Biomedical subjects

R Sahli

Publications and source records attributed to R Sahli.

31 records · Page 2Linked to original sources

PubMed1992

Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line.

A wild-type p53 gene under control of the metallothionein MT-1 promoter was stably transfected into human colon tumor-derived cell line EB. Repeated inductions of the metallothionein wild-type p53 gene with zinc chloride results in progressive detachment of wild-type p53 cells grown on culture dishes. Examination at both the light and electron microscopic level revealed that cells expressing wild-type p53 developed morphological features of apoptosis. DNA from both attached and detached cells was degraded into a ladder of nucleosomal-sized fragments. Expression of wild-type p53 inhibited colony formation in soft agar and tumor formation in nude mice. Furthermore, established tumors in nude mice underwent regression if wild-type p53 expression was subsequently induced. Regressing tumors showed histological features of apoptosis. Thus, regression of these tumors was the result of apoptosis occurring in vivo. Apoptosis may be a normal part of the terminal differentiation program of colonic epithelial cells. Our results suggest that wild-type p53 could play a critical role in this process.

Base Sequence↗

PubMed1992

Development of polymerase chain reaction assays for detection of Listeria monocytogenes in clinical cerebrospinal fluid samples.

In order to improve the diagnosis of Listeria meningitis or meningoencephalitis, especially in patients who have received antibiotics before their cerebrospinal fluid (CSF) has been examined, two assays for the detection of Listeria monocytogenes based on the polymerase chain reaction (PCR) were evaluated. After a standard PCR, the amplified DNA was detected either by a second round of PCR with internal primers followed by gel electrophoresis and ethidium bromide staining (nested PCR) or by dot blot hybridization to an internal digoxigenin-labeled probe (PCR-dot blot). For PCR, two sets of primers within the invasion-associated protein gene (iap gene) were chosen. They allowed for the highly specific detection of all L. monocytogenes reference strains tested (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, and 7). These primers did not detect amplification products from various other gram-positive or gram-negative bacterial DNAs or human DNA. The sensitivities of both assays were assessed on sterile CSF samples that were artificially seeded with serial dilutions of L. monocytogenes serotype 4b cells. By both methods the limit of detection was less than 10 cells in the initial reaction. Since the nested PCR is more prone to contamination because of manipulation of the amplified products, a standard PCR assay followed by dot blot hybridization was applied to 52 CSF samples in a retrospective study. Of 28 CSF samples which were sterile or positive for bacteria other than Listeria species, 24 were PCR negative. In contrast, from 17 patients with culture-proven Listeria meningitis, 14 of 17 initial CSF samples were PCR positive, as were 3 of 7 culture-negative followup CSF samples taken after patients received antibiotics. These results support the usefulness of this approach in the diagnosis of Listeria meningitis, in particular, when antibiotic administration precedes culture of CSF.

Anti-Bacterial Agents↗

PubMed1992

Significance of the detection of Epstein-Barr virus DNA in lymph nodes in patients with Hodgkin's disease.

Epstein-Barr virus (EBV) DNA is frequently identified in benign and malignant lymphoproliferative conditions. As shown by in situ hybridization studies viral DNA is localized within malignant cells as well as benign lymphocytes. Clonal and nonclonal EBV genomes are present in Hodgkin's disease (HD), lymphomas of the immunocompromised host and reactive lymph node hyperplasia. Lytic infection with formation of linear genomes is observed in the same conditions but appears to be infrequent in HD as shown by quantitation of mRNA coding for viral capsid antigen. Expression of the oncogene LMP (latent membrane protein) is seen in Sternberg-Reed (SR) cells and immunoblasts of AIDS-related lymphoma and infectious mononucleosis (IM). In HD, the region of the BNLF1 oncogene coding for the amino terminal and transmembrane domains (associated with oncogenic function) of LMP appears to be homogeneous whereas the region coding for the intracytoplasmic (carboxy terminal) domain of LMP is heterogeneous. Cytological similarities between SR cells and immunoblasts of IM and AIDS-related lymphomas are consistent with the hypothesis that the BNLF1 oncogene is one possible inducer of morphological features of SR cells. Whether chromosomal integration of EBV DNA is an important factor in activation of such a transforming activity remains to be elucidated. EBV DNA positive and negative HD cases with numerous SR cells lack significant mRNA expression of the two recombinase activating genes (RAG-1 and RAG-2). Therefore the SR cells appear to be derived from lymphocytes beyond the pre-B-cell or common thymocyte stage which may or may not subsequently become infected by EBV.

