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PubMed2005

[Methods of experimental validation of potential target proteins for creation of new drugs].

Review is devoted to the description of the main existing and developing technologies for experimental validation of potential targets predicted in silico by comparative genomics and bioinformatics methods. Since this problem has not been solved yet, the description of a wide set of methods, suitable for the validation of potential targets, is given. The following questions have been considered: (1) applications of proteomic technologies (control of potential targets expression and their variability, analysis of protein-protein interactions); (2) use of genomics technologies in experimental validation of targets (inactivation of the target genes, suppression of transcription, inactivation of the target mRNA, suppression of translation); (3) methods of direct inactivation of target proteins (monoclonal antibodies, light-inactivation, one-chain antibodies, intrabodies, aptamers); (4) high-throughput technologies; (5) targets validations in vivo.

Animals↗
PubMed1995

Increased number of CD4-CD8+ MHC class II-specific T cells in MHC class II-deficient mice.

Targeted disruption of the A beta-encoding gene of H2b mice abolishes major histocompatibility complex (MHC) class II expression and results in a failure to develop CD4+8- T cells. Besides this major effect, the lack of class II expression affects the level of T-cell receptor (TCR) and CD4 expression on differentiating thymocytes. Moreover, there is no class II-mediated negative selection of thymocytes. All this could result in TCR repertoire changes of the CD4-8+ T-cell subpopulation, which apparently develops normally in these mice. To test this hypothesis, the class II reactivity of CD4-8+ T cells from class II-deficient (class II0) mice was analysed. It was found that CD4-8+ T cells from class II0 but not from class II-expressing mice developed a significant level of cytotoxicity against class II-expressing target cells. These results demonstrate an influence of MHC class II molecules on the TCR repertoire of CD4-8+ T cells.

Animals↗
PubMed2006

[Construction of RNA interference expression vectors of human neuropathy target esterase and its inhibition for expression of NTE in mammalian cells].

OBJECTIVE: To construct the RNA interference expression vector for expression of human neuropathy target esterase (NTE) gene in mammalian cells. METHODS: Spe I and Xho I-digested insert from pSUPER, which comprised H1 RNA polymerase III promoter and the multiple cloning sites, were cloned into the compatible in the pcDNA3.1 (+) to generate pSUPER/neo that could express small interfering RNA in mammalian cells. The annealed oligos targeting the expression of NTE were ligated into pSUPER/neo vector digested with Bgl II and Hind III to generate pSUPER/neo-NTE, which was transfected into COS7 and SH-SY5Y cells. The inhibitory effect of the expression of NTE was detected by western blot analysis and the enzyme activity assay. RESULTS: pSUPER/neo-NTE could stably express double-stranded RNA of NTE. The expression of pSUPER/neo-NTE in COS7 and SH-SY5Y cells could efficiently inhibit the activity of NTE in the mammalian cells. CONCLUSION: Stable eukaryotic expression vector of double-stranded RNA of NTE, pSUPER/neo-NTE, has been constructed successfully with promoter substitution strategy.

Animals↗
PubMed1996

High-level expression of recombinant human fibrinogen in the milk of transgenic mice.

Fibrinogen is a complex plasma protein composed of two each of three different polypeptide chains. We have targeted expression of r-human fibrinogen to the mammary gland of transgenic mice. Three expression cassettes, each containing the genomic sequence for one of the three human fibrinogen chains controlled by sheep whey protein beta-lactoglobulin promoter sequences, were coinjected into fertile mouse eggs. Southern blot analysis demonstrated that more than 80% of the transgenic founders contained all three fibrinogen genes. Reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis of milk from the highest producing founder animal demonstrated the presence of human fibrinogen subunits at concentrations of 2000 micrograms/ml. In several animals with a balanced ratio of the individual fibrinogen subunits, up to 100% of the protein was incorporated into fully assembled fibrinogen hexamers. Incubation of the transgenic milk with thrombin and factor XIII resulted in a cross-linked fibrin clot, indicating that a major portion of the secreted fibrinogen was functional. These studies represent the first report of high-level biosynthesis and secretion of a functional, complex, hexameric protein in the milk of a transgenic animal.

