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PubMed2004

[What place for DNA microarray in inflammatory diseases?].

PURPOSE: DNA chip is a recently developed technique allowing analysis of thousands of genes at the same time in multiple biological samples. In few years it has become an obligatory step in massive gene expression study. The enormous quantity of results generated and the new way of thinking allowed make this kind of study a true revolution. KEY MESSAGE AND RECENT FACTS: The enormous discovery potential permitted by the accomplishment of multiple genomes sequencing and the advent of technologies allowing massive gene expression analyses have totally modified the diseases approach. Considering the obtainment of a real full picture of the transcriptional activity in an organ, tissue or cell is now legitimate. DNA microarray is obviously not the only technique allowing such type of analysis but it is without contest the technology which is the most popular and the one which has been recently the subject of the most important developments. It is certainly the technology which brought the main advances in tumour classification and discovery of new biomarkers. The first results based on this technology in inflammatory diseases have recently been reported. PERSPECTIVE AND PROJECTS: The optimal use of DNA microarrays will necessitate a powerful statistical analysis and an high quality biological experimentation. Strict standard and quality criteria are developing. Obviously, the DNA chips have a role to play in multifactorial inflammatory diseases mainly through their potential to bring new answers to diagnostic and pathophysiological problems. One potential development of the technique in such diseases will be the definition of disease specific gene profiles and the generation of chips allowing the detection of few targeted genes with all the known mutations of these genes. The correlation of global or targeted gene expression with clinical and pathological data will allow a new step forward in the understanding and taking care of inflammatory diseases.

Animals↗
PubMed2000

Targeted modification of the domestic animal genome.

While the technique of homologous recombination, or gene targeting, has led to the generation of transgenic mice of great value to biomedical research, similar approaches are only being developed in other species. With the exception of recent reports on the generation of gene-targeted sheep, the technology in domestic animals is still in its infancy (45). The development of techniques for generating large animals with deleted or modified genes will result in the generation of animals of great value to society. While the technical difficulties to achieve gene targeting in domestic species are significant, they are not insurmountable. Potential applications in both the bovine and porcine species are described with particular emphasis on the generation of cattle resistant to bovine spongiform encephalopathy (BSE) and pigs that can be of use in xenotransplantation.

Animals↗
PubMed1997

Is function of the Drosophila homeotic gene Ultrabithorax canalized?

Genetic variation affecting the expressivity of an amorphic allele of the homeotic gene Ultrabithorax, (Ubx1) was characterized after 11 generations of introgression into 29 different isofemale lines. Heterozygotes display a range of haploinsufficient phenotypes, from overlap with wild-type halteres to dramatic transformations such as a 50% increase in area and the presence of over 20 bristles on the anterior margin of each haltere. In both the wild-type and mutant genetic backgrounds, there is moderate genetic variance and low environmental variance/developmental asymmetry, as expected of a trait under stabilizing selection pressure. Surprisingly, there is little evidence that mutant halteres are more variable than wild-type ones, so it is unclear that haltere development is also canalized. The correlation between wild-type and Ubx haltere size is very low, indicating that interactions among modifiers of Ubx are complex, and in some cases sex-specific. The potential quantitative genetic contributions of homeotic genes to appendage morphology are discussed, noting that population-level effects of variation in key regulatory genes may be prevalent and complex but cannot be readily extrapolated to macroevolutionary diversification.

Animals↗
PubMed2002

Differential regulation of active zone density during long-term strengthening of Drosophila neuromuscular junctions.

In this study we established a transgenic Ca2+ imaging technique in Drosophila that enabled us to target the Ca2+ sensor protein yellow Cameleon-2 specifically to larval neurons. This noninvasive method allowed us to measure evoked Ca2+ signals in presynaptic terminals of larval neuromuscular junctions (NMJs). We combined transgenic Ca2+ imaging with electrophysiological recordings and morphological examinations of larval NMJs to analyze the mechanisms underlying persistently enhanced evoked vesicle release in two independent mutants. We show that persistent strengthening of junctional vesicle release relies on the recruitment of additional active zones, the spacing of which correlated with the evoked presynaptic Ca2+ dynamics of individual presynaptic terminals. Knock-out mutants of the postsynaptic glutamate receptor (GluR) subunit DGluR-IIA, which showed a reduced quantal size, developed NMJs with a smaller number of presynaptic boutons but a strong compensatory increase in the density of active zones. This resulted in an increased evoked vesicle release on single action potentials and larger evoked Ca2+ signals within individual boutons; however, the transmission of higher frequency stimuli was strongly depressed. A second mutant (pabp(P970)/+), which showed enhanced evoked vesicle release triggered by elevated subsynaptic protein synthesis, developed NMJs with an increased number of presynaptic boutons and active zones; however, the density of active zones was maintained at a value typical for wild-type animals. This resulted in wild-type evoked Ca2+ signals but persistently strengthened junctional signal transmission. These data suggest that the consolidation of strengthened signal transmission relies not only on the recruitment of active zones but also on their equal distribution in newly grown boutons.

