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PubMed1999

Retroviral-mediated transfer and expression of the common gamma chain into human hematopoietic progenitors.

The common gamma chain (gammac) of cytokine receptors is mutated in X-linked severe combined immunodeficiency, a lethal disorder characterized by the absence of both humoral and cellular immune defenses. Allogeneic bone marrow transplantation from HLA-identical siblings usually results in complete reconstitution of the immune system and is the current treatment of choice. Genetic correction and reinfusion of autologous hematopoietic stem cells represents an alternative therapeutic approach for those patients who lack suitable marrow donors. In this study, we show that retroviral-mediated transfer of the gammac gene results in efficient expression in CD34+ cells and high transduction rate of colony-forming progenitors.

Antigens, CD34↗
PubMed1996

Development of antigen-delivery systems, based on the Escherichia coli hemolysin secretion pathway.

We describe the development of plasmid vectors carrying the expression sites, an hlyA cassette and the secretion genes of Escherichia coli hemolysin. These allow the synthesis and secretion of heterologous microbial antigens in E. coli and attenuated Salmonella aroA strains. Genes or gene fragments encoding microbial antigens are inserted in-frame into a residual part of the hlyA gene which essentially encodes the HlyA secretion signal (HlyAs). In general, the fused genes, carrying the hlyAs sequence at the 3' terminus, are efficiently expressed, and the synthesized antigens are secreted into the culture supernatant of the producing strain. Attenuated Salmonella strains synthesizing either HlyAs-fused listeriolysin or p60 of Listeria monocytogenes were constructed by this procedure and shown to provide protective immunity against L. monocytogenes in mice. The most effective protection was obtained when these microbial antigens were secreted by the attenuated Salmonella strains. We further present new approaches which may allow the application of this antigen-delivery system to any microbial antigen.

Animals↗
PubMed2000

Induction of dwarfism in transgenic Solanum dulcamara by over-expression of a gibberellin 20-oxidase cDNA from pumpkin.

The gibberellin (GA) 20-oxidase (CmGA20ox1) from immature pumpkin seed produces predominantly inactive tricarboxylic acid GAs. We expressed CmGA20ox1 under the control of the CaMV 35S promoter in Solanum dulcamara to assess the usefulness of this gene for reducing GA content in transgenic plants. All transgenic plants obtained were semi-dwarfs with smaller, deep-green leaves and highly pigmented stems compared to the wild-type. Such transformants flowered earlier than the wild-type plants and produced more fruit and more seeds per fruit. The transgene was efficiently expressed, producing high levels of CmGA20ox1 transcript and protein. Furthermore, the concentration of GA(1) was reduced in leaves of the transformants to approximately 20% or less of that in the wild-type and to about 40% or less in stems. The concentrations of other 13-hydroxylated GAs were also reduced, except for the tricarboxylic acid, GA(17), which accumulated in the transformants due to 13-hydroxylation of GA(25). By contrast, the concentrations of non-13-hydroxylated GAs, GA(4) and GA(34), were not consistently reduced, indicating that the effect of expressing the pumpkin gene may not be predictable. Transcript abundance for a native GA 20-oxidase gene was higher in the leaves and stems of S. dulcamara transformed with the pumpkin gene than in wild-type, reflecting the feedback control of 20-oxidase gene expression that serves as a homeostatic mechanism for GAs.

Base Sequence↗
PubMed1997

Expression of bovine beta-lactoglobulin/human erythropoietin fusion protein in the milk of transgenic mice and rabbits.

