Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pseudomonas syringae pv. tabaci BR2 produces tabtoxin and causes wildfire disease on tobacco and bean plants. Approximately 2,700 Tn5 insertion mutants of a plasmid-free strain, PTBR 2.024, were generated by using suicide plasmid pGS9. Of these Tn5 mutants, 8 were no longer pathogenic on tobacco plants and 10 showed reduced symptoms. All of the eight nonpathogenic mutants caused typical wildfire disease symptoms on bean plants. Two of the nonpathogenic mutants failed to produce tabtoxin. The eight nonpathogenic mutants have Tn5 insertions into different EcoRI and SalI restriction fragments. The EcoRI fragments containing Tn5 from the eight nonpathogenic mutants were cloned into vector pTZ18R or pLAFR3. A genomic library of the parent strain was constructed in the broad-host-range cosmid pLAFR3. Three different cosmid clones that hybridized to the cloned Tn5-containing fragment from one of the nonpathogenic mutants, PTBR 4.000, were isolated from the genomic library. These clones contained six contiguous EcoRI fragments (a total of 57 kilobases [kb]). A 7.2-kb EcoRI fragment common to all three restored pathogenicity to mutant PTBR 4.000. None of the six EcoRI fragments hybridized to Tn5-containing fragments from the other seven mutants. The 7.2-kb fragment was conserved in P. syringae pv. tabaci and P. syringae pv. angulata, but not in other pathovars or strains. Because the mutants retained pathogenicity on bean plants and because of the conservation of the 7.2-kb EcoRI fragment only in pathovars of tobacco, we suggest that genes on the fragment might be related to host specificity.
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PMID:Isolation and characterization of pathogenicity genes of Pseudomonas syringae pv. tabaci. 283 63

Replication-defective adenovirus vectors were generated in which the gene of interest (lacZ, luciferase or HSV-tk) is driven by the adenovirus major late promoter (MLP) or the human cytomegalovirus immediate-early gene promoter/enhancer (CMV). In vitro experiments with rat (II-45) and human (MERO 25) mesothelioma cell lines revealed that the CMV promoter was stronger than the MLP promoter regarding levels of expression of the luciferase reporter gene and ganciclovir (GCV) killing efficiency after tk gene transfer. Following administration of IG.Ad.CMV.lacZ recombinant adenovirus (Introgene, IG) into the pleural cavity of Fischer rats with established mesothelioma, a widespread distribution of infectious virus particles through the thorax contents was demonstrated. However, a relatively small proportion of tumor cells were transduced. Nevertheless, a strong tumor growth inhibition was observed following treatment with IG.Ad.CMV.TK recombinant adenovirus and GCV. Separate groups of rats inoculated on day 0 with 10(5) II-45 cells in the pleural cavity, received 7 x 10(9) infectious particles of IG.Ad. CMV.TK on day 1, day 2, day 4 or day 8. One day after virus administration, 25 mg/kg GCV or PBS (controls) was injected i.p. (intraperitoneally) twice daily. On day 15, all animals were killed. Significant tumor regression, equivalent to 5 log cell kill, occurred in the treated rats suggesting an impressive bystander effect. In a survival study, animals were treated 9 days after inoculation of 10(5) tumor cells with IG.Ad.CMV.TK and a 14 days course of GCV. This treatment prolonged symptom-free survival time from 19 days in the controls to 33 days in the treated group. These responses can be best explained by assuming continued tk expression in or around the tumor tissue during GCV treatment. Our results confirm and extend earlier findings with the same model and demonstrate the potential of the herpes simplex virus thymidine kinase suicide gene therapy as a local treatment for malignant mesothelioma.
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PMID:Gene therapy of experimental malignant mesothelioma using adenovirus vectors encoding the HSVtk gene. 917 12

