Effects of ozone treatment on the infectivity of hepatitis A virus

The inactivation of a large-focus-forming variant of hepatitis A virus (HM-175) by ozone was investigated. Experiments using mainly single-particle virus preparations suspended in phosphate-carbonate buffer were conducted over a range of pH levels (6-8) at 4 degrees C. Viral enumerations involved the use of a radioimmunofocus assay. While some tolerance to lower (i.e., 0.1-0.5 mg/L) ozone residuals was noted, the exposure of virus particles to ozone concentrations of 1 mg/L or greater at all pH levels resulted in their complete (5 log) inactivation within 60 s. The pH-related effects that were observed were not considered to be significant.     

Gaseous ozone treatment inactivates Listeria innocua in vitro

AIMS: To investigate the effects of ozone on inactivation of Listeria innocua on solid media. METHODS AND RESULTS: Suspensions of L. innocua ranging from 4.5 x 10(4 )- 6.4 x 10(4) CFU ml(-1) were inoculated onto potato dextrose agar (PDA, pH 5.6 and 6.8) and nutrient agar (NA, pH 6.0 and 6.8), then exposed to gaseous ozone. Variable factors included postinoculation standing time at 20 degrees C before exposure to ozone, ozone concentration, treatment duration and treatment temperature (5 or 20 degrees C). The interaction among ozone concentration, treatment duration, media and temperature in effecting changes in colony-forming units (CFU) was significant. The 100 nl l(-1) ozone treatment for 2 h reduced the microbial populations by 2-3 log CFU ml(-1). Cell viability decreased more rapidly on PDA than on NA. The average time to obtain a 2 log CFU ml(-1) reduction was 1.3 h at 20 degrees C and 2.5 h at 5 degrees C (P < 0.001). CONCLUSIONS: Gaseous ozone effectively inactivates L. innocua at concentrations of 50 and 100 nl l(-1) during short exposure times at both 5 and 20 degrees C. The Gompretz model can be utilized for determining the response of L. innocua to ozone over time. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information on ozone inactivating Listeria spp., which may be imposed on ensuring quality and safety of horticultural produce and food products.     

Effects of cold storage on field and laboratory performance of Trichogramma carverae (Hymenoptera: Trichogrammatidae) and the response of three Trichogramma spp. (T. carverae, T. nr. brassicae, and T. funiculatum) to cold

Delaying emergence of Trichogramma spp. is critical for commercial production. Here, diapause induction was considered for three species (Trichogramma nr. brassicae Bezdenko, Trichogramma carverae Oatman & Pinto, and Trichogramma funiculatum Carver), and the effect of storage temperature (4 degrees C, 8 degrees C, and 10 degrees C) and time (1-8 wk) was investigated for T. carverae. For all species, percentage of emergence was lowered after an initial diapause induction period (28 d at 14 degrees C and a photoperiod of 8:16 [L:D] h) and lowered further after 1-mo storage at 3 degrees C and a photoperiod of 0:24 (L:D) h. No wasps emerged after 2 mo of storage, suggesting that true diapause was not induced. The effect of 1-8-wk storage on wasp quality was investigated for T. carverae both in the laboratory and the field. Initial fieldwork suggested that this species could be successfully stored at 10 degrees C under continuous light (after 5-d development at 25 degrees C and a photoperiod of 16:8 [L:D] h) without reducing the ability of wasps to parasitize eggs in the field. In a second experiment, storage temperatures lower than 10 degrees C and storage times 3 wk or longer had a negative impact on emergence and longevity, and effects were not additive. Negative effects may partly reflect size changes, because size decreased in response to storage time, and there was an interaction between time and temperature effects on size. Storage time was the major factor influencing fecundity and field success; both fitness measures were reduced after storage of 3 wk or longer. T. carverae can therefore be successfully stored for up to 2 wk without detrimental effects, and 10 degrees C is the preferred storage temperature. T. carverae seems to survive unfavorable temperature conditions by entering a state of quiescence.     

