Inactivation of Escherichia coli O157:H7, salmonellae, and Campylobacter jejuni in raw ground beef by gamma irradiation.

Raw ground beef patties inoculated with stationary-phase cells of Escherichia coli O157:H7, salmonellae, or Campylobacter jejuni were subjected to gamma irradiation (60Co) treatment, with doses ranging from 0 to 2.52 kGy. The influence of two levels of fat (8 to 14% [low fat] and 27 to 28% [high fat]) and temperature (frozen [-17 to -15 degrees C] and refrigerated [3 to 5 degrees C]) on the inactivation of each pathogen by irradiation was investigated. In ascending order of irradiation resistance, the D10 values ranged from 0.175 to 0.235 kGy (C. jejuni), from 0.241 to 0.307 kGy (E. coli O157:H7), and from 0.618 to 0.800 kGy (salmonellae). Statistical analysis revealed that E. coli O157:H7 had a significantly (P < 0.05) higher D10 value when irradiated at -17 to -15 degrees C than when irradiated at 3 to 5 degrees C. Regardless of the temperature during irradiation, the level of fat did not have a significant effect on the D10 value. Salmonellae behaved like E. coli O157:H7 in low-fat beef, but temperature did not have a significant effect when the pathogen was irradiated in high-fat ground beef. Significantly higher D10 values were calculated for C. jejuni irradiated in frozen than in refrigerated low-fat beef. C. jejuni was more resistant to irradiation in low-fat beef than in high-fat beef when treatment was at -17 to -15 degrees C. Regardless of the fat level and temperature during inactivation, these pathogens were highly sensitive to gamma irradiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of Air Dried Radiation Processed Amniotic Membranes under Different Storage Conditions

Amniotic membranes collected from the placentae of screened donors were processed, air dried and sterilized by gamma irradiation at 25 kGy. Effect of storage under different temperature and humidity conditions (10 degrees C, RH 80-90%; 10 degrees C, RH 40-50%; 40 degrees C, RH 50-60% and 40 degrees C, RH 10-20%) on the properties of the membrane were examined. Infrared (IR) spectral scanning was carried out to examine degradation or change if any in the tissue under different storage conditions. The degradation of amnion on irradiation with gamma rays or during storage after irradiation would tend to produce the relative variation in IR absorption troughs. This kind of addendum was absent in all the samples indicating no qualitative change in the material property of amnion. Water absorption and water vapour transmission rate (WVTR) of the membrane remained unchanged even after 6 months. No effect on the microbial permeability of membrane was observed during storage. The amniotic membranes were found to be impermeable to different strains of bacteria - Bacillus, Escherichia coli, Pseudomonas, Citrobacter, Flavimonas and Staphylococcus. The results indicate that amniotic membranes processed by air-drying are stable and can be stored under different environmental conditions without compromise to their clinical performance.     

DNA damage and biological expression of adenovirus: a comparison of liquid versus frozen conditions of exposure to gamma rays

Human adenovirus type 2 (Ad 2) was irradiated with 137Cs gamma rays in the liquid state at 0 degree C. DNA breaks were correlated with the inactivation of several viral functions and compared to results obtained previously for irradiation of Ad 2 under frozen conditions at -75 degrees C. Irradiation at 0 degree C induced 170 +/- 20 single-strand breaks and 2.6 +/- 0.4 double-strand breaks/Gy/10(12) Da in the viral DNA. Viral adsorption to human KB cells was inactivated with a D0 of 9.72 +/- 1.18 kGy, whereas the inactivation of Ad 2 plaque formation had a D0 of 0.99 +/- 0.14 or 1.1 +/- 0.29 kGy when corrected for the effect of radiation on virus adsorption. For the adsorbed virus, an average of 4.3 +/- 1.7 single-strand and 0.065 +/- 0.02 double-strand breaks were induced in the viral DNA per lethal hit. In contrast, irradiation of Ad 2 at -75 degrees C results in 2.6- to 3.4-fold less DNA breakage per Gy and a 5.6-fold increase in D0 for plaque formation of the adsorbed virus. Furthermore, although host cell reactivation (HCR) of Ad 2 viral structural antigen production for irradiated virus was substantially reduced in the xeroderma pigmentosum fibroblast strain (XP25RO) compared to normal strains for irradiation at -75 degrees C (57% HCR), it was only slightly reduced compared to normal for irradiation at 0 degree C (88% HCR). These results indicate that the spectrum of DNA damage is both quantitatively and qualitatively different for the two conditions of irradiation.     

