Cytokine-mediated induction of human xylosyltransferase-I in systemic sclerosis skin fibroblasts.

Systemic sclerosis (SSc) is an inflammatory fibrotic disease characterized by an excessive extracellular matrix deposition in the skin and internal organs. One fibrotic key event remains the fibroblast-to-myofibroblast differentiation that is controlled by a combination of mechanical and soluble factors, such as transforming growth factor-ß1 (TGF-ß1) and interleukin-1ß (IL-1ß). One important myofibroblast biomarker is human xylosyltransferase-I (XT-I), the initial enzyme in proteoglycan biosynthesis. Increased serum XT activity was quantified in SSc, but the underlying cellular mechanisms remain elusive. This study aims to determine the cellular basis of XT-I induction in SSc by using a myofibroblast cell culture model with SSc fibroblasts (SScF) and healthy control fibroblasts. We found that SScF exhibit a higher extracellular XT-I activity compared to control fibroblasts. This increased XT-I activity in SScF was demonstrated to be mediated by an enhanced autocrine TGF-ß signaling. Upon IL-1ß treatment, SScF showed an increased mRNA expression level of XT-I and TGF-ß receptor II (TGFBR2), while healthy control fibroblasts did not, pointing towards an involvement of IL-1ß in the cytokine-mediated XT-I induction. Performing microRNA (miRNA) inhibition experiments in the presence of TGF-ß1, we showed that the pro-fibrotic effect of IL-1ß may be mediated by a miRNA-21/TGF-ß receptor II axis, enhancing the autocrine TGF-ß signaling in SScF. Taken together, this study improves the mechanistic understanding of fibrotic XT-I induction in SSc by identifying a hitherto unknown IL-1ß-mediated miRNA-21/TGFBR2 regulation contributing to the enhanced XYLT1 expression and XT-I activity in SScF.

microRNA-145 mediates xylosyltransferase-I induction in myofibroblasts via suppression of transcription factor KLF4.

Remodelling of the extracellular matrix by myofibroblasts is crucial for wound repair, but if deregulated, it might contribute to the development of fibrosis. Fibroblast-to-myofibroblast differentiation is promoted by aberrant microRNA-145-5p (miR-145) expression in response to transforming growth factor ß1 (TGFß1). One of several myofibroblast markers is human xylosyltransferase-I (XT-I), which is the initial and rate-limiting enzyme of proteoglycan biosynthesis. Increased serum XT activity was quantified in patients with systemic sclerosis (SSc), but the underlying cellular mechanism of this disease remains unknown. This study aims to determine the underlying molecular basis of XT-I induction by considering the miR-mediated regulation of XT-I. We found that miR-145 is upregulated in TGFß1-treated dermal fibroblasts and correlates with an increased cellular XYLT1 expression and XT activity. Overexpression of miR-145 in dermal fibroblasts induced XYLT1 expression and XT activity and enhanced TGFß1-promoted XT activity increase. Since direct XYLT1 3'-UTR targeting by miR-145 could be experimentally excluded, an indirect effect of miR-145 on XT-I regulation was indicated. We identified six transcription factor-binding sites for Krueppel-like factor 4 (KLF4), a zinc-finger transcription regulator and putative miR-145 target, in the XYLT1 promoter in silico. A suppressive role of KLF4 on XYLT1 expression was confirmed by targeted gene silencing in dermal fibroblasts and the quantification of KLF4 expression in SSc fibroblasts. Taken together, this study improves the mechanistic understanding of fibrotic remodelling in SSc by identifying a hitherto unknown miR-145/KLF4 pathway mediating the fibrogenic XT-I induction. This knowledge on XYLT1 may lead to the development of novel approaches in the therapy of fibrosis.

Quinolinate Phosphoribosyltransferase Promotes Invasiveness of Breast Cancer Through Myosin Light Chain Phosphorylation.

