👤 David I Smith

Plasma triglyceride (TG) and apoAV levels are reported to be positively correlated, yet SNPs defining haplotype APOA52 have consistently shown association with elevated plasma triglyceride (TG) but not plasma apoAV levels. We previously reported that individually -1131T>C, -3A>G and +1891T>C did not influence luciferase activity or in vitro translation efficiency. To investigate the combined effect of these SNPs additional constructs were examined. Compared to the wildtype -1131T/-3A/+1891T (TAT), the triple rare allele construct -1131C/-3G/+1891C (CGC) conferred 46% lower luciferase activity (p<0.0001), showing these SNPs are acting co-operatively. Although only these two combinations occur in vivo, we experimentally altered the TAT construct one site at a time; -3G (TGT) had the largest effect (94% lower luciferase), with lesser effects from CAT (-77%) and TAC (-70.3%) (all p<0.0001). Deletion constructs excluding one site at a time showed that -3G/1891C ( -GC) in combination, compared to -AT, was having the largest effect on luciferase activity (-59%, p=0.055). Using sequence homology and EMSA analysis no transcription factor binding at -1131 or +1891 was identified, though +1891 lies within a putative mRNA stability motif. Taken together, these data identify -3A>G in the Kozak sequence as functional, affecting translation initiation and driving the haplotype effects, while showing interaction with +1891T>C and to a lesser extent -1131T>C. A paradox arises since these results predict that APOA52 will lead to reduced apoAV with concomitant reduced LPL activation or lipoprotein-receptor interaction, resulting in higher plasma TG levels. We conclude that APOA5 expression, and not circulating plasma apoAV levels, is causatively associated with plasma TG levels. Show less

Schizophrenic patients who are treated with antipsychotics, especially second generation antipsychotics, such as clozapine and olanzapine, manifest an increase in cholesterol and triglycerides as well as other changes associated with diabetes or the metabolic syndrome. Previous studies have shown that polymorphisms in several genes that regulate lipid metabolism can influence the levels of these lipids and response to drug treatment. We have investigated in an exploratory study whether polymorphisms in the apolipoprotein C-III (ApoC3), apolipoprotein A-V gene (ApoA5) and lipoprotein lipase genes influence differential lipid response to treatment with three second generation antipsychotics-olanzapine, clozapine and risperidone-or treatment with a first generation antipsychotic. A total of 189 patients with schizophrenia or schizoaffective disorder who were being treated with a single antipsychotic were studied in a cross-sectional study design in which fasting serum cholesterol and triglycerides and selected single-nucleotide polymorphosms (SNPs) in the three lipid metabolism genes were assessed. The treatment with antipsychotic monotherapy makes drug haplotype ascertainment less complex. Our analyses showed several nominally significant drug x gene and drug x haplotype interactions. The rarer C allele or the ApoA5₁₁₃₁ (T/C) SNP was associated with higher cholesterol levels in patients treated with first generation antipsychotics and lower cholesterol levels in patients treated with olanzapine or clozapine. The rarer C allele of the ApoA5_SW19 (G/C) SNP was associated with higher cholesterol in risperidone-treated patients. An ApoA5 CG haplotype was associated with decreased cholesterol in olanzapine- or clozapine-treated patients and higher cholesterol in patients treated with first generation antipsychotics. The presence of the rarer T allele of the ApoC3₁₁₀₀ (C/T) SNP or the presence of the ApoC3 TG haplotype was associated with decreased triglyceride levels in patients treated with olanzapine or clozapine and a nonsignificant trend for increased triglycerides in patients treated with first generation antipsychotics. The presence of the ApoC3 CC haplotype was associated with increased triglycerides in patients treated with olanzapine or clozapine. The overall magnitude of the effects was not large. These results provide a potential initial step toward a pharmacogenetic approach to selection of antipsychotic treatment which may help minimize the side effect of increases in serum lipids. Show less

The chronological lifespan of eukaryotic organisms is extended by the mutational inactivation of conserved growth-signaling pathways that regulate progression into and through the cell cycle. Here we show that in the budding yeast S. cerevisiae, these and other lifespan-extending conditions, including caloric restriction and osmotic stress, increase the efficiency with which nutrient-depleted cells establish or maintain a cell cycle arrest in G1. Proteins required for efficient G1 arrest and longevity when nutrients are limiting include the DNA replication stress response proteins Mec1 and Rad53. Ectopic expression of CLN3 encoding a G1 cyclin downregulated during nutrient depletion increases the frequency with which nutrient depleted cells arrest growth in S phase instead of G1. Ectopic expression of CLN3 also shortens chronological lifespan in concert with age-dependent increases in genome instability and apoptosis. These findings indicate that replication stress is an important determinant of chronological lifespan in budding yeast. Protection from replication stress by growth-inhibitory effects of caloric restriction, osmotic and other stresses may contribute to hormesis effects on lifespan. Replication stress also likely impacts the longevity of higher eukaryotes, including humans. Show less

