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Enzyme Digestion (enzyme + digestion)
Kinds of Enzyme Digestion Selected AbstractsThe prevalence of the mitochondrial DNA 16189 variant in non-diabetic Korean adults and its association with higher fasting glucose and body mass indexDIABETIC MEDICINE, Issue 8 2002J. H. Kim Abstract Aims To evaluate the prevalence of the 16189 variant of mitochondrial DNA in Korean adults and its association with insulin resistance. Methods We investigated 160 non-diabetic subjects from a community-based diabetes survey conducted in Yonchon County, Korea in 1993. We extracted the DNA from peripheral blood and examined the 16189 variant by polymerase chain reaction and restrictive enzyme digestion. We compared body mass index (BMI), blood pressure, fasting plasma glucose, 2-h plasma glucose after 75 g glucose load, fasting insulin, cholesterol, and homeostasis model assessment of insulin resistance and ,-cell function between the subjects with 16189 variant and wild type. Results The prevalence of the 16189 variant in Korean adults was 28.8% (46 of 160). Subjects with the 16189 variant had higher fasting glucose and BMI than those with wild type, but fasting insulin, homeostasis model assessment of insulin resistance and ,-cell function, cholesterol, and blood pressure were not different between two groups. Conclusion Our results provide evidence for an association of a frequent mitochondrial polymorphism with higher fasting glucose and the risk factors of diabetes mellitus. [source] PON1 L55M polymorphism is not a predictor of coronary atherosclerosis either alone or in combination with Q192R polymorphism in an Italian populationEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 1 2002M. Arca Abstract Background, The present study evaluated the role of the PON1 L55M polymorphism independently and in conjunction with the Q192R polymorphism on the risk of coronary atherosclerosis in an Italian population. Materials and methods, Three hundred and ninety-one subjects with significant coronary stenosis (> 50%) (coronary artery disease-positive; CAD+), 196 subjects with normal coronary arteries (< 10% stenosis) (CAD,) and 178 healthy controls were screened using a combination of polymerase chain reaction and restriction enzyme digestion. Results, In the pooled population, the frequencies of L and M alleles were 0·63 and 0·37, respectively; the most common haplotypes were QQ/LM (24·2%) and QR/LL (21·8%) and a strong linkage disequilibrium between L/55 and R/192 alleles was observed (D, = ,0·91; P < 0·0001). CAD+ subjects did not show any significant differences in the distribution of PON1,55 genotypes as compared to CAD, subjects and population controls (,2 = 1·5, P = 0·8). After controlling for other risk factors, the low-concentration M allele was not associated with a significant change of CAD risk (OR 1·02; 95% CI 0·80,1·29; P = 0·87). Moreover, the L55M polymorphism did not show any interaction with other risk factors such as smoking, diabetes, hypertension, low levels of high-density lipoprotein (HDL) or high ratios of low-density to high-density lipoproteins. The combination of L55M with the Q192R polymorphism did not show any effect on CAD risk. However, a marginal decrease in myocardial infarction risk was detected when QQ/MM carriers (OR 0·51; 95% CI 0·26,0·99; P = 0·048), but not LL/RR carriers, were compared with subjects not homozygous for an L or R allele. Conclusions, These findings did not indicate a major effect of the PON1 L55M polymorphism, either alone or in combination with the Q192R polymorphism, on CAD risk. Additional studies are needed for a better evaluation of the role of the 55/192 PON1 genotypes in combination on myocardial infarction risk. [source] A novel mutation (p.Thr198Ser) in the 1A helix of keratin 5 causes the localized variant of Epidermolysis Bullosa SimplexEXPERIMENTAL DERMATOLOGY, Issue 7 2009Paul E. Bowden Abstract:, A novel missense mutation (p.Thr198Ser) in the 1A helix of keratin 5 (K5) has been identified in a four-generation family with a history of the localized variant of epidermolysis bullosa simplex (EBS-loc), a genetic skin fragility disorder caused by K5 or K14 mutations. Genomic DNA was isolated from blood samples of patients and their healthy relatives, and all exons of the genes encoding K5 and K14 (KRT5 and KRT14) were amplified by PCR and directly sequenced. The identified mutation was confirmed by mismatch allele-specific (MM-AS)-PCR and restriction enzyme digestion with RsaI. K5 p.Thr198Ser lies at the C-terminal end of the 1A helical domain and is considered to be outside of the main mutation