Purification and partial characterization of cytochrome b5 from Tetrahymena pyriformis

With the use of detergents and successive column chromatographies, Tetrahymena b-type cytochrome was purified from microsomes to a specific content of 36.0 nmol per mg of protein. The purified form showed a single band on SDS-polyacrylamide gel with molecular weight of 22,000. The spectral properties of the reduced b-type cytochrome, the α-peak of which is situated at 560 nm and asymmetric with a shoulder at 556 nm, was different from that of rat liver microsomal cytochrome b₅. However, it was reducible by NADH in the presence of NADH-cytochrome b₅ reductase purified from rat liver microsomes.The results indicated that the microsomal b-type cytochrome should be designated as cytochrome b₅ of a ciliated protozoan, Tetrahymena pyriformis.     

43Ca NMR studies of Ca2+-Tetrahymena calmodulin complexes

⁴³Ca NMR spectra of Ca²⁺-Tetrahymena calmodulin(Tet. CaM.) complexes have been observed under various conditions. Off-rate of Ca²⁺ from Tet. CaM. is estimated to be approx. 2.7 × 10³ s^?1 under a certain assumption. Relaxation rates of ⁴³Ca NMR of Ca²⁺-Tet. CaM. are remarkably increased(by one order in magnitude) by adding trifluoperazine(TFP), a potent calmodulin antagonist. Relaxation parameters estimated suggest that Ca²⁺ mobility is reduced by the TFP binding. A stoichiometry of TFP is two moles per Tet. CaM. molecule. The relaxation rates of ⁴³Ca NMR signals are increased by adding excessive Mg²⁺ to the Ca²⁺-Tet. CaM. solutions. The addition of Mg²⁺ to the Ca²⁺-Tet. CaM. complex decreases apparent pKa value of the complex as well.     

Detection of brain-specific gene expression in brain cells in primary culture: a novel promoter assay based on the use of a retrovirus vector.

Promoter activities of the brain-specific genes for glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were investigated in brain cells in primary culture with the use of a novel retrovirus vector, pIP200. With this vector, promoter activity can be expressed in terms of beta-galactosidase activity. Differentiation of the primary brain cells to mature glial cells was not affected by treatment with the pIP200 virus vector. The 256-bp 5'-flanking region of the GFAP gene directed astrocyte-specific expression of lacZ. It was silent in fibroblasts, even in multiple copies. The 1.3-kb 5'-flanking region of the MBP gene exhibited strict tissue (oligodendrocyte) specificity under the present assay method but showed some leakiness when integrated into the chromosome in multiple copies. Promoter regions conferring cell type specificity in brain were effectively identified by the present method.     

Neurotensin and Neuromedin N Are Differently Metabolized in Ventral Tegmental Area and Nucleus Accumbens

Whole homogenates and membrane-bound and cytosoluble fractions prepared from rat ventral tegmental area (VTA) and nucleus accumbens were examined for their content of peptidasic activities and for their ability to metabolize neurotensin and its natural related hexapeptide neuromedin N. No qualitative differences were observed between these two brain regions concerning the presence and the subcellular distribution of a series of activities able to hydrolyze various specific fluorimetric enzymatic substrates. However, aminopeptidase B, endopeptidase 24-15, and endopeptidase 24-11 were significantly lower in the VTA than in the nucleus accumbens membrane preparations, while proline endopeptidase was detected in significantly higher amount only in the cytosolic fraction prepared from nucleus accumbens. Both neurotensin and neuromedin N were metabolized more rapidly in the nucleus accumbens than in the VTA. Furthermore, the degradation rate of neuromedin N was considerably faster than that of neurotensin whatever the cerebral area examined. Studies carried out with highly specific peptidase inhibitors revealed that endopeptidase 24-15 mainly contributed to the catabolism of neurotensin in homogenates and membrane-bound preparations of nucleus accumbens and VTA, while aminopeptidase B appeared predominantly responsible for the rapid disappearance of neuromedin N in both cerebral tissues. The possibility that the different metabolic processes of the two peptide congeners could explain their distinct pharmacological profiles observed after their microinjection in the nucleus accumbens and in the VTA is discussed.     

Effects of Hypoxia on the Activity of the Dopaminergic Neuron System in the Rat Striatum as Studied by In Vivo Brain Microdialysis

The purpose of the present study is to clarify the effects of hypoxia on the activity of the dopaminergic neurons in the brain and its mechanism of action. For this purpose, the effects of hypoxia on the extracellular levels of 3,4-dihy-droxyphenylethylamine (dopamine) were examined in the rat Striatum using in vivo brain microdialysis in the presence or absence of pretreatment with either tetrodotoxin (a blocker of voltage-dependent sodium channels) or nomifensine (a blocker of dopamine reuptake). Exposure to various degrees of hypoxia (15, 10, and 8% O₂ in N₂) increased dopamine levels in striatal dialysates to 200, 400, and 1,100%, respectively, of the control value. On reoxygenation, dopamine levels in the dialysates rapidly returned to the control level. Reexposure to hypoxia increased the dopamine levels to the same extent as during the first exposure. After addition of tetrodotoxin (40 mUM) to the perfusion fluid or pretreatment with nomifensine (100 mg/kg, i.p.), exposure to hypoxia no longer increased the dopamine levels. These results suggest that although hypoxia induces an increase in the extracellular dopamine levels (hence, an apparent increase in the activity of the dopaminergic neurons), this increase is not the result of an increase in dopamine release itself, but rather the result of inhibition of the dopamine reuptake mechanism.     

