Effect of cooling of equine spermatozoa before freezing on post-thaw motility: preliminary results

The ability to ship cooled stallion semen to a facility that specializes in cryopreservation of spermatozoa would permit stallions to remain at home while their semen is cryopreserved at facilities having the equipment and expertise to freeze the semen properly. To accomplish this goal, methods must be developed to freeze cooled shipped semen. Three experiments were conducted to determine the most appropriate spermatozoal extender, package, time of centrifugation, spermatozoal concentration and length of time after collection that spermatozoa can be cooled before cryopreservation. In the first experiment, spermatozoa were centrifuged to remove seminal plasma, resuspended in either a skim milk extender, a skim milk-egg yolk-sugar extender or a skim milk-egg yolk-salt extender, cooled to 5 degreesC and frozen in 0.5- or 2.5-mL straws either 2.5 or 24 h after cooling. Samples frozen 2.5 h after cooling had higher percentages of progressively motile (PM) spermatozoa (27%) than samples frozen 24 h after cooling (10%; P < 0.05). Samples frozen 2.5 h after cooling in skim milk extenders containing egg yolk had higher percentages of PM spermatozoa (average 32%) than did spermatozoa frozen in extender containing skim milk alone (average 16%; P < 0.05). The percentages of PM spermatozoa frozen in 0.5- or 2.5-mL straws were similar (21 and 28%, respectively; P > 0.05). In the second experiment, spermatozoa were centrifuged to remove seminal plasma either before (25 degreesC) or after cooling (5 degreesC), and spermatozoa were frozen after being cooled to 5 degreesC for 2, 6, or 12 h. The percentages of PM spermatozoa were higher (P < 0.05) for spermatozoa centrifuged before cooling (30%) than for spermatozoa centrifuged after cooling (19%). Spermatozoa centrifuged at 25 degreesC then cooled for 12 h to 5 degreesC had higher (P < 0.05) post-thaw progressive motility (23%) compared to spermatozoa cooled for 12 h and centrifuged at 5 degreesC (13%). In the third experiment, spermatozoa were centrifuged for seminal plasma removal, resuspended at spermatozoal concentrations of 50,250 or 500 x 10(6)/mL, cooled to 5 degreesC for 12 h and then frozen. Samples with spermatozoa packaged at 50 or 250 x 10(6)/mL had higher (P < 0.05 percentages of PM spermatozoa (25 and 23%) after freezing than did samples packaged at 500 x 10(6) spermatozoa/mL (17%). We recommend that semen be centrifuged at 25 degreesC to remove seminal plasma, suspended to 250 x 10(6) spermatozoa/ml and held at 5 degreesC for 12 h prior to freezing.     

Cooperativity of alpha- and beta-subunits of group II chaperonin from the hyperthermophilic archaeum Aeropyrum pernix K1

alpha- and beta-subunits (ApCpnA and ApCpnB) are group II chaperonins from the hyperthermophilic archaeum Aeropyrum pernix K1, specialized in preventing the aggregation and inactivation of substrate proteins under conditions of transient heat stress. In the present study, the cooperativity of alpha- and beta-subunits from the A. pernix K1 was investigated. The ApCpnA and ApCpnB chaperonin genes were overexpressed in E. coli Rosetta and Codonplus (DE3), respectively. Each of the recombinant alpha- and beta- subunits was purified to 92% and 94% by using anionexchange chromatography. The cooperative activity between purified alpha- and beta-subunits was examined using citrate synthase (CS), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) as substrate proteins. The addition of both alpha- and beta-subunits could effectively protect CS and ADH from thermal aggregation and inactivation at 43 degreesC and 50 degreesC, respectively, and MDH from thermal inactivation at 80 degreesC and 85 degreesC. Moreover, in the presence of ATP, the protective effects of alpha- and beta-subunits on CS from thermal aggregation and inactivation, and ADH from thermal aggregation, were more enhanced, whereas cooperation between chaperonins and ATP in protection activity on ADH and MDH (at 85 degreesC) from thermal inactivation was not observed. Specifically, the presence of both alpha- and beta- subunits could effectively protect MDH from thermal inactivation at 80 degreesC in an ATP-dependent manner.     

