Inactivation of polygalacturonase during growth ofSaccharomyces pastorianus in culture

The effect of exogenous glucose addition on polygalacturonase (EC 3.2.1.15) activity in the culture medium ofSaccharomyces pastorianus was studied. A rapid but transient decrease in the enzyme activity was observed after 9–12 h after adding glucose to the culture medium. This effect was not associated with protein degradation or modification of the spectrum of secreted proteins. Ethyl acetate appeared in the culture medium durine this period.     

In vitro analysis of the role of glucose oxidase from Talaromyces flavus in biocontrol of the plant pathogen Verticillium dahliae.

Culture filtrates from Talaromyces flavus grown on glucose contained high levels of glucose oxidase activity, while culture filtrates from T. flavus grown on xylan contained negligible glucose oxidase activity. Culture filtrates from T-flavus grown on both media contained complex protein profiles. However, only culture filtrates from T. flavus grown on glucose inhibited germination of microsclerotia of Verticillium dahliae in in vitro inhibition assays. A polyclonal antiserum preparation, pABGO-1, raised against purified glucose oxidase from T. flavus was highly specific for glucose oxidase. Only one protein band in culture filtrates (from glucose medium), migrating at 71 kDa, was detected in Western blots (immunoblots) with this antiserum. This band comigrated with purified glucose oxidase. No bands were detected in culture filtrates from the xylan medium. Glucose oxidase was removed via immunoprecipitation from culture filtrates of T. flavus grown in glucose medium, resulting in filtrates which no longer inhibited in vitro microsclerotial germination. When glucose oxidase-depleted filtrates were amended with purified glucose oxidase from T. flavus, the ability to kill microsclerotia in vitro was restored to original levels. We conclude that glucose oxidase is the only protein in culture filtrates of T. flavus responsible for inhibition of germination of microsclerotia of V. dahliae.     

Preservation of the effects of pregnancy on rat islets of Langerhans in tissue culture.

Islets of Langerhans, isolated from normal or 19-day pregnant rats, were cultured for 20 h at 37 degrees C in tissue culture medium 199. When islets were cultured in medium containing low glucose (5.5 mM), the higher adenylate cyclase activity and insulin secretory responses characteristic of islets from pregnant rats were maintained during the test period of 29 h. Islets from normal and pregnant rats were also cultured for 20 h in medium containing a very high glucose concentration (83.3 mM) in order to load the B cells with glycogen. It was found, after glycogen loading, that, while adenylate cyclase activity increased to a greater extent in islets from pregnant rats than controls, this activity was not increased in proportion to the striking changes in insulin release rate observed in pregnant rat islets. The results show that the difference in insulin secretory response between islets from normal and pregnant rats may be preserved when the islets are cultured for 20 h, and that these differences are enhanced for a variety of reasons after culture of islets in 83.3 mM glucose.     

Influence of cytokinins on growth of Phycomyces blakesleeanus and on the activities of the glyoxylate cycle enzymes isocitrate lyase and malate synthase

Treatment of the 1 + strain of Phycomyces blakesleeanus Bgff. with various cytokinins resulted in a stimulation of growth. The magnitude of growth stimulation depended on both the structure of the hormone used and the carbon source in the culture medium. Most of the cytokinin derivatives were active effect in glucose and oleic acid cultures. Benzyladenine (BA) and benzyladenosine stimulated the fungal growth only when oleic acid was the sole carbon source, while they had no effect in glucose cultures within the tested range of concentrations. [¹⁴C]-BA was accumulated by the mycelium of oleic acid cultures. Therefore, differences in BA uptake between glucose and oleic acid cultures could account mainly for the specific growth-promoting effect of BA. In oleic acid cultures isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) activities were enhanced by 40 and 34%, respectively, in the presence of BA. A time course of the hormone effect suggests that BA is not involved in induction, but in the regulation of the mentioned enzymes in Phycocmyces. In contrast, acetate when presented as the sole carbon source or after addition to a glucose culture medium, induced isocitrate lyase activity. This enzyme induction was prevented by simultaneous addition of cycloheximide.     

