Bacterial test system for the primary screening of substances with potential antitumor activity

A fundamental possibility of antitumor activity testing in the bacterial system represented by Escherichia coli of the wild type and its MS2-induced mutant has been shown. The initial bacterial strain is an indicator of toxic properties of tested substances and the mutant strain is a specific test culture modeling a tumor cell. The comparison of the data described for eukaryotes with the data obtained using the proposed bacterial test system confirms an adequate response of both strains to substances with proved antitumor properties. The data obtained are considered as very promising for the further improvement of this test system towards its application for primary screening of antitumor substances.     

Wheat inoculation with Azospirillum brasilense Sp6 and some mutants altered in nitrogen fixation and indole-3-acetic acid production

Abstract Inoculation of wheat seedlings with Azospirillum brasilense Sp6 produced an increase in the number and length of the lateral roots as a plant response. Inoculation with a Nif⁻ mutant, A. brasilense SpF103, which is producer of indole-3-acetic acid (IAA), yielded a very similar plant response. However, inoculation with a Nif⁻ mutant, A. brasilense SpF57, which is a low producer of IAA, did not elitic any response from the plant. The data suggest that the root system response of wheat seedlings to bacterial inoculation is due mainly to production of auxin-type substances by the microorganism.     

Antimicrobial activities of daunorubicin and adriamycin derivatives on bacterial and protoplast type L-form cells of Bacillus subtilis 170, Escherichia coli B, and Proteus mirabilis VI. Structure--activity relationship

The antibacterial activity of ten N-alkylated derivatives of daunorubicin and adriamycin as well as of 5-iminodaunorubicin has been tested by using Bacillus subtilis 170, Escherichia coli B, and Proteus mirabilis VI and their stable protoplast type L-forms in an agar diffusion test. Eight of the substances showed similar activities against B. subtilis and the L-forms of all test organisms, but no activity against the bacterial forms of E. coli and P. mirabilis. The cell wall of these gram-negative bacteria is responsible for this resistance by not allowing the antibiotics to enter the cells. The piperidino compound N-(CH2)5 daunorubicin shows 2-4 times higher activity against B. subtilis and all L-forms in comparison to daunorubicin and the other derivatives. Five of the substances were inactive against all test strains. Their inactivity seems to be associated with the larger substituents at the C-3' position. Relations between molecular structure and activity are discussed considering data about the interaction with DNA and the antitumor activity. Stable protoplast type L-forms and their bacterial forms represent a suitable and effective test system to screen for more effective substances and to get more information about their mode of action.     

Towards a structure-function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides.

The iron-binding protein lactoferrin is a multifunctional protein that has antibacterial, antifungal, antiviral, antitumour, anti-inflammatory, and immunoregulatory properties. All of these additional properties appear to be related to its highly basic N-terminal region. This part of the protein can be released in the stomach by pepsin cleavage at acid pH. The 25-residue antimicrobial peptide that is released is called lactoferricin. In this work, we review our knowledge about the structure of the peptide and attempt to relate this to its many functions. Microcalorimetry and fluorescence spectroscopy data regarding the interaction of the peptide with model membranes show that binding to net negatively charged bacterial and cancer cell membranes is preferred over neutral eukaryotic membranes. Binding of the peptide destabilizes the regular membrane bilayer structure. Residues that are of particular importance for the activity of lactoferricin are tryptophan and arginine. These two amino acids are also prevalent in "penetratins", which are regions of proteins or synthetic peptides that can spontaneously cross membranes and in short hexapeptide antimicrobial peptides derived through combinatorial chemistry. While the antimicrobial, antifungal, antitumour, and antiviral properties of lactoferricin can be related to the Trp/Arg-rich portion of the peptide, we suggest that the anti-inflammatory and immunomodulating properties are more related to a positively charged region of the molecule, which, like the alpha- and beta-defensins, may act as a chemokine. Few small peptides are involved in as wide a range of host defense functions as bovine and human lactoferricin.     

