A genetic overhaul of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 424A(LNH-ST) to improve xylose fermentation

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD⁺-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.     

Development of isoform selective PI3-kinase inhibitors as pharmacological tools for elucidating the PI3K pathway

Using a parallel synthesis approach to target a non-conserved region of the PI3K catalytic domain a pan-PI3K inhibitor 1 was elaborated to provide alpha, delta and gamma isoform selective Class I PI3K inhibitors 21, 24, 26 and 27. The compounds had good cellular activity and were selective against protein kinases and other members of the PI3K superfamily including mTOR and DNA-PK.     

Genomewide linkage scan for split-hand/foot malformation with long-bone deficiency in a large Arab family identifies two novel susceptibility loci on chromosomes 1q42.2-q43 and 6q14.1

Split-hand/foot malformation with long-bone deficiency (SHFLD) is a rare, severe limb deformity characterized by tibia aplasia with or without split-hand/split-foot deformity. Identification of genetic susceptibility loci for SHFLD has been unsuccessful because of its rare incidence, variable phenotypic expression and associated anomalies, and uncertain inheritance pattern. SHFLD is usually inherited as an autosomal dominant trait with reduced penetrance, although recessive inheritance has also been postulated. We conducted a genomewide linkage analysis, using a 10K SNP array in a large consanguineous family (UR078) from the United Arab Emirates (UAE) who had disease transmission consistent with an autosomal dominant inheritance pattern. The study identified two novel SHFLD susceptibility loci at 1q42.2-q43 (nonparametric linkage [NPL] 9.8, P=.000065) and 6q14.1 (NPL 7.12, P=.000897). These results were also supported by multipoint parametric linkage analysis. Maximum multipoint LOD scores of 3.20 and 3.78 were detected for genomic locations 1q42.2-43 and 6q14.1, respectively, with the use of an autosomal dominant mode of inheritance with reduced penetrance. Haplotype analysis with informative crossovers enabled mapping of the SHFLD loci to a region of approximately 18.38 cM (8.4 Mb) between single-nucleotide polymorphisms rs1124110 and rs535043 on 1q42.2-q43 and to a region of approximately 1.96 cM (4.1 Mb) between rs623155 and rs1547251 on 6q14.1. The study identified two novel loci for the SHFLD phenotype in this UAE family.     

Three flavonoid glycosides containing acetylated allose from stachys recta

By means of ¹³C and ¹H NMR spectroscopy three flavone glycosides, obtained from Stachys recta, were identified as 7-O-(2″-O-6″′-O-acetyl-β-D-allopyranosyl-β-D-glucopyranosides) of 4′-O-methylisoscutellarein, isoscutellarein and 3′-hydroxy-4′-O-methylisoscutellarein. The latter two compounds are isolated for the first time. Only mannose and glucose have been reported previously as sugar components of flavonoids of the genus Stachys.     

Neutralizing a Surface Charge on the FMN Subdomain Increases the Activity of Neuronal Nitric-oxide Synthase by Enhancing the Oxygen Reactivity of the Enzyme Heme-Nitric Oxide Complex

