[Low-Molecular Thiols as a Factor Improving the Sensitivity of Escherichia coli Mutants with Impaired ADP-Heptose Synthesis to Antibiotics].

Low molecular-weight thiols as glutathione and cysteine are an important part of the cell's redox regulation system. Previously, we have shown that inactivation of ADP-heptose synthesis in Escherichia coli with a gmhA deletion induces the oxidative stress. It is accompanied by rearrangement of thiol homeostasis and increased sensitivity to antibiotics. In our study, we found that restriction of cysteine metabolism (ΔcysB and ΔcysE) and inhibition of glutathione synthesis (ΔgshAB) lead to a decrease in the sensitivity of the ΔgmhA mutant to antibiotics but not to its expected increase. At the same time, blocking of the export of cysteine (ΔeamA) or increasing import (Ptet-tcyP) into cells of the oxidized form of cysteine-cystine leads to an even greater increase in the sensitivity of gmhA-deleted cells to antibiotics. In addition, there is no correlation between the cytotoxic effect of antibiotics and the level of reactive oxygen species (ROS), the total pool of thiols, or the viability of the initial cell population. However, a correlation between the sensitivity to antibiotics and the level of oxidized glutathione in cells was found in our study. Apparently, a decrease in the content of low-molecular-weight thiols saves NADPH equivalents and limits the processes of protein redox modification. This leads to increasing of resistance of the ΔgmhA strain to antibiotics. An increase in low-molecular-weight thiols levels requires a greater expenditure of cell resources, leads to an increase in oxidized glutathione and induces to greater increase in sensitivity of the ΔgmhA strain to antibiotics.

Enhancement of the Bactericidal Effect of Antibiotics by Inhibition of Enzymes Involved in Production of Hydrogen Sulfide in Bacteria.

Counteraction of the origin and distribution of multidrug-resistant pathogens responsible for intra-hospital infections is a worldwide issue in medicine. In this brief review, we discuss the results of our recent investigations, which argue that many antibiotics, along with inactivation of their traditional biochemical targets, can induce oxidative stress (ROS production), thus resulting in increased bactericidal efficiency. As we previously showed, hydrogen sulfide, which is produced in the cells of different pathogens protects them not only against oxidative stress but also against bactericidal antibiotics. Next, we clarified the interplay of oxidative stress, cysteine metabolism, and hydrogen sulfide production. Finally, demonstrated that small molecules, which inhibit a bacterial enzyme involved in hydrogen sulfide production, potentiate bactericidal antibiotics including quinolones, beta-lactams, and aminoglycosides against bacterial pathogens in in vitro and in mouse models of infection. These inhibitors also suppress bacterial tolerance to antibiotics by disrupting the biofilm formation and substantially reducing the number of persister bacteria, which survive the antibiotic treatment. We hypothesise that agents which limit hydrogen sulfide biosynthesis are effective tools to counteract the origin and distribution of multidrug-resistant pathogens.

[Enhancement of the Bactericidal Effect of Antibiotics by Inhibition of Enzymes Involved in Production of Hydrogen Sulfide in Bacteria].

Counteraction of the origin and distribution of multidrug-resistant pathogens responsible for intra-hospital infections is a worldwide issue in medicine. In this brief review, we discuss the results of our recent investigations, which argue that many antibiotics, along with inactivation of their traditional biochemical targets, can induce oxidative stress (ROS production), thus resulting in increased bactericidal efficiency. As we previously showed, hydrogen sulfide, which is produced in the cells of different pathogens protects them not only against oxidative stress but also against bactericidal antibiotics. Next, we clarified the interplay of oxidative stress, cysteine metabolism, and hydrogen sulfide production. Finally, demonstrated that small molecules, which inhibit a bacterial enzyme involved in hydrogen sulfide production, potentiate bactericidal antibiotics including quinolones, beta-lactams, and aminoglycosides against bacterial pathogens in in vitro and in mouse models of infection. These inhibitors also suppress bacterial tolerance to antibiotics by disrupting the biofilm formation and substantially reducing the number of persister bacteria, which survive the antibiotic treatment. We hypothesise that agents which limit hydrogen sulfide biosynthesis are effective tools to counteract the origin and distribution of multidrug-resistant pathogens.

