Reversal of coenzyme specificity of 2,3‐butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis

Saccharomyces cerevisiae NAD(H)‐dependent 2,3‐butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3‐butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)‐dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu²²¹, Ile²²², and Ala²²³) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site‐directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration. Biotechnol. Bioeng. 2009; 104: 381–389 © 2009 Wiley Periodicals, Inc.     

A Comparative Study on In Vitro Osteogenic Priming Potential of Electron Spun Scaffold PLLA/HA/Col,PLLA/HA,and PLLA/Col for Tissue Engineering Application

A comparative study on the in vitro osteogenic potential of electrospun poly-L-lactide/hydroxyapatite/collagen (PLLA/HA/Col, PLLA/HA, and PLLA/Col) scaffolds was conducted. The morphology, chemical composition, and surface roughness of the fibrous scaffolds were examined. Furthermore, cell attachment, distribution, morphology, mineralization, extracellular matrix protein localization, and gene expression of human mesenchymal stromal cells (hMSCs) differentiated on the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA were also analyzed. The electrospun scaffolds with a diameter of 200–950 nm demonstrated well-formed interconnected fibrous network structure, which supported the growth of hMSCs. When compared with PLLA/H%A and PLLA/Col scaffolds, PLLA/Col/HA scaffolds presented a higher density of viable cells and significant upregulation of genes associated with osteogenic lineage, which were achieved without the use of specific medium or growth factors. These results were supported by the elevated levels of calcium, osteocalcin, and mineralization (P<0.05) observed at different time points (0, 7, 14, and 21 days). Furthermore, electron microscopic observations and fibronectin localization revealed that PLLA/Col/HA scaffolds exhibited superior osteoinductivity, when compared with PLLA/Col or PLLA/HA scaffolds. These findings indicated that the fibrous structure and synergistic action of Col and nano-HA with high-molecular-weight PLLA played a vital role in inducing osteogenic differentiation of hMSCs. The data obtained in this study demonstrated that the developed fibrous PLLA/Col/HA biocomposite scaffold may be supportive for stem cell based therapies for bone repair, when compared with the other two scaffolds.     

Exercise training prevents decline in stroke volume during exercise in young healthy subjects.

Stroke volume (SV) increases above the resting level during exercise and then declines at higher intensities of exercise in sedentary subjects. The purpose of this study was to determine whether an attenuation of the decline in SV at higher exercise intensities contributes to the increase in maximal cardiac output (Qmax) that occurs in response to endurance training. We studied six men and six women, 25 +/- 1 (SE) yr old, before and after 12 wk of endurance training (3 days/wk running for 40 min, 3 days/wk interval training). Cardiac output was measured at rest and during exercise at 50 and 100% of maximal O2 uptake (Vo2max) by the C2H2-rebreathing method. VO2max was increased by 19% (from 2.7 +/- 0.2 to 3.2 +/- 0.3 l/min, P less than 0.001) in response to the training program. Qmax was increased by 12% (from 18.1 +/- 1 to 20.2 +/- 1 l/min, P less than 0.01), SV at maximal exercise was increased by 16% (from 97 +/- 6 to 113 +/- 8 ml/beat, P less than 0.001) and maximal heart rate was decreased by 3% (from 185 +/- 2 to 180 +/- 2 beats/min, P less than 0.01) after training. The calculated arteriovenous O2 content difference at maximal exercise was increased by 7% (14.4 +/- 0.4 to 15.4 +/- 0.4 ml O2/100 ml blood) after training. Before training, SV at VO2max was 9% lower than during exercise at 50% VO2max (P less than 0.05). In contrast, after training, the decline in SV between 50 and 100% VO2max was only 2% (P = NS). Furthermore, SV was significantly higher (P less than 0.01) at 50% VO2max after training than it was before. Left ventricular hypertrophy was evident, as determined by two-dimensional echocardiography at the completion of training. The results indicate that in young healthy subjects the training-induced increase in Qmax is due in part to attenuation of the decrease in SV as exercise intensity is increased.     

