Amount Of Inactivation Research Articles

A Comparison of Three Methods for Determining Thermal Inactivation Kinetics: A Case Study on Salmonella enterica in Whole Milk Powder

Different methods for determining the thermal inactivation kinetics of microorganisms can result in discrepancies in thermal resistance values. In this study, thermal resistance of Salmonella in whole milk powder was determined with three methods: thermal death time (TDT) disk in water bath, pouches in water bath, and the TDT Sandwich system. Samples from three production lots of whole milk powder were inoculated with a five-strain Salmonella cocktail and equilibrated to a water activity of 0.20. The samples were then subjected to three isothermal treatments at 75, 80, or 85°C. Samples were removed at six time points and cultures were enumerated for survivors. The inactivation data were fitted to two consolidated models: two primary models (log linear and Weibull) and one secondary model (Bigelow). Normality testing indicated that all the model parameters were normally distributed. None of the model parameters for both consolidated models were significantly different (α = 0.05). The amount of inactivation during the come-up time was also not significantly different among the methods (α = 0.05). However, the TDT Sandwich resulted in less inactivation during the come-up time and overall less variation in model parameters. The survivor data from all three methods were combined and fitted to both consolidated models. The Weibull had a lower root mean square error and a better fit, according to the corrected Akaike's information criterion. The three thermal treatment methods produced results that were not significantly different; thus, the methods are interchangeable, at least for Salmonella in whole milk powder. Comparisons with more methods, other microorganisms, and larger varieties of food products using the same framework presented in this study could provide guidance for standardizing thermal inactivation kinetics studies for microorganisms in foods.

Corrigendum to “Analysis of the variation in use-dependent inactivation of high-threshold tetrodotoxin-resistant sodium currents recorded from rat sensory neurons”

On page 931, Fig. 6, in the Tripathi et al. (2006) study, there was a glitch in the pClamp 8 protocol used to acquire data on inactivation from the closed state of high-threshold tetrodotoxin-resistant Na currents recorded from acutely isolated rat DRG cells. The glitch made it appear as if there was a fast phase of inactivation, which was complete before the first time point of 5 ms included in the protocol. Subsequent experiments with a revised data acquisition protocol demonstrated that no inactivation from the closed state occurred over the first 5 ms at 50 mV, and that overall inactivation was well fitted with a single exponential function. This new information was used to correct the data presented in the results and in Fig. 6 on inactivation from the closed state in the Tripathi et al. (2006) study. The correction method was to subtract out the artifactual inactivation corresponding to the 5 ms time point from that observed at the other time points. The resulting corrected Fig. 6 is presented below. For the corrected data, the time constant for inactivation from the closed state averaged 2667 827 ms, and there was a significant correlation between the amount of inactivation from the closed state observed after 10 s, and use-dependent inactivation (see part D below). Other data acquired in the Tripathi et al. (2006) study indicated that the high threshold tetrodotoxin-resistant Na channels expressed by individual DRG cells were homogeneous regarding inactivation properties regardless of the amount of use-dependent inactivation exhibited (see Fig. 2, Tripathi et al., 2006). Thus, we believe that the method of correction employed is reasonably accurate. The correct Fig. 6 appears here. (Available online 12 May 2011)

Membrane Damage and Viability Loss of Escherichia coli K-12 and Salmonella Enteritidis in Liquid Egg by Thermal Death Time Disk Treatment

