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PubMed2000

Inhibition of agonist-induced p42 and p38 mitogen-activated protein kinase phosphorylation and CD40 ligand/P-selectin expression by cyclic nucleotide-regulated pathways in human platelets.

Platelet activation and adhesion to endothelial cells and extracellular matrix proteins are crucial events in the development of arterial cardiovascular diseases. Platelet activation is initiated by stimulation of intracellular signaling cascades, including the p42 mitogen-activated protein kinase (MAPK) and p38 MAPK pathways, followed by major changes in the platelet cytoskeleton and expression and activation of platelet surface receptors, such as P-selectin (CD62P) and CD40 ligand (CD40L). Activated platelets directly bind to vascular endothelial cells via CD40L/CD40 interactions and induce inflammatory reactions that initiate or aggravate atherosclerotic lesions. The aim of this study was to investigate effects of two known platelet inhibitors-the cAMP-elevating prostaglandin E(1) (PG-E(1)) and the cGMP-elevating sodium nitroprusside (SNP)-on platelet p42 MAPK and p38 MAPK activation as well as on surface expression of CD62P and CD40L. MAPK activation was analyzed by Western blot experiments using phosphorylation-specific antibodies, and surface CD40L and CD62P expression was determined by flow cytometry analysis. PG-E(1) and SNP strongly inhibited p42 and p38 MAPK phosphorylation as well as CD40L and CD62P expression in response to thrombin, a thromboxane A(2) analog, and ADP. These data indicate that adenosine and guanosine 3',5'-cyclic monophosphate-dependent protein kinases not only inhibit platelet pathways leading to activation and aggregation, but also those resulting in enhanced surface expression of protein ligands involved in inflammation. Expression of CD40L and CD62P was found to be independent of MAPK activation, since it was not inhibited by specific MAPK inhibitors. Inhibition of platelet-induced inflammatory responses including CD62P- and CD40L-mediated interaction of platelets with leukocytes and endothelial cells, respectively, is suggested to be an important component of the long-term vasoprotective effects of cyclic nucleotide-elevating prostaglandins and NO donors.

Alprostadil↗
PubMed2004

Multiple marker analysis at the promoter region of the DRD4 gene and ADHD: evidence of linkage and association with the SNP -616.

Abnormalities of dopamine neurotransmission have been hypothesized to play an important role in the pathophysiology of attention deficit hyperactivity disorder (ADHD). Support for this has come from numerous association studies on candidate genes including the dopamine D4 receptor gene (DRD4). One of the most replicated associations between ADHD and the dopaminergic system is the 7-repeat allele of the VNTR polymorphism of this gene. A lack of association between this marker and ADHD has also been reported in several investigations. In the absence of a firm link between the number of the VNTR repeats and the function of the gene, we sought to investigate several additional markers at the 5'-end of the gene with potential influence on the expression of the DRD4. We observed a significant over transmission of single nucleotide polymorphism (SNP) (chi(2) = 7.45, P = 0.008, OR = 1.63). In addition, an excess transmission of the A allele of the -521 SNP was observed, although it did not attain statistical significance (chi(2) = 2.14, P = 0.17, OR = 1.25). Linkage disequilibrium (LD) analysis demonstrated a weak level of D' between any of the tested markers implying that this may be a region of high recombination. It also raises the possibility that the new association with ADHD may be independent of the 7-repeat allele. Further analyses, preferably in samples demonstrating association with the VNTR, or in other ethnic groups, are required to confirm these observations.

Adult↗
PubMed2004

High-throughput single nucleotide polymorphism typing by fluorescent single-strand conformation polymorphism analysis with capillary electrophoresis.

In this study, we performed high-throughput and precise single nucleotide polymorphism (SNP) typing by fluorescent capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis. A system composed of a multicapillary DNA analyzer, a newly developed sieving matrix, four different colors of fluorescent labels, and a multiplex polymerase chain reaction (PCR) enabled low-cost and highly reliable SNP typing. Moreover, this system enabled the estimation of SNP allele frequencies using pooled DNA samples, which should be beneficial for large-scale association studies. Thus, fluorescent CE-SSCP analysis is a useful method for large-scale SNP typing.

DNA Fingerprinting↗
PubMed2006

Application of machine learning in SNP discovery.

