Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.     

Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography.

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.     

Stabilization of heat-induced changes in plant peroxidase preparations by ClpX, a bacterial heat shock protein

Peroxidases (PODs) are known to be quite stable at elevated temperatures. Moreover, partially denatured peroxidases are able to regain their catalytic activity during incubation at room temperature. In this paper, we describe the effects of some heat shock proteins on the self-reactivation of plant peroxidase preparations. Horseradish and artichoke peroxidases (HRP and ARP, respectively) were first heated (at 60 °C or 90 °C), then incubated at a slightly elevated temperature (30 °C). The heat-treatment resulted in a considerable loss of activity of both enzymes but the subsequent incubation allowed their reactivation. However, no reactivation could be detected when incubation was carried out in the presence of the molecular chaperone ClpX. Other chaperones that were tested (DnaK, DnaJ and GrpE) did not show the inhibitory effect. Electrophoretic analyses further indicated that the heat-treated horseradish peroxidase, but not the native enzyme, binds to ClpX eliminating the possibility of undesirable protein refolding that would result in aggregation.     

Bindings of toxic shock syndrome toxin-1 and staphylococcal enterotoxins A, B, and C to rabbit spleen cells

Toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins (SE) A, B, and C were studied on binding to rabbit spleen cells. The toxins showed remarkable mitogenic effects on the cells. Among them, SEA and TSST-1 had much stronger mitogenic activities than SEB and SEC. Binding study showed that labeled TSST-1 and SEA bound considerably to cells, but that labeled SEB or SEC was not observed to bind at a detectable level under the same conditions as TSST-1 and SEA. Competitive binding analysis between toxins to cells proved that TSST-1 and SEA clearly competed with each other in binding. Scatchard plots for TSST-1 and SEA in binding were linear at the doses used. The Scatchard analysis for TSST-1 and SEA gave a dissociation constant of 2.5 X 10(-9) M and 7.6 X 10(-8) M and the number of binding sites per cell of 5.3 X 10(3) and 1.0 X 10(5), respectively.     

Combined effects of sulfites, temperature, and agitation time on production of glycerol in grape juice by Saccharomyces cerevisiae.

Analysis of variance was used to evaluate the simultaneous effects of strain, incubation temperature (15 to 25 degrees C), agitation time (0 to 24 h), and initial sulfite concentration (100 to 300 ppm) on glycerol production in grape juice by Saccharomyces cerevisiae. Fourteen strains were studied to determine their growth patterns in the presence of sulfites and ethanol. Baker's yeast strains were more sensitive to sulfite than wine strains, and little growth occurred at initial sulfite levels greater than 150 ppm. Sensitivity to sulfite increased with increasing levels of ethanol. Three strains exhibiting the best growth in the presence of sulfites and ethanol were selected for interaction studies. Fermentations were carried out until the solids content had decreased to less than 6 degrees Brix, which was the point that glycerol content became stable. For the three strains used, the greatest level of glycerol production was observed in the presence of 300 ppm of sulfite for most incubation temperatures and agitation times. There was significant interaction between the strain, incubation temperature, and agitation time parameters for glycerol synthesis, and a response surface method was used to predict the optimal conditions for glycerol production. Under static conditions, the highest level of glycerol production was observed at 20 degrees C, while incubation at 25 degrees C gave the best results when the cultures were agitated for 24 h. Response surface equations were used to predict that the optimum conditions for glycerol production by S. cerevisiae Y11 were a temperature of 22 degrees C, an initial sulfite concentration of 300 ppm, and no agitation, which yielded 0.68 g of glycerol per 100 ml.     

Staphylococcal Enterotoxin B Initiates Protein Kinase C Translocation and Eicosanoid Metabolism While Inhibiting Thrombin-Induced Aggregation in Human Platelets

Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10μ g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC.The views of the authors do not purport to reflect the position of the Department of the Army or the Department of Defense (Para, 4–3) AR360-5.     