The effect of pH value and temperature on the stability of L-aminoacidoxidase from the venom of the sand viper]

The stability of highly purified L-amino acid oxidase from the sand viper venom remains practically unaffected by the pH-value at 4degreesC between pH 5 and 8, whereas a sharp activity fall was observed on both sides of this range. At temperatures above 30 degreesC the enzyme is stable only at pH 5.0--5.5. The inactivation pH values above 5.5 follows a first-order rate equation with characteristic changes in the absorption and emission spectra of the enzyme. The stability of the enzyme is dependent on the temperature of storage. At pH 7.5 there is a stability minimum at --10 degrees and -- 30 degreesC. At -- 72 degreesC the enzyme is stable practically for an unlimited period of time; temperatures exceeding 50 degrees C rapidly lead to complete inactivation. Also in the cold, the L-amino acid oxidase is most stable at pH 5.5. There are characteristic changes in absorption and emission spectra in the temperature-stability minimum (--15 degreesC) and at temperatures above 30degreesC. The inactivations follow a first-order rate equation. The cold inactivation is reversible. The stability of the enzyme is diminished by some anions and cations at 37 degreesC. The cold inactivation is promoted by several inorganic anions; organic anions and ammonium sulfate prevent cold inactivation.     

Geldanamycin inhibits trichostatin A-induced cell death and histone H4 hyperacetylation in COS-7 cells

As widely believed treating cells with trichostatin A (TSA), an inhibitor of histone deacetylase, results in histone H4 hyperacetylation and cell cycle arrest. This compound is often compared with other potential anticancer drugs in cell cycle, proliferation and differentiation research. Furthermore, geldanamycin (GA), a 90-kDa heat shock protein (HSP90) specific inhibitor, is a well-known potential anticancer agent. This study examines whether GA can affect the cellular functions induced by TSA. When using TSA treatment, although caused COS-7 cell death, pretreatment of 0.5 microg/ml GA for 30 min and an addition of 50 ng/ml TSA (GA + TSA) apparently averted cell death. Our results indicated that the cell survival rate was only approximately 20% when prolonged treatment was undertaken with 50 ng/ml TSA (TSA) alone for 24 h. In contrast, the cell survival rate was enhanced by two folds when treating with GA + TSA. Furthermore, DNA fragmentation assay revealed that fragmented DNA was produced 8 h after prolonged treatment with TSA alone. Within 16 h, the apoptotic percentages of TSA-treated cells were between 15-25%. In contrast, the other treatments did not exceed 6%. Furthermore, GA inhibited TSA-induced histone H4 hyperacetylation. Western blotting analysis further demonstrated that the HSP70 levels did not significantly increase in TSA-treated cells. However, the accumulated 70-kDa heat shock protein (HSP70) markedly increased up to 2 to 3 folds at 8 h in GA- and GA + TSA-treated cells, and the maximum amount up to 5 to 7 folds at 20 h. Conversely, HSP90 did not markedly increase in all treatments. Based on the results in this study, we suggest that apoptosis induced by TSA can be prevented by GA-induced increment of heat shock proteins, particularly HSP70.     

Trichostatin A induces cell death at the concentration recommended to differentiate the RGC-5 cell line