Perturbed Nicotinamide adenine dinucleotide (NAD+) homeostasis is involved in cancer progression and metastasis. Quinolinate phosphoribosyltransferase (QPRT) is the rate-limiting enzyme in the kynurenine pathway participating in NAD+ generation. In this study, we demonstrated that QPRT expression was upregulated in invasive breast cancer and spontaneous mammary tumors from MMTV-PyVT transgenic mice. Knockdown of QPRT expression inhibited breast cancer cell migration and invasion. Consistently, ectopic expression of QPRT promoted cell migration and invasion in breast cancer cells. Treatment with QPRT inhibitor (phthalic acid) or P2Y11 antagonist (NF340) could reverse the QPRT-induced invasiveness and phosphorylation of myosin light chain. Similar reversibility could be observed following treatment with Rho inhibitor (Y16), ROCK inhibitor (Y27632), PLC inhibitor (U73122), or MLCK inhibitor (ML7). Altogether, these results indicate that QPRT enhanced breast cancer invasiveness probably through purinergic signaling and might be a potential prognostic indicator and therapeutic target in breast cancer.

First description of a compensatory xylosyltransferase I induction observed after an antifibrotic UDP-treatment of normal human dermal fibroblasts.

Fibrosis is a serious health problem often leading to accompanying organ failure. During the manifestation of the disease, an accumulation of different extracellular matrix (ECM) molecules, such as proteoglycans, takes place. There is no appropriate therapeutic option available to heal fibrosis to date. Current research focuses primarily on targets such as the cytokine transforming growth factor-ß1 (TGF-ß1), which is assumed to be one of the key mediators of fibrosis. Both xylosyltransferase isoforms, XT-I and XT-II, catalyze the rate-limiting step of the proteoglycan biosynthesis. Consequently, inhibiting XT activity could be a promising approach to treat fibrosis. It was shown in earlier studies that nucleotides and nucleosides have anti-fibrotic properties and decrease XT activity in cell-free systems. In contrast, we evaluated the mechanisms beyond an UDP-mediated induction of intracellular XT-activity in normal human dermal fibroblasts (NHDF). The observed pseudo-fibrotic XT increasement could be attributed to a compensation of decreased UDP-glucuronate decarboxylase 1 (UXS1) mRNA expression as well as a diminished intracellular UDP-xylose concentration. In summary, our results describe a so far unknown XT-inductive pathway and show that UDP could be a promising molecule for the development of an anti-fibrotic therapy. Nevertheless, XT activity has to be inhibited in parallel intracellularly.

Fibronectin fragment inhibits xylosyltransferase-1 expression by regulating Sp1/Sp3- dependent transcription in articular chondrocytes.

OBJECTIVE: We investigated the effects of 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) on xylosyltransferase-1 (XT-1), an essential anabolic enzyme that catalyzes the initial and rate-determining step in glycosaminoglycan chain synthesis, in human primary chondrocytes. METHODS: Proteoglycan and XT-1 expression in cartilage tissue was analyzed using safranin O staining and immunohistochemistry. The effects of 29-kDa FN-f on XT-1 expression and its relevant signaling pathway were analyzed by quantitative real-time-PCR, immunoblotting, chromatin immunoprecipitation, and immunoprecipitation assays. The receptors for 29-kDa FN-f were investigated using small interference RNA and blocking antibodies. RESULTS: The expression of XT-1 was significantly lower in human osteoarthritis cartilage than in normal cartilage. Intra-articular injection of 29-kDa FN-f reduced proteoglycan levels and XT-1 expression in murine cartilage. In addition, in 29-kDa FN-f-treated cells, XT-1 expression was significantly suppressed at both the mRNA and protein levels, modulated by the transcription factors specificity protein 1 (Sp1), Sp3, and activator protein 1 (AP-1). The 29-kDa FN-f suppressed the binding of Sp1 to the promoter region of XT-1 and enhanced the binding of Sp3 and AP-1. Inhibition of mitogen-activated protein kinase and nuclear factor kappa B signaling pathways restored the 29-kDa FN-f-inhibited XT-1 expression, along with the altered expression of Sp1 and Sp3. Blockading toll-like receptor 2 (TLR-2) and integrin α5ß1 via siRNA and blocking antibodies revealed that the effects of 29-kDa FN-f on XT-1 expression were mediated through the TLR-2 and integrin α5ß1 signaling pathways. CONCLUSION: These results demonstrate that 29-kDa FN-f negatively affects cartilage anabolism by regulating glycosaminoglycan formation through XT-1.

