Toxin production by Clostridium botulinum type A under various fermentation conditions.

The time of appearance and the quantity of toxin produced by the Hall strain of Clostridium botulinum type A were examined under various conditions. A 70-liter fermentor and a complex medium consisting of 2% casein hydrolysate and 1% yeast extract plus an appropriate concentration of glucose were employed. Optimal conditions for toxin production were as follows: a nitrogen overlay at a rate of 5 liters/min, an agitation rate of 50 rpm, a temperature of 35 degrees C, and an initial glucose concentration of 1.0% with the pH uncontrolled. Under these conditions, the maximum toxin concentration (6.3 x 10(5) mouse median lethal doses/ml) was attained within 24 h. Cell lysis was apparently not required to obtain maximum toxin concentrations under the fermentation conditions described.     

Toxin production by Clostridium botulinum type A under various fermentation conditions.

The time of appearance and the quantity of toxin produced by the Hall strain of Clostridium botulinum type A were examined under various conditions. A 70-liter fermentor and a complex medium consisting of 2% casein hydrolysate and 1% yeast extract plus an appropriate concentration of glucose were employed. Optimal conditions for toxin production were as follows: a nitrogen overlay at a rate of 5 liters/min, an agitation rate of 50 rpm, a temperature of 35 degrees C, and an initial glucose concentration of 1.0% with the pH uncontrolled. Under these conditions, the maximum toxin concentration (6.3 x 10(5) mouse median lethal doses/ml) was attained within 24 h. Cell lysis was apparently not required to obtain maximum toxin concentrations under the fermentation conditions described.     

The effect of dissolved oxygen on the metabolic profile of a murine hybridoma grown in serum-free medium in continuous culture

The murine B-lymphocyte hybridoma, CC9C10 was grown at steady state under serum-free conditions in continuous culture at dissolved oxygen (DO) concentrations in the range of 10% to 150% of air saturation. Cells could be maintained with this range at high viability in a steady state at a dilution rate of 1 d(-1), although with lower cell concentrations at higher DO. A higher specific antibody production measured at higher DO was matched by a decrease in the viable cell concentration at steady state, so that the volumetric antibody titre was not changed significantly. An attempt to grow cells at 250% of air saturation was unsuccessful but the cells recovered to normal growth once the DO was decreased.There was a requirement for cellular adaptation at each step-wise increase in dissolved oxygen. Adaptation to a DO of 100% was associated with an increase in the specific activities of glutathione peroxidase (x18), glutathione S-transferase (x11) and superoxide dismutase (x6) which are all known antioxidant enzymes. At DO above 100%, the activities of GPX and GST decreased possibly as a result of inactivation by reactive oxygen radicals.The increase in dissolved oxygen concentration caused changes in energy metabolism. The specific rate of glucose uptake increased at higher dissolved oxygen concentrations with a higher proportion of glucose metabolized anaerobically. Short-term radioactive assays showed that the relative flux of glucose through glycolysis and the pentose phosphate pathway increased whereas the flux through the tricarboxylic acid cycle decreased at high DO. Although the specific glutamine utilization rate increased at higher DO, there was no evidence for a change in the pattern of metabolism. This indicates a possible blockage of glycolytic metabolites into the TCA cycle, and is compatible with a previous suggestion that pyruvate dehydrogenase is inhibited by high oxygen concentrations.Analysis of the oxygen uptake rate of cell suspensions at steady state under all conditions showed a pronounced Crabtree effect which was manifest by a decrease (up to 40%) in oxygen consumption on addition of glucose. This indicates that the degree of aerobic metabolism in these cultures is highly sensitive to the glucose concentration. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 153-164, 1997.     

