The activation of urease

1. It has been shown that the activity of solutions of twice recrystallized urease is reversibly increased by moderate heating and reversibly decreased by storage in the cold, even in the frozen state. 2. Crude extracts of jack bean meal containing potent urease undergo this same type of reversible activation by heating and inactivation by cooling. Dilution has the same potentiating effect on the activity as moderate heating. As much as a fivefold increase in activity can be obtained when a sample previously inactivated by storage for 24 hours at -10 degrees C. is heated for 5 minutes at 60 degrees C. 3. Solutions of crystalline urease protected by serum albumin and preserved in the cold give a constant "potential" activity over a period of more than 30 days if heated 5 minutes at 60 degrees C. before assay. 4. The data presented have been interpreted to mean that an association between urease molecules (or between urease and other proteins) might occur, resulting in inactivation of the enzyme which would be reversed on dissociation. 5. It has been postulated that the same forces are responsible for the reversible inactivation brought about by standing at temperatures above or below the freezing point.     

Sensitivity to bile salts of Shigella flexneri sublethally heat stressed in buffer or broth.

Batch cultures of Shigella flexneri M4243 were grown at 37 degrees C in broth to early stationary phase, washed, and heated at 50 degrees C in 0.1 M phosphate buffer (pH 7.0). Cells were surface plated on a tryptic phytone glucose agar (TPGA), TPGA with 0.15 or 0.85% bile salts no. 3 (TPGA-BS 0.15 or TPGA-BS 0.85), or TPGA with 0.25 or 0.50% sodium deoxycholate (TPGA-DC 0.25 or TPGA-DC 0.50). Cells sampled after no heating produced colony counts on TPGA-BS 0.85 or on TPGA-DC 0.50 that were no more than about 0.5 log lower than for unheated cell samples plated on TPGA. Cells heated at 50 degrees C for 30 min produced colony counts on TPGA-DC 0.50 or on TPGA-BS 0.85 that were about 1.5 logs lower than on TPGA. Cells heated for 30 min and shifted to TPG broth at 37 degrees C to allow resuscitation required about 2 h to regain tolerance to 0.85% BS. However, heated cells resuscitated on solid TPGA at 35 degrees C before being challenged with overlays of TPGA-BS 0.85 or TPGA-DC 0.50 required 6 to 8 h on TPGA to regain tolerance to 0.85% BS or 0.50% DC. To regain tolerance to overlays of 0.15% BS or 0.25% DC, heated cells required resuscitation periods on TPGA of about 2 or 2 to 6 h, respectively. Cells heated in TPG broth and sampled after no heating produced colony counts on TPGA that were about 1.5 logs lower than for unheated cell suspensions, suggesting greater apparent injury when heat stressed in broth than in buffer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitivity to bile salts of Shigella flexneri sublethally heat stressed in buffer or broth

Batch cultures of Shigella flexneri M4243 were grown at 37 degrees C in broth to early stationary phase, washed, and heated at 50 degrees C in 0.1 M phosphate buffer (pH 7.0). Cells were surface plated on a tryptic phytone glucose agar (TPGA), TPGA with 0.15 or 0.85% bile salts no. 3 (TPGA-BS 0.15 or TPGA-BS 0.85), or TPGA with 0.25 or 0.50% sodium deoxycholate (TPGA-DC 0.25 or TPGA-DC 0.50). Cells sampled after no heating produced colony counts on TPGA-BS 0.85 or on TPGA-DC 0.50 that were no more than about 0.5 log lower than for unheated cell samples plated on TPGA. Cells heated at 50 degrees C for 30 min produced colony counts on TPGA-DC 0.50 or on TPGA-BS 0.85 that were about 1.5 logs lower than on TPGA. Cells heated for 30 min and shifted to TPG broth at 37 degrees C to allow resuscitation required about 2 h to regain tolerance to 0.85% BS. However, heated cells resuscitated on solid TPGA at 35 degrees C before being challenged with overlays of TPGA-BS 0.85 or TPGA-DC 0.50 required 6 to 8 h on TPGA to regain tolerance to 0.85% BS or 0.50% DC. To regain tolerance to overlays of 0.15% BS or 0.25% DC, heated cells required resuscitation periods on TPGA of about 2 or 2 to 6 h, respectively. Cells heated in TPG broth and sampled after no heating produced colony counts on TPGA that were about 1.5 logs lower than for unheated cell suspensions, suggesting greater apparent injury when heat stressed in broth than in buffer.(ABSTRACT TRUNCATED AT 250 WORDS)     

