RNA Polymerase II transcription independent of TBP in murine embryonic stem cells.

Transcription by RNA Polymerase II (Pol II) is initiated by the hierarchical assembly of the pre-initiation complex onto promoter DNA. Decades of research have shown that the TATA-box binding protein (TBP) is essential for Pol II loading and initiation. Here, we report instead that acute depletion of TBP in mouse embryonic stem cells has no global effect on ongoing Pol II transcription. In contrast, acute TBP depletion severely impairs RNA Polymerase III initiation. Furthermore, Pol II transcriptional induction occurs normally upon TBP depletion. This TBP-independent transcription mechanism is not due to a functional redundancy with the TBP paralog TRF2, though TRF2 also binds to promoters of transcribed genes. Rather, we show that the TFIID complex can form and, despite having reduced TAF4 and TFIIA binding when TBP is depleted, the Pol II machinery is sufficiently robust in sustaining TBP-independent transcription.

The curvature sensitivity of a membrane-binding amphipathic helix can be modulated by the charge on a flanking region.

Membrane-induced amphipathic helices (m-AH) can act as membrane curvature sensors by binding preferentially to hydrophobic lipid packing defects enriched in curved surfaces. Reliance on hydrophobicity and membrane curvature for binding is enhanced when electrostatic interactions are weak. We probed the role of modifying membrane and protein charge on the curvature sensing of two m-AH-containing proteins, CTP:phosphocholine cytidylyltransferase (CCT) and α-synuclein (α-syn). The m-AH domains in both proteins are flanked by disordered tails with multiple phosphoserines (CCT) or acidic residues (α-syn), which we mutated to glutamate or serine to modify protein charge. Analysis of binding to vesicles of varying curvature showed that increasing the negative charge of the tail region decreased the binding strength and augmented the curvature dependence, especially for CCT. We attribute this to charge repulsion. Conversely, increasing the membrane negative charge dampened the curvature dependence. Our data suggest that discrimination of curved versus flat membranes with high negative charge could be modulated by phosphorylation.

The amphipathic helix of an enzyme that regulates phosphatidylcholine synthesis remodels membranes into highly curved nanotubules.

CTP:phosphocholine cytidylyltransferase (CCT) is an amphitropic protein regulating phosphatidylcholine synthesis. Lipid-induced folding of its amphipathic helical (AH) membrane-binding domain activates the enzyme. In this study we examined the membrane deforming property of CCT in vitro by monitoring conversion of vesicles to tubules, using transmission electron microscopy. Vesicle tubulation was proportional to the membrane density of CCT and proceeded either as growth from a pre-formed surface bud, or as a global transformation of roughly spherical vesicles into progressively thinner tubules. The tubulation pathway depended on the lipid compositional heterogeneity of the vesicles, with heterogeneous mixtures supporting the bud-extension pathway. Co-existence of vesicles alongside thick and thin tubules suggested that CCT can discriminate between flat membrane surfaces and those with emerging curvature, binding preferentially to the latter. Thin tubules had a limiting diameter of ~12nm, likely representing bilayer cylinders with a very high density of 1 CCT/50 lipids. The AH segment was necessary and sufficient for tubulation. AH regions from diverse CCT sources, including C. elegans, had tubulation activity that correlated with α-helical length. The AH motifs in CCT and the Parkinson's-related protein, α-synuclein, have similar features, however the CCT AH was more effective in its membrane remodeling function. That CCT can deform vesicles of physiologically relevant composition suggests that CCT binding to membranes may initiate deformations required for organelle morphogenesis and at the same time stimulate synthesis of the PC required for the development of these regions.

Nutraceutical effects of garlic oil, its nonpolar fraction and a Ficus flavonoid as compared to vitamin E in CCl4 induced liver damage in rats.

