Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease

Little is known about the potential for engraftment of autologous hematopoietic stem cells in human adults not subjected to myeloablative conditioning regimens. Five adult patients with the p47phox deficiency form of chronic granulomatous disease received intravenous infusions of autologous CD34+ peripheral blood stem cells (PBSCs) that had been transduced ex vivo with a recombinant retrovirus encoding normal p47phox. Although marrow conditioning was not given, functionally corrected granulocytes were detectable in peripheral blood of all five patients. Peak correction occurred 3–6 weeks after infusion and ranged from 0.004 to 0.05% of total peripheral blood granulocytes. Corrected cells were detectable for as long as 6 months after infusion in some individuals. Thus, prolonged engraftment of autologous PBSCs and continued expression of the transduced gene can occur in adults without conditioning. This trial also piloted the use of animal protein-free medium and a blood-bank-compatible closed system of gas-permeable plastic containers for culture and transduction of the PBSCs. These features enhance the safety of PBSCs directed gene therapy.

Decreased GA1 Content Caused by the Overexpression of OSH1 Is Accompanied by Suppression of GA 20-Oxidase Gene Expression

We previously reported that overexpression of the rice homeobox gene OSH1 led to altered morphology and hormone levels in transgenic tobacco (Nicotiana tabacum L.) plants. Among the hormones whose levels were changed, GA1 was dramatically reduced. Here we report the results of our analysis on the regulatory mechanism(s) of OSH1 on GA metabolism. GA53 and GA20, precursors of GA1, were applied separately to transgenic tobacco plants exhibiting severely changed morphology due to overexpression of OSH1. Only treatment with the end product of GA 20-oxidase, GA20, resulted in a striking promotion of stem elongation in transgenic tobacco plants. The internal GA1 and GA20 contents in OSH1-transformed tobacco were dramatically reduced compared with those of wild-type plants, whereas the level of GA19, a mid-product of GA 20-oxidase, was 25% of the wild-type level. We have isolated a cDNA encoding a putative tobacco GA 20-oxidase, which is mainly expressed in vegetative stem tissue. RNA-blot analysis revealed that GA 20-oxidase gene expression was suppressed in stem tissue of OSH1-transformed tobacco plants. Based on these results, we conclude that overexpression of OSH1 causes a reduction of the level of GA1 by suppressing GA 20-oxidase expression.

The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake.

The CCC2 gene of the yeast Saccharomyces cerevisiae is homologous to the human genes defective in Wilson disease and Menkes disease. A biochemical hallmark of these diseases is a deficiency of copper in ceruloplasmin and other copper proteins found in extracytosolic compartments. Here we demonstrate that disruption of the yeast CCC2 gene results in defects in respiration and iron uptake. These defects could be reversed by supplementing cells with copper, suggesting that CCC2 mutant cells were copper deficient. However, cytosolic copper levels and copper uptake were normal. Instead, CCC2 mutant cells lacked a copper-dependent oxidase activity associated with the extracytosolic domain of the FET3-encoded protein, a ceruloplasmin homologue previously shown to be necessary for high-affinity iron uptake in yeast. Copper restored oxidase activity both in vitro and in vivo, paralleling the ability of copper to restore respiration and iron uptake. These results suggest that the CCC2-encoded protein is required for the export of copper from the cytosol into an extracytosolic compartment, supporting the proposal that intracellular copper transport is impaired in Wilson disease and Menkes disease.

The upstream Variable Number Tandem Repeat polymorphism of the monoamine oxidase type A gene influences trigeminal pain-related evoked responses

