Calorimetric and stoichiometric analysis of growth of Kluyveromices fragilis in continuous culture: nitrogen limitation imposed upon carbon-limited growth

The calorimetric response of the yeast Kluyveromices fragilis was investigated for growth in continuous culture where nitrogen limitation was imposed on a carbon-limited culture. Calorimetric measurements were combined with off gas analysis, measurements of biomass, substrate and product concentrations, elemental biomass composition, and heat production to study the physiological response of K. fragilis. Regions where both carbon and nitrogen limited growth, were found over a broad range of dilution rates and feed carbon-to-nitrogen ratios. The principle mechanism by which K. fragilis accommodated regions of dual carbon and nitrogen limitation was by partial decoupling of the anabolic and catabolic pathways. When the culture was only nitrogen-limited, increased decoupling of the two pathways was observed. The principal effect of the decoupling was an increased catabolic consumption of glucose, generating an increased heat yield. The preferred way to process the excess glucose was through respiration but the cells were also capable of fermenting a small percentage of the excess glucose in specific cases where the dissolved oxygen partial pressure approached zero. In addition, these results were qualitatively compared to similar studies on Saccharomices cerevisiae. The two yeasts were similar in their ability to accommodate dual limitation by uncoupling anabolic biomass formation from substrate consumption. The two yeasts were dissimilar in how the catabolic substrate was processed. For S. cerevisiae the presence of a bottleneck in the respiration pathway dictated that the majority of the catabolic glucose consumption was by fermentation. For K. fragilis, the lack of a bottleneck in the respiration pathway dictated that the majority of catabolic glucose substrate consumption was by respiration.     

Deterministic interpretation of the temperature response of microbial growth

A deterministic approximation of the thermal deactivation process is undertaken by structuring growth kinetics of cell populations on the activity level. Growth data of the green microalga Scenedesmus obliquus under forced periodic conditions of light intensity have been used for model verification. The model considers a straightforward extension of normal growth kinetics in such a way that different growth activities can be adjusted in the context of a thermal damage and repair mechanism. The nonlinear fitting of the measured response renders parameter values as a function of the light availability per unit biomass.     

Effect of amino acids on the heat production and growth efficiency of Streptococcus bovis: balance of anabolic and catabolic rates.

Streptococcus bovis JB1 grew nearly twice as fast (0.9 versus 1.6 h-1) and had a 40% greater growth yield (18 versus 12.5 mg of protein per mmol of glucose) when an ammonia-based medium was supplemented with amino acids, but the glucose consumption rate (88 mumol mg of protein-1 h-1) and specific rate of heat production (2.1 mW/mg of protein) were unaffected. Amino acid availability had little effect on the catabolic rate, but the specific heat decreased 40% (8.8 to 5.2 J/mg of protein). These growth rate-dependent changes in metabolic efficiency were fivefold greater than the maintenance energy. Chloramphenicol (100 mg/l), an inhibitor of protein synthesis, caused a gradual decrease in anabolic (growth) rate, but there was little change in the rate of glucose consumption and the specific heat increased. When growth was inhibited by iodoacetate, the catabolic and anabolic rates both declined and there was not increase in specific heat. On the basis of these results, the benefit of amino acid supplementation was largely explained by the balance of anabolic and catabolic rates. When amino acids were available, the anabolic and catabolic rates were more closely matched and less energy was spilled as heat.     

The unified metabolism of Gluconacetobacter entanii in continuous and batch processes

The capabilities of Gluconacetobacter spp. to grow on polyol carbon sources through partial oxidation and to synthesize carbohydrate polymers have been noted early in microbiology, yet this genus is now the object of renewed research attention. We address the complex physiology of a species recently classified into this genus, Gluconacetobacter entanii, in transient and steady state experiments. The interdependence between the two main catabolic routes and their links to central metabolism and anabolic routes are supported by our experiments. The disperse accounts of the various catabolic, central metabolism and anabolic pathways are assembled into an integral metabolic structure. A lumped bioreaction scheme is proposed and the rate of change of key internal metabolite concentrations is obtained through a simple stoichiometric model. This model helps interpret our experimental results and also to analyze the findings of other researchers. This work shows for the first time an account of the abilities of G. entanii to synthesize cellulose and a hydrophilic polymer (possibly acetan).     

