ODC Gene Research Articles

Virulence, antibiotic resistance and biogenic amines of bacteriocinogenic lactococci and enterococci isolated from goat milk

The present study aimed to investigate the virulence, antibiotic resistance and biogenic amine production in bacteriocinogenic lactococci and enterococci isolated from goat milk in order to evaluate their safety. Twenty-nine bacteriocinogenic lactic acid bacteria (LAB: 11 Lactococcus spp., and 18 Enterococcus spp.) isolated from raw goat milk were selected and subjected to PCR to identify gelE, cylA, hyl, asa1, esp, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc genes. The expression of virulence factors (gelatinase, hemolysis, lipase, DNAse, tyramine, histamine, putrescine) in different incubation temperatures was assessed by phenotypic methods, as well as the resistance to vancomycin, gentamicin, chloramphenicol, ampicillin and rifampicin (using Etest®). The tested isolates presented distinct combinations of virulence related genes, but not necessarily the expression of such factors. The relevance of identifying virulence-related genes in bacteriocinogenic LAB was highlighted, demanding for care in their usage as starter cultures or biopreservatives due to the possibility of horizontal gene transfer to other bacteria in food systems.

Role of efflux pumps and intracellular thiols in natural antimony resistant isolates of Leishmania donovani.

BackgroundIn view of the recent upsurge in the phenomenon of therapeutic failure, drug resistance in Leishmania , developed under natural field conditions, has become a great concern yet little understood. Accordingly, the study of determinants of antimony resistance is urgently warranted. Efflux transporters have been reported in Leishmania but their role in clinical resistance is still unknown. The present study was designed to elucidate the mechanism of natural antimony resistance in L. donovani field isolates by analyzing the functionality of efflux pump(s) and expression profiles of known genes involved in transport and thiol based redox metabolismMethodology/Principal FindingsWe selected 7 clinical isolates (2 sensitive and 5 resistant) in addition to laboratory sensitive reference and SbIII resistant mutant strains for the present study. Functional characterization using flow cytometry identified efflux pumps that transported substrates of both P-gp and MRPA and were inhibited by the calmodulin antagonist trifluoperazine. For the first time, verapamil sensitive efflux pumps for rhodamine 123 were observed in L. donovani that were differentially active in resistant isolates. RT-PCR confirmed the over-expression of MRPA in isolates with high resistance index only. Resistant isolates also exhibited consistent down regulation of AQP1 and elevated intracellular thiol levels which were accompanied with increased expression of ODC and TR genes. Interestingly, γ-GCS is not implicated in clinical resistance in L. donovani isolates.Conclusions/SignificanceHere we demonstrate for the first time, the role of P-gp type plasma membrane efflux transporter(s) in antimony resistance in L. donovani field isolates. Further, decreased levels of AQP1 and elevated thiols levels have emerged as biomarkers for clinical resistance.

Association of SNPs in ODC and PRDM16 with Body Weight Traits in Korean Native Chicken

Both ODC and PRDM16 genes were known to be associated with body weight traits in chicken. These two genes were located on GGA3 and GGA21, respectively, where the QTLs of body weights are located. Therefore, the objectives of this study were to identify the SNPs in these two genes and their associations with body weight traits in Korean native chicken. Fluidigm Dynamic Array integrated fluidic circuits (IFCs) assay was used to genotype 7 SNPs consisting g.-353C>T, g.2136A>G, g.2524T>C, g.3607C>T SNPs of the ODC gene, and g.182216C>T, g.182290A>T, g.182491A>T SNPs of the PRDM16 gene. Statistical analysis showed that g.2136A>G SNP of the ODC was associated with body weight at 20 weeks of age and slaughter weight, and g.3607C>T SNP of the ODC was associated with body weight at 2 weeks of age. Association between g.182216C>T SNP of the PRDM16 and body weight at 12 weeks of age has also been revealed. In addition, g.182491A>T SNP of PRDM16 has significant correlation with body weight (BW) at 8 weeks, BW at 10 weeks and BW at 14 weeks of age. These results suggested that both ODC and PRDM16 could be strong candidate genes for body weight traits in Korean native chicken.

