Study of stringency conditions for human papillomavirus DNA detection on cell lines,frozen and paraffin-embedded tissue sections by in situ hybridization with biotinylated probes

Summary In situ hybridization was mainly used for typing human papillomavirus (HPV) in paraffin-embedded or frozen sections under stringent conditions (SC). We tested 5 different conditions of stringency with biotinylated HPV 1, 2, 16 and 18 probes on 3 cell lines (Siha and CaSki with HPV16, HeLa with HPV18) by varying the concentration of formamide in the hybridization mixture and washings in order to determine the stringency conditions to be used to assess the presence of HPV and its typing: A-low stringency, hybridization at 35° C below the melting temperature of DNA (Tm-35° C) and washings without formamide; B-low stringency, hybridization and washings at Tm-35° C; C-medium stringency, hybridization at Tm-35° C and washings at Tm-12° C; D-high stringency, hybridization at Tm-12° C and washing without formamide; E-very high stringency, hybridization and washings at –12° C. This study showed that HPV typing required a high stringency. On the contrary, under non stringent conditions (NSC), each cell line was positive with the heterologous probes.When 3 to 5 stringency conditions were assayed on 4 frozen samples, similar results were obtained. Typing required high stringency conditions whereas NSC allowed HPV detection. Furthermore, this study demonstrated the specificity of the reaction in lesions positive with more than one type.Stringent (Tm-12° C) and non stringent (Tm-35° C) conditions of hybridization were further applied to 57 biopsy sections (17 frozen and 40 paraffin-embedded specimens) from typical wart lesions and lesions suspected of HPV. Nineteen samples were totally negative under both NSC and SC, and considered as non-infected by HPV. In 22 specimens positive, under both NSC and stringent conditions (SC), the HPV type was identified. Ten specimens reacted with 1, 2 or 3 HPV types under NSC but the HPV DNA was not typed with the probes used. Six lesions were negative under NSC but were typed under SC. Most paraffin sections were labeled only with one HPV probe under NSC, whereas frozen sections were often labeled with 2 or 3 HPV probes. The HPV probe positive under SC was usually positive under NSC in both frozen and paraffin sections. HPV type 1 probe was more frequently positive under NSC in paraffin- embedded sections than the others and the 4 probes tested were equally positive in frozen sections.These findings show the interest of in situ hybridization in low stringency conditions since 17% of our lesions (10/57) were positive only under NSC: HPV DNA was detected but not typed with the probes used. Frozen sections were more frequently positive than paraffin sections, suggesting a loss of DNA accessibility in the latter, due to the fixation or processing before hybridization.     

Three-dimensional structure of the regular surface glycoprotein layer of Halobacterium volcanii from the Dead Sea

A three-dimensional reconstruction from electron micrographs of negatively stained cell envelopes of Halobacterium volcanii has revealed the structure of the surface glycoprotein to a resolution of 2 nm. The glycoprotein is arranged on a p6 lattice with a lattice constant of 16.8 nm. It forms 4.5 nm high, dome-shaped, morphological complexes with a narrow pore at the apex opening into a `funnel' towards the cell membrane. The polarity of the structure was derived from freeze-etching experiments and `edge' views. Six radial protrusions emanate from each morphological complex and join around the 3-fold axis to provide lateral connectivity. Using the primary structure of the surface glycoprotein of the closely related species Halobacterium halobium (Lechner and Sumper, 1987) and the cell envelope profile from a previous X-ray analysis of the same species (Blaurock et al., 1976) we have integrated our reconstruction into a model of halobacterial cell envelope.     

Stretch-activated ion channels modulate the resting membrane potential during early embryogenesis

By using the patch-clamp technique, stretch-activated ionic channels were found in the membrane of cleaving freshwater fish embryos at the early stages of embryogenesis (2-256 cells). The application of negative pressure to the pipette increased the frequency of activation and the duration of bursts. This type of channel has a preferential K+ selectivity. When bathed on both membrane surfaces with 140 mM KCl the channel conductance was 71 pS. The kinetic behaviour did not depend markedly on either membrane potential (in the range from -70 to +70 mV) or calcium concentration on the cytoplasmic side of the membrane. On continuous recording, the probability of the channel being open was found to change periodically over a 5- to 20-fold range for different cells. These variations correlated with changes in resting potential and membrane conductance during the cell cycle. These results suggest that the oscillation of resting potential within the cell cycle is associated with the operation of stretch-activated ion channels.     

