Expression of matrix metalloproteinase matrilysin (mmp-7) was induced by activated ki-ras via ap-1 activation in sw1417 colon cancer cells

The matrix metalloproteinase matrilysin (MMP-7) is a member of the matrix metallo-proteinase gene family, which is believed to play an important role in tumor invasion and metastasis. We have previously found that matrilysin mRNA is specifically expressed in colorectal cancers and adenomas and that its message is localized in the tumor cells themselves. We examined the effects of activated Ki-ras oncogene on the expression of matrilysin in colon cancer cells. We showed that both mRNA and the enzymatic activity of matrilysin were induced by the introduction of activated Ki-ras into SW1417 colon cancer cells. To understand the mechanisms regulating this induction, we analyzed alterations of AP-1 activity induced by activated Ki-ras, using the chloramphenicol acetyltransferase assay. AP-1 activity in SW1417 cells expressing activated Ki-ras was higher than that in control cells. The gel-shift assay also showed higher levels of AP-1 binding protein in SW1417 cells expressing activated Ki-ras than those in control cells. Our results suggest that activated Ki-ras may play a role in inducing expression of matrilysin through an AP-1-dependent pathway in colon cancer cells.     

Effect of S-8666 on Cl- transport in the rabbit connecting tubule perfused in vitro.

S-8666, [6, 7-dichloro-5-(N, N-dimethylsulfamoyl)-2, 3-dihydrobenzofurancarboxylic acid] is a potent diuretic with uricosuric action. Although the major site of action of S-8666 has been proven by the in vitro microperfusion study to be the thick ascending limb of Henle's loop, clearance studies in the rat suggested that this drug has an additional thiazide-like action. To provide direct evidence that S-8666 acts also on distal nephron segments, we examined effect of S-8666 on Cl- flux across the rabbit connecting tubule perfused in vitro. The drug suppressed the lumen-to-bath Cl- flux by 96 +/- 41 (S.E.)pmol.mm-1.min-1 (n = 9) without affecting transmural voltage. To demonstrate that S-8666 acts on the connecting tubule cell, the target of thiazide diuretics, we compared effects of S-8666 and trichlormethiazide on the basolateral membrane voltage of the connecting tubule cell. Both drugs added to the lumen caused a small but significant hyperpolarization of the basolateral membrane without affecting transmural voltage. We conclude that S-8666 is a unique uricosuric diuretic having actions on both thick ascending limb of Henle's loop and connecting tubule.     

Purification of 45 KDa estramustine binding protein (EMBP) and preparation of its antibody

The purification of 45 KDa EMBP and the production of monospecific anti-serum is described. 45 KDa EMBP was purified by relatively simple methods using ion exchange HPLC (TSK-GEL DEAE-5PW column) and size exclusion HPLC (TSK-GEL G3000SW column). The results clearly demonstrated the speed and simplicity of the method using these columns, compared to previously-described methods for purification of 45 KDa EMBP.     

Reduction in Internal Carotid Arterial Blood Flow Velocity in Children During Antiepileptic Drug Therapy with Clinical Dosages

Summary: We studied the effect of antiepileptic drugs (AEDs) on internal carotid artery (ICA) blood flow velocity, as an index of total cerebral blood flow (CBF). The subjects were 45 newly diagnosed children with febrile convulsion or epilepsy who were seizure-free for a period long enough not to affect the results. They had no neurologic deficit, received fixed monotherapy, and were examined by a noninvasive Doppler ultrasound method, in comparisonwith 13 age-matched normal volunteers with no AED. In 30 patients, the measurements were performed before and after AED administration [10 with phenobarbital (PB), 10 with carbamazepine (CBZ), and 10 with valproate (VPA)], and performed before and after AED discontinuation in the remaining 15 patients (all with PB). Normal volunteers underwent the two consecutive examinations with a mean interval equal to that of the entire patient group, and there was no difference in velocity values between the measurements. In patients receiving CBZ or VPA, a significant reduction was noted in blood flow velocity after drug administration. Although velocity values in the patients receiving PB did not change after drug administration, they were significantly increased after complete discontinuation. In the present study, a slight but significant reduction in CBF caused by AED administration at therapeutic doses in children was suggested.     

Characterization and comparison of two newly established Epstein-Barr virus (EBV)-negative and EBV-positive Burkitt's lymphoma cell lines. EBV-negative cell line with a low level of expression of ICAM-1 molecule and EBV-positive cell line with a high level of expression of ICAM-1 molecule.

Two human Burkitt's lymphoma cell lines (HBL-4 and HBL-5) were established individually from two patients with small noncleaved cell lymphoma (Burkitt's type). The HBL-4 cell line is Epstein-Barr virus (EBV)-negative, and the HBL-5 cell line is EBV-positive. Cytogenetically, both cell lines had the same chromosomal translocation, t(8;14)(q24;q32) as those observed in the primary malignant cells from individual patients. Morphologic, immunophenotypic, cytogenetic, and molecular studies confirmed that both cell lines were derived from the primary lymphoma cells in vivo. HBL-4 cells lacked CD23(H107), CD11a(LFA-1), and latent membrane protein (LMP) but expressed CD54(ICAM-1) at low levels, whereas HBL-5 cells showed the high level of expression of CD54 and faint expression of LMP but lacked CD11a. In addition, the EBV-positive lymphoblastoid cell line (LCL) expressed CD11a, CD23, CD54, and LMP at high levels. Therefore, an HBL-5 phenotype with expression of CD54 and LMP tends toward an LCL phenotype, and the augmentation of CD54 on the HBL-5 cells in comparison with primary lymphoma cells is likely to be upregulated by LMP, probably resulting from the EBV infection. There was little difference in the BrdUrd uptake in vivo and in vitro, doubling time, tumorigenicity, and dynamics of tumor growth in athymic nude mice between both cell lines. These findings indicate that the potentiality of cell growth and tumorigenicity of these two cell lines are unlikely to be related with EBV.     