To increase the antitumor effects of cytosine deaminase (AdCD) gene therapy and induce more potent antitumor immunity, Th1 cytokine interleukin-18 encoded adenovirus (AdIL18) was combined with adenovirus encoding CD (AdCD) for the therapy of established murine B16 melanoma. Combination therapy of the tumor-bearing mice with AdIL 18 and AdCD/5FC inhibited the growth of the subcutaneous B16 tumors more significantly, compared with AdIL 18 or AdCD/5FC alone. In vivo depletion analysis with anti-CD4, anti-CD8 or anti-NK 1.1 McAb illustrated that both CD8+ T cells and CD4+ T cells played key roles in the augmented antitumor response of the combined therapy. Peptide/MHC tetramer represents a powerful and general tool for rapid, highly sensitive, and direct analysis of antigen-specific T cells. In this study, we prepared H-2Kb/TRP-2180-188 tetramer, which was demonstrated to bind H-2Kb-restricted, B16 melanoma-specific CD8+ T cells. B16 specific H-2Kb/TRP2180-188 tetramer was used to stain the tumor-specific CD8+ T cells and the results showed that CD8+ tetramer+ T cells were about 3-5% of the splenic CD8+ T cells derived from tumor-bearing mice after combined therapy. The CTL cytotoxicity was markedly induced in mice after combined therapy, suggesting efficient induction of tumor-specific CD8+ T cells after combined gene therapy with AdCD/5FC/AdIL18. IL-18 gene transfer could significantly augment the cytotoxicity of NK cells and macrophages, and increase the production of interleukin-2 and interferon-gamma, as compared with treatments with AdCD/5FC, AdlacZ/5FC or PBS. These data suggested that in vivo IL-18 gene transfer could augment the antitumor effects of CD suicide gene therapy through efficient induction of antitumor immunity.
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PMID:Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitumor immunity. 1108 76

We evaluated the interaction between oncolytic, replication-competent adenoviral vectors and the herpes simplex virus-1 thymidine kinase (HSV1-tk) gene/ganciclovir (GCV) suicide system for the treatment of malignant gliomas. We constructed a panel of replication-competent adenoviral vectors in which the luciferase (IG.Ad5E1(+). E3Luc) or HSV1-tk gene (IG.Ad5E1(+).E3TK) replace the M(r) 19,000 glycoprotein (gp19K) coding sequence in the E3 region. IG.Ad5E1. IG.Ad5.ClipLuc and IG.AdApt.TK are E1-deleted viruses that contain the luciferase or the HSV1-tk gene in the former E1 region driven by the human cytomegalovirus promoter. IG.Ad5. Sarcoma 1800HSA.E3Luc contains an irrelevant gene in the E1 region, whereas the gp19K coding sequence in the E3 region is replaced by the luciferase gene as in the replicating virus IG.Ad5E1(+).E3Luc. For in vitro experiments, we used a panel of human glioma cell lines (U87 MG, T98G, A172, LW5, and U251), a rat gliosarcoma cell line (9 L), and human lung (A549) and prostate carcinoma (P3) cell lines. In vitro, GCV sensitivity (10 microg/ml) was studied in U87 MG cells after infection at a multiplicity of infection of 1 and 10. A s.c. U87 MG glioma xenograft model was established in NIH-bg-nu-xid mice. Tumors of 100-150 mm(3) were treated with a single injection of adenovirus 10(9) IU suspended in 100 microl of PBS, and GCV 100 mg/kg was administered i.p. twice daily for 7 days. The cytopathic effect of all three replication-competent adenoviral vectors was similar to the cytopathic effect of wild-type adenovirus 5 on all human cell lines tested, indicating that deletion of the E3 gp19K sequences did not affect the oncolytic effect of the vectors. In vitro, luciferase expression was the same for both E1-deleted vectors (IG.Ad5.ClipLuc and IG.Ad5. Sarcoma 1800HSA.E3Luc), demonstrating the strength of the internal E3 promoter even in the absence of E1A. However, in vitro expression levels obtained with replication-competent IG.Ad5E1(+). E3Luc were 3 log higher (allowing infection with a 2-3-log lower multiplicity of infection) in the human cell lines. In U87 MG glioma cells, the oncolytic effect of replication-competent IG.Ad5E1(+).E3TK was significantly enhanced by the addition of GCV and greatly exceeded the cytotoxicity of replication-incompetent IG.AdApt.TK combined with GCV. In established s.c. U87 MG glioma xenografts, a single injection of IG.Ad5E1(+).E3TK resulted in a significant slowing of tumor growth and prolonged survival compared with injection of IG.AdApt.TK. Addition of GCV slowed tumor growth, further adding to survival. In conclusion, the oncolytic effect of replicating adenoviral vectors and HSV1-tk/GCV have potent antitumor effects in gliomas. When combined, these two approaches are complementary, resulting in a significantly improved treatment outcome. In addition, replication-competent adenoviral vectors missing the E3 gp19K coding sequences, have oncolytic efficacy comparable with wild type. In combination with high expression levels obtained with the natural E3 promoter, such vectors are promising new anticancer agents.
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PMID:Treatment of malignant gliomas with a replicating adenoviral vector expressing herpes simplex virus-thymidine kinase. 1175 94

