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Y Kohara

Publications and source records attributed to Y Kohara.

At least 55 records · Page 3Linked to original sources
PubMed1996

A multi-well version of in situ hybridization on whole mount embryos of Caenorhabditis elegans.

We report an efficient procedure for in situ hybridization with a multi-well format on Caenorhabditis elegans embryos for large scale screening of gene expression patterns in this organism. Each hybridization well contains embryos at various stages throughout embryogenesis. The validity of the method was confirmed through results with control genes whose expression patterns have been reported; glp-1 in very early embryos, myo-2 in pharyngeal muscle and unc-54 in body wall muscle. Several collagen genes and a pepsinogen gene were also examined to establish a set of lineage-specific markers. As a pilot project, we examined approximately 100 unique cDNA species classified by our cDNA project, finding that approximately 10% of the cDNA groups were expressed in specific cells and at specific stages.

Animals↗
PubMed1996

Selecting and amplifying one fragment from a DNA fragment mixture by polymerase chain reaction with a pair of selective primers.

A new method for selecting and amplifying a single DNA fragment from a mixture is proposed. This method is applicable for the rapid classification of DNA fragments from a mixture and for preparation of sequencing templates. DNAs of several to tens of kilobases (kb) are digested with a four-base recognition restriction enzyme to produce smaller fragments. The complementary strand extension reactions are then carried out to produce fluorophore-labeled DNA fragments from the digestion products. These fragments can be rapidly classified according to their terminal-base sequences and their sizes are analyzed by capillary-array gel electrophoresis (CAGE). Electropherograms are used to characterize the fragments and to select polymerase chain reaction (PCR) primers. Any fragment in a digestion mixture can be amplified by PCR with a pair of primers selected from a primer pool by referring to the electropherograms of the fragments. This method was successfully used to compare the electropherograms of two different DNA strands and to sequence a several-kb DNA fragment without subcloning. Combined with CAGE, this method could be used to dramatically simplify DNA fragment analysis.

DNA↗
PubMed1996

Two genes controlling acute phase responses by the antitumor polysacch aride, lentinan.

Lentinan, a beta-1,6;1,3-glucan, is tumor-specific for transplantable mouse solid-type tumors and it also stimulates the production of acute phase proteins (APPs). The APP response to lentinan is of the delayed type (DT-APR) and differs from that to lipopolysaccharide, which is acute. We found that the responses were genetically controlled in mice and that low responsiveness is dominant (Maeda et al. 1991). Using 123 segregants of crosses between SWR/J (a high responder) and Mus spretus (a low responder), we analyzed the linkage between DT-APR responsiveness and the DNA polymerase chain reaction-simple sequence length polymorphism (PCR-SSLP) phenotype using 80 chromosome-specific microsatellite markers. We identified two loci (ltn1.1 and ltn1.2) responsible for DT-APR. ltn1.1 is closely linked to D3Mit11 on chromosome 3 and ltn1.2 to D11Nds9 on chromosome 11 (P <0.001). The linkage analysis also suggested that ltn1.2 is the major determinant for DT-APR. Correlation between lentinan-specific IL-6 mRNA expression (the late expression) controlled recessively and DT-APR induction suggests that the ltn1 loci control some process(es) of IL-6 expression in the regulation step before NF-IL6.

Acute-Phase Reaction↗
PubMed1995

Meiotic recombination, noncoding DNA and genomic organization in Caenorhabditis elegans.

The genetic map of each Caenorhabditis elegans chromosome has a central gene cluster (less pronounced on the X chromosome) that contains most of the mutationally defined genes. Many linkage group termini also have clusters, though involving fewer loci. We examine the factors shaping the genetic map by analyzing the rate of recombination and gene density across the genome using the positions of cloned genes and random cDNA clones from the physical map. Each chromosome has a central gene-dense region (more diffuse on the X) with discrete boundaries, flanked by gene-poor regions. Only autosomes have reduced rates of recombination in these gene-dense regions. Cluster boundaries appear discrete also by recombination rate, and the boundaries defined by recombination rate and gene density mostly, but not always, coincide. Terminal clusters have greater gene densities than the adjoining arm but similar recombination rates. Thus, unlike in other species, most exchange in C. elegans occurs in gene-poor regions. The recombination rate across each cluster is constant and similar; and cluster size and gene number per chromosome are independent of the physical size of chromosomes. We propose a model of how this genome organization arose.

Animals↗
PubMed1994

Targeted mutations in the Caenorhabditis elegans POU homeo box gene ceh-18 cause defects in oocyte cell cycle arrest, gonad migration, and epidermal differentiation.

