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.     

Glucagon-like peptide I enhances the insulinotropic effect of glibenclamide in NIDDM patients and in the perfused rat pancreas

OBJECTIVE: To investigate the acute effects of glibenclamide and glucagon-like peptide I (GLP-I) and their combination in perfused isolated rat pancreas and in patients with secondary failure to sulfonylureas. RESEARCH DESIGN AND METHODS: Rat islets were perfused with 10 nmol/l GLP-I in combination with 2 mumol/l glibenclamide. In human experiments, GLP-I (0.75 pmol. kg-1.min-1) was given as a continuous infusion during 240 min, while glibenclamide (3.5 mg) was administered orally. Eight patients participated in the study (age 57.6 +/- 2.7 years, BMI 28.7 +/- 1.5 kg/m2, mean +/- SE). In all subjects, blood glucose was first normalized by insulin infusion administered by an artificial pancreas (Biostator). RESULTS: GLP-I increased the insulinotropic effect of glibenclamide fourfold in the perfused rat pancreas. In human experiments, treatment with GLP-I alone and in combination with glibenclamide significantly decreased basal glucose levels (5.1 +/- 0.4 and 4.5 +/- 0.1 vs. 6.0 +/- 0.3 mmol/l, P < 0.01), while with only glibenclamide, glucose concentrations remained unchanged. GLP-I markedly decreased total integrated glucose response to the meal (353 +/- 60 vs. 724 +/- 91 mmol.l-1. min-1, area under the curve [AUC] [-30-180 min], P < 0.02), whereas glibenclamide had no effect (598 +/- 101 mmol.l-1. min-1, AUC [-30-180 min], NS). The combined treatment further enhanced the glucose lowering effect of GLP-I (138 +/- 24 mmol. l-1.min, AUC [-30-180 min], P < 0.001). GLP-I, glibenclamide, and combined treat-stimulated meal-induced insulin release as reflected by insulinogenic indexes (control 1.44 +/- 0.4; GLP-I 6.3 +/- 1.6, P < 0.01; glibenclamide 6.8 +/- 2.1, P < 0.01; combination 20.7 +/- 5.0, P < 0.001). GLP-I inhibited basal but not postprandial glucagon responses. Using paracetamol as a marker for gastric emptying rate of the test meal, treatment with GLP-I decreased gastric emptying at 180 min by approximately 50% compared with the control subjects (P < 0.01). CONCLUSIONS: In acute experiments on overweight patients with NIDDM, GLP-I exerted a marked antidiabetogenic action on the basal and postprandial state. The peptide stimulated insulin, suppressed basal glucagon release, and prolonged gastric emptying. The glucose-lowering effect of GLP-I was further enhanced by glibenclamide. This action may be at least partially accounted for by a synergistic effect of these two compounds on insulin release. Glibenclamide per se enhanced postprandial but not basal insulin release and exerted a less pronounced antidiabetogenic effect compared with GLP-I.     

Oculomotor paralysis. The perspective of a neuro-ophthalmology consultation

Ocular paresis is a usual diagnosis in neuro-ophthalmologic consultation, and its aetiology is indeed a challenge. The authors studied 3400 records concerning this consultation, between 1982 and the third quarter of 1993. During this period 221 cases with ocular paresis were selected. During this retrospective study we point out the following items: the cranial nerve affected; the neuro-ophthalmologic semiology; the aetiology and clinical evolution, according to the age groups. From 221 cases selected, 111 were paresis from the VIth pair (50.2%), 88 from the IIIrd pair (39.8%), 14 multiple paresis (6.4%) and 8 paresis from the IVth pair (3.6%). The most frequent complaint was diplopia (> 90%). As far as the aetiological diagnosis is concerned, this was easier to establish in patients > 50 years of age. In this age group the most usual aetiology was vascular and traumatic pathologies. In younger patients the most frequent pathologies were traumatic and tumoral. The prognostic was better in the vascular group, the paresis recovery being > 50% in all other pathologies, except the tumoral one.     

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)     

Comparison of three different methods for measurement of plaque-pH in humans after consumption of soft bread and potato chips

Three different techniques for measurement of plaque-pH--the sampling, the microtouch, and the telemetric methods--were compared after subjects had consumed different starch products. Ten volunteers, equipped with partial lower prostheses, incorporating a miniature glass pH electrode, refrained from toothbrushing for 3 days. Four products were tested: (1) soft bread, (2) potato chips, (3) 5% starch, and (4) 5% sucrose. The pH of plaque was measured for 45 min by means of all three of the methods. The results showed that the mean pH at 10 min was 1.5 units lower with the telemetric than with the sampling method and 1.0 unit lower with the telemetric than with the microtouch method. Relatively small differences were found among the effects of the four test products for all three methods, with the clearest distinctions among the pH curves being with the microtouch and telemetric methods. The main conclusions from the present investigation are: (1) that there were large differences in pH levels measured with the sampling, the microtouch, and the telemetric methods, even though they ranked the test products in about the same order, and (2) that the two starchy foods, bread and potato chips, were both easily fermented by dental plaque.