Nitric-oxide synthases (NOSs) are calmodulin-dependent flavoheme enzymes that oxidize l-Arg to nitric oxide (NO) and l-citrulline. Their catalytic behaviors are complex and are determined by their rates of heme reduction (k_r), ferric heme-NO dissociation (k_d), and ferrous heme-NO oxidation (k_ox). We found that point mutation (E762N) of a conserved residue on the enzyme''s FMN subdomain caused the NO synthesis activity to double compared with wild type nNOS. However, in the absence of l-Arg, NADPH oxidation rates suggested that electron flux through the heme was slower in E762N nNOS, and this correlated with the mutant having a 60% slower k_r. During NO synthesis, little heme-NO complex accumulated in the mutant, compared with ∼50–70% of the wild-type nNOS accumulating as this complex. This suggested that the E762N nNOS is hyperactive because it minimizes buildup of an inactive ferrous heme-NO complex during NO synthesis. Indeed, we found that k_ox was 2 times faster in the E762N mutant than in wild-type nNOS. The mutational effect on k_ox was independent of calmodulin. Computer simulation and experimental measures both indicated that the slower k_r and faster k_ox of E762N nNOS combine to lower its apparent K_m,O2 for NO synthesis by at least 5-fold, which in turn increases its V/K_m value and enables it to be hyperactive in steady-state NO synthesis. Our work underscores how sensitive nNOS activity is to changes in the k_ox and reveals a novel means for the FMN module or protein-protein interactions to alter nNOS activity.Nitric oxide (NO)² is a biological mediator that is produced in animals by three NO synthase isozymes (NOS, EC 1.14.13.39): inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) (1, 2). The NOS are modular enzymes composed of an N-terminal oxygenase domain and a C-terminal flavoprotein domain, with a calmodulin (CaM)-binding site connecting the two domains (3). During NO synthesis, the flavoprotein domain transfers NADPH-derived electrons through its FAD and FMN cofactors to a heme located in the oxygenase domain. The FMN-to-heme electron transfer enables heme-dependent oxygen activation and a stepwise conversion of l-Arg to NO and citrulline (4, 5). Heme reduction also requires that CaM be bound to NOS and is rate-limiting for NO biosynthesis (6–9).NOS enzymes operate under the constraint of having their newly made NO bind to the ferric heme before it can exit the enzyme (10). How this intrinsic heme-NO binding event impacts NOS catalytic cycling is shown in Fig. 1 and has previously been discussed in detail (10–13). The l-Arg to NO biosynthetic reaction (Fe^III to Fe^IIINO in Fig. 1) is limited by the rate of ferric heme reduction (k_r), because all biosynthetic steps downstream are faster than k_r. However, once the ferric heme-NO complex forms at the end of each catalytic cycle, it can either dissociate to release NO into the medium (at a rate k_d as shown in Fig. 1) or become reduced by the flavoprotein domain (at a rate k′_r in Fig. 1; equal to k_r) to form the enzyme ferrous heme-NO species (Fe^IINO), which releases NO very slowly (11, 12). Consequently, two cycles compete during steady-state NO synthesis (Fig. 1); NO dissociation from the ferric heme (k_d) is part of a “productive cycle” that releases NO and is essential for NOS bioactivity, whereas reduction of the ferric heme-NO complex (k_r′) channels the enzyme into a “futile cycle” that actually represents a NO dioxygenase activity. The rate of futile cycling is also determined by the rate of O₂ reaction with the ferrous heme-NO complex (at a rate k_ox in Fig. 1), which regenerates the ferric enzyme. Surprisingly, NOS enzymes have evolved to have a broad range of k_r (varies 40×), k_ox (varies 15×), and k_d (varies 30×) values (Table S1) (12). This causes each NOS to distribute quite differently during steady-state NO synthesis and gives each NOS a unique catalytic profile (12).Open in a separate windowFIGURE 1.Global kinetic model for NOS catalysis. Ferric enzyme reduction (k_r) is rate-limiting for the biosynthetic reactions (central linear portion). k_cat1 and k_cat2 are the conversion rates of the enzyme Fe^IIO₂ species to products in the l-Arg and N^ω-hydroxy-l-arginine (NOHA) reactions, respectively. The ferric heme-NO product complex (Fe^IIINO) can either release NO (k_d) or become reduced (k′_r) to a ferrous heme-NO complex (Fe^IINO), which reacts with O₂ (k_ox) to regenerate ferric enzyme. Enzyme partitioning and NO release are determined by the relative rates of k_r, k_ox, and k_d. This figure is adapted from Ref. 12.The enzyme physical and electronic factors that may set and regulate each of the three kinetic parameters (k_r, k_ox, and k_d) in NOS enzymes remain to be fully described. At present, the composition of the NOS flavoprotein domain and CaM appear to be primarily responsible for determining the k_r (14–17), whereas the composition of the NOS oxygenase domain is presumed to determine the k_d and k_ox (18, 19). Indeed, our recent point mutagenesis study identified a patch of electronegative residues on the FMN subdomain that are required to maintain a normal k_r and NO synthesis activity in nNOS, suggesting that subdomain electrostatic interactions are important in the process (20). We found particularly large effects when the negative charge at Glu⁷⁶² was neutralized via mutation to Asn. Remarkably, the NO synthesis activity of E762N nNOS was double that of wild-type nNOS, despite the mutant displaying a slow k_r that was half of wild type. In the current report, we show that the E762N mutation has an additional, unsuspected effect on the k_ox kinetic parameter of nNOS. How this effect alters distribution of the nNOS enzyme during steady-state catalysis, impacts the apparent K_m,O2, and leads to hyperactive NO synthesis is described. Our finding that the nNOS flavoprotein domain can tune a key kinetic parameter that defines the rate of a heme-based reaction in the nNOS oxygenase domain is unusual and suggests a means by which protein-protein interactions could regulate the catalytic behavior of nNOS.     

