Fluorescence study of the interaction of Suwannee River fulvic acid with metal ions and Al3+-metal ion competition

In this study emission and synchronous-scan fluorescence spectroscopy have been used to investigate the interaction of the class A (oxygen seeking 'hard acid') metal Al(3+), with Suwannee River fulvic acid (SRFA), as well as competition between Al(3+) and several other metal ions (Ca(2+), Mg(2+), Cu(2+), Pd(2+), La(3+), Tb(3+) and Fe(3+)) for binding sites on SRFA. Of the four metal ions possessing very similar (and relatively low) ionic indices (Ca(2+), Mg(2+), Cu(2+) and Pd(2+)) only the latter two paramagnetic ions significantly quenched SRFA fluorescence emission intensity. Of the four metal ions possessing very similar (and relatively low) covalent indices (Ca(2+), Mg(2+), La(3+) and Tb(3+)) only the last paramagnetic ion (Tb(3+)) significantly quenched SRFA fluorescence. None of these metals was able to significantly compete with SRFA-bound Al(3+).Fe(3+), which differs substantially from all of the other metals examined in this study in that it possesses a relatively high ionic index (but not as high as Al(3+)) and a relatively low covalent index (but not as low as Al(3+)), was able not only to quench SRFA fluorescence but also to compete (at least to some extent) with SRFA-bound Al(3+). Synchronous-scan fluorescence SRFA spectra taken in the absence and presence of Fe(3+) and/or Al(3+) support the view that these two metal ions can compete for sites on SRFA. The results of these fluorescence experiments further confirm the Al(3+), and metal ions that have electronic properties somewhat similar to Al(3+) (such as Fe(3+)) are somewhat unique in their ability to interact strongly with binding sites on fulvic acids.     

Analysis of ATP binding to CheA containing tryptophan substitutions near the active site

Signal transduction in the chemotaxis system of Escherichia coli involves an autophosphorylating protein histidine kinase, CheA. At the active site of CheA, phenylalanine residues 455 and 459 occupy positions near the ATP-binding pocket, immediately adjacent to one of the hinge regions of a loop that undergoes an ATP-induced conformational change ("lid closure") that has been characterized previously in X-ray crystal structures [Bilwes et al. (2001) Nat. Struct. Biol. 8, 353-360]. We generated versions of CheA carrying F455W and F459W replacements and investigated whether the fluorescence properties of the introduced tryptophan side chains were affected by nucleotide binding in a manner that would provide a signal for investigating the dynamics of active site events, such as ATP binding and lid closure. Our results indicate that CheA(F455W) is useful in this regard, but CheA(F459W) is not. CheA(F455W) retained full catalytic activity and exhibited easily monitored fluorescence changes upon binding nucleotide: we observed a 25-30% decrease in CheA(F455W) fluorescence emission intensity at 330 nm upon binding ATP in the absence of Mg(2+); in the presence of Mg(2+), the emission spectrum of the CheA(F455W):ATP complex was red-shifted by 5 nm and exhibited an increased intensity (approximately 20% higher at 345 nm relative to that of uncomplexed CheA(F455W)). Different fluorescence changes were observed when two ATP analogues (ADPNP and ADPCP) were used instead of ATP and when Mn(2+) or Ca(2+) was used in place of Mg(2+). We exploited the fluorescence changes induced by Mg(2+)-ATP to explore the kinetics and mechanism of nucleotide binding by CheA(F455W). In the absence of Mg(2+), binding appears to involve a simple one-step equilibrium (k(assn) = 0.7 microM(-1) s(-1) and k(dissn) = 270 s(-1) at 4 degrees C). In the presence of Mg(2+), the binding mechanism involves at least two steps: (i) rapid, relatively weak binding followed by (ii) a rapid, reversible step (k(forward) = 300 s(-1) and k(reverse) =15 s(-1) at 4 degrees C) that enhanced the overall affinity of the complex and generated an increase in W455 fluorescence. This second step could reflect a conformational change at the CheA active site, such as lid closure. These results provide the first insight into the dynamics of nucleotide binding and substrate-induced conformational changes at the active site of a protein histidine kinase.     

