Parasympathetic reflex vasodilatation in rat submandibular gland

The present study was designed to investigate 1) whether parasympathetic reflex vasodilatation occurs in the submandibular gland (SMG) in deeply urethan-anesthetized, cervically vagotomized, and sympathectomized rats when the central cut end of the lingual nerve (LN) is electrically stimulated and 2) to what extent the neural mechanisms underlying such responses are the same as those involved in the response to direct stimulation of the chorda-LN (CLN). Stimulation of each nerve separately elicited a marked blood flow increase in SMG. Section of the chorda tympani abolished the SMG blood flow response but had no effect on the lip blood flow increase evoked by LN stimulation. Section of the CLN abolished the SMG blood flow increases evoked by stimulation of either nerve. The SMG blood flow increases (regardless of whether they were evoked by LN or CLN stimulation) were markedly reduced by the autonomic cholinergic ganglion blocker hexamethonium. The present study demonstrates that a parasympathetic reflex vasodilator mechanism is present in the rat SMG and that it can express its effects under deep general anesthesia.     

Neurodegeneration in mnd2 mutant mice is not prevented by parkin transgene

Parkinson’s disease (PD) is a common neurodegenerative disorder. The motor neuron degeneration 2 mutant (mnd2) mouse is considered to be an animal model of PD, and exhibits striatal neuron loss, severe muscle wasting, weight loss and death before 40 days of age. We found for the first time that parkin expression was decreased in the mnd2 mouse brain. Since parkin is a crucial protein for PD, the neurodegenerative disorder in mnd2 mice may be caused by parkin protein loss. We therefore examined whether compensation of parkin protein prevents neurodegenerative disorders in mnd2 mice by generating parkin-transgenic (parkin-Tg) mnd2 mice. However, both parkin-Tg mnd2 mice and mnd2 mice were smaller than wild type mice. In muscle strength and survival rate, parkin-Tg mnd2 mice showed similar values to mnd2 mice. Our data suggest that repression of parkin protein does not play a major role in neurodegeneration of mnd2 mice and administration of parkin protein does not rescue mnd2 mice.     

Recessive Mutations in the Putative Calcium-Activated Chloride Channel Anoctamin 5 Cause Proximal LGMD2L and Distal MMD3 Muscular Dystrophies

The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies.     

The development of plumage polymorphism in male Madagascar paradise flycatcher Terpsiphone mutata

The development of plumage polymorphism in male Madagascar paradise flycatcher Terpsiphone mutata (Linnaeus, 1766) was investigated in north‐western Madagascar. Four distinct morphs were observed, namely, white‐coloured with long tails (WL), rufous‐coloured with long tails (RL), rufous‐coloured with tails of middle length (RM), and rufous‐coloured with short tails (RS). Females were rufous and had short tails. Indivudual males were marked and re‐observed during 1994–98. RS males changed to RM in the following seasons. Among RM males, some individuals retained RM, others changed to RL or WL. RM males which changed to WL in the next season had possessed white patches on their belly, whereas RM males which changed to RL had not had such patches. Neither WL nor RL males changed their morphs. Nestlings were rufous regardless of their father's morph. One nestling changed to RS in the next season. From these results and the difference of body size among four morphs, it is deduced that RS males are one year old, and change to RM males in the next season. RM males change to RL or WL males after one or two years. RL and WL are two stable terminal plumages, and they may be genetically determined morphs.     

Molecular cloning and expression of gelatinases (MMP-2 and MMP-9) in the pufferfish Takifugu rubripes

To determine the metabolism location of the extra-cellular matrix proteins in fugu (Takifugu rubripes), we cloned the cDNAs of the fugu gelatinases, matrix metalloproteinase-2 (MMP-2) and MMP-9, and examined their expressions in various adult tissues using a quantitative real-time PCR. The expression profiles of fugu gelatinases were different among tissues. FgMMP-9 mRNA was abundant in tissues that contain blood cells abundantly where fgMMP-2 mRNA was little expressed. We also examined the expression of these genes in fugu embryos during development using a whole mount in situ hybridization. Fugu MMP-2 mRNA was expressed in the pharyngeal area and mesenchyme in embryos at 80 hours post fertilization (hpf). While fugu MMP-9 mRNA was expressed in the vent at 140 hpf and the caudal end of the fin fold at 172 hpf. Although fugu MMP-2 mRNA was expressed in the pectoral fin bud at 120 hpf, fugu MMP-9 mRNA did not appear in this tissue until 10 days post fertilization (dpf). These data show expression profiles differ between the fugu gelatinases and suggest expressions of these genes are controlled at the matrix protein degradation site in fugu embryos during development.     