Cytoskeleton in preimplantation mouse development

This paper reviews the constituents of the cytoskeleton in the cells of the preimplantation mouse embryo and how they change as the development proceeds. The cytoskeleton can be divided into two distinct groups, that in the cytosplasm and that associated with the membrane. The first and better-known group contains microfilaments, microtubules and intermediate filaments, the second such components of the cell and nuclear membrane as spectrin-like protein and nuclear lamin. The filamentous components of the cytoplasmic cytoskeleton adhere to the nuclear and cell membrane at attachment points where specific proteins such as vinculin may mediate the interaction. Each cell of the early embryo has all of these components, but their morphological organization and molecular constitution alter as the embryo develops. These modifications are especially pronounced when the cleavage-stage embryo compacts and when the blastocysts forms and differentiates. These events represent the most critical stages of morphogenesis and cytodifferentiation in the preimplantation embryo. The cytoskeleton may thus have an important role in the control of the early mammalian development.     

Antigenic polysaccharides of bacteria. 35. Establishment of the structure of polysaccharide chains of lipopolysaccharides of Pseudomonas cepacia IMV 4176 and IMV 4202 (Serotype 3)

On mild acid degradation of the Pseudomonas cepacia strain IMV 4176 lipopolysaccharide, two polysaccharides were obtained, one of which is a homopolymer of N-acetyl-D-galactosamine and the other is composed of equal amounts of N-acetyl-D-galactosamine and D-ribose. Partial hydrolysis with aqueous oxalic acid caused depolymerization of the heteropolysaccharide, and the homopolysaccharide was isolated in the individual state. On the basis of methylation and 13C NMR analysis, it was concluded that both polysaccharides are built up of disaccharide repeating units having the following structures: ----4)-alpha-D-GalpNAc-(1----4)-beta-D-GalpNAc-(1---- and ----4)-alpha-D-GalpNAc-(1----2)-beta-D-Ribf-(1----. The heteropolysaccharide from P. cepacia strain 4176 is identical by the structure of the repeating unit to the O-specific polysaccharide of P. cepacia strain IMV 4202 (serotype 3), Pseudomonas aeruginosa O12 and Serratia marcescens O14.     

Genes coding for RNA polymerase in bacteria. III. The use of modified Sanger's method for sequencing the C-terminal region of rpoB gene, N-terminal region of rpoC gene and intercistron region of RNA polymerase in Pseudomonas putida

The Sanger method was modified and the primary structure of the SalI-C fragment of the Pseudomonas putida rpoBC operon was elucidated.     

Immunological relevance of malonic dialdehyde (MDA): II. Precipitation reactions of human sera with MDA-crosslinked proteins

Using precipitation reactions in agarose gels (Bidimensional double diffusion: Ouchterlony. Counterimmunoelectrophoresis: CEP), we showed that: a) sera from normal human subjects contain components able to bind and precipitate with MDA-crosslinked lysozyme (ML) and not with native lysozyme, which indicates that the chemical structures involved in such bindings arise from reaction of MDA with lysozyme and probably include 1-amino-3-iminopropene (AIP) bridges. b) some if not all of these seric components are immunoglobulins. c) the F(ab')2 regions of these immunoglobulins are involved in their binding and precipitating properties. These results lead us to assume that sera from normal human subjects contain immunoglobulins with antibody-like specificity for MDA-crosslinked proteins. Nevertheless, this assumption remains to be assessed by further studies, especially about the "epitopes" involved in such reactions.     

Determination of the intracellular protein thiol distribution of hepatocytes using monobromobimane derivatisation of intact cells and isolated subcellular fractions

The derivatisation of intact rat hepatocytes with monobromobimane resulted in rapid labelling of accessible protein thiols in several subcellular fractions. The derivatisation procedure did not cause acute cytotoxicity, nor did it alter the buoyant densities of the fractions or their gross protein compositions. Quantitation of the fluorescence irreversibly associated with the fractions demonstrated considerable intracellular heterogeneity in this pool of thiols. Values were highest in cytosol (ca. 90 nmol/mg protein), intermediate in microsomes (ca. 65 nmol/mg protein) and mitochondria (ca. 45 nmol/mg protein) and lowest in a crude fraction containing both nuclei and plasma membrane (ca. 35 nmol/mg protein). Similar values were obtained from microsomes and cytosol derivatised after fractionation but there were significant increases of ca. 100% in corresponding values from isolated mitochondria and the nuclear/plasma membrane fraction. These results are discussed in terms of the dynamic fluxes in monobromobimane protein thiols during fractionation and the applicability of this noninvasive method to studies of the mechanism(s) of toxicity of reactive xenobiotics and the role(s) of protein thiols in normal cellular function.     

Electrical stability of erythrocytes in the presence of divalent cations

Erythrocytes suspended in a medium of low ionic strength lyse under the effect of an exponential electrical pulse. The percentage of haemolysed cells decreases several-fold in the presence of divalent cations. The protective action of the ions studied increases in the following order: Ca⁺⁺, Mg⁺⁺, Zn⁺⁺. It is assumed that divalent ions bind to the negative charges of the lipid and protein molecules and reduce their electrostatic repulsion, which results in stabilization of the membranes.