Stability of rifamycin B in aqueous solutions

Stability of rifamycin B in aqueous solutions at various values of pH and temperature was studied. It was shown that inactivation of the antibiotic in neutral and alkaline solutions proceeded according to the first order equation. In acid solutions rifamycin B was oxidized with air oxygen to form rifamycin O.     

Stability of bilirubin oxidase in organic solvent media: a comparative study on two low-water systems

The storage stability of bilirubin oxidase was studied in water-in-oil CTAB microemulsions with a chloroformrich continuous organic phase. The kinetics of the inactivation process were best described by a double exponential equation. Approximately half of enzymatic activity was lost during a "fast" phase with a half life of ca. 50 min, whereas the remaining activity was lost much more slowly (half life ca. 1000 min). Rates of inactivation were not affected significantly by variation of either solvent composition or concentration of water droplets, but inactivation was more rapid when droplet size was very small. Steady-state enzyme kinetics were studied at various stages in the inactivation process, and it was shown that inactivation occurred without change in the K(m) of the enzyme for bilirubin. Stability was also studied in a liquid/solid two-phase system; it was found that the inactivation process in this system; it was found that the inactivation process in this system was best described by a single exponential term. The rate was similar to the "fast" phase rate observed in the water-in-oil microemulsion system. Inactivation of the enzyme slow. Addition of the surfactant CTAB to the aqueous environment increased the rate of inactivation to levels comparable to those of the "slow" phase observed in water-in-oil microemulsions. (c) 1993 Wiley & Sons, Inc.     

Comparative study of the relationship between the blood concentration of gentamicin and streptomycin and their muscle-relaxing activity]

A mathematical model of relation between the miorelaxant effect value of aminocyclitol antibiotics and their blood levels is proposed. The kinetics of the reduction of the neuromuscle transmission damaged under the action of the drugs was studied in detail in acute experiments with cats treated with gentamycin or streptomycin administered intravenously. The changes in the effect value in time were satisfactorily described by the logistic function. Parallel determination of the blood levels of the antibiotics in the animals provided construction of an adequate model of their pharmacokinetics. Tabulation of the corresponding biexponential equations and logistic functions for the same time intervals during reduction of the neuromuscle transmission provided necessary information for plotting the "effect: concentration" curves. Comparison of the drug levels at which the effect of inhibition of the neuromuscle transmission was equal to 50% of the maximum one, revealed a statistically reliable difference in the miorelaxant activity of gentamycin and streptomycin. A high miorelaxant activity of gentamycin in comparison to streptomycin was also shown on comparison of the average "effect: concentration" curves plotted on the basis of tabulation of the general equations presenting a combination of the logistic and biexponential equations.     

Microbiological study of gentamiycin]

Gentamycin prepared at the All-Union Research Institute of Antibiotics did not differ by its antibacterial spectrum and the activity level from gentamycin samples from other countries. By its activity against clinical strains of Ps. aeruginosa gentamycin was somewhat inferior than polymyxin but much more superior than carbenicillin. An agar-diffusion method using Bac. pumilus NTCC 8241 as the test microbe was developed for determination of gentamycin activity. The gentamycin sulfate complex and the components of gentamycin had the same activity levels, antibacterial spectrum and diffusion capacity.     

Exploiting the combined effects of high pressure and moderate heat with nisin on inactivation of Clostridium botulinum spores

In the present work, we studied the combined effects of pressure (300.0-700.0 MPa), temperature (30-70 degrees C) and the presence of nisin (0-333 IU/ml) on the inactivation of Clostridium botulinum 33A spores at various pressure holding times (7.5-17.5 min). Moreover, response surface methodology (RSM) was employed and a quadratic equation for HPP and nisin-induced inactivation was built with RSM. By analyzing the response surface plots and their corresponding contour plots as well as solving the quadratic equation, the experimental values were shown to be significantly in good agreement with predicted values because the adjusted determination coefficient (R(Adj)(2)) was 0.9261 and the level of significance was P<0.0001. The optimum process parameters for a six-log cycle reduction of C. botulinum spores were obtained as: pressure, 545.0 MPa; temperature, 51 degrees C; pressure holding time, 13.3 min; and nisin concentration, 129 IU/ml. The adequacy of the model equation for predicting the optimum response values was verified effectively for 10 test points. Compared to conventional high pressure processing (HPP) techniques, the main process advantages of HPP-nisin combination sterilization in the UHT milk are, lower pressure, natural preservative (nisin), and temperature in a shorter treatment time.     