BACKGROUND: Single nucleotide polymorphisms (SNP) constitute more than 90% of the genetic variation, and hence can account for most trait differences among individuals in a given species. Polymorphism detection software PolyBayes and PolyPhred give high false positive SNP predictions even with stringent parameter values. We developed a machine learning (ML) method to augment PolyBayes to improve its prediction accuracy. ML methods have also been successfully applied to other bioinformatics problems in predicting genes, promoters, transcription factor binding sites and protein structures. RESULTS: The ML program C4.5 was applied to a set of features in order to build a SNP classifier from training data based on human expert decisions (True/False). The training data were 27,275 candidate SNP generated by sequencing 1973 STS (sequence tag sites) (12 Mb) in both directions from 6 diverse homozygous soybean cultivars and PolyBayes analysis. Test data of 18,390 candidate SNP were generated similarly from 1359 additional STS (8 Mb). SNP from both sets were classified by experts. After training the ML classifier, it agreed with the experts on 97.3% of test data compared with 7.8% agreement between PolyBayes and experts. The PolyBayes positive predictive values (PPV) (i.e., fraction of candidate SNP being real) were 7.8% for all predictions and 16.7% for those with 100% posterior probability of being real. Using ML improved the PPV to 84.8%, a 5- to 10-fold increase. While both ML and PolyBayes produced a similar number of true positives, the ML program generated only 249 false positives as compared to 16,955 for PolyBayes. The complexity of the soybean genome may have contributed to high false SNP predictions by PolyBayes and hence results may differ for other genomes. CONCLUSION: A machine learning (ML) method was developed as a supplementary feature to the polymorphism detection software for improving prediction accuracies. The results from this study indicate that a trained ML classifier can significantly reduce human intervention and in this case achieved a 5-10 fold enhanced productivity. The optimized feature set and ML framework can also be applied to all polymorphism discovery software. ML support software is written in Perl and can be easily integrated into an existing SNP discovery pipeline.

Algorithms↗
PubMed2004

Reconstituting the frequency spectrum of ascertained single-nucleotide polymorphism data.

Most of the available SNP data have eluded valid population genetic analysis because most population genetical methods do not correctly accommodate the special discovery process used to identify SNPs. Most of the available SNP data have allele frequency distributions that are biased by the ascertainment protocol. We here show how this problem can be corrected by obtaining maximum-likelihood estimates of the true allele frequency distribution. In simple cases, the ML estimate of the true allele frequency distribution can be obtained analytically, but in other cases computational methods based on numerical optimization or the EM algorithm must be used. We illustrate the new correction method by analyzing some previously published SNP data from the SNP Consortium. Appropriate treatment of SNP ascertainment is vital to our ability to make correct inferences from the data of the International HapMap Project.

Alleles↗
PubMed2006

Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays.

Recurrent genomic aberrations are important prognostic parameters in chronic lymphocytic leukemia (CLL). High-resolution 10k and 50k Affymetrix SNP arrays were evaluated as a diagnostic tool for CLL and revealed chromosomal imbalances in 65.6% and 81.5% of 70 consecutive cases, respectively. Among the prognostically important aberrations, the del13q14 was present in 36 (51.4%), trisomy 12 in 9 (12.8%), del11q22 in 9 (12.8%), and del17p13 in 4 cases (5.7%). A prominent clustering of breakpoints on both sides of the MIRN15A/MIRN16-1 genes indicated the presence of recombination hot spots in the 13q14 region. Patients with a monoallelic del13q14 had slower lymphocyte growth kinetics (P=.002) than patients with biallelic deletions. In 4 CLL cases with unmutated VH genes, a common minimal 3.5-Mb gain of 2p16 spanning the REL and BCL11A oncogenes was identified, implicating these genes in the pathogenesis of CLL. Twenty-four large (>10 Mb) copy-neutral regions with loss of heterozygosity were identified in 14 cases. These regions with loss of heterozygosity are not detectable by alternative methods and may harbor novel imprinted genes or loss-of-function alleles that may be important for the pathogenesis of CLL. Genomic profiling with SNP arrays is a convenient and efficient screening method for simultaneous genome-wide detection of chromosomal aberrations.

Adult↗
PubMed2004

Single nucleotide polymorphism in CTH associated with variation in plasma homocysteine concentration.

