Secretion of N-terminal domain of α-dystroglycan in cerebrospinal fluid

α-Dystroglycan (α-DG) plays crucial roles in maintaining the stability of cells. We demonstrated previously that the N-terminal domain of α-DG (α-DG-N) is secreted by cultured cells into the culture medium. In the present study, to clarify its function in vivo, we generated a monoclonal antibody against α-DG-N and investigated the secretion of α-DG-N in human cerebrospinal fluid (CSF). Interestingly, we found that a considerable amount of α-DG-N was present in CSF. α-DG-N in CSF was a sialylated glycoprotein with both N- and O-linked glycan. These observations suggest that secreted α-DG-N may be transported via CSF and have yet unidentified effects on the nervous system.     

The genetic structure of endangered indigenous populations of the amago salmon, <Emphasis Type="Italic">Oncorhynchus</Emphasis><Emphasis Type="Italic">masou</Emphasis><Emphasis Type="Italic">ishikawae</Emphasis>, in Japan

The amago salmon, Oncorhynchus masou ishikawae, is an endemic subspecies of O. masou in Japan. Owing to the extensive stocking of hatchery fish throughout Japan, indigenous populations of O. m. ishikawae are now on the verge of extinction. We examined the genetic effects of stocking hatchery fish on wild populations in the River Koza, Japan, using microsatellite and mitochondrial DNA (mtDNA) markers. For mtDNA, haplotype mt1, which is common in wild populations, was present exclusively in isolated wild populations assumed to be unaffected by previous stocking, while it was never observed in hatchery fish. Genetic diversity was much higher in wild populations in the stocked area, which shared many mtDNA haplotypes with hatchery fish, than in isolated wild populations with haplotype mt1. Pairwise F _ST estimates based on microsatellites showed significant differentiation among the isolated populations with many microsatellite loci monomorphic. Significant deviation from Hardy–Weinberg equilibrium was observed in wild populations in the area subject to stocking, where a Bayesian-based assignment test showed a high level of introgression with hatchery fish. These results suggest that wild populations with haplotype mt1, which became isolated through anthropogenic environmental change in the 1950–1960s, represent indigenous populations of O. m. ishikawae in the River Koza. They have low genetic diversity, most likely caused by genetic bottlenecks following damming and environmental deterioration, while stocking of hatchery fish over the past 30 years apparently had a large impact on the genetic structure of wild populations in the main channel of the River Koza.     

EPR signals of cytochrome b558 purified from porcine neutrophils.

Cytochrome b558 of pig blood neutrophils was partially purified, and its EPR spectra were measured. The cytochrome b558 was solubilized from membranes with the detergent n-heptyl-beta-thioglucoside and purified by DEAE-Sepharose and heparin-Sepharose chromatographies. The small and large subunits of cytochrome b558 were detected on gel by immunoblotting. A solution of the purified, undenatured cytochrome b558 at 85-108 microM concentration was obtained. The concentrated cytochrome b558 showed an EPR signal at a g value of 3.26 with a bandwidth of 100 G at 10 K. Addition of 2 mM KCN had no effect on the low spin signal at g = 3.26 but caused disappearance of a minor high spin signal. The cyanide-insensitive signal at g = 3.26 disappeared completely on reduction with Na2S2O4. These results suggest that the g = 3.26 signal is characteristic of the low spin heme in cytochrome b558 of neutrophils.     

Differential and Isomer-Specific Modulation of Vitamin A Transport and the Catalytic Activities of the RBP Receptor by Retinoids

Retinoids are vitamin A derivatives with diverse biological functions. Both natural and artificial retinoids have been used as therapeutic reagents to treat human diseases, but not all retinoid actions are understood mechanistically. Plasma retinol binding protein (RBP) is the principal and specific carrier of vitamin A in the blood. STRA6 is the membrane receptor for RBP that mediates cellular vitamin A uptake. The effects of retinoids or related compounds on the receptor’s vitamin A uptake activity and its catalytic activities are not well understood. In this study, we dissected the membrane receptor-mediated vitamin A uptake mechanism using various retinoids. We show that a subset of retinoids strongly stimulates STRA6-mediated vitamin A release from holo-RBP. STRA6 also catalyzes the exchange of retinol in RBP with certain retinoids. The effect of retinoids on STRA6 is highly isomer-specific. This study provides unique insights into the RBP receptor’s mechanism and reveals that the vitamin A transport machinery can be a target of retinoid-based drugs.     

Herpes Simplex Virus-Induced Expression of 70 kDa Heat Shock Protein (HSP70) Requires Early Protein Synthesis But Not Viral DNA Replication

The major 70 kDa heat shock protein (HSP70), which is scarcely expressed in unstressed rodent cells, was apparently induced by infection with herpes simplex virus (HSV). Infection with HSV types 1 and 2 elevated HSP70 mRNA levels within 4 hr post-infection. HSP70 synthesis and accumulation increased in HSV-infected cells. Irradiation of HSV with UV-light abolished the ability to induce HSP70 mRNA. Inhibitors of viral DNA synthesis did not affect the induction of HSP70 in infected cells. Protein synthesis within 2 hr after infection was necessary for HSP70 induction.     

Design and synthesis of pyrrolo[3,2-d]pyrimidine HER2/EGFR dual inhibitors: Improvement of the physicochemical and pharmacokinetic profiles for potent in vivo anti-tumor efficacy

During the course of our studies on a novel HER2/EGFR dual inhibitor (TAK-285), we found an alternative potent pyrrolo[3,2-d]pyrimidine compound (1a). To enhance the pharmacokinetic (PK) profile of this compound, we conducted chemical modifications into its N-5 side chain and conversion of the chemically modified compounds into their salts. Among them, 2cb, the tosylate salt of compound 2c, showed potent HER2/EGFR kinase inhibitory activity (IC₅₀: 11/11 nM) and cellular growth inhibitory activity (BT-474 cell GI₅₀: 56 nM) with a good drug metabolism and PK (DMPK) profile. Furthermore, 2cb exhibited significant in vivo antitumor efficacy in both mouse and rat xenograft models with transplanted 4-1ST gastric cancer cell lines (mouse, T/C = 0%, 2cb po bid at 100 mg/kg; rat, T/C: -1%, 2cb po bid at 25 mg/kg).     

Effect of Theanine,r-Glutamylethylamide,on Brain Monoamines and Striatal Dopamine Release in Conscious Rats

Theanine, r-glutamylethylamide, is one of the major components of amino acids in Japanese green tea. Effect of theanine on brain amino acids and monoamines, and the striatal release of dopamine (DA) was investigated. Determination of amino acids in the brain after the intragastric administration of theanine showed that theanine was incorporated into brain through blood-brain barrier via leucine-preferring transport system. The concentrations of norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole acetic acid (5HIAA) in the brain regions were unaffected by the theanine administration except in striatum. Theanine administration caused significant increases in serotonin and/or DA concentrations in the brain, especially in striatum, hypothalamus and hippocampus. Direct administration of theanine into brain striatum by microinjection caused a significant increase of DA release in a dose-dependent manner. Microdialysis of brain with calcium-free Ringer buffer attenuated the theanine-induced DA release. Pretreatment with the Ringer buffer containing an antagonist of non-NMDA (N-methyl-D-aspartate) glutamate receptor, MK-801, for 1 hr did not change the significant increase of DA release induced by theanine. However, in the case of pretreatment with AP-5, (±)-2-amino-5-phosphonopentanoic acid; antagonist of NMDA glutamate receptor, the theanine-induced DA release from striatum was significantly inhibited. These results suggest that theanine might affect the metabolism and/or the release of some neurotransmitters in the brain, such as DA.