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PubMed2002

Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase.

Metabolic engineering studies have generally focused on manipulating enzyme levels through either the amplification, addition, or deletion of a particular pathway. However, with cofactor-dependent production systems, once the enzyme levels are no longer limiting, cofactor availability and the ratio of the reduced to oxidized form of the cofactor can become limiting. Under these situations, cofactor manipulation may become crucial in order to further increase system productivity. Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. However, cofactor manipulations can potentially become a powerful tool for metabolic engineering. Nicotinamide adenine dinucleotide (NAD) functions as a cofactor in over 300 oxidation-reduction reactions and regulates various enzymes and genetic processes. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. This paper investigates a genetic means of manipulating the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase. More specifically, it explores the effect on the metabolic patterns in Escherichia coli under anaerobic and aerobic conditions of substituting the native cofactor-independent formate dehydrogenase (FDH) by and NAD(+)-dependent FDH from Candida boidinii. The over-expression of the NAD(+)-dependent FDH doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed, increased the final cell density, and provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically. Under anaerobic conditions, the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol-to-acetate ratio. Even more interesting is the observation that during aerobic growth, the increased availability of NADH induced a shift to fermentation even in the presence of oxygen by stimulating pathways that are normally inactive under these conditions.

Aerobiosis↗

PubMed2002

The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures.

It is generally known that cofactors play a major role in the production of different fermentation products. This paper is part of a systematic study that investigates the potential of cofactor manipulations as a new tool for metabolic engineering. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ and producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. We have previously investigated a genetic means of increasing the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase and have demonstrated that this manipulation provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically (Berríos-Rivera et al., 2002, Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase, Metabolic Eng. 4, 217-229). The current work explores further the effect of substituting the native cofactor-independent formate dehydrogenase (FDH) by an NAD(+)-dependent FDH from Candida boidinii on the NAD(H/+) levels, NADH/NAD+ ratio, metabolic fluxes and carbon-mole yields in Escherichia coli under anaerobic chemostat conditions. Overexpression of the NAD(+)-dependent FDH provoked a significant redistribution of both metabolic fluxes and carbon-mole yields. Under anaerobic chemostat conditions, NADH availability increased from 2 to 3 mol NADH/mol glucose consumed and the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol to acetate ratio and a decrease in the flux to lactate. It was also found that the NADH/NAD+ ratio should not be used as a sole indicator of the oxidation state of the cell. Instead, the metabolic distribution, like the Et/Ac ratio, should also be considered because the turnover of NADH can be fast in an effort to achieve a redox balance.

Anaerobiosis↗

PubMed1985

Dependence of protein synthesis on aortic pressure and calcium availability.

Increased aortic pressure accelerated protein synthesis in control-beating and arrested-drained hearts supplied with either glucose or pyruvate. Elevation of perfusion pressure from 60 to 120 mm Hg increased oxygen consumption in control-beating but not in arrested-drained preparations. Energy availability, as assessed by adenylate energy charge or creatine phosphate/creatine ratio, or both, was increased in arrested-drained hearts supplied with glucose and perfused at 60 and 120 mm Hg aortic pressure. In control-beating or arrested-drained hearts supplied with pyruvate, energy availability was not improved by elevation of aortic pressure from 60 to 120 mm Hg. An increase of perfusate calcium concentration from 0.5 to 5.0 mM in control-beating Langendorff preparations supplied with glucose and perfused at an aortic pressure of 90 mm Hg doubled oxygen consumption and decreased energy availability, but had no effect on the rate of protein synthesis. In arrested-drained hearts supplied with either glucose or pyruvate and calcium concentrations ranging from 0.5 to 5.0 mM, the rates at 120 mm Hg aortic pressure were 11-25% higher than at 60 mm Hg. These findings provide no evidence to implicate increased oxidative metabolism, energy availability, or extracellular calcium concentration as important factors in the mechanism that accounts for the effects of increased aortic pressure on protein synthesis.

Animals↗

PubMed1989

Triiodothyronine bound to red blood cells is not available for transport through the blood-brain barrier.

Many steroid and thyroid hormones and some drugs are bound by circulating red cells. Red cell-bound ligand may not be physiologically inert, as recent studies show that red cell-bound drug is available for uptake by brain. To investigate whether triiodothyronine (T3) is available for uptake by brain in vivo from the circulating red cell pool, the present investigations measure the effects of human erythrocytes on rat brain uptake of [125I]T3 in vivo. The fraction of circulating T3 available for uptake in vivo in the presence of 0, 2, 5, 10, 22, or 44% red cells was essentially identical to the fraction of [125I]T3 unbound in vitro. Therefore, [125I]T3 bound to red cells obtained from normal volunteers is not available for uptake by brain in vivo.