Plasma total homocysteine (tHcy) concentration, an independent risk factor of atherosclerosis, has numerous genetic and environmental determinants. While the thermolabile polymorphism in MTHFR encoding methylenetetrahydrofolate reductase is the best-studied genetic factor associated with variation in plasma tHCy, other candidate genes are being evaluated. Recently, we discovered that cystathioninuria was caused by mutations in the CTH gene encoding cystathionine gamma-lyase, an enzyme that converts cystathionine to cysteine in the trans-sulfuration pathway. We also identified a common single nucleotide polymorphism (SNP), namely c.1364G>T (S403I) in exon 12 of CTH. In the current analysis, we studied the association of genotypes of this SNP with plasma tHcy concentrations in 496 Caucasian subjects. CTH 1364T/T homozygotes had significantly higher mean plasma tHcy concentration than subjects with other genotypes, and the effect sizes of CTH and MTHFR genotypes were similar. The findings suggest that common variation in CTH may be a determinant of plasma tHcy concentrations.

Amino Acid Substitution↗
PubMed2006

Two-temperature LATE-PCR endpoint genotyping.

BACKGROUND: In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay. RESULTS: Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the same alleles. CONCLUSION: SNP genotyping via Two-Temperature LATE-PCR takes place in a homogeneous closed-tube format and uses a single hybridization probe per SNP site. These assays are convenient, rely on endpoint analysis, improve the options for construction of multiplex assays, and are suitable for SNP genotyping, mutation scanning, and detection of DNA duplication or deletions.

Alleles↗
PubMed2003

Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26).

Retinitis pigmentosa (RP), the main cause of adult blindness, is a genetically heterogeneous disorder characterized by progressive loss of photoreceptors through apoptosis. Up to now, 39 genes and loci have been implicated in nonsyndromic RP, yet the genetic bases of >50% of the cases, particularly of the recessive forms, remain unknown. Previous linkage analysis in a Spanish consanguineous family allowed us to define a novel autosomal recessive RP (arRP) locus, RP26, within an 11-cM interval (17.4 Mb) on 2q31.2-q32.3. In the present study, we further refine the RP26 locus down to 2.5 Mb, by microsatellite and single-nucleotide polymorphism (SNP) homozygosity mapping. After unsuccessful mutational analysis of the nine genes initially reported in this region, a detailed gene search based on expressed-sequence-tag data was undertaken. We finally identified a novel gene encoding a ceramide kinase (CERKL), which encompassed 13 exons. All of the patients from the RP26 family bear a homozygous mutation in exon 5, which generates a premature termination codon. The same mutation was also characterized in another, unrelated, Spanish pedigree with arRP. Human CERKL is expressed in the retina, among other adult and fetal tissues. A more detailed analysis by in situ hybridization on adult murine retina sections shows expression of Cerkl in the ganglion cell layer. Ceramide kinases convert the sphingolipid metabolite ceramide into ceramide-1-phosphate, both key mediators of cellular apoptosis and survival. Ceramide metabolism plays an essential role in the viability of neuronal cells, the membranes of which are particularly rich in sphingolipids. Therefore, CERKL deficiency could shift the relative levels of the signaling sphingolipid metabolites and increase sensitivity of photoreceptor and other retinal cells to apoptotic stimuli. This is the first genetic report suggesting a direct link between retinal neurodegeneration in RP and sphingolipid-mediated apoptosis.

Amino Acid Sequence↗
PubMed2005

[Genotype combination distribution of 5-site SNPs in 3 subtypes of beta adrenoceptor].

DNA was extracted from the peripheral venous blood of 338 subjects using BLOOD DNA MINI KIT. The 5 site SNP in 3 subtypes of Beta-AR were genotyped by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) and allele-specific primer PCR techniques. The genotypes combination distribution of SNP at 5 sites in the 3 subtypes of Beta-AR were determined by clustering analysis technique. The natural combination distribution characteristics for SNP at 5 sites in the 3 subtypes of Beta-AR in 338 subjects were obtained. Sixty-seven combinations types were found. The preceding 5 combinations in the natural combination distribution of the SNP were: (1) The genotype combination of forty subjects was B1-AR S/S49+B1-AR R/R389+B2-AR R/G16+B2-AR Q/E27+B3-AR W/W64, its probability was 11.83%. (2) The genotype combination of thirty-three subjects was B1-AR S/S49+B1-AR R/R389+B2-AR R/G16+B2-AR Q/Q27+B3-AR W/W64, its probability was 9.76%. (3) The genotype combination of nineteen subjects was B1-AR S/S49+B1-AR R/G389+B2-AR R/G16+B2-AR Q/Q27+B3-AR W/W64, its probability was 5.62%. (4) The genotype combination of sixteen subjects was B1-AR S/S49+B1-AR R/G389+B2-AR R/G16+B2-AR Q/E27+B3-AR W/W64, its probability was 4.74%. (5) The genotype combination of thirteen subjects was B1-AR S/G49+B1-AR R/R389+B2-AR R/G16+B2-AR Q/E27+B3-AR W/W64, its probability was 3.85%. The obvious correlations exist among full sample and female or male subgroup, and between female and male subgroups.