We have generated several transgenic mouse lines and rabbits expressing efficiently (up to 0.3 mg/ml in mice and up to 0.5 mg/ml in rabbits) human erythropoietin in their milk as bovine beta-lactoglobulin fusion protein. Human erythropoietin cDNA was inserted in frame into exon 5 of the bovine beta-lactoglobulin gene with a linker oligonucleotide encoding the cleavage site for bacterial IgA protease. RNA analysis performed on one lactating transgenic mouse and one transgenic rabbit revealed that the fusion gene was expressed almost exlusively in the mammary gland, although low amounts of transgene-derived RNA were detectable in salivary glands and uterus or in the kidney. The fusion protein was specifically cleaved with IgA protease. The erythropoietin part obtained upon digestion had a lower molecular mass than recombinant erythropoietin produced in Chinese hamster ovary cells. By deglycosylation analysis it was shown that the difference in size was due to a different type of glycosylation. Biological activity of the fusion protein, as determined by growth stimulation of TF-1 erythroleukemia cells, was less than 15% of that of human recombinant erythropoietin. Upon digestion of the fusion protein with IgA protease, biological activity comparable to that of the recombinant erythropoietin was recovered. Transgenic males and virgin females did not show signs of enhanced erythropoiesis, but lactating females expressing the transgene displayed transient increases in their hematocrit values.

Amino Acid Sequence↗
PubMed1993

Dynamic effects and pharmacokinetics of lemakalim in the isolated guinea-pig heart.

Myocardial effects of lemakalim were studied in the isolated retrogradely perfused and spontaneously beating guinea-pig heart. Pharmacokinetic analysis of drug accumulation and disposition showed two-compartment characteristics with phasic half-times of about 0.27 and 2.1 min., respectively. Myocardial clearance was 6.8 ml min.-1, and myocardial drug accumulation was 5-fold. Dynamic parameters were studied during increasing lemakalim concentrations from 1.4 nM up to 10 microM. Dynamic steady-states developed within 3-4 min. Increase of coronary flowrate showed an Emax of 185% with an EC50 of 60 nM. Lemakalim produced biphasic inhibitory actions on contraction velocity and amplitude. Emax and EC50 for the two phases were: 48% and 47% and 100 nM and 65 nM, respectively, for the first phase and 100% in both cases and 1.7 microM and 1.6 microM for the second phase. Heart frequency increased to a maximum of 15% above baseline at 0.3 microM. Oxygen consumption increased progressively at concentrations above 7 nM and reached a maximum of 175% at 0.3 microM. The frequency-corrected QT-interval was biphasically shortened to a maximal extent of 30% at the highest concentration. Myocardial efficiency expressed as the ratio of amplitude times frequency to oxygen consumption exhibited a progressive decline to about 20% of control values. PQ- and QRS-intervals showed only minor or no changes, respectively. No arrythmogenic effects were observed. The study demonstrated a rapid and very moderate accumulation of lemakalim in the guinea-pig heart accompanied by a marked increase in coronary flowrate and a progressive negative inotropic effect followed by reduced myocardial efficiency.

Animals↗
PubMed1990

Herpesvirus saimiri U RNAs are expressed and assembled into ribonucleoprotein particles in the absence of other viral genes.

Marmoset T lymphocytes transformed by herpesvirus saimiri contain a set of five virally encoded U RNAs called HSUR1 through HSUR5. HSUR genes have been individually transfected into a nonlymphoid, nonsimian cell line (HeLa cells) in the absence of any other coding regions of the herpesvirus saimiri genome. The levels of HSUR1 through HSUR4 in HeLa transient-expression systems are comparable to those found in virally transformed T cells (23 to 91%). In contrast, HSUR5 is expressed at ninefold-higher levels in transfected HeLa cells. Immunoprecipitation experiments show that HSURs expressed in transfected cells bind proteins with Sm determinants and acquire a 5' trimethylguanosine cap structure, as they do in transformed T cells. HSUR1 or HSUR4 particles from transfected HeLa cells migrate between 10S and 15S in velocity gradients, identical to the sedimentation of "monoparticles" produced in virally transformed lymphocytes. We conclude from these transfection experiments that no other herpesvirus saimiri or host-cell-specific gene products appear to be required for efficient expression of the HSUR genes or for subsequent assembly of the viral U RNAs into small nuclear ribonucleoprotein particles. In lymphocytes transformed by herpesvirus saimiri, HSUR small nuclear ribonucleoprotein particles are involved in higher-order complexes that sediment between 20S and 25S. HSUR1, HSUR2, and HSUR5 dissociate from such complexes upon incubation at 30 degrees C, whereas the complex containing HSUR4 is stable to incubation.

