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PubMed2006

Octaplex PCR and fluorescence-based capillary electrophoresis for identification of human diarrheagenic Escherichia coli and Shigella spp.

A multiplex PCR assay, amplifying seven specific virulence genes and one internal control gene in a single reaction, was developed to identify the five main pathotypes of diarrheagenic Escherichia coli and Shigella spp. The virulence genes selected for each category were Stx1, Stx2, and eaeA for enterohemorrhagic E. coli (EHEC), eaeA for enteropathogenic E. coli (EPEC), STIb and LTI for enterotoxigenic E. coli (ETEC), ipaH for enteroinvasive E. coli (EIEC) and Shigella spp., and aggR for enteroaggregative E. coli (EAEC). Each forward primer was labelled with a fluorochrome and the PCR products were separated by multicolour capillary electrophoresis on an ABI PRISM310 Genetic Analyzer (Applied Biosystems). If present, several gene variants of each virulence gene were identified. The internal control gene rrs, encoding 16S rRNA, was amplified in all 110 clinical strains analyzed. Virulence genes were demonstrated in 103 (94%) of these strains. In the majority of the cases (98/103, 95%), classification obtained by the novel multiplex PCR assay was in agreement with that previously determined by phenotypic assays combined with other molecular genetic approaches. Numerous multiplex PCR assays have been published, but only a few of them detect all five E. coli pathotypes within a single reaction, and none of them has used multicolour capillary electrophoresis to separate the PCR products. The octaplex PCR assay followed by capillary electrophoresis presented in the present paper provides a simple, reliable, and rapid procedure that in a single reaction identifies the five main pathotypes of E. coli, and Shigella spp. This assay will replace the previous molecular genetic methods used in our laboratory and work as an important supplement to the more time-consuming phenotypic assays.

Adhesins, Bacterial↗
PubMed2006

Functional dyspepsia--a multicausal disease and its therapy.

Functional dyspepsia is a common chronic disorder with non-specific upper abdominal symptoms which can not be explained by organic or biochemical abnormalities. The dyspeptic symptoms are very compromising and bothersome and result in a substantial reduction of quality of life. The substantial direct and indirect medical and economical costs induce a high socioeconomic interest in the pathogenesis and the treatment options of this disease. Over the past 30 years several theories about the etiology of the symptoms in functional dyspepsia patients have been put forward. These include disorders of gastrointestinal motility, acid secretion, visceral hypersensitivity, adaptation and accommodation, Hp-infection, mucosal inflammation and finally genetic predisposition. There is increasing evidence that functional dyspepsia is a multi-causal disorder, which leads to altered processing of afferent information from the gastrointestinal tract to the CNS. Autonomic hypersensitivity and altered central processing could be a common phenomenon whereas motility changes, inflammation or altered secretion could increase neural afferent inputs. Treatment of this complex disorder could and should involve these different levels of symptom generation. Thus different approaches with anti-secretory, spasmolytic, prokinetic and anti-inflammatory effects and most preferably reduction of visceral hypersensitivity seem logical. This could explain the variety of drugs which show a positive symptomatic response. This could also offer the conclusion, whether a combination of these drugs could be clinically superior which remains to be proven. And this could offer a logical approach for the use of substances with a multi-target action, e.g. STW 5.

Diagnosis, Differential↗
PubMed2004

Molecular, cellular and functional imaging of atherothrombosis.

Recent years have seen a dramatic expansion in our knowledge of the events of atherogenesis and in the availability of drugs that can retard the progression - and even induce the regression - of this disease process. Our understanding has been advanced considerably by developments in genetics and molecular biology and by the use of genetically modified mouse models that have provided key mechanistic insights. Increasingly sophisticated imaging techniques will capitalize on these advances by bringing forward diagnosis, enhancing disease characterization and providing more precise evaluation of the effects of treatment. In this review, techniques for imaging atherosclerosis and thrombosis will be discussed. Particular attention will be given to magnetic resonance imaging techniques that enable lesion characterization and allow the targeted imaging of cells, molecules and biological processes. Emphasis is given to the potential contribution of magnetic resonance imaging methods to therapeutic monitoring, drug delivery and drug discovery.

