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Hypoxia-inducible factor 1 a (Hif1α) plays a crucial role in brain development. To study the function of Hif1α in early brain development, we generated neuroepithelial cell-specific Hif1α-knockout mice. Hif1α-knockout mice died soon after birth; these mice exhibited an abnormal head shape, indicating the presence of brain defects. Morphological analysis revealed that Hif1α ablation reduced the overall size of the brain, especially affecting the telencephalon. Neuronal apoptosis predominantly occurred in deep-layer neurons, consequently the alignment of cortical layers was severely disorganized in Hif1α knockout mice. Furthermore, we demonstrated that Vegf signaling contributes to the survival of deep-layer neurons as a downstream effector of Hif1α-dependent hypoxia signaling. Taken together, our findings demonstrate that Hif1α plays a critical role in the early stages of telencephalon development.

The effect of plant extracts on D-glucose uptake by Ehrlich ascites tumour cells was examined. Among the 23 extracts of medicinal plants, five samples inhibited, and six samples activated, the uptake significantly. From one of the active plants, Lagerstroemia speciosa, two triterpenoids, colosolic acid and maslinic acid were isolated. Colosolic acid was shown to be a glucose transport activator. Since this compound was known to have hypoglycemic activity, our simple in vitro bioassay method can at least be used as a first screening for anti-diabetic activity.

An aqueous extract of mesocarps of the fruits of Balanites aegyptiaca exhibited a prominent antidiabetic activity by oral administration in streptozotocin induced diabetic mice. From one of the active fractions of this extract, two new steroidal saponins were isolated, and their structures were determined as 26-O-beta-D-glucopyranosyl-(25R)-furost-5-ene-3 beta,22,26-triol 3-O-[alpha-L-rhamnopyranosyl-(1----2)]-[beta-D-xylopyranosyl-(1---- 3)]-[alpha-L-rhamnopyranosyl-(1----4)]-beta-D-glucopyranoside and its 22-methyl ether. In addition, two known saponins, 26-O-beta-D-glucopyranosyl-(25R)-furost-5-ene-3 beta,22,26-triol 3-O-(2,4-di-O-alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside and its methyl ether were isolated and identified. It was revealed that the individual saponins did not show antidiabetic activity, while the recombination of these saponins resulted in significant activity. From an ethanolic extract of the epicarps, two known flavonol glycosides, isorhamnetin-3-O-robinobioside and isorhamnetin-3-O-rutinoside were isolated and identified.

Guanylate cyclase activity was examined in inflammatory granuloma tissue induced by subcutaneous injection of carrageenin solution in the rat. Enzymological properties of guanylate cyclase in this tissue, such as pH optimum, metal ion requirement, etc., were examined, with special reference to the intracellular distribution of the enzyme. Differences were found between guanylate cyclase in the soluble fraction and that in the particulate fraction in the response to Mn2+ and Triton X-100 in this tissue.

Treatment of rat reticulocytes with isoproterenol caused about 50, 25, and 25% decreases in beta-adrenergic agonist-, fluoride-, and guanine nucleotide-stimulated adenylate cyclase activities, respectively. The desensitization was also induced by dibutyryl adenosine 3',5'-cyclic monophosphate (cyclic AMP) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) prevented the isoproterenol-induced desensitization, suggesting the involvement of cyclic AMP in the desensitization. Time course studies revealed that the desensitization to NaF-AlCl3 occurred faster than that to isoproterenol. Furthermore, the rate of the resensitization to NaF-AlCl3 by removal of isoproterenol was also faster than that to isoproterenol. Thus, it is likely that both guanine nucleotide-binding stimulatory regulatory protein, Ns, and beta-adrenergic receptor are sequentially involved in both desensitization and resensitization of the adenylate cyclase system in rat reticulocytes to isoproterenol.

The effect of dan-shen extract, the root of Salvia miltiorrhiza, on adenylate cyclase was investigated in both rat brain and rat erythrocytes. The EtOAc fraction of the MeOH extract was proved to have significant inhibitory activity. Potent inhibitory principles in the EtOAc fraction were isolated and identified as 4 polyphenolic acids, rosemarinic acid, lithospermic acid, and their methyl ester derivatives.

The transport function and orientation of the reconstituted human erythrocyte glucose transporter was studied with liposomes made with bovine brain lipid or Escherichia coli lipid. Reconstitution was achieved by a simple octyl glucoside dilution method. The reconstituted transporters with either lipid showed identical counterflow transport activity, the same response to various inhibitors, and characteristic cytochalasin B (CB) labeling. Functional location and purification of the glucose transporter was performed by using gel-permeation high-performance liquid chromatography with octyl glucoside-containing buffer. The reconstituted transport activity was associated only with band 4.5 protein (preactin) and not with band 3 protein. Both CB binding and transport function of the reconstituted transporters were resistant to trypsin but susceptible to chymotrypsin digestion. However, both trypsin and chymotrypsin treatment of unsealed ghosts completely eliminated the CB labeling and transport function of the glucose transporter. In our reconstitution system the glucose transporters maintained a normal asymmetrical (right-side-out) orientation and good transport function. A specific monoclonal antibody against the glucose transporter inhibited CB labeling of the transporters on unsealed ghosts. This was not found with the reconstituted system; however, after freeze-thawing there was a significant inhibition of CB binding by the antibody. These findings suggest that the CB-binding site of the reconstituted transporter is on the inner side of the proteoliposomes.