Adult↗
PubMed2006

Allelic homogeneity in Avellino corneal dystrophy due to a founder effect.

Avellino corneal dystrophy (ACD) is a common corneal dystrophy that shows allelic homogeneity, R124H mutation in the transforming growth factor beta-induced (TGFBI) gene. There are distinct phenotypes of homozygous Avellino corneal dystrophy, termed types I and II. To investigate if the difference is caused by a modifier mutation, we sequenced the entire coding region of TGFBI of two types of ACDs. The sequences obtained from each type were identical, and we could not find any nucleotide alternations. Instead, we found seven single nucleotide polymorphisms (SNPs) compared with the normal control. Primer extension analysis revealed that all 14 homozygous patients were homozygotes in each SNP, which meant that all the patients shared the same disease haplotype. Subsequent analysis of 45 heterozygous ACD patients showed strong linkage disequilibrium between disease alleles of each SNP and ACD. These results strongly suggest that the allelic homogeneity of TGFBI associated corneal dystrophies (ACD, lattice corneal dystrophy types I and III, granular corneal dystrophy and Reis-Bucklers dystrophy) might not be caused by mutation hot spots but by the founder effects.

Alleles↗
PubMed2003

Identification of three single nucleotide polymorphisms in the chicken insulin-like growth factor 1 and 2 genes and their associations with growth and feeding traits.

The chicken insulin-like growth factor (IGF)1 and IGF2 genes have been partially sequenced in six individuals of each of two chicken strains of the Black Penedesenca breed (PN and MN). These two strains are genetically diverse for growth traits. Sequence alignment revealed the existence of three single nucleotide polymorphisms (SNP) (IGF1-SNP1, IGF2-SNP2, and IGF2-SNP3). These three SNP and a fourth IGF1 polymorphism (IGF1-SNP4) were typed in 60 individuals from each strain by using PCR-RFLP or primer extension analysis. No significant associations among these four SNP, growth traits, and plasma IGF1 concentration were identified. In contrast, suggestive associations (P < or = 0.05) were found between IGF1-SNP1 and average daily gain at 107 d and feed efficiency at 44, 73, and 107 d. However, these associations were not simultaneously found in both strains suggesting that they might have been produced by linkage disequilibrium with another mutation located in the IGF1 locus or another linked gene. Since the PN and MN strains differ very markedly on their feed intake, the chicken leptin gene was included in the sequence analysis. Unfortunately, attempts to amplify several regions of this gene were unsuccessful. Even when primers complementary to highly conserved regions were used, the PCR consistently failed. Other authors have reported similar problems when trying to amplify avian leptin sequences.

Animals↗
PubMed2004

Single nucleotide polymorphism genotyping by two colour melting curve analysis using the MGB Eclipse Probe System in challenging sequence environment.

Probe and primer design for single nucleotide polymorphism (SNP) detection can be very challenging for A-T DNA-rich targets, requiring long sequences with lower specificity and stability, while G-C-rich DNA targets present limited design options to lower GC-content sequences only. We have developed the MGB Eclipse Probe System, which is composed of the following elements: MGB Eclipse probes and primers, specially developed software for the design of probes and primers, a unique set of modified bases and a Microsoft Excel macro for automated genotyping, which ably solves, in large part, this challenge. Fluorogenic MGB Eclipse probes are modified oligonucleotides containing covalently attached duplex-stabilising dihydrocyclopyrroloindole tripeptide (DPI3), the MGB ligand (MGB is a trademark of Epoch Biosciences, Bothell, WA), which has the combined properties of allowing the use of short sequences and providing great mismatch discrimination. The MGB moiety prevents probe degradation during polymerase chain reaction (PCR), allowing the researcher to use real time data; alternatively, hybridisation can be accurately measured by a post-PCR two-colour melt curve analysis. Using MGB Eclipse probes and primers containing modified bases further enhances the analysis of difficult SNP targets. G- or C-rich sequences can be refractory to analysis due to Hoogsteen base pairing. Substitution of normal G with Epoch's modified G prevents Hoogsteen base pairing, allowing both superior PCR and probe-based analysis of GC-rich targets. The use of modified A and T bases allows better stabilisation by significantly increasing the Tm of the oligonucleotides. Modified A creates A-T base pairs that have a stability slightly lower than a G-C base pair, and modified T creates T-A base pairs that have a stability about 30 per cent higher than the unmodified base pair. Together, the modified bases permit the use of short probes, providing good mismatch discrimination and primers that allow PCR of refractory targets. The combination of MGB Eclipse probes and primers enriched with the MGB ligand and modified bases has allowed the analysis of refractory SNPs, where other methods have failed.