Action Potentials↗
PubMed2000

Attenuation of interleukin-8 production by inhibiting nuclear factor-kappaB translocation using decoy oligonucleotides.

Interleukin-8 (IL-8), a monocyte-derived neutrophil chemoattractant factor, is a polymorphonuclear neutrophil chemotaxin that is involved in a number of inflammatory disorders. Transcription of the IL-8 gene is controlled by regulatory proteins, including nuclear factor-kappaB (NF-kappaB), a family of proteins that is important in the transcriptional control of a number of genes. When cells are activated, NF-kappaB translocates from the cytoplasm to the nucleus, where it activates transcription by binding to a specific sequence within the 5' untranslated region of the gene. During translocation, NF-kappaB is potentially susceptible to diversion by oligonucleotides that contain the binding sequence for this protein. In the current study, we produced phosphorothioate-modified oligonucleotides containing the specific DNA sequence that NF-kappaB binds within the IL-8 gene. We then investigated the effects of transfection of monocytes with these oligonucleotides on interleukin-1beta (IL-1beta)-stimulated IL-8 production, IL-8 mRNA expression, and NF-kappaB binding activity. We found that transfection with these oligonucleotides significantly inhibited monocyte IL-8 production. A single-stranded oligonucleotide with two copies of the NF-kappaB-binding sequence was the most potent of those tested. This single-stranded oligonucleotide also inhibited IL-1beta-induced translocation of NF-kappaB to the nucleus and reduced IL-8 mRNA expression. These studies demonstrated that monocyte production of IL-8 can be attenuated using a single-stranded oligonucleotide that binds a transcriptional activating protein before it translocates to the cell nucleus. This approach ultimately may be useful in the control of inflammation involved in a number of diseases.

Binding, Competitive↗
PubMed1989

Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodospirillum rubrum.

Nitrogen fixation activity in the photosynthetic bacterium Rhodospirillum rubrum is controlled by the reversible ADP-ribosylation of the dinitrogenase reductase component of the nitrogenase enzyme complex. This report describes the cloning and characterization of the genes encoding the ADP-ribosyltransferase (draT) and the ADP-ribosylglycohydrolase (draG) involved in this regulation. These genes are shown to be contiguous on the R. rubrum chromosome and highly linked to the nifHDK genes. Sequence analysis revealed the use of TTG as the initiation codon of the draT gene as well as a potential open reading frame immediately downstream of draG. The mono-ADP-ribosylation system in R. rubrum is the first in which both the target protein and modifying enzymes as well as their structural genes have been isolated, making it the model system of choice for analysis of this post-translational regulatory mechanism.

Adenosine Diphosphate Ribose↗
PubMed2001

Receptor-targeted gene delivery using multivalent phagemid particles.

Although growth factor- and antibody-targeted filamentous phage have recently been demonstrated to transduce mammalian cells, there is a significant need to increase transduction efficiency so as to improve the usefulness of targeted phage vectors for gene therapy and ligand discovery. Here, we describe the use of multivalent phagemid vectors that are specifically designed for ligand-targeted mammalian cell transduction. This phagemid system has certain advantages over phage vectors, such as larger insert size and vector stability, and it retains the multivalent display necessary for efficient cell binding and internalization. Immunoblotting revealed that the most efficient multivalent display (exceeding that of a phage vector) was achieved in the phagemid system when epidermal growth factor (EGF) was fused to the C-terminal domain of the pIII coat protein. We compared phagemid particles displaying EGF at high or low valence by rescuing the vector with R408d3 (pIII deleted) or wild-type R408 helper phage, respectively. More efficient display of EGF correlated with increased internalization, vector potency, and transduction efficiency ( approximately 9%). The findings described here support our original hypothesis that phage-based vectors can be modified for more efficient gene transfer and suggest that directed evolution may be applied to increase their potential even further.