The properties of the extracellular alkaline phosphatase of the causative agent of melioidosis]

After growing P. pseudomallei VPA on solid medium extracellular alkaline phosphatase with a molecular weight of 93,000 AMU was isolated, and practically purified from the extract of this medium by precipitation with ammonium sulfate, subsequent gel chromatography and concentration on membrane filters. The optimum conditions for enzymatic reaction were found to be pH 9.0 and a temperature of 50 degrees C. The enzyme was resistant to freezing and to heating at a temperature of up 60 degrees C for 30 minutes, as well as to the action of pH 3.0-10.5, but became completely inactivated after heating at 90 degrees C for 10 minutes and incubation at pH 2.0 for 20 hours.     

Adenylate cyclase of the causative agent of plague: its purification and properties

Three forms of adenylate cyclase have been detected in Y. pestis: membrane-bound, cytoplasmic and extracellular. Extracellular adenylate cyclase has been purified so as to achieve a homogeneous state, and some of its physicochemical parameters have been investigated. In the process of purification the initial preparation of this enzyme has been subjected to heating at 100 degrees C for 15 minutes, fractionation with ammonium sulfate, and gel filtration on Sephadex G-100. The homogeneity of adenylate cyclase has been confirmed by electrophoresis in 7.5% polyacrylamide gel and precipitation by the plague agglutinating serum. The enzyme has been found to have a molecular weight of 30,000 daltons and to show the optimum activity at pH 7.0-7.2 and at a temperature between 37 and 40 degrees C. Monospecific rabbit serum to the homogeneous preparation of adenylate cyclase has been obtained.     

Immunogenicity of the surface polysaccharide of serotype A meningococci possessing different physico-chemical properties

The influence of changes in the physico-chemical parameters of serogroup A meningococcal polysaccharide on its immunogenicity for mice was studied by means of passive local hemolysis in gel and the passive hemagglutination test. The polysaccharide was depolymerized by heating at 100 degrees C for 5, 30 and 120 minutes; during this process the progressing decrease of the molecular weight and the content of O-acetyl groups in the preparation could be observed. Mice showed high sensitivity to changes in the above-mentioned physico-chemical parameters, which was manifested by a sharp drop in the intensity of the immune response of the animals to the heated samples of the antigen. The role of the parameters under study, i. e. the molecular weight of the antigen and the presence of O-acetyl groups, in the manifestations of the immunogenicity of polysaccharide A is discussed.     

Destructive effect of heating against toxoplasma oocysts.

Heating was examined for destructive effect against Toxoplasma oocysts, both unsporulated and sporulated, of the O-1 and O-3 strains. Sporulation-inhibition rates were measured by counting sporulated and unsporulated oocysts in each examination. As a result, the sporulation of Toxoplasma oocysts was completely inhibited by exposure to 60 or 70 degrees C for 10 seconds, 55 degrees C for 30 seconds, 50 degrees C for 2.5 minutes. 45 degrees C for 1 hour, or 37 degrees C for 48 hours. When examined by the mouse inoculation method, the infectivity of sporulated oocysts became extinct after heating at 90 degrees C for 30 seconds, 80 degrees C for 1 minute, 70 degrees C for 2 minutes, 55 or 60 degrees C for 15 minutes, or 50 degrees C for 30 minutes. It was confirmed that heating was effective for the sterilization of Toxoplasma oocysts.     