A case of mitral valve replacement with autologous pulmonic valve in congenital mitral stenosis

A one-year-old infant with congenital mitral stenosis and pulmonary hypertension underwent by mitral valve replacement with his pulmonic valve autograft and pulmonary tract reconstruction with heterogeneous pericardial conduit. His postoperative hemodynamics data showed that left atrial pressure decreased and pulmonary hypertension continued. Echocardiography showed that the implanted autograft functioned properly. On the seventh postoperative day, he died of pulmonary hypertension. In case with congenital mitral stenosis with the small mitral annulus and the small left ventricular cavity, it is difficult to perform mitral valve replacement by commercially available mechanical or tissue valves. Because these valves are not suitable for the small mitral annulus. The mitral valve replacement with pulmonic valve autograft is recommended in such a case with the congenital mitral stenosis.     

Modification of the Na,K-pump of glial cells within cobalt-induced epileptogenic cortex of rat

The characteristics of a glial Na⁺,K⁺-pump dependent on extracellular K⁺ within epileptogenic cortex were studied electrophysiologically, biochemically and histochemically in vitro using slices from cobalt-induced epileptogenic cortex of rat. When the extracellular K⁺ concentration ([K⁺]_o) was varied between 4 and 40 mM, the mean slope of membrane potential plotted against [K⁺]_o was about 57 mV in glia from the normal cortex (tissue A) and about 44 mV in glia from the epileptogenic cortex (tissue B); whereas no significant difference in the resting membrane potential of these tissues was observed. In glia from tissue B, a marked transient hyperpolarization above control level was caused by replacement of elevated [K⁺]_o with the normal medium. Ouabain abolished these phenomena observed in glia from tissue B, but had no effect on the membrane potential during normal [K⁺]_o. Reduction of extracellular Na⁺, Ca²⁺ and Cl⁻ did not significantly affect the membrane potential of glia from either tissue. In tissue A, the cells marked by intracellular injection of horseradish peroxidase after intracellular recording were protoplasmic astrocytes; in tissue B, fibrous astrocytes with abnormal processes predominated. K⁺-dependent stimulation of Na⁺,K⁺-ATPase activity of the astrocyte-enriched fraction and its membrane preparation from tissue B was much larger than that from tissue A. A certain amount of the reaction product of K⁺-pNPPase activity was seen on glial plasma membrane within tissue B but not on that from tissue A. The above findings suggest that a glial Na⁺,K⁺-pump within actively firing epileptogenic cortex may be modified to increase in its activity.     

Polyclonal and monoclonal antibodies monospecific to MMTV LTR orf protein produced in E. coli.

Monoclonal and polyclonal antibodies specific to an open reading frame of the mouse mammary tumor virus long terminal repeat were generated using an open reading frame-beta-galactosidase fusion protein produced in E. coli. Both antibodies reacted with the open reading frame-beta-galactosidase fusion protein but not with beta-galactosidase alone using an immunoblotting technique. It is concluded that these antibodies were specific for the protein encoded by the open reading frame of the mouse mammary tumor virus long terminal repeat.     

Induction of nitric oxide synthase by cyclic AMP in rat vascular smooth muscle cells.

By measurements of NO2-/NO3- (NOx) production and Northern blot analysis, we studied the effects of a membrane-permeable cAMP derivative, 8-bromo-cAMP, on the expression of inducible nitric oxide synthase (iNOS) gene and the synthesis of NOx in cultured rat vascular smooth muscle cells (VSMCs). 8-bromo-cAMP stimulated NOx production and increased steady-state levels of iNOS mRNA in rat VSMC in a time- and dose-dependent manner. NG-monomethyl-L-arginine, a NOS inhibitor, completely blocked the 8-bromo-cAMP-induced NOx production, whose effect was partially, but significantly reversed by an excess L-arginine, but not by D-arginine. Compounds that increase intracellular cAMP levels (cholera toxin, forskolin, and 3-isobutyl-1-methylxanthine), all stimulated NOx production. Dexamethasone inhibited the stimulated NOx production, as well as the induction of iNOS mRNA by cAMP. Both actinomycin D and cycloheximide completely blocked the stimulated NOx production by cAMP. Actinomycin D abolished the cAMP-induced iNOS mRNA, whereas cycloheximide remarkably increased iNOS mRNA levels in the presence and absence of 8-bromo-cAMP (superinduction). Actinomycin D, but not dexamethasone, completely abolished the cycloheximide-induced iNOS mRNA. The half-life of cAMP-induced iNOS mRNA was approximately 2 h, whereas no decay in the cycloheximide-induced iNOS mRNA was observed during 12 h. These results demonstrate that iNOS gene is upregulated by cAMP and the superinduction of iNOS mRNA is attributable to increased mRNA stability in rat VSMC.