Bacterial injury, including leakage of intracellular substance and viability loss, of Escherichia coli K-12 (ATCC 23716) and Salmonella Enteritidis (ATCC 13076) inoculated in liquid egg white and liquid whole egg was determined by thermal death time disk. E. coli K-12 and Salmonella Enteritidis were inoculated in liquid egg white and liquid whole egg to a final count of 7.8 log CFU/ml and were thermally treated with thermal death time disks at room temperature (23°C), 54, 56, 58, and 60°C from 0 to 240 s. Sublethal injury, leakage of intracellular substances, and viability loss of E. coli K-12 and Salmonella Enteritidis was investigated by plating 0.1 ml on selective trypticase soy agar containing 3% NaCl, 5% NaCl, sorbitol MacConky agar, and xylose lysine sodium tetradecylsulfate and nonselective trypticase soy agar. No significant (P >0.05) differences on percent injury or viability loss for E. coli K-12 and Salmonella populations were determined in all samples treated at 23°C. Sublethal injury occurred in E. coli and Salmonella populations at 54°C or above for 120 s. Viability losses for both bacteria averaged 5 log at 54°C or above for 180 s, and the surviving populations were below detection (<10 CFU/ml). Thermal treatment at 40°C and above led to membrane damage, leakage, and accumulation of intracellular ATP from 2 to 2.5 log fg/ml and UV-absorbing substances of 0.1 to 0.39 in the treated samples. These results indicate similar thermal injury/damage on both E. coli and Salmonella membranes as determined by the amount of inactivation, viability loss, and leakage of intracellular substances of bacteria.

Cysteine-scanning Mutagenesis of Serotonin Transporter Intracellular Loop 2 Suggests an α-Helical Conformation

Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT) in intact cells expressing these mutants, but it inactivated beta-CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an alpha-helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity.

Thermal inactivation of polyphenoloxidase in pineapple puree

Prevention of browning in pineapple puree by thermal inactivation of enzyme, Polyphenoloxisase (PPO), was examined between 40 and 90 °C and in relation to exposure time. The amount of inactivation was measured as a function of time and temperature under isothermal conditions. Reaction rate constant and activation energy ( E a) as well as Decimal reduction time ( D) and z-value of thermal inactivation, were determined. The rate of inactivation varied with temperatures and follows a logarithmic law. Kinetic studies showed that the thermal inactivation (40–90 °C) of the PPO followed first-order kinetics.

Oligomerization of serotonin transporter and its functional consequences

Two forms of serotonin transporter (SERT) were prepared with different epitope tags. When co-expressed in HeLa cells, the form containing a FLAG tag (Res-FLAG) was associated with the form containing a c-myc tag (Sens-myc). Antibody against c-myc precipitated Res-FLAG from detergent extracts of cells expressing both forms, but not when Res-FLAG was expressed alone. The specificity of the interaction was demonstrated by the observation that anti-myc antibodies did not precipitate the unrelated vesicular stomatitis virus coat glycoprotein when it was co-expressed with Sens-myc. Sens-myc contained a reactive cysteine at position 172, which reacted with both (2-aminoethyl)methanethiosulfonate and N-biotinylaminoethyl methanethiosulfonate on the surface of intact cells. Sens-myc, but not Res-FLAG, was inactivated by these reagents. When co-expressed with Sens-myc, functionally active Res-FLAG was precipitated by immobilized streptavidin from digitonin-solubilized cells that had been treated with N-biotinylaminoethyl methanethiosulfonate. In cells co-expressing mixtures of Sens-myc and Res-FLAG, the amount of inactivation by (2-aminoethyl)methanethiosulfonate was less than expected if the two forms were independent. The results are consistent with a dimeric form of SERT with functional interactions between subunits, and with association of dimers into a higher order complex, possibly a tetramer.

Oligomerization of serotonin transporter and its functional consequences.

Two forms of serotonin transporter (SERT) were prepared with different epitope tags. When co-expressed in HeLa cells, the form containing a FLAG tag (Res-FLAG) was associated with the form containing a c-myc tag (Sens-myc). Antibody against c-myc precipitated Res-FLAG from detergent extracts of cells expressing both forms, but not when Res-FLAG was expressed alone. The specificity of the interaction was demonstrated by the observation that anti-myc antibodies did not precipitate the unrelated vesicular stomatitis virus coat glycoprotein when it was co-expressed with Sens-myc. Sens-myc contained a reactive cysteine at position 172, which reacted with both (2-aminoethyl)methanethiosulfonate and N-biotinylaminoethyl methanethiosulfonate on the surface of intact cells. Sens-myc, but not Res-FLAG, was inactivated by these reagents. When co-expressed with Sens-myc, functionally active Res-FLAG was precipitated by immobilized streptavidin from digitonin-solubilized cells that had been treated with N-biotinylaminoethyl methanethiosulfonate. In cells co-expressing mixtures of Sens-myc and Res-FLAG, the amount of inactivation by (2-aminoethyl)methanethiosulfonate was less than expected if the two forms were independent. The results are consistent with a dimeric form of SERT with functional interactions between subunits, and with association of dimers into a higher order complex, possibly a tetramer.