Complex phylogeography in Rhinoclemmys melanosterna: conflicting mitochondrial and nuclear evidence suggests past hybridization (Testudines: Geoemydidae).

We examined differentiation within the Colombian wood turtle Rhinoclemmys melanosterna, and among R. melanosterna and the closely allied species R. diademata, R.funerea and R. punctularia, based on 1060 base pairs of the mitochondrial cyt b gene. We also assessed the phylogenetic relationships among these species using 2050 bp of mtDNA (partial cyt b, 12S and 16S genes) and 3620 bp of nuclear DNA (partial Rag 1, Rag 2, C-mos, R35 and ODC genes). There is considerable phylogeographic structuring within R. melanosterna, with seven distinct clades distributed across the species' range. These clades correlate to some extent with previously described differences in the dorsal pattern of head coloration. Individual and combined analyses of mitochondrial and nuclear DNA indicated contradictory relationships among R. melanosterna, R. diademata, R. funerea and R. punctularia. Mitochondrial DNA sequences revealed R. melanosterna to be non-monophyletic with respect to R. diademata, R. funerea and R. punctularia. In contrast, R. melanosterna constituted a well-supported monophyletic clade using nuclear DNA. This conflict between mitochondrial and nuclear data suggests past gene flow among the allopatrically and parapatrically distributed species R. melanosterna, R. diademata, R. funerea and R. punctularia. Compared to the other Rhinoclemmys species, the taxa under study are weakly differentiated. To assess their taxonomic status, further research is warranted using additional nuclear markers and additional samples of R. diademata, R. funerea and R. punctularia. For the time being, a continued classification of R. melanosterna, R. diademata, R. funerea and R. punctularia as distinct species is justified owing to their allopatric and parapatric distributions, and to conserve the established usage of names that is based on morphological and karyotypic differentiation.

Chemically induced oxidative stress increases polyamine levels by activating the transcription of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase in human hepatoma HUH7 cells

Biogenic polyamines spermine and spermidine participate in numerous cellular processes including transcription, RNA processing and translation. Specifically, they counteract oxidative stress, an alteration of cell redox balance involved in generation and progression of various pathological states including cancer. Here, we investigated how chemically induced oxidative stress affects polyamine metabolism, specifically the expression and activities of enzymes catalyzing polyamine synthesis (ornithine decarboxylase; ODC) and degradation (spermidine/spermine-N(1)-acetyltransferase; SSAT), in human hepatoma cells. Oxidative stress induced the up-regulation of ODC and SSAT gene transcription mediated by Nrf2, and in case of SSAT, also by NF-κB transcription factors. Activation of transcription led to the elevated intracellular activities of both enzymes. The balance in antagonistic activities of ODC and SSAT in the stressed hepatoma cells was shifted towards polyamine biosynthesis, which resulted in increased intracellular levels of putrescine, spermidine, and spermine. Accumulation of putrescine is indicating for accelerated degradation of polyamines by SSAT - acetylpolyamine oxidase (APAO) pathway generating toxic products that promote carcinogenesis, whereas accelerated polyamine synthesis via activation of ODC is favorable for proliferation of cells including those sub-lethally damaged by oxidative stress.

Molecular cloning, heterologous expression, and characterization of Ornithine decarboxylase from Oenococcus oeni.

Ornithine decarboxylase (ODC) is responsible for the production of putrescine, the major biogenic amine found in wine. Oenococcus oeni is the most important lactic acid bacterium in the winemaking process and is involved in malolactic fermentation. We report here the characterization of ODC from an O. oeni strain isolated from wine. Screening of 263 strains isolated from wine and cider from all over the world revealed that the presence of the odc gene appears to be strain specific in O. oeni. After cloning, heterologous expression in Escherichia coli, and characterization, the enzyme was found to have a molecular mass of 85 kDa and a pI of 6.2 and revealed maximal activity at pH 5.5 and an optimum temperature of 35°C. Kinetic studies showed that O. oeni ODC is specific for L-ornithine with a K(m) value of 1 mM and a V(max) of 0.57 U·mg(-1). The hypothesis that cadaverine, which results from lysine decarboxylation, may be linked to putrescine production is not valid since O. oeni ODC cannot decarboxylate L-lysine. As no lysine decarboxylase was detected in any of the O. oeni genomes sequenced, cadaverine synthesis may result from another metabolic pathway. This work is the first characterization of an ODC from a lactic acid bacterium isolated from a fermented product.

