Exploitation of the electro-optical characteristics of microbial suspensions for determining p-nitrophenol and microbial degradative activity

The effect of p-nitrophenol metabolism on the electrophysical properties of a microbial cell suspension of Acinetobacter calcoaceticum was investigated. The dependence of the suspension's optical density changes due to the orientation of the electric field on the frequency of an orienting electric field (orientational spectra) over the range 10–10 000 kHz was used. Upon optimization of cell culture conditions, followed by orientational spectrum measurements, it was found that the most dramatic changes in orientation spectrum associated with p-nitrophenol metabolism took place over the frequency range 10–1000 kHz. Additionally, a linear dependence of the magnitude of the electrooptical effect on p-nitrophenol concentrations of 0.1–0.8 mM was established. The minimum p-nitrophenol concentration detectable by this calibration was 0.1 mM.     

Changes in electrophysical characteristics of Azospirillum brasilense Sp7 cells during their interaction with polyclonal antibodies

Electrooptical characteristics of Azospirillum brasilense Sp7 cells during their specific interaction with polyclonal rabbit antibodies were studied. Dependence of optical density of cell suspension during electroorientation of cells from frequency of orienting field in interval 10, 100, 250, and 500 kHz was evaluated. Itwas shown that electrooptical (EO) characteristics of bacterial suspensions change during interaction of A. brasilense cells with antibodies, and maximal changes occur when frequency of oriented field amounts 100-250 kHz. During interaction of A. brasilense Sp7 with strain-specific polyclonal antibodies in the presence of Escherichia coli K-12 and Pseudomonas putida C-11 decrease of amplitude of analytic signal was observed but detection of A. brasilense Sp7 cells was possible. Possibility of detection of microorganisms by EO analysis during their interaction with antibodies was shown.     

The respiratory activity of Rhodococcus rhodochrous M8 cells producing nitrile-hydrolyzing enzymes]

The respiratory activity of Rhodococcus rhodochrous M8 cells containing nitrile hydratase and amidase was studied in the presence of nitriles and amides of carbonic acids. Culturing of cells with acrylonitrile and acrylamide yielding their maximum respiratory activity was studied. The optimum conditions for measurements and maintenance of respiratory activity were found. Curves for the linear concentration dependence of cell respiratory activity on 0.01-0.5 mM acrylonitrile, 0.025-1.0 mM acetonitrile, and 0.01-0.1 mM acrylamide were plotted. The selectivity of cell respiratory activity for some substrates was analyzed.     

Association of nicotinic acetylcholine receptors with central respiratory control in isolated brainstem-spinal cord preparation of neonatal rats

Nicotine exposure is a risk factor in several breathing disorders Nicotinic acetylcholine receptors (nAChRs) exist in the ventrolateral medulla, an important site for respiratory control. We examined the effects of nicotinic acetylcholine neurotransmission on central respiratory control by addition of a nAChR agonist or one of various antagonists into superfusion medium in the isolated brainstem-spinal cord from neonatal rats. Ventral C4 neuronal activity was monitored as central respiratory output, and activities of respiratory neurons in the ventrolateral medulla were recorded in whole-cell configuration. RJR-2403 (0.1-10 mM), alpha4beta2 nAChR agonist induced dose-dependent increases in respiratory frequency. Non-selective nAChR antagonist mecamylamine (0.1-100 mM), alpha4beta2 antagonist dihydro-beta-erythroidine (0.1-100 mM), alpha7 antagonist methyllycaconitine (0.1-100 mM), and a-bungarotoxin (0.01-10 mM) all induced dose-dependent reductions in C4 respiratory rate. We next examined effects of 20 mM dihydro-beta-erythroidine and 20mM methyllycaconitine on respiratory neurons. Dihydro-beta-erythroidine induces hyperpolarization and decreases intraburst firing frequency of inspiratory and preinspiratory neurons. In contrast, methyllycaconitine has no effect on the membrane potential of inspiratory neurons, but does decrease their intraburst firing frequency while inducing hyperpolarization and decreasing intraburst firing frequency in preinspiratory neurons. These findings indicate that alpha4beta2 nAChR is involved in both inspiratory and preinspiratory neurons, whereas alpha7 nAChR functions only in preinspiratory neurons to modulate C4 respiratory rate.     