Functional Consequences of Sulfhydryl Modification of the γ-Aminobutyric Acid Transporter 1 at a Single Solvent-Exposed Cysteine Residue

The aims of this study were to optimize the experimental conditions for labeling extracellularly oriented, solvent-exposed cysteine residues of ??-aminobutyric acid transporter 1 (GAT1) with the membrane-impermeant sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) and to characterize the functional and pharmacological consequences of labeling on transporter steady-state and presteady-state kinetic properties. We expressed human GAT1 in Xenopus laevis oocytes and used radiotracer and electrophysiological methods to assay transporter function before and after sulfhydryl modification with MTSET. In the presence of NaCl, transporter exposure to MTSET (1?C2.5?mM for 5?C20?min) led to partial inhibition of GAT1-mediated transport, and this loss of function was completely reversed by the reducing reagent dithiothreitol. MTSET treatment had no functional effect on the mutant GAT1 C74A, whereas the membrane-permeant reagents N-ethylmaleimide and tetramethylrhodamine-6-maleimide inhibited GABA transport mediated by GAT1 C74A. Ion replacement experiments indicated that MTSET labeling of GAT1 could be driven to completion when valproate replaced chloride in the labeling buffer, suggesting that valproate induces a GAT1 conformation that significantly increases C74 accessibility to the extracellular fluid. Following partial inhibition by MTSET, there was a proportional reduction in both the presteady-state and steady-state macroscopic signals, and the functional and pharmacological properties of the remaining signals were indistinguishable from those of unlabeled GAT1. Therefore, covalent modification of GAT1 at C74 results in completely nonfunctional as well as electrically silent transporters.     

Neuron survival in vitro is more influenced by the developmental age of the cells than by glucose condition

The objective of this study was to determine whether the sensitivity to varying glucose conditions differs for the peripheral and central nervous system neurons at different developmental stages. Ventral horn neurons (VHN) and dorsal root ganglion neurons (DRG) from rats of different postnatal ages were exposed to glucose-free or glucose-rich culture conditions. Following 24 h at those conditions, the number of protein gene product 9.5 positive (PGP⁺) DRG neurons and choline acetyltransferase positive (ChAT⁺) VHN were counted and their neurite lengths and soma diameters were measured. For both DRG and VHN, the highest number of cells with and without neurite outgrowth was seen when cells from postnatal day 4 donors were cultured, while the lowest cell numbers were when neurons were from donors early after birth and grown under glucose-free conditions. The length of the neurites and the soma diameter for VHN were not affected by either glucose level or age. DRG neurons, however, exhibited the shortest neurites and smallest soma diameter when neurons were obtained and cultured early after birth. Our results indicate that survival of neurons in vitro is more influenced by the developmental stage than by glucose concentrations.     

Engineering of 2,3-Butanediol Dehydrogenase To Reduce Acetoin Formation by Glycerol-Overproducing,Low-Alcohol Saccharomyces cerevisiae