Antigens, Viral↗

PubMed1991

Structure of simian virus 40 at 3.8-A resolution.

The crystallographically determined structure of simian virus 40 shows that the 72 pentamers of viral protein VP1, which form the outer shell, have identical conformations except for the C-terminal arms of their subunits. Five arms emerge from each pentamer and insert into neighbouring pentamers. This tying together of standard building blocks allows for the required variability in packing geometry without sacrificing specificity.

Amino Acid Sequence↗

PubMed1991

Frequent detection of Epstein-Barr virus DNA by the polymerase chain reaction in lymph node biopsies from patients with Hodgkin's disease without genomic evidence of B- or T-cell clonality.

This study of 52 Swiss patients with Hodgkin's disease (HD), including 17 cases with a high content of Sternberg-Reed (SR) and Hodgkin (H) cells, was performed to determine the percentage of cases harboring Epstein-Barr virus (EBV) DNA and/or clonal rearrangements of Ig and T-cell antigen receptor (TcR) genes in diagnostic lymph node biopsies. Special attention was drawn to the heavily infiltrated cases to detect a possible relationship between clonality and EBV DNA identification. EBV DNA was detected by the polymerase chain reaction (PCR) using three different sets of specific primers. The viral origin of the amplification products was confirmed by hybridization with a radiolabeled internal probe or demonstration of a specific Sma I restriction site. Genomic rearrangement of Ig and TcR genes was studied by Southern blot analysis. EBV DNA was identified by PCR in 38 of 48 cases (79%). Clonal rearrangements were identified in only 4 of 52 cases (Ig genes) and were independent of the degree of infiltration by SR cells and the presence of EBV DNA. The absence of EBV DNA in three cases with numerous SR cells (only one of them showed clonal rearrangement) and the presence of only a few viral copies in four further cases with numerous SR cells (semiquantitative analysis of viral DNA by PCR was performed in 26 EBV-positive cases) suggests that this virus is modulating rather than an etiologic agent in a considerable proportion of HD cases.

B-Lymphocytes↗

PubMed1991

Squamous cell carcinoma of the lung in a nonsmoking, nonirradiated patient with juvenile laryngotracheal papillomatosis. Evidence of human papillomavirus-11 DNA in both carcinoma and papillomas.

Malignant transformation of laryngeal juvenile papillomatosis most often occurs in patients with previous radiation therapy or smoking histories. We report the case of a 35-year-old, nonsmoking, nonirradiated man who developed squamous cell carcinoma of the lung with a 33-year history of laryngotracheal juvenile papillomatosis. Postmortem examination showed pulmonary cavitating papillomatosis and chest wall, vertebrae, and peribronchial lymph node involvement by tumor. Molecular studies (Southern blot, polymerase chain reaction) showed extrachromosomal human papillomavirus-11 (HPV11) DNA in both carcinoma and two laryngotracheal squamous cell papillomas, including one excised 20 years previously. Our observation is analogous to the previously reported cases of spontaneous (not related to irradiation or smoking) malignant transformation of juvenile laryngotracheal papillomatosis. Although HPV11 viral infection likely played a role in the malignant transformation, other less likely factors, such as drugs given for treatment and radiography performed throughout the illness, should be considered. Repeated pulmonary infections and the host immune response are additional considerations.