We used targeted gene inactivation to analyze the function of a Caenorhabditis elegans POU gene, ceh-18, and to dissect its functional domains in vivo. In ceh-18 mutants, oocytes exhibit an incompletely penetrant failure to arrest in diakinesis of meiotic prophase I and instead undergo multiple rounds of DNA replication without cytokinesis. ceh-18 is expressed in the gonadal sheath cells that signal the oocyte, but not in the oocyte. This suggests that ceh-18 affects, directly or indirectly, a sheath cell signal that causes oocytes to maintain diakinesis arrest. ceh-18 also participates in directing gonad migration and in specifying the differentiated phenotypes of epidermal cells during postembryonic development. Analysis of targeted deletions that disrupt half of the POU domain selectively by deleting either the POUhd or the POUsp alone, indicates that each CEH-18 POU subdomain is sufficient for partial activity in vivo.

Amino Acid Sequence↗
PubMed1994

Antihypertensive effects of a highly potent and long-acting angiotensin II subtype-1 receptor antagonist, (+-)-1-(cyclohexyloxycarbonyloxy)ethyl 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H- benzimidazole-7-carboxylate (TCV-116), in various hypertensive rats.

The antihypertensive effects of (+-)-(cyclohexyloxycarbonyloxy)ethyl2-ethoxy-1-[[2'-(1H- tetrazol-5- yl)biphenyl-4-yl]methyl]-1-H-benzimidazole-7-carboxylate (TCV-116), an angiotensin II (AII) subtype-1 receptor antagonist, were studied in various hypertensive and normotensive rats, using 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)bip hen yl-4- yl)methyl]-imidazole, potassium salt (losartan) as a reference compound. TCV-116 is a prodrug, which is converted in vivo to the active component, 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl)]methyl]-1H- benzimidazole-7-carboxylic acid (CV-11974). In spontaneously hypertensive rats (SHR) p.o. TCV-116 (0.1 mg/kg) demonstrated a sustained antihypertensive effect that lasted for more than 10 hr and the dose that reduced the blood pressure by an average of 25 mm Hg for 24 hr (ED25), was 0.68 mg/kg. Intravenous CV-11974 reduced the blood pressure with an ED25 of 0.0027 mg/kg. Repeated p.o. administration of TCV-116 (1 mg/kg) to SHR once daily for 2 weeks reduced the blood pressure by 30 to 50 mm Hg over 24 hr without any heart rate changes. The antihypertensive effects of TCV-116 correlated well with the inhibition of angiotensin II-induced contractile responses of aortic strips prepared ex vivo after p.o. administration of TCV-116. Oral TCV-116 had a sustained antihypertensive effect with ED25 of 0.03 and 0.23 mg/kg in two-kidney, one-clip and one-kidney, one-clip hypertensive rats, respectively, and was much more potent in SHR and renal-hypertensive rats than losartan.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin Receptor Antagonists↗
PubMed1993

Nonpeptide angiotensin II receptor antagonists. Synthesis and biological activity of potential prodrugs of benzimidazole-7-carboxylic acids.

In order to improve the oral bioavailability (BA) of 2-butyl-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimid azole - 7-carboxylic acid (3: CV-11194) and 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4- yl]methyl]-1H-benzimidazole-7-carboxylic acid (4: CV-11974), novel angiotensin II (AII) receptor antagonists, chemical modification to yield prodrugs has been examined. After selective tritylation of the tetrazole rings in 3 and 4, treatment of N-tritylated benzimidazole-7-carboxylic acids (6, 7) with a variety of alkyl halides, followed by deprotection with hydrochloric acid, afforded esters of 3 and 4. Mainly 1-(acyloxy)alkyl esters and 1-[(alkoxycarbonyl)oxy]alkyl esters, double ester derivatives, were synthesized. Their inhibitory effect on AII-induced pressor response in rats and oral BA were investigated. (Pivaloyloxy)methyl and (+/-)-1-[[(cyclohexyloxy)-carbonyl]oxy]ethyl esters of 3 and 4 showed marked increases in oral bioavailability which significantly potentiated the inhibitory effect of the parent compounds on AII-induced pressor response. Among them, (+/-)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl 2- ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimida zole- 7-carboxylate (10s, TCV-116) was selected as a candidate for clinical evaluation.

Administration, Oral↗
PubMed1993

Nonpeptide angiotensin II receptor antagonists. Synthesis and biological activity of benzimidazolecarboxylic acids.