Base Sequence↗
PubMed2003

Modeling and E-M estimation of haplotype-specific relative risks from genotype data for a case-control study of unrelated individuals.

The US National Cancer Institute has recently sponsored the formation of a Cohort Consortium (http://2002.cancer.gov/scpgenes.htm) to facilitate the pooling of data on very large numbers of people, concerning the effects of genes and environment on cancer incidence. One likely goal of these efforts will be generate a large population-based case-control series for which a number of candidate genes will be investigated using SNP haplotype as well as genotype analysis. The goal of this paper is to outline the issues involved in choosing a method of estimating haplotype-specific risk estimates for such data that is technically appropriate and yet attractive to epidemiologists who are already comfortable with odds ratios and logistic regression. Our interest is to develop and evaluate extensions of methods, based on haplotype imputation, that have been recently described (Schaid et al., Am J Hum Genet, 2002, and Zaykin et al., Hum Hered, 2002) as providing score tests of the null hypothesis of no effect of SNP haplotypes upon risk, which may be used for more complex tasks, such as providing confidence intervals, and tests of equivalence of haplotype-specific risks in two or more separate populations. In order to do so we (1) develop a cohort approach towards odds ratio analysis by expanding the E-M algorithm to provide maximum likelihood estimates of haplotype-specific odds ratios as well as genotype frequencies; (2) show how to correct the cohort approach, to give essentially unbiased estimates for population-based or nested case-control studies by incorporating the probability of selection as a case or control into the likelihood, based on a simplified model of case and control selection, and (3) finally, in an example data set (CYP17 and breast cancer, from the Multiethnic Cohort Study) we compare likelihood-based confidence interval estimates from the two methods with each other, and with the use of the single-imputation approach of Zaykin et al. applied under both null and alternative hypotheses. We conclude that so long as haplotypes are well predicted by SNP genotypes (we use the Rh2 criteria of Stram et al. [1]) the differences between the three methods are very small and in particular that the single imputation method may be expected to work extremely well.

Algorithms↗
PubMed2006

Genome-wide searching of single-nucleotide polymorphisms among eight distantly and closely related rice cultivars (Oryza sativa L.) and a wild accession (Oryza rufipogon Griff.).

We searched the genomes of eight rice cultivars (Oryza sativa L. ssp. japonica and ssp. indica) and a wild rice accession (Oryza rufipogon Griffith) for nucleotide polymorphisms, and identified 7805 polymorphic loci, including single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels), in predicted intergenic regions. Polymorphisms are useful as DNA markers for genetic analysis or positional cloning with segregating populations of crosses. Pairwise comparison between cultivars and a neighbor-joining tree calculated from SNPs agreed very well with relationships between rice strains predicted from pedigree data or calculated with other DNA markers such as p-SINE1 and simple sequence repeats (SSRs), suggesting that whole-genome SNP information can be used for analysis of evolutionary relationships. Using multiple SNPs to identify alleles, we drew a map to illustrate the alleles shared among the eight cultivars and the accession. The map revealed that most of the genome is mono- or di-allelic among japonica cultivars, whereas alleles well conserved among modern japonica paddy rice cultivars were often shared with indica cultivars or wild rice, suggesting that the genome structure of modern cultivars is composed of chromosomal segments from various genetic backgrounds. Use of allele-sharing analysis and association analysis were also tested and are discussed.

Alleles↗
PubMed2006

High-resolution genomic profiling of chromosomal aberrations using Infinium whole-genome genotyping.

