Decay of incorporated radioactive phosphorus during reproduction of bacteriophage T2

The multiplication of vegetative T2 bacteriophage in B/r bacteria has been followed by studying the lethal effects of decay of incorporated radiophosphorus P³² at various stages of the eclipse period. Experiment I. Non-radioactive B/r bacteria were infected with highly radioactive (i.e. P³²-unstable) T2 and infection allowed to proceed at 37°C. for various numbers of minutes before freezing the infected cells and storing them in liquid nitrogen. The longer development had been allowed to proceed at 37°C. before freezing, the slower the inactivation of the frozen infective centers by P³² decay. Samples which were frozen after incubation for 9 minutes were completely stable. Experiment II. Radioactive B/r bacteria in radioactive growth medium were infected with non-radioactive (i.e. stable) T2 and incubated for various lengths of time before being frozen and stored in liquid nitrogen, like those of Experiment I. In this case, the infective centers were stable to P³² decay as long as they were frozen before the end of the eclipse period. The T2 progeny phages issuing from the infected bacteria were P³²-unstable. Experiment III. Radioactive B/r bacteria in radioactive medium were infected with radioactive (i.e. P³²-unstable) T2 and otherwise incubated and frozen like those of the first two experiments. In this case, the same progressive stabilization, of the infective centers towards inactivation by P³² decay was observed as that found in Experiment I. The ability to yield infective progeny of infected bacteria incubated for 10 minutes at 37°C. before freezing could no longer be destroyed by P³² decay. The progeny issuing from the infected cells were as unstable as the parental phage. These results could be explained by one of three general hypotheses. As vegetative phage begins to multiply, it is possible that: (a) there is a high probability that any part of the vegetative phage already duplicated can be saved after its destruction by P³² decay through a process analogous to multiplicity reactivation or, (b) there occurs a change in state of the deoxyribonucleic acid (DNA) preliminary to or in the course of its replication that renders it refractory to destruction by P³² decay, or, finally (c) there occurs a transfer of the genetic factors from the DNA of the infecting phage to another substance not sensitive to destruction by P³² decay.     

Studies of Chloroplast Development in Euglena: XIV. Sequential Interactions of Ultraviolet Light and Photoreactivating Light in Green Colony Formation

Photoreactivation (PR) of green colony-forming ability in Euglena is pH-insensitive from pH 6.0 to 8.0 and temperature-sensitive with a maximum rate at 35°C. There is no PR at 0°C. The rate of PR varies with the growth stage of the cells; PR of exponential phase cells is slower than that of stationary phase cells. The reciprocity rule holds for PR over a 6-fold range of intensity. The shape of PR curves is a function of the UV dose; there appears to be a progressive increase in multiplicity until a limiting multiplicity is reached as indicated by the fact that curves for high doses are superposable. Dark-grown and light-grown cells give the same PR response for comparable UV doses. UV inactivation of cells which have been treated with UV and then with PR light shows that, if the PR dose is sufficiently large, the same UV-inactivation curve is obtained as for nonpretreated control cells. Doses of PR lower than the saturating dose produce UV-inactivation curves, the ultimate slopes of which are parallel to the slope of the control curve, but which show reduced multiplicity. The multiplicity of these curves increases with increasing PR dose. The UV inactivation of green colony-forming ability in Euglena is completely photoreactivable at the doses studied, in contrast with the UV inactivation of colony-forming ability, which occurs at considerably higher UV doses and behaves like most other photoreactivable systems, showing a photoreactivable sector of 0.32.     

Tyrosinase inactivation in its action on dopa

Under aerobic or anaerobic conditions, tyrosinase undergoes a process of irreversible inactivation induced by its physiological substrate l-dopa. Under aerobic conditions, this inactivation occurs through a process of suicide inactivation involving the form oxy-tyrosinase. Under anaerobic conditions, both the met- and deoxy-tyrosinase forms undergo irreversible inactivation. Suicide inactivation in aerobic conditions is slower than the irreversible inactivation under anaerobic conditions. The enzyme has less affinity for the isomer d-dopa than for l-dopa but the velocity of inactivation is the same. We propose mechanisms to explain these processes.     

