An endogenous inhibitor of the ADP-ribosylation of GTP-binding proteins by pertussis toxin is present in bovine brain

The ADP-ribosylation of GTP-binding proteins (G-proteins) catalyzed by pertussis toxin was inhibited by endogenous inhibitor activity in the membrane extract of bovine brain. Most of the activity appeared in the fractions eluted from a DEAE-Sephacel column by 0.5 M NaCl. The activity was heat-stable and sensitive to pronase K. The results suggest the presence of an endogenous inhibitor of pertussis toxin in bovine brain.     

Fructose 2,6-bisphosphate-dependent regulation of phosphofructokinase in rat submandibular gland

1. Regulation of phosphofructokinase in rat submandibular gland was non-Michaelis-Menten type at physiological pH. 2. At pH 7.3, ATP played a dual role on phosphofructokinase acting as a substrate and inhibitor at high concentration of ATP. 3. The activator of phosphofructokinase was present in cytosol fraction, and its properties were resemble to those of fructose 2,6-bisphosphate. 4. Both the activator and authentic fructose 2,6-bisphosphate relieved the inhibition of phosphofructokinase by ATP, and increased the affinity for fructose 6-phosphate. 5. Concentration of fructose 2,6-bisphosphate in rat submandibular gland was 8.22 nmol/g tissue, and which was about the half of that in liver. 6. Phosphofructokinase in rat submandibular gland was found to be regulated synergistically by ATP, fructose 6-phosphate and fructose 2,6-bisphosphate.     

Complex gangliosides as cell surface inhibitors for the ecto-NAD+ glycohydrolase of CD38

Leukocyte cell surface antigen CD38 is a single-transmembrane protein whose extracellular domain has catalytic activity for NAD(+) glycohydrolase (NADase). We previously reported that b-series gangliosides inhibit the NADase activity of the extracellular domain of CD38 expressed as a fusion protein [Hara-Yokoyama, M., Kukimoto, I., Nishina, H., Kontani, K., Hirabayashi, Y., Irie, F., Sugiya, H., Furuyama, S., and Katada, T. (1996) J. Biol. Chem. 271, 12951-12955]. In the present study, we examined the effect of exogenous gangliosides on the NADase activity of CD38 on the surface of retinoic acid-treated human leukemic HL60 cells and CD38-transfected THP-1 cells. After incubation of the cells with G(T1b), inhibition of NADase activity was observed. The time course of inhibition was slower than that of the incorporation of G(T1b) into the cells, suggesting that incorporation into the cell membranes is a prerequisite for inhibition. Inhibition occurred efficiently when G(T1b) and CD38 were present on the same cells (cis interaction) rather than on different cells (trans interaction). Although gangliosides may affect localization of cell surface proteins, indirect immunofluorescence intensity due to CD38 was not affected after G(T1b) treatment. Comparison of the effect of G(T1b) and G(D1a) indicates that the tandem sialic acid residues linked to the internal galactose residue of the gangliotetraose core are crucial to the inhibition. These results suggest a novel role of complex gangliosides for the first time as cell surface inhibitors of CD38 through specific and cis interaction between the oligosaccharide moiety and the extracellular domain.     

Hepatocyte growth factor ameliorates dermal sclerosis in the tight-skin mouse model of scleroderma

The tight-skin (TSK/+) mouse, a genetic model of systemic sclerosis (SSc), develops cutaneous fibrosis and defects in pulmonary architecture. Because hepatocyte growth factor (HGF) is an important mitogen and morphogen that contributes to the repair process after tissue injury, we investigated the role of HGF in cutaneous fibrosis and pulmonary architecture defects in SSc using TSK/+ mice. TSK/+ mice were injected in the gluteal muscle with either hemagglutinating virus of Japan (HVJ) liposomes containing 8 mug of a human HGF expression vector (HGF-HVJ liposomes) or a mock vector (untreated control). Gene transfer was repeated once weekly for 8 weeks. The effects of HGF gene transfection on the histopathology and expression of tumor growth factor (TGF)-beta and IL-4 mRNA in TSK/+ mice were examined. The effect of recombinant HGF on IL-4 production by TSK/+ CD4+ T cells stimulated by allogeneic dendritic cells (DCs) in vitro was also examined. Histologic analysis revealed that HGF gene transfection in TSK/+ mice resulted in a marked reduction of hypodermal thickness, including the subcutaneous connective tissue layer. The hypodermal thickness of HGF-treated TSK/+ mice was decreased two-fold to three-fold compared with untreated TSK/+ mice. However, TSK/+ associated defects in pulmonary architecture were unaffected by HGF gene transfection. HGF gene transfection significantly inhibited the expression of IL-4 and TGF-beta1 mRNA in the spleen and skin but not in the lung. We also performed a mixed lymphocyte culture and examined the effect of recombinant HGF on the generation of IL-4. Recombinant HGF significantly inhibited IL-4 production in TSK/+ CD4+ T cells stimulated by allogeneic DCs. HGF gene transfection inhibited IL-4 and TGF-beta mRNA expression, which has been postulated to have a major role in fibrinogenesis and reduced hypodermal thickness, including the subcutaneous connective tissue layer of TSK/+ mice. HGF might represent a novel strategy for the treatment of SSc.     