Hypothalamic neuronal histamine is involved in the central regulation of energy expenditure through the activation of sympathetic nerves innervating brown adipose tissue (BAT). The present study examined the effect of L-histidine, a precursor of neuronal histamine, on BAT sympathetic nerve activity in rats. Infusion of histamine at a dose of 1 nmol/rat into the third cerebroventricle significantly increased BAT sympathetic nerve activity as compared with the effect of phosphate buffered saline (P < 0.05). Intraperitoneal (i.p.) injection of L-histidine (0.3 mmol/rat) also significantly increased BAT sympathetic nerve activity as compared with the effect of PBS (P < 0.05). Pretreatment with an i.p. bolus injection of 224 micromol/kg alpha-fluoromethylhistidine, a suicide inhibitor of the histamine synthesizing enzyme histidine decarboxylase, blocked the stimulatory effect of l-histidine on BAT sympathetic nerve activity. These results indicate that L-histidine regulates BAT sympathetic nerve activity through its conversion into neuronal histamine in the hypothalamus.
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PMID:L-histidine stimulates sympathetic nerve activity to brown adipose tissue in rats. 1519 56

To investigate a novel suicide gene for nasopharyngeal carcinoma (NPC) therapy, the yCDglyTK gene was constructed by fusing yeast cytosine deaminase (CD) and herpes simplex type 1 thymidine kinase. The expression of the yCDglyTK gene was detected by RT-PCR and Western blotting, and its bioactivity was demonstrated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. An animal study was carried out in which BALB/C nude mice bearing yCDglyTK gene-modified tumors were treated with prodrugs and radiation. Our results revealed that the yCDglyTK gene could be expressed in CNE-2 cells in vitro. In MTT analysis, at the transfection rate of 10%, 66% cells were killed. The synergistic effect of CD and TK showed 91% of yCDglyTK-transfected cells were killed with the treatment of 5-fluorocytosine (5-FC) alone, 60% killed with ganciclovir (GCV) alone, and 75% killed with 5-FC and GCV together. In vivo, the tumor volume in all of the four prodrugs and/or radiation-treated groups were significantly different from that in the PBS-controlled group (P<.01); also yCDglyTK+prodrug+radiation group was different from the other three groups (P<.05). Our findings suggested there was a synergistic antitumor effect when combining suicide gene therapy and radiation, and yCDglyTK has potent antitumor efficacy and may be a candidate suicide gene for cancer therapy.
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PMID:A novel fusion suicide gene yeast CDglyTK plays a role in radio-gene therapy of nasopharyngeal carcinoma. 1549 80

To improve the effectiveness of herpes simplex virus (HSV) thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy, the replication-defective HSV vector TOIkappaB expressing both HSV-TK and a mutant form of the NF-kappaB inhibitor IkappaBalpha (IkappaBalphaM) was developed. TOIkappaB was constructed by recombining the IkappaBalphaM gene into the U(L)41 locus of a replication-defective lacZ expression vector, TOZ.1. Expression of IkappaBalphaM was confirmed by Western blotting, and the ability of the mutant protein to inhibit NF-kappaB nuclear translocation was examined by electrophoretic mobility shift assay. In human glioblastoma U-87MG cells, the p50/p50 dimer of NF-kappaB was already translocated to the nucleus without receptor-dependent signaling by TNF-alpha. Following infection with TOIkappaB, nuclear translocation of NF-kappaB in U-87MG cells was significantly inhibited and caspase-3 activity increased compared with TOZ.1-infected cells. The cytotoxicity of TOIkappaB for U-87MG cells was investigated by colorimetric MTT assay. At an MOI of 3, TOIkappaB infection killed 85% of the cells compared to 20% killed by TOZ.1 infection. In the presence of GCV, these numbers increased to 95-100% for TOIkappaB and 80-85% for TOZ.1. TOIkappaB neurotoxicity measured on cultured murine neurons was relatively low and similar to that of TOZ.1. The survival of nude mice implanted into the brain with U-87MG tumor cells was markedly prolonged by intratumoral TOIkappaB injection and GCV administration. Survival of TOIkappaB+GCV group was significantly longer (P<.02, Wilcoxon test) than for the control groups (TOZ.1 or TOIkappaB only, PBS or PBS+GCV). These results suggest that IkappaBalphaM expression may be a safe enhancement of replication-defective HSV-based suicide gene therapy in vitro and in vivo.
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PMID:Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant IkappaBalpha and HSV thymidine kinase. 1569 8

