Isoelectric focusing of subtilisin inhibitors: detection and partial characterization of cereal inhibitors of chymotrypsin and microbial proteases.
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Bacillus↗
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Results for “microbial detection”
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Physiochemical characterization of substituted chromeno[4,3-b][1,5]benzodiazepine stereoisomers designed as cell membrane active antitumor agents.
As an alternative to naturally occurring pyrrolo[2,1-c][1,4]benzodiazepines (e.g., antramycin) which possess properties of DNA alkylation, we have designed several antileukemic chromeno[4,3-b][1,5]benzodiazepine derivatives with potential activity toward leukemia cell membranes and the cyclic nucleotide system. The cis and trans diastereoisomers were characterized by NMR. The absolute configurations of the enantiomers were established by X-ray diffraction and circular dichroism (CD) measurements. By means of absorption spectroscopy and determinations of fluorescence and fluorescence decay, it was found that the cancerostatically active compound (+)(6aR, 13aS)-3,4-dimethoxy-10,11-dimethyl-6,6a,7,8,13, 13a-hexahydrochromeno[4,3-b][1,5]benzodiazepine (ZIMET 54/79) and its biologically inactive (-) enantiomer (ZIMET 55/79) interact with liposomal membranes. At pH values of 6.0 and 7.3 the long-wave absorption bands of these agents showed weak bathochromic and hypochromic effects upon addition of neutral, and positively and negatively charged phosphatidylcholine and phosphatidylcholine/cholesterol liposomes. Such spectral changes are interpreted as resulting from the binding of both agents to phospholipid bilayers. Steady-state determinations using the membrane probe 1-anilino-8-naphthalenesulfonic acid (1,8-ANS) led to the observation of a small decrease in fluorescence intensity in the presence of either agent. Time-resolved measurements demonstrate that the mechanism of action of the agents occurs mainly through the partial displacement of probe molecules from regions of hydrophobic binding to areas of greater solvent accessibility. No significant differences in binding between the cancerostatically active and inactive enantiomers with liposomes (archiral systems) were detectable on the basis of spectrophotometric and fluorescence determinations. Cell membrane bound adenylate cyclase is stimulated by ZIMET 54/79, resulting in an increase of 103% in the level of cAMP in mouse L1210 leukemia cells. On examination of structure-activity relationships, it was found that the biological activity (leukemia L1210, P388, Lewis lung carcinoma, melanoma B16, increase in cAMP) is correlated with the particular configuration (6aR,13aS) and type of substituent at positions 3 and 4 of the benzo ring in the case of alkoxy groups and positions 10 and 11 for methyl groups. No activity was detected toward DNA/RNA using microbial test systems.
Animals↗
Unraveling anaerobic indole degradation in an acclimated sludge consortium: Candidate pathways and microbial division of labor inferred from metagenomic and metatranscriptomic analyses.
Indole is a widespread nitrogen-containing heterocyclic compound in manure, sludge, and wastewater systems, yet the enzymes and microbial populations involved in its anaerobic transformation remain poorly resolved. Here, we established a long-term acclimated anaerobic sludge consortium and combined degradation assays, metabolite profiling, metatranscriptomics, and genome-resolved metagenomics to investigate the functional basis of anaerobic indole degradation. After 120 days of acclimation, the consortium stably degraded 100 mg/L indole, whereas skatole was not effectively removed under the same strategy, indicating substrate-specific adaptation of the microbial community. Metabolite profiling detected oxindole, dioxindole, isatin, and anthranilic acid, supporting a putative transformation route involving pyrrole-ring oxidation and ring cleavage toward anthranilate-like intermediates. Metatranscriptomic analysis identified 16,660 differentially expressed genes after indole addition, with strong transcriptional responses involving oxidoreductases, hydrolases, cofactor-dependent redox metabolism, aromatic-CoA-related metabolism, and methane metabolism-associated pathways. Transcriptional responses highlighted the xanthine dehydrogenase-like molybdenum-enzyme system and isatin hydrolase as candidate contributors to upstream indole transformation, whereas those of abmG-like, bcrC, and oah genes were consistent with possible anthranilic acid activation and downstream CoA-type processing. MAG-resolved analysis further suggested that these candidate functions may be distributed among populations affiliated with Bacteroidota, Chloroflexota, Desulfobacterota, and Methanobacterium. Together, these findings establish a stable anaerobic indole-degrading consortium and provide a testable functional framework for syntrophic interactions linking upstream indole transformation, aromatic-CoA metabolism, and methanogenesis-associated carbon flow.
Anaerobic degradation↗
Detection of intestinal bacterial translocation in subclinical ischemia-reperfusion using the polymerase chain reaction technique.