The common fragile sites are regions of profound genomic instability found in all individuals. The full size of each region of instability ranges from under one megabase (Mb) to greater than 10 Mbs. At least half of the CFS regions have been found to span extremely large genes that spanned from 600 kb to greater than 2.0 Mbs. The large CFS genes are also very interesting from a cancer perspective as several of them, including FHIT and WWOX, have already demonstrated the capacity to function as tumor suppressor genes, both in vitro and in vivo. We estimate that there may be 40-50 large genes localized in CFS regions. The expression of a number of the large CFS genes has been previously shown to be lost in many different cancers and this is frequently associated with a worse clinical outcome for patients. To determine if there was selection for the inactivation of different large CFS genes in different cancers, we examined the expression of 13 of the 20 known large CFS genes: FHIT, WWOX, PARK2, GRID2, NBEA, DLG2, RORA isoforms 1 and 4, DAB1, CNTNAP2, DMD, IL1RAPL1, IMMP2L and LARGE in breast, ovarian, endometrial and brain cancers using real-time RT-PCR analysis. Each cancer had a distinct profile of different large CFS genes that were inactivated. Interestingly, in breast, ovarian and endometrial cancers there were some cancers that had inactivation of expression of none or only one of the tested genes, while in other specimens there was inactivation of multiple tested genes. Brain cancers had inactivation of many of the tested genes, a number of which function in normal neurological development. We find that there is no relationship between the frequency that any specific CFS is expressed and the frequency that the gene from that region is inactivated in different cancers. Instead, it appears that different cancers select for the inactivation of different large CFS genes. Show less

MAP kinase phosphatase 3 (MKP3, also known as DUSP6 and PYST1) is involved in extracellular signal receptor kinase (ERK) regulation and functions as a specific phosphatase to the activated (phosphorylated) forms of ERK1 and ERK2. MKP3 displays allosteric activation, which aids in tightly regulating its function to ERK substrates, but not other related MAPKs. Due to MKP3's specificity for the ERK signaling pathway, the development of specific activators or inhibitors to the enzyme have been suggested in order to expressly influence the ERK1 and ERK2 pathways. To produce the high yields of MKP3 protein necessary for physico-chemical characterization of MKP3 and for high throughput screening of its small-molecule activators and inhibitors, we have cloned, purified and, and identified refolding conditions suitable for producing full-length, human MKP3 from Escherichia coli inclusion bodies. Furthermore, we demonstrate the use of a 96-well plate format refolding assay in which the ERK-induced activity of MKP3 is simulated by 33% DMSO. The assay allowed for rapid detection of MKP3's function following a refolding screen in the absence of ERK and thus provides quick and inexpensive testing of MKP3 activity. Following screening, the refolded product was confirmed to be correctly folded by steady-state kinetic analysis and by the CD spectroscopy-determined secondary structure content. CD data were consistent with 36% helix and 14% sheet, which compared to an expected 32.9% helix and 12.4% sheet. These data indicated that MKP3 was properly folded, making it a suitable protein for use in functional studies. Show less

Monocarboxylates are primary energy substrates in the retina. Recently, the authors identified two sodium-coupled monocarboxylate transporters (SMCTs), SMCT1 (a high-affinity transporter) and SMCT2 (a low-affinity transporter). Expression of SMCT1 and SMCT2 has been studied in several tissues; however, little is known about their expression in retina. In the present study, the authors asked whether SMCT1 and SMCT2 are also expressed in retina and, if so, in which particular retinal cell types. SMCT1 and SMCT2 expression was analyzed in intact mouse retina and cultured retinal cells (ganglion, Müller, RPE) by RT-PCR, in situ hybridization, and immunofluorescence. Uptake assays were performed to demonstrate SMCT1 (RGC-5 and ARPE-19 cells) and SMCT2 (rMC-1 cells) expression at the functional level. SMCT1 mRNA and protein were detected in the ganglion cell layer, inner nuclear layer, inner/outer plexiform layers, photoreceptor inner segments, and RPE. In RPE, the expression of SMCT1 was restricted to the basolateral membrane. SMCT2 mRNA and protein were detected only in neural retina, with a pattern of protein localization consistent with labeling of Müller cells. In vitro studies confirmed the cell type-specific expression of SMCT1 and SMCT2. Uptake assays demonstrated Na(+)-coupled monocarboxylate transport in RGC-5, ARPE-19, and rMC-1 cells. These data provide the first evidence for the expression of SMCT1 and SMCT2 in the retina and for the cell-type specific distribution of these transporters within the retina. These studies suggest that SMCT1 and SMCT2 play a differential role in monocarboxylate transport in the retina in a cell type-specific manner. Show less

