Decigram quantities of pure foot-and-mouth disease virus from cell cultures

Baby-hamster kidney (BHK) cell cultures grown in rolling 2-liter Baxter bottles are used for the production of foot-and-mouth disease virus (FMDV) which is subsequently purified. The bottles are held securely in round wire racks (19 per rack) and rotated on a three-tiered roller mill. The use of a strongly buffered growth medium makes changes of the medium unnecessary. A sheet of aluminum foil is used to seal the cultures. It is pressed tightly over all the bottles in a rack by means of a polyurethane foam sheeting bonded to the underside of a rigid snap-on cover. Special equipment eliminates removing the bottles from the racks at any stage in their use. The loaded racks fit directly onto headers of a glassware washer. Spent cell growth media and virus fluids are collected by inverting an entire rack of bottles-Current production is 400 BHK cultures (21 racks) containing 8 × 10⁸ cells each after 6 days growth. About 344 of these cultures (18 racks) are used to grow virus. The purification process yields about 113 mg of pure FMDV per week; the overall recovery based on infectivity is 32%. The projected maximum production of purified virus in the present facilities is approximately 2.5 times greater than this amount.     

Device for Turbidity Standardizing of Cultures for Antibiotic Sensitivity Testing

A specially designed metal rack which is illuminated by a modified Rh-typing view box is described. This device facilitates the handling and reduces the time involved in standardizing cultures.     

Modeling bacterial contamination of fuel ethanol fermentation

The emergence of antibiotic‐resistant bacteria may limit the effectiveness of antibiotics to treat bacterial contamination in fuel ethanol plants, and therefore, new antibacterial intervention methods and tools to test their application are needed. Using shake‐flask cultures of Saccharomyces cerevisiae grown on saccharified corn mash and strains of lactic acid bacteria isolated from a dry‐grind ethanol facility, a simple model to simulate bacterial contamination and infection was developed. Challenging the model with 10⁸ CFU/mL Lactobacillus fermentum decreased ethanol yield by 27% and increased residual glucose from 6.2 to 45.5 g/L. The magnitude of the effect was proportional to the initial bacterial load, with 10⁵ CFU/mL L. fermentum still producing an 8% decrease in ethanol and a 3.2‐fold increase in residual glucose. Infection was also dependent on the bacterial species used to challenge the fermentation, as neither L. delbrueckii ATCC 4797 nor L. amylovorus 0315‐7B produced a significant decrease in ethanol when inoculated at a density of 10⁸ CFU/mL. In the shake‐flask model, treatment with 2 µg/mL virginiamycin mitigated the infection when challenged with a susceptible strain of L. fermentum (MIC for virginiamycin ≤2 ppm), but treatment was ineffective at treating infection by a resistant strain of L. fermentum (MIC = 16 ppm). The model may find application in developing new antibacterial agents and management practices for use in controlling contamination in the fuel ethanol industry. Biotechnol. Bioeng. 2009;103: 117–122. Published 2008 Wiley Periodicals, Inc.     

Increased sensitivity to NMDA is involved in alcohol-withdrawal induced cytotoxicity observed in primary cultures of cortical neurones chronically pre-treated with ethanol