Studies on the Biochemistry and Fine Structure of Silica Shell Formation in Diatoms. Photosynthesis and Respiration in Silicon-Starvation Synchrony of Navicula pelliculosa

Rates of photosynthesis, measured by oxygen electrode or by ¹⁴CO₂ fixation, dark respiration and ³²P-phosphate incorporation are reported for the silicon-starvation synchrony of the fresh water diatom Navicula pelliculosa. During late exponential growth the rates were consistent with increase in carbon mass. During silicon starvation, rates of carbon dioxide fixation, oxygen evolution and ³²P incorporation fell, and the saturating light intensity decreased from 27,000 lux to 5000 lux. Reintroduction of silicon led to immediate transients in all parameters studied, followed by a prolonged increase in rate of dark respiration and a gradual increase in apparent photosynthesis. During release of daughter cells, the rates of dark respiration decreased as photosynthesis and ³²P incorporation increased. These results are discussed in relation to effects of silicon on the energy metabolism of the diatom.     

Characterization of essential arginyl residues in sheep kidney (Na+ + K+) -ATPase.

Treatment of highly purified sheep kidney medulla (Na⁺ + K⁺)-ATPase with 2,3-butanedione results in a rapid inactivation of the enzyme. Contrary to a previous report using rabbit kidney enzyme (DePont et al., Biochim. Biophys. Acta (1977) 482, 213), the inactivation is biphasic under a variety of experimental conditions, with a rapid, initial inactivation which is followed by a slower loss of activity. The second, slower phase of the inhibition obeys pseudo-first order kinetics, with a second order rate constant for inhibition of 20 min^?1M^?1. ATP and ADP provide no protection in the initial phase of the inhibition, but protect the enzyme completely from the second phase of the inhibition. AMP, while less effective than ATP and ADP, provides a partial protection of the enzyme activity from inhibition by 2,3-butanedione. Inorganic phosphate provides partial protection in both phases of the inactivation. Adenosine alone is without effect, but adenosine plus inorganic phosphate provides a greater protection than phosphate alone. The results indicate that either (1) two or more active site residues or (2) a single arginine, experiencing different reactivities in two different active site conformations, are essential to (Na⁺ + K⁺)-ATPase activity.     

Relationship between efflux of ionic calcium and phosphorus during excitation of pancreatic islets with glucose

Simultaneous rates of [³²P]orthophosphate and ⁴⁵Ca²⁺ efflux from prelabeled rat pancreatic islets have been evaluated to assess whether these ions move in concert throughout all phases of “stimulus-secretion coupling”. Perifusion with stimulatory concentrations of glucose elicited immediate but transitory increases in ³²P outflow accompanied by initial retardations and subsequent augmentations in net ⁴⁵Ca²⁺ outflows. These monophasic ³²P and biphasic ⁴⁵Ca²⁺ responses to secretory stimulation were abolished completely by membrane stabilization with tetracaine. However, certain manipulations enabled individual components to be modified separately. During stimulation with glucose, inhibition of insulin release by Ni²⁺ abolished the late increases in ⁴⁵Ca²⁺ outflow without affecting the initial retentions of ⁴⁵Ca²⁺ or the increased releases of ³²P. Under basal conditions, the ionophore A23187 “triggered” increased releases of ⁴⁵Ca²⁺ and insulin without prior retentions of ⁴⁵Ca²⁺ or enhancements of ³²P efflux. Thus, the immediate retardations of ⁴⁵Ca²⁺ outflow and heightened efflux of ³²P may reflect early events in stimulus-secretion coupling which can be dissociated from the augmented release of ⁴⁵Ca²⁺ accompanying activated emiocytosis.     