D-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for D-serine in retinal Müller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for D-serine, is expressed in the rMC-1 Müller cell line and primary cultures of mouse Müller cells (1 degrees MCs). The relative contributions of different amino acid transport systems to d-serine uptake were determined based on differential substrate specificities and ion dependencies. D-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for D-serine in Müller cells. The Na+:substrate stoichiometry for the transport process was 1:1. D-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that D-serine transport in Müller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in Müller cells. Immunoblotting detected ASCT2 in neural retina and in 1 degrees MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1 degrees MCs. The efflux of D-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a Müller cell line and in 1 degrees MCs and suggest that D-serine, synthesized in Müller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons. Show less

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly and is also considered a progeroid genetic syndrome. The etiology of AD is complex and the mechanisms underlying its pathophysiology remain to be clarified. It has been suggested that a high serum cholesterol level is a risk factor for (AD), and that some polymorphisms of genes encoding proteins regulating cholesterol metabolism are associated with AD development. APOA5 is a recently discovered apolipoprotein involved primarily with triglyceride (TG) metabolism disorder. This study investigates the association of AD with the APOA5 gene -1131T>C polymorphisms in samples of 106 patients with Alzheimer's disease (AD), 76 elderly healthy controls and 93 young healthy controls. DNA samples were isolated from blood cells, amplified by PCR and digested with Tru1l. We observed that the genotype distributions of APOA5 variants were within Hardy-Weinberg equilibrium in all subject samples. Furthermore, chi-square test comparison for genotype distributions and allele frequencies did not reveal any significant difference among the three groups of subjects P>0.05). These results support the idea that these variants are not involved as a risk factor for developing AD. Show less

Expression of the gene encoding the MKP-3/Pyst1 protein phosphatase, which inactivates ERK MAPK, is induced by FGF. However, which intracellular signalling pathway mediates this expression is unclear, with essential roles proposed for both ERK and PI(3)K in chick embryonic limb. Here, we report that MKP-3/Pyst1 expression is sensitive to inhibition of ERK or MAPKK, that endogenous MKP-3/Pyst1 co-localizes with activated ERK, and expression of MKP-3/Pyst1 in mice lacking PDK1, an essential mediator of PI(3)K signalling. We conclude that MKP-3/Pyst1 expression is mediated by ERK activation and that negative feedback control predominates in limiting the extent of FGF-induced ERK activity. Show less

Papillary thyroid carcinomas are associated with nonoverlapping activating mutations of RET, NTRK, RAS and BRAF, which altogether are present in approximately 70% of cases. We postulated that compounds that inhibit a distal effector in the mitogen-activated protein kinase (MAPK) pathway would inhibit growth and tumorigenicity of human thyroid cancer cell lines with mutations of RET or BRAF. We first examined the effects of AAL-881 and LBT-613, two inhibitors of RAF kinase activity, on RAF-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK activation in thyroid PCCL3 cells after conditional induction of expression of H-RAS(G12V) or BRAF(V600E). Both compounds blocked RAS and RAF-dependent MEK and ERK phosphorylation. They also potently blocked MEK phosphorylation in human thyroid cancer cell lines with either RET/PTC1 (TPC1) or BRAF(V600E) (NPA, ARO, and FRO) mutations. Inhibition of ERK phosphorylation was transient in TPC1 and ARO cells, with recovery of ERK phosphorylation associated with concomitant down-regulation of the MAPK phosphatases MKP-3 and DUSP5. Both compounds inhibited growth of all cell lines, with LBT-613 being approximately 10-fold more potent than AAL-881. TPC1 cells were more sensitive to growth inhibition (IC50 0.1-0.25 and approximately 0.05 micromol/L for AAL-881 and LBT-613, respectively) than BRAF + lines (IC50 2.5-5 and 0.1-0.5 micromol/L, respectively). Growth inhibition was associated with G1 arrest, and induction of cell death. Growth of ARO and NPA tumor xenografts was inhibited by LBT-613 or AAL-881. MEK and ERK phosphorylation was inhibited by both compounds in ARO but not in NPA cell xenografts. Compounds that inhibit kinase activity are effective growth inhibitors for poorly differentiated thyroid cancer cell lines with either RET or RAF mutations, and hold promise for treatment of most forms of papillary thyroid carcinoma. Show less