Adult↗

PubMed1994

Increased pineal melatonin content coupled to restricted water availability in a Pavlovian conditioning paradigm in rats.

The objective of this study was to assess whether rat pineal melatonin content could be modified in a classical conditioning paradigm. In rats kept under light (200 lux) from 06.00 to 18.00 h daily, the time of lights off was selected as the unconditioned stimulus (US). Restricted water availability (from 10 min before to 10 min after light-dark, LD, transition) was the conditioned stimulus (CS). The conditioned and unconditioned responses were measured as the changes in pineal melatonin levels 4 h after LD transition. In animals under regular lighting conditions, lights out at 18.00 h (the US) caused a 4.4-7.8-fold increase of pineal melatonin concentration 4 h after later, when compared to animals maintained under light for the 4 h-period. After a training period of 7 days of restricted water availability (the CS), significantly augmented pineal melatonin levels were found in rats that were exposed to water but were maintained under light for the 4 h period after expected LD transition. The control animals for this experiment, i.e., rats which had undergone the training period, were kept for 4 h under light after expected LD transition, and did not receive water at LD transition, exhibited very low pineal melatonin levels. The conditioned increase of pineal melatonin content attained lower values than those in rats exposed to normal lighting conditions. It also fulfilled the contingency criterion, that is, it caused at trial a significant elevation of pineal melatonin content only when water availability was applied from 10 min previously to LD transition during training, and not 20 min after LD transition. After a training period of 7 days, restricted water availability applied 4 h before lights off (at 14.00 h), caused an enhanced production of melatonin 4 h later, regardless of the animals being exposed either to a dark or to a light environment. The results indicate that pineal melatonin production can be manipulated in a classical conditioning paradigm, when an appropriate CS stimulus is used.

Animals↗

PubMed1991

[Content of available lysine in gamma-irradiated soybean products].

The content in available lysine of soya protein products irradiated in a 60Co-gamma cell were investigated. The results indicate that available lysine content and crude protein content at irradiation doses of 1, 3 and 5 kGy were unaffected at the 95% significance level. During 3-month storage the irradiated samples showed no significant (p = 0,05) changes of available lysine content. In samples with high content of lecithin, which were irradiated at 1 kGy as well as non-irradiated the content of available lysine was significantly (p = 0,05) reduced after 8-month storage; the other products as well as the at 3 and 5 kGy irradiated samples showed no significant decrease. Immediately after irradiation the microflora was significantly (p = 0,05) reduced. Under the effect of radiation treatment at 3 and 5 kGy the microflora was reduced to nearly 100% after 3-month storage.

Bacteria↗

PubMed1979

Plasma amino acid response to single test meals in humans. VI. Determination of available lysine.

The plasma amino acid response to single test meals in young adults was used to determine the amount of physiologically available lysine in heat-treated milk samples. The plasma amino acid responses were evaluated as PAA ratios according to a modified calculation procedure based on the method proposed by Longenecker and Hause. The amount of available lysine was determined by two separate series of single-meal tests. In the first, a relationship was established between the plasma amino acid response (PAA ratio) and the dietary level of lysine (given as synthetic amino acid mixtures). This relationship could be described by linear regression (r = 0.90). This regression line was then used to quantitatively evaluate the lysine PAA ratios obtained from single test meals with heat-treated milk samples. The heat-treatment of the milk samples caused a marked reduction of the amount of physiologically available lysine. The quantitative estimates obtained by the present plasma amino acid technique were in good agreement with determinations of available lysine in the same milk samples based on rat bioassay and chemical analysis.

Adult↗

PubMed1994

Effects of concurrent saccharin availability and buprenorphine pretreatment on demand for smoked cocaine base in rhesus monkeys.

The effects of saccharin and the opioid partial agonist buprenorphine on cocaine base smoking were evaluated in five male rhesus monkeys. Monkeys completed a sequence of responding consisting of lever-press responses maintained under a fixed-ratio (FR) schedule followed by inhalation responses (FR5) on a smoking spout to gain access to a single delivery of volatilized cocaine base (1.0 mg/kg per delivery). Monkeys could receive a maximum of ten smoke deliveries per session. In the first experiment, either saccharin (0.03% wt/vol) or water was concurrently available under an FR1 schedule through a lip-operated drinking device. As lever FR values increased from 128 to 256, 512, 1024 and 2048, the number of cocaine smoke deliveries decreased. Cocaine intake was not statistically different when water versus saccharin was concurrently available. However, as cocaine consumption decreased, saccharin intake increased demonstrating that under these conditions, saccharin was substituting for cocaine as a reinforcer. On the first day that lidocaine replaced cocaine, all of the monkeys received the maximum number of smoke deliveries (ten) and saccharin intake increased. Lever-press responding gradually extinguished over days when lidocaine (1.0 mg/kg per delivery) was available with concurrent saccharin. In the second experiment, water was concurrently available with cocaine and buprenorphine (0.01 or 0.1 mg/kg) was administered intramuscularly (IM) 30 min before the start of the session. Although pretreatment with the lower dose of buprenorphine (0.01 mg/kg) had little effect on cocaine intake overall, individual differences in cocaine intake occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

