Subtype-specific trafficking of endothelin receptors

We investigated the subcellular localization of two endothelin receptors (ET(A)R and ET(B)R). To visualize these receptors directly, the C terminus of each receptor was fused to the N terminus of enhanced green fluorescent protein (designated as ETR-EGFP). When transiently expressed in various mammalian cell lines, ET(A)R-EGFP was predominantly localized on the plasma membrane. By contrast, ET(B)R-EGFP was, independent of ligand stimulation, predominantly localized on the intracellular vesicular structures containing Lamp-1. Immunoblot analyses revealed that at steady state ET(B)R-EGFP was highly degraded, and its degradation was inhibited by bafilomycin A(1). Antibody uptake experiments suggested that the ET(B)R-EGFP molecules were internalized from the plasma membrane. It is therefore likely that ET(B)R is first transported to the plasma membrane and then internalized, irrespective of ligand stimulation, to lysosomes where it undergoes proteolytic degradation. Exchanging the C-terminal cytoplasmic tails of the two ETRs revealed that the cytoplasmic tail is responsible for both the intracellular localization and the degradation of the receptors. Deletion of the extreme C-terminal 35 amino acids from both receptors allowed the receptor proteins to localize predominantly in the intracellular vesicles and to degrade. These observations indicate that the cytoplasmic tail of ET(A)R determines its plasma membrane localization. Stimulation with endothelin-1 increased the amount of intact ETR-EGFP fusion proteins without increasing their de novo synthesis, suggesting that binding of endothelin-1 stabilizes the ETRs.     

Multivariate analysis of hematological and serum biochemical values in female cynomolgus monkeys (Macaca fascicularis) aged less than a year

Hematological and serum biochemical data obtained from 91 laboratory-bred cynomolgus monkeys (Macaca fascicularis) aged 11 to 362 days were analyzed by the discriminant analysis. All animals used had been bred and reared under uniform environmental conditions at Tsukuba Primate Center for Medical Science, N. I. H., Japan. The examination items were as follows: red blood cell count (RBC), hematocrit value (Ht), hemoglobin concentration (Hb), mean corpuscular volume (MCV), white blood cell count (WBC), glutamic oxaloacetic transaminase activity (GOT), glutamic pyruvic transaminase activity (GPT), total protein concentration (TP), albumin concentration (ALB), albumin-globulin ratio (A/G), blood urea nitrogen (BUN), glucose concentration (GLU), total cholesterol concentration (TCHO), free cholesterol concentration (FCHO), triglyceride concentration (TG), alkaline phosphatase activity (ALP) and calcium concentration (Ca). The animals were divided into four groups (A: Suckling infants, less than 176 days old. B, C, D: Weanlings and juveniles, 121 to 220 days old, 221 to 280 days old, and 281 to 362 days old, respectively). Discrimination was possible among these four groups on the basis of the Mahalanobis' generalized distance. Regarding the canonical discriminant analysis (discriminant analysis with reduction of dimensionality), discrimination was possible. The suckling infant group could be discriminated from the juvenile groups by the first canonical variate. Concerning the juvenile groups, age was highly correlated to the value of the second canonical variate.2+ Judging from an approximate relative eigenvector value for the second canonical variate, the effective discriminant variables were WBC, TP, ALB, A/G, FCHO, TG, and ALP. It can be concluded that these eight parameters are important and useful for monitoring the physiologicals conditions of growing juvenile monkeys.     

Decrease in the levels of NGF and BDNF in brains of mice fed a tryptophan-deficient diet

The roles of dietary tryptophan (Trp) were evaluated in regulation of production of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 in the various brain regions in ddY mice. Feeding the mice a Trp-deficient diet for 2 weeks significantly decreased in the hippocampal level of NGF but not those of BDNF and NT-3, as compared with feeding an adequate Trp diet. The mice fed excess Trp did not have different levels of any of these neurotrophins than in the mice fed an adequate Trp diet. The levels of BDNF in the cerebral cortex were also significantly lower in the mice fed on a Trp-deficient diet, while the levels of NGF and NT-3 in the region were not modulated upon feeding of the diet. The dietary Trp level had no significant effect on the levels of NGF, BDNF, or NT-3 in the entorhinal cortex nor septum of the mice. These results demonstrate that the brain levels of NGF and BDNF are dependent on the dietary content of tryptophan.     