Supplementation with Trichostatin A (TSA) has been described as the method of choice for differentiating the RGC-5 cell line into cells with neuronal properties. However, TSA is known to induce apoptosis. We therefore investigated whether TSA at the recommended concentration for differentiation (500 nM) and at three additional concentrations (40, 150 and 2000 nM) induces apoptosis or cell death in the RGC-5 cell line. Morphological changes of the RGC-5 cells occurred after 24 and 48 hours (h) of treatment with 500 and 2000 nM TSA. Differentiation of RGC-5 cell began at 150 nM. A decrease in the cell count was observed from 150 nM TSA onwards compared to controls. Five hundred nanomolar of TSA reduced the amount of cells to 51% (p<0.005) after 24h and to 24% (p<0.005) after 48 h compared to controls on crystal violet staining. At 500 nM TSA a massive induction of apoptosis after 24 and 48 h was noted. Supplementation of 500 nM TSA increased caspase 3/7 activity 5.0-fold (p<0.005). Furthermore, 27× more TUNEL-positive cells were found and the cleaved caspase 3/caspase 3 ratio was 1.8-fold (p<0.1) higher 24h after the addition of 500 nM TSA. The Bax/Bcl-2 ratio was 3.4-fold (p<0.05) higher after 48 h. Cell viability decreased to 70% (p<0.005) and to 35% (p<0.005) after 24 and 48 h, respectively. Moreover, 103× (p<0.05) more dead cells (via propidium iodide staining) were found after 48 h of treatment with 500 nM TSA. In conclusion, TSA induces cell death and apoptosis at the concentration recommended for differentiation. The induction of apoptosis occurred dose and time dependently and already at even lower concentrations of TSA which did not lead to differentiation induced apoptosis. Thus, studies with RGC-5 cells should not be performed within the first 48 h after supplementation with TSA.     

Growth of Desulfovibrio on the Surface of Agar Media

Growth of Desulfovibrio desulfuricans (API strain) was found to take place in an atmosphere of hydrogen on the agar surface of complex media, including yeast extract (Difco), and Trypticase Soy Agar (BBL) without any added reducing agents. For growth on a 2% yeast extract-agar surface in the absence of hydrogen (nitrogen atmosphere), sodium lactate was required in the medium. Growth on the surface of Trypticase Soy Agar (TSA) under nitrogen took place readily in the absence of an added hydrogen donor. A medium (TSA plus salts) is described based upon the addition of sodium lactate (4 ml per liter), magnesium sulfate (2 g per liter), and ferrous ammonium sulfate (0.05%) to TSA, which appears suitable for the isolation and growth of Desulfovibrio on the surface of agar plates in an atmosphere of hydrogen. Sodium lactate does not appear to be essential in this medium for good growth and sulfate reduction in a hydrogen atmosphere, but is essential in a nitrogen atmosphere. Growth of Desulfovibrio (hydrogen atmosphere) on the agar surface of media commonly used for its cultivation as well as on an inorganic medium containing bicarbonate as a source of carbon is poor and erratic unless inoculated (Desulfovibrio) plates of TSA plus salts are incubated in the same container with plates of these media. This stimulatory effect of incubation with inoculated plates of TSA plus salts medium appears to be due to as yet unidentified volatile material produced by D. desulfuricans when growing on this medium. Another volatile material, or possibly the identical material, appears to act similarly to a hydrogen donor.     

Multi-temperature effects on Hill reaction activity of barley chloroplasts.

1. The relationship between temperature and Hill reaction activity has been investigated in chloroplasts isolated from barley (Hordeum vulgare L. cv. Abyssinian). 2. An Arrhenius plot of the photoreduction of 2,6-dichlorophenolindophenol (DCIP) showed no change in slope over the temperature range 2--38degreesC. The apparent Arrhenius activation energy (Ea) for the reaction was 48.1 kJ/mol. 3. In the presence of an uncoupler of photophosphorylation, methylamine, the Ea for DCIP photoreduction went through a series of changes as the temperature was increased. Changes were found at 9, 20, 29 and 36degreesC. The Ea was highest below 9degreesC at 63.7 kJ/mol. Between 9 and 20degreesC the Ea decreased to 40.4 kJ/mol and again to 20.2 kJ/mol between 20 and 29degreesC. Between 29 and 36degreesC there was no further increase in activity with increasing temperature. The temperature-induced changes at 9, 20 and 29degreesC were reversible. At temperatures above 36degreesC (2 min) a thermal and largely irreversible inactivation of the Hill reaction occurred. 4. Temperature-induced changes in Ea were also found when ferricyanide was substituted for DCIP or gramicidin D for methylamine. The addition of an uncoupler of photophosphorylation was not required to demonstrate temperature-induced changes in DCIP photoreduction following the exposure of the chloroplasts to a low concentration of cations. 5. The photoreduction of the lipophilic acceptor, oxidized 2, 3, 5, 6-tetramethyl-p-phenylenediamine, also showed changes in Ea in the absence of an uncoupler. 6. The temperature-induced changes in Hill activity at 9 and 29degreesC coincided with temperature-induced changes in the fluidity of chloroplast thylakoid membranes as detected by measurements of electron spin resonance spectra. It is suggested that the temperature-induced changes in the properties and activity of chloroplast membranes are part of a control mechanism for regulation of chloroplast development and photosynthesis by temperature.     