BACKGROUND/PURPOSE: The purpose of this study was to evaluate the detection of bacterial translocation after subclinical ischemia reperfusion injuries in rats with the polymerase chain reaction (PCR) technique. METHODS: Six-week-old weaning rats were divided into 3 groups. (1) Experiment rats (n = 20) were gavaged with 10(10) Escherichia coli followed by superior mesentery artery occluded for 10 minutes, then reperfused for 30 minutes. (2) Control rats (n = 20) received bacterial gavage. (3) Group 3 were sham rats (n = 20). After the procedure, 3 mL of blood was obtained from the portal vein. The terminal ileum and mesenteric lymph node (MLN) near the terminal ileum were removed. E. coli DNA was detected in blood and MLN samples by PCR, and histological changes were examined. RESULTS: E. coli DNA detection in ischemia-reperfusion (I/R) group animals was 6 of 20 (30%) in the MLN and 2 of 20 (10%) in the blood. PCR was negative in all the rats in the control group and in the sham group (P < .05). There were no significant differences in the histological examination of rat intestines. CONCLUSION: These data suggest that subclinical intestinal I/R injury results in bacterial translocation. Also, PCR is a highly sensitive and rapid method to detect the presence of microbial DNA.
Animals↗
Ancient metalloenzymes as possible markers in molecular archaeology.
The successful preparation of an active remnant of Cu,Zn-superoxide dismutase from mummified brain tissue stimulated the isolation of both biochemically and immunologically active alkaline Zn2Mg-phosphatase from antique bone samples of different archaeological sites and age. In particular, specimens from pharaonic Egypt being up to 4000 years of age were used. Gel filtration, ion exchange and affinity chromatographies were employed to optimise the preparation of the ancient enzyme. Compared to the specific activity of alkaline phosphatase from modern autopsy some 50% for a Ptolemaic and 10% for the Old Kingdom enzyme was detectable. The possibility of microbial contamination was checked by employing specific monoclonal antibodies directed against the human bone enzyme. Fortunately, ubiquitously present specified microorganisms on the respective ancient bones did not cross-react with these antibodies while the ancient metalloenzyme reacted with high specificity. Alkaline phosphatase mimicks could be excluded as in the presence of the inhibitors 1,10-phenanthroline and L-homoarginine the enzyme activity was diminished. The presence of ortho-vanadate as a substrate analogon abolished the catalytic function of the enzyme. Likewise, heating to 100 degrees C and replacement of zinc(II) by cadmium(II) resulted in a dramatic loss of activity. In conclusion, alkaline phosphatase appears to be a useful marker enzyme in molecular archaeology.
Alkaline Phosphatase↗
Improved selectivity of microbial biosensor using membrane coating. Application to the analysis of ethanol during fermentation.
A ferricyanide mediated microbial biosensor for ethanol detection was prepared by surface modification of a glassy carbon electrode. The selectivity of the whole Gluconobacter oxydans cell biosensor for ethanol determination was greatly enhanced by the size exclusion effect of a cellulose acetate (CA) membrane. The use of a CA membrane increased the ethanol to glucose sensitivity ratio by a factor of 58.2 and even the ethanol to glycerol sensitivity ratio by a factor of 7.5 compared with the use of a dialysis membrane. The biosensor provides rapid and sensitive detection of ethanol with a limit of detection of 0.85 microM (S/N=3). The selectivity of the biosensor toward alcohols was better compared to previously published enzyme biosensors based on alcohol oxidase or alcohol dehydrogenases. The biosensor was successfully used in an off-line monitoring of ethanol during batch fermentation by immobilized Saccharomyces cerevisiae cells with an initial glucose concentration of 200 g l(-1).
Biofilms↗
Molecular approaches to the study of aquatic microbial communities
Molecular methods for studying microbial communities are still under development. Enormous sequence catalogues can be collected; they must now be related to microbial activities. Messenger RNA detection, fluorescent in situ hybridization, cell sorting, and oligonucleotide array technology are currently being explored. Biases are associated with all these methods, but combined approaches offer checks and balances.
Journal Article↗
Bacterial "competence" genes: signatures of active transformation, or only remnants?
An exhaustive review published ten years ago reported natural genetic transformation, a potential mechanism for intra- and interspecies gene transfer, in approximately 40 species belonging to different taxonomic and trophic groups. Since then, considerable progress has been made in characterizing DNA-uptake machineries and regulatory circuits controlling their expression in cells competent for genetic transformation. In this article, in light of the recent description of a Group A streptococcal isolate capable of DNA transfer in mixed cultures, we discuss whether the detection in completely sequenced microbial genomes of intact homologues of key competence-regulatory and/or DNA-uptake proteins enables the prediction of new transformable species.