Effect and aftereffect of gamma radiation pretreatment on enzymatic hydrolysis of wheat straw

Irradiation pretreatment of wheat straw was carried out at different doses by using Co-60 gamma radiation. The weight loss and fragility of wheat straw after irradiation, the combination effect of irradiation and mechanical crushing on enzymatic hydrolysis of wheat straw as well as the aftereffect of irradiation were examined. It is shown that irradiation can cause significant breakdown of the structure of wheat straw. The weight loss of wheat straw increased and the size distribution after crushing moved to fine particles at elevated irradiation doses. The glucose yield of enzymatic hydrolysis of wheat straw increased with increasing doses and achieved the maximum (13.40%) at 500 kGy. A synergistic effect between irradiation and crushing was observed, with a glucose yield of 10.24% at a dose of 500 kGy with powder of 140 mesh. The aftereffect of irradiation had important impact on enzymatic hydrolysis of wheat straw. The aftereffect (at 22nd day) of 400 kGy irradiation accounted for 20.0% of the initial effect for glucose production, and the aftereffects of 50, 100, 200 (at 9th day) and 300 kGy (at 20th day) accounted for 12.9%, 14.9%, 8.9% and 9.1%, respectively, for reducing sugar production.     

Elimination of Escherichia coli O157:H7 in meats by gamma irradiation.

Undercooked and raw meat has been linked to outbreaks of hemorrhagic diarrhea due to the presence of Escherichia coli O157:H7; therefore, treatment with ionizing radiation was investigated as a potential method for the elimination of this organism. Response-surface methods were used to study the effects of irradiation dose (0 to 2.0 kGy), temperature (-20 to +20 degrees C), and atmosphere (air and vacuum) on E. coli O157:H7 in mechanically deboned chicken meat. Differences in irradiation dose and temperature significantly affected the results. Ninety percent of the viable E. coli in chicken meat was eliminated by doses of 0.27 kGy at +5 degrees C and 0.42 kGy at -5 degrees C. Small, but significant, differences in radiation resistance by E. coli were found when finely ground lean beef rather than chicken was the substrate. Unlike nonirradiated samples, no measurable verotoxin was found in finely ground lean beef which had been inoculated with 10(4.8) CFU of E. coli O157:H7 per g, irradiated at a minimum dose of 1.5 kGy, and temperature abused at 35 degrees C for 20 h. Irradiation is an effective method to control this food-borne pathogen.     

Effect of gamma irradiation on microbial load,aflatoxins and phytochemicals present in Trigonella foenum-graecum

The effects of gamma irradiation on microbial load, total aflatoxins and phytoconstituents content of Trigonella foenum-graecum have been studied. Gamma irradiation at a dose of 2.5 kGy resulted in 2 log reduction of the total aerobic microbial count. A complete sterilization was, however, observed at 10 kGy. The total aflatoxin level decreased gradually with increase in gamma irradiation dose as compared to its un-irradiated counterparts, whereas the high performance liquid chromatography (HPLC) profile showed no change in the levels of phytochemicals up to the gamma irradiation dose of 10 kGy. HPLC profiles, however, differed in peak areas, and retention times of the components. These results suggest that gamma irradiation at a dose of 5.0 kGy was very effective for microbial decontamination because it did not adversely affect the active components of T. foenum-graecum.     