Natural incidence of endophytic bacteria in pea cultivars under field conditions

Pea plants grown in the field were used to study the natural incidence of endophytic bacteria in the stem. Eleven pea cultivars at the flowering stage were screened for the presence of endophytic bacteria using a printing technique with surface disinfested stem cross-sections on 5% Trypticase Soy Agar (TSA). Five cultivars showed colonization. Cultivar Twiggy showed the highest and most consistent colonization and was further investigated. Stems of cv. Twiggy at the pod stage were analyzed for endophytic bacterial types and populations. Cross-sections of surface disinfested stems were printed on 5% TSA. Endophytic bacterial populations decreased from the lower to the upper part of the stem. One section from the third and the fourth internode was surface disinfested, homogenized, and spiral plated on the media 5% TSA, R2A, and SC (Davis et al. 1980). Over a series of 30 samples, 5% TSA gave significantly better recovery of bacterial endophytes compared with R2A and SC media. For most stems, populations ranged from 10(4) to 10(5) CFU/g except in one of the field blocks in which endophyte populations were uniformly higher. Comparison of colony counts by spiral plating and printing showed a positive correlation. The most frequently recovered bacterial types were Pantoea agglomerans and Pseudomonas fluorescens. Less frequently isolated were Pseudomonas viridiflava and Bacillus megaterium.     

Radiation resistance and injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25 degrees C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and -30 degrees C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at -20 degrees C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at -20 degrees C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at -20 degrees C, nor did storage at -20 degrees C alter the cell's resistance to irradiation at 25 degrees C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36 degrees C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36 degrees C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5 degrees C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36 degrees C for 1 day than at 5 degrees C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

The in vitro development of cloned sheep embryos treated with Scriptaid and Trichostatin (A)

Introduction and aimsAlthough, it has been success in the generation of animal clones from somatic cells in various animal species, the information related to nuclear reprogramming of cloned embryos is found to be limited. This study aims to compares the effect of both Scriptaid (SCR) and Trichostatin (A) treatments in improving cloning efficiency, and embryos developmental rate of cloned sheep embryos in vitro. Three groups were formed, i.e., one SCR group, second TSA group, with both treatment concentrations of 5 nM, 50 nM, and 500 nM, respectively, and third were control group with 0 nM. Methods: Ovaries of slaughtered sheep were collected and oocytes were recovered from antral follicles using aspiration method and in vitro maturation of oocytes were done. Then zona dissecting with micropipettes and oocyte enucleation were carried out under the micromanipulator. Later nuclear transfer, cell fusion and activation were done via cell fusion machine. Finally the embryo cultured in incubating chamber at the CO2 incubator up to 9 days. The result: In general the results showed that when the concentration increases the cleavage rate increased. The cleavage rates of the SCNT embryos treated with SCR at different concentrations are closely related to cleavage rate of embryos treated with TSA at same concentration; such as 39.47% for 500 nM TSA, 38.09% for 500 nM SCR; 18.6% for 50 nM TSA, 19.17% for 50 nM SCR, and 22.64% for 5 nM TSA, 17.18% for 5 nM SCR. As for the control group, the cleavage rate of the SCNT embryos cleavage ratewere27.47%., 30% and 30.85% respectively for bothtreatments. While there is a significant difference in TSA treatments at an eight-cell stage at the concentration (5 and 50 nM TSA) compared to the all other cleavage cell stages of (500 nM TSA and control). Also their were a differences between (50 nM of TSA) compared to the (50 nM SCR). Also there were a significant differences between the 16 cell stage at the (500 nM TSA) compared to other treatment (5 nM, 50 nM TSA and control). Regarding the SCR there were a significant difference at 8 cell stage between (5 nM SCR), compared to the other treatment (50 nM, 500 nM SCR and control). Also there were a significant difference at 16 cell stage between (50 nM, and 500 nM SCR), compared to the other treatment (5 nM SCR and control). While in the development of the embryos reach to blastocyst stage the SCR and the control group show a higher rate, in compered to TSA that did not show any development to blastocyst stage. The total SCR treatment showed (3/41 = 7.31%), and the total control showed (4/89 = 4.49%) blastula stage. It concludes that SCR improve the final development blastula stage compared to the TSA treatments that did not improved embryos reach to final developmental blastula stages may be due to spices differences or to the toxicity of TSA, especially at higher concentrations.     

