A novel GIP receptor splice variant influences GIP sensitivity of pancreatic beta-cells in obese mice

Gastric inhibitory polypeptide (GIP) is an incretin that potentiates insulin secretion from pancreatic beta-cells by binding to GIP receptor (GIPR) and subsequently increasing the level of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). We have identified a novel GIPR splice variant in mouse beta-cells that retains intron 8, resulting in a COOH-terminal truncated form (truncated GIPR). This isoform was coexpressed with full-length GIPR (wild-type GIPR) in normal GIPR-expressing tissues. In an experiment using cells transfected with both GIPRs, truncated GIPR did not lead to cAMP production induced by GIP but inhibited GIP-induced cAMP production through wild-type GIPR (n = 3-4, P < 0.05). Wild-type GIPR was normally located on the cell surface, but its expression was decreased in the presence of truncated GIPR, suggesting a dominant negative effect of truncated GIPR against wild-type GIPR. The functional relevance of truncated GIPR in vivo was investigated. In high-fat diet-fed obese mice (HFD mice), blood glucose levels were maintained by compensatory increased insulin secretion (n = 8, P < 0.05), and cAMP production (n = 6, P < 0.01) and insulin secretion (n = 10, P < 0.05) induced by GIP were significantly increased in isolated islets, suggesting hypersensitivity of the GIPR. Total GIPR mRNA expression was not increased in the islets of HFD mice, but the expression ratio of truncated GIPR to total GIPR was reduced by 32% compared with that of control mice (n = 6, P < 0.05). These results indicate that a relative reduction of truncated GIPR expression may be involved in hypersensitivity of GIPR and hyperinsulinemia in diet-induced obese mice.     

Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus

Background information. The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe ( 2004 ) Cell Struct. Funct. 29 , 85–90]. Results. In the present study, we cloned an AQP gene from A. proteus [ApAQP (A. proteus AQP)] that encodes a 295‐amino‐acid protein. The protein has six putative TMs (transmembrane domains) and two NPA (Asn‐Pro‐Ala) motifs, which are conserved among various AQPs and are thought to be involved in the formation of water channels that span the lipid bilayer. Using Xenopus oocytes, we have demonstrated that the ApAQP protein product can function as a water channel. Immunofluorescence microscopy with anti‐ApAQP antibody revealed that ApAQP is detected on the CV membrane and on the vesicles around the CV. The presence of V‐ATPase (vacuolar H⁺‐ATPase) on the vesicle membrane around the CV was also detected. Conclusions. Our data on ApAQP allow us to provide the first informed explanation of the high water permeability of the CV membrane in amoeba. Moreover, the results suggest that vesicles possessing V‐ATPase are involved in generating an osmotic gradient. Based on our findings, we propose a new hypothesis for the mechanism of CV function.     

Analyses of functional interaction between RECQL1, RECQL5, and BLM which physically interact with DNA topoisomerase IIIalpha

RECQL1 and RECQL5 as well as BLM reportedly interact with TOP3alpha whose defect is lethal for the cell. Therefore in this study, we characterized recql5/recql1/blm triple mutants from DT40 cells to determine whether the triple mutants show a top3alpha disrupted cell-like phenotype. The triple mutants are viable. Moreover, both blm/recql1 and recql5/blm cells, and recql5/recql1/blm cells grew slightly slower than blm cells, that is, triple mutant cells grew almost the same rate as either of the double mutant cells. The blm cells showed sensitivity to methyl methanesulfonate (MMS) and ultraviolet light (UV), about a 10-fold increase in sister chromatid exchange (SCE), and about a 3-fold increase in damage-induced mitotic chiasma compared to wild-type cells. The triple mutants showed the same sensitivity to MMS or UV and the same frequency of damage-induced mitotic chiasma compared to those of blm cells, indicating that unlike BLM, RECQL1 and RECQL5 play a little role in the repair of or tolerance to DNA damages. However, recql5/blm cells showed higher frequency of SCE than blm cells, whereas the RECQL1 gene disruption had no effect on SCE in blm cells and even in recql5/blm cells.     

