Oyster-associated outbreaks of Norovirus gastroenteritis in Singapore.

Outbreaks of gastroenteritis associated with the consumption of raw imported half-shelled frozen oysters occurred in Singapore between 16 Dec 2003 and 04 Jan 2004. A total of 305 cases were reported with clinical symptoms of diarrhoea (94%), abdominal cramps (72%), vomiting (69%) and fever (54%). The median incubation period was 30.8h and the duration of illness was 2-3 days. The overall relative risk of oyster consumption was 14.1 (95% CI: 8.3-24.0, P<0.001). Stool and oyster samples tested negative for common bacterial pathogens, including Vibrio parahaemolyticus. However, stool samples were positive for the presence of Norovirus group II RNA via RT PCR while oyster samples indicated the presence of Norovirus particles by electron microscopy. The clinical and epidemiological features were suggestive of Norovirus gastroenteritis and were subsequently confirmed by laboratory tests of stools and implicated oysters. Steps have been taken to ensure that food outlets do not thaw frozen oysters and serve them raw.

Localization of ligand-binding domains of human corticotropin-releasing factor receptor: a chimeric receptor approach.

Two CRF receptors, CRFR1 and CRFR2, have recently been cloned and characterized. CRFR1 shares 70% sequence identity with CRFR2, yet has much higher affinity for rat/human CRF (r/hCRF) than CRFR2. As a first step toward understanding the interactions between rat/human CRF and its receptor, the regions that are involved in receptor-ligand binding and/or receptor activation were determined by using chimeric receptor constructs of the two human CRFR subtypes, CRFR1 and CRFR2, followed by generating point mutations of the receptor. The EC50 values in stimulation of intracellular cAMP of the chimeric and mutant receptors for the peptide ligand were determined using a cAMP-dependent reporter system. Three regions of the receptor were found to be important for optimal binding of r/hCRF and/or receptor activation. The first region was mapped to the junction of the third extracellular domain and the fifth transmembrane domain; substitution of three amino acids of CRFR1 in this region (Val266, Tyr267, and Thr268) by the corresponding CRFR2 amino acids (Asp266, Leu267, and Val268) increased the EC50 value by approximately 10-fold. The other two regions were localized to the second extracellular domain of the CRFR1 involving amino acids 175-178 and His189 residue. Substitutions in these two regions each increased the EC50 value for r/hCRF by approximately 7- to 8-fold only in the presence of the amino acid 266-268 mutation involving the first region, suggesting that their roles in peptide ligand binding might be secondary.

Localization of agonist- and antagonist-binding domains of human corticotropin-releasing factor receptors.

The CRF receptors, CRFR1 and CRFR2, are members of the G protein-coupled receptor superfamily. Despite their considerable sequence similarity, CRFR1 and CRFR2 have quite different affinities for the peptide ligand rat/human CRF. Previous studies using chimeric receptors between human CRFR1 and CRFR2 have identified three potentially important regions in the second and third extracellular domains of CRF receptor for the binding of rat/human CRF. The present report further demonstrates that these same three regions also affect the binding of urocortin and sauvagine, two other members of the CRF peptide family, albeit to different extents. We also show that a fourth region in the third extracellular domain, Asp254, has been identified to be important for sauvagine but not CRF or urocortin binding. Thus, the three peptide ligands not only interact with a different set of regions on CRFR1 and CRFR2 but also differentially interact with some of the same regions. These data could, at least in part, account for the much higher affinity of CRFR2 for urocortin and sauvagine compared with rat/human CRF. We have also identified two amino acid residues, His199 in the third transmembrane domain and Met276 in the fifth transmembrane domain, that are important for binding the non-peptide high-affinity CRFR1 antagonist NBI 27914. Mutations of His199 and Met276 to the corresponding amino acids in CRFR2 each decreased the binding affinity of NBI 27914 for CRFR1 by 40- and 200-fold, respectively. This suggests that the transmembrane regions are critically important in forming the binding pocket for the nonpeptide antagonist.

Cloning and characterization of the human corticotropin-releasing factor-2 receptor complementary deoxyribonucleic acid.