Adult↗

PubMed1991

A single-amino-acid substitution in polyomavirus VP1 correlates with plaque size and hemagglutination behavior.

The plaque size and hemagglutination characteristics of five cloned wild-type strains of polyomavirus were determined. The strains fell into two groups, those with large or small plaques, each with distinctive hemagglutination behavior at different temperatures and pHs. The nucleotide sequence of VP1, the major capsid protein of the virus, was determined for each of the viral strains. The PTA (large-plaque) and RA (small-plaque) strains differed only at residue 92 of VP1, where there is a glutamic acid or glycine, respectively (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991). The same amino acid difference in VP1 correlated with plaque size and hemagglutination properties of the other sequenced viruses. Mutagenesis converting amino acid 92 from glutamic acid to glycine converted the plaque size and hemagglutination behavior of the large-plaque PTA strain to that of a small-plaque strain. Furthermore, PTA and RA VP1 proteins produced in Escherichia coli behaved as their parental viruses did in hemagglutination assays. These results demonstrate that amino acid residue 92 of VP1 is involved in determining the plaque size and hemagglutination behavior of polyomavirus and strongly suggest that this region of the VP1 polypeptide interacts directly with cell receptors.

Amino Acid Sequence↗

PubMed1990

Detection of Epstein-Barr virus DNA by polymerase chain reaction in lymph node biopsies from patients with angioimmunoblastic lymphadenopathy.

Epstein-Barr virus DNA was detected by the polymerase chain reaction (PCR) in five lymph node biopsies from eight patients with diagnosis of angioimmunoblastic lymphadenopathy (AILD). Three pairs of specific primers detected EBV DNA sequences near the 5' end (Bam W region), the middle (BMRF 1 region) and the 3' end (Eco RI D region) of the viral genome with equal accuracy when 1 microgram of DNA and 30 amplification cycles were used. When only 100 ng of DNA were screened with the BMRF 1 set of primers, a specific amplification product was still identifiable. The 593 base pair amplification product obtained using the Eco RI D set of primers was shown to contain an expected Sma I site at position 215, confirming the viral origin of the amplified sequence [corrected]. Our findings indicate that lymph nodes of AILD patients frequently harbour the entire EBV genome at a high percentage of at least 1 viral copy per 15,000 human cells.

Base Sequence↗

PubMed1988

Characterization of the cell type-specific determinant in the genome of minute virus of mice.

Two strains of minute virus of mice (MVM) show different host cell specificities. The prototype strain MVM(p) grows in fibroblasts, whereas the immunosuppressive variant MVM(i) grows in T lymphocytes. In this study, we have mapped on the viral genome a cell type-specific determinant: it is located between 69 and 85 map units in a region coding for the viral capsid proteins. The DNA of MVM(p) does not replicate in lymphocytes. MVM(i) cannot help MVM(p) grow in lymphocytes; thus the determinant acts in a cis fashion. We did not detect viral mRNA during a restrictive infection of lymphocytes with MVM(p). However, when the same cells were transfected with cloned DNA, both MVM(p) and MVM(i) DNAs were transcribed with the same efficiency from both promoters and the RNA was processed normally. Therefore, the specificity determinant is not a cell type-specific enhancer.

Cell Line↗

PubMed1986

A precise map of splice junctions in the mRNAs of minute virus of mice, an autonomous parvovirus.