Reversible, positive cooperative interaction of 11 beta-chloromethyl-[3H]estradiol-17 beta with the calf uterine estrogen receptor

The binding of 11 beta-chloromethyl-[3H]estradiol-17 beta [3H]CME2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4-1.5. This positive cooperative interaction of [3H]CME2 was indistinguishable from the typical cooperative interaction of [3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics of [3H]CME2 from the receptor was biphasic, composed of a fast dissociating component (15%, t1/2 = 4 min at 0 degrees C; 9%, t1/2 = 4 min at 28 degrees C) and a slow dissociating component (85%, t1/2 greater than 50 h at 0 degrees C; 91%, t1/2 greater than 50 h at 28 degrees C). The dissociation kinetics of [3H]estradiol was also biphasic: the t1/2 of the fast dissociating component was 4 min at 0 and 28 degrees C and approximately 200 min for the slow dissociating component at both temperatures. The fraction of the slow [3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [3H]CME2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME2 are discussed.     

Effect of relative humidity and air temperature on survival of hepatitis A virus on environmental surfaces.

Stainless steel disks (diameter, 1 cm) were contaminated with fecally suspended hepatitis A virus (HAV; strain HM-175) and held at low (25% +/- 5%), medium (55% +/- 5%), high (80% +/- 5%), or ultrahigh (95% +/- 5%) relative humidity (RH) at an air temperature of 5,20, or 35 degrees C. HAV survival was inversely proportional to the level of RH and temperature, and the half-lives of the virus ranged from greater than 7 days at the low RH and 5 degrees C to about 2 h at the ultrahigh RH and 35 degrees C. In parallel tests with fecally suspended Sabin poliovirus (PV) type 1 at the low and ultrahigh RH, all PV activity was lost within 4 h at the low RH whereas at the ultrahigh RH it remained detectable up to 12 h. HAV could therefore survive much better than PV on nonporous environmental surfaces. Moreover, the ability of HAV to survive better at low levels of RH is in direct contrast to the behavior of other enteroviruses. These findings should help in understanding the genesis of HAV outbreaks more clearly and in designing better measures for their control and prevention.     

Effect of the method of preparing monochloramine upon inactivation of MS2 coliphage, Escherichia coli, and Klebsiella pneumoniae.

Monochloramine prepared in situ by first adding chlorine to a suspension of microorganisms, followed by subsequent addition of ammonia, inactivated the MS2 coliphage more rapidly than did exposure of phage to monochloramine prepared either by adding chlorine to ammonia or by adding chlorine and ammonia simultaneously. The rapid viral inactivation was apparently due to the exposure of MS2 to free chlorine before the addition of ammonia. The average 99% CT value of MS2 when exposed to free chlorine was 1.3 and 1.1 at 5 and 15 degrees C, respectively. The average 99% CT values of MS2 briefly exposed to the combined action of free chlorine followed by the addition of ammonia to form monochloramine in situ were 19.3 and 1.5 at 5 and 15 degrees C, respectively. No 99% CT values were calculated for the inactivation of MS2 with preformed monochloramine because less than 1 log (90%) of inactivation occurred during a 4-h contact time. Inactivation of MS2 by monochloramine was more rapid at 15 than at 5 degrees C and when the chlorine to nitrogen weight ratio was 5:1 compared with 3:1. Monochloramine was a more efficient inactivating agent for the coliforms Escherichia coli and Klebsiella pneumoniae than it was for the MS2 coliphage.     

Effect of relative humidity and air temperature on survival of hepatitis A virus on environmental surfaces.

Stainless steel disks (diameter, 1 cm) were contaminated with fecally suspended hepatitis A virus (HAV; strain HM-175) and held at low (25% +/- 5%), medium (55% +/- 5%), high (80% +/- 5%), or ultrahigh (95% +/- 5%) relative humidity (RH) at an air temperature of 5,20, or 35 degrees C. HAV survival was inversely proportional to the level of RH and temperature, and the half-lives of the virus ranged from greater than 7 days at the low RH and 5 degrees C to about 2 h at the ultrahigh RH and 35 degrees C. In parallel tests with fecally suspended Sabin poliovirus (PV) type 1 at the low and ultrahigh RH, all PV activity was lost within 4 h at the low RH whereas at the ultrahigh RH it remained detectable up to 12 h. HAV could therefore survive much better than PV on nonporous environmental surfaces. Moreover, the ability of HAV to survive better at low levels of RH is in direct contrast to the behavior of other enteroviruses. These findings should help in understanding the genesis of HAV outbreaks more clearly and in designing better measures for their control and prevention.     

