Molecular cloning of rat transmembrane domain protein of 40 kDa regulated in adipocytes and its expression in H9c2 cells exposed to ischemic hypoxia and reoxygenation.

We clone a 1230 bp complementary DNA encoding rat transmembrane domain protein of 40 kDa regulated in adipocytes (TPRA40), an orphan receptor, by reverse transcription-polymerase chain reaction using H9c2 cells derived from embryonic rat heart. The deduced amino acid sequence of rat TPRA40 consists of 369 amino acids and has a longer carboxyl terminus than that of the mouse protein. The level of TPRA40 mRNA decreases significantly throughout ischemic hypoxia and reoxygenation.

Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H(2)O(2) stimulation.

Cytokines and various cellular stresses are known to activate c-Jun N-terminal kinase-1 (JNK1), which is involved in physiological function. Here, we investigate the activation of JNK1 by oxidative stress in H9c2 cells derived from rat cardiomyocytes. H(2)O(2) (100 microM) significantly induces the tyrosine phosphorylation of JNK1 with a peak 25 min after the stimulation. The amount of JNK1 protein remains almost constant during stimulation. Immunocytochemical observation shows that JNK1 staining in the nucleus is enhanced after H(2)O(2) stimulation. To clarify the physiological role of JNK1 activation under these conditions, we transfected antisense JNK1 DNA into H9c2 cells. The antisense DNA (2 microM) inhibits JNK1 expression by 80% as compared with expression in the presence of the sense DNA, and significantly blocks H(2)O(2)-induced cell death. Consistent with the decrease in cell number, we detected condensation of the nuclei, a hallmark of apoptosis, 3 h after H(2)O(2) stimulation in the presence of the sense DNA for JNK1. The antisense DNA of JNK1 inhibits the condensation of nuclei by H(2)O(2). Under these conditions, the H(2)O(2)-induced phosphorylation of proteins with molecular masses of 55, 72, and 78 kDa is blocked by treatment with the antisense DNA for JNK1 as compared with the sense DNA for JNK1. These findings suggest that JNK1 induces apoptotic cell death in response to H(2)O(2), and that the cell death may be involved in the phosphorylations of 55, 72, and 78 kDa proteins induced by JNK1 activation.

Isolation of 88F actin mutants of Drosophila melanogaster and possible alterations in the mutant actin structures.

The 88F actin (act88F) gene of Drosophila, melanogaster encodes an actin isoform that is expressed exclusively in the indirect flight muscle. In order to isolate a large number of act88F mutants, an efficient screening method was used to obtain dominant flightless mutants. Genetic analyses revealed that 25 mutations were located near or at the act88F locus. From each mutant strain, the DNA fragments including the coding region of the act88F gene were asymmetrically amplified by the polymerase chain reaction method, and the amplified fragments were directly sequenced. Eighteen of them were found to have point mutations within their coding regions. Of these, 13 were novel alleles of this gene. We have characterised these mutations in detail. First, their flight abilities were tested after introducing two normal alleles of this gene. Second, two-dimensional gel electrophoresis was used to examine actin isoforms and whole thorax proteins. Third, morphological anomalies of indirect flight muscle fibres and myofibrils were examined with an optical microscope. On the basis of these phenotypes and the known atomic structure of actin, possible alterations in the structure of actin brought about by these mutations are discussed.

Disorder of cerebellar foliation in Walker's lissencephaly and neu-laxova syndrome.

A diffuse disorder of cerebellar foliation was found in eight infants and one fetus with Walker's lissencephaly. The cerebellar cortex consisted of fused and irregularly distorted folia. In the white matter, trilaminated rings of cortex were concentrically arranged around blood vessels and mesenchymal tissue. The normal relative position of the different classes of cortical nerve cells was preserved. Cells of the external granular layer invaded the meninges and migrated along penetrating blood vessels. We believe that this foliation disorder is caused by a defect in the external basal lamina that allows adjacent folia to be fused and sulci obliterated by intrameningeal ectopias of external granule layer cells. Physical forces applied during development probably contribute to the distortion of the gyral pattern. There was a volumetric reduction of the neocerebellum, which might also be a consequence of the basal lamina defect. The cerebellum of a fetus with the Neu-Laxova syndrome showed the same abnormalities as in Walker's lissencephaly. It is postulated that these two conditions belong to a class of prenatal developmental disorders that involves a defect of the extracellular matrix.