The protective effect of immunisation against diphtheria, pertussis and tetanus (DPT) in relation to sudden infant death syndrome

Epidemiological evidence indicates infants immunised against diphtheria, pertussis and tetanus (DPT) are at decreased risk of sudden infant death syndrome (SIDS). Asymptomatic whooping cough and pyrogenic toxins of Staphylococcus aureus have been implicated in the aetiology of SIDS. The objectives of the present study were: (1) to determine if the DPT vaccine induced antibodies cross-reactive with the staphylococcal toxins; (2) to determine if antibodies to the pertussis toxin (PT) and the staphylococcal toxins were present in the sera of women during late pregnancy; (3) to examine the effects of infant immunisation on levels of antibodies to PT and the staphylococcal toxins; (4) to assess the effects of changes in immunisation schedules in the UK on the incidence and age distribution of SIDS. Enzyme-linked immunosorbent assays (ELISA) were used to measure binding of rabbit or human IgG to the DPT vaccine, PT, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins A (SEA), B (SEB) and C (SEC). Neutralisation activity of anti-DPT serum was assessed by a bioassay for induction of nitric oxide from human monocytes by the staphylococcal toxins. Anti-DPT serum bound to the DPT vaccine, PT and each of the staphylococcal toxins. It also reduced the ability of the four toxins to induce nitric oxide from monocytes. In pregnant women, levels of IgG to PT, SEC and TSST-1 decreased significantly in relation to increasing weeks of gestation while antibodies to SEA and SEB increased. In infants' sera there were significant correlations between levels of IgG bound to DPT and IgG bound to PT, TSST-1 and SEC but not SEA or SEB. Antibody levels to the toxins in infants declined with age; sera from infants < or = 2 months of age had higher levels of IgG bound to the toxins than those older than 2 months. This pattern was observed for infants whose immunisation schedules began at 2 months of age or 3 months of age. The decrease in IgG bound to the toxins was, however, less for those immunised at 2 months. The decrease in SIDS deaths after the change in immunisation schedules was greatest in the 4-6-month age range. While DPT immunisation might prevent some unexplained infant deaths due to asymptomatic whooping cough, these data indicate that immunisation with DPT also induces antibodies cross-reactive with pyrogenic staphylococcal toxins implicated in many cases of SIDS. Passive immunisation of infants who have low levels of these antibodies might reduce further the numbers of these infant deaths.     

Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods.

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.     

Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods.

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.     

Staphylococcal enterotoxin-B (SEB) alters [14C]-choline transport and phosphatidylcholine metabolism in cultured human kidney proximal tubular cells

We studied the effects of SEB on [¹⁴C]-choline transport and metabolism of choline containing phospholipids in cultured human kidney proximal tubular (PT) cells. SEB increased the uptake of [¹⁴C]-choline in PT cells as a function of toxin concentration, incubation time, and pH. The maximum increase in uptake (3.5–5-fold compared to control) was observed at a toxin concentration of 10 ug/10⁴ cells, at 4 h and at pH 7.4. Two toxins structurally related to SEB, Staphylococcal enterotoxin-A and toxic shock toxin (TST-1) failed to alter [¹⁴C]-choline uptake in PT cells, a finding which indicates that SEB-mediated alteration in choline uptake in PT cells has high specificity.We found that SEB markedly and significantly increased the incorporation of [¹⁴C]-choline into phosphatidylcholine, Iysophosphatidylcholine and sphingomyelin, but not into phosphatidylethanolamine. Maximum increase in the incorporation of [¹⁴C]-choline into phosphatidlycholine (3-fold compared to control) was observed at 4 h after incubation with toxin. In contrast, SEB did not alter the incorporation of [¹⁴C]-choline in phosphatidylethanolamine. The cellular level of phosphatidylcholine was also increased (2-fold compared to control) in PT cells incubated with SEB. This was accompanied by a 3-to-4-fold increase in CTP: phosphocholine, cytidyltransferase activity.In sum, SEB specifically stimulates phosphatidylcholine synthesis in PT cells by increasing choline uptake or by activating CTP: phosphocholine, cytidyltransferase, or both. We believe this is the first-ever report indicating that a toxin can increase phosphatidylcholine synthesis. This high order of specificity may be in part due to the presence of a glycosphingolipid receptor in PT cells that specifically binds SEB but not SEA or TST-1. Accordingly, it is tempting to speculate that the receptor may somehow be involved in the SEB-mediated regulation of phosphatidylcholine synthesis.Abbreviations SEB Staphylococcal entertoxin-B - SEA Staphylococcal enterotoxin-A - TST-1 Toxic shock syndrome toxin-1 - PT Proximal tubular - PC Phosphatidylcholine - SM Sphingomyelin - LPC Lysophosphatidyl-choline - CT Cytidyltransferase     