Temporal separation of insulin-stimulated GLUT4/IRAP vesicle plasma membrane docking and fusion in 3T3L1 adipocytes

Examination of the time and temperature dependence of insulin-stimulated GLUT4/IRAP-containing vesicle trafficking demonstrated an approximate 7-fold increase in the half-time for plasma membrane translocation at 23 degrees C (t((1)/(2)) = approximately 30 min) compared with 37 degrees C (t((1)/(2)) = approximately 4 min) without a significant change in the extent of either GLUT4 or IRAP translocation. Localization of the endogenous GLUT4 and expressed GLUT4-enhanced green fluorescent protein fusion protein in intact 3T3L1 adipocytes demonstrated that at 23 degrees C there was a time-dependent accumulation of discrete GLUT4-containing vesicles adjacent to the inner face of the cell surface membrane but that was not contiguous and/or physically incorporated into the plasma membrane. Together, these data demonstrate that the temperature-dependent decrease in the rate of GLUT4 and IRAP translocation results from a reduction in GLUT4/IRAP-containing vesicle fusion and not trafficking or docking to the plasma membrane.     

A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-beta-xylanase II

We have successfully engineered a disulphide bridge into the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII) by substituting Thr-2 and Thr-28 with cysteine. The T2C:T28C mutational changes increased the half-life in thermal inactivation of this mesophilic enzyme from approximately 40 s to approximately 20 min at 65 degrees C, and from less than 10 s to approximately 6 min at 70 degrees C. Therefore, the N-terminal disulphide bridge enables the use of XYNII at substantially higher temperatures than permitted by its native mesophilic counterpart. Altogether, thermostability increased by about 15 degrees C. The kinetic properties of the mutant XYNII were maintained at the level of the wild type enzyme. Our findings demonstrated that a properly designed disulphide bridge, here within the N-terminal region of XYNII, can be very effective in resisting thermal inactivation.     

Activation of thymocyte glucocorticoid receptors to the steroid binding form. The roles of reduction agents, ATP, and heat-stable factors.

The specific glucocorticoid binding capacity in cytosol preparations of rat thymocytes decays with a half-life of 4 h at 0 degrees C or 20 min at 25 degrees C. Phosphatase inhibitors (molybdate, fluoride, glucose 1-phosphate) added alone do not prevent this inactivation. Dithiothreitol (2 mM) has a large stabilizing effect on the binding capacity at 0 degrees C but only a small effect at 25 degrees C. Addition of 10 mM molybdate plus 2 mM dithiothreitol totally prevents inactivation for at least 8 h at 25 degrees C as well as at 0 degrees C. Fluoride (100 mM) also retards the inactivation if added with dithiothreitol. Addition of dithiothreitol at 25 degrees C to inactivated cytosol receptors results in partial activation of the binding capacity. Addition of dithiothreitol to receptors inactivated at 25 degrees C in the presence of molybdate allows total reactivation of the binding capacity to the maximum zero time value. If binding capacity is inactivated by preincubation of the cytosol at 25 degrees C, addition of ATP with dithiothreitol enhances the activation observed with only dithiothreitol. This ATP stimulated activation is optimal at 1 to 3 mM. ATP (10 mM) is required when molybdate is added to prevent simultaneous inactivation. ADP, GTP, CTP, and UTP have some activating capacity but the effects of all nucleotides are inhibited by the ATP analog, adenyl-5'-yl (beta, gamma-methylene)diphosphonate. ATP-dependent activation can also be prevented with 50 mM EDTA, and addition of magnesium partially overcomes the EDTA inhibition. Dithiothreitol activation of thymocyte glucocorticoid binding capacity can also be enhanced by addition of a heat-stable preparation from thymocytes, L cells, or liver. Sephadex G-25 chromatography, assay of ATP, and inhibition of the activation with adenyl-5'-yl (beta, gamma-methylene)diphosphonate suggest that these preparations contain varying amounts of endogenous reducing equivalents and ATP as well as a larger heat stable factor. Maximum activation is obtained by adding dithiothreitol, ATP, molybdate, and the larger heat-stable factor. These results suggest that stabilization and activation of glucocorticoid binding capacity in thymocytes requires phosphorylation as well as reduction of the receptor itself or of some other component required for the steroid binding reaction.     

