Functional analysis of titin/connectin N2-B mutations found in cardiomyopathy

Hypertrophic cardiomyopathy and dilated cardiomyopathy are two major clinical phenotypes of “idiopathic” cardiomyopathy. Recent molecular genetic analyses have now revealed that “idiopathic” cardiomyopathy is caused by mutations in genes for sarcomere components. We have recently reported several mutations in titin/connectin gene found in patients with hypertrophic cardiomyopathy or dilated cardiomyopathy. A hypertrophic cardiomyopathy-associated titin/connectin mutation (Arg740Leu) was found to increase the binding to actinin, while other dilated cardiomyopathy-associated titin/connectin mutations (Ala743Val and Val54Met) decreased the binding to actinin and Tcap/telethonin, respectively. We also reported several other mutations in the N2-B region of titin/connectin found in hypertrophic cardiomyopathy and dilated cardiomyopathy. Since the N2-B region expresses only in the heart, it was speculated that functional alterations due to the mutations cause cardiomyopathies. In this study, we investigated the functional changes caused by the N2-B region mutations by using yeast-two-hybrid assays. It was revealed that a hypertrophic cardiomyopathy-associated mutation (Ser3799Tyr) increased the binding to FHL2 protein, whereas a dilated cardiomyopathy-associated mutation (Gln4053ter) decreased the binding. In addition, another TTN mutation (Arg25618Gln) at the is2 region was found in familial DCM. Because FHL2 protein is known to tether metabolic enzymes to N2-B and is2 regions of titin/connectin, these observations suggest that altered recruitment of metabolic enzymes to the sarcomere may play a role in the pathogenesis of cardiomyopathies.     

Pharmacological and Pharmacokinetic Characteristics of YM435, a Novel Dopamine DA1-Receptor Agonist,in Anaesthetized Dogs

Time-course of plasma concentration of unchanged drug of the dopamine DA₁-receptor agonist (–)-(S)-4-(3,4-dihydroxyphenyl)-7,8-dihydroxy-1,2,3,4-tetrahydroisoquinoline hydrochloride hydrate (YM435), and its effects on blood pressure and renal blood flow were investigated in anaesthetized dogs. Continuous intravenous infusion of YM435 (0.1–3 μg kg^? min^?) rapidly increased renal blood flow and lowered blood pressure in a dose-dependent manner. These effects remained generally stable throughout the infusion period. Following the start of infusion, plasma concentration of unchanged drug also rose rapidly and dose-dependently and remained virtually constant throughout the infusion period. A significant correlation was observed between log YM435 plasma concentration and the increase in renal blood flow (r = 0.93, P < 0.0001) and between the former and the reduction in blood pressure (r = 0.93, p < 0.0001). The present results indicate that YM435 produces renal vasodilatation and lowering of blood pressure in a dose-dependent manner and with rapid onset following continuous intravenous infusion, and that these effects are generally stable throughout the period of infusion. These haemodynamic effects of YM435 were in good agreement with the time-course of plasma concentration of unchanged drug.     

Susceptibility genes for schizophrenia

Abstract  It is well known that genetic factors contribute to the susceptibility for schizophrenia. Recent advances in the molecular genetics of schizophrenia strongly suggest several susceptibility genes (e.g. dysbindin, neuregulin-1, DISC1, COMT, G72, RGS4 and Akt1). We discuss the evidence and biology of these genes. As glutamate transmission is especially implicated in these genes, neurobiological basis of schizophrenia might be elucidated by investigation of functional interactions between susceptibility genes for schizophrenia and the glutamatergic system.     

Detection of the preneoplastic lesions of small hepatocellular carcinoma in cirrhotic livers