[Inactivation of Terminal Oxidase bd-I Leads to Supersensitivity of E. coli to Quinolone and Beta-Lactam Antibiotics].

In cells of Escherichia coli, terminal oxidase bd-I encoded by the cydAB gene catalyzes the reduction of O2 to water using hydroquinone as an electron donor. In addition to the cydAB operon, two other genes, cydC and cydD, encoding the heterodimeric ATP-binding cassette-type transporter are essential for the assembly of cytochrome bd-I. It was shown that inactivation of cytochrome bd-I by the introduction of cydB or cydD deletions into the E. coli chromosome leads to supersensitivity of the bacteria to antibiotics of the quinolone and beta-lactam classes. The sensitivity of these mutants to antibiotics is partially suppressed by introduction of a constitutively expressed gene katG under the control of the Ptet promoter into their genome. The increased level of hydrogen sulfide resulting from the introduction of the mstA gene, encoding 3-mercaptopyruvate sulfurtransferase, under the control of the Ptet promoter, leads to the same effect. These data demonstrate the important role of cytochrome bd-I in the defense of bacteria from oxidative stress and bactericidal antibiotics.

[Biotechnological Potential of the Bacillus subtilis 20 Strain].

Bacillus subtilis bacteria play an important role in veterinary medicine, medicine, and biotechnology, and the permanently growing demand for biotechnological products fuels the improvement of the properties of biotechnological strains. B. subtilis strains with improved characteristics maybe obtained by rational design and the directed evolution technologies, or be found among newly described strains. In the course of the long-term microbiome composition studies in the Russian segment of the International Space Station, the B. subtilis 20 strain was isolated, this strain shows the capacity for rapid growth and considerable biomass accumulation, as well as increased resistance to acidification of the environment in comparison to the "terrestrial" B. subtilis 168 strain. What is more, B. subtilis 20 is hyperresistant to the DNA and protein damaging factors that are linked to the overexpression of the genes controlling DNA repair, hydrogen sulfide production, and reactive oxygen species neutralization. The described properties of B. subtilis 20 are indicative of its considerable potential as a promising producer of biologically active compounds.

Possible Function of the ribT Gene of Bacillus subtilis: Theoretical Prediction, Cloning, and Expression.

The complete decipherment of the functions and interactions of the elements of the riboflavin biosynthesis operon (rib operon) of Bacillus subtilis are necessary for the development of superproducers of this important vitamin. The function of its terminal ribT gene has not been established to date. In this work, a search for homologs of the hypothetical amino acid sequence of the gene product through databases, as well as an analysis of the homolgs, was performed; the distribution of secondary structure elements was theoretically predicted; and the tertiary structure of the RibT protein was proposed. The ribT gene nucleotide sequence was amplified and cloned into the standard high-copy expression vector pET15b and then expressed after induction with IPTG in E. coli BL21 (DE3) strain cells containing the inducible phage T7 RNA polymerase gene. The ribT gene expression was confirmed by SDS-PAGE. The protein product of the expression was purified by affinity chromatography. Therefore, the real possibility of RibT protein production in quantities sufficient for further investigation of its structure and functional activity was demonstrated.

[Butanol as a regulatory factor of ompC gene expression in E. coli cells].

The influence of butanol on the expression of ompC gene encoding synthesis of OmpC porin in the MG 1655 strain of E. coli and butanol-tolerant mutant ButR was studied. It was shown that in the case of wild bacteria, the addition of butanol to the growth medium results in an increased level of ompC transcription. However, OmpC porin is not detected in the membrane fraction of cells. ButR mutant exhibits a higher level of ompC gene expression. A direct correlation is observed between the level of OmpC porin expression and its content in the membrane fraction of ButR mutant cells. In the regulatory region of the ompC gene of the ButR mutant, three nucleotide substitutions located in the binding sites of OmpR and CpxR activator proteins were identified. It was shown that mutations in the regulatory region of the ompC gene in the ButR mutant are responsible for the decreased level of OmpC porin expression under normal growth conditions. However, these mutations lead to an increased level of OmpC porin synthesis in the presence of butanol. These data suggest an additional mechanism of ompC gene regulation with the participation of butanol as a positive transcription effector.