Bacteriophages↗
PubMed1996

Evaluation of the thymidine kinase (tk) locus as an insertion site in the highly attenuated vaccinia MVA strain.

The highly attenuated 'modified vaccinia Ankara' (MVA) strain is a potential live vaccine vector. Insertional inactivation of the tk-gene resulted in viruses difficult to purify. Co-integration of a functional fowlpox virus tk-gene allowed easy generation of recombinants, indicating that the genetically stable tk-gene region is a suitable insertion site, if tk-gene activity is substituted.

Animals↗
PubMed2005

Cell microarrays on photochemically modified polytetrafluoroethylene.

We studied the adhesion, proliferation, and viability of human umbilical vein endothelial cells (HUVEC) and human embryonic kidney cells (HEK) on modified spots at polytetrafluoroethylene (PTFE) surfaces. The viability of the cells was assessed using an aqueous non-radioactive cell proliferation assay. Round spots with a diameter of 100 microm were modified by exposure to the ultraviolet (UV) light of a Xe(2)(*)-excimer lamp at a wavelength of 172 nm in an ammonia atmosphere employing a contact mask. The spots were arranged in a quadratic pattern with 300 microm center-to-center spot distances. With optimized degree of modification, the cells adhered to the modified spots with a high degree of selectivity (70-90%). The adhered cells on the spots proliferated. This resulted in a significant increase in the number of adhering HUVECS or HEK cells after seeding and in the formation of confluent cell clusters after 3-4 days. With higher start seeding density, these clusters were not only confined to the modified spots but extended several micrometer to the neighborhood. The high potential of the cell microarrays for gene analysis in living cells was demonstrated with HEK cells transfected by yellow fluorescent protein (YFP).

Biocompatible Materials↗
PubMed2006

Familial aggregation of insulin resistance in first-degree relatives of patients with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: An association between nonalcoholic fatty liver disease (NAFLD) and the insulin resistance (IR) syndrome exists. Familial clustering of IR may support a genetic predisposition to NAFLD or cryptogenic cirrhosis (CC). METHODS: Patients with NAFLD (n = 20) and healthy controls (n = 20) matched for age, sex, and body mass index with at least 4 living relatives and 2 generations of lineage participated in a familial aggregation study. A medical history was obtained from each subject and confirmed with first-degree family members. RESULTS: NAFLD patients were more likely to have cirrhosis (odds ratio [OR] = 12.0), cardiac disease (OR, 3.0), hyperlipidemia (OR, 12.1), diabetes mellitus (DM) (OR, 9.1), renal stones (OR, 4.1), and arthritis (OR, 6.1). IR (P = .042) (the primary dependent variable) and DM (P = .013) were noted in their first-degree relatives. A trend for familial clustering of NAFLD or CC (P = .059) with a maternal linkage for disease expression also may exist. Cholelithiasis (P = .047), presumed NAFLD and/or CC (P = .049), and a trend toward IR (P = .07) were noted in the mothers, but not the fathers, of patients with NAFLD. An increased risk of DM (OR, 4.2; 95% confidence interval, 1.26-18.7; P = .013), IR (OR, 2.86; 95% confidence interval, 1.02-9.38; P = .042), and smoking (OR, 3.41; 95% confidence interval, 1.43-9.07; P = .003) was observed in first-degree relatives of NAFLD patients. No significant survival difference was observed between family cohorts. CONCLUSIONS: Familial clustering and a potential maternal linkage for IR support a genetic predisposition for NAFLD. Alternatively, the presence of suppressor genes modifying the expression of IR in paternal lineage warrants further investigation.

Adult↗
PubMed2004

Bio-fermentation of modified flavonoids: an example of in vivo diversification of secondary metabolites.

