Skeletal FGFR1 signaling is necessary for regulation of serum phosphate level by FGF23 and normal life span

Fibroblast growth factor (FGF) 23 produced by the bone is the principal hormone to regulate serum phosphate level. Serum FGF23 needs to be tightly regulated to maintain serum phosphate in a narrow range. Thus, we hypothesized that the bone has some phosphate-sensing mechanism to regulate the production of FGF23. Previously we showed that extracellular phosphate induces the phosphorylation of FGF receptor 1 (FGFR1) and FGFR1 signaling regulates the expression of Galnt3, whose product works to increase FGF23 production in vitro. In this study, we show the significance of FGFR1 in the regulated FGF23 production and serum phosphate level in vivo. We generated late-osteoblast/osteocyte-specific Fgfr1-knockout mice (Fgfr1^fl/fl; Ocn^Cre/+) by crossing the Ocn-Cre and the floxed Fgfr1 mouse lines. We evaluated serum phosphate and FGF23 levels, the expression of Galnt3 in the bone, the body weight and life span. A selective ablation of Fgfr1 aborted the increase of serum active full-length FGF23 and the enhanced expression of Galnt3 in the bone by a high phosphate diet. These mice showed more pronounced hyperphosphatemia compared with control mice. In addition, these mice fed with a control diet showed body weight loss after 23 weeks of age and shorter life span. These results reveal a novel significance of FGFR1 signaling in the phosphate metabolism and normal life span.     

A MEK inhibitor, PD98059 enhances IL-1-induced NF-kappaB activation by the enhanced and sustained degradation of IkappaBalpha

Interleukin-1 (IL-1) mediates numerous host responses through rapid activation of nuclear factor-kappaB (NF-kappaB), but signal pathways leading to the NF-kappaB activation appear to be complicated and multiplex. We propose a novel regulatory system for NF-kappaB activation by the extracellular signal-related kinase (ERK) pathway. In a human glioblastoma cell line, T98G, IL-1-induced NF-kappaB activation was significantly augmented by the pretreatment of a specific MEK inhibitor, PD98059. In contrast, ectopic expression of a constitutive activated form of Raf (v-Raf) reduced IL-1-induced NF-kappaB activation, and this inhibition was completely reversed by PD98059. Interestingly, PD98059 sustained IL-1-induced NF-kappaB DNA binding activity by an electrophoretic mobility shift assay and also IkappaBalpha degradation, presumably by augmenting and sustaining the proteasome activation. Concomitantly, two NF-kappaB dependent genes, A20 and IkappaBalpha expression were prolonged with PD98059. These data suggested that MEK-ERK pathway exerts a regulatory effect on NF-kappaB activation, providing a novel insight on the role of MEK-ERK pathway.     

Reidentification of the keratinase-producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans

Alkaliphilic Bacillus sp. AH-101 was characterized in terms of physiological and biochemical characteristics, and 16S rDNA sequence homology and DNA–DNA hybridization analyses were performed. Phylogenetic analysis of strain AH-101 based on comparison of 16S rDNA sequences revealed that this strain is closely related to Bacillus halodurans. DNA–DNA hybridization of AH-101 and related Bacillus reference strains showed that the highest level of DNA–DNA relatedness (88%) was found between strain AH-101 and the B. halodurans type strain (DSM497). Our findings demonstrate that strain AH-101 is a member of the species B. halodurans. Received: June 10, 1999 / Accepted: August 6, 1999     

Sulfated sialic acid-polymers inhibit the cytotoxic action of bee and snake venom

Colominic acid is an 2,8-linked sialic acid polymer produced by Escherichia coli. We found that synthetic sulfated-colominic acids (SC) remarkably inhibited the cytotoxicity of bee and snake venom toward mouse fibroblast cells, but colominic acids showed no inhibition themselves, indicating the important role of sulfate groups in the inhibitory activity of SC. Other sulfated carbohydrates such as chondroitin sulfates, heparin and heparan sulfate showed no inhibition. SC also exhibited potent inhibition of melittin, a highly basic peptide, which is a major cytotoxic component of bee venom. SC did not inhibit phospholipase A₂ activity in bee venom. This suggests that the inhibition of bee and snake venom by SC is due to inhibition of melittin and cardiotoxin, which is a cytolytic peptide in snake venom, respectively. SC with a higher sulfur content and a larger molecular mass showed more potent activity. The interaction between SC and melittin basically seems an ionic one, however, the conformation of SC is also likely important. For the binding of SC to melittin leading loss of its cytotoxic activity, the sulfate groups of SC must be properly arranged to interact with lysine and arginine residues of melittin molecules, which play an important role in the cytolytic activity. A higher molecular mass of SC substituted with more sulfate groups is required for more obvious inhibition of the cytotoxic activity.     