Cytotoxic effect of antineoplastic substances and their effect on DNA synthesis and cGMP level in tumor and normal tissues

The cytostatic effect of antitumour agents line on the level of malignantly changed actively proliferating system (tumour growth) and normal actively proliferating system (splin) was investigated on the basis of DNA synthesis and cGMP rate. Studies of these antitumour agents were carried out on three tumour growth models (Pliss lymphosarcoma, Yablonovskaya glyoblastoma and human glyoblastoma heterografts (subcapsular test). It has been demonstrated that chlofiden, brotheophine, vincristine, adriablastine, natulan, phthorafur have a marked and stable effect of DNA synthesis inhibition, resulting in their marked cytostatic effect and antitumour action stability under the therapy conditions. The experimental data have shown cGMP rates decrease in Pliss lymphosarcoma in the case of high antitumour activity.     

Use of a Titrimetric Method to Assess the Bacterial Spoilage of Fresh Beef

A new method of determining bacterial spoilage in fresh beef is presented. The technique is based upon the fact that as beef undergoes refrigerator spoilage, there is a gradual increase in the production of alkaline substances by the spoilage flora. The level of these substances was measured by titrating meat homogenates to a pH 5.00 end point, employing 0.02 n HCl and an autotitrator. When 23 samples of ground beef from retail stores were tested, an average of 1.32 ml of acid was required for titration of 1 g of fresh beef to pH 5.00, whereas 2.58 ml was required for the same meat at the onset of spoilage. Preliminary data indicate that beef which requires more than 2 ml of 0.02 n HCl/g to lower its pH to 5.00 under the conditions of the test is in some state of incipient spoilage. The statistical correlation between titration values, log bacterial numbers, and extract-release volume was high (P < 0.001). The technique is simple to execute and is highly reproducible, and duplicate samples can be run within 15 min.     

Antibacterial Mechanism of Curcumin: A Review

Curcumin is a plant‐derived polyphenolic active substance with broad‐spectrum antibacterial properties. Curcumin blocks bacterial growth owing to its structural characteristics and the generation of antioxidation products. Curcumin can inhibit bacterial virulence factors, inhibit bacterial biofilm formation and prevent bacterial adhesion to host receptors through the bacterial quorum sensing regulation system. As a photosensitizer, curcumin acts under blue light irradiation to induce phototoxicity and inhibit bacterial growth. Moreover, it can exert a synergistic antibacterial effect with other antibacterial substances. In this review, we summarize the research progress on the antibacterial mechanism of curcumin based on five targeting structures and two modes of action. Our discussion provides a theoretical basis and technical foundation for the development and application of natural antibacterial agents.     

Mutagenic activity of dioxydine]

A previous evaluation of mutagenic activity of some drugs and perspective substances is carried out using indicator microorganisms. The mutagenicity of dioxydine, a drag with discovered antibacterial activity, is investigated. Dioxydine is shown to induce reversions in mutant of Salmonella typhimurium TA-1950, the indicator strain which demonstrates mutagenic activity of agents, producing mutations of base pair substitution type. Dioxydine proved to affect logariphmiically growing bacterial culture with great activity. Mutageni effect of dioxydine is not modified itself in microsomal oxidation system in vitro. Some data concerning participation of excision reparation enzyme (uvr-B+ gene product) in repair of lethal damages induced by dioxydine, have been obtained. The dioxydine ability to cause bacterial gene mutations in host mediated assay as well as dominant and recessive sex-linked lethal mutations in Drosophila is demonstrated. Dioxydine is capable of inducing chromosome aberrations in bone marrow cells and dominant lethal mutations in mouse germ cells.     

Anaerobic microflora of the female reproductive tract

The microflora of the female reproductive tract is very diverse and plays an important role in both normal and pathological states. The data on the mechanisms of colonization resistance which involve the vaginal microbios (the production of H2O2, organic acids, bacteriocin-like substances, competition for adhesion sites) are presented. The data on the role of individual antagonistically active substances of anaerobic bacteria in suppressing gonococci, fungi, microorganisms, associated with bacterial vaginosis, etc. are given. The leading role of anaerobic microorganisms in the appearance of microecological disturbances, including bacterial vaginosis, is emphasized. The role of the pathogenic properties of anaerobic bacteria for the development of different pathological processes, such as premature birth, postnatal and postoperative purulent septic diseases, inflammation of pelvic organs, cancer of the neck of uterus, is discussed.     