Eicosapentaenoic acid (EPA) induces Ca(2+)-independent activation and translocation of endothelial nitric oxide synthase and endothelium-dependent vasorelaxation.

Eicosapentaenoic acid (EPA), but not its metabolites (docosapentaenoic acid and docosahexaenoic acid), stimulated nitric oxide (NO) production in endothelial cells in situ and induced endothelium-dependent relaxation of bovine coronary arteries precontracted with U46619. EPA induced a greater production of NO, but a much smaller and more transient elevation of intracellular Ca(2+) concentration ([Ca(2+)]i), than did a Ca(2+) ionophore (ionomycin). EPA stimulated NO production even in endothelial cells in situ loaded with a cytosolic Ca(2+) chelator 1,2-bis-o-aminophenoxythamine-N',N',N'-tetraacetic acid, which abolished the [Ca(2+)]i elevations induced by ATP and EPA. The EPA-induced vasorelaxation was inhibited by N(omega)-nitro-L-arginine methyl ester. Immunostaining analysis of endothelial NO synthase (eNOS) and caveolin-1 in cultured endothelial cells revealed eNOS to be colocalized with caveolin in the cell membrane at a resting state, while EPA stimulated the translocation of eNOS to the cytosol and its dissociation from caveolin, to an extent comparable to that of the eNOS translocation induced by a [Ca(2+)]i-elevating agonist (10 microM bradykinin). Thus, EPA induces Ca(2+)-independent activation and translocation of eNOS and endothelium-dependent vasorelaxation.

Sphingosylphosphorylcholine induces Ca(2+)-sensitization of vascular smooth muscle contraction: possible involvement of rho-kinase.

Sphingosylphosphorylcholine (SPC), a sphingolipid, concentration-dependently (1-50 microM) induced contraction and slight elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in smooth muscle of the pig coronary artery, the result being a marked increase in the force/[Ca(2+)](i) ratio. In alpha-toxin- or beta-escin-permeabilized, but not Triton X-100-permeabilized, vascular strips, SPC induced contraction at constant [Ca(2+)](i) (pCa 6.3) in the absence of GTP, whereas a G-protein-coupled receptor agonist, histamine, required the presence of GTP to induce the contraction. The Rho-kinase blocker, Y-27632 (10 microM) abolished the SPC-induced Ca(2+)-sensitization, without affecting the Ca(2+)-induced contraction. These results suggest that SPC induces Ca(2+)-sensitization of force in vascular smooth muscle, presumably through the activation of Rho-kinase (or a related kinase).

Sphingosylphosphorylcholine induces cytosolic Ca(2+) elevation in endothelial cells in situ and causes endothelium-dependent relaxation through nitric oxide production in bovine coronary artery.

Sphingosylphosphorylcholine (SPC) increased intracellular Ca(2+) concentration ([Ca(2+)]i) and nitric oxide (NO) production in endothelial cells in situ on bovine aortic valves, and induced endothelium-dependent relaxation of bovine coronary arteries precontracted with U-46619. The SPC-induced vasorelaxation was inhibited by N(omega)-monomethyl-L-arginine, an inhibitor of both constitutive and inducible NO synthase (NOS), but not by 1-(2-trifluoromethylphenyl) imidazole, an inhibitor of inducible NOS (iNOS). Immunoblotting revealed that endothelial constitutive NOS, but not iNOS, was present in endothelial cells in situ on the bovine aortic valves. We propose that SPC activates [Ca(2+)]i levels and NO production of endothelial cells in situ, thereby causing an endothelium-dependent vasorelaxation.

