective control of the lateral organisation of the artificial arrays of neuronal and glia cells.     

GroEL chaperone binding to beetle luciferases and the implications for refolding when co-expressed

The folding of many proteins including luciferase in vivo requires the assistance of molecular chaperone proteins. To understand how a chaperone targets luciferase, we took three luciferases that give different bioluminescence with the same luciferin substrate and with differences in homology. The three luciferase genes, firefly luciferase (FF-Luc) (from Pyrocoelia miyako), and red (RE-Luc) and green (GR-Luc) bioluminescence-emitting luciferases (from Phrixothrix railroad-worms), were expressed in Escherichia coli to produce fusion proteins with predicted molecular masses. Subsequently, we observed that DnaK and GroEL were co-purified along with recombinant luciferase. Although the amount of co-purified DnaK was almost the same compared to FF-Luc, GroEL was 25 and 32 times higher in GR-Luc and RE-Luc respectively. Furthermore, co-expression of GroEL/GroES along with luciferase substantially refolded RE-Luc and GR-Luc compared to FF-Luc.     

Pressure activates rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) play a central role in development of pancreatic fibrosis. In chronic pancreatitis, pancreatic tissue pressure is higher than that of the normal pancreas. We here evaluate the effects of pressure on the activation of rat PSCs. PSCs were isolated from the pancreas of Wistar rat using collagenase digestion and centrifugation with Nycodenz gradient. Pressure was applied to cultured rat PSCs by adding compressed helium gas into the pressure-loading apparatus to raise the internal pressure. Cell proliferation rate was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation. MAPK protein levels and alpha-smooth muscle actin (alpha-SMA) expression were evaluated by Western blot analysis. Concentration of activated transforming growth factor-beta1 (TGF-beta1) secreted from PSCs into culture medium was determined by ELISA. Collagen type I mRNA expression and collagen secretion were assessed by quantitative PCR and Sirius red dye binding assay, respectively. Application of pressure significantly increased BrdU incorporation and alpha-SMA expression. In addition, pressure rapidly increased the phosphorylation of p44/42 and p38 MAPK. Treatment of PSCs with an MEK inhibitor and p38 MAPK inhibitor suppressed pressure-induced cell proliferation and alpha-SMA expression, respectively. Moreover, pressure significantly promoted activated TGF-beta1 secretion, collagen type I mRNA expression, and collagen secretion. Our results demonstrate that pressure itself activates rat PSCs and suggest that increased pancreatic tissue pressure may accelerate the development of pancreatic fibrosis in chronic pancreatitis.