Ehrlich ascites tumor-bearing mice were treated with colchi-cine or mitomycin C. Under colchicine, adenylate cyclase activity in tumor cells decreased to about a third of the untreated control level, whereas 5'-nucleotidase activity increased to almost twice the control level. Colchicine treatment resulted in about a 30 % reduction of 3H-thymidine incorporation into DNA. Mitomycin C inhibited the incorporation of 3H-thymidine much more mark-edly but changes in the two enzyme activities were less than under colchicine. The two enzyme activities and 3H-thymidine incorporation into DNA showed parallel changes with lapse of time after inoculation of tumor cells into mice. Thus, the colchicine effects on the two enzyme activities seemed not due simply to inhibition of cell proliferation. The two enzymes seemed to be associated differently with the microtubular system in Ehrlich ascites tumor cells.

Involvement of divalent cations was examined in the activation of catecholamine sensitive adenylate cyclase in Ehrlich ascites tumor cells. In the presence of various concentrations of either Mg2+ or Mn2+, both basal and norepinephrine-stimulated, adenylate cyclase activities were measured. Both activities were gradually increased with the increase in Mg2+ concentration. On the other hand, with Mn2+, both activities were maximum at the low (2.5 mM) concentration, and decreased at the highest concentrations, the decrease being marked in the norepinephrine-stimulated activity. Similar difference between the two divalent cations was also observed in the effect on the binding of the beta-adrenergic ligand to the membrane receptor.

The effect of barbiturates on an adenylate cyclase system in rat brain was examined. The activity of the catalytic unit of this system isolated from the synaptic membrane was inhibited by phenobarbital in dose- and time-dependent manners. The mode of the inhibition was non-competitive with respect to Mg-adenosine triphosphate (ATP). The activity of the synaptic membrane-bound adenylate cyclase was also inhibited by phenobarbital in a similar manner. The inhibitory effect of phenobarbital was more potent on the activation of the enzyme by 5'-guanylylimidodiphosphate (GppNHp) than on the basal enzyme activity. The inhibitory effect, however, was not observed in the synaptic membrane preparation in which the guanine nucleotide-binding stimulatory regulatory protein (Ns) and the catalytic unit of adenylate cyclase system had been functionally coupled by pretreatment with GppNHp. Similar results were obtained with other pharmacologically active barbiturates. These findings indicate that barbiturates primarily affect the activation of the catalytic unit by an interaction with Ns resulting in the inhibition of the enzyme activity.

The D-glucose transporter in the human erythrocyte membranes was photoaffinity-labeled with [3H]cytochalasin B and solubilized with n-octyl beta-D-glucopyranoside (octyl glucoside). [3H]Cytochalasin B-bound proteins were further isolated by using Sephadex G-50 chromatography. The amount of [3H]cytochalasin B associated with the membrane proteins was approximately 10% of the total radioactivity in the octyl glucoside extract. The solubilized photoaffinity-labeled D-glucose transporter was isolated and found to consist of two major peaks by DEAE-Sephacel chromatography. The radioactivity of peak II was considerably greater than that of peak I. The incorporation of [3H]cytochalasin B into both peaks was blocked by the presence of D-glucose during photolysis. With preparative NaDod-SO4/polyacrylamide gel electrophoresis, the radioactivity of peak I could be released, but that of peak II remained with the D-glucose transporter. These results indicate that [3H]cytochalasin B was covalently bound to the D-glucose transporter only in peak II and that peak II could be generated by the photoaffinity labeling of peak I. However, the D-glucose transport activity was associated only with peak I. These findings suggest that the anionic domain of the D-glucose transporter becomes exposed because of conformational changes of the protein as a result of covalent binding with [3H]cytochalasin B by photoaffinity labeling.

Forskolin-activated adenylate cyclase activity in rat erythrocyte membranes was markedly inhibited by methanol in a concentration dependent manner. On the contrary, no change was observed in the basal cyclase activity in the presence of methanol. The methanol inhibition forskolin-activated cyclase activity was protected by the stimulation of Gs in the presence of NaF in a concentration dependent manner. These data indicate that low- and high-affinity binding sites of forskolin in rat erythrocyte membranes have different sensitivities to methanol.

The effect of pentobarbital on the adenylate cyclase system was examined in synaptosomal membranes from rat brain. Pentobarbital inhibited forskolin-stimulated enzyme activity more effectively than the basal and Mn2(+)-stimulated enzyme activities. The degree of inhibition of the enzyme activity by pentobarbital was increased by the presence of forskolin in a concentration-dependent manner. No significant difference is observed in the degree of the inhibition by pentobarbital between the basal and forskolin-stimulated activities in the membranes prepared from the peripheral tissues.

Rat brain adenylate cyclase was solubilized with a combination of 0.7% sodium cholate and 0.6 M ammonium sulfate, and fractionated by addition of solid ammonium sulfate. The precipitate at 35% ammonium sulfate saturation contained neither guanine nucleotide-binding regulatory protein (G protein) nor calmodulin, and was used as the catalytic unit of the enzyme system. This catalytic unit was activated synergistically by forskolin and Mn2+. An apparent Km value for Mg-adenosine triphosphate (ATP) of the catalytic unit was about 80 microM in the basal state, while it increased in concurrence with the increase in the enzyme activity when forskolin was added to the assay system. The increase in the Km value depended on the forskolin concentration up to 1 microM, above which the value converged on ca. 200 microM. Furthermore, activation of the catalytic unit by forskolin was more marked at higher concentration of Mg-ATP. On the other hand, Mn2+ suppressed the increase in the Km value for Mg-ATP by forskolin, though the value in the basal state was not changed by Mn2+ alone. These findings indicate that the activation of the catalytic unit by forskolin is accompanied by the change in the affinity for Mg-ATP and Mn2+ modifies the change.