Identification of Drosophila indirect flight muscle myofibrillar proteins by means of two-dimensional electrophoresis.

When proteins of whole Drosophila thorax were analyzed by two-dimensional gel electrophoresis, 186 spots were detected by protein staining with Coomassie brilliant blue R-250. Two methods were developed to identify proteins which exist in indirect flight muscle (IFM) and its myofibrils. 1) A whole fly was freeze-dried in a dry ice-acetone mixture, and indirect flight muscle fibers were cleanly dissected out from the thorax. The muscle cells and the rest of the thorax were analyzed separately. The muscle contained 146 polypeptides, of which 12 were not detected elsewhere. 2) Flies were frozen in liquid nitrogen and shaken vigorously so that their thoraces broke off from heads and abdomens. The thoraces were separated from the rest by sieving and centrifugation. After homogenization of the thorax, myofibrils were prepared by centrifugation in a discontinuous sucrose density gradient. The myofibril fraction contained at least 20 proteins. There were two types of actin (II and III), myosin heavy chain, tropomyosin and paramyosin. Nine of the other myofibrillar proteins were specific to this muscle.

Heat shock gene activation by mutant actin is independent of myofibril degeneration in Drosophila muscle.

Artificially mutagenized Drosophila Act88F actin genes with triple and double mutations were expressed in the indirect flight muscles of transgenic flies. The triple mutant actin, GD245T (Gly-36----Glu, Glu-83----Asp, and Gly-245----Asp), induced heat shock protein (hsp) synthesis without affecting flight ability. On the other hand, the double mutation, GD245D (Gly-36----Glu and Glu-83----Asp), disrupted myofibrils but induced little hsp synthesis. These results demonstrate that myofibril degeneration is not the primary cause of the anomalous heat shock gene activation by mutant actins.

Actin with tumor-related mutation is antimorphic in Drosophila muscle: two distinct modes of myofibrillar disruption by antimorphic actins.

A mutant beta-actin with an amino acid substitution from Gly-245 to Asp has been shown to be related to tumorigenic transformation of a human fibroblast cell line (Leavitt, J. et al. (1987) Mol. Cell. Biol. 7, 2467-2476). To examine the effects of this mutation, we artificially introduced the same amino acid change into the Act88F actin gene of Drosophila melanogaster. The gene (Act88FGD245) was inserted in the Drosophila genome to make transgenic adult flies which synthesize the mutant actin in the indirect flight muscles. The mutant actin was found to be antimorphic with regard to flight and also to cause myofibrillar disruption in transformants even in the presence of two normal alleles. It was initially incorporated into myofibrils and later induced their degeneration from center to periphery. This mode of myofibrillar disruption is distinct from that of previously reported Act88F mutations, where defects are found only in the peripheral region of myofibrils. This indicates that actin functions are altered differently in the two classes of antimorphic mutations.

Hepatic actinomycosis: case report and review of the literature in Japan.

Hepatic actinomycosis is rare. We report an 86-year-old Japanese man with a 3-day history of high fever and anorexia who had an actinomycotic liver abscess complicated by disseminated intravascular coagulation (DIC). A definitive diagnosis was made when an Actinomyces species was cultured from aspirated pus. The clinical course was satisfactory. Treatment included prompt percutaneous drainage coupled with long-term intravenous administration of high-dose minocycline and piperacillin, combined with therapy for DIC. We reviewed 11 cases in Japan of Actinomyces involving the liver, including the case reported here. In most patients, there were no predisposing factors. Common symptoms and laboratory findings included fever, abdominal pain, leukocytosis, and elevated C-reactive protein. In 6 of the 11 patients a partial hepatectomy was performed because hepatic tumor was suspected. Five patients presented with a liver abscess. Hepatic actinomycosis should be considered in the differential diagnoses of pyogenic liver abscess and space-occupying lesions of the liver.

