Transport of L-alanine in cultured human fibroblasts: Evidence for two kinetically distinguishable systems

The transport of L-alanine in human diploid fibroblasts was investigated. Transport measurements were performed on subcultures between the third and eighth passages with subconfluent cells growing on glass coverslips. Kinetic analysis of approximate initial rates of transport at substrate concentrations from 0.05 to 10 mmole/liter indicate the presence of two distinguishable systems. The high affinity system has a K_m of 0.24 mmole/liter and a V_max of 6.4 nmole/100 μg protein/2 min. For the low affinity system, the contribution of the high affinity system to the uptake must absolutely be taken into account. The K_m and V_max values, obtained by using a computer program, are a K_m of 15.0 mmole/liter and a V_max of 14.7 nmole/100 μg protein/2 min. For alanine concentrations below 1 mmole/liter, the contribution of the Na⁺-independent uptake is less than 10%, and the kinetic constants of the high affinity system are in the same range if this contribution is taken into account. On the contrary the influence of a diffusion-like process is more significant on the low affinity system whose K_m is about 49 mmole/liter after subtraction of the Na⁺-independent uptake from the experimental velocities. Inhibition studies were performed with NCH₃-alanine. They permitted us first to confirm the existence of system A in cultured human fibroblasts in agreement with two recent works and second to show how this system contributes to L-alanine uptake. This contribution seems very small in low concentrations but it rises as the concentrations increase.     

Functional differences in the catabolism of branched-chain L-amino acids in cultured normal and maple syrup urine disease fibroblasts

Possible functional differences in the catabolism of the four branched-chain L-amino acids in maple syrup urine disease were assessed using cultured human skin fibroblast stains. Transamination and oxidative decarboxylation were comparatively studied in 90-min incubations with 1 mmole/liter of 1-14C-labeled substrates. In normal cell strains (n = 5), apparent transamination rates (sum of branched-chain 2-oxo[14C]acid and 14CO2 release; means expressed in nmole/90 min/mg of cell protein) were in the order L-leucine (32) greater than L-valine (17) greater than or equal to L-isoleucine (14) greater than L-allo-isoleucine (8); 14CO2 production was in the order L-valine (9) greater than L-isoleucine (6) greater than or equal to L-leucine (5) greater than L-allo-isoleucine (2). In variant (n = 5) as well as classical (n = 2) MSUD cell lines, branched-chain 2-oxo-[14C]acid release rates were generally comparable to the control values. As compared to the 14CO2 release in controls (= 100%), branched-chain 2-oxo acid dehydrogenase activity in MSUD fibroblasts was individually reduced and varied considerably between strains (residual activity 2-38%). Within individual strains, only small differences in the residual decarboxylation activity were observed in incubations with L-valine, L-leucine, and L-isoleucine. It was remarkably high, however, when L-allo-isoleucine was applied as a substrate. With the exception of L-allo-isoleucine, apparent total transamination rates of branched-chain L-amino acids were therefore distinctly lower in MSUD cells than in normal cells.     

Detection of heterozygotes in maple-syrup-urine disease: measurements of branched-chain alpha-ketoacid dehydrogenase and its components in cell cultures.

To detect heterozygotes for maple-syrup-urine disease (MSUD), activities of branched-chain-alpha-ketoacid (BCKA) dehydrogenase and its components in skin fibroblasts of two obligatory heterozygotes and amnion cells of a fetus at risk were measured. Intact heterozygous cells were found to decarboxylate [1-14C] alpha-ketoisovalerate at rates equal to or only slightly lower than normal subjects. The inability to differentiate heterozygotes from normals with the intact cell assay confirms earlier studies with intact leukocytes using [1-14C]leucine as substrate. By contrast, measurements of BCKA dehydrogenase activity with disrupted cell suspensions showed MSUD heterozygotes with 30%--60% of normal activity. Moreover, biphasic kinetics in heterozygous cells were observed with increasing substrate concentrations. The altered biphasic kinetics probably reflect expression of the normal allele in the early hyperbolic portion of the curve of the mutant allele in the later secondary rise at high substrate concentrations. Assays of component activities showed concordant E1 decarboxylase deficiency in both heterozygous- and homozygous-affected cells, whereas the E3, dihydrolipoyl dehydrogenase-component, activity was normal. The above results taken together appear to provide an approach to detection of the heterozygote in MSUD.     