Sigma receptors (sigmaRs) are nonopioid, nonphencyclidine binding sites with robust neuroprotective properties. Type 1 sigmaR1 (sigmaR1) is expressed in brain oligodendrocytes, but its expression and binding capacity have not been analyzed in retinal glial cells. This study examined the expression, subcellular localization, binding activity, and regulation of sigmaR1 in retinal Müller cells. Primary mouse Müller cells (MCs) were analyzed by RT-PCR, immunoblotting, and immunocytochemistry for the expression of sigmaR1, and data were compared with those of the rat Müller cell line (rMC-1) and the rat ganglion cell line (RGC-5). Confocal microscopy was used to determine the subcellular sigmaR1 location in primary mouse MCs. Membranes prepared from these cells were used for binding assays with [3H]-pentazocine (PTZ). The kinetics of binding, the ability of various sigmaR1 ligands to compete with sigmaR1 binding, and the effects of donated nitric oxide (NO) and reactive oxygen species (ROS) on binding were examined. sigmaR1 is expressed in primary mouse MCs and is localized to the nuclear and endoplasmic reticulum membranes. Binding assays showed that in primary mouse MCs, rMC-1, and RGC-5, the binding of PTZ was saturable. [3H]-PTZ bound with high affinity in RGC-5 and rMC-1 cells, and the binding was similarly robust in primary mouse MCs. Competition studies showed marked inhibition of [3H]-PTZ binding in the presence of sigmaR1-specific ligands. Incubation of cells with NO and ROS donors markedly increased sigmaR1 binding activity. MCs express sigmaR1 and demonstrate robust sigmaR1 binding activity, which is inhibited by sigmaR1 ligands and is stimulated during oxidative stress. The potential of Müller cells to bind sigmaR1 ligands may prove beneficial in retinal degenerative diseases such as diabetic retinopathy. Show less

The identification of genetic polymorphisms as risk factors for complex diseases can be relevant for their prevention, diagnosis, and prognosis. The apolipoprotein A-IV: 360 Gln/His polymorphism was investigated in 383 elderly individuals, who were participants of a longitudinal study commenced in 1991. The major morbidities that affect elderly people, such as cardiovascular diseases, diabetes, low cognitive function, depression, and obesity, were extensively investigated. DNA was isolated from blood cells, amplified by PCR, and digested with Fnu4HI. In this population the frequency of the His allele was 0.056 and the genotypes were distributed according to Hardy-Weinberg equilibrium. Logistic regression analysis showed a significant association between the presence of His allele and cerebrovascular disease and/or transitory ischemic attack (odds ratio) (OR = 3.070, P = 0.027), obesity (OR = 2.241, P = 0.047), and depression (OR = 2.879, P = 0.005). This study indicates that the presence of the rare allele in elderly people can play a significant role in the occurrence of multifactorial diseases. This is the first study analyzing this polymorphism in elderly people in Brazil. More studies should be encouraged to elucidate the mechanisms involved in these diseases. Show less

The PSD-95 family of membrane- associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD-95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD-95, PSD-93, and two isoforms of SAP-97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan-MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD-95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism. Show less

Retinoids induce growth arrest, differentiation, and cell death in many cancer cell types. One factor determining the sensitivity or resistance to the retinoid anticancer signal is the transcriptional response of retinoid-regulated target genes in cancer cells. We used cDNA microarray to identify 31 retinoid-regulated target genes shared by two retinoid-sensitive neuroblastoma cell lines, and then sought to determine the relevance of the target gene responses to the retinoid anticancer signal. The pattern of retinoid responsiveness for six of 13 target genes (RARbeta2, CYP26A1, CRBP1, RGS16, DUSP6, EGR1) correlated with phenotypic retinoid sensitivity, across a panel of retinoid-sensitive or -resistant lung and breast cancer cell lines. Retinoid treatment of MYCN transgenic mice bearing neuroblastoma altered the expression of five of nine target genes examined (RARbeta2, CYP26A1, CRBP1, DUSP6, PLAT) in neuroblastoma tumour tissue in vivo. In retinoid-sensitive neuroblastoma, lung and breast cancer cell lines, direct inhibition of retinoid-induced RARbeta2 expression blocked induction of only one of eight retinoid target genes (CYP26A1). DNA demethylation, histone acetylation, and exogenous overexpression of RARbeta2 partially restored retinoid-responsive CYP26A1 expression in RA-resistant MDA-MB-231 breast, but not SK-MES-1 lung, cancer cells. Combined, rather than individual, inhibition of DUSP6 and RGS16 was required to block retinoid-induced growth inhibition in neuroblastoma cells, through phosphorylation of extracellular-signal-regulated kinase. In conclusion, sensitivity to the retinoid anticancer signal is determined in part by the transcriptional response of key retinoid-regulated target genes, such as RARbeta2, DUSP6, and RGS16. Show less

RET/PTC rearrangements represent key genetic events involved in papillary thyroid carcinoma (PTC) initiation. The aim of the present study was to identify the early changes in gene expression induced by RET/PTC in thyroid cells. For this purpose, microarray analysis was conducted on PCCL3 cells conditionally expressing the RET/PTC3 oncogene. Gene expression profiling 48 h after activation of RET/PTC3 identified a statistically significant modification of expression of 270 genes. Quantitative PCR confirmation of 20 of these demonstrated 90% accuracy of the microarray. Functional clustering of genes with greater than or less than 1.75-fold expression change (86 genes) revealed RET/PTC3-induced regulation of genes with key functions in apoptosis (Ripk3, Tdga), cell-cell signaling (Cdh6, Fn1), cell cycle (Il24), immune and inflammation response (Cxcl10, Scya2, Il6, Gbp2, Oas1, Tap1, RT1Aw2, C2ta, Irf1, Lmp2, Psme2, Prkr), metabolism (Aldob, Ptges, Nd2, Gss, Gstt1), signal transduction (Socs3, Nf1, Jak2, Cpg21, Dusp6, Socs1, Stat1, Stat3, Cish) and transcription (Nr4a1, Junb, Hfh1, Runx1, Foxe1). Genes coding for proteins involved in the immune response and in intracellular signal transduction pathways activated by cytokines and chemokines were strongly represented, indicating a critical role of RET/PTC3 in the early modulation of the immune response. Show less