Temperature dependence of gonadal regression in Syrian hamsters exposed to short day lengths

We sought to determine whether ambient temperature (T(a)) affects gonadal function by altering the rate at which circadian rhythms entrain to short day lengths. Syrian hamsters were housed in cages where they received 14 h of light per day ("long days," 14L) at 22 degrees C. Hamsters were then transferred to cages to receive 10 h of light per day ("short days," 10L) and kept at 5, 22, or 28 degrees C or were maintained in 14L at 22 degrees C. Body mass and estimated testis volume as well as duration of nocturnal locomotor activity (alpha), previously established as a reliable indicator of the duration of nocturnal melatonin secretion, were determined over the course of 24 wk. Testicular regression in short days was accelerated by 4 wk at 5 degrees C and delayed by 3 wk at 28 degrees C relative to 22 degrees C. The interval between alpha-expansion and initiation of testicular regression was markedly affected by T(a) with delays of 0, 3, and 6 wk at 5, 22, and 28 degrees C, respectively. All hamsters held at 5 and 22 degrees C underwent testicular regression, but 25% of those maintained at 28 degrees C failed to do so. We suggest that T(a) modulates testicular regression primarily by affecting responsiveness of neuroendocrine target tissues to long melatonin signals.     

Environmental factors influencing the inactivation of Listeria monocytogenes by pulsed electric fields

AIMS: To investigate the influence of the growth phase, growth temperature, storage time, pH and aw of the treatment medium on the resistance of Listeria monocytogenes to pulsed electric fields (PEF). METHODS AND RESULTS: Square wave pulses of 2 micros at a frequency of 1 Hz and 25 and 28 kV cm(-1) were used. Cells were more PEF resistant in the stationary than in the exponential phase at both incubation temperatures investigated (4 and 35 degrees C). Cells grown at 4 degrees C were more PEF sensitive than cells grown at 35 degrees C independent of the growth phase. After a treatment of 25 kV cm(-1) and 800 micros, 1.48, 3.86 and 5.09 log10 cycles of inactivation were obtained at pH 7.0, 5.4 and 3.8, respectively. A reduction in the aw of the treatment medium protected cells against PEF treatments. CONCLUSIONS: The PEF resistance of L. monocytogenes depended on different environmental factors. The influence of growth conditions and treatment medium characteristics should be known and controlled to obtain reproducible and reliable PEF inactivation data. SIGNIFICANCE AND IMPACT OF THE STUDY: Erroneous conclusions and misinterpretation of results are possible if factors affecting the PEF resistance of L. monocytogenes are not considered during PEF inactivation studies.     