Enhancement of Sf-9 cell growth and longevity through supplementation of culture medium with hemolymph

The benefits of insect cell culture medium supplementation with hemolymph of Lonomia obliqua were investigated. The addition of hemolymph to the medium induced high levels of cell growth, and the viability was maintained for longer periods. The maximum cell yield increased almost 3-fold after hemolymph supplementation. Cultures in their stationary phase were rescued through hemolymph supplementation, also reaching high cell concentrations. These actions were much dependent on the concentration of hemolymph; low hemolymph concentration had a positive effect in cell growth, whereas high hemolymph concentration showed a deleterious effect. Fractionation of hemolymph by gel filtration chromatography showed the presence of three factors with different activity in insect cell culture: an potential anti-apoptotic factor, a growth-promoting factor, and an enzyme that hydrolyzes sucrose. Addition of hemolymph to the medium induced high levels of glucose production. The sucrose to glucose conversion was also linearly dependent upon the hemolymph concentration. Therefore, we conclude that cell growth and longevity can be increased by supplementation of the culture medium with hemolymph.     

Decreasing glycolysis increases sensitivity to mitochondrial inhibition in primary cultures of renal proximal tubule cells

Summary We have previously shown that shaking the culture plates (SHAKE) of rabbit renal proximal tubule cells (RPTC) to maintain adequate aeration increased aerobic metabolism and decreased the induction of glycolysis compared to RPTC cultured under standard conditions (STILL). However, glycolysis in SHAKE RPTC remained elevated compared to glycolysis in proximal tubules in vivo. In the present study the contribution of culture medium sugar composition and concentration to glycolytic metabolism was assessed in RPTC. SHAKE and STILL RPTC cultured in 5 mM glucose contained lactate levels equivalent to the respective SHAKE and STILL RPTC cultured in standard culture medium which contains 17.5 mM glucose. Similarly, the activity of lactate dehydrogenase was unchanged by lowering the medium glucose concentration. Substituting 5 mM galactose for 5 mM glucose in the culture medium significantly reduced the lactate content of both SHAKE and STILL RPTC but had no effect on lactate dehydrogenase activity. Cell growth was equivalent under all culture conditions. Sensitivity to mitochondrial inhibition was determined for each culture condition by measuring cell death after exposure to the respiratory inhibitor antimycin A. The results showed a hierarchy of sensitivity to antimycin A (5 mM galactose SHAKE >5 mM glucose SHAKE >17.5 mM glucose SHAKE = 17.5 mM glucose STILL), which was generally inversely correlated with the level of glycolysis as measured by lactate content (17.5 mM glucose STILL >17.5 mM glucose SHAKE = 5 mM glucose SHAKE >5 mM galactose SHAKE).     

Unmasking of the inhibitory action of glucose on insulin release after alpha 2-adrenergic activation

Insulin release in response to glucose was measured after culture of islets from ob/ob-mice in a Ca2+-deficient medium. The stimulatory effect of 20 mM glucose disappeared after addition of 1 microM L-epinephrine, and it was reversed into inhibition when the medium contained 0.1 to 10 microM clonidine. Glucose inhibited insulin release also after activation of the alpha 2-adrenoceptors with B-HT 933, whereas blocking of these receptors with idazoxan removed glucose inhibition in the presence of clonidine. It is concluded that alpha 2-adrenergic activation provides an efficient means of unmasking the inhibitory component in the action of glucose on insulin release.     

Effect of the culture medium and incubation time on auxins production by bacteria isolated from the roots of pine seedlings (Pinus silvestris l.).

Studies were performed on the effect of culture medium and incubation time on the production of auxins by bacteria. The bacteria studied produced more auxins in the mineral medium containing glucose and tryptophan than in that enriched with casamino acids and yeast extract. The amount of auxins elaborated depended both upon the strain and the age of the culture. Some strains produced the largest amounts of these substances after 7 days of incubation while others required a longer period. Most of the substances showing auxin activity were located on the chromatograms at Rf 0.3-0.4 and 0.8-1.0.     

