Biomedical subjects

E Altman

Publications and source records attributed to E Altman.

At least 19 recordsLinked to original sources

PubMed2006

Homolactate fermentation by metabolically engineered Escherichia coli strains.

We report the homofermentative production of lactate in Escherichia coli strains containing mutations in the aceEF, pfl, poxB, and pps genes, which encode the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, and phosphoenolpyruvate synthase, respectively. The process uses a defined medium and two distinct fermentation phases: aerobic growth to an optical density of about 30, followed by nongrowth, anaerobic production. Strain YYC202 (aceEF pfl poxB pps) generated 90 g/liter lactate in 16 h during the anaerobic phase (with a yield of 0.95 g/g and a productivity of 5.6 g/liter . h). Ca(OH)(2) was found to be superior to NaOH for pH control, and interestingly, significant succinate also accumulated (over 7 g/liter) despite the use of N(2) for maintaining anaerobic conditions. Strain ALS961 (YYC202 ppc) prevented succinate accumulation, but growth was very poor. Strain ALS974 (YYC202 frdABCD) reduced succinate formation by 70% to less than 3 g/liter. (13)C nuclear magnetic resonance analysis using uniformly labeled acetate demonstrated that succinate formation by ALS974 was biochemically derived from acetate in the medium. The absence of uniformly labeled succinate, however, demonstrated that glyoxylate did not reenter the tricarboxylic acid cycle via oxaloacetate. By minimizing the residual acetate at the time that the production phase commenced, the process with ALS974 achieved 138 g/liter lactate (1.55 M, 97% of the carbon products), with a yield of 0.99 g/g and a productivity of 6.3 g/liter . h during the anaerobic phase.

Aerobiosis↗

PubMed2006

Increased recombinant protein production in Escherichia coli strains with overexpressed water-forming NADH oxidase and a deleted ArcA regulatory protein.

Glycolytic flux is increased and acetate production is reduced in Escherichia coli by the expression of heterologous NADH oxidase (NOX) from Streptococcus pneumoniae coupled with the deletion of the arcA gene, which encodes the ArcA regulatory protein. In this study, we examined the overproduction of a model recombinant protein in strains of E. coli expressing NOX with or without an arcA mutation. The presence of NOX or the absence of ArcA reduced acetate by about 50% and increased beta-galactosidase production by 10-20%. The presence of NOX in the arcA strain eliminated acetate production entirely in batch fermentations and resulted in a 120% increase in beta-galactosidase production.

Acetates↗

PubMed2006

Overflow metabolism in Escherichia coli during steady-state growth: transcriptional regulation and effect of the redox ratio.

Overflow metabolism in the form of aerobic acetate excretion by Escherichia coli is an important physiological characteristic of this common industrial microorganism. Although acetate formation occurs under conditions of high glucose consumption, the genetic mechanisms that trigger this phenomenon are not clearly understood. We report on the role of the NADH/NAD ratio (redox ratio) in overflow metabolism. We modulated the redox ratio in E. coli through the expression of Streptococcus pneumoniae (water-forming) NADH oxidase. Using steady-state chemostat cultures, we demonstrated a strong correlation between acetate formation and this redox ratio. We furthermore completed genome-wide transcription analyses of a control E. coli strain and an E. coli strain overexpressing NADH oxidase. The transcription results showed that in the control strain, several genes involved in the tricarboxylic acid (TCA) cycle and respiration were repressed as the glucose consumption rate increased. Moreover, the relative repression of these genes was alleviated by expression of NADH oxidase and the resulting reduced redox ratio. Analysis of a promoter binding site upstream of the genes which correlated with redox ratio revealed a degenerate sequence with strong homology with the binding site for ArcA. Deletion of arcA resulted in acetate reduction and increased the biomass yield due to the increased capacities of the TCA cycle and respiration. Acetate formation was completely eliminated by reducing the redox ratio through expression of NADH oxidase in the arcA mutant, even at a very high glucose consumption rate. The results provide a basis for studying new regulatory mechanisms prevalent at reduced NADH/NAD ratios, as well as for designing more efficient bioprocesses.

