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The Journal of Experimental Medicine Sep 1945The time of blending the rickettsial inoculum, as also the strain of hen's egg employed, influenced the degree of infection which developed in fertile eggs after the...
The time of blending the rickettsial inoculum, as also the strain of hen's egg employed, influenced the degree of infection which developed in fertile eggs after the injection of typhus rickettsiae into the yolk sac. By varying these factors, maximal or minimal infections could be obtained. Eggs incubated at 40 degrees C. developed only minimal rickettsial infection, whereas control eggs incubated at 37.5 degrees C. became heavily infected. Potassium cyanide markedly enhanced rickettsial growth in experiments in which the control eggs developed only minimal infection. Under circumstances such that the control eggs became heavily infected, KCN had no appreciable effect. Toluidin blue and methylene blue delayed the development of rickettsial infection in the yolk sac, but their rickettsiostatic action under the conditions of these experiments was less marked than that of penicillin and para-aminobenzoic acid. The rickettsiostatic action resulting from temperature elevation was neutralized by KCN, and hence is believed to be due to the increased activity of the cyanide-sensitive respiratory enzyme (cytochrome oxidase) in the entodermal cells in which the rickettsiae multiply. The rickettsiostatic action of toluidin blue and methylene blue, though probably also resulting from increased metabolic activity in the entodermal cells, was not neutralized by KCN. This observation is in harmony with the reported observation that dyes of this type furnish an alternative mechanism for intracellular oxidation which is cyanide-insensitive. The rickettsiostatic action of para-aminobenzoic acid was not neutralized by KCN. No conclusions can be reached at present concerning the mechanism of action of this compound. Dinitrophenol and several compounds related to para-aminobenzoic acid gave negative results.
PubMed: 19871494
DOI: 10.1084/jem.82.3.193 -
Military Medicine Jun 2009Currently, no reliable noninvasive methods exist for monitoring the severity of in vivo cyanide (CN) toxicity, treatment, and resulting physiological changes. We...
Currently, no reliable noninvasive methods exist for monitoring the severity of in vivo cyanide (CN) toxicity, treatment, and resulting physiological changes. We developed a broadband diffuse optical spectroscopy (DOS) system to measure bulk tissue absorption and scattering. DOS was used to optically monitor CN toxicity and treatment with sodium nitrite (NaNO2). To perform experiments, the DOS probe was placed on the hind leg of rabbits. A sodium CN solution was infused intravenously. DOS and concurrent physiologic measurements were obtained. After completion of CN infusion, NaNO2 was infused to induce methemoglobinemia (MetHb). During infusion of CN, blood gas measurements showed an increase in venous partial pressure of oxygen (pO2), and following reversal, venous pO2 values decreased. DOS measurements demonstrated corresponding changes in hemoglobin oxygenation states and redox states of cytochrome-c oxidase (CcO) during CN infusion and NaNO2 treatment. Therefore, DOS enables detection and monitoring of CN toxicity and treatment with NaNO2.
Topics: Animals; Blood Gas Analysis; Electron Transport Complex IV; Feasibility Studies; Hematinics; Hemoglobins; Hydroxocobalamin; Indicators and Reagents; Male; Methemoglobin; Models, Animal; Oximetry; Oxyhemoglobins; Poisoning; Potassium Cyanide; Rabbits; Sodium Nitrite; Spectrophotometry, Infrared; Spectrum Analysis; Statistics as Topic; Toxicity Tests, Acute
PubMed: 19585775
DOI: 10.7205/milmed-d-02-7408 -
Journal of Photochemistry and... Apr 2009Reduced nicotinamide adenine dinucleotide, NADH, is a major electron donor in the oxidative phosphorylation and glycolytic pathways in cells. As a result, there has been...