An investigation into the electro-physical characteristics of microbial cells during the metabolism of toxic compounds under conditions of limited O2 availability

The purpose of the work reported here was to experimentally clarify the interconnection between changes in the electro-physical characteristics of microbial suspensions and processes of the metabolism of certain toxic compounds (acrylamide and p-nitrophenol (PNP)) in cells containing enzyme systems of the initial metabolism of these compounds. In this work, we used cells of two strains, Acinetobacter calcoaceticum A-122 and Brevibacterium sp. 13PA, which are capable of utilising PNP and acrylamide, respectively, as the sole source of carbon. Suspensions of these cells exhibited appreciable decreases in the magnitude of the electro-optical signal when the microbial metabolism of PNP and acrylamide was inhibited under conditions of limited O2 supply. This attests to a relationship between the electro-physical characteristics of microbial suspensions and cellular metabolic reactions and also to the negligibly small effect that the non-specific interaction of substrate with the cells has on the suspensions' electro-optical properties.     

Determination of organophosphorus aromatic nitro insecticides and p-nitrophenol by microbial-cell respiratory activity

We examined the possibility of measuring the organophosphorus aromatic nitro insecticides metaphos and sumithion as well as their hydrolysis product p-nitrophenol (PNP) by the specific respiratory activity (SRA) of Pseudomonas putida C-11, P. putida BA-11, and Acinetobacter calcoaceticum A-122. The plots of cellular SRA against the two insecticides and PNP were linear over the ranges of 0.5-2.5 microM for P. putida C-11 and BA-11 and 0.5-1.0 microM for A. calcoaceticum A-122. P. putida BA-11 showed the greatest respiratory-response selectivity in the determination of the test substrates. We made comparison studies of the SRA of cells immobilised by two methods: carrier-surface adsorption and inclusion in various gels. We discuss the feasibility of developing a microbial sensor system for the determination of metaphos, sumithion, and PNP in aqueous media.     

Microcalorimetric studies of hybridoma cells

In the present study, overall metabolism has been estimated in hybridoma cells by microcalorimetric measurement. Heat production rate was found to be 30-50 pW/cell at cell concentrations 0.65-4.5 x 10(5)/ml. High cell concentrations (1 x 10(6) cells/ml) caused unstable power-curves with an initial high peak and a rapid declining phase, whereas low cell concentrations (0.6-4.5 x 10(5) cells/ml) produced steady-state power-curves. Oxygen consumption was found to range between 1.5-6.1 x 10(-5) mol 02/cell/min, corresponding to about 80% of the total metabolic activity. The metabolic inhibitors sodium fluoride (50 nM), sodium azide (160 mM) and rotenone (0.1 mM) caused a reduction in overall cell metabolism of 60, 55 and 40% respectively.     

Cyanide sensitive and insensitive bioenergetics in a clonal neuroblastoma x glioma hybrid cell line

The primary mechanism of cyanide (CN) intoxication is the inhibition of metabolism in the central nervous system. We determined the effects of CN on several biochemical processes in neuroblastoma x glioma hybrid NG108-15 cells, which possess numerous neuronal properties. These cells were not sensitive to a high concentration (1 mM) of NaCN, but became sensitive in the presence of the anaerobic glycolysis inhibitors sodium iodoacetate (IA) and 2-deoxyglucose (2-DG): cellular metabolic processes (e.g., DNA, RNA and protein synthesis) decreased, to about 40% of control due to treatment with 0.5 mM NaCN+0.05 mM IA and 0.1 mM NaCN+20 mM 2-DG. ATP in cells exposed to 0.01 or 0.1 mM NaCN+20 mM 2-DG was reduced 75% and 100%, respectively within one min. Pretreatment of cells with the CN antidote cobalt (II) chloride (CoCl₂) (0.06–0.18 mM) for 5 min prevented the depression of both [³H]leucine incorporation and ATP synthesis due to 1 mM NaCN+20 mM 2-DG in a concentration-dependent manner. A proposed CN antidote alpha-ketoglutaric acid (disodium salt) also prevented the depression of cellular metabolism due to NaCN plus 2-DG. These results indicate that blocking anaerobic glycolysis makes NG108-15 cells sensitive to a low concentration of CN. Thus NG108-15 cells should be useful to study the mechanisms of neurotoxicity of CN and to test antidotes.     

Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy

Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150+/-10 kDa, regardless of the conditions investigated-protein concentration, 0.18-1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4-20 degrees C. The parameters obtained from the self-associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 microg/mL-1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s(0)20,w=7.7+/-0.3 and 8.3+/-0.4 S, respectively. The molecular mass of the sedimenting particle (146+/-30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single-step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.     

Measurement of myosin adenosinetriphosphatase and myosin content in cultured heart cells

An assay specific for myosin ATPase in whole-cell extracts of cultured heart cells has been developed. Myosin ATPase is measured by the production of Pi from ATP in the presence of high ionic strength (0.5 M KCl) at pH 9.1. Enzyme activity is maximal with 10 mM CaCl2 and completely inhibited with 5 mM MgCl2. Spontaneously beating myocytes grown in the presence of 10% newborn calf serum and 0.1 mM 5-bromo-2'-deoxyuridine show a significant rise in myosin ATPase between Days 1 and 4 in culture. The measurement of myosin ATPase allows for the quantitation of cellular myosin content, and can be used to assess changes in myosin content that occur during growth, development, and cellular repair.     

Thymbra capitata essential oil has a significant antimicrobial activity against methicillin-resistant Staphylococcus aureus pre-formed biofilms

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant opportunistic pathogen with a great ability to form biofilms. Herein, the antimicrobial potential of Thymbra capitata essential oil (EO) against MRSA biofilms was investigated. The determination of the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC) of the T. capitata EO was first investigated on a group of clinical isolates from septicaemias, diabetic foot ulcers and osteomyelitis. Biofilms were incubated with the EO at the MLC and its anti-biofilm potential was investigated. A strong antimicrobial activity was observed, with MIC and MLC values between 0·32 and 0·64 mg l⁻¹. However, the concentration of EO necessary for the eradication of planktonic cells was insufficient to significantly reduce the biofilm biomass of some isolates. Nevertheless, cell culturability and overall cellular metabolism was strongly reduced in all biofilms tested, only when the EO was tested. Contrary to the tested antibiotics, T. capitata EO showed a significant antimicrobial activity against MRSA biofilms, by reducing cellular metabolism and cellular culturability.     

Dynamics of the changes of electrophysical properties of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7 cells at their binding with wheat germ agglutinin

The electrooptical properties of Azospirillum brasilense Sp7 cell suspensions, have been studied at a specific interaction with wheat germ agglutinin (WGA), using the dependences between the changes of optical densities of cell suspensions at the electric orientation of cells and the orienting field frequencies of 740, 1000, 1450, 2000, and 2800 kHz. It was shown that the electrooptical (EO) properties of cell suspensions changed at the interaction of A. brasilense Sp7 cells with WGA and that the EO signal value changed irrespective of the cultivation conditions. At the same time, the dynamics of the changes of the EO properties of microbial suspensions was different for microbial cells grown under different conditions. It may be evidence of the differences in the cell surface properties of microbial cells, and of the dependence, between bacterial response to lectin and growth conditions. The possibility of using the EO analysis of bacterial suspensions for the study of the high-specific binding of polypeptide molecular signals with the bacterial target cells and for assessment of the dynamics of this process has been demonstrated.     

Electrooptic analysis of Escherichia coli cell suspension for assessment of antibacterial activity of levomycetin

Influence of chloramphenicol on electrophysiologic charateristics of Escherichia coli strains susceptible (K-12 strain) and resistant (pBR-325 strain) to it has been studied. It has been shown that incubation of susceptible bacteria with chloramphenicol leads to significant change of magnitute of electrooptic (EO) signal. Significant changes in orientantional spectra of suspensions of susceptible to chloramphenicol cells incubated with different concentrations of antibiotic were observed only on first five frequencies of orienting electric field (10 - 1000 kHz). Maximal change of EO signal occurred at chloramphenicol concentration 35 mg/ml and it didn't depend on the time of antibiotic exposure. Incubation of resistant strain pBR-325 with chloramphenicol did not lead to change of EO parameters of cell suspension. Potential for use of electrophysical analytic methods for assessment of antibacterial activity of chloramphenicol to control effect of antibiotics on microorganisms has been proposed.     

