Assessment of the ferret as an in vivo model for mumps virus infection.

Humans are the sole reservoir for mumps virus (MuV), the causative agent of mumps. No animal model currently exists; therefore, in vivo knowledge of the virus is limited. Ferrets were assessed for their susceptibility to MuV based on their success as a model for influenza. We infected ferrets with clinical or attenuated vaccine MuVs by the nasal route and demonstrated evidence of immunogenicity in these animals with generation of a serum antibody response specific to MuV infection and cytokine production consistent with infection. However, no live virus or viral RNA was detected in nasal washes, oral swabs, urine, faeces or tissue homogenates, and no animals exhibited clinical signs. We suggest results to be obtained from ferrets are limited in fundamental in vivo MuV research and that they may not be a suitable animal model for this virus.

Inhibition of atherosclerosis in apolipoprotein-E-deficient mice following muscle transduction with adeno-associated virus vectors encoding human apolipoprotein-E.

Apolipoprotein E (apoE) is a multifunctional plasma glycoprotein involved in lipoprotein metabolism and a range of cell signalling phenomena. ApoE-deficient (apoE(-/-)) mice exhibit severe hypercholesterolaemia and are an excellent model of human atherosclerosis. ApoE somatic gene transfer and bone marrow transplantation in apoE(-/-) mice results in reversal of hypercholesterolaemia, inhibition of atherogenesis and regression of atherosclerotic plaque density. Replication defective adeno-associated virus vectors (rAAVs) are an attractive system currently in clinical trial for muscle-based heterologous gene therapy to express secreted recombinant plasma proteins. Here we have applied rAAV transduction of skeletal muscle to express wild-type (epsilon3) and a defective receptor-binding mutant (epsilon2) human apoE transgene in apoE(-/-) mice. In treated animals, apoE mRNA was present in transduced muscles and, although plasma levels of recombinant apoE fell below the detection levels of our ELISA (ie <10 ng/ml), circulating antibodies to human apoE and rAAV were induced. Up to 3 months after a single administration of rAAV/apoE3, a significant reduction in atherosclerotic plaque density in aortas of treated animals was observed (approximately 30%), indicating that low-level rAAV-mediated apoE3 expression from skeletal muscle can retard atherosclerotic progression in this well-defined genetic model.

Identification of a gene selectively expressed in the brain, which encodes a putative transmembrane protein and a soluble cytoplasmic isoform.

Subtractive cloning procedures led to the identification of a variety of transcripts expressed in mammalian brain. However, little is known about the encoded proteins and the regulation of gene expression. Here, we describe the isolation and characterisation of a single-copy gene (83.5) of 21.7 kb which is specifically expressed in porcine brain. In situ hybridisation and immunohistochemistry experiments showed a distinct pattern of gene expression in neuronal cell types in different parts of the brain. The gene contains two mini exons, confirming neural-specific expression. cDNA cloning experiments revealed two species of mRNA differing in their 5'-regions. These transcripts are generated by two distinct transcription start sites that are under the control of different potential promoter regions as shown by primer-extension experiments. The amino acid sequences of the deduced proteins predict that one mRNA species encodes a novel type-I transmembrane protein, whereas the other transcript encodes only a part of its cytoplasmic domain. In Western-blot experiments, we detected two proteins of the predicted size and cellular localisation in porcine brain. The precise function of these proteins remains to be determined. However, our findings suggest that they may be generated by alternative promoter usage, leading to the expression of a membrane protein and its truncated cytoplasmic isoform.

Measles virus RNA is not detected in inflammatory bowel disease using hybrid capture and reverse transcription followed by the polymerase chain reaction.

Recent epidemiological and immunohistochemical studies have indicated a possible link between measles virus and inflammatory bowel disease (IBD). The aim of this study was to use a sensitive and robust method for the detection of measles virus RNA in IBD and control clinical samples. Peripheral blood mononuclear cells and intestinal resection tissue from IBD and control patients were studied. Two methods were used to determine the presence of measles virus RNA: hybrid capture, using measles virus-specific oligonucleotides linked to paramagnetic solid-phase supports, was carried out on total cellular RNA to enrich for measles virus RNA sequences. Reverse transcription followed by the polymerase chain reaction (RT-PCR) using rTth DNA polymerase was employed for amplification of measles virus N-gene sequences amongst the enriched species. Total RNA was also used for RT-PCR of a housekeeping mRNA species to assess RNA quality. RT-PCR for another region of the measles genome (the haemagglutinin (H) gene) was also undertaken in order to confirm the results obtained using N-gene primers for analysis of these samples. None of the samples were positive for measles N- or H-gene RNA using RT-PCR. Positive control samples confirmed the sensitivity of the methods employed. These results show that either measles virus RNA was not present in the samples, or was present below the sensitivity limits known to have been achieved.