Characterization of an atypical, thermostable, organic solvent- and acid-tolerant 2'-deoxyribosyltransferase from Chroococcidiopsis thermalis.

In our search for thermophilic and acid-tolerant nucleoside 2'-deoxyribosyltransferases (NDTs), we found a good candidate in an enzyme encoded by Chroococcidiopsis thermalis PCC 7203 (CtNDT). Biophysical and biochemical characterization revealed CtNDT as a homotetramer endowed with good activity and stability at both high temperatures (50-100 °C) and a wide range of pH values (from 3 to 7). CtNDT recognizes purine bases and their corresponding 2'-deoxynucleosides but is also proficient using cytosine and 2'-deoxycytidine as substrates. These unusual features preclude the strict classification of CtNDT as either a type I or a type II NDT and further suggest that this simple subdivision may need to be updated in the future. Our findings also hint at a possible link between oligomeric state and NDT's substrate specificity. Interestingly from a practical perspective, CtNDT displays high activity (80-100%) in the presence of several water-miscible co-solvents in a proportion of up to 20% and was successfully employed in the enzymatic production of several therapeutic nucleosides such as didanosine, vidarabine, and cytarabine.

Tissue-specific and time-dependent clonal expansion of ENU-induced mutant cells in gpt delta mice.

DNA mutations play a crucial role in the origins of cancer, and the clonal expansion of mutant cells is one of the fundamental steps in multistage carcinogenesis. In this study, we correlated tumor incidence in B6C3F1 mice during the period after exposure to N-ethyl-N-nitrosourea (ENU) with the persistence of ENU-induced mutant clones in transgenic gpt delta B6C3F1 mice. The induced gpt mutations afforded no selective advantage in the mouse cells and could be distinguished by a mutational spectrum that is characteristic of ENU treatment. The gpt mutations were passengers of the mutant cell of origin and its daughter cells and thus could be used as neutral markers of clones that arose and persisted in the tissues. Female B6C3F1 mice exposed for 1 month to 200 ppm ENU in the drinking water developed early thymic lymphomas and late liver and lung tumors. To assay gpt mutations, we sampled the thymus, liver, lung, and small intestine of female gpt delta mice at 3 days, 4 weeks, and 8 weeks after the end of ENU exposure. Our results reveal that, in all four tissues, the ENU-induced gpt mutations persisted for weeks after the end of mutagen exposure. Clonal expansion of mutant cells was observed in the thymus and small intestine, with the thymus showing larger clone sizes. These results indicate that the clearance of mutant cells and the potential for clonal expansion during normal tissue growth depends on tissue type and that these factors may affect the sensitivity of different tissues to carcinogenesis. Environ. Mol. Mutagen. 58:592-606, 2017. © 2017 Wiley Periodicals, Inc.

Cytotoxic effect of co-expression of human hepatitis A virus 3C protease and bifunctional suicide protein FCU1 genes in a bicistronic vector.

