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Results for “Potential modifier genes”

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PubMed2005

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus.

Vibrio parahaemolyticus isolates display variation in colony morphology, alternating between opaque (OP) and translucent (TR) cell types. Phase variation is the consequence of genetic alterations in the locus encoding the quorum sensing output regulator OpaR. Here, we show that both cell types form stable, but distinguishable biofilms that differ with respect to attachment and detachment profiles to polystyrene, pellicle formation and stability at the air/medium interface, and submerged biofilm architecture and dispersion at a solid/liquid interface. The pellicle, which is a cohesive mat of cells, was exploited to identify mutants having altered or defective biofilm formation. Transposon insertion mutants were obtained with defects in genes affecting multiple cell surface characteristics, including extracellular polysaccharide, mannose-sensitive haemagglutinin type 4 pili and polar (but not lateral) flagella. Other insertions disrupted genes coding for potential secreted proteins or transporters of secreted proteins, specifically haemolysin co-regulated protein and an RTX toxin-like membrane fusion transporter, as well as potential modifiers of cell surface molecules (nagAC operon). The pellicle screen also identified mutants with lesions in regulatory genes encoding H-NS, a CsgD-like repressor and an AraC-like protein. This work initiates the characterization of V. parahaemolyticus biofilm formation in the OP and TR cell types and identifies a diverse repertoire of cell surface elements that participate in determining multicellular architecture.

Bacterial Proteins↗
PubMed1991

Intracerebral delivery of growth factors: potential application of genetically modified fibroblasts.

To date, a number of different growth factors (e.g. nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor, and fibroblast growth factor) have been shown to act as a neurotrophic and/or neurotrophic agents on distinct neuronal populations within the peripheral and central nervous system. Knowledge as to how most of these factors influence the development and regeneration of growth factor-sensitive neurons has been obtained from in vitro examination. A new approach that can be used to assess the effects of growth factors on neuronal groups in vivo is the combined use of gene transfer and intracerebral grafting techniques. The present article explores the potential use of grafting genetically modified fibroblasts within the nervous system as a delivery method for growth factors.

Animals↗
PubMed2003

Gene therapy applications for intervertebral disc degeneration.

STUDY DESIGN: Review of current approaches to gene therapy as a potential treatment modality for regeneration of the degenerating intervertebral disc. OBJECTIVES: Describe recent advances in the field of gene therapy pertaining to treatment of the degenerating intervertebral disc. SUMMARY OF BACKGROUND DATA: The potential application of gene therapy to the treatment of degenerative disc disease has been an area of exciting and active research. Several in vitro studies have documented the capacity of gene therapy to favorably modify the biologic functions of the intervertebral disc cells with the delivery of the cDNA for various growth factors, whereas in vivo studies have reinforced this potential in animal models. Currently, investigators are exploring the therapeutic potential of gene delivery of the cDNA for several promising factors. METHODS: Literature review. RESULTS/CONCLUSIONS: The application of gene therapy to the treatment of the degenerating intervertebral disc holds much clinical promise and continues to expand as an area of active research. The therapeutic potential of multiple agents, as well as possible vectors, are currently being investigated with encouraging results.

Animals↗
PubMed2005

Conotoxins and the posttranslational modification of secreted gene products.

The venoms of predatory cone snails (genus Conus) have yielded a complex library of about 50-100,000 bioactive peptides, each believed to have a specific physiological target (although peptides from different species may overlap in their target specificity). Conus has evolved the equivalent of a drug development strategy that combines the accelerated evolution of toxin sequences with an unprecedented degree of posttranslational modification. Some Conus venom peptide families are the most highly posttranslationally modified classes of gene products known. We review the variety and complexity of posttranslational modifications documented in Conus peptides so far, and explore the potential of Conus venom peptides as a model system for a more general understanding of which secreted gene products may have modified amino acids. Although the database of modified conotoxins is growing rapidly, there are far more questions raised than answers provided about possible mechanisms and functions of posttranslational modifications in Conus.

Amino Acid Sequence↗
PubMed1999

Modifying human thymidylate kinase to potentiate azidothymidine activation.

