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PubMed2005

Genome-wide transcriptional changes associated with enhanced activity in the Drosophila nervous system.

Neuronal plasticity is an important feature of the developing brain and requires neuronal circuits to reconfigure their functional connectivity depending upon activity patterns. To explore changes in neuronal function that occur downstream of altered activity, we performed an expression analysis in Drosophila mutants with acute or chronic alterations in neuronal activity. We find that seizure induction leads to an overproliferation of synaptic connections, indicating that activity-dependent neuronal rewiring occurs in Drosophila. To analyze transcriptional recoding during altered neuronal activity, we performed genome-wide DNA microarray analysis following multiple seizure induction and recovery paradigms. Approximately 250 genes implicated in cell adhesion, membrane excitability, and cellular signaling are differentially regulated, including the Kek 2 neuronal cell adhesion protein, which, as we demonstrate, functions as a regulator of synaptic growth. These data identify a collection of activity-regulated transcripts that may link changes in neuronal firing patterns to transcription-dependent modulation of brain function, including activity-dependent synaptic rewiring.

Action Potentials↗
PubMedDate not supplied

Animal models of mental retardation: from gene to cognitive function.

About 2-3% of all children are affected by mental retardation, and genetic conditions rank among the leading causes of mental retardation. Alterations in the information encoded by genes that regulate critical steps of brain development can disrupt the normal course of development, and have profound consequences on mental processes. Genetically modified mouse models have helped to elucidate the contribution of specific gene alterations and gene-environment interactions to the phenotype of several forms of mental retardation. Mouse models of several neurodevelopmental pathologies, such as Down and Rett syndromes and X-linked forms of mental retardation, have been developed. Because behavior is the ultimate output of brain, behavioral phenotyping of these models provides functional information that may not be detectable using molecular, cellular or histological evaluations. In particular, the study of ontogeny of behavior is recommended in mouse models of disorders having a developmental onset. Identifying the role of specific genes in neuropathologies provides a framework in which to understand key stages of human brain development, and provides a target for potential therapeutic intervention.

Animals↗
PubMed2000

Bacterial toxin-antitoxin gene system as containment control in yeast cells.

The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae. Expression of the relE gene was highly toxic to yeast cells. However, expression of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae. Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial fermentation processes in which the escape of genetically modified cells would be considered highly risky.

Antitoxins↗
PubMed2005

AAV serotype-dependent apolipoprotein A-I Milano gene expression.

Recent evidence from a double-blind, randomized study showed that treatment with apolipoprotein A-I Milano (ApoA-I Milano) in a complex with phospholipids produced significant regression of the coronary atheroma burden in patients with acute coronary syndromes. We previously showed similar regression of atherosclerosis in an animal model. Here, we examined a viral vector-based gene delivery system as a basis for ApoA-I Milano gene therapy. Comparing levels of expression using combinations of the cytomegalovirus (CMV) promoter in a recombinant serotype 2 adeno-associated virus (rAAV2) linked to ApoA-I Milano or the enhanced green fluorescent protein (EGFP) genes, we found that a promoter construct of two CMV core promoters sharing a CMV enhancer was more active than other combinations or a single CMV promoter. In vivo assessment of this optimal CMV construct using rAAV2 virus particles for intravenous (IV) or intramuscular (IM) routes of delivery produced high circulating levels of ApoA-I Milano protein for extended periods (up to 220 ng/ml at 22 weeks p.i.) by IV delivery while the IM route resulted in a relatively short period of very low-level ApoA-I Milano expression. Since there was no difference in the immune response between the two routes of delivery, we reasoned that tissue tropism might be responsible for this differential gene expression. To explore this possibility, we investigated the effect of different AAV serotypes on ApoA-I Milano gene expression in vivo. It found that rAAV1-mediated expression of ApoA-I Milano was approximately 15- and 9-fold higher than rAAV2 and rAAV5, respectively when IM injection routes were compared while all three AAV serotypes produced substantial levels of ApoA-I Milano expression from IV injection. These studies demonstrate that by modifying the promoter and serotype, increases in the efficiency of AAV-directed transgene expression could be achieved and support the potential of AAV-mediated gene therapy.

Adenoviridae↗
PubMed2005

Multi-functional drugs for various CNS targets in the treatment of neurodegenerative disorders.