Carbon tetrachloride feeding (3.2g/kg/72hr) for one month increased significantly the serum and tissue lipid profile and deranged the enzyme levels viz; alkaline phosphatase, alanine transaminase, aspartate transaminase, glutathionze reductase, HMGCoA reductase, catalase, gluc.6.PDH and malic enzyme in rats. Simultaneously the lipid peroxidation level in liver was also raised. On administration of garlic oil and its major nonpolar fraction (NPFGO) and a flavonoid isolated from the bark of Ficus bengalensis Linn, viz; leucopelargonin derivative respectively to different groups(100mg/kg/day) the deleterious effects of CCl4 were significantly ameliorated. The liver damage by CCl4 was satisfactorily prevented by these samples as effectively as Vit. E (50 mg/kg/day). The results prove that important nutraceuticals (phytonutrients) like bioflavonoids and theols i.e. allylic sulphide rich fractions give protection from toxins like CCl4. The order of beneficial effects of the drugs are Leucopelargonin > NPFGO > Garlic oil and their effects are comparable to that of vitamin E used at a minimal dose.

Detection of rinderpest virus using N-protein monoclonal antibodies.

A panel of monoclonal antibodies (mAbs) was generated against the RBOK strain of rinderpest virus (RPV). All of them bound to the N protein of RPV. The antigen capture ELISA using the mAbs could detect the virus in crude viral preparations. The mAb 12BF8.1.1 showed higher reactivity with cell-associated (CA) virus, whereas the mAbs 12AD10.1.1, 12BD7.1.1 and 12DG7.1.1 showed higher reactivity with extracellular virus (hereafter referred to as cell-free (CF) virus). The mAbs 12BF8.1.1 and 12AD10.1.1 could detect the virus in infected Vero cell culture supernatants (CCS) as early as 24 h post-cytopathic effect (CPE) initiation. Detergent treatment (Triton X-100) of RPV preparations enhanced the binding of the mAbs to the virus. All the seven mAbs showed specific fluorescence in virus-infected cell cultures. The immunofluorescence (IFA) using mAbs was found to be more sensitive and reliable than the immunoperoxidase test (IPT) for detection of rinderpest.

Development of a thermoresistant tissue culture rinderpest vaccine virus.

The currently used Plowright's tissue culture rinderpest vaccine (RBOK strain) gives full protection and lifelong immunity, but it is highly thermolabile and requires maintenance of cold chain from vaccine production till delivery. Keeping in view the need for a thermostabile vaccine in tropical developing countries with limited refrigeration facilities, we passaged serially the RBOK strain of rinderpestvirus (RPV) at gradually elevated temperature up to 40 degrees C to obtain a thermoresistant RPV (TR-RPV) mutant. The thermoresistance (thermostability) and antigenicity of TR-RPV were compared with those of the vaccine virus by various methods, confirming the acquired properties. Thus, the infectivity titres of the TR-RPV mutant and vaccine virus were determined after incubation for various times at 37 degrees C. Regression analysis indicated that TR-RPV had a half-life of 1.81 hr and a degradation constant of 0.1656, while the parent vaccine virus had a half-life of 1.11 hr and a degradation constant of 0.2686. In capture ELISA with four different monoclonal antibodies (MAbs) to the N protein of RPV, TR-RPV showed a 10-fold higher reactivity with one MAb as compared to the vaccine virus. Although TR-RPV did react also with the other three MAbs, its reactivity was only 4-5 times higher than that of the vaccine virus. A treatment of the virus with Triton X-100 resulted in 2-4 times higher reactivity with the MAbs. The 35S-methionine-labeled vaccine virus-and TR-RPV-infected Vero cell lysates showed 6 polypeptide bands with identical pattern of migration in polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Radioimmunoprecipitation assay (RIPA) of the TR-RPV and vaccine virus with a rabbit anti-RPV immune serum (RHIS) and bovine anti-RPV hyperimmune serum (BHIS) showed the presence of four identical antigenic proteins, namely H, N, F and M, for both viruses. It can be concluded that TR-RPV has indeed retained the antigenic properties of the parental vaccine virus besides acquiring thermoresistance.

Flexible fiberoptic bronchoscopy.