Probing the role of two hydrophobic active site residues in the human dihydrofolate reductase by site-directed mutagenesis

In the x-ray structure of the human dihydrofolate reductase, phenylalanine 31 and phenylalanine 34 have been shown to be involved in hydrophobic interactions with bound substrates and inhibitors. Using oligonucleotide-directed mutagenesis and a bacterial expression system producing the wild-type and mutant human dihydrofolate reductases at levels of 10% of the bacterial protein, we have constructed, expressed, and purified a serine 31 (S31) mutant and a serine 34 (S34) mutant. Fluorescence titration experiments indicated that S31 bound the substrate H2folate 10-fold tighter and the coenzyme NADPH 2-fold tighter than the wild-type human dihydrofolate reductase. The serine 31 mutation had little effect on the steady-state kinetic properties of the enzyme but produced a 100-fold increase in the dissociation constant (Kd) for the inhibitor methotrexate. The serine 34 mutant had much greater alterations in its properties than S31; specifically, S34 had a 3-fold reduction in the Km for NADPH, a 24-fold increase in the Km for H2folate, a 3-fold reduction in the overall reaction rate kcat, and an 80,000-fold increase in the Kd for methotrexate. In addition, the pH dependence of the steady-state kinetic parameters of S34 were different from that of the wild-type enzyme. These results suggest that phenylalanine 31 and phenylalanine 34 make very different contributions to ligand binding and catalysis in the human dihydrofolate reductase.     

Evaluation of the properties of potassium ferrate used for water purification by luminescence bioassay

Characteristics of four natural water samples from urban and rural areas and the efficiency of a new purifying agent, potassium ferrate K₂FeO₄, were studied by bacterial luminescence bioassay for 30 minutes. It was revealed that two samples of water from the urban areas are toxic, while the other two samples (one from urban and one from rural environment) are nontoxic. Numerous data obtained on the increase in toxicity index with time allow reasonable conclusions to be made about the chemical nature of substances present in the test water samples. Toxic natural water samples were likely to contain heavy metals and were well purified using potassium ferrate, including via their adsorption. In nontoxic natural water samples, toxic complexes with organic compounds present in water could form at the addition of potassium ferrate. The obtained data call for further studying the properties of potassium ferrate complexes with organic compounds. Bacterial luminescence bioassay is a promising method for the rapid assessment of properties of various water sources (their integral toxicity and presumable chemical composition) and new reagents for their purification (effective concentrations, bactericidal properties, and mechanisms of interacting with heavy metals and organic substances in water).     

Electron donor properties of the antitumour drug amsacrine as studied by fluorescence quenching of DNA-bound ethidium

The effect of the antitumour acridine derivative amsacrine [4'-(9-acridinylamino)methanesulphon-m-anisidide] on the fluorescence lifetime of DNA-bound ethidium has been investigated using a synchronously pumped cavity dumped dye laser producing picosecond pulses for sample excitation and a time-correlated single photon counting detection system. As the proportion of DNA-bound amsacrine on the synthetic DNA polymer poly[deoxyadenylic-thymidylic acid] is increased, the fluorescence decay curve of ethidium can be accurately resolved into two exponential components. The short lifetime component, whose proportion increases with increasing proportions of DNA-bound amsacrine, has a lifetime of between 3 and 4 ns, significantly longer than that of ethidium in aqueous solution (1.63 ns). The magnitude of the long lifetime component decreases from 25.4 to 14 ns with increasing proportions of bound amsacrine. It is concluded that a new fluorescence state of ethidium (lifetime 3-4 ns) is present, probably resulting from reversible electron transfer between ethidium and amsacrine. The ability of various 9-anilinoacridine derivatives to quench the fluorescence of DNA-bound ethidium appears to be related to the electron donor properties of the substituents on the anilino ring, as well as to experimental antitumour activity. The electron donor properties of DNA-bound amsacrine may therefore be relevant to its antitumour action.     

An illusion of hormesis in the Ames test: statistical significance is not equivalent to biological significance

A recent report (Calabrese et al., Mutat. Res. 726 (2011) 91-97) concluded that an analysis of Ames test mutagenicity data provides evidence of hormesis in mutagenicity dose-response relationships. An examination of the data used in this study and the conclusions regarding hormesis reveal a number of concerns regarding the analyses and possible misinterpretations of the Salmonella data. The claim of hormesis is based on test data from the National Toxicology Program using Salmonella strain TA100. Approximately half of the chemicals regarded as hormetic, and the majority of the specific dose-responses identified as hormetic, were actually nonmutagenic. We conclude that the data provide no evidence of hormetic effects. The Ames test is an excellent measure of bacterial mutagenicity, but the numbers of revertant (mutant) colonies on the plate are the result of a complex interaction between mutagenicity and toxicity, which renders the test inappropriate for demonstrating hormesis in bacterial mutagenicity experiments.     