Maple syrup urine disease: branched-chain keto acid decarboxylation in fibroblasts as measured with amino acids and keto acids.

Branched-chain keto acid decarboxylase activity in skin fibroblasts from control subjects and from patients with classical and variant forms of maple syrup urine disease (MSUD) was measured with leucine and alpha-ketoisocaproic acid. When the keto acid was used as substrate in high concentrations (more than 5 mM), the three groups overlapped extensively, even classical cases of MSUD exhibiting decarboxylase activity. With leucine as substrate, decarboxylase activity plateaued at about 1.5 mM, and the three groups could be clearly differentiated. Classical cases of MSUD had minimal or no decarboxylase activity.     

Maple syrup urine disease: Branched-chain amino acid concentrations and metabolism in cultured human lymphoblasts

The intracellular concentration of free leucine, isoleucine, and valine and their metabolism were studied in lymphoblast cultures established from peripheral blood of an individual with maple syrup urine disease (MSUD) and a control subject. Branched-chain -keto acid decarboxylase activity in the MSUD cells was 10% or less of the control value as measured by the ability of the cells to release ¹⁴CO₂ from the corresponding [1-¹⁴C]labeled branched-chain amino acid. The intracellular concentrations of free leucine and isoleucine were increased three-fold in MSUD lymphoblasts as compared to control cells. Free valine was present in only trace amounts of less than 0.1 mMin both cell lines. Exposure of normal and mutant cells to a 10 mMload of leucine, isoleucine, and valine resulted in a comparable concentration within cells after 24 hr. Concentrations returned to base values in normal cells 12 hr after removal of load, but leucine remained elevated in MSUD cells after 3 days. Leucine and its keto acid, -ketoisocaproic acid, added to the culture medium gave significant growth inhibition of MSUD lymphoblasts but not of normal cells, in the millimolar range. Isoleucine, valine, and their keto acids had no effect.This investigation was supported in part by Grants AM-13622, AM-05646, and GM-17702 from the United States Public Health Service, Veterans Administration Grant M.R.I.S. No. 3181 to Dr. Nathan Gochman, and grants from the National Foundation and the Kroc Foundation. S. D. S. is a Postdoctoral Research Fellow supported by United States Public Health Service Training Grant AM-05646.     

Capillaries phosphorylate glucose in a concentration-dependent manner through a glucokinase-like enzyme: a study in the eel

Glucose phosphorylation was studied in a pure capillary preparation obtained from the rete mirabile of the eel swimbladder. In the 3000g supernatant of capillary homogenates, the glucose phosphorylating activity did not reach the maximum at low glucose concentration (1 mmole/liter), as it occurs in most tissues, but increased with the increase in glucose concentration and approached the maximum at very high (300 mmole/liter) glucose levels, with values (mean +/- SEM, n = 10) of 5.85 +/- 0.94 nmole.min-1.mg-1 protein and 19.97 +/- 1.89 at 1 and 300 mmole/liter glucose, respectively. The apparent Km value for glucose was about 50 mmole/liter, i.e., at supraphysiological glucose concentration, like the enzyme glucokinase, typically present in the liver but absent from most other tissues. This new enzyme did not phosphorylate fructose (similar to glucokinase from liver, which is rather specific for glucose) but was not inhibited by N-acetyl-glucosamine (in contrast to hepatic glucokinase). Thus, capillaries phosphorylate glucose in a concentration-dependent manner, which suggests that they are equipped with a glucokinase-like enzyme. This may explain the reported increase in glucose uptake during capillary exposure to high glucose concentrations and would suggest that the hyperglycemia of the diabetic state may be associated with increased glucose utilization, which may play a role in the development of microangiopathy.     