Low Concentrations of Tetracycline Enhance the Photophosphorylation and P/O Ratio of Chloroplasts

This study investigated the effects of tetracycline on photophosphorylation, electron transport and P/O ratio of spinach chloroplasts. When chloroplast preparations were treated with low concentrations of tetracycline, non-cyclic and cyclic photophosphorylation activities increased, electron transport rates and P/O ratios improved, chloroplast ms-DLE also improved, and the Mg²⁺-ATPase activity of CF₁ increased in comparison to the control. These results indicate that spinach chloroplasts are sensitive to tetracycline. Next, we used the fluorescence emission spectra of CF₁ to examine the possible binding sites for tetracycline. The fluorescence emission spectra of CF₁ treated with glutaraldehyde, NEM and TNBS, which interact with CF₁ across its whole structure, at the γ subunit and at the β subunit, respectively, were compared with that of control CF₁. The peak sites of the various fluorescence emission spectra were the same, but the peak values for CF₁ treated with glutaraldehyde, NEM and TNBS were lower than that of control CF₁. The peak value of CF₁ treated with 50 μM tetracycline was very similar to that of CF₁ treated with NEM. The above results indicate that the acting site of tetracycline may be at or near the γ subunit of CF₁, and allows the creation of a model in which tetracycline binding strengthens the subunit interactions of ATP synthase, enlarges the proton motive force across the thylakoid membrane, and allows the excess proton motive force to increase ATP formation and improve the P/O ratio. This revised version was published online in June 2006 with corrections to the Cover Date.     

Kinetics of conformational changes induced by the binding of various metal ions to bovine alpha-lactalbumin.

The kinetic effects of the binding of various metal ions (Ca(2+), Cd(2+), Co(2+), Mg(2+), Mn(2+), Sr(2+) and Zn(2+)) to apo bovine alpha-lactalbumin has been monitored by means of stopped-flow fluorescence spectroscopy. Our results show that the measured rate constant for the binding of metal ions to the Ca(2+)-site increases with increasing binding constant. This is, however, not the case for metal ions binding to the Zn(2+)-site. The binding experiments performed at different temperatures allowed us to calculate the activation energy for the transition from the metal-free to the metal-loaded state of the protein. These values do not depend on the nature of the metal ion but are correlated with the type of binding site. As a result, we were able to demonstrate that Mg(2+), a metal ion which was thought to bind to the Ca(2+)-site, shows the same binding characteristics as Co(2+) and Zn(2+) and therefore most likely interacts with the residues belonging to the Zn(2+)-binding site.     

Analytical methods for heavy metals in herbal medicines

Introduction – It is estimated that about 70–80% of the world's population relies on non‐conventional medicine, mainly of herbal origin. However, owing to the nature and sources of herbal medicines, they are sometimes contaminated with toxic heavy metals such as lead, arsenic, mercury and cadmium, which impose serious health risks to consumers. It is critical to analyse source materials for heavy metals in order to ensure that their concentrations meet the related standards or regulations limiting their concentrations in herbal medicines. In this review, different analytical methods for analysis of heavy metals in herbal medicines are discussed. Objective – To provide a comprehensive review of the current state of the art in analytical methods used to detect heavy metals in herbal medicines. Methodology – We systematically searched and reviewed the research articles regarding analytical methods for heavy metals in herbal medicine from various databases, such as Medline/PubMed, ScienceDirect, SciFinder, Google Scholar, EBSCO, Gale InfoTrac, Ingenta, Ovid, ProQuest and ISI Web of Knowledge. Results – In this review, we discuss in detail several commonly used and sensitive analytical techniques, including atomic absorption spectrometry, inductively coupled plasma optical emission spectrometry or mass spectrometry, X‐ray fluorescence spectrometry, high‐performance liquid chromatography, differential pulse polarography, neutron activation analysis and anodic stripping voltammetry. We also provide some application examples of these analytical techniques for heavy metals in herbal medicines. Copyright © 2011 John Wiley & Sons, Ltd.     