Early effect of glucose and oxygen deprivation on the spontaneous acetylcholine release from the myenteric plexus of the guinea pig ileum

The early effects of glucose and oxygen deprivation on the spontaneous acetylcholine output from the myenteric plexus - longitudinal muscle preparation of the guinea pig ileum were studied using an incubation chamber that permitted rapid sample collection in 2-min intervals. Glucose deprivation or hypoxia resulted in a gradual decline in rate of spontaneous acetylcholine collection in 2-min intervals. Glucose deprivation or hypoxia resulted in a gradual decline in rate of spontaneous acetylcholine output. However, glucose deprivation plus hypoxia caused an acceleration in acetylcholine output within 10-15 min, which attained a rate seven times greater than observed under normal conditions. Recovery of low resting rates was obtained by reintroduction of oxygen and glucose into the bath medium. Neither morphine (2.7 x 10(-5) M) nor tetrodotoxin (1.6 x 10(-6) M) prevented the increase in acetylcholine output induced by energy deprivation. The substitution of Ca2+ by Mg2+, in the presence of EGTA, greatly reduced the acetylcholine output induced by energy deprivation. However, a small transitory output of acetylcholine was observed under these conditions which was resistant to tetrodotoxin and ot affected by depolarizing amounts of K+. The transitory output was repeatable by reintroduction of glucose and oxygen to the Ca2+-free medium with subsequent return to conditions of hypoxia and glucose deprivation. These results suggest that energy deprivation initially stimulates normal acetylcholine secretion by (a) increasing Ca2+ influx across the plasma membrane and (b) mobilizing an intracellular Ca2+ poll. This implies that processes involved in maintenance of normal low transmitter release are more sensitive to energy lack than the neurosecretion process itself.     

Effects of chemical anoxia on adrenergic responses of goldfish hepatocytes and the contribution of alpha- and beta-adrenoceptors

Adrenergic responses during normoxia and chemical anoxia were investigated in anoxia-tolerant hepatocytes from the goldfish, Carassius auratus. Epinephrine-stimulated glucose release was unaltered after 1 hr of chemical anoxia, the concentration of epinephrine required for half maximal stimulation of glucose release (K0.5(GLU)) ranging from 0.62 x 10(-8) to 2.05 x 10(-8) M. Similarly, the maximum rate of glucose release caused by hormonal stimulation was not affected by chemical anoxia. In anoxic goldfish hepatocytes [Ca2+](i) remained constant in nonstimulated cells but could be elevated by addition of epinephrine. The magnitude of this [Ca(2+)](i)-increase was dependent on the concentration of the catecholamine and this dependency was similar under normoxia (K0.5(Ca2+) = 1.17 x 10(-8) M) and chemical anoxia (K0.5(Ca2+) = 1.15 x 10(-8) M), as was the percentage of cells responding (77%) and displaying oscillatory [Ca2+]i response patterns (60%) after epinephrine addition, although the frequency of [Ca2+]i oscillations was significantly lower in anoxic cells. To analyze a possible shift in the importance of alpha- and beta-adrenoceptors during chemical anoxia, the effect of phentolamine and propranolol, alpha- and beta-adrenergic antagonists respectively, on epinephrine-stimulated glucose release was studied. Application of the alpha-antagonist caused a dose-dependent reduction of glucose-release which was similar under both conditions, whereas the sensitivity to the beta-antagonist was lowered after chemical anoxia. Taken together these results provide evidence that during chemical anoxia goldfish hepatocytes remain responsive to adrenergic stimulation and that there is a partial shift regarding the contribution of alpha- and beta-adrenergic pathways to the induction of cellular glucose release stimulated by epinephrine.     

Regulation of glycogen synthesis in rat-hepatocyte cultures by glucose, insulin and glucocorticoids.

The changes in glycogen content and in its rate of synthesis in two-day-old primary cultures of rat hepatocytes were assessed under various conditions. Hepatocytes cultivated in serum-free and hormone-free medium switch from glycogen degradation to glycogen deposition at 10.3 mM glucose. After pretreatment of the cells with glucocorticoids this threshold was reduced, in the absence or presence of insulin, to 5.4 or 1.2 mM glucose, respectively. The rate of glycogen synthesis in the presence of 10 mM glucose was amplified from 5 nmol x h-1 x mg protein-1 to 20 nmol glucose x h-1 x mg protein-1 after pretreatment with triamcinolone. Glucagon pretreatment also significantly increased the subsequent glycogen synthesis rate. Insulin addition accelerated glycogen synthesis about twofold regardless of the pretreatment. The dose-response relationship between insulin concentration and glycogen synthesis rate showed half-maximal effect at 0.62 +/- 0.22 nM (mean +/- S.D.) insulin. Pretreatment of hepatocytes with glucocorticoids, glucagon, insulin or combinations of these hormones did not significantly change the concentration which gives the half-maximal effect.     