A comparison of a Bayesian population method with two methods as implemented in commercially available software.

In this paper we describe and discuss three specific estimation procedures that are available within commercially available population software packages. The first version of NONMEM (l) was released in 1979 and later versions are the standard analysis tools in both industry and academia. Recently, two commercially available pieces of software have become available. PPHARM was released during 1994 and POPKAN was released in 1995. We provide descriptions and critique the FOCE method within NONMEM, the two-step algorithm within PPHARM and the Markov chain Monte Carlo method that is utilized by POPKAN. We use simulated data generated from a monoexponential model to evaluate the parameter estimation capabilities of these methods within the three software tools. In particular we investigate the effect on parameter estimation of increasing both interindividual and intraindividual variability.

Bayes Theorem↗

PubMedDate not supplied

Impact of the availability of a prior electrocardiogram on the triage of the patient with acute chest pain.

STUDY OBJECTIVE: To determine whether information from a prior electrocardiogram (ECG) improves diagnostic accuracy in the emergency department (ED) evaluation of patients with acute chest pain. DESIGN: Analysis of prospectively collected data from a cohort study. SETTING: Emergency departments of four community and three university hospitals. PATIENTS: 5,673 patients aged greater than or equal to 30 years who presented to the EDs of participating hospitals for evaluation of acute chest pain, including 772 (14%) with acute myocardial infarction (AMI). MEASUREMENTS AND MAIN RESULTS: After adjusting for clinical characteristics, no significant difference was found in the sensitivities of admission to the hospital or to the coronary care unit (CCU) between AMI patients with and without prior ECGs available for review. However, non-AMI patients with prior ECGs available for review were more likely to avoid CCU admission than were non-AMI patients without prior ECGs. This improvement in specificity was most marked in the 2,024 patients whose current ED ECGs had changes consistent with ischemia or infarction: when a prior ECG was available, non-AMI patients were more than twice as likely to be discharged (26% vs. 12%) and about 1.5 times as likely to avoid CCU admission (39% vs. 27%) (both p less than 0.0001). Admission rates of AMI patients with and without prior ECGs were similar. CONCLUSION: When the current ECG is consistent with ischemia or infarction, the availability of a prior ECG for comparison to determine whether the ECG changes are old or new improves diagnostic accuracy and triage decisions by reducing the admission of patients without AMI or acute ischemic heart disease (increased specificity) without reducing the admission of patients with these diagnoses (unchanged sensitivity).

Aged↗

PubMed2003

Measuring quality of life in patients with breast cancer: a systematic review of reliable and valid instruments available in Japan.

BACKGROUND: Little is known about the availability and psychometric properties of instruments to measure quality of life (QOL) in clinical research on Japanese patients with breast cancer. The purpose of this systematic review is to find reliable and valid instruments available in Japan, and to summarize their characteristics. METHODS: Instruments available in Japan were found through a systematic search of the literature. Each instrument identified was evaluated for item development, reliability, validity, interpretability and utility. RESULTS: Six questionnaires to measure health-related QOL (the QOL-ACD, the EORTC QLQ-C30, the EORTC QLQ-BR23, the FACT-B, the SF-36, the WHO/QOL-26) and five scales to quantify the psychological burden (the STAI, the POMS, the SDS, the HADS, the GHQ), for which reliability and validity have been documented, are available in Japanese. All instruments were developed in foreign countries except for the QOL-ACD. Two of the QOL questionnaires were specific to breast cancer (the EORTC QLQ-BR23, the FACT-B). Though the measurements can be interpreted in some manner, the meaning of change scores over time has been documented for only three instruments (the EORTC QLQ-C30, the FACT-B, and the GHQ). CONCLUSIONS: The review provides grounds for designing and implementing quantitative research on QOL of breast cancer patients in Japan. Methodological challenges, however, continue, particularly for validating instruments with regard to various study populations of Japanese people and demonstrating the clinical importance of change scores.