Monoamines have an important role in neural plasticity, a key factor in cortical pain processing that promotes changes in neuronal network connectivity. Monoamine oxidase type A (MAOA) is an enzyme that, due to its modulating role in monoaminergic activity, could play a role in cortical pain processing. The X-linked MAOA gene is characterized by an allelic variant of length, the MAOA upstream Variable Number Tandem Repeat (MAOA-uVNTR) region polymorphism. Two allelic variants of this gene are known, the high-activity MAOA (HAM) and low-activity MAOA (LAM). We investigated the role of MAOA-uVNTR in cortical pain processing in a group of healthy individuals measured by the trigeminal electric pain-related evoked potential (tPREP) elicited by repeated painful stimulation. A group of healthy volunteers was genotyped to detect MAOA-uVNTR polymorphism. Electrical tPREPs were recorded by stimulating the right supraorbital nerve with a concentric electrode. The N2 and P2 component amplitude and latency as well as the N2-P2 inter-peak amplitude were measured. The recording was divided into three blocks, each containing 10 consecutive stimuli and the N2-P2 amplitude was compared between blocks. Of the 67 volunteers, 37 were HAM and 30 were LAM. HAM subjects differed from LAM subjects in terms of amplitude of the grand-averaged and first-block N2-P2 responses (HAM>LAM). The N2-P2 amplitude decreased between the first and third block in HAM subjects but not LAM subjects. The MAOA-uVNTR polymorphism seemed to influence the brain response in a repeated tPREP paradigm and suggested a role of the MAOA as a modulator of neural plasticity related to cortical pain processing. Monoamines have an important role in neural plasticity, a key factor in cortical pain processing that promotes changes in neuronal network connectivity. Monoamine oxidase type A (MAOA) is an enzyme that, due to its modulating role in monoaminergic activity, could play a role in cortical pain processing. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Isolation and characterization of plastid terminal oxidase gene from carrot and its relation to carotenoid accumulation

Carrot (Daucus carota L.) is a biennial plant that accumulates considerable amounts of carotenoid pigments in the storage root. To better understand the molecular mechanisms for carotenoid accumulation in developing storage roots, plastid terminal oxidase (PTOX) cDNA was isolated and selected for reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Present in photosynthetic species, PTOX is a plastid-located, nucleus encoded plastoquinone (PQ)-O2 oxidoreductase (plastioquinol oxidase). The enzyme is known to play a role as a cofactor for phytoene desaturase, and consequently plays a key role in the carotenoid biosynthesis pathway. A single PTOX gene was identified (DcPTOX) in carrot. DcPTOX encodes a putative protein with 366 amino acids that contains the typical structural features of PTOXs from higher plants. The expression of DcPTOX was analysed during the development of white, yellow, orange, red, and purple carrot roots, along with five genes known to be involved in the carotenoid biosynthesis pathway, PSY2, PDS, ZDS1, LCYB1, and LCYE. Expression analysis revealed the presence of DcPTOX transcripts in all cultivars, and an increase of transcripts during the time course of the experiment, with differential expression among cultivars in early stages of root growth. Our results demonstrated that DcPTOX showed a similar profile to that of other carotenoid biosynthetic genes with high correlation to all of them. The preponderant role of PSY in the biosynthesis of carotenoid pigments was also confirmed.

Genomic organisation, activity and distribution analysis of the microbial putrescine oxidase degradation pathway

The catalytic action of putrescine specific amine oxidases acting in tandem with 4-aminobutyraldehyde dehydrogenase is explored as a degradative pathway in Rhodococcus opacus. By limiting the nitrogen source, increased catalytic activity was induced leading to a coordinated response in the oxidative deamination of putrescine to 4-aminobutyraldehyde and subsequent dehydrogenation to 4-aminobutyrate. Isolating the dehydrogenase by ion exchange chromatography and gel filtration revealed that the enzyme acts principally on linear aliphatic aldehydes possessing an amino moiety. Michaelis-Menten kinetic analysis delivered a Michaelis constant (KM=0.014mM) and maximum rate (Vmax=11.2μmol/min/mg) for the conversion of 4-aminobutyraldehyde to 4-aminobutyrate. The dehydrogenase identified by MALDI-TOF mass spectrometric analysis (E value=0.031, 23% coverage) belongs to a functionally related genomic cluster that includes the amine oxidase, suggesting their association in a directed cell response. Key regulatory, stress and transport encoding genes have been identified, along with candidate dehydrogenases and transaminases for the further conversion of 4-aminobutyrate to succinate. Genomic analysis has revealed highly similar metabolic gene clustering among members of Actinobacteria, providing insight into putrescine degradation notably among Micrococcaceae, Rhodococci and Corynebacterium by a pathway that was previously uncharacterised in bacteria.