Overexpression of odc (ornithine decarboxylase) in Datura innoxia enhances the yield of scopolamine

Scopolamine is widely used for its anticholinergic properties. Because of higher physiological activity and less side effects the world demand of scopolamine is estimated to be ten times greater than other anticholinergic agents, hyoscyamine and atropine. Since natural production is limited, alternatives are required to boost the production. We report the introduction of mouse odc gene of polyamine biosynthesis pathway which is also the primary pathway of tropane alkaloids in Datura innoxia. Polyamines, mainly putrescine, serve as the common metabolite for tropane alkaloids and nicotine. We have overexpressed odc gene to modulate the metabolic flux downstream and eventually achieved higher accumulation of scopolamine in transgenic plants. Among six independent transformed lines one line (O10) produced scopolamine (0.258 μg/g dry weight) almost six times higher than that produced by control plants (0.042 μg/g DW). To our knowledge, this is the first report of odc overexpression in D. innoxia leading to higher scopolamine yield.

LPA Induced Activation of NHE3 via LPA5 Receptor is Apical EGF Receptor Dependent

G A A b st ra ct s the mechanisms by which polyamines regulate RBPs remain elusive. microRNAs (miRNAs) generally base-pair to sequences in the 3'-untranslated regions (UTR) of labile mRNAs and regulate gene expression posttranscriptionally. This study determined if polyamines regulate CUGBP1 expression through miRNA-503 (miR-503), thereby contributing to the control of intestinal mucosal growth. Methods: Studies were conducted in mice and cultured intestinal epithelial cells (IEC-6 line). CUGBP1 and miR-503 expression was examined by real-time PCR andWestern blotting analyses; CUGBP1 translation was measured by chimeric CUGBP1 3'-UTR luciferease reporter and newly synthesized CUGBP1 protein assays. Interaction of miR-503 with the CUGBP1 mRNA was examined by pmir-Glo reporter system and biotin labeled miR-503 pull-down assays. Polyamine levels were depleted by DFMO (a specific inhibitor of polyamine biosynthesis) but increased by ODC gene overexpression. The functions of miRNA-503 were examined by miR-503-directed siRNA and ectopic overexpression of its precursor. Results: Polyamine depletion by DFMO and fasting increased CUGBP1 protein expression (by ~2.6-fold) but decreased miR-503 in the small intestinal mucosa, which was associated with an inhibition of intestinal mucosal growth as indicated by a decrease in DNA synthesis and villous height. In cultured IECs, polyamine depletion also increased CUGBP1 protein (by ~3.5-fold) but decreased miR-503 (by ~70%), whereas elevated polyamines reduced CUGBP1 (by >85%) and increased miR-503 (by ~2-fold), neither intervention changed CUGBP1 mRNA levels. miR-503 directly bound the CUGBP1 mRNA via CUGBP1 coding region but not its 3'-UTR; this interaction was increased by increasing polyamines but it was repressed by polyamine depletion. miR-503 silencing reduced [miR-503/CUGBP1 mRNA] complex, stimulated CUGBP1 translation, increased its expression (by ~1.5-fold), and inhibited IEC proliferation. Conversely, miR-503 overexpression repressed CUGBP1 translation, decreased its protein levels (by >80%), and enhanced IEC growth. Ectopic CUGBP1 overexpression also repressed cell proliferation by ~65% when measured 6 days after the transfection. Conclusions: These results indicate that 1) polyamines repress CUGBP1 mRNA translation by increasing miR-503 and 2) induced CUGBP1 plays an important role in inhibition of intestinal mucosal growth after polyamine depletion.