Neurotrophic effect of Semaphorin 4D in PC12 cells

Semaphorins provide crucial attractive and repulsive cues involved in axon guidance during neural development. Out of them, Semaphorin 4D (Sema4D) is enriched in the nervous and immune tissues, and acts as proliferative and survival factors of peripheral lymphocytes in the immune system, but is poorly understood in the nervous system. By using PC12 cells which are well known to differentiate into neural cells in response to nerve growth factor (NGF), we found that soluble forms of Sema4D had neurotrophic effects which were inhibited by neutralizing antibodies to Sema4D. Sema4D strikingly potentiated neurite outgrowth in the presence of 50 ng/ml NGF and increased sensitivity to NGF. Cells responded to very low concentrations of NGF in the presence of 1 nM Sema4D. Activation of following signal proteins, protein kinase C (PKC), L-type of voltage-dependent Ca(2+) channel, and phosphatidylinositol (PI) 3-kinase mediated neurotrophic neurite-outgrowth action of Sema4D. These findings suggest a new function of Sema4D as a neurotrophic signal in PC12 cells.     

Linkage mapping of the locus responsible for congenital multiple ocular defects in cattle on bovine Chromosome 18

Congenital multiple ocular defects (MOD) in Japanese black cattle is a hereditary ocular disorder with an autosomal recessive manner of inheritance, showing developmental defects of the lens, retina, and iris, persistent embryonic eye vascularization, and microphthalmia. In the present study, we mapped the locus responsible for the disorder by linkage analysis using 240 microsatellite markers covering the entire bovine genome and an inbred pedigree obtained from commercial herds. The linkage analysis demonstrated a significant linkage between the disorder locus and markers on the proximal region of bovine Chromosome (BTA) 18 with the maximum LOD score of 5.1. Homozygosity mapping using the haplotype of the linked markers further refined the critical region. The results revealed the localization of the locus responsible for MOD in an approximately 6.6-cM region of BTA18. Comparison of published linkage and radiation hybrid (RH) maps of BTA18 with its evolutionary ortholog, human Chromosome (HSA) 16, revealed several potential candidate genes for the disorder including the MAF and FOXC2 genes.     

Hepatocyte growth factor inhibits the formation of the basement membrane of alveolar epithelial cells in vitro

Hepatocyte growth factor (HGF) is a pulmotrophic factor for the regeneration of injured pulmonary tissue. We investigated the role of HGF in basement membrane formation during wound healing by immortalized alveolar type II epithelial cells that could form a continuous basement membrane when they were cultured on collagen fibrils in the presence of entactin-contaminated laminin-1. Cells cultured with 5.0 ng/ml HGF neither formed a continuous basement membrane on collagen fibrils nor maintained a continuous basement membrane architecture on a basement membrane substratum. The cells showed increased secretion of matrix metalloproteinase-9 and urokinase-type plasminogen activator, and the HGF-induced inhibition of basement membrane formation was attenuated by addition of 200 ng/ml tissue inhibitor of matrix metalloproteinase-1. Cells sequentially exposed to HGF and 1.0 ng/ml transforming growth factor-beta1 had enhanced basement membrane formation compared with those receiving these reagents in the reverse order or concurrently. HGF simultaneously stimulated proliferation and migration of the cells so that it advanced wound closure on the basement membrane substratum. The present results indicate that the role of HGF in wound healing is the stimulation of reepithelization, but this factor may also contribute to the degradation of the basement membrane.     

Phosphoinositide 3-kinase in nitric oxide synthesis in macrophage: critical dimerization of inducible nitric-oxide synthase

Phosphoinositide 3-kinase (PI3K) has important functions in various biological systems, including immune response. Although the role of PI3K in signaling by antigen-specific receptors of the adaptive immune system has been extensively studied, less is known about the function of PI3K in innate immunity. In the present study, we demonstrate that macrophages deficient for PI3K (p85alpha regulatory subunit) are impaired in nitric oxide (NO) production upon lipopolysaccharide and interferon-gamma stimulation and thus vulnerable for intracellular bacterial infection such as Chlamydophila pneumoniae. Although expression of inducible nitric-oxide synthase (iNOS) is induced normally in PI3K-deficient macrophages, dimer formation of iNOS protein is significantly impaired. The amount of intracellular tetrahydrobiopterin, a critical stabilizing cofactor for iNOS dimerization, is decreased in the absence of PI3K. In addition, induction of GTP cyclohydrolase 1, a rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, is greatly reduced. Our current results demonstrate a critical role of class IA type PI3K in the bactericidal activity of macrophages by regulating their NO production through GTP cyclohydrolase 1 induction.