Changes in FAD and NADH Fluorescence in Neurosecretory Terminals Are Triggered by Calcium Entry and by ADP Production

We measured changes in the intrinsic fluorescence (IF) of the neurosecretory terminals of the mouse neurohypophysis during brief (1-2 s) trains of stimuli. With fluorescence excitation at either 350 +/- 20 or 450 +/- 50 nm, and with emission measured, respectively, at 450 +/- 50 or > or = 520 nm, DeltaF/F(o) was approximately 5-8 % for a 2 s train of 30 action potentials. The IF changes lagged the onset of stimulation by approximately 100 ms and were eliminated by 1 microM tetrodotoxin (TTX). The signals were partially inhibited by 500 microM Cd(2+), by substitution of Mg(2+) for Ca(2+), by Ca(2+)-free Ringer's with 0.5 mM EGTA, and by 50 microM ouabain. The IF signals were also sensitive to the mitochondrial metabolic inhibitors CCCP (0.3 microM), FCCP (0.3 microM), and NaN(3) (0.3 mM), and their amplitude reflected the partial pressure of oxygen (pO(2)) in the bath. Resting fluorescence at both 350 nm and 450 nm exhibited significant bleaching. Flavin adenine dinucleotide (FAD) is fluorescent, while its reduced form FADH(2) is relatively non-fluorescent; conversely, NADH is fluorescent, while its oxidized form NAD is non-fluorescent. Thus, our experiments suggest that the stimulus-coupled rise in [Ca(2+)](i) triggers an increase in FAD and NAD as FADH(2) and NADH are oxidized, but that elevation of [Ca(2+)](i), alone cannot account for the totality of changes in intrinsic fluorescence.     

利用栽培稻C_0t-1 DNA和基因组DNA比较分析宽叶野生稻基因组

利用AA染色体组栽培稻的中高度重复序列C0t-1 DNA和基因组DNA作为探针,通过荧光原位杂交技术对宽叶野生稻(Oryza latifolia)(CCDD染色体组)进行了比较基因组分析。结果显示,在宽叶野生稻染色体上,C0t-1 DNA的杂交信号没有基因组DNA的杂交信号明显;杂交信号主要分布在着丝粒、近着丝粒及端粒区域;随着洗脱严谨度的不同,杂交信号呈现出较高的种特异性。本研究以不同洗脱严谨度下的荧光原位杂交结果为依据,对宽叶野生稻进行的核型分析,可进一步提高稻属染色体识别的准确性。     

Differential determination of phospholipase A(2) and PAF-acetylhydrolase in biological fluids using fluorescent substrates

The purpose of the present study was the development and evaluation of a fluorimetric method for the screening and differential determination of phospholipase A(2) and PAF-acetylhydrolase in bronchoalveolar lavage (BAL) fluid and serum. Phospholipase A(2) was determined using C(12)-NBD-PC in the presence of Ca(2+), from the slope of the fluorescence enhancement due to the formation of C(12)-NBD-fatty acid. PAF-acetylhydrolase was determined using C(6)-NBD-PC, from the slope of the curve due to C(6)-NBD-fatty acid formation in the absence of Ca(2+). The results were confirmed after TLC analysis. The method's selectivity was evaluated by comparing to radiometric measurements. Light scattering did not interfere and inner filter effects was not observed under our experimental conditions. The effects of pH, temperature, and Ca(2+) were investigated. Protein caused an increase in the background fluorescence of both NBD-PCs. The standard curves of both NBD-fatty acids exhibited the same slope. Linearity extended at least up to 4. 5 nmoles per ml of reaction mixture at the normal pH 7.4. The fluorescence of the NBD-fatty acids remained stable for increasing concentrations of BAL fluid and serum and for BSA up to 100 microg/ml of reaction mixture. Porcine pancreatic PLase A(2) showed preference for C(12)-NBD-PC in the presence of Ca(2+), while without Ca(2+), serum PAF-AcH hydrolyzed only C(6)-NBD-PC. The method is highly sensitive, accurate, and reproducible and can be applied for the differential determination of phospholipase A(2) and PAF-acetylhydrolase activities in BAL fluid and serum.     