A series of 2-substituted-1-[(biphenyl-4-yl)methyl]-1H-benzimidazole-7- carboxylic acids was prepared from the key intermediate 3-amino-2-[[(biphenyl-4- yl)methyl]amino]benzoate (6a-c) in order to clarify the structure-activity relationships of various analogues of 2-butyl-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-ben zimidazole- 7-carboxylic acid (CV-11194), a potent and long acting angiotensin II (AII) receptor antagonist. The AII antagonistic activity of the benzimidazoles was investigated by in vitro assays, which included an AII receptor binding assay and AII-induced vasocontraction assay, as well as by in vivo assays such as an AII-induced pressor response in rats. Most of the benzimidazoles showed high affinity for the AII receptor (IC50 value, 10(-6)-10(-7) M) and inhibited the AII-induced pressor response at 1 or 3 mg/kg po, and the effects were more potent than those of CV-11194 and DuP 753. The structure-activity relationship studies on the binding affinity and the inhibition of AII-induced pressor response suggested that straight chains of a certain length (e.g., ethoxy groups, ethyl groups) were the best as substituents at the 2-position and that their steric factors, lipophilicity, and electronic effects affected the potency of the AII antagonistic action. Both a carboxyl group at the 7-position and a tetrazole ring at the 2'-position were particularly important for potent and orally active AII antagonistic activity and a long-acting hypotensive effect. The representative compound, 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H- benzimidazole-7-carboxylic acid (26b, CV-11974), inhibited the specific binding of [125I]AII to bovine adrenal cortical membrane with an IC50 value of 1.1 x 10(-7) M. The AII-induced contraction of rabbit aortic strips was antagonized by CV-11974 (IC50 value, 3.0 x 10(-10) M). Oral administration of CV-11974 to conscious normotensive rats at 1 mg/kg resulted in long-lasting inhibition of the AII-induced pressor response. CV-11974 at 0.1-1 mg/kg iv reduced blood pressure dose-dependently in spontaneously hypertensive rats.

Angiotensin Receptor Antagonists↗
PubMed1993

Inhibition of rabbit aortic angiotensin II (AII) receptor by CV-11974, a new nonpeptide AII antagonist.

The angiotensin II (AII) antagonistic action of CV-11974 (2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl] benzimidazole-7-carboxylic acid) was investigated in an AII-receptor binding assay using rabbit aortic membranes and an AII-induced contraction assay using rabbit aortic strips. A single class of [125I]AII-(Sar1,Ile8) binding sites was found in the membranes with a dissociation constant (Kd) of 0.15 nM and a receptor concentration (Bmax) of 86.9 fmol/mg protein. CV-11974 markedly reduced Kd without affecting Bmax. The specific binding of [125I]AII-(Sar1,Ile8) in this preparation was inhibited completely by CV-11974 [the inhibition constant (Ki) = 0.64 nM], DuP 753 [an angiotensin II type I (AT1) receptor-selective antagonist] (Ki = 51 nM) and EXP3174 (an active metabolite of DuP 753) (Ki = 6.8 nM), but was not affected by PD123177 (an AT2 receptor-selective antagonist). These results suggest that the single binding site in rabbit aortic membranes is an AT1 receptor subtype. The affinity of CV-11974 to these AT1 receptors was approximately 80 and 10 times higher than that of DuP 753 and EXP3174, respectively. CV-11974 showed no appreciable affinity for the AT2 receptors found in bovine cerebellum. In the in vitro functional study, CV-11974 markedly reduced the AII-induced maximal contractile response of rabbit aortic strips (pD'2 = 9.97). In contrast, Compound 7-H, which lacks the carboxyl group at the benzimidazole ring of CV-11974, inhibited the contraction in a competitive manner. The inhibition by CV-11974 was long lasting. These results suggest that CV-11974 is a potent and long-acting AT1 receptor-selective, competitive antagonist. The carboxyl group at the benzimidazole ring plays an important role in the interaction between CV-11974 and the AT1 receptor.

Angiotensin II↗
PubMed1993

Nonpeptide angiotensin II receptor antagonists. Synthesis and biological activity of benzimidazoles.

A series of substituted 2-butylbenzimidazoles bearing a biphenylylmethyl moiety at the 1-position was prepared via three synthetic routes and evaluated for angiotensin II (AII) receptor antagonistic activity (in vitro and in vivo). Binding affinity was determined using bovine adrenal cortical membrane. Substitution at the 4-, 5-, or 6-position reduced the affinity relative to that of the unsubstituted compound (13a). However, most of the compounds with a substituent at the 7-position showed binding affinity comparable to that of DuP 753 (losartan). In functional studies, a carboxyl group was found to be very important for antagonistic activity against AII. Comparison of 2-butyl-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]- 1H-benzimidazole-4-, -5-, -6-, and -7-carboxylic acids (15a-d) in an AII-induced rabbit aortic ring contraction assay clearly demonstrated the importance of the substitutional position of the carboxyl group. In an in vivo assay, oral administration of benzimidazole-7-carboxylic acids caused long-lasting inhibition of the AII-induced pressor response in rats. The optimum substituent at the 7-position of the benzimidazole ring was found to be a carboxyl or an ester group. The representative compound, 2-butyl-1-[[2'- (1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxy lic acid (15d, CV-11194), inhibited the specific binding of [125I]AII to bovine adrenal cortical membrane with an IC50 value of 5.5 x 10(-7) M. The AII-induced contraction of rabbit aortic strips was antagonized by CV-11194 (IC50 value, 5.5 x 10(-11) M), while the compound had no effect on the contraction induced by norepinephrine or KCl. Orally administered CV-11194 at doses of 0.3-10 mg/kg dose-dependently inhibited the AII-induced pressor response in rats and dogs. CV-11194 at 1 mg/kg po reduced blood pressure in spontaneously hypertensive rats (SHR). The three-dimensional molecular structure of CV-11194 was determined by X-ray diffraction.