Based on the knowledge of the crystal structures of yeast and Escherichia coli thymidylate kinases (TmpKs) and the observation that TmpK from E. coli can phosphorylate azidothymidine monophosphate (AZT-MP) much more efficiently than either the yeast or the highly homologous human enzyme, we have engineered yeast and human TmpKs to obtain enzymes that have dramatically improved AZT-MP phosphorylation properties. These modified enzymes have properties that make them attractive candidates for gene therapeutic approaches to potentiating the action of AZT as an inhibitor of human immunodeficiency virus (HIV) replication. In particular, insertion of the lid domain of the bacterial TmpK into the human enzyme results in a pronounced change of the acceptance of AZT-MP such that it is now phosphorylated even faster than TMP.

Amino Acid Sequence↗
PubMed2005

Hypertension genes are genetic markers for insulin sensitivity and resistance.

Insulin resistance is a determinant of blood pressure variation and risk factor for hypertension. Because insulin resistance and blood pressure cosegregate in Mexican American families, we thus investigated the association between variations in 9 previously reported hypertension genes (ACE, AGT, AGTRI, ADDI, NPPA, ADDRB2, SCNN1A, GNB3, and NOS3) and insulin resistance. Families were ascertained via a coronary artery disease proband in the Mexican American Coronary Artery Disease Project. Individuals from 100 Mexican American families (n=656) were genotyped for 14 polymorphisms in the 9 genes and all adult offspring and offspring spouses were phenotyped for insulin sensitivity by hyperinsulinemic euglycemic clamp (n=449). AGT M235T and NOS3 A(-922)G and E298D polymorphisms were significantly associated with insulin sensitivity (P=0.018, 0.036, 0.039) but were not significant after adjusting for body mass index. ADD1 G460W was associated with insulin sensitivity only after adjusting for body mass index. The NPPA T2238C and SCNN1A A663T were associated with decreased fasting insulin levels after adjusting for body mass index (P=0.015 and 0.028). In conclusion, AGT, NOS3, NPPA, ADRB2, ADD1, and SCNN1A may well be genetic markers for insulin resistance, and adiposity was a potential modifier for only some gene/trait combinations. Our data support the hypothesis that genes in the blood pressure pathway may play a role in insulin resistance in Mexican Americans.

Adult↗
PubMed2005

Genome-wide identification of genes expressed in Arabidopsis pistils specifically along the path of pollen tube growth.

Plant reproductive development is dependent on successful pollen-pistil interactions. In crucifers, the pollen tube must breach the stigma surface and burrow through the extracellular matrix of the stigma epidermal cells and transmitting tract cells before reaching its ovule targets. The high degree of specificity in pollen-pistil interactions and the precision of directional pollen tube growth suggest that signals are continually being exchanged between pollen/pollen tubes and cells of the pistil that line their path. However, with few exceptions, little is known about the genes that control these interactions. The specialized functions of stigma epidermal cells and transmitting tract cells are likely to depend on the activity of genes expressed specifically in these cells. In order to identify these genes, we used the Arabidopsis (Arabidopsis thaliana) ATH1 microarray to compare the whole-genome transcriptional profiles of stigmas and ovaries isolated from wild-type Arabidopsis and from transgenic plants in which cells of the stigma epidermis and transmitting tract were specifically ablated by expression of a cellular toxin. Among the 23,000 genes represented on the array, we identified 115 and 34 genes predicted to be expressed specifically in the stigma epidermis and transmitting tract, respectively. Both gene sets were significantly enriched in predicted secreted proteins, including potential signaling components and proteins that might contribute to reinforcing, modifying, or remodeling the structure of the extracellular matrix during pollination. The possible role of these genes in compatible and incompatible pollen-pistil interactions is discussed.

Arabidopsis↗
PubMed2004

Human cytokine-induced killer cells have enhanced in vitro cytolytic activity via non-viral interleukin-2 gene transfer.

Modulation of the immune system by genetically modified immunological effector cells is of potential therapeutic value in the treatment of malignancies. Interleukin-2 (IL-2) is a crucial cytokine which induces potent antitumor response. Cytokine-induced killer cells (CIK) have been described as highly efficient cytotoxic effector cells capable of lysing tumor cell targets and are capable of recognizing these cells in a non-MHC restricted fashion. Dendritic cells (DC) are the major antigen presenting cells. This study evaluated the antitumor effect of CIK cells which were non-virally transfected with IL-2 and co-cultured with pulsed and unpulsed DC. Human CIK cells generated from peripheral blood were transfected in vitro with plasmid encoding for the human IL-2. Transfection involved a combination of electrical parameters and a specific solution to deliver plasmid directly to the cell nucleus by using the Nucleofector® electroporation system. Nucleofection resulted in the production of IL-2 with a mean of 478.5 pg/106 cells (range of 107.6-1079.3 pg /106 cells/24 h) compared to mock transfected CIK cells (31 pg/106 cells) (P = 0.05). After co-culturing with DC their functional ability was assessed in vitro by a cytotoxicity assay. On comparison with non-transfected CIK cells co-cultured with DCs (36.5 ± 5.3 %), transfected CIK cells co-cultured with DC had a significantly higher lytic activity of 58.5 ± 3.2% (P = 0.03) against Dan G cells, a human pancreatic carcinoma cell line.

CIK↗
PubMed2006

RNA Interference with chemically modified siRNA.

siRNA has become an indispensable tool for functional characterization of genes. It has also demonstrated tremendous potential as a new generation of drug candidates. Although the technology works very well to a great panel of cells in vitro, it is still a challenge to translate the success into in vivo target validation easiness and, even more difficult, into therapeutic applications. With a number of chemically modified compounds under initial clinical trial from several commercial entities, the interests in chemical modification of siRNA have become heightened. In this review we have tried to touch on most of the chemical modifications of RNA that have been tested in the siRNA landscape, but maintained a focus on the backbone modifications, and 2'-modifications on the ribose ring. It is anticipated that more modifications and more systematic comparisons between different modifications will be performed to draw more educated conclusions over some of the modifications.

Hydroxylation↗
PubMed1999

Systemic correction of the muscle disorder glycogen storage disease type II after hepatic targeting of a modified adenovirus vector encoding human acid-alpha-glucosidase.

This report demonstrates that a single intravenous administration of a gene therapy vector can potentially result in the correction of all affected muscles in a mouse model of a human genetic muscle disease. These results were achieved by capitalizing both on the positive attributes of modified adenovirus-based vectoring systems and receptor-mediated lysosomal targeting of enzymes. The muscle disease treated, glycogen storage disease type II, is a lysosomal storage disorder that manifests as a progressive myopathy, secondary to massive glycogen accumulations in the skeletal and/or cardiac muscles of affected individuals. We demonstrated that a single intravenous administration of a modified Ad vector encoding human acid alpha-glucosidase (GAA) resulted in efficient hepatic transduction and secretion of high levels of the precursor GAA proenzyme into the plasma of treated animals. Subsequently, systemic distribution and uptake of the proenzyme into the skeletal and cardiac muscles of the GAA-knockout mouse was confirmed. As a result, systemic decreases (and correction) of the glycogen accumulations in a variety of muscle tissues was demonstrated. This model can potentially be expanded to include the treatment of other lysosomal enzyme disorders. Lessons learned from systemic genetic therapy of muscle disorders also should have implications for other muscle diseases, such as the muscular dystrophies.

Adenoviridae↗
PubMed2004

In vitro autoradiographic localization of calcitonin and amylin binding sites in monkey brain.

Calcitonin (CT) and amylin are related peptides with potent central actions, including suppression of appetite and gastric acid secretion. Little is known about the distribution and binding characteristics of amylin receptors in species other than rat; therefore, in this study, by using in vitro autoradiography, we have mapped the distribution of 125I-rat amylin binding sites in the monkey brain and compared this distribution to that of binding sites for 125I-salmon CT (125I-sCT). Highest densities of 125I-amylin binding were in the hypothalamus, including the arcuate nucleus and parts of the ventromedial hypothalamic nuclei, and the solitary nucleus. Rostrally, moderate to high density binding was present in parts of the preoptic area, bed nucleus of the stria terminalis, amygdala and accumbens nucleus (Acb). Caudally, binding of amylin was more restricted, with moderate to high density binding present only in dorsal raphe, and area postrema. The primary visual cortex displayed strong and periodic CT binding in layer 4. The subcortical pattern of distribution of amylin and CT receptors in the monkey was similar to that seen previously in the rat, although the relative densities of binding to different brain structures were not always conserved. As with rat, monkey amylin receptors were a subset of the sites labeled with 125I-sCT. Analysis of receptor specificity indicated a greater relative potency of CT peptides in competing for 125I-amylin binding in monkey, when compared to rat, while, there was a decrease in the relative potency of CT gene-related peptides, potentially due to differences the level of receptor activity modifying proteins (RAMPs) in monkey versus rat brain. Amylin receptors in primates are likely to perform a similar role to those in rats; however, the interaction of the receptors with related peptides may differ.

Amyloid↗
PubMed2005

A novel genomic approach identifies bacterial DNA-dependent RNA polymerase as the target of an antibacterial oligodeoxynucleotide, RBL1.

Rapid emergence of antibiotic-resistant bacterial pathogens limits the applicability of existing drugs, which has created an urgent need for novel antibiotics preferably with entirely new mechanisms of action. Oligodeoxynucleotides (ODNs) and their modified forms have been shown to inhibit bacterial gene expression, representing a potential for developing highly specific and efficacious antibacterial agents. In this study, a tetracycline (Tet)-inducible, randomized single-stranded DNA (ssDNA) expression library was constructed and screened for conditional growth-defective or lethal phenotypes in an Escherichia coli system. From approximately 5000 transformants screened, 12 bacterial colonies were identified with either growth-defective or lethal phenotypes. One clone, CY01, with a lethal phenotype was selected and sequenced, and the ODN sequence that it generates was designated as RBL-1. Because RBL-1 shows no significant homologies to any bacterial gene sequence, a potential RBL-1 targeting protein was isolated by affinity purification. Using mass spectrometry analysis, this protein was identified as bacterial DNA-dependent RNA polymerase (RNAP). RBL-1 was further shown to effectively inhibit RNA polymerase activity in vitro. The usage of this randomized ssDNA expression library screening technology to selectively modulate production and/or function of proteins may provide a powerful strategy in both identifying novel gene targets for antibiotic discovery and developing novel antibacterial agents.

Amino Acid Sequence↗
PubMed2005

Identification and isolation of a hyperphosphorylated, conformationally changed intermediate of human protein tau expressed in yeast.

Hyperphosphorylation and aggregation of protein tau are typical for neurodegenerative tauopathies, including Alzheimer's disease (AD). We demonstrate here that human tau expressed in yeast acquired pathological phosphoepitopes, assumed a pathological conformation, and formed aggregates. These processes were modulated by yeast kinases Mds1 and Pho85, orthologues of GSK-3beta and cdk5, respectively. Surprisingly, inactivation of Pho85 increased phosphorylation of tau-4R, concomitant with increased conformational change defined by antibody MC1 and a 40-fold increase in aggregation. Soluble protein tau, purified from yeast lacking PHO85, spontaneously and rapidly formed tau filaments in vitro. Further fractionation of tau by anion-exchange chromatography yielded a hyperphosphorylated monomeric subfraction, termed hP-tau/MC1, with slow electrophoretic mobility and enriched with all major epitopes, including MC1. Isolated hP-tau/MC1 vastly accelerated in vitro aggregation of wild-type tau-4R, demonstrating its functional capacity to initiate aggregation, as well as its structural stability. Combined, this novel yeast model recapitulates hyperphosphorylation, conformation, and aggregation of protein tau, provides insight in molecular changes crucial in tauopathies, offers a source for isolation of modified protein tau, and has potential for identification of modulating compounds and genes.

Antibodies↗
PubMed2002

Chemically modified ribozyme targeting TNF-alpha mRNA regulates TNF-alpha and IL-6 synthesis in synovial fibroblasts of patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is chronic polyarthritis in which a variety of inflammatory cytokines play a role. Since tumor necrosis factor-alpha (TNF-alpha) is one of the most important cytokines in the pathogenesis of RA, we evaluated the feasibility of ribozymes as a therapeutic agent to control the inflammatory process of RA synovium. A hammerhead ribozyme against TNF-alpha was chemically modified to increase nuclease resistance and added to RA fibroblastlike cell cultures without using a delivery system. The cellular uptake of fluorescent-labeled ribozyme into synovial cells was found to last at least 48 hr by confocal laser scanning microscopy. The ribozyme targeting TNF-alpha gene inhibited both the expression of TNF-alpha mRNA and the secretion of TNF-alpha and IL-6. The cytotoxic effect by the ribozyme on synovial cells was negligible when determined by an alamar blue assay. Chemically modified ribozymes designed to suppress the TNF-alpha gene may be potential as a therapeutic agent for rheumatoid arthritis.

Arthritis, Rheumatoid↗
PubMed2003

Bone marrow-derived, endothelial progenitor-like cells as angiogenesis-selective gene-targeting vectors.

Human and mouse proliferation-competent, bone marrow or peripheral circulation derived endothelial progenitor-like cells (EPCs) were isolated, expanded and genetically engineered ex vivo to express the beta galactosidase (beta-gal), green fluorescence protein or thymidine kinase (TK) genes using retrovirus-mediated gene transfer. Genetically labeled EPCs were transplanted into sublethally irradiated tumor-bearing mice and were found to migrate to and incorporate into the angiogenic vasculature of growing tumors while maintaining transgene expression. Ganciclovir treatment resulted in significant tumor necrosis in animals previously administered TK-expressing EPCs with no systemic toxicity. These results demonstrate the feasibility of using genetically modified EPCs as angiogenesis-selective gene-targeting vectors and the potential of this approach to mediate non-toxic and systemic antitumor responses.

Animals↗
PubMed2002

Tissue-specific resistance to cancer development in the rat: phenotypes of tumor-modifier genes.

Resistance to carcinogenesis in the rat is both strain- and tissue-specific. The phenotypic characteristics of resistance in the mammary gland, liver and peripheral nervous system (PNS) are strikingly similar. In all three tissues, initiation is intact with subsequent formation of preneoplastic cells and lesions. In the mammary gland and PNS, activation of the Ha-ras and neu proto-oncogenes, respectively, takes place. A number of different modifier genes are involved in resistance, many of which appear to be tissue-specific in their action with no overlap between strains. A single resistance phenotype, however, involving the formation, growth and subsequent loss of preneoplastic lesions is common to all three tissues of resistant strains. In the PNS, there is evidence that preneoplastic cells are eliminated by apoptosis or immunosurveillance. In the mammary gland and liver, the immune system is not involved in the loss of preneoplastic lesions and there are no clear differences between susceptible and resistant strains in the kinetics of proliferation and apoptosis of preneoplastic cells. The evidence to date favors a mechanism in which preneoplastic cells from these tissues undergo a process of remodeling/redifferentiation to yield cells with a normal phenotype. Identification of human homologues of rodent tumor-modifier genes will result in a better understanding of cancer development and potentially provide new strategies for prevention and therapy.

Animals↗
PubMed2006

Influences on gene expression in vivo by a Shine-Dalgarno sequence.

The Shine-Dalgarno (SD+: 5'-AAGGAGG-3') sequence anchors the mRNA by base pairing to the 16S rRNA in the small ribosomal subunit during translation initiation. We have here compared how an SD+ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD+ is confirmed. A downstream SD+ gives decreased gene expression. This effect is also valid for appropriately modified natural Escherichia coli genes. If an SD+ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD+ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD+ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD+ located between them. A minor positive contribution to upstream initiation resulting from 3' to 5' ribosomal diffusion along the mRNA is suggested. Analysis of the E. coli K12 genome suggests that the SD+ or SD-like sequences are systematically avoided in the early coding region suggesting an evolutionary significance.

Base Sequence↗
PubMed2006

A locus on chromosome 13 influences levels of TAFI antigen in healthy Mexican Americans.

When activated, thrombin activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis by modifying fibrin, depressing its plasminogen binding potential. Polymorphisms in the TAFI structural gene (CPB2) have been associated with variation in TAFI levels, but the potential occurrence of influential quantitative trait loci (QTLs) located elsewhere in the genome has been explored only in families ascertained in part through probands affected by thrombosis. We report the results of the first genome-wide linkage screen for QTLs that influence TAFI phenotypes. Data are from 635 subjects from 21 randomly ascertained Mexican American families participating in the San Antonio Family Heart Study. Potential QTLs were localized through a genome-wide multipoint linkage scan using 417 highly informative autosomal short tandem repeat markers spaced at approximately 10-cM intervals. We observed a maximum multipoint LOD score of 3.09 on chromosome 13q, the region of the TAFI structural gene. A suggestive linkage signal (LOD = 2.04) also was observed in this region, but may be an artifact. In addition, weak evidence for linkage occurred on chromosomes 17p and 9q. Our results suggest that polymorphisms in the TAFI structural gene or its nearby regulatory elements may contribute strongly to TAFI level variation in the general population, although several genes in other regions of the genome may also influence variation in this phenotype. Our findings support those of the Genetic Analysis of Idiopathic Thrombophilia (GAIT) project, which identified a potential TAFI QTL on chromosome 13q in a genome-wide linkage scan in Spanish thrombophilia families.

Adult↗