TGF-beta mediated G1 arrest in a human melanoma cell line lacking p15INK4B: evidence for cooperation between p21Cip1/WAF1 and p27Kip1

We have studied TGF-beta mediated G1 arrest in WM35, an early stage human melanoma cell line. These cells have lost p15INK4B expression through loss of one chromosome 9 and rearrangement of the other. In asynchronously growing WM35, TGF-beta caused reductions in cyclin D1, cyclin A and cdk4 proteins and their associated kinase activities and an increase in both p21Cip1/WAF1 and p27Kip1. These findings were confirmed in cells released from quiescence in the presence of TGF-beta, in which TGF-beta inhibited or delayed the reduction in the cdk inhibitors that normally occurs in late G1. In contrast to observations in other cell types, there was an increased association of both p21Cip1/WAF1 and p27Kip1 with cyclin D1/cdk4 and with cyclin E/cdk2 during TGF-beta mediated arrest of asynchronously growing cells. Upregulation of p21Cip1/WAF1 preceded that of p27Kip1. Furthermore, p21Cip1/WAF1 and p27Kip1 were not present in the same cdk complexes but bound distinct populations of target cdk molecules. Both p21Cip1/WAF1 and p27Kip1 immunoprecipitates from asynchronously growing cells contained active kinase complexes. These KIP-associated kinase activities were reduced in TGF-beta arrested cells. It has been proposed that in TGF-beta arrested epithelial cells, up-regulation of p15INK4B and of p15INK4B binding to cdk4 serves to destabilize the association of p27Kip1 with cyclin D1/cdk4, promoting p27Kip1 binding and inhibition of cyclin E/cdk2. Our findings demonstrate that this is not a universal mechanism of G1 arrest by TGF-beta. In TGF-beta arrested WM35, which lack p15INK4B, the increased p21Cip1/WAF1 may serve a similar function to that of p15INK4B: initiating kinase inhibition and providing an additional mechanism to supplement the effect of p27Kip1 on G1 cyclin/cdks.     

Placental and fetal cardiac laminin are targets for cross-reacting autoantibodies from mothers of children with congenital heart block

The association of congenital heart block (CHB) with maternal autoantibodies to the Ro and La ribonucleoprotein antigens may be due to cross-reactions between maternal anti-La antibodies and fetal cardiac specific antigens. One of the major components of cardiac myocytes, laminin, is accessible for binding by maternal autoantibodies and we have previously reported cross-reactivity of mouse laminin with anti-La antibodies affinity purified from the sera of patients with primary Sj?gren's syndrome. Affinity purified anti-La antibodies from ten women who had at some time given birth to a child with CHB were examined for cross-reactivity with human placental laminin, which shares structural similarities with cardiac laminin. All ten anti-La antibodies bound to the surface of cryosections of normal full term placental trophoblasts. Binding could be inhibited by pre-incubation of antibodies with either La or placental laminin. Eight anti-La antibodies also reacted with placental laminin by ELISA and La inhibited up to 82% of binding to laminin while laminin inhibited up to 85% of binding to La in a dose dependent manner. Eight anti-La antibodies also bound to the surface of fetal cardiac myocytes at 10.3 weeks of gestation and five showed lower levels of reactivity with the surface of fetal cardiac myocytes at 16.5 weeks of gestation. None showed any surface staining of normal adult heart. These data confirm the cross-reactivity of anti-La antibodies with laminin and may support a placental role in preventing the majority of potentially pathogenic antibodies from reaching the fetal circulation.     

