Expression of the GABAB receptor in Xenopus oocytes and inhibition of the response by activation of protein kinase C.

The functional GABAB receptor was expressed in Xenopus oocytes by injecting mRNA obtained from the cerebellum of the rat. Application of GABA in the presence of bicuculline induced a hyperpolarization under current-clamp conditions and an outward current under voltage-clamp conditions. Baclofen mimicked the effect of GABA in the presence of bicuculline, and the effect of baclofen was antagonized by phaclofen. The GABA-induced outward current was slightly inhibited by treatment with GDP-beta-S and was completely inhibited by treatment with GTP-gamma-S. The activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA), but not 4 alpha-phorbol-12,13-didecanoate, suppressed the GABAB receptor-mediated hyperpolarization, and the effect of TPA was antagonized by sphingosine. Thus, activation of protein kinase C inhibits the expressed GABAB receptor-mediated response.     

Effects of aldose reductase inhibitor (ONO-2235) on human erythrocyte sorbitol concentrations in 75 g oral glucose tolerance tests

In order to evaluate the effects of the aldose reductase inhibitor, ONO-2235, on the short-term response of human erythrocyte sorbitol to hyperglycemia in vivo, eleven diet-treated Type 2 (non-insulin-dependent) diabetic patients were studied twice in 75 g oral glucose tolerance tests - with and without ONO-2235 (200 mg p.o.) premedication. The erythrocyte sorbitol concentrations increased with the increments of blood glucose and erythrocyte glucose concentrations in the test performed without ONO-2235. The erythrocyte sorbitol response in the test performed with administration of ONO-2235 30 min prior to glucose load was lower than that in the test performed without ONO-2235 (F = 5.782, P less than 0.05). No significant differences were found between the two tests in blood glucose and erythrocyte glucose concentrations (F = 0.092, P = 0.761; F = 0.029, P = 0.860, respectively). It is concluded that human erythrocyte sorbitol concentrations change promptly in response to rapid changes in erythrocyte glucose concentrations and that administered ONO-2235 is effective in inhibiting the human erythrocyte sorbitol pathway in man.     

A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.     

Recognition of a 56 kDa protein in partially purified rat hepatic nuclear thyroid hormone receptor by anti-human c-erb A beta antibody.

Human beta thyroid hormone receptor (c-erb A beta protein) produced by an Escherichia coli expression system was purified by sequential column chromatography followed by electroelution from an electrophoresis gel and an antibody was prepared. The antibody recognized a 56 kDa protein band in a partially purified rat hepatic nuclear thyroid hormone receptor fraction on Western blotting. Although multiple bands appeared on Western blotting of crude rat hepatic receptor preparations, a 56 kDa band was the most prominent and preadsorption of the antibody by purified c-erb A protein resulted in almost complete disappearance of the 56 kDa band, indicating that the 56 kDa band was formed by a specific antigen-antibody interaction. Furthermore, the 56 kDa protein appeared to co-elute with 3, 5, 3'-triiodo-L-thyronine binding activity in hydroxylapatite, Sephacryl S-200, and DNA-cellulose column chromatography of rat hepatic nuclear receptor, and sequential column purification resulted in selective enrichment of the 56 kDa band. These results suggest that the 56 kDa protein may be the major component of the rat hepatic thyroid hormone receptor.     

Autoregulation of androgen receptor expression in rodent prostate: immunohistochemical and in situ hybridization analysis

Autoregulation of androgen receptor mRNA and protein was investigated by immunohistochemical and in situ hybridization techniques. In both mouse and rat prostate, the epithelial cell nuclei were stained with the monoclonal or polyclonal antibodies raised against human androgen receptor. It was observed that 3 days after castration, nuclear staining of the epithelium was greatly reduced, while androgen treatment restored the staining intensity to a normal level. In situ hybridization using an androgen receptor cDNA fragment as probe demonstrated that the change in androgen receptor mRNA level correlated with the change in antibody staining intensity. These data suggested an up-regulation of androgen receptor expression by androgen.     

Presence of 2-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and 1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, novel endogenous amines, in parkinsonian and normal human brains

2-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and 1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline were identified for the first time as novel endogenous amines in parkinsonian and normal human brains by gas chromatography-mass spectrometry. It is of interest that these tetrahydroisoquinolines are analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which produces Parkinson's disease.     