Two CRF receptor subtypes (CRF1 and CRF2 receptors) with distinct brain localizations and pharmacological profiles have recently been cloned and characterized. For the CRF2 receptor subtype, at least 2 splice forms with different 5'-coding sequences (CRF2 alpha and CRF2 beta) have been identified in rat. In this article, we report the genomic structure and the corresponding complementary DNA (cDNA) sequence of the human CRF2 receptor. The gene coding for human CRF2 receptor consists of at least 12 exons and spans approximately 30 kilobases. The cDNA sequence in the protein-coding region is 94% identical to that of the reported rat CRF2 alpha receptor. At present, there is no evidence for the existence of a CRF2 beta receptor homolog in humans. The encoded receptor is 411 amino acids in length and is 70% identical to the human CRF1 receptor, with least sequence homology in the N-terminal extracellular domain (47% identical). Cells transfected with the full-length human CRF2 receptor cDNA responded to rat/human CRF and sauvagine by increasing the intracellular cAMP level, with EC50 values of approximately 20 and 1 nM, respectively. The CRF- and sauvagine-induced accumulation of intracellular cAMP could be competitively inhibited by the CRF receptor antagonist D-Phe-CRF. This pharmacological profile was comparable to that of the rat CRF2 alpha receptor. The relative abundance of the CRF2 receptor messenger RNA appears to be lower in humans than in rats for the tissues studied thus far.

Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain.

The present study reports the isolation of a cDNA clone that encodes a second member of the corticotropin-releasing factor (CRF) receptor family, designated as the CRF2 receptor. The cDNA was identified using oligonucleotides of degenerate sequence in a PCR paradigm. A PCR fragment obtained from rat brain was utilized to isolate a full-length cDNA from a rat hypothalamus cDNA library that encoded a 411-amino acid protein with approximately 70% identity to the known CRF1 receptor over the entire coding region. When expressed in mouse Ltk- cells, this receptor stimulates cAMP production in response to CRF and known CRF-like agonists. CRF and the nonmammalian CRF-related peptides sauvagine and urotensin I stimulate adenylate cyclase activity in a dose-dependent manner with a rank order of potency different from that of the CRF1 receptor: sauvagine > urotensin > or = rat/human CRF > ovine CRF. Tissue distribution analysis of the mRNAs by reverse transcriptase-PCR shows CRF2 receptor mRNA is present in rat brain and detectable in lung and heart. In situ hybridization studies indicate specific expression within the brain in the ventromedial nuclei of the hypothalamus, the lateral septum, the amygdala, and entorhinal cortex, but there is unremarkable expression in the pituitary. An additional splice variant of the CRF2 receptor with a different N-terminal domain has been identified by PCR, encoding a putative protein of 431 amino acids. Thus, the data demonstrate the presence of another functional CRF receptor, with significant differences in the pharmacological profile and tissue distribution from the CRF1 receptor, which would predict important functional differences between the two receptors.

Colorimetric assay for rapid screening of corticotropin releasing factor receptor ligands.

The corticotropin releasing factor (CRF) receptor is known to be coupled to Gs and transduces its signal through stimulation of cyclic AMP (cAMP) production. Here we describe the characterization of several stable CRF receptor-expressing LVIP2.0Zc cell lines that also contain an exogenous cAMP-responsive beta-galactosidase reporter gene construct. The CRF receptor activity was assayed by measuring the induction of beta-galactosidase in response to CRF. Rat/human and bovine CRF stimulated beta-galactosidase activity in a dose-dependent manner with EC50 values of approximately 0.1 nM; the biologically weak deamidated analog of bovine CRF was approximately 500-fold less potent. The CRF receptor antagonist, [d-Phe12,Nle21,38,Ala32]r/hCRF(12-41) produced a dose-dependent inhibition of CRF-stimulated beta-galactosidase activity, further demonstrating the pharmacological specificity of the interaction. The magnitude of the maximal response to CRF varied among individual cell lines. This variation was independent of the level of CRF receptor expression, but reflected differences in the intrinsic activity of adenylate cyclase. In contrast to most cAMP assay systems, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine decreased the CRF-induced beta-galactosidase activity when used in the context of the assay regimen described here. Since the assay can be easily performed in a high-throughput 96-well plate format, these cell lines provide an efficient way for the identification of CRF receptor agonists and antagonists.

A cell culture model of the blood-brain barrier.

Endothelial cells that make up brain capillaries and constitute the blood-brain barrier become different from peripheral endothelial cells in response to inductive factors found in the nervous system. We have established a cell culture model of the blood-brain barrier by treating brain endothelial cells with a combination of astrocyte-conditioned medium and agents that elevate intracellular cAMP. These cells form high resistance tight junctions and exhibit low rates of paracellular leakage and fluid-phase endocytosis. They also undergo a dramatic structural reorganization as they form tight junctions. Results from these studies suggest modes of manipulating the permeability of the blood-brain barrier, potentially providing the basis for increasing the penetration of drugs into the central nervous system.

Identification and cloning of two species of cadherins in bovine endothelial cells.

