Hiroaki Ohkubo

Molecular cloning and sequence analysis of human genomic DNA encoding A novel membrane protein which exhibits a slowly activating potassium channel activity

The amino acid sequence for a novel human membrane protein that induces selective potassium permeation by membrane depolarization was deduced by molecular cloning and sequence analysis of its genomic DNA. This protein consists of 129 amino acid residues and shares several structural characteristics with the rat counterpart. These include a single putative transmembrane domain surrounded by many charged amino acid residues, two potential N-glycosylation sites at the amino-terminal portion and a single cysteine residue at the carboxyl-terminal portion. The transmembrane domain and its flanking carboxyl-terminal sequence are highly conserved between the human and rat sequences. Because the slowly activating potassium current elicited by the human protein on its expression in Xenopus oocytes is indistinguishable from that induced by the rat protein, the sequence conserved at the transmembrane domain and its following sequence should play an essential role in the induction of selective K+ permeation.

Measurement of multidrug-resistance messenger RNA in urogenital cancers; elevated expression in renal cell carcinoma is associated with intrinsic drug resistance.

We measured the levels of messenger RNA of the human multidrug-resistance gene (MDR1) in 25 urogenital tumors before chemotherapy. Many of the renal cell carcinomas continued to express MDR1 gene at high levels, reflecting the increased expression of MDR1 RNA in normal kidneys. In other urogenital tumors, the MDR1 RNA levels were low reflecting low MDR1 RNA levels in normal bladder, prostate and testis. For comparative purposes, we performed in vitro chemosensitivity testing on many tumor samples using soft agar culture techniques. Vinblastine sensitivity in vitro inversely correlated with MDR1 RNA levels (p less than 0.01). Moreover, mean sensitivity of seven renal cell carcinomas to vinblastin was significantly lower than that of the other seven cancers (p less than 0.05). As for doxorubicin, mean sensitivity of six renal cell carcinomas was lower than the others (p less than 0.1). These results suggest that the high MDR1 RNA levels in renal cell carcinomas are associated with intrinsic multidrug-resistance.

Cellular localization of rat Isk protein in the stria vascularis by immunohistochemical observation.

A novel rat membrane protein, termed Isk protein, that exhibits a voltage-dependent potassium channel activity was first reported through molecular cloning combined with an electrophysiological assay (Takumi et al., 1988). In the present study, we made an attempt to identify the cellular localization of the rat Isk protein in the stria vascularis using two types of antibodies that specifically react with the distinct parts of the rat Isk protein. Immunohistochemical analysis showed that the rat Isk protein was present only on the endolymphatic surface of the marginal cell. The possibility that the Isk protein is involved in potassium permeation in the luminal membrane of the marginal cell will be also discussed.

Augmented expression of atrial natriuretic polypeptide gene in ventricles of spontaneously hypertensive rats (SHR) and SHR-stroke prone.

Tissue levels of atrial natriuretic polypeptide (ANP) and ANP messenger RNA (mRNA) in the atrium and ventricle were measured simultaneously in spontaneously hypertensive rats (SHR) and its substrain, SHR-stroke prone (SHRSP), and these levels were compared with those in control Wistar-Kyoto rats (WKY). At 27 weeks of age with established hypertension and ventricular hypertrophy, ANPmRNA levels in ventricles from SHR and SHRSP were markedly increased, and total contents of ventricular ANPmRNA in SHR and SHRSP were approximately 50% and 250%, respectively, of the corresponding atrial contents. However, levels and total contents of atrial ANPmRNA in SHR and SHRSP were similar to those of WKY, and the total content of ventricular ANPmRNA in WKY was only 6% of the content of atrial ANPmRNA. ANP concentrations in ventricles of SHR and SHRSP were increased in association with the augmentation of ANPmRNA levels. During the prehypertensive stage at 6 weeks of age, slight increases in levels and total contents of ANPmRNA and ANP in the ventricle were observed only in SHRSP. These results demonstrate that the expression of the ANP gene is markedly augmented in ventricles of SHR and SHRSP, especially of SHRSP, at the stage of established hypertension and ventricular hypertrophy, and they also suggest that these genetically hypertensive rats are one of the best animal models to investigate the biosynthesis, storage, and secretion of ventricular ANP.