Acetates↗

PubMed2005

Superfluid-insulator transition in a moving system of interacting bosons.

We analyze the stability of superfluid currents in a system of strongly interacting ultracold atoms in an optical lattice. We show that such a system undergoes a dynamic, irreversible phase transition at a critical phase gradient that depends on the interaction strength between atoms. At commensurate filling, the phase boundary continuously interpolates between the classical modulation instability of a weakly interacting condensate and the equilibrium quantum phase transition into a Mott insulator state at which the critical current vanishes. We argue that quantum fluctuations smear the transition boundary in low dimensional systems. Finally we discuss the implications to realistic experiments.

Journal Article↗

PubMed2005

Physiological response of central metabolism in Escherichia coli to deletion of pyruvate oxidase and introduction of heterologous pyruvate carboxylase.

We studied the physiological response of Escherichia coli central metabolism to the expression of heterologous pyruvate carboxylase (PYC) in the presence and absence of pyruvate oxidase (POX). These studies were complemented with expression analysis of central and intermediary metabolic genes and conventional in vitro enzyme assays to evaluate glucose metabolism at steady-state growth conditions (chemostats). The absence of POX activity reduced nongrowth-related energy metabolism (maintenance coefficient) and increased the maximum specific rate of oxygen consumption. The presence of PYC activity (i.e., with POX activity) increased the biomass yield coefficient and reduced the maximum specific oxygen consumption rate compared to the wildtype. The presence of PYC in a poxB mutant resulted in a 42% lower maintenance coefficient and a 42% greater biomass yield compared to the wildtype. Providing E. coli with PYC or removing POX increased the threshold specific growth rate at which acetate accumulation began, with an 80% reduction in acetate accumulation observed at a specific growth rate of 0.4 h-1 in the poxB-pyc+ strain. Gene expression analysis suggests utilization of energetically less favorable glucose metabolism via glucokinase and the Entner-Doudoroff pathway in the absence of functional POX, while the upregulation of the phosphotransferase glucose uptake system and several amino acid biosynthetic pathways occurs in the presence of PYC. The physiological and expression changes resulting from these genetic perturbations demonstrate the importance of the pyruvate node in respiration and its impact on acetate overflow during aerobic growth.

Acetates↗

PubMed2004

Aerobic production of alanine by Escherichia coli aceF ldhA mutants expressing the Bacillus sphaericus alaD gene.

Alanine was produced from glucose in an Escherichia coli aceF ldhA double mutant strain that contained the pTrc99A- alaD plasmid expressing Bacillus sphaericus alanine dehydrogenase. The aceF gene encodes one of the proteins of the pyruvate dehydrogenase complex, and therefore this strain required acetate as an additional carbon source. The ldhA gene encodes fermentative lactate dehydrogenase, a competitor of alanine dehydrogenase for the substrate pyruvate. Fermentations included an oxygenated growth phase followed by an oxygen-limited alanine production phase. The lowest value for the mass transfer coefficient ( k(L)a) studied during the production phase yielded the greatest alanine. With feeding of glucose and NH(4)Cl, 32 g/l alanine accumulated in 27 h with a yield of 0.63 g alanine generated per gram glucose consumed.

Aerobiosis↗

PubMed2004

Helicobacter pylori with a truncated lipopolysaccharide O chain fails to induce gastritis in SCID mice injected with splenocytes from wild-type C57BL/6J mice.