Trypanosoma brucei: trypanocidal effect of salicylhydroxamic acid plus glycerol in infected rats.

Rats inoculated with Trypanosoma brucei brucei EATRO 427 and having a high degree of parasitemia were treated with a series of intra-peritoneal injections of Salicylhydroxamic acid (SHAM) plus glycerol. Permanent cures were obtained with 380 mg/kg SHAM plus 3.8 g/kg glycerol, a dosage regime which was just sublethal. Using a regime with which permanent cure was obtained, the SHAM concentration in the blood plasma remained above 2 mmole/liter for about 20 min, while the glycerol concentration remained above 22 mmole/liter for about 1 hr. The brain concentration of SHAM was close to the plasma concentration. The concentration of glycerol in the brain remained far below the plasma concentration, reaching 6 to 8 mmole/liter between 1 and 2 hr after the beginning of treatment. Treatment with glycerol did not affect the mobility of the trypanosomes nor the survival of infected rats after treatment with suramin.     

Activities of branched-chain 2-oxo acid dehydrogenase and its components in skin fibroblasts from normal and classical-maple-syrup-urine-disease subjects.

1. Comparisons of the activity and kinetics of the branched-chain 2-oxo acid dehydrogenase in cultured skin fibroblasts from normal and classical maple-syrup-urine-disease (MSUD) subjects provide a kinetic explanation for the enzyme defect. 2. In the intact cell assays, normal fibroblasts demonstrated hyperbolic kinetics with 3-methyl-2-oxo[1-14C]butyrate as a substrate. Intact fibroblasts from four classical MSUD patients showed no decarboxylation over a substrate concentration range of 0.25 to 5.0 mM, and thiamin (4 mM) was without effect. 3. The overall reaction of the multienzyme complex was efficiently reconstituted by using a disrupted-cell system. Normals again showed typical hyperbolic kinetics at the 2-oxo acid concentrations of 0.1 to 5 mM. The Vmax. and apparent Km values were 0.10 +/- 0.02 m-unit/mg of protein and 0.05-0.1 mM respectively, with 3-methyl-2-oxobutyrate. In contrast, classical MSUD patients exhibited sigmoidal kinetics (Hill coefficient, 2.5) with activity approaching 40-60% of the normal value at 5 mM substrate. The K0.5 values from the Hill plots for MSUD patients were 4-7 mM. 4. The E1 (branched-chain 2-oxo acid decarboxylase) component of the multienzyme complex was measured in disrupted-particulate preparations. Normals again showed hyperbolic kinetics with the 2-oxo acid, whereas MSUD preparations exhibited sigmoidal kinetics with the activity of E1 strictly dependent on substrate concentration. Apparent Km or K0.5 were 0.1 and 1.0 mM for normal and MSUD subjects respectively. 5. Measurements of E2 (dihydrolipoyl transacylase) and E3 (dihydrolipoyl dehydrogenase) in MSUD preparations showed them to be in the normal range. 6. The above data suggest a defect in the E1 step of branched-chain 2-oxo acid dehydrogenase in classical MSUD patients.     

Effects of Related Anionic Detergents on Flagellation, Motility, Swarming, and Growth of Proteus

The effects of a series of sodium alkyl sulfates (C(4) to C(16)) on flagellation, motility, swarming, and growth of Proteus were examined. The concentrations of the various sodium alkyl sulfates completely inhibiting the swarming phenomenon (on solid medium) and motility (in liquid medium) were in the same order of magnitude. The inhibiting effect of the detergents examined increased from sodium hexyl sulfate (inhibitory concentration, 20 to 30 mmoles per liter) to sodium tetradecyl sulfate (inhibitory concentration, 0.1 to 0.5 mmoles per liter). Flagella were produced neither in liquid nor on solid medium at these concentrations as could be observed by electron microscopy. At concentrations where motility was not impaired, intact flagellation could be observed. At a concentration of 0.1 mmole per liter, sodium tetradecyl sulfate completely inhibited the motility of Proteus in the liquid medium employed without impairing growth.     