Severe cellular damage and neuronal cell loss were previously observed in cultures of primary cortical neurones after chronic ethanol pre-treatment followed by ethanol-withdrawal. In this study, we investigated the circumstances and the possible cellular changes leading to alcohol-withdrawal induced neuronal cell death. When cultures were pre-treated with ethanol (25-200mM) once for 24 or 72h, the amount of the subsequent 24h alcohol-withdrawal induced cell death-estimated by measuring the release of lactate dehydrogenase (LDH)-was elevated only in cultures pre-treated with 200mM ethanol for 72h. On the contrary, as little as 50mM ethanol produced significant (P<0.01) increase in the withdrawal induced LDH-release in cultures pre-treated repeatedly with ethanol once daily for three consecutive days. When ethanol was re-added to the cultures during the withdrawal period, the LDH-release was dose-dependently reduced to the level of control. In ethanol pre-treated cultures N-methyl-D-aspartate (NMDA) (0.01-1mM) induced excitotoxicity as well as NMDA evoked elevation of cytosolic calcium ion concentration was increased. In contrast, the depolarising agent veratridine (0.01-1mM) produced similar extent of neuronal injury and elevation in cytosolic calcium ion concentration in control as in ethanol pre-treated cultures. According to these observations, repeated ethanol treatment appears to cause more robust adaptive changes in cultured neurones leading to more pronounced withdrawal induced cellular damage than chronic but single treatment does. In addition, the glutamatergic neurotransmission, especially the NMDA receptor system seems to be highly involved in the adaptive changes and in the cytotoxic effect of alcohol-withdrawal.     

Comparison of Differences in Chemical Composition and Related Antioxidant Activity of Snow Lotus from Different Origins

The snow lotus is an endangered traditional Chinese medicinal herb. Saussurea involucrata, Saussurea laniceps, and Saussurea medusa, the three main snow lotus species (five herbs and two S. involucrata cell cultures), were selected for this study. Snow lotus (XLs) was extracted using 75 % (v/v) ethanol. Two reversed phase-high performance liquid chromatography-diode array detector methods were developed and validated for the determination of 10 representative components in XLs. The antioxidant efficacy of XLs and their components was investigated using DPPH, ABTS free radical scavenging, and ROS inhibition experiments. The results showed that the IC₅₀ for DPPH scavenging ranged from 0.06–0.29 mg/mL for XLs and from 0.13–0.63 mg/mL for ABTS, and could downregulate ROS to varying degrees. The results of the antioxidant activity showed that rutin, quercetin, and isochlorogenic acid A contributed to the antioxidant capacity of XLs. The high content and activity of the cell cultures indicate that they can serve as an effective alternative to snow lotus, thus providing a theoretical basis for the selection of herbs and cell cultures to fulfil various needs.     

The accumulation of succinate by the yeast Brettanomyces bruxellensis.

The metabolism of Brettanomyces bruxellensis was investigated to determine the metabolic block responsible for the accumulation of acetate seen in cultures of this yeast. In glucose-grown cultures the major non-volatile intracellular organic acide was succinic acid. These cultures also had low levels of succinic dehydrogenase (succinate dehydrogenase, EC 1.3.99.1) and did not produce CO2 from the carbons of ethanol. It was concluded that a block in the oxidation of ethanol occurred at the level of succinic dehydrogenase. If glucose-grown cultures were transferred to ethanol medium, the block in the metabolism of ethanol was partially overcome; the level of succinic dehydrogenase increased, the concentration of the intracellular succinate decreased, and CO2 could be produced from C-1 of ethanol.     

Calorimetric control of fed-batch cultures of Saccharomyces cerevisiae

Calorimetry has been used to control the glucose feeding in fed-batch cultures of S. cerevisiae in order to avoid ethanol formation and maintain a fully respiratory metabolism. Comparisons between batch and fed-batch cultivations showed that the former had a much lower growth yield. The growth yields for fed-batch cultivations were more than 30% higher than for batch cultures. However, energy balance calculations showed that a large part of the increase could be explained by the evaporation of ethanol during batch cultivations. When the growth yields obtained from the batch cultures were corrected for the evaporation of ethanol, the increase in growth yield for fed-batch cultures was about 10%.     