Recent reports on various cancer models demonstrate a great potential of cytosine deaminase/5-fluorocytosine suicide system in cancer therapy. However, this approach has limited success and its application to patients has not reached the desirable clinical significance. Accordingly, the improvement of this suicide system is an actively developing trend in gene therapy. The purpose of this study was to explore the cytotoxic effect observed after co-expression of hepatitis A virus 3C protease (3C) and yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein (FCU1) in a bicistronic vector. A set of mono- and bicistronic plasmid constructs was generated to provide individual or combined expression of 3C and FCU1. The constructs were introduced into HEK293 and HeLa cells, and target protein synthesis as well as the effect of 5-fluorocytosine on cell death and the time course of the cytotoxic effect was studied. The obtained vectors provide for the synthesis of target proteins in human cells. The expression of the genes in a bicistronic construct provide for the cytotoxic effect comparable to that observed after the expression of genes in monocistronic constructs. At the same time, co-expression of FCU1 and 3C recapitulated their cytotoxic effects. The combined effect of the killer and suicide genes was studied for the first time on human cells in vitro. The integration of different gene therapy systems inducing cell death (FCU1 and 3C genes) in a bicistronic construct allowed us to demonstrate that it does not interfere with the cytotoxic effect of each of them. A combination of cytotoxic genes in multicistronic vectors can be used to develop pluripotent gene therapy agents.

Unravelling the potential of a new uracil phosphoribosyltransferase (UPRT) from Arabidopsis thaliana in sensitizing HeLa cells towards 5-fluorouracil.

In silico studies with uracil phosphoribosyltransferase from Arabidopsis thaliana (AtUPRT) revealed its lower binding energies for uracil and 5-fluorouracil (5-FU) as compared to those of bacterial UPRT indicating the prospective of AtUPRT in gene therapy implications. Hence, AtUPRT was cloned and stably expressed in cervical cancer cells (HeLa) to investigate the effect of prodrug 5-FU on these transfected cancer cells. The treatment of AtUPRT-expressing HeLa (HeLa-UPP) cells with 5-FU for 72h resulted in significant decrease in cell viability. Moreover, 5-FU was observed to induce apoptosis and perturb mitochondrial membrane potential in HeLa-UPP cells. While cell cycle analysis revealed significant S-phase arrest as a result of 5-FU treatment in HeLa-UPP cells, quantitative gene expression analysis demonstrated simultaneous upregulation of important cell cycle related genes, cyclin D1 and p21. The survival fractions of non-transfected, vector-transfected and AtUPRT-transfected HeLa cells, following 5-FU treatment, were calculated to be 0.425, 0.366 and 0.227, respectively.

Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus.

Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis.

Identification of sensory hair-cell transcripts by thiouracil-tagging in zebrafish.

BACKGROUND: Sensory hair cells are exquisitely sensitive to mechanical stimuli and as such, are prone to damage and apoptosis during dissections or in vitro manipulations. Thiouracil (TU)-tagging is a noninvasive method to label cell type-specific transcripts in an intact organism, thereby meeting the challenge of how to analyze gene expression in hair cells without the need to sort cells. We adapted TU-tagging to zebrafish to identify novel transcripts expressed in the sensory hair cells of the developing acoustico-lateralis organs. METHODS: We created a transgenic line of zebrafish expressing the T.gondii uracil phospho-ribosyltransferase (UPRT) enzyme specifically in the hair cells of the inner ear and lateral line organ. RNA was labeled by exposing 3 days post-fertilization (dpf) UPRT transgenic larvae to 2.5 mM 4-thiouracil (4TU) for 15 hours. Following total RNA isolation, poly(A) mRNA enrichment, and purification of TU-tagged RNA, deep sequencing was performed on the input and TU-tagged RNA samples. RESULTS: Analysis of the RNA sequencing data revealed the expression of 28 transcripts that were significantly enriched (adjusted p-value < 0.05) in the UPRT TU-tagged RNA relative to the input sample. Of the 25 TU-tagged transcripts with mammalian homologs, the expression of 18 had not been previously demonstrated in zebrafish hair cells. The hair cell-restricted expression for 17 of these transcripts was confirmed by whole mount mRNA in situ hybridization in 3 dpf larvae. CONCLUSIONS: The hair cell-restricted pattern of expression of these genes offers insight into the biology of this receptor cell type and may serve as useful markers to study the development and function of sensory hair cells. In addition, our study demonstrates the utility of TU-tagging to study nascent transcripts in specific cell types that are relatively rare in the context of the whole zebrafish larvae.