Determination of the equilibrium association constant between Tet repressor and tetracycline at limiting Mg2+ concentrations: a generally applicable method for effector-dependent high-affinity complexes.

An analytical method for determining very high binding constants at equilibrium for reactions requiring an effector is proposed and applied to study the interaction of tetracycline with the repressor of the tetracycline resistance gene from Tn10. In this method complex formation is limited by low concentrations of the effector, which is Mg2+ for the interaction of tetracycline and Tet repressor. The binding of Mg2+ to tetracycline and subsequent formation of the ternary repressor-Mg(2+)-tetracycline complex are coupled reactions yielding a dependence of repressor-tetracycline-Mg2+ complex formation on the concentration of free Mg2+. The binding constants can be determined from the quantitative analysis of ternary complex formation with increasing Mg2+ concentrations. This method allows the determination of very high association constants at equilibrium in a large range of protein concentrations. In the case of repressor and tetracycline, the same affinity constant of 3 +/- 2 x 10(9) M-1 was found in the range of 0.1 to 5 microM of repressor. This result indicates that no association or dissociation of the repressor subunits occurs upon binding of tetracycline. Furthermore, the results show that a repressor dimer binds two effector molecules without significant cooperativity.     

Simultaneous determination of various aromatic amines and metabolites of aromatic nitro compounds in urine for low level exposure using gas chromatography-mass spectrometry

A newly developed method permits the simultaneous quantitative determination of various aromatic amines (or metabolites of aromatic nitro compounds, respectively) in human urine in one analytical run. Applying this method it is possible to determine aniline, toluidines, 4-isopropylaniline, o-anisidine, 3- and 4-chloroaniline, 4-bromoaniline, aminonitrotoluenes, aminodinitrotoluenes, 3,5- and 3,4-dichloroaniline, alpha- and beta-naphtylamine and 4-aminodiphenyl. After separation from the urinary matrix by a simple liquid-liquid extraction at pH 6.2-6.4 the analytes are converted into their pentafluoropropionic acid amides. Separation and quantitative analysis is carried out by capillary gas chromatography and mass-selective detection in the single ion monitoring mode. The limits of detection were within the range from 0.05 microg/l (4-aminobiphenyl, o-anisidine, 3,5-dichloroaniline) to 2 microg/l urine (4-amino-2,6-dinitrotoluene). The relative standard deviation of the within-series imprecision (determined at spiked concentrations of 2.0 microg/l and 10 microg/l) was between 2.9 and 13.6% depending on analyte and concentration. The relative recovery rates were in the range of 70-121%. The analytes that do not contain a nitro function showed better performance regarding the analytical reliability criteria. In order to determine the suitability of this new method for biological monitoring we analysed 20 12-h urine samples of persons without known exposure to aromatic amines, nitroaromatics or precursors in a pilot study. In these samples various aromatic amines could be clearly identified. The general population renally excretes aniline (median: 3.5 microg/l; 95th percentile: 7.9 microg/l), o- (0.12 microg/l; 2.7 microg/l), m- (0.17 microg/l; 2.2 microg/l) and p-toluidine (0.11 microg/l; 0.43 microg/l), and o-anisidine (0.22 microg/l; 0.68 microg/l). Additionally, we found that the persons investigated also excrete 3- (<0.05 microg/l; 0.55 microg/l) and 4-chloroaniline (0.11 microg/l; 0.57 microg/l) as well as 3,5-dichloroaniline (0.18 microg/l; 1.5 microg/l). 3,4-Dichloroaniline was found in some specimens (20%) in concentrations near the limit of detection (<0.05 microg/l; 0.12 microg/l). We did not detect alpha- or beta-naphtylamine, 4-aminobiphenyl or metabolites of explosives in the samples.     

