A putative GDP–GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc-5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc-5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc-5 interacts genetically with mig-2, which encodes Rho GTPase. These results suggest that EXC-5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.     

Introduction of amino acid residues at the N-terminus of the zeocin-resistance protein increases its expression in Saccharomyces cerevisiae

Nucleotide sequences proximal to the initiation codon of a gene are known to affect the expression efficiency of that gene. We screened 10-bp random sequences upstream of the initiation codon of the zeocin-resistance gene to identify sequences that could enhance its expression in Saccharomyces cerevisiae. Of the isolated sequences, 20 sequences exhibited a common feature, i.e. ATG at the position −9 through −7, which resulted in the incorporation of three amino acids at the N-terminus of the protein. The introduction of these sequences upstream of the initiation codon increased the expression levels of zeocin-resistance protein by 2.2–6.5-fold. One of these sequences increased the expression levels of three out of four human proteins, thereby suggesting that this sequence may also enhance the expression efficiency of mammalian proteins in yeast.     

Identification of human ST2 protein in the sera of patients with autoimmune diseases.

Soluble human ST2 protein (IL1RL1-a) in the sera of patients with various autoimmune diseases was identified by a newly developed procedure using specific monoclonal antibodies. After immunoprecipitation and subsequent immunoblotting, a glycosylated protein of about 60 kDa was detected in the sera of SLE patients, but not in the sera of healthy controls. The experiments using gel filtration and SDS-PAGE under a nonreducing condition indicated the existence of the ST2 multimer in serum. The mobility of the natural protein was slower than that of the recombinant human ST2 protein produced by COS7 cells in SDS-PAGE, suggesting a difference of glycosylation between humans and monkeys. The identification of the natural human ST2 protein should be important both to fundamental researches and the further clarification of the clinical implications of the ST2 protein.     

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.     

Two phosphorylation reactions induced by murine beta interferon in BALB/c-3T3 cells.

Treatment with 1000 units/ml of murine beta-interferon enhanced an adenylate kinaselike activity and markedly increased the level of L-alpha-phosphatidyl inositol 4-monophosphate in quiescent BALB/c-3T3 cells. The addition of platelet-derived growth factor (22 units/ml) or poly(I).poly(C) (0.3-1 microgram/ml) to the phosphorylation reaction mixture did not alter this interferon action.     

N-type Calcium Channel in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca²⁺ channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca²⁺ channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca²⁺ channel α_1B-deficient (α_1B^−/−) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35–55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α_1B^−/− mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α_1B^−/− mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α_1B^−/− mice and was significantly inhibited by a selective N-type Ca²⁺ channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca²⁺. These results suggest that the N-type Ca²⁺ channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.     

Supra-annual variation in the influence of Myrica rubra fruit on the behavior of a troop of Japanese macaques in Yakushima

Observations were made on a well-habituated natural troop of Japanese macaques (Macaca fuscata yakui), living in warm-temperate, lowland forest in Yakushima. Between mid-May and the end of June the macaques feed on the fruit of the evergreen tree Myrica rubra (Myricaceae). The fruit of this species are abundant in some years and scarce in others. Data were compared for two heavy-fruiting years (1988 and 1990) and one poor-fruiting year (1991) to examine the influence of fruit availability on patterns of foraging, ranging, and the frequency of inter-troop encounters. In both heavy-fruiting years M. rubra fruit accounted for a maximum of over 70% of foraging time, compared with a maximum of <5% in the poor-fruiting year. Heavy fruiting was also associated with a marked decrease in the overall time spent foraging. In early May of all three years troop movements were largely confined to northern parts of the home range. By early June of both heavy-fruiting years ranging had shifted to the south-west, and included an area with a high concentration of M. rubra trees. This area was rarely visited at other times, and was not visited during the study period in the poor-fruiting year. The overlap in range-use between the two heavy-fruiting years was significantly greater than that between the heavy-fruiting years and the poor-fruiting year. Heavy fruiting was also associated with an increase in the frequency of inter-troop encounters. © 1995 Wiley-Liss, Inc.     

Transgenic Expression of the Formin Protein Fhod3 Selectively in the Embryonic Heart: Role of Actin-Binding Activity of Fhod3 and Its Sarcomeric Localization during Myofibrillogenesis

Fhod3 is a cardiac member of the formin family proteins that play pivotal roles in actin filament assembly in various cellular contexts. The targeted deletion of mouse Fhod3 gene leads to defects in cardiogenesis, particularly during myofibrillogenesis, followed by lethality at embryonic day (E) 11.5. However, it remains largely unknown how Fhod3 functions during myofibrillogenesis. In this study, to assess the mechanism whereby Fhod3 regulates myofibrillogenesis during embryonic cardiogenesis, we generated transgenic mice expressing Fhod3 selectively in embryonic cardiomyocytes under the control of the β-myosin heavy chain (MHC) promoter. Mice expressing wild-type Fhod3 in embryonic cardiomyocytes survive to adulthood and are fertile, whereas those expressing Fhod3 (I1127A) defective in binding to actin die by E11.5 with cardiac defects. This cardiac phenotype of the Fhod3 mutant embryos is almost identical to that observed in Fhod3 null embryos, suggesting that the actin-binding activity of Fhod3 is crucial for embryonic cardiogenesis. On the other hand, the β-MHC promoter-driven expression of wild-type Fhod3 sufficiently rescues cardiac defects of Fhod3-null embryos, indicating that the Fhod3 protein expressed in a transgenic manner can function properly to achieve myofibril maturation in embryonic cardiomyocytes. Using the transgenic mice, we further examined detailed localization of Fhod3 during myofibrillogenesis in situ and found that Fhod3 localizes to the specific central region of nascent sarcomeres prior to massive rearrangement of actin filaments and remains there throughout myofibrillogenesis. Taken together, the present findings suggest that, during embryonic cardiogenesis, Fhod3 functions as the essential reorganizer of actin filaments at the central region of maturating sarcomeres via the actin-binding activity of the FH2 domain.     

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using H NMR

¹H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events folowing exocytosis, activate glycolysis.