Reasons for the apparent difference in the effects of produced and added ethanol on culture viability during rapid fermentation by Saccharomyces cerevisiae

By feeding ethanol at various high rates to low cell density cultures of Saccharomyces cerevisiae it was shown that the sharp fall in viability when ethanol is produced during rapid fermentations is in part a direct consequence of the high rate of change of extracellular ethanol concentration. Nevertheless, the fall in viability in high cell density rapid fermentations which produced 98 g L(-1) ethanol in 3 h considerably exceeded that of control low cell density cultures to which ethanol was added at the same rate. This difference was shown to be not due to intracellular ethanol accumulation or to differences in glucose concentration between the cultures. The concentrations of a range of potentially toxic fatty acids, higher alcohols, and esters were measured during rapid fermentations, but when added at these concentrations to control cultures in the presence of ethanol they had no significant toxic effect. However, when rapid fermentations were conducted in rich medium containing 80 g L(-1) yeast extract, the apparent difference in toxicity of produced and added ethanol virtually disappeared. Magnesium was shown to be the component of yeast extract primarily responsible for this effect. The high rate of fall of viability when ethanol is rapidly produced is suggested to be partly due to the inability of the cells to adapt quickly enough to the rising ethanol concentration and partly to an increased demand for magnesium at higher ethanol concentrations which cannot be met in Mg-unsupplemented high cell density fermentations.     

Fermentation kinetics of Zymomonas mobilis near zero growth rate

The fermentation kinetics Zymomonas mobilis were studied near zero growth rate in fed-batch cultures and continuous cultures with complete cell recycle. The results show the ethanol enhances that specific substrate conversion rate under these conditions. The maximum achievable ethanol concentration in continuous cultures with cell recycle (66 g/L) was significantly lower than in fed-batch cultures (100 g/L). The results indicate that growth-rate-independent metabolism is not instantaneous and can lag behind steadily increasing ethanol concentrations in fed-batch fermentations. A model is proposed to account for this slow adaptation.     

Effect of various factors on ethanol yields from lignocellulosic biomass by Thermoanaerobacterium AK₁₇

The ethanol production capacity from sugars and lignocellulosic biomass hydrolysates (HL) by Thermoanaerobacterium strain AK(17) was studied in batch cultures. The strain converts various carbohydrates to, acetate, ethanol, hydrogen, and carbon dioxide. Ethanol yields on glucose and xylose were 1.5 and 1.1 mol/mol sugars, respectively. Increased initial glucose concentration inhibited glucose degradation and end product formation leveled off at 30 mM concentrations. Ethanol production from 5 g L(-1) of complex biomass HL (grass, hemp, wheat straw, newspaper, and cellulose) (Whatman paper) pretreated with acid (0.50% H(2) SO(4)), base (0.50% NaOH), and without acid/base (control) and the enzymes Celluclast and Novozyme 188 (0.1 mL g(-1) dw; 70 and 25 U g(-1) of Celluclast and Novozyme 188, respectively) was investigated. Highest ethanol yields (43.0 mM) were obtained on cellulose but lowest on hemp leafs (3.6 mM). Chemical pretreatment increased ethanol yields substantially from lignocellulosic biomass but not from cellulose. The influence of various factors (HL, enzyme, and acid/alkaline concentrations) on end-product formation from 5 g L(-1) of grass and cellulose was further studied to optimize ethanol production. Highest ethanol yields (5.5 and 8.6 mM ethanol g(-1) grass and cellulose, respectively) were obtained at very low HL concentrations (2.5 g L(-1)); with 0.25% acid/alkali (v/v) and 0.1 mL g(-1) enzyme concentrations. Inhibitory effects of furfural and hydroxymethylfurfural during glucose fermentation, revealed a total inhibition in end product formation from glucose at 4 and 6 g L(-1), respectively.     

