Analysis of HPV-1 E4 gene expression using epitope-defined antibodies.

Six monoclonal antibodies (mAbs) have been raised against the E4 proteins of HPV-1. Five of these were found to recognize denaturation-resistant epitopes as determined by Western blotting--and their binding sites were identified by determining their reactivity against a panel of bacterial E4--beta-galactosidase fusion proteins which contained progressive deletions at the C-terminal end of the E4 region. The five mAbs were found to bind to four distinct sites. By using these epitope-defined mAbs, along with anti-peptide antibodies raised against putative N- and C-terminal E4 sequences, we have determined the relationships between the eight distinct polypeptides (mol. wt 10/11 kd, 16/17 kd, 21/23 kd and 32/34 kd) previously shown to be expressed from the E4 gene of HPV-1 in productively infected papillomas. The 17 kd E4 polypeptide appears to be the product of a spliced mRNA encoding five amino acids from open reading frame (ORF) E1 joined onto 120 from the E4 ORF. The 16 kd and 10/11 kd proteins, which may be derived from this, lack sequences (approximately 15 and 70 amino acids respectively) encoded by the 5' end of the E4 gene. The 32/34 kd proteins were detected by all antibodies which reacted with the 16/17 kd polypeptides, suggesting that they represent dimers of the latter species. The 21/23 kd polypeptides, however, do not appear to be simple dimers of the 10/11 kd protein as previously predicted, and reacted with antibodies whose epitopes mapped in the N-terminal half of the E4 protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil

The conjugative plasmid pIJ101 and its conjugative nondeletion derivatives pIJ303 and pIJ211 were tested for their transferability between strains of Streptomyces on laboratory media and in the soil environment. Their roles in the mobilization of the cloning vector plasmid pIJ702, a nonconjugative deletion derivative of pIJ101, were also examined. Biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor Streptomyces lividans and several recipient Streptomyces strains previously isolated from soil. Conjugative plasmids were transferred to seven recipients in slant crosses and to three recipients in soil. Plasmids isolated from recipients showed restriction fragment patterns identical to that of the original plasmid in S. lividans. Plasmid pIJ303 was transferred less frequently in soil than on slants, and the frequency of transfer was higher at 30 degrees C than at the other temperatures examined. Transconjugant Streptomyces strains differed in their ability to maintain pIJ303. The nonconjugative plasmid pIJ702 was mobilized on agar slants into S. coelicolor 2708, which already contains a self-transmissible plasmid. Plasmid pIJ702 was also mobilized into S. flavovirens, Streptomyces sp. strain 87A, and S. parvulus on slants and in sterile soil after triparental crosses with two donors, one containing pIJ702 and the other containing either pIJ101 or pIJ211. The presence of a conjugative plasmid donor was required for the transfer of pIJ702 to S. parvulus 1234, S. flavovirens 28, and Streptomyces sp. strain 87A. Plasmid pIJ702 was always transferred in its normal, autonomous form. Chromosomal recombination also occurred in transconjugants after the transfer of pIJ702. This is the first report of gene transfer between Streptomyces strains in soil.     

Introduction of antibody (PL/IM 430) to a 100 kDa protein into permeabilised platelets inhibits intracellular sequestration of Ca2+

A monoclonal antibody (PL/IM 430), previously found to inhibit the uptake of Ca²⁺ into highly purified platelet intracellular membrane vesicles (Hack, N., Wilkinson, J. M. and Crawford, N. 1988,Biochem. J. 250, 355–361) has been introduced into saponin-permeabilised platelets. At a saponin concentration (20–25 g/ml) commensurate with total LDH release, sequestration of Ca²⁺ into intracellular non-mitochondrial stores is inhibited by the antibody (50% inhibition at 20 g/ml IgG). At higher saponin concentrations when intracellular binding of¹²⁵I-labelled mAb is maximum, inhibition of Ca²⁺ sequestration approaches 70%. The inhibition is specific, control studies with non-platelet directed mouse IgG and mAbs which immunoblot platelet antigens other than the 100 kDa protein did not affect the Ca²⁺ sequestration.No effect of the antibody were observed against IP₃-induced release of prestored Ca²⁺, either in permeabilised platelets or with isolated intracellular membrane vesicles. The mAb PL/IM 430 appears to bind only to the Ca²⁺ translocating channel protein associated with the intracellular membrane (Ca²⁺+Mg²⁺) ATPase and not to Ca²⁺ channels responsive to IP₃.Abbreviations mAb monoclonal antibody - PBS phosphate buffered saline - LDH lactate dehydrogenase     