Transduction of hematopoietic stem cells with a retroviral vector expressing the neomycin phosphotransferase gene.

Transduction of stem cells with a marking gene holds promise to determine if tissue repair or regeneration is derived from the adult hematopoietic stem cell and if relapse of an autoimmune disease should occur whether relapse arises from the stem cell compartment or from lymphocytes surviving the conditioning regimen. New safety concerns about gene-modified stem cell would entail new safety testing such as documentation of the insertional site prior to release.

Cellular suicide therapy of malignant disease.

Adoptive cellular therapy is developing as a supplement or alternative to chemotherapy and/or radiation for malignant disease. Our focus is two ongoing clinical studies with transgeneic (genetically altered) cellular therapy; one uses allogeneic (from another person) lymphocytes to treat leukemia, and the second uses xenogeneic (from another species) fibroblast cells genetically altered to contain a toxin-producing suicide gene to treat ovarian cancer. Allogeneic donor lymphocyte infusions (DLI) are known to induce remission of hematologic malignancies. However, the toxicity associated with DLI is related to graft-versus-host-disease, which is due to donor lymphocytes attacking normal tissue in the recipient. Therefore, we have taken the approach of infusing DLI that have been modified to contain a latent suicide gene to treat leukemia. To treat ovarian cancer, we used xenogeneic nonimmune fibroblast-derived cells to deliver a tumor-directed cytotoxic gene to carcinoma cells. These cells release HStk transgene retroviruses that in turn transduce replicating tumor cells but not quiescent epithelium, rendering the tumor selectively susceptible to ganciclovir-mediated killing. These initial trials summarize the early stage of allogeneic/xenogeneic adoptive cellular therapy for cancer, and although the data are limited, it is encouraging to see some patients with evidence of antitumor responses. Advances in our understanding of the basic science of these treatments, together with improvements in the technology of vector design, will be required to streamline these methodologies into broader application.

Cellular suicide therapy of malignant disease.

Adoptive cellular therapy is developing as a supplement or alternative to chemotherapy and/or radiation for malignant disease. Our focus is two ongoing clinical studies with transgeneic (genetically altered) cellular therapy; one uses allogeneic (from another person) lymphocytes to treat leukemia, and the second uses xenogeneic (from another species) fibroblast cells genetically altered to contain a toxin-producing suicide gene to treat ovarian cancer. Allogeneic donor lymphocyte infusions (DLI) are known to induce remission of hematologic malignancies. However, the toxicity associated with DLI is related to graft-versus-host-disease, which is due to donor lymphocytes attacking normal tissue in the recipient. Therefore, we have taken the approach of infusing DLI that have been modified to contain a latent suicide gene to treat leukemia. To treat ovarian cancer, we used xenogeneic nonimmune fibroblast-derived cells to deliver a tumor-directed cytotoxic gene to carcinoma cells. These cells release HStk transgene retroviruses that in turn transduce replicating tumor cells but not quiescent epithelium, rendering the tumor selectively susceptible to ganciclovir-mediated killing. These initial trials summarize the early stage of allogeneic/xenogeneic adoptive cellular therapy for cancer, and although the data are limited, it is encouraging to see some patients with evidence of antitumor responses. Advances in our understanding of the basic science of these treatments, together with improvements in the technology of vector design, will be required to stream-line these methodologies into broader application.

Murine retroviral vector producer cells survival and toxicity in the peritoneal cavity of dogs.

