Crk-associated substrate lymphocyte type regulates transforming growth factor-beta signaling by inhibiting Smad6 and Smad7.

Crk-associated substrate lymphocyte type (Cas-L) is a 105 kDa docking protein with diverse functional properties, including regulation of cell division, proliferation, migration and adhesion. Cas-L is also involved in beta1 integrin- or antigen receptor-mediated signaling in B and T cells. In the present study, we demonstrate that Cas-L potentiates transforming growth factor-beta (TGF-beta) signaling pathway by interacting with Smad6 and Smad7. Immunoprecipitation experiments reveal that single domain deletion of full-length Cas-L completely abolishes its docking function with Smad6 and Smad7, suggesting that the natural structure of Cas-L is necessary for its association with Smad6 and Smad7. On the other hand, both N-terminal and C-terminal deletion mutants of Smad6 and Smad7 still retain their docking ability to Cas-L, suggesting that Smad6 and Smad7 possess several binding motifs to Cas-L. Moreover, Cas-L interaction with Mad-homology (MH)2 domain, but not with MH1 domain of Smad6 or Smad7, ameliorates TGF-beta-induced signaling pathway. Finally, depletion of Cas-L by small-interfering RNA oligo attenuates TGF-beta-induced growth inhibition of Huh-7 cells, with a concomitant reduction in phosphorylation of Smad2 and Smad3. These results strongly suggest that Cas-L is a potential regulator of TGF-beta signaling pathway.

[Assessment of anti-tremorogenic drugs--nicotine-induced tail-tremor model].

The repeated administration of nicotine at small doses, which do not produce whole body tremor or convulsion, causes tremor only in the tail (tail-tremor) of rats. The tremor is accompanied by locomotor hyperactivity without rigidity and immobility of the whole body, suggesting that the nicotine-induced tail-tremor model is useful for studying the mechanism underlying tremor associated with movement. The tail-tremor induced by nicotine was suppressed by mecamylamine, a nicotinic antagonist, but not by atropine or scopolamine, muscalinic antagonists. Moreover, the tail-tremor was suppressed by the beta-blockers propranolol and pindolol, as well as the benzodiazepines diazepam and clonazepam. Tremor at rest is observed only in Parkinson's disease, which is improved with anti-muscalinic drugs. Essential tremor is one of the typical tremors connected with movement (postural and kinetic tremor) and is improved with beta-blocker. These findings and results suggest that nicotine-induced tail-tremor is useful for the study of essential tremor in animal models.

Comparison of serum 7S fragment of type IV collagen and serum central triple-helix of type IV collagen for assessment of liver fibrosis in patients with chronic viral liver disease.

BACKGROUND/AIMS: A competitive radioimmunoassay for serum 7S fragment of type IV collagen (7S collagen) using a polyclonal antibody against 7S collagen and a sandwich enzyme immunoassay for serum central triple-helix of type IV collagen (IV collagen) using two monoclonal antibodies against the pepsin-solubilized type IV collagen may be used as diagnostic aids for liver fibrosis in clinical medicine. We compared the clinical usefulness for assessing liver fibrosis of serum 7S collagen and IV collagen tests in chronic viral liver diseases, and also examined the elution pattern of 7S collagen- and IV collagen-related antigens in serum by gel filtration analysis. METHODS: Serum 7S collagen and IV collagen levels were assayed in 151 patients with chronic viral liver disease and 30 healthy control subjects. RESULTS: Gel filtration on the Sephacryl S400HR column revealed that the 7S collagen antigenicity in serum was heterogeneous, whereas the IV collagen antigen in serum was uniform in size. Serum levels of 7S collagen and IV collagen showed increases closely correlated with the severity of liver disease. The abnormal percentage of 7S collagen in three patient groups was similar to that of IV collagen in the corresponding groups. Serum 7S collagen and IV collagen levels were strongly correlated with the histological degree of liver fibrosis; the correlation coefficients were r = +0.675 for 7S collagen and r = +0.665 for IV collagen. When we assessed the ability of each test to detect cirrhosis with a receiver operating curve, the serum 7S collagen test was a slightly better marker than the serum IV collagen test. For the detection of cirrhosis, serum 7S collagen was 83% sensitive and 88% specific at a cutoff value of 9 ng/ml, and serum IV collagen was 80% sensitive and 81% specific at a cutoff value of 160 ng/ml. CONCLUSIONS: These findings suggested that serum 7S collagen and IV collagen tests are similarly useful for assessing liver fibrosis in patients with chronic viral liver disease, although the former is slightly better for diagnosing cirrhosis than the latter.

