Effects of mebendazole on the absorption of low molecular weight nutrients by Ascaris suum

Van Den Bossche H. and De Nollin S. 1973. Effects of mebendazole on the absorption of low molecular weight nutrients by Ascaris suum. International Journal for Parasitology3: 401–407. The effect of the anthelmintic drug, mebendazole, on the uptake and/or transport of glucose, fructose, 3-O-methylglucose, glycine, proline, methionine and palmitic acid was studied on in vitro incubated Ascaris suum. The experiments presented indicate that mebendazole inhibits the uptake and/or transport of glucose by A. suum. This inhibition is followed by a marked decrease in the glycogen content of the ascaris muscle. The addition of glucose to the incubation medium significantly enhanced the rate of uptake and/or transport of 3-O-methylglueose, glycine, methionine, proline and palmitic acid indicating that the absorption mechanisms depend on energy.Therefore, the inhibitory effect of mebendazole on the glucose uptake also results in a decreased uptake of 3-O-methylglucose and of the amino acids and fatty acid studied. The fructose uptake was not affected by the addition of glucose.Although mebendazole decreased the uptake of the hexoses and of the amino acids whether or not glucose was added, the uptake of palmitic acid was not affected when glucose was omitted from the medium. Mebendazole failed to exhibit an effect on the uptake, transport and/or utilization of glucose in rat.     

Copper uptake inAspergillus niger during batch growth and in nongrowing mycelial suspensions

Copper accumulation by the filamentous fungusAspergillus niger from a glucose mineral salts medium containing copper in the concentration range 16 to 157 μM was maximal in the lag phase of growth. In the subsequent linear growth phase, the mycelial copper contents were dramatically reduced on a per gram dry weight basis. The fungal mycelium exhibited pelleted morphology and exponential growth was not apparent. The medium pH was reduced during growth in flask cultures, but this was not responsible for the reduction in copper uptake as indicated by the similar effect in cultures grown in a stirred-tank fermenter with electronic maintenance of pH at 5.5. Voltammetric analysis of medium which had supported growth of the fungus showed that copper added at a final concentration of 40 μM was complexed. Energy-dependent copper uptake from 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.5 containing 40 μM copper could not be demonstrated in nongrowing mycelium. Incubation at 4°C reduced copper uptake while the presence of 10 mM glucose or preincubation of the mycelium in 1 mM sodium azide had no effect on copper uptake.     

Mechanism of proline uptake by Chlorella vulgaris

An amino acid uptake system specific for glycine, alanine, serine and proline was induced by glucose in Chlorella vulgaris. The uptake system translocated the zwitterionic form of the amino acid. There was more than 100-fold accumulation which indicated a coupling to metabolic energy. The depolarization of the membrane potential during proline uptake and the sensitivity of its uptake rate to the membrane potential point to coupling with an ion flow. Inhibitors of plasmalemma-bound H⁺-ATPase inhibit proline uptake. These data are interpreted to mean that proline is taken up as a proton symport. In some Chlorella strains the proline-coupled H⁺ uptake could be measured with electrodes, but not in Chlorella vulgaris. There is evidence that the transport of amino acids rapidly stimulates the proton-translocating ATPase of Chlorella vulgaris, so that the proline-coupled proton uptake is immediately neutralized.     

Carbohydrate utilisation by cell suspension cultures of Phaseolus vulgaris

Uptake of sugar by Phaseolus vulgaris cell suspension cultures from a sucrose supplemented medium is predominantly in the hexose form. This is due to a rapid cleavage of the sucrose by an apoplastic acid invertase activity and an apparent very low demand for and uptake of carbon from the medium prior to induction of cell growth and division. Glucose is preferentially taken up, leading to an accumulation of fructose in the medium. However, when the glucose is depleted the cells do take up the fructose at a rate similar to that of glucose. When glucose or fructose is supplied individually to cell cultures, both are utilised very efficiently with growth slightly better on the fructose medium. Hexose uptake is largely an active process with diffusion uptake even at the highest concentrations (> 50 m M ) contributing less than 30%. The hexose uptake system of the cells has a greater affinity for glucose (K_m= 240 µ M ) than for fructose (K_m= 960 µ M ) but the maximum uptake (V_max) is similar. The major difference in the kinetic properties of hexose uptake is that glucose is a strong inhibitor of fructose uptake, while fructose has little effect on glucose uptake. The differences in the kinetic properties of the uptake system for the two hexoses can largely explain the observed pattern of hexose utilisation when both glucose and fructose are present in the medium.     