Engineered Saccharomyces cerevisiae strains overexpressing GPD1, which codes for glycerol-3-phosphate dehydrogenase, and lacking the acetaldehyde dehydrogenase Ald6 display large-scale diversion of the carbon flux from ethanol toward glycerol without accumulating acetate. Although GPD1 ald6 strains have great potential for reducing the ethanol contents in wines, one major side effect is the accumulation of acetoin, having a negative sensory impact on wine. Acetoin is reduced to 2,3-butanediol by the NADH-dependent 2,3-butanediol dehydrogenase Bdh1. In order to investigate the influence of potential factors limiting this reaction, we overexpressed BDH1, coding for native NADH-dependent Bdh1, and the engineered gene BDH1_221,222,223, coding for an NADPH-dependent Bdh1 enzyme with the amino acid changes 221 EIA 223 to 221 SRS 223, in a glycerol-overproducing wine yeast. We have shown that both the amount of Bdh1 and the NADH availability limit the 2,3-butanediol dehydrogenase reaction. During wine fermentation, however, the major limiting factor was the level of synthesis of Bdh1. Consistent with this finding, the overproduction of native or engineered Bdh1 made it possible to redirect 85 to 90% of the accumulated acetoin into 2,3-butanediol, a compound with neutral sensory characteristics. In addition, the production of diacetyl, a compound causing off-flavor in alcoholic beverages, whose production is increased in glycerol-overproducing yeast cells, was decreased by half. The production of higher alcohols and esters, which was slightly decreased or unchanged in GPD1 ald6 cells compared to that in the control cells, was not further modified in BDH1 cells. Overall, rerouting carbons toward glycerol and 2,3-butanediol represents a new milestone in the engineering of a low-alcohol yeast with desirable organoleptic features, permitting the decrease of the ethanol contents in wines by up to 3°.A large number of quality wines produced by modern winemaking practices, which favor harvesting fully ripened grapes, frequently contain an excessive ethanol content. This tendency is observed in the majority of the world''s wine-producing areas, and reducing the alcohol levels in wines has become a major concern of the wine industry. Consequently, numerous attempts have been made to engineer Saccharomyces cerevisiae yeast strains with reduced ethanol yields, which would offer faster and less expensive biological alternatives to the current physical processes available for the production of low- and reduced-alcohol wines (29). The biological approaches used so far are all based on diverting sugar metabolism toward by-products other than ethanol by metabolic engineering strategies (7, 8, 18, 19, 21, 22, 30). However, these strategies have so far not satisfied the need to obtain a significant reduction in the ethanol yield without causing the accumulation of undesirable secondary products and/or without affecting yeast physiology. Among these various advances, an efficient strategy is based on the rerouting of the carbon flux toward the production of glycerol. This polyol is a relatively neutral compound from an olfactory perspective, and it is has been demonstrated previously to contribute positively to wine quality through enhanced sweetness and viscosity (27). In yeast, glycerol plays a major role as an osmolyte under osmotic stress conditions and also functions as an essential redox sink in the absence of oxygen, when the reoxidation of excess cytosolic NADH is required (1, 2, 42, 44, 46). This compound is formed by the reduction of dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenase (encoded by GPD1 and GPD2), followed by dephosphorylation by glycerol-3-phosphatase, which exists as two isoforms: Gpp1 and Gpp2p (see Fig. ​Fig.1).1). By overexpressing GPD1 or GPD2, the production of glycerol has been greatly enhanced, making it possible to decrease the ethanol yield as the result of carbon diversion and reduced NADH availability for the alcohol dehydrogenase reaction. This strategy has been used previously to reduce the ethanol yields in wine and brewer''s yeast (4, 7, 23, 25, 26, 31). For wine, it was shown that these glycerol-overproducing strains have the potential to reduce the ethanol content by 1 to 2°. Nevertheless, major modifications in the production levels of other metabolites, in particular acetate and acetoin (23, 31), which are undesirable at high concentrations in wine, are generated. The production of acetate in glycerol-overproducing wine yeasts has been reduced to a normal level by the deletion of ALD6, coding for an acetaldehyde dehydrogenase (4, 30). The major problem of the accumulation of acetoin, which was shown to accumulate at several grams per liter in commercial GPD1 ald6 wine yeast strains (4), remains to be overcome. At usual concentrations in wine, which vary from undetectable levels to 80 mg/liter (32, 33, 40), this compound has no negative organoleptic influence. However, at concentrations higher than its threshold level (around 150 mg/liter [11]), acetoin can confer an unpleasant buttery flavor on wines. In contrast, the reduced form of acetoin, 2,3-butanediol (2,3-BD), has neutral sensory qualities (data not shown). It is found in wines at concentrations ranging from 0.2 to 3 g/liter (14, 41).Open in a separate windowFIG. 1.Schematic representation of metabolic pathways implicated in our design strategy for a low-alcohol yeast. GP, glycerol phosphatase, encoded by GPP1 and GPP2; GPDH, glycerol phosphate dehydrogenase, encoded by GPD1 and GPD2; PDC, pyruvate decarboxylase, encoded by PDC1, PDC5, and PDC6; ACDH, acetaldehyde dehydrogenase, encoded by ALD4, ALD5, and ALD6; ADH, alcohol dehydrogenase, encoded by ADH1; BDH, Bdh1, encoded by BDH1 (other BDHs exist; however, no other identified gene has been associated with BDH activity); ALS, acetolactate synthase, encoded by ILV2; DS, diacetyl synthetase; DR, diacetyl reductase; G3P, glycerol-3-phosphatase; DHAP, dihydroxyacetone phosphate; acetyl CoA, acetyl coenzyme A; TPP, thiamine PP_i.Bdh1, encoded by BDH1, is the only identified enzyme in yeast catalyzing the reduction of acetoin into 2,3-BD (12). This enzyme has strict stereospecificity for the OHs of carbons in R configuration and acts preferentially as a reductase rather than as a dehydrogenase (11, 12). It is essentially responsible for the formation of (2R,3R)-2,3-BD and part of meso-2,3-BD from (3R)-acetoin and (3S)-acetoin, respectively.The accumulation of acetoin in strains engineered for glycerol overproduction has been attributed to several factors (4). On one hand, it was assumed that the amount of Bdh1 is a rate-limiting factor in the conversion of acetoin into 2,3-BD. On the other hand, it is possible that the Bdh1 reaction is limited by the low level of available NADH since this coenzyme is preferentially used for glycerol synthesis in these strains.The aim of the present study was to investigate in detail the metabolic prerequisites for reducing accumulated acetoin in S. cerevisiae overproducing glycerol and exhibiting reduced acetate formation by promoting the conversion of acetoin into the compound 2,3-BD, which has neutral sensory characteristics. In this study, we first determined the role of Bdh1 in the reduction of acetoin into 2,3-BD during wine fermentation. Next, we studied the impact of the overproduction of NADH-dependent Bdh1 or an engineered NADPH-dependent form of Bdh1 in a model wine yeast, V5, overexpressing GPD1 and lacking ALD6 during fermentation in synthetic must with various sugar concentrations. The NADPH-dependent Bdh1 has been obtained previously by the replacement of three amino acids involved in the NADH binding domain, resulting in the complete reversal of the coenzyme specificity from NADH to NADPH (6). The effects on the growth and fermentation properties of the engineered strains and the levels of by-products and key aromatic compounds formed by the strains were determined.     