Calcium-dependent changes in the flexibility of the regulatory domain of troponin C in the troponin complex

With the recent advances in structure determination of the troponin complex, it becomes even more important to understand the dynamics of its components and how they are affected by the presence or absence of Ca(2+). We used NMR techniques to study the backbone dynamics of skeletal troponin C (TnC) in the complex. Transverse relaxation-optimized spectroscopy pulse sequences and deuteration of TnC were essential to assign most of the TnC residues in the complex. Backbone amide (15)N relaxation times were measured in the presence of Ca(2+) or EGTA/Mg(2+). T(1) relaxation times could not be interpreted precisely, because for a molecule of this size, the longitudinal backbone amide (15)N relaxation rate due to chemical shift anisotropy and dipole-dipole interactions becomes too small, and other relaxation mechanisms become relevant. T(2) relaxation times were of the expected magnitude for a complex of this size, and most of the variation of T(2) times in the presence of Ca(2+) could be explained by the anisotropy of the complex, suggesting a relatively rigid molecule. The only exception was EF-hand site III and helix F immediately after, which are more flexible than the rest of the molecule. In the presence of EGTA/Mg(2+), relaxation times for residues in the C-domain of TnC are very similar to values in the presence of Ca(2+), whereas the N-domain becomes more flexible. Taken together with the high flexibility of the linker between the two domains, we concluded that in the absence of Ca(2+), the N-domain of TnC moves independently from the rest of the complex.     

Determination of MDMA, MDEA and MDA in urine by high performance liquid chromatography with fluorescence detection

This paper describes the development and validation of analytical methodology for the determination of the use of MDMA, MDEA and MDA in urine. After a simple liquid extraction, the analyses were carried out on a high performance liquid chromatography (HPLC) in an octadecyl column, with fluorescence detection. The mobile phase using a sodium dodecyl sulfate ion-pairing reagent allows good separation and efficiency. The method showed good linearity and precision. Recovery was between 85 and 102% and detection limits were 10, 15 and 20 ng/ml for MDA, MDMA and MDEA, respectively. No interfering substances were detected with fluorescence detection.     

An integrated portable apparatus for the simultaneous field measurement of photosynthetic CO2 and water vapour exchange, light absorption and chlorophyll fluorescence emission of attached leaves

Abstract. A portable apparatus has been constructed to measure simultaneously the quantum yield of CO₂ assimilation, light absorption, chlorophyll fluorescence emission and water vapour exchange of attached intact leaves in the field. The core of the instrument is a light-integrating spherical leaf chamber which includes ports for a light source, photosynthetically active radiation sensor, fluorescence probes and gas inlet and outlet manifolds. Measurement of the quantum flux inside the empty chamber and with a leaf present allows determination of leaf absorptance. An open gas exchange system is employed using an infra-red analyser to measure leaf CO₂ exchange. Using a DC white light source the quantum yield of CO₂ assimilation based on absorbed light (φ_abs) may be determined rapidly in either ambient air or artificial gas mixtures. Inclusion of capacitance humidity probes into the gas inlet and outlet ports allows simultaneous determination of water vapour exchange and subsequent estimation of stomatal conductance to CO₂ and intercellular CO₂ concentration. Measurement of fluorescence emission by the sample leaf exposed to white light is achieved by a modulated fluorescence detection system. In addition to determination of the minimal, maximal and variable fluorescence levels, a further analysis allows the photochemical and non-photochemical components of fluorescence quenching, to be estimated. The theory and design of this apparatus is described in detail. The use of the apparatus in the field is demonstrated through a study of the photosynthetic performance of a maize and bean crop during the growing season and by analysis of the photosynthetic performance of crops subjected to nitrogen-stress and a herbicide treatment.     

A selective 19F nuclear magnetic resonance spectroscopic method for the assay of the neuroleptic drug cis(Z)-flupentixol in human serum

This investigation was carried out to evaluate 19F nuclear magnetic resonance as an analytical tool for the measurement of the cis(Z) and trans(E) stereoisomers of the antipsychotic drug flupentixol in human serum. The method is based on the integration of appropriate signals of both analytes and an internal standard. The proposed method was applied to the analysis of real samples without any interference, manipulation of large samples, and lengthy instrument time. Experimental parameters were selected to optimize accuracy, precision, and analysis time. The calibration curves in human serum matrix were linear for cis(Z)- and trans(E)-flupentixol over the ranges 4.0-50.0 and 2.6-25.0 microg/mL, respectively, with respective minimum detectable limits (S/N=3) of 1.67 and 1.72 microg/mL. The method was validated through spike and recovery for the two isomers of flupentixol from a human serum matrix.     