Taurine enhances low density lipoprotein binding. Internalization and degradation by cultured Hep G2 cells

To evaluate the impact of taurine on hepatic cholesterol catabolism low density lipoprotein (LDL) binding, internalization and degradation were measured in cultured Hep G2 cells. Preincubation of cells with 0.1-10 mM taurine for 24 h stimulated LDL receptor activity by as much as 100%. Only the high affinity LDL receptor activity (specific) was increased by taurine preincubation, whereas the low affinity receptor activity (nonspecific) remained unchanged. Scatchard analysis of the binding data revealed that taurine doubled the number of LDL receptors without affecting receptor affinity. Taurine-enhanced LDL receptor activity was most pronounced when LDL concentrations exceeded 100 micrograms/ml, but was noted at taurine concentrations as low as 0.1 mM (plasma level). Interestingly, taurine had no effect on LDL receptor activity when it was added simultaneously with 125I-LDL to Hep G2 cells, or when non-bile acid-producing human skin fibroblasts were tested. Stimulation of LDL receptor activity was also obtained with 10 mM cysteine, a taurine precursor, but not with glycine. Increased cellular concentrations of taurine and cysteine were associated with an elevated rate of bile acid synthesis and a reduced cellular free cholesterol concentration. The data suggest that taurine enhanced LDL receptor activity by sparing cysteine, a known sulfhydryl group donor and stimulator of 7 alpha-hydroxylase activity, and that the latter stimulated bile acid production leading to increased utilization of cellular free cholesterol and enhanced LDL uptake.     

Modulation of the cough reflex by antitussive agents within the caudal aspect of the nucleus tractus solitarii in the rabbit

We have previously shown that ionotropic glutamate receptors in the caudal portion of the nucleus tractus solitarii (NTS), especially in the commissural NTS, play a prominent role in the mediation of tracheobronchial cough and that substance P potentiates this reflex. This NTS region could be a site of action of some centrally acting antitussive agents and a component of a drug-sensitive gating mechanism of cough. To address these issues, we investigated changes in baseline respiratory activity and cough responses to tracheobronchial mechanical stimulation following microinjections (30-50 nl) of centrally acting antitussive drugs into the caudal NTS of pentobarbitone-anesthetized, spontaneously breathing rabbits. [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and baclofen decreased baseline respiratory frequency because of increases in the inspiratory time only at the higher concentration employed (5 mM and 1 mM, respectively). DAMGO (0.5 mM) and baclofen (0.1 mM) significantly decreased cough number, peak abdominal activity, peak tracheal pressure, and increased cough-related total cycle duration. At the higher concentrations, these agents suppressed the cough reflex. The effects of these two drugs were counteracted by specific antagonists (10 mM naloxone and 25 mM CGP-35348, respectively). The neurokinin-1 (NK1) receptor antagonist CP-99,994 (10 mM) abolished cough responses, whereas the NK2 receptor antagonist MEN 10376 (5 mM) had no effect. The results indicate that the caudal NTS is a site of action of some centrally acting drugs and a likely component of a neural system involved in cough regulation. A crucial role of substance P release in the mediation of reflex cough is also suggested.     

The electro-optical investigation of suspensions of Escherichia coli K-12 cells metabolizing glucose,lactose, and galactose

The electro-optical characteristics of suspensions of Escherichia coli K-12 cells metabolizing glucose, lactose, and galactose were studied my measuring the suspension turbidity as a function of cell alignment in an orienting electric field whose frequency was varied from 10 kHz to 10 MHz. In a frequency range of 10 kHz to 1 MHz, the orientational spectra of E. coli K-12 cells grown on glucose and lactose considerably changed after their incubation in the presence of the sugars. These changes likely reflect alterations in the polarizability of the cells induced by sugar metabolism.     