Animals↗
PubMed1986

Incorporation of 1-beta-D-arabinofuranosylcytosine into DNA and mutagenesis of herpes simplex virus type 1.

To investigate the association between incorporation of 1-beta-D-arabinofuranosylcytosine (ara-C) into herpes simplex virus type 1 (HSV-1) DNA and mutagenesis at selected genetic loci, we grew virus in the presence or absence of ara-C and compared the mutation frequencies. Both the forward mutation frequency of HSV-1 (KOS) to the tk- phenotype and the reversion frequency of a temperature-sensitive mutant to the non-temperature-sensitive (ts+) phenotype were significantly increased following growth in ara-C. When the same viruses were grown in an inhibitor of the HSV-1 DNA polymerase that is not incorporated into DNA, no significant increase in the frequency of tk- or ts+ particles was observed. Furthermore, HSV-1 araAr, a mutant resistant to ara-C on the basis of reduced incorporation of the analog into viral DNA, did not demonstrate an increase in the number of tk- particles following growth in ara-C. These results suggest that mutagenesis of HSV-1 following growth in ara-C correlates with incorporation of the nucleoside analog into viral DNA.

Cytarabine↗
PubMed2006

Rapid prenatal diagnosis of aneuploidy for chromosomes 21, 18, 13, and X by quantitative fluorescence polymerase chain reaction.

OBJECTIVE: Quantitative fluorescence polymerase chain reaction (QF-PCR) is a rapid method for detection of chromosome copy number by amplification of repeat sequences at polymorphic loci. Our objective was to assess the performance of QF-PCR in detecting common aneuploidies in prenatal diagnosis. STUDY DESIGN: The study group consisted of pregnant women referred for amniocentesis or chorionic villus sampling (CVS) due to increased risk of fetal aneuploidy. Samples were collected from known affected and normal pregnancies. These were blindly screened for trisomy of chromosomes 21, 18, 13, and sex chromosome abnormalities, using QF-PCR. DNA from uncultured amniocytes was directly extracted using a modified alkaline lysis method. DNA from CVS was extracted by the phenol-chloroform procedure. Ten short tandem repeat (STR) markers were used for detection of fetal aneuploidy and gender. The STRs were selected for high heterozygosity rates and efficiency of the PCR amplification. The forward primer of each pair was labeled with a unique fluorescent dye. Amplified products were detected by an ABI Prism 310 Genetic Analyzer and results were analyzed using GeneScan Analysis Software. RESULTS: A total of 65 amniotic fluid and CVS samples were collected from affected and normal pregnancies. Two samples were contaminated with blood and were therefore excluded from the analysis. All 29 cases of aneuploidy were correctly diagnosed by QF-PCR, including 17 cases of trisomy 21, 7 cases of trisomy 18, and 5 cases with trisomy 13. The 34 normal samples were also correctly diagnosed as such. Thus, all results were in agreement with the standard cytogenetic results. There were no false-positive or false-negative results. CONCLUSION: We conclude that QF-PCR is a rapid, reliable, and reproducible method that may be used to provide rapid results in prenatal diagnosis of aneuploidy.

Amniocentesis↗
PubMed1998

Individual growth in Class III malocclusions and its relationship to the chin cap effects.

Individual growth characteristics of the maxilla and the mandible in Class III malocclusions were investigated in terms of growth amount, growth direction, and timing of growth; chin cap effects were considered in the context of growth characteristics of the jaws. Longitudinal cephalograms of six untreated Japanese Class III subjects were used for the analysis of jaw growth from 8 to 14 years of age. The facial patterns were classified into the five groups with different effects of therapy by discriminant functions derived from our previous chin cap study. The results obtained were as follows: (1) Among the six subjects, inhibition of maxillary forward growth was found in four subjects at ages before the maximum pubertal growth in body height occurred, resulting from occlusal interference of anterior cross bite. The growth potential of the jaws appeared genetically normal, and the facial patterns were classified into groups in which chin caps have been found to be effective to some extent. (2) A subject with growth characteristics of strong mandibular downward growth was classified in a group showing backward growth of the mandible by chin caps. (3) One of the remaining sample showing the strongest mandibular forward growth was classified in a group where chin caps are not effective. (4) It was concluded that effects of chin cap therapy are closely related to growth characteristics of the mandible in Class III treatment.