hotspot region. This is the first reported mutation to affect position 30 of the 1A helix (1A:T30S) in any of the 54 known keratins. [source] 5-HT2A T102C receptor polymorphism and neuropsychiatric symptoms in Alzheimer's diseaseINTERNATIONAL JOURNAL OF GERIATRIC PSYCHIATRY, Issue 6 2004Linda Chiu Wa Lam Abstract Objective To investigate the association between 5-HT2A receptor polymorphism and neuropsychiatric (NP) symptoms in Chinese elderly with Alzheimer's disease (AD). Methods This case-control study evaluated Chinese subjects with AD first presented to an university affiliated psychogeriatric clinic. Eighty-seven subjects with NINCDS-ADRDA diagnosis for probable and possible AD were recruited consecutively from the psychogeriatric clinics of the Prince of Wales Hospital in Hong Kong. 5-HT2A receptor polymorphisms were examined by polymerase chain reaction (PCR), enzyme digestion and gel electrophoresis. NP symptoms were assessed by the Chinese version of the Neuropsychiatric Inventory (NPI). Results The genotype frequencies were significantly different in subjects with regards to the presentation of delusions, aggression, aberrant motor behavior and apathy (Pearson Chi Squares, p,<,0.05). If only homozygote states were included, there were significantly fewer subjects of CC genotype with delusion (Pearson chi square, p,<,0.05). Conclusions Specific NP symptoms in AD were significantly associated with 5-HT2A receptor polymorphisms. Possible ethnic differences in the behavioral expression of 5-HT2A receptor polymorphisms are worthy of further exploration. Copyright © 2004 John Wiley & Sons, Ltd. [source] Sequestosome 1 Mutations in Paget's Disease of Bone in Australia: Prevalence, Genotype/Phenotype Correlation, and a Novel Non-UBA Domain Mutation (P364S) Associated With Increased NF-,B Signaling Without Loss of Ubiquitin Binding,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2009Sarah L Rea Abstract Previously reported Sequestosome 1(SQSTM1)/p62 gene mutations associated with Paget's disease of bone (PDB) cluster in, or cause deletion of, the ubiquitin-associated (UBA) domain. The aims of this study were to examine the prevalence of SQSTM1 mutations in Australian patients, genotype/phenotype correlations and the functional consequences of a novel point mutation (P364S) located upstream of the UBA. Mutation screening of the SQSTM1 gene was conducted on 49 kindreds with PDB. In addition, 194 subjects with apparently sporadic PDB were screened for the common P392L mutation by restriction enzyme digestion. HEK293 cells stably expressing RANK were co-transfected with expression plasmids for SQSTM1 (wildtype or mutant) or empty vector and a NF-,B luciferase reporter gene. GST-SQSTM1 (wildtype and mutant) proteins were used in pull-down assays to compare monoubiquitin-binding ability. We identified SQSTM1 mutations in 12 of 49 families screened (24.5%), comprising 9 families with the P392L mutation and 1 family each with the following mutations: K378X, 390X, and a novel P364S mutation in exon 7, upstream of the UBA. The P392L mutation was found in 9 of 194 (4.6%) patients with sporadic disease. Subjects with SQSTM1 mutations had more extensive disease, but not earlier onset, compared with subjects without mutations. In functional studies, the P364S mutation increased NF-,B activation compared with wildtype SQSTM1 but did not reduce ubiquitin binding. This suggests that increased NF-,B signaling, but not the impairment of ubiquitin binding, may be essential in the pathogenesis of PDB associated with SQSTM1 mutations. [source] Construction and identification of a recombinant live attenuated Salmonella typhimurium vaccine strain carrying Helicobacter pylori heat shock protein subunit B geneJOURNAL OF DIGESTIVE DISEASES, Issue 4 2003Guo Qing LI OBJECTIVE: Because Helicobacter pylori is the principal cause of chronic active gastritis and peptic ulcer disease, the present study explored the possibility of constructing a recombinant live attenuated S. typhimurium vaccine strain carrying H. pylori heat shock protein subunit B (HspB) gene. METHODS: A 1640-bp HspB gene was cloned into a prokaryotic expression plasmid pTrc99A. After sequence analysis, the result was compared with the sequence of H. pylori -HspB gene and protein provided by the Genebank using BLAST analysis. The identified recombinant plasmid was then introduced into a live attenuated S. typhimurium strain SL3261. RESULTS: Using polymerase chain reaction (PCR) and restriction enzyme digestion, a recombinant prokaryotic expression plasmid pTrc99A-HspB harboring the HspB gene was constructed and successfully introduced into a live