Inactivation of glutathione transferase zeta by dichloroacetic acid and other fluorine-lacking alpha-haloalkanoic acids.

Dichloroacetic acid (DCA) is a contaminant of chlorinated drinking water supplies, is carcinogenic in rats and mice, and is a therapeutic agent used for the treatment of congenital lactic acidosis. The biotransformation of DCA to glyoxylic acid is catalyzed by glutathione transferase zeta (GSTZ). Treatment of rats and human subjects with DCA increases its plasma elimination half-life and reduces the extent of DCA biotransformation in rat hepatic cytosol. In the investigation presented here, the kinetics of the DCA-induced inactivation of GSTZ, the turnover of GSTZ, and the susceptibility of GSTZ to inactivation by a panel of alpha-haloacids were studied. DCA rapidly inactivated GSTZ in both rat hepatic cytosol and intact Fischer 344 rats. The time course of inactivation in vivo was mirrored by a concomitant loss of immunoreactive GSTZ protein. The turnover of GSTZ in rat liver was 0.21 day(-1), which corresponded to a half-life of 3.3 days. The degree of GSTZ inactivation after daily administration of DCA could be predicted from the amount of inactivation after a single treatment. Other fluorine-lacking dihaloacetic acids also inactivated GSTZ, whereas alpha-monohaloacids and fluorine-containing dihaloacetic acids failed to inactivate GSTZ. These data show that the observed DCA-induced decrease in the level of DCA metabolism is caused by the inactivation of GSTZ.

Multiple components of voltage-dependent potassium current in normal rat anterior pituitary cells.

1. Voltage-dependent K+ currents were studied in normal rat anterior pituitary cells using the patch-clamp technique. To obtain cultures enriched for lactotrophs, density gradient centrifugation was performed on pituitary cells isolated from lactating rats. 2. Depolarizations to about -30 mV from a holding potential of -80 mV activate a rapidly inactivating [time constant (tau) approximately 15-20 ms at -20 mV]K+ current. This transient current activated at low voltages (termed IA) is abolished by 5 mM external 4-aminopyridine (4-AP) but is largely resistant to external tetraethylammonium (TEA) (< or = 30 mM). 3. Recovery from inactivation of IA is fast, with a tau of 100-200 ms at -80 mV. Deactivation is also fast (tau approximately 2.2 ms at -50 mV). The voltage of half-activation of IA is approximately -20 mV. The current is completely inactivated at a holding potential of -40 mV. 4. Voltage-dependent K+ current activated by depolarizations from a holding potential of -40 mV was first detectable at about -20 mV (high voltage-activated) and had a time course that varied among cells. 5. Deactivation of high voltage-activated K+ current was best described by the sum of two exponentials, with tau of about 3.7 and 30 ms at -50 mV. Both components reversed close to the equilibrium potential for K+. 6. The amplitudes of the two tail currents were independent of each other when variable-duration commands were used to activate current. The amplitude of the fast component was largest with 10- to 20-ms commands to +40 mV and was reduced (< or = 50%) with 136-ms commands. The slow component amplitude reached a peak by 40 ms and remained constant for commands < or = 136 ms at +40 mV. 7. The contribution of each component to the total high voltage-activated tail current was variable among cells, with the amount of fast component correlating with the amount of inactivation produced by commands to +40 mV. 8. The two components of tail current activated by depolarizations from the -40 mV holding potential were abolished by external TEA (10 mM). 4-AP (5 mM externally) selectively abolished the fast component of high voltage-activated tail current while only partially reducing the slow component. 9. These results suggest that normal rat anterior pituitary cells possess at least three distinct types of voltage-dependent K+ current: a low voltage-activated, transient current (IA) and two high voltage-activated currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Site of freeze-thaw damage and cryoprotection by amino acids of the calcium ATPase of sarcoplasmic reticulum