The goal of this study was to determine whether Helicobacter pylori lipopolysaccharide (LPS) O-chain polysaccharide contributes to gastritis in a mouse model. C57BL/6J or C57BL/6-Prkdc(scid) (severe combined immunodeficient [SCID]) mice were inoculated with H. pylori strain SS1 or SS1::0826kan, in which a beta-1,4-galactosyltransferase (HP0826), an LPS biosynthetic enzyme, had been disrupted. H. pylori strain SS1::0826kan expresses truncated LPS lacking O chain. Recipient SCID mice were given C57BL/6J splenocytes by intraperitoneal injection. Bacterial colonization, gastric lesions (gastritis, neutrophilic infiltration, and gastric epithelial metaplasia), cellular (delayed-type hypersensitivity) and humoral immune responses to H. pylori sonicate, and gastric gamma interferon (IFN-gamma) mRNA expression were quantified. Recipient SCID mice colonized by H. pylori strain SS1 developed extensive gastritis with loss of normal fundic gland morphology. In contrast, gastric mucosa of recipient SCID mice colonized by H. pylori strain SS1::0826kan was not statistically distinguishable from that of uninfected recipient mice. Delayed-type hypersensitivity and humoral immune responses were detected in infected mice inoculated with wild-type SS1, but not with SS1::0826kan. IFN-gamma transcription was lower in mice infected with SS1::0826kan than in mice infected with SS1. In this model of rapidly progressive gastritis due to H. pylori, the O chain contributed to the extent of gastritis and to the host immune response. These data support a role for H. pylori LPS O chain in direct induction of the host immune response leading to gastritis and gastric damage and are in contrast to protein antigens, such as urease and cag products which do not contribute to gastritis in mice.

Animals↗

PubMed2003

Optimization of recombinant aminolevulinate synthase production in Escherichia coli using factorial design.

The production of recombinant Rhodobacter sphaeroides aminolevulinate (ALA) synthase was optimized in two strains of Escherichia coli: the wild-type strain MG1655, and a ptsG mutant AFP111. The effects of initial succinate, glucose and isopropyl-beta-d-thiogalactopyranoside (IPTG) concentrations and the time of induction on enzyme activity were studied. One-way analysis was used to approximate the optimal ranges for these factors, followed by a full factorial design to quantify the effects of each factor and the interactions between the factors. Initial succinate, glucose, and IPTG concentration were observed to be the key factors affecting ALA synthase activity with the optimal levels determined to be above 6 g/l succinate, 0 g/l glucose, and 0.10 mM IPTG. ALA synthase activity was generally lower with AFP111 than with MG1655, and the effect of these three key factors was also lower with AFP111 than with MG1655. Based on the full factorial design results, a fermentation was completed that yielded 296 mU/mg protein with a final ALA concentration of 5.2 g/l (39 mM).

5-Aminolevulinate Synthetase↗

PubMed2003

The effect of acetate pathway mutations on the production of pyruvate in Escherichia coli.

We compared pyruvate accumulation in six strains of Escherichia coli and their corresponding ppc mutants. Each strain contained a mutation of a gene involved in the pathway to acetate synthesis. Strains with mutations in genes encoding the pyruvate dehydrogenase complex generally exhibited the greatest pyruvate accumulation of which CGSC6162 (an aceF mutant) and CGSC6162 Delta ppc were studied in greater detail in controlled fermenters. Both CGSC6162 and CGSC6162 Delta ppc accumulated greater than 35 g/l pyruvate in a medium supplemented with acetate. We observed pyruvate mass yields from glucose of 0.72 in CGSC6162, with volumetric productivities above 1.5 g l(-1) h(-1). For CGSC6162 Delta ppc, we observed pyruvate yields of 0.78 and volumetric productivities above 1.2 g l(-1) h(-1). CGSC6162 consumed all initially supplied acetate, while CGSC6162 Delta ppc first consumed and then generated acetate during the course of a 36 h fermentation. Acetate generation and pyruvate oxidase activity was pH- and temperature-dependent, with a pH of 7.0 and the lowest temperature studied (32 degrees C) favoring the greatest pyruvate generation. Lactate was an unexpected by-product even though measured lactate dehydrogenase (LDH) activity was very low.

Acetates↗

PubMed2003

Production of 5-aminolevulinic acid by an Escherichia coli aminolevulinate dehydratase mutant that overproduces Rhodobacter sphaeroides aminolevulinate synthase.

