Disruptions of Genes Encoding Ribose-5-Phosphate Isomerases in Increases Sensitivity of Bacteria to Antibiotics.

In cells, the main enzymes involved in pentose interconversion are ribose-5-phosphate isomerases RpiA and RpiB and ribulose-5-phosphate epimerase Rpe. The inactivation of limits ribose-5-phosphate (R5P) synthesis via the oxidative branch of the pentose phosphate pathway (PPP) and unexpectedly results in antibiotic supersensitivity. This type of metabolism is accompanied by significant changes in the level of reducing equivalents of NADPH and glutathione, as well as a sharp drop in the ATP pool.

Physicochemical and Biological Properties of Vancomycin-Containing Antibacterial Polysaccharide Gels for Biocomposite Bone Implant Impregnation.

Polymeric drugs containing up to 60% by weight of the antibiotic vancomycin were synthesized based on dextran carriers activated with epichlorohydrin. Vancomycin was covalently bound, involving the primary amino group of the molecule through the hydroxypropyl radical to the C6 position of the anhydroglucose units of the dextran main chain. Covalent binding is necessary to prevent spontaneous release of the antibiotic from the gel, thereby reducing the risk of bacterial multiresistance.

Novel Hydroxamic Acids Containing Aryl-Substituted 1,2,4- or 1,3,4-Oxadiazole Backbones and an Investigation of Their Antibiotic Potentiation Activity.

UDP-3-(-3-hydroxymyristoyl)--acetylglucosamine deacetylase (LpxC) is a zinc amidase that catalyzes the second step of the biosynthesis of lipid A, which is an outer membrane essential structural component of Gram-negative bacteria. Inhibitors of this enzyme can be attributed to two main categories, non-hydroxamate and hydroxamate inhibitors, with the latter being the most effective given the chelation of Zn in the active site. Compounds containing diacetylene or acetylene tails and the sulfonic head, as well as oxazoline derivatives of hydroxamic acids, are among the LpxC inhibitors with the most profound antibacterial activity.

[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.

Activation of Purine Biosynthesis Suppresses the Sensitivity of Mutant to Antibiotics.

Inactivation of enzymes responsible for biosynthesis of the cell wall component of ADP-glycero-manno-heptose causes the development of oxidative stress and sensitivity of bacteria to antibiotics of a hydrophobic nature. The metabolic precursor of ADP-heptose is sedoheptulose-7-phosphate (S7P), an intermediate of the non-oxidative branch of the pentose phosphate pathway (PPP), in which ribose-5-phosphate and NADPH are generated. Inactivation of the first stage of ADP-heptose synthesis () prevents the outflow of S7P from the PPP, and this mutant is characterized by a reduced biosynthesis of NADPH and of the Glu-Cys-Gly tripeptide, glutathione, molecules known to be involved in the resistance to oxidative stress.

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.

[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.

The Inactivation of LPS Biosynthesis Genes in Cells Leads to Oxidative Stress.

Impaired lipopolysaccharide biosynthesis in Gram-negative bacteria results in the "deep rough" phenotype, which is characterized by increased sensitivity of cells to various hydrophobic compounds, including antibiotics novobiocin, actinomycin D, erythromycin, etc. The present study showed that mutants carrying deletions of the ADP-heptose biosynthesis genes became hypersensitive to a wide range of antibacterial drugs: DNA gyrase inhibitors, protein biosynthesis inhibitors (aminoglycosides, tetracycline), RNA polymerase inhibitors (rifampicin), and β-lactams (carbenicillin). In addition, it was found that inactivation of the , , , and genes led to dramatic changes in the redox status of cells: a decrease in the pool of reducing NADPH and ATP equivalents, the concentration of intracellular cysteine, a change in thiol homeostasis, and a deficiency in the formation of hydrogen sulfide.

[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.

CydDC functions as a cytoplasmic cystine reductase to sensitize to oxidative stress and aminoglycosides.

l-cysteine is the source of all bacterial sulfurous biomolecules. However, the cytoplasmic level of l-cysteine must be tightly regulated due to its propensity to reduce iron and drive damaging Fenton chemistry. It has been proposed that in the component of cytochrome -I terminal oxidase, the CydDC complex, shuttles excessive l-cysteine from the cytoplasm to the periplasm, thereby maintaining redox homeostasis.

[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.

Mechanism of HS-mediated protection against oxidative stress in .

Endogenous hydrogen sulfide (HS) renders bacteria highly resistant to oxidative stress, but its mechanism remains poorly understood. Here, we report that 3-mercaptopyruvate sulfurtransferase (3MST) is the major source of endogenous HS in Cellular resistance to HO strongly depends on the activity of , a gene that encodes 3MST. Deletion of the ferric uptake regulator (Fur) renders ∆ cells hypersensitive to HO Conversely, induction of chromosomal from a strong pLtetO-1 promoter (P -) renders ∆ cells fully resistant to HO Furthermore, the endogenous level of HS is reduced in ∆ or ∆ ∆ cells but restored after the addition of an iron chelator dipyridyl.