Hereditary multiple exostosis (HME) is an autosomal dominant disorder characterized by the development of benign cartilage-capped tumors at the juxta-epiphyseal regions of long bones. HME is usually caused by mutations of EXT1 or EXT2. The objective of this study was to investigate a three-generation Austrian kindred with HME for EXT1 and EXT2 mutations and for abnormalities of bone mineral density (BMD). DNA sequence and mRNA analyses were used to identify the mutation and its associated consequences. Serum biochemical and radiological investigations assessed bone metabolism and BMD. HME-affected members had a lower femoral neck BMD compared with nonaffected members (z-scores, -2.98 vs. -1.30; P = 0.011), and in those less than 30 yr of age, the lumbar spine BMD was also low (z-scores, -2.68 vs. -1.42; P = 0.005). However, they had normal mobility and normal serum concentrations of calcium, phosphate, alkaline phosphatase activity, creatinine, PTH, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, osteocalcin, and beta-crosslaps. DNA sequence analysis of EXT1 revealed a heterozygous g-->c transversion that altered the invariant ag dinucleotide of the intron 8 acceptor splice site. RT-PCR analysis using lymphoblastoid RNA showed that the mutation resulted in skipping of exon 9 with a premature termination at codon 599. DNA sequence abnormalities of the osteoprotegerin gene, which is in close proximity to the EXT1 gene, were not detected. A novel heterozygous acceptor splice site mutation of EXT1 results in HME that is associated with a low peak bone mass, indicating a possible additional role for EXT1 in bone biology and in regulating BMD. Show less

The expression and function of the glutamine transporters ATA1 and ATA2 (isoforms of system A), SN1 and SN2 (isoforms of system N), and LAT1 and LAT2 (isoforms of system L) were investigated in Müller cells in a rat Müller cell line (rMC1) and primary cultures of mouse Müller cells. Glutamine uptake in rMC1 cells and primary Müller cells was measured. The relative contributions of various transport systems to glutamine uptake were determined based on the differential substrate specificities and Na(+) dependence of individual transport systems. RT-PCR was used to analyze the expression of transporter-specific mRNAs. Three different transport systems participated in glutamine uptake in rMC1 cells: system L (Na(+)-independent), system A (Na(+)-dependent and alpha-(methylamino)isobutyric acid [MeAIB]-sensitive), and system N (Na(+)-dependent and MeAIB-insensitive). System N was the principal contributor (approximately 70%); the contributions of systems A and L were relatively lesser (20% and <10%, respectively). The functional features of Na(+)-dependent and MeAIB-insensitive glutamine uptake were similar to the known characteristics of clones of SN1 and SN2. Glutamine uptake in primary Müller cells behaved in a manner similar to that in rMC1 cells. mRNA transcripts specific for ATA1, ATA2, SN1, SN2, LAT1, and LAT2 were expressed in Müller cells. System N (SN1 as well as SN2) is responsible for most of the glutamine uptake in Müller cells. Because system N is capable of mediating the release of glutamine from the cells, its abundant expression in Müller cells is of importance in the handling of glutamine in the retina. Show less

While there is considerable appeal to the idea of selecting a few SNPs to represent all, or much, of the DNA sequence variability in a local chromosomal region, it is also important to quantify what detail is lost in adopting such an approach. To address this issue, we compared high- and low-resolution depictions of sequence diversity for the same genomic region, the APOA1/C3/A4/A5 gene cluster on chromosome 11. First, extensive re-sequencing identified all nucleotide and sequence haplotype variation of the linked apolipoprotein genes in 72 individuals from three populations: African-Americans from Jackson, Miss., Europeans from North Karelia, Finland, and European-Americans from Rochester, Minn. We identified 124 SNPs in 17.7 kb and significant differences in variation among genes. APOC3 gene diversity was particularly distinctive at high resolution, showing large allele frequency differences ( F(ST) values >0.250) between Jackson and the other two samples, and divergent population-specific haplotype lineages. Next, we selected haplotype-tagging SNPs (htSNPs) for each gene, at a density of approximately one SNP per kb, using an algorithm suggested by Stram et al. (2003). The 17 htSNPs identified were then used to reconstruct low-resolution haplotypes, from which inferences about the structure of variation were also drawn. This comparison showed that while the htSNPs successfully tagged common haplotype variation, they also left much underlying sequence diversity undetected and failed, in some cases, to co-classify groups of closely related haplotypes. The implications of these findings for other haplotype-based descriptions of human variation are discussed. Show less

