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At least 145 records · Page 8Linked to original sources
PubMed2005

HCV NS3 serine protease-neutralizing single-chain antibodies isolated by a novel genetic screen.

Hepatitis C virus (HCV) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer. The high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for HCV. The HCV NS3 is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. Here, we report the development of a novel bacterial genetic screen for inhibitors of NS3 catalysis and its application for the isolation of single-chain antibody-inhibitors. Our screen is based on the concerted co-expression of a reporter gene, of recombinant NS3 protease and of fusion-stabilized single-chain antibodies (scFvs) in Escherichia coli. The reporter system had been constructed by inserting a short peptide corresponding to the NS5A/B cleavage site of NS3 into a permissive site of the enzyme beta-galactosidase. The resulting engineered lacZ gene, coding for an NS3-cleavable beta-galactosidase, is carried on a low copy plasmid that also carried the NS3 protease-coding sequence. The resultant beta-galactosidase enzyme is active, conferring a Lac+ phenotype (blue colonies on indicator 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal) plates), while induction of NS3 expression results in loss of beta-galactosidase activity (transparent colonies on X-gal plates). The identification of inhibitors, as shown here by isolating NS3-inhibiting single-chain antibodies, expressed from a compatible high copy number plasmid, is based on the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active). Our source of inhibitory scFvs was an scFv library that we prepared from spleens of NS3-immunized mice and subjected to limited affinity selection. Once isolated, the inhibitors were validated as genuine and specific NS3 binders by an enzyme-linked immunosorbent assay and as bone fide NS3 serine protease inhibitors by an in vitro catalysis assay. We further show that upon expression as cytoplasmic intracellular antibodies (intrabodies) in NS3-expressing mammalian cells, three of the scFvs inhibit NS3-mediated cell proliferation. Although applied here for the isolation of antibody-based inhibitors, our genetic screen should be applicable for the identification of candidate inhibitors from other sources.

Amino Acid Sequence↗
PubMed2005

A genetic screen for mutations that affect cranial nerve development in the mouse.

Cranial motor and sensory nerves arise stereotypically in the embryonic hindbrain, act as sensitive indicators of general and region-specific neuronal development, and are directly or indirectly affected in many human disorders, particularly craniofacial syndromes. The molecular genetic hierarchies that regulate cranial nerve development are mostly unknown. Here, we describe the first mouse genetic screen that has used direct immunohistochemical visualization methods to systematically identify genetic loci required for cranial nerve development. After screening 40 pedigrees, we recovered seven new neurodevelopmental mutations. Two mutations model human genetic syndromes. Mutation 7-1 causes facial nerve anomalies and a reduced lower jaw, and is located in a region of conserved synteny with an interval associated with the micrognathia and mental retardation of human cri-du-chat syndrome. Mutation 22-1 is in the Pax3 gene and, thus, models human Waardenburg syndrome. Three mutations cause global axon guidance deficits: one interferes with initial motor axon extension from the neural tube, another causes overall axon defasciculation, and the third affects general choice point selection. Another two mutations affect the oculomotor nerve specifically. Oculomotor nerve development, which is disrupted by six mutations, appears particularly sensitive to genetic perturbations. Phenotypic comparisons of these mutants identifies a "transition zone" that oculomotor axons enter after initial outgrowth and in which new factors govern additional progress. The number of interesting neurodevelopmental mutants revealed by this small-scale screen underscores the promise of similar focused genetic screens to contribute significantly to our understanding of cranial nerve development and human craniofacial syndromes.

Amino Acid Sequence↗
PubMedDate not supplied

Ethical aspects of genetic screening in Israel.

Advances in genetic research make it possible to identify carriers of a growing number of genetic diseases. The World Health Organization (WHO) published several preconditions for community carrier screening. This paper aims to present some of the dilemmas about screening in Israel and the difficulties in following the WHO's helpful criteria. Some of the genetic diseases that are rare in the world are relatively common among several communities in Israel, and one of the dilemmas is for which of them screening is justified. Consensus exists among professionals and among the public about screening for a severe disease like Tay-Sachs, but the justification for screening for a disease with a widely variable expression, like Gaucher disease, is debatable. Another dilemma is whether to offer screening to the general population when the options to solve the problem are still not clear, as is the case of screening for carriers of BRCA1 and BRCA2. The author also explains why geneticists in Israel cooperate with a screening program in the ultra-orthodox community, although it is done according to unusual rules.