Iron chelator deferiprone rescues memory deficits,hippocampal BDNF levels and antioxidant defenses in an experimental model of memory impairment

Brain-derived neurotrophic factor (BDNF) plays a key role in neural development and physiology, as well as in pathological states. Post-mortem studies demonstrate that BDNF is reduced in the brains of patients affected by neurodegenerative diseases. Iron accumulation has also been associated to the pathogenesis of neurodegenerative diseases. In rats, iron overload induces persistent memory deficits, increases oxidative stress and apoptotic markers, and decreases the expression of the synaptic marker, synaptophysin. Deferiprone (DFP) is an oral iron chelator used for the treatment of systemic iron overload disorders, and has recently been tested for Parkinson’s disease. Here, we investigated the effects of iron overload on BDNF levels and on mRNA expression of genes encoding TrkB, p75^NTR, catalase (CAT) and NQO1. We also aimed at investigating the effects of DFP on iron-induced impairments. Rats received iron or vehicle at postnatal days 12–14 and when adults, received chronic DFP or water (vehicle). Recognition memory was tested 19 days after the beginning of chelation therapy. BDNF measurements and expression analyses in the hippocampus were performed 24 h after the last day of DFP treatment. DFP restored memory and increased hippocampal BDNF levels, ameliorating iron-induced effects. Iron overload in the neonatal period reduced, while treatment with DFP was able to rescue, the expression of antioxidant enzymes CAT and NQO1.     

An analytical study of in vivo survival of limited populations of animal red blood cells tagged with radio-iron

Animal red blood cell in vivo survival curves, obtained by the radioiron tagging of populations of approximately the same age followed by the administration of non-radioactive iron to suppress radioiron reutilization, have been subjected to mathematical analysis on the basis of the three following assumptions:— (A) Red blood cells disappear from the circulation as the result of senescence: there is an average life span around which the life spans of individual cells are distributed in the usual way. (B) Red blood cells may be removed from the circulation by a process of random destruction which continuously removes a constant fraction of the cells present at any moment irrespective of age or other characteristics. (C) Under the conditions of the experiments described, a fraction of the radioiron, constant for each animal, is reutilized in new red cell formation when released by red cell destruction. This mathematical analysis indicates the following average life spans with the respective standard errors of the mean: dog 107 days ± 1.14; rabbit 67.6 days ± 1.94; cat 68.4 ± 1.50. The mathematical treatment presented has permitted a consideration of the theoretical variation of red cell life spans which was found in these experiments to be relatively small for all three species studied. In the rabbit and cat 2.5 per cent of tagged populations of red cells of the same age would theoretically have disappeared by senescence 17 days before the average life span was reached. The variation of red cell life in the dog was slightly less. Animals of the three species studied, in spite of apparently normal health, exhibited varying degrees of random destruction of both autogenous and transfused fresh normal homologous red cells. As yet, we have no explanation for this random loss of cells occurring in apparently healthy normal animals. The method of mathematical analysis presented is applicable to animal red cell survival studies employing radioiron in which differing rates of random destruction are operating in the removal of red cells.     

The determination of the radiochemical purity of phosphorus-32 and tritium-labeled diisopropylphosphorofluoridate (DFP).

A method is described for the determination of the radiochemical purity of labeled diisopropylphosphorofluoridate (DFP), based on the irreversible inhibition reaction with the enzyme α-chymotrypsin. The nature of the impurities in commercially available ³²P- and ³H-labeled DFP is discussed.     

Circular dichroism spectra of some lower homologs of bradykinin potentiating peptide 9 alpha

The destruction of cytochrome P-450 by allylisopropylacetamide (2-isopropyl-4-pentenamide) in microsomes from phenobarbital-pretreated rats has been shown to require oxygen, to be inhibited by NADP through inhibition of cytochrome P-450 reductase, and to be slightly stimulated by NADH. Glutathione (1 mm) does not inhibit destruction, but methyl 4,5-epoxy-2-isopropylpentanoate (5 mm), an analog of the epoxide of allylisopropylacetamide, does. The inactivation of cytochrome P-450 is both time dependent and saturable, although no more than approximately 40% of the microsomal enzyme appears to be normally destructible. However, mechanical perturbation of the microsomal suspension by rehomogenization initiates renewed destruction. Kinetic analysis shows that the destructive process is pseudo-first-order, with an apparent inactivation rate constant of 1.4 × 10^?3 s^?1 and an apparent K_m of 1.14 mm. Approximately 230 molecules of substrate are turned over for each destructive event. These results, in conjunction with previously reported data, clearly and unambiguously establish that inactivation of cytochrome P-450 by allylisopropylacetamide is a suicidal process.     