Isolation of a fosfomycin-hypersensitive mutant and production of α-d-glucose-l-phosphate from Bacillus sp. BA-3796 screened by its use

The development of a very sensitive and highly specific screening method for detection of new cell wall inhibitors of the fosfomycin type is described. A fosfomycin-hypersensitive mutant, f-360, was isolated from Staphylococcus aureus Newman by selection with fosfomycin, an antibiotic that inhibits synthesis of the bacterial cell wall. The mutant f-360 was 50-fold more sensitive than the parent strain to fosfomycin. The mutant was constitutive for the hexose phosphate transport system. Using the organism in screening, BA-3796, which had an antibacterial activity against mutant f-360 was found to be produced by a bacterium designated Bacillus sp. BA-3796. Starch and beef extract were the most suitable carbon and nitrogen sources for BA-3796 production and the amount of BA-3796 reached 3 g/l at a maximum level. The purified BA-3796 was identified as α-d-glucose-l-phosphate by its various physiochemical properties. α-d-Glucose-1-phosphate showes an antibacterial activity against Staphylococci in the presence of a slight amount pf α-d-glucose-6-phosphate.     

Mutation of pfm affects the adherence of Pseudomonas aeruginosa to host cells and the quorum sensing system

The Pseudomonas aeruginosa quorum sensing (QS) system is controlled by the signal molecules acyl homoserine lactones (AHLs) that are synthesized from acyl enoyl-acyl carrier proteins (acyl-ACPs) provided by the fatty acid biosynthesis cycle. Pfm (PA2950), an enoyl-CoA reductase, has previously been shown to affect swimming mobility and fatty acid biosynthesis. In this report, we further show that pfm influences bacterial adherence to human cells. Microarray assay results suggest that pfm affects bacterial adherence through its influence on the QS system. Further experiments confirmed that the pfm mutant strain produces significantly less QS signal molecules than the corresponding wild-type strain. Using strains Escherichia coli DH5α(pECP64, lasB'-lacZ) and E.?coli DH5α(pECP61.5, rhlA'-lacZ), biosensors for N-(3-oxododecanoyl) homoserine lactone (3O-C(12) -HSL) and N-butyryl homoserine lactone (C(4) -HSL), respectively, we found that pfm mutant strain produces decreased amounts of both signal molecules. Elastase activity and pyocyanin measurements further confirmed the reduced levels of 3O-C(12) -HSL and C(4) -HSL in the pfm mutant. Finally, bacterial virulence, as assessed by the Caenorhabditis elegans worm killing assay, is decreased in the pfm mutant. Taken together, these data indicate that pfm can be an important target for the control of P.?aeruginosa infectivity.     

Selective isolation of bioactive soil actinomycetes belonging to the Streptomyces violaceusniger phenotypic cluster

AIMS: To devise and evaluate a strategy for isolating members of the Streptomyces violaceusniger phenotypic cluster, which are known to be a promising source of bioactive metabolites. METHODS AND RESULTS: The treatment of four soil samples with 1.5% phenol (30 degrees C, 30 min) prior to inoculation on humic acid-vitamin (HV) agar eliminated most of the streptomycetes and other bacterial populations. The surviving streptomycetes on the HV isolation plates were subcultured, and species-group identification was made according to the probabilistic identification system of Williams et al. (1989). Of the 133 streptomycetes subcultured, 102 (77%), were assigned to the S. violaceusniger cluster. A test with an overlay technique revealed that all of these S. violaceusniger-cluster isolates had broad antimicrobial spectra, as they inhibited the growth of all test Gram-positive bacteria, yeasts and filamentous fungi. Antitumour activity against colon carcinoma cells was found among 68 or 67%, of these S. violaceusniger-cluster isolates, following growth in submerged culture. CONCLUSIONS: Chemical pretreatment of soil samples with phenol reduces the growth of ubiquitous Streptomyces species, thereby facilitating the recovery of less-abundant S. violaceusniger-cluster strains that are characterized by high antimicrobial and antitumour activities. SIGNIFICANCE AND IMPACT OF THE STUDY: The development and application of new methodologies with which to selectively isolate rare, bioactive streptomycete groups is important for discovering novel secondary metabolites with bioactive properties.     