Monoclonal antibody against platelet thrombospondin decreases erythrocyte aggregation rate.

The effect of thrombospondin, a major glycoprotein in the platelet alpha-granule, on the erythrocyte aggregation rate was investigated. Venous blood was sampled from 8 healthy male volunteers and anticogulated with 1.1 mg/ml EDTA(K2). The erythrocyte aggregation rate of each blood sample was measured with a whole-blood erythrocyte aggregometer before and after incubation with murine monoclonal antibody against human platelet thrombospondin. After 15 min incubation, the erythrocyte aggregation rate exhibited a significant decrease to 0.055 +/- 0.022/s, representing 71.9 +/- 8.7% of the control value (0.075 +/- 0.028/s) (p less than 0.0005). The results obtained suggest that thrombospondin may participate in the control of erythrocyte aggregability in the circulating blood.     

Evidence that glycine and GABA mediate postsynaptic inhibition of bulbar respiratory neurons in the cat.

Experiments were carried out on decerebrate cats to identify transsynaptic mediators of spontaneous postsynaptic inhibition of bulbar inspiratory and postinspiratory neurons. Somatic membrane potentials were recorded through the central micropipette of a coaxial multibarreled electrode. Blockers of type A gamma-aminobutyric acid (GABA-A) and glycine receptors were iontophoresed extracellularly from peripheral micropipettes surrounding the central pipette. Effective antagonism was demonstrated by iontophoresis of agonists with antagonists; application of strychnine antagonized the action of glycine but not GABA, and application of bicuculline antagonized the action of GABA but not glycine. In both types of neurons, iontophoresis of either antagonist depolarized the somatic membrane and increased input resistance throughout the respiratory cycle. Bicuculline preferentially depolarized the somatic membrane in both types of neurons during inactive phases. Strychnine increased the firing rate of inspiratory neurons during inspiration despite maintenance of somatic membrane potential at preiontophoresis levels. Tetrodotoxin reduced the effects of iontophoresed bicuculline and strychnine, suggesting that the action of the antagonists required presynaptic axonal conduction. The present results suggest that presynaptic release of both GABA and glycine contributes to tonic postsynaptic inhibition of bulbar respiratory neurons. GABA-A receptors appear to contribute to inhibition during inactive phases in inspiratory and postinspiratory neurons, whereas glycinergic mechanisms appear to contribute to inspiratory inhibition in inspiratory neurons.     

Primary structure of a precursor for the delta-subunit of sweet potato mitochondrial F1-ATPase deduced from full length cDNA.

A cDNA library constructed from poly(A)-rich RNA of the sweet potato tuberous root using a newly developed plasmid vector carrying tac-SP6 promoters was used to identify full length cDNAs for the nuclear-encoded delta-subunit of mitochondrial F1-ATPase by oligonucleotide-hybridization selection. Selected clones contained cDNA insert which carry the entire coding capacity for the pre-delta-subunit, since the RNA transcribed in vitro from SP6 promoter on the vector directed the synthesis of pre-delta-subunit polypeptide in a wheat germ in vitro translation assay. The nucleotide sequence of one of these cDNAs indicates that it can code for the pre-delta-subunit of 244 amino acids of which 199 amino acids encode the mature subunit. The amino acid sequence of the mature delta-subunit shows similarities of about 18-25% amino acid positional identity with the delta-subunits of bacterial F1-ATPases, about 26% with the delta-subunit of chloroplast CF1-ATPase, and about 32-37% with oligomycin sensitivity conferring proteins of animal and fungal mitochondria. The N-terminal presequence of the precursor composed of maximum of 45 amino acids does not show any obvious sequence homology with either the transit peptide of the nuclear-encoded pre-delta-subunit of chloroplast CF1 or the presequence of the nuclear-encoded pre-oligomycin sensitivity conferring proteins. At least two types of the delta-subunit cDNAs with very similar structures were identified from the library, and the presence of multiple copies of the delta-subunit gene in the hexaploid genome of the sweet potato is also suggested by genomic Southern blot hybridization.