Xylosyltransferase-1 expression is refractory to inhibition by the inflammatory cytokines tumor necrosis factor α and IL-1ß in nucleus pulposus cells: novel regulation by AP-1, Sp1, and Sp3.

We investigated whether expression of xylosyltransferase-1 (XT-1), a key enzyme in glycosaminoglycan biosynthesis, is responsive to disk degeneration and to inhibition by the inflammatory cytokines tumor necrosis factor α and IL-1ß in nucleus pulposus (NP) cells. Analysis of human NP tissues showed that XT-1 expression is unaffected by degeneration severity; XT-1 and Jun, Fos, and Sp1 mRNA were positively correlated. Cytokines failed to inhibit XT-1 promoter activity and expression. However, cytokines decreased activity of XT-1 promoters containing deletion and mutation of the -730/-723 bp AP-1 motif, prompting us to investigate the role of AP-1 and Sp1/Sp3 in the regulation of XT-1 in healthy NP cells. Overexpression and suppression of AP-1 modulated XT-1 promoter activity. Likewise, treatment with the Sp1 inhibitors WP631 and mithramycin A or cotransfection with the plasmid DN-Sp1 decreased XT-1 promoter activity. Inhibitors of AP-1 and Sp1 and stable knockdown of Sp1 and Sp3 resulted in decreased XT-1 expression in NP cells. Genomic chromatin immunoprecipitation analysis showed AP-1 binding to motifs located at -730/-723 bp and -684/-677 bp and Sp1 binding to -227/-217 bp and -124/-114 bp in XT-1 promoter. These results suggest that XT-1 expression is refractory to the disease process and to inhibition by inflammatory cytokines and that signaling through AP-1, Sp1, and Sp3 is important in the maintenance of XT-1 levels in NP cells.

NAMPT and NAPRT1: novel polymorphisms and distribution of variants between normal tissues and tumor samples.

Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase domain containing 1 (NAPRT1) are the main human NAD salvage enzymes. NAD regulates energy metabolism and cell signaling, and the enzymes that control NAD availability are linked to pathologies such as cancer and neurodegeneration. Here, we have screened normal and tumor samples from different tissues and populations of origin for mutations in human NAMPT and NAPRT1, and evaluated their potential pathogenicity. We have identified several novel polymorphisms and showed that NAPRT1 has a greater genetic diversity than NAMPT, where any alteration can have a greater functional impact. Some variants presented different frequencies between normal and tumor samples that were most likely related to their population of origin. The novel mutations described that affect protein structure or expression levels can be functionally relevant and should be considered in a disease context. Particularly, mutations that decrease NAPRT1 expression can predict the usefulness of Nicotinic Acid in tumor treatments with NAMPT inhibitors.

Mesenchymal stem cells expressing therapeutic genes induce autochthonous prostate tumour regression.

Mesenchymal stem cells (MSC) as vehicles of therapeutic genes represent a unique tool to activate drugs within a neoplastic mass due to their property to home and engraft into tumours. In particular, MSC expressing the cytosine deaminase::uracil phosphoribosyltransferase (CD-MSC) have been previously demonstrated to inhibit growth of subcutaneous prostate cancer xenografts thanks to their ability to convert the non-toxic 5-fluorocytosine into the antineoplastic 5-fluorouracil. Since both the immune system and the tumour microenvironment play a crucial role in directing cancer progression, in order to advance towards clinical applications, we tested the therapeutic potential of this approach on animal models that develop autochthonous prostate cancer and preserve an intact immune system. As cell vectors, we employed adipose-tissue and bone-marrow MSC. CD-MSC toxicity on murine prostate cancer cells and tumour tropism were verified in vitro and ex-vivo before starting the preclinical studies. Magnetic Resonance Imaging was utilised to follow orthotopic tumour progression. We demonstrated that intravenous injections of CD-MSC cells, followed by intraperitoneal administration of 5-fluorocytosine, caused tumour regression in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, which develops aggressive and spontaneous prostate cancer. These results add new insights to the therapeutic potential of specifically engineered MSC in prostate cancer disease.