A bio-fermentation technique was used for the in vivo diversification of flavonoid structures based on expression in Escherichia coli of six O-methyltransferases (OMTs) from Mentha x piperita and one O-glucosyltransferase (GT) each from Arabidopsis thaliana and Allium cepa. Enzymes were shown to be regio-specific in in vitro experiments and modified a broad range of flavonoid substrates at various positions. Using the flavonol quercetin as a model substrate, we show that the product spectrum produced with the in vivo approach is identical to that found in vitro. Additionally, using mixed cultures of E. coli expressing different classes of modifying genes (OMTs and GTs), the production of polymethylated flavonoid glucosides was observed. This report demonstrates the potential to increase the structural diversity of plant secondary metabolites using a multi-enzyme, bio-fermentation approach.

Amino Acid Sequence↗
PubMed2002

Substantial equivalence--an appropriate paradigm for the safety assessment of genetically modified foods?

Safety assessment of genetically modified food crops is based on the concept of substantial equivalence, developed by OECD and further elaborated by FAO/WHO. The concept embraces a comparative approach to identify possible differences between the genetically modified food and its traditional comparator, which is considered to be safe. The concept is not a safety assessment in itself, it identifies hazards but does not assess them. The outcome of the comparative exercise will further guide the safety assessment, which may include (immuno)toxicological and biochemical testing. Application of the concept of substantial equivalence may encounter practical difficulties: (i) the availability of near-isogenic parental lines to compare the genetically modified food with; (ii) limited availability of methods for the detection of (un)intended effects resulting from the genetic modification; and (iii) limited information on natural variations in levels of relevant crop constituents. In order to further improve the methodology for identification of unintended effects, new 'profiling' methods are recommended. Such methods will allow for the screening of potential changes in the modified host organism at different integration levels, i.e. at the genome level, during gene expression and protein translation, and at the level of cellular metabolism.

Animals↗
PubMed2006

Mosaic vectors comprised of modified AAV1 capsid proteins for efficient vector purification and targeting to vascular endothelial cells.

Vascular-targeted gene therapies have the potential to treat many of the leading causes of mortality in the western world. Unfortunately, these therapies have been ineffective due to poor vascular gene transfer. The use of alternative virus serotypes and the incorporation of vascular targeting ligands into vectors has resulted in only modest increases in vascular gene transfer. Adeno-associated virus (AAV) 1 has shown the most promise among the AAV vectors for the transduction of vascular endothelial cells. However, no straightforward small-scale purification strategy exists for AAV1 as it does for AAV2 making it difficult to quickly produce AAV1 vector for analysis. Here we have combined two AAV1 capsid protein modifications to enhance vascular gene transfer and allow easy purification of vector particles. Mosaic vector particles have been produced comprised of capsid proteins containing the well-characterized RGD4C modification to target integrins present on the vasculature, and capsid proteins containing a modification that permits metabolic biotinylation and efficient purification of mosaic particles by avidin affinity chromatography. We show that the RGD modification results in a 50-100-fold enhancement in endothelial cell gene transfer that is maintained in biotinylated mosaic AAV1 particles. These results suggest that mosaic virions hold significant promise for targeted gene delivery to the vasculature.

Cell Line↗
PubMed2005

Association between genetic polymorphisms of CYP1A2, arylamine N-acetyltransferase 1 and 2 and susceptibility to cholangiocarcinoma.

Human CYP1A2 and arylamine N-acetyltransferases, which are encoded by the polymorphic CYP1A2 and NAT genes respectively, have been shown to have wide interindividual variations in metabolic capacity and may be potential modifiers of an individual's susceptibility to certain types of cancers. The present study aimed to evaluate the relationship between CYP1A2, NAT1 and NAT2 polymorphisms and cholangiocarcinoma (CCA), the most prevalent cancer in the north-east of Thailand. A total of 216 CCA patients and 233 control subjects were genotyped by polymerase chain reaction with restriction fragment length polymorphism based assays. Two CYP1A2 alleles (CYP1A2*1A wild-type and *1F), six NAT1 alleles (NAT1*4 wild-type, *3, *10, *11, *14A and *14B) and seven NAT2 alleles (NAT2*4 wild-type, *5, *6A, *6B, *7A, *7B and *13), which are the major alleles found in most populations, were analysed. Although CYP1A2*1A allele, NAT1*10 allele, and the NAT2 slow acetylator alleles were not associated with CCA risk, among the male subjects, the genotype CYP1A2*1A/*1A conferred a decreased risk of the cancer (adjusted odds ratio (OR) 0.28, 95% confidence interval (CI) 0.08-0.94) compared with CYP1A2*1F/1*F. Frequency distributions of rapid NAT2*13 and two slow alleles (*6B and *7A), but not the other major alleles, were associated with lower CCA risk. Adjusted OR of the genotypes consisting of at least one of these alleles significantly decreased the cancer risk compared with none of them (OR 0.26, 95% CI 0.15-0.44). This study suggests that the NAT2 polymorphism may be a modifier of individual risk to CCA.