To identify the preneoplastic lesions of hepatocellular carcinoma and the fine structure of preneoplastic hepatocytes, we studied proliferative conditions in cirrhosis of the liver. In all, 46 foci of cellular alteration (FCA), three regions of adenomatous hyperplasia (ADH), and 21 small hepatocellular carcinomas (sHCC) were studied by published criteria for sHCC and by the proliferative activity of the lesions as examined with monoclonal antibodies against DNA polymerase alpha and proliferating cell nuclear antigen. The four patients with FCA composed of basophilic hepatocytes were classified by the criteria as having sHCC; cells had features similar to those of sHCC. Two of these four patients with FCA were found to have HCC several years later. The number of hepatocytes stained for proliferating cell nuclear antigen was 72 and 81 per 1000 hepatocyte nuclei in the two patients who developed HCC. In one of the three patients with ADH, a sHCC was found 1 year later, and dysplastic hepatocytes from the region of ADH in this patient had features similar to those of HCC cells by light and electron microscopy. In this patient, the number of hepatocytes stained for DNA polymerase alpha was 452 per 1000 nuclei. Therefore, FCA and ADH might be preneoplastic lesions of sHCC in cirrhosis of the liver. Preneoplastic hepatocytes seem to be small cells with basophilic cytoplasm, with a large nucleus to cytoplasm ratio, finely indented nuclei with a smaller amount of condensed chromatin than normal, and poorly to moderately developed organelles.     

Proliferation and cytotoxicity of lectin-induced lymphokine-activated killer (LILAK) cells

More than 10(11) killer cells are needed for adoptive immunotherapy, but it is difficult to obtain so many from patients. Peripheral blood lymphocytes (PBL) treated with lectin and then with recombinant interleukin-2 (rIL-2) give many lectin-induced lymphokine-activated killer (LILAK) cells, studied here for proliferation, cytotoxicity, IL-2 receptors (IL-2R) and subsets. PBL obtained from hepatoma patients or healthy adults were incubated with phytohaemagglutinin (PHA) or concanavalin A (ConA) for 3 days and with rIL-2 for 4 days. Then the medium was replaced with fresh medium containing rIL-2 every 3 or 4 days, with the volume increased as cells proliferated. Cytotoxicity was expressed as the percentage lysis of target cells by 4 h 51Cr release. LILAK cells from healthy adults increased 120-fold in 2 weeks when incubated with ConA; the lymphokine-activated killer (LAK) cells increased 7-fold. The percentage of IL-2R+ cells increased more with ConA than with rIL-2 alone. ConA induced more suppressor T cells than PHA. LILAK cells obtained from patients by PHA treatment increased 180-fold in 2 weeks. Their cytotoxicity to Daudi cells was 1% before culture and 91% in 2 weeks; that of LAK cells was 60%. LILAK cells were cytotoxic to the tumour target cells, but not to allogeneic PBL. Adoptive immunotherapy may become more practical if many LILAK cells can be obtained at once by large-scale culture, such as by a hollow-fibre system.     

Significance of blood flow in the inferior and superior mesenteric veins for the formation of oesophageal varices

The degree of involvement of blood flow in the superior mesenteric vein and inferior mesenteric vein in the formation of oesophageal varices is not known. We have developed a method by which the contributions of these veins to portal blood flow can be evaluated simultaneously in a relatively non-invasive way. An enteric-coated capsule containing [123I]iodoamphetamine (IMP) is given by mouth and 3 h later [123I]IMP is instilled into the rectum. The data obtained are treated by computer to calculate the portal shunt index via the inferior and superior mesenteric veins. In chronic hepatitis and cirrhosis, when varices were absent, the difference in these indices was not significant. In the presence of varices, the portal shunt index via the inferior mesenteric vein was significantly higher than that via the superior mesenteric vein. It was suggested that the contribution of blood flow in the inferior mesenteric vein the portal/splenic axis is important in the formation of varices.     

Portal circulation in monkeys and humans studied after ingestion of a capsule containing a radionuclide

Capsules (8 x 30 mm) of technetium-99m pertechnetate were designed for measurement of portal blood flow. Most of the radionuclide entered the superior mesenteric vein. The capsule was taken orally and monitored with a collimator for scintigraphy until it reached the small intestine, when a magnetic field completed an electrical circuit in a sensor, burning a thread, releasing a spring, and discharging the preparation. A study in crab-eating monkeys (Macaca fascicularis) showed that the radionuclide in the small intestine circulated through the superior mesenteric vein to the portal vein and liver. Portal scintigraphy through the small intestine could be analysed in the same way as per-rectal portal scintigraphy, in which blood flow mostly from the inferior mesenteric vein is evaluated. A study of four volunteers showed that, after the radionuclide was released, it circulated through the superior mesenteric vein to the portal vein and liver. Use of a capsule enclosing a radioisotope was possible, and the procedure seemed to be safe. The use of the per-small intestine method plus the per-rectal method should give more accurate results than either method used alone, because the haemodynamics of both the superior and inferior mesenteric vein would be reflected.