Adolescent↗
PubMed1995

Obesity, diabetes, and insulin resistance: implications from molecular biology, epidemiology, and experimental studies in humans and animals. Synopsis of the American Diabetes Association's 29th Research Symposium and Satellite Conference of the 7th International Congress on Obesity, Boston, Massachusetts.

The number of genetic features potentially contributing to the development of obesity and of NIDDM continues to expand, even while evidence from epidemiological studies and prospective longitudinal evaluations of risk factors continue to argue strongly for nongenetic factors in the expression of these disorders. Even as the intensive search goes on for the diabesity gene(s), it is imminently clear that treatment initiatives can and must be oriented to reducing the well-established risk factors, most likely through multiple simultaneous interventions and alterations. Clearly restriction of calories is the most immediately effective of the various tools currently available. The recent results of long-term minimal restriction suggest extraordinary benefits to health can result for those predisposed to the diabesity or insulin resistance syndrome. Changes in diet composition may facilitate maintenance of a lower calorie intake, but in themselves they are not likely to significantly alter the risks of the syndrome, with the exception of lower cholesterol diets for reducing elevated cholesterol levels. Exercise and increased general physical activity must be seen as potentially beneficial; however, because of the magnitude of the physical training activities likely to be effective in lowering insulin resistance on a chronic basis, the expected outcomes from the usual types of relatively limited exercise regimen are less likely to have powerful preventive effects. Nevertheless, the combination of exercise and moderate alterations in caloric intake is likely to have a synergistic beneficial effect. Pharmacological interventions in the insulin resistance syndrome are in their early developmental phases, and we look forward to new developments in this area. Effective anti-obesity agents are greatly needed, and new understanding of the excess risk carried by obesity for many features of the diabesity or insulin resistance syndrome suggest the urgency of greater investment in research toward new modes of therapy. We look forward with great anticipation to the next Obesity, Diabetes, and Insulin Resistance Conference, where new progress in prediction, new understanding of pathogenesis, new genetic markers and modifiers, and new treatment candidates are sure to be forthcoming.

Animals↗
PubMed1991

Yeast mutants with increased bacterial transposon Tn5 excision.

Five complementing recessive mutations that exhibit increased bacterial transposon Tn5 precise excision in yeast Saccharomyces cerevisiae were obtained by ethylmethanesulfonate treatment. One of these mutations (tex1) was submitted to extensive genetic analysis. tex1 is a recessive temperature-sensitive mutation resulting in a 20-100-fold increase in Tn5 excision. It also has increased frequencies of ochre mutation reversion, of forward mutation to canavanine resistance, and loss of chromosome III or its right arm. The possible mechanism of tex1 effects is discussed.

Alleles↗
PubMed1985

[Danger to the human caused by animal poxvirus following discontinuation of mandatory vaccination against smallpox].

The discontinuation of smallpox vaccination will lead to a gradual decrease or disappearance of immunity to poxviruses of the genus Orthopoxvirus. It is discussed whether orthopoxviruses of animals may then constitute a potential danger to man, with respect to their mutagenic and adaptation capabilities as well as their possible genetic interactions. Infection of man with these viruses is generally possible, but at present, not of acute importance. Monkeypox and cowpox viruses, as well as their variants occurring in carnivores and rodents, are of primary interest for individuals who have not been vaccinated against smallpox. Suggestions are put forward on how the human population, as well as domestic and laboratory animals, may be protected against infection with orthopoxviruses originating from animals. In particular, it is recommended that endangered groups of people should receive voluntary vaccination with genetically stable, attenuated vaccinia virus strains.

