Early intracellular signalling pathway of ethanol in vascular smooth muscle cells

1. ERKs belong to MAP kinase family and are activated by several growth and stress factors. Although ethanol has been shown to modulate ERK1 and ERK2 (p44(mapk) and p42(mapk)) activity, it can also act as an antiproliferative agent in various mammalian cells. Since the nature of the antiproliferative effect of ethanol in VSMCs has not been defined, we examined its effects on growth and on early intracellular events normally induced by growth factors in VSMCs. 2. Measurement of cytosolic Ca(2+) and pH in cell monolayers was performed using fura-2/AM and BCECF/AM, respectively. The effect of ethanol on VSMCs growth was assessed by [(3)H]-thymidine incorporation, by cell counting and by determination of the caspase 3 activity. Stimulation of ERK1 and ERK2 was examined by the chemiluminescence Western blotting method. The expression of c-fos was quantitated by Northern blotting. Determination of inositolphosphates was performed after labelling of VSMCs with myo-[2-(3)H]-inositol and separation of inositolphosphates by HPLC. 3. Ethanol (0.3 - 1.0% v v(-1), 17 - 170 mM) induced a dose-dependent maximal stimulation of p44(mapk)/p42(mapk) at 30 min and expression of c-fos mRNA with a maximum at 120 min. Intracellular events upstream to MAP kinase, like an increase in [Ca(2+)](i), activation of the Na(+)/H(+) exchanger and formation of phosphoinositol metabolites were also markedly activated by ethanol. Treatment of VSMCs with ethanol for 3 - 5 min induced an increase in DNA synthesis whereas treatment of the cells for more than 30 min was toxic. Caspase 3 activity was not modulated by ethanol treatment of VSMCs. 4. We may postulate that the activation of these mitogenic signals including the elevation of DNA synthesis reflects a cell effort to protect itself against the toxic effects of ethanol.     

On the contractile function of protein phosphatases in isolated human coronary arteries

It is unknown whether protein phosphatases types 1 and 2A are present in and can regulate the tone of human vascular tissue. The expression and possible function of serine/threonine protein phosphatases (PP) type 1 (PP1) and type 2A (PP2A) were studied in isolated human coronary arteries. Catalytic subunits of PPI and PP2A were identified by means of phosphatase activity measurement in tissue homogenates, by separation of enriched extracts through affinity column chromatography, by immunoblotting with specific antibodies, by hybridization of mRNA with specific DNA probes and PCR of reverse transcribed mRNA. Based on these methods, the catalytic subunits of PP1(alpha,beta,gamma) and PP2A(alpha,beta) were identified. Appropriately, cantharidin, an inhibitor of PP1 and PP2A, increased basal tone of human isolated coronary artery rings with an EC50 of about 16 micromol/l by increasing the phosphorylation state of the regulatory light chains of myosin. In summary, PP1 and PP2A are expressed in human coronary arteries and they can alter vascular tone.     

Expression of endothelial cell-derived nitric oxide synthase (eNOS) is increased during gastric adaptation to chronic aspirin intake in humans

BACKGROUND: Gastric adaptation to aspirin is well-documented. However, the mechanisms underlying the reduction of aspirin-induced mucosal damage despite continued ingestion of the drug remain poorly understood. METHODS: Eight healthy volunteers who received aspirin 1 g b.d. for 14 days were compared with eight placebo-dosed controls. Gastroscopy with mucosal biopsy was performed, and gastric mucosal blood flow was measured before and following 3, 7 and 14 days of aspirin treatment. At the same time points, tissue concentration and the content of prostaglandin E2 in the gastric juice were determined and expression of endothelial cell-derived nitric oxide synthase (eNOS) in mucosal biopsies was measured using Western blot analysis. RESULTS: Aspirin-induced mucosal damage that reached a maximum on day 3, declining significantly by day 14. Concomitantly, mucosal blood flow significantly increased on day 3 and returned to initial values on day 14. Aspirin intake led to a significant decrease in prostaglandin E2 concentration in the gastric mucosa and in gastric juice during the whole period of aspirin consumption. eNOS expression started to increase on day 7 in oxyntic mucosa and on day 3 in antral mucosa, reaching its highest values at the end of the consumption of aspirin. CONCLUSIONS: The human gastric mucosa adapts to prolonged aspirin intake, and this is accompanied by an increase in mucosal blood flow and reduced prostaglandin synthesis. Increase of mucosal eNOS expression might compensate for reduced prostaglandin synthesis and be responsible for gastric adaptation to chronic aspirin intake in humans.     

Quality Determining Parameters of Balloon Angioplasty Catheters

The investigations carried out on balloon angioplasty catheters using a parameter test unit allow an objective comparison and a qualitative assessment of catheter brands with respect to their stenosis passability. The design of the test unit allows the measure of other parameters important for clinical practice, such as pushability and trackability. Altogether 8 over-the-wire and 10 monorail-catheters from 7 different manufacturers were investigated by our study. Between modern over-the-wire and monorail systems there exists no fundamental difference in passage capability. In some brands of catheter the stenosis model shows no increase in the amount of thrust required, even after repeated inflation; this is accountable to the good folding properties of the balloon. Angioplasty balloons made of less overstretchable or thinner material yielded the best results. The fact that changes in balloon quality may result from pre-inflation should carry weight in discussions concerning the reuse of catheters. The measurement results are useful for the correct selection of a catheter appropriate to the therapy task at hand.     