Sporulation of Clostridium perfringens (welchii) in Four Laboratory Media

S ummary . Sporulation of 7 strains of Clostridium perfringens ( welchii ) was investigated in 4 laboratory media. A method to induce rapid and simultaneous sporulation was attempted which involved obtaining a purely vegetative culture to inoculate the test media. Heat resistance of spores produced in the individual media by each of 4 selected strains was investigated. The clean spores for the heating tests were obtained by a special procedure which included chilling to 6° for a minimum of 1 week immediately following the usual incubation period, then centrifuging, resuspending to volume in 0.85% NaCl solution and pasteurizing at 75° for 20 min before subjecting to the heating tests. Morphology of each strain was studied using stained microscopic preparations from the 24 h sporulating cultures.
In the Ellner medium spore counts approaching 10⁷/ml were recorded and this medium appeared to be the most efficient when judged in terms of numbers of spores produced. In other media the counts were in the range 10⁴-10⁵ spores/ml. Cooked meat medium yielded slightly higher spore counts than did either SEC broth or modified Wagenaar & Dack medium, the latter contained in a dialysis sac apparatus. A period of chilling to 6° for a minimum of 1 week following incubation enhanced maturation in all cultures except those grown in SEC broth for 24 h or 15 days and those grown 15 days in the modified Wagenaar & Dack medium.
Considerable heat resistance, expressed as percentage spore survival, was recorded for spores of 4 strains when heated at 80°, and heat resistance generally increased with lengthening of incubation time for the culture. Survival of spores heated at 100° for 10 min was usually less than 0.01% but spores in SEC broth after 15 days showed a somewhat greater heat resistance than the others. In no instance did total destruction of spores occur at 100°.     

Gelatinization mechanism of potato starch

The non-Newtonian behavior and dynamic viscoelasticity of potato starch (Jaga kids red ’90, 21.0% amylose content) solutions after storage at 25 and 4°C for 24 h were measured with a rheogoniometer. The flow curves, at 25°C, of potato starch showed plastic behavior >1.0% (w/v) after heating at 100°C for 30 min. A gelatinization of potato starch occurred above 1.0% at room temperature. A very large dynamic viscoelasticity was observed when potato starch solution (3.0%) was stored at 4°C for 24 h and stayed at a constant value with increasing temperature. A small dynamic modulus of potato starch was observed upon addition of urea (4.0 M) at low temperature (0°C) even after storage at 25 and 4°C for 24 h. A small dynamic modulus was also observed in 0.05 M NaOH solution. Possible models of gelatinization and retrogradation mechanism of potato starch were proposed.     

The effect of Staphylococcus aureus carriage in late pregnancy on antibody levels to staphylococcal toxins in cord blood and breast milk

We investigated the effect of carriage of Staphylococcus aureus in the later stages of pregnancy on levels of antibody specific to the S. aureus toxins, staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC) and toxic shock syndrome toxin-1 (TSST-1), in cord blood and breast milk and also explored the relationship between levels of antibody in antenatal serum and cord blood. Nasopharyngeal swabs and stool samples were collected on two occasions, from 96 women, during the last 6 weeks of pregnancy. Samples were cultured and S. aureus isolates were identified. Antenatal and cord blood samples from the same women and their infants were analysed for IgG antibody to SEB, SEC and TSST-1 by enzyme-linked immunosorbent assay. Breast milk samples were analysed for IgA antibody to the same toxins. We found that S. aureus carriage in pregnancy is common and exposure to a toxin-producing isolate boosts immunity. Over 89% of women and infants have some protective antibody to the toxins, and antitoxin IgG levels are higher in cord blood samples compared with antenatal samples. Levels of cord blood IgG and breast milk IgA specific for the staphylococcal toxins vary. Some infants lack protection and could be at risk of toxin-induced disease.     