Individuals with neurodegenerative diseases such as Parkinson's disease or Alzheimer's disease are benefiting from drugs developed to act on a single molecular target. However, current pharmacological approaches are limited in their ability to modify significantly the course of the disease, and offer incomplete and transient benefit to patients. New therapeutic strategies comprise drug candidates designed specifically to act on multiple neural and biochemical targets for the treatment of cognition impairment, motor dysfunction, depression and neurodegeneration. Examples include the development of single molecular entities that combine two or more of the following properties: (i) cholinesterase inhibition; (ii) activation or inhibition of specific subtypes of acetylcholine receptors or alpha-adrenoceptors; (iii) anti-inflammatory activity; (iv) monoamine oxidase inhibition; (v) catechol-O-methyl transferase inhibition; (vi) nitric oxide production; (vii) neuroprotection; (viii) anti-apoptotic activity; and (ix) activation of mitochondrial-dependent cell-survival genes and proteins. These bi- or multi-functional compounds might provide greater symptomatic efficacy, and better utility as potential neuroprotective disease-modifying drugs.

Animals↗
PubMed2004

Conditional expression in the malaria mosquito Anopheles stephensi with Tet-On and Tet-Off systems.

We report successful conditional gene expression in the malaria vector, Anopheles stephensi, on the basis of binary systems consisting of gene driver and responder transgenic lines generated by Minos-mediated germline transformation. An A. gambiae tissue-specific enhancer derived from a serpin (SRPN10) gene was utilized to control the temporal and spatial expression of doxycycline (dox)-sensitive transcriptional regulators in the driver lines. The "Tet-Off" driver utilized the tetracycline-controlled transcriptional activator (tTA) that is unable to bind and activate transcription from tetracycline operators (TetO) in the presence of dox; the "Tet-on" driver utilized the reverse tTA (rtTA) that, conversely, binds and activates TetO operators in the presence of dox. The responder lines carried insertions encompassing a LacZ reporter gene, cis-regulated by a TetO-P-element hybrid promoter. The progeny of crosses between driver and responder lines expressed beta-galactosidase under dual, tissue-specific and dox-mediated regulation. In adult rtTA/TetOPlacZ progeny, dox treatment rapidly induced beta-galactosidase activity throughout the midgut epithelium and especially in malaria parasite-invaded epithelial cells. Transactivator-dependent, dox-mediated regulation was observed in hemocytes and pericardial cells using both systems. Conditional tissue-specific regulation is a powerful tool for analyzing gene function in mosquitoes and potentially for development of strategies to control disease transmission.

Animals↗
PubMed1999

Prospects for antisense agents in the therapy of bacterial infections.

Antisense agents have received widespread interest as potential therapeutic agents for a number of diseases, including cancer, inflammatory conditions and viral infections. However, less emphasis has been placed on their potential application in the therapy of bacterial infections. This review considers the reported effects of backbone modified oligonucleotides (phosphorothioate and methyl phosphonate analogues) as well as peptide nucleic acids (PNAs) on gene expression and bacterial growth. In addition to suppressing bacterial growth by decreasing the expression of essential genes, it is also evident that antisense agents can be specifically targeted to genes that control expression of antibiotic resistance mechanisms, thereby potentially restoring an antibiotic-sensitive phenotype to the cell. Despite observations from several studies that antisense agents can interfere with bacterial gene expression in a sequence specific manner, their uptake into bacteria is poor. At present this is a limiting factor in their potential application as therapeutic agents for bacterial infections.

Journal Article↗
PubMed2005

Modified direct-double-differential PCR for gene dosage quantification of HER2.

HER2 amplification and/or overexpression in breast cancer are adverse prognostic factors and can predict the response to trastuzumab therapy. As assessment of HER2 status in breast cancer is of great importance for clinical decision-making, it is crucial to have reliable methods to quantify HER2. In the present project, we have developed a modification of the direct-double-differential PCR method (dddPCR) for gene dosage quantification of HER2 in breast cancer samples. The study was also undertaken to establish the potential application of the newly developed dddPCR method for HER2 testing in a diagnostic laboratory. The dddPCR validity for determining HER2 amplification in comparison to IHC-based HercepTest for the detection of protein levels was performed. Modified dddPCR was proven to be highly reproducible and reliable. A statistically significant correlation between protein expression and gene dosage of HER2 was found in the examined material. The obtained results indicate that dddPCR provides a fast and reliable diagnostic tool for HER2 quantification and may be considered as a supplementary method to verify equivocal results obtained with IHC-based HercepTest. Notably, dddPCR can be performed in every basic molecular biology laboratory, and is inexpensive and time-saving.