We cloned the streptolysin O gene from the Streptococcus pyogenes genome and tested the possibility of using it as an anticancer reagent. Transient transfection of the streptolysin O gene efficiently killed 293T cells after 12 hours of transfection as determined by lactate dehydrogenase release and propidium iodide uptake. No caspase activity was observed and necrosis was prominent during streptolysin O-induced cell death. Biochemical analysis of streptolysin O protein revealed that the deletion of only 5 amino acids from the COOH-terminal region of streptolysin O, which is essential for cholesterol binding activity, abolished its cell-killing activity, whereas the NH2-terminal region was more resilient, i.e., up to 115 amino acids could be deleted without changing its cell-killing activity. We generated a streptolysin O-expressing adenovirus and injected it into human cervical cancer cell-derived tumors grown in a nude mouse model. Twenty-one days postinjection, the average size of tumors in the streptolysin O adenovirus-injected group was 29.3% of that of the control PBS-treated group. Our results show that the genes of pore-forming toxins, like streptolysin O protein, have the potential to establish a novel class of suicide gene therapeutic reagents.
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PMID:Suicide cancer gene therapy using pore-forming toxin, streptolysin O. 1681 21

Gene therapy with adenoviral vectors can eliminate neoplasic cells through selective replication and/or through pro-apoptotic, immunogenic or suicide gene expression. However, an adenoviral vector may provide anti-cancerous effects even in the absence of replication or therapeutic gene expression. The present study evaluates the therapeutic effects caused by the administration of an adenoviral vector, alone, in HPV-dependent neoplasias (HPV-N). In vivo trials were carried out in two HPV-N mouse models. One model was immunocompetent and the other was immunodeficient. In both models, the effect of intratumoral administration of saline solution (PBS) was compared with administration of an adenoviral vector that had no replicative capacity or therapeutic gene (Ad-BGal). In the immunocompetent mice, Ad-BGal adenoviral vector administration significantly reduced tumor growth, compared with PBS. No differences were observed in the immunodeficient mice. In conclusion, the present study lends support to the use of adenoviral vectors in HPV-N treatment since they are capable of generating an antitumoral effect in immunocompetent individuals, even in the absence of a therapeutic gene or viral vector replication.
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PMID:Preclinical evaluation of the therapeutic effect of adenoviral vectors in human papillomavirus-dependent neoplasias. 1863 68

The prevalence of depression and suicide is increased in patients with mesial temporal lobe epilepsy (MTLE); however, the underlying mechanism remains unknown. Anhedonia, a core symptom of depression that is predictive of suicide, is common in patients with MTLE. Glutamine synthetase, an astrocytic enzyme that metabolizes glutamate and ammonia to glutamine, is reduced in the amygdala in patients with epilepsy and depression and in suicide victims. Here, we sought to develop a novel model of anhedonia in MTLE by testing the hypothesis that deficiency in glutamine synthetase in the central nucleus of the amygdala (CeA) leads to epilepsy and comorbid anhedonia. Nineteen male Sprague-Dawley rats were implanted with an osmotic pump infusing either the glutamine synthetase inhibitor methionine sulfoximine [MSO (n=12)] or phosphate buffered saline [PBS (n=7)] into the right CeA. Seizure activity was monitored by video-intracranial electroencephalogram (EEG) recordings for 21days after the onset of MSO infusion. Sucrose preference, a measure of anhedonia, was assessed after 21days. Methionine sulfoximine-infused rats exhibited recurrent seizures during the monitoring period and showed decreased sucrose preference over days when compared with PBS-infused rats (p<0.01). Water consumption did not differ between the PBS-treated group and the MSO-treated group. Neurons were lost in the CeA, but not the medial amygdala, lateral amygdala, basolateral amygdala, or the hilus of the dentate gyrus, in the MSO-treated rats. The results suggest that decreased glutamine synthetase activity in the CeA is a possible common cause of anhedonia and seizures in TLE. We propose that the MSO CeA model can be used for mechanistic studies that will lead to the development and testing of novel drugs to prevent seizures, depression, and suicide in patients with TLE.
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PMID:Inhibition of glutamine synthetase in the central nucleus of the amygdala induces anhedonic behavior and recurrent seizures in a rat model of mesial temporal lobe epilepsy. 2626 37


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