Purpose: Previously, we showed that lactate promoted the proliferation and mobility of hepatocellular carcinoma (HCC) cells by increasing the expression of ornithine decarboxylase 1 (ODC1). In this study, we determined the relationship between ODC1 and pyruvate kinase M2 (PKM2, a key lactate metabolism enzyme), and determined the combined effects of difluoromethylornithine (DFMO; an ODC1 inhibitor) and compound 3k (a PKM2 inhibitor) on HCC cells.

Methods: First, the relationship between PKM2 and ODC1 was analyzed using Western blotting, Cell Counting Kit (CCK)-8 assays, transwell assays, bioinformatics, quantitative real-time fluorescent PCR (qRT-PCR), and immunohistochemical staining. Thereafter, the ODC1 inhibitor DFMO and the PKM2 inhibitor compound 3k were employed. Their combined effects on HCC cell proliferation and mobility were evaluated via CCK-8 assay, flow cytometry, a subcutaneous xenograft tumor model in mice, wound healing assays, and transwell assays. Additionally, the effects of DFMO and compound 3k on the epithelial-mesenchymal transition phenotype and the AKT/GSK-3β/β-catenin pathway were explored using Western blotting and immunofluorescence.

Results: PKM2 knockdown significantly decreased the ODC1 expression, and the proliferation and invasion of HCC cells, while ODC1 overexpression reversed the inhibitory effects of PKM2 knockdown. Similarly, inhibition of ODC1 also decreased the expression of PKM2 via reducing the c-myc-induced transcription. PKM2 was co-expressed with ODC1 in HCC samples, while simultaneously upregulated PKM2 and ODC1 led to the poorest survival outcome. DFMO and compound 3k synergistically inhibited HCC cell proliferation, induced apoptosis, and suppressed cell mobility, as well as the EMT phenotype and the AKT/GSK-3β/β-catenin pathway. The AKT activator SC79 reversed the inhibitory effects.

Conclusion: PKM2/ODC1 are involved in a positive feedback loop. The simultaneous inhibition of ODC1 and PKM2 using DFMO and compound 3k exerts synergistic effects against HCC cells via the AKT/GSK-3β/β-catenin pathway. Thus, DFMO combined with compound 3k may be a novel effective strategy for treating HCC.

Keywords: DFMO; compound 3k; hepatocellular carcinoma; ornithine decarboxylase 1; pyruvate kinase M2; synergistic effect.

Previous studies have proposed that the human papillomavirus (HPV) E6 oncoproteins modify the transcriptional activity of eIF4E through mechanisms dependent on p53 degradation. However, the effect of these oncoproteins on pathways regulating the activity of the eIF4E protein remains poorly understood. Hence, we investigated the mechanisms whereby E6 proteins regulate the activity of the eIF4E protein and its effect on target genes. Overexpression of E6 constructs (HPV-6, HPV-16, HPV-18, and HPV52) showed that E6 oncoproteins increased phosphorylation of the eIF4E protein (Serine-209). This result was mainly mediated by phosphorylation of the 4EBP1 protein via the PI3K/AKT pathway. Additionally, the pharmacological inhibition of eIF4E phosphorylation in cervical cancer cell lines substantially reduced the protein levels of CCND1 and ODC1, indicating that E6 of the high-risk genotypes may modify protein synthesis of the eIF4E target genes by increasing the activity of the AKT and ERK pathways.

Keywords: E6 oncoprotein; Human Papillomavirus; eIF4E; post-transcriptional regulation.

Quantitative real-time polymerase chain reaction is a widely used technique that relies on reference genes for the normalisation of gene expression. These reference genes are constitutively expressed and must remain stable across all samples and treatments. Stability of housekeeping genes may vary and must be optimised for a specific tissue, sample or cell line. Here we present a study screening for possible reference gene candidates, eef1a1, rpl8, sub1.L, clta, H4 and odc1, in the Xenopus laevis (A6) kidney cell line. Quantification cycle results were analysed using geNorm to calculate the average expression stability and the coefficient of variation (CV) for each candidate reference gene. All of the tested genes met the guidelines for stable reference genes, namely an average expression stability of < 0.5 and a CV value of < 0.2, with eef1a1 > sub1.L > rpl8 > clta > odc1 > H4. By using pairwise variation analysis, the optimal number of reference targets was determined to be 2. As such, we report that the reference genes eef1a1 and sub1.L should be used to achieve optimal normalisation in A6 cells.

