Molecular cloning and biochemical characterization of two novel Neu3 sialidases, neu3a and neu3b, from medaka (Oryzias latipes)

Mammalian Neu3 sialidases are involved in various biological processes, such as cell death and differentiation, through desialylation of gangliosides. The enzymatic profile of Neu3 seems to be highly conserved from birds to mammals. In fish, the functional properties of Neu3 sialidase are not clearly understood, with the partial exception of the zebrafish form. To cast further light on the molecular evolution of Neu3 sialidase, we identified the encoding genes in the medaka Oryzias latipes and investigated the properties of the enzyme. PCR amplification using medaka brain cDNA allowed identification of two novel medaka Neu3 genes, neu3a and neu3b. The YRIP, VGPG motif and Asp-Box, characteristic of consensus motifs of sialidases, were well conserved in the both medaka Neu3 sialidases. When each gene was transfected into HEK293 to allow cell lysates for the use of enzymatic characterization, two Neu3 sialidases showed strict substrate specificity toward gangliosides, similar to mammalian Neu3. The optimal pH values were at pH 4.2 and pH 4.0, respectively, and neu3b in particular showed a broad optimum. Immunofluorescence assays indicated neu3a localization at plasma membranes, while neu3b was found in cytosol. The tissue distribution of two genes was then investigated by estimation of mRNA expression and sialidase activity, both being dominantly expressed in the brain. In neu3a gene-transfected neuroblastoma cells, the enzyme was found to positively regulate retinoic acid-induced differentiation with the elongation of axon length. On the other hand, neu3b did not affect neurite formation. These results and phylogenetic analysis suggested that the medaka neu3a is an evolutionally conserved sialidase with regard to enzymatic properties, whereas neu3b is likely to have originally evolved in medaka.     

The hypothalamus is involved in the anorexic effect of glucagon-like peptide-1 in chicks

The present study was carried out to investigate whether the hypothalamus is involved in the anorexic effect of glucagon-like peptide-1 (GLP-1) in chicks. To examine this, Fos expression in the chick hypothalamus were immunohistochemically detected after intracerebroventricular (ICV) injection of 30-pmol GLP-1. ICV injection of GLP-1 stimulated the expression of Fos-like immunoreactive (FLI) cells in the ventromedial hypothalamic nucleus (VMN). When 15-pmol GLP-1 was directly injected into the chick VMN, the chick's food intake was significantly decreased compared with the control treatment. Microinjection of GLP-1 into the (LHA) also inhibited feeding in chicks, although ICV injection of GLP-1 did not stimulate FLI expression in the brain area. These results suggest that VMN and some brain regions are involved in the anorexic effect of GLP-1 in chicks.     

Transgenic pigs expressing human decay-accelerating factor regulated by porcine MCP gene promoter

Porcine membrane cofactor protein (pMCP) is abundantly expressed throughout the body with particularly strong expression on the vascular endothelia. Previous studies demonstrated that the promoter of the pMCP gene induced efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55), in transgenic mice. In the present study, we tried to produce transgenic pigs with two hybrid genes, 0.9/hDAF and 5.4/hDAF, which were composed of human DAF (hDAF) gene regulated under pMCP promoters of different lengths (0.9 and 5.4 kb). Five live founder transgenic pigs were obtained only with the 0.9/hDAF construct. Although, four founder pigs transmitted the transgene to the second generation, the transmission rates varied among founders. We examined the expression of hDAF in tissues of descendants of two lines (Dm1 and Dm4). Human DAF specific RNAs were confirmed by an RT-PCR analysis in all organs examined. Levels of hDAF protein in the organs from the descendants of Dm1 line were higher than those in the corresponding human organs as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies showed that the tissue distribution of hDAF in the descendants of both lines was similar to that of endogenous pMCP. The expression level of hDAF on the vascular endothelial cells in Dm1 line was twice that on the corresponding human cells. We tested whether proinflammatory cytokines upregulate an efficiency of pMCP promoter on hDAF expression in transgenic pigs. Although the expression of hDAF on the human endothelial cells increased with a combination of cytokines, tumor necrosis factor alpha and interferon-gamma, no cytokine-induced upregulation was seen in the cells of transgenic pigs. The endothelial cells from transgenic pigs exhibited high resistance to the human serum-mediated cytolysis.     