Optimal gamma-ray dose and irradiation conditions for producing low-molecular-weight chitosan that retains its chemical structure

This study focuses on the optimal conditions for gamma irradiation to reduce the molecular weight of chitosan but still retain its chemical structure. Chitosan was irradiated under various conditions, i.e. flake solid state (condition 1), flake dispersed in water (condition 2), flake dispersed in 0.05, 0.1, 1 and 2% aqueous K(2)S(2)O(8) solution (conditions 3a, 3b, 3c and 3d, respectively), flake dispersed in 0.5, 1 and 2% aqueous H(2)O(2) solution (conditions 4a, 4b and 4c, respectively), and chitosan acetic acid solution (condition 5). Comparative studies were done using three types of chitosans with molecular weights of the order of 10(5) Da with degrees of deacetylation of 0.80, 0.85 and 0.90%. For all conditions, after irradiation, there were two regions of molecular weight reduction. A severe degradation occurred in the first region with decreases in the molecular weight of 80% for radiation doses up to 50 kGy for conditions 1, 2 and 3 (3a-3c) and 20 kGy for condition 4. In the second region, a slow degradation occurred, which resembled a plateau stage. The results for conditions 3d and 5 were the most dramatic, since the primary structure of chitosan was changed after the irradiation. The degradation of chitosan by gamma rays was found to be most effective for the amorphous structure. The retention of the structure of chitosan after gamma irradiation makes it possible to produce a low-molecular-weight chitosan that retains its functionality, as demonstrated by its activity in the coupling reaction with N,N'-carbonyldiimidazole.     

In vitro release of cytotoxic nucleoside analogs from lactide-caprolactone and lactide-glycolide copolymers

The aims of our study were to assess the release of cytotoxic nucleoside analogs 5-fluorouracil and 2-chloro-2'-deoxyadenosine from different lactide-glycolide or lactide-caprolactone biodegradable copolymers and the effects of sterilization on this release. The polymers were sterilized either with ethylene oxide at 37 degrees C, or with gamma radiation (15 kGy, 20 kGy, or 25 kGy). The kinetics of nucleoside release from the copolymers were measured over 50 days. Four copolymers exhibited relatively constant release of nucleosides in micromolar concentrations during the entire observation period. Sterilization with either ethylene oxide or gamma radiation only slightly influenced nucleoside release. Further development of these copolymers as an intracerebral nucleoside delivery system for local treatment of brain tumors is indicated.     

Temperature affects the production,activity and stability of ligninolytic enzymes in<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> and<Emphasis Type="Italic">Trametes versicolor</Emphasis>

Enzyme activity was determined in cultures of Pleurotus ostreatus and Trametes versicolor with cellulose as a sole C source and high C/N ratio. The fungi were able to grow and produce laccase and Mn-peroxidase (MnP) at 5-35 degrees C, the highest production being recorded at 25-30 degrees C in P. ostreatus and at 35 degrees C in T. versicolor. Production of both enzymes at 10 degrees C accounted only for 4-20% of the maximum value. Temperature optima for enzyme activity were 50 and 55 degrees C for P. ostreatus and T. versicolor laccases, respectively, and 60 degrees C for MnP. Temperatures causing 50% loss of activity after 24 h were 32 and 47 degrees C for laccases and 36 and 30 degrees C for MnP from P. ostreatus and T. versicolor, respectively.     

Inactivation of Coxiella burnetii by gamma irradiation

The gamma radiation inactivation kinetics for Coxiella burnetii at -79 degrees C were exponential. The radiation dose needed to reduce the number of infective C. burnetii by 90% varied from 0.64 to 1.2 kGy depending on the phase of the micro-organism, purity of the culture and composition of suspending menstruum. The viability of preparations containing 10(11) C. burnetii ml-1 was completely abolished by 10 kGy without diminishing antigenicity or ability to elicit a protective immune response in vaccinated mice. Immunocytochemical examinations using monoclonal antibodies and electron microscopy demonstrated that radiation doses of 20 kGy did not alter cell-wall morphology or cell-surface antigenic epitopes.     

Role of the competitive microbial flora in the radiation-induced enhancement of ochratoxin production by Aspergillus alutaceus var. alutaceus NRRL 3174.