Saccharomyces cerevisiae Gis2 interacts with the translation machinery and is orthogonal to myotonic dystrophy type 2 protein ZNF9

The myotonic dystrophy type 2 protein ZNF9/CNBP is a small nucleic acid binding protein proposed to act as a regulator of transcription and translation. The precise functions and activity of this protein are poorly understood. Previous studies suggested that ZNF9 regulates translation and facilitates the process of cap-independent translation through interactions with mRNA and the translating ribosome. To help determine the role played by ZNF9 in the activation of translation initiation, we combined genetic and biochemical analysis of the putative ZNF9 ortholog GIS2, in the budding yeast Saccharomyces cerevisiae. Purification of the Gis2p protein followed by mass spectrometry based-proteomic analysis identified a large number of co-purifying ribosomal subunits and translation factors, strongly suggesting that Gis2p interacts with the protein translation machinery. Polysome profiling and ribosome isolation experiments confirm that Gis2p physically interacts with the translating ribosome. Interestingly, expression of yeast Gis2p in HEK293T cells activates cap-independent translation driven by the 5′UTR of the ODC gene. These data suggest that Gis2 is functionally orthologous to ZNF9 and acts as a cap-independent translation factor.

Staphylococcus lugdunensis Is the Likely Origin of the Ornithine Decarboxylase Operon in Staphylococcus epidermidis 2015B

We read with interest the recent paper by Coton and colleagues (1) on the origin of the putrescine-producing ability of Staphylococcus epidermidis 2015B. As noted in the paper, Gram-positive bacteria generally do not exhibit ornithine decarboxylation. However, we would like to draw attention to a major exception, Staphylococcus lugdunensis, which is related to S. epidermidis but not mentioned in the original article. Ornithine decarboxylase (ODC) activity is present in virtually all S. lugdunensis strains (2, 4) and represents one of the key tests for identification of the species (3). This example is distinct from the other examples, S. epidermidis and Oenococcus oeni, for which ODC activity is present in only a small minority of strains. As our group had recently completed the first reference genome sequence of S. lugdunensis (5), the presence of the odc and potE genes was examined in light of the findings reported by Coton et al. The two genes (locus tags SLGD_00246 and SLGD_00247; GenBank accession no. {type:entrez-nucleotide,attrs:{text:NC_013893,term_id:289549371,term_text:NC_013893}}NC_013893) were arranged in an operon with structural similarity to those found in S. epidermidis 2015B and O. oeni RM83. However, unlike the other two bacteria, S. lugdunensis shows no evidence of recent horizontal gene transfer in the form of transposases, integrases, or other remnants of mobile genetic elements around the ODC operon, which argues against the acquisition of the operon through recent horizontal gene transfer (HGT). This argument is also reflected in the ubiquity of ODC activity in S. lugdunensis strains. We also updated the phylogenetic analysis of the putrescine-ornithine antiporter (PotE) with the inclusion of additional S. lugdunensis sequences, which showed phylogenetic relatedness to PotE of S. epidermidis 2015B (Fig. ​(Fig.1).1). Whereas S. epidermidis 2015B ODC and PotE showed only 65 and 69% sequence identities to the homologues in O. oeni RM83, they showed 83 and 88% sequence identities to the homologues in S. lugdunensis HKU09-01. Further analysis revealed additional similarities between the putrescine production pathways of the two Staphylococcus species, like the presence of a cep gene homologue downstream of the ODC operon and the lack of an N-terminal extension compared to the O. oeni ODC. FIG. 1. Neighbor-joining trees showing the phylogenetic relationships of ornithine decarboxylase homologues (A) and putrescine-ornithine antiporter homologues (B) in selected bacteria. Isolates 1 to 11 are additional S. lugdunensis clinical isolates included ... Taken together, these results strongly suggested that the putrescine production pathway of S. epidermidis 2015B originated from S. lugdunensis and that this pathway had been conserved in the genome of the latter for a significantly longer period. Identifying the biological role of the pathway in S. lugdunensis may help elucidate its significance in S. epidermidis and other Gram-positive bacteria.