attenuated S. typhimurium strain SL3261. Most of the H. pylori -HspB in the recombinant plasmid pTrc99A-HspB was consistent with the H. pylori -HspB sequence provided by the Genebank. The exchange of A/T or C/G in the cloned sequence hardly changed the encoded amino acids; the homology for both the genes and proteins of H. pylori SS1 strain was 97%; the homology with other common H. pylori strains J99 and 26695 was 96% for both. CONCLUSION: A recombinant live attenuated S. typhimurium vaccine strain harboring H. pylori -HspB gene was successfully constructed and verified, which may help in the development of an oral vaccine against H. pylori infection. [source] 145 Preliminary Results of Cytoskeletal Components in Various Red Algae Using Confocal Laser Scanning MicroscopyJOURNAL OF PHYCOLOGY, Issue 2003W. E. Schmidt Data concerning the cytoskeletal components of red algae are scant. The goal of this ongoing comparative survey is to develop a more complete characterization of the red algal cytoskeleton, and subsequent elucidation of its function, using representative taxa belonging to major evolutionary lineages within the Rhodophyta. Preliminary data were obtained using enzyme digestion of cell walls and detergent rinses with direct (phalloidin) and indirect (monoclonal antibodies) labeling methods for microfilaments and tubulin, respectively. Samples were viewed using confocal laser scanning microscopy. Results will be discussed in light of the more thoroughly understood cytoskeletal system reported for green algae. [source] Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cellsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2010Fei Yu To study self-renewal, genetic modification, and differentiation of avian spermatogonial stem cells (SSCs), we isolated chicken SSCs from fetal testes on the 16th hatching day via enzyme digestion, and then cultured the SSCs over 2 months after purification in vitro. SSCs were identified by alkaline phosphatase staining and SSEA-1 fluorescence. The EGFP gene was transfected into SSCs by three different methods: electroporation, liposome transfer and calcium acid phosphate precipitation. The transfection rate and cell survival rate using electroporation were higher than when using liposomes or calcium acid phosphate (20.52% vs. 9.75% and 5.61%; 69.86% vs. 65.00% and 51.16%, respectively). After selection with G418 for 8 days, the transgenic SSCs were transplanted into the testes of cocks treated with busulfan. Twenty-five days after transplantation, the recipients' semen was light ivory in color, and the density of spermatozoa was 3.87 (×107/ml), with 4.25% expressing EGFP. By 85 days after transplantation, the number of spermatozoa increased to 32.7 (×107/ml) and the rate of EGFP expression was 16.25%. Frozen sections of the recipients' testes showed that transgenic SSCs were located on the basal membrane of the seminiferous tubules and differentiated into spermatogenic cells at different stages. The EGFP gene was successfully amplified from the DNA of all recipients' semen samples. Mol. Reprod. Dev. 77: 340,347, 2010. © 2010 Wiley-Liss, Inc. [source] Site-independently integrated transgenes in the elite restorer rice line Minghui 63 allow removal of a selectable marker from the gene of interest by self-segregationPLANT BIOTECHNOLOGY JOURNAL, Issue 3 2003Jumin Tu Summary In this study, we have demonstrated that two independent loci are involved in the integration of the insecticidal protein gene cryIAb/cryIAc and selectable marker gene hph in the recipient genome of the elite Chinese CMS restorer line Minghui 63. We have also documented the structural organization of these transgenes in each locus by restriction enzyme digestion and Southern blot analysis. The independent locus integration of different transgenes allowed us to remove the selectable marker gene hph from the gene of interest simply by self-segregation. Not having the selectable marker gene will enhance the commercial value of our transgenic line TT51-1, which showed a consistently high level of resistance against repeated infestations of yellow stem borers and natural outbreaks of leaf-folders, without a reduction in yield potential. [source] Prenatal diagnosis of sickle syndromes in India: dilemmas in counsellingPRENATAL DIAGNOSIS, Issue 5 2005Roshan Colah Abstract Objectives The sickle gene is prevalent in the scheduled caste and tribal populations in India. The clinical presentation of sickle cell disease is extremely variable, and there are no neonatal screening programmes. This is the first report on prenatal diagnosis of sickle syndromes in 85 couples at risk (sickle cell anemia-69; sickle thalassemia-16) from different regions in India. Most of the couples were from a low socioeconomic group and their decisions were entirely dependent on the local counselling given. We have evaluated the acceptability of prenatal diagnosis and the dilemmas faced in counselling these families. Methods Chorion villus sampling was done in the first trimester and DNA analysis using reverse dot blot hybridization or restriction enzyme digestion with Dde1 in 65 cases. Cordocentesis was done in the second trimester and fetal blood analyses by automated HPLC in 20 cases who came late. Results 32.9% of couples came prospectively for diagnosis. 