Induction of auxin-nonrequiring tobacco calluses and its reversal by treatments with auxins

Auxin-nonrequiring calluses were induced with high frequenciesby short treatments with auxins from auxin-requring ones. Auxin-requiringcalluses T22 and XD6S2, subcultured on a medium containing 1mg/liter IAA as plant growth regulator, required quite differenttreatment for induction from auxin-nonrequiring calluses; fromcalluses T22 and XD6S2, auxin-nonrequiring calluses were inducedby treatments with low concentrations of auxins (0.01–0.1mg/liter of IAA or NAA) and high concentrations of syntheticauxins (10–100 mg/liter of NAA or 1–10 mg/literof 2,4-D), respectively. No auxin-nonrequiring calluses wereobtained when they were transferred directly to the basal medium(hormone-free medium). The transformation of auxin-requiring into auxin-nonrequiringcalluses was fully reversible by treatment with 1 mg/liter ofIAA at an early stage of subculturing on the basal medium, butnot after prologed subculturing. ¹Part XXI in the series, ‘Studies on Plant Tissue Cultures’.Part XX, see Proceedings of the IVth International FermentationSymposium: Ferment. Technol. Today 697 (1972). (Received May 29, 1973; )     

High and low dietary potassium effects on rat vascular sodium pump activity

Studies of renal and other tissues suggest that chronic elevation or reduction of dietary potassium intake could affect vascular smooth muscle sodium pump (Na-pump) activity. To examine this possibility, the effects of 3 weeks of low (LK: 4 mmole KCl/kg chow), normal (NK; 162 mmole/kg), and high (HK; 1350 mmole/kg) dietary potassium intake on Na-pump activity, the Na-pump activity response to changes in extracellular potassium concentration, and Na-pump site density were determined in tail arteries of rats. Plasma potassium concentration was elevated by 21% in HK rats and reduced by 45% in LK rats. When incubated in autologous plasma, compared to arteries from NK rats, Na-pump activity was decreased in the tail arteries from LK rats but not altered in those from HK rats. When arteries from NK and LK rats were incubated in autologous plasma with the potassium concentration increased to equal that of the HK rats, Na-pump activity exceeded that of HK rat arteries: Na-pump activity of arteries incubated in autologous plasma did not differ from that of arteries incubated in Krebs-Henseleit buffer with the potassium concentration adjusted to equal that of the plasma. Tail artery Na-pump activity for all three dietary potassium groups increased as potassium concentration of the incubation medium was increased from 1 to 12 mM; Na-pump activity was similar for the NK and LK rats at all potassium concentrations, but Na-pump activity of HK rat arteries was less than that of NK arteries at high extracellular potassium concentrations. Na-pump site density was not altered by either HK or LK diet. It is concluded that in tail arteries of rats fed the LK diet, chronically decreased extracellular potassium results in chronically decreased Na-pump activity. In contrast, an adaptive change occurs in tail arteries of rats fed HK diet, such that Na-pump activity remains at normal levels despite elevated extracellular potassium; this adaptive response to chronically increased dietary potassium does not appear to be the result of decreased Na-pump site density.     

Protein synthesis in central nervous tissue: studies on retina in vitro

Rabbit retinas were maintained in vitro in medium that resembled CSF but with leucine varied from 2 to 1000 microM. Both leucine and threonine were isotopically labelled. When leucine in the medium was 100-1000 microM, leucine was incorporated into protein at 2.03 +/- 0.04 (S.E.M.) mumol/g dry wt./h, a turnover per h of 0.55% of the leucine in retinal protein. Incorporation was constant for at least 7 h. It was reduced 34% when the other amino acids were omitted from the medium and 24% when they were increased 15 fold above physiological levels. When medium leucine was reduced to 2 microM with other amino acids constant, 14C-leucine incorporation fell 70% without significant change in 3H-threonine incorporation, indicating a fall in intracellular specific activity of leucine. The intracellular/extracellular concentration ratio of labelled leucine was 4:1 with medium leucine 23 microM. It fell markedly when medium leucine was reduced to 2 microM or increased to 1000 microM. The concentration ratio of labelled threonine was 15:1 with medium leucine at physiological levels but fell to 6:1 when medium leucine was increased to 1000 microM. Decarboxylation removed 1.5% of free intracellular leucine per min and, at physiological concentrations, was 7.7% the rate of protein incorporation. The ratio of protein synthesis/breakdown, estimated from changes in leucine and 7 other essential amino acids in the medium, was nearly unity. The potential of this preparation for study of CNS protein metabolism is discussed.     