Population dynamics of lactic acid bacteria during spontaneous malolactic fermentation in industrial cider

A deeper knowledge on the complex microbial ecology of malolactic fermentation (MLF) in spontaneous cider production is essential to understand the cider making process, thus, applicable to develop adequate malolactic starters. Trends in food fermentation focussed on the isolation of proper wild-type strains from traditional products to be used as starter cultures, with the aim of conducting industrial production processes without losing their unique flavour and product characteristics. A mixed inoculum consisted of samples taken from the four main areas of the most traditional cider making region in Spain was used to carry out MLF under controlled laboratory conditions. A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) by molecular tools. By monitoring MLF, population dynamics involved in the fermentation process were assessed. Interactions of different microorganisms adapted to a common environment, competition and resistance during the process, give rise to dominant species showing selective advantages. The main aim of this work was the identification of dominant species during the process in order to obtain potential starter cultures with a clear industrial utility. The fermentative capacity of a representative strain belonging to each isolated species, after analysis of interspecific polymorphisms in 16S rRNA sequencing of 84 isolates, was compared. Molecular characterization for the presence of gene coding for exopolysaccharide production ( dps gene amplification), acrolein production ( pdu gene amplification) and biogenic amine production ( tyrdc , hdc and odc genes amplification) was carried out to verify the suitability of the selected strains.

Translational Regulation of CUG‐BP1 by Polyamines through the RNA‐binding Protein HuR

All mammalian cells depend on polyamines for growth and division, but the exact roles of polyamines at the molecular level remain unclear. CUG‐BP1 protein interacts with CG‐rich element of many labile mRNAs and regulates decay and translation of its target transcripts. HuR modulates mRNA turnover and translation and its activity is tightly regulated by cellular polyamines. This study tests if polyamines regulate CUG‐BP1 expression through HuR in normal intestinal epithelial cells (IEC‐6 line).MethodsPolyamine levels were depleted by inhibiting ODC (key enzyme for polyamine biosynthesis) with DFMO but increased by overexpressing ODC gene. CUG‐BP1 expression was examined by newly translated protein and reporter assay using chimeric CUG‐BP1 3′‐UTR. RNA‐binding was examined by biotin pull‐down and RNP‐IP assays.ResultsDecreased polyamines by DFMO increased CUG‐BP1 protein, whereas elevated polyamines through ODC overexpression reduced CUG‐BP1 protein, neither intervention changed CUG‐BP1 mRNA levels. Polyamine depletion also increased CUG‐BP1 translation, but it failed to alter CUG‐BP1 protein stability. HuR bound the CUG‐BP1 mRNA and this association increased following polyamine depletion. HuR silencing repressed CUG‐BP1 translation in polyamine‐deficient cells, thus reducing CUG‐BP1 expression levels.ConclusionsThese results indicate that polyamines repress CUG‐BP1 translation by reducing HuR interaction with the CUG‐BP1 mRNA.

EVALUATION OF BIOGENIC AMINES FORMATION BY PROTEOLYTIC ENTEROCOCCI STRAINS ISOLATED FROM RAW DROMEDARY MILKS FROM SOUTHERN ALGERIA

ABSTRACT Twenty‐three strains of lactic acid enterococci isolated from dromedary raw milks in Southern Algeria were investigated for their proteolytic activity and ability to produce biogenic amines by decarboxylating amino acids: histidine, tyrosine and ornithine. All strains tested displayed proteolytic activities, but none of them were able to produce detectable biogenic amines in the usual adequate laboratory media, namely, Joosten and Northolt medium and Leuschner medium. Polymerase chain reaction experiments indicate a positive correlation between the absence of the hdc and odc genes and the lack of histamine and putrescine production for all tested strains. By contrast, all strains appeared to possess tdc gene, but no correlation was found with a negative expression of tyrosine decarboxylase.PRACTICAL APPLICATIONSIn foods, biogenic amines are mainly generated by decarboxylation of the corresponding amino acids through substrate‐specific enzymes of the microorganisms present in this environment, particularly histidine, tyrosine, lysine, ornithine and arginine, and the capability of enterococci and some other lactic acid bacteria to produce biogenic amines has been described. For this purpose, several qualitative and quantitative methods to determine biogenic amines production by these microorganisms have been developed. There is great interest to identify and to characterize the bacteria strains intended for an industrial use and to verify if they are potentially able to produce biogenic amines in order to prevent it's synthesis and limit the poisoning risk.