We have determined the exact splicing patterns of the mRNAs of the minute virus of mice by a combination of cDNA sequencing and S1 nuclease protection analysis. There are four virus-specific mRNA species, each coding for one of the four polypeptides identified by in vitro translation. The R1 mRNA comprises sequences from nucleotide approximately 200 to 2281 and from 2378 to approximately 4800 and codes for the NS1 protein. The R2 mRNA is derived from nucleotides approximately 200 to 515, 1991 to 2281, and 2378 to approximately 4800 and codes for the NS2 protein. Between nucleotides 1991 and 2281, the coding sequence for NS2 overlaps that of NS1, but in a different reading frame. R3 covers nucleotides approximately 2007 to 2281 and 2378 to approximately 4800 and codes for VP2. The fourth species, R3', differs from R3 by using an alternative splice donor and acceptor in the region around 47 map units (nucleotide 2400); it extends from nucleotide approximately 2007 to 2317 and from 2400 to approximately 4800 and almost certainly codes for VP1. The R2 transcript is unusual in that the intron that was removed from it (nucleotides 516 to 1990) starts with GC rather than the canonical GU. With the exception of the splice acceptor at position 2378, which is found only in rodent parvoviruses, the splice junctions are highly conserved among autonomous parvoviruses. These results show that minute virus of mice, like other small DNA viruses, uses multiple strategies to compress the coding information for several viral proteins into a short (5,104 nucleotide) genome.

Base Sequence↗

PubMed1985

DNA sequence comparison between two tissue-specific variants of the autonomous parvovirus, minute virus of mice.

We have determined the complete nucleotide sequence of the DNA of the immunosuppressive variant of the parvovirus minute virus of mice (MVMi) and compared it to the published sequence (12) of the fibroblast-specific strain (MVMp). We have found 175 differences between the two viruses, most of which affect single nucleotides. Despite these differences, the genomic organization of MVMp and MVMi is identical. There are 29 amino-acid changes between the putative viral gene products of MVMi and MVMp, 16 of which are conservative. We discuss the possibility that the differential tissue-specificity of the two variants is linked to differences within the non-transcribed region near the 5' end of the viral genomes.

Animals↗

PubMed1985

Ornithodoros moubata: spermateleosis and secretory activity of the sperm.

Cytological aspects of spermateleosis in the tick Ornithodoros moubata were studied by electron microscopy. During spermateleosis, detachment of the operculum from the outer sheath of the prospermium results from the fusion of the plasma and the cisternal membranes. The fusion occurs between the shoulder of the acrosomal vesicle and the electron-dense layer of the operculum. A factor inducing vitellogenesis and egg-laying is secreted by the sperm cell after spermateleosis, and begins after the cell is almost completely devaginated. In vitro, fully devaginated spermiophores secrete most of this factor during the first 12 hr of incubation. The vitellogenesis-inducing activity of the secretion is sensitive to proteinase K (EC 3.4.21.14) digestion and correlates with the presence of two high-molecular-weight proteins in the sperm cell incubation medium.

Animals↗

PubMedDate not supplied

Adenovirus-mediated gene transfer into selected liver segments using a vascular exclusion technique.

Adenovirus-mediated gene therapy is hampered by severe virus-related toxicity, especially to the liver. The aim of the present study was to test the ability of a vascular exclusion technique to achieve transgene expression within selected liver segments, thus minimizing both viral and transgene product toxicity to the liver. An E1-E3-deleted replication-deficient adenovirus expressing a green fluorescent protein (GFP) reporter gene was injected into the portal vein of BDIX rats, with simultaneous clamping of the portal vein tributaries to liver segments II, III, IV, V, and VIII. GFP expression and inflammatory infiltrate were measured in the different segments of the liver and compared with those of the livers of animals receiving the viral vector in the portal vein without clamping. The GFP expression was significantly higher in the selectively perfused segments of the liver as compared with the non-perfused segments (p < 0.0001) and with the livers of animals that received the vector in the portal vein without clamping (p < 0.0001). Accordingly, the inflammatory infiltrate was more intense in the selectively perfused liver segments as compared with all other groups (p < 0.0001). Fluorescence was absent in lungs and kidneys and minimal in spleen. The clinical usefulness of adenovirus-mediated gene transfer to the liver largely depends on the reduction of its liver toxicity. Clamping of selected portal vein branches during injection allows for delivery of genes of interest to targeted liver segments. Transgene expression confined to selected liver segments may be useful in the treatment of focal liver diseases, including metastases.

Adenoviridae↗