23.5% of fetuses were affected (sickle cell anemia-18, sickle thalassemia-2). The ,-thalassemia mutation in both cases was IVS 1,5(G- > C). All the couples with an unfavourable diagnosis opted for termination of pregnancy. Conclusion Sickle cell anemia has a relatively benign clinical course in some tribal groups in India. This raises a dilemma whether we are justified in advising prenatal diagnosis in all such cases. Copyright © 2005 John Wiley & Sons, Ltd. [source] A Survey of Polymerase Chain Reaction (PCR) Amplification Studies of Unicellular Protists Using Single-Cell PCRTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 5 2009DENIS H. LYNN ABSTRACT. We surveyed a variety of studies that have used single-cell polymerase chain reaction (SC-PCR) to examine the gene sequences of a diversity of unicellular protists. Representatives of all the Super-Groups of eukaryotes have been subjected to SC-PCR with ciliates and dinoflagellates being most commonly examined. The SC-PCR was carried out either by directly amplifying a single lysed cell or by first extracting DNA and following this with amplification of the DNA extract. Cell lysis methods included heating, freezing, mechanical rupture, and enzyme digestion. Cells fixed or preserved with ethanol, methanol, and Lugol's have also been used successfully. Heminested or seminested PCR might follow the initial PCR, whose products were then directly sequenced or cloned and then sequenced. The methods are not complicated. This should encourage protistologists to use SC-PCR in the description of new or revised taxa, especially rare and unculturable forms, and it should also enable the probing of gene expression in relation to life history stages. [source] Genetic variation of chloroplast DNA in Zingiberaceae taxa from Myanmar assessed by PCR,restriction fragment length polymorphism analysisANNALS OF APPLIED BIOLOGY, Issue 1 2009D. Ahmad Abstract We examined genetic variation in 22 accessions belonging to 11 species in four genera of the Zingiberaceae, mainly from Myanmar, by PCR,restriction fragment length polymorphism analysis to investigate their relationships within this family. Two of 10 chloroplast gene regions (trnS-trnfM and trnK2,trnQr) showed differential PCR amplification across the taxa. Restriction enzyme digestion of the PCR products revealed interspecific variability. The restriction patterns were used to classify the regions as either highly conserved or variable across the taxa. None of the regions was highly conserved across the four genera, and the level of conservation varied. The gene region trnS-trnfM appeared to display interspecific variability among most of the species. However, the relative efficiency of different restriction enzymes depended on the gene regions and genera investigated. Cluster analysis revealed interspecific discrimination among the taxa. The two Curcuma species (Curcuma zedoaria and Curcuma xanthorrhiza) appeared to be identical, thus supporting their recent classification as synonyms. The results provide the basis for selecting specific combinations of restriction enzymes and gene regions of chloroplast DNA (cpDNA) to identify interspecific variation in the Zingiberaceae and to identify both highly conserved and variable regions. Overall, cpDNA depicted comparatively diverse genetic profile of the studied germplasm. The genetic information revealed here can be applied to the conservation and future breeding of Zingiber and Curcuma species. [source] SNP Discovery and Haplotype Analysis in the Segmentally Duplicated DRD5 Coding RegionANNALS OF HUMAN GENETICS, Issue 3 2009Donna J. E. Housley Summary The dopamine receptor 5 gene (DRD5) holds much promise as a candidate locus for contributing to neuropsychiatric disorders and other diseases influenced by the dopaminergic system, as well as having potential to affect normal behavioral variation. However, detailed analyses of this gene have been complicated by its location within a segmentally duplicated chromosomal