Isoform-specific expression of the Coxsackie and adenovirus receptor (CAR) in neuromuscular junction and cardiac intercalated discs

BACKGROUND: The Coxsackie and adenovirus receptor (CAR) has a restricted expression pattern in the adult. In skeletal muscle, although CAR is expressed in immature fibers, its transcript levels are barely detectable in mature muscle. This is in contrast to the robust expression observed in the heart. However, both heart and skeletal muscle are susceptible to infection with the Coxsackie B virus which utilizes primarily CAR for cellular internalization. The specific point of viral entry in skeletal and heart muscle remains unknown. RESULTS: Using antibodies directed against the extracellular and the cytoplasmic domains of CAR, we show CAR in normal human and mouse skeletal muscle to be a novel component of the neuromuscular junction. In cardiac muscle, CAR immunoreactivity is observed at the level of intercalated discs. We demonstrate a single isoform of CAR to be expressed exclusively at the human neuromuscular junction whereas both predominant CAR isoforms are expressed at the intercalated discs of non-diseased human heart. CONCLUSION: The localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus

Essential Role of the Coxsackie - and Adenovirus Receptor (CAR) in Development of the Lymphatic System in Mice.

Peer reviewe

Multiple Phenotypes in Adult Mice following Inactivation of the Coxsackievirus and Adenovirus Receptor (Car) Gene

To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhibited a complete atrio-ventricular block and abnormal thymopoiesis. These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo

Loss of the coxsackie and adenovirus receptor contributes to gastric cancer progression

Loss of the coxsackie and adenovirus receptor (CAR) has previously been observed in gastric cancer. The role of CAR in gastric cancer pathobiology, however, is unclear. We therefore analysed CAR in 196 R0-resected gastric adenocarcinomas and non-cancerous gastric mucosa samples using immunohistochemistry and immunofluorescence. Coxsackie and adenovirus receptor was found at the surface and foveolar epithelium of all non-neoplastic gastric mucosa samples (n=175), whereas only 56% of gastric cancer specimens showed CAR positivity (P<0.0001). Loss of CAR correlated significantly with decreased differentiation, increased infiltrative depths, presence of distant metastases, and was also associated with reduced carcinoma-specific survival. To clarify whether CAR impacts the tumorbiologic properties of gastric cancer, we subsequently determined the role of CAR in proliferation, migration, and invasion of gastric cancer cell lines by application of specific CAR siRNA or ectopic expression of a human full-length CAR cDNA. These experiments showed that RNAi-mediated CAR knock down resulted in increased proliferation, migration, and invasion of gastric cancer cell lines, whereas enforced ectopic CAR expression led to opposite effects. We conclude that the association of reduced presence of CAR in more severe disease states, together with our findings in gastric cancer cell lines, suggests that CAR functionally contributes to gastric cancer pathogenesis, showing features of a tumour suppressor

Molecular evolution of the LNX gene family

<p>Abstract</p> <p>Background</p> <p>LNX (Ligand of Numb Protein-X) proteins typically contain an amino-terminal RING domain adjacent to either two or four PDZ domains - a domain architecture that is unique to the LNX family. LNX proteins function as E3 ubiquitin ligases and their domain organisation suggests that their ubiquitin ligase activity may be targeted to specific substrates or subcellular locations by PDZ domain-mediated interactions. Indeed, numerous interaction partners for LNX proteins have been identified, but the <it>in vivo </it>functions of most family members remain largely unclear.</p> <p>Results</p> <p>To gain insights into their function we examined the phylogenetic origins and evolution of the <it>LNX </it>gene family. We find that a <it>LNX1/LNX2</it>-like gene arose in an early metazoan lineage by gene duplication and fusion events that combined a RING domain with four PDZ domains. These PDZ domains are closely related to the four carboxy-terminal domains from multiple PDZ domain containing protein-1 (MUPP1). Duplication of the <it>LNX1/LNX2</it>-like gene and subsequent loss of PDZ domains appears to have generated a gene encoding a LNX3/LNX4-like protein, with just two PDZ domains. This protein has novel carboxy-terminal sequences that include a potential modular LNX3 homology domain. The two ancestral <it>LNX </it>genes are present in some, but not all, invertebrate lineages. They were, however, maintained in the vertebrate lineage, with further duplication events giving rise to five LNX family members in most mammals. In addition, we identify novel interactions of LNX1 and LNX2 with three known MUPP1 ligands using yeast two-hybrid asssays. This demonstrates conservation of binding specificity between LNX and MUPP1 PDZ domains.</p> <p>Conclusions</p> <p>The <it>LNX </it>gene family has an early metazoan origin with a LNX1/LNX2-like protein likely giving rise to a LNX3/LNX4-like protein through the loss of PDZ domains. The absence of LNX orthologs in some lineages indicates that LNX proteins are not essential in invertebrates. In contrast, the maintenance of both ancestral <it>LNX </it>genes in the vertebrate lineage suggests the acquisition of essential vertebrate specific functions. The revelation that the LNX PDZ domains are phylogenetically related to domains in MUPP1, and have common binding specificities, suggests that LNX and MUPP1 may have similarities in their cellular functions.</p

