Cystine Deprivation Induces Oligodendroglial Death: Rescue by Free Radical Scavengers and by a Diffusible Glial Factor

Abstract: In this study we examined the effect on oligodendroglial survival of exogenous cystine deprivation. Oligodendroglia isolated from mixed glial primary cultures derived from brains of 1-day-old rats, and then grown for 3 days, were markedly dependent on extracellular cystine for survival. The EC₅₀ values for cystine for a 24-h exposure ranged from 2 to 65 µ M . After 6 h of cystine deprivation, the cellular glutathione level decreased to 21 ± 13% of the control. Free radical scavengers (α-tocopherol, ascorbate, idebenone, and N-tert -butyl-α-phenylnitrone) were protective against cystine deprivation but had no effect on the glutathione level. An iron chelator, desferrioxamine mesylate, also was protective. These findings suggest that intracellular hydroxyl radicals are important for this toxicity. In contrast to the observations in 3-day-old cultures, the dependence on exogenous cystine for cell viability was not observed consistently in oligodendroglia cultured for 6 days before the onset of cystine deprivation. Several observations suggested that this loss of cystine dependence was due to a diffusible factor. Sensitivity to the toxicity of cystine deprivation in day 6 cultures increased as the volume of medium was increased from 0.3 to 2 ml. Furthermore, preincubation of cystine-depleted medium with astrocyte cultures eliminated the toxicity of the cystine deprivation. HPLC assay of the conditioned cystine-depleted medium showed no significant change in cystine or cysteine concentration. We conclude that oligodendroglia are highly susceptible to cystine deprivation in day 3 cultures and that this susceptibility is due to the accumulation of intracellular free radicals in the setting of glutathione depletion. The resistance of day 6 oligodendroglial cultures is caused at least in part by a diffusible factor.     

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Characterization of translucent segments observed in an smbA mutant of Escherichia coli

Abstract The smbA gene of Escherichia coli is essential for cell proliferation. The smbA2 mutant shows cold-sensitive colony formation at 22°C. A novel morphological phenotype, formation of a translucent segment at midcell or at a cell pole, was observed by phase-contrastt microscopy at a high frequency in the smbA2 mutant cells incubated in L medium lacking NaCl at 22°C, but not observed in L medium containing 1% NaCl or 20% sucrose at the same temperature. No translucent segment was observed in the wild-type cells in any of the media used. Electron microscopic observation revealed that the translucent segments resulted from the enlargement of a periplasmic space by separation of the inner membrane from the peptidoglycan layer and the outer membrane.     

Distribution among tissues and intracellular localization of cofilin, a 21kDa actin-binding protein

Cofilin, a 21kDa actin-binding protein, binds to F-actin in a 1:1 molar ratio of cofilin to actin molecule (Nishida, E., S. Maekawa, and H. Sakai, Biochemistry, 23, 5307-5313, 1984) and is capable of controlling actin polymerization and depolymerization in vitro in a pH-sensitive manner (Yonezawa, N., E. Nishida, and H. Sakai, J. Biol. Chem., 260, 14410-14412, 1985). In this study, immunoblot analysis using monospecific antibodies against cofilin showed that cofilin is ubiquitously distributed in a variety of bovine and rat organs and tissues. Cofilin is also present in various cultured cell lines. Indirect immunofluorescence staining of mouse fibroblastic cells and human epidermoid carcinoma cells indicated that cofilin is distributed nearly uniformly in the cytoplasm and is concentrated in ruffling membranes where F-actin is also concentrated as revealed by staining with rhodamine-phalloin. Stress fiber structures were not strongly stained with the anti-cofilin antibody, although stress fiber staining was sometimes observed near the cell periphery in mouse 3T3 cells. These results suggest that the bulk of cofilin may not be associated with F-actin bundles in vivo.     

Decreased intracellular free magnesium in erythrocytes of spontaneously hypertensive rats

Using 31p-NMR (the phosphorus nuclear magnetic resonance) spectroscopy, we measured intracellular free Mg levels in the erythrocytes of untreated (n = 7) and diltiazem-treated spontaneously hypertensive rats (SHR) (n = 8), and compared them with age-matched Wistar-Kyoto rats (WKY) (n = 10). The intracellular free Mg levels were significantly (p less than 0.01) decreased in untreated SHR compared with those in control WKY. A successful antihypertensive treatment with diltiazem increased the intracellular free Mg levels compared with untreated SHR (p less than 0.05). Furthermore, an inverse correlation was observed between intracellular free Mg levels and blood pressure levels in all groups (r = -0.48, p less than 0.01, n = 25). These observations suggest that abnormalities of intracellular Mg metabolism may be, in part, related to the development or the maintenance of hypertension in SHR.     

Spacing and kinship in the Formosan squirrel living in different habitats

Summary Spacing and kinship of the Formosan squirrel, Callosciurus erythraeus thaiwanensis, were studied in two different habitats. One, native habitat in the woods of Kenting, southern Formosa, was rich in available food throughout the year and had several species of predators. The other, a site in Kamakura, central Japan where squirrels had been introduced, had relatively scanty food and few potential predators. 1. Home ranges among males and between sexes overlapped extensively in both habitats. 2. Females occupied exclusive home ranges in Kamakura but had small overlapping home ranges in Ken-ting. 3. Most males disappeared from their natal areas at 1 year old in both habitats (86% in Kamakura and 93% in Ken-ting), but less females disappeared (36% in Kamakura and 35% in Ken-ting). 4. In Kamakura, daughters settled adjacent to the mother or inherited the home range of the mother, but never shared the mother's home range. In Ken-ting, 35% of daughters shared the home range with their mothers. 5. Tolerance among female kin in Ken-ting was probably facilitated by the richness of available food throughout the year, and functioned to reduce predation risk via alarm calling and mobbing.     