Protective effect of heparin on renal glomerular anionic sites of streptozotocin-injected rats.

Albuminuria at the rate of 500 micrograms/24 h was observed in rats treated with 50 mg/kg body wt. i.v. streptozotocin (STZ). When STZ was combined with heparin, administered twice daily (250 IU/kg subcutaneous injection) after 24 h following STZ injection, the daily urinary albumin excretion (U-AE) was less than half the amount found in non-heparinized rats (280.3 micrograms/24 h). The observation period in both instances was 8 weeks. When animals were permitted to develop albuminuria over the first 4 weeks, subsequent heparin administration for another 4 weeks lowered U-AE from 500 micrograms/24 h to less than half the amount (227.8 micrograms/24 h). A significantly negative correlation (P < 0.001) existed between U-AE and the number of anionic sites (AS) in the lamina rara externa of glomerular basement membranes, as visualized by electron microscopy. The number of AS per 1000 nm in STZ-injected rats (15.5 +/- 0.2) was lower than that in control rats (23.1 +/- 0.6); however, in heparinized animals, regardless of STZ, the values were not significantly different from normal. Heparin by itself had no effect on the number of AS in normal rats. All STZ-injected animals became hyperglycemic (400-550 mg/dl), but received no insulin. Heparin had no effect on plasma glucose levels. From these results, it is concluded that heparin suppressed both of an increase in U-AE and a decrease in AS count of glomerular basement membranes in STZ-injected rats.

Antifungal nickel(II) complexes derived from amino sugars against pathogenic yeast, Candida albicans.

Nickel(II) complexes containing N-glycosides derived from D-glucosamine (D-GlcN) and ethylenediamine (en) and trimethylenediamine (tn), [Ni(D-GlcN-en)2]Cl2.H2O (1) (D-GlcN-en = 1-¿(2-aminoethyl)amino¿-2-amino-1,2-dideoxy-D-glucose) and [Ni(D-GlcN-tn)2]Cl2.4H2O (2) (D-GlcN-tn = 1-¿(3-aminopropyl)amino¿-2-amino-1,2-dideoxy-D-glucose), are fairly stable in water at room temperature and showed effective antifungal activity against pathogenic yeast, Candida albicans, with the MIC (minimal concentration of inhibition) values of the complexes being 0.25 mM. The results obtained enzyme assays by using preparations of C. albicans chitinase fraction suggested that the sugar complexes 1 and 2 played a role of novel chitinase (chitin-degradation enzyme) inhibitor, where the modes of inhibition were competitive (Ki = 1.3 mM for 1, Ki = 1.8 mM for 2). The newly prepared nickel(II) complex 2 was characterized by elemental analysis, magnetic susceptibility, electronic absorption and circular dichroism spectroscopies, and an X-ray crystallographic analysis.

Relationship between Mn-induced contractile inhibition and cyclic AMP content in the longitudinal muscle of estrogen-treated rat uterus.

In longitudinal myometrial strips of estrogen-treated rats, nifedipine (0.03 microM) induced the gradual inhibition of phasic contractions evoked by repetitive electrical stimulations, with inhibition reaching a plateau level (43.4 +/- 2.6% of the control group, n = 6) within 20 min. In contrast, 0.5 mM induced a rapid and transient suppression of the phasic contractions (58.3 +/- 8.9% of the control group, n = 10, at 4 min) followed by sustained inhibition of the contractions, the level of which was slightly lower than that of the control group (84.2 +/- 5.0%, n = 10, at 20 min). The Mn also induced a rapid and transient increase in cellular cAMP level, which reached a peak (201.2 +/- 20.8% of the control group, n = 5) at 2 min and decreased to the prestimulation level ( = 100%) within 15 min. Pretreatment with 1 nM isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, potentiated the effect of Mn on the cAMP level. The transient effects of Mn, but not of nifedipine, on the contractions and cAMP levels (and their potentiation by IBMX) do not support the simple action of Mn as a Ca antagonist, but are compatible with the intracellular dual action of Mn on adenylate cyclase.

