Attenuation of contractility in rat epididymal vas deferens by Rho kinase inhibitors.

The involvement of Ca(2+) sensitization mediated through Rho kinase in the contractility of rat epididymal vas deferens was investigated using Rho kinase inhibitors, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinilcyclohexanecarboxamide dihydrochloride (Y-27632) and 1-(5-isoquinolinesulphonyl)homopiperazine (HA 1077), in comparison with myosin light chain kinase (MLCK) inhibitors, wortmannin and 1-(5-chloronaphthalenesulphonyl)homopiperazine (ML-9) and agents that affect protein kinase C (PKC) and non-receptor tyrosine kinase intracellular signalling. 2 In Ca(2+)-free/ethyleneglycol-bis-(beta-aminoethylether)N,N,N('),N(')-tetraacetic acid (EGTA) (1 mM) medium, noradrenaline evoked sustained contractions. Y-27632 and HA 1077 caused a concentration-dependent inhibition and complete relaxation (IC(50), 1.08 and 1.75 microM respectively). The Ca(2+)-free contraction was reduced by wortmannin (10 microM) or ML-9 (10 microM) but not by inhibitors of diacylglycerol metabolism, 3-[2-[4[bis(4-Fluoropheny)methylene]-1-piperidinyl]-2,3-dihydro-2-thioxi-4(H)-quinazolinone (R59949) (10 microm) or 1,6-bis(cyclohexyloximinocarbonylamino)hexane (RHC-80267) (10 microM) or by the phospholipase A(2) (PLA(2)) inhibitor, quinacrine (up to 100 microM) or tyrosine kinase inhibitor, genistein (30 microM). 3 In the presence of Ca(2+) (2.5 mM), noradrenaline (100 microM) evoked rhythmic activity and biphasic tonic contractions. Y-27632 (1-10 microM) or HA 1077 (1-10 microM) reduced the amplitude of rhythmic activity and tonic contractions. ML-9 (10 microM) attenuated the occurrence of rhythmic activity and modestly reduced the tonic contractions. ML-9 (10 microM) combined with Y-27632 (10 microM) significantly reduced the tonic contractions. ML-9 (30 microM) alone (or combined with Y-27632 10 microM) suppressed the rhythmic activity and substantially reduced (or abolished) the tonic contractions. 4 Contractions evoked by high [K(+)](o) (120 mM) or alpha,beta-methylene ATP (10 microM) were reduced significantly by Y-27632 (1-3 microM) indicating that the Rho kinase signalling pathway is activated by direct tissue depolarization or by stimulation of ligand-gated P(2X) purinoceptors. 5 Collectively, these results indicate that Ca(2+)-sensitization mediated by Rho kinase is involved in agonist- or depolarization-induced contraction of rat epididymal vas deferens. It is the major contractile mechanism underlying noradrenaline-induced Ca(2+)-free responses. It contributes to Ca(2+)-dependent rhythmic contractility and optimizes the development of full contractile tension triggered through calmodulin/MLCK activation by stimulated influx of Ca(2+).

Expression of cyclin D1, cyclin E, cdk4 and loss of heterozygosity of 8p, 13q, 17p in hepatocellular carcinoma: comparison study of childhood and adult hepatocellular carcinoma.

AIMS/BACKGROUND: In hepatitis B virus-related hepatocellular carcinoma (HCC), at least 20-40 years of continuous necro-inflammation is necessary for the hepato-carcinogenesis to occur. However, HCC in childhood shows an unusually short latent period and rapid progression. In our previous report, mutation of c-met was found only in childhood HCC, but not in adult HCC. In order to determine the specific biological tumorous features of childhood HCC, a comparison study of childhood and adult HCC was performed. METHODS: Eighteen cases of HBV positive HCC (nine children and nine adults aged more than 40 years) were selected. The expression of G1 phase regulatory proteins (cyclin D1, cyclin E and cdk4) was studied using immunohistochemical methods. Loss of heterozygosity (LOH) on chromosomal arms 8p, 13q and 17p was analyzed. RESULTS: Cyclin D1 expression was significantly lower in childhood HCC than in adult HCC (cases of cyclin D1 expression under 3+: childhood 5/9 vs. adult 1/9, p=0.046). No difference in cyclin E and cdk4 expression was found between childhood and adult HCC. LOH frequency on 13q was relatively higher in childhood than in adult HCC (66.7% vs. 22.2%, p=0.058). LOH frequency on 8p and 17p was 44.4% and 33.3% in childhood HCC and 44.4% and 75% in adult HCC with no statistical significance between the two groups. CONCLUSION: Our data suggest that childhood HCC may be less dependent on cyclin D1 protein for tumor growth and progression than adult HCC, and that the LOH on 13q may be an important feature of childhood HCC.

Loss of p16 and p27 is associated with progression of human gastric cancer.

We performed the immunohistochemical staining for six G1 check point cell cycle proteins to study their expression patterns and roles in the gastric carcinogenesis. We studied 76 cases of paraffin blocks that included the sections of 18 tubular adenomas (TA), 38 early gastric carcinomas (EGC) (20 cases of mucosal type, nine cases of submucosal type with no nodal metastasis, nine cases of submucosal type with nodal metastasis), 20 advanced gastric carcinomas (AGC) (ten cases with no nodal metastasis, ten cases with nodal metastasis). We found that abnormal expression of p16 and p27 increased with the progression of tubular adenomas to advanced gastric cancers. Inverse relationship between pRb and p16 proteins was found in a small portion of the gastric tumors. Expressions of pRb and cdk4 were consistently high in benign and malignant gastric tumors. Expression of cyclin D1 and cyclin E rather decreased with the tumor progression. In conclusion, losses of p16 and p27 seem to play a significant role during the gastric carcinogenesis, and the G1 checkpoint cell cycle proteins such as pRb, cdk4, cyclin D1, and cyclin E variably participate in the gastric carcinogenesis and metastasis by the mechanisms which are yet unknown; thus, further studies need to be performed to elucidate the mechanisms.

Low-cost wind tunnel for aerosol inhalation studies.

A low-cost wind tunnel for aerosol studies has been designed, constructed, and evaluated for aerosol uniformity with 2- and 0.46-micron particles. A commercial nebulizer was used to produce the suspended test particles, and a custom-made, four-hole injector was used to introduce the aerosol into the wind tunnel. A commercially available optical particle counter measured the particle concentration. Performance tests of the velocity profile and particle concentration distribution at two flow rates showed that the system performs well for small particles.