Co-expression of phosphoenolpyruvate carboxykinase and nicotinic acid phosphoribosyltransferase for succinate production in engineered Escherichia coli.

Succinate is not the dominant fermentation product from xylose in wild-type Escherichia coli K12. E. coli BA 203 is a lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), and phosphoenolpyruvate (PEP)-carboxylase (ppc) deletion strain. To increase succinate accumulation and reduce byproduct formation, engineered E. coli BA204, in which ATP-forming PEP-carboxykinase (PEPCK) is overexpressed in BA203, was constructed and produced 2.17-fold higher succinate yield. To further improve the biomass and the consumption rate of xylose, nicotinic acid phosphoribosyltransferase (NAPRTase), a rate limiting enzyme in the synthesis of NAD(H), was also overexpressed. Thus, co-expression of PEPCK and NAPRTase in recombinant E. coli BA209 was investigated. In BA209, the pck gene and the pncB gene each have a trc promoter, hence, both genes are well expressed. During a 72-h anaerobic fermentation in sealed bottles, the total concentration of NAD(H) in BA209 was 1.25-fold higher than that in BA204, and the NADH/NAD+ ratio decreased from 0.28 to 0.11. During the exclusively anaerobic fermentation in a 3-L bioreactor, BA209 consumed 17.1 g L⁻¹ xylose and produced 15.5 g L⁻¹ succinate. Furthermore, anaerobic fermentation of corn stalk hydrolysate contained 30.1 g L⁻¹ xylose, 2.1 g L⁻¹ glucose and 1.5 g L⁻¹ arabinose, it produced a final succinate concentration of 17.2 g L⁻¹ with a yield of 0.94 g g⁻¹ total sugars.

[Effect of co-expression of nicotinic acid phosphoribosyl transferase and pyruvate carboxylase on succinic acid production in Escherichia coli BA002].

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.

[Enhanced biohydrogen production by homologous over-expression of fnr, pncB, fdhF in Klebsiella sp. HQ-3].

To enhance biohydrogen production of Klebsiella sp. HQ-3, the global transcriptional factor (Fnr), formate dehydrogenase H (FDH1) and the pncB gene encoding the nicotinic acid phosphoribosyltransferase (NAPRTase) were for the first time over-expressed in Klebsiella sp. HQ-3. The fnr, fdhF, pncB genes were cloned from the genomic DNA of Klebsiella sp. HQ-3 by 3 pairs of universal primers, and introduced into the corresponding sites of the modified pET28a-Pkan, resulting in the plasmids pET28a-Pkan-fnr, pET28a-Pkan-fdhF and pET28a-Pkan-pncB. The 4 plasmids were then electroported into wild Klebsiella sp. HQ-3 to create HQ-3-fnr, HQ-3-fdhF, HQ-3-pncB and HQ-3-C, respectively. Hydrogen production was measured using a gas chromatograph and the metabolites were analyzed with a high-performance liquid chromatograph (HPLC). The results indicate that over-expression of fnr, fdhF and pncB significantly enhanced hydrogen production in the three recombinant strains. Hydrogen production per mol glucose for HQ-3 fnr, HQ-3 pncB, HQ-3 fdhF was 1.113, 1.106 and 1.063 mol of hydrogen/mol glucose, which was respectively increased by 12.26%, 11.62% and 7.28% compared with that of the control strain HQ-3-C (0.991 mol of hydrogen/mol glucose). Moreover, the analysis of HPLC showed that the concentrations of formate and lactate were markedly decreased, but succinate remained unchanged in culture media compared with those of the control strain HQ-3-C.