Change of Carbon Metabolic Activity of Rhizobium Under Carbon Starvation

One fast growing strain of Rhizobium sp (Vigna mungo) VBS 1 was tested for its metabolic activities under carbon starvation. Specific activities of the catabolic enzymes like phosphofructokinase, fructose-1,6-bisphosphate aldolase, iso-citrate dehydrogenase and malate dehydrogenase decreased remarkably whereas, induction of two anapleurotic enzymes like fructose-1,6-bisphosphatase and iso-citrate lyase took place in the cell-free extract of the strain. Almost unchanged specific activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase indicated its key role in maintaining a balance between catabolic and anabolic activities under carbon starvation.     

Charges of nicotinamide adenine nucleotides and adenylate energy charge as regulatory parameters of the metabolism in Escherichia coli.

Methods for measurements of catabolic reduction charge (defined as NADH/(NADH+NAD+)) and anabolic reduction charge (defined as NADPH/(NADPH + NADP+)) are described using [14C]nicotinamide labeling of Escherichia coli cultures. Together with these parameters the adenylate energy charge (ATP + 1/2ADP)/(ATP + ADP + AMP) was measured using labeling with [2-3H]adenine. These three charges were found under different exponential growth conditions to have values independent of the growth conditions: catabolic reduction charge, 0.05; anabolic reduction charge, 0.45; and adenylate energy charge, 0.9. The charges were examined during interruption of growth primarily affecting catabolism, respiration, or anabolism, leading to changes of the charges. The changes of charges are evaluated as a possible regulation of the metabolic rates utilizing or producing the nucleotides by their respective charges.     

Multifunctional role of plant cysteine proteinases

Cysteine proteinases also referred to as thiol proteases play an essential role in plant growth and development but also in senescence and programmed cell death, in accumulation of storage proteins such as in seeds, but also in storage protein mobilization. Thus, they participate in both anabolic and catabolic processes. In addition, they are involved in signalling pathways and in the response to biotic and abiotic stresses. In this review an attempt was undertaken to illustrate these multiple roles of cysteine proteinases and the mechanisms underlying their action.     

Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.     

Sex hormonal regulation and hormesis in aging and longevity: role of vitagenes

Aging process is accompanied by hormonal changes characterized by an imbalance between catabolic hormones, such as cortisol and thyroid hormones which remain stable and hormones with anabolic effects (testosterone, insulin like growth factor-1 (IGF-1) and dehydroepiandrosterone sulphate (DHEAS), that decrease with age. Deficiencies in multiple anabolic hormones have been shown to predict health status and longevity in older persons.Unlike female menopause, which is accompanied by an abrupt and permanent cessation of ovarian function (both folliculogenesis and estradiol production), male aging does not result in either cessation of testosterone production nor infertility. Although the circulating serum testosterone concentration does decline with aging, in most men this decrease is small, resulting in levels that are generally within the normal range. Hormone therapy (HT) trials have caused both apprehension and confusion about the overall risks and benefits associated with HT treatment. Stress-response hormesis from a molecular genetic perspective corresponds to the induction by stressors of an adaptive, defensive response, particularly through alteration of gene expression. Increased longevity can be associated with greater resistance to a range of stressors. During aging, a gradual decline in potency of the heat shock response occur and this may prevent repair of protein damage. Conversely, thermal stress or pharmacological agents capable of inducing stress responses, by promoting increased expression of heat-shock proteins, confer protection against denaturation of proteins and restoration of proteome function. If induction of stress resistance increases life span and hormesis induces stress resistance, hormesis most likely result in increased life span. Hormesis describes an adaptive response to continuous cellular stresses, representing a phenomenon where exposure to a mild stressor confers resistance to subsequent, otherwise harmful, conditions of increased stress. This biphasic dose–response relationship, displaying low-dose stimulation and a high-dose inhibition, as adaptive response to detrimental lifestyle factors determines the extent of protection from progression to metabolic diseases such as diabetes and more in general to hormonal dysregulation and age-related pathologies. Integrated responses exist to detect and control diverse forms of stress. This is accomplished by a complex network of the so-called longevity assurance processes, which are composed of several genes termed vitagenes. Vitagenes encode for heat shock proteins (Hsps), thioredoxin and sirtuin protein systems. Nutritional antioxidants, have recently been demonstrated to be neuroprotective through the activation of hormetic pathways under control of Vitagene protein network. Here we focus on possible signaling mechanisms involved in the activation of vitagenes resulting in enhanced defense against functional defects leading to degeneration and cell death with consequent impact on longevity processes.     