Retroviral vector producer cells (VPC) can effectively transfer genes in vivo. To develop a safe method to target gene delivery into intraperitoneal tumors, we have examined the toxicity of intraperitoneal (i.p.) infusion of retroviral VPC in a xenogeneic canine model. Mongrel dogs were injected intraperitoneally (i.p.) with 2 x 10(9) murine LTKOSN.2 VPC. The animals did not demonstrate acute toxicity and tolerated the i.p. infusion of the cells without difficulty. Starting 7 days after i.p. injection, the dogs received intravenous injections of ganciclovir (GCV) twice daily (5 mg/kg) for 7 days. The treatment dogs underwent peritoneal washings on days 3, 7 and 14 after their initial infusion of cells to study the persistence of the VPC. GCV treatment did not cause significant toxicities. Dogs underwent serial blood tests to evaluate bone marrow, renal, liver and immunological function. Complete blood counts, electrolytes and renal function remained normal throughout the study. Although, transient mild elevations occurred of serum alkaline phosphate, the remaining hepatic enzymes remained normal. Histologic examination of tissues from animals sacrificed after the i.p. administration of the VPC revealed no tissue destruction of the normal peritoneal lining. The dogs mounted an antibody response to the murine VPC that was first observed 7 days post injection. PCR analysis of selected tissues after GCV administration did not reveal persistent vector sequences. These results demonstrated that the injection of xenogeneic VPC is not accompanied by significant adverse effects over a 1 month period following administration into the canine peritoneal cavity. These data support the potential clinical application of the VPC in Phase I clinical trials in humans.

Murine retroviral vector producer cells survival and toxicity in the dog liver.

To develop a safe method to target gene delivery into intrahepatic tumors, we examined the toxicity of intrahepatic (IH) injection of retroviral vector producer cells (VPC) into the canine liver. VPC have been demonstrated to effectively transfer genes in vivo. To evaluate for adverse effects form xenogeneic cell transplantation, mongrel dogs were injected IH with 1 x 10(9) murine LTKOSN.2 VPC divided into three aliquots. The animals were then monitored for acute toxicity induced by the VPC. The intraoperative IH injections of the cells were tolerated without difficulty. Starting 7 days after IH injection, the dogs then received intravenous ganciclovir (GCV) twice daily (5 mg/kg) for 7 days. GCV treatment did not cause significant toxicities. Dogs underwent serial blood tests to evaluate bone marrow, renal, liver and immunological function. Complete blood counts, electrolytes, liver function and renal function tests remained normal except for mild elevations of alkaline phosphatase. Histologic examination of liver tissues from the IH injection site revealed no apparent normal tissue destruction induced by the VPC. Two of the four treated dogs underwent liver biopsy on day 3. These biopsy specimens were cultured and persistent, viable VPC were recovered. The dogs mounted an antibody response to the murine VPC that was first demonstrated 5 days post injection. PCR analysis demonstrated low level gene transfer into dog liver tissue. Overall, our results demonstrate that IH xenogeneic VPC injections are not accompanied by significant adverse effects over a 1 month period following administration into the canine liver. These data support the safety aspects of using murine VPC in Phase I clinical trials.

Adoptive immunotherapy for leukemia: donor lymphocytes transduced with the herpes simplex thymidine kinase gene.

The overall goal of adoptive immunotherapy with genetically modified lymphocytes is to decrease the morbidity and mortality associated with allogeneic bone marrow transplantation. The initial data reviewed here suggest that the behavior of the allogeneic HStk transgenic cells can be modified after administration to patients. Further study is needed to identify the response rates and risks associated with this procedure. In particular, larger studies will be needed with appropriate randomization to determine if the response rate to genetically modified cells is equivalent to the response rates with unmodified cells. Wider application of these techniques in the initial setting of allogeneic transplantation will undoubtedly occur and such trials have been initiated at several institutions. Careful attention to vector, suicide gene, selectable marker, efficiency of transduction, and cell dose will be necessary when comparing different trials since these variables will probably affect transgenic cell survival and response rates. [figure: see text]

Eliciting hyperacute xenograft response to treat human cancer: alpha(1,3) galactosyltransferase gene therapy.