Animals↗
PubMed1993

Human immunodeficiency virus type 1 tat gene enhances human papillomavirus early gene expression.

To investigate the possible direct/indirect role of Human immunodeficiency virus (HIV) as a cofactor in human papillomavirus (HPV) oncogenesis, cotransfection experiments were carried out in which a recombinant plasmid containing the HPV16 long control region (LCR) linked to the chloramphenicol acetyltransferase (CAT) gene was cotransfected into cultured cells with a plasmid expressing HIV-1 Tat protein. Tat expression efficiency and transactivation activity were evaluated in different cell lines by cotransfecting plasmids containing the HIV tat gene and HIV LTR-driven CAT-coding sequences. HeLa and CaSki cell lines represented the most appropriate recipient cells for Tat-directed transactivation of both the HIV LTR and the HPV LCR promoters. Furthermore, HIV tat was transfected into HeLa cells (containing 10-20 copies per cell of HPV18), and HPV18 E7 protein expression was evaluated by a radioimmunoprecipitation assay using polyclonal antibodies against the E7 protein. Our results show that the Tat protein can transactivate the HPV LCR and increase HPV18 E7 expression in HeLa cells.

Gene Expression Regulation, Viral↗
PubMed2005

[Studies on fermentation conditions and purification of mutant human interleukin-2 expressed in Pichia pastoris].

Interleukin-2 (IL-2) was initially isolated as a T cell growth factor and had been shown to direct the expansion and differentiation of several hematopoietic cell types. Clinical studies using IL-2 in the treatment of AIDS have been encouraging, due to its critical role as a proliferative signal for activated T-lymphocytes. IL-2 has also undergone trials in the treatment of several types of cancer, based on its stimulation of cytotoxic, antitumor cells. Today, human IL-2 is produced completely by genetically engineered method, and it has been proved that genetically engineered recombinant human IL-2 has almost the same function and clinical effect as wild IL-2. In the former study, recombinant human IL-2 usually comes from E. coli, in this paper the mutant IL-2 was successfully expressed and purified in Pichia pastoris for the first time. As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression level. Expression conditions of human mutant interleukin-2(the codon for cysteine-125 of human IL-2 with alanine; the codon for leucine-18 with methionine; the codon for leucine-19 with serine) in the recombinant Pichia pastoris strain were optimized via test of some factors such as the rate of aeration, the inductive duration, the initial pH and the concentration of methanol. The results from tests showed that the most important parameter for efficient expression of interleukin-2 in recombinant Pichia pastoris strain is adequate aeration during methanol induction, and the optimum inductive condition for interleukin-2 expression was: more than 80% aeration, 2 days for induction, the initial pH of 6.0, the final methanol concentration of 1.0%. With this condition, the expressed IL-2 was secreted into fermentation broth and reached a yield of 30%, approximately 200 mg/L. Expressed interleutin-2 (MvIL-2) was isolated and purified by centrifugation, millipore filtration to concentration, Econo-PacS strongly acidic cation exchanger cartridge and molecular sieve chromatography and the yield of MvIL-2 was 27%. MvIL-2 was purified to electrophoretic purity by SDS-PAGE and only one peak being loaded on HPLC. Purified MvIL-2 protein had stimulating activity similar to the wild type of IL-2 as assayed by IL-2-dependent CTLL-2 cells. However, the stability of MvIL-2 was superior than that of IL-2 at different temperatures. The activity of obtained MvIL-2 was 4 - 5 times of the wild type of IL-2, So MvIL-2 had an advantage over wild type of rhIL-2 in storage stability and activity.

Fermentation↗
PubMed1999

Identification of the canarypox virus thymidine kinase gene and insertion of foreign genes.