Culture Conditions Affect the Molecular Weight Properties of Hyaluronic Acid Produced by Streptococcus zooepidemicus

The effect of five culture variables on the molecular weight properties of hyaluronic acid (HA) produced by Streptococcus zooepidemicus was studied in batch culture with a complex medium containing glucose and 10 g of yeast extract per liter. Neither the culture pH (pH 6.3 to 8.0) nor the agitation speed (300 to 1,000 rpm) affected the weight-average molecular weight (M(infw)) of HA under anaerobic conditions at 37(deg)C when 20 g of glucose per liter was used initially. M(infw) was in the narrow range of 1.5 x 10(sup6) to 2.3 x 10(sup6), and polydispersity (P) was between 1.8 and 2.5. When S. zooepidemicus was grown at lower temperatures or with aeration, higher-molecular-weight polymer and increased yields were observed. The polydispersity, however, remained unaffected. Anaerobically, the mean M(infw) (based on three samples taken within 4 h of glucose exhaustion) was (2.40 (plusmn) 0.10) x 10(sup6) and (1.90 (plusmn) 0.05) x 10(sup6) at 32 and 40(deg)C respectively. Aeration of the culture at 1 vol/vol/min produced HA with mean M(infw) of (2.65 (plusmn) 0.05) x 10(sup6) compared with (2.10 (plusmn) 0.10) x 10(sup6) under equivalent anaerobic conditions. The initial glucose concentration had the most pronounced effect on polymer characteristics. Increasing this concentration from 20 to 40 g/liter produced HA with mean M(infw) of (3.1 (plusmn) 0.1) x 10(sup6) at 1-vol/vol/min aeration. The molecular weight of HA also exhibited time dependency, with smaller chains (M(infw), ca. 2.5 x 10(sup6)) detected early in the culture time course, rising to a maximum (M(infw), 3.2 x 10(sup6)) in the late exponential phase of growth. The mean polydispersity was also greater (2.7 (plusmn) 0.1) under these conditions. Replicate experiments performed under conditions resulting in the lowest (40(deg)C, anaerobic) and highest (40 g of glucose per liter, 1-vol/vol/min aeration)-M(infw) polymer demonstrated excellent experimental reproducibility.     

Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells

The effects of cell density as well as the concentration levels of glucose and glutamine on the specific respiration rate of a hybridoma cell line were investigated. The experimental oxygen consumption rate was found to be constant over a wide range of dissolved oxygen levels if the suspension medium contained glutamine. In glutamine-free medium, however, the rate of oxygen consumption decreased slowly with time.In a stationary flask batch culture, the specific respiration rate decreased from about 7 to 2.9 mumol/min per 10(9) cells as the cell density increased exponentially from 1 x 10(5) to 1.2 x 10(6)/mL. To isolate the effect of cell density, cells were re suspended in fresh culture medium so that nutrient concentrations were the same for all experiments. The specific respiration rate decreased with increasing cell density in the same manner as in the stationary flask culture, falling from 8 to 4 mumol/min per 10(9) cells as the cell density increased from 10(5) to 10(6) cells/mL, then declining to 2 mumol/min per 10(9) cells when the cell density reached 10(7) cells/mL.Cells suspended in Hanks balanced sale solution (HBSS) were used to elucidate the effect of glucose and glutamine levels on respiration. The addition of glucose in concentrations of 0.25, 0.50, and 0.75 g/L had no observable effect on the specific oxygen uptake rate; however, a glucose concentration of 1 g/L reduced the uptake rate by 22%. Glutamine in a concentration of 0.30 g/L increased the specific respiration rate in HBSS containing 0 and 1 g/L glucose by approximately 13%.     