Animals↗
PubMed2000

Sequential CD4-coreceptor interactions in human immunodeficiency virus type 1 Env function: soluble CD4 activates Env for coreceptor-dependent fusion and reveals blocking activities of antibodies against cryptic conserved epitopes on gp120.

We devised an experimental system to examine sequential events by which the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) interacts with CD4 and coreceptor to induce membrane fusion. Recombinant soluble CD4 (sCD4) activated fusion between effector cells expressing Env and target cells expressing coreceptor (CCR5 or CXCR4) but lacking CD4. sCD4-activated fusion was dose dependent, occurred comparably with two- and four-domain proteins, and demonstrated Env-coreceptor specificities parallel to those reported in conventional fusion and infectivity systems. Fusion activation occurred upon sCD4 preincubation and washing of the Env-expressing effector cells but not the coreceptor-bearing target cells, thereby demonstrating that sCD4 exerts its effects by acting on Env. These findings provide direct functional evidence for a sequential two-step model of Env-receptor interactions, whereby gp120 binds first to CD4 and becomes activated for subsequent functional interaction with coreceptor, leading to membrane fusion. We used the sCD4-activated system to explore neutralization by the anti-gp120 human monoclonal antibodies 17b and 48d. These antibodies reportedly bind conserved CD4-induced epitopes involved in coreceptor interactions but neutralize HIV-1 infection only weakly. We found that 17b and 48d had minimal effects in the standard cell fusion system using target cells expressing both CD4 and coreceptor but potently blocked sCD4-activated fusion with target cells expressing coreceptor alone. Both antibodies strongly inhibited sCD4-activated fusion by Envs from genetically diverse HIV-1 isolates. Thus, the sCD4-activated system reveals conserved Env-blocking epitopes that are masked in native Env and hence not readily detected by conventional systems.

3T3 Cells↗
PubMed1989

Class I gene regulation of haplotype preference may influence antiviral immunity in vivo.

The lymphocytic choriomeningitis virus (LCMV)-specific Tc response in (C3 X D2) F1 hybrids (k X d) is markedly biased in favor of the H-2d haplotype. Adoptive transfer experiments established that this haplotype preference also applied to T cell function in vivo. Using different mouse strain combinations we were unable to detect an influence of sex, non-H-2 background, maternal genotype, or route of priming on the preference pattern. In other haplotype combinations tested (k and b, b and d) no distinct haplotype preference was observed. A comparison of the LCMV-specific Tc response of (C X C3) F1 and (C-H-2dm2 X C3) F1 hybrids revealed that the dominance of the H-2d haplotype was controlled by H-2Ld. The ability of this gene to down-regulate the generation of an H-2k-restricted response did not seem to reflect antigenic mimicry since H-2k-restricted LCMV-specific Tc did not lyse H-2d expressing targets. In regard to the in vivo significance of haplotype preference it was found that (C X C3) F1 mice expressed an earlier and stronger virus-specific delayed type hypersensitivity response and exerted a more efficient virus control than did (C-H-2dm2 X C3) F1. Taken together these findings suggest that haplotype preference reflects a selection process favoring the restriction element associated with the most efficient immune response in vivo. The implications of this are discussed.

Animals↗
PubMed2000

Genomic approaches to plant stress tolerance.

Past efforts to improve plant tolerance to drought, high salinity and low-temperature through breeding and genetic engineering have had limited success owing to the genetic complexity of stress responses. Progress is now anticipated through comparative genomics studies of an evolutionarily diverse set of model organisms, and through the use of techniques such as high-throughput analysis of expressed sequence tags, large-scale parallel analysis of gene expression, targeted or random mutagenesis, and gain-of-function or mutant complementation. The discovery of novel genes, determination of their expression patterns in response to abiotic stress, and an improved understanding of their roles in stress adaptation (obtained by the use of functional genomics) will provide the basis of effective engineering strategies leading to greater stress tolerance.

Disasters↗
PubMed1998

Tumors of the retinal pigment epithelium metastasize to inguinal lymph nodes and spleen in tyrosinase-related protein 1/SV40 T antigen transgenic mice.