Influence of intracellular Mn on the contractile inhibition caused by db cAMP, forskolin, and porcine relaxin in the circular muscle of the estrogen-treated rat uterus.

Whether Mn ion permeates into circular muscle cells of the estrogen-treated rat uterus and how contractile inhibitions caused by db cAMP, forskolin, and relaxin are affected by treatment with Mn were examined. By exposing the muscle strip to 0.35 mM Mn added to Mg-free Krebs solution, the magnitude of phasic contractions evoked by electrical stimulation was reduced to 21.8 +/- 14.7% (n = 22) of control. Mn influx was measured by fura-2 fluorescence quenching at the Ca isosbestic excitation wavelength (360 nm). The contractile inhibitions caused by 30 microM db cAMP and 0.2 microM forskolin were enhanced after pretreatment with 0.35 mM Mn for 10 min, whereas the inhibition caused by 100 mU relaxin underwent enhancement or attenuation. These results are discussed in relation to those reported previously for the longitudinal muscle in which the influences of treatment with Mn were somewhat different.

[Novel mechanisms for increasing Ca2+ sensitivity of contractile apparatus in smooth muscle].

Smooth muscle contraction is primarily regulated not only by changes in cytosolic Ca2+ concentrations ([Ca2+]i) but also by changes in the force/[Ca2+]i ratio. The use of membrane-permeabilization technique facilitated demonstration of an increase in the level of force at constant [Ca2+]i (Ca2+ sensitization). It was clarified that Rho-associated kinase (Rho-kinase) is a novel mediator of Ca2+ sensitization of the smooth muscle contraction, by introducing the recombinant catalytic domain of Rho-kinase into the cytosol of vascular smooth muscle permeabilized with beta-escin. This review article focuses on novel mechanisms, by which activation of receptor-coupled G-protein(s) increases Ca2+ sensitivity of the contractile apparatus in smooth muscle: Rho-kinase and protein kinase C.

Isolation of Drosophila flightless mutants which affect myofibrillar proteins of indirect flight muscle.

A large number of dominant flightless mutants of Drosophila were chemically induced, and their thorax proteins were examined by chemically induced, and their thorax proteins were examined by means of two-dimensional gel electrophoresis (O'Farrell 1975). Among them, 26 lines were found to have deficiency or reduction of some of myofibrillar proteins in indirect flight muscle (IFM). The gel patterns of the mutants could be classified into eleven groups. In general, more than a few polypeptides were either absent or reduced in each mutant line. Although the mutations affect myofibrillar proteins in apparently complex and diverse ways, logical correlations were found among the changes. There are pairs of proteins which always change together when a number of mutants are compared. There are also many pairs in which presence of one protein is necessary, but not sufficient for presence of the other. This suggests that absence of one component leads to disappearance or reduction of others which are either spatially or functionally related to the former. The correlation is possibly due to a hierarchy of the proteins in the myofibrillar assembly processes. Chromosomal loci of eleven typical mutants were examined, and it was found that most of them are located in two small regions of the second and the third chromosomes. IFM myofibrils of these mutants are either abnormal or absent in homozygotes as well as in heterozygotes.

An impairment of barrier size and charge selectivity of glomerular basement membrane in streptozotocin-induced diabetes and prevention by pharmacological therapy.

We examined barrier size (pore size) and charge selectivity (anionic sites) of the glomerular basement membrane (GBM) in experimental diabetes. For estimation of the pore size we employed a new method, tissue negative staining. In diabetic rats, enlarged pores and decreased numbers of anionic sites of GBM were observed. Both insulin treatment and angiotensin-converting enzyme inhibitors prevented these changes. The renoprotective effect of the two drugs is discussed in the article.