We mapped the canarypox virus (CaPV) thymidine kinase (TK) gene within a 5.8-kbp XbaI fragment of the genome by Southern blotting using the fowlpox virus (FPV) TK gene as a probe. Nucleotide sequence analysis of the fragment revealed seven open reading frames (ORFs) showing gene organization similar to that of FPV. The TK gene contained in this region had an ORF of 179 amino acids encoding a polypeptide with a putative molecular mass of 20.0 kDa. An A/T-rich region and a transcription termination signal, TTTTTAT, were found upstream and at the end of the ORF, which is consistent with poxvirus early gene regulation. The consensus sequence of the late promoter TAAAT also overlapped with the initiation codon of the ORF. The amino acid sequence similarity between the TK genes of CaPV and FPV, avipoxviruses, was 64.2%, which was lower than the similarities between vaccinia and variola orthopoxviruses (97.2%) and between Shope fibroma and myxoma leporipoxviruses (82.6%). However, the monophyly of avian clades of CaPV and FPV was supported by phylogenetic analysis. We then inserted the genes encoding lacZ, luciferase (luci), and envelope of human T-lymphotropic virus type 1 (HTLV-1 env) into the TK gene of CaPV to evaluate its suitability as an expression vector. The recombinant viruses obtained were unstable, although the foreign genes were expressed efficiently in the mammalian cells infected with the viruses.

Amino Acid Sequence↗
PubMed2003

Development of a gene therapy strategy for the restoration of survival motor neuron protein expression: implications for spinal muscular atrophy therapy.

Spinal muscular atrophy (SMA) is a motor neuron degeneration disorder, and manifests itself in patients as muscle weakness and paralysis that ultimately leads to death. Currently, there is no effective treatment for this disease. As a first step in developing a treatment for SMA, we are examining whether delivery of the gene encoding survival motor neuron (SMN) protein to primary fibroblast cell lines derived from SMA patients can lead to restoration of nuclear-staining foci, called gems, which are absent in patients with severe SMA. Using adenovirus-mediated gene delivery, we show that SMN can be efficiently expressed in patient fibroblasts, and leads to restoration of nuclear gems, which are thought to be important for the functional rescue of the SMA phenotype. The number of gems per cell is equal to or greater than those found in fibroblasts of normal individuals. Furthermore, ectopic expression of SMN also caused relocalization of Gemin2, an SMN-interacting protein, to gems. Overall, this work is the first demonstration of the feasibility of virus-based delivery of the SMN-coding gene to restore the normal SMN expression pattern in SMA patient-derived cells, and holds promise for gene therapy of SMA, as a potential long-term therapy for this devastating childhood disease.

Adenoviridae↗
PubMed2000

Ex vivo gene delivery using an adenovirus vector in treatment for cartilage defects.

OBJECTIVE: To realize local selective gene expression in grafted chondrocytes for cartilage defect, we investigated the usefulness of an ex vivo gene delivery method using an adenovirus vector. METHODS: Beta-galactosidase gene (LacZ) was transfected using an adenovirus vector to chondrocytes isolated from rat joints. The cells were then embedded into collagen gel, and LacZ expression in the gel was examined using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining; beta-galactosidase activity was also measured. The collagen gel containing transfected chondrocytes was grafted to the experimental cartilage defects, and the expression of delivered gene was histologically examined after X-gal staining of the tissue containing the grafted area. RESULTS: X-gal positive chondrocytes in the gel accounted for 82% at one week and 55% at 8 weeks after gene delivery. Beta-galactosidase activity decreased with time, but its expression was maintained even at 8 weeks after gene delivery. Chondrocytes used in the allograft maintained their morphology, and the expression of delivered gene continued during the 8 week period. CONCLUSION: In this ex vivo method, delivered gene can be expressed efficiently for a long time; this method would be useful in allografts for cartilage defects.

Adenoviridae↗
PubMed1996

Rapid detection of recombinant antibody fragments directed against cell-surface antigens by flow cytometry.