Transport of leucine and sodium in central nervous tissue: studies on retina in vitro

Unidirectional leucine fluxes were measured in isolated rabbit retina maintained under steady state conditions in medium resembling CSF but with leucine varied from 2 to 20,000 microM. At physiological leucine concentration (11 microM), 1/2 time for outward transport was 88 s and intracellular fluid was cleared of isotopically labelled leucine at 2.3 ml/g dry wt./min; 1/2 time for inward transport was 16 s and interstitial fluid was cleared at 7.5 ml/g dry wt./min. The rate of leucine influx corresponded quite well with its rate of disappearance from the intracellular fluid, over a wide range of concentrations. Exchange diffusion was demonstrated for transport in both directions. There was competition by other amino acids, but no interaction between Na+ and leucine transport could be demonstrated. Kinetic analysis indicated the presence of more than one transport system for leucine. There was an unexpected fall in the efflux coefficient, with reduction in leucine concentration at the lower end of the concentration range, for which an explanation is proposed. Under control conditions, 1/2 time for efflux of intracellular 24Na+ was about 0.9 min. With intracellular Na+ increased 4 fold, 1/2 time for efflux was slightly reduced. Problems encountered in measuring fluxes in organized tissue are discussed.     

Selection of a chemically defined medium for submerged cultivation of Streptomyces aureofaciens with high extracellular caseinolytic activity

A chemically defined medium was developed for the submerged cultivation of Streptomyces aureofaciens with a high secretion of caseinolytic activity. The medium composition is: 40 g/liter maltose; 1.640 g/liter L-leucine (0.0125M); 1.765 g/liter L-lysine (0.0125M); 6.976 g/liter K2HPO4 (0.04M); 4 g/liter CaCO3; 0.2 g/liter MgSO4.7H2O; 0.01 g/liter ZnSO4.7H2O; 0.01 g/liter FeSO4.7H2O: 0.01 g/liter MnSO4H2O, and 0.005 g/liter CoSO4.7H2O. Quantitative correlations were established between the concentrations of nutrients in the medium and the secretion of proteolytic activity. In this medium the secretion of proteolytic activity parallels growth, reaching a maximum after 70 hr at 30 degrees C in shaker cultures. The secretion appears to be an active process and to require aerobic conditions.     

Probing the role of endogenous ethylene in the degreening of citrus fruit with ethylene antagonists

The ethylene antagonists, 2,5-norbornadiene (NBD) and silver nitrate, were used to probe the involvement of endogenous ethylene in the natural degreening of citrus fruit. Mature-green, detached Shamouti orange (Citrus sinensis L. Osbeck) fruit were treated with NBD vapor or dipped in solutions of silver nitrate. More than 80% of the chlorophyll was lost from control fruit after 8 days. NBD (0.11 mmole/liter) inhibited the loss of chlorophyll by 60%. NBD also antagonized the degreening induced by exogenous ethylene by 50%. Silver nitrate (0.1 mM) inhibited the loss of chlorophyll by 55%. Ethylene evolution of mature, green detached fruit was <2 nl.fruit^-1.h^-1 (ca. 13.5 nl.Kg^-1FW.h^-1) and did not change significantly for 7 days after harvest. NBD concentrations up to 0.22 mmole/liter did not enhance ethylene evolution. Not with-standing the extremely low amounts of ethylene evolved, the inhibition of degreening by NBD and silver nitrate suggests that endogenous ethylene is involved in the control of this process in mature citrus fruit.     

