Isolation of cDNA for a Plasma Membrane H+-ATPase from Guard Cells of Vicia faba L.

cDNA encoding the plasma membrane H⁺-ATPase of guard cells ofVicia faba L. was isolated. The clone encoded a 105-kDa polypeptide(956 amino acids) that was 79–85% identical in terms ofamino acid sequence to other plant H⁺-ATPases. High levels ofmRNA explain the high H⁺-ATPase activity of these plasma membranes. (Received December 24, 1994; Accepted April 12, 1995)     

Sex-related spatial kin structure in a spring population of grey-sided voles Clethrionomys rufocanus as revealed by mitochondrial and microsatellite DNA analyses

Polymerase chain reaction-directed mitochondria (mt) and microsatellite DNA analyses were performed to examine the kin structure in a spring population of grey-sided voles Clethrionomys rufocanus in Hokkaido, Japan. The spatial distribution of 81 voles in a trapping grid (about 1 ha) was estimated by using the catch-mark-release method. DNA samples were extracted from the toes clipped for individual identification. Maternal lineages of voles were unequivocally determined by the mtDNA haplotypes, as identified by nucleotide sequencing of the control region. Relatedness between individuals was estimated based on the genotype and allele frequencies at several microsatellite loci. Although the distribution of voles was uniform within the grid, neighbouring females were frequently from the same maternal lineage. Relatedness values between females correlated negatively with geographical distances. Combination of the two molecular markers revealed four clusters of closely related, matrilineal females in the population, whereas no such cluster was apparent in males. The present study first demonstrated a sex-related spatial kin structure in a natural population of the grey-sided vole.     

Protective Effects of Pyridoxal Phosphate Against Glucose Deprivation-Induced Damage in Cultured Hippocampal Neurons

Abstract: When hippocampal cultures were deprived of glucose, massive release of lactate dehydrogenase (LDH), an indicator of neuronal death, occurred via NMDA receptor activation. Addition of pyridoxal phosphate (PLP; 1 and 10 µ M ) inhibited this LDH release in a concentration-dependent manner. Prior exposure to PLP evoked more potent inhibitory effects on LDH release compared with those treated at the onset of glucose deprivation. Furthermore, PLP inhibited the reduction of intracellular content of pyruvate induced by glucose deprivation, which was accompanied by the reversal of intracellular ATP depletion. A noteworthy elevation of extracellular glutamate in response to glucose deprivation was completely reversed by addition of PLP. Aminooxyacetic acid, a potent inhibitor of PLP-dependent enzymes, antagonized the effects of PLP on LDH release, pyruvate production, and ATP formation. These results suggest that PLP protects neurons from glucose deprivation-induced damage by enhancing the formation of energy-yielding products and relieving extracellular load of glutamate. The observed phenomena further indicate that PLP might be used prophylactically against neuronal death induced by metabolic disorders.     

Localization of immuno-analogues of erythrocyte protein 4.1 and spectrin in epidermis of psoriasis vulgaris

The presence and localization of immuno-analogues of human erythrocyte protein 4.1 and spectrin were examined in the epidermis of psoriasis vulgaris. Immunoblot analysis with antibodies against human erythrocyte protein 4.1 revealed that psoriatic epidermis contains a 4.1-like protein of 80 kDa, and also minor immunoreactive polypeptides, including a 45-kDa polypeptide. The 45-kDa band was not detected in non-lesional epidermis. Lesional epidermis of psoriasis contains spectrin-like proteins of 240 kDa. Analysis with immunofluorescence microscopy revealed that 4.1-like proteins were detected mainly in the cytoplasm of the suprabasal cells in lesional epidermis and in the peripheral cytoplasm of the basal cells in non-lesional epidermis. On the other hand, spectrin-like proteins were localized to the peripheral cytoplasm of basal keratinocytes in both lesional and non-lesional psoriatic epidermis. The present results indicate that proteins related to protein 4.1 and spectrin are consistently detected within epidermal cells of psoriasis, a chronic skin disease characterized by epidermal hyperplasia; the expression and distribution of protein 4.1 in lesional epidermis of psoriasis differs from that in non-lesional epidermis. These membrane skeletal proteins may be of significance in the hyperproliferative epidermis of psoriasis.     