Critical factors influencing the occurrence of Vibrio cholerae in the environment of Bangladesh

The occurrence of outbreaks of cholera in Africa in 1970 and in Latin America in 1991, mainly in coastal communities, and the appearance of the new serotype Vibrio cholerae O139 in India and subsequently in Bangladesh have stimulated efforts to understand environmental factors influencing the growth and geographic distribution of epidemic Vibrio cholerae serotypes. Because of the severity of recent epidemics, cholera is now being considered by some infectious disease investigators as a "reemerging" disease, prompting new work on the ecology of vibrios. Epidemiological and ecological surveillance for cholera has been under way in four rural, geographically separated locations in Bangladesh for the past 4 years, during which both clinical and environmental samples were collected at biweekly intervals. The clinical epidemiology portion of the research has been published (Sack et al., J. Infect. Dis. 187:96-101, 2003). The results of environmental sampling and analysis of the environmental and clinical data have revealed significant correlations of water temperature, water depth, rainfall, conductivity, and copepod counts with the occurrence of cholera toxin-producing bacteria (presumably V. cholerae). The lag periods between increases or decreases in units of factors, such as temperature and salinity, and occurrence of cholera correlate with biological parameters, e.g., plankton population blooms. The new information on the ecology of V. cholerae is proving useful in developing environmental models for the prediction of cholera epidemics.     

A Critical Tryptophan and Ca2+ in Activation and Catalysis of TPPI,the Enzyme Deficient in Classic Late-Infantile Neuronal Ceroid Lipofuscinosis

Background

Tripeptidyl aminopeptidase I (TPPI) is a crucial lysosomal enzyme that is deficient in the fatal neurodegenerative disorder called classic late-infantile neuronal ceroid lipofuscinosis (LINCL). It is involved in the catabolism of proteins in the lysosomes. Recent X-ray crystallographic studies have provided insights into the structural/functional aspects of TPPI catalysis, and indicated presence of an octahedrally coordinated Ca²⁺.

Methodology

Purified precursor and mature TPPI were used to study inhibition by NBS and EDTA using biochemical and immunological approaches. Site-directed mutagenesis with confocal imaging technique identified a critical W residue in TPPI activity, and the processing of precursor into mature enzyme.

Principal Findings

NBS is a potent inhibitor of the purified TPPI. In mammalian TPPI, W542 is critical for tripeptidyl peptidase activity as well as autocatalysis. Transfection studies have indicated that mutants of the TPPI that harbor residues other than W at position 542 have delayed processing, and are retained in the ER rather than transported to lysosomes. EDTA inhibits the autocatalytic processing of the precursor TPPI.