Expression of c-Myc is related to host cell death following Salmonella typhimurium infection in macrophage

It has been known that ornithine decarboxylase (ODC) induced by the binding of c-Myc to odc gene is closely linked to cell death. Here, we investigated the relationship between their expressions and cell death in macrophage cells following treatment with Salmonella typhimurium or lipopolysaccharide (LPS). ODC expression was increased by bacteria or LPS and repressed by inhibitors against mitogen-activated protein kinases (MAPKs) in Toll-like receptor 4 (TLR4) signaling pathway. In contrast, c-Myc protein level was increased after treatment with bacteria, but not by treatment with LPS or heat-killed bacteria although both bacteria and LPS increased the levels of c-myc mRNA to a similar extent. c-Myc protein level is dependent upon bacterial invasion because treatment with cytochalasin D (CCD), inhibitors of endocytosis, decreased c-Myc protein level. The cell death induced by bacteria was significantly decreased after treatment of CCD or c-Myc inhibitor, indicating that cell death by S. typhimurium infection is related to c-Myc, but not ODC. Consistent with this conclusion, treatment with bacteria mutated to host invasion did not increase c-Myc protein level and cell death rate. Taken together, it is suggested that induction of c-Myc by live bacterial infection is directly related to host cell death.

A review of gene-drug interactions for nonsteroidal anti-inflammatory drug use in preventing colorectal neoplasia

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to be effective chemopreventive agents for colorectal neoplasia. Polymorphisms in NSAID targets or metabolizing enzymes may affect NSAID efficacy or toxicity. We conducted a literature review to summarize current evidence of gene-drug interactions between NSAID use and polymorphisms in COX1, COX2, ODC, UGT1A6 and CYP2C9 on risk of colorectal neoplasia by searching OVID and PubMed. Of 134 relevant search results, thirteen investigated an interaction. One study reported a significant interaction between NSAID use and the COX1 Pro17Leu polymorphism (P=0.03) whereby the risk reduction associated with NSAID use among homozygous wild-type genotypes was not observed among NSAID users with variant alleles. Recent pharmacodynamic data support the potential for gene-drug interactions for COX1 Pro17Leu. Statistically significant interactions have also been reported for ODC (315G>A), UGT1A6 (Thr181Ala+Arg184Ser or Arg184Ser alone), and CYP2C9 (*2/*3). No statistically significant interactions have been reported for polymorphisms in COX2; however, an interaction with COX2 -765G>C approached significance (P=0.07) in one study. Among seven remaining studies, reported interactions were not statistically significant for COX1, COX2 and ODC gene polymorphisms. Most studies were of limited sample size. Definitions of NSAID use differed substantially between studies. The literature on NSAID-gene interactions to date is limited. Reliable detection of gene-NSAID interactions will require greater sample sizes, consistent definitions of NSAID use and evaluation of clinical trial subjects of chemoprevention studies.

RNA‐directed RNA polymerase 1 (RdR1) mediates the resistance of Nicotiana attenuata to herbivore attack in nature