浸泡条件下孔雀石绿及其代谢物隐色孔雀石绿在斑点叉尾 组织中分布及消除规律研究

以7 mg/L的孔雀石绿浸泡斑点叉尾 苗种5min后将其饲养于池塘的网箱中, 研究了在养殖模式下孔雀石绿及其代谢物隐色孔雀石绿在斑点叉尾 苗种各组织中的分布及消除规律。采用高效液相色谱串联质谱法(HPLC-MS/MS)分析孔雀石绿及其代谢物隐色孔雀石绿在斑点叉尾 血液、肌肉、皮肤、肝脏、肾脏组织中的浓度水平。采用药代动力学分析软件3p97对血药浓度时间数据进行分析。结果表明, 孔雀石绿和隐色孔雀石绿血药浓度时间曲线符合有吸收二室模型, 动力学方程分别为: C孔雀石绿 =683.063 e-0.248 t+ 11.176 e-0.006 t- 694.239e-0.333 t, C隐色孔雀石绿 =757.240 e-0.222 t + 14.474 e-0.007 t 771.714 e-0.382 t。血液中孔雀石绿和隐色孔雀石绿达峰时间Tpeak分别为3.480和3.623h, 峰浓度值Cmax分别为81.560和159.619 ng/mL, 表观分布容积Vd/F分别为37.689和21.125 L/kg, 分布相的一级速率常数分别为0.248和0.222/h, 消除相的一级速率常数分别为 0.006和0.007/h, 吸收半衰期T(1/2) 分别为2.794和3.124h, 消除半衰期T(1/2)分别为113.068和105.841h, 中央室向周边室转运的一级速率常数K12分别为0.020和0.015/h, 周边室向中央室转运的一级速率常数K21分别为0.159和0.121/h, 药-时曲线下面积AUC分别为2493.944和3601.863 ngh/mL。肌肉、皮肤、肝脏和肾脏组织中孔雀石绿和隐色孔雀石绿浓度水平的结果表明, 孔雀石绿在斑点叉尾 4种组织中浓度由高到低的顺序是皮肤肌肉肾脏肝脏, 其中斑点叉尾 皮肤组织易蓄积孔雀石绿, 其残留时间最长, 肝脏组织由于对孔雀石绿有极强的代谢转化功能而浓度较低。孔雀石绿在肌肉、皮肤、肝脏和肾脏组织中的消除方程分别为C=5.570 e-0.009t、C=6.302 e-0.007t、C=4.791 e-0.006t和C=4.591 e-0.002t, 相关系数r20.773, 消除半衰期T1/2肌肉、皮肤、肝脏和肾脏分别为3.2、4.1、4.8和14.4d。肌肉、皮肤、肝脏和肾脏组织中孔雀石绿分别在45、60、30和60d才未被检测到; 隐色孔雀石绿在斑点叉尾 4种组织中浓度由高到低的顺序是肝脏皮肤肌肉肾脏, 残留时间最长的组织也是皮肤组织。隐色孔雀石绿在肌肉、皮肤、肝脏和肾脏组织中的消除方程分别为C=6.491 e-0.004t、C=6.958 e-0.003t、C=6.722 e-0.007t和C=6.162 e-0.002t, 相关系数r20.673, 消除半衰期T1/2肌肉、皮肤、肝脏和肾脏分别为7.2、9.6、4.1和14.4d。肌肉、皮肤、肝脏和肾脏组织中隐色孔雀石绿分别在90、90、60和90d才未被检出。试验期间(2011年5月17日至7月15日)平均水温为26.4℃, 孔雀石绿和隐色孔雀石绿90d后在各组织中才未检测到, 因此, 使用7 mg/L孔雀石绿浸泡2龄斑点叉尾 苗种孔雀石绿及其代谢物隐色孔雀石绿至少应经过2376℃d后才能消除。       

Growth of Listeria monocytogenes and Yersinia enterocolitica colonies under modified atmospheres at 4 and 8 degrees C using a model food system

The growth of Listeria monocytogenes and Yersinia enterocolitica colonies was studied on solid media at 4 and 8 degrees C under modified atmospheres (MAs) of 5% O2: 10% CO2: 85% N2 (MA1), 30% CO2: 70% N2 (MA2) and air (control). Colony radius, determined using computer image analysis, allowed specific growth rates (mu) and the time taken to detect bacterial colonies to be estimated, after colonies became visible. At 4 degrees C both MAs decreased the growth rates of L. monocytogenes by 1.5- and 3.0-fold under MA1 (mu = 0.02 h(-1)) and MA2 (mu = 0.01 h(-1)), respectively, as compared with the control (mu = 0.03 h(-1)). The time to detection of bacterial colonies was increased from 15 d (control) to 24 (MA1) and 29 d (MA2). At 8 degrees C MA2 decreased the growth rate by 1.5-fold (mu = 0.04 h(-1)) as compared with the control (mu = 0.06 h(-1)) and detection of colonies increased from 7 (control) to 9 d (MA2). At 4 degrees C both MAs decreased the growth rates of Y. enterocolitica by 1.5- and 2.5-fold under MA1 (mu = 0.03 h(-1)) and MA2 (mu = 0.02 h(-1)), respectively, as compared with the control (mu = 0.05 h(-1)). At 8 degrees C identical growth rates were obtained under MA1 and the control (mu = 0.07 h(-1)) whilst a decrease in the growth rate was obtained under MA2 (mu = 0.04 h(-1)). The detection of colonies varied from 6 (8 degrees C, aerobic) to 19 d (4 degrees C, MA2). Refrigerated modified atmosphere packaged foods should be maintained at 4 degrees C and below to ensure product safety.     