Studies on two transcription regulatory proteins : cellular C-jun and adenovirus E1A

STUDIES ON TWO TRANSCRIPTION REGULATORY PROTEINS: CELLULAR C-JUN AND ADENOVIRUS ElA. KERSTIN SOLLERBRANT Departrnent of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, S-171 77 Stockholm, Sweden. Adenoviruses cause Iytic infections in a range of mammalian cells, but can in certain cases also transform the host cell. The first adenovirus gene to be expressed upon infection is EIA. The ElA gene encodes two major proteins of 243 and 289 amino acids, respectively, both of which serve as key regulators during virus infection. The ElA proteins can redirect transcription and also reprogram the cell cycle to suit the need of the virus. In these processes different regulatory proteins from the host cell are bound by the ElA proteins via its conserved regions (CRl, CR2 and CR3). Studies of ElA function have thus proven very useful for learning about regulation of transcription and cell cycle control in the cell. The EIA proteins can both activate and repress transcription of specific cellular,and viral genes The repressing function of EIA has previously been shown only for RNA polymerase II transcribed genes. Using transient transfection experiments, we here demonstrate that EIA can repress transcription also of the polymerase III-driven VA RNA genes E IA deletion studies showed that CRI and the first five amino acids of CR2 were required for this repression. We also discovered that another adenovirus encoded protein, EIB 19K, could counteract the ElA-mediated repression by activating transcription of the VA RNA genes The importance of ElB l9K for VA RNA expression was demonstrated also during a viral infection In a parallel project, we identified a previously undescribed transcription regulatory region located near the C-terminus of the ElA proteins. This region exactly corresponds to the binding site for the cellular protein CtBP. Binding of CtBP to ElA has previously been suggested to suppress the ability of ElA to transform host cells. We show that transcription mediated by the El A-CR I domain is regulated by the CtBP binding region. Moreover, we show that the CtBP-binding region is needed for efficient induction of some, but not all, ElA243R-responsive cellular genes. We therefore propose a novel mechanistic model for ElA243R-mediated transactivation. Studies on transcription activation domains in mammalian cells are often done by fusing them to a heterologous DNA binding domain (DBD). Such fusion proteins expressing the DNA binding domain of the yeast transcription factor Gal4 can activate transcription from synthetic promoters with binding sites for the Gal4 DBD. This Gal4-fusion system is widely used since mammalian genes do not contain binding sites for Gal4. As a spin-off result from our studies with the CR1 activation domain, we however found that Gal4 DBD can bind to the cellular transcription factor c-Jun This resulted in efficient transactivation in vivo of promoters containing binding sites for the c-Jun containing APl factor. This finding has important consequences for everyone working with Gal4 fusions in mammalian cells, since transactivation is not restricted to promoters containing binding sites for Gal4, as was previously thought. In summary, the work presented in this thesis reveals new insight into the mechanisms used by the ElA proteins to modulate transcription. It also describes a previously unknown interaction between the yeast Gal4 and mammalian c-Jun transcription factors and the consequences of this interaction for the study of transcription initiation is discussed. Keywords: adenovirus, EIA, EIB, transcription, CtBP, c-Jun, Gal4 ISBN: 91-628-2190-

Comparison and evaluation of FastQ B*27 direct and LAMP Human HLA-B27 direct detection KIT for HLA-B27 allele detection

Autoimmunitet är ett tillstånd där kroppens immunsystem felaktigt attackerar och skadar sina egna vävnader och celler. HLA-B27 är en genvariation som kan kopplas till autoimmun sjukdom som ankyloserande spondylit med en prevalens på 2-4% i världens befolkning. Denna studie syftade till att utvärdera och jämföra två kit för HLA-B27 alleler mot den nuvarande metoden på Länssjukhuset Ryhov i Region Jönköpings län. De metodprinciper som användes var realtids-PCR samt LAMP.  Totalt analyserades 37 avidentifierade blodprover med vardera av kiten samt med nuvarande metod. Resultatet visade en överensstämmelse med avseende på förväntade positiva och negativa resultat för HLA-B27 för de två kiten jämfört med nuvarande metoden. De tre metoderna/kiten detekterar de vanligaste HLA-B27 allelerna. Utifrån studiens resultat visade sig båda kiten vara effektiva, lättanvända samt ha stabila reagenser. Dessutom uppnådde båda kiten de IVD-krav som ställs inom EU.  Autoimmunity is a condition where the body's immune system mistakenly attacks and damages its own tissues and cells. HLA-B27 is a genetic variation that can be linked to autoimmune diseases such as ankylosing spondylitis, with a prevalence of 2-4% in the world’s population. This study aimed to evaluate and compare two kits for HLA-B27 alleles against the current method at Ryhov County Hospital in Region Jönköping County. The methodological principles used were real-time PCR and LAMP. A total of 37 anonymized blood samples were analyzed using each of the kits and the routine method. The results showed concordance with the expected positive and negative results for HLA-B27 between the two kits compared to the current method. The three methods/kits detect the most common HLA-B27 alleles.  Based on the study’s results, both kits proved to be effective, user friendly, and have stable reagents. Additionally, both kits met the IVD requirements set within the EU