A kinetic approach to the evaluation of alkaline phosphatase inactivation by urea

A kinetic approach is described which enables the measurement of the enzyme inactivation rate constant during the reaction course. A mathematical analysis is presented and it is shown that a time-dependent step may be postulated to exist. Reaction kinetics follow an exponential rule with time as the independent variable and enzymatic activity as the dependent variable. A simple procedure of graphical analysis is reported and the influence on the inactivation rate constant of various conditions (temperature and inhibitor concentration) is evaluated. The method is illustrated by an experimental model: the inactivation of bovine kidney alkaline phosphatase by urea.     

Estimation of the overall kinetic parameters of enzyme inactivation using an isoconversional method

An isoconversional method is proposed in order to calculate the kinetic parameters of enzyme inactivation. The method provides an efficient and low-cost procedure to describe both operational and thermal inactivation. Unlike the ordinary kinetic assays performed at constant enzyme concentration and at various substrate concentrations, the isoconversional method requires several extended kinetic curves for constant initial substrate concentration and different enzyme concentrations. The procedure was tested and validated using simulated data obtained for several kinetic models frequently discussed in the literature. After the validation, the isoconversional method was used for the investigation of the thermoinactivation of urease during urea hydrolysis in self buffered medium and the operational inactivation (destructive oxidation by excess peroxide) of catalase at high concentration of hydrogen peroxide. The results showed that the isoconversional method gives good results of global inactivation constant for both simple and more complex models.     

Study of the antiviral action of gentamicin]

Experimental data on the effect of various concentrations of gentamycin on reproduction of VEE and Sindbis viruses in tissue culture are presented. It was found that gentamycin had no cytotoxic effect on the primary tripsinized chick embryon fibroblasts (CEF) when used in doses of 10, 20 or 30 mg/ml and only when used in a dose of 50 mg/ml it induced 50 percent destruction of the cell layer. Multiplication of the VEE and Sindbis viruses in the culture of CEF was inhibited in the presence of gentamycin by 1.5--3.5 lg PFU/ml. Two stages in the virus inhibiting effect of gentamycin were determined on the model of VEE, i. e. the stage of inhibition in the absence of visible damages of the cells and the stage associated with their destruction. The doses of gentamycin higher than 3 mg/ml inhibited in parallel the virus specific synthesis and synthesis of the cell proteins and nucleic acids. At the same time, when gentamycin was used in a dose of 10 mg/ml, no impairement of the cell viability was observed and the cell capacity to produce high titers of the model virus was reduced after incubation without the antibiotic for 24 hours. The antiviral activity of gentamycin were therefore determined by revers inhibition of the cell metabolic activity.     

Factors influencing virus inactivation and retention of platelet properties following treatment with aminomethyltrimethylpsoralen and ultraviolet A light.

A wide variety of viruses are inactivated by psoralen compounds in the presence of ultraviolet A light (UVA). Use of aminomethyltrimethylpsoralen (AMT) and UVA is being evaluated as a method to inactivate viruses that may be present in platelet suspensions prepared for transfusion. Studies have been conducted to assess how variation in various environmental parameters influences the extent of viral inactivation and the retention of platelet properties. Most notably, it was determined that increasing levels of plasma progressively inhibited the inactivation of model viruses. As a result, experiments were routinely conducted at a plasma level of approximately 14.5%, using 40 micrograms/ml AMT, which was determined to be optimal when using this reduced plasma level. The reduced plasma level was achieved by dilution with a nonplasma medium that has been shown to be satisfactory for storage of platelets. Under these conditions, about 5 logs of vesicular stomatitis virus (VSV), pseudorabies, and phi 6 inactivation were achieved. Variation of platelet and leukocyte counts, within normal levels, had a minimal effect on extent of viral inactivation. Although oxygen level (mean levels, 97.9 mm Hg versus 19.2 mm Hg) had only a small influence on viral inactivation with 2.4, 4.8, and 7.2 J/cm2 of UVA (equivalent to 1-3 minutes of exposure), in vitro platelet properties, such as medium pH, morphology characteristics, and aggregation response, were better retained with a longer exposure time at the reduced oxygen level. With normal oxygen (97.9 mm Hg), platelet properties declined substantially relative to untreated controls (no UVA, no AMT) on exposure to 4.8 J/cm2. Our studies have identified two sets of conditions that provide about 5 logs of virus inactivation without extensively altering platelet in vitro properties.     

Analysis of the mechanism and kinetics of thermal inactivation of enzymes: Critical assessment of isothermal inactivation experiments

The study deals with information obtained from conventional isothermal inactivation experiments. When the lumped character of enzyme activity was analysed, identical activity-time profiles could be obtained from several mechanisms when the number of equivalent mechanisms increased exponentially with the number of parameters in the corresponding kinetic equations. The assessment of isothermal evaluation of inactivation kinetics was demonstrated using selected experiments from the literature exhibiting a deviation from first-order kinetics. Experiments were classified, according to the shape of the inactivation curve, into biphasic, grace-period and activation-period inactivations. A unified evaluation procedure, based on the discrimination among a few kinetic models derived from a general unimolecular series mechanism, was employed for all experiments. It was shown that regardless of the shape of the inactivation curve, only a few experiments needed more than a simple series mechanism, represented by a biexponential curve, for a satisfactory fit. Carrying out inactivation experiments at several temperatures was shown to be very effective in checking the consistency of kinetic equations obtained by isothermal evaluation. The results of isothermal evaluation could always be questioned in this way and no consequent conclusions on inactivation mechanism could be drawn.     