Angiotensinogen gene expression in extrahepatic rat tissues: Application of a solution hybridization assay

SummaryAngiotensin II has numerous biological effects in a hitherto unsuspected variety of tissues. The generation of angiotensin in tissue requires the local presence of its high molecular weight precursor angiotensinogen and is best tested by investigating angiotensinogen gene expression. A quantitative solution hybridization assay for rapid and sensitive measurement of angiotensinogen mRNA was therefore established to study the extrahepatic expression of the angiotensinogen gene. We used a 714 bases BamHI angiotensinogen cDNA fragment cloned into vector pSPT18 and developed a sensitive and rapid assay with a detection limit of 0.5 pg RNA. Quantification of angiotensinogen mRNA from male Sprague-Dawley rats resulted in the following tissue levels (n = 10 for all tissues, except pituitary where n = 5), was expressed as fg mRNA per jig total RNA, in descending order: liver (9950), hypothalamus (6050), midbrain (4450), brainstem (3950), total brain (2325), aorta (625), kidney (338), adrenal gland (170), and heart atrium (140). The high sensitivity of the assay in addition also allowed for the first time measurement of angiotensinogen mRNA in the low gene expression tissues pituitary (70), heart ventricle (30), and testis (30). This assay will allow detailed studies on the regulation of tissue angiotensinogen and the pathophysiological role of the tissue renin angiotensin systems.

Immunohistochemical Study of a Rat Membrane Protein which Induces a Selective Potassium Permeation: Its Localization in the Apical Membrane Portion of Epithelial Cells

SummaryWe previously reported a novel rat membrane protein that exhibits a voltage-dependent potassium channel activity on the basis of molecular cloning combined with an electrophysiological assay. This protein, termedIsK protein, is small and different from the conventional potassium channel poroteins but induces selective permeation of potassium ions on its expression inXenopus oocytes. In this investigatiion, we examined cellular localization of ratIsK protein by preparing three different types of antibody that specifically reacts with a distinct part of ratIsK protein. Immunohistochemical analysis using these antibody preparations demonstrated that ratIsK protein is confined to the apical membrane portion of epithelial cells in the proximal tubule of the kidney, the submandibular duct and the uterine endometrium. The observed tissue distribution of ratIsK protein was consistent with that of theIsK protein mRNA determined by blot hybridization analysis. In epithelial cells the sodium, potassium-ATPase pump in the basolateral membrane generats a sodium gradient acrossthe epithelial cell and allows sodium ions to entere the cell through the apical membrane. Thus, taking into account the cellular localization of theIsK protein, together with its electrophysiological properties, we discussed a possible function of theIsK protein, namely that this protein is involved in potassium permeation in the apical membrane of epithelial cells through the depolarizing effect of sodium entry.

Induction of rat liver angiotensinogen mRNA following acute inflammation

Inflammatory responses of the angiotensinogen mRNA in rat liver and brain were examined by RNA blot-hybridization analysis with use of a cDNA probe specific for rat angiotensinogen. The angiotensinogen mRNA in the liver increased rapidly during the first 5 h following the administration of Escherichia coli lipopolysaccharide, and at maximum level of induction, the mRNA increased approximately 5-fold over its normal level. The levels of the mRNA increased with increasing doses of lipopolysaccharide, the half-maximal dose being approximately 1 microgram/100 g body weight. In contrast, no such increase was observed in the brain angiotensinogen mRNA. Thus, the expression of the rat angiotensinogen mRNA is regulated in a tissue-specific manner in response to induction of acute inflammation.