The radiation sensitivity and the toxigenic potential of conidiospores of the fungus Aspergillus alutaceus var. alutaceus were determined after irradiation with 60Co gamma rays and high-energy electrons. Over the pH range of 3.6 to 8.8, the doses required for a 1 log10 reduction in viability based on the exponential portion of the survival curve ranged from 0.21 to 0.22 kGy, with extrapolation numbers (extrapolation of the exponential portion of the survival curve to zero dose) of 1.01 to 1.33, for electron irradiation, and from 0.24 to 0.27 kGy, with extrapolation numbers of 2.26 to 5.13, for gamma irradiation. Nonsterile barley that was inoculated with conidia of the fungus and then irradiated with either electrons or gamma rays and incubated for prolonged periods at 28 degrees C and at a moisture content of 25% produced less ochratoxin A with increasing doses of radiation. Inoculation of barley following irradiation resulted in enhanced ochratoxin levels compared with unirradiated controls. In these experiments, inoculation with 10(2) spores per g produced greater radiation-induced enhancement than inoculation with 10(5) spores per g. There was no radiation-induced enhancement when the barley was surface sterilized by chemical means prior to irradiation. These results are consistent with the hypothesis that a reduction in the competing microbial flora by irradiation is responsible for the enhanced mycotoxin production observed when nonsterile barley is inoculated with the toxigenic fungus A. alutaceus var. alutaceus after irradiation.     

Role of the competitive microbial flora in the radiation-induced enhancement of ochratoxin production by Aspergillus alutaceus var. alutaceus NRRL 3174.

The radiation sensitivity and the toxigenic potential of conidiospores of the fungus Aspergillus alutaceus var. alutaceus were determined after irradiation with 60Co gamma rays and high-energy electrons. Over the pH range of 3.6 to 8.8, the doses required for a 1 log10 reduction in viability based on the exponential portion of the survival curve ranged from 0.21 to 0.22 kGy, with extrapolation numbers (extrapolation of the exponential portion of the survival curve to zero dose) of 1.01 to 1.33, for electron irradiation, and from 0.24 to 0.27 kGy, with extrapolation numbers of 2.26 to 5.13, for gamma irradiation. Nonsterile barley that was inoculated with conidia of the fungus and then irradiated with either electrons or gamma rays and incubated for prolonged periods at 28 degrees C and at a moisture content of 25% produced less ochratoxin A with increasing doses of radiation. Inoculation of barley following irradiation resulted in enhanced ochratoxin levels compared with unirradiated controls. In these experiments, inoculation with 10(2) spores per g produced greater radiation-induced enhancement than inoculation with 10(5) spores per g. There was no radiation-induced enhancement when the barley was surface sterilized by chemical means prior to irradiation. These results are consistent with the hypothesis that a reduction in the competing microbial flora by irradiation is responsible for the enhanced mycotoxin production observed when nonsterile barley is inoculated with the toxigenic fungus A. alutaceus var. alutaceus after irradiation.     

The reduction of artificial electron acceptors at sub-zero temperatures by chloroplasts suspended in fluid media.

1. Chloroplasts can be suspended in aqueous/organic mixtures which are liquid at sub-zero temperatures with a good retention of the ability to reduce artificial electron acceptors. The reduction of ferricyanide and 2,6-dichlorophenolindophenol at temperatures above 0 degrees C is about 50% inhibited by 50% (v/v) ethylene glycol. Higher concentrations cause more extensive inhibition. 2. Different solvents were compared on the basis of their ability to cause a given depression of the freezing point of an aqueous solution. Ethylene glycol caused less inhibition of electron transport than glycerol, which in turn was found to be superior to methanol. 3. The reduction of oxidised 2,3,5,6-tetramethyl-p-phenylenediamine could be measured at -25 degrees C in 40% (v/v) ethylene glycol. Using an acceptor with a high extinction coefficient, methyl purple (a derivative of 2,6-dichlorophenolindophenol) it was possible to observe electron flow at temperatures as low as -40 degrees C in 50% (v/v) ethylene glycol. 4. From studies of the effects of the inhibitors 3(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone it is suggested that electron flow from the donor side of Photosystem II to the acceptor side of Photosystem I can occur at temperatures at least as low as -25 degrees C. The ultimate electron donor is presumably water but it was not possible to demonstrate this directly.     