Bioanalysis and pharmacokinetics of chitosan ester in rabbit serum by HPLC with postcolumn fluorescence derivatization

Interest in antiatherosclerotic activity of chitosan ester (PS916) with a new form of sulfate amino polysaccharide derived from marine chitin has necessitated the development of a sensitive and specific method to study its pharmacokinetics. A sensitive and reproducible high-performance liquid chromatography (HPLC) with postcolumn fluorescence derivatization method was developed and validated for the determination of PS916 in rabbit serum. Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of methanol-water (20:80, v/v) at a flow rate of 0.2 ml/min. The derivatization procedure involved postcolumn reaction with guanidine hydrochloride in an alkaline medium at 110 degrees C. The fluorometric detector was operated at 250 nm (excitation) and 435 nm (emission). The assay was linear over the concentration range of 5-100 microg/ml. The lower limit of detection (LLOD) was found to be 1.0 microg/ml. The proposed method was successfully applied for a pharmacokinetic study of PS916 in rabbits.     

Liquid chromatographic method for the quantitative determination of Nepsilon-carboxymethyllysine in human plasma proteins

The modification of the lysine moieties of proteins to Nepsilon-carboxymethyllysine (CML) is supposed to play a major role in the development of long-term complications in patients with diabetes mellitus. This paper presents an analytical method for the quantitative determination of CML in plasma proteins, which could be used for studying the development of diabetic complications. The method is based on isolating proteins from plasma by precipitation with trichloroacetic acid and hydrolysing these under acidic conditions (6M hydrochloric acid at 110 degrees C for 20 h) to the individual amino acids. After hydrolysis, CML is derivatised along with the other amino acids to 9-fluorenylmethoxycarbonyl (FMOC) derivatives, which are subsequently separated by reversed-phase column liquid chromatography using a 150 mm x 4.6 mm C8 column and a mobile phase of 25 mM potassium phosphate buffer (pH 2.0) and acetonitrile (80:20 (v/v)) and detected using fluorescence detection (excitation at 260 nm and emission at 310 nm). Quantification of the protein-bound CML content of a plasma sample is achieved using standard addition. The impact of several aspects of the sample preparation and chromatography on method performance is discussed. Method evaluation results are reported and show that this method is capable of determining CML with good accuracy and precision (below 10%) in the relevant concentration range (1-10 microg/ml), with a limit of detection of 0.2 microg/ml.     