To determine whether taurine transporter (TauT) activity and expression are regulated by hyperosmolarity in RPE, ganglion, and Müller cells. Uptake of taurine was measured in ARPE-19 cells cultured in DMEM-F12 medium without or with the addition of 50 mM NaCl or 100 mM mannitol. The kinetics of the transport were analyzed. RT-PCR and Northern and Western blot analyses were used to assess TauT mRNA and protein levels. The influence of hyperosmolarity on the uptake of taurine, myo-inositol, and gamma-aminobutyric acid GABA was studied in RPE, RGC-5, and rMC1 cells. TauT activity was abundant in RPE and was stimulated (3.5-fold) when the cells were exposed to hyperosmolar conditions (DMEM-F12 culture medium plus 50 mM NaCl or 100 mM mannitol). Peak stimulation of taurine uptake occurred after 17 hours of exposure to hyperosmolar medium. Kinetic analysis revealed that the hyperosmolarity-induced stimulation was associated with an increase in V(max) of TauT with no change in K(m). TauT mRNA and protein levels increased in RPE cells exposed to hyperosmolar conditions. Hyperosmolarity also stimulated the uptake of myo-inositol ( approximately 15-fold); GABA uptake was influenced less markedly. Immunofluorescence and functional studies showed that TauT is present in cultured RGC-5 and rMC1 cells. TauT activity was robust in these cells in normal osmolar conditions and increased by approximately twofold in hyperosmolar conditions. These studies provide the first evidence that hyperosmolarity regulates TauT activity and expression in RPE and that TauT is present in ganglion and Müller cells and is regulated by hypertonicity. The data are relevant to diseases such as diabetes, macular degeneration, and neurodegeneration, in which retinal cell volumes may fluctuate dramatically. Show less

Activation of Wnt signaling through beta-catenin mutations contributes to the development of hepatocellular carcinoma (HCC) and hepatoblastoma (HB). To explore the contribution of additional Wnt pathway molecules to hepatocarcinogenesis, we examined beta-catenin, AXIN1 and AXIN2 mutations in 73 HCCs and 27 HBs. beta-catenin mutations were detected in 19.2% (14 out of 73) HCCs and 70.4% (19 out of 27) HBs. beta-catenin mutations in HCCs were primarily point mutations, whereas more than half of the HBs had deletions. AXIN1 mutations occurred in seven (9.6%) HCCs and two (7.4%) HBs. The AXIN1 mutations included seven missense mutations, a 1 bp deletion, and a 12 bp insertion. The predominance of missense mutations found in the AXIN1 gene is different from the small deletions or nonsense mutations described previously. Loss of heterozygosity at the AXIN1 locus was present in four of five informative HCCs with AXIN1 mutations, suggesting a tumor suppressor function of this gene. AXIN2 mutations were found in two (2.7%) HCCs but not in HBs. Two HCCs had both AXIN1 and beta-catenin mutations, and one HCC had both AXIN2 and beta-catenin mutations. About half the HCCs with AXIN1 or AXIN2 mutations showed beta-catenin accumulation in the nucleus, cytoplasm or membrane. Overall, these data indicate that besides the approximately 20% of HCCs and 80% of HBs with beta-catenin mutations contributing to hepatocarcinogenesis, AXIN1 and AXIN2 mutations appear to be important in an additional 10% of HCCs and HBs. Show less

Intravitreal injection of ciliary neurotrophic factor (CNTF) is known to induce glial intermediate filament protein (GFAP) expression in retinal Müller cells. Because CNTF binding can activate multiple signaling kinases, we have examined the involvement of JAK/STAT pathway in GFAP induction in Müller cells. CNTF was injected intravitreally into mouse eyes. Immunocytochemistry and immunoblotting were used to study GFAP and STAT3-p (phosphorylated STAT3) levels either in mouse eyes, retinal explant cultures or in a Müller cell line, rMC-1. In protein extracts of CNTF-injected eyes, retinal explants and the Müller cells, there was a substantial increase in STAT3-p level. Immunocytochemistry showed that STAT3-p was now present in many cell bodies in the INL and the GCL. To prove that CNTF acted via the JAK-STAT pathway, rMC-1 cells were transfected with a dominant-negative STAT3 mutant prior to treatment with CNTF. In the immunoblots of transfected cells, there was decrease in GFAP level. The results establish that CNTF can induce GFAP expression in retinal Müller cells through the JAK/STAT signaling pathway. Show less

