A genomic-based approach identifies FXYD domain containing ion transport regulator 2 (FXYD2)gammaa as a pancreatic beta cell-specific biomarker.

AIMS/HYPOTHESIS: Non-invasive imaging of the pancreatic beta cell mass (BCM) requires the identification of novel and specific beta cell biomarkers. We have developed a systems biology approach to the identification of promising beta cell markers. METHODS: We followed a functional genomics strategy based on massive parallel signal sequencing (MPSS) and microarray data obtained in human islets, purified primary rat beta cells, non-beta cells and INS-1E cells to identify promising beta cell markers. Candidate biomarkers were validated and screened using established human and macaque (Macacus cynomolgus) tissue microarrays. RESULTS: After a series of filtering steps, 12 beta cell-specific membrane proteins were identified. For four of the proteins we selected or produced antibodies targeting specifically the human proteins and their splice variants; all four candidates were confirmed as islet-specific in human pancreas. Two splice variants of FXYD domain containing ion transport regulator 2 (FXYD2), a regulating subunit of the Na(+)-K(+)-ATPase, were identified as preferentially present in human pancreatic islets. The presence of FXYD2gammaa was restricted to pancreatic islets and selectively detected in pancreatic beta cells. Analysis of human fetal pancreas samples showed the presence of FXYD2gammaa at an early stage (15 weeks). Histological examination of pancreatic sections from individuals with type 1 diabetes or sections from pancreases of streptozotocin-treated Macacus cynomolgus monkeys indicated a close correlation between loss of FXYD2gammaa and loss of insulin-positive cells. CONCLUSIONS/INTERPRETATION: We propose human FXYD2gammaa as a novel beta cell-specific biomarker.

DNA microarrays for comparison of gene expression profiles between diagnosis and relapse in precursor-B acute lymphoblastic leukemia: choice of technique and purification influence the identification of potential diagnostic markers.

Microarrays for gene expression profiling are rapidly becoming important research tools for the identification of novel markers, for example, for novel classification of leukemias and lymphomas. Here, we review the considerations and infrastructure for microarray experiments. These considerations are illustrated via a microarray-based comparison of gene expression profiles of paired diagnosis-relapse samples from patients with precursor-B acute lymphoblastic leukemia (ALL), who relapsed during therapy or after completion of treatment. Initial experiments showed that several seemingly differentially expressed genes were actually derived from contaminating non-leukemic cells, particularly myeloid cells and T-lymphocytes. Therefore, we purified the ALL cells of the diagnosis and relapse samples if their frequency was lower than 95%. Furthermore, we observed in earlier studies that extra RNA amplification leads to skewing of particular gene transcripts. Sufficient (non-amplified) RNA of purified and paired diagnosis-relapse samples was obtained from only seven cases. The gene expression profiles were evaluated with Affymetrix U95A chips containing 12 600 human genes. These diagnosis-relapse comparisons revealed only a small number of genes (n=6) that differed significantly in expression: mostly signaling molecules and transcription factors involved in cell proliferation and cell survival were highly upregulated at relapse, but we did not observe any increase in drug-resistance markers. This finding fits with the observation that tumors with a high proliferation index have a poor prognosis. The genes that changed between diagnosis and relapse are currently not in use as diagnostic or disease progression markers, but represent potential new markers for such applications. Leukemia (2003) 17, 1324-1332. doi:10.1038/sj.leu.2402974

Loss of heterozygosity on 8p in prostate cancer implicates a role for dematin in tumor progression.

