Presynaptic α2 Adrenoceptors Inhibit Glutamate Release from Rat Spinal Cord Synaptosomes

Abstract: The presynaptic regulation of amino acid release from nerve terminals was investigated using synaptosomes prepared from the rat spinal cord. The basal releases of endogenous glutamate (Glu), aspartate (Asp), and γ-amino-butyric acid (GABA) were 34.6, 21.5, and 10.0 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCl (30 m M ) evoked 2.7-, 1.5-, and 2.9-fold increases in Glu, Asp, and GABA release, respectively. Clonidine reduced the K⁺-evoked overflow of Glu to 56% of the control overflow with a potency (IC₅₀) of 17 n M , but it did not affect K⁺-evoked overflow of Asp, GABA, and their basal releases. Similarly, noradrenaline inhibited the K⁺-evoked overflow of Glu, although phenylephrine and isoproterenol showed no effect. The inhibitory effect of clonidine was counteracted by α₂-adrenoceptor antagonists, rauwolscine, yohimbine, and idazoxan, regardless of the imidazoline structures. Because Glu is considered a neurotransmitter of primary afferents that transmit both nociceptive and nonnociceptive stimuli in the spinal cord, these data suggest that part of Glu release may be regulated by the noradrenergic system through α₂ adrenoceptors localized on the primary afferent terminals.     

RAD51 homologues in Xenopus laevis: two distinct genes are highly expressed in ovary and testis

The RAD51 gene is a eukaryotic counterpart of the Escherichia coli recA gene which is involved in genetic recombination. Two distinct Xenopus laevis RAD51 cDNA clones (XRAD51.1 and XRAD51.2) were isolated from an oocyte cDNA library using the human RAD51 cDNA (HsRAD51) as a probe. Sequence analysis revealed that 98.2% of the amino-acid residues were identical between XRAD51.1 and XRAD51.2, and that both were 95% identical to HsRAD51. Both of the XRAD51 genes were expressed at a higher level in ovary and testis than in other somatic tissues, suggesting their involvement in meiotic recombination. The expression of XRAD51.1 was about eightfold in excess of that of XRAD51.2 in all of the tissues examined. Analysis of the rates of synonymous substitution in the coding sequences of the two XRAD51 suggests that these two genes diverged about 50 million years ago. The structural similarities of the XRAD51 proteins to RecA in E. coli and Rad51 in yeasts or vertebrates are discussed.     

Contribution of spermatozoal centrosomes to the microtubule-organizing centre in Antarctic minke whale ( Balaenoptera bonaerensis )

Using an interspecies microinsemination assay with bovine oocytes, it was examined whether centrosomes of Antarctic minke whale spermatozoa function as the microtubule-organizing centre (MTOC). Bull and rat spermatozoa were used as positive and negative controls, respectively. Vitrified-warmed bovine mature oocytes were subjected to immunostaining against alpha-tubulin 4-6 h after intracytoplasmic injection (ICSI) of 5 mM dithiothreitol-treated spermatozoa. Aster formation occurred from whale spermatozoa (33%) and bull spermatozoa (33%), but very little from rat spermatozoa (3%). Activation treatment for the microinseminated oocytes with 7% ethanol + 2 mM 6-dimethylaminopurine resulted in a similar proportion of oocytes forming a whale sperm aster (35% vs 27% in the non-treated group; 4 h after ICSI) but a significantly larger aster (ratio of aster diameter to oocyte diameter, 0.57 vs 0.30 in the non-treated group). These results indicate that the centrosome introduced into bovine oocytes by whale spermatozoa contributes to the MTOC and that assembly of the microtubule network is promoted by oocyte activation.     

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.     

Adipose‐derived mesenchymal stem cells and regenerative medicine

Adipose tissue‐derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β‐cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow‐derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.     

Changes of Lipid Composition with Growth Phase of Cryptococcus neoformans

Qualitative and quantitative profiles of phospholipids, neutral lipids, and fatty acid composition in Cr. neoformans during the growth phase were investigated in relation to pyrophosphatidic acid. A marked increase of the total lipid content, which depended on the accumulation of triglyceride in yeast cells with the growth, was observed. The total phospholipid contents in yeast cells remained almostly constant during the exponential phase and slightly decreased in the stationary phase. The major phospholipids of this yeast were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and cardiolipin, the next groups being pyrophosphatidic acid, phosphatidic acid, lysophos-phatidylcholine, and unidentified components. The amounts of phosphatidylcholine, phosphatidylinositol, and cardiolipin were fairly constant throughout the growth phase, but the amount of phosphatidylethanolamine increased and that of phosphatidylserine decreased with progressive growth. The pyrophosphatidic acid contents were 0.9~0.7% for total phospholipid during the growth phase. The major fatty acids of pyrophosphatidic acid were C_16:0, C_18:1, and C_18:2 acids. The changing patterns of fatty acid composition in pyrophosphatidic acid through the growth phase closely resembled that of phosphatidic acid, which contained larger amounts of C_18:1 acid (35~45%) than C_16:0 acid (30~25%) and C_18:2 acid (30~25%). Phosphatidylserine and phosphatidylinositol contained considerable amounts of saturated fatty acid (C_16:0 acid, more than 55%). On the other hand, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin contained extremely large amounts of unsaturated fatty acid (C_18:1 and C_18:2 acid, 85ç90%).     

Anticancer and superoxide scavenging activities of p-alkylaminophenols having various length alkyl chains

A series of p-alkylaminophenols including 3, p-butylaminophenol; 4, p-hexylaminophenol; 5, p-octylaminophenol; and 6, N-(p-methoxybenzyl)aminophenol were synthesized based on the structure of fenretinide, N-(4-hydroxyphenyl)retinamide (1). This latter agent is a synthetic amide of all-trans-retinoic acid (RA), which is a cancer chemopreventive and antiproliferative agent. It was found that elongation of the alkyl chain length in these compounds increased antioxidative activity and inhibition of lipid peroxidation. These findings led us to investigate whether antiproliferative activity against cancer cells was effected by the length of alkyl chains linked to the aminophenol residue. All p-alkylaminophenols inhibited growth of HL60 and HL60R cells in a dose-dependent manners. The HL60R line is a resistant clone against RA. Growth of various cancer cell lines (HL60, HL60R, MCF-7, MCF-7/Adr(R), HepG2, and DU-145) was suppressed by p-alkylaminophenols in a fashion dependent on the aminophenol alkyl chain length (5>4>3>p-methylaminophenol (2)), with 5 being the most potent inhibitor of cell growth against HL60R, MCF-7/Adr(R), and DU-145 cells among p-alkylaminophenols tested, including 1. In particular, with the exception of compound 2, antiproliferative activity against DU-145 cells by these p-alkylaminophenols was greater than by 1. In HL60 cells, growth inhibition was associated with apoptosis. On the other hand, elongation of the alkyl chain length reduced superoxide trapping capability (2>3>4>5) in contrast to the effects on inhibition of lipid peroxidation. These results indicate that anticancer activity of p-alkylaminophenols correlated with the inhibitory activity of lipid peroxidation, but not with the superoxide scavenging activity.     

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb^−/−). Rheb^−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb^−/− was lower than that in the control (Rheb^+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb^+/+ mice but not in Rheb^−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb^−/− hearts during the neonatal period. Rheb^−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb^−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb^−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.