Binding and aggregation of the 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis to cell membranes and alteration by monoclonal antibodies and amino acid modifiers

The 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis binds irreversibly to Aedes albopictus cells, Choristoneura fumiferana cells, and erythrocytes. The binding to cells increased with both toxin concentration and time and when the cells were first preincubated with unlabeled toxin. Binding data indicated a two- to threefold increase in the rate of binding after the amount of the membrane-bound toxin reached approximately 3.5 fmol/3 x 10(5) A. albopictus cells or 3.3. fmol/2 x 10(5) C. fumiferana cells. When this level of bound toxin was reached, the toxins also began forming aggregates at the cell membrane. The toxin aggregates were extracted with 10% Triton X-100 and separated from the monomers with a 5 to 20% sucrose density gradient. The toxin aggregates isolated from A. albopictus and C. fumiferana cell membranes were ca. 400 kilodaltons, while those isolated from human erythrocytes were significantly smaller. The proportion of the toxin found in aggregate form increased rapidly with the amount of toxin bound; however, the molecular size of the aggregates remained constant. Eleven monoclonal antibodies raised against the native form of the toxin blocked 80 to 97% of the toxin binding to cells. The epitope of one of these monoclonal antibodies was mapped to a domain which included the cysteine, suggesting the importance of the domain around this amino acid to binding. Toxin binding and cell lysis were also inhibited by treating the toxin with HgCl2, further indicating the importance of the C-terminal hydrophobic cysteine-containing domain in cytolytic activity of the 25-kilodalton protein.     

Binding and aggregation of the 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis to cell membranes and alteration by monoclonal antibodies and amino acid modifiers.

The 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis binds irreversibly to Aedes albopictus cells, Choristoneura fumiferana cells, and erythrocytes. The binding to cells increased with both toxin concentration and time and when the cells were first preincubated with unlabeled toxin. Binding data indicated a two- to threefold increase in the rate of binding after the amount of the membrane-bound toxin reached approximately 3.5 fmol/3 x 10(5) A. albopictus cells or 3.3. fmol/2 x 10(5) C. fumiferana cells. When this level of bound toxin was reached, the toxins also began forming aggregates at the cell membrane. The toxin aggregates were extracted with 10% Triton X-100 and separated from the monomers with a 5 to 20% sucrose density gradient. The toxin aggregates isolated from A. albopictus and C. fumiferana cell membranes were ca. 400 kilodaltons, while those isolated from human erythrocytes were significantly smaller. The proportion of the toxin found in aggregate form increased rapidly with the amount of toxin bound; however, the molecular size of the aggregates remained constant. Eleven monoclonal antibodies raised against the native form of the toxin blocked 80 to 97% of the toxin binding to cells. The epitope of one of these monoclonal antibodies was mapped to a domain which included the cysteine, suggesting the importance of the domain around this amino acid to binding. Toxin binding and cell lysis were also inhibited by treating the toxin with HgCl2, further indicating the importance of the C-terminal hydrophobic cysteine-containing domain in cytolytic activity of the 25-kilodalton protein.     

Interactions between oxygen tension and glucose concentration that modulate actions of heat shock on bovine oocytes during in vitro maturation

Exposure of oocytes to elevated temperature (i.e. heat shock) during maturation can reduce fertilization rate and development of the resultant embryos. Given the possible role of free radicals in actions of heat shock on cellular function, we tested the hypothesis that a high oxygen environment exacerbates the magnitude of deleterious effects of heat shock on in vitro maturation of bovine oocytes. A preliminary experiment was performed to establish conditions for oocyte maturation that would be independent of oxygen concentration. Oocytes were matured in a modified tissue culture medium-199 (mTCM-199) or modified synthetic oviduct fluid (mSOF) containing 5.6 or 20 mM glucose and under either high (atmospheric oxygen, approximately 21%, v/v) or low oxygen (5%, a value approximating oxygen content of the follicle). For oocytes matured in mTCM-199, development was greater in high oxygen than in low oxygen, whereas development was unaffected by oxygen using mSOF (mediumxoxygen, P<0.05). Accordingly, mSOF was used as the maturation medium in a second study to test the effect of oxygen concentration on the magnitude of actions of heat shock during maturation. Maturation was at 38.5 degrees C for 22 h (control) or 41 degrees C for 12h and 38.5 degrees C for 10h (heat shock). Heat shock slightly decreased cleavage rate, regardless of the maturation conditions, and decreased blastocyst development under all maturation conditions except for the group matured under high oxygen and high glucose (temperature x glucose for oocytes under low oxygen, P<0.05). The percentage of oocytes becoming blastocysts for control and heat shocked oocytes was 25.9% versus 22.5% (low oxygen -- 5.6 mM glucose), 41.6% versus 34.9% (low -- 20 mM), 41.7% versus 35.0% (high -- 5.6 mM), and 37.6% versus 37.5% (high -- 20 mM). In conclusion, under an oxygen tension that approached physiological conditions, heat shock during in vitro maturation reduced oocyte competence for fertilization and subsequent development.     