Evidence for sex-dependent anabolic response to androgenic steroids mediated by muscle glucocorticoid receptors in the rat

The muscle anabolic/anti-catabolic activity of the androgenic steroids testosterone and trenbolone was studied in rats to investigate whether such steroids act as agonists via muscle androgen receptors, or as antagonists that oppose the catabolic effects of endogenous glucocorticoids via their interaction with muscle glucocorticoid receptors. For comparison, the effects of the potent glucocorticoid antagonist RU486 were also examined. The parameters measured included growth rate, muscle weight, serum growth hormone and corticosterone levels, and receptor binding parameters in muscle cytosol. Females responded better than males to anabolic treatment with the androgenic steroids. Ovariectomy or adrenalectomy abolished this response. Neither the sex difference nor the requirement for ovaries or adrenals could be explained in terms of muscle receptor parameters or serum growth hormone levels. The muscle anabolic activity of androgenic steroids was restored when castrated males were treated with oestradiol and when adrenalectomized females were treated with corticosterone. RU486 also prevented the catabolic/anti-anabolic activity of exogenous corticosterone in adrenalectomized rats. Testosterone and RU486 behaved as anti-glucocorticoids in vivo since they inhibited glucocorticoid-induced liver tyrosine aminotransferase activity. The results suggest that anabolic steroids can act via muscle glucocorticoid receptors, thereby antagonizing the catabolic activity of endogenous glucocorticoids, rather than via muscle androgen receptors.     

Enhanced thermotolerance and temperature-induced changes in protein composition in the hyperthermophilic archaeon ES4.

The hyperthermophilic archaeon ES4, a heterotrophic sulfur reducer isolated from a deep-sea hydrothermal vent, is capable of protecting itself from thermal stress at temperatures above its optimum for growth. The thermotolerance of ES4 was determined by exposing log-phase cells to various lethal high temperatures. When ES4 was shifted from 95 to 102 degrees C, it displayed recovery from an exponential rate of death, followed by transient thermotolerance. When ES4 was shifted directly from 95 to either 105 or 108 degrees C, only exponential death occurred. However, a shift from 95 to 105 degrees C with an intermediate incubation at 102 degrees C also gave ES4 transient thermotolerance to 105 degrees C. The protein composition of ES4 was examined at temperatures ranging from 75 to 102 degrees C by one-dimensional electrophoresis. Two proteins with molecular masses of approximately 90 and 150 kDa significantly decreased in abundance with increasing growth temperature, while a 98-kDa protein, present at very low levels at normal growth temperatures (76 to 99 degrees C), was more abundant at higher temperatures. The enhanced tolerance to hyperthermal conditions after a mild hyperthermal exposure and the increased abundance of the 98-kDa protein at above-optimal temperatures imply that ES4 is capable of a heat shock-like response previously unseen in hyperthermophilic archaea.     

Quantitative comparison of transient growth of Saccharomyces cerevisiae,Saccharomyces kluyveri,and Kluyveromyces lactis