The Ca2+,Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) is irreversibly inactivated by a freeze-thaw (FT) cycle. The membrane does not become more permeable to calcium after a FT cycle, suggesting that the reduced uptake is due to damage to the Ca2+,Mg(2+)-ATPase. Several amino acids, in addition to standard cryoprotectants provide good protection of calcium uptake against FT damage. The amount of protection given by the amino acids is generally inversely proportional to a measure of hydrophobicity, the mean fractional area loss upon incorporation in globular proteins of the amino acid side chain. Unlike the case for cells, glutamine and dimethyl sulfoxide do not act independently as cryoprotectants for SR calcium ATPase. When the protein is exposed to multiple FT cycles, the amount of inactivation is exponentially proportional to the number of FT cycles. This is true for both protected and unprotected samples. Some SR vesicles fuse during FT. Fusion of vesicles cannot account for the observed inactivation of the enzyme. Fluorescence studies, using intrinsic tryptophan and extrinsic FITC and NCD-4, suggest that FT does not damage the transmembrane region of the Ca2+,Mg(2+)-ATPase or the calcium binding sites, but only the mechanism coupling ATPase activity to calcium translocation. Differential scanning calorimetry (DSC) studies suggest that this region comprises less than 15% of the whole enzyme.

Properties of the transient outward current in rabbit atrial cells.

1. Whole-cell and patch clamp techniques have been used to study the steady-state voltage dependence and the kinetics of a transient outward current, It, in single cells from rabbit atrium. 2. The steady-state voltage dependence of both activation and inactivation of It are well described by Boltzmann functions. Inactivation is fully removed at potentials negative to -70 mV and it is complete near 0 mV. The threshold for activation of It is near -30 mV and it is fully activated at +30 mV. The region of overlap between the activation and inactivation curves indicates that a steady non-inactivating current will be recorded over a membrane potential range from approximately -30 to 0 mV. 3. In general, the time course of inactivation at potentials in the range 0 to +50 mV is best described as a sum of two exponential functions. The kinetic parameters controlling these processes exhibit only very weak voltage dependence. 4. Comparison of the time course of the development of inactivation in response to long depolarizing voltage clamp steps with the development of inactivation in response to trains of brief depolarizing pulses indicates that inactivation develops very quickly and decays relatively slowly at potentials near the resting potential (e.g. -70 mV). Thus, in response to (i) a train of voltage-clamp pulses or (ii) a series of action potentials, the magnitude of It decreases due to a progressive increase in the amount of inactivation. 5. A simple model of channel gating is presented: it can account for the major aspects of the voltage dependence and kinetics of It (cf. Aldrich, 1981). 6. Cell-attached patch clamp recordings have been used to identify the single-channel or unitary events underlying the current, It. In general, only one active channel is present per patch. The single-channel conductance in normal Tyrode solution is approximately 14 pS and the current-voltage relationship is approximately linear between +50 and +150 mV with respect to rest. This information, in combination with the fully activated current-voltage characteristics from the whole-cell data, can be used to estimate the number and density of It channels per cell: these are 1600 and one per 3-4 micron 2, respectively. 7. Ensemble averages obtained from patch recordings are very similar in time course to the macroscopic or whole-cell current itself: the ensemble current rises to a peak within approximately 5 ms and decays with a biexponential time course in response to depolarizations to approximately +50 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Inactivation of calcium current in bull-frog atrial myocytes.