Breast Neoplasms↗
PubMedDate not supplied

The feasibility of premarital genetic screening laws.

Advances in medical science are occurring at an astounding rate. Genetics has seen many recent breakthroughs. It is now possible to determine whether an individual has genes which predispose him to a genetic disease or whether a person is a carrier of a genetic disease. This information can be very useful, as it aids individuals in determining the likelihood that their children will be afflicted with a genetic disease. Because these medical advances enable people to predict the incidence of genetic defects and consequently to choose whether or not to reproduce, it has been proposed that genetic screening tests should be required for all individuals before they are issued a marriage license. This proposal, however, presents many legal, ethical and moral problems which need to be considered before such a requirement is instituted.

Civil Rights↗
PubMedDate not supplied

[Genetic screenings: state of the art and perspectives for public health].

Management of genetic data needs the fulfillment of technical requirements, involving ethics, law, and social issues. Introduction of genetic information in preventive medicine implies complex consequences and specialized expertise. Genetic screenings represent a promising tool, but further advances are needed to provide their effective and appropriate use in the respect of the privacy. Application areas, tests validation procedures and availability of effective laboratory and qualified service networks are considered. The Authors present main laws and bioethics guidelines in the field.

Genetic Testing↗
PubMed1997

Genetic screening--uses, potential abuses and ethical issues.

Potential uses, abuses and control of genetic screening techniques are discussed and the ethical issues arising from their use are compared with those for existing health screening practices. The main emphasis is upon issues affecting employment but more wide ranging implications are also touched upon. Although the current policy of various bodies is described, the views expressed are personal.

Advisory Committees↗
PubMed1975

Some influences on public participation in a genetic screening program.

To identify the psychosocial factors associated with voluntary cooperation in mass genetic testing, stratified random samples of 500 participants and 500 nonparticipants were drawn from an identified at-risk population for Tay-Sachs disease. Participants were relatively younger and better educated, reported higher levels of perceived susceptibility to being a carrier, and also stated more often that the impact of learning of being a carrier would be low. Participants were also more likely to indicate they would not alter plans for future progeny. Recommendations are made for enhancing participation in future genetic screening programs of this type.

Adolescent↗
PubMed2005

Pragmatic approaches to genetic screening.

Pragmatic approaches to genetic testing are discussed and appraised. Whilst there are various schools of pragmatism, the Deweyan approach seems to be the most appreciated in bioethics as it allows a historical approach indebted to Hegel. This in turn allows the pragmatist to specify and balance principles in various contexts. There are problems with where to draw a line between what is referred to here as the micro- and macro-level of doing bioethics, unless one is simply to be classified as a principlist. Whilst most discussions on genetics occur at a macro level, most specifying must be done also at a micro level - the clinical encounter. Whilst pragmatism encourages us to understand better social and scientific factors and puts into perspective statements like 'playing God', doubts are raised about the 'consensus' process and how one can put aside fundamental values such as the moral status of the embryo on which there is general disagreement. If those doing pragmatism do not endorse these values, there seems to be little ground for process and compromise with those who do. It seems therefore that pragmatism cannot ignore values, even those which are not endorsed by everyone.

Abortion, Eugenic↗
PubMed2004

Cost-effectiveness analysis of the genetic screening program for familial hypercholesterolemia in The Netherlands.

Familial hypercholesterolemia (FH) is associated with pronounced atherosclerosis leading to premature cardiovascular disease and untimely death. Despite the availability of effective preventative drug treatments, many affected individuals remain undiagnosed and untreated until they become symptomatic with cardiovascular disease. To assess the cost-effectiveness of systematic genetic screening of family members of persons diagnosed with FH, an analysis was conducted using data from a nationwide screening program for the identification of individuals with FH, instituted in The Netherlands in 1994, and from other sources. There was DNA testing of families with a known genetic defect to identify new cases of FH in the presymptomatic stage of the disease. After identification, most newly identified patients were started on cholesterol-lowering statin treatment. On average, new cases diagnosed by the screening program gained 3.3 years of life each. Twenty-six myocardial infarctions would be avoided for every 100 persons treated with statins between the ages of 18 and 60 years. The average total lifetime incremental costs, over all age ranges and both sexes, including costs for screening and testing, lifetime drug treatment, and treatment of cardiovascular events, was US dollars 7500 per new case identified. Cost per life-year gained was US dollars 8700. Therefore, systematic genetic screening of family members of persons diagnosed with FH is cost-effective in The Netherlands and should be considered for other settings.

