Vesicular Integral-Membrane Protein 36 Is Involved in the Selective Secretion of Fucosylated Proteins into Bile Duct-like Structures in HepG2 Cells.

Fucosylated proteins are widely used as biomarkers of cancer and inflammation. Fucosylated alpha-fetoprotein (AFP-L3) is a specific biomarker for hepatocellular carcinoma. We previously showed that increases in serum AFP-L3 levels depend on increased expression of fucosylation-regulatory genes and abnormal transport of fucosylated proteins in cancer cells. In normal hepatocytes, fucosylated proteins are selectively secreted in the bile duct but not blood. In cases of cancer cells without cellular polarity, this selective secretion system is destroyed. Here, we aimed to identify cargo proteins involved in the selective secretion of fucosylated proteins, such as AFP-L3, into bile duct-like structures in HepG2 hepatoma cells, which have cellular polarity like, in part, normal hepatocytes. α1-6 Fucosyltransferase (FUT8) is a key enzyme to synthesize core fucose and produce AFP-L3. Firstly, we knocked out the FUT8 gene in HepG2 cells and investigated the effects on the secretion of AFP-L3. AFP-L3 accumulated in bile duct-like structures in HepG2 cells, and this phenomenon was diminished by FUT8 knockout, suggesting that HepG2 cells have cargo proteins for AFP-L3. To identify cargo proteins involved in the secretion of fucosylated proteins in HepG2 cells, immunoprecipitation and the proteomic Strep-tag system experiments followed by mass spectrometry analyses were performed. As a result of proteomic analysis, seven kinds of lectin-like molecules were identified, and we selected vesicular integral membrane protein gene VIP36 as a candidate of the cargo protein that interacts with the α1-6 fucosylation (core fucose) on N-glycan according to bibliographical consideration. Expectedly, the knockout of the VIP36 gene in HepG2 cells suppressed the secretion of AFP-L3 and other fucosylated proteins, such as fucosylated alpha-1 antitrypsin, into bile duct-like structures. We propose that VIP36 could be a cargo protein involved in the apical secretion of fucosylated proteins in HepG2 cells.

Increased lipid absorption and transport in the small intestine of zucker obese rats.

The aim of this study was to compare the potency of intestinal lipid absorption in Zucker obese rats compared with Sprague-Dawley (SD) rats. Six male Zucker obese (fa/fa) and six male SD rats baring intestinal lymph fistulae were used in this study. After 24 h recovery, rats were infused inraduodenally with a lipid emulsion containing 40 micromol triolein (labeled with glycerol tri[(3)H-oleate]), 7.8 micromol phosphatidyl choline in 3 ml phosphate buffered saline at a rate of 3 ml/h for 8 h. Lymph samples were collected and the radioactive lipid content determined. Apolipoprotein B (apo B) level in the lymph was evaluated. The Zucker obese rats transported more radioactive lipid into the lymph compared with the SD rats, particularly in the early phase of lipid absorption. Lymph apo B levels in the intestinal mucosa were significantly increased compared with the SD rats. In conclusion, this study indicated that lipid absorption and transport in Zucker obese rats is concomitant with increased apo B levels in the mesenteric lymph, indicating that the increase in lipid absorption may be responsible, at least in part, for obesity progression and hyperlipidemia.

Establishment and characterization of a fucosylated α-fetoprotein-specific monoclonal antibody: a potential application for clinical research.

The Lens culinaris agglutinin (LCA)-reactive fraction of α-fetoprotein (AFP-L3) is a well-known cancer biomarker for hepatocellular carcinoma (HCC) with very high specificity. Because LCA recognizes only bi-antennary N-glycans with a core fucose, some of fucosylated AFP in HCC patients may not be detected. Then glycan antibodies, which recognize both specific glycan and protein, are desired for glycobiology. Here, we successfully established a novel glycan antibody for fucosylated AFP and demonstrated its potential clinical application. After immunization with a fucosylated AFP peptide, positive screening was performed for fucosylated AFP peptides using solid-phase enzyme-linked immunosorbent assay (ELISA). The newly developed antibody was designated: fucosylated AFP-specific mAb (FasMab). Western blot analysis showed that FasMab reacted with AFP produced by HepG2 cells, but not with AFP produced by α-1,6-fucosyltransferase deficient HepG2 cells. The specific binding of FasMab to fucosylated AFP was confirmed with ELISA as well as western blot analysis. A preliminary high sensitivity chemiluminescence enzyme immunoassay kit showed increased levels of fucosylated AFP in the sera of patients with HCC, but not in the sera of normal patients, or patients with chronic liver diseases. Thus, the novel glycan antibody, FasMab, is a promising tool to study fucosylated AFP with clinical and basic research applications.