Acyl migration in 1,2-dipalmitoyl-sn-glycerol

The acyl migration of 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) to 1,3-dipalmitoylglycerol (1,3-DPG) in different states, neat, in the presence of egg yolk lecithin (sonicated and unsonicated) and on silica gel was studied. The isomerization was quantitated by scanning densitometry of charred TLC plates, at different temperatures and for varying periods of time. At equilibrium the amount of 1,3-DPG was found to be 56%. The rates of initial isomerization, and the time required to isomerize to half the equilibrium quantity (i.e., t1/2 eq. = 1,3-DPG 28%) under the above conditions was estimated. In the case of neat melt at 74 degrees C and in an organic solvent the time required to t1/2 eq. is 18 h and a few days, respectively. However, at 62 degrees C in the presence of a polar solvent (sodium phosphate buffer at pH 7.0) the t1/2 eq. is 1-2 h. On dry silica gel (TLC plate) at 24 degrees C the t1/2 eq. is reached in less than 1 h.     

Oxidative inactivation of an extramitochondrial acetyl-CoA hydrolase by autoxidation of L-ascorbic acid

The activity of acetyl-CoA hydrolase (dimeric form) purified from the supernatant fraction of rat liver was shown to have a half-life (t1/2) of 3 min at 0 degree C, but to stable at 37 degrees C (t1/2 = 34 h) [Isohashi, F., Nakanishi, Y. & Sakamoto, Y. (1983) Biochemistry 22, 584-590]. Incubation of the purified enzyme with L-ascorbic acid (AsA) at 37 degrees C resulted in inactivation of the enzyme (t1/2 = 90 min at 2 mM AsA). The extent of inactivation was greatly enhanced by addition of transition metal ions (Cu2+, Fe2+, and Fe3+). Thiol reducing agents, such as reduced glutathione and DL-dithiothreitol, protected the hydrolase from inactivation by AsA. However, these materials did not restore the catalytic activity of the enzyme inactivated by AsA. When AsA solution containing Cu2+ was preincubated under aerobic conditions at 37 degrees C for various times in the absence of enzyme, and then aliquots were incubated with the enzyme solution for 20 min, remaining activity was found to decrease with increase in the preincubation time, reaching a minimum at 60 min. However, further preincubation reduced the potential for inactivation. Catalase, a hydrogen peroxide (H2O2) scavenger, almost completely prevented inactivation of the enzyme by AsA plus Cu2+. Superoxide dismutase and tiron, which are both superoxide (O2-) scavengers, also prevented inactivation of the enzyme. A high concentration of mannitol, a hydroxyl radical (OH) scavenger, partially protected the enzyme from inactivation. These results suggest that inactivation of the enzyme by AsA in the presence of Cu2+ was due to the effect of active oxygen species (H2O2, O2-, OH) that are known to be autoxidation products of AsA. Valeryl-CoA, a competitive inhibitor of acetyl-CoA hydrolase, greatly protected the enzyme from inactivation by AsA plus Cu2+, but ATP and ADP, which are both effectors of this enzyme, had only slight protective effects. These results suggest that inactivation of this enzyme by addition of AsA plus Cu2+ was mainly due to attack on its active site.     