region. Microsatellites and SNPs upstream from the coding region have been used for association studies, but we find, using bioinformatics resources, that these markers all lie within a previously unrecognized second segmental duplication (SD). In order to accurately analyze the DRD5 locus for polymorphisms in the absence of contaminating pseudogene sequences, we developed a fast and reliable method for sequence analysis and genotyping within the DRD5 coding region. We employed restriction enzyme digestion of genomic DNA to eliminate the pseudogenes prior to PCR amplification of the functional gene. This approach allowed us to determine the DRD5 haplotype structure using 31 trios and to reveal additional rare variants in 171 unrelated individuals. We clarify the inconsistencies and errors of the recorded SNPs in dbSNP and HapMap and illustrate the importance of using caution when choosing SNPs in regions of suspected duplications. The simple and relatively inexpensive method presented herein allows for convenient analysis of sequence variation in DRD5 and can be easily adapted to other duplicated genomic regions in order to obtain good quality sequence data. [source] Increased prevalence of M694V in patients with ankylosing spondylitis: Additional evidence for a link with familial mediterranean feverARTHRITIS & RHEUMATISM, Issue 10 2010Nurullah Akkoc Objective To assess whether there is a statistically significant difference in the frequency of common MEFV allele variants in patients with ankylosing spondylitis (AS) as compared with control patients with rheumatoid arthritis (RA) and with healthy control subjects. Methods Sixty-two patients with AS, 50 healthy control subjects, and 46 patients with RA were assessed for the presence of MEFV variants. Exon 10 was analyzed by direct sequencing. E148Q was analyzed by restriction endonuclease enzyme digestion (REED) or by direct sequencing when REED analysis failed. Results The allele frequency of all MEFV variants in the AS group was significantly higher than that in the pooled control group of healthy subjects plus RA patients (15.3% versus 6.8%; P = 0.021). M694V was the only variant that was significantly more common in the AS group than in the combined or individual control groups (P = 0.026 for AS patients versus healthy controls, P = 0.046 for AS patients versus RA patient controls, and P = 0.008 for AS patients versus healthy and RA patient control groups). The carriage rate of M694V was also significantly higher in the AS patient group than in the combined control group (odds ratio 7.0, P = 0.014). Neither M694V nor any other MEFV variant showed a correlation with most of the disease-related measures examined. Conclusion We found an increased frequency of MEFV variants in AS patients as compared with healthy controls and with RA patient controls. This was primarily due to the presence of M694V. The roles of other exon 10 variants, as well as the relationship between the variant status and the severity and clinical course of the disease, need to be explored in further studies that include sufficiently large sample sizes. [source] Development of an ex vivo cellular model of rheumatoid arthritis: Critical role of cd14-positive monocyte/macrophages in the development of pannus tissueARTHRITIS & RHEUMATISM, Issue 9 2007Toshiko Nozaki Objective To establish an ex vivo cellular model of pannus, the aberrant overgrowth of human synovial tissue (ST). Methods Inflammatory cells that infiltrated pannus tissue from patients with rheumatoid arthritis (RA) were collected without enzyme digestion, and designated as ST-derived inflammatory cells. Single-cell suspensions of ST-derived inflammatory cells were cultured in medium alone. Levels of cytokines produced in culture supernatants were measured using enzyme-linked immunosorbent assay kits. ST-derived inflammatory cells were transferred into the joints of immunodeficient mice to explore whether these cells could develop pannus. CD14 and CD2 cells were depleted by negative selection. Results Culture of ST-derived inflammatory cells from 92 of 111 patients with RA resulted in spontaneous reconstruction of inflammatory tissue in vitro within 4 weeks. Ex vivo tissue contained fibroblasts, macrophages, T cells, and tartrate-resistant acid phosphatase,positive multinucleated cells. On calcium phosphate,coated slides, ST-derived inflammatory cell cultures showed numerous resorption pits. ST-derived inflammatory cell cultures continuously produced matrix metalloproteinase 9 and proinflammatory cytokines associated with osteoclastogenesis, such as tumor necrosis factor ,, interleukin-8, and macrophage