Localization of the beta(beta)3-adrenoceptor in the human gastrointestinal tract: an immunohistochemical study.

BACKGROUND: Activation of human and non-human colonic beta(beta)3-adrenoceptors causes smooth muscle relaxation. beta3-Adrenoceptor agonists protect against experimental indomethacin-induced jejunal ulceration. The mechanism of protection may involve spasmolytic/vasodilatory agonist activity. The precise localization of beta3-adrenoceptors in the human gut is not known. AIM: To localize the beta3-adrenoceptor within the human gastrointestinal tract using the immunohistochemical technique. METHODS: Human beta3-adrenoceptors were immuno-localized in paraffin sections of human oesophagus (OS), stomach (ST), duodenum (DU), ileum (IL), sigmoid colon (SC), rectum (R) and gall-bladder (GB) using the rabbit polyclonal antibody anti-P12. Staining was graded , ++, + and 0. Immunostaining of SC was also done with pre-incubation of anti-P12 with P12 peptide. Western blotting of anti-P12 on human and murine IL and SC isolated membrane proteins was performed. RESULTS: All epithelia, vascular endothelial cells and ganglia scored 0. Smooth muscle of the vasculature, muscularis propria, muscularis mucosae and mucosa was graded, respectively, as follows; OS ( , , ,-), ST (++, , ++, ++), DU (++, , , +), IL (++, ++, ++, +), SC ( , ++, ++, ++), R (++, ++, +, +), GB ( , , -, 0). Pre-incubation of anti-P12 with P12 peptide almost abolished SC smooth muscle positivity. Western blot analysis using anti-P12 on human, but not murine, IL and SC membrane proteins revealed a single 5 5 kDa band, a size consistent with the predicted size of a partially glycosylated form of the human beta3-adrenoceptor. CONCLUSIONS: This immunohistochemical study has localized the beta3-adrenoceptor to vascular and nonvascular smooth muscle in the human gastrointestinal tract. These findings support a role for the beta3-adrenoceptor in the control of blood flow and motility in the human gastrointestinal tract.

Cloning and characterization of a porcine protein kinase gene and relationship to a class of heat shock proteins.

We have determined the genomic sequence of a porcine protein kinase (PPK) gene, including 1,844 bp upstream of the transcription initiation site. The gene spans over 19 kb and consists of 18 exons and 17 introns. The 5' regulatory region contains a characteristic heat shock element in the first intron, a weak heat shock element 1,464 bp upstream of the transcription initiation site, an atypical TATA box, and further consensus sequences typical for eukaryotic promoters such as an SP-1 binding site. Southern blot analysis indicates that PPK exists as a single-copy gene in the porcine haploid genome. The PPK gene is transcribed in all investigated tissues as shown by Northern blotting and reverse transcriptase polymerase chain reaction. Comparison of the protein and cDNA sequences of PPK to other sequences in DNA and protein databases indicates significant homology to a class of heat shock proteins, the glucose-regulated proteins (GRP94). In addition, nucleotide sequences at the 5' terminus of the PPK gene show strong homology to the GRP94 family. Domains highly conserved with human tumor rejection antigen (GP96) or glucose-regulated protein (GRP94) genes are identified within the 5' terminus and the first intron of the PPK gene. These findings suggest that these proteins are either identical or represent a family of closely related proteins.

Organization of the 5'-End of the porcine gamma-glutamyl transpeptidase gene and identification of three different mRNAs in the kidney.

Three different mRNAs coding for the porcine gamma-glutamyl transpeptidase (GGT) in the kidney were identified by 5'-RACE-PCR. These differ in their 5'-noncoding region. Genomic Southern blot analysis has demonstrated the existence of a single GGT gene in the porcine genome. Thus, the existence of multiple mRNAs can only be explained by the use of different promoters or alternative splicing. Four GGT-specific genomic clones containing the complete 5'-end of the gene were isolated and characterized, revealing six exons common to all three mRNAs. Four of these exons were located in the coding region comprising the codons for amino acids 1 to 138. Two exons and an intervening sequence were identified upstream from these six common exons representing the unique 5'-ends of the three mRNAs. The coding exons show a significant sequence homology to mouse, rat, and human GGT cDNA, whereas exons 1 and 3 display no homology.