The role of aeration and agitation in the production of glucose oxidase in submerged culture

The influence of agitation and aeration on growth and on production of glucose oxidase of Asp. niger has been studied. It was found that both rate of growth and glucose oxidase production was higher at an agitation speed of 700 rpm than at 460 rpm. Further increase in speed of agitation resulted in neither a higher rate of growth nor a higher glucose oxidase activity. Total glucose oxidase activity was highest in a medium containing 5% sugar (at an agitation speed of 700 rpm) and did not get higher when the sugar concentration of the medium was increased to 7%. When pure oxygen was bubbled through the culture the rate of growth of the culture (in the linear phase) was 95 mg. mycelial dry wt./100 ml./hr., and only 61 mg. when air was applied. The glucose oxidase activity of oxygenated culture was double the activity of aerated culture. Viscosity of the homogenized culture became higher with higher concentration of mycelia. The viscosity of oxygenated culture was found to be lower than that of aerated culture.     

Requirement of carbohydrates for cell fusion induced by vesicular stomatitis virus

The fusion of BHK-21-KB cells by vesicular stomatitis virus was not induced in Eagle's minimal essential medium without glucose. In medium containing glucose, the rate of polykaryocyte formation decreased as the concentration of glucose was reduced below 5 mM. However, no reduction in virus production 24 hr after infection was seen under this condition. Addition of pyruvate or mannose to the culture medium caused a reversal of cell fusing activity. Cell fusion and virus growth were significantly suppressed by sodium azide and 2,4-dinitrophenol.     

The effect of glucosamine concentration on the development and sex ratio of bovine embryos

Glucosamine is a component of hyaluronic acid and an alternative substrate to glucose for the extracellular matrix synthesis of COCs. Its addition to an IVM medium reduces the glucose consumption of bovine COCs. Glucosamine is also metabolized to UDP-N-acetyl glucosamine (UDP-GlcNAc) via the hexosamine biosynthesis pathway and is utilized for O-linked glycosylation by the X-linked enzyme, O-linked GlcNAc transferase (OGT). Moreover, the inactivation of the second X chromosome in female embryos is influential in producing the sex ratio bias observed in vitro when embryos are cultured in the presence of glucose above 2.5mM. Accordingly, the aim of this study is to examine whether the presence of glucosamine during maturation or embryo culture causes a sex ratio bias in bovine blastocysts. Glucosamine was added to the medium in three different embryo developmental periods: in vitro maturation, the one-cell to eight-cell stage (before the maternal-zygotic transition, MZT), and the eight-cell to blastocyst stage (after MZT). When glucosamine was added during in vitro maturation, the developmental competence of oocytes was severely compromised. However, the sex ratio of embryos was not influenced. When glucosamine was added to embryo culture medium during development from one-cell to eight-cell stage (before MZT), it affected neither the development nor the sex ratio of bovine embryos. Finally, when glucosamine was added after MZT, the development rate of embryos was severely decreased, and the sex ratio was skewed toward males. Moreover, an inhibitor of OGT, benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (BADGP), negated the effect of glucosamine on the sex ratio when it was added to embryo culture medium from the eight-cell to blastocyst stage (after MZT). These results suggest that, like glucose, the supplementation of glucosamine into the medium skewed the sex ratio to males and that OGT, an X-linked enzyme, was involved in this phenomenon. Moreover, this effect of glucosamine was limited only to when it was present in the embryo culture medium after MZT.     