The value of flexible fiberoptic bronchoscopy in children are increasing day by day, but still underutilized even in many advanced institutions. 630 Fiberoptic bronchoscopy procedures under local anaesthesia were performed for various clinical conditions for diagnosis and therapy. Nasal route is preferred than oral route. Therapeutic indications are more in children than adults. Nearly one third of children with collapse, consolidation have shown radiological clearance after repeated bronchoscopy.

Postmarketing surveillance study of permethrin creme rinse.

BACKGROUND: An observational, epidemiological study was undertaken to evaluate the safety of permethrin 1% creme rinse (Nix) for treatment of head lice infestations. METHODS: Thirty-seven local public health departments enrolled a total of 38,160 patients for 47,578 treatments with permethrin or other pediculicides from September 1, 1986, through January 31, 1988. Follow-up safety information was collected between 7 and 14 days following treatment via return visit or telephone contact. RESULTS: One hundred three adverse events were reported among 41,955 evaluable treatments. The rates of reported adverse events were 2.2 per 1000 treatments among permethrin treatments, 3.4 per 1000 treatments among lindane treatments, and 1.5 per 1000 treatments among other over-the-counter treatments. No serious, unexpected adverse events were detected in the 18,950 patients treated with permethrin. CONCLUSIONS: This study confirmed the safety profile of permethrin in conditions of general use, as seen in clinical trials. Postmarketing safety monitoring in public health departments of drugs used to treat public health conditions was shown to be feasible.

Surgical risk factors in total anomalous pulmonary venous connection.

Eighty-three patients underwent surgical correction of total anomalous pulmonary venous connection (TAPVC) between 1973 and 1986. There were 46 boys and 37 girls. Median age at operation was 60 days (1 to 240) and median weight 3.9 kg (1 to 22). The anatomic types encountered included infracardiac connection (16 patients), supracardiac connection (32) and pulmonary venous drainage connected directly to the coronary sinus (27). Mixed anomalous drainage or pulmonary venous return connected directly to the right atrium occurred in 8 patients. Diagnosis was established by cardiac catheterization and angiography (56 patients), clinical examination (3) and cross-sectional echocardiography alone in 24 of the last consecutive 28 patients. Pulmonary hypertension was present in 26 (55%) of those who underwent cardiac catheterization. The median pulmonary vascular resistance was 4.2 units/m2 (body surface area) for all the patients, whereas in those with infracardiac pulmonary venous connection the median value was 10 units/m2. The median interval between admission and operation was 72 hours. Surgical correction was performed using profound hypothermia and circulatory arrest in 68; for the remainder, conventional cardiopulmonary bypass with profound to moderate hypothermia was used. Ten patients developed 1 or more pulmonary hypertensive crises during the early postoperative period. These were diagnosed in 8 by direct pulmonary artery pressure measurement and in 2 by clinical examination. Late reoperation was necessary in 6 patients (10%). Analyses of risk factors for 30-day survival for all patients showed that the risk of early death was associated with the type of anomaly (infradiaphragmatic), occurrence of pulmonary hypertensive crises, year of the operation, set of the patient and pressure of preoperative congestive heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Embolisation procedures in congenital heart disease.

Eight therapeutic embolisation procedures were performed by the transcutaneous catheter technique in seven patients with congenital heart disease. After surgical correction of tetralogy of Fallot (four patients), catheter embolisation was used to occlude two large aortopulmonary collaterals (one patient), three small aortopulmonary collaterals (one patient), and two Blalock-Taussig shunts (two patients). In two patients congenital coronary anomalies were occluded--a coronary arteriovenous malformation and a coronary artery/bronchial artery anastomosis. In one patient a pulmonary arteriovenous malformation was embolised. Detachable balloons were used to occlude six large arteries, the three small arteries were occluded with small gelfoam fragments, and the pulmonary arteriovenous malformation was occluded with multiple steel coils and large gelfoam pieces. Successful occlusion was achieved in all cases. No complications were encountered and the procedure was well tolerated even in the two patients receiving postoperative intensive care. Therapeutic embolisation in suitable cases is a safe and effective alternative to surgery and the detachable balloon technique is effective in occluding high flow vessels.