Central,but not peripheral,glucagon-like peptide-1 inhibits crop emptying in chicks

We investigated the effect of central and peripheral glucagon-like peptide-1 (GLP-1) on crop emptying in growing chicks. Intracerebroventricular injection of two concentrations of GLP-1 (15 and 60 pmol) similarly suppressed crop emptying, compared with control chicks. The delay in crop emptying induced by GLP-1 (15 pmol) was partly attenuated by co-administration with exendin (5-39) (600 pmol), a GLP-1 receptor antagonist, although exendin (5-39) alone did not affect crop emptying. On the other hand, intraperitoneal administration of several doses of GLP-1 (120, 300 and 3000 pmol) did not alter crop emptying. The present study revealed that central, but not peripheral, GLP-1 inhibits crop emptying in chicks.     

Salt intake and mental distress among rural community-dwelling Japanese men

Background

Activated mineralocorticoid receptors influence the association between daily salt intake and blood pressure. A relatively low mineralocorticoid receptor function is reported to be a risk for mental distress such as depression. Since mental distress is also a known risk for hypertension and cardiovascular disease, understanding of the association between estimated daily salt intake and mental distress contributing to hypertension is important for risk estimation for cardiovascular disease. However, no single study has reported this association.

Methods

We conducted a cross-sectional study of 1014 Japanese men undergoing general health check-ups. Mental distress was diagnosed as a Kessler 6 scale score ≥5. We also classified mental distress by levels of hypertension. Estimated daily salt intake was calculated from a causal urine specimen.

Results

Independent from classical cardiovascular risk factors and thyroid disease, we found a significant inverse association between estimated daily salt intake and mental distress. When we analyzed for mental distress and hypertension, we also found a significant association. With the reference group being the lowest tertiles of estimated daily salt intake, the multivariable odds ratios (ORs) of mental distress and mental distress with hypertension for the highest tertiles were 0.50 (0.29–0.88) and 0.46 (0.22–0.96).

Conclusions

Lower estimated daily salt intake is a significant risk of mental distress for rural community-dwelling Japanese men. Since depression is reported to be associated with cardiovascular disease, risk estimation for the lower intake of salt on mental distress, especially for mental distress with hypertension, may become an important tool to prevent cardiovascular disease.     

Establishment of a Patient-Derived Orthotopic Xenograft (PDOX) Model of HER-2-Positive Cervical Cancer Expressing the Clinical Metastatic Pattern

Squamous cell carcinoma of the cervix, highly prevalent in the developing world, is often metastatic and treatment resistant with no standard treatment protocol. Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) nude mouse model with the technique of surgical orthotopic implantation (SOI). Unlike subcutaneous transplant patient-derived xenograft (PDX) models, PDOX models metastasize. Most importantly, the metastasis pattern correlates to the patient. In the present report, we describe the development of a PDOX model of HER-2-positive cervical cancer. Metastasis after SOI in nude mice included peritoneal dissemination, liver metastasis, lung metastasis as well as lymph node metastasis reflecting the metastatic pattern in the donor patient. Metastasis was detected in 4 of 6 nude mice with primary tumors. Primary tumors and metastases in the nude mice had histological structures similar to the original tumor and were stained by an anti-HER-2 antibody in the same pattern as the patient’s cancer. The metastatic pattern, histology and HER-2 tumor expression of the patient were thus preserved in the PDOX model. In contrast, subcutaneous transplantation of the patient’s cervical tumors resulted in primary growth but not metastasis.