Cloning the correct genes coding for antibody variable domains (especially VL kappa) from hybridomas is often complicated by the presence of several immunoglobulin transcripts, some of them arising from the myeloma cell line. Indeed, four different VL genes were obtained after the amplification of immunoglobulin genes by PCR from the hybridoma HD37, which produces an antibody against the human CD19 B cell differentiation antigen. Most of the variants (eight out of 15) were derived from the kappa chain of the myeloma MOPC-21. For the rapid functional evaluation of recombinant antibody fragments against cell surface antigens, we established an efficient expression and detection system. First, deleted and mutated genes were eliminated by a colony screening procedure. Bacteria from picked colonies were then induced and grown in the presence of 0.4 M sucrose to increase the accumulation of soluble scFv in the periplasm (5-10 micrograms per ml of bacterial shake-tube culture). Finally, the cell-specific binding of scFv in crude periplasmic extracts was detected by flow cytometry. This procedure facilitated the efficient cloning of a functional anti-CD19 VH/VL combination from the hybridoma cDNA.

Amino Acid Sequence↗
PubMed1986

Undermethylation of interferon-gamma gene in human T cell lines and normal T lymphocytes.

The relative levels of DNA methylation at CCGG sequences within and around the interferon-gamma (IFN-gamma) gene in normal human tissues and cell lines were examined by Southern blot analysis using isoschizomeric restriction enzymes, HpaII and MspI. On the test of normal tissues, the IFN-gamma gene was undermethylated only in a small population of T lymphocyte, whereas the gene was fully methylated in T cell-depleted lymphocytes and uterus cells. In TCL-Fuj cell line which is a T cell line producing a high level of IFN-gamma spontaneously, the IFN-gamma gene was undermethylated. Moreover, the extent of DNA methylation was inversely correlated to the level of expression of the IFN-gamma gene in several T cell lines including sublines derived from TCL-Fuj cells. However, partial or complete unmethylation at the CCGG sites of IFN-gamma gene was observed in a promyelocytic leukemia cell line and two epithelial cell lines that fail to produce IFN-gamma irrespective of induction. These results suggest that undermethylation of IFN-gamma gene is necessary but not sufficient for its efficient expression.

Cell Line↗
PubMed2003

Specific transgene expression in human and mouse CD4+ cells using lentiviral vectors with regulatory sequences from the CD4 gene.

Achieving cell-specific expression of a therapeutic transgene by gene transfer vectors represents a major goal for gene therapy. To achieve specific expression of a transgene in CD4(+) cells, we have generated lentiviral vectors expressing the enhanced green fluorescent protein (eGFP) reporter gene under the control of regulatory sequences derived from the CD4 gene--a minimal promoter and the proximal enhancer, with or without the silencer. Both lentiviral vectors could be produced at high titers (more than 10(7) infectious particles per milliliter) and were used to transduce healthy murine hematopoietic stem cells (HSCs). On reconstitution of RAG-2-deficient mice with transduced HSCs, the specific vectors were efficiently expressed in T cells, minimally expressed in B cells, and not expressed in immature cells of the bone marrow. Addition of the CD4 gene-silencing element in the vector regulatory sequences led to further restriction of eGFP expression into CD4(+) T cells in reconstituted mice and in ex vivo-transduced human T cells. Non-T CD4(+) dendritic and macrophage cells derived from human CD34(+) cells in vitro expressed the transgene of the specific vectors, albeit at lower levels than CD4(+) T cells. Altogether, we have generated lentiviral vectors that allow specific targeting of transgene expression to CD4(+) cells after differentiation of transduced mice HSCs and human mature T cells. Ultimately, these vectors may prove useful for in situ injections for in vivo gene therapy of HIV infection or genetic immunodeficiencies.

Adult↗
PubMed1998

Protection of metabolic and exercise capacity in unselected weight-losing cancer patients following treatment with recombinant erythropoietin: a randomized prospective study.