Kinetics and mechanism of oxidation of d-fructose and l-sorbose by chromium(VI) and vanadium(V) in perchloric acid medium

Kinetic data for the oxidations of d-fructose and l-sorbose by chromium(VI) and vanadium(V) in perchloric acid medium are reported. The addition of perchloric acid and sodium perchlorate increases the pseudo-first-order rate constants. Change of the reaction medium from water to deuterium oxide appreciably affects the rates of chromium(VI) oxidations, but does not affect those of vanadium(V) oxidations. The activation parameters are ΔH3 = 46.6 ±3.4 (fructose) and 50.6 ±6.3 (sorbose) kJ.mol^?1, and ΔS3 = ?105 ±11 (fructose) and ?100 ±20 (sorbose) J.deg^?1.mol^?1 for chromium(VI) oxidations, and, for the other reactions, ΔH3 = 53.2 ±4.2 (fructose) and 52.3 ±6.3 (sorbose) kJ.mol^?1, and ΔS3 = ?139.0 ±14 (fructose) and ?137 ±20 (sorbose) J.deg^?1.mol^?1. The kinetics of the oxidations of ketohexoses by chromium(VI) indicate no intermediate-complex formation, whereas those for vanadium(V) indicate the formation of a 1:1 intermediate complex between ketohexoses and vanadium(V).     

Oxidation of 2-keto-4-hydroxyglutarate by pig heart and Escherichia coli alpha-ketoglutarate dehydrogenase complex.

Enzyme preparations from pig heart and Escherichia coli have been found to catalyze a NAD⁺- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate. Several independent lines of evidence indicate that 2-keto-4-hydroxyglutarate is a substrate for the well-known α-ketoglutarate dehydrogenase complex of the citric acid cycle. The evidence includes (a) a constant ratio of specific activity values for the two substrates throughout purification, (b) identical elution profiles from a Ca₃(PO₄)₂ gel-cellulose column, (c) the same sucrose density sedimentation patterns, (d) similar responses in controlled heat inactivation studies, and (e) identical pH-activity curves.     

Calcium-dependent regulation of glycogen synthase activity in a muscle glycogen particle

The calcium-dependent inactivation of glycogen synthase in an isolated glycogen-protein complex (glycogen pellet) from rabbit skeletal muscle has been investigated. Addition of 1 mm Ca²⁺, 10 mm Mg²⁺, and 1 mm ATP-γ-S to a concentrated suspension of glycogen pellet resulted in a rapid activation of glycogen phosphorylase concomitant with an inactivation of glycogen synthase. These conversion reactions were blocked by ethylene glycol bis(β-aminoethyl ether) N, N′-tetraacetic acid or by pretreatment of the complex with an antiserum to purified phosphorylase kinase. These data suggest that in the glycogen-protein complex, which may be a functional unit of glycogen metabolism in vivo, phosphorylase kinase can catalyze a Ca²⁺-dependent activation of glycogen phosphorylase synchronized with an inactivation of glycogen synthase. If under similar conditions phosphoprotein phosphatase activity was assayed using exogenous [³²P]phosphorylase, there was an apparent inactivation of the phosphatase. Evidence is presented that this apparent inactivation of phosphatase was due to an accumulation of endogenous phosphorylase a which acted as an inhibitor to the exogenous [³²P]-phosphorylase.     

Specific Growth Rate Determines the Sensitivity of Escherichia coli to Thermal, UVA, and Solar Disinfection

