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Biomedical subjects

S Belkin

Publications and source records attributed to S Belkin.

At least 19 recordsLinked to original sources
PubMed2004

Biological laser printing of genetically modified Escherichia coli for biosensor applications.

One of the primary requirements of cell- or tissue-based sensors is the placement of cells and cellular material at or near the sensing elements of the device. The ability to achieve precise, reproducible and rapid placement of cells is the focus of this study. We have developed a technique, biological laser printing or BioLP, which satisfies these requirements and has advantages over current technologies. BioLP is capable of rapidly depositing patterns of active biomolecules and living cells onto a variety of material surfaces. Unlike ink jet or manual spotting techniques, this process delivers small volume (nl to fl) aliquots of biomaterials without the use of an orifice, thus eliminating potential clogging issues and enabling diverse classes of biomaterials to be deposited. This report describes the use of this laser-based printing method to transfer genetically-modified bacteria capable of responding to various chemical stressors onto agar-coated slides and into microtiter plates. The BioLP technology enables smaller spot sizes, increased resolution, and improved reproducibility compared to related technologies.

Biological Assay↗
PubMed2001

Monitoring of phosphorus bioavailability in water by an immobilized luminescent cyanobacterial reporter strain.

Massive growth of cyanobacteria, known as "algal blooms", has become a major concern for water monitoring. It has been observed that environmental factors like temperature, light, and certain patterns of availability of nutrients such as P, N, Fe influence cyanobacterial proliferation and toxin production. In order to monitor nutrients in aquatic ecosystems, an assay for monitoring phosphorus bioavailability to cyanobacteria was developed. The test consists of an immobilized luminescent reporter strain of Synechococcus PCC 7942, designated APL. The reporter strain harbours the gene coding the reporter protein luciferase from Vibrio harveyi under control of the inducible alkaline phosphatase promoter from Synechococcus PCC 7942, and can be induced under phosphorus limitation. The resultant CyanoSensor detects PO(3-)(4)-P in a concentration range of 0.3-8 microM after a sample incubation time of 8 h under continuous illumination (50 microE m(-2) s(-1)). The sensor also responded to a variety of organic phosphorus sources and was storable for 3 weeks at 4 degrees C. It could be demonstrated that the CyanoSensor for bioavailability monitoring is an improvement to conventional phosphorus detection methods.

Biological Availability↗
PubMed2001

Survival of enteric bacteria in seawater.

Enteric bacteria exposed to the marine environment simultaneously encounter a variety of abiotic and biotic challenges. Among the former, light appears to be critical in affecting seawater survival; previous growth history plays a major part in preadaptation of the cells, and stationary phase cells are generally more resistant than exponentially growing ones. Predation, mostly by protozoa, is probably the most significant biotic factor. Using Escherichia coli as a model, a surprisingly small number of genes was found that, when mutated, significantly affect seawater sensitivity of this bacterium. Most prominent among those is rpoS, which was also dominant among genes induced upon transfer to seawater.

Enterobacteriaceae↗
PubMed2000

Improved bacterial SOS promoter∷lux fusions for genotoxicity detection.

Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter-operator region were previously shown to be potentially useful for detecting genotoxicants. In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: (1) modifying the host cell's toxicant efflux capacity via a tolC mutation; (2) incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; (3) changing the reporter element to a different lux system (Photorhabdus luminescens), with a broader temperature range; (4) using Salmonella typhimurium instead of an E. coli host. A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed. Generally, fastest responses were exhibited by Sal94, a S. typhimurium strain harboring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by DPD3063, an E. coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant. Overall, the two latter strains appeared to perform better and seemed preferable over the others. The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response.

Alginates↗
PubMed2000

Cationic peptide antimicrobials induce selective transcription of micF and osmY in Escherichia coli.

Cationic antimicrobial peptides, such as polymyxin and cecropin, activated transcription of osmY and micF in growing Escherichia coli independently of each other. The micF response required the presence of a functional rob gene. It is intriguing that in this and other assays an identical response profile was also seen with hyperosmotic salt or sucrose gradient, two of the most commonly used traditional food preservatives. The osmY and micF transcription was not induced by hypoosmotic gradient, ionophoric peptides, uncouplers, or with other classes of membrane perturbing agents. The antibacterial peptides did not promote transcription of genes that respond to macromolecular or oxidative damage, fatty acid biosynthesis, heat shock, or depletion of proton or ion gradients. These and other results show that the antibacterial cationic peptides induce stasis in the early growth phase, and the transcriptional efficacy of antibacterial peptides correlates with their minimum inhibitory concentration, and also with their ability to mediate direct exchange of phospholipids between vesicles. The significance of these results is developed as the hypothesis that the cationic peptide antimicrobials stress growth of Gram-negative organisms by making contacts between the two phospholipid interfaces in the periplasmic space and prevent the hyperosmotic wrinkling of the cytoplasmic membrane. Broader significance of these results, and of the hypothesis that the peptide mediated contacts between the periplasmic phospholipid interfaces are the primary triggers, is discussed in relation to antibacterial resistance.