Reduced nicotinamide adenine dinucleotide, NADH, is a major electron donor in the oxidative phosphorylation and glycolytic pathways in cells. As a result, there has been recent resurgence in employing intrinsic NADH fluorescence as a natural probe for a range of cellular processes that include apoptosis, cancer pathology, and enzyme kinetics. Here, we report on two-photon fluorescence lifetime and polarization imaging of intrinsic NADH in breast cancer (Hs578T) and normal (Hs578Bst) cells for quantitative analysis of the concentration and conformation (i.e., free-to-enzyme-bound ratios) of this coenzyme. Two-photon fluorescence lifetime imaging of intracellular NADH indicates sensitivity to both cell pathology and inhibition of the respiratory chain activities using potassium cyanide (KCN). Using a newly developed non-invasive assay, we estimate the average NADH concentration in cancer cells (168+/-49 microM) to be approximately 1.8-fold higher than in breast normal cells (99+/-37 microM). Such analyses indicate changes in energy metabolism and redox reactions in normal breast cells upon inhibition of the respiratory chain activity using KCN. In addition, time-resolved associated anisotropy of cellular autofluorescence indicates population fractions of free (0.18+/-0.08) and enzyme-bound (0.82+/-0.08) conformations of intracellular NADH in normal breast cells. These fractions are statistically different from those in breast cancer cells (free: 0.25+/-0.08; bound: 0.75+/-0.08). Comparative studies on the binding kinetics of NADH with mitochondrial malate dehydrogenase and lactate dehydrogenase in solution mimic our findings in living cells. These quantitative studies demonstrate the potential of intracellular NADH dynamics (rather than intensity) imaging for probing mitochondrial anomalies associated with neurodegenerative diseases, cancer, diabetes, and aging. Our approach is also applicable to other metabolic and signaling pathways in living cells, without the need for cell destruction as in conventional biochemical assays.
Topics: Anisotropy; Cell Line; Fluorescent Dyes; Humans; L-Lactate Dehydrogenase; Malate Dehydrogenase; Mammary Glands, Human; Microscopy, Fluorescence, Multiphoton; NAD; Oxidation-Reduction; Potassium Cyanide
PubMed: 19179090
DOI: 10.1016/j.jphotobiol.2008.12.010 -
Journal of Bacteriology May 1984Purified Rickettsia prowazekii cells were able to transport L-proline. The influx of this amino acid had a Kt of 14 microM and a Vmax of about 64 pmol/min per mg of...
Purified Rickettsia prowazekii cells were able to transport L-proline. The influx of this amino acid had a Kt of 14 microM and a Vmax of about 64 pmol/min per mg of protein. Proline could not be transported by heat-killed or metabolically poisoned rickettsiae or at 0 degrees C. The uptake of proline was linear for almost 2 h. More than 90% of the accumulated intracellular radioactivity was proline. This intracellular pool could not be chased out of the cell by excess non-radioactive proline and did not exit into a proline-free medium. These results indicate that intracellular proline was bound or that the cell had a very limited efflux component for proline transport. The influx of proline was specific: among various analogs tested, only 3,4-dehydro-D,L-proline was effective in inhibiting proline uptake. R. prowazekii cells were unable to utilize proline as an energy source to drive hemolysis, and no measurable evolution from the rickettsiae of CO2 derived from proline occurred. The activities of the enzymes pyrroline-5-carboxylate-reductase and pyrroline-5-carboxylate dehydrogenase were not detectable. These enzymes are important in anabolism and catabolism of proline, respectively, and, if present in R. prowazekii have activities less than 1% of those in Escherichia coli.
Topics: 1-Pyrroline-5-Carboxylate Dehydrogenase; 2,4-Dinitrophenol; Biological Transport, Active; Dinitrophenols; Ethylmaleimide; Hemolysis; Kinetics; Oxidoreductases Acting on CH-NH Group Donors; Potassium Cyanide; Proline; Pyrroline Carboxylate Reductases; Rickettsia prowazekii
PubMed: 6427180
DOI: 10.1128/jb.158.2.460-463.1984 -
FEBS Letters May 1986The relationship between the rate of glutathione disulfide (GSSG) export and the energy state was studied in isolated perfused rat heart. The intracellular GSSG level...
The relationship between the rate of glutathione disulfide (GSSG) export and the energy state was studied in isolated perfused rat heart. The intracellular GSSG level was maintained at saturation for transport (7.5 nmol GSSG X min-1 X g heart-1) by continuous perfusion with 20 microM t-butyl hydroperoxide. GSSG release was substantially restricted upon the addition of inhibitors of mitochondrial respiration such as KCN, antimycin A or rotenone. In contrast, no effect was observed on GSSG release during potassium-induced cardiac arrest, although changes in oxygen consumption and coronary flow were similar to those observed with KCN. The dependence of the GSSG transport rate on the cytosolic free ATP/ADP ratio reveals that GSSG transport is half-maximal at (ATP/ADP)free approximately equal to 10. The capacity of GSSG transport was unchanged by infusion of epinephrine, norepinephrine or dibutyryl cyclic AMP.