Expression and distribution of fugu TIMP-2s (fgTIMP-2a and fgTIMP-2b) mRNAs in tissues and embryos

In teleosts, two distinct types of TIMP-2s occur, TIMP-2a and TIMP-2b, but little is known about their locations and quantitative expressions. Here, we examined pufferfish (Takifugu rubripes) TIMP-2a (fgTIMP-2a) and TIMP-2b (fgTIMP-2b) quantities and locations in fugu adult tissues and embryos. To compare the quantitative expression of fgTIMP-2s, we performed a quantitative real-time PCR (qPCR). FgTIMP-2a mRNA was constitutively expressed and significant differences in expression were not observed among adult tissues. Whereas, fgTIMP-2b mRNA was significantly differently expressed in ordinary muscle and gill compared to the expression level in whole blood (P<0.05). Although significant difference was not observed between brain and other tissues, both fgTIMP-2s mRNAs were abundant in the brain. In addition, we examined embryos during development using qPCR. Both fgTIMP-2s mRNAs gradually increased during embryonic development from 48 hpf. However, fgTIMP-2b mRNA was obviously abundant compared to fgTIMP-2a mRNA in embryos. We also examined the specific mRNA distribution in embryos. The fgTIMP-2s mRNAs showed the same distribution during development. Both fgTIMP-2s are expressed in adult fugu tissues and embryos but their expression levels clearly differ, suggesting that there is a predominance of fgTIMP-2b over fgTIMP-2a in vivo.     

The floor plate is sufficient for development of the sclerotome and spine without the notochord

Danforth'sshort-tail (Sd) mouse is a semi-dominant mutation affecting the development of the vertebral column. Although the notochord degenerates completely by embryonic day 9.5, the vertebral column exists up to the lumber region, suggesting that the floor plate can substitute for notochord function. We previously established the mutant mouse line, Skt(Gt), through gene trap mutagenesis and identified the novel gene, Skt, which was mapped 0.95cM distal to the Sd locus. Taking advantage of the fact that monitoring notochordal development and genotyping of the Sd locus can be performed using the Skt(Gt) allele, we assessed the development of the vertebra, notochord, somite, floor plate and sclerotome in +-+/+-Skt(Gt), Sd-+/+-+, Sd-Skt(Gt)/+-+, Sd-Skt(Gt)/+-Skt(Gt), Sd-+/Sd-+ and Sd-Skt(Gt)/Sd-Skt(Gt) embryos. In Sd homozygous mutants with a C57BL/6 genetic background, the vertebral column was truncated in the 6th thoracic vertebra, which was more severe than previously reported. The floor plate and sclerotome developed to the level of somite before notochord degeneration and the number of remaining vertebrae corresponded well with the level of development of the floor plate and sclerotome. Defects to the sclerotome and subsequent vertebral development were not due to failure of somitogenesis. Taken together, these results suggest that the notochord induced floor plate development before degeneration, and that the remaining floor plate is sufficient for maintenance of differentiation of the somite into the sclerotome and vertebra in the absence of the notochord.     

The prevalence of Toxoplasma gondii antibodies in wild and captive cetaceans from Japan

The seroprevalence of Toxoplasma gondii was investigated in wild and captive cetaceans from Japan. Antibodies against T. gondii were examined by both latex agglutination test (LAT) and indirect hemagglutination test (IHAT) for 77 serum or plasma samples obtained from 59 individuals of 6 species, including 2 hybrids. Antibody titers greater than 1:64 in LAT and greater than 1:640 in IHAT, indicative of the presence of T. gondii, were found in 11.9% of 59 individuals. In 7 samples that showed a positive reaction by IHAT, T. gondii titers were examined for each immunoglobulin (Ig) fraction separated by sucrose gradient centrifugation. The antibody peaks in each fraction were divided into 3 types, thought to be a reaction of IgM (type 1), IgG (type 2), and IgM with IgG (type 3). Type 1 was found in serum from a bottle-nosed dolphin (Tursiops truncatus) and a killer whale (Orcinus orca) sampled soon after capture off the Japanese coast in 1988; it was concluded that infection in the wild had occurred less than 15 yr before the study was performed. The prevalence of putative IgM and IgG antibodies from a captive-bred T. truncatus suggested that T. gondii infection also occurred in the aquarium.     

Expression and distribution of Gpr119 in the pancreatic islets of mice and rats: predominant localization in pancreatic polypeptide-secreting PP-cells

The GPR119 was recently shown to be activated by oleoylethanolamide (OEA), a naturally occurring bioactive lipid with hypophagic and anti-obesity effects. In this study, we have cloned and characterized its murine counterpart, Gpr119. The full-length cDNA contained an open reading frame of 1008bp encoding a 335-amino acid protein. The genomic organization of Gpr119 was unique, having a 3'-untranslated second exon that was also involved in an alternative splicing event. Gene expression analyses confirmed its specific expressions in pancreatic islets and two endocrine cell-lines, MIN6 and alphaTC1. Immunohistochemistry and double-immunofluorescence studies using a specific antibody revealed the predominant Gpr119 localization in pancreatic polypeptide (PP)-cells of islets. No definitive evidence of Gpr119-immunoreactivity in adult beta- or alpha-cells was obtained. The Gpr119 mRNA levels were elevated in islets of obese hyperglycemic db/db mice as compared to control islets, suggesting a possible involvement of this receptor in the development of obesity and diabetes.