Two Escherichia coli strains, a wild-type strain and a hemB mutant, which contained the hemA gene from Rhodobacter sphaeroides produced aminolevulinate at 4 g l(-1). The hemB mutant did not increase the accumulation of aminolevulinate, an observation attributed to its 50% lower aminolevulinate synthase activity in than the wild-type. Growth was limited for both strains probably due to the accumulation of 0.5 g aminoacetone l(-1).

5-Aminolevulinate Synthetase↗

PubMed2003

Using protein-based motifs to stabilize peptides.

While the use of synthetically derived novel inhibitor peptides as a source of new therapeutics for medicine remains incredibly promising, there is a major problem with implementing this technology, as many synthetic peptides have proven to be unstable and are degraded by peptidases in the host cell. In this study, we have investigated methods by which peptides can be stabilized using protein-based motifs in order to prevent the action of peptidases. Using an in vivo approach our laboratory developed to screen for synthetic peptides which can inhibit the growth of Escherichia coli, we found that protecting the amino or carboxyl terminus of the peptides via fusion to the very stable Rop protein, or the incorporation of two proline residues, increased the frequency at which potent inhibitor peptides could be isolated. Using an in vitro degradation assay in which extracts from several different cell types were tested, we demonstrated that peptides stabilized with multiple proline residues were more resistant to degradation than peptides stabilized by amidation or acetylation, two approaches that are routinely utilized to improve the stability of peptide drugs.

Amino Acid Motifs↗

PubMed2002

Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions.

We examined succinic acid production in Escherichia coli AFP111 using dual-phase fermentations, which comprise an initial aerobic growth phase followed by an anaerobic production phase. AFP111 has mutations in the pfl, ldhA, and ptsG genes, and we additionally transformed this strain with the pyc gene (AFP111/pTrc99A-pyc) to provide metabolic flexibility at the pyruvate node. Aerobic fermentations with these two strains were completed to catalog physiological states during aerobic growth that might influence succinate generation in the anaerobic phase. Activities of six key enzymes were also determined for these aerobic fermentations. From these results, six transition times based on physiological states were selected for studying dual-phase fermentations. The final succinate yield and productivity depend greatly on the physiological state of the cells at the time of transition. Using the best transition time, fermentations achieved a final succinic acid concentration of 99.2 g/l with an overall yield of 110% and productivity of 1.3 g/l h.

Aerobiosis↗

PubMed2002

Expression of an anaplerotic enzyme, pyruvate carboxylase, improves recombinant protein production in Escherichia coli.

Anaplerotic enzyme reactions are those which replenish tricarboxylic acid intermediates that are withdrawn for the synthesis of biomass. In this study, we examined recombinant protein production in Escherichia coli containing activity in an additional anaplerotic enzyme, pyruvate carboxylase. In batch fermentations, the presence of pyruvate carboxylase resulted in 68% greater production of the model protein, beta-galactosidase, 41% greater cell yield, and 57% lower acetate concentration. We discuss why these results indicate that acetate concentration does not limit cell growth and protein synthesis, as predicted by other researchers, and suggest instead that the rate of acetate formation represents an inefficient consumption of glucose carbon, which is reduced by the presence of pyruvate carboxylase.

Biotechnology↗

PubMed2002

Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli.

Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.

Aerobiosis↗

PubMed2001

A comparative evaluation of three self-rating scales for acute mania.

This study compared the performance of three self-rating mania scales, The Internal State Scale (ISS), the Self-Report Manic Inventory (SRMI), and the Altman Self-Rating Mania Scale (ASRM), in a group of patients with acute mania. Forty-four adult inpatients with bipolar disorder, manic or mixed, completed all scales shortly after admission, and 31 patients completed them again after 4-6 weeks of pharmacotherapy. Patients also were rated by clinicians on the Clinician-Administered Rating Scale for Mania (CARS-M). At baseline, scores on the ASRM and the ISS well-being subscale were significantly correlated with CARS-M scores. Posttreatment scores were significantly decreased for the ASRM, SRMI, and the ISS activation subscale. The sensitivities for each scale to correctly identify patients with acute symptoms was 45% for the ISS, 86% for the SRMI, and 93% for the ASRM. Specificities were 73%, 46.6%, and 33%, respectively. The ASRM and SRMI were more sensitive than the ISS in screening patients with acute mania. All three measures were sensitive to treatment effects; however, the item content of the SRMI and the poor sensitivity of the ISS may limit their utility in inpatient settings.