colony-stimulating factor. More importantly, transferring ST-derived inflammatory cells into the joints of immunodeficient mice resulted in the development of pannus tissue and erosive joint lesions. Both in vitro development and in vivo development of pannus tissue by ST-derived inflammatory cells were inhibited by depleting CD14-positive, but not CD2-positive, cells from ST-derived inflammatory cells. Conclusion These findings suggest that overgrowth of inflammatory cells from human rheumatoid synovium simulates the development of pannus. This may prove informative in the screening of potential antirheumatic drugs. [source] Understanding the key factors for enzymatic conversion of pretreated lignocellulose by partial least square analysisBIOTECHNOLOGY PROGRESS, Issue 2 2010Renliang Huang Abstract The relationship between the physicochemical properties of lignocellulosic substrates and enzyme digestion is still not well known. After different pretreatments, cellulase hydrolysis and measurements of physicochemical characteristics by column solute exclusion, particle size analysis, X-ray diffraction, Fourier transform infrared spectroscopy and solid state 13C nuclear magnetic resonance were performed in this study. Partial least squares was then applied to seek the key factors limiting the rate and extent of cellulose digestion. According to the PLS results, the most important factor for cellulose digestion was accessible interior surface area, followed by delignification and the destruction of the hydrogen bonds. The cellulose digestion at 2 and 24 hr were improved with the increased accessibility of interior surface area to the reporter molecules of 5.1-nm diameter. Removal of lignin and breaking of hydrogen bonds were also found to significantly promote cellulose conversion. Other properties, including the breakdown of intramolecular hydrogen bonds, cellulose crystallinity, and hemicellulose content, had less effect on the efficiency of enzymatic hydrolysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Evaluation of infant methylenetetrahydrofolate reductase genotype, maternal vitamin use, and risk of high versus low level spina bifida defects,BIRTH DEFECTS RESEARCH, Issue 3 2003Kelly A. Volcik BACKGROUND Several studies have suggested that homozygosity for the C677T 5,10-methylenetetrahydrofolate reductase (MTHFR) variant is a potential risk factor for neural tube defects (NTDs), as individuals homozygous for the C677T allele have slightly elevated homocysteine concentrations under conditions of low folic acid intake. It has been hypothesized that maternal folic acid supplementation prevents NTDs by partially correcting reduced MTHFR activity associated with the variant form of the enzyme. METHODS Genomic DNA was extracted from newborn screening blood spots obtained from 145 infants with spina bifida (SB) and 260 nonmalformed control infants. The MTHFR C677T genotype was determined by restriction enzyme digestion of PCR amplification products with Hinf1. We investigated whether infant MTHFR genotype influenced the risk for the anatomic level of the SB lesion (high vs. low); we also explored whether maternal vitamin use influenced this risk. RESULTS Compared to controls, the frequency of SB infants with the homozygous 677 TT genotype was greatest in those infants with high level SB defects (26%; odds ratio [OR] = 2.9; 95% confidence interval [CI] = 0.9,10.1) than for those with low level SB defects (22%; OR = 1.8; 95% CI = 0.9,3.2). Furthermore, homozygous 677TT infants whose mothers did not use vitamins containing folic acid had a modestly increased risk of SB (OR = 1.8; 95% CI = 0.8,3.9), with this risk increasing more than three-fold (OR = 5.5; 95% CI = 0.8,28.1) for those infants with high level SB defects whose mothers did not use vitamins. CONCLUSIONS Based upon our observations, it is suggested that the association between the infant MTHFR homozygous variant genotype and spina bifida risk may be conditional upon both lesion level and maternal vitamin use. Birth Defects Research (Part A) 67:154,157, 2003. © 2003 Wiley-Liss, Inc. [source] Apolipoprotein E and apolipoprotein B genotypes and risk for spina bifidaBIRTH DEFECTS RESEARCH, Issue 5 2002Kelly A. Volcik Background Altered cholesterol metabolism and defects in cholesterol biosynthesis may influence abnormal central nervous system (CNS) development. During early stages of embryonic development, high levels of cholesterol are needed by rapidly proliferating cells that utilize cholesterol as a key cell membrane component. Alterations in cholesterol levels are influenced by variations in the apolipoprotein E (apoE) and apolipoprotein