PAM14, a novel MRG- and Rb-associated protein, is not required for development and T-cell function in mice

PAM14 has been found to associate in complexes with the MORF4/MRG family of proteins as well as Rb, the tumor suppressor protein. This suggested that it might be involved in cell growth, immortalization, and/or senescence. To elucidate the in vivo function of PAM14, we characterized the expression pattern of mouse Pam14 and generated PAM14-deficient (Pam14(-/-)) mice. Pam14 was widely expressed in all mouse tissues and as early as 7 days during embryonic development. Despite this ubiquitous expression in wild-type mice, Pam14(-/-) mice were healthy and fertile. Response to mitogenic stimulation and production of interleukin-2 were the same in stimulated splenic T cells from Pam14(-/-) mice as in control littermates. Cell growth rates of mouse embryonic fibroblasts (MEFs) from all three genotypes were the same, and immortalized cells were obtained from all cell cultures during continuous culture. There was also no difference in expression of growth-related genes in response to serum stimulation in the null versus control MEFs. These data demonstrate that PAM14 is not essential for normal mouse development and cell cycle control. PAM14 likely acts as an adaptor protein in nucleoprotein complexes and is probably compensated for by another functionally redundant protein(s).     

Regulatory molecules for coronary expressions of VEGF and its angiogenic receptor KDR in hypoestrogenic middle-aged female rats

We studied the effects of estrogen deprivation and replacement on the protein and gene expression levels molecules that can be considered to be essential for coronary angiogenesis in middle-aged female rats. The animals were subjected to sham operation, ovariectomy, or ovariectomy with estrogen replacement therapy (ERT). Following ovariectomy, protein and gene expressions of vascular endothelial growth factor (VEGF) and its angiogenic receptor (KDR) showed a marked decline in coronary vessels, as determined by immunohistochemistry and in situ hybridization. ERT resulted in restoration of the ovariectomy-induced changes to intact levels. The coronary expression level of basic fibroblast growth factor was unaffected by estrogen deprivation or treatment. The changes in VEGF and KDR expressions were strongly associated with those in endothelial nitric oxide synthase (eNOS) expression in coronary vessels. Moreover, the age- and gender-dependent accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) protein appeared to be a determinant molecule of VEGF expression in middle-aged female rats. We reached a conclusion that the VEGF-KDR system plays a key role in coronary angiogenesis in hypoestrogenic elderly women and is critically regulated by estrogen, eNOS and HIF-1alpha.     

Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from the novel marine isolate, JAMB-A94

A gene, agaA, for a novel beta-agarase from the marine bacterium JAMB-A94 was cloned and sequenced. The 16S rDNA of the isolate had the closest match, of only 94.8% homology, with that from Microbulbifer salipaludis JCM11542(T). The agaA gene encoded a protein with a calculated molecular mass of 48,203 Da. The deduced amino acid sequence showed 37-66% identity to those of known agarases in glycoside hydrolase family 16. A carbohydrate-binding module-like amino acid sequence was found in the C-terminal region. The recombinant enzyme was hyper-produced extracellularly when Bacillus subtilis was used as a host. The purified enzyme was an endo-type beta-agarase, yielding neoagarotetraose as the main final product. It was very thermostable up to 60 degrees C. The optimal pH and temperature for activity were around 7.0 and 55 degrees C respectively. The activity was not inhibited by EDTA (up to 100 mM) and sodium dodecyl sulfate (up to 30 mM).     

Effect of high-NaCl or high-KCl diet on hepatic Na+- and K+-receptor sensitivity and NKCC1 expression in rats

We previously reported that the bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter (NKCC1) is involved in the hepatic Na+ and K+ sensor mechanism. In the present study, we examined the effects of a high-NaCl or high-KCl diet on hepatic Na+ and K+ receptor sensitivity and NKCC1 expression in the liver of Sprague-Dawley rats. RT-PCR and Western blots were used to measure NKCC1 mRNA and protein expression, respectively. Infusion of hypertonic NaCl or isotonic KCl + NaCl solutions into the portal vein increased hepatic afferent nerve activity (HANA) in a Na+ or K+ dose-dependent manner. After 4 wk on a high-NaCl or high-KCl diet, HANA responses were attenuated compared with animals fed a normal diet, and NKCC1 expression was reduced. These results show that a high-NaCl or high-KCl diet decreases NKCC1 expression in the liver, and it might cause a reduction in hepatic Na(+)- and K(+)-receptor sensitivity.     