Atrial natriuretic polypeptide (ANP) in human ventricle increased gene expression of ANP in dilated cardiomyopathy

Tissue levels of atrial natriuretic polypeptide (ANP) messenger RNA (ANPmRNA) and ANP in the human atrium and ventricle were measured simultaneously by the blot hybridization technique and the specific radioimmunoassay for ANP. Hearts were obtained from two patients without cardiac complications and from a patient with dilated cardiomyopathy (DCM) at autopsy. Total RNA extracted from ventricles contained a hybridizing RNA band of the same size as atrial ANPmRNA in both control and DCM hearts. The ANPmRNA level in the control ventricle was 0.2% of that in the atrium. The ANPmRNA level in the DCM ventricle increased to about 7% of that in the corresponding atrium and was approximately 40 times higher than that in the control ventricle, although the ANPmRNA level in the DCM atrium was comparable to that in the control atrium. The total content of ANPmRNA in the DCM ventricle reached about 30% of that in the corresponding atrium and was much the same as that in the control atrium. The ANP level in the DCM ventricle was approximately 1.0 microgram/g and much higher than that in the control ventricle (0.02 microgram/g).

Pre- and postsynaptic features of the central angiotensin systems. Indications for a role of angiotensin peptides in volume transmission and for interactions with central monoamine neurons.

The transmitter receptor matches and mismatches in the angiotensin (ANG) immunoreactive (IR) neuronal systems of the rat CNS have been characterized in various regions by means of ANG II immunocytochemistry and 125I-angiotensin II receptor autoradiography. By means of in situ hybridization the distribution of angiotensinogen mRNA has been mapped out and related to the distribution of ANG IR. In some areas, high densities of ANG IR nerve terminals and ANG II receptors (e.g. paraventricular hypothalamic nucleus, locus coeruleus and nucleus tractus solitarius) or high densities of ANG II receptors alone (e.g. medial geniculate body, subthalamic nucleus and superficial layer of the superior collicle) were often associated with high levels of angiotensinogen mRNA, suggesting the existence of an extracellular formation of ANG II, mediating biological responses. These results underline a role of ANG peptides in volume transmission in addition to transmitter function. Other areas, such as nuc. n. hypoglossi, practically lacking ANG IR terminals, pericarya and receptors, also contained high levels of angiotensinogen mRNA, suggesting a different role of angiotensinogen in these areas. Evidence for presynaptic (turnover changes) and post-synaptic (receptor-receptor crosstalk) interactions with CA neuronal systems has been obtained especially in cardiovascular centers. Thus, ANG II reduces in a concentration related way the affinity of 3H-paraminoclonidine binding sites in the dorsomedial medulla without influencing the Bmax value. These results indicate the existence of intramembrane interactions between ANG II and alpha 2 adrenergic receptors. Finally paraventricular but not perifornical ANG immunoreactive nerve cells costore nuclear glucocorticoid receptor IR, suggesting that some ANG neurons may be directly regulated by glucocorticoids.

Cloning of a full-length cDNA encoding bovine thymus poly(ADP-ribose) synthetase: evolutionarily conserved segments and their potential functions.

The primary structure of bovine thymus poly(ADP-ribose) synthetase, as deduced from the nucleotide sequence of a cloned cDNA, indicated that this enzyme is composed of 1016 amino acids (aa) with an Mr of 113,481. An abundance of Lys and Arg residues was in accord with the known basic nature of this protein. A comparison with reported sequences of human counterparts revealed: (1) three functional domains separated by partial proteolysis, i.e., DNA-binding (N-terminal), automodification (central), and NAD-binding (C-terminal) domains, have, in this order, increasing degrees of homology; (2) the DNA-binding domain is composed of two distinct regions: one, less conserved, containing zinc-binding fingers and the other, more conserved, containing helix-turn-helix motifs; (3) all Glu and Asp residues in the automodification domain are conserved; and (4) a 78-aa stretch encompassing the nucleotide-binding fold in the NAD-binding domain is completely conserved. These results are compatible with specific features of each domain, i.e., complex DNA-enzyme interactions, multiple automodification at acidic aa residues, and a stringent specificity for the substrate, NAD.