Isolation and properties of glucose-6-phosphate dehydrogenase from human cultivated cells]

A new procedure for purification of glucose-6-phosphate dehydrogenase resulting in an electrophoretically homogenous preparation made up of 5.10(8) cells (390 mg of protein) is proposed. The enzyme yield is more than 20%. The molecular weights of a subunit and a native enzyme are 55000 and 220000, respectively. The isoelectric point for the protein lies at 4,8. The kinetics of the enzyme thermal inactivation obey the first order equation with the inactivation rate constant of 6.10(-3) min-1.     

Kinetic modeling of thermal inactivation of the Bacillus sp. protease P7

Data from thermal stability of a keratinolytic protease produced by the Amazon isolate Bacillus sp. P7 was fitted to various mathematical models. Kinetic modeling showed that Weibull distribution was the best equation to describe the residual activity of protease P7 after heat treatment. The effects of temperature on equation parameters and on characteristics of the inactivation curves were evaluated. As expected, faster inactivation was observed at higher temperatures. The critical temperature to accelerate protease decomposition was about 70 °C. The reliable life (t _R) of the enzyme, analogous to the D value, ranged from 1,824 to 8 min at 45–65 °C. Within these temperatures, an increase of 8.81 °C was needed to lower enzyme t _R in one-log unit. Protease P7 is a potentially useful biocatalyst for various industrial bioprocesses, and therefore, kinetic modeling of thermal inactivation addresses an important topic aiming enzyme characterization and applications.     

Effect of modifications of a series of amino acid radicals on the enzymatic activity of glucoamylase from Aspergillus awamori

The effect of chemical modification of various amino acid residues on the enzymatic activity of glucoamylase from Asp. awamori was studied. Modification of the carboxyl groups by taurine in the presence of water-soluble carbodiimide results in complete inactivation of the enzyme. The inactivation process includes two steps, namely non-specific modification and modification of the active center carboxyls. The rate constants of inactivation at both steps were measured in the presence and absence of the substrate, i. e. maltose. It was shown that the enzyme is inactivated by N-bromosuccinimide. Based on the data on the protection of the enzyme active center by the substrates (maltooligosaccharides of various lengths), it was concluded that the essential tryptophane residue(s) is localized in the fourth subsite. Ethoxycarbonylation, nitration and acetylation of glucoamylase do not change the catalytic activity of the enzyme. The protein was shown to contain no SH-groups.     

Stability of 6-beta-[(hexahydro-1H-azepin-l-yl)methyleneamino]-penicillanic acid in aqueous solutions]

Stability of acqueous solutions of 6-beta-[(hexahydro-IH-azepin-I-yl)methylenamino] penicillanic acid at various values of pH and temperature was studied. It was found that inactivation of the antibiotic in both the acid and the alkaline medium proceeded according to the equation of the 1st order. At pH 1.3 and a temperature of 35 degrees the half life of the antibiotic was 7 hours. The activation energy calculated according to the Arrenius equation was 13.5 kcal/mol at pH 1.3 and 22.2 kcal/mol at pH 10.5. The antibiotic was inactivated in glycol and phosphate buffers. Its qualitative analysis was performed according to an improved iodometric method.     

Experimental and numerical study of heterogeneous pressure-temperature-induced lethal and sublethal injury of Lactococcus lactis in a medium scale high-pressure autoclave

The present contribution is dedicated to experimental and theoretical assessment of microbiological process heterogeneities of the high-pressure (HP) inactivation of Lactococcus lactis ssp. cremoris MG 1363. The inactivation kinetics are determined in dependence of pressure, process time, temperature and absence or presence of co-solutes in the buffer system namely 4 M sodium chloride and 1.5 M sucrose. The kinetic analysis is carried out in a 0.1-L autoclave in order to minimise thermal and convective effects. Upon these data, a deterministic inactivation model is formulated with the logistic equation. Its independent variables represent the counts of viable cells (viable but injured) and of the stress-resistant cells (viable and not injured). This model is then coupled to a thermo-fluiddynamical simulation method, high-pressure computer fluid dynamics technique (HP-CFD), which yields spatiotemporal temperature and flow fields occurring during the HP application inside any considered autoclave. Besides the thermo-fluiddynamic quantities, the coupled model predicts also the spatiotemporal distribution of both viable (VC) and stress-resistant cell counts (SRC). In order to assess the process non-uniformity of the microbial inactivation in a 3.3-L autoclave experimentally, microbial samples are placed at two distinct locations and are exposed to various process conditions. It can be shown with both, experimental and theoretical models that thermal heterogeneities induce process non-uniformities of more than one decimal power in the counts of the viable cells at the end of the treatment.     