Xenograft hyperacute rejection in humans occurs as a secondary response to a cellular glycosylation incompatibility with most non-human mammalian species. A key component of hyperacute rejection, alpha(1,3)galactosyl (agal) epitopes present on the surface of most non-human mammal cells, is bound by host anti-agal IgG antibodies leading to the activation of complement and, cellular lysis (1). The enzyme causing specific glycosylation patterns, alpha(1,3)galactosyltransferase [alpha(1,3)GT], directs the addition of agal to N-acetyl glucosamine residues in the trans Golgi apparatus in most mammalian species including Mus musculus, but not old world primates, apes or humans. In this report, we cloned both a truncated and full length murine alpha(1,3)GT gene into a retroviral vector backbone in order to transfer alpha(1,3)galactosyl epitopes into human A375 melanoma cells. Expression of agal epitopes on A375 cells after alpha(1,3)GT gene transfer was demonstrated using FITC-labeled ligand and FACS analysis. These cells were exposed to human serum for 30 minutes and > 90% of the agal expressing cells were killed by this treatment. These pretreated cells failed to establish tumors after implantation into athymic nude mice. This is the first report of retroviral vector transfer of the alpha(1,3)GT gene into human tumor cells in an attempt to elicit hyperacute rejection as a novel anti-cancer gene therapy strategy.

Adoptive immunotherapy for leukemia: donor lymphocytes transduced with the herpes simplex thymidine kinase gene for remission induction. HGTRI 0103.

This study will evaluate the safety and efficacy of allogenic donor lymphocyte infusions in patients who have relapsed hematologic malignancies after allogeneic bone marrow transplantation (BMT). Donor lymphocyte transfusions have resulted in the cure of some patients with relapsed leukemia or lymphoproliferative disorder after allogeneic BMT, but has been complicated by the development of graft versus host disease (GvHD). We hypothesize that a retroviral vector containing the Herpes simplex thymidine kinase (HStk) gene will allow for retention of the anti-leukemia response of transfused donor lymphocytes while allowing for the adverse effects of GVHD to be mitigated. Patients with relapsed hematologic malignancies after allogeneic BMT will be infused with ex vivo gene modified donor lymphocytes. The Herpes Simplex thymidine kinase (HStk) gene will be transduced into the cells ex vivo using LTKOSN. 1 vector supernate. Insertion of the HStk gene into lymphocytes confers a sensitivity to the anti-herpes drug ganciclovir (GCV). This selective destruction of donor lymphocytes in situ will be used to abrogate the effect of graft versus host disease, if it develops.

[The repair of DNA single-stranded breaks and degradation under the action of novobiocin in gamma-irradiated HeLa cells]. / Reparatsiia odnonitevykh razryvov i degredatsiia DNK pri deistvii novobiotsina v gamma-obluchennykh kletkakh HeLa.

A method of sedimentation in alkaline sucrose gradients was used to study repair of gamma-induced DNA single strand breaks (SSB) and DNA degradation in HeLa cells treated with novobiocin (Nb), an inhibitor of topoisomerase II. After irradiation in a dose of 150 Gr, Nb in a concentration of 1 mM does not affect the effectivity of SSB repair and DNA molecular mass in the irradiated cells treated with Nb for 60-180 min before irradiation. Besides, it does not lead to additional DNA degradation in cells treated with Nb for 60-180 min before gamma-rays, as well as following a postirradiation incubation during 60-180 min. Nb in a concentration of 4 mM, much exceeding the Nb concentration when DNA synthesis and cell transit through the cycle are inhibited, causes the following changes. It does not affect DNA molecular mass in non-irradiated cells, inhibits repair of DNA SSB, causes partial DNA degradation, if cells are treated for 60-180 min before gamma-rays and during the following postirradiation incubation (60-180 min). Taking into account the Nb-mediated DNA degradation, the inhibition of DNA repair by Nb appears not significant. Since in a concentration, which inhibits topoisomerase II, Nb does not affect repair of gamma-induced DNA SSB, one may assume the lack of involvement of topoisomerase II into repair of these DNA lesions. Inhibition of DNA repair by 4 mM Nb may result from its effect on the number of proteins, including reparative DNA polymerase, rather than from a selective effect on topoisomerase II.