Dematin is a cytoskeletal protein that bundles actin filaments in a phosphorylation-dependent manner. The primary structure of dematin is organized into an N-terminal core domain of unknown function and a C-terminal domain that is homologous to the "headpiece" domain of villin. We have previously localized the dematin gene on human chromosome 8p21.1, a region distal to the ankyrin locus for hereditary spherocytosis. Radiation hybrid mapping now places dematin between D8S258 and D8S137, two microsatellite markers frequently deleted in prostate cancer. The 8p21.1 region is also deleted in prostate, breast, colon, and bladder cancers, suggesting the presence of a tumor suppressor gene(s). Using laser-capture microdissection technique and fluorescence in situ hybridization (FISH), we demonstrate loss of heterozygosity (LOH) of the dematin gene in a majority of chromosomal region 8p21-linked prostate tumors. One allele of dematin was also deleted in the established prostate adenocarcinoma cell line PC-3, which displays a classic oncogenic phenotype. Overexpression of wild-type dematin in PC-3 cells resulted in the restoration of a more polarized, epithelial-like phenotype. Conversely, the heterologous expression of dominant negative mutants of dematin perturbed normal cell morphology of NIH 3T3 fibroblasts. These results suggest a biological function of dematin in the regulation of cell shape, with implications in the pathobiology of prostate tumorigenesis.

Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.

Mutations of the gene Lps selectively impede lipopolysaccharide (LPS) signal transduction in C3H/HeJ and C57BL/10ScCr mice, rendering them resistant to endotoxin yet highly susceptible to Gram-negative infection. The codominant Lpsd allele of C3H/HeJ mice was shown to correspond to a missense mutation in the third exon of the Toll-like receptor-4 gene (Tlr4), predicted to replace proline with histidine at position 712 of the polypeptide chain. C57BL/10ScCr mice are homozygous for a null mutation of Tlr4. Thus, the mammalian Tlr4 protein has been adapted primarily to subserve the recognition of LPS and presumably transduces the LPS signal across the plasma membrane. Destructive mutations of Tlr4 predispose to the development of Gram-negative sepsis, leaving most aspects of immune function intact.

Genetic and physical mapping of the Lps locus: identification of the toll-4 receptor as a candidate gene in the critical region.

On the basis of 2093 meioses analyzed in two separate intraspecific backcrosses, the location of the mouse Lpsd mutation was circumscribed to a genetic interval 0.9 cM in size. A total of 19 genetic markers that lie in close proximity to the mutation were examined in mapping. Most of these were previously unpublished polymorphic microsatellites, identified by fragmentation of YAC and BAC clones spanning the region of interest. Lpsd was found to be inseparable from the microsatellite marker D4MIT178, and from three novel polymorphic microsatellites identified near D4MIT178. The mutation was confined between two novel microsatellite markers, herein designated "B" and "83.3." B lies centromeric to the mutation, and was separated by four crossovers in a panel of 1600 mice; 83.3 lies distal to the mutation and was separated by three crossovers in a panel of 493 mice. 66 BAC clones and one YAC clone were assembled to cover > 95% of the critical region. Estimates based on pulsed field gel electrophoresis and fluorescence in situ hybridization indicate that the The B-->83.3 interval is about 3.2 Mb in length. A minimal area of zero recombinational distance from Lpsd was also assigned, and found to occupy approximately 1.2 Mb of physical size. To identify gene candidates, nearly 40,000 sequencing runs were performed across the critical region. Selective hybridization and exon trapping were also employed to identify genes throughout the "zero" region. Only a single intact gene was identified within the entire critical region. This gene encodes the Toll-4 receptor, a member of the IL-1 receptor family.

Limatin (LIMAB1), an actin-binding LIM protein, maps to mouse chromosome 19 and human chromosome 10q25, a region frequently deleted in human cancers.

LIM domains, found in over 60 proteins, play key roles in the regulation of developmental pathways. They were first identified as cysteine-rich motifs found in the three proteins Lin-11, Isl-1, and Mec-3. LIM proteins frequently contain DNA-binding homeodomains, allowing these proteins to activate transcription. LIM domains also function as protein-binding interfaces, mediating specific protein-protein interactions. Limatin is a novel LIM protein that binds to actin filaments via a domain that is homologous to erythrocyte dematin. Here we report the murine and human chromosomal localizations of limatin (LIMAB1). Limatin was mapped to mouse Chromosome 19 by restriction fragment length polymorphism analysis and to human chromosome region 10q25 by fluorescence in situ hybridization. Radiation hybrid mapping placed LIMAB1 in a 37-cR interval between markers D10S554 and D10S2390. Interestingly, 10q25 is a region of frequent loss of heterozygosity in human tumors, thus identifying limatin as a candidate tumor suppressor gene.