Cloning, expression, characterisation and mutational analysis of l-aspartate oxidase from Pseudomonas putida

L-Amino acid oxidases (LAAOs) are useful catalysts for the deracemisation of racemic amino acid sub-strates when combined with abiotic reductants. The gene nadB encoding the L-aspartate amino acid oxidase from Pseudomonas putida (PpLASPO) has been cloned and expressed in E. coli. The purified PpLASPO enzyme displayed a K M for l-aspartic acid of 2.26 mM and a k cat = 10.6 s −1 , with lower activity also displayed towards L-asparagine, for which pronounced substrate inhibition was also observed. The pH optimum of the enzyme was recorded at pH 7.4. The enzyme was stable for 60 min at up to 40 • C, but rapid losses in activity were observed at 50 • C. A mutational analysis of the enzyme, based on its sequence homology with the LASPO from E. coli of known structure, appeared to confirm roles in substrate binding or catalysis for residues His244, His351, Arg386 and Arg290 and also for Thr259 and Gln242. The high activity of the enzyme, and its promiscuous acceptance of both L-asparagine and L-glutamate as substrates, if with low activity, suggests that PpLASPO may provide a good model enzyme for evolution studies towards AAOs of altered or improved properties in the future.

High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris

By combining gene design and heterologous over-expression of Rhodotorula gracilis D-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.

Mitochondrial DNA sequence analysis from multiple gene fragments reveals genetic heterogeneity of Crassostrea ariakensis in East Asia

The native Asian oyster, Crassostrea ariakensis is one of the most common and important Crassostrea species that occur naturally along the coast of East Asia. Molecular species diagnosis is a prerequisite for population genetic analysis of wild oyster populations because oyster species cannot be discriminated reliably using external morphological characters alone due to character ambiguity. To date there have been few phylogeographic studies of natural edible oyster populations in East Asia, in particular this is true of the common species in Korea C. ariakensis. We therefore assessed the levels and patterns of molecular genetic variation in East Asian wild populations of C. ariakensis from Korea, Japan, and China using DNA sequence analysis of five concatenated mtDNA regions namely; 16S rRNA, cytochrome oxidase I, cytochrome oxidase II, cytochrome oxidase III, and cytochrome b. Two divergent C. ariakensis clades were identified between southern China and remaining sites from the northern region. In addition, hierarchical AMOVA and pairwise UST analyses showed that genetic diversity was discontinuous among wild populations of C. ariakensis in East Asia. Biogeographical and historical sea level changes are discussed as potential factors that may have influenced the genetic heterogeneity of wild C. ariakensis stocks across this region.

Detecting quantitative trait loci affecting beef tenderness on bovine chromosome 7 near calpastatin and lysyl oxidase

From a study of 3 large half-sib families of cattle, we describe linkage between DNA polymorphisms on bovine chromosome 7 and meat tenderness. Quantitative trait loci (QTL) for Longissimus lumborum peak force (LLPF) and Semitendonosis adhesion (STADH) were located to this map of DNA markers, which includes the calpastatin ( CAST) and lysyl oxidase (LOX) genes. The LLPF QTL has a maximum lodscore of 4.9 and allele substitution of approximately 0.80 of a phenotypic standard deviation, and the peak is located over the CAST gene. The STADH QTL has a maximum lodscore of 3.5 and an allele substitution of approximately 0.37 of a phenotypic standard deviation, and the peak is located over the LOX gene. This suggests 2 separate likelihood peaks on the chromosome. Further analyses of meat tenderness measures in the Longissimus lumborum, LLPF and LL compression (LLC), in which outlier individuals or kill groups are removed, demonstrate large shifts in the location of LLPF QTL, as well as confirming that there are indeed 2 QTL on bovine chromosome 7. We found that both QTL are reflected in both LLPF and LLC measurements, suggesting that both these components of tenderness, myofibrillar and connective tissue, are detected by both measurements in this muscle.