Characterization of cellobiose fermentations to ethanol by yeasts

Twenty-two different yeasts were screened for their ability to ferment both glucose and cellobiose. The fermentation characteristics of Candida lusitaniae (NRRL Y-5394) and C. wickerhamii (NRRL Y-2563) were selected for further study because their initial rate of ethanol production from cellobiose was faster than the other test cultures. C. lusitaniae produced 44 g/L ethanol from 90 g/L cellobiose after 5-7 days. When higher carbohydrate concentrations were employed, fermentation ceased when the ethanol concentration reached 45-60 g/L. C. lusitaniae exhibited barely detectable levels of beta-glucosidase, even though the culture actively fermented cellobiose. C. wickerhamii produced ethanol from cellobiose at a rate equivalent to C. lusitaniae; however, once the ethanol concentration reached 20 g/L, fermentation ceased. Using p-nitrophenyl-beta-D-glucopyranoside (pNPG) as substrate, beta-glucosidase (3-5 U/mL) was detected when C. wickerhamii was grown anaerobically on glucose or cellobiose. About 35% of the beta-glucosidase activity was excreted into the medium. The cell-associated activity was highest against pNPG and salicin. Approximately 100-fold less activity was detected with cellobiose as substrate. When empolying these organisms in a simultaneous saccharification-fermentation of avicel, using Trichoderma reesei cellulase as the saccharifying agent, 10-30% more ethanol was produced by the two yeasts capable of fermenting cellobiose than by the control, Saccharomyces cerevisiae.     

Direct Effects of Chronic Ethanol Exposure on β-Adrenergic and Adenosine-Sensitive Adenylate Cyclase Activities and Cyclic AMP Content in Primary Cerebellar Cultures

The direct effects of chronic ethanol exposure on adenylate cyclase activity and cyclic AMP content were investigated in primary cerebellar cultures. By morphological criteria these cultures mainly contain granule cells with some astrocytes, and each cell type appears to contain both beta-adrenergic and adenosine-sensitive adenylate cyclase systems. Chronic treatment of the primary cerebellar cultures with 120 mM ethanol for 6 days caused a reduction in the stimulation of cyclic AMP content by isoproterenol and by the adenosine analogue 2-chloroadenosine. Kinetic analysis indicated that the chronic ethanol treatment decreased maximal activation of adenylate cyclase, as well as increased the EC50 values for norepinephrine and 2-chloroadenosine. Activation of norepinephrine-stimulated adenylate cyclase activity by in vitro ethanol was significantly enhanced after the chronic ethanol exposure. However, the chronic treatment did not alter activation of the 2-chloroadenosine-stimulated enzyme by in vitro ethanol. A similar difference in the response to in vitro ethanol after the chronic treatment was observed when cyclic AMP content of the intact cells was measured. The present data indicate that chronic ethanol exposure causes a selective increase in the sensitivity of adenylate cyclase to ethanol in some brain cells and a more generalized desensitization of receptor-stimulated cyclic AMP production.     

中药提取物对酵母菌抗真菌活性研究

目的探讨6味中药2种方法提取成分对酵母菌的抑菌和杀菌作用。方法采用药基琼脂稀释法,测定6味中药水提和醇提成分对白念珠菌和糠秕马拉色菌的MIC和MFC。结果对白念珠菌：水提黄连、醇提黄柏、醇提土槿皮MIC范围分别为0．625—1．25mg／mL、0．625～1．25mg/mL、0．313—0．625mg／mL;均值均为0．625mg／mL;对糠秕马拉色菌：水提和醇提黄连MIC范围分别为0．625～1．25mg／mL和1．25mg／mL,均值均为1．25mg／mL。对白念珠菌：醇提土槿皮MFC范围0．625～2．5mg/mL,均值0．625rag／mL。结论水提黄连、醇提黄柏和土槿皮对白念珠菌有较强抑菌作用,其中醇提土槿皮有较强杀菌作用。水提和醇提黄连对糠秕马拉色菌有较强抑菌作用。     

Effect of ethanol on activity of the plasma-membrane ATPase in, and accumulation of glycine by, Saccharomyces cerevisiae