Although hepatoblastoma is the most common pediatric liver cancer, its genetic heterogeneity and therapeutic targets are not well elucidated. Therefore, we conducted a multiomics analysis, including mutatome, DNA methylome, and transcriptome analyses, of 59 hepatoblastoma samples. Based on DNA methylation patterns, hepatoblastoma was classified into three clusters exhibiting remarkable correlation with clinical, histological, and genetic features. Cluster F was largely composed of cases with fetal histology and good outcomes, whereas clusters E1 and E2 corresponded primarily to embryonal/combined histology and poor outcomes. E1 and E2, albeit distinguishable by different patient age distributions, were genetically characterized by hypermethylation of the HNF4A/CEBPA-binding regions, fetal liver-like expression patterns, upregulation of the cell cycle pathway, and overexpression of NQO1 and ODC1. Inhibition of NQO1 and ODC1 in hepatoblastoma cells induced chemosensitization and growth suppression, respectively. Our results provide a comprehensive description of the molecular basis of hepatoblastoma and rational therapeutic strategies for high-risk cases.

This study examined the effects of buffalo oocyte extracts (BOE) on donor cells reprogramming and molecular characterisation of oocytes screened via brilliant cresyl blue (BCB) staining and comparison of gene expression profiles of developmentally important genes in blastocysts from IVF and cloned derived from BOE treated donor cells with BCB selected recipient cytoplasts. Relative abundance (RA) of OCT4 and NANOG was increased (P < 0.05) and HDAC-1, DNMT-1, and DNMT-3A decreased (P < 0.05) in extract treated cells (ETCs). This ETCs dedifferentiated into neuron-like lineage under appropriate induction condition. The RA of NASP, EEF1A1, DNMT1, ODC1 and RPS27A was increased (P < 0.05) in BCB+ oocytes, whereas ATP5A1 and S100A10 increased (P < 0.05) in BCB- oocytes. Total cell number and RA of OCT4, NANOG, SOX2, DNMT1, IGF2, IGF2R, MNSOD, GLUT1, BAX and BCL2 in cloned blastocysts derived from BCB+ oocytes with ETC more closely followed that of IVF counterparts compared to BCB+ oocytes with extract untreated cell and BCB- oocytes with ETC derived blastocysts. In conclusion, BOE influenced epigenetic reprogramming of buffalo fibroblasts making them suitable donors for nuclear transfer (NT). BCB staining can be effectively used for selection of developmentally competent oocytes for NT. The combined effects of epigenetic reprogramming of donor nuclei by BOE and higher nuclear reprogramming capacity of BCB+ oocytes improve developmentally important gene expression in cloned blastocysts. Whether these improvements have long-term effects on buffalo calves born following embryo transfer remains unknown.

Scale-down of bioreactors is currently done based on matching one or more measurable parameters such as k(L) a and P/V, which could result in insufficient process comparability. Currently, there is a lack of genomic translational studies in cell culture scale-down, which could help delineate measurable cellular attributes for improved scale-down. In this study, we scaled-down from a typical bench-scale 5-L bioreactor to a novel high-throughput 35-mL minibioreactor based on matching oxygen transfer rate, which resulted in cell growth and product-related discrepancies using Sp2/0 cells. Performing DNA microarrays on time-course samples from both systems, we identified ∼200 differentially expressed transcripts, presumably because of bioreactor aeration and mixing differences with scale-down. Evaluating these transcripts for bioreactor-relevant cellular functions such as oxidative stress response and DNA damage response, we chose 18 sentinel genes based on their degree of difference and functionality, which we further verified by quantitative real-time polymerase chain reaction (qRT-PCR). Tracking the differential expression of Sod1, Apex1, and Odc1 genes, we were able to correlate sparging-related damage and poor mixing, as possible causes for physiological changes such as prolonged culture in minibioreactors. Additionally, to verify our sentinel gene findings, we performed follow-up improved scale-down studies based on gene analysis and measured transcriptomic changes. As a result, qRT-PCR-based genomic profiles and cell growth profiles showed better convergence between the improved minibioreactor conditions and the model 5-L bioreactor. Our results broadly show that based on the knowledge from transcriptomic changes of sentinel gene profiles, it is possible to improve bioreactor scale-down for more comparable processes.

