[Antigenic retrieval using a pressure cooker]. / Restauration antigénique à l'aide d'un autocuiseur.

This technical note describes antigen retrieval procedures using a pressure cooker. These procedures are perfectly adapted to routine immunohistochemistry and improve the overall quality of immunostaining.

Physical mapping and nucleotide sequence of a herpes simplex virus type 1 gene required for capsid assembly.

In this report, we describe some phenotypic properties of a temperature-sensitive mutant of herpes simplex type 1 (HSV-1) and present data concerning the physical location and nucleotide sequence of the genomic region harboring the mutation. The effect of shifts from the permissive to the nonpermissive temperature on infectious virus production by the mutant A44ts2 indicated that the mutated function is necessary throughout, or late in, the growth cycle. At the nonpermissive temperature, no major differences were detected in viral DNA or protein synthesis with respect to the parent A44ts+. On the other hand, electron microscopy of mutant-infected cells revealed that neither viral capsids nor capsid-related structures were assembled at the nonpermissive temperature. Additional analyses employing the Hirt extraction procedure showed that A44ts2 is also unable to mature replicated viral DNA into unit-length molecules under nonpermissive conditions. The results of marker rescue experiments with intact A44ts2 DNA and cloned restriction fragments of A44ts+ placed the lesion in the coordinate interval 0.553 to 0.565 (1,837 base pairs in region UL) of the HSV-1 physical map. No function has previously been assigned to this region, although it is known to be transcribed into two 5' coterminal mRNAs which code in vitro for a 54,000-molecular-weight polypeptide (K. P. Anderson, R. J. Frink, G. B. Devi, B. H. Gaylord, R. H. Costa, and E. K. Wagner, J. Virol. 37:1011-1027, 1981). We sequenced the interval 0.551 to 0.565 and found an open reading frame (ORF) for a 50,175-molecular-weight polypeptide. The predicted product of this ORF exhibits strong homology with the product of varicella-zoster virus ORF20 and lower, but significant, homology with the product of Epstein-Barr virus BORF1. For the three viruses, the corresponding ORFs lie just upstream of the gene coding for the large subunit of viral ribonucleotide reductase. The ORF described here corresponds to the ORF designated UL38 in the recently published nucleotide sequence of the HSV-1 UL region (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988).

Genome structure of cottontail rabbit herpesvirus.

The genome structure of a herpesvirus isolated from primary cultures of kidney cells from the cottontail rabbit Sylvilagus floridanus was elucidated by using electron microscopy and restriction enzyme analysis. The genome, which was about 150 kilobase pairs long and which had an average G + C composition of 45%, consisted of two regions with unique base sequences (54 and 47 kilobase pairs) enclosed by reiterations of a 925-base-pair sequence with a variable copy number. The internal repeats were in opposite polarity with respect to the terminal repeats, and both unique regions underwent inversion. The nucleotide sequence of the repeat unit was determined, and virion DNA termini were precisely localized within this sequence. Elements showing homology with the cleavage-packaging signals common to other herpesviruses were detected. The data indicate that this virus is different from the previously described herpesvirus sylvilagus.

Genome structure and virion polypeptides of the primate herpesviruses Herpesvirus aotus types 1 and 3: comparison with human cytomegalovirus.

Two serologically distinguishable primate herpesviruses, Herpesvirus aotus type 1 and type 3, were examined with regard to their genomes and structural polypeptides. The duplex DNA genomes of these two viruses were found to be essentially identical in molecular weight (Mr approximately equal to 145 X 10(6)) and guanine plus cytosine composition (55%). Both contained unique and inverted repeat nucleotide sequences of the same size and arrangement, which, as judged by DNA-DNA hybridization and restriction enzyme analyses, were at least 95% homologous. In addition, no differences were observed in electrophoretic profiles of virion polypeptides. Because of their great similarity with respect to these criteria, the two viruses ought to be considered independent isolates (or strains) of a single virus, which should be designated H. aotus type 1. The elevated molecular weight and presence of two sets of inverted repeat sequences closely resemble the structure of the human cytomegalovirus genome. However, no sequence homology (less than 5%) nor similarity in virion polypeptides was detected between H. aotus type 1 and human cytomegalovirus.