Refined Global Analysis of Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae) Mitochondrial Cytochrome Oxidase 1 to Identify Species Level Genetic Boundaries.

Identifying species boundaries within morphologically indistinguishable cryptic species complexes is often contentious. For the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae), the lack of a clear understanding about the genetic limits of the numerous genetic groups and biotypes so far identified has resulted in a lack of consistency in the application of the terms, the approaches use to apply them and in our understanding of what genetic structure within B. tabaci means. Our response has been to use mitochondrial gene cytochrome oxidase one to consider how to clearly and consistently define genetic separation. Using Bayesian phylogenetic analysis and analysis of sequence pairwise divergence we found a considerably higher to number of genetic groups than had been previously determined with two breaks in the distribution, one at 11% and another at 3.5%. At >11% divergence, 11 distinct groups were resolved, whereas at >3.5% divergence 24 groups were identified. Consensus sequences for each of these groups were determined and were shown to be useful in the correct assignment of sequences of unknown origin. The 3.5% divergence bound is consistent with species level separations in other insect taxa and Suggests that B. tabaci is it cryptic species composed of at least 24 distinct species. We further show that the placement of Bemesia atriplex (Froggatt) within the B. tabaci in, group adds further weight to the argument for species level separation within B. tabaci. This new analysis, which constructs consensus sequences and uses these its a standard against which unknown sequences call be compared, provides for the first time it consistent means of identifying the genetic hounds of each species with it high degree of certainty.

Characterisation, cloning and production of industrially interesting enzymes: gluconolactone oxidase of Penicillium cyaneo-fulvum and gluconate 5-dehydrogenase of Gluconobacter suboxydans

The work covered in this thesis is focused on the development of technology for bioconversion of glucose into D-erythorbic acid (D-EA) and 5-ketogluconic acid (5-KGA). The task was to show on proof-of-concept level the functionality of the enzymatic conversion or one-step bioconversion of glucose to these acids. The feasibility of both studies to be further developed for production processes was also evaluated. The glucose - D-EA bioconversion study was based on the use of a cloned gene encoding a D-EA forming soluble flavoprotein, D-gluconolactone oxidase (GLO). GLO was purified from Penicillium cyaneo-fulvum and partially sequenced. The peptide sequences obtained were used to isolate a cDNA clone encoding the enzyme. The cloned gene (GenBank accession no. AY576053) is homologous to the other known eukaryotic lactone oxidases and also to some putative prokaryotic lactone oxidases. Analysis of the deduced protein sequence of GLO indicated the presence of a typical secretion signal sequence at the N-terminus of the enzyme. No other targeting/anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence supports this analysis, as near complete secretion of GLO was observed in two different yeast expression systems. Highest expression levels of GLO were obtained using Pichia pastoris as an expression host. Recombinant GLO was characterised and the suitability of purified GLO for the production of D-EA was studied. Immobilised GLO was found to be rapidly inactivated during D-EA production. The feasibility of in vivo glucose - D-EA conversion using a P. pastoris strain co-expressing the genes of GLO and glucose oxidase (GOD, E.C. 1.1.3.4) of A. niger was demonstrated. The glucose - 5-KGA bioconversion study followed a similar strategy to that used in the D-EA production research. The rationale was based on the use of a cloned gene encoding a membrane-bound pyrroloquinoline quinone (PQQ)-dependent gluconate 5-dehydrogenase (GA 5-DH). GA 5-DH was purified to homogeneity from the only source of this enzyme known in literature, Gluconobacter suboxydans, and partially sequenced. Using the amino acid sequence information, the GA 5-DH gene was cloned from a genomic library of G. suboxydans. The cloned gene was sequenced (GenBank accession no. AJ577472) and found to be an operon of two adjacent genes encoding two subunits of GA 5-DH. It turned out that GA 5-DH is a rather close homologue of a sorbitol dehydrogenase from another G. suboxydans strain. It was also found that GA 5-DH has significant polyol dehydrogenase activity. The G. suboxydans GA 5-DH gene was poorly expressed in E. coli. Under optimised conditions maximum expression levels of GA 5-DH did not exceed the levels found in wild-type G. suboxydans. Attempts to increase expression levels resulted in repression of growth and extensive cell lysis. However, the expression levels were sufficient to demonstrate the possibility of bioconversion of glucose and gluconate into 5-KGA using recombinant strains of E. coli. An uncharacterised homologue of GA 5-DH was identified in Xanthomonas campestris using in silico screening. This enzyme encoded by chromosomal locus NP_636946 was found by a sequencing project of X. campestris and named as a hypothetical glucose dehydrogenase. The gene encoding this uncharacterised enzyme was cloned, expressed in E. coli and found to encode a gluconate/polyol dehydrogenase without glucose dehydrogenase activity. Moreover, the X. campestris GA 5-DH gene was expressed in E. coli at nearly 30 times higher levels than the G. suboxydans GA 5-DH gene. Good expressability of the X. campestris GA-5DH gene makes it a valuable tool not only for 5-KGA production in the tartaric acid (TA) bioprocess, but possibly also for other bioprocesses (e.g. oxidation of sorbitol into L-sorbose). In addition to glucose - 5-KGA bioconversion, a preliminary study of the feasibility of enzymatic conversion of 5-KGA into TA was carried out. Here, the efficacy of the first step of a prospective two-step conversion route including a transketolase and a dehydrogenase was confirmed. It was found that transketolase convert 5-KGA into TA semialdehyde. A candidate for the second step was suggested to be succinic dehydrogenase, but this was not tested. The analysis of the two subprojects indicated that bioconversion of glucose to TA using X. campestris GA 5-DH should be prioritised first and the process development efforts in future should be focused on development of more efficient GA 5-DH production strains by screening a more suitable production host and by protein engineering.