Adolescent↗
PubMed2004

Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation.

Genetic selection provides the most powerful method to assay large libraries of biomolecules for function. However, harnessing the power of genetic selection for the detection of specific, nonendogenous small-molecule targets in vivo remains a significant challenge. The ability to genetically select for small molecules would provide a reaction-independent mechanism to clone biosynthesis genes from large DNA libraries and greatly facilitate the exploration of large libraries of mutant enzymes for improved synthetic capabilities including altered substrate specificities and enhanced regio- or stereoselectivities. While remarkable progress has been made in developing genetic methods to detect small molecules in vivo, many of these methods rely on engineering small-molecule-protein interactions which remains a difficult problem, and the potential for some of these systems to assay large libraries is limited by the low transformation efficiency and long doubling time of yeast relative to bacteria. Herein, we demonstrate that synthetic riboswitches that activate protein translation in response to a specific small molecule can be used to perform sensitive genetic screens and selections for the presence of small molecules in Escherichia coli. We further demonstrate that the exquisite molecular discrimination properties of aptamers selected in vitro translate directly into an in vivo genetic selection system. Finally, we demonstrate that a cell harboring a synthetic riboswitch with a particular ligand specificity can be selectively amplified from a million-fold larger pool of cells containing mutant riboswitches that respond to a closely related ligand, suggesting that it is possible to use genetic selection in E. coli to discover synthetic riboswitches with new ligand specificities from libraries of mutant riboswitches.

5' Untranslated Regions↗
PubMed1987

The effects of genetic screening and assortative mating on lethal recessive-allele frequencies and homozygote incidence.

The widespread use of genetic screening, along with mating and reproductive patterns reflecting that information, can significantly alter the genetic structure of populations. Both allele frequencies and mortality could be significantly reduced if carriers of lethal recessive alleles were withdrawn from the mating pool. But schemes to mask deleterious alleles in heterozygous condition could significantly increase the deleterious-allele frequencies while resulting in only a slight reduction in mortality. The immediate and equilibrium consequences of such mating strategies may be quite disparate.

Alleles↗
PubMed1999

Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling.

We describe genetic screens in Saccharomyces cerevisiae designed to identify mammalian nonreceptor modulators of G-protein signaling pathways. Strains lacking a pheromone-responsive G-protein coupled receptor and expressing a mammalian-yeast Galpha hybrid protein were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen). Mammalian cDNAs that conferred plasmid-dependent growth under restrictive conditions were identified. One of the cDNAs identified from the activator screen, a human Ras-related G protein that we term AGS1 (for activator of G-protein signaling), appears to function by facilitating guanosine triphosphate (GTP) exchange on the heterotrimeric Galpha. A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5.

Cloning, Molecular↗
PubMed2000

Genetic screening in a large family with juvenile onset primary open angle glaucoma.

AIMS: A number of genetic loci have been implicated in the pathogenesis of primary open angle glaucoma (POAG). The aim of this study was to identify the genetic cause of POAG in a large Scottish family and, if possible, offer genetic screening and advice to family members. METHODS: Family members were examined to determine their disease status. Base excision sequence scanning was carried out in order to test for the presence of a POAG causing mutation at known genetic loci. Direct DNA sequencing was performed in order to determine the mutation sequence. RESULTS: All family members of known affected disease status and two family members of unknown disease status were found to have a mutation in the TIGR gene. The mutation resulted in the substitution of a glycine residue with an arginine residue at codon 252 (Gly252Arg). No other sequence variations were present in any members of the family. CONCLUSION: The Gly252Arg mutation in the TIGR gene results in the development of POAG in this family. It was possible to identify younger, currently unaffected, members of the family who carry the mutation and who are therefore at a very high risk of developing POAG themselves. This is the first demonstration that Gly252Arg can be a disease causing mutation rather than a benign polymorphism. The possible pathogenic mechanisms and wider implications of the mutation are considered.