A multitude of metabolic regulations occur in yeast, particularly under dynamic process conditions, such as under sudden glucose excess. However, quantification of regulations and classification of yeast strains under these conditions have yet to be elucidated, which requires high-frequency and consistent quantification of the metabolic response. The present study aimed at quantifying the dynamic regulation of the central metabolism of strains Saccharomyces cerevisiae, S. kluyveri, and Kluyveromyces lactis upon sudden glucose excess, accomplished by a shift-up in dilution rate inside of the oxidative region using a small metabolic flux model. It was found that, under transient growth conditions, S. kluyveri behaved like K. lactis, while classification using steady-state conditions would position S. kluyveri close to S. cerevisiae. For transient conditions and based on the observation whether excess glucose is initially used for catabolism (energy) or anabolism (carbon), we propose to classify strains into energy-driven, such as S. cerevisiae, and carbon-driven, such as S. kluyveri and K. lactis, strains. Furthermore, it was found that the delayed onset of fermentative catabolism in carbon-driven strains is a consequence of low catabolic flux and the initial shunt of glucose in non-nitrogen-containing biomass constituents. The MFA model suggests that energy limitation forced the cell to ultimately increase catabolic flux, while the capacity of oxidative catabolism is not sufficient to process this flux oxidatively. The combination of transient experiments and its exploitation with reconciled intrinsic rates using a small metabolic model could corroborate earlier findings of metabolic regulations, such as tight glucose control in carbon-driven strains and transient changes in biomass composition, as well as explore new regulations, such as assimilation of ethanol before glucose. The benefit from using small metabolic flux models is the richness of information and the enhanced insight into intrinsic metabolic pathways without a priori knowledge of adaptation kinetics. Used in an online context, this approach serves as an efficient tool for strain characterization and physiological studies.     

The Opposing Actions of Target of Rapamycin and AMP-Activated Protein Kinase in Cell Growth Control

Cell growth is a highly regulated, plastic process. Its control involves balancing positive regulation of anabolic processes with negative regulation of catabolic processes. Although target of rapamycin (TOR) is a major promoter of growth in response to nutrients and growth factors, AMP-activated protein kinase (AMPK) suppresses anabolic processes in response to energy stress. Both TOR and AMPK are conserved throughout eukaryotic evolution. Here, we review the fundamentally important roles of these two kinases in the regulation of cell growth with particular emphasis on their mutually antagonistic signaling.An efficient homeostatic response to maintain cellular energy despite a noncontinuous supply of nutrients is crucial for the survival of organisms. Cells have, therefore, evolved a host of molecular pathways to sense both intra- and extracellular nutrients and thereby quickly adapt their metabolism to changing conditions. The target of rapamycin (TOR) and AMP-activated protein kinase (AMPK) signaling pathways control growth and metabolism in a complementary manner with TOR promoting anabolic processes under nutrient- and energy-rich conditions, whereas AMPK promotes a catabolic response when cells are low on nutrients and energy. Both pathways are highly conserved from yeast to human. This review summarizes the cross talk between TOR and AMPK in different organisms.     

Glucocorticoid-induced lymphoma cell death: the good and the evil

Glucocorticoid hormones and their synthetic derivatives are widely used in therapy due to their strong anti-inflammatory and immunosuppressive potential. While the molecular basis of the anti-inflammatory action is to date at least partially understood, knowledge regarding the mechanism underlying glucocorticoid effects on the immune system is rather fragmentary. The immunosuppression could be attributed to at least two distinct processes: inhibition of the production of growth mediators and glucocorticoid-induced cell death. The mechanism of glucocorticoid-induced cell death can be divided into two steps, a reversible growth inhibition and cell lysis. The first step is characterized by many metabolic alterations typical of the catabolic potential of corticosteroids. After a delay of several hours activation of an endonuclease appears to initiate the lytic phase. By the action of this endonuclease the DNA is fragmented. In opposition to the chromatin damage, poly(ADP-ribosyl)ation is activated in order to stabilize the chromatin structure until the antagonistic potential is exhausted and the cells die. Therefore it can be speculated that the lethal event in glucocorticoid-induced cell death is a destruction of the regular chromatin structure.     

Anabolic ornithine carbamoyltransferase of Escherichia coli and catabolic ornithine carbamoyltransferase of Pseudomonas putida. Steady-state kinetic analysis.