Fatty Acid Profiles of Erwinia amylovora as Influenced by Growth Medium, Physiological Age and Experimental Conditions

Total cellular fatty acids for 20 strains of Erwinia amylovora grown on trypticase soy agar (TSA) for 3 days at 27°C, and for 3 strains grown on nutrient agar (NA), King's B (KB), glucoseyeast extract carbonate (GYCA) and TSA for 1, 3 and 6 days were analyzed by gas-liquid chromatography. Forty one percent of total fatty acids were saturated even-carbon straight chains, 6 % were saturated odd-carbon chains, 34 % unsaturated acids, 6 % hydroxy-substituted acids, 1 % branched chains, 7 % cyclic acids, and 4 % unidentified components. Composition was affected by growth medium, physiological age of cells, and chromatograph sensitivity. On TSA and NA saturated odd-carbon and cyclic acids of the 3 strains (combining 1, 3 and 6-day data) were higher than on KB and GYCA. Cyclic acids increased with physiological age on GYCA and KB medium. Even-carbon straight chain and unsaturated fatty acids (major components) constituted a significantly lower percentage of total fatty acids in chromatograms of high sensitivity (30–50 components) than of low instrument sensitivity (15–20 components).     

Effect of concentration and environmental form of tetradecenyl succinic acid on its mineralization in soil

Tetradecenyl succinic acid (TSA) is the major component of a detergent builder (C12-C14 alkenyl succinic acid), which is inherently biodegradable. ¹⁴C-TSA was dosed as a component of sewage sludge into a soil with a history of sludge amendment at final added concentrations of 1.5 and 30 mg (kg soil)^-1. In addition, it was dosed to the soil in an aqueous solution to a final added concentration of 30 mg (kg soil)^-1. Dose and form were found to have a pronouced effect on the mineralization kinetics. When dosed in a realistic form and concentration (i.e. 1.5 mg (kg soil)^-1 as a component of sludge), TSA was mineralized at its highest rate and to its greatest extent, and the mineralization half-life was 2.4 days. When dosed at 30 mg (kg soil)^-1 as a component of sludge, mineralization began immediately, and the half-life was 23 days. In contrast, when dosed at this concentration in aqueous solution, the onset of mineralization was preceded by a 13 day lag period and the mineralization half-life was 69 days. Primary biodegradation and mineralization rates of TSA were very similar. Approximately, half the radioactivity was evolved as ¹⁴CO₂, while the remaining radioactivity became non-extractable, having presumably been incorporated into biomass or natural soil organic matter (humics). This study demonstrated that TSA is effectively removed from sludge-amended soils as a result of biodegradation. Furthermore, it showed the effect that dose form and concentration have on the biodegradation kinetics and the importance of dosing a chemical not only at a relevant concentration but also in the environmental form in which it enters the soil environment.     