The pigment epithelium of the retina (RPE) is derived from the optic cup and is essential for function and development of the eye. We produced a transgenic mouse line that expresses simian virus (SV40) transforming sequences under control of the 1.4 kb tyrosinase-related protein 1 (TRP-1) promoter, targeting expression of T antigen (Tag) to the RPE. In transgenic embryos, RPE cells proliferated in the anterior part of the eye and near the optic nerve. This resulted in formation of tumors, which were pigmented and of epithelial origin. In 3 months-old mice, pigmented cells were detected in spleen and inguinal lymph nodes. In spleen, tyrosinase, TRP-1 and SV40 Tag were expressed and tyrosinase was enzymatically active. Pigmented regions were positive for an epithelial marker, cytokeratin. Cell lines were established from tumor and metastases and kept in culture for more than 2 months. These were pigmented, and maintained expression of tyrosinase, TRP-1, cytokeratin and SV40 Tag. This demonstrates that RPE tumor cells metastasize to lymph node and spleen. In conclusion, the metastasis from TRP-1/Tag RPE tumors towards spleen and lymph nodes serves as potential tool to investigate biology and metastasis of tumors derived from the pigment epithelium.

Animals↗
PubMed2004

DREF is required for EGFR signalling during Drosophila wing vein development.

The DNA replication-related element binding factor (DREF) has been suggested as being involved in regulation of DNA replication- and proliferation-related genes in Drosophila. Recently, by searching the Drosophila genome database, we also found DRE-like sequences in the 5'-flanking regions of many genes with other functions. In addition, immunostaining of polytene chromosomes with an anti-DREF monoclonal antibody revealed that DREF can bind to a hundred regions of polytene chromosomes, suggesting regulation of multiple genes and multiple roles in vivo. When we over-expressed DREF protein or inverted repeat RNA of the DREF gene in wing imaginal discs using the GAL4-UAS targeted expression system in Drosophila, the results were veins of increased width and a loss of veins, respectively. With DREF over-expression, Rolled, a Drosophila MAPK homologue, was ectopically activated. Furthermore, half reduction of the D-raf gene dose suppressed this DREF-induced vein of increased width phenotype. In addition, when DREF transcripts were reduced by introducing double-stranded RNA of the DREF gene into S2 cells, the D-raf gene promoter activity was diminished to 4%. These data indicate that DREF is involved in regulation of vein formation through the activation of EGFR signalling in the Drosophila wing imaginal discs.

Animals↗
PubMed2003

Deoxyribonucleic acid (DNA) encoding a pan-major histocompatibility complex class II peptide analogue augmented antigen-specific cellular immunity and suppressive effects on tumor growth elicited by DNA vaccine immunotherapy.

Vaccine immunotherapy must induce helper and cytotoxic cell-mediated immunity to generate the powerful antitumor immune responses needed to suppress cancer progression. We reported previously that a 16-amino acid peptide analogue derived from pigeon cytochrome c can bind broad ranges of MHC class II types and activate helper T cells in mice. To determine whether DNA encoding the Pan-MHC class II IA peptide (Pan-IA) can increase the efficacy of tumor suppression by DNA vaccine immunotherapy targeting tumor antigens, Pan-IA DNA was administered with ovalbumin (OVA) DNA to C57BL/6 mice bearing the OVA-expressing tumor cell line E.G7. Specific proliferative responses to and cytotoxic activities against OVA-expressing targets were induced in mice vaccinated with both OVA and Pan-IA DNA but not in those vaccinated with OVA DNA alone or control DNA plus Pan-IA DNA. Growth of E.G7 cells was suppressed only by combined vaccination with OVA and Pan-IA DNA, and tumors in five of the nine mice that received this combined vaccination were eradicated completely. In those mice, the frequency of CD8-positive T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased in the tumor site. Furthermore, immunofluorescent study of the inoculated tumors revealed increased accumulation of both CD4- and CD8-positive T cells producing IFN-gamma in the tumor only by this vaccine protocol. The data suggest that Pan-IA DNA can augment suppressive effects of DNA vaccines on tumor growth by increasing numbers of antigen-specific CTLs and helper T cells. This is the first study in which established tumors have been eradicated successfully by vaccination with DNA corresponding to CTL epitopes and helper T cell epitopes. Our animal model may contribute to the development of therapeutic DNA vaccines against cancer.