The effects of shortening lactoferrin derived peptides against tumour cells, bacteria and normal human cells.

A number of shortened derivatives of the lactoferrin model peptide L12, PAWRKAFRWAKRMLKKAA, were designed in order to elucidate the structural basis for antitumour activity of lactoferrin derivatives. Three tumour cell lines were included in the study and toxicity determined by measuring lysis of human red blood cells and fibroblasts. The results demonstrated a strong correlation between antitumour activity and net positive charge, in which a net charge close to +7 was essential for a high antitumour activity. In order to increase the antitumour activity of the shortest peptide with a net charge less than +7, the hydrophobicity had to be increased by adding a bulky Trp residue. None of the peptides were haemolytic, but toxicity against fibroblasts was observed. However, modifications of the peptides had a higher effect on reducing fibroblast toxicity than antitumour activity and thereby resulted in peptides displaying an almost 7-fold selectivity for tumour cells compared with fibroblasts. The antimicrobial activity against the Gram-negative bacteria Escherichia coil and the Gram-positive bacteria Staphylococcus aureus was also included in order to compare the structural requirements for antitumour activity with those required for a high antimicrobial activity. The results showed that most of the peptides were highly active against both bacterial strains. Less modification by shortening the peptide sequences was tolerated for maintaining a high antitumour activity and selectivity compared with antimicrobial activity. The order of the amino acid residues and thereby the conformation of the peptides was highly essential for antitumour activity, whereas the antimicrobial activity was hardly influenced by changes in this parameter. Thus, in addition to a certain net positive charge and hydrophobicity, the ability to adopt an amphipathic conformation was a more critical structural parameter for antitumour activity than for antimicrobial activity, and implied that a higher flexibility or number of active conformations was tolerated for the peptides to exert a high antimicrobial activity.     

Complementation studies with human ClpP in Bacillus subtilis

ATP-dependent intracellular proteolysis is essential for all living organisms. ClpP, the proteolytic subunit of the ATP-dependent Clp proteases, shares 56% protein identity between B. subtilis and man. The aim of this study was to verify, whether human ClpP (HClpP) is able to substitute the bacterial pendant, BClpP, irrespectively of the huge evolutionary distance. For this reason hclpP was expressed from the natural B. subtilis promoters at the original chromosomal site. Growth at 37 °C as well as sporulation in the presence of hclpP depict an intermediate phenotype between wild type and clpP mutant suggesting a partial functional substitution of BClpP by HClpP. Northern as well as Western blot analyses show a similar induction pattern of both, bclpP and hclpP during heat stress on the mRNA as well as on the protein levels. Co-immunoprecipitation experiments imply specific interaction of HClpP with bacterial ClpC, ClpX and ClpE during control as well as heat stress conditions. Radioactive pulse-chase labeling and immunoprecipitation revealed that a ClpXP substrate, the short-living regulatory protein MgsR, is degraded by HClpP, although with an extremely slower rate in comparison to BClpP. The occurrence of an exceptional thickened cell wall of a clpP mutant can be almost fully reversed by the complementation with HClpP. The utilization of the HClpP expressing strain as a test system for new biological or synthetic active substances targeting BClpP is discussed.     

Cancer despite immunosurveillance: immunoselection and immunosubversion

Numerous innate and adaptive immune effector cells and molecules participate in the recognition and destruction of cancer cells, a process that is known as cancer immunosurveillance. But cancer cells avoid such immunosurveillance through the outgrowth of poorly immunogenic tumour-cell variants (immunoselection) and through subversion of the immune system (immunosubversion). At the early stages of carcinogenesis, cell-intrinsic barriers to tumour development seem to be associated with stimulation of an active antitumour immune response, whereas overt tumour development seems to correlate with changes in the immunogenic properties of tumour cells. The permanent success of treatments for cancer might depend on using immunogenic chemotherapy to re-establish antitumour immune responses.     

Study of the mechanism of mutagenesis directed by phosphotriester analogs of oligonucleotides. The role of repair in mutagenesis

The mutation system has been suggested in an effort to test insertion and deletion mutants by changing the Lac-phenotype of bacterial colonies transformed by mutant DNA. This system also makes possible to determine heterozygotes and homozygotes among the mutants. The yield of mutants in shown to depend on the structure of the DNA heteroduplex region. The yield of deletion mutants is greater than that of insertion mutants. Heterozygotes prevail in mutant colonies (greater than 90%).