A recombinant vesicular stomatitis virus encoding CCR5-tropic HIV-1 receptors targets HIV-1-infected cells and controls HIV-1 infection.

Anti-retroviral therapy is useful to treat human immunodeficiency virus type 1 (HIV-1)-infected individuals, but has some major problems, such as the generation of multidrug-resistant viruses. To develop a novel supplemental or alternative therapeutic for CCR5-tropic (R5) HIV-1 infection, we generated a recombinant vesicular stomatitis virus (rVSV) in which the gene encoding its envelope glycoprotein (G) was replaced with the genes encoding R5 HIV-1 receptors (human CD4 and CCR5), designated VSVΔG-CC5. Our present data demonstrate that this rVSV specifically infects cells that are transiently expressing R5 HIV-1 envelope glycoproteins, but does not infect those expressing CXCR4-tropic HIV-1 envelope glycoproteins. Notably, after a CD4+CCR5+ T cell line or primary cells initially infected with R5 HIV-1 were inoculated with G-complemented VSVΔG-CC5, the rVSV significantly reduced the number of HIV-1-infected cells, probably through direct targeting of the rVSV and VSV-mediated cytolysis and/or through syncytium formation- or cell-cell fusion-dependent killing, and markedly inhibited HIV-1 production. Furthermore, G-complemented VSVΔG-CC5 also efficiently inhibited HIV-1 infection in R5 HIV-1-infected humanized immunodeficient mice. Taken together, our findings indicate that a cytolytic rVSV that targets and eliminates R5 HIV-1-infected cells potentially has therapeutic value for controlling R5 HIV-1 infection.

Glucagon-like peptide-1, corticotropin-releasing hormone, and hypothalamic neuronal histamine interact in the leptin-signaling pathway to regulate feeding behavior.

Glucagon-like peptide-1 (GLP-1), corticotropin-releasing hormone (CRH), and hypothalamic neuronal histamine suppress food intake, a target of leptin action in the brain. This study examined the interactions of GLP-1, CRH, and histamine downstream from the leptin-signaling pathway in regulating feeding behavior. Infusion of GLP-1 into the third cerebral ventricle (i3vt) at a dose of 1 mug significantly decreased the initial 1 h cumulative food intake in rats as compared with phosphate-buffered saline (PBS) controls. The GLP-1-induced suppression of feeding was partially attenuated by intraperitoneal pretreatment with alpha-fluoromethylhistidine (FMH), a specific suicide inhibitor of histidine decarboxylase, which depletes hypothalamic neuronal histamine. Pretreatment with alpha-helical CRH (10 microg/rat, i3vt), a nonselective CRH antagonist, abolished the GLP-1-induced suppression of feeding completely. I3vt infusion of GLP-1 increased the CRH content and histamine turnover assessed using the pargyline-induced accumulation of tele-methyl histamine (t-MH), a major metabolite of neuronal histamine, in the hypothalamus. The central infusion of CRH also induced the increase of histamine turnover and CRH receptor type 1 was localized on the cell body of histamine neuron. Pretreatment with exendin(9-39), a GLP-1 receptor antagonist, attenuated the leptin-induced increase in CRH content of the hypothalamus. Finally, i3vt infusion of leptin also increased histamine turnover in the hypothalamus. Pretreatment with exendin(9-39), alpha-helical CRH or both antagonists attenuated the leptin-induced responses of t-MH levels in the hypothalamus. These results suggest that CRH or hypothalamic neuronal histamine mediates the GLP-1-induced suppression of feeding behavior, that CRH mediates GLP-1 signaling to neuronal histamine and that a functional link from GLP-1 to neuronal histamine via CRH constitutes the leptin-signaling pathway regulating feeding behavior.

Development of an infectious surrogate hepatitis C virus based on a recombinant vesicular stomatitis virus expressing hepatitis C virus envelope glycoproteins and green fluorescent protein.

