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Colon cancers have been shown to develop after accumulation of multiple genetic and epigenetic alterations with changes in global gene expression profiles, contributing to the establishment of widely diverse phenotypes. Transcriptional and posttranscriptional regulation of gene expression by small RNA species, such as the small interfering RNA and microRNA and the RNA-induced silencing complex (RISC), is currently drawing major interest with regard to cancer development. SND1, also called Tudor-SN and p100 and recently reported to be a component of RISC, is among the list of highly expressed genes in human colon cancers. In the present study, we showed remarkable up-regulation of SND1 mRNA in human colon cancer tissues, even in early-stage lesions, and also in colon cancer cell lines. When mouse Snd1 was stably overexpressed in IEC6 rat intestinal epithelial cells, contact inhibition was lost and cell growth was promoted, even after the cells became confluent. Intriguingly, IEC6 cells with high levels of Snd1 also showed an altered distribution of Ecadherin from the cell membrane to the cytoplasm, suggesting loss of cellular polarity. Furthermore, the adenomatous polyposis coli (Apc) protein was coincidentally down-regulated, with no significant changes in the Apc mRNA level. Immunohistochemical analysis using chemically induced colonic lesions developed in rats revealed overexpression of Snd1 not only in colon cancers but also in aberrant crypt foci, putative precancerous lesions of the colon. Up-regulation of SND1 may thus occur at a very early stage in colon carcinogenesis and contribute to the posttranscriptional regulation of key players in colon cancer development, including APC and B-catenin. [Cancer Res 2007;67(19):9568-76]

Glycogen-storage disease type 1 (GSD-1), also known as "von Gierke disease," is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase) activity. There are four distinct subgroups of this autosomal recessive disorder: 1a, 1b, 1c, and 1d. All share the same clinical manifestations, which are caused by abnormalities in the metabolism of glucose-6-phosphate (G6P). However, only GSD-1b patients suffer infectious complications, which are due to both the heritable neutropenia and the functional deficiencies of neutrophils and monocytes. Whereas G6Pase deficiency in GSD-1a patients arises from mutations in the G6Pase gene, this gene is normal in GSD-1b patients, indicating a separate locus for the disorder in the 1b subgroup. We now report the linkage of the GSD-1b locus to genetic markers spanning a 3-cM region on chromosome 11q23. Eventual molecular characterization of this disease will provide new insights into the genetic bases of G6P metabolism and neutrophil-monocyte dysfunction.

Methionine adenosyltransferase (MAT) I/III deficiency, caused by mutations in the MAT1A gene, is characterized by persistent hypermethioninemia without elevated homocysteine or tyrosine. Clinical manifestations are variable and poorly understood, although a number of individuals with homozygous null mutations in MAT1A have neurological problems, including brain demyelination. We analyzed MAT1A in seven hypermethioninemic individuals, to provide insight into the relationship between genotype and phenotype. We identified six novel mutations and demonstrated that mutations resulting in high plasma methionines may signal clinical difficulties. Two patients-a compound heterozygote for truncating and severely inactivating missense mutations and a homozygote for an aberrant splicing MAT1A mutation-have plasma methionine in the 1,226-1,870 mM range (normal 5-35 mM) and manifest abnormalities of the brain gray matter or signs of brain demyelination. Another compound heterozygote for truncating and inactivating missense mutations has 770-1,240 mM plasma methionine and mild cognitive impairment. Four individuals carrying either two inactivating missense mutations or the single-allelic R264H mutation have 105-467 mM plasma methionine and are clinically unaffected. Our data underscore the necessity of further studies to firmly establish the relationship between genotypes in MAT I/III deficiency and clinical phenotypes, to elucidate the molecular bases of variability in manifestations of MAT1A mutations.

We investigated the numbers of planktonic and biofilm cells and the expression levels of genes encoding efflux pumps and biofilm-related proteins in 10 clinical isolates of multi-drug resistant Acinetobacter baumannii (MDRA) as well as in its standard strain ATCC 19606 in the presence of colistin (CST), polymyxin B (PMB), minomycin (MIN), and tigecycline (TGC) at their respective sub-MICs. The number of planktonic and biofilm cells of ATCC 19606 decreased in the presence of all aforementioned antibiotics in a dose-dependent manner. Cell number also decreased in two representative MDRA strains, R2 and R3, in the presence of MIN and TGC in a dose-dependent manner. In contrast, the number of biofilm cells in these two strains increased in the presence of CST, while they increased significantly in the presence of PMB in R2 only. Pearson correlation analysis revealed that the number of biofilm cells was positively and significantly correlated with the mRNA levels of genes encoding efflux pumps (adeB and adeG) and autoinducer synthase (abaI) in strain R2 and adeB, adeG, adeJ, poly-acetyl-glucosamine-porin (pgaA), and abaI in strain R3 in the presence of CST. It was positively and significantly correlated with the mRNA levels of genes encoding adeB in strain R2 and an outer membrane protein A (ompA) and biofilm-associated protein (bap) in strain R3 in the presence of PMB. These results provide valuable insights into the biofilm formation potency of clinical isolates of MDRA that depends on efflux pumps and biofilm-related genes and its regulation by antibiotics.

Methionine adenosyltransferase (MAT) is a key enzyme in transmethylation, transsulfuration, and the biosynthesis of polyamines. Genetic deficiency of ea/,B-MAT causes isolated persistent hypermethioninemia and, in some cases, unusual breath odor or neural demyelination. However, the molecular mechanism(s) underlying this deficiency has not been clearly defined. In this study, we characterized the human a/,B-MAT transcription unit and identified several mutations in the gene of patients with enzymatically confirmed diagnosis of MAT deficiency. Site-directed mutagenesis and transient expression assays demonstrated that these mutations partially inactivate MAT activity. These results establish the molecular basis of this disorder and allow for the development of DNA-based methodologies to investigate and diagnose hypermethioninemic individuals suspected of having abnormalities at this locus.

In the present study we have established novel intermittent protocols featuring a high fat (HF) diet for efficient induction of large intestinal tumors with a relatively small amount of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In protocol 1, F344 male rats were first fed a diet containing 400 p.p.m. PhIP for 2 weeks, followed by continuous administration of a HF diet without PhIP for 108 weeks. In protocol 2, 2 week PhIP treatments were repeated three times with 4 week intervals on the HF diet alone, followed by continuous feeding of the HF diet for 42 weeks. At termination of the experiments, 16 (3 of 19) and 45% (9 of 20) of the rats had developed a total of three and 13 large intestinal tumors with protocols 1 and 2, respectively. The tumor incidence in protocol 2 was comparable with that observed with continuous feeding of 400 p.p.m. PhIP for 52 weeks, after exposure to only approximately 10% of the amount of carcinogen. Five of nine (55%) tumors harbored mutations in either the beta-catenin or Apc gene, while all demonstrated accumulation of beta-catenin protein in the cytoplasm and nucleus. This suggests that other unknown genetic alterations in the Wnt-Apc-beta-catenin signaling pathway could have been involved in the development of tumors. By further modifying this intermittent protocol with HF diet, one could expect more efficient induction of lesions with much smaller amounts of PhIP in a shorter period. In addition, this model could provide a means to elucidate genetic alterations in large intestinal tumors induced by relatively low levels of carcinogenic insult, mimicking the cases of human colon carcinogenesis induced by exposure to environmental carcinogens.

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