Part I. Synthesis of L-amino acid oxidase by a serine- or glycine-requiring strain of neurospora. Part II. Studies concerning multiple electrophoretic forms of tyrosinase in neurospora

Part I: Synthesis of L-Amino Acid Oxidase by a Serine- or Glycine-Requiring Strain of Neurospora

Wild-type cultures of Neurospora crassa growing on minimal medium contain low levels of L-amino acid oxidase, tyrosinase, and nicotinarnide adenine dinucleotide glycohydrase (NADase). The enzymes are derepressed by starvation and by a number of other conditions which are inhibitory to growth. L-amino acid oxidase is, in addition, induced by growth on amino acids. A mutant which produces large quantities of both L-amino acid oxidase and NADase when growing on minimal medium was investigated. Constitutive synthesis of L-amino acid oxidase was shown to be inherited as a single gene, called P110, which is separable from constitutive synthesis of NADase. P110 maps near the centromere on linkage group IV.

L-amino acid oxidase produced constitutively by P110 was partially purified and compared to partially purified L-amino acid oxidase produced by derepressed wild-type cultures. The enzymes are identical with respect to thermostability and molecular weight as judged by gel filtration.

The mutant P110 was shown to be an incompletely blocked auxotroph which requires serine or glycine. None of the enzymes involved in the synthesis of serine from 3-phosphoglyceric acid or glyceric acid was found to be deficient in the mutant, however. An investigation of the free intracellular amino acid pools of P110 indicated that the mutant is deficient in serine, glycine, and alanine, and accumulates threonine and homoserine.

The relationship between the amino acid requirement of P110 and its synthesis of L-amino acid oxidase is discussed.

Part II: Studies Concerning Multiple Electrophoretic Forms of Tyrosinase in Neurospora

Supernumerary bands shown by some crude tyrosinase preparations in paper electrophoresis were investigated. Genetic analysis indicated that the location of the extra bands is determined by the particular T allele present. The presence of supernumerary bands varies with the method used to derepress tyrosinase production, and with the duration of derepression. The extra bands are unstable and may convert to the major electrophoretic band, suggesting that they result from modification of a single protein. Attempts to isolate the supernumerary bands by continuous flow paper electrophoresis or density gradient zonal electrophoresis were unsuccessful.