Aminoguanidine inhibits the development of retinopathy in diabetic animals, but the mechanism remains unclear. Inasmuch as aminoguanidine is a relatively selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), we have investigated the effects of hyperglycemia on the retinal nitric oxide (NO) pathway in the presence and absence of aminoguanidine. In vivo studies utilized retinas from experimentally diabetic rats treated or without aminoguanidine for 2 months, and in vitro studies used bovine retinal endothelial cells and a transformed retinal glial cell line (rMC-1) incubated in 5 mm and 25 mm glucose with and without aminoguanidine (100 microg/mL). NO was detected as nitrite and nitrate, and nitrotyrosine and iNOS were detected using immunochemical methods. Retinal homogenates from diabetic animals had greater than normal levels of NO and iNOS (p < 0.05), and nitrotyrosine was greater than normal, especially in one band immunoprecipitated from retinal homogenates. Oral aminoguanidine significantly inhibited all of these increases. Nitrotyrosine was detected immunohistochemically only in the retinal vasculature of non-diabetic and diabetic animals. Retinal endothelial and rMC-1 cells cultured in high glucose increased NO and NT, and aminoguanidine inhibited both increases in rMC-1 cells, but only NT in endothelial cells. Hyperglycemia increases NO production in retinal cells, and aminoguanidine can inhibit this abnormality. Inhibition of diabetic retinopathy by aminoguanidine might be mediated in part by inhibition of sequelae of NO production. Show less

Tankyrase 1, a human telomeric poly(ADP-ribose) polymerase, was originally identified through its interaction with TRF1, a negative regulator of telomere length. Tankyrase 1 ADP-ribosylates TRF1 in vitro, and its overexpression induces telomere elongation in human cancer cells. In addition to its telomeric localization, tankyrase 1 resides at multiple subcellular sites, suggesting additional functions for this protein. Here we identify TAB182, a novel tankyrase 1-binding protein of 182 kDa. TAB182 displays a complex pattern of subcellular localization. TAB182 localizes to the nucleus in a heterochromatic staining pattern and to the cytoplasm, where it co-stains with the cortical actin network. TAB182 coimmunoprecipitates with tankyrase 1 from human cells and serves as an acceptor of poly(ADP-ribosyl)ation by tankyrase 1 in vitro. Like TRF1, TAB182 binds to the ankyrin domain (comprising 24 ankyrin repeats) of tankyrase 1. Surprisingly, dissection of this domain reveals multiple discrete and overlapping binding sites for TRF1 and TAB182. Thus, we demonstrate five well conserved ankyrin repeat clusters in tankyrase 1. Although each of the five ankyrin repeat clusters independently binds to TRF1, only three of the five bind toTAB182. These findings suggest that tankyrase 1 may act as a scaffold for large molecular mass complexes made up of multiple binding proteins. We discuss potential roles for tankyrase 1-mediated higher order complexes at telomeres and at other subcellular sites. Show less

To describe the pathophysiologic features of retinal degeneration in Batten disease (juvenile neuronal ceroid lipofuscinosis [JNCL]) caused by mutations in the CLN3 gene. Comparative human tissue study. The retina and other ocular tissues of a 22-year-old man with JNCL were compared with the same tissues of a healthy 30-year-old man. DNA from whole blood and RNA from retina were used for genotype analysis. The retinas, corneas, conjunctiva, and ciliary body were processed for histopathologic and immunofluorescence analysis. Genomic DNA was subjected to polymerase chain reaction (PCR) and nucleotide sequence analyses. Reverse transcriptase/PCR and sequence analysis were performed on retinal RNA. The JNCL donor was heterozygous for a approximately 1 kb deletion in CLN3, as found in most JNCL patients. The other allele had a single base pair deletion in exon 6 that resulted in a frame shift. Gross pathology of the JNCL retina resembled that in retinitis pigmentosa, including deposits of bone spicule pigment. Histopathologic studies revealed loss of neurons from all retinal layers. Immunofluorescence labeling with antibodies to rhodopsin, recoverin, and cone opsin demonstrated degenerate rods and cones with short outer segments in the far periphery. Autofluorescent lipopigment granules were prominent in ganglion cells and some cells of the inner nuclear layer, but not in the photoreceptors. The retinal pigment epithelium (RPE) had fewer lipofuscin granules than the control specimen. Increased numbers of lipofuscin granules were found in the epithelia of the ciliary body and conjunctiva, but not in the cornea of the JNCL eye. Immunofluorescence studies revealed degenerate rods and cones in the far periphery. Lipofuscin granules were decreased in the RPE, consistent with loss of photoreceptor outer segments. The novel finding that degenerate photoreceptors did not contain autofluorescent inclusions suggests that granule accumulation may not precede photoreceptor degeneration in JNCL. The presence of normal photoreceptor proteins in the degenerate rods and cones suggests that these cells may be capable of functional regeneration if a therapy for Batten disease is developed. Show less