Modulation of Neuronal Signal Transduction Systems by Extracellular ATP

The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 microM. The present study examined the effects of extracellular ATP in the concentration range of 0.1-1.0 mM on second-messenger-generating systems in cultured neural cells of the clones NG108-15 and N1E-115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin-2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCl. Extracellular ATP induced a concentration-dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin-2 signals. At the same concentration range (0.1-1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1-stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15-fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG108-15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half-maximal effects on Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oligomeric structure of mammalian purine nucleoside phosphorylase in solution determined by analytical ultracentrifugation

The influence of phosphate, ionic strength, temperature and enzyme concentration on the oligomeric structure of calf spleen purine nucleoside phosphorylase (PNP) in solution was studied by analytical ultracentrifugation methods. Sedimentation equilibrium analysis used to directly determine the enzyme molecular mass revealed a trimeric molecule with Mr = (90.6 +/- 2.1) kDa, regardless the conditions investigated: protein concentration in the range 0.02-1.0 mg/ml, presence of up to 100 mM phosphate and up to 200 mM NaCl, temperature in the range 4-25 degrees C. The sedimentation coefficient (6.04 +/- 0.02) S, together with the diffusion coefficient (6.15 +/- 0.11) 10(-7) cm2/s, both values obtained from the classic sedimentation velocity method at 1.0 mg/ml PNP concentration in 20 mM Hepes, pH 7.0, yielded a molecular mass of (90.2 +/- 1.6) kDa as expected for the trimeric enzyme molecule. Moreover, as shown by active enzyme sedimentation, calf spleen PNP remained trimeric even at low protein concentrations (1 microg/ml). Hence in solution, similar like in the crystalline state, calf spleen PNP is a homotrimer and previous suggestions for dissociation of this enzyme into more active monomers, upon dilution of the enzyme or addition of phosphate, are incorrect.     

In vitro modulation of proliferation and melanization of melanoma cells by citrate

B16/F10 murine melanoma cells were grown for 24 and 36 h in Dulbecco's modified Eagle medium in presence of 10-20 mM trisodium citrate. The intracellular melanin concentration and the melanin secreted in the extracellular medium was estimated. It is observed that 20 mM citrate stimulates extracellular melanin secretion in B16/F10 melanoma cells by 200% at 36 h treatment. The intracellular melanin content increased by 90%. This stimulatory effect of citrate was totally abolished when these cells were grown in presence of 1 mM phenyl thiourea, a specific inhibitor of tyrosinase activity. Citrate (0.1-5 mM) had no effect on dopa oxidase activity either at pH 5.0 or at pH 6.8. There was no increase in the tyrosinase specific activity in presence of citrate. The increased melanin synthesis was shown to be due to stimulation of cellular tyrosine hydroxylase activity by citrate. It has been suggested that enhanced melanin synthesis results in an increased production of metabolites that are toxic to the growth of melanoma cells. We have studied the effect of citrate on cellular proliferation. Following 24 and 36 h treatment with citrate, the cells exhibited a dose-dependent decrease in proliferation. In presence of 20 mM citrate the cell number was only up to 50% of the control cultures after 36 h of incubation. The growth retardation was not due to cytotoxicity. Citrate, a natural metabolite, is a unique molecule which may be involved in the regulation of melanin biosynthetic pathway, since it enhances melanogenesis by increasing the hydroxylase activity of tyrosinase which is the regulatory enzyme of this pathway. These observations add further support to the critical role of intramelanosomal pH in regulation of melanogenesis.     

L-Carnitine alters nitric oxide synthase activity in fibroblasts depending on the peroxisomal status

Fibroblast cellular models are widely used for research on fatty acid metabolism. Due to the importance of L-carnitine in intermediary metabolism we studied the effects of L-carnitine on healthy human skin fibroblasts and fibroblasts without functional peroxisomes (Zellweger Syndrome) cultivated under carnitine deficiency, which is caused by standard media compositions. The application of physiological (0.1mM) or super-physiological (1mM) doses of L-carnitine causes a significant decrease of the specific activity of nitric oxide synthase (NOS, 2.25+/-0.10 to 1.36 pmol/(minmg)+/-0.09 pmol/(minmg) at 0.1mM), proliferation and a tendentious decrease of the antioxidant defence potential against hydrogen peroxide only in control cells. Simultaneous application of L-carnitine and 100 micro M N-acetylcysteine (NAC) prevents the alterations in control cells. Thus, L-carnitine alters the cellular regulation of the NOS probably by reactive oxygen species (ROS), which suggests that carnitine deficient media neither reflect physiological conditions for cellular models for fatty acid metabolism nor for the regulation of NOS.