Epstein-Barr virus strain variation in nasopharyngeal carcinoma from the endemic and non-endemic regions of China.

Nasopharyngeal carcinoma (NPC) occurs with a striking geographic incidence and is endemic in parts of southern China, where it is the major cause of cancer death. Epstein-Barr virus (EBV) is detected in all cells of the majority of NPC cases regardless of geographic origin. A small subset of EBV genes is expressed in NPC, including the latent membrane protein (LMP-1). LMP-1 is essential for transformation of B lymphocytes and is considered to be the EBV oncogene. This analysis of the DNA sequence variation within the LMP-1 gene reveals a consensus sequence for a strain, denoted China1, which predominates in East Asia where NPC is endemic. The China1 strain is characterized by nucleotide changes at 13 loci in the amino terminal portion of the LMP-1 gene when compared with the B95-8 prototype, including a point mutation resulting in the loss of an Xho1 restriction site. This strain was present in 9 of 15 NPC biopsy specimens from the endemic region and in 7 of 13 from northern China, where NPC is non-endemic. A second strain, China2, was detected in 4 of 15 endemic isolates and in 2 of 13 non-endemic isolates; this strain was characterized by a cluster of 5 nucleotide changes in the amino terminal portion of LMP-1 in addition to those seen in China1. It was also marked by distinct changes in the carboxy terminal region of LMP-1 including the retention of amino acids 343-352. All China1 isolates were EBV type 1, whereas the China2 isolates did not correlate with EBV type. Phylogenetic relationships between these 2 strains were determined, as were signature amino acid alterations that discriminate between them.

Purification, cDNA sequence, and tissue distribution of rat uroguanylin.

Guanylin, a peptide purified from rat jejunum, is thought to regulate water and electrolyte balance in the intestine. We show here, using a combination of Northern blots, Western blots, and functional assays, that guanylin and its receptor (GCC) are not distributed in parallel within the rat intestine. To investigate the possibility that there might be a second intestinal peptide that serves as a ligand for GCC, we assayed tissue extracts for the ability to stimulate cyclic GMP synthesis in a GCC-expression cell line. Duodenal extracts display a peak of biological activity that is not present in colon and that does not comigrate with guanylin or proguanylin. The activity co-purifies with a novel peptide (TIATDECELCINVACTGC) that has high homology with uroguanylin, a peptide initially purified from human and opossum urine. A rat uroguanylin cDNA clone was found to encode a propeptide whose C-terminus corresponds to our purified peptide. Northern blots with probes generated from this clone reveal that prouroguanylin mRNA is strongly expressed in proximal small intestine, but virtually absent from colon, corroborating our biochemical measurements. Taken together, these studies demonstrate an intestinal origin for uroguanylin, and show that within the intestine its distribution is complementary to that of guanylin.

Low salt intake down-regulates the guanylin signaling pathway in rat distal colon.

BACKGROUND & AIMS: Guanylin, an endogenous gastrointestinal peptide, causes the translocation of NaCl from interstitial fluid to the intestinal lumen. The aim of this study was to examine whether changes in dietary salt intake lead to compensatory changes in expression of the guanylin signaling pathway. METHODS: Rats received low-, normal-, or high-sodium diets for 1 week. Colonic guanylin expression was evaluated by Western and Northern blotting, rates of guanylin secretion by measuring biologically active guanylin released into the medium from colon explants, and expression of the guanylin receptor (C-type guanylate cyclase) by Northern blotting and bioassay. RESULTS: By every criterion, the low-salt diet reduced expression of guanylin to 30%-40% of the level found in control animals. Guanylin receptor expression was also decreased, although less dramatically and with a lower statistical significance. For both guanylin and guanylin receptor, the high-salt diet had no significant effect on expression. CONCLUSIONS: The data support the hypothesis that the guanylin pathway is down-regulated as an adaptive response to salt restriction.

The Epstein-Barr virus EBNA-2 gene in oral hairy leukoplakia: strain variation, genetic recombination, and transcriptional expression.

Oral hairy leukoplakia (HLP) lesions frequently contain defective Epstein-Barr virus (EBV) genomes with deletions in the EBNA-2 gene that abundantly replicate and persist within the lesion. To characterize these viral strains and recombinant variants, the EBNA-2 gene in EBV DNA from several different HLP biopsy specimens was analyzed. Amplification of EBNA-2 coding sequences by PCR demonstrated the presence in HLP of intact EBNA-2 genes as well as a variety of internally deleted variants of both EBNA-2A and EBNA-2B. Some of the deletion variants evolved within the HLP lesion from intact EBNA-2 genes, while other variants appeared to be transmissible strains that directly infected the lesion. Intrastrain recombination within the HLP lesion also generated variation within the EBNA-2 polyproline region. Cloning and sequencing of HLP cDNA demonstrated transcription from the internally deleted EBNA-2 open reading frame, indicating that these variant genes are expressed in HLP. Comparative analysis of the HLP EBNA-2 sequences confirmed previous findings of EBV coinfection with multiple types and strains. Sequence variation of these wild-type genes demonstrated that EBNA-2A sequences distinguish at least two separate strains and a variety of substrains of EBV type 1. Two of the HLP EBNA-2A sequences contained amino acid changes in a cytotoxic T-cell epitope within an otherwise highly conserved region of the gene. These data indicate that EBV coinfection, strain variation, and recombination within the EBNA-2 gene are common features of HLP and suggest that the expression of internally deleted EBNA-2 variants could contribute to EBV pathogenesis in permissive infection.