Knowledge about the sensitivity of the test organism is essential for the evaluation of any disinfection method. In this work we show that sensitivity of Escherichia coli MG1655 to three physical stresses (mild heat, UVA light, and sunlight) that are relevant in the disinfection of drinking water with solar radiation is determined by the specific growth rate of the culture. Batch- and chemostat-cultivated cells from cultures with similar specific growth rates showed similar stress sensitivities. Generally, fast-growing cells were more sensitive to the stresses than slow-growing cells. For example, slow-growing chemostat-cultivated cells (D = 0.08 h⁻¹) and stationary-phase bacteria from batch culture that were exposed to mild heat had very similar T₉₀ (time until 90% of the population is inactivated) values (T_{90, chemostat} = 2.66 h; T_{90, batch} = 2.62 h), whereas T₉₀ for cells growing at a μ of 0.9 h⁻¹ was 0.2 h. We present evidence that the stress sensitivity of E. coli is correlated with the intracellular level of the alternative sigma factor RpoS. This is also supported by the fact that E. coli rpoS mutant cells were more stress sensitive than the parent strain by factors of 4.9 (mild heat), 5.3 (UVA light), and 4.1 (sunlight). Furthermore, modeling of inactivation curves with GInaFiT revealed that the shape of inactivation curves changed depending on the specific growth rate. Inactivation curves of cells from fast-growing cultures (μ = 1.0 h⁻¹) that were irradiated with UVA light showed a tailing effect, while for slow-growing cultures (μ = 0.3 h⁻¹), inactivation curves with shoulders were obtained. Our findings emphasize the need for accurate reporting of specific growth rates and detailed culture conditions in disinfection studies to allow comparison of data from different studies and laboratories and sound interpretation of the data obtained.     

A Bioluminescence Assay Using Nitrosomonas europaea for Rapid and Sensitive Detection of Nitrification Inhibitors

An expression vector for the luxAB genes, derived from Vibrio harveyi, was introduced into Nitrosomonas europaea. Although the recombinant strain produced bioluminescence due to the expression of the luxAB genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin. When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity. The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O₂ uptake and NO₂⁻ production rates. The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase. These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways. We conclude that the assay system using luminous Nitrosomonas can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants.     

Chilling-Induced Inactivation and Its Recovery of Tonoplast H-ATPase in Mung Bean Cell Suspension Cultures

The processes involved in adaptation to cold temperature were examined by growing suspension cultured cells of mung bean (Vigna radiata [L.] Wilczek) at 2°C for various periods of time and assaying the activities of various membrane-bound enzymes in vitro. The tonoplast H⁺-ATPase activity and the ATP-proton transport extracted from cells incubated at 2°C declined rapidly and reached a minimum level after 10 hours. The inactivation was reversible within 24 hours of chilling. The recovery of the cold-inactivated H⁺-ATPase was found to proceed in two steps, a faster recovery of ATP hydrolysis activity and a slower recovery of the proton transport. The recovery was markedly inhibited by the presence of azide, but not affected by 0.578 millimolar cycloheximide. This suggested the involvement of an energy process that had no requirement for de novo synthesis of protein. The cold-induced inactivation of the H⁺-ATPase may be due to a structural alteration of the enzyme. The slower recovery of proton transport relative to ATP hydrolysis during warming suggests that the protogenic domains in the enzyme may be affected differently by chilling.     

Inactivation of bacteria by decay of incorporated radioactive phosphorus

Cultures of Escherichia coli will not grow in media containing very high specific activities of radiophosphorus P³², the inhibition of growth being due to the decay of assimilated P³² atoms. Experiments with a differentially labeled thymineless strain of E. coli show that the P³² disintegrations which occur in the bacterial deoxyribonucleic acid, i.e. in the nucleus, are mainly responsible for the inactivation of the cell. The kinetics with which radioactive bacterial populations are inactivated indicate that the function of several nuclei per bacterial cell must be eliminated by P³² decay before the ability to generate a colony is lost. The efficiency with which each P³² disintegration inactivates the nucleus in which it has occurred is calculated to be 0.02 (at –196°), i.e., similar in magnitude to the killing efficiency of P³² decay in bacteriophages. P³² decay and thymine starvation cooperate in bringing about the death of individuals of the thymineless strain, from which observation it is inferred that "thymineless death" is likewise a nuclear inactivation. The descendants of a non-radioactive bacterial culture grown for several generations in the presence of P³² and the descendants of a radioactive culture grown in the absence of P³² are inactivated by P³² decay in a manner which indicates that the phosphorus atoms of bacterial nuclei are dispersed among the progeny nuclei in their line of descendance.     