Evaluation of different procedures for the dissociation of retinal pigmented epithelium into single viable cells

Retinal pigmented epithelium (RPE) from 7-day-old chicken embryos (stages 29 to 31) was isolated and dissociated into single cells using different procedures. The results were assessed in two ways. (1) The yield of single RPE cells per embryo was determined, and their ability to form pigmented colonies in clonal culture was tested. The most efficient and gentle procedure included isolation of the RPE in EDTA solution, trypsinization at low temperature and low enzyme concentration in the presence of EDTA, followed by incubation in culture medium for up to 4 hr. The completely dissociated cells thus obtained had a much higher plating efficiency and more uniform pattern of colony growth and differentiation than those obtained under any other conditions tested. (2) The effects of different treatments on cell junctions and morphological integrity of the cells were determined by transmission electron microscopy. EDTA solution yielded excellent separation of the epithelial sheet from the mesenchyme by dissociating it from Bruch's membrane, but had little effect on the junctions between adjacent RPE cells. Trypsinization of the epithelium under various conditions separated the basal lateral cell borders and caused loss of gap junctions, but left many cells still joined by apical tight junctions. Final disruption of the tight junctions occurred during recovery of the trypsinized cells in culture medium and was accompanied by dedifferentiation of the RPE cells.     

Flow characteristics of red cell containing fluids through pores. The effect of filter plugging, a mathematical model

A mathematical model was developed that describes the effects of filter plugging on flow through 3 micron pore polycarbonate filters as a function of time, pressure, and cell concentration, both under stirring and nonstirring conditions. The mathematical constants for the model were derived from experimental data generated with a filtration apparatus, and were tested by using various concentrations of cells that are able to plug filter pores. A computer simulation program was written to test the model over a wide range of nonfilterable cell concentrations.     

Enzyme regulation in C4 photosynthesis: Mechanism of activation of NADP-malate dehydrogenase by reduced thioredoxin

The mechanism of activation of thioredoxin-linked NADP-malate dehydrogenase was investigated by using ¹⁴C-iodoacetate and ¹⁴C-dansylated thioredoxin m, and Sepharose affinity columns (thioredoxin m, NADP-malate dehydrogenase) as probes to monitor enzyme sulfhydryl status and enzyme-thioredoxin interaction. The data indicate that NADP-malate dehydrogenase, purified to homogeneity from corn leaves, is activated by a net transfer of reducing equivalents from thioredoxin m, reduced by dithiothreitol, to enzyme disulfide groups, thereby yielding oxidized thioredoxin m and reduced enzyme. The appearance of new sulfhydryl groups that accompanies the activation of NADP-malate dehydrogenase appears to involve a structural change that is independent of the formation of a stable complex between the enzyme and reduced thioredoxin m. The data are consistent with the conclusion that oxygen promotes deactivation of NADP-malate dehydrogenase through oxidation of SH groups on reduced thioredoxin and on the reduced (activated) enzyme.     

Kinetics of p-cresol degradation by an immobilized Pseudomonas sp.