Animals↗
PubMed1999

Use of base excision sequence scanning for detection of genetic variations in St. Louis encephalitis virus isolates.

Twenty-two isolates of St. Louis encephalitis (SLE) virus of various geographical origins (Brazil, Argentina, Panama, Texas, Missouri, Maryland, California, and Florida) were examined for genetic variation by the base excision sequence scanning (BESS T-scan) method. A fragment was amplified in the envelope gene with the forward primer labeled in the PCR. The BESS T-scan method determined different clusters according to the profiles generated for the isolates and successfully grouped the isolates according to their geographical origins. Two major clusters, the North American cluster (cluster A) and the South and Central American cluster (cluster B), were defined. Two subgroups, the Texas-California subgroup (subgroup A1) and the Missouri-Maryland-Florida subgroup (subgroup A2), were distinguished within group A. Similarly, group B strains were subclustered to a South American subgroup (subgroup B1) and a Central American subgroup (subgroup B2). These results were consistent with those obtained by DNA sequencing analysis. The ability of the BESS T-scan method to discriminate between strains that present with high degrees of nucleotide sequence similarity indicated that this method provides reliable results and multiple applications for other virus families. The method has proven to be suitable for phylogenetic comparison and molecular epidemiology studies and may be an alternative to DNA sequencing.

Animals↗
PubMed2005

Eating disorders: from twin studies to candidate genes and beyond.

Substantial effort has been put into the exploration of the biological background of eating disorders, through family, twin and molecular genetic studies. Family studies have shown that anorexia (AN) and bulimia nervosa (BN) are strongly familial, and that familial etiologic factors appear to be shared by both disorders. Twin studies often focus on broader phenotypes or subthreshold eating disorders. These studies consistently yielded moderate to substantial heritabilities. In addition, there has been a proliferation of molecular genetic studies that focused on Diagnostic and Statistical Manual of Mental Disorders (4th ed.; DSM-IV; American Psychiatric Association, 1994) AN and BN. Seven linkage regions have been identified in genome-wide screens. Many genetic association studies have been performed, but no consistent association between a candidate gene and AN or BN has been reported. Larger genetic association studies and collaborations are needed to examine the involvement of several candidate genes and biological pathways in eating disorders. In addition, twin studies should be designed to assist the molecular work by further exploring genetic determinants of endophenotypes, evaluating the magnitude of contribution to liability of measured genotypes as well as environmental risk factors related to eating disorders. In this manner twin and molecular studies can move the field forward in a mutually informative way.

Anorexia↗
PubMed2001

A way forward.

Explore the source record for details and available documents.

Cloning, Organism↗
PubMed1995

Comparison of the mutagenicity and mutagen specificity of ethylenimine with triethylenemelamine in the ad-3 region of heterokaryon 12 of Neurospora crassa.

Studies have been performed to compare the mutagenicity and mutagenic specificity of the trifunctional alkylating agent, triethylenemelamine (TEM), and a closely related monofunctional agent, ethylenimine (EI), in the adenine-3 (ad-3) region of a 2-component heterokaryon (H-12) of Neurospora crassa. The primary objective of our studies was to characterize the genetic damage produced by each agent with regard to (1) mutagenic potency, and (2) the spectrum of specific-locus mutations induced in a lower eukaryotic organism. As in higher eukaryotes, specific-locus mutations in the ad-3 region of H-12 result from gene/point mutations, multilocus deletion mutations, and multiple-locus mutations. Specific-locus mutations resulting from gene/point mutation and multilocus deletion mutation can be detected in higher eukaryotes, but multiple-locus mutations can be detected only with difficulty or not at all. Our experiments with the ad-3 forward-mutation assay have demonstrated that TEM is a strong mutagen (maximum forward-mutation frequency between 100 and 1000 ad-3 mutations per 10(6) survivors) and EI is a moderate mutagen (maximum forward-mutation frequency between 10 and 100 ad-3 mutations per 10(6) survivors) for the induction of specific-locus mutations in the ad-3 region. Classical genetic tests were used to identify the different genotypic classes and subclasses among the EI- and TEM-induced ad-3 mutations from each experiment. The overall data base demonstrates that both EI- and TEM-induced ad-3 mutations result predominantly from gene/point mutations at the ad-3A and ad-3B loci (97.3% and 95.5%, respectively), and infrequently from multilocus deletion mutations (2.7% and 4.5%, respectively). Heterokaryon tests for allelic complementation on TEM- and EI-induced ad-3B mutations, however, have revealed a difference between the percentages showing allelic complementation (63.1% and 40.9%, respectively). Based on the specific revertibility of complementing and noncomplementing ad-3B mutations induced by other agents, this difference in the percentages of ad-3B mutations showing allelic complementation results from a difference between the spectrum of genetic alterations at the molecular level. In addition, comparison of the ratio of TEM-induced ad-3A and ad-3B mutations with those induced by EI has revealed a difference between the ad-3B/ad-3A ratios. Additional comparisons are made of the mutagenic effects of TEM and EI with those of other chemical mutagens and carcinogens in the ad-3 specific-locus assay in Neurospora.