Exposure to pretreatment hypothermia as a determinant of heat killing

When Chinese hamster ovary (CHO) cells were exposed to 22 degrees C for 2 hr prior to 42.4 degrees C hyperthermia, neither the shoulder region of the survival curve nor the characteristic development of thermotolerance after 3-4 hr of heating were observed. Absolute cell survival after 4 hr at 42.4 degrees C was decreased by a factor of between 10 and 100 (depending on the rate of heating of nonprecooled controls). Conditioning at 30 degrees C for 2 hr, 26 degrees C for 2 hr, or 22 degrees C for 20 min followed by heating to 42.4 degrees C over 30 min did not result in sensitization. Prolonged (16 hr) conditioning at 30 degrees C, however, increased the cytotoxicity of immediate exposure to 41.4 or 45 degrees C with maximum sensitization to 45 degrees C occurring after 6 hr at 30 degrees C. Both 3- and 18-hr pretreatments at 30 degrees C similarly increased the cytotoxicity of 45-41.5 degrees C step-down heating (D0 = 28 min in precooled versus 40 min in nonprecooled cells).     

Identification of a putative ribosomal protein mRNA in Chironomus riparius and its response to cadmium, heat shock, and actinomycin D

A putative ribosomal protein (rp) mRNA in Chironomus riparius has been found using differential display (DD). Its sequence has 84.8% identity with mosquito rp L8, Aedes albopictus, and is approximately 0.9 kb. Studies were undertaken in order to evaluate rp as a control for environmentally relevant genes. Responses of Drosophila heat shock 70 gene (hsp70) were used to establish heat shock temperatures and cadmium (Cd) concentrations for Chironomus experiments and to validate DD. Expression of hsp70 was induced over control by 28 degrees C at 30 minutes and 1 mM Cd at 24 hours (p< or =0.05). For Chironomus, DD, Northern blot, and nuclease sensitivity were used to measure responses to two stressors: heat shock for 30 minutes and Cd for 24 or 48 hours. Differential display and nuclease sensitivity assays found expression of rp mRNA at 37 degrees C and 16 mM Cd to be similar to controls. Northern blots indicated statistically significant effects for heat shock (p = 0.046) but not Cd (p = 0.406). However, mRNA levels at 37 degrees C were increased only 1.72-fold over controls. A concentration of 24 nM actinomycin D suppressed rp expression as measured by nuclease sensitivity assays. Stressors should not affect rp mRNA levels below their LC-50s.     

Thermal inactivation and injury of Bacillus stearothermophilus spores.

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively. Recovery on AAMS-S resulted in reduced decimal reduction time. The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C. The rates of inactivation and injury were similar. Injury (judged by salt sensitivity) was a linear function of the heating temperature. At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time). Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation.     

Thermal inactivation and injury of Bacillus stearothermophilus spores

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively. Recovery on AAMS-S resulted in reduced decimal reduction time. The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C. The rates of inactivation and injury were similar. Injury (judged by salt sensitivity) was a linear function of the heating temperature. At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time). Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation.     

The N intermediate of bacteriorhodopsin at low temperatures: stabilization and photoconversion

In aqueous suspensions of purple membranes (pH 10.2, 0.4 M KCl) an intermediate having an absorption maximum at 570-575 nm (at -196 degrees C) was produced by first heating the M intermediate up to -30 degrees C and then stabilizing it by subsequent cooling to -60 degrees C. We suggest that this species is the intermediate N (or P or R) found and characterized earlier near room temperature. Upon illumination at -196 degrees C N is transformed into a bathochromically absorbing species KN which has an absorption maximum near 605 nm and an extinction 1.35 times that of N. This light reaction is photoreversible. The quantum yield ratio for the forward and back reaction is 0.18 +/- 0.02. The maximum photo steady state concentration of KN is about 0.24. The N intermediate was also trapped in water suspensions of purple membranes at neutral pH and low salt concentration by illumination at lambda greater than 620 nm during cooling. In addition to N another intermediate absorbing in the red (maximum at 610-620 nm) was accumulated in smaller amounts. It is not photoactive at -196 degrees C and apparently is the O intermediate or a photoproduct of N.     