1. A single-microelectrode technique has been used to study the voltage dependence and the kinetics of inactivation and reactivation of a tetrodotoxin-resistant inward current (ICa) in single cells from bull-frog atrium. 2. In most cases the kinetics of both inactivation and reactivation can be well described as a single-exponential process. 3. Several different observations indicate that inactivation of ICa in these cells is controlled by both voltage-dependent and current-dependent processes, as has been demonstrated previously in heart (Kass & Sanguinetti, 1984; Lee, Marban & Tsien, 1985) and in other tissues (Hagiwara & Byerly, 1981; Tsien, 1983; Eckert & Chad, 1984). 4. Evidence in favour of a voltage-dependent inactivation mechanism included: (a) In paired-pulse measurements of steady-state inactivation ('f infinity') a 'conventional' steady-state f infinity vs. membrane potential (Vm) relationship was obtained in the range of membrane potentials from -60 to 0 mV. (b) Increasing [Ca2+]o from 2.5 to 7.5 mM, which resulted in a 2-3-fold increase in ICa, did not produce any significant increase in the amount of inactivation. (c) Using a 'gapped' double-pulse protocol non-monotonic U-shaped inactivation relationships were obtained, i.e. positive to approximately +20 mV some removal of inactivation occurred. However, f never approached a value near 1.00 at very depolarized potentials; it reached a maximum between 0.5 and 0.6. (d) In constant [Ca2+]o and at fixed Vm, the kinetics of ICa inactivation were independent of peak size of ICa. This was demonstrated by: (i) varying the holding potential (-90 to -30 mV), (ii) using paired-pulse 'recovery' protocols, and (iii) partial block by La3+ (1-10 microM) and Cd2+ (0.1 mM). (e) Influx of Ca2+ ions was not an obligatory prerequisite for development of inactivation. In all ionic conditions (Ca2+, Sr2+, Ba2+, Na+-free and Ca2+-free Ringer solutions) currents displayed inactivation phenomena, although the extent and kinetics of inactivation were dependent upon ionic conditions. Outward currents recorded above the reversal potential for ICa exhibited time- and voltage-dependent inactivation, and could be inactivated by brief depolarizing pre-pulses that produced no net inward current flow. Evidence against a possible role of the electrogenic Na+-Ca2+ exchanger in producing inactivation of these outward currents was obtained.(ABSTRACT TRUNCATED AT 400 WORDS)

Inactivation of particle-associated coliforms by chlorine and monochloramine

Sieves and nylon screens were used to separate primary sewage effluent solids into particle fractions of less than 7- or greater than 7-micron size. The efficiency of separation was determined by using a particle counter. Indigenous coliforms associated with the particle fractions were tested for their resistance to chlorine and monochloramine. Coliforms associated with the less than 7-microns fraction were inactivated more rapidly by 0.5 mg of chlorine per liter at 5 degrees C and pH 7 than coliforms associated with the greater than 7-microns fraction. Homogenization of the greater than 7-microns fraction not only resulted in an increase in the number of less than 7-microns particles, but also increased the rate of inactivation to a rate similar to that of the less than 7-microns fraction. With 1 mg of monochloramine per liter at 5 degrees C and pH 7, particle size had no appreciable effect on the rate of inactivation. At pH 8, however, the less than 7-micron fraction was inactivated more rapidly than the greater than 7-micron fraction. The time required for 99% inactivation of the particle fractions with monochloramine at pH 7 or 8 was 20- to 50-fold greater than the time required for the same amount of inactivation with chlorine at pH 7. The results indicate that coliforms associated with sewage effluent particles are inactivated more rapidly with 0.5 mg of chlorine per liter than with 1.0 mg of monochloramine per liter. However, greater than 7-micron particles can have a protective effect against the disinfecting action of chlorine.

Separation of current- and voltage-dependent inactivation of calcium current in frog sensory neuron

The inactivation process of the calcium current was studied in frog dorsal root ganglion cells using a newly developed 'concentration clamp' technique, and an inactivation component independent of Ca influx was separated from the Ca-dependent component. The neuron was first placed in Ca-free solution, and pre-pulses of various amplitude were applied, thereby inducing depolarization without the Ca influx. Successive test pulses were applied immediately after a rapid exchange to a solution containing normal Ca. The amount of inactivation under this condition was much smaller than that measured when both pre- and test pulses were applied in the normal solution, and reached a steady level on pre-pulses over +50 mV. We suggest that the inactivation process of the Ca current consists of two components, i.e. a dominant component which is dependent on Ca influx, and a smaller one which is presumably voltage-dependent.