Futile substrate cycles in the glycolytic pathway of boar and rat spermatozoa and the effect of alpha-chlorohydrin

In boar spermatozoa incubated with 0.1 mM-glucose about 20 nmol glucose were converted to lactate and CO2 and the rate of futile substrate cycling between glucose and glucose 6-phosphate was about 6 nmol/10(8) spermatozoa/30 min. Futile cycling was increased in the presence of 0.05 or 1 mM-alpha-chlorohydrin but not to an extent sufficient to account for the rapid decline in ATP concentration observed under these conditions. These estimates include a substantial rate of fructose formation from fructose phosphates. The addition of 10 mM-L-lactate plus 1 mM-pyruvate protected the spermatozoa against the effect of alpha-chlorohydrin and glucose on the ATP concentration but increased futile substrate cycling. Substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate could not be measured in boar spermatozoa but in rat spermatozoa its rate (nmol/10(8) spermatozoa/30 min) was about 10 under control condition and about 25 in the presence of 1 mM-alpha-chlorohydrin. This increase was insufficient to account for the decline in ATP concentration. In both species futile substrate cycling consumed a significant proportion of the ATP synthesis during lactate production but only about 5% of that produced in the oxidation of glucose to acetyl carnitine and CO2.     

Biosynthesis of novel exopolymers by Aureobasidium pullulans

Aureobasidium pullulans ATCC 42023 was cultured under aerobic conditions with glucose, mannose, and glucose analogs as energy sources. The exopolymer extracts produced under these conditions were composed of glucose and mannose. The molar ratio of glucose to mannose in the exopolymer extract and the molecular weight of the exopolymer varied depending on the energy source and culture time. The glucose content of exopolymer extracts formed with glucose and mannose as the carbon sources was between 91 and 87%. The molecular weight decreased from 3.5 x 10(6) to 2.12 x 10(6) to 0.85 x 10(6) to 0.77 x 10(6) with culture time. As the culture time increased, the glucose content of the exopolymer extract formed with glucosamine decreased from 55 +/- 3 to 29 +/- 2 mol%, and the molecular weight increased from 2.73 x 10(6) to 4.86 x 10(6). There was no evidence that glucosamine was directly incorporated into exopolymers. The molar ratios of glucose to mannose in exopolymer extracts ranged from 87 +/- 3:13 +/- 3 to 28 +/- 2:72 +/- 2 and were affected by the energy source added. On the basis of the results of an enzyme hydrolysis analysis of the exopolymer extracts and the compositional changes observed, mannose (a repeating unit) was substituted for glucose, which gave rise to a new family of exopolymer analogs.     

Studies on nonidet P40 lysis of murine lymphoid cells. I. Use of cholera toxin and cell surface Ig to determine degree of dissociation of the plasma membrane.

Lymphoid cells from A/J mice were iodinated (125I) by the lactoperoxidase lysed with the non-ionic detergent NP-40. The plasma membrane glycolipid receptor for cholera toxin and cell surface immunoglobulin were utilized in immune precipitation systems to characterize the degree of dissociation of the plasma membrane under various conditions. It was found that at 0.1% NP-40 and at cell concentration from 5 to 10 times 10(7) cells/ml, lipid-protein and protein-lipid-protein complexes formed in NP-40 which were soluble after centrifugation at 10(5) times G. Column chromatography of 125I-cell lysates on agarose A-0.5 M in 0.1% or 0.5% NP-40/PBS indicated that the majority of iodinated cell surface material existed as aggregates in detergent micelles. The availability of the oligosaccharide moiety of the glycolipid to interact with the cholera toxin was dependent on both the detergent concentration and the cell concentration used for cell lysis. However, the cell surface immunoglobulin was immunoprecipitable under all conditions of lysis tested.     