Harmful or Not: Trichostatin A treatment of embryos generated by ICSI or ROSI

Trichostatin A (TSA), a histone deacetylase inhibitor, is a known teratogen causing malformations such as vertebral fusions when applied during the postimplantation period; TSA also causes developmental arrest when applied during the preimplantation period. Regardless of these hindrances, we have succeeded in the establishment of an efficient somatic cloning method for the mouse where reconstructed embryos are treated with TSA. To elucidate this apparent discrepancy, we treated fertilized mouse embryos generated either by intracytoplasmic sperm injection (ICSI) or round spermatid injection (ROSI) with 50 nM TSA for 20 h after fertilization as well as parthenogenetic embryos and found that TSA treatment inhibited the preimplantation development of ICSI embryos but not ROSI or parthenogenetic embryos. And, although we often observed hypomorphism following TSA treatment in embryos grown to full term produced by both ICSI (av. of body weight: 1.7 g vs. 1.5 g) and ROSI (1.6 g vs. 1.2 g), TSA treatment reduced the offspring production rate for ICSI from 57% to 34% but not for ROSI from 30% to 36%. Thus, these data indicate that the effects, harmful or not, of TSA treatment on embryonic development depend on their nuclear derivations. Also, the resulting hypomorphism after TSA treatment is a caveat for this procedure in current Assisted Reproductive Technologies.     

Evaluation of hydrophobic properties of Yersinia enterocolitica strains isolated from humans and pigs]

The aim of the study was to evaluate the hydrophobic properties of Yersinia enterocolitica and to determine the influence of the culture conditions, such as: type of medium, temperature, and duration of the culture on the manifestation of these properties. The subject of the study were 117 of Y. enterocolitica strains isolated from humans and pigs. The ammonium sulphate salt aggregation test according to Lindahl modified method was used to evaluate the hydrophobic properties of Y. enterocolitica strains. Strains of Y. enterocolitica were cultured for 24 h at 25 degrees C on TSA (Difco) medium. During investigation of the influence of the culture conditions the chosen strains were incubated for 24 h and 48 h at 25 degrees C and 37 degrees C on TSA (Difco), LB (Difco), enrichment agar (Biomed), and enrichment agar with 5% sheep blood (Graso). A total of 44.5%, 17.9%, 9.4%, and 28.2% strains of Y. enterocolitica showed very strong hydrophobic properties, strong hydrophobic properties, some hydrophobic properties, and were non-hydrophobic, respectively when strains of Y. enterocolitica were cultured for 24 h at 25 degrees C on TSA medium. A total of 75.5% strains isolated from humans showed very strong hydrophobic properties and 13.5% strains were non-hydrophobic. Among strains isolated from pigs 30% showed very strong hydrophobic properties but 35% were non-hydrophobic. The hydrophobic properties of Y. enterocolitica depended on the temperature, duration of the culture and the type of media. The highest number of strains with very strong hydrophobic properties (89.6%) was obtained after 48 h of the incubation at 37 degrees C on the enrichment agar with 5% sheep blood. The highest number of non-hydrophobic strains of Y. enterocolitica (28.5%) was obtained after 24 h at 25 degrees C on TSA medium.     

Injury to Staphylococcus aureus during sausage fermentation.

Staphylococcus aureus 196E added to a beef sausage containing starter culture and 0.5 to 2.0% glucose and incubated at 35 degrees C was unable to grow when plated on tryptic soy agar (TSA) containing 7.5% NaCl. The injury, presumed to be due to the lactic acid produced during fermentation, was more pronounced at the lower concentrations of glucose (and lower acid levels). In the absence of glucose and/or starter culture, no injury was observed. When sausages containing S. aureus injured by fermentation at 35 degrees C were incubated at 5 degrees C, the counts on TSA (measures both injured and uninjured cells) and TSA containing 7.5% NaCl (measures uninjured cells only) remained constant; however, upon reincubation of the cold-stored sausage at 35 degrees C, the staphylococcus counts on TSA and TSA containing 7.5% NaCl and were similar to the counts of S. aureus present in fermenting sausages that had never been subjected to 5 degrees C. The demonstration of acid injury indicated that the injury phenomenon must be considered when determining numbers of viable S. aureus in fermented sausages.     