Adaptation, Physiological↗
PubMed1999

Reactive oxygen species are partially involved in the bacteriocidal action of hypochlorous acid.

Hypochlorous acid (HOCl) is probably the most widely used disinfectant worldwide and has an important role in inflammatory reaction and in human resistance to infection. However, the nature and mechanisms of its bactericidal activity are still poorly understood. Bacteria challenged aerobically with HOCl concentrations ranging from 9.5 to 76 microM exhibit higher ability to form colonies anaerobically than aerobically. Conversely, aerobic plating greatly increased lethality after an anaerobic HOCl challenge, although anaerobic survival did not depend on whether HOCl exposure was aerobic or anaerobic. Even a short transient exposure to air after anaerobic HOCl challenge reduced anaerobic survival, indicative of immediate deleterious effects of oxygen. Exposure to HOCl can cause lethal DNA damage as judged by the fact that recA sensitivity to HOCl was oxygen dependent. Antioxidant defenses such as reduced glutathione and glucose-6-phosphate dehydrogenase were depleted or inactivated at 10 microM HOCl, while other activities, such as superoxide dismutase, dropped only above 57 microM HOCl. Cumulative deficiencies in superoxide dismutase and glucose-6-phosphate dehydrogenase rendered strains hypersensitive to HOCl. This indicates that part of HOCl toxicity on Escherichia coli is mediated by reactive oxygen species during recovery.

DNA Damage↗
PubMed1999

Combinations of chlorocatechols and heavy metals cause DNA degradation in vitro but must not result in increased mutation rates in vivo.

Chlorocatechols introduced into the environment directly or as a result of degradation processes are highly toxic, particularly when combined with heavy metals. With in vitro DNA degradation assays, the high reactivity of chlorocatechols combined with heavy metals could be shown, whereby copper was shown to be more active than iron. Structure-activity analysis showed that the degradation potential of the chlorocatechols decreased with an increasing number of chloratoms. The addition of reactive oxygen species scavengers allowed the identification of hydrogen peroxide as an important agent leading to DNA damage in this reaction. The potential of other reactive compounds, however, can neither be determined nor excluded with this approach. Exposure of Escherichia coli and Salmonella typhimurium cultures to the same mixtures of chlorocatechols and copper surprisingly did not lead to an enhanced mutation rate. This phenomenon was explained by doing marker gene expression measurements and toxicity tests with E. coli mutants deficient in oxidative stress defense or DNA repair. In catechol-copper-exposed cultures an increased peroxide level could indeed be demonstrated, but the highly efficient defense and repair systems of E. coli avoid the phenotypical establishment of mutations. Increased mutation rates under chronic exposure, however, cannot be excluded.

Catalase↗
PubMed1997

Detection of DNA damage by use of Escherichia coli carrying recA'::lux, uvrA'::lux, or alkA'::lux reporter plasmids.

Plasmids were constructed in which DNA damage-inducible promoters recA, uvrA, and alkA from Escherichia coli were fused to the Vibrio fischeri luxCDABE operon. Introduction of these plasmids into E. coli allowed the detection of a dose-dependent response to DNA-damaging agents, such as mitomycin and UV irradiation. Bioluminescence was measured in real time over extended periods. The fusion of the recA promoter to luxCDABE showed the most dramatic and sensitive responses. lexA dependence of the bioluminescent SOS response was demonstrated, confirming that this biosensor's reports were transmitted by the expected regulatory circuitry. Comparisons were made between luxCDABE and lacZ fusions to each promoter. It is suggested that the lux biosensors may have use in monitoring chemical, physical, and genotoxic agents as well as in further characterizing the mechanisms of DNA repair.

Adenosine Triphosphatases↗
PubMed1996

Hypochlorous acid activates the heat shock and soxRS systems of Escherichia coli.

A series of plasmids, containing fusions of different stress promoters to lux reporter genes, was used in an attempt to monitor the defense circuits activated upon exposure of Escherichia coli to sublethal doses of free chlorine. A significant level of activation was exhibited by promoters of three heat shock genes (grpE, dnaK, and lon), in an rpoH-dependent manner. The promoter of micF, a gene under the control of the soxRS regulon, was also strongly induced, but not in a soxR mutant. This induction was not affected by sodA and sodB mutations, implying that it did not involve oxygen radical activity. Free-chlorine activation of both heat shock and soxRS regulons required an exposure of less then I s in duration. The oxyR or the SOS regulons were apparently not induced by free chlorine (as judged by lack of activation of katG and recA, respectively), and neither was the universal stress (uspA) protein.