Formation of higher alcohols and phenol by strains of zymomonas sp.

Strains of the bacteria Zymomonas sp. were studied for their ability to form higher alcohols. In a complex growth medium, six strains were shown to produce significant amounts of 1-propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol, pentanols, secondary hexyl-alcohols, and trace amounts of n-hexanol. When resting cells of these organisms were placed into a fermentation medium containing glucose and Tris-buffer, Z. mobilis 8938 produced increased levels of 1-butanol, and secondary hexyl-alcohols at concentrations of 13.5 mg/liter and 5.8 mg/liter, respectively. Another strain, Z. mobilis subsp. mobilis B 806, stimulated the formation of 1-propanol and 1-butanol at concentrations of 14.9 mg/liter and 23.52 mg/liter, respectively. Amino acids or amino acid precursors were then added to the fermentation medium. The presence of threonine and α-ketobutyric acid stimulated Z. mobilis 8938 to produce 82.6 mg/liter secondary hexyl-alcohols and 8.0 mg/liter n-hexanol, respectively. Isoleucine and valine increased the production of 2-methyl-1-butanol (394.0 mg/liter) and 3-methyl-1-butanol (113.4 mg/liter), respectively, by Z. mobilis subsp. mobilis B 806. Glutamine enhanced the formation of 2-methyl-2-butanol production to concentrations 38.8 mg/liter in Zymomonas strain B 806. Additional experiments suggested that higher alcohol production could also be accomplished in the absence of glucose when cells were allowed to metabolize the precursors only. The effect of aromatic amino acids on phenol production was determined using resting cells of Zymomonas sp. The maximum yield of phenol (111.6 mg/liter) was found by Zymomonas strain 8938 in the presence of tyrosine. The addition of phenylalanine also stimulated this strain to form 71.4 mg/liter of phenol.     

Growth and ultrastructure of the marine unicellular alga <Emphasis Type="Italic">Dunaliella salina</Emphasis> (Chlorophyta) after chronic selenium intoxication

The effects of selenium (0.01, 0.5, 1, 5 and 10 mg/liter) on the growth and ultrastructure of the microalga Dunaliella salina were investigated following its transfer into clean water. Selenium concentrations of 5 and 10 mg/liter were toxic to D. salina, and reinoculation of microalga into clean water did not prevent it from total mortality. When reinoculated from medium with 0.01 mg Se/liter, the cell population density of D. salina was restored in 14 days. The number of ultrastructural alterations in cells was the same as in the control, while the excretory activity of microalga between days 4 and 10 of this experiment was higher. Cell population growth of D. salina transferred from 0.5 and 1 mg Se/liter was lower than in the control. No ultrastructural defects were observed in microalga reinoculated from medium with a selenium concentration of 0.5 mg/liter and the excretion level corresponded to that at 0.01 mg/liter. Various types of ultrastructural damage were found in microalga from medium with 1 mg Se/liter, which was previously reported to be threshold for D. salina; however, the number of cell injuries decreased with increasing time in clean medium. Excretory activity was decreased at the beginning of experiment; but after 7 days, it was restored to the control level. Though there were no ultrastructural alterations in microalgal cells from medium with 0.5 mg Se/liter, we assume that they had molecular defects that could inhibit the cell population growth. The study of microalgae following their reinoculation from medium containing toxicants into clean medium can be a useful method for evaluating algal survival after toxic exposure.     

