Cystathionine beta-synthase (CBS) deficiency is a recessive genetic disorder in humans characterized by elevated levels of total plasma homocysteine (tHcy) and frequent thrombosis in humans. The I278T mutation is the most common mutation found in human CBS-deficient patients. The T424N mutation was identified as a mutation in human CBS that could restore function to I278T in Saccharomyces cerevisiae. In this report, we have engineered mice that express human I278T and I278T/T424N proteins from a metallotheinein-driven transgene. These transgene-containing mice were then bred to CBS knockout animals (Cbs-) to generate mice that express only human I278T or I278T/T424N protein. Both the I278T and the I278T/T424N transgenes are able to entirely rescue the previously described neonatal mortality phenotype despite the animals having a mean tHcy of 250 microm. The transgenic Cbs-/- animals exhibit facial alopecia, have moderate liver steatosis and are slightly smaller than heterozygous littermates. In contrast to human CBS deficiency, these mice do not exhibit extreme methioninemia. The mutant proteins are stable in the liver, kidney and colon, and liver extracts have only 2-3% of the CBS enzyme activity found in wild-type mice. Surprisingly, the I278T/T424N enzyme had exactly the same activity as the I278T enzyme indicating that T424N is unable to suppress I278T in mice. Our results show that elevated tHcy per se is not responsible for the neonatal lethality observed in Cbs-/- animals and suggests that CBS protein may have a function in addition to its role in homocysteine catabolism. These transgenic animals should be useful in the study of homocysteine related human disease.

A quarter century ago, Toohey (10) first recognized that certain murine malignant hematopoietic cell lines lacked methylthioadenosine phosphorylase (MTAP) 1 activity. MTAP is a key enzyme in the methionine salvage pathway (see Fig. 1). This pathway functions to salvage methylthioadenosine (MTA), which is formed as a by-product of polyamine metabolism. Phosphorolysis of MTA by MTAP results in the conversion of MTA into adenine and methylthioribose 1-phosphate. A series of reactions then salvages the methyl-thio group from methylthioribose 1-phosphate to form methionine. This pathway has been most extensively studied in Klebsiella pneumoniae (11-14) but has also been shown to exist in rat liver (15-17) and in Saccharomyces cerevisiae (18,19).Loss of MTAP activity through gene deletion is common in many kinds of human cancers including non-small cell lung cancer, glioma, T-cell acute leukemia, bladder cancer, osteosarcoma, and endometrial cancer (2-5, 20 -22). Recently, we found that expression of MTAP in an MTAP-deleted breast adenocarcinoma cell line (MCF-7) resulted in a dramatic inhibition of tumorigenicity in vitro and in vivo, showing that MTAP can function as a tumor suppressor gene (1). We also found that MTAP expression causes a significant decrease in intracellular polyamine levels and alters the ratio of putrescine to total polyamines. Consistent with this observation, the polyamine biosynthesis inhibitor ␣-difluoromethylornithine inhibits the ability of MTAP-deficient MCF-7 cells to form colonies in soft agar, whereas the addition of the polyamine putrescine stimulates colony formation in MTAP-expressing MCF-7 cells. These results indicate that the tumor suppressor activity of MTAP may be mediated by its effect on the intracellular polyamine pools.Polyamines are small, aliphatic amines involved in a variety of cellular processes including transcription and apoptosis (8). The rate-limiting enzyme in the production of polyamines is ornithine decarboxylase (ODC). Elevated ODC activity has been observed in a wide variety of human and animal tumors, and overexpression of ODC in NIH/3T3 cells is sufficient to cause transformation in vitro (7,23). Transgenic mice overexpressing ODC in skin develop skin tumors at a high frequency (9, 24). These observations show that overexpression of ODC is tumorigenic.Examination of the S. cerevisiae genome for MTAP homologues suggests that the MEU1 gene, with 35% identity and 53% similarity over 275 amino acids, may encode the yeast MTAP homologue. Furthermore, recent studies show that cells lacking MEU1, in combination with a mutation that allows yeast to take up methylthioadenosine, are unable to grow on medium containing MTA as the sole sulfur source (25). MEU1 was initially identified in a screen for genes that regulate expression of the yeast ADH2 gene (26). Overexpression of MEU1 increased ADH2 expression, whereas deletion of MEU1 resulted in reduced ADH2 expression. At the time these experiments were published, the MTAP gene had not yet been identified, so the relatio...

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The role of quantitative viral load in development of hepatocellular carcinoma (HCC) among chronic hepatitis B virus (HBV) carriers was evaluated using real-time PCR (TaqMan PCR), a highly sensitive method for quantitative detection of HBV DNA. Serum samples collected at study entry from HCC cases and matched controls were chosen separately from ongoing prospective cohort studies in Senegal, West Africa, and Haimen City, China. For 14 HCC cases and 28 controls from Senegal, the relative risk (RR, 95% CI) of HCC was 15.6 (2.0-124.3) for those positive by the TaqMan PCR assay. Average length of follow-up (study entry to death from HCC) among cases was 2.8 (+/-1.6) years. The paired median difference between cases and controls was 3.8 x 10(4) virions/ml, with cases higher (P = 0.09). In order to clarify the relationship with lower-titer viremia, we selected 55 cases and 55 matched controls from the Chinese cohort all negative for serum HBV DNA by conventional dot blot hybridization. In this group, the RR associated with HBV DNA positivity by TaqMan PCR was 3.1 (1.1-9.2), with an average duration of follow-up of 3.3 (+/-2.1) years. The median difference in quantitative viremia between cases and controls was 6.0 x 10(4) virions/ml, with cases higher (P < 0.0001). Increased risk appeared to be confined to subjects with viral loads >2.3 x 10(4) virions/ml. In conclusion, HBV viremia, except perhaps at extremely low levels, is associated with increased risk for HCC in prospective studies of chronic carriers in two disparate populations.

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