Amino Acid Sequence↗
PubMed1991

Induction of CD4+, human T lymphotropic virus type-1-specific cytotoxic T lymphocytes from patients with HAM/TSP. Recognition of an immunogenic region of the gp46 envelope glycoprotein of human T lymphotropic virus type-1.

Although the humoral response to human T lymphotropic virus type-1 (HTLV-I) has been well characterized in patients with HTLV-I-associated neurologic disease (HAM/TSP), little is known about a functional HTLV-I-specific human T cell response, such as CTL, in these patients. To define both the phenotype of the responding CTL and the fine specificity of this response, long term T cell lines were generated from two HAM/TSP patients who were from two different countries. Patient's peripheral blood lymphocytes were repeatedly stimulated in vitro with an HTLV-I expressing autologous T cell line. The resultant long term T cell culture was shown to be CD4+ and cytotoxic for targets expressing HTLV-I Ag. Using a panel of synthetic peptides that span hydrophilic regions of the HTLV-I gp46 envelope glycoprotein, the CTL lines generated from both patients were shown to recognize the same region of the HTLV-I envelope between amino acids 196-209 as defined by the synthetic peptide sp4a1. Interestingly, this sequence overlaps a region of HTLV-I envelope that had also been shown to elicit a strong B cell response in HAM/TSP patients (amino acids 190-203). One CTL line recognized this HTLV-I epitope in the context of HLA DQ5 whereas the other CTL line was restricted by HLA DRw16. The generation of two independent CTL lines from two HAM/TSP patients from different geographic areas that recognize the same region of the HTLV-I envelope glycoprotein highlights the immunogenic nature of this envelope region.

Amino Acid Sequence↗
PubMed1998

Conditional lineage ablation to model human diseases.

Cell loss contributes to the pathogenesis of many inherited and acquired human diseases. We have developed a system to conditionally ablate cells of any lineage and developmental stage in the mouse by regulated expression of the diphtheria toxin A (DTA) gene by using tetracycline-responsive promoters. As an example of this approach, we targeted expression of DTA to the hearts of adult mice to model structural abnormalities commonly observed in human cardiomyopathies. Induction of DTA expression resulted in cell loss, fibrosis, and chamber dilatation. As in many human cardiomyopathies, transgenic mice developed spontaneous arrhythmias in vivo, and programmed electrical stimulation of isolated-perfused transgenic hearts demonstrated a strikingly high incidence of spontaneous and inducible ventricular tachycardia. Affected mice showed marked perturbations of cardiac gap junction channel expression and localization, including a subset with disorganized epicardial activation patterns as revealed by optical action potential mapping. These studies provide important insights into mechanisms of arrhythmogenesis and suggest that conditional lineage ablation may have wide applicability for studies of disease pathogenesis.

Action Potentials↗
PubMed2000

Abnormal turning behavior in Drosophila larvae. Identification and molecular analysis of scribbler (sbb).

Our genetic dissection of behavior has isolated scribbler (sbb), a vital gene that encodes a novel protein expressed in the embryonic and larval nervous systems and in the imaginal discs. Larvae with mutations in sbb exhibit abnormally high amounts of turning behavior in the absence of food. sbb is a large gene spanning >50 kb of genomic DNA with four major developmentally regulated transcripts. Transgenic rescue of scribbler behavior was demonstrated by targeting expression of a normal sbb transgene (sbb(+)) expressing one of the major transcripts to the nervous system. The vital function of sbb was restored by ubiquitous expression of this transgene throughout development.

Amino Acid Sequence↗
PubMed2006

p21 and mTERT are novel markers for determining different ischemic time periods in renal ischemia-reperfusion injury.