Accumulation of Polyphosphates and Expression of High Molecular Weight Exopolyphosphatase in the Yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The effect of cultivation time and concentration of inorganic phosphate (P(i)) in the culture medium on the accumulation of polyphosphates (polyP) and the activity of two cytosolic exopolyphosphatases of the yeast Saccharomyces cerevisiae was studied: an exopolyphosphatase of 40 kD encoded by PPX1 and a high molecular weight exopolyphosphatase encoded by another gene. Depletion of polyP in the cells on P(i) starvation is a signal factor for the accumulation of polyP after the subsequent addition of 5-20 mM P(i) and glucose to the cells or spheroplasts. A high activity of both exopolyphosphatases does not prevent the accumulation of polyP. The expression of the high molecular weight exopolyphosphatase is due to the acceleration of metabolism in cells that have reached the stage of growth deceleration on the addition of P(i) and glucose or complete culture medium. This process may occur independently from the accumulation of polyP. The activity of exopolyphosphatase PPX1 depends less on the mentioned factors, decreasing 10-fold only under conditions of phosphate surplus at the stationary growth stage.     

The Utilization of Glucose and Proline by Culture Forms of Trypanosoma brucei*

Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2-deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose-containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline-containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.     

Effects of Rapeseed Oil on Activity of Methylmalonyl-CoA Carboxyltransferase in Culture of Streptomyces fradiae

To investigate why more tylosin was produced when Streptomyces fradiae T1558 was cultured in a rapeseed oil medium than in a glucose or starch medium, we measured the activity of methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) and intracellular propionic acid. The activity of the enzyme, which catalyzes the formation of the precursor of tylosin, protylonolide, was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which was 2.5- and 1.3-fold that with the glucose or starch medium, respectively. The intracellular propionic acid concentration was 1.2 g/g of dry weight, which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively. The addition of propionic acid increased tylosin production in batch culture: when 0.2 g/l (final concentration) propionic acid was added to the glucose medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the amount without propionic acid. These findings suggest that in glucose medium, intracellular propionic acid is a limiting factor because of the low activity of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.     

Glucose deprivation induces Akt-dependent synthesis and incorporation of GLUT1, but not of GLUT4, into the plasma membrane of 3T3-L1 adipocytes

Reduction of the glucose concentration in the culture medium of 3T3-L1 adipose cells below 1.25 mM produces a 4-8-fold stimulation of 2-deoxyglucose uptake which starts after a lag phase of 2 h and is maximal after 10-16 h. In the present study, we employed the 'membrane sheet assay' in order to re-assess the contribution of the transporter isoforms GLUT1 and GLUT4 to this effect. Immunochemical assay of glucose transporters in membranes prepared with the 'sheet assay' revealed that the effect reflected a marked increase of GLUT1 in the plasma membrane with no effect on GLUT4. Glucose deprivation increased the total cellular GLUT1 protein in parallel with the transport activity, whereas GLUT4 was unaltered. The specific PI 3-kinase inhibitor wortmannin inhibited the effect of glucose deprivation on transport activity and also on GLUT1 synthesis. Glucose deprivation produced a moderate, biphasic increase in the activity of the protein kinase Akt/PKB that was inhibitable by wortmannin. When wortmannin was added after stimulation of cells in order to assess the internalization rate of transporters, the effect of insulin was reversed considerably faster (T1/2 = 18 min) than that of glucose deprivation (T1/2 > 60 min). These data are consistent with the conclusion that the effect of glucose deprivation reflects a specific, Akt-dependent de-novo synthesis of GLUT1, and not of GLUT4, and its insertion into a plasma membrane compartment which is distinct from that of the insulin-sensitive GLUT1.     

Factors affecting the accumulation of exocellularexo-β-d-galactofuranosidase and other enzymes fromPenicillium charlesii

exo-β-d-galactofuranosidase and β-d-glucosidase first appeared in culture filtrates of Day 14 cultures ofPenicillium charlesii and after the medium was depleted of glucose. Acid phosphatase, ribonuclease, and protease activities appeared about 7 days earlier. The appearance of the glycohydrolases also occurred in Day 7 culture filtrates if the growth medium of Day 3 cultures was titrated to 4 with alkali. This treatment of the growth medium did not change the initial appearance of acid phosphatase, ribonuclease, or protease activities. Addition of protease inhibitors to the modified growth medium further enhanced galactofuranosidase activity and the maximum activity attained was severalfold greater than that obtained in untreated cultures. These data suggest that galactofuranosidase activity is destroyed by acid protease(s) and that galactofuranosidase activity appears in the medium only after it is depleted of glucose and sufficient organic acid has been taken up to increase the pH of the medium above 4. A possible role of organic acids in regulating the onset of release of glycohydrolases into the medium is discussed.     