Regulation by thyrotropin-releasing hormone (TRH) of TRH receptor mRNA degradation in rat pituitary GH3 cells.

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) down-regulates TRH receptor (TRH-R) mRNA (Fujimoto, J., Straub, R.E., and Gershengorn, M.C. (1991) Mol. Endocrinol. 5, 1527-1532), at least in part, by stimulating its degradation (Fujimoto, J., Narayanan, C.S., Benjamin, J.E., Heinflink, M., and Gershengorn, M.C. (1992) Endocrinology 130, 1879-1884). Here we show that TRH regulates RNase activity in GH3 cells and that specific mRNA sequences are needed for in vivo regulation of TRH-R mRNA by TRH. TRH affected RNase activity in a biphasic manner with rapid stimulation (by 10 min) followed by a decrease to a rate slower than in control lysates within 6 h. This time course paralleled the effects of TRH on degradation of TRH-R mRNA in vivo. The regulated RNase activity was in a polysome-free fraction of the lysates and was not specific for TRH-R RNA. A truncated form of TRH-R RNA that was missing the entire 3'-untranslated region (TRHR-R5) was more stable than full-length TRH-R RNA (TRHR-WT). In contrast to TRHR-WT mRNA, TRHR-R5 mRNA and TRHR-D9 mRNA, which was missing the 143 nucleotides 5' of the poly(A) tail, were not down-regulated by TRH in stably transfected GH3 cells as their rates of degradation were not increased. These data show that TRH regulates RNase activity in GH3 cells, that the 3'-untranslated region bestows decreased stability on TRH-R mRNA and that the 3' end of the mRNA is necessary for regulation by TRH of TRH-R mRNA degradation. We present an hypothesis that explains specific regulation of TRH-R mRNA degradation by TRH in GH3 pituitary cells.     

Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae

Activation of various receptors by extracellular ligands induces an influx of Ca2+ through the plasma membrane, but its molecular mechanism remains elusive and seems variable in different cell types. In the present study, we utilized mAbs generated against the cerebellar type I inositol 1,4,5-trisphosphate (InsP3) receptor and performed immunocytochemical and immunochemical experiments to examine its localization in several non-neuronal cells. By immunogold electron microscopy of ultrathin frozen sections as well as permeabilized tissue specimens, we found that a mAb to the type I InsP3 receptor (mAb 4C11) labels the plasma membrane of the endothelium, smooth muscle cell and keratinocyte in vivo. Interestingly, the labeling with the antibody was confined to caveolae, smooth vesicular inpocketings of the plasma membrane. The reactive protein, with an M(r) of 240,000 by SDS-PAGE, could be biotinylated with a membrane-impermeable reagent, sulfo-NHS-biotin, in intact cultured endothelial cells, and recovered by streptavidin-agarose beads, which result further confirmed its presence on the cell surface. The present findings indicate that a protein structurally homologous to the type I InsP3 receptor is localized in the caveolar structure of the plasma membrane and might be involved in the Ca2+ influx.     

Non-isopeptide-selective endothelin receptors in human Girardi heart cells.

We characterized the endothelin (ET) receptor in Girardi heart (GH) cells derived from human atrium. The ET isopeptides ET-1, ET-2 and ET-3 induced the monotonous and long-lasting rise in cytosolic free Ca2+ concentration [( Ca2+]i) with almost the same potency in GH cells. Scatchard analysis of [125I]ET-1 and [125I]ET-3 binding revealed that GH cells have almost the same number of binding sites for either labeled ligand. All ET isopeptides displaced either [125I]ET-1 or [125I]ET-3 binding in GH cells almost equipotently. These results reveal that the functional ET receptors in GH cells are of the ETB-type. GH cells are the first cell line to be found to express the functional ETB-receptor.     

Purification of achatin-I from the atria of the African giant snail, Achatina fulica, and its possible function.

Achatin-I previously purified from the ganglia of the African giant snail Achatina fulica was isolated from the atria of this snail. Achatin-I appeared to enhance the cardiac activity in two ways; centrally this peptide increased impulse frequency and produced spike broadening of the identified heart excitatory neuron, PON, and peripherally it enhanced amplitude and frequency of the heart beat. Achatin-I showed excitatory actions not only on the heart but on several other muscles.