By taking advantage of the extensive homology found in the cytoplasmic domains of several cloned cadherin molecules, we were able to identify two species of cadherins in bovine aortic endothelial cells using the polymerase chain reaction (PCR). The two species of PCR products were subsequently used as DNA probes to isolate the corresponding cDNA clones from bovine adrenal microvascular endothelial cells. Sequence comparison with other characterized cadherin molecules indicates that the major cDNA species encodes a cadherin molecule highly homologous to chicken and mouse N-cadherins, while the minor species is most homologous to mouse and human P-cadherins. Northern blot analysis with the corresponding cDNA probe showed a wide distribution of bovine N-cadherin among non-neuronal, as well as neuronal tissues, while P-cadherin was most abundant in kidney among all the bovine tissues tested, but was undetectable in placenta.

Structure, sequence, and polymorphism of the Lyt-2 T cell differentiation antigen gene.

We have determined the nucleotide sequence and genomic organization of the mouse Lyt-2 T lymphocyte differentiation antigen gene. This gene consists of five exons and four introns, and the organization roughly parallels the protein domains. Alternative splicing to include or exclude exon IV (encoding part of the cytoplasmic tail) results in two forms of mRNA and accounts for the difference in size between the alpha- and alpha'-chains of Lyt-2. The gene structure provides further evidence for the evolutionary relationship between Lyt-2 and immunoglobulin genes. Comparison of the nucleotide sequence of the Lyt-2.1 and Lyt-2.2 alleles shows a high degree of conservation, but indicates that a single nucleotide change and consequent amino acid substitution in the variable region-like domain accounts for the serologic difference between these two alleles.

Two Lyt-2 polypeptides arise from a single gene by alternative splicing patterns of mRNA.

The Lyt-2/3 molecule is a glycoprotein expressed on T lymphocytes and has classically been considered a marker for the cytotoxic/suppressor T cell subset. It has been postulated to be a receptor for class I major histocompatibility complex proteins. We have used a cDNA clone encoding the analogous human protein, Leu-2/T8, to isolate mouse cDNA clones, which were used as probes to isolate mouse genomic clones. By transfection we have shown that the mouse homologue of Leu-2/T8 is Lyt-2 and not Lyt-3. We have further demonstrated that two Lyt-2 polypeptide chains are encoded by a single gene and result from alternative modes of mRNA splicing. The nucleotide sequence of cDNA clones encoding each of these polypeptide chains has been determined and shows the difference between the two Lyt-2 polypeptide chains to be in the lengths of their cytoplasmic tails.

Rapid induction of a specific nuclear mRNA precursor by thyroid hormone.

Administration of thyroid hormone to the thyroidectomized rat results in a rapid and dramatic increase in the relative amount of hepatic mRNA coding for spot 14--a translational product with an approximate Mr of 17,500 and isoelectric point of 4.9. We have now isolated a cDNA clone containing sequences homologous to this thyroid hormone-responsive mRNA. Two distinct mRNA species that differed by 200 nucleotides in length were found to be capable of hybridizing to the cDNA probe. Both mRNA species were proportionally elevated in relative concentration in rats with increasing plasma levels of thyroid hormone. The earliest change in the levels of mature mRNA occurred at 20 minutes following thyroid hormone treatment of the thyroidectomized rat. Analysis of nuclear RNA revealed a single higher molecular weight species that was homologous to spot 14 mRNA. An increase in the nuclear level of this putative precursor occurred by 10 minutes following thyroid hormone administration, a time preceding the earliest change in mature mRNA. Thus, thyroid hormone appears to act at least in part at a nuclear level in altering the cellular concentration of this mRNA species. The rapidity of this change suggests that it may reflect a direct response to the binding of thyroid hormone to its nuclear receptor.

Characterization of a thyroid hormone-responsive gene from rat.

We have isolated and characterized a rat gene coding for spot 14 mRNA: a hepatic product induced rapidly by thyroid hormone. This gene is present in a single copy/haploid genome, but encodes two mRNA species differing by 170 nucleotides in length. Through S1 nuclease analyses, the difference was mapped to the 3'-end of the mRNA with one species extending 170 nucleotides beyond the 3'-end of the other mRNA. The putative poly(A) addition signals AUUAAA and AAUAAA are found to precede the sites of polyadenylation in the shorter and longer mRNA, respectively. The mRNA codes for a protein of 150 amino acids with a molecular weight of 17,010. The protein-coding region is located closer to the 5'-end of the mRNA, leaving a relatively long 3'-untranslated region, which contains the only intron of 3,150 base pairs within the gene. At approximately 27 base pairs upstream from the start site of the mRNA, a sequence homologous to the TATA box, TAGAAAT , was found.