Studies on staphylococcal enterotoxin B. II. Production and regulation (author's transl)]

Enterotoxigenic strain of Staphylococcus aureus (ATCC 14458) was grown under various conditions with constant shaking to determine the requirements for maximum toxin production. It was evident that 3% tryptic soy broth, 3% NZ-Amine NAK + 3% casein hydrolysate, 3% NZ-Amine NAK + 1% yeast extract, and 3% NZ-Amine NAK + 1% yeast extract + 0.2% glucose are most available toxin production media. But concentration of glucose could strictly triggered the enterotoxin producing efficiency. When glucose concentration was less than 0.5%, although with higher yield, the toxin production was delayed for certain period of time. However, if glucose concentration was up to more than 0.5%, the enterotoxin production was almost inhibited. Some metabolites of glucose to elucidate the inhibitory effect have also investigated. Our results indicated that glycerol and citric acid inhibited the toxin production directly, while the inhibitory effect of lactic acid and acetic acid were due to those acidic metabolites, decreased the pH value of media, and adversely suppressed the bacterial growth.     

Regulation of Staphylococcal Enterotoxin B

Several factors influenced the formation of enterotoxin B by Staphylococcus aureus strain S-6. In the standard casein hydrolysate medium, toxin was not produced in detectable quantities during exponential growth; it was produced during the post-exponential phase when total protein synthesis was arithmetic. The rate of toxin synthesis was much greater than the rate of total protein synthesis. The appearance of enterotoxin was inhibited by chloramphenicol; thus, the presence of toxin was dependent on de novo protein synthesis. When low concentrations of glucose (<0.30%) were added to the casein hydrolysate medium, growth was diauxic; glucose was completely metabolized during the first growth period. During the second growth period, enterotoxin was synthesized. In unbuffered casein hydrolysate medium containing excess glucose, toxin synthesis was completely repressed. The absence of toxin production under such conditions might be explained by the low (4.6) pH resulting from the acid end products of glucose metabolism. At pH <5.0, little or no toxin was produced. Toxin synthesis was initiated in the presence of glucose when the medium were buffered at any pH above 5.6. In such media, the differential rates of toxin synthesis, with respect to the rates of total protein synthesis, were lower than the differential rates in casein hydrolysate medium alone. Addition of glucose to a culture synthesizing toxin resulted in an immediate decrease in the differential rate without any change in pH. Thus, toxin synthesis appeared to be regulated by catabolite repression.     

Binding of latrotoxin to synaptosomes and synaptic membranes of the rat brain

The binding of [125I] alpha-latrotoxin to synaptosomes from the rat brain is studied. It is shown that the constant rate of toxin association with the synaptosome receptor at 37 degrees C is equal to 8.2 +/- 1.3 x 10(7) M-1.s-1, while that of synaptosomal membrane -7.6 +/- 2.7 x 10(6) M-1 s-1. Depolarization of the synaptosome membrane induced by 55 mM KCl decreases the binding rate of toxin to the receptor, the rate constant being equal to 3.9 +/- 1.5 x 10(7) m-1 s-1. The pattern of the dissociation process of the toxin-receptor complex of synaptosomes and of synaptosomal membrane is different. In the first case dissociation follows two stages with the rate constants 3.6 x 10(-3) s-1 and 1.2/10(-4) s-1, in the second case it follows one stage with the constant equalled 2.0 x 10(-5) s-1. The quantity of the toxin binding sites on synaptosomes may vary under the action of agents modifying the activity of calcium fluxes which are induced by alpha-latrotoxin. It is supposed that a decrease in the ATP level in synaptosomes as well as deenergy of the surface membrane leads to a change in the state of the alpha-latrotoxin receptor.     