Pathogenesis of malignant ascites formation: initiating events that lead to fluid accumulation

Initiating events leading to the accumulation of malignant ascites in the peritoneal cavity were investigated in two syngeneic transplantable murine ascites-producing tumors, MOT mouse ovarian tumor and the TA3/St mammary carcinoma. The transport of two tracers, 125I-labeled human serum albumin (125I-HSA) and 51Cr-labeled red blood cells (51Cr-RBC), into and out of the peritoneal cavity was studied at early times after i.p. tumor cell injection, prior to abundant fluid accumulation, and at intervals of 5 to 360 min after i.v. or i.p. tracer injection. Tracer influx and efflux rates were estimated from the mass of tracer passing into or out of the peritoneal cavity following a bolus injection of tracer into either the blood or the peritoneal cavity. Efflux of 125I-HSA from the peritoneal cavity was markedly reduced (3- to 5-fold) within 1 day of i.p. injection of either type of tumor cell. Significantly reduced efflux preceded any increase in tumor cell number and by itself did not induce peritoneal fluid accumulation. 125I-HSA tracer influx from plasma to peritoneal fluid did not increase detectably until 5 to 7 days after tumor cell injection, when the tumor cell number had increased by 10- to 100-fold. Only at relatively late stages of ascites tumor growth, when the flow rate into the peritoneal cavity had increased relative to the flow rate out of the peritoneum, was there net peritoneal fluid accumulation. Thus, increased influx, in addition to impaired efflux, were required for malignant ascites accumulation. Following i.p. injection, the efflux rates of 125I-HSA always exceeded those of 51Cr-RBC, even in ascites tumor-bearing animals. Furthermore, 125I-HSA tracer disappeared from the peritoneal cavity more rapidly than it appeared in the plasma, suggesting that 125I-HSA moves more rapidly through the channels by which 51Cr-RBC egress from the peritoneum (primarily diaphragmatic lymphatics) and/or has access to additional pathways not open to 51Cr-RBC. Finally, flow rates into and out of the blood and peritoneum were used to obtain kinetic parameters that characterized tracer transport: k1, the rate constant for tracer transport from the blood to the peritoneum; k2, the rate constant for tracer transport from the peritoneal cavity to the blood; and k6, the rate constant for tracer transport from the peritoneal cavity to surrounding interstitial tissue.(ABSTRACT TRUNCATED AT 400 WORDS)     

Survey of minor surgery in general practice in the west of Scotland

BACKGROUND: Noncutaneous psammomatous melanotic schwannoma has recently been reported as an unusual component of Carney's complex (myxomas, spotty pigmentation, and endocrinopathy). The most common locations for this lesion include peripheral nerve roots and the gastrointestinal tract. OBSERVATIONS: A cutaneous psammomatous melanotic schwannoma is reported in a patient with known Carney's complex. This neoplasm was pseudoencapsulated, with epithelioid to spindle-shaped cells and no nuclear atypia. Immunostaining was positive for S100 protein and vimentin, as well for HMB-45 antibody. Electron microscopy showed melanosomes in cytoplasmic processes of cells that were ensheathed by layers of reduplicated basal lamina. CONCLUSIONS: Location in the superficial soft tissues is extremely unusual for psammomatous melanotic schwannomas. Recognition of this new cutaneous marker as a part of this complex may aid in identification of individuals at risk for cardiac myxomas. It is also important that this lesion is not mistaken for melanoma, given the strong HMB-45 positivity.     

The kappa opioid receptor expressed on the mouse R1.1 thymoma cell line down-regulates without desensitizing during chronic opioid exposure

The R1.1 mouse thymoma cell line expresses a single class of kappa opioid receptors that is negatively coupled to adenylyl cyclase through a Bordetella pertussis toxin-sensitive inhibitory guanine nucleotide-binding protein. The aim of the present study was to determine whether chronic opioid treatment of R1.1 cells altered either the binding properties or the functional response associated with the kappa opioid receptor. Culturing of R1.1 cells with the kappa-selective agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U50,488) for 3 hr and longer, followed by extensive washing of R1.1 cell membranes, produced a concentration- and time-dependent reduction in the binding of the kappa-selective ligand (5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1- oxaspiro(4,5)dec-8-yl) benzeneacetamide ([3H]U69,593). Culturing of R1.1 cells with 100 nM U50,488 for 24 hr produced approximately a 50% reduction in the Bmax value for [3H]U69,593 and [3H]naloxone binding. In contrast to the reduction in binding, there was no change in the inhibition of adenylyl cyclase activity by (-)-U50,488. To determine whether kappa opioid receptor function was maintained by spare receptors after agonist-induced down-regulation, membranes from untreated R1.1 cells were incubated with 400 nM of the irreversible opioid antagonist beta-chlornaltrexamine (beta-CNA) followed by extensive washing. beta-CNA produced a 50% reduction in the [3H]U69,593 binding and a 6-fold increase in the IC50 value for (-)-U50,488 inhibition of adenylyl cyclase activity, with no change in the maximal inhibition of cyclic AMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apoptosis as a cause of death in measles virus-infected cells