In many clinical settings, the duration of renal ischemia and therefore the outcome of acute renal failure cannot be determined adequately. Renal ischemia reperfusion injury is known to shorten telomeres and upregulate stress-induced genes, such as the cyclin-dependent kinase (CDK) inhibitor p21. So far, the expression and role of CDK inhibitors, as well as mouse telomerase reverse transcriptase (mTERT), has not been investigated in a model with variable lasting ischemic periods. Male C57Bl/6 mice were subjected to renal ischemia reperfusion injury by clamping both renal pedicles for 10, 20, 30, and 45 min, and the kidneys were allowed to be reperfused for 3, 24, and 48 h. Expression of different CDK inhibitors and mTERT was evaluated. Mice developed signs of acute renal failure linear to the duration of the ischemic period. Real-time PCR revealed that mTERT was only significantly upregulated in kidneys after short ischemic periods (20 min). In contrast, p21 was constantly upregulated in kidneys after long ischemic intervals (30 and 45 min), but not in kidneys, which were clamped for shorter periods. Mainly, tubular cells contributed to the observed increase in p21 expression. Targeting p21 via the selective p53 inhibitor pifithrin-alpha was able to prevent acute renal failure when administered immediately before ischemia. The expression of another CDK inhibitor, namely p16, was differentially regulated, depending on the time of reperfusion. Taken together, we detected mTERT and p21 as "indicator" genes for short and long ischemic intervals, respectively. These two proteins might also be possible new therapeutic targets in the treatment and prevention of acute renal failure.

Animals↗
PubMed1996

Paradoxical tumor inhibitory effect of p53 loss in transgenic mice expressing epidermal-targeted v-rasHa, v-fos, or human transforming growth factor alpha.

To investigate the effect of p53 tumor suppressor gene loss in the mouse skin model of multistage carcinogenesis, p53 knockout mice, generated by gene targeting (p53 -/-), were mated to transgenic mice expressing v-rasHa (HK1.ras), v-fos (HK1.fos), or human transforming growth factor alpha+HK1.TGFalpha) exclusively in the epidermis, by means of a keratin K1-based targeting vector (HK1). HK1-p53 transgenic progeny expressing wild-type p53 alleles (p53 +/+) or hemizygous for the p53 knockout allele (p53+/-) were identical to parental HK1 lines and exhibited neonatal epidermal hyperplasia or wound-associated hyperplasia in adults, together with spontaneous or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced benign papillomas. Mating to p53-/- did not lead to the expected tumorigenesis in adults. Instead, whereas HK1.ras or HK1.TGFalpha transgenic mice null for p53 (HK1.ras-p53-/- and HK1.TGFalpha-p53-/-, respectively) retained the neonatal epidermal hyperplasia phenotype, in adults, spontaneous and TPA-promoted papilloma formation was blocked. Similarly, wound-associated epidermal hyperplasia/hyperkeratosis, a hallmark of adult HK1.fos phenotypes, was completely absent in HK1.fos-p53 -/- mice. Histological, immunofluorescence, and bromodeoxyuridine labeling analysis of neonatal or adult epidermis in HK1-p53 transgenic genotypes +/+, +/-, and -/- for p53 revealed no obvious differences in morphology, expression of keratinocyte differentiation markers, or mitotic index attributed to p53 loss. To determine whether the paradoxical absence of papillomas centered on up-regulation of p53 target genes, WAF1/CIP1/p21 RNA expression levels were examined in TPA promotion experiments. WAF1/CIP1/p21 expression increased in response to TPA promotion in all HK1-p53 transgenic genotypes regardless of p53 status. However, in HK1-p53 null genotypes, although TPA-induced, p53-independent WAF1/CIP1/p21 expression was observed, no large increase in expression was associated with the observed paradoxical tumorigenesis block. These data suggest that epidermis is somewhat resistant to the neoplastic effects of p53 loss, possibly possessing several compensatory systems. Alternatively, there may be a requirement forp53 expression in response to TPA or a wound-promotion stimulus in mouse epidermis.

Animals↗
PubMed2004

Generation of kidney cancer-specific antitumor immune responses using peripheral blood monocytes transduced with a recombinant adenovirus encoding carbonic anhydrase 9.