Breast Neoplasms↗

PubMed1998

Serotonin-immune interactions in elderly volunteers and in patients with Alzheimer's disease (DAT): lower plasma tryptophan availability to the brain in the elderly and increased serum interleukin-6 in DAT.

The aims of this study were to examine the plasma availability of tryptophan, the precursor of 5-hydroxytryptamine (5-HT), and serum cytokines, such as interleukin-6 (IL-6) and IL-8, in normal elderly volunteers and in patients with Alzheimer's disease (DAT). Elderly normal volunteers (mean age = 78.3 +/- 5.7 years) had a significantly lower tryptophan/competing amino acids (valine + leucine + isoleucine + phenylalanine + tyrosine) ratio than younger subjects (mean age = 32.9 +/- 8.1 years). In normal volunteers, there were significant and inverse relationships between age and either plasma tryptophan or the tryptophan/competing amino acids ratio, and between the availability of tryptophan to the brain and serum IL-6 or IL-8. DAT patients had significantly higher serum IL-6, but not IL-8, than age-matched normal volunteers. There were no significant differences in the availability of tryptophan to the brain between DAT patients and age-matched normal volunteers. The results suggest that: 1) in normal humans, the availability of plasma tryptophan to the brain decreases with age, and with activation of the immune system; and 2) increased production of IL-6 may play a role in the pathogenesis of DAT.

Adult↗

PubMed1998

Relationship between steady-state activation and availability of cardiac sodium channel: evidence of uncoupling.

The coupling between steady-state activation and availability from inactivation was characterized for the cardiac Na+ channel. To evaluate this coupling, we plotted the relationship between the conductance and availability curve midpoint potentials measured in 92 rat ventricular cardiomyocytes and applied a correlation analysis. We found a high correlation between the midpoints (correlation coefficient = 0.86, slope = 0.95) within the availability midpoint potential range positive to -100 mV. In contrast, the midpoints were not correlated in the myocytes (37 of 92 cells) having midpoint potential negative to -100 mV, indicating an uncoupling between activation and availability.

Animals↗

PubMed2004

Characterization of humus microbial communities in adjacent forest types that differ in nitrogen availability.

To address the link between soil microbial community composition and soil processes, we investigated the microbial communities in forest floors of two forest types that differ substantially in nitrogen availability. Cedar-hemlock (CH) and hemlock-amabilis fir (HA) forests are both common on northern Vancouver Island, B.C., occurring adjacently across the landscape. CH forest floors have low nitrogen availability and HA high nitrogen availability. Total microbial biomass was assessed using chloroform fumigation-extraction and community composition was assessed using several cultivation-independent approaches: denaturing gradient gel electrophoresis (DGGE) of the bacterial communities, ribosomal intergenic spacer analysis (RISA) of the bacterial and fungal communities, and phospholipid fatty acid (PLFA) profiles of the whole microbial community. We did not detect differences in the bacterial communities of each forest type using DGGE and RISA, but differences in the fungal communities were detected using RISA. PLFA analysis detected subtle differences in overall composition of the microbial community between the forest types, as well as in particular groups of organisms. Fungal PLFAs were more abundant in the nitrogen-poor CH forests. Bacteria were proportionally more abundant in HA forests than CH in the lower humus layer, and Gram-positive bacteria were proportionally more abundant in HA forests irrespective of layer. Bacterial and fungal communities were distinct in the F, upper humus, and lower humus layers of the forest floor and total biomass decreased in deeper layers. These results indicate that there are distinct patterns in forest floor microbial community composition at the landscape scale, which may be important for understanding nutrient availability to forest vegetation.

Bacteria↗

PubMed2004

Effect of different levels of NADH availability on metabolite distribution in Escherichia coli fermentation in minimal and complex media.

A range of intracellular NADH availability was achieved by combining external and genetic strategies. The effect of these manipulations on the distribution of metabolites in Escherichia coli was assessed in minimal and complex medium under anoxic conditions. Our in vivo system to increase intracellular NADH availability expressed a heterologous NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii in E. coli. The heterologous FDH pathway converted 1 mol formate into 1 mol NADH and carbon dioxide, in contrast to the native FDH where cofactor involvement was not present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 mol to 4 mol NADH/mol glucose consumed. In the current study, we found that yields of greater than 4 mol NADH were achieved when carbon sources more reduced than glucose were combined with our in vivo NADH regeneration system. This paper demonstrates experimentally that different levels of NADH availability can be achieved by combining the strategies of feeding the cells with carbon sources which have different oxidation states and regenerating NADH through the heterologous FDH pathway. The general trend of the data is substantially similar for minimal and complex media. The NADH availability obtained positively correlates with the proportion of reduced by-products in the final culture. The maximum theoretical yield for ethanol is obtained from glucose and sorbitol in strains overexpressing the heterologous FDH pathway.