Adult↗
PubMed2006

Design and preclinical evaluation of a multigene human immunodeficiency virus type 1 subtype C DNA vaccine for clinical trial.

In this study, the design and preclinical development of a multigene human immunodeficiency virus type 1 (HIV-1) subtype C DNA vaccine are described, developed as part of the South African AIDS Vaccine Initiative (SAAVI). Genetic variation remains a major obstacle in the development of an HIV-1 vaccine and recent strategies have focused on constructing vaccines based on the subtypes dominant in the developing world, where the epidemic is most severe. The vaccine, SAAVI DNA-C, contains an equimolar mixture of two plasmids, pTHr.grttnC and pTHr.gp150CT, which express a polyprotein derived from Gag, reverse transcriptase (RT), Tat and Nef, and a truncated Env, respectively. Genes included in the vaccine were obtained from individuals within 3 months of infection and selection was based on closeness to a South African subtype C consensus sequence. All genes were codon-optimized for increased expression in humans. The genes have been modified for safety, stability and immunogenicity. Tat was inactivated through shuffling of gene fragments, whilst maintaining all potential epitopes; the active site of RT was mutated; 124 aa were removed from the cytoplasmic tail of gp160; and Nef and Gag myristylation sites were inactivated. Following vaccination of BALB/c mice, high levels of cytotoxic T lymphocytes were induced against multiple epitopes and the vaccine stimulated strong CD8+ gamma interferon responses. In addition, high titres of antibodies to gp120 were induced in guinea pigs. This vaccine is the first component of a prime-boost regimen that is scheduled for clinical trials in humans in the USA and South Africa.

AIDS Vaccines↗
PubMed2001

Transformation of an oral bacterium via chromosomal integration of free DNA in the presence of human saliva.

Transformation of Streptococcus gordonii DL1 by free DNA was studied in human saliva. Competent S. gordonii could be transformed in vitro with plasmid DNA that had been taken into the human mouth. Transformation also occurred with a plasmid that cannot replicate in S. gordonii, but that has a region of chromosomal homology, by integration into the bacterial chromosome, although linearised plasmid DNA gave no transformants. Linear chromosomal DNA fragments did however transform S. gordonii/Tn916 efficiently in saliva when regions of homology with the recipient chromosome flanked the marker gene. These findings are discussed in relation to the potential for acquisition of DNA sequences, including genetically modified DNA, by gut and oral bacteria.

Bacterial Proteins↗
PubMed2006

A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region.

The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States.

Animals↗
PubMed2003

Renin similar to the submaxillary gland form is expressed in mouse mesangial cells: subcellular localization and all generation under control and glucose-stimulated conditions.

It was analyzed the forms of renin produced by a mouse immortalized mesangial cell line (MIC) and their ability to generate angiotensin II (AII). The synthesis, localization and secretion of renin and AII by MIC were evaluated under conditions of normal (10 mM) or high (30 mM) glucose concentration. Two major bands of 35 kDa and 70 kDa were observed in SDS-PAGE. The amino-terminal sequencing revealed the presence of prorenin and renin in these bands with higher homology with the submaxillary gland form of renin. Renin and AII were detected in cell lysate and in culture medium, indicating that MIC synthesize and secrete these peptides. Renin was localized in the cytoplasm while AII was seen predominantly inside the nucleus. High glucose induced an increase in the synthesis and secretion of renin and AII. Results suggest that MIC produce AII and a renin form similar to the submandibular. Intracellular AII may be directed at the nucleus and/or be secreted, indicating that AII may directly influences gene expression in these cells. The mechanisms of synthesis and secretion of renin and AII are potentially modified by high glucose concentration, suggesting a possible role of AII produced by mesangial cells in diabetic nephropathy.

Amino Acid Sequence↗