Adenine↗
PubMed2006

Genetic epidemiology of type 1 diabetes.

The evidence that there is clinical heterogeneity of type 1 diabetes is reviewed and the implications for genetic studies are discussed. In the past year, genome-wide linkage analysis of 1435 multiplex families was reported. Additionally, confirmed evidence for association of specific markers at two loci (PTPN22, OAS1) as well as failure to replicate three others (IL12B, SUMO4, PAX4) is discussed. Some common themes are identified and suggestions for improvements are made. We look forward to the results from genome-wide association studies.

Age of Onset↗
PubMed2005

Genetic studies in rat models: insights into cardiovascular disease.

PURPOSE OF REVIEW: Limited to 2003-2004 publications, this review focuses on 'big picture' concepts learned from rat genetic studies of cardiovascular disease. RECENT DEVELOPMENTS: Analysis reveals insights into pathogenic paradigms, as well as experimental perspectives into rat-based systems of analyses of complex cardiovascular disease. Key concepts are forwarded. Multiple susceptibility genes underlie several quantitative trait loci for blood pressure suggesting a 'quantitative trait loci cluster' concept; hypertension end-organ disease quantitative trait loci are distinct from blood pressure quantitative trait loci indicating differential susceptibility paradigms for hypertension and each complication (stroke, renal disease, cardiac hypertrophy); distinct blood pressure quantitative trait loci are found in males and females indicating gender-specific susceptibility; and genetic subtypes comprise polygenic hypertension in rat models suggesting a genetic basis for clinical heterogeneity of human essential hypertension. Gender specific genetic susceptibility plays a key role in coronary artery disease susceptibility; multiple distinct quantitative trait loci underlie hyperlipidemia and type-2 diabetes, indicating multiple susceptibilities in risk factors for cardiovascular disease. Studies in transgenic inbred rat-strain models demonstrate value for serial, complex, cardiovascular pathophysiological analyses within a genetic context. SUMMARY: Cognizant of the limitations of animal model studies, observations from rat genetic studies provide insight into respective modeled human cardiovascular diseases and risk factor susceptibility, as well as systematically dissect the multifaceted complexities apparent in human complex cardiovascular disease. Given the recapitulation of many features of human cardiovascular disease, the value of rat model-based genetic studies for complex cardiovascular disease is unequivocal, thus mandating the expansion of resources for maximization of rat-based genetic studies.

Animals↗
PubMed2003

[Ecological studies on genetically engineered microorganism in environment].

This paper discussed the main problems which should be considered on the environmental release of genetically engineered microorganism (GEM), including GEM construction, gene transfer, fitness, diffusion, translocation, potential eco-influence and so on. Moreover, aiming at the special ecological characteristic and eco-influence of GEM, this paper brought forward the policy of "respective analysis on different problems", and postulated the corresponding projects of GEM ecological research to apply GEM safely and effectively in nature.

Ecology↗