Topics: Adenine Nucleotides; Animals; Antimycin A; Energy Metabolism; Glutathione; Glutathione Disulfide; Heart Arrest; Hydrogen-Ion Concentration; Kinetics; Magnetic Resonance Spectroscopy; Male; Mitochondria, Heart; Myocardium; Perfusion; Peroxides; Potassium Cyanide; Rats; Rats, Inbred Strains; tert-Butylhydroperoxide
PubMed: 3699157
DOI: 10.1016/0014-5793(86)80524-5 -
American Journal of Physiology.... Jan 2002To identify specific transporters that drive xenobiotics from the central nervous system to blood, the accumulation of fluorescent drugs was studied in isolated...
To identify specific transporters that drive xenobiotics from the central nervous system to blood, the accumulation of fluorescent drugs was studied in isolated capillaries from killifish and dogfish shark brain using confocal microscopy and quantitative image analysis. In killifish brain capillaries, luminal accumulation of fluorescent derivatives of cyclosporin A and verapamil was concentrative, specific, and energy dependent (inhibition by KCN). Transport was reduced by PSC-833, but not by leukotriene C4, indicating the involvement of P-glycoprotein. The ability of capillaries to transport the cyclosporin A derivative was unchanged over 20 h, demonstrating the long-term viability of the preparation. Luminal accumulation of the fluorescent organic anions sulforhodamine 101 and fluorescein-methotrexate was also concentrative, specific, and energy dependent. Transport of these compounds was reduced by leukotriene C4, but not by PSC-833, indicating the involvement of a multidrug resistance-associated protein (Mrp). Similar results were obtained for isolated capillaries from dogfish shark. Immunostaining localized P-glycoprotein and Mrp2 to the luminal surface of the killifish brain capillary endothelium. These findings validate a new and long-lived comparative model for studying drug transport across the blood-brain barrier and, as in mammals, implicate P-glycoprotein and Mrp2 in transport from the central nervous system to blood in fish.
Topics: 4-Chloro-7-nitrobenzofurazan; ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Blood-Brain Barrier; Boron Compounds; Brain; Calcium Channel Blockers; Capillaries; Cyclosporine; Cyclosporins; Dogfish; Enzyme Inhibitors; Fluorescent Dyes; Fundulidae; In Vitro Techniques; Indicators and Reagents; Leukotriene C4; Membrane Transport Proteins; Microscopy, Confocal; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Potassium Cyanide; Verapamil; Xenobiotics
PubMed: 11742838
DOI: 10.1152/ajpregu.00305.2001 -
The Journal of Cell Biology Nov 1969Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3'-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium...
Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3'-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium tetroxide. Electron microscopic observation of treated tissues revealed pronounced deposition of a highly electron-opaque material in microbodies but not in other organelles. The coarsely granular reaction product is presumably osmium black formed by reaction of oxidized DAB with osmium tetroxide. Reaction of the microbodies with DAB was completely inhibited by 0.02 M 3-amino-1,2,4-triazole and was considerably reduced by 0.01 M potassium cyanide. These results, when considered in light of recent biochemical studies, strongly suggest that catalase is responsible for the reaction. Sharp localization of this enzyme in microbodies establishes that they are identical to the catalase-rich "peroxisomes" recently isolated from leaf cell homogenates. A browning reaction that occurred in leaves during the incubation step was inhibited by cyanide but not by aminotriazole and therefore could not have been caused by the same enzyme. This reaction and a slight deposition of dense material within primary and secondary walls are ascribed to oxidation of DAB by soluble and wall-localized peroxidases.
Topics: Catalase; Cyanides; Histocytochemistry; Hydrogen Peroxide; Microscopy, Electron; Microsomes; Osmium; Peroxidases; Plant Cells; Plants; Plants, Toxic; Nicotiana; Triazoles; p-Dimethylaminoazobenzene
PubMed: 4981071
DOI: 10.1083/jcb.43.2.343 -
Proceedings of the National Academy of... Oct 1993The bacterio-opsin gene was introduced into a "blind" Halobacterium salinarium mutant that (i) lacked all the four retinal proteins [bacteriorhodopsin (BR),...