Injury to Staphylococcus aureus during sausage fermentation.

Staphylococcus aureus 196E added to a beef sausage containing starter culture and 0.5 to 2.0% glucose and incubated at 35 degrees C was unable to grow when plated on tryptic soy agar (TSA) containing 7.5% NaCl. The injury, presumed to be due to the lactic acid produced during fermentation, was more pronounced at the lower concentrations of glucose (and lower acid levels). In the absence of glucose and/or starter culture, no injury was observed. When sausages containing S. aureus injured by fermentation at 35 degrees C were incubated at 5 degrees C, the counts on TSA (measures both injured and uninjured cells) and TSA containing 7.5% NaCl (measures uninjured cells only) remained constant; however, upon reincubation of the cold-stored sausage at 35 degrees C, the staphylococcus counts on TSA and TSA containing 7.5% NaCl and were similar to the counts of S. aureus present in fermenting sausages that had never been subjected to 5 degrees C. The demonstration of acid injury indicated that the injury phenomenon must be considered when determining numbers of viable S. aureus in fermented sausages.     

Development of new plasmid DNA vaccine vectors with R1-based replicons

ABSTRACT: BACKGROUND: There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA) in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. RESULTS: In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30degreesC to 42degreesC. However, using Escherichia coli DH5alpha as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30degreesC, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42degreesC. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5alpha[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42degreesC. CONCLUSIONS: Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.     

In vitro ageing of pig oocytes: effects of the histone deacetylase inhibitor trichostatin A.

After in vitro maturation, the unfertilized pig oocytes underwent the process called ageing. This process involves typical events such as fragmentation, spontaneous parthenogenetic activation or lysis. Inhibition of histone deacetylase, using its specific inhibitor trichostatin A (TSA), significantly delayed the maturation of pig oocytes cultured in vitro. The ageing of oocytes matured under the effect of TSA is the same as the ageing in oocytes matured without TSA. The inhibition of histone deacetylase during oocyte ageing significantly reduced the percentage of fragmented oocytes (from 30% in untreated oocytes to 9% in oocytes aged under the effect of 100 nM of TSA). Oocytes matured in vitro and subsequently aged for 1 day under the effects of TSA retained their developmental capacity. After parthenogenetic activation, a significantly higher portion (27% vs. 15%) of oocytes developed to the blastocyst stage after 24 h ageing under 100 nM TSA when compared with oocytes activated after 24 h ageing in a TSA-free medium. The parthenogenetic development in oocytes aged under TSA treatment is similar to the development of fresh oocytes (29% of blastocyst) artificially activated immediately after in vitro maturation.     

Biochemical Alternations in Bacillus megaterium as Produced by Aflatoxin B1

Bacillus megaterium NRRL B-1368 cells and spores were produced on Trypticase Soy Broth (TSB) and Agar (TSA) containing 3.8 μg of aflatoxin B₁ per ml, analyzed for selected chemical constituents, and compared to cells and spores of B. megaterium produced on nontoxic Trypticase Soy Media. There was an initial 30% kill of cells after inoculation into toxic TSB and during the first 3.5 hr of incubation followed by a logarithmic growth phase in which the generation time was 75 min as compared to 20 min for the control culture. Chemical analyses revealed an increase in protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) on both a per cell basis and a per cent dry weight basis when B. megaterium was grown in toxic TSB. There was a concurrent decrease in the total amounts of cellular protein, DNA, and RNA synthesized in toxic TSB. Amino acid analyses of control and test cell walls showed little, if any, difference in cell wall composition. About 97% sporulation of B. megaterium occurred after 3 days on nontoxic TSA although 6 days were required to attain 65% sporulation on toxic TSA. Germination of spores was not inhibited by 4.0 μg of aflatoxin per ml but outgrowth was. No significant differences were observed in the heat resistance, protein, DNA, RNA, or dipicolinic acid content of spores formed on toxic TSA and nontoxic TSA.