B (apoB) genes. The purpose of our study was to explore the possible association between infant genetic variations in the apoE and apoB genes and spina bifida (SB) risk. Methods Genomic DNA was extracted from newborn screening blood spots obtained from 26 infants with SB and 73 non-malformed control infants. ApoE and apoB genotypes were determined by restriction enzyme digestion of PCR amplification products. Results Genotype frequencies for the apoE and apoB polymorphisms were not statistically different between case and control infants. For each apoB polymorphism, however, the frequency of the wild-type allele was higher in SB infants as compared to controls. Additionally, the apoE genotype E2/E3 was observed more frequently in the controls than in SB infants [15% in controls compared to 4% in cases; OR = 0.2 (0,1.6)]. Conclusions Results from this study suggest that genetic variations in the apoE and apoB genes, known to regulate cholesterol metabolism, do not substantially contribute to the risk of SB in infants. Teratology 66:257,259, 2002. © 2002 Wiley-Liss, Inc. [source] Assembly of DNA Nanostructures with Branched Tris-DNACHEMISTRY - AN ASIAN JOURNAL, Issue 4 2006Takahiro Kuroda Abstract Branched tris-DNA, in which two oligonucleotides of the same sequence and one other oligonucleotide of a different sequence are connected with a rigid central linker, was prepared chemically by using a DNA synthesizer. Two branched tris-DNA molecules with complementary DNA sequences form dimer and tetramer as well as linear and spherical oligomer complexes. The complex formation was studied by UV/thermal denaturation, enzyme digestion, gel electrophoresis, and AFM imaging. [source] A GENETIC VARIANT OF APOLIPOPROTEIN M INCREASES SUSCEPTIBILITY TO CORONARY ARTERY DISEASE IN A CHINESE POPULATIONCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2008Wei-Wei Xu SUMMARY 1High-density lipoprotein (HDL) is widely accepted as a lipoprotein that protects against coronary artery and other atherosclerotic diseases. Recently, a new apolipoprotein encoded by the APOM gene, which plays an important role in affecting the intrinsic properties of HDL, has been reported. Genetic variations exist in the APOM gene, but their significance is presently unclear. The aim of the present study was to elucidate whether the APOM T-855C mutant allele is implicated in coronary artery disease (CAD). 2In the present study, 418 patients with CAD and 372 controls were studied, all of whom were Han Chinese from Jiangsu Province, China. Plasma levels of triglycerides (TG), total cholesterol (TC), HDL,cholesterol (HDL-C) and low-density lipoprotein,cholesterol (LDL-C) were evaluated. Genomic DNA from the whole blood from these subjects was subjected to polymerase chain reaction amplification and restriction enzyme digestion to determine genotype with respect to the APOM T-855C polymorphism. 3The allelic frequencies were in Hardy,Weinberg equilibrium. Plasma HDL levels were significantly lower in subjects with CAD than in control subjects (1.08 ± 0.31 vs 1.25 ± 0.32, respectively; P < 0.001) and the distribution of genotypes and allelic frequencies was significantly different in the two groups (P = 0.013 and 0.005, respectively). Multiple logistic regression analysis after adjustment for age, gender, smoking, body mass index, hypertension and serum glucose showed that, compared with the wild-type TT genotype, carriers of the C allele had an increased risk of CAD (odds ratio = 1.819, 95% confidence interval 1.142,2.898; P = 0.012). 4In conclusion, the results of the present study suggest that the APOM T-855C polymorphism carries an increased risk for CAD in this Chinese population. [source] The influence of human leucocyte antigen (HLA) genes on autoimmune thyroid disease (AITD): results of studies in HLA-DR3 positive AITD familiesCLINICAL ENDOCRINOLOGY, Issue 1 2002Yoshiyuki Ban Summary objective Population-based, case,control studies have consistently shown association of Graves' disease (GD) with human leucocyte antigen (HLA)-DR3 in Caucasian populations. HLA association studies in Hashimoto's thyroiditis (HT) have also suggested an association with DR3, as well as with other HLA alleles. In contrast, HLA linkage studies in autoimmune thyroid disease (AITD) have been largely negative. The aim of the present study was to investigate the role of HLA in AITD and to explain the observed associations, but lack of linkage, by examining only AITD families with the associated allele, DR3. patients We studied 99 probands (60 with GD and 39 with HT) from 99 multiplex, multigenerational Caucasian AITD families, and 135 age- and