Background and objectives: Assessment of drugs toxicity and associated biomarker genes is one of the most important tasks in the pre-clinical phase of drug development pipeline as well as in toxicogenomic studies. There are few statistical methods for the assessment of doses of drugs (DDs) toxicity and their associated biomarker genes. However, these methods consume more time for computation of the model parameters using the EM (expectation-maximization) based iterative approaches. To overcome this problem, in this paper, an attempt is made to propose an alternative approach based on hierarchical clustering (HC) for the same purpose. Methods and materials: There are several types of HC approaches whose performance depends on different similarity/distance measures. Therefore, we explored suitable combinations of distance measures and HC methods based on Japanese Toxicogenomics Project (TGP) datasets for better clustering/co-clustering between DDs and genes as well as to detect toxic DDs and their associated biomarker genes. Results: We observed that Word's HC method with each of Euclidean, Manhattan, and Minkowski distance measures produces better clustering/co-clustering results. For an example, in the case of the glutathione metabolism pathway (GMP) dataset LOC100359539/Rrm2, Gpx6, RGD1562107, Gstm4, Gstm3, G6pd, Gsta5, Gclc, Mgst2, Gsr, Gpx2, Gclm, Gstp1, LOC100912604/Srm, Gstm4, Odc1, Gsr, Gss are the biomarker genes and Acetaminophen_Middle, Acetaminophen_High, Methapyrilene_High, Nitrofurazone_High, Nitrofurazone_Middle, Isoniazid_Middle, Isoniazid_High are their regulatory (associated) DDs explored by our proposed co-clustering algorithm based on the distance and HC method combination Euclidean: Word. Similarly, for the peroxisome proliferator-activated receptor signaling pathway (PPAR-SP) dataset Cpt1a, Cyp8b1, Cyp4a3, Ehhadh, Plin5, Plin2, Fabp3, Me1, Fabp5, LOC100910385, Cpt2, Acaa1a, Cyp4a1, LOC100365047, Cpt1a, LOC100365047, Angptl4, Aqp7, Cpt1c, Cpt1b, Me1 are the biomarker genes and Aspirin_Low, Aspirin_Middle, Aspirin_High, Benzbromarone_Middle, Benzbromarone_High, Clofibrate_Middle, Clofibrate_High, WY14643_Low, WY14643_High, WY14643_Middle, Gemfibrozil_Middle, Gemfibrozil_High are their regulatory DDs. Conclusions: Overall, the methods proposed in this article, co-cluster the genes and DDs as well as detect biomarker genes and their regulatory DDs simultaneously consuming less time compared to other mentioned methods. The results produced by the proposed methods have been validated by the available literature and functional annotation.

The aim of the present study was to investigate variation in the expression pattern of ornithine decarboxylase (ODC1), spermine (SPM), spermidine (SPD) and antizyme inhibitor (AZIN1) in hypothalamus, ovary and uterus during the estrous cycle of rats. Further, to understand any correlation between polyamines and GnRH I expression in hypothalamus; effect of putrescine treatment on GnRH I expression in hypothalamus and progesterone and estradiol levels in serum were investigated. The study also aims in quantifying all the immunohistochemistry images obtained based on pixel counting algorithm to yield the relative pixel count. This algorithm uses a red green blue (RGB) colour thresholding approach to quantify the intensity of the chromogen present. The result of the present study demonstrates almost similar expression pattern of polyamine and polyamine related factors, ODC1, SPD, SPM and AZIN1, with that of hypothalamic GnRH I, all of which mainly localized in the medial preoptic area (MPA) of the hypothalamus, during the proestrus, estrus and diestrus. This suggest that hypothalamic GnRH I expression is under regulation of polyamines. The study showed significant increase in hypothalamic GnRH I expression for both the doses of putrescine treatment to adult female rats. Further, it was shown that in ovary expression pattern of ODC1, SPM, SPD and AZIN1 were similar with that of steroidogenic factor, StAR during the estrous cycle, and putrescine supplementation increased significantly estradiol and progesterone levels in serum, all suggesting ovarian polyamines are involved in regulation of ovarian steroidogenesis. Localization of these factors in the theca and granulosa cells suggest involvement of polyamines in the process of folliculogenesis and luteinization; and ODC1, SPD, SPM and AZIN1 in oocyte further suggests polyamine role in maintenance of oocyte physiology. Finally, in uterus SPM and AZIN1 were localized throughout the estrous cycle, being comparatively more during the metestrus phase. There was intense immunostaining of SPD in the luminal and glandular epithelium during the metestrus and diestrus phases of the estrous cycle suggesting these all the three polyamines as such play important role in regulation of uterine physiology.