Bacteria↗
Nucleic Acid-based Pathogen Discovery Techniques: Potential Application to Xenozoonoses.
Pathogen discovery techniques based on detection and characterization of microbial nucleic acids have potential application to the field of xenotransplantation. Techniques such as broad-range polymerase chain reaction and representational difference analysis may make it possible to define the zoonotic flora present within donor animal species to an extent never before possible. This review describes the current practice of these techniques, with an emphasis on the use of these methods for identifying zoonotic agents. Identification of potential pathogens in colony-reared donors may lead to isolation and selective breeding practices that could ultimately eliminate these agents from the donor pool.
Journal Article↗
Uracil as an index of lactic acid bacteria contamination of tomato products.
The aim of this research was to evaluate the suitability of uracil as an hygienic quality index of tomato products. Whereas uridine was naturally present throughout tomato fruits' ripening, uracil appeared only after microbial contamination. In tomato pulp inoculated with nine different microbial strains, all five lactic acid bacteria (LAB) studied released relevant quantities of uracil (150-1040 mg/kg of dm), with a correlated partial or total decrease of uridine. Uracil production by yeasts and molds was very low or nonexistent; the starting uridine concentration (approximately 960 mg/kg of dm) remained constant or increased. Uracil thermostability was also verified. Twenty-six samples of tomato paste (30 degrees Brix) were collected from bag-in-drums produced in an industrial processing plant, some with evident swelling symptoms. All of the samples with high microbial count presented uracil. Uracil was also present in samples with microbial contamination under the detection limit and Howard mold count below legislation limits, implying the reprocessing, at least partial, of altered tomato product. The results indicate that uracil presence in tomato products is an index of LAB contamination that has occurred before heat treatment.
Drug Stability↗
Analysis of Arabidopsis genome sequence reveals a large new gene family in plants.
A detailed analysis of the currently available Arabidopsis thaliana genomic sequence has revealed the presence of a large number of open reading frames with homology to the stigmatic self-incompatibility (S) genes of Papaver rhoeas. The products of these potential genes are all predicted to be relatively small, basic, secreted proteins with similar predicted secondary structures. We have named these potential genes SPH (S-protein homologues). Their presence appears to have been largely missed by the prediction methods currently used on the genomic sequence. Equivalent homologues could not be detected in the human, microbial, Drosophila or C. elegans genomic databases, suggesting a function specific to plants. Preliminary RT-PCR analysis indicates that at least two members of the family (SPH1, SPH8) are expressed, with expression being greatest in floral tissues. The gene family may total more than 100 members, and its discovery not only illustrates the importance of the genome sequencing efforts, but also indicates the extent of information which remains hidden after the initial trawl for potential genes.
Arabidopsis↗
Differences in inflammatory responses and cytotoxicity in RAW264.7 macrophages induced by Streptomyces Anulatus grown on different building materials.
Streptomyces anulatus, an indicator microbe of mold in buildings, was grown on different building materials in order to study the impact of growth conditions on the ability of the spores of this microbe to induce toxicity and inflammatory responses. The microbes were grown for 2 months on sterilized and unsterilized wood, chipboard, concrete, plaster board and mineral wool in tight glass vessels under humid conditions. The highest microbial spore concentration was detected on the sterilized mineral wool followed by the sterilized plaster board and the unsterilized mineral wool. Mouse RAW264.7 macrophages were exposed in vitro for 24 h to the spores of S. anulatus and the production of the inflammatory mediators, nitric oxide (NO), tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), interleukin-10 (IL-10) and cytotoxicity, were measured. The dose equivalent to 5 x 10(5) spores/ml of medium was used to compare the different materials. The most intense production of NO (11.6 microM), TNF alpha (560 pg/ml) and IL-6 (2800 pg/ml) in macrophages was induced by the spores grown on sterilized plaster board. They also caused the greatest loss of cell viability (39%). The spores grown on sterilized concrete induced significant production of NO (1.5 microM) and decreased cell viability (22%), and the spores grown on unsterilized and sterilized mineral wool increased production of NO (4.1 microM and 0.8 microM, respectively). The spores did not stimulate production of the anti-inflammatory cytokine IL-10. These results indicate that the ability of S. anulatus to induce inflammatory responses and cytotoxicity in macrophages is dependent on the growth conditions provided by different building materials.
Air Pollution, Indoor↗
Intracellular localization of Toll-like receptor 9 prevents recognition of self DNA but facilitates access to viral DNA.