Attenuation of Cf-mediated defense responses at elevated temperatures correlates with a decrease in elicitor-binding sites

The interaction between the fungal pathogen Cladosporium fulvum and its only host, tomato, is a well-described gene-for-gene system and several resistance (Cf) genes of tomato and matching fungal avirulence (Avr) genes have been characterized. Transgenic tobacco suspension cells expressing Cf genes respond to matching elicitors with typical defense responses, such as medium alkalization and an oxidative burst. We found that this response is attenuated at elevated ambient temperatures. Tomato seedlings expressing both a Cf and the matching Avr gene rapidly die as a result of systemic necrosis at normal temperatures, but are rescued at 33 degrees C. We demonstrate that, at 33 degrees C, the Cf/Avr-mediated induction of defense-related genes is reversibly suppressed. Furthermore, in cell suspensions, the AVR-induced medium alkalization response is slowly suppressed upon incubation at 33 degrees C, but is quickly restored after transfer to lower temperatures. A high-affinity binding site (HABS) for AVR9 is present on plasma membranes isolated from solanaceous plants and has been suggested to act as a co-receptor for AVR9. The amount of AVR9-HABS is 80% reduced in tobacco cell suspensions incubated at 33 degrees C, as compared with cell suspensions incubated at 20 degrees C. Our data suggest that the temperature sensitivity of Cf-mediated defense responses resides at the level of perception of the fungal avirulence factors.     

Continuous enzymatic cooking and liquefaction of starch using the technique of direct resistive heating

Continuous cooker prototypes of very simple design, using electricity as a primary energy source, were developed for the process of cooking and liquefaction of starch suspensions. Previous work on equipment using microwave dielectric heating has already been reported. Results of energy consumption as low as 330 kcal/kg based on starch content were achieved. Considering these results and looking for new solutions or engineering concepts, the authors have been investigating the possibility of using electric energy at 60 Hz for direct resistive heating, in which the starch suspension is the proper "resistor."The most important results of energetic yield obtained until now, working in a continuous process of cooking-liquefaction, are not larger than 235 kcal (272 Wh)/kg based on starch content. These results were obtained using a commercial grade alpha-amylase from B. subtillis, working with temperatures ranging from 70 to 75 degrees C, and with residence times in the reactor not greater than 1.5 min. The experiments of saccharification and fermentation accomplished as a test for the efficiency of this heating technique gave good results (as with a conventional technique) and thus enabled us to proceed with the studies.     

Kinetics and in vitro origin of the temperature-dependent transition of the estrogen receptor monomer

Partitioning of estrogen receptors in aqueous two-phase polymer systems has provided the basis for a detailed kinetic analysis of the effects of temperature on estrogen receptor (ER) structure in vitro. Exposure to temperatures of 0-30 degrees C increased the rate of change in ER partition coefficients by up to 100-fold but did not affect the final extent of the process. The temperature-dependent change in ER partition coefficients was characterized by a linear Arrhenius plot and an activation energy of 25 kcal/mol. The rate of the temperature-dependent ER transition (28 degrees C) was found to be unaffected by greater than 50-fold changes in receptor concentration, which indicates that the temperature-dependent change in partition coefficients reflects a first-order process. The partition coefficients of heated ER were unaffected by subsequent 18-h incubations at 0 degree C, indicating that the temperature-dependent ER transition is irreversible in vitro. Direct heating of the unoccupied ER resulted in both a change in ER partition coefficients and a loss of ER binding sites. The temperature-dependent change in unoccupied ER partition coefficients was complete within 30 min at 28 degrees C and yielded a first-order rate constant that was the same as that obtained for heating the receptor-estradiol complex at 28 degrees C. In contrast, the loss of unoccupied ER binding sites that occurred during 28 degrees C incubations did not reach completion after 150 min of heating and was found to behave as a second-order process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bactericidal action of high-power Nd:YAG laser light on Escherichia coli in saline suspension

Infra-red light (1064 nm) from a high-power Nd:YAG laser caused more than 90% loss of viability of Escherichia coli during exposures that raised the temperature of PBS suspensions of the bacteria to 50 C in a thermocouple-equipped cuvette. In contrast, there was minimal loss of viability after heating the same suspensions to 50 degrees C in a water-bath, or in a PCR thermal cycler. The mechanism of laser killing at 50 degrees C was explored by differential scanning calorimetry, by laser treatment of transparent and turbid bacterial suspensions, and by optical absorbency studies of E. coli suspensions at 1064 nm. Taken together, the data suggested that the bactericidal action of Nd:YAG laser light at 50 degrees C was due partly to thermal heating and partly to an additional, as yet undefined, mechanism. Scanning electron microscopy revealed localized areas of surface damage on laser-exposed E. coli cells.     

Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes

Four strains of Listeria monocytogenes were examined for catalase (CA) and superoxide dismutase (SOD) activities. The two strains having the highest CA activities (LCDC and Scott A) also possessed the highest SOD activities. The CA activity of heated cell extracts of all four strains examined decreased sharply between 55 and 60 degrees C. SOD was more heat labile than CA. Two L. monocytogenes strains demonstrated a decline in SOD activity after heat treatment at 45 degrees C, whereas the other two strains demonstrated a decline at 50 degrees C. Sublethal heating of the cells at 55 degrees C resulted in increased sensitivity to 5.5% NaCl. Exogenous hydrogen peroxide was added to suspensions of L. monocytogenes; strains producing the highest CA levels showed the greatest H2O2 resistance.     

The sensitivity of the thoracolumbar spinal cord of the mouse to hyperthermia

Twelve millimeters of the thoracolumbar spinal cord of mice has been treated with a radiofrequency heating system which has been shown previously to produce localized and controllable elevation of temperature. The severity of neurological damage was assessed by measuring the reduction in the reflex leg extension of the hind legs of the mice from video-recorded images and by scoring the performance of the mice by a negative geotaxis test. The response to treatment was rapid with maximum paralysis occurring within a few days after treatment. Only minor symptoms were observed in those animals which had not developed paralysis within 2 weeks. A 40% reduction in the reflex leg extension was chosen as an end point, and the percentage of mice having reached the end point for different thermal doses was determined in groups of nine mice. The ED50 for heating for 1 h was 43.1 degrees C and for heating at 45 degrees C was 10.8 min. An increase in temperature by 1 degree C required a decrease in time by a factor of 2.25 to produce the same effect. Thermotolerance was observed 24 h after preheating at 45 degrees C for 1.9 min with a thermotolerance ratio of 1.7. The rapid response and high sensitivity of the spinal cord will have to be taken into consideration in the clinical application of hyperthermia.     

Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes.

Four strains of Listeria monocytogenes were examined for catalase (CA) and superoxide dismutase (SOD) activities. The two strains having the highest CA activities (LCDC and Scott A) also possessed the highest SOD activities. The CA activity of heated cell extracts of all four strains examined decreased sharply between 55 and 60 degrees C. SOD was more heat labile than CA. Two L. monocytogenes strains demonstrated a decline in SOD activity after heat treatment at 45 degrees C, whereas the other two strains demonstrated a decline at 50 degrees C. Sublethal heating of the cells at 55 degrees C resulted in increased sensitivity to 5.5% NaCl. Exogenous hydrogen peroxide was added to suspensions of L. monocytogenes; strains producing the highest CA levels showed the greatest H2O2 resistance.     

Acid tolerance of Shigella sonnei and Shigella flexneri

AIMS: Determination of the behaviour of Shigella sonnei and Sh. flexneri under acid conditions. METHODS AND RESULTS: The growth and survival of Shigella spp. (9 isolates) in acidified Brain Heart Infusion (BHI) (pH 5.0-3.25 with pH intervals of 0.25) was determined after 6, 24 and 30 h incubation at 37 degrees C. Subsequently, survival of shigellae was studied in apple juice and tomato juice stored at 7 degrees C and 22 degrees C for up to 14 days and in strawberries and a fresh fruit salad, kept at 4 degrees C for 4 and 48 h. CONCLUSIONS: The minimum pH for growth in acidified BHI for Sh. flexneri and Sh. sonnei was, respectively, pH 4.75 and pH 4.50. Survival in fruit juices and fresh fruits depended upon their pH, the type of strain and the incubation temperature. Shigella spp. Survived for up to 14 days in tomato juice and apple juice stored at 7 degrees C. The shortest survival time (2-8 d) was observed in apple juice at 22 degrees C. Sh. sonnei but not Sh. flexneri was recovered after 48 h from strawberries and fruit salad kept at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Acid foods, especially if kept at refrigeration temperatures, support survival of Shigella spp. and may cause Shigella food poisoning.