Survival of Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) serum in vitro

Virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes were examined for survival and growth in non-immune and immune rainbow trout serum, in vitro. A majority of the examined strains consumed complement of non-immune serum, but the complement cascade was not able to cause an immediate (after 3 h incubation) notable reduction in viability of the inoculated cells. After 24 h incubation a more pronounced reduction in the number of viable bacteria was observed in untreated serum as well as in serum heated at 45 degrees C. In serum heated at 56 degrees C this reduction in cell number was not observed, but an increase in cell number did not occur either. The serum survival of one of the examined strains was different from the others in showing cell multiplication after 24 h incubation in normal as well as heat-treated (45 and 56 degrees C) serum. In immune serum no immediate reduction in viability of inoculated cells, of all tested strains, was observed. The number of viable cells showed a slow decrease or remained almost unchanged for up to 72 h post-inoculation in untreated serum, at 5 degrees C as well as 15 degrees C. In heat-treated serum (45 degrees C) the number of viable cells decreased slowly at 5 degrees C and 15 degrees C for up to 72 h. The results suggest that the examined strains were unaffected by the alternative complement reaction present in fish serum as well as by antibodies against F. psychrophilum. However, some unknown component(s) in the fish sera, or lack of nutrients or essential growth factors, inhibited the growth of most of the examined strains in the tested fish sera.     

Heat resistance of Listeria monocytogenes by two recovery media used with and without cold preincubation

S. SÖRQVIST. 1993. Three strains of Listeria monocytogenes were heat-treated at three temperatures in physiological saline by a capillary tube method. Recovery of heat-treated bacteria was performed on blood agar and on tryptose phosphate agar with ferric citrate and aesculin (TPA-FE). Both media were used in two ways: (1) incubation at 37C for 7 d, and (2) preincubation at 4C for 5 d in order to obtain repair of heat-injured bacteria, followed by incubation at 37C for 1 d. D and z values were determined. In both incubation procedures, better average recovery was obtained on blood agar than on TPA-FE. Thus, higher D values were recorded when blood agar was used. In most cases the differences were statistically significant. Repair at 4C of heat-injured bacteria occurred on both media but the proportions of repaired bacteria were higher on blood agar. The repair on this medium was generally reflected in higher D values for preincubated samples. Some significant differences in heat resistance were noted between the strains.     

Effect of heat treatment on the antimicrobial properties of tef dough,injera, kocho and aradisame and the fate of selected pathogens

A known population from each of a 24h culture of Bacillus cereus, Pseudomonas aeruginosa, Salmonella spp., Shigella spp., Klebsiella spp. and Staphylococcus aureus was inoculated into tef flour–water/kocho–water mixtures in screw-capped flasks and allowed to ferment for 30h at room temperature (18–21°C). The flasks were then heat-treated. Cultures of the test bacteria were inoculated into tubes containing graded volumes of 30-h-fermented tef dough/kocho extracts which had been heat-treated at 45, 61 and 80°C in assay broth containing aqueous extracts from injera and aradisame. They were incubated for 24h at 32°C and optical densities determined. Populations of the major indigenous bacteria, yeasts and moulds in fermented tef dough (30h), kocho samples, injera and aradisame were determined from other control portions of the same samples. Higher temperature (80°C) heat-treatment promoted the inhibitory potential of extracts from doughs of both foods as compared with lower temperature heat-treatments (45 and 61°C). Asporogenous test bacteria were affected more than the spore-formers. Better efficacy of extracts from injera and aradisame suggested improved antimicrobial properties of the baked products than in doughs. Heat of baking inactivated all vegetative cells although spores of B.cereus, the yeasts and moulds survived the heat (100°C) applied for 5min. The c.f.u./g of food for B. cereus was below the disease-causing level (0.5×101 and 1.5×103, in injera and aradisame, respectively). Actual baking temperatures in homes are higher than the ones used here; if post-baking contamination is minimized or prevented, the products would be microbiologically safe with respect to the asporogenous pathogens when served fresh. Further studies on aflatoxins and improved storage conditions for kocho are recommended.     