Toll-like receptors (TLRs) sense infection by detecting molecular structures of microbial origin. TLR3, TLR7 and TLR9 recognize nucleic acids and are localized to intracellular compartments where they normally respond to viral nucleic acids. The purpose for this intracellular localization, however, is not clear. Here we describe a chimeric TLR9 receptor that localized to the cell surface and responded normally to synthetic TLR9 ligands but not to viral nucleic acids. However, the 'relocated' chimeric TLR9 receptor was able to recognize self DNA, which does not stimulate wild-type TLR9. These data demonstrated that intracellular localization of TLR9 was not required for ligand recognition. Instead, localization of the nucleic acid-sensing TLRs is critical in discriminating between self and nonself nucleic acid.
Animals↗
The Walkerton tragedy --issues for water quality monitoring.
One of the most significant weaknesses in modern day water quality management is the detection of microbiological indicators. Microbial tests are normally conducted off-site, often resulting in long turn-around time, risk of contamination, cross contamination and adulteration. Here Stephen Brown and Moe Hussain present a thought provoking case study where limitations in current analytical technologies for water monitoring had fatal results and discuss new approaches to microbiological monitoring that might prevent similar disasters occurring in the future.
Environmental Monitoring↗
Microbiologic profile of intra-abdominal infections at Belo Horizonte, Brazil.
Intra-abdominal infections (IAIs) represent one of the most common clinical problems in hospital practice, especially in surgical areas and centers of intensive care. The treatment of IAIs generally involves the draining of abscesses and empirical antimicrobial therapy. In this study, among 150 patients suffering from IAI, 106 (70.7%) yielded samples that presented microbial growth. Polyinfection was detected in 51.9% of the cases and varied from 2 to 9 distinct microbes per specimen. The overall mean number of micro-organisms isolated per patient was 2.17. Aerobic bacteria (as strict aerobes and facultative anaerobes), strict anaerobic bacteria, and fungi of the genus Candida represented 93.4%, 30.2%, and 13.2% of the cases positive for micro-organisms, respectively. The most common aerobic bacteria were those of the genera Staphylococcus, Escherichia, Proteus, and Streptococcus. Despite the frequent prior use (52%) with antimicrobials of recognized action against strict anaerobes, these micro-organisms constituted 30.9% of the total isolates, and the most frequently found were of the Bacteroides fragilis group and Prevotella species. The high prevalence of anaerobes in the specimens obtained from IAI demonstrates the need to give greater importance to these micro-organisms by making available material and human resources to carry out culture of the anaerobes as part of routine hospital procedures.
Abdominal Abscess↗
Oxygen isotope ratios of PO4: an inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life.
The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (delta(18)O(p)) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO(4)-H(2)O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that delta(18)O(P) values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of delta(18)O(p) as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, delta(18)O(p) may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars.
Biomarkers↗
Toll-like receptor 4 plays a crucial role in the immune-adrenal response to systemic inflammatory response syndrome.
Sepsis and septic shock are leading killers in the noncoronary intensive care unit, and they remain worldwide health concerns. The initial host defense against bacterial infections involves Toll-like receptors (TLRs), which detect and respond to microbial ligands. In addition, a coordinated response of the adrenal and immune systems is crucial for survival during severe inflammation. Previously, we demonstrated a link between the innate immune system and the endocrine stress response involving TLR-2. Like TLR-2, TLR-4 is also expressed in human and mouse adrenals. In the present work, by using a low dose of LPS to mimic systemic inflammatory response syndrome, we have revealed marked cellular alterations in adrenocortical tissue and an impaired adrenal corticosterone response in TLR-4-/- mice. Our findings demonstrate that TLR-4 is a key mediator in the crosstalks between the innate immune system and the endocrine stress response. Furthermore, TLR polymorphisms could contribute to the underlying mechanisms of impaired adrenal stress response in patients with bacterial sepsis.
Adrenal Glands↗
Cytoplasmic domain-mediated dimerizations of toll-like receptor 4 observed by beta-lactamase enzyme fragment complementation.
Toll-like receptors (TLRs) detect the presence of microbial challenge and initiate innate immune defensive responses. In this work we have explored the mechanism and role of TLR dimerization in signal transduction using the newly developed technique of beta-lactamase protein fragment complementation, among others. We observed that TLR4 interactions with itself, with MyD88, or with TLR2 were accurately reported by the enzyme complementation technique. That technique, as well as co-immunoprecipitation, transfection-initiated cell activation, and site-directed mutagenesis all suggest an important role for TLR intracellular domains in receptor dimerization. These findings broaden our understanding of TLR self-interactions as well as heterodimer formation.
Adaptor Proteins, Signal Transducing↗