Growth characteristics and nutrient depletion of Miscanthus × ogiformis Honda ‘Giganteus’ suspension cultures

The growth characteristics and nutrient depletion in suspension cultures of Miscanthus × ogiformis Honda ‘Giganteus’ grown in media containing either Murashige and Skoog or N6 basal nutrient salts were studied during a culture period of 15 days. Proline was added to both media in concentrations from 0 to 300 mM. The fresh and dry weights of the suspension aggregates and the concentrations of ammonium, nitrate, proline and sugar remaining in the medium were measured at different points in time during the culture period. The results showed an almost total depletion of ammonium but a limited nitrate depletion of only 15 mM in both media. Proline at 12.5 to 50 mM was totally depleted during the culture period whereas approximately half the concentration remained in media with 100 to 300 mM proline. Higher fresh weight increases were obtained by the proline additions to the N6 as compared to the MS suspension cultures. Sucrose was hydrolysed into its monosaccharide components in the culture medium. Glucose was depleted faster than fructose indicating a preference for glucose as a carbohydrate source of the M. × ogiformis cultures. The high water uptake by the suspension aggregates 12 to 15 days after subculture indicate that the cultures should be subcultured more frequently to prevent the formation of nonembryogenic cells in the cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Proline metabolism in procyclic Trypanosoma brucei is down-regulated in the presence of glucose

Proline metabolism has been studied in procyclic form Trypanosoma brucei. These parasites consume six times more proline from the medium when glucose is in limiting supply than when this carbohydrate is present as an abundant energy source. The sensitivity of procyclic T. brucei to oligomycin increases by three orders of magnitude when the parasites are obliged to catabolize proline in medium depleted in glucose. This indicates that oxidative phosphorylation is far more important to energy metabolism in this latter case than when glucose is available and the energy needs of the parasite can be fulfilled by substrate level phosphorylation alone. A gene encoding proline dehydrogenase, the first enzyme of the proline catabolic pathway, was cloned. RNA interference studies revealed the loss of this activity to be conditionally lethal. Proline dehydrogenase defective parasites grew as wild-type when glucose was available, but, unlike wild-type cells, they failed to proliferate using proline. In parasites grown in the presence of glucose, proline dehydrogenase activity was markedly lower than when glucose was absent from the medium. Proline uptake too was shown to be diminished when glucose was abundant in the growth medium. Wild-type cells were sensitive to 2-deoxy-D-glucose if grown using proline as the principal carbon source, but not in glucose-rich medium, indicating that this non-catabolizable glucose analogue might also stimulate repression of proline utilization. These results indicate that the ability of trypanosomes to use proline as an energy source can be regulated depending upon the availability of glucose.     

Persistence of Streptococcus mutans in Stationary-Phase Batch Cultures and Biofilms

Streptococcus mutans is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of S. mutans strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation; BacLight staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (±8) days, and an average of 2.7 × 10⁵ CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.     

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.     

Selection of lactose-adapted cells in vinca minor and Dantura innoxia cultures; location and characterization of β-galactosidase and lactase activities

Lactose-adapted cells were obtained from Datura innoxia sucrose growing calli cultures and from Vinca minor glucose growing calli cultures. Lactose adaptation process points out the homogeneity of the cell population towards lactose uptake in V. minor cultures while it reveals the presence of heterogeneous population in D. innoxia cultures.In both species, lactose hydrolysis was only occurring in the cells; no lactase activity was detected in the culture medium. An intermittent lactase activity was determined in a cell-free extract during the culture period. Lactase activity was detected in Vinca glucose grown cells as well in Datura lactose-adapted cells cultured in absence of lactose; so lactase is a constitutive enzyme. Galactose liberated during lactose hydrolysis was not toxic for thecells; it was released into the culture medium and not metabolized in Vinca cultures while it was metabolized in Datura cultures at the end of the culture period.     