Some Properties of Unpurified Chitinase from the Crayfish Plague Fungus, Aphanomyces astaci

The culture filtrate of the crayfish plague fungus, Aphanomyces astaci (Saprolegniaceae), incubated in a peptone glucose medium was tested for chitinase activity under different conditions. The activities were assayed turbidimetrically using low-polymerized chitin as a substrate. Adsorption of chitinase was found to occur on chitin and probably on cellulose and sulphomethyl cellulose but not at all or only a little on some other cellulose derivatives. The pH optimum of the enzyme activity was found to lie at about pll 5.0–5.5. The stability was greatest near pH 6.5 and the highest degree of adsorption occurred at still higher pH values. Enzyme adsorption on the substrate seemed to protect the enzyme against inactivation by heating, shaking, and extreme pH-conditions. The chitinase activity was positively affected by the rest of the culture filtrate. Mercury, cobalt, and copper chlorides, and to a lesser degree some other metal salts, lowered the enzyme activity when present in the test medium. Cellobiose, but neither glucose nor N-acetyl glucosamine had a pronounced inhibiting effect on the activity. Neither cellobiose nor N-acetyl glucosamine seemed to affect chitinase adsorption on chitin. Some chelating and reducing compounds inactivated the culture filtrate. This activity-reducing effect of chelators was strongly prevented by EDTA in some cases.     

效应面法优化CHO细胞培养基中几种主要成分的含量

目的:考察培养基中葡萄糖、谷氨酰胺、血清、碳酸氢钠含量对CHO细胞生长繁殖的影响。方法:在CHO细胞培养基中添加不同成分的葡萄糖、谷氨酰胺、血清、碳酸氢钠,通过单因素实验结果结合Box-Behnken效应面法,根据二次回归模型的分析结果,以细胞表达蛋白体外活性为指标进行实验,考察培养基中葡萄糖、谷氨酰胺、血清、碳酸氢钠含量对细胞生长繁殖的影响。结果:根据回归方程分析结果,作出相应的曲面图和等高线图,优选出培养基中各组分的最佳配比为:葡萄糖2.54 g/L、谷氨酰胺0.59 g/L、血清8.3%,碳酸氢钠2.96 g/L。结论:Box-Behnken实验设计法用于细胞培养过程中考察培养基中各组分的优选是可行的,数学模型的预测值与实验观察值相符。通过对CHO细胞培养基成分的优化,使CHO细胞蛋白表达量高,有利于提高产品质量和降低生产成本。     

Enzyme activity of the formate hydrogenlyase complex in Citrobacter freundii

Citrobacter freundii 62 can grow in the absence of oxygen in media containing glucose, peptone, fumarate or malate. When the medium contained fumarate or malate, the culture could grow under anaerobic conditions only in the presence of molecular hydrogen, formate or nitrate. The highest activity of formatehydrogenlyase and hydrogenase was found when C. freundii grew in a medium with glucose and formate. The activity was lower in media with other organic substrates, particularly, in the absence of formate or H2. The activity of hydrogenase was very low in cells grown under aerobic conditions or in the presence of nitrates while the activity of formatehydrogenlyase was not found at all for all practical purposes. The activity of formate dehydrogenase assessed in the presence of methylene blue was rather high irrespective of the conditions under which the culture was grown. However, when the activity of formate dehydrogenase was determined in the presence of benzyl viologen, it was high only in cells grown in the medium with glucose and formate.