Structural investigation of the thymidine phosphorylase from Salmonella typhimurium in the unliganded state and its complexes with thymidine and uridine.

Highly specific thymidine phosphorylases catalyze the phosphorolytic cleavage of thymidine, with the help of a phosphate ion, resulting in thymine and 2-deoxy-α-D-ribose 1-phosphate. Thymidine phosphorylases do not catalyze the phosphorolysis of uridine, in contrast to nonspecific pyrimidine nucleoside phosphorylases and uridine phosphorylases. Understanding the mechanism of substrate specificity on the basis of the nucleoside is essential to support rational drug-discovery investigations of new antitumour and anti-infective drugs which are metabolized by thymidine phosphorylases.

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.

Crystallization and preliminary X-ray study of Vibrio cholerae uridine phosphorylase in complex with 6-methyluracil.

Uridine phosphorylase catalyzes the phosphorolysis of ribonucleosides, with the nitrogenous base and ribose 1-phosphate as products. Additionally, it catalyzes the reverse reaction of the synthesis of ribonucleosides from ribose 1-phosphate and a nitrogenous base. However, the enzyme does not catalyze the synthesis of nucleosides when the substrate is a nitrogenous base substituted at the 6-position, such as 6-methyluracil (6-MU).

[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.

Expression, purification, crystallization and preliminary X-ray structure analysis of Vibrio cholerae uridine phosphorylase in complex with thymidine.

A high-resolution structure of the complex of Vibrio cholerae uridine phosphorylase (VchUPh) with its physiological ligand thymidine is important in order to determine the mechanism of the substrate specificity of the enzyme and for the rational design of pharmacological modulators. Here, the expression and purification of VchUPh and the crystallization of its complex with thymidine are reported. Conditions for crystallization were determined with an automated Cartesian Dispensing System using The Classics, MbClass and MbClass II Suites crystallization kits.

Effective treatment of preexisting melanoma with whole cell vaccines expressing alpha(1,3)-galactosyl epitopes.

The hyperacute immune response in humans is a potent mechanism of xenograft rejection mediated by complement-fixing natural antibodies recognizing alpha(1,3)-galactosyl epitopes (alphaGal) not present on human cells. We exploited this immune mechanism to create a whole cell cancer vaccine to treat melanoma tumors. B16 melanoma vaccines genetically engineered to express alphaGal epitopes (B16alphaGal) effectively treated preexisting s.

Complete protection against melanoma in absence of autoimmune depigmentation after rejection of melanoma cells expressing alpha(1,3)galactosyl epitopes.

The major barrier for xenotransplantation in humans is the presence of alpha(1-3) Galactosyl epitopes (alphaGal) in xenogeneic tissue and the vast quantities of natural antibodies (Ab) produced by humans against this epitope. The binding of anti-alphaGal Ab to cells expressing alphaGal triggers a complement-mediated hyperacute rejection of target cells. The hyperacute rejection of whole cancer cells, modified to express alphaGal epitopes, could be exploited as a new cancer vaccine to treat human cancers.

Rat sodium iodide symporter for radioiodide therapy of cancer.

Design and development of new approaches for targeted radiotherapy of cancer and improvement of therapeutic index by more local radiation therapy are very important issues. Adenovirus-mediated delivery of the sodium iodide symporter (NIS) gene to cancer cells is a powerful technique to concentrate lethal radiation in tumor cells and eradicate tumors with increased therapeutic index. A replication-defective adenoviral vector expressing the rat NIS gene (Ad-rNIS) was used for in vitro gene delivery and into human prostate cancer xenografts to study antitumor effect.

Effective suppression of class I major histocompatibility complex expression by the US11 or ICP47 genes can be limited by cell type or interferon-gamma exposure.

An impediment encountered in many viral-based gene therapy clinical trials has been the rapid destruction of the transgene by the host's immune response. The processing and presentation of antigens through the class I major histocompatibility complex (MHC) pathway is the initial specific response to viral infection. Disruption of the class I MHC pathway by herpes simplex virus (HSV) or the human cytomegalovirus (HCMV) results in a decrease of the CD8(+) cytotoxic T lymphocyte (CTL) response and prolongs survival of infected cells in the host.

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.

Sodium iodide symporter/radioactive iodine system has more efficient antitumor effect in three-dimensional spheroids.

The sodium iodide symporter (NIS) is a plasma membrane protein that mediates active uptake of inorganic iodide from plasma into thyroid cells. Expression of the NIS gene in tumor cells may provide a novel mechanism for treating cancer. Previously, we cloned cDNA of rat NIS into the retroviral vector LXSN, transduced human and murine tumor cells, and demonstrated 50-60% killing effect of 131I in cells expressing NIS.