This study was aimed at evaluating whether anemia could be prevented in unselected weight-losing cancer patients on anti-inflammatory treatment by early and prophylactic treatment with recombinant human erythropoietin (rhEPO) and whether such a benefit could be translated into improved physical function and metabolic efficiency. One hundred eight cancer patients who experienced progressive cachexia due to solid, mainly gastrointestinal tumors were randomized to receive twice daily a cyclo-oxygenase inhibitor (controls; indomethacin, 50 mg twice a day) or indomethacin and erythropoietin, provided on individual basis to prevent development of progressive anemia (study patients; indomethacin, 50 mg twice a day plus rhEPO; range, 12,000-30,000 units per week). All patients were treated and followed up until death or to preterminal stage. Biochemical tests (blood, liver, kidney, and thyroid), nutritional state assessment (food intake and body composition), and exercise testing with simultaneous measurements of respiratory gas exchanges before and during exercise were performed before institution of treatments and then at regular intervals during the treatment period (2-30 months after start). Study and control patients did not differ in survival. rhEPO prevented development of anemia during the entire observation period. This was associated with a significantly more preserved maximum exercise capacity in study patients compared to control patients during the follow-up period (101 +/- 10 versus 66 +/- 6 W; P < 0.0001), based on more effective ventilation and whole-body respiratory gas exchanges. These improvements were also evident when exercise performance was normalized to lean body mass, an indirect measure of the skeletal muscle mass. The metabolic efficiency, expressed as oxygen uptake per watt produced, was also significantly preserved in rhEPO-treated patients compared to controls (14.1 +/- 1.1 versus 16.3 +/- 0.9 ml O2/W, P < 0.05). Our results demonstrate that institution of early and prophylactic rhEPO treatment to patients with progressive cancer prevents development of tumor-induced anemia. This achievement was associated with a better preserved exercise capacity, which is explained in part by improved whole-body metabolic and energy efficiency during work load.

Aged↗
PubMed2005

Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice.

Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthe grisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.

Animals↗
PubMed1991

Characterization of the opposite-strand genes from the mouse bidirectionally transcribed HTF9 locus.

The mouse HTF9 locus contains two genes that are bidirectionally transcribed with opposite polarity from a shared CpG-rich island. Both genes were previously shown to be expressed in a housekeeping fashion in mouse. We have now determined the molecular organization of the genes over 12 kb surrounding the island. In addition, we show that the HTF9 locus resides in the proximal region of mouse chromosome 16. We have sequenced the cDNAs corresponding to both divergent transcripts. Both genes appear to code for novel proteins that are structurally unrelated to each other. Finally, we show that both genes are highly conserved and efficiently expressed in human cells.

Amino Acid Sequence↗
PubMed1993

High-level cryIVD and cytA gene expression in Bacillus thuringiensis does not require the 20-kilodalton protein, and the coexpressed gene products are synergistic in their toxicity to mosquitoes.

Interactions among the 20-kDa protein gene and the cytA and cryIVD genes located in a 9.4-kb HindIII fragment were studied. A series of plasmids containing a combination of these different genes was constructed by using the Escherichia coli/Bacillus thuringiensis shuttle vector pHT3101. The plasmids were then used to transform an acrystalliferous strain, cryB, derived from B. thuringiensis subsp. kurstaki. The results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses suggest that although the 20-kDa protein is required for the efficient CytA protein production in E. coli, it is not required in B. thuringiensis. With or without the truncated 20-kDa protein gene, the CtyA and/or CryIVD proteins are produced and form parasporal inclusions in B. thuringiensis cells. However, more-efficient expression is obtained when a second protein, probably acting as a chaperonin, is present. In addition, the time course studies show that the CytA and CryIVD proteins are coordinately produced. Both the crude B. thuringiensis culture and purified inclusions from each recombinant B. thuringiensis strain are toxic to Culex quinquefasciatus larvae. The parasporal inclusions formed in B. thuringiensis cells are mosquitocidal, with CytA synergizing CryIVD toxicity.

Animals↗