Adaptation of Mycobacterium smegmatis to Stationary Phase

Mycobacterium tuberculosis can persist for many years within host lung tissue without causing clinical disease. Little is known about the state in which the bacilli survive, although it is frequently referred to as dormancy. Some evidence suggests that cells survive in nutrient-deprived stationary phase. Therefore, we are studying stationary-phase survival of Mycobacterium smegmatis as a model for mycobacterial persistence. M. smegmatis cultures could survive 650 days of either carbon, nitrogen, or phosphorus starvation. In carbon-limited medium, cells entered stationary phase before the carbon source (glycerol) had been completely depleted and glycerol uptake from the medium continued during the early stages of stationary phase. These results suggest that the cells are able to sense when the glycerol is approaching limiting concentrations and initiate a shutdown into stationary phase, which involves the uptake of the remaining glycerol from the medium. During early stationary phase, cells underwent reductive cell division and became more resistant to osmotic and acid stress and pool mRNA stabilized. Stationary-phase cells were also more resistant to oxidative stress, but this resistance was induced during late exponential phase in a cell-density-dependent manner. Upon recovery in fresh medium, stationary-phase cultures showed an immediate increase in protein synthesis irrespective of culture age. Colony morphology variants accumulated in stationary-phase cultures. A flat colony variant was seen in 75% of all long-term-stationary-phase cultures and frequently took over the whole population. Cryo scanning electron microscopy showed that the colony organization was different in flat colony strains, flat colonies appearing less well organized than wild-type colonies. Competition experiments with an exponential-phase-adapted wild-type strain showed that the flat strain had a competitive advantage in stationary phase, as well a providing evidence that growth and cell division occur in stationary-phase cultures of M. smegmatis. These results argue against stationary-phase M. smegmatis cultures entering a quiescent state akin to dormancy but support the idea that they are a dynamic population of cells.     

Genetic control of transport loss during development of Aspergillus nidulans.

The transport of glucose by spore-originated liquid cultures of Aspergillus nidulans varied with culture age. At early times after conidial inoculation, the uptake rate increases, reaches a maximum at about 11 hr, and subsequently declines exponentially. This decline in uptake rate with age is also observed for sucrose, fructose, alanine, and the nonmetabolizable glucose analog, 2-deoxyglucose. Conidiation of liquid-grown Aspergillus nidulans can be induced (by transfer to solid medium) only after a certain developmental stage, called competence, is attained. Two mutants, selected for precocious conidiation on solid medium, differ from wild-type and from each other in the rate of decline of glucose uptake with culture age: The rate of decline is inversely related to the time of conidiation. The precocious development of these mutants is due to a premature acquisition of competence rather than an acceleration of the events that follow induction. We postulate that an internal clock controls the time of acquisition of developmental competence and suggest that this clock is related to changes in a membrane transport system.     

Trypanosoma cruzi Proline Transport Presents a Cell Density‐dependent Regulation

Trypanosoma cruzi, the etiological agent of Chagas disease, uses proline as its main carbon source, essential for parasite growth and stage differentiation in epimastigotes and amastigotes. Since proline is mainly obtained from extracellular medium by transport proteins, in this work we studied the regulation of the T. cruzi proline transporter TcAAAP069. Proline uptake and intracellular concentration presented oscillations during epimastigote growth phases, increasing during the early exponential phase (322 pmol/min) and decreasing to undetectable levels during the late exponential phase. Transporter expression rate correlated with proline uptake, and its subcellular localization alternated from both, the plasma membrane and close to the flagellar pocket, when the transport is higher, to only the flagellar pocket region, when the transport decreased until proline uptake and TcAAAP069 protein became undetectable at the end of the growth curve. Interestingly, when parasites were treated with conditioned medium or were concentrated to artificially increase the culture density, the proline transport was completely abolished resembling the effects observed in late exponential phase. These data highlight for the first time the existence of a density‐associated regulation of relevant physiological processes such as proline metabolism.     

Proline excretion by Escherichia coli K12

Proline excretion from proline overproducing strains of E. coli K12 has been studied as a model chemical production system. We have isolated proline overproducing mutants of E. coli and have shown that uncontrolled synthesis is not sufficient to cause excretion of this amino acid. An episomal mutation causing proline over production has been introduced into a series of otherwise isogenic strains that bear well defined, chromosomal lesions affecting the active uptake and catabolism of L-proline. A syntropism test reveals that L-proline is excreted by overproducing strains only if transport and/or catabolism are impaired. Dansyl derivatization and chromatographic analysis of culture supernatants shows that proline is the only amino acid excreted. Batch cultures of an excreting strain in an amino acid production medium yield culture supernatants containing 1 g proline/L, whereas no proline is detectable in supernatants derived from cultures of an overproducing strain with normal transport and catabolic activities. These data reveal that genetic lesions eliminating active uptake can be used to specifically enhance metabolite excretion.     