Removal of Ca current inactivation in dialysed guinea-pig atrial cardioballs by Ca chelators

Ca currents flowing during voltage clamp depolarizations were studied in cultured guinea-pig atrial cardioballs by means of single low resistance patch clamp pipettes. The pipettes were filled with solutions containing Cs+ as major cation in order to block K+ currents and high concentrations of various Ca chelating agents (EGTA, nitrilotriacetic acid, citrate, dipicolinic acid) to prevent rises of the intracellular Ca-activity by Ca-entry. Ca currents of myocytes loaded with 20 mM of either EGTA [(ethylenedioxy)-diethylenedinitrilo)tetra-acetic acid] or NTA (nitrilotriacetic acid) display a biphasic time course of inactivation at membrane potentials between -25 and +45 mV. The fast phase is reduced with increasingly positive membrane potentials. In cells loaded with either citrate or DPA (dipicolinic acid, pyridine-2,6-dicarboxylic acid) inactivation is negligible or absent for small depolarizations. In the range of membrane potentials where maximum current flows (0-+10 mV) a monophasic slow time course of inactivation is observed. At more positive membrane potentials inactivation is slowed. The amount of inactivation under this condition is related to the current density of the cell. Conditions, which for a given membrane potential reduce the amplitude of ICa such as extracellular application of blocking ions (Co2+, Cd2+), a conditioning depolarization, or 'rundown' of Ca-channels lead to a slowing or a complete removal of inactivation in cells dialysed with citrate or DPA respectively. Cells loaded with these Ca chelators did not show any symptom of voltage dependent inactivation of ICa. Under the conditions described action potentials were recorded in the current clamp mode. Upon dialysis with EGTA the typical 'triangular shaped' atrial action potential develops a plateau of 500 to 800 ms in duration. With citrate-containing pipette solutions the action potential duration usually is several seconds. The results for the first time demonstrate that inactivation of cardiac ICa can be considerably slowed or even removed. They provide further strong support for the hypothesis that inactivation of this current depends on Ca entry rather than membrane potential. The fast phase of inactivation observed with EGTA (NTA) possibly reflects the slow kinetics of the binding reaction of this type of Ca chelators.

Inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine.

The kinetics of inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine were studied at 5 degrees C with a purified preparation of single virions and a preparation of cell-associated virions. Inactivation of the virus preparations with chlorine and chlorine dioxide was studied at pH 6 and 10. The monochloramine studies were done at pH 8. With 0.5 mg of chlorine per liter at pH 6, more than 4 logs (99.99%) of the single virions were inactivated in less than 15 s. Both virus preparations were inactivated more rapidly at pH 6 than at pH 10. With chlorine dioxide, however, the opposite was true. Both virus preparations were inactivated more rapidly at pH 10 than at pH 6. With 0.5 mg of chlorine dioxide per liter at pH 10, more than 4 logs of the single-virus preparation were inactivated in less than 15 s. The cell-associated virus was more resistant to inactivation by the three disinfectants than was the preparation of single virions. Chlorine and chlorine dioxide, each at a concentration of 0.5 mg/liter and at pH 6 and 10, respectively, inactivated 99% of both virus preparations within 4 min. Monochloramine at a concentration of 10 mg/liter and at pH 8 required more than 6 h for the same amount of inactivation.

Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals.

Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E. coli wild-type spheroplasts. On the other hand, using spheroplasts prepared from E. coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found. Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage. The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals. Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair. Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions. We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins.