Within the core histone octamer each histone H4 interacts with each H2A-H2B dimer subunit through two binding surfaces. Tyrosines play a central role in these interactions with H4 tyrosines 72 and 88 contacting one H2A-H2B dimer subunit, and tyrosine 98 contacting the other. To investigate the roles of these interactions in vivo, we made site-directed amino acid substitutions at each of these tyrosine residues. Elimination of either set of interactions is lethal, suggesting that binding of the tetramer to both dimers is essential. Temperature-sensitive mutants were obtained through single amino acid substitutions at each of the tyrosines. The mutants show both strong positive and negative effects on transcription. Positive effects include Spt- and Sin-phenotypes resulting from mutations at each of the three tyrosines. One allele has a strong negative effect on the expression of genes essential for the G1 cell cycle transition. At restrictive temperature, mutant cells fail to express the CLN1, CLN2, SWI4 and SWI6 genes, and have reduced levels of CLN3 mRNA. These results demonstrate the critical role of histone dimer-tetramer interactions in vivo, and define their essential role in the expression of genes regulating G1 cell cycle progression. Show less

Mitogen-activated protein (MAP) kinase phosphatases constitute a growing family of dual specificity phosphatases thought to play a role in the dephosphorylation and inactivation of MAP kinases and are therefore likely to be important in the regulation of diverse cellular processes such as proliferation, differentiation, and apoptosis. For this reason it has been suggested that MAP kinase phosphatases may be tumor suppressors. We have determined the chromosomal locations of three human dual specificity phosphatase genes by fluorescence in situ hybridization and radiation hybrid mapping. The genes were localized to three different chromosomes, MKP2 (DUSP4) to 8p11-p12, MKP3 (DUSP6) to 12q22-q23, and MKPX (DUSP7) to 3p21. This will allow the potential roles of these genes in disease processes to be evaluated. Show less

The discs-large family is a collection of proteins that have a common structural organization and are thought to be involved in signal transduction and mediating protein-protein interactions at the cytoplasmic surface of the cell membrane. The defining member of this group of proteins is the gene product of the Drosophila lethal (1) discs large (dlg) 1 locus, which was originally identified by the analysis of recessive lethal mutants. Germline mutations in dlg result in loss of apical-basolateral polarity, disruption of normal cell-cell adhesion, and neoplastic overgrowth of the imaginal disc epithelium. We have isolated and characterized a novel human gene, DLG3, that encodes a new member of the discs-large family of proteins. The putative DLG3 gene product has a molecular weight of 66 kDa and contains a discs-large homologous region, a src oncogene homology motif 3, and a domain with homology to guanylate kinase. The DLG3 gene is located on chromosome 17, in the same segment, 17q12-q21, as the related gene, DLG2. The products of the DLG2 and DLG3 genes show 36% identity and 58% similarity to each other, and both show nearly 60% sequence similarity to p55, an erythroid phosphoprotein that is a component of the red cell membrane. We suggest that p55, DLG2, and DLG3 are closely related members of a gene family, whose protein products have a common structural organization and probably a similar function. Show less

Matrix metalloproteases are a family of enzymes that play critical roles in the physiological and pathological degradation of the extracellular matrix. These enzymes may be important therapeutic targets for the treatment of various diseases where tissue degradation is part of the pathology, such as cancer and arthritis. Matrilysin is the smallest member of this family of enzymes, all of which require zinc for catalytic activity. The first X-ray crystal structures of human matrilysin are presented. Inhibitors of metalloproteases are often characterized by the chemical group that interacts with the active site zinc of the protein. The structures of matrilysin complexed with hydroxamate (maximum resolution 1.9 A), carboxylate (maximum resolution 2.4 A), and sulfodiimine (maximum resolution 2.3 A) inhibitors are presented here and provide detailed information about how each functional group interacts with the catalytic zinc. Only the zinc-coordination group is variable in this series of inhibitors. Examination of these inhibitor-matrilysin complexes emphasizes the dominant role the zinc-coordinating group plays in determining the relative potencies of the inhibitors. The structures of these matrilysin-inhibitor complexes also provide a basis for comparing the catalytic mechanism of MMPs and other metalloproteins. Show less

We have isolated a novel cDNA that maps distal to BRCA1 at 17q12-q21. The total sequence predicts a protein of 576 amino acids with three conserved regions: a 90-amino-acid repeat domain, a SH3 (src homology region 3) motif, and a guanylate kinase domain. These conserved regions are shared among members of the discs-large family of proteins that include human p55, a membrane protein expressed in erythrocytes, rat PSD-95/SAP90, a synapse protein expressed in brain, Drosophila dIg-A, a septate junction protein expressed in various epithelia, and human and mouse ZO-1 and canine ZO-2, two tight junction proteins. dIg-A has been shown to act as a tumor suppressor, and the other members may all be involved in signal transduction through specialized membrane domains with highly organized cytoskeletons and thus are potential tumor suppressors. Since allelic loss has been reported in the 17q12-q21 region in breast and ovarian cancer and it appears that BRCA1 is not the target of the losses, we looked for somatic alterations in DLG2 in sporadic breast tumors. No evidence for mutation was found, making it unlikely that DLG2 is involved in sporadic breast cancer. Show less