Tet repressor-tetracycline interaction

Previous studies [Wasylewskiet al. (1996),J. Protein Chem. 15, 45–58] have shown that the W43 residue localized within the helix-turn-helix structure domain of Tet repressor can exist in the ground state in two conformational states. In this paper we investigate the fluorescence properties of W43 of TetR upon binding of tetracycline inducer and its chemical analogs such as anhydro- and epitetracycline. Binding of the drug inducer to the protein indicates that the W43 residue still exists in two conformational states; however, its environment changes drastically, as can be judged by the changes in fluorescence parameters. The FQRS (fluorescence-quenching-resolved spectra) method was used to decompose the total emission spectrum. The resolved spectra exhibit maxima of fluorescence at 346 and 332 nm and the component quenchable by KI (346 nm) is shifted 9 nm toward the blue side of the spectrum upon inducer binding. The observed shift does not result from the changes in the exposure of W43, since the bimolecular quenching rate constant remains the same and is equal to about 2.7×10⁹M^–1sec^–1. The binding of tetracycline leads to drastic decrease of the W43 fluorescence intensity and increase of the tetracycline intensity as well as the decrease of fluorescence lifetime, especially of the W43 component characterized by the emission at 332 nm. The observed energy transfer from W43 to tetracycline is more efficient for the state characterized by the fluorescence emission at 332 nm (88%) than for the component quenchable by iodide (53%) Tetracycline and several of its derivatives were also used to observe how chemical modifications of the hydrophilic groups in tetracycline influence the mechanism of binding of the antibiotic to Tet repressor. By use of pulsed-laser photoacoustic spectroscopy it is shown that the binding of tetracyclines to Tet repressor leads to significant increase of tetracycline fluorescence quantum yields. Steady-state fluorescence quenching of tetracycline analogs in complexes with Tet repressor using potassium iodide as a quencher allowed us to determine the dependence of the exposure of bound antibiotic on the modifications of hydrophilic substituents of tetracycline. Circular dichroism studies of the TetR-[Mg · tc]⁺ complex do not indicate dramatic changes in the secondary structure of the protein; however, the observed small decrease in the TetR helicity may occur due to partial unfolding of the DNA recognition helix of the protein. The observed changes may play an important role in the process of induction in which tetracycline binding results in the loss of specific DNA binding.Abbreviations FQRS fluorescence-quenching-resolved spectra - HTH helix-turn-helix motif - tc tetracycline - TetR tetracycline repressor from Escherichia coli - TetR WT wild-type TetR - TetR W43 single point mutant with phenylalanine substituted for tryptophan at position 75 in both subunits     

R-factor in Proteus vulgaris from ulcerative disease of fish,Channa punctatus

A Proteus vulgaris isolated from external ulcers of the fresh water fish Channa punctatus showed multidrug resistance and heavy metal tolerance. The isolate from the ulcer showed resistance to chloramphenicol (Ch), nalidixic acid (Nx), streptomycin (Str) and tetracycline (Tet) with minimum inhibitory concentration (MIC) values of 750, 150, 75 and 125 microg/ml, respectively. The isolate showed growth in medium containing cadmium (Cd2+), up to a concentration of 2.5 mM indicating its heavy metal tolerance. Resistance to Ch, Str, Tet and Cd2+ of the isolate was lost after plasmid curing. Presence of plasmid DNA in the wild type and its absence in the cured P. vulgaris suggested that the resistance were plasmid mediated.     

Cytoplasmic membrane polarization in Gram-positive and Gram-negative bacteria grown in the absence and presence of tetracycline

The ability of numerous diverse compounds and ions to cross the bacterial cytoplasmic membrane by diffusion and active transport is highly dependent on cytoplasmic membrane fluidity, which can be measured using fluorescent probes to estimate membrane polarization values. However, membrane polarization data are lacking for most bacterial species. The cytoplasmic membrane polarization values for Arthrobacter sp. ATCC 21908, Bacillus cereus NRC 3045, Pseudomonas fluorescens R2F, Pseudomonas putida NRC 2986 and Escherichia coli C600 bacterial cells were spectrofluorometrically measured over a temperature range from 10 to 50 degrees C, and in the absence and presence of 1 microg/ml tetracycline, using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to obtain new information on their membrane fluidity. At an assay temperature of 10 degrees C, E. coli cells grown in the absence of tetracycline exhibited the highest cytoplasmic membrane polarization value (least fluid membrane) of 0.446, followed by values of 0.392, 0.371, 0.344 and 0.293, respectively, for B. cereus, Arthrobacter sp., P. fluorescens and P. putida. At an assay temperature of 30 degrees C, the polarization values ranged from 0.357 to 0.288 for cells grown in the absence of tetracycline, regardless of the species. B. cereus grown in the presence of 1 microg/ml tetracycline had lower polarization values than when grown in the absence of this antibiotic at all assay temperatures. Regardless of the absence or presence of 1 microg/ml tetracycline in the growth medium, all bacterial species generally exhibited a more fluid membrane as the assay temperature increased from 10 to 50 degrees C. To our knowledge, these are some of the first cytoplasmic membrane polarization values reported for these Gram-negative and Gram-positive bacteria over a broad temperature range and also for cells grown in the presence of tetracycline.