Biosynthesis of nucleoside analogues via thermostable nucleoside phosphorylase.

Biocatalyzed synthesis of nucleoside analogues was carried out using two thermostable nucleoside phosphorylases from the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix K1. The synthesis of the 2,6-diaminopurine nucleoside and 5-methyluridine was used as a reaction model to test the process. Both the purine nucleoside phosphorylase (apPNP) and uridine phosphorylase (apUP) were functionally expressed in Escherichia coli. The recombinant enzymes were characterized after purification, and both enzymes showed high thermostability and broad substrate specificity. Both enzymes retained 100 % of their activity after 60 min at high temperature, and the optimum temperature for the enzymes was 90-100 °C. The nucleoside phosphorylases obtained from A. pernix are valuable industrial biocatalysts for high-temperature reactions that produce nucleoside drugs in high yields.

Purification and comparison of native and recombinant tRNA-guanine transglycosylases from Methanosarcina acetivorans.

Many archaeal tRNAs have archaeosine (G(+)) at position 15 in the D-loop and this is thought to strengthen the tertiary interaction with C48 in the V-loop. In the first step of G(+) biosynthesis, archaeosine tRNA-guanine transglycosylase (ArcTGT)(1) catalyzes the base exchange reaction from guanine to 7-cyano-7-deazaguanine (preQ(0)). ArcTGT is classified into full-size or split types, according to databases of genomic information. Although the full-size type forms a homodimeric structure, the split type has been assumed to form a heterotetrameric structure, consisting of two kinds of peptide. However, there has been no definitive evidence for this presented to date. Here, we show that native ArcTGT could be isolated from Methanosarcina acetivorans and two peptides formed a robust complex in cells. Consequently, the two peptides function as actual subunits of ArcTGT. We also overexpressed recombinant ArcTGT in Escherichia coli cells. Product was successfully obtained by co-overexpression of the two subunits but one subunit alone was not adequately expressed in soluble fractions. This result suggests that interaction between the two subunits may contribute to the conformational stability of split ArcTGT. The values of the kinetic parameters for the recombinant and native ArcTGT were closely similar. Moreover, tRNA transcript with preQ(0) at position 15 was successfully prepared using the recombinant ArcTGT. This tRNA transcript is expected to be useful as a substrate for studies seeking the enzymes responsible for G(+) biosynthesis.

Chemovirotherapy for head and neck squamous cell carcinoma with EGFR-targeted and CD/UPRT-armed oncolytic measles virus.

First-line treatment of recurrent and/or refractory head and neck squamous cell carcinoma (HNSCC) is based on platinum, 5-fluorouracil (5-FU) and the monoclonal antiEGFR antibody cetuximab. However, in most cases this chemoimmunotherapy does not cure the disease, and more than 50% of HNSCC patients are dying because of local recurrence of the tumors. In the majority of cases, HNSCC overexpress the epidermal growth factor receptor (EGFR), and its presence is associated with a poor outcome. In this study, we engineered an EGFR-targeted oncolytic measles virus (MV), armed with the bifunctional enzyme cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT). CD/UPRT converts 5-fluorocytosine (5-FC) into the chemotherapeutic 5-FU, a mainstay of HNSCC chemotherapy. This virus efficiently replicates in and lyses primary HNSCC cells in vitro. Arming with CD/UPRT mediates efficient prodrug activation with high bystander killing of non-infected tumor cells. In mice bearing primary HNSCC xenografts, intratumoral administration of MV-antiEGFR resulted in statistically significant tumor growth delay and prolongation of survival. Importantly, combination with 5-FC is superior to virus-only treatment leading to significant tumor growth inhibition. Thus, chemovirotherapy with EGFR-targeted and CD/UPRT-armed MV is highly efficacious in preclinical settings with direct translational implications for a planned Phase I clinical trial of MV for locoregional treatment of HNSCC.