A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability

Mouse morulae were exposed to solutions containing 30-50% of permeable agents (ethylene glycol, glycerol, propylene glycol) in modified phosphate-buffered saline (PB1 medium) at 20 degrees C for 20 min. A high percentage of them developed to expanded blastocysts in culture, after exposure to 30% and 40% ethylene glycol (98 and 84%, respectively), or 30% glycerol (88%). Ethylene glycol and glycerol were diluted to 30 and 40% with PB1 medium or with PB1 containing 30% Ficoll or 30% Ficoll + 0.5 M-sucrose, immersed in liquid nitrogen in straws and warmed in 20 degrees C water. Solutions containing 40% of a permeable agent with Ficoll did not crystallize during cooling or warming. Mouse morulae were exposed to 40% ethylene glycol in PB1 medium containing 30% Ficoll (EF) or PB1 medium + 30% Ficoll + 0.5 M-sucrose (EFS) for 5-20 min at 20 degrees C. EFS solution was non-toxic to the embryos during 5 min of exposure. When embryos, equilibrated in EFS solution for 2 or 5 min at 20 degrees C, were vitrified at -196 degrees C and were warmed rapidly, nearly all embryos developed in culture (97-98%), and 51% developed to live young at term after transfer. This method, which results in virtually no decrease in embryonic viability, may be of practical use for embryo preservation.     

Effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin. I. Change in the biological activity of nonpurified cholera exotoxin as affected by ionizing radiation

Crude cholera exotoxin (filtrate toxin) was irradiated with increasing doses of gamma radiation. A significant drop in enterotoxicity, in the activity of the permeation factor and a decrease in toxicity were shown to occur as radiation doses increased. Radiation doses of 50-70 kGy were found to completely inactivate enterotoxicity in liquid toxic preparations. A higher radioresistance of dried preparations in comparison with liquid ones was registered: inactivation occurred at 150-200 kGy. Different batches of the initial filtrate toxin had varying radiosensitivity. The sterilizing effect of gamma radiation was achieved at doses of 20 kGy for liquid preparations and 30 kGy for dried preparations. During the prolonged storage of the irradiated preparations of crude toxin (the term of observation being 1.5 years) at different temperatures no reversion of toxicity was found to occur, while their immunogenic properties remained unchanged.     

Enhancement in furanocoumarin content and phenylalanine ammonia lyase activity in developing seedlings of Psoralea corylifolia L. in response to gamma irradiation of seeds

Gamma irradiation of seeds is known to be an important factor in stimulating biochemical and physiological processes. The aim of the present study was to investigate phenylpropanoids and associated enzymes responsible for the production of active metabolites. Furanocoumarin content was estimated in seeds of Psoralea corylifolia L. during two successive generations (G(1) and G(2)) where as phenylalanine ammonia lyase (PAL) activity was measured in leaves at different developmental stages of P. corylifolia L. raised from seeds irradiated with variable doses of gamma rays. Maximum accumulation of psoralen and isopsoralen was observed at 15 and 20 kGy doses during G(1) and G(2) generations, respectively. Psoralen proved to be the dominating metabolite in terms of its concentration, while isopsoralen was accumulated at relatively lower concentrations in successive generations. PAL activity was induced maximally following 15 and 20 kGy in G(1) plants and was preceded by psoralen and isopsoralen accumulation which peaked at the same dose rates in both generations. These effects were transmitted and prevalent in the next generation, that is, G(2) (indirectly irradiated). These long-term changes in plant metabolomics demonstrate genomic instability induced by gamma irradiation. However, no detrimental effects were seen at any irradiation dose in seeds. Furanocoumarin concentrations were also enhanced at 15 and 20 kGy. The present study further points out the persistence of changes in the biosynthesis of coumarin derivatives in the next generation. However, accumulation of these metabolites does not lead to any lethal effects.     

Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101

AIMS: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production. METHODS AND RESULTS: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions. CONCLUSIONS: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease. SIGNIFICANCE AND IMPACT OF THE STudy: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.     