Bacterial Proteins↗
PubMed1996

Oxidative stress detection with Escherichia coli harboring a katG'::lux fusion.

A plasmid containing a transcriptional fusion of the Escherichia coli katG promoter to a truncated Vibrio fischeri lux operon (luxCDABE) was constructed. An E. coli strain bearing this plasmid (strain DPD2511) exhibited low basal levels of luminescence, which increased up to 1,000-fold in the presence of hydrogen peroxide, organic peroxides, redox-cycling agents (methyl viologen and menadione), a hydrogen peroxide-producing enzyme system (xanthine and xanthine oxidase), and cigarette smoke. An oxyR deletion abolished hydrogen peroxide-dependent induction, confirming that oxyR controlled katG'::lux luminescence. Light emission was also induced by ethanol by an unexplained mechanism. A marked synergistic response was observed when cells were exposed to both ethanol and hydrogen peroxide; the level of luminescence measured in the presence of both inducers was much higher than the sum of the level of luminescence observed with ethanol and the level of luminescence observed with hydrogen peroxide. It is suggested that this construction or similar constructions may be used as a tool for assaying oxidant and antioxidant properties of chemicals, as a biosensor for environmental monitoring and as a tool for studying cellular responses to oxidative hazards.

Bacterial Proteins↗
PubMed1995

Localization of multiple human dihydrodiol dehydrogenase (DDH1 and DDH2) and chlordecone reductase (CHDR) genes in chromosome 10 by the polymerase chain reaction and fluorescence in situ hybridization.

Multiple human dihydrodiol dehydrogenases and human chlordecone reductase belong to the aldoketo reductase superfamily. These two enzymes are involved in the metabolism of xenobiotics, such as polycyclic aromatic hydrocarbons and pesticides. Recently we have isolated three closely related genes encoding two dihydrodiol dehydrogenases (DDH1 and DDH2) and the chlordecone reductase (CHDR). Mapping of the location of the genes was performed using the polymerase chain reaction using gene-specific primers to amplify gene sequences in human/hamster hybrid DNA. All three genes were found to be located on chromosome 10. In situ hybridization using a lambda clone as the probe further confirmed regional localization at 10p14-p15.

Alcohol Oxidoreductases↗
PubMed1995

Nucleoside diphosphate kinase from Escherichia coli.

Nucleoside diphosphate (NDP) kinase from Escherichia coli was purified to homogeneity and was crystallized. Gel filtration analysis of the purified enzyme indicated that it forms a tetramer. The enzyme was phosphorylated with [gamma-32P]ATP, and the pH stability profile of the phosphoenzyme indicated that two different amino acid residues were phosphorylated. Both a histidine residue and serine residues, including Ser-119 and Ser-121, appear to be phosphorylated. A Ser119Ala/Ser121Ala double mutant (i.e., with a Ser-to-Ala double mutation at positions 119 and 121), as well as Ser119Ala and Ser121Ala mutants, was isolated. All of these retained NDP kinase activity; also, both the Ser119Ala and Ser121Ala mutants could still be autophosphorylated. In the case of the double mutant, a slight autophosphorylation activity, which was resistant to acid treatment, was still detected, indicating that an additional minor autophosphorylation site besides His-117 exists. These results are discussed in light of the recent report of N. J. MacDonald et al. on the autophosphorylation of human NDP kinase (J. Biol. Chem. 268:25780-25789, 1993).

Adenosine Triphosphate↗
PubMed1993

Bromoalkane-degrading Pseudomonas strains.

Two Pseudomonas isolates, named ES-1 and ES-2, were shown to possess a wide degradative spectrum for haloalkanes in general and bromoalkanes in particular but did not degrade nonsubstituted alkanes. The utilization of water-insoluble haloalkanes, such as 1-bromooctane, appeared to consist of three phases: (i) extracellular emulsification by a constitutively excreted, broad-spectrum surface-active agent, (ii) dehalogenation by an inducible hydrolytic dehalogenase (possibly periplasmic), and (iii) intracellular degradation of the residual carbon skeleton. Several observations suggest the existence of more than one dehalogenase in strain ES-2.

Alkanes↗
PubMed1989

Electron spin resonance measurements of the effect of ionophores on the transmembrane pH gradient of an acidophilic bacterium.

The delta pH in ionophore-treated cells of an acidophile has been determined by electron spin resonance spectroscopy. The values obtained were comparable to those obtained using the more conventional techniques involving radiolabeled probes. No binding of the spin-labeled probe was observed as determined by two independent control experiments and by the characteristics of the probe signal. These results led us to conclude that the delta pH measured in protonophore/ionophore-treated cells is a result of a Donnan potential, which may be a physical property of all intact bacterial cells at low pH values.

Bacillus↗