Radial and axial variations of bacteria within the cecum and proximal colon of guinea pigs revealed by PCR-DGGE

We investigated the local variability of bacterial species within the lumen of the cecum and proximal colon of guinea pigs by using PCR-denaturing gradient gel electrophoresis (DGGE). The DGGE banding profiles revealed radial and axial variation of the bacteria. This variation of bacteria within the large intestinal lumen might be of physiological importance for the degradation of dietary fiber and interaction between bacteria and the mucosal immune system.     

Population structure and gene flow among anadromous arctic lamprey (Lethenteron camtschaticum) populations deduced from polymorphic microsatellite loci

Information on migration ecology is important for effective conservation and management of fishery resources, particularly diadromous animals. We investigated the genetic population structure and gene flow pattern of anadromous Arctic lamprey Lethenteron camtschaticum by analyzing polymorphic microsatellite loci. Bayesian population structure analysis was used to estimate the existence of two genetically divergent ancestral sources and latitudinal gradual changes of their frequencies among the present populations. When hypotheses of historical migration models were tested using coalescent-based methods, the best model was found to be an n-island model assuming equal population size and reciprocal migration among all populations. These results suggest that L. camtschaticum has considerable ability to migrate long distances in the sea and has lower homing abilities. For resource management of L. camtschaticum, unconstrained gene flow across all populations should be maintained. The present findings might be beneficial for conservation and contribute the sustainable use of L. camtschaticum resource.     

The HA and NS Genes of Human H5N1 Influenza A Virus Contribute to High Virulence in Ferrets

Highly pathogenic H5N1 influenza A viruses have spread across Asia, Europe, and Africa. More than 500 cases of H5N1 virus infection in humans, with a high lethality rate, have been reported. To understand the molecular basis for the high virulence of H5N1 viruses in mammals, we tested the virulence in ferrets of several H5N1 viruses isolated from humans and found A/Vietnam/UT3062/04 (UT3062) to be the most virulent and A/Vietnam/UT3028/03 (UT3028) to be avirulent in this animal model. We then generated a series of reassortant viruses between the two viruses and assessed their virulence in ferrets. All of the viruses that possessed both the UT3062 hemagglutinin (HA) and nonstructural protein (NS) genes were highly virulent. By contrast, all those possessing the UT3028 HA or NS genes were attenuated in ferrets. These results demonstrate that the HA and NS genes are responsible for the difference in virulence in ferrets between the two viruses. Amino acid differences were identified at position 134 of HA, at positions 200 and 205 of NS1, and at positions 47 and 51 of NS2. We found that the residue at position 134 of HA alters the receptor-binding property of the virus, as measured by viral elution from erythrocytes. Further, both of the residues at positions 200 and 205 of NS1 contributed to enhanced type I interferon (IFN) antagonistic activity. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals.     

Vasodilative effect of perillaldehyde on isolated rat aorta

The vasodilative effect of perillaldehyde, one of the major oil components in Perilla frutescens BRITTON, was studied using isolated rat aorta. Perillaldehyde at final concentrations of 0.01 to 1 mM showed dose-dependent relaxation of the aorta contracted by treatment with prostaglandin F2alpha or norepinephrine. Neither the presence of NG-nitro-L-arginine methyl ester nor removal of the aortic endothelium affected the vasodilatation, suggesting that perillaldehyde exerts a direct effect on vascular smooth muscle cells. The vasodilative effect of perillaldehyde was not inhibited by pretreatment with a beta-adrenergic receptor blocker (propranolol), an inhibitor of phosphodiesterase (theophylline), a delayed rectifier K+ channel blocker (tetraethylammonium chloride), or an ATP-sensitive K+ channel blocker (glibenclamide). However, perillaldehyde showed contrasting effects on vasodilatation of the aorta contracted by an influx of extracellular Ca2+ - perillaldehyde caused little vasodilatation on the aorta contracted by the Ca2+ ionophore A23187, while it inhibited the vasoconstriction induced by treatment with high-concentration K+, which dominantly opened the voltage-dependent Ca2+ channel. These results suggest that the vasodilative effect of perillaldehyde is derived from blocking the Ca2+ channels.