Changes in protein phosphorylation in guinea pig polymorphonuclear leukocytes by treatment with membrane-perturbing agents which stimulate superoxide anion production

Phosphorylation of proteins was examined in guinea pig polymorphonuclear leukocytes in relation to the effects of membrane-perturbing agents, which stimulate superoxide anion production, and their inhibitors. The phosphorylation was detected by 32P autoradiography after separation by two-dimensional electrophoresis of proteins phosphorylated in 32P-preloaded cells. Though phosphorylation of various proteins was stimulated by each of the membrane-perturbing agents, the stimulation was especially marked in six proteins. Phorbol myristate acetate and digitonin enhanced the phosphorylation of the six proteins, while myristate and concanavalin A increased the phosphorylation of five and three proteins, respectively, out of the six proteins. p-Bromophenacyl bromide, an inhibitor of phospholipase A2, inhibited the stimulatory effect of phorbol myristate acetate on both superoxide anion production and protein phosphorylation. Trifluoperazine, a calmodulin inhibitor, also inhibited the effect of phorbol myristate acetate on both, except for an increase in the phosphorylation of one out of the six proteins. alpha-Methylmannoside, an inhibitor of concanavalin A binding, inhibited the stimulation of the phosphorylation of the three proteins by concanavalin A. The results indicate that the activation of superoxide anion production by the membrane-perturbing agents in guinea pig polymorphonuclear leukocytes is accompanied by the phosphorylation of, at least some of, these six proteins.     

Rat alpha1-acid glycoprotein. Purification and immunological estimation of its serum concentration.

A rapid method is described for the preparation of serum alpha1-acid glycoprotein from rats with inflammation induced with turpentine oil injection. The protein obtained by two purification steps, batchwise adsorption with DEAE-cellulose followed by chromatography on CM-cellulose, was proved to be native alpha1-acid glycoprotein in a high degree of purity by electrophoretical, immunological, ultracentrifugal and carbohydrate analysis. The monospecific and potent antiserum to this protein was prepared by immunizing rabbits with the desialyzed material emulsified with Freund's incomplete adjuvant. Using purified alpha1-acid glycoprotein and its specific antiserum, the concentration of alpha1-acid glycoprotein in rat serum was determined by single radial immunodiffusion. Abnormally high levels of its concentration (5-6 times higher than the control) were observed in inflammatory and tumor bearing rats.     

Modifying effect of vinblastine on superoxide anion production by membrane perturbing agents in polymorphonuclear leukocytes

Superoxide anion production in peritoneal polymorphonuclear leukocytes obtained from guinea pigs was stimulated by in vitro treatment with membrane-perturbing agents, such as cytochalasin D, concanavalin A, phorbol myristate acetate, myristate, digitonin, and NaF. Vinblastine modified these stimulating effects on the superoxide anion production, but its modifying effect was not uniform. The effect of cytochalasin D was stimulated by vinblastine at the concentration of 10(-5)-10(-7) M, whereas it was inhibited at the concentration of 10(-4) M. At 10(-4)-10(-5) M, vinblastine was inhibitory to the effect of concanavalin A, and lower concentrations had no significant effect. Stimulation of the superoxide anion production by phorbol myristate acetate and myristate was further enhanced by vinblastine at any concentration in the range of 10(-4)-10(-8) M with peaks at 10(-6) and 10(-5) M, respectively. Vinblastine had little effect on the stimulation of the superoxide anion production by digitonin and NaF throughout the concentration range examined. The mechanism of the interaction of these membrane-perturbing stimulants and vinblastine is discussed.     

Epinephrine-stimulated phosphorylation of pyruvate kinase in hepatocytes

Incubation of rat liver parenchymal cells with 10^?5m epinephrine or norepinephrine resulted in a rapid incorporation of ³²P into pyruvate kinase. Inclusion of α-adrenergic blocking agents (phenoxybenzamine or phentolamine) in the hepatocyte incubation medium prior to addition of epinephrine suppressed the subsequent phosphorylation of pyruvate kinase. On the other hand, inclusion of the β-adrenergic antagonist, propranolol, in the hepatocyte incubation medium prior to addition of epinephrine did not suppress the epinephrine-elicited phosphorylation of pyruvate kinase. Exogenous addition of either cyclic AMP or cyclic GMP to the hepatocyte incubation medium also resulted in increased phosphorylation of pyruvate kinase. To investigate whether the same amino acid residue(s) of liver pyruvate kinase was being phosphorylated in each instance, ³²P-labeled pyruvate kinase was isolated from hepatocytes after incubation in the presence or absence of either glucagon or epinephrine. In addition, purified liver pyruvate kinase was phosphorylated in vitro with a rat liver cyclic AMP-dependent protein kinase. Each ³²P-labeled pyruvate kinase was then subjected to tryptic digestion, two-dimensional thin-layer peptide mapping, and autoradiography. Each ³²P-labeled pyruvate kinase sample yielded 44 to 48 tryptic peptides upon staining with ninhydrin and 4 peptides that contain ³²P as detected by autoradiography. Furthermore, the same 4 peptides of pyruvate kinase were radiolabeled in each instance. Thus phosphorylation of pyruvate kinase in vitro with [γ-³²P]ATP or upon addition of either glucagon or epinephrine to hepatocytes incubated with ³²P_i resulted in phosphorylation of the same amino acid residues.