This study evaluated the effects of bisphenol A (BPA) on proliferation of ovine trophectoderm (oTr1) cells, as well as expression of genes for transport of arginine and synthesis of polyamines. BPA reduced proliferation of oTr1 cells at concentrations of 1 × 10-6, 1 × 10-5, 1 × 10-4 M compared to concentrations of 0, 1 × 10-9, and 1 × 10-8 M at 24 and 96 h of culture. Lower concentrations of BPA significantly increased expression of mRNAs for agmatinase (AGMAT), arginine decarboxylase (ADC), ornithine decarboxylase (ODC1) and solute carrier family 7 member 1 (SLC7A1). Similarly, synthesis of polyamines by oTr1 cells was greatest at lower concentrations of BPA and decreased as the dose of BPA increased. Expression of mRNAs for interferon tau (IFNT) and insulin-like growth factor 2 (IGF2) by oTr1 cells was greater than for controls at 1 × 10-9 M BPA. Overall, the effects of BPA on proliferation and gene expression by oTr1 cells were highly dose-dependent.

Background: Administration of progesterone (P4) to ewes during the first 9 to 12 days of pregnancy accelerates blastocyst development by day 12 of pregnancy, likely due to P4-induced up-regulation of key genes in uterine epithelia responsible for secretion and transport of components of histotroph into the uterine lumen. This study determined if acceleration of blastocyst development induced by exogenous P4 during the pre-implantation period affects fetal-placental development on day 125 of pregnancy. Suffolk ewes (n = 35) were mated to fertile rams and assigned randomly to receive daily intramuscular injections of either corn oil vehicle (CO, n = 18) or 25 mg progesterone in CO (P4, n = 17) for the first 8 days of pregnancy. All ewes were hysterectomized on day 125 of pregnancy and: 1) fetal and placental weights and measurements were recorded; 2) endometrial and placental tissues were analyzed for the expression of candidate mRNAs involved in nutrient transport and arginine metabolism; and 3) maternal plasma, fetal plasma, allantoic fluid, and amniotic fluid were analyzed for amino acids, agmatine, polyamines, glucose, and fructose.

Results: Treatment of ewes with exogenous P4 did not alter fetal or placental growth, but increased amounts of aspartate and arginine in allantoic fluid and amniotic fluid, respectively. Ewes that received exogenous P4 had greater expression of mRNAs for SLC7A1, SLC7A2, SLC2A1, AGMAT, and ODC1 in endometria, as well as SLC1A4, SLC2A5, SLC2A8 and ODC1 in placentomes. In addition, AZIN2 protein was immunolocalized to uterine luminal and glandular epithelia in P4-treated ewes, whereas AZIN2 localized only to uterine luminal epithelia in CO-treated ewes.

Conclusions: This study revealed that exogenous P4 administered in early pregnancy influenced expression of selected genes for nutrient transporters and the expression of a protein involved in polyamine synthesis on day 125 of pregnancy, suggesting a 'programming' effect of P4 on gene expression that affected the composition of nutrients in fetal-placental fluids.

Keywords: Amino acids; Endometrium; Fructose; Gene expression; Glucose; Placenta; Polyamines; Progesterone.

BACKGROUND

Dietary arginine and meat consumption are implicated in colorectal cancer (CRC) progression via polyamine-dependent processes. Polymorphism in the polyamine-regulatory gene, ornithine decarboxylase 1 (Odc1, rs2302615) is prognostic for CRC-specific mortality. Here, we examined joint effects of meat consumption and Odc1 polymorphism on CRC-specific mortality.