Adrenal Cortex↗
PubMed1993

Pharmacological profile of a highly potent and long-acting angiotensin II receptor antagonist, 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4- yl]methyl]-1H-benzimidazole-7-carboxylic acid (CV-11974), and its prodrug, (+/-)-1-(cyclohexyloxycarbonyloxy)-ethyl 2-ethoxy-1-[[2'-(1H-tetrazol-5- yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylate (TCV-116).

The angiotensin II (AII) antagonistic action of 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl-1H-benzi mid azole-7 - carboxylic acid (CV-11974) was examined in in vitro assay systems, including AII receptor binding assay using membrane fractions of bovine adrenal cortex or rabbit aorta and AII-induced contraction assay using rabbit aortic strips, and CV-11974 and its prodrug, (+/-)1-(cyclohexyloxycarbonyloxy)ethyl 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H- benzimidazole-7-carboxylate (TCV-116), were examined in an in vivo system of AII-induced pressor response in conscious rats. DuP 753 or EXP3174 (the main active metabolite of DuP 753) was used as the reference compound. CV-11974 inhibited the binding of [125I] AII to the bovine adrenal cortical membrane and rabbit aortic membrane with IC50 values of 1.12 x 10(-7) and 2.86 x 10(-8) M, respectively. Similar results were obtained with EXP3174. CV-11974 interacted with AII in these membrane fractions with subtype 1 receptor in a competitive manner. CV-11974 at 10(-5) M did not affect the binding of [125I]AII to subtype 2 (AT2) receptor in bovine cerebellum. CV-11974 selectively inhibited the AII-induced contraction of rabbit aortic strips in a noncompetitive manner (pD' 2, 9.97); it had no effects on the contraction induced by norepinephrine, KCl, serotonin, prostaglandin F2 alpha or endothelin. EXP3174 showed a pD'2 value of 8.95 for the AII-induced contraction. CV-11974 given intravenously and TCV-116 given orally inhibited the AII-induced pressor response in rats with ID50 values of 0.033 mg/kg and 0.069 mg/kg, respectively. These effects of CV-11974 and TCV-116 were 12 and 48 times more potent than those of EXP3174 and DuP 753, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1992

Identification of a homeobox-containing gene located between lin-45 and unc-24 on chromosome IV in the nematode Caenorhabditis elegans.

Using two primers corresponding to helix 1 and helix 3 regions in the homeodomain, we subjected genomic DNA from Caenorhabditis elegans to amplification by the polymerase chain reaction. Sequence analysis of the amplified products revealed a new homeobox-containing gene, designated ceh-19. This gene was located between lin-45 and unc-24 on chromosome IV where no homeogene has previously been mapped.

Amino Acid Sequence↗
PubMed1992

Correlation of a subset of the pLC plasmids to the physical map of Escherichia coli K-12.

We determined map positions of the Escherichia coli K-12 portions of a subset of the hybrid E. coli-ColE1 plasmids constructed by Clarke and Carbon. The probe DNA of pLC plasmids was labeled with digoxigenine-dUTP, hybridized to the 476 phage clones of the E. coli ordered clone bank miniset, which was adsorbed on a strip of nylon membrane filters, and detected by enzyme-linked immunoassay and a subsequent enzyme-catalyzed color reaction. The total number of Clarke-Carbon plasmids we analyzed was 518, for which chromosomal locations of 297 clones were newly determined in the present study. Another 180 plasmids gave results that agreed with those reported previously, and the remaining 41 plasmids gave map positions different from those described in the previous report. A chromosome map of E. coli which shows the locations of 518 pLC plasmids on it is presented, as well as a table which correlates the pLC plasmids with the clones of the E. coli ordered clone bank miniset on the basis of the hybridization data. We estimate that approximately one-half of the entire genome of E. coli was covered by the pLC plasmids used in this study.

Chromosome Mapping↗