Osteoplastic frontal sinusotomy and extradural microsurgical repair of frontobasal cerebrospinal fluid fistulas

Summary The choice of the surgical approach and operative technique for the management of cerebrospinal fluid (CSF) fistulas of the anterior cranial fossa are still a controversially discussed topic. Although extracranial approaches through the paranasal sinuses are becoming increasingly more popular among otolaryngologists and maxillo-facial surgeons, most neurosurgeons traditionally prefer the intracranial repair of CSF fistulas by a craniotomy.We present an approach through the frontal sinus for the repair of dural defects behind the posterior wall of the frontal sinus and at the floor of the anterior cranial fossa. The operative procedure comprises the following main steps: 1) exposure of the anterior wall of the frontal sinus by a bicoronal incision; 2) excision of the anterior wall without frontal burr holes; 3) bilateral removal of the posterior wall of the fronal sinus; 4) extradural inspection of the dura behind the frontal sinus and above the cribriform plate, ethmoidal roof, and orbital roof bilaterally; 5) closure of dural tears by direct suture and a periosteal graft; 6) reinsertion of the anterior wall of the frontal sinus and fixation with titanium micro plates.Twenty-five patients operated upon using this technique are described. The aetiology of the frontobasal lesion was traumatic in 23, and an ethmoid carcinoma in two. In all patients, the dural fistulas were successfully repaired during the initial procedure. One patient died from sudden circulatory arrest after an uneventful postoperative course of nine days. Otherwise, there were no postoperative complications.This technique affords atraumatic extradural inspection and repair of dural fistulas bilaterally behind the frontal sinus, and above the cribriform plate and the ethmoidal and orbital roofs with none or minimal brain retraction. It therefore allows early repair of CSF fistulas also in patients with severe brain injury. Although we consider the extradural closure of fistulas the method of choice, this approach also allows for a combined extradural-intradural procedure, thus enabling the surgeon to treat associated intradural pathologies, such as traumatic lesions or tumours of the frontal cranial base.     

The 1990 United States-Japan seminar on bioorganic marine chemistry, meeting report.

The second U.S.-Japan Seminar on "Bioorganic Marine Chemistry" was held in Honolulu, Hawaii, 3-7 December, 1990. Twenty-one invited lecturers and fourteen observers from the two countries attended. Recent results in the areas of (a) new bioactive natural products, (b) biological and pharmacological activity of marine natural products, and (c) biosynthesis of marine natural products were presented and discussed. Summaries of the presentations and relevant chemical structures are presented in this report.     

Isolation and characterization of propagable cell lines (HUNC) from the androgen-sensitive Dunning R3327H rat prostatic adenocarcinoma

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC- E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.      

Zinc replenishment reduces esophageal cell proliferation and N- nitrosomethylbenzylamine (NMBA)-induced esophageal tumor incidence in zinc-deficient rats

Previous work has shown that sustained increased and decreased cell proliferation, induced by dietary zinc deficiency and caloric restriction respectively, influence the course of N- nitrosomethylbenzylamine (NMBA)-induced esophageal carcinogenesis in rats. The present study considered whether the increased cell proliferation and esophageal tumor incidence induced by zinc deficiency are reversed upon zinc replenishment. Weanling rats were maintained initially on a deficient diet containing 4 p.p.m. zinc. After 5 weeks, carcinogen-treated animals were given six intragastric doses of NMBA (2 mg/kg twice weekly). Controls were untreated. After the second NMBA dose, the rats were divided into three dietary groups. One group was continued on the deficient diet, while the other two groups were switched to diets containing either 75 or 200 p.p.m. zinc, with half of the members in each group fed ad libitum and half pair-fed with deficient rats. NMBA-untreated controls were similarly replenished. At various time points, esophageal cell proliferation was assessed in five animals from each group by immunohistochemical detection of cells in S phase, with in vivo 5-bromo-2'deoxyuridine labeling. At 11 weeks after the first dose, esophageal tumor incidence was greatly reduced, from 100% in the deficient group to 26 and 14% respectively in the replenished groups fed ad libitum 75 and 200 p.p.m. zinc and to 14 and 11% respectively in the replenished groups pair-fed 75 and 200 p.p.m. zinc. In addition, the number of tumors per esophagus was reduced from 9.93 +/- 4.25 in deficient rats, to a range of 0.11 +/- 0.31-0.30 +/- 0.54 in replenished animals. Following zinc replenishment, esophageal cell proliferation, as measured by labeling index (LI), the number of labeled cells and the total number of cells, was markedly decreased in NMBA-untreated and -treated esophagi as compared with those in corresponding deficient esophagi. Thus, the esophageal cell proliferation induced by zinc deficiency is reversed by zinc replenishment and replenished animals have a markedly lower incidence of esophageal tumors.