Orientation of Wall Microfibrils in Avena Coleoptiles and Mesocotyls and in Pisum Epicotyls

Microfibrils (MFs) on the inner surface of the walls of Avenacoleoptile and mesocotyl cells and of Pisum epicotyl cells wereexamined by a replica method. In the elongating epidermis ofthese three organs, cells having MFs that were transverse, obliqueor longitudinal to the elongation axis were intermingled. Inthe elongating parenchymal tissues, all cells deposited MFstransversely. In non-elongating cells of Avena coleoptiles andPisum epicotyls, the orientation of MFs on the inner wall surfaceof both epidermal and parenchymal cells was more longitudinalthan in elongating cells. These observations on the orientationsof MFs are compatible with those our previously reported observationson the orientations of microtubules (MT) (Iwata and Hogetsu1988). Disruption of MTs of Avena coleoptiles by treatment withamiprophosmethyl caused changes in the orientation of depositionof MFs. These results support the idea that MFs are usuallyco-aligned with MTs in organ cells and that the orientationof MFs is controlled by MTs. The averaged direction of MFs, visualized under polarized light,showed a clear difference between the epidermal and inner-tissuecell walls in the elongating regions of the three organs. Inalmost all elongating and non-elongating epidermal cells, theaveraged direction of MFs was longitudinal, while it was transversein all inner-tissue cells. (Received December 16, 1988; Accepted April 28, 1989)     

Identification of the cell surface molecule involved in sexual cell fusion of Dictyostelium discoideum

Dictyostelium discoideum was used as a model system for elucidating the molecular mechanism of sexual cell fusion. In heterothallic strains NC4 and HM1 of D. discoideum, complements in mating type, amoeboid cells acquire fusion competence only under certain environmental conditions, such as the presence of excess water and a certain period of darkness, to fuse sexually. The surface of cells which acquired fusion competence was found to possess specific antigens. Monovalent antibodies prepared from rabbit antiserum against fusion-competent NC4 cells inhibit the sexual cell fusion of these cells completely. Two specific antigenic proteins, 39 and 138 k Da in relative molecular mass and specific for fusion-competent cells, were detected. Only one, the 138-k Da protein, was capable of neutralizing the fusion-inhibitory activity of the monovalent antibody. These results show that the 139-k Da protein is the one involved in the sexual cell fusion of NC4 and HM1 strains in D. discoideum.     

Species-specific TNF induction of thymocyte proliferation

Summary Recombinant murine (rMu) tumor necrosis factor (TNF), in a standard comitogenic assay with phytohemagglutinin, induced murine thymocyte proliferation, while up to 10,000-fold higher concentrations of recombinant human TNF did not. The induction of thymocyte proliferation was dependent upon TNF concentration in a biphasic manner. Thus, 100 to 1000 units/ml TNF were near optimal while concentrations 1,000 units/ml caused apparent down regulation. The effect was abrogated by neutralizing antibody to rMu-TNF but not by neutralizing antibody to rMu-interleukin 1 or . The rMu-TNF did not induce proliferation of the mature murine T-helper cell line, D10.G4.1, in the presence of mitogen. Taken together the results indicate that TNF, in a strictly species-specific manner, can regulate thymocyte proliferation independently of interleukin 1.Supported in part by Asahi Chemical Industry Co., Inc. and by USPHS Grants CA-24538, CA-15142 and CA-09072 awarded by the National Cancer Institute, Department of Health and Human Services     

Cloning and characterization of porcine brain cofilin cDNA. Cofilin contains the nuclear transport signal sequence

Cofilin is a widely distributed, pH-sensitive, actin-modulating protein with an apparent molecular mass of 21 kDa, which forms intranuclear and/or cytoplasmic actin/cofilin rods in cultured fibroblastic cells under specific conditions. In this study, a cDNA library from porcine brain mRNA was constructed, and full-length brain cofilin cDNA clones were isolated by screening with oligonucleotide probes. The deduced amino acid sequence of cofilin is 166 residues long and contains a sequence of Lys-Lys-Arg-Lys-Lys which is very similar to the nuclear transport signal sequence (Pro-Lys-Lys-Lys-Arg-Lys-Val) of SV40 large T antigen. The sequence may act as a signal capable of inducing nuclear accumulation of cofilin in cells exposed to heat shock or dimethyl sulfoxide. The cofilin sequence contains a hexapeptide (Asp-Ala-Ile-Lys-Lys-Lys) identical to the amino-terminal sequence (residues 2-7) of muscle and nonmuscle tropomyosin. Cofilin also has in the carboxyl-terminal portion a region homologous to the sequence shared by gelsolin, fragmin, and Acanthamoeba profilin. Furthermore, the overall amino acid sequence of cofilin shows weak homology with the rod portion of myosin and suggests a high alpha-helical content.