To develop surrogate viruses for hepatitis C virus (HCV), we previously produced recombinant vesicular stomatitis viruses (rVSVs) lacking glycoprotein G but instead expressing chimeric HCV E1/E2 fused to G. These rVSVs were not infectious in HCV-susceptible hepatoma cells. In this study, to develop an infectious surrogate HCV based on an rVSV (vesicular stomatitis virus [VSV]/HCV), we generated a novel rVSV encoding the native E1/E2 (H77 strain) and green fluorescent protein (GFP) instead of G. Here, we showed that this VSV/HCV efficiently infected human hepatoma cells, including Huh7 human hepatoma cells, expressed GFP in these cells, and propagated, but did not do so in nonsusceptible BHK-21 cells. The infectivity of VSV/HCV, measured as the number of foci of GFP-positive cells, was specifically reduced by the addition of chimpanzee anti-HCV serum, anti-E2 antibody, or anti-CD81 antibody to the cultures. When sera obtained from HCV-infected or uninfected patients were added, infection was selectively inhibited only by the sera of HCV-infected patients. These data together suggest that this infectious GFP-expressing VSV/HCV could be a useful tool for studying the mechanisms of HCV entry into cells and for assessing potential inhibitors of viral entry, including neutralizing antibodies.

Prepro-TRH 178-199 inhibits histamine- or restraint stress-induced activation of corticotropin releasing hormone production in rat hypothalamus.

Under restraint stress conditions, prepro-thyrotropin releasing hormone (TRH) 178-199 suppresses adrenocorticotropic hormone (ACTH) secretion from the rat pituitary, which indicates that prepro-TRH 178-199 is a candidate endogenous corticotropin releasing inhibitory factor (CRIF). Restraint stress also activates the release of hypothalamic neuronal histamine, which increases both the expression of CRH mRNA in the paraventricular nucleus (PVN) and plasma concentrations of ACTH. The aim of this study was to determine whether prepro-TRH 178-199 modulates histamine- or restraint stress-induced activation of corticotropin releasing hormone (CRH) in the rat hypothalamus. Infusion of prepro-TRH 178-199 into the third cerebroventricle (i3vt) at a dose of 6 µg/kg significantly decreased the amount of CRH in the PVN, as compared to vehicle-treated controls (p < 0.05), but did not affect the CRH amount in other hypothalamic regions. Restraint stress increased the amount of CRH in the PVN and ventromedial hypothalamic nucleus (VMH), as compared to non-restrained controls (p < 0.05); this was attenuated by pretreatment with i3vt infusion of prepro-TRH 178-199 (p < 0.05). I3vt infusion of histamine (270 nmol/rat) suppressed cumulative food consumption over 24 h, increased plasma ACTH concentrations, and increased the content of CRH in the PVN, as compared to vehicle-treated controls (p < 0.05 for each); these effects were attenuated by pretreatment with prepro-TRH 178-199 (p < 0.05). These results suggest that prepro-TRH 178-199 may regulate ACTH secretion by affecting basal and histamine- or stress-induced synthesis and/or secretion of CRH and ACTH by modulating histaminergic input to the PVN and VMH.:

Hypothalamic neuronal histamine mediates the thyrotropin-releasing hormone-induced suppression of food intake.

We examined the involvement of thyrotropin-releasing hormone (TRH) and TRH type 1 and 2 receptors (TRH-R1 and TRH-R2, respectively) in the regulation of hypothalamic neuronal histamine. Infusion of 100 nmol TRH into the rat third cerebroventricle (3vt) significantly decreased food intake (p < 0.05) compared to controls infused with phosphate- buffered saline. This TRH-induced suppression of food intake was attenuated partially in histamine-depleted rats pre-treated with alpha-fluoromethylhistidine (a specific suicide inhibitor of histidine decarboxylase) and in mice with targeted disruption of histamine H1 receptors. Infusion of TRH into the 3vt increased histamine turnover as assessed by pargyline-induced accumulation of tele-methylhistamine (t-MH, a major metabolite of neuronal histamine in the brain) in the tuberomammillary nucleus (TMN), the paraventricular nucleus, and the ventromedial hypothalamic nucleus in rats. In addition, TRH-induced decrease of food intake and increase of histamine turnover were in a dose-dependent manner. Microinfusion of TRH into the TMN increased t-MH content, histidine decarboxylase (HDC) activity and expression of HDC mRNA in the TMN. Immunohistochemical analysis revealed that TRH-R2, but not TRH-R1, was expressed within the cell bodies of histaminergic neurons in the TMN of rats. These results indicate that hypothalamic neuronal histamine mediates the TRH-induced suppression of feeding behavior.