sex-matched Caucasian controls in association studies. In addition, a dataset of 34 Caucasian AITD families (out of the 99 families) with HLA-DR3 positive probands were analysed in linkage studies. design HLA typing was performed using the technique of group-specific polymerase chain reaction-amplification with restriction enzyme digestion. Whole genome screening was performed using the ABI microsatellite panels. For fine mapping of the HLA region, we used the following markers: D6S276, D6S464, D6S439, D6S273, tumour necrosis factor , and D6S1610. LOD scores were calculated using the LIPED and GeneHunter programs. results Case,control association analyses using the probands from our 99 Caucasian families showed an association of GD with DRB1*03 [P = 0·00032, relative risk (RR) = 3·4]. Linkage analysis for the HLA region in the 34 DR3 positive AITD families showed negative LOD scores throughout the region. The two-point LOD score at marker D6S273 (the closest to HLA-DRB1) was ,3·0, and the multipoint LOD score was ,7·6, demonstrating strong evidence against linkage to the HLA region in the subset of DR3 positive families. Whole genome screening in the subset of 34 DR3 positive families revealed one locus showing evidence for linkage to AITD: D3S1580 on chromosome 3q27 with a maximum two-point LOD score of 2·1. conclusions The HLA locus did not cosegregate with disease in DR3 positive families, suggesting that HLA genes are not major genes for AITD expression even within DR3 positive families; Hence, although HLA-DR3 was associated with GD in the probands, it was most likely a modulating gene and not causative; and, as the DR3 positive families showed evidence for linkage with D3S1580, it may imply that the DR3 gene modulated the effect of a susceptibility gene within the D3S1580 locus. [source] Canine COL1A2 Mutation Resulting in C-Terminal Truncation of Pro-,2(I) and Severe Osteogenesis ImperfectaJOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2001Bonnie G. Campbell Abstract RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the proband's RNA had a unique cleavage pattern in the region of COL1A2 encoding the C-propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991-3994 ("CTAG") were replaced with "TGTCATTGG." The first seven bases of the inserted sequence were identical to nucleotides 4002-4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse-transcription polymerase chain reaction (RT-PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Increased density of pC-,2(I) suggested comigration with the similarly sized pro-,2(I) derived from the mutant allele. Furthermore, ,-chains were overhydroxylated and the ratio of ,1(I):,2(I) was 3.2:1, consistent with the presence of ,1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro-,2(I) C-propeptide and confirmed a diagnosis of OI. [source] Polymorphism of renin-angiotensin system genes in IgA nephropathyNEPHROLOGY, Issue 5 2004KENG-THYE WOO SUMMARY: Background and Aims: Individuals are prone to disease because of certain disease-susceptible genes. The angiotensin I-converting enzyme (ACE) gene insertion/deletion (I/D), the angiotensinogen (AGT) gene, M235T, and the angiotensin II type 1 receptor (ATR) gene, A1166C, polymorphisms have been associated with IgA nephropathy (IgAN) and its progression. Several studies on Caucasians and Japanese patients have reported contradictory results. We determined these polymorphisms in 118 Chinese patients with IgAN and 94 healthy Chinese subjects to assess their clinical impact. Methods: Genotyping was performed with DNA isolated from peripheral leucocytes, polymerase chain reaction amplification of the polymorphic sequence, restriction enzymes digestion, and separation and identification of DNA fragments. Clinical data at renal biopsy and final status on renal function were determined from patients' records. Results: Comparing all IgAN patients with controls, AGT and ATR genotype distributions were similar, whereas there was a significant increase in the ACE DD genotype (P < 0.05). When comparing patients with end-stage renal failure (IgAN-ESRF) and those without (IgAN-nonESRF), there was no difference among the three gene polymorphisms. In contrast, there were significant differences in higher male prevalence (P < 0.05), increased serum creatinine at presentation (P < 0.05), more sclerosis (P < 0.01) and higher tubulointerstitial lesion score (P < 0.001) in the IgAN-ESRF group. Conclusion: Among the ACE, AGT and ATR gene polymorphisms, only the DD genotype may predispose the individual to IgAN in the Chinese population. None are significant for prognosticating ESRF. [source] | |||||||||||||