Transient Responses of Glucose-Limited Cultures of Cytophaga johnsonae to Nutrient Excess and Starvation

Cells from glucose-limited chemostat cultures of Cytophaga johnsonae were subjected to a sudden relaxation of substrate limitation by injecting the cells into fresh batch cultures. Starvation experiments were carried out by injecting glucose-limited cells into batch cultures lacking glucose. Transient responses of biomass, glucose uptake and mineralization, ATP content, and viability on different agar media were monitored during these nutrient-shift experiments. Cells reacted differently depending on growth rate and time spent in the chemostat. Fast-growing cells showed an immediate adaptation to the new growth conditions, despite some initial overshoot reactions in ATP and uptake potential. In contrast, slowly growing cells and long-term-adapted cells showed extensive transient growth responses. Glucose uptake and mineralization potentials changed considerably during the transient growth phase before reaching new levels. During the starvation experiments, all cell types displayed a fast decrease in ATP, but the responses of the substrate uptake and mineralization potentials were strongly dependent upon the previous growth rate. Both potentials decreased rapidly in cells with high growth rates. On the other hand, cells with low growth rates maintained 80% of their uptake and mineralization potentials after 8 h of starvation. Thus, slowly growing cells are much better adapted for starvation than are fast-growing cells.     

Osmoprotection of Escherichia coli by Peptone Is Mediated by the Uptake and Accumulation of Free Proline but Not of Proline-Containing Peptides

The effect of meat peptone type I (Sigma) on the growth of Escherichia coli cells under hyperosmotic stress has been investigated. Peptone is a complex mixture of peptides with a small content of free amino acids, which resembles nutrients found in natural environments. Our data showed that peptone enhances the growth of E. coli cells in high-osmolarity medium to levels higher than those achieved with the main compatible solute in bacteria, glycine betaine. The mechanism of osmoprotection by peptone comprises the uptake and accumulation of the compatible solute, proline. The main role of the peptides contained in peptone is the provision of nutrients rather than the intracellular accumulation of osmolytes. In contrast to Listeria monocytogenes (M. R. Amezaga, I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth, Microbiology 141:41–49, 1995), E. coli does not accumulate exogenous peptides for osmoprotection and peptides containing proline do not lead to the accumulation of proline as a compatible solute. In late-logarithmic-phase cultures of E. coli growing at high osmolarity plus peptone, proline becomes the limiting factor for growth, and the intracellular pools of proline are not maintained. This is a consequence of the low concentration of free proline in peptone, the catabolism of proline by E. coli, and the inability of E. coli to utilize proline-containing peptides as a source of compatible solutes. Our data highlight the role that natural components in food such as peptides play in undermining food preservation regimes, such as high osmolarity, and also that the specific mechanisms of osmoprotection by these compounds differ according to the organism.     

Effects of particulate materials and osmoprotectants on very-high-gravity ethanolic fermentation by Saccharomyces cerevisiae.

The effects of osmoprotectants (such as glycine betaine and proline) and particulate materials on the fermentation of very high concentrations of glucose by the brewing strain Saccharomyces cerevisiae (uvarum) NCYC 1324 were studied. The yeast growing at 20 degrees C consumed only 15 g of the sugar per 100 ml from a minimal medium which initially contained 35% (wt/vol) glucose. Supplementing the medium with a mixture of glycine betaine, glycine, and proline increased the amount of sugar fermented to 30.5 g/100 ml. With such supplementation, the viability of the yeast cells was maintained above 80% throughout the fermentation, while it dropped to less than 12% in the unsupplemented controls. Among single additives, glycine was more effective than proline or glycine betaine. On incubating the cultures for 10 days, the viability decreased to only 55% with glycine, while it dropped to 36 and 27%, respectively, with glycine betaine and proline. It is suggested that glycine and proline, known to be poor nitrogen sources for growth, may serve directly or indirectly as osmoprotectants. Nutrients such as tryptone, yeast extract, and a mixture of purine and pyrimidine bases increased the sugar uptake and ethanol production but did not allow the population to maintain the high level of cell viability. While only 43% of the sugar was fermented in unsupplemented medium, the presence of particulate materials such as wheat bran, wheat mash insolubles, alumina, and soy flour increased sugar utilization to 68, 75, 81, and 82%, respectively.     