The pH optimum of the ATPase activity in plasma membranes from Saccharomyces cerevisiae NCYC 431 from 8 h cultures was around 6.5 and that in membranes from organisms from 16 h cultures near 6.0. The Km[ATP] of the enzyme was virtually unaffected by the age of the culture from which organisms were harvested, although the Vmax of the enzyme in membranes from organisms from 8 h cultures was higher than that for organisms from 16 h cultures. Ethanol non-competitively inhibited ATPase activity in membranes, although the inhibition constant for the enzyme from organisms from 8 h cultures was lower than that from organisms from 16 h cultures. Glycine accumulation by the general amino acid permease was non-competitively inhibited by ethanol. Inhibition constants were virtually the same for glycine uptake by deenergized organisms from 8 h and 16 h cultures, but under energized conditions the value was greater for organisms from 16 h rather than 8 h cultures. The data indicate that inhibition of plasma-membrane ATPase activity by ethanol could account, at least in part, for inhibition of glycine accumulation by ethanol.     

Physiological characterisation of a pyruvate-carboxylase-negative Saccharomyces cerevisiae mutant in batch and chemostat cultures

A prototrophic pyruvate-carboxylase-negative (Pyc-) mutant was constructed by deleting the PYC1 and PYC2 genes in a CEN.PK strain of Saccharomyces cerevisiae. Its maximum specific growth rate on ethanol was identical to that of the isogenic wild type but it was unable to grow in batch cultures in glucose-ammonia media. Consistent with earlier reports, growth on glucose could be restored by supplying aspartate as a sole nitrogen source. Ethanol could not replace aspartate as a source of oxaloacetate in batch cultures. To investigate whether alleviation of glucose repression allowed expression of alternative pathways for oxaloacetate synthesis, the Pyc- strain and an isogenic wild-type strain were grown in aerobic carbon-limited chemostat cultures at a dilution rate of 0.10 h-1 on mixtures of glucose and ethanol. In such mixed-substrate chemostat cultures of the Pyc- strain, steady-state growth could only be obtained when ethanol contributed 30% or more of the substrate carbon in the feed. Attempts to further decrease the ethanol content of the feed invariably resulted in washout. In Pyc- as well as in wild-type cultures, levels of isocitrate lyase, malate synthase and phospho-enol-pyruvate carboxykinase in cell extracts decreased with a decreasing ethanol content in the feed. Nevertheless, at the lowest ethanol fraction that supported growth of the Pyc- mutant, activities of the glyoxylate cycle enzymes in cell extracts were still sufficient to meet the requirement for C4-compounds in biomass synthesis. This suggests that factors other than glucose repression of alternative routes for oxaloacetate synthesis prevent growth of Pyc-mutants on glucose.     

Involvement of a cell size control mechanism in the induction and maintenance of oscillations in continuous cultures of budding yeast

Spontaneous oscillations occur in glucose-limited continuous cultures of Saccharomyces cerevisiae under aerobic conditions. The oscillatory behavior is detectable as a periodic change of many bioparameters such as dissolved oxygen, ethanol production, biomass concentration, as well as cellular content of storage carbohydrates and is associated to a marked synchronization of the yeast population. These oscillations may be related to a periodic accumulation of ethanol produced by yeast in the culture medium.The addition of ethanol to oscillating yeast cultures supports this hypothesis: indeed, no effect was observed if ethanol was added when already present in the medium, while a marked phase oscillation shift was obtained when ethanol was added at any other time. Moreover, the addition of ethanol to a nonoscillating culture triggers new oscillations. An accurate analysis performed at the level of nonoscillating yeast populations perturbed by addition of ethanol showed that both the growth rate and the protein content required for cell division increased in the presence of mixed substrate (i.e., ethanol plus limiting glucose). A marked synchronization of the yeast population occurred when the added ethanol was exhausted and the culture resumed growth only on limiting glucose. A decrease of protein content required for cell division was also apparent. These experimental findings support a new model for spontaneous oscillations in yeast cultures in which the alternative growth on limiting glucose and limiting glucose plus ethanol modifies the critical protein content required for cell division.     