Expression of an adenovirally encoded lymphotoxin-beta inhibitor prevents clearance of Listeria monocytogenes in mice.

The lymphotoxin (LT)-beta heterotrimer was recently identified as a molecule containing LT-alpha subunits, tethered to the cell through non-covalent association with an integral plasma membrane protein, derived from the LT-beta gene. Since knockout mutations of the LT-alpha gene yield animals that lack lymph nodes, whereas animals lacking either or both of the receptors for tumor necrosis factor (TNF) and LT-alpha homotrimers have normal lymph nodes, it has been inferred that the association between the LT-beta heterotrimer and its cognate receptor is required for lymph node ontogeny. Similarly, LT-beta and its receptor are thought to be important for development of the spleen. Since LT-alpha deficient mice lack lymph nodes, it is difficult to assess the extradevelopmental contribution of LT-beta to immune competence. To this end, we employed a strategy for the conditional blockade of LT-beta heteromer activity in normal mice. The interaction between LT-beta and its receptor is essential for the destruction of intracellular Listeria monocytogenes.

Cloning and sequencing of Schizosaccharomyces pombe car1 gene encoding arginase. Expression of the arginine anabolic and catabolic genes in response to arginine and related metabolites.

We report here the cloning and sequencing of the gene encoding arginase (car1) from Schizosaccharomyces pombe. Since no arginase-less strain exists in this organism, we cloned the gene by functional complementation of a car1 mutant strain from Saccharomyces cerevisiae. The S. pombe car1 gene encodes a 323 amino acids polypeptide sharing identity with arginases from different organisms. Measurements of arg3, arg11 and car1 mRNA under different growth conditions confirm the very weak repression by arginine of the two anabolic genes and show that the induction of arginase synthesis operates at a transcriptional level. The promoter of S. pombe car1 gene does not contain the 'arginine boxes' defined as the target of the ARGR-MCM1 proteins in the promoters of the arginine co-regulated genes in S. cerevisiae. The heterologous expression of S. pombe car1 gene in S. cerevisiae is independent of the ARGRII gene product (ArgRIIp/Arg81p). Determination of arginine, ornithine and citrulline intracellular concentrations shows the efficiency of the different controls operating in S. cerevisiae, and also indicates that in S. pombe enzyme compartmentation is not always sufficient to control the arginine metabolic flux.

Cloning and sequencing of arg3 and arg11 genes of Schizosaccharomyces pombe on a 10-kb DNA fragment. Heterologous expression and mitochondrial targeting of their translation products.

The Schizosaccharomyces pombe arginine anabolic genes encoding ornithine carbamoyltransferase (arg3) and acetylglutamate kinase/acetylglutamyl-phosphate reductase (arg11) were cloned by functional complementation of S. pombe arg3 and arg11 mutant strains from S. pombe DNA genomic libraries. Restriction analysis and sequencing of the two clones showed that both genes are located on a common DNA fragment. The arg3 gene encodes a 327-amino-acid polypeptide presenting a strong identity to Saccharomyces cerevisiae and human ornithine carbamoyltransferases. The arg11 gene encodes a 884-amino-acid polypeptide. The acetylglutamate kinase and acetylglutamate-phosphate reductase domains have been defined by their identity with the S. cerevisiae ARG5,6 protein. The cloned arg11 gene from S. pombe does not complement an arg5,6 mutation in S. cerevisiae, nor does the ARG5,6 gene complement the S. pombe arg11- mutation. In contrast, both ornithine-carbamoyltransferase-encoding genes function in S. pombe. However, the S. pombe arg3 gene complements only weakly an arg3 S. cerevisiae strain, which is in agreement with the low level of expression of the S. pombe gene in S. cerevisiae. The subcellular localization of both ornithine carbamoyltransferases in the two yeasts indicates that, in contrast to the S. pombe enzyme, more than 95% of the S. cerevisiae enzyme remains in the S. pombe cytoplasm. The low expression of S. pombe ornithine carbamoyltransferases in S. cerevisiae did not allow its localization. The promoters of S. pombe arg3 and arg11 genes do not present striking similarities among themselves nor with the promoters of the equivalent genes of S. cerevisiae.