Adult↗
PubMed2001

In vitro prevention of cell-mediated xeno-graft rejection via the Fas/FasL-pathway in CrmA-transducted porcine kidney cells.

Cell-mediated cytotoxicity may be involved in delayed and/or chronic xenograft rejection in which apoptosis is induced in the grafted cells via the Fas/Fas-ligand (FasL) and perforin/granzyme pathways. One barrier to the potential use of xeonogenic grafts for humans may be Fas/FasL-mediated apoptosis, which would be blocked by the gene expression of cytokine response modifier A (CrmA), a cowpox virus gene product. The purpose of this study is to explore whether crmA is an effective candidate gene for inhibiting apoptosis in an in vitro model of xenograft rejection, using Fas-expressing non-primate cells cultured with a soluble recombinant human FasL (sFasL). A recombinant adenovirus vector expressing CrmA (AxCALNLCrmA) was successfully generated with a Cre-mediated switching system. PK15 cells, derived from a porcine kidney and infected with AxCALNLCrmA and/or AxCANCre at a multiplicity of infection (MOI) ranging from 0.1 to 100, were cultured with human sFasL derived from KFL74.18, a human FasL-overexpressed cell line. The gene-expression level of the PK15 cells was confirmed by CrmA-immune staining. Approximately 70% of the control PK15 cells showed induced apoptosis when cultured with sFasL. In contrast, the apoptosis was dramatically reduced in crmA-gene-transduced PK15 cells. The inhibitory effect of apoptosis increased with an increase in the infection dose of AxCANCre. In addition, the activity of caspases 3 and 8 was significantly inhibited in the crmA-transduced cells. These results indicate that CrmA is an effective gene product for inhibiting Fas/FasL-mediated apoptosis, which suggests the potential therapeutic use of its gene transduction to protect against graft damage due to delayed and/or chronic xenograft rejection.

Animals↗
PubMed2006

Large-scale gene expression profiling reveals major pathogenetic pathways of cartilage degeneration in osteoarthritis.

OBJECTIVE: Despite many research efforts in recent decades, the major pathogenetic mechanisms of osteoarthritis (OA), including gene alterations occurring during OA cartilage degeneration, are poorly understood, and there is no disease-modifying treatment approach. The present study was therefore initiated in order to identify differentially expressed disease-related genes and potential therapeutic targets. METHODS: This investigation consisted of a large gene expression profiling study performed based on 78 normal and disease samples, using a custom-made complementary DNA array covering >4,000 genes. RESULTS: Many differentially expressed genes were identified, including the expected up-regulation of anabolic and catabolic matrix genes. In particular, the down-regulation of important oxidative defense genes, i.e., the genes for superoxide dismutases 2 and 3 and glutathione peroxidase 3, was prominent. This indicates that continuous oxidative stress to the cells and the matrix is one major underlying pathogenetic mechanism in OA. Also, genes that are involved in the phenotypic stability of cells, a feature that is greatly reduced in OA cartilage, appeared to be suppressed. CONCLUSION: Our findings provide a reference data set on gene alterations in OA cartilage and, importantly, indicate major mechanisms underlying central cell biologic alterations that occur during the OA disease process. These results identify molecular targets that can be further investigated in the search for therapeutic interventions.

Aged↗
PubMed2002

Strain-dependent variation in vascular responses to nitric oxide in the isolated murine heart.

Numerous studies in the literature have employed gene-modified mice to investigate vascular function. However, only very limited information exists on baseline murine vascular physiology or on potential variations between different strains. We therefore compared coronary and aortic vascular responses to endothelium-derived vasodilators and exogenous nitric oxide (NO) in three commonly used mouse strains and correlated these data with expression of eNOS, NADPH oxidase subunits, gp91(phox) and p67(phox), and superoxide production. Isolated perfused hearts from MF1, 129sv and C57BL/6J mice were subjected to: (a) increasing doses of bradykinin, acetylcholine and sodium nitroprusside, and (b) bolus doses of adenosine and the NO synthase inhibitor, N(G)-monomethyl- L -arginine. Vascular responses of thoracic aortic rings were assessed for comparison. Expression of eNOS and NADPH oxidase subunits was assessed by immunoblotting, and superoxide production by lucigenin-enhanced chemiluminescence. Coronary vasodilator responses to bradykinin, acetylcholine and sodium nitroprusside were significantly attenuated in MF1 compared with C57BL/6J and 129sv hearts. Similarly, aortic relaxation to acetylcholine was significantly impaired in MF1 aortic rings compared with in C57BL/6J aortae; these differences were reversed by Tiron. N(G)-monomethyl- L -arginine induced significantly less vasoconstriction in MF1 and 129sv hearts compared with C57BL/6J. No differences in aortic relaxation to A23187 or sodium nitroprusside were observed. Cardiac and aortic superoxide production and cardiac expression of p67(phox) and gp91(phox) were significantly greater in MF1 mice compared with the other strains. There is significant strain-dependent variation in coronary and aortic vascular responsiveness in mice, which may reflect differences in the balance between NO and superoxide generation.