The anabolic and catabolic ornithine carbamoyltransferases of Pseudomonas putida display an undirectional catalytic specialization: in citrulline synthesis for the anabolic enzyme, in citrulline phosphorolysis for the catabolic one. The irreversibility of the anabolic enzyme in vitro has been previously explained by its kinetic properties, whereas the irreversibility of the catabolic transferase in vivo was shown to be due to its allosteric behaviour. In this work a steady-state kinetic analysis has been carried out on the catabolic ornithine carbamoyltransferase at pH 6.8 in the presence of the allosteric activator, phosphate. The kinetic mechanism of Escherichia coli ornithine carbamoyltransferase serving as a reference was also determined. For the E. coli enzyme in the reverse direction, the initial velocity patterns converging on the abscissa were obtained with either citrulline or arsenate as variable substrate. The inhibition by the product ornithine was linear competitive with respect to citrulline and linear non-competitive with respect to arsenate. In the forward direction phosphate and its analogs induce an inhibition by ornithine which is partial and competitive with respect to carbamoylphosphate. Together with the results of thermo-inactivation studies in the presence of each reactant, this observation suggests a random kinetic mechanism, but with most of the reaction flux following the path where carbamoylphosphate adds before ornithine, when substrates are present at Km levels. The allosteric catabolic ornithine carbamoyltransferase of Pseudomonas displays qualitatively the same pattern as the E. coli enzyme.     

Thyroid Hormones and Activities of Lysosomal Proteolytic Enzymes in Organs of the Arctic Fox Alopex lagopus

Age-related changes of content of thyroid hormones in the blood serum and of activities of lysosomal proteinases in organs were studied in the arctic fox Alopex lagopus L. in early postnatal ontogenesis. The existence has been shown of a negative correlation between the age changes in the serum thyroxin content and lysosomal proteinase activities in some organs of these animals. The results obtained indicate predominance of anabolic function of thyroid hormones in arctic fox cubs. The problem of anabolic and catabolic effects of thyroid hormones on protein metabolism is discussed.     

Effects of diacerein on biosynthesis activities of chondrocytes in culture

The maintenance of articular cartilage integrity requires a balance between anabolic and catabolic processes which are under the control of chondrocytes. These cells are living in an anaerobic environment and normally do not divide. They are responsible for the continuous maintenance of the cartilage extracellular matrix (ECM). Although multiple factors are involved in the dynamic homeostasis of cartilage, increases in cytokines such as interleukin-1 (IL-1) are associated with a decrease in synthesis and an increase in degradation of the proteoglycans and collagens. Conversely, growth factors such as transforming growth factor-beta (TGF-beta) stimulate chondrocyte synthesis of collagens and proteoglycans, and reduce the activity of IL-1 stimulated metalloproteases, thus opposing the inhibitory and catabolic effects of IL-1. By its capability to reduce IL-1 effects and to stimulate TGF-beta expression in cultured articular chondrocytes, diacerein could favour anabolic processes in the OA cartilage and, hence may contribute to delay the progression of the disease.     

The metabolic response of Saccharomyces cerevisiae to continuous heat stress

A study has been initiated to integrate molecular and physiological responses of Saccharomyces cerevisiae to heat stress conditions. We focus our research on a quantification of the energetics of the stress response. A series of continuous heat stresses was applied to exponentially growing cells of the strain X2180-1A at 28°C, by increasing the growth temperature to 37, 39, 40, 41, 42, or 43°C. Here, the results on cell growth and viability, as well as on anabolic and catabolic rates are presented. We observed a surprisingly thin line for the cells between growing, surviving, and dying, with regard to growth temperature. The heat stress showed a dual effect on catabolism: immediately after the temperature increase a strong peak was seen, after which a new, steady level was reached. In addition, the yield on glucose decreased with increasing temperature. Our results indicate that life at elevated temperatures is energetically unfavourable and a non-lethal heat stress invokes a redistribution of catabolic and anabolic fluxes.     

Acid phosphatase deactivation by a series mechanism

Acid phosphatase (E.C.3.1.3.2.) thermal deactivation at pH 3.77 has been investigated by monitoring the enzyme activity as a function of time in the hydrolysis of p-nitrophenyl phosphate. The experimental curves obtained show a two-slope behavior in a log (activity)versus-time plot, which indicates that deactivation occurs via a complex mechanism. From the dependence of the kinetic parameters on both deactivation and hydrolysis temperatures, it is inferred that the deactivation mechanism involves intermediate, temperature-dependent, less-active forms of the enzyme. This interpretation is confirmed by the results of additional tests in which the temperature was suddenly changed during the deactivation process.