Inverted repeat nucleotide sequences in the genomes of Marek disease virus and the herpesvirus of the turkey.

The DNAs of two herpesvirus, the oncogenic Marek disease virus and the serologically related herpesvirus of the turkey, were studied by electron microscopy. On the basis of fold-back molecules observed in single-stranded DNA from both viruses, structures have been derived from the overall nucleotide sequence arrangement in their genomes. Although differing in molecular weight, the genomes of Marek disease virus and turkey herpesvirus are both constructed according to the same plan--two regions of unique nucleotide sequence, each enclosed by inverted repeat sequence. The genome structure of these viruses therefore closely resembles that of herpes simplex virus rather than the biologically more similar herpesvirus Epstein--Barr virus, H. saimiri, and H. ateles.

Characterization of simian virus 40 transcriptional intermediates in infected CV1 cell nuclei.

Simian Virus (SV40) transcriptional intermediates (T.I.) were isolated from infected cell nuclei incubated in vitro in the presence of the four ribonucleoside triphosphates. The nascent mRNA strands in the viral DNA-RNA hybrid molecules were hydrogen bonded to their template by 200-250 nucleotides on the average, as judged from the extent of their RNase resistance and the aspect of T.I. under electron microscope after treatment with 50 per cent formamide. The RNA polymerase involved (RNA polymerase II) synthesized up to full length transcripts at a rate of approximately 150 nucleotides/min. at 25 degrees C. Each SV40 infected cell was found to contain about 200 active T.I. molecules at the peak of late transcription. The DNA in the T.I. molecules was exclusively form I DNA only in cell infected with the tsA30 mutant of SV40 that had been transferred to non-permissive temperature in order to arrest DNA replication, but both form I DNA and molecules behaving as replicative intermediates (R.I.) in wild type infected cells.

Circular adenovirus DNA-protein complexes from infected HeLa cell nuclei.

Adenovirus (Ad) DNA was extracted by the use of Sarkosyl from Ad5-infected HeLa cell nuclei at 15 hr after infection. Mature and replicating viral DNA molecules in the Sarkosyl extract were purified by sucrose gradient centrifugation followed by equilibrium centrifugation in preformed metrizamide gradients. Part of the material was digested with the Hpa I restriction endonuclease and analyzed by electrophoresis through 1% agarose gels. Both terminal fragments D and E did not enter the gel unless first treated with Pronase, suggesting that intracellular viral DNA molecules were linked to proteins by their termini. Examination under the electron microscope of mature intracellular Ad2 or Ad5 DNA molecules extracted with Sarkosyl showed the presence of a majority of circular unit-length molecules. Most of these were attached to a prominent globular structure which disappeared, together with the circularization of the molecule, after treatment with Pronase. We interpret these structures as protein links responsible for the circularization of the molecule. The mature linear DNA molecules observed also showed evidence for linkage structures attached to one or both of their ends. Examination of replicating Ad5 DNA molecules extracted with Sarkosyl revealed the presence of various types of forked, circular molecules, most of which showed evidence for the presence of linkage structures. It is suggested that replicating and newly made Ad DNA molecules are circular inside the cell, perhaps through linkage with proteins, and the possible role of circularization in the replication of viral DNA is discussed.

[Late dyskinesia caused by neuroleptics. Survey on 1,660 psychiatric hospital patients]. / Les dyskinésies tardives des neuroleptiques Enquête sur 1 660 malades d'hôpital psychiatrique

Among 1 660 patients of 2 psychiatric hospitals (980 + 680), most of them chronically treated by neuroleptics, we found 120 tardive dyskinesia (7,2%). 64% were more of 50 years old. 85 of 923 women (9,1%), 36 of 737 men (4,8%), but most of the men are younger. In 67 cases oral dyskinesia is isolated, in 53 cases it is associated with choreic syndrom. 13 patients are free of neuroleptics for more than 6 months and the dyskinesia is probably irreversible (mean age = 74). The interest is double: 1. Practical: prevention by moderated and adapted prescription of neuroleptics, specially for long term treatments and oldest patients; anticholinergic correctors must be cautiously prescribed. 2. Theoretical: this human experimentation is a model of iatrogenic pathology for the study of mental illnesses, extrapyramidal troubles, and neuromediators...