Pressure measurement to evaluate ethanol or lactic acid production during glucose fermentation by yeast or heterofermentative bacteria in pure and mixed culture

A rapid and simple technique to follow CO2 release during fermentation of glucose by heterofermentative bacteria or yeasts was used in order to evaluate ethanol and lactate production in pure and mixed cultures of yeast and bacteria. In pure cultures, good correlations were found between gas pressure variations (deltaP) and ethanol or lactate production by yeasts or heterofermentative bacteria, and ratios between deltaP and ethanol or lactate produced could be established. In mixed cultures, ratios between maximal deltaP and total amount of glucose consumed were determined. It was thus possible to evaluate the amount of glucose that was consumed by each strain and then deduce the bacterial lactate production. Good results were obtained for mixed cultures of yeast and homofermentative bacteria. This technique may be useful to evaluate the activity of strains in mixed cultures of yeast and lactic acid bacteria.     

The effects of ethanol on cellular calcium content in primary myocardial cell cultures from offspring of sedentary and swim-trained pregnant rats

Primary myocardial cell cultures obtained from offspring of swim-trained (T) and sedentary (S) Sprague-Dawley rat mothers (dams) were used to evaluate the influence of exercise training and ethanol on cellular calcium (45Ca) content. The pregnant rats swam in water maintained at 37 degrees C 6 days/week during gestation. The dams swam continuously for 30 min on the first day of gestation. The swimming time was increased by 5 min until the rats swam continuously for 1.5 hr. After the cultures had been in incubation for 4 days, the cells were treated with 600, 800, and 1000 mg% ethanol for 30 min and 1 hr. 45Ca content (nmol/mg protein) of the untreated controls and all but one of the ethanol treated groups from the T cultures (1000 mg%) were significantly elevated over the comparable groups from the S cultures for both 30 min and 1 hr of incubation (p less than or equal to 0.05). The data suggest that exercise during pregnancy induces adaptations in myocardial cell cultures from the offspring such that 45Ca content levels are elevated which may provide protection against ethanol toxicity.     

Differential alterations in the expression of NMDA receptor subunits following chronic ethanol treatment in primary cultures of rat cortical and hippocampal neurones

In our previous experiments, severe cellular damages and neuronal cell loss were observed following 24h of alcohol withdrawal in primary cultures of rat cortical neurones pre-treated with ethanol (50-200 mM) repeatedly for 3 days. Increased NMDA induced cytosolic calcium responses and excitotoxicity were also demonstrated in the ethanol pre-treated cultures. Thus, the enhancement in functions of NMDA receptors was supposed to be involved in the adaptive changes leading to the neurotoxic effect of alcohol-withdrawal. In this study, we investigated the effect of the 3-day repeated ethanol (100 mM) treatment on the function and subunit composition of the NMDA receptors. Here, we demonstrate that the maximal inhibitory effect of ethanol was significantly increased after ethanol pre-treatment. Similarly, the inhibitory activity of the NR2B subunit selective antagonists threo-ifenprodil, CP-101,606 and CI-1041 was also enhanced. On the contrary, the efficiency of the channel blocker agent MK-801 and the glycine-site selective antagonist 5,7-dichlorokynurenic acid was the same as in control cultures. According to these observations, a shift in subunit expression in favour for the NR2B subunit was suggested. Indeed, we provided evidence for increased expression of the NR2B and the C1 and C2' cassette containing splice variant forms of the NR1 subunit proteins in ethanol pre-treated cultures in further experiments using a flow cytometry based immunocytochemical method. These changes may constitute the basis of the increased NMDA receptor functions and subsequently the enhanced sensitivity of ethanol pre-treated cortical neurones to excitotoxic insults resulting in increased neuronal cell loss after ethanol withdrawal. Such alterations may play a role in the neuronal adaptation to ethanol as well as in the development of alcohol dependence, and might cause neuronal cell loss in certain areas of the brain during alcohol withdrawal.