A p-cresol (PCR)-degrading Pseudomonas sp. was isolated from creosote-contaminated soil and shown to degrade PCR by conversion to protocatechuate via p-hydroxybenzaldehyde (PBA) and p-hydroxybenzoate (PHB). Cells of the Pseudomonas sp. were immobilized in calcium alginate beads and in polyurethane foam. The relationship between the PCR concentration and the PCR transformation rate was investigated in batch and continuous culture bioreactors. The biodegradation kinetics of PBA and PHB also were investigated. In batch culture reactors, the maximum PCR degradation rate (Vmax) for the alginate-immobilized Pseudomonas sp. cells was 1.5 mg of PCR g of bead-1 h-1 while the saturation constant (Ks) was 0.22 mM. For PHB degradation, the Vmax was 0.62 mg of PHB g of bead-1 h-1 while the Ks was 0.31 mM. For polyurethane-immobilized Pseudomonas sp. cells, the Vmax of PCR degradation was 0.80 mg of PCR g of foam-1 h-1 while the Ks was 0.28 mM. For PHB degradation, the Vmax was 0.21 mg of PHB g of foam-1 h-1 and the Ks was 0.22 mM. In a continuous column alginate bead reactor, the Vmax for PCR transformation was 2.6 mg g of bead-1 h-1 while the Ks was 0.20 mM. The Vmax and Ks for PBA transformation in the presence of PCR were 0.93 mg g of bead-1 h-1 and 0.063 mM, respectively. When PHB alone was added to a reactor, the Vmax was 1.48 mg g of bead-1 h-1 and the Ks was 0.32 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidural anaesthesia and long term backache after childbirth.

OBJECTIVE--To examine the relation between epidural anaesthesia and long term backache after childbirth. DESIGN--Data from postal questionnaire on morbidity after childbirth sent to women who had delivered in one maternity hospital between 1978 and 1985 were linked to maternity case notes for each woman. SETTING--Maternity hospital in Birmingham. SUBJECTS--11,701 Women who had delivered their most recent baby at the maternity hospital during the defined period and who returned their completed questionnaires. MAIN OUTCOME MEASURES AND RESULTS--Of the 1634 women who reported backache, 1132 (69%) had had it for over a year. A significant association was found between backache and epidural anaesthesia (relative risk = 1.8); 903 of 4766 women (18.9%) who had had epidural anaesthesia reported this symptom, compared with 731 of the 6935 women (10.5%) who had not had epidural anaesthesia. This association was consistent in both "normal" and "abnormal" deliveries, the only exception being after an elective caesarean section when no excess backache occurred after epidural anaesthesia. CONCLUSIONS--The relation between backache and epidural anaesthesia is probably causal. It seems to result from a combination of effective analgesia and stressed posture during labour. Further investigations on the mechanisms causing backache after epidural anaesthesia are required.     

Purification and Characterization of a Central Cholinergic Enhancing Factor from Rat Brain: Its Identity as Phosphoethanolamine

A compound that can enhance the apparent synthesis of acetylcholine in cultured explants of the medial septal nucleus has been purified from rat brain and identified as phosphoethanolamine. Acetylcholine synthesis is stimulated two- to threefold in cultures grown for 5 days in the presence of phosphoethanolamine, ethanolamine, or cytidine 5'-diphosphoethanolamine at concentrations above 100 microM. This effect appears to result from an increase in the accumulation of choline via the high-affinity, sodium-dependent uptake mechanism. The development of choline acetyltransferase activity is not affected. Phosphoethanolamine and ethanolamine seem to enhance the ability of developing cholinergic neurons to utilize choline accumulated via the sodium-dependent high-affinity choline uptake mechanism for the preferential production of acetylcholine without increasing the general metabolism of the cultures. Choline itself and its related derivatives are not stimulatory for these effects.     

Cloning and nucleotide sequence of the simian rotavirus gene 6 that codes for the major inner capsid protein.

The nucleotide sequence of the gene that codes for the major inner capsid protein of the simian rotavirus SA11 has been determined. A DNA copy of mRNA from gene 6 was cloned in the E. coli plasmid pBR322. The full-length gene is 1357 nucleotides long with a 5'-noncoding region of 23 nucleotides and a 3'-noncoding region of 140 nucleotides. The gene contains a single, long, open reading-frame of 1194 nucleotides capable of coding for a protein of 397 amino acids with a molecular weight of 44,816. The predicted protein product is relatively proline-rich with a net charge at neutral pH of -3.5. One stretch of 53 amino acids (encoded by nucleotides 327-485) is basic.