To determine the mechanism of measles virus-induced cell death, we studied the infection of Vero cells and monocytic cell lines with wild-type (Chicago-1) and vaccine (Edmonston) strains of measles virus. DNA fragmentation indicative of apoptosis was apparent by flow cytometry, agarose gel electrophoresis, and electron microscopy. Within syncytia, DNA strand breaks were demonstrated by end labeling with terminal transferase and then by visualization.     

Laser prostatectomy: initial experience and urodynamic follow-up

OBJECTIVES: An evolving technology for the treatment of bladder outlet obstruction due to benign prostatic hyperplasia (BPH) is the use of the side-firing neodymium: yttrium-aluminum-garnet (Nd:YAG) laser to achieve prostatic tissue ablation. The purpose of this study was to determine the short-term efficacy of this procedure in both an objective and subjective manner. METHODS: We examined this technique by carefully evaluating our first 25 men undergoing the procedure. Each patient was subjected to careful symptom score analysis using the American Urological Association symptom index and multichannel urodynamics, including pressure-flow studies both preoperatively and at 3 months postoperatively. RESULTS: At the 3-month follow-up, symptom scores improved from a preoperative mean of 11.4 to 7.2 and the mean maximum flow rate improved from 6.1 to 14.5 cc/s. These are both significant at P < 0.001. Statistically similar improvement was seen in detrusor pressure at opening and at maximum flow. Eighty percent of the men studied had at least a 50% reduction in symptom score and a 50% improvement in flow rate. CONCLUSIONS: We conclude that laser prostatectomy is a promising minimally invasive treatment for bladder outlet obstruction secondary to BPH and deserves further evaluation at longer terms of follow-up.     

Prevention of bleeding after cardiopulmonary bypass with high-dose tranexamic acid. Double-blind, randomized clinical trial

This prospective, double-blind, randomized trial assessed the effectiveness of high-dose tranexamic acid given in the preoperative period on blood loss in patients undergoing cardiopulmonary bypass. One hundred fifty patients scheduled to undergo cardiac operations with cardiopulmonary bypass were randomized into three groups of equal size. The first group received 10 gm of tranexamic acid intravenously over 20 minutes before sternotomy and a placebo infusion over 5 hours. The second group received 10 gm of tranexamic acid over 20 minutes and then another 10 gm infused intravenously over 5 hours. The control group received a placebo bolus and a placebo infusion over 5 hours (0.9% normal saline solution). The blood loss after the operation was measured at 6 hours and 24 hours. The homologous blood and blood products given during and up to 48 hours after operation were recorded. Eighteen percent of the control group patients shed more than 750 ml blood in 6 hours compared with only 2% in both tranexamic acid groups. Patients who shed more than 750 ml blood required 93% more red blood cell transfusions than patients without excessive bleeding. Tranexamic acid (10 gm) given intravenously in the period before cardiopulmonary bypass reduced blood loss over 6 hours by 50% and over 24 hours by 35%. Continued tranexamic acid infusion (10 gm over 5 hours) did not reduce bleeding further. There was no difference in the coagulation profile before operation between patients with and without excessive bleeding. However, coagulation tests done in the postoperative period indicated ongoing fibrinolysis and platelet dysfunction in patients with excessive bleeding.