Assessment of in vitro growth potential of hepatocytes expressing hepatocyte growth factor in an autocrine fashion.

We recently developed transgenic mice expressing hepatocyte growth factor (HGF) specific to hepatocytes. Hepatocytes of HGF transgenic mice showed a 2-fold increase of DNA labeling indices in vivo compared with those of wild type mice. To assess in vitro growth potential of hepatocytes from HGF transgenic mice, we studied the effects of epidermal growth factor (EGF), insulin or transforming growth factor beta (TGF beta) on DNA synthesis of hepatocytes derived from HGF transgenic or wild type mice, respectively. We found that DNA synthesis of hepatocytes from HGF transgenic mice was significantly enhanced, compared with that from wild type mice, respectively, and that its effect was additive with EGF or insulin. Further, growth-inhibitory effects of TGF beta on hepatocytes was greatly depressed in transgenic mice-derived hepatocytes, compared with that in wild type hepatocytes. These data suggest that the autocrine action of HGF is a potent stimulus for hepatocyte growth, and stress its importance as regulator of liver regeneration.

Teratological assessment of glutaraldehyde in rats by gastric intubation.

Pregnant rats were given glutaraldehyde (GA) by gastric intubation at a dose of 0, 25, 50 or 100 mg/kg on days 6-15 of pregnancy. Maternal toxicity occurred in the 100 mg/kg group as evidenced by a significant increase in maternal death and a significant decrease in maternal body weight gain and food consumption. A significantly lowered fetal weight was also found in the 100 mg/kg group. No significant change induced by GA was detected in the incidence of postimplantation loss. Morphologic examinations of fetuses revealed no evidence of teratogenicity of GA. It could be concluded that GA has no teratogenic effects on rat offspring even at a dose which induced severe maternal toxicity.

Teratological assessment of diiodomethyl p-tolyl sulfone in rats.

Pregnant rats were given diiodomethyl p-tolyl sulfone (DIMPTS) at a dose of 0, 0.125, 0.25, 0.5 or 1.0% in the diet on days 6-15 of pregnancy. Maternal body weight gain and food consumption during the administration period were significantly lowered in the 0.25, 0.5 and 1.0% groups. No significant changes induced by DIMPTS were detected in the number of resorptions and dead fetuses, and body weight of live fetuses. Morphological examinations of fetuses revealed no evidence of teratogenesis. It could be concluded that DIMPTS has no teratogenic effects on rat offspring, even at doses which induced maternal toxicity.

[Application of non-invasive methods to assessment of left ventricular function in cardiomyopathy (author's transl)].

Left ventricular function in cardiomyopathy was studied by non-invasive methods. Various indices of left ventricular function were measured in patients with cardiomyopathy by mechanocardiography and echocardiography and were compared with indices in normal subjects and the following conclusions were obtained. 1) Patients with Congestive cardiomyopathy had high PEP/LVET, low mVcf, low EF, and low mPWV, suggesting depressed cardiac function. 2) Patients with Hypertrophic obstructive cardiomyopathy had characteristic findings, such as low DDR, high IVST/PWT, and SAM. 3) Patients with Hypertrophic non-obstructive cardiomyopathy had no characteristic changes in indices, however in some of the findings transition to Hypertrophic obstructive cardiomyopathy was suggested.