Anaerobiosis↗

PubMed2001

Equipment availability and diagnostic strategies for suspected pulmonary embolism in Austria.

The aim of this study was to investigate equipment availability and current diagnostic strategies for suspected pulmonary embolism (PE) in Austrian hospitals. A questionnaire was sent to the medical directors of all Austrian hospitals with emergency and/or surgical, orthopedic, and medical departments. The questionnaire contained questions regarding the available equipment suitable for the imaging diagnosis of PE, the first-line and second-line imaging tests for patients with suspected PE, and additional lower extremity venous imaging and laboratory tests that complement the diagnostic armamentarium. The return rate for questionnaires was 81% (127 of 157 hospitals). There were 97% of hospitals that had the equipment to perform sonography, 59% could perform pulmonary angiography, 54% spiral CT, 19% ventilation/perfusion (V/P) scintigraphy, and 4% perfusion scintigraphy alone. Spiral-CT angiography (SCTA) was the first-line imaging study for suspected PE in 56% of hospitals, followed by echocardiography and V/P scintigraphy. Lower extremity venous imaging (47%) and, interestingly, V/P scintigraphy (43%), served as second-line imaging tests. D-dimer tests were included in the diagnostic strategy in 74% of hospitals. Spiral-CT angiography is the most commonly used primary method for suspected PE in Austrian hospitals. The V/P scintigraphy is available only in a minority of hospitals to investigate patients with suspected PE. When V/P scintigraphy is available, however, it is employed in a large number of patients per annum.

Austria↗

PubMed2003

The effect of metabolic fuel availability on thermoregulation and torpor in a marsupial hibernator.

The physiological signal for torpor initiation appears to be related to fuel availability. Studies on metabolic fuel inhibition in placental heterotherms show that glucose deprivation via the inhibitor 2-deoxy-D-glucose (2DG) initiates a torpor-like state, whereas fatty acid deprivation via mercaptoacetate (MA) does not. As previous studies using inhibitors were limited to quantifying body temperature in placentals, we investigated whether inhibition of glucose or fatty acids for cellular oxidation induces torpor in the marsupial hibernator Cercartetus nanus, and how the response of metabolic rate is related to body temperature. Glucoprivation initiated a torpor-like state in C. nanus, but animals had much higher minimum body temperatures and metabolic rates than those of torpid food-deprived animals and arousal rates were slower. Moreover, 2DG-treated animals were thermoregulating at ambient temperatures of 20 and 12 degrees C, whereas food-deprived torpid animals were thermo-conforming. We suggest that glucoprivation reduces the hypothalamic body temperature set point, but only by about 8 degrees C rather than the approximately 28 degrees C during natural torpor. Reduced fatty acid availability via MA also induced a torpor-like state in some C. nanus, with physiological variables that did not differ from those of torpid food-deprived animals. We conclude that reduced glucose availability forms only part of the physiological trigger for torpor initiation in C. nanus. Reduced fatty acid availability, unlike for placental heterotherms, may be an important cue for torpor initiation in C. nanus, perhaps because marsupials lack functional brown adipose tissue.

Adaptation, Physiological↗

PubMed2005

Effects of magnesium availability on the activity of plasma membrane ion transporters and light-induced responses from broad bean leaf mesophyll.

Considering the physiological significance of Mg homeostasis in plants, surprisingly little is known about the molecular and ionic mechanisms mediating Mg transport across the plasma membrane and the impact of Mg availability on transport processes at the plasmalemma. In this study, a non-invasive ion-selective microelectrode technique (MIFE) was used to characterize the effects of Mg availability on the activity of plasma membrane H+, K+, Ca2+, and Mg2+ transporters in the mesophyll cells of broad bean (Vicia faba L.) plants. Based on the stoichiometry of ion-flux changes and results of pharmacological experiments, we suggest that at least two mechanisms are involved in Mg2+ uptake across the plasma membrane of bean mesophyll cells. One of them is a non-selective cation channel, also permeable to K+ and Ca2+. The other mechanism, operating at concentrations below 30 microM, was speculated to be an H+/Mg+ exchanger. Experiments performed on leaves grown at different levels of Mg availability (from deficient to excessive) showed that Mg availability has a significant impact on the activity of plasma-membrane transporters for Ca2+, K+, and H+. We discuss the physiological significance of Mg-induced changes in leaf electrophysiological responses to light and the ionic mechanisms underlying this process.

Biological Transport↗