Effects of potassium ion concentrations on the antimicrobial activities of ionophores against ruminal anaerobes

The antimicrobial activities of monensin and lasalocid against representative strains of ruminal bacteria were evaluated in medium containing three different concentrations of potassium (1.3, 7.9, or 23.3 mM). The growth of Eubacterium ruminantium was inhibited by low concentrations of ionophores (less than or equal to 0.16 mg/liter), while the strain of Streptococcus bovis tested was resistant to high concentrations of ionophores (40 mg/liter) at all potassium concentrations tested. The MICs of the ionophores for strains of Bacteroides succinogenes, Butyrivibrio fibrisolvens, Ruminococcus albus, and Ruminococcus flavefaciens and for one strain of Bacteroides ruminicola increased with increasing potassium concentrations in the medium. High concentrations of ionophores (40 mg/liter) decreased the maximum cell yields or increased the lag times or both in cultures of one strain of Bacteroides ruminicola and two strains of Selenomonas ruminantium but did not completely inhibit the growth of these organisms. Increased potassium concentrations in the medium (from 7.9 to 23.3 mM) decreased the lag times or increased the cell yields or both when these three strains were grown in ionophore-containing medium, while the activities of lasalocid and monensin against these organisms were enhanced in the medium containing low potassium concentrations (1.3 mM). The data from this study suggest that extracellular potassium concentrations may influence the antimicrobial activities of ionophores in the rumen.     

Effects of potassium ion concentrations on the antimicrobial activities of ionophores against ruminal anaerobes.

The antimicrobial activities of monensin and lasalocid against representative strains of ruminal bacteria were evaluated in medium containing three different concentrations of potassium (1.3, 7.9, or 23.3 mM). The growth of Eubacterium ruminantium was inhibited by low concentrations of ionophores (less than or equal to 0.16 mg/liter), while the strain of Streptococcus bovis tested was resistant to high concentrations of ionophores (40 mg/liter) at all potassium concentrations tested. The MICs of the ionophores for strains of Bacteroides succinogenes, Butyrivibrio fibrisolvens, Ruminococcus albus, and Ruminococcus flavefaciens and for one strain of Bacteroides ruminicola increased with increasing potassium concentrations in the medium. High concentrations of ionophores (40 mg/liter) decreased the maximum cell yields or increased the lag times or both in cultures of one strain of Bacteroides ruminicola and two strains of Selenomonas ruminantium but did not completely inhibit the growth of these organisms. Increased potassium concentrations in the medium (from 7.9 to 23.3 mM) decreased the lag times or increased the cell yields or both when these three strains were grown in ionophore-containing medium, while the activities of lasalocid and monensin against these organisms were enhanced in the medium containing low potassium concentrations (1.3 mM). The data from this study suggest that extracellular potassium concentrations may influence the antimicrobial activities of ionophores in the rumen.     

Combining endocytic and freezing‐induced trehalose uptake for cryopreservation of mammalian cells

Fibroblasts take up trehalose during freezing and thawing, which facilitates cryosurvival of the cells. The aim of this study was to investigate if trehalose uptake via fluid‐phase endocytosis prefreeze increases cryosurvival. To determine endocytic trehalose uptake in attached as well as suspended fibroblasts, intracellular trehalose concentrations were determined during incubation at 37°C using an enzymatically based trehalose assay. In addition, freezing‐induced trehalose uptake of extracellularly added trehalose was determined. Cryosurvival rates were determined via trypan blue staining. Intracellular trehalose contents of attached as well as suspended cells were found to increase linearly with time, consistent with fluid‐phase endocytosis. Furthermore, the intracellular trehalose concentration increased with increasing extracellular trehalose concentration (0–100 mM) in a linear fashion. Prefreeze loading of cells with trehalose via fluid‐phase endocytosis only showed increased cryosurvival rates at extracellular trehalose concentrations lower than 50 mM in the cryopreservation medium. To obtain satisfactory cryosurvival rates after endocytic preloading, extracellular trehalose is needed to prevent efflux of trehalose during freezing and thawing and for freezing‐induced trehalose uptake. At trehalose concentrations greater than 100 mM, cryosurvival rates were similar or slightly higher if cells were not loaded with trehalose prefreeze. Cells that were grown in the presence of trehalose showed a tendency to aggregate after harvesting. It is concluded that it is particularly freezing‐induced trehalose uptake that facilitates cryosurvival when trehalose is used as the sole cryoprotectant for cryopreservation of fibroblasts. Preloading with trehalose does not increase cryosurvival rates if trehalose is also added as extracellular protectant. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:229–230, 2017