Mutual adjustment of glucose uptake and metabolism in Trypanosoma brucei grown in a chemostat.

The mutual adjustment of glucose uptake and metabolism in the insect stage of the protozoan parasite Trypanosoma brucei was studied. T. brucei was preadapted in the chemostat to conditions in which either glucose or proline served as the major carbon and energy source. Cells were grown and adapted to either energy or non-energy limitation at a low dilution rate (0.5 day-1) or a high dilution rate (1 day-1). The cells were then used in short- to medium-term uptake experiments with D-[14C]glucose as a tracer. In time course experiments a steady state was reached after 15 min regardless of the preadaptation conditions. This steady-state level increased with increasing glucose availability during preadaptation. The rate of glucose uptake and the hexokinase activity were linearly correlated. In short-term 5- to 90-s) uptake experiments a high transport rate was measured with cultures grown in excess glucose, an intermediate rate was measured with proline-grown cultures, and a low rate was measured in organisms grown under glucose limitation. Glucose metabolism and proline metabolism did not affect each other during the 15-min incubations. Glucose uptake, as a function of the external glucose concentration, did not obey simple Michaelis-Menten kinetics but could be described by a two-step mechanism: (i) transport of glucose by facilitated diffusion and (ii) subsequent metabolism of glucose. The respective rates of the two steps were adjusted to each other. It is concluded that T. brucei is capable of adjusting the different metabolic processes in a way that gives maximum energy efficiency at the cost of short-term flexibility.     

Characteristics and osmoregulatory roles of uptake systems for proline and glycine betaine in Lactococcus lactis.

Lactococcus lactis subsp. lactis ML3 contains high pools of proline or betaine when grown under conditions of high osmotic strength. These pools are created by specific transport systems. A high-affinity uptake system for glycine betaine (betaine) with a Km of 1.5 microM is expressed constitutively. The activity of this system is not stimulated by high osmolarities of the growth or assay medium but varies strongly with the medium pH. A low-affinity proline uptake system (Km, > 5 mM) is expressed at high levels only in chemically defined medium (CDM) with high osmolarity. This transport system is also stimulated by high osmolarity. The expression of this proline uptake system is repressed in rich broth with low or high osmolarity and in CDM with low osmolarity. The accumulated proline can be exchanged for betaine. Proline uptake is also effectively inhibited by betaine (Ki of between 50 and 100 microM). The proline transport system therefore probably also transports betaine. The inhibition of proline transport by betaine results in low proline pools in cells grown in high-osmotic-strength, betaine-containing CDM. The energy and pH dependency and the influence of ionophores on the activity of both transport systems suggest that these systems are not proton motive force driven. At low osmolarities, proline uptake is low but significant. This low proline uptake is also inhibited by betaine, although to a lesser extent than in cells grown in high-osmotic-strength CDM. These data indicate that proline uptake in L. lactis is enzyme mediated and is not dependent on passive diffusion, as was previously believed.     

Inhibition by glucose of the synthesis of proline from glutamic acid

While in the absence of glucose, proline is not a required amino acid, in the presence of glucose the growth of Micrococcus pyogenes var. aureus in amino acid medium is proportional to the concentration of proline when all other amino acids and growth factors are present in amounts adequate for optimal growth. The data presented here and the ideas prevailing in the literature indicate that glutamic acid is a precursor of proline. Glucose inhibits the conversion of glutamic acid into proline, which in turn causes failure of growth. Thus, 1 μg. and 10 μg. glucose/ ml. cause 50% and 100% inhibition, respectively, of the growth dependent on the synthesis of proline. One μg. proline antagonizes completely the inhibition in the presence of 5,000 μg. glucose/ml.One μg. glycerol, 100 μg. pyruvate, 250 μg. lactate, or 100 μg. α-glycerophosphate/ml., individually, cause from 25 to 50% inhibition of the growth dependent on the synthesis of proline from glutamic acid. Five thousand μg./ml. either of malic, succinic, fumaric, α-keto-glutaric, cis-aconitic acid, or dihydroxyacetone, or 500 μg. citric acid/ml. fails to cause inhibition.Pyrrolidone carboxylic acid was found to substitute for glutamic acid but not for proline in tests with M. pyogenes var. aureus. Also, seven proline-less mutant strains of Escherichia coli were unable to utilize pyrrolidone carboxylic acid in place of proline. No evidence was obtained to indicate that pyrrolidone carboxylic acid could serve as a direct precursor of proline.