Effect of methylguanidine and guanidinosuccinic acid on pyruvate kinase activity in human red cells

An effect of methylguanidine and guanidinosuccinic acid on pyruvate kinase activity in human red cells was determined in vitro following a 3-hour incubation at 37 degrees C. The obtained results have shown that methylguanidine in the concentration of 1.8 x 10(-5) M/l inhibits pyruvate kinase activity by 20.8%. Pyruvate kinase activity was statistically significantly inhibited on addition of methylguanidine in the concentration of 5.4 x 10(-5) M/l whereas higher concentrations have no such an effect Guanidinesuccinic acid exerted similar but weaker effect on the activity of pyruvate kinase in human red cells. Mixture of methylguanidine (5.4 x 10(-5) m/l) and guanidinesuccinic acid (2.8 x 10(-5) M/l) does not affect pyruvate kinase activity in normal human red cells under identical experimental conditions.     

Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor

To improve rabies vaccine production by Vero cells, we have developed a strategy based on high cell density culture and optimization of virus yield. We have first optimized cell growth in spinner flask using a Taguchi's L8 experimental design. We analyzed the effects of the following factors: initial glucose and glutamine concentrations, Cytodex 1 concentration and the regulation of glucose level at 1 g l(-1). We have also investigated the effect of the following factor interactions: Cytodex 1 concentration/glutamine concentration, Cytodex 1 concentration/glucose concentration and glucose concentration/glutamine concentration. Statistical analysis of the collected data pointed to the initial glucose concentration, the regulation of glucose level at 1 g l(-1) and the interactions between Cytodex 1 concentration/initial glucose concentration and Cytodex 1 concentration/initial glutamine concentration as the parameters that affected cell growth. Using the optimal conditions determined earlier, we have studied Vero cell growth in a 7-l bioreactor and in batch culture, and obtained a cell density level equal to 3.6 +/- 0.2 x 10(6) cells ml-1. Cell infection with rabies virus (LP 2061/Vero strain) at a multiplicity of infection (MOI) of 0.3 using M199 medium supplemented with 0.2% bovine serum albumin (BSA), yielded a maximal virus titer equal to 8 +/- 1.6 x 10(7) Fluorescent Focus Units (FFU) ml-1. We have also studied Vero cell growth in a 7-l bioreactor using recirculation as a perfusion culture mode during cell proliferation step and perfusion for virus multiplication phase. In comparison to batch culture, we reached a higher cell density level that was equal to 10.1 +/- 0.5 x 10(6) cells ml-1. Cell infection under the conditions previously indicated, yielded 14l of virus harvest that had a virus titer equal to 2.6 +/- 0.5 x 10(7) FFU ml-1. The activity of the inactivated virus harvest showed a protective activity that meets WHO requirements.     

Starch industry wastewater as a substrate for antagonist, Trichoderma viride production

Starch industry wastewater was investigated to assess and improve its potential as a raw material for the conidia production of biocontrol fungi, Trichoderma viride. The wastewater was tested with and without supplements of glucose, soluble starch, meat peptone and probable conidiation inducer chemicals in shake flask culture. Addition of complex carbon source (soluble starch, 1% and 2% w/v) produced maximum conidia ( approximately 3.02 and 4.2 x 10(10)CFU/mL, respectively). On the other hand, glucose addition as a simpler carbon source was either ineffective or, reduced conidia production (from 1.6 x 10(8) in control to 3.0 x 10(7)CFU/mL in 5% w/v glucose supplement). Supplement of nitrogen source showed a small increase of conidia concentration. Propionic, maleic and humic acids, EDTA, pyridine, glycerol and CaCO(3) were examined as probable conidiation inducers and showed effect only on initial rate of conidiation with no increase in final conidia concentration. Intra and extracellular ATP correlation with spore production showed dependence on growth media used and conidia concentration at the end of fermentation. Addition of carbon and nitrogen sources showed an increase in protease activity (from 0.4985 to 2.43 IU/mL) and entomotoxicity (from 10448 to 12335 spruce budworm unit (SBU)/microL). Entomotoxicity was improved by 11% in fermenter over shake flask when starch industry wastewater was supplemented with meat peptone.