Percutaneous balloon pulmonary valvuloplasty.

Percutaneous pulmonary valvuloplasty was performed in 27 patients with congenital pulmonary valve stenosis. A fall in the transvalve gradient of at least 15 mm Hg occurred in 22 patients. In five there was little change in the severity of the stenosis; in three of these the pulmonary valve was dysplastic. None of the successfully treated patients had a dysplastic valve. The two other failures, early in the series, were probably due to inadequate balloon size. In one patient the procedure was performed twice, with a successful result from the second dilatation with a larger balloon. Follow up studies in a further six patients showed no evidence of restenosis in those who had been successfully treated and no late improvement in the remainder. There were no important complications. Percutaneous pulmonary valvuloplasty should be the initial treatment for congenital pulmonary valve stenosis, although when the valve is dysplastic the result is less likely to be satisfactory.

Radionuclide measurement of left ventricular ejection fraction in tricuspid atresia.

In 15 patients with tricuspid atresia and one with tricuspid stenosis the left ventricular ejection fraction was measured by equilibrium gated radionuclide angiography and the results compared with those from a control group of 16 patients. The patients with tricuspid atresia had a significantly depressed ejection fraction. Those who had a surgical shunt or who had had pulmonary artery banding had significantly lower ejection fractions than the remainder. There was no significant correlation between the ejection fraction and age, the arterial oxygen saturation, or the haemoglobin concentration. Five patients were also studied during isometric exercise; three had an abnormal response. Volume overload of the ventricle is identified as one cause of the dysfunction, but other factors may be important. Radionuclide angiography offers a non-invasive method of studying ventricular function in this condition.

Balloon atrial septostomy in the neonatal intensive care unit.

In eight infants with complete transposition of the great arteries balloon atrial septostomy was performed in the neonatal intensive care unit solely under cross sectional echocardiographic control. One infant developed necrotising enterocolitis; otherwise there were no complications. This technique eliminates the need for radiographic screening during the procedure, is quick, and causes little disturbance to the infant who does not have to be removed from the incubator.

M-mode echocardiography in the developing human fetus.

The M-mode echocardiogram can be obtained in the developing human fetus from around the 16th week of gestation until term. Errors in interpretation of the M-mode echocardiogram can be avoided by concurrent two dimensional fetal cardiac examination. The pattern of motion of the mitral, tricuspid, aortic, and pulmonary valves and ventricular wall motion can be studied. Motion of the foramen ovale flap, which is characteristic in fetal life, can also be observed. Using the M-mode tracing, measurement data for six variables were made and growth charts constructed. The variables recorded were septal and left ventricular wall thickness, right and left ventricular internal dimension in diastole, aortic root dimension, and left atrial internal dimension. The measurements were made in normal pregnancies where the fetal heart was structurally normal. Confidence limits for each measurement were derived for gestational ages between 16 and 39 weeks.

Recognition of imperforate atrioventricular valves by two dimensional echocardiography.

The majority of hearts in which selective right or left atrial angiography shows no direct communication between one atrium and a ventricular chamber are considered to be examples of atresia of the right or left atrioventricular valves. Most patients presenting with the clinical features of tricuspid atresia have an absent right atrioventricular connection, while those with features of mitral atresia and a normal aortic root frequently have an absent left atrioventricular connection. By studying 67 patients using two dimensional echocardiography we have identified 12 in whom there was an imperforate atrioventricular valve and not an absent atrioventricular connection. Thus, in 44 hearts with the angiographic features of tricuspid atresia, 36 had absence of the right atrioventricular connection and six had an imperforate right atrioventricular valve. In the latter, four were examples of atrioventricular concordance and two were univentricular hearts of left ventricular morphology in which the mode of atrioventricular connection was through one perforate and one imperforate valve. Similarly, 17 out of 23 cases of mitral atresia had absence of the left atrioventricular connection. The remaining six had an imperforate left atrioventricular valve which was associated with atrioventricular concordance in two patients and with double inlet univentricular heats in four patients.