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Biomedical subjects

R L Slobbe

Publications and source records attributed to R L Slobbe.

10 recordsLinked to original sources
PubMed1997

Widespread presence of cytomegalovirus DNA in tissues of healthy trauma victims.

AIMS: To determine the localisation of human cytomegalovirus (CMV) DNA in abdominal aorta, spleen, and transplantable organs, such as kidney, pancreas, and liver, obtained from healthy individuals; to characterise the cell type(s) in these tissues that serve as a reservoir for latent CMV. METHODS: CMV DNA was detected by dot blot DNA hybridisation and in situ DNA hybridisation with a probe for CMV major immediate early sequences (UL123) and nested PCR with primers derived from the CMV major immediate early (IE) gene exon 4 (UL123ex4). Samples of liver, abdominal aorta, spleen, kidney, and pancreas were obtained at necropsy or from donor kidneys from healthy subjects. RESULTS: CMV DNA was detected in most tissue samples using dot blot hybridisation and nested PCR. In situ hybridisation demonstrated that, in addition to smooth muscle cells in the arterial wall, hepatocytes, tubular and glomerular kidney cells, splenic red pulp cells, and pancreatic acinar cells also harboured CMV DNA. CMV DNA was detected in seropositive and in some seronegative subjects. CONCLUSION: CMV DNA is widely distributed in organs of healthy subjects. CMV DNA was found in various cell types in several organs, suggesting that during latency, CMV DNA is present thoughout the body.

Adolescent↗
PubMed1996

Detection and sequence analysis of the major immediate early and PP150 gene of latent human cytomegalovirus in spleen, liver, and kidney tissues of trauma victims.

The presence of human cytomegalovirus (HCMV) DNA in liver, spleen, and kidney samples of HCMV-seropositive trauma victims during latency was demonstrated by polymerase chain reaction (PCR), using primers reactive with the major immediate early gene exon 4 and the structural gene pp150. Sequence analysis of the PCR amplificates showed more than 95% homology with the reference HCMV strain AD169. The few mutations observed were mostly distributed randomly. In one subject two types of the MIE-4 gene were detected, and in another subject two types of the pp150 gene were found, suggesting that different strains of HCMV can be found in organs of the same patient during latency.

Adolescent↗
PubMed1992

Ro ribonucleoprotein assembly in vitro. Identification of RNA-protein and protein-protein interactions.

The human Y RNAs, small RNAs with an unknown function, are complexed with at least three proteins: the 60,000 M(r) Ro protein (Ro60), the 52,000 M(r) Ro protein (Ro52) and the La protein (La). In this study we examined the intermolecular interactions between the components of these so-called Ro ribonucleoprotein (Ro RNP) complexes. Incubation of 32P-labelled hY1 RNA in HeLa S100 extract allows the reconstitution of Ro RNP complexes, which were analysed by immunoprecipitation with monospecific antisera. By immunodepletion of HeLa S100 extracts for either Ro60, Ro52 or La, followed by supplementation with recombinant Ro60 or La, it was demonstrated that both Ro60 and La bind to hY1 RNA directly without being influenced by one of the other proteins. However, binding of Ro52 to hY1 RNA required the presence of Ro60, which strongly suggests that the association of Ro52 with Ro RNPs is mediated by protein-protein interactions between Ro60 and Ro52.

Autoantigens↗
PubMed1991

Analysis of protein--RNA interactions within Ro ribonucleoprotein complexes.

The interactions between Ro and La proteins and hY RNAs have been analysed. The binding site for the 60 kDa Ro protein on hY RNAs is shown to be the terminal part of the base paired stem structure, which contains the most highly conserved sequence among hY RNAs. The bulged C-residue within this region plays an important role in the recognition by this protein. The same regions of hY RNAs are essential for the association of the 52 kDa Ro protein with the RNAs, strongly suggesting that the 60 kDa Ro protein is required for the 52 kDa Ro protein to bind, presumably via protein-protein interactions, to Ro RNPs. The binding site for the La protein on hY RNAs is shown to be the oligouridylate stretch near the 3'-end of the RNAs, which is also recognized when additional nucleotides flank this motif at the 3'-side. Additional sequence elements in hY3 and hY5, but not in hY1, are bound by the La protein as well. Deletion mutagenesis showed that the RNP motif, previously identified in many ribonucleoprotein (RNP) proteins and in some cases shown to be almost sufficient for the interaction with RNA, of both the 60 kDa Ro and the La protein are not sufficient for the interaction with hY RNAs. Substantial parts of these proteins flanking the RNP motif are needed as well. It is likely that they stabilize the correct conformation of the RNP motif for RNA binding.

Base Composition↗
PubMed1991

Detection and occurrence of the 60- and 52-kD Ro (SS-A) antigens and of autoantibodies against these proteins.

The simultaneous detection of anti-La, anti-60-kD Ro and anti-52-kD Ro antibodies by immunoblotting is greatly improved by changing the crosslinking level in the gel to an acrylamide/bisacrylamide ratio of 19:1. Using this method for the analysis of a number of systemic lupus erythematosus (SLE) and Sjögren's syndrome patient sera it was observed that antibody to the 52-kD Ro protein without anti-60-kD Ro antibody was restricted to Sjögren's syndrome patients (9/26), whereas antibody to the 60-kD Ro protein without contaminating anti-52-kD Ro antibody was only found in SLE patients (8/38). Moreover, in Sjögren's syndrome patient sera anti-Ro antibody was found only in combination with anti-La antibody (20/26), whereas in SLE patient sera anti-Ro antibody could be found without detectable anti-La specificity (4/38). Double immunofluorescence microscopy revealed that the 52-kD Ro and the 60-kD Ro proteins co-localize in the cytoplasm as well as in the nucleus, whereas immunoprecipitation of [32P]-labelled HeLa cell extract with monospecific anti-52-kD Ro and anti-60-kD Ro sera showed that both proteins are associated with the Ro RNAs. These data suggest the presence of both the 52-kD and the 60-kD Ro proteins in the same ribonucleoprotein complexes. To study the evolutionary conservation of the 52-kD Ro, the 60-kD Ro and the La proteins, extracts of cell lines derived from various mammalian species were analysed on Western blots using monospecific human antibodies. In contrast to the 60-kD Ro and the La antigens which are well conserved in evolution, the 52-kD Ro antigen could be detected in primate cells only by this immunological approach.

Antibody Specificity↗
PubMed1987

Upstream regions of the hamster desmin and vimentin genes regulate expression during in vitro myogenesis.

Varying lengths of the hamster desmin and vimentin promoter regions were fused to the bacterial chloramphenicol acetyl-transferase gene. These constructs were transfected into two different myogenic cell lines, T984 and C2C12. In both cell lines an increase in endogenous desmin expression takes place upon myogenesis. A region between -89 and +25 bp relative to the desmin transcription initiation site directs high-level tissue- and stage-specific expression upon in vitro myogenesis. At the myoblast stage, C2C12 cells appeared to express both desmin and vimentin, whereas in T984 myoblasts only vimentin expression was detected. Although vimentin is expressed during all stages of myogenesis, a strong decrease in vimentin expression occurs during differentiation of C2C12 cells. Vimentin--CAT constructs followed the endogenous expression pattern, showing that this down-regulation is mediated by 5' flanking sequences. Vimentin promoter activity is modulated by at least two separate regions, both in myogenic and in non-myogenic cell lines.

Acetyltransferases↗