METHODS

The analytic cohort was comprised of 329 incident stage I-III CRC cases diagnosed 1994-1996 with follow- up through March 2008. Odc1 genotyping was conducted using primers that amplify a 172-bp fragment containing the polymorphic base at +316. Dietary questionnaires were administered at cohort entry. Multivariate Cox proportional hazards regression analysis for CRC-specific mortality was stratified by tumor, node, metastasis (TNM) stage, and adjusted for clinically relevant variables, plus meat consumption (as a continuous variable, i.e., the number of medium-sized servings/week), Odc1 genotype, and a term representing the meat consumption and Odc1 genotype interaction. The primary outcome was the interaction of Odc1 and meat intake on CRC-specific mortality, as assessed by departures from multiplicative joint effects.

RESULTS

Odc1 genotype distribution was 51% GG, 49% GA/AA. In the multivariate model, there was a significant interaction between meat consumption and Odc1 genotype, P-int = 0.01. Among Odc1 GA/AA CRC cases in meat consumption Quartiles 1-3, increased mortality risk was observed when compared to GG cases (adjusted hazards ratio (HR) = 7.06 [95% CI 2.34-21.28]) - a difference not found among cases in the highest dietary meat consumption Quartile 4.

CONCLUSIONS

Effects of meat consumption on CRC-specific mortality risk differ based on genetic polymorphism at Odc1. These results provide further evidence that polyamine metabolism and its modulation by dietary factors such as meat may have relevance to CRC outcomes.

Polyamines are low molecular weight, organic cations that play a critical role in many major cellular processes including cell cycle regulation and apoptosis, cellular division, tissue proliferation, and cellular differentiation; however, the functions of polyamines in regulating the storage of metabolic fuels such as triglycerides and glycogen is poorly understood. To address this question, we focused on the Drosophila homolog of ornithine decarboxylase (Odc1), the first rate-limiting enzyme in the synthesis of polyamines. Mutants in Odc1 are lethal, but heterozygotes were viable to adulthood. Odc1 heterozygotes appeared larger than their genetic background control flies and consistent with this observation, weighed more than the controls. However, the increased weight was not due to increased food consumption as heterozygotes ate less than the controls. Interestingly, Odc1 heterozygous flies had augmented triglyceride storage, and this lipid phenotype was due to increased triglyceride storage per cell and an increase in the number of fat cells produced. Odc1 heterozygous flies also displayed increased expression of the lipid synthesis genes fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), suggesting increased lipid synthesis was the cause of the augmented triglyceride phenotype. These results provide a link between the expression of Odc1 and triglyceride storage suggesting that the polyamine pathway plays a role in regulating lipid metabolism.

Zanthoxylum limoncello is a native plant from southern Mexico which is used as a timber source, condiment and as a traditional medicine. Herein, we report on the volatile content of the leaf essential oil and its biological activities. The annual essential oils (2015-2018) contained volatile organic compounds which exhibited a moderate growth inhibitory activity against H. pylori ATCC 53504 (MIC 121.4-139.7 μg mL^-1 ), 26695 (MIC 85.5-94.9 μg mL^-1 ) and J99 (MIC 94.7-110.4 μg mL^-1 ). These hydrodistillates contained 2-undecanone (31.6-36.8 %; MIC 185.3-199.2 μg mL^-1 ) and 2-undecenal (25.1-35.7 %; MIC 144.8-111.3 μg mL^-1 ) as the most abundant compounds which were partially involved in the anti-H. pylori activity. The human ornithine decarboxylase enzyme (ODC1), which shows increased activity in several cancer types, was non-competitively inhibited (V_max 2.7>0.8 K_cat s^-1 ) by the essential oil of Z. limoncello as well as by 2-undecanone and 2-undecenal in accordance to in vitro kinetic studies. In silico calculations strongly suggest that the carbonyl group of these oxygenated hydrocarbons interacts with both Asn319 and Ala39 at the subunit A of ODC1. Considering that Ala39 is located close to Asn44, a crucial amino acid of the ODC's allosteric site, the non-competitive inhibition of the enzyme by 2-undecanone and 2-undecenal is endorsed. Finally, the essential oil of Z. limoncello and its main volatiles showed a significant (p<0.01) and prolonged repellent effect against Aedes aegypti.

Polyamines