Effects of Ultraviolet Light on Melanocyte Differentiation: Studies with Mouse Neural Crest Cells and Neural Crest‐derived Cell Lines

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L ‐3,4‐dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT‐ or DOPA‐positive cells between the UV‐irradiated cultures and the non‐irradiated cultures. We then examined the effects of UV light on KIT‐positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase‐positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with α‐melanocyte‐stimulating hormone (α‐MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase‐negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal‐regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as α‐MSH and/or endothelin‐1.     

Spermosin, a trypsin-like protease from ascidian sperm: cDNA cloning, protein structures and functional analysis.

We have previously reported that two trypsin-like enzymes, acrosin and spermosin, play key roles in sperm penetration through the vitelline coat of the ascidian (Urochordata) Halocynthia roretzi [Sawada et al. (1984), J. Biol. Chem. 259, 2900-2904; Sawada et al. (1984), Dev. Biol. 105, 246-249]. Here, we show the amino-acid sequence of the ascidian preprospermosin, which is deduced from the nucleotide sequence of the isolated cDNA clone. The isolated ascidian preprospermosin cDNA consisted of 1740 nucleotides, and an open reading frame encoding 388 amino acids, which corresponds to a molecular mass of 41 896 Da. By sequence alignment, it was suggested that His178, Asp230 and Ser324 make up a catalytic triad and that ascidian spermosin be classified as a novel trypsin family member. The mRNA of preprospermosin is specifically expressed in ascidian gonads but not in other tissues. Purified spermosin consists of 33- and 40-kDa bands as determined by SDS/PAGE under nonreducing conditions. The 40-kDa spermosin consists of a heavy chain (residues 130-388) and a long light chain designated L1 (residues 23-129), whereas the 33-kDa spermosin includes the same heavy chain and a shorter light chain designated L2 (residues 97-129). The L1 chain contains a proline-rich region, designated L1(DeltaL2) which is lacking in L2. Investigation with the glutathione-S-transferase (GST)-spermosin-light-chain fusion proteins, including GST-L1, GST-L2, and GST-L1(DeltaL2), revealed that the proline-rich region in the L1 chain binds to the vitelline coat of ascidian eggs. Thus, we propose that sperm spermosin is a novel trypsin-like protease that binds to the vitelline coat and also plays a part in penetration of sperm through the vitelline coat during ascidian fertilization.     

Basic fibroblast growth factor protects cardiac myocytes from iNOS-mediated apoptosis.

Basic fibroblast growth factor (bFGF) is an important angiogenic factor produced by hearts subjected to ischemia. However, the direct effects of bFGF on myocardial cells are unknown. Primary cultured cardiac myocytes from neonatal rats were stimulated with lipopolysaccharide (LPS), a potent inducer of inducible nitric oxide synthase (iNOS), in the presence or the absence of bFGF. LPS induced the expression of iNOS in cardiac myocytes, demonstrated at both mRNA and protein levels. We showed that LPS activated the apoptotic pathway, evidenced by TUNEL staining, DNA ladder formation, and morphologic features. LPS-induced apoptosis was blocked by the administration of L-NAME, an inhibitor of NOS. This indicates that LPS induces apoptosis via an iNOS-dependent pathway. Administration of bFGF completely inhibited myocardial cell apoptosis induced by hydrogen peroxide or acidic medium as well as LPS. To determine signaling pathways for this inhibitory effect, we utilized PD098059, an MEK-1-specific inhibitor. PD098059 blocked bFGF-induced activation of ERK (extracellularly responsive kinase)-1/2 and neutralized the apoptotic inhibitory effect of bFGF. These findings demonstrate that LPS induces myocardial cell apoptosis in an iNOS-dependent manner. The results also suggest that bFGF is a protective factor against myocardial cell apoptosis and that this protection requires the MEK-1-ERK pathway.