PURPOSE: Carbonic anhydrase 9 (CA9) is the most promising molecular marker described for renal cell carcinoma (RCC) to date. We investigated whether transduction of monocytes from peripheral blood with adenovirus encoding the CA9 gene (AdV-CA9) could stimulate a T-cell mediated immune response against cancer cells expressing CA9. The ability to consistently generate a T-cell response is an important step toward the development of a CA9-specific RCC vaccine. EXPERIMENTAL DESIGN: AdV-CA9 was generated using the AdEasy system. AdV-CA9-transduced peripheral blood mononuclear cell (PBMC)-derived monocytes were used to raise CTLs from autologous peripheral blood lymphocytes (PBLs). The ability of CTLs to lyse targets expressing CA9 was assessed by (51)Cr-release. RESULTS: Monocytes were efficiently transduced with AdV-CA9. In five of six experiments, AdV-CA9-transduced monocytes were able to induce a population of CTLs from bulk PBLs. CTLs were capable of lysing autologous, but not allogeneic monocytes expressing CA9. Furthermore, CTLs were able to lyse autologous RCC tumor cells expressing CA9. The ability of CTLs to lyse relevant targets was blocked by anti-CD3, anti-CD8, and anti-MHC class I antibodies demonstrating a MHC class I restricted response. CONCLUSIONS: These results suggest that PBMC-derived monocytes transduced with AdV-CA9 can generate RCC-specific MHC class I restricted CTLs capable of lysing CA9-expressing cancer cells. Transduction of PBMC-derived monocytes with adenovirus provides a simple and effective alternative to the use of dendritic cells for the induction of antigen-specific CTL.

Adenoviridae↗
PubMed2002

Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes.

Incorporation of an internal ribosome entry site (IRES) into the gene therapy vector represents a promising strategy to efficiently co-express several gene products from the same promoter. However, vector systems that utilize the encephalomyocarditis virus IRES express the downstream gene much less efficiently than the upstream gene. In this study, we compared four IRESes isolated from human genes against the EMCV IRES, using beta-galactosidase and chloramphenicol acetyl transferase genes as reporters, to evaluate their potential for providing better expression of the downstream gene. We found that an IRES from the eukaryotic initiation factor 4G gene mediates greater than 100-fold higher expression of the downstream gene compared with the EMCV IRES in four different cell lines tested. Other mammalian IRESes displayed more variable results and smaller enhancement of downstream gene expression in three different cell lines tested. Furthermore, while the efficiency of the IRES from the vascular endothelium growth factor gene was not significantly higher than the EMCV IRES under normoxic conditions, expression was significantly increased under hypoglycemic conditions, suggesting that the VEGF IRES could be exploited in cancer gene therapy to preferentially target expression of therapeutic genes at the relatively hypoglycemic cores of tumors.

Encephalomyocarditis virus↗
PubMed2003

Virus load correlates inversely with the expression of cytotoxic T lymphocyte activation markers in HIV-1-infected/AIDS patients showing MHC-unrestricted CTL-mediated lysis.

Cytotoxic T lymphocytes (CTL) are key players to suppress viral load (VL) but CTL responses become compromised with progression of HIV-infection/AIDS. Some progressors develop MHC-unrestricted CTL with anti-CD4+ cytocidal activity. Immune activation status of these CTL and its significance in disease progression are unknown. To determine the relationship between VL and T cell activation, a cross-sectional study was carried out using blood samples from 13 HIV-1-infected/AIDS patients at various stages of progression and seven age-matched seronegative controls. We examined expression of HLA-DR and CD38 activation markers on purified CTL. MHC-unrestricted killing by these CTL was also evaluated against uninfected, allogeneic CD4+ T cells as well as several human cell lines. The expression of activation markers correlated inversely (rs = - 0.91, P < 0.0001) with VL of the subjects. CTL effectors of these patients killed targets expressing or lacking CD4+, independently of MHC class I recognition. Interestingly, the patients with higher VL showed an increased number of gammadeltaTCR-bearing CTL in blood and their MHC-unrestricted killing activity was blocked significantly (P < 0.01) by gammadeltaTCR-specific monoclonal antibody. CD3+ T counts of these patients were also consistently subnormal. Inverse correlation between VL and CD8+ T cell activation markers seems to be an indicator of CTL-associated immunopathogenesis in HIV patients with elevated gammadeltaCTL in the peripheral blood.

Acquired Immunodeficiency Syndrome↗