Small RNAs are important regulators of plant development and resistance to viruses. To determine whether small RNAs mediate defense responses to herbivore attack, we silenced the expression of three RNA-directed RNA polymerases (RdRs) in the native tobacco Nicotiana attenuata by virus-induced gene silencing. Larvae of the leaf-chewing solanaceous specialist Manduca sexta grew faster on the RdR1-silenced plants than on empty vector (EV) controls; silencing RdR3 and 2 had little to no effect on larval performance. NaRdR1 transcripts were strongly elicited when puncture wounds were treated with M. sexta oral secretions (OS) to simulate herbivore attack, and with SA and JA, phytohormones that are elicited by herbivore attack. Stably silencing RdR1 by transforming N. attenuata with an inverted-repeat RdR1 construct produced plants (irRdR1) that grew normally but were highly susceptible to both M. sexta larvae and the cell-content-feeder Tupiocoris notatus. When irRdR1 lines were planted into N. attenuata's native habitat in the Great Basin Desert (Utah, USA), they were highly susceptible to herbivore attack, due to deficiencies in direct rather than indirect defenses. Microarray analysis revealed the downregulation of ADC and ODC genes, which supply substrates for synthesizing the chemical defense compound nicotine; irRdR1 lines failed to accumulate nicotine after attack. We conclude that RdR1 mediates herbivore resistance, and infer that the small RNAs produced by RdR1 are probably involved in orchestrating some of the rapid metabolic adjustments required for plants to survive herbivore attack in their natural habitats. The experiment highlights the value of carrying out 'real-world' tests of gene function early in the discovery process.

PCR detection of oxidative base modifications in promoters of pulmonary artery endothelial cell (PAEC) genes differentially regulated by hypoxia

Oxygen radicals generated during hypoxia cause oxidative base modifications in the VEGF promoter that may be linked to induction of VEGF mRNA expression (FASEB J. 19: –394, 2005). To explore this idea, we searched for oxidative modifications in functionally relevant sequences of genes that are up-, down-, or non-regulated by hypoxia. PCR was used to amplify selected DNA sequences before and after treatment with formamidopyrimidine-DNA glycosylase (Fpg), an N-glycosylase and AP-lyase that releases damaged bases from DNA, under the premise that if oxidation products are present, then Fpg will reduce PCR amplification efficiency. Hypoxic, but not normoxic, PAECs displayed Fpg-induced reductions in PCR amplification of the hypoxic response elements of the VEGF and HO-1 promoters but not in functionally insignificant promoter sequences or in intronic or exonic coding sequences. Fpg-detectable modifications were related in time to changes in VEGF and HO-1 mRNA expression. Neither promoter nor coding sequences of the hypoxia down-regulated ODC gene displayed Fpg sensitive modifications. Similarly, neither promoter nor coding sequences of the constitutively active actin gene were modified in hypoxia. These observations suggest that hypoxia-induced oxidative base modifications are targeted to functionally significant sequences of hypoxia-inducible genes.

Evidence for horizontal gene transfer as origin of putrescine production in Oenococcus oeni RM83.

The nucleotide sequence of a 17.2-kb chromosomal DNA fragment containing the odc gene encoding ornithine decarboxylase has been determined in the putrescine producer Oenococcus oeni RM83. This DNA fragment contains 13 open reading frames, including genes coding for five transposases and two phage proteins. This description might represent the first evidence of a horizontal gene transfer event as the origin of a biogenic amine biosynthetic locus.

Mouse Ornithine Decarboxylase-like Gene Encodes an Antizyme Inhibitor Devoid of Ornithine and Arginine Decarboxylating Activity

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).

The ornithine decarboxylase gene of Trypanosoma brucei: Evidence for horizontal gene transfer from a vertebrate source

Kinetoplastid protozoans in the family Trypanosomatidae are parasites, many of them responsible for serious diseases in humans and domestic animals. Ornithine decarboxlyase (ODC), a protein at the core of polyamine metabolism, is a potential target for therapies to overcome these diseases. Eukaryotic phylogenies were constructed from full-length genes for ODC to determine the origin of ODC in the kinetoplastid protozoans. The Odc genes from Trypanosoma brucei and two other African trypanosomes, T. congolense and T. vivax, clustered with Odc genes from vertebrates rather than with Odc genes from other kinetoplastids and other protozoans, making this gene a candidate for horizontal gene transfer from a vertebrate source. This result is unique to the Odc gene from the African trypanosomes as four other genes produced phylogenies consistent with the expected taxonomic relationships for the organisms. Analysis of the genomic regions around the Odc genes in Leishmania major, T. brucei, and Trypanosoma cruzi supports the hypothesis of loss of the Odc gene in the Trypanosoma lineage followed by acquisition of a new copy from a vertebrate host in the African branch of the genus.