Array-CGH is a powerful tool for the detection of chromosomal aberrations. The introduction of high-density SNP genotyping technology to genomic profiling, termed SNP-CGH, represents a further advance, since simultaneous measurement of both signal intensity variations and changes in allelic composition makes it possible to detect both copy number changes and copy-neutral loss-of-heterozygosity (LOH) events. We demonstrate the utility of SNP-CGH with two Infinium whole-genome genotyping BeadChips, assaying 109,000 and 317,000 SNP loci, to detect chromosomal aberrations in samples bearing constitutional aberrations as well tumor samples at sub-100 kb effective resolution. Detected aberrations include homozygous deletions, hemizygous deletions, copy-neutral LOH, duplications, and amplifications. The statistical ability to detect common aberrations was modeled by analysis of an X chromosome titration model system, and sensitivity was modeled by titration of gDNA from a tumor cell with that of its paired normal cell line. Analysis was facilitated by using a genome browser that plots log ratios of normalized intensities and allelic ratios along the chromosomes. We developed two modes of SNP-CGH analysis, a single sample and a paired sample mode. The single sample mode computes log intensity ratios and allelic ratios by referencing to canonical genotype clusters generated from approximately 120 reference samples, whereas the paired sample mode uses a paired normal reference sample from the same individual. Finally, the two analysis modes are compared and contrasted for their utility in analyzing different types of input gDNA: low input amounts, fragmented gDNA, and Phi29 whole-genome pre-amplified DNA.

Cell Line, Tumor↗
PubMed2005

Effect of nitric oxide on mitochondrial respiratory activity of human articular chondrocytes.

OBJECTIVE: To investigate the effect of nitric oxide (NO) on mitochondrial activity and its relation with the apoptosis of human articular chondrocytes. MATERIALS AND METHODS: Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities, and mitochondrial membrane potential (Delta psi m). The activities of the mitochondrial respiratory chain (MRC) complexes (complex I: NADH CoQ(1) reductase, complex II: succinate dehydrogenase, complex III: ubiquinol cytochrome c reductase, complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from normal cartilage. The Delta psi m was measured by 5,5',6,6'-tetracholoro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide (JC-1) using flow cytometry. Apoptosis was analysed by flow cytometry. The mRNA expression of caspases was analysed by ribonuclease protection analysis and the detection of protein synthesis by western blotting. Sodium nitroprusside (SNP) was used as an NO compound donor. RESULTS: SNP at concentrations higher than 0.5 mmol/l for 24 hours induced cellular changes characteristic of apoptosis. SNP elicited mRNA expression of caspase-3 and caspase-7 and down regulated bcl-2 synthesis in a dose and time dependent manner. Furthermore, 0.5 mM SNP induced depolarisation of the mitochondrial membrane at 5, 12, and 24 hours. Analysis of the MRC showed that at 5 hours, 0.5 mM SNP reduced the activity of complex IV by 33%. The individual inhibition of mitochondrial complex IV with azide modified the Delta psi m and induced apoptosis. CONCLUSIONS: This study suggests that the effect of NO on chondrocyte survival is mediated by its effect on complex IV of the MRC.

Adult↗
PubMed2006

Candidate gene analysis of IP-10 gene in patients with Alzheimer's disease.

Interferon-gamma-inducible Protein-10 (IP-10) is supposed to play a role in Alzheimer's disease (AD) development, as demonstrated by increased levels in cerebrospinal fluid from patients with AD. A mutation scanning of IP-10 exonic region was carried out in 10 patients with AD and 10 age-matched controls, demonstrating the presence of two previously reported single nucleotide polymorphisms (SNPs) in exon 4 (G-->C and T-->C) as well as a novel SNP in exon 2 (C-->T). Exon 4 G-->C and T-->C allelic variants were next evaluated in a population of 279 AD patients and 251 controls, in order to determine whether their presence could influence the susceptibility towards the development of the disease. These two SNPs were in complete linkage disequilibrium. No differences in haplotype frequencies were found in AD patients as compared with controls, even stratifying according to the presence of Apolipoprotein E varepsilon4 allele, gender or age at onset. A new protocol was developed to easily determine the C-->T SNP in exon 2. A preliminary analysis revealed a very low frequency of this allelic variant (1%). Therefore, the complete association study was not carried out because the size of our population was not sufficient to draw reliable conclusions. According to these results, IP-10 does not seem to be a risk factor for AD. However, a novel rare polymorphism has been identified, which could exert a role in AD susceptibility. Thus, further studies on larger populations are needed before confidently excluding IP-10 as a susceptibility gene for AD.

Age of Onset↗