Modeling of pathogen survival during simulated gastric digestion

The objective of the present study was to develop a mathematical model of pathogenic bacterial inactivation kinetics in a gastric environment in order to further understand a part of the infectious dose-response mechanism. The major bacterial pathogens Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. were examined by using simulated gastric fluid adjusted to various pH values. To correspond to the various pHs in a stomach during digestion, a modified logistic differential equation model and the Weibull differential equation model were examined. The specific inactivation rate for each pathogen was successfully described by a square-root model as a function of pH. The square-root models were combined with the modified logistic differential equation to obtain a complete inactivation curve. Both the modified logistic and Weibull models provided a highly accurate fitting of the static pH conditions for every pathogen. However, while the residuals plots of the modified logistic model indicated no systematic bias and/or regional prediction problems, the residuals plots of the Weibull model showed a systematic bias. The modified logistic model appropriately predicted the pathogen behavior in the simulated gastric digestion process with actual food, including cut lettuce, minced tuna, hamburger, and scrambled egg. Although the developed model enabled us to predict pathogen inactivation during gastric digestion, its results also suggested that the ingested bacteria in the stomach would barely be inactivated in the real digestion process. The results of this study will provide important information on a part of the dose-response mechanism of bacterial pathogens.     

Kinetics of protein-modification reactions. Determination of the fractional concentration of enzyme protein groups, or group reactivities, essential for catalytic function.

A mathematical treatment of modification-induced enzyme protein inactivation is presented, and it is shown that, at initial reaction conditions, the ratio of the first derivative of the equation describing enzyme activity loss to the first derivative of the equation describing protein groups modification is equal to the fractional concentration of enzyme protein reactive groups, or group reactivities, essential for catalytic function.     

Sophoraflavanone G from sophora pachycarpa enhanced the antibacterial activity of gentamycin against Staphylococcus aureus

In this study the enhancement effect of Sophora pachycarpa roots' acetone extract on the antibacterial activity of gentamycin was evaluated against Staphylococcus aureus. Disc diffusion and broth dilution methods were used to determine the antibacterial activity of gentamycin in the absence and presence of plant extract and its various fractions separated by TLC. A clinical isolate of S. aureus was used as test strain. The active component of the plant extract involved in enhancement of gentamycin's activity had Rf = 0.72 on a TLC plate. The spectral data (1H NMR, 13C NMR) of this compound revealed that this compound was 5,7,2',4'-tetrahydroxy-8-lavandulylflavanone (sophoraflavanone G), previously isolated from Sophora exigua. In the presence of 0.03 microg/ mL of sophoraflavanone G the MIC value of gentamycin for S. aureus decreased from 32 to 8 microg/mL (a four-fold decrease). These results signify that the ultra-low concentration of sophoraflavanone G potentiates the antimicrobial action of gentamycin suggesting a possible utilization of this compound in combination therapy against Staphylococcus aureus.     

庆大霉素单克隆抗体的制备及试剂盒的配制

目的建立庆大霉素直接竞争酶联免疫吸附分析方法。方法应用戊二醛法制备庆大霉素完全抗原,通过杂交瘤技术筛选分泌特异性庆大霉素抗体的杂交瘤细胞株,并建立庆大霉素竞争酶联免疫吸附分析检测方法。结果获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,建立了庆大霉素竞争酶联免疫吸附分析检测方法,该方法操作简单具有良好的线性、特异性和精密度;庆大霉素质量浓度在1.5625～50.0000 ng/mL范围内,呈现良好的线性,r2=0.9913,50%抑制浓度为(IC50)为7.37 ng/mL,检测限(LOD)为1.54 ng/mL,该试剂盒与链霉素等8种药物无交叉反应。结论获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,研制的庆大霉素竞争ELISA检测试剂盒具有良好的线性、特异性和精密度。     

Study of the inactivation of antibiotics by bacterial colonies

A procedure of bacteria application to disks from the colonies was used for determining antibiotic inactivation in the disks by the bacteria colonies after the disk direct contact with the colonies. Changes in the antibiotic activity in the disks were registered after incubation at 37 degrees C for 2 hours. It was shown that ampicillin resistant strains of E. coli K12 carrying R plasmids and strains of S. typhimurium and S. aureus inactivated the antibiotics in the disks and their population were homogenous in this respect. It is advisable to use the procedure in assaying drug resistance of bacterial populations.