Effects of pulsed electric fields on inactivation kinetics of Listeria innocua

The effects of pulsed electric field (PEF) treatment and processing factors on the inactivation kinetics of Listeria innocua NCTC 11289 were investigated by using a pilot plant PEF unit with a flow rate of 200 liters/h. The electric field strength, pulse length, number of pulses, and inlet temperature were the most significant process factors influencing the inactivation kinetics. Product factors (pH and conductivity) also influenced the inactivation kinetics. In phosphate buffer at pH 4.0 and 0.5 S/m at 40 degrees C, a 3. 0-V/microm PEF treatment at an inlet temperature of 40 degrees C resulted in > or = 6.3 log inactivation of strain NCTC 11289 at 49.5 degrees C. A synergistic effect between temperature and PEF inactivation was also observed. The inactivation obtained with PEF was compared to the inactivation obtained with heat. We found that heat inactivation was less effective than PEF inactivation under similar time and temperature conditions. L. innocua cells which were incubated for a prolonged time in the stationary phase were more resistant to the PEF treatment, indicating that the physiological state of the microorganism plays a role in inactivation by PEF. Sublethal injury of cells was observed after PEF treatment, and the injury was more severe when the level of treatment was increased. Overall, our results indicate that it may be possible to use PEF in future applications in order to produce safe products.     

Chromosome number and cytogenetics of Euphorbia heterophylla L

Euphorbia heterophylla L. (Euphorbiaceae) is a herbaceous species of great economic importance due to its invasive potential and consequent damage to agriculture and pasture land. For the first time, we provide information on its chromosome number, morphology, and behavior of mitotic chromosomes. Seeds were germinated and submitted to four treatments to obtain metaphases: 0.5% colchicine for 2 to 5 h, at ambient temperature; 0.5% colchicine for 16 to 24 h; 0.0029 M 8-hydroxyquinoline (8-HQ) for 2 to 5 h at ambient temperature, and 0.0029 M 8-HQ for 16 to 24 h at 4 degrees C. The material was then fixed in methanol:acetic acid (3:1) and kept at -20 degrees C for 24 h. Roots were macerated in the enzyme solution of Flaxzyme (NOVO FERMENT)-distilled water (1:40) at 34 degrees C for 2 h and later fixed again. Chromosome preparations were obtained by the dissociation of the apical meristems. The best chromosome preparations were obtained with the use of 8-HQ for 21 h 30 min at 4 degrees C. E. heterophylla showed 2n = 28 chromosomes. The short arm of the largest pair of chromosomes of the complement (pair number 1) displayed a secondary constriction while the nucleolus was observed in the interphasic cell. Structural rearrangements were also observed in the E. heterophylla L. genome. The genomic instability associated with polyploidy may be the result of selection shaped by environmental adaptations and/or human-induced manipulation through agricultural practices.     

Resistance of heat-shocked cells of Listeria monocytogenes to mano-sonication and mano-thermo-sonication

Heat shocks did not increase the resistance of Listeria monocytogenes to an ultrasonication treatment under pressure (Mano-Sonication; MS). While heat-shocked cells (180 min, 45 degrees C) became sixfold more heat resistant than native cells (D62 = 1.8 min vs D62 = 0.24 min), the resistance of native and heat-shocked cells to MS (200 kPa, 117 microns) was the same (DMS = 1.6 min). The inactivation rate of non-heat-shocked cells of L monocytogenes by a combined heat/ultrasonication treatment under pressure (Mano-Thermo-Sonication; MTS) was an additive effect. On the contrary, on heat-shocked cells, the inactivation rate of MTS was greater than that of heat added to the inactivation rate of MS (synergistic effect) in the range 62-68 degrees C.     

Biochemical and thermostability features of acetyl esterase Aes from Escherichia coli

Previously we characterized an acetyl-esterase from Escherichia coli, formally Aes, from a thermodynamic point of view in comparative studies with thermophilic homologs. Since the enzyme appeared unusually resistant to the thermal denaturation we analysed the kinetic behaviour with respect to the temperature. The enzyme displays a surprising optimal temperature at 65 degrees C, showing a specific activity of 250 U/mg using pNP-butanoate as substrate, but a low kinetic stability at the same temperature (t(1/2) of inactivation=5 min). By a random mutagenesis approach we searched for mutated versions of Aes with increased thermostability. We found the mutant T74A, which shows the same specific activity of wild type but a t(1/2) of inactivation of 30 min at 65 degrees C.