Conclusions/Significance

We propose that W542 and Ca²⁺ are critical for maintaining the proper tertiary structure of the precursor proprotein as well as the mature TPPI. Additionally, Ca²⁺ is necessary for the autocatalytic processing of the precursor protein into the mature TPPI. We have identified NBS as a potent TPPI inhibitor, which led in delineating a critical role for W542 residue. Studies with such compounds will prove valuable in identifying the critical residues in the TPPI catalysis and its structure-function analysis.     

Tablet formulation containing meloxicam and beta-cyclodextrin: mechanical characterization and bioavailability evaluation

The purpose of this research was to evaluate beta-cyclodextrin (beta-CD) as a vehicle, either singly or in blends with lactose (spray-dried or monohydrate), for preparing a meloxicam tablet. Aqueous solubility of meloxicam in presence of beta-CD was investigated. The tablets were prepared by direct compression and wet granulation techniques. The powder blends and the granules were evaluated for angle of repose, bulk density, compressibility index, total porosity, and drug content. The tablets were subjected to thickness, diameter, weight variation test, drug content, hardness, friability, disintegration time, and in vitro dissolution studies. The effect of beta-CD on the bioavailability of meloxicam was also investigated in human volunteers using a balanced 2-way crossover study. Phase-solubility studies indicated an A(L)-type diagram with inclusion complex of 1:1 molar ratio. The powder blends and granules of all formulations showed satisfactory flow properties, compressibility, and drug content. All tablet formulations prepared by direct compression or wet granulation showed acceptable mechanical properties. The dissolution rate of meloxicam was significantly enhanced by inclusion of beta-CD in the formulations up to 30%. The mean pharmacokinetic parameters (C(max), K(e), and area under the curve [AUC](0-infinity)) were significantly increased in presence of beta-CD. These results suggest that beta-CD would facilitate the preparation of meloxicam tablets with acceptable mechanical properties using the direct compression technique as there is no important difference between tablets prepared by direct compression and those prepared by wet granulation. Also, beta-CD is particularly useful for improving the oral bioavailablity of meloxicam.     

Oxidative metabolism and protoplast culture

Summary Barley leaf blade protoplasts accumulate malonaldehyde, a product of lipid peroxidation, during culture. In addition, glutathione levels fall after protoplast isolation and the proportion of glutathione in the oxidized state rises. These data indicate oxidative stress after protoplast isolation and during culture. The cause of this phenomenon is revealed by data showing that the activities of enzymes associated with antioxidative processes including glutathione reductase and ascorbate peroxidase decrease after barley protoplast isolation. In contrast, protoplasts isolated from suspension cultured cells of bromegrass and soybean exhibit little evidence for oxidative stress and increased activities of glutathione reductase and ascorbate peroxidase. We suggest that an antioxidative response is associated with mitosis and colony formation from protoplasts, as exhibited by bromegrass and soybean. Conversely, failure of an antioxidative response is associated with low viability and absence of mitosis, as in barley. Increased viability of barley leaf protoplasts cultured on feeder layer cells is correlated with increased glutathione content and higher glutathione reductase activity.     

Molecular genetics of met 17 and met 25 mutants of Saccharomyces cerevisiae: intragenic complementation between mutations of a single structural gene

Summary A method for transposon mutagenesis in Azospirillum lipoferum 29708 is reported with transposon Tn5. The suicide plasmid pSUP2021 was used to deliver Tn5 in A. lipoferum using Escherichia coli SM10 as the donor. Neomycin-resistant transconjugants were detected at a frequency of 6x10^-6 per recipient. Different types of mutants were isolated, e.g. auxotrophic, coloured, IAA-negative, and IAA-overproducers. Among the auxotrophic mutants, cysteine and methionine requirers prevailed. Random Tn5-insertion with only one copy per mutant was demonstrated by Southern blotting and hybridization. Tn5-induced mutants are relatively stable, with reversion rates of 2–20×10^-8. A gene which is a part of the carotenoid pathway is closely linked to the histidine genes. The existence of two pathways for IAA production in A. lipoferum is discussed.