Tracking the dynamic interplay between bacterial and host factors during pathogen‐induced vacuole rupture in real time

Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen‐induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram‐negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin‐3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high‐content and high‐throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.     

Influence of comorbidities on therapeutic progression of diabetes treatment in Australian veterans: a cohort study

Background

This study assessed whether the number of comorbid conditions unrelated to diabetes was associated with a delay in therapeutic progression of diabetes treatment in Australian veterans.

Methodology/Principal Findings

A retrospective cohort study was undertaken using data from the Australian Department of Veterans'' Affairs (DVA) claims database between July 2000 and June 2008. The study included new users of metformin or sulfonylurea medicines. The outcome was the time to addition or switch to another antidiabetic treatment. The total number of comorbid conditions unrelated to diabetes was identified using the pharmaceutical-based comorbidity index, Rx-Risk-V. Competing risk regression analyses were conducted, with adjustments for a number of covariates that included age, gender, residential status, use of endocrinology service, number of hospitalisation episodes and adherence to diabetes medicines. Overall, 20134 veterans were included in the study. At one year, 23.5% of patients with diabetes had a second medicine added or had switched to another medicine, with 41.4% progressing by 4 years. The number of unrelated comorbidities was significantly associated with the time to addition of an antidiabetic medicine or switch to insulin (subhazard ratio [SHR] 0.87 [95% CI 0.84–0.91], P<0.001). Depression, cancer, chronic obstructive pulmonary disease, dementia, and Parkinson''s disease were individually associated with a decreased likelihood of therapeutic progression. Age, residential status, number of hospitalisations and adherence to anti-diabetic medicines delayed therapeutic progression.

Conclusions/Significance

Increasing numbers of unrelated conditions decreased the likelihood of therapeutic progression in veterans with diabetes. These results have implications for the development of quality measures, clinical guidelines and the construction of models of care for management of diabetes in elderly people with comorbidities.