Comparison of aminoglycoside assays

Aminoglycosides are an important group of antibiotics but possess significant potential for toxicity. Serum levels are useful in making appropriate dosage alterations to avoid toxicity and obtain therapeutic concentrations. Rapid estimations are particularly helpful in patients with compromised renal function. In this respect we evaluated two methods of assaying serum gentamicin, high pressure liquid chromatography (HPLC-E) and Emit, and compared them with bioassay. HPLC-E with post-column derivatization requires sample preparation utilizing a cation exchange column, CM-Sephadex (G 25) to extract aminoglycoside from serum, followed by 15 μ1 injection of the prepared sample onto a C18 uBondapak reverse phase column. The post-column derivatization utilizes ortho-phthaldialdehyde as the fluorescent label and the reaction occurs in a 2 m delay coil. Fluorescence is measured at 340 nm excitation and 418 nm emission. The Emit-gentamicin assay is an immunoassay in which anti-gentamicin antibodies inactivate a gentamicin-labelled enzyme. Free gentamicin in the serum sample competes with the gentamicin-enzyme complex for binding to the anti-gentamicin antibody, thereby decreasing the amount of inactivation of enzyme-labelled gentamicin. The unbound gentamicin-enzyme complex will act upon the substrate glucose-6-phosphate with the reduction of NAD to NADH resulting in a spectrophotometric change when measured at 340 nm. The bioassay was performed in duplicate by standard disc diffusion technique using Oxoid agar No. 5 with B. cereus or E. hafnia as the test organisms. The diameter of each zone of inhibition was measured after incubation at 35°Cfor 16 h. Comparing the methods there was good correlation between the following: HPLC-E and bioassay; r = 0.92, Y = 1.06x +0.08; Emit and bioassay; r = 0.90, Y = 0.98x +0.59; and HPLC-E and Emit; r = 0.90, Y = 0.94x + 0.13. Results were obtained within 1 h for Emit; HPLC-E required 3 h. Within-day precision (CV) for the rapid tests was 5% for Emit and 3% for HPLC-E. The Emit and HPLC-E systems did not show any interference from tobramycin, amikacin, co-trimoxazole, cephalosporins and penicillins. Further development of HPLC-E has allowed assays for tobramycin and amikacin.

Maturation of evoked climbing fiber input to rat cerebellar Purkinje cells (I.)

An analysis of evoked responses of Purkinje cells in developing rat cerebellum to climbing fiber input was conducted to determine which identifying properties of this afferent system are established early in development and which specific features mature with age. Rat pups at various ages were anesthetized with 0.5% halothane and unit recordings made with glass micropipettes. By the third postnatal day, electrical stimulation of the sensorimotor cortex and limbs at low stimulation rates (less than 1/sec) could elicit distinct burst responses appearing at long latencies (180 msec), indicating that pathways of both ascending and descending climbing fiber systems are intact early in cerebellar cortical development. A distinctive feature maturing over the first 1-1.5 weeks was the characteristic of the all-or-none burst response since before about day 11 the interspike interval, amount of inactivation, and number of spikes in evoked burst responses all varied from stimulation to stimulation. Mean latencies decreased from 180 msec at dat 3 to 50 msec by day 10, but did not achieve the adult value of 20 msec until the fourth week. Typically, climbing fiber responses could only follow at stimulation rates of less than 0.2/sec at day 3, but by day 12 could follow up to 10/sec, which is the same as in the adult. The data indicate that the climbing fiber system establishes connectivity from diverse sources and exhibits identifying characteristics similar to the adult early in cerebellar development. Most aspects of the maturation of transmission can be explained if there were a decrease in the time scale of function of the synapses involved, mainly those in the inferior olive.

The role of histidine residues in yeast hexokinase. Enzymic and structural studies.

Specific acylation of the histidine residues of yeast hexokinase with diethylpyrocarbonate at pH 6.1 or 7.5 leads to a partial and reversible inactivation of the enzyme.The carboethoxylation of the histidine residues at pH 7.5 was faster than at 6.1 and the loss in activity was also higher.However the difference in the amount of inactivation was related to a greater instability of the carboethoxylated enzyme at pH 7.5.When the carboethoxylation was carried out at pH 7.5 and at 0 °C, only the histidines residues were modified (nine residues per enzyme subunit) and still 40% of the activity was left. d‐Glucose protects the enzyme activity in the course of the carboethoxylation at pH 7.5, but has no effect at pH 6.1. However the rate and number of histidines acylated were the same as when the experiments were carried in absence of the substrate.All these results clearly indicate that the histidine residues are not implicated either in the catalytic or in the substrate binding site of yeast hexokinase.This was also confirmed by showing that the 60% inactivated enzyme had the same Km as the native enzyme for both substrates d‐glucose and Mg · ATP. Only v was affected.The inactivation effect of the carboethoxylation was not related to a displacement of the pH optimum of the activity, since the enzymes species having five and eight histidine residues modified‐respectively in absence and presence of d‐glucose at pH 7.5 gave the same pH profile of activity as the native enzyme.Absorption spectroscopy, fluorescence and ultracentrifugation studies have shown that the loss of enzymic activity in the course of the modification of the histidine residues is only related to changes of the protein conformation.