The bacterio-opsin gene was introduced into a "blind" Halobacterium salinarium mutant that (i) lacked all the four retinal proteins [bacteriorhodopsin (BR), halorhodopsin, and sensory rhodopsins (SRs) I and II] and the transducer protein for SRI and (ii) showed neither attractant response to long wavelength light nor repellent response to short wavelength light. The resulting transformed cells acquired the capability to sense light stimuli. The cells accumulated in a light spot, demonstrating the BR-mediated orientation in spatial light gradients. As in wild-type cells, a decrease in the intensity of long wavelength light caused a repellent response by inducing reversals of swimming direction, but, in contrast to wild-type cells, a decrease in the intensity of short wavelength light also repelled the cells. An increase in light intensity evoked an attractant response (i.e., a transient suppression of spontaneous reversals). Signal processing times and adaptation kinetics were similar to the SRI-mediated reactions. However, compared to SR-mediated photoresponses, higher light intensities were necessary to induce the BR-mediated responses. The light sensitivity of the transformant was increased by adding 1 mM cyanide and decreased by the addition of arginine, agents that respectively reduce and increase the light-independent generation of the electrochemical potential difference of H+ ions (delta mu H+). A decrease in irradiance to an intensity that was still high enough to saturate BR-initiated delta mu H+ changes failed to induce the repellent effect, but the addition of a protonophorous uncoupler sensitized the cell to these light stimuli. The BR D96N mutant (Asp-96 is replaced by Asn) with decreased proton pump activity showed strongly reduced BR-mediated responses. Azide, which increases this mutant's H+ pump efficiency, increased the photosensitivity of the mutant cells. Moreover, azide diminished (i) the membrane potential decreasing and (ii) repellent effects of blue light added to the orange background illumination in this mutant. We conclude that the BR-mediated photoreception is due to the light-dependent generation of delta mu H+. Our data are consistent with the assumption that the H. salinarium cell monitors the membrane energization level with a "protometer" system measuring total delta mu H+ changes or its electric potential difference component.
Topics: Bacteriorhodopsins; Halobacterium salinarum; Hydrogen-Ion Concentration; In Vitro Techniques; Light; Membrane Potentials; Mutagenesis; Photoreceptor Cells; Potassium Cyanide; Transformation, Genetic
PubMed: 8415720
DOI: 10.1073/pnas.90.20.9446 -
Neurotoxicity Research Oct 2013Cyanate is formed mostly during nonenzymatic urea biodegradation. Its active form isocyanate reacts with protein -NH2 and -SH groups, which changes their structure and...
Cyanate is formed mostly during nonenzymatic urea biodegradation. Its active form isocyanate reacts with protein -NH2 and -SH groups, which changes their structure and function. The present studies aimed to investigate the effect of cyanate on activity of the enzymes, which possess -SH groups in the active centers and are implicated in anaerobic cysteine transformation and cyanide detoxification, as well as on glutathione level and peroxidative processes in different brain structures of the rat: cortex, striatum, hippocampus, and substantia nigra. In addition, we examined whether a concomitant treatment with lipoate, a dithiol that may act as a target of S-carbamoylation, can prevent these changes. Cyanate-inhibited sulfurtransferase activities and lowered sulfide level, which was accompanied by a decrease in glutathione concentration and elevation of reactive oxygen species level in almost all rat brain structures. Lipoate administered in combination with cyanate was able to prevent the above-mentioned negative cyanate-induced changes in a majority of the examined brain structures. These observations can be promising for chronic renal failure patients since lipoate can play a double role in these patients contributing to efficient antioxidant defense and protection against cyanate and cyanide toxicity.
Topics: Animals; Brain; Glutathione; Male; Potassium Cyanide; Rats; Rats, Wistar; Reactive Oxygen Species; Thioctic Acid
PubMed: 23625581
DOI: 10.1007/s12640-013-9395-2 -
Acta Poloniae Pharmaceutica 2015The oxidation of cyanide (CN-) to a much less toxic thiocyanate (SCN-) is the main in vivo biochemical pathway for CN- detoxification. SCN- is excreted mainly in urine....
The oxidation of cyanide (CN-) to a much less toxic thiocyanate (SCN-) is the main in vivo biochemical pathway for CN- detoxification. SCN- is excreted mainly in urine. This study was performed to investigate the effect of lipoic acid (LA) on the urinary excretion of thiocyanate (SCN-; rhodanate) in rats. Groups of the animals were treated intraperitoneally (i.p.) as follows: group 1: potassium cyanide (KCN) (1 mg/kg); group 2: KCN (1 mg/kg) + LA (100 mg/kg). Urine was collected for 24 h and the pooled samples were examined for SCN- levels. The obtained results indicated that the treatment of animals with potassium cyanide and LA in combination significantly increased the urinary excretion of SCN- in comparison with the respective values in the KCN-alone-treated group. It indicates that LA increased the rate of CN- detoxification in rats.
Topics: Animals; Male; Potassium Cyanide; Rats; Rats, Wistar; Thioctic Acid; Thiocyanates
PubMed: 25850200
DOI: No ID Found