QUANTITATIVE STUDIES ON ENZYMES IN STRUCTURES IN STRIATED MUSCLES BY LABELED INHIBITOR METHODS : I. The Number of Acetylcholinesterase Molecules and of Other DFP-Reactive Sites at Motor Endplates, Measured by Radioautography

Di-isopropylfluorophosphate (DFP) labeled with phosphorus-32 was applied to fragments of the diaphragm and sternomastoid muscles of the mouse, in conditions in which it saturated all available sites at the motor endplates. After adequate washing and exchange with unlabeled DFP, single endplates were obtained by microdissection and their radioactivity was found by beta track radioautography. The number of sites phosphorylated by DFP-³²P per endplate was relatively constant for each muscle: in the sternomastoid, about 9 x 10⁷ sites per endplate, in the diaphragm, about 3 x 10⁷. Reaction with DFP-³²P was abolished by prior treatment with unlabeled DFP. Labeling was unaffected by prior fixation in formaldehyde, but was inversely proportional to the time of incubation in the Koelle staining medium, when this preceded labeling. The contribution of acetylcholinesterase (AChase) to this total number of DFP-reactive sites was determined by three methods. The first involved reactivation of the phosphorylated AChase by pyridine-2-aldoxime methiodide (2-PAM), in conditions in which the reactivation of other enzymes would be insignificant. The other two methods involved protection of the active centers of AChase from phosphorylation by labeled DFP by use of 284C51, an inhibitor highly specific for this enzyme, or by use of eserine. Each of these methods indicated that about 35% of the DFP-reactive sites at endplates of the sternomastoid and diaphragm are AChase. The mean number of AChase molecules was thus found to be 3.1 x 10⁷ and 1.1 x 10⁷per endplate in sternomastoid and diaphragm, respectively. No significant reaction of labeled DFP with muscle and nerve was observed. Mast cells in the muscle had a concentration of DFP-reactive sites far higher than the endplates.     

Biphasic Thermal Inactivation Kinetics in Salmonella enteritidis PT4

The thermal inactivation kinetics of Salmonella enteritidis PT4 between 49 and 60°C were investigated. Using procedures designed to eliminate methodological artifacts, we found that the death kinetics deviated from the accepted model of first-order inactivation. When we used high-density stationary-phase populations and sensitive enumeration, the survivor curves at 60°C were reproducibly biphasic. The decimal reduction time at 60°C (D_60°C) of the tail subpopulation was more than four times that of the majority population. This difference decreased with decreasing temperature; i.e., the survivor curves became more linear, but the proportion of tail cells remained a constant proportion of the initial population, about 1 in 10⁴ to 10⁵. Z plots (log D versus temperature) for the two populations showed that the D values coincided at 51°C, indicating that the survivor curves should be linear at this temperature, and this was confirmed experimentally. Investigations into the nature of the tails ruled out genotypic differences between the populations and protection due to leakage from early heat casualties. Heating of cells at 59°C in the presence of 5 or 100 μg of chloramphenicol per ml resulted in reductions in the levels of tailing. These reductions were greatest at the higher chloramphenicol concentration. Our results indicate that de novo protein synthesis of heat shock proteins is responsible for the observed tailing. Chemostat-cultured cells heated at 60°C also produced biphasic survivor curves in all but one instance. Cells with higher growth rates were more heat sensitive, but tailing was comparable with batch cultures. Starved cells (no dilution input) displayed linear inactivation kinetics, suggesting that during starvation a rapid heat shock response cannot be initiated.     

Serine hydrolases in differentiating Rhodomicrobium vannielii swarmer cells

³H-diisopropylfluorophosphate (DFP) was used to specifically label serine hydrolases in differentiating Rhodomicrobium vannielii swarmer cells. Fluorography of SDS-polyacrylamide gels revealed several changes in the pattern of ³H-DFP-labelled polypeptides, including the appearance of a new species and apparent increases in the amounts of others. Most of these changes occurred late in differentiation and may be associated with cell division events.Abbreviations DFP diisopropylfluorophosphate - SDS sodium dodecyl sulphate