Purification and characterization of myofibril-bound serine protease from lizard fish (Saurida undosquamis) muscle

Myofibril-bound serine protease (MBSP) from lizard fish (SAURIDA UNDOSQUAMIS: Synodontidae) skeletal muscle was purified to homogeneity with higher purification (1260-fold) and higher recovery (7%) than our previous report in lizard fish (Saurida wanieso). The new purification method combines a heat-treatment for dissociation from washed myofibrils, acid-treatment at pH 5.0 before and after lyophilization, and alcohol-treatment, followed by two column chromatographies. The molecular mass of the enzyme was estimated to be 50 kDa under non-reducing conditions and 28 kDa under reducing conditions by SDS-PAGE. The N-terminal amino acid sequence of the MBSP was determined to be 22 residues (IVGGYEXEAYSKPYQVSINLGY) and the sequence showed high homology to carp and other fish trypsins (64-77%), but did not show high homology to carp MBSP (41%). The enzyme activity was inhibited by serine protease inhibitors such as Pefabloc SC, leupeptin, TLCK and native protein inhibitors (soybean trypsin inhibitor, alpha(1)-antitrypsin and aprotinin). The purified enzyme specifically hydrolyzed at the carboxyl side of the arginine residue of synthetic 4-methyl-coumaryl-7-amide substrate. When purified MBSP was stored at -35 degrees C in the presence of 50% ethylene glycol (V/V), the enzyme activity was entirely preserved over 6 months and stable against freezing and thawing. Activities for both casein and the synthetic substrate were most active at pH 9.0, and the enzyme was most active approximately 55 degrees C with casein and between 35 and 45 degrees C for synthetic substrate. When myofibrils were incubated with purified MBSP, myosin heavy chain was mostly degraded approximately 55 degrees C, but the degradation of actin was very slow.     

Enzyme activity down to -100 degrees C

The activities of two enzymes, beef liver catalase (EC 1.11.1.6) and calf intestine alkaline phosphatase (EC 3.1.3.1), have been measured down to -97 degrees C and -100 degrees C, respectively. Enzyme activity has not previously been measured at such low temperatures. For catalase, the cryosolvents used were methanol:ethylene glycol:water (70:10:20) and DMSO:ethylene glycol:water (60:20:20). For alkaline phosphatase, methanol:ethylene glycol:water (70:10:20) was used. All of the Arrhenius plots were linear over the whole of the temperature range examined. Since the lowest temperatures at which activity was measured are well below the dynamic transition observed for proteins, the results indicate that the motions which cease below the dynamic transition are not essential for enzyme activity. In all cases the use of cryosolvent led to substantial increases in Arrhenius activation energies, and this imposed practical limitations on the measurement of enzyme activity below -100 degrees C. At even lower temperatures, enzyme activity may be limited by the effect of solvent fluidity on substrate/product diffusion, but overall there is no evidence that any intrinsic enzyme property imposes a lower temperature limit for enzyme activity.     

Rejoining of gamma-ray-induced DNA damage in Cryptosporidium parvum measured by the comet assay

Cryptosporidium parvum is a well-known waterborne intracellular protozoan that causes severe diarrheal illness in immunocompromised individuals. This organism is highly resistant to harsh environmental conditions and various disinfectants, and it exhibits one of the highest known resistances to gamma irradiation. We investigated rejoining of gamma-ray-induced DNA damage in C. parvum by neutral comet assay. Oocysts were gamma irradiated at various doses (1, 5, 10, and 25 kGy) and were incubated for various periods (6-96 h) after exposure to 10 kGy. The comet tail moment showed that the number of DNA double-strand breaks increased concomitantly with the gamma irradiation dose. When investigating rejoining after irradiation at 10 kGy, double-strand breaks peaked at 6 h postirradiation, and rejoining was highest at 72 h postirradiation. The observed rejoining pattern suggests that repair process occurs slowly even when complex DNA double-strand breaks in C. parvum were induced by high dose irradiation, 10 kGy.