Acute Disease↗

PubMed2001

The physiological effects and metabolic alterations caused by the expression of Rhizobium etli pyruvate carboxylase in Escherichia coli.

Oxaloacetate (OAA) plays an important role in the tricarboxylic acid cycle and for the biosynthesis of a variety of cellular compounds. Some microorganisms, such as Rhizobium etli and Corynebacterium glutamicum, are able to synthesize OAA during growth on glucose via either of the enzymes pyruvate carboxylase (PYC) or phosphoenolpyruvate carboxylase (PPC). Other microorganisms, including Escherichia coli, synthesize OAA during growth on glucose only via PPC because they lack PYC. In this study we have examined the effect that the R. etli PYC has on the physiology of E. coli. The expressed R. etli PYC was biotinylated by the native biotin holoenzyme synthase of E. coli and displayed kinetic properties similar to those reported for alpha4 PYC enzymes from other sources. R. etli PYC was able to restore the growth of an E. coli ppc null mutant in minimal glucose medium, and PYC expression caused increased carbon flow towards OAA in wild-type E. coli cells without affecting the glucose uptake rate or the growth rate. During aerobic glucose metabolism, expression of PYC resulted in a 56% increase in biomass yield and a 43% decrease in acetate yield. During anaerobic glucose metabolism, expression of PYC caused a 2.7-fold increase in succinate concentration, making it the major product by mass. The increase in succinate came mainly at the expense of lactate formation. However, in a mutant lacking lactate dehydrogenase activity, expression of PYC resulted in only a 1.7-fold increase in succinate concentration. The decreased enhancement of succinate formation in the /dh mutant was hypothesized to be due to accumulation of pyruvate and NADH, metabolites that affect the interconversion of the active and inactive form of the enzyme pyruvate formate-lyase.

Acetates↗

PubMed2001

Modified Seldinger technique for the insertion of standard chest tubes.

Closed tube thoracostomy is a standard procedure for the evacuation of air, blood, or other materials from the pleural space. This paper describes a modification of the Seldinger technique that facilitates chest tube insertion. Either a Nelaton or Thieman catheter is threaded into the side drainage hole and out the tip of a standard Argyle-type chest tube. After using the clamp to insert the catheter into the pleural space through a previously dissected tract, the catheter serves as a guide over which the chest tube is inserted. The technique is simple to use, effective, and safe. It employs standard, inexpensive materials to insert chest tubes in such a way as to minimize the potential traumatic complications inherent in other techniques.

Chest Tubes↗

PubMed2001

An in vivo study of novel bioactive peptides that inhibit the growth of Escherichia coli.

We have created a system in which synthetically produced novel bioactive peptides can be expressed in vivo in Escherichia coli. Twenty thousand of these peptides were screened and 21 inhibitors were found that could inhibit the growth of E. coli on minimal media. The inhibitors could be placed into one of two groups, 1-day inhibitors, which were partially inhibitory, and 2-day inhibitors, which were completely inhibitory. Sequence analysis showed that two of the most potent inhibitors were actually peptide-protein chimeras in which the peptides had become fused to the 63 amino acid Rop protein which was also contained in the expression vector used in this study. Given that Rop is known to form an incredibly stable structure, it could be serving as a stabilizing motif for these peptides. Sequence analysis of the predicted coding regions from the next 10 most inhibitory peptides showed that four of the 10 peptides contained one or more proline residues either at or very near the C-terminal end of the peptide which could act to prevent degradation by peptidases. Collectively, based on what we observed in our screen of synthetic bioactive peptides that could prevent the growth of E. coli and what has been learned from structural studies of naturally occurring bioactive peptides, the presence of a stabilizing motif seems to be important for small peptides, if they are to be biologically active.

Anti-Bacterial Agents↗