Effect of heat treatment on the antimicrobial properties of tef dough, injera, kocho and aradisame and the fate of selected pathogens

A known population from each of a 24h culture of Bacillus cereus, Pseudomonas aeruginosa, Salmonella spp., Shigella spp., Klebsiella spp. and Staphylococcus aureus was inoculated into tef flour–water/kocho–water mixtures in screw-capped flasks and allowed to ferment for 30h at room temperature (18–21°C). The flasks were then heat-treated. Cultures of the test bacteria were inoculated into tubes containing graded volumes of 30-h-fermented tef dough/kocho extracts which had been heat-treated at 45, 61 and 80°C in assay broth containing aqueous extracts from injera and aradisame. They were incubated for 24h at 32°C and optical densities determined. Populations of the major indigenous bacteria, yeasts and moulds in fermented tef dough (30h), kocho samples, injera and aradisame were determined from other control portions of the same samples. Higher temperature (80°C) heat-treatment promoted the inhibitory potential of extracts from doughs of both foods as compared with lower temperature heat-treatments (45 and 61°C). Asporogenous test bacteria were affected more than the spore-formers. Better efficacy of extracts from injera and aradisame suggested improved antimicrobial properties of the baked products than in doughs. Heat of baking inactivated all vegetative cells although spores of B.cereus, the yeasts and moulds survived the heat (100°C) applied for 5min. The c.f.u./g of food for B. cereus was below the disease-causing level (0.5×101 and 1.5×103, in injera and aradisame, respectively). Actual baking temperatures in homes are higher than the ones used here; if post-baking contamination is minimized or prevented, the products would be microbiologically safe with respect to the asporogenous pathogens when served fresh. Further studies on aflatoxins and improved storage conditions for kocho are recommended.     

Nucleolar vacuolation in soybean root meristematic cells during recovery after chilling

The nucleolar vacuole formation in soybean root meristematic cells from seedlings grown 3 d at temperature 25 °C (control), 3 d at temperature 25 °C and then transferred to 10 °C (chilling) for 4 d, and after recovery for 1.5, 3, 6, 12 and 24 h at 25 °C were observed on semi-thin sections. Simultaneously, autoradiographic studies with ³H-uridine on squashed preparations were carried out. During recovery of plants, the number of vacuolated nucleoli increased gradually from 24 % after 1.5 h up to 40 % after 24 h, while in the control there were 18 % of nucleoli with vacuoles and after 4-d chilling only 5 %. Labelling of cells during 20-min incubation in ³H-uridine and during 80-min post-incubation in non-radioactive medium was increased in recovered plants in comparison with the control and chilled plants. The conclusion has been drawn that nucleolar vacuoles in soybean plants are formed as a result of migration of granular component accumulated in nucleolus during 4-d chilling.     

Influence of hyperthermia on gamma-ray-induced mutation in V79 cells

Asynchronously growing V79 cells were assayed for mutation induction following exposure to hyperthermia either immediately before or after being irradiated with 60Co gamma rays. Hyperthermia exposures consisted of either 43.5 degrees C for 30 min or 45 degrees C for 10 min. Each of these heat treatments resulted in a survival level of 42%. For all sequences of combined treatment with hyperthermia and radiation, cell killing by gamma rays was enhanced. Mutation induction by gamma rays was enhanced when heat preceded gamma irradiation, but no increase was observed when heat was given after gamma exposures. Treatment at 45 degrees C for 10 min gave a higher yield in mutants at all gamma doses studied compared to treatment at 43.5 degrees C for 30 min. When heat-treated cells were incubated for different periods before being exposed to gamma rays, thermal enhancement of radiation killing was lost after 24 h. In contrast, only 5-6 h incubation was needed for loss of mutation induction enhancement.