Monocyte chemotactic protein-1 regulates leukocyte recruitment during gastric ulcer recurrence induced by tumor necrosis factor-alpha

TNF-alpha has numerous biological activities, including the induction of chemokine expression, and is involved in many gastric injuries. C-C chemokines [monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha] and C-X-C chemokines [MIP-2 and cytokine-induced neutrophil chemoattractant (CINC)-2alpha] mediate chemotaxis of monocytes and neutrophils, respectively. We examined the roles of TNF-alpha and dynamics of chemokine expression in gastric ulceration including ulcer recurrence and indomethacin-induced injury. Rats with healed chronic gastric ulcers received intraperitoneal TNF-alpha to induce ulcer recurrence. Some rats were given neutralizing antibodies against neutrophils or MCP-1 together with TNF-alpha. In a separate experiment, rats were orally administered 20 mg/kg indomethacin with or without pretreatment with pentoxifylline (an inhibitor of TNF-alpha synthesis) or anti-MCP-1 antibody. TNF-alpha (1 microg/kg) induced gastric ulcer recurrence after 48 h, which was completely prevented by anti-neutrophil antibody. TNF-alpha increased the number of macrophages and MCP-1 mRNA expression in scarred mucosa from 4 h, whereas it increased MPO activities (marker of neutrophil infiltration) and mRNA expression of MIP-2 and CINC-2alpha from 24 h. Anti-MCP-1 antibody inhibited leukocyte infiltration with reduction of the levels of C-X-C chemokines and prevented ulcer recurrence. Indomethacin treatment increased TNF-alpha/chemokine mRNA expression from 30 min and induced macroscopic erosions after 4 h. Pentoxifylline inhibited the indomethacin-induced gastric injury with reduction of neutrophil infiltration and expression of chemokine (MCP-1, MIP-2, and CINC-2alpha). Anti-MCP-1 antibody also inhibited the injury and these inflammatory responses but did not affect TNF-alpha mRNA expression. In conclusion, increased MCP-1 triggered by TNF-alpha may play a key role in gastric ulceration by regulating leukocyte recruitment and chemokine expression.     

Effects of nonylphenol and phytoestrogen-enriched diet on plasma vitellogenin, steroid hormone, hepatic cytochrome P450 1A, and glutathione-S-transferase values in goldfish (Carassius auratus)

The effects of nonylphenol (NP) on plasma vitellogenin (VTG) and steroid hormone values, as well as hepatic cytochrome P450 1A (CYP1A) and glutathione-S-transferase (GST) activities, were measured in goldfish (Carassius auratus) fed a diet with a low (formulated diet, FD) or high (commercial diet, CD) content of phytoestrogens, including genistein and daidzein. Male goldfish with secondary sexual characteristics were exposed to nominal NP concentrations of 0.1, 1.0, 10, and 100 microg/L in the water for 28 days while being fed either the FD or CD diet at 1.0% of body weight daily. Plasma VTG concentration in male goldfish exposed to 100 microg of NP/L and fed FD was significantly higher than that in the FD-fed control fish at seven, 21, and 28 days. However, fish of the CD-fed group exposed to 100 microg of NP/ L had significantly higher plasma VTG concentration than did fish of the CD-fed control group at 28 days only. Moreover, plasma VTG concentration in fish of the CD-fed control group was about 100-fold higher than that in fish of the FD-fed control group. Although the estrogenic effects of a phytoestrogen-enriched diet caused a decrease in testosterone and/or 11-ketotestosterone values in the CD-fed fish, there was no dose-response relationship between androgen and amount of NP to which the FD-fed fish were exposed. Nonylphenol does not have appreciable effects on hepatic CYP1A and GST activities in male goldfish at concentrations as low as 100 microg/L. These results suggest that NP has estrogenic activity in male goldfish at the nominal concentration of 100 microg/L, and that phytoestrogens, such as genistein and daidzein, in the CD inhibit an aspect(s) of steroid release and/or synthesis common to testosterone and 11-ketotestosterone. However, results of in vivo screening assays for endocrine-disrupting chemicals may be seriously affected by phytoestrogens in the diet, depending on content or potency of estrogenic activity; therefore, we recommend use in research of a standardized, open-formula diet in which estrogenic substances have been reduced to amounts that do not alter the results of studies that are influenced by exogenous estrogens.