Adolescent↗
PubMed2004

Advances in the Genetic Screening, Work-up, and Treatment of Pancreatic Cancer.

Familiarity with the updated results in genetic screening and work-up presented here is essential to early diagnosis and possible cure. In the metastatic setting, we most frequently begin with the GTX regimen, consisting of Gemcitabine, Taxotere, and Xeloda. The regimen is based on our laboratory data demonstrating a synergistic increase in cell killing of pancreatic cancer cell lines. The combination takes advantage of the selective cell cycle effects of each of the three drugs. In our initial experience, we have seen a response rate of 40% at metastatic sites and 31% at the primary site after nine cycles of GTX. We are now conducting a formal phase II protocol to confirm these results. The median survival of this group of patients (at least 10.4 months) is as long as, or longer than other currently used regimens. In those patients who do not tolerate GTX or progress despite the regimen, we have found that a regimen of the same three drugs, administered on a different schedule, can produce responses. In the neoadjuvant (unresectable) setting, we treat with GTX initially and then follow with radiation; gemcitabine is used as a radiosensitizer during this treatment. An aggressive surgical approach with a team of surgeons were able to resect for cure 12 of the 16 patients who were initially unresectable; one year survival of these 12 was 100%; 2 year survival was 50%. Future work in this disease should focus on targeted agents such as bevacizumab.

Journal Article↗
PubMed2005

A genetic screen for candidate tumor suppressors identifies REST.

Tumorigenesis is a multistep process characterized by a myriad of genetic and epigenetic alterations. Identifying the causal perturbations that confer malignant transformation is a central goal in cancer biology. Here we report an RNAi-based genetic screen for genes that suppress transformation of human mammary epithelial cells. We identified genes previously implicated in proliferative control and epithelial cell function including two established tumor suppressors, TGFBR2 and PTEN. In addition, we uncovered a previously unrecognized tumor suppressor role for REST/NRSF, a transcriptional repressor of neuronal gene expression. Array-CGH analysis identified REST as a frequent target of deletion in colorectal cancer. Furthermore, we detect a frameshift mutation of the REST gene in colorectal cancer cells that encodes a dominantly acting truncation capable of transforming epithelial cells. Cells lacking REST exhibit increased PI(3)K signaling and are dependent upon this pathway for their transformed phenotype. These results implicate REST as a human tumor suppressor and provide a novel approach to identifying candidate genes that suppress the development of human cancer.

Cell Line, Tumor↗
PubMed2003

Utility of genetic screening in hypertrophic cardiomyopathy: prevalence and significance of novel and double (homozygous and heterozygous) beta-myosin mutations.

Genetic screening of the beta-myosin heavy chain gene (MYH7) was evaluated in 100 consecutive unrelated patients with hypertrophic cardiomyopathy (HCM) and 200 normal unrelated subjects. Seventeen beta-myosin mutations were identified in 19 patients. Notably, 13, or 76%, were novel. Mutations were detected in both alleles in two patients: homozygous for Lys207Gln in one, and heterozygous for Pro211 Leu and Arg663His in another. No mutation was detected in the controls. MYH7-associated HCM was associated with more marked left atrial enlargement and syncope than non-MYH7-related HCM. Our findings indicate that: (1) screening methods should allow identification of novel mutations; and (2) more than one sarcomeric mutation may be present in a patient more commonly than is appreciated. Further studies are necessary to ascertain the clinical consequences of the novel and compound gene abnormalities, and to determine whether correlating functional domain to phenotype provides more useful information about the clinical significance of the molecular defects.

Adolescent↗
PubMed2002

The art and design of genetic screens: Drosophila melanogaster.

The success of Drosophila melanogaster as a model organism is largely due to the power of forward genetic screens to identify the genes that are involved in a biological process. Traditional screens, such as the Nobel-prize-winning screen for embryonic-patterning mutants, can only identify the earliest phenotype of a mutation. This review describes the ingenious approaches that have been devised to circumvent this problem: modifier screens, for example, have been invaluable for elucidating signal-transduction pathways, whereas clonal screens now make it possible to screen for almost any phenotype in any cell at any stage of development.

Animals↗