Animals↗
PubMed2004

Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis.

Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.

Animals↗
PubMed2001

The human G-protein beta3 subunit C825T polymorphism is associated with coronary artery vasoconstriction.

AIMS: Abnormal coronary vasomotion plays a role in the clinical expression of coronary artery disease. We hypothesized that the functional C825T polymorphism located in the ubiquitous G-protein beta3 subunit, implicated in the cellular signal transduction of many receptors, could modify artery coronary vasomotion. We assessed the potential association of the pertussis toxin-sensitive G protein beta3 subunit (GNB3) gene C825T polymorphism on coronary vasomotion in humans. METHODS AND RESULTS: We examined the response of angiographically normal human coronary arteries (n=131) after intravenous injection of methylergonovine maleate, a vasoconstrictor, followed by injection of isosorbide dinitrate, a vasodilator, according to GNB3 genotypes. Coronary vasomotion was assessed with quantitative coronary angiography. Subjects bearing at least one T allele had greater susceptibility to vasoconstriction in response to methylergonovine maleate than CC subjects, whereas vasodilation in response to isosorbide dinitrate did not differ among the different genotypes. CONCLUSION: The C825T polymorphism of the G-protein beta3 subunit may be a genetic determinant of coronary artery vasomotion in humans.

Coronary Angiography↗
PubMed2000

SOCS-3 is an insulin-induced negative regulator of insulin signaling.

The SOCS proteins are induced by several cytokines and are involved in negative feedback loops. Here we demonstrate that in 3T3-L1 adipocytes, insulin, a hormone whose receptor does not belong to the cytokine receptor family, induces SOCS-3 expression but not CIS or SOCS-2. Using transfection of COS-7 cells, we show that insulin induction of SOCS-3 is enhanced upon Stat5B expression. Moreover, Stat5B from insulin-stimulated cells binds directly to a Stat element present in the SOCS-3 promoter. Once induced, SOCS-3 inhibits insulin activation of Stat5B without modifying the insulin receptor tyrosine kinase activity. Two pieces of evidence suggest that this negative regulation likely results from competition between SOCS-3 and Stat5B binding to the same insulin receptor motif. First, using a yeast two-hybrid system, we show that SOCS-3 binds to the insulin receptor at phosphotyrosine 960, which is precisely where Stat5B binds. Second, using confocal microscopy, we show that insulin induces translocation of SOCS-3 from an intracellular compartment to the cell membrane, leading to colocalization of SOCS-3 with the insulin receptor. This colocalization is dependent upon phosphorylation of insulin receptor tyrosine 960. Indeed, in cells expressing an insulin receptor mutant in which tyrosine 960 has been mutated to phenylalanine, insulin does not modify the cellular localization of SOCS-3. We have thus revealed an insulin target gene of which the expression is potentiated upon Stat5B activation. By inhibiting insulin-stimulated Stat5B, SOCS-3 appears to function as a negative regulator of insulin signaling.

3T3 Cells↗
PubMed2002

A novel approach to tumor suppression with microencapsulated recombinant cells.

A novel approach to cancer gene therapy is to implant microcapsules containing nonautologous cells engineered to secrete molecules with antineoplastic properties. The efficacy of this treatment is now tested in a mouse model bearing HER-2/neu-positive tumors. Nonautologous mouse myoblasts (C(2)C(12)) were genetically modified to secrete interleukin-2 linked to the Fv region of a humanized antibody with affinity to HER-2/neu. The resulting fusion protein, sFvIL-2, would encompass immune-stimulatory cytokine activity now targeted to the HER-2/neu-expressing tumor. These recombinant cells were then immunoprotected with alginate-poly-L-lysine-alginate microcapsules before implantation into tumor-bearing mice. Treatment with these encapsulated cells led to a delay in tumor progression and prolonged survival of the animals. The long-term efficacy was limited by an inflammatory reaction against the implanted microcapsules probably because of the secreted cytokine and antigenic response against the xenogeneic fusion protein itself. However, over the short term (initial 2 weeks), efficacy was confirmed when a significant amount of biologically active interleukin-2 was detected systemically, and targeting of the fusion protein to the HER-2/neu-expressing tumor was shown immunohistochemically. The tumor suppression in the treated animals was associated with increased apoptosis and necrosis in the tumor tissue, thus demonstrating successful targeting of the antiproliferative effect to the tumors by this delivery paradigm. In conclusion, this new approach to systemic cancer gene therapy needs to be modified to provide long-term delivery, but has demonstrated short-term efficacy and potential to become a cost-effective, benign, and non-viral-based adjunct to the current armory of anticancer strategies.

Animals↗
PubMed2005

CAM and cell fate targeting: molecular and energetic insights into cell growth and differentiation.

Evidence-based medicine is switching from the analysis of single diseases at a time toward an integrated assessment of a diseased person. Complementary and alternative medicine (CAM) offers multiple holistic approaches, including osteopathy, homeopathy, chiropractic, acupuncture, herbal and energy medicine and meditation, all potentially impacting on major human diseases. It is now becoming evident that acupuncture can modify the expression of different endorphin genes and the expression of genes encoding for crucial transcription factors in cellular homeostasis. Extremely low frequency magnetic fields have been found to prime the commitment to a myocardial lineage in mouse embryonic stem cells, suggesting that magnetic energy may direct stem cell differentiation into specific cellular phenotypes without the aid of gene transfer technologies. This finding may pave the way to novel approaches in tissue engineering and regeneration. Different ginseng extracts have been shown to modulate growth and differentiation in pluripotent cells and to exert wound-healing and antitumor effects through opposing activities on the vascular system, prompting the hypothesis that ancient compounds may be the target for new logics in cell therapy. These observations and the subtle entanglement among different CAM systems suggest that CAM modalities may deeply affect both the signaling and transcriptional level of cellular homeostasis. Such a perception holds promises for a new era in CAM, prompting reproducible documentation of biological responses to CAM-related strategies and compounds. To this end, functional genomics and proteomics and the comprehension of the cell signaling networks may substantially contribute to the development of a molecular evidence-based CAM.

Journal Article↗
PubMed1994

Therapeutic promise of molecular genetics.

The application of molecular genetics to medicine is based on the observation that even common diseases have both genetic and environmental components. Conventional medicines are often effective in managing environmental components of disease but are generally ineffective in managing genetic diseases or manipulating the genetic component of multifactorial diseases. The development of therapies aimed at the genetic component of disease will require non-conventional medicinal applications of molecular genetics. Various approaches have been proposed such as diagnosing the propensity for disease to facilitate early intervention with conventional therapies, selectively eliminating mutant genes from human populations, correcting mutations in human chromosomes, and using genes as medicines to modify the genetic components of disease. Of these, it is the development of gene medicines that has the greatest practical potential. The combination of conventional medicines, focused on the environmental components of disease, and gene medicines, focused on the genetic components, will provide the clinician with broad options for managing health and disease. The challenge to molecular biology is to develop gene medicines that are effective, safe, and socially acceptable, and therapies that map well to established clinical practice and may be employed efficaciously alongside conventional medicines.

Genetic Diseases, Inborn↗
PubMed2005

Basal cell carcinoma: from host response and polymorphic variants to tumour suppressor genes.

The molecular factors and events that characterize susceptibility and outcome in cutaneous basal cell carcinoma (BCC) have been the focus of much research interest. As a result, we are beginning to understand the complex relationships between exposure to ultraviolet radiation (UVR), host response and the resulting damage to key genes that characterize these tumours. In this review, we will focus on genetic factors that influence susceptibility and outcome. While the search for susceptibility genes has generally resulted in the identification of low penetrance allelic variants, studies on modifier genes influencing outcome variables such as tumour number, age of onset and tumour subtype have identified factors with higher potential impact. Here we will briefly describe some recent work on the genetic basis of the immune response to UVR, the effect of UVR on the generation of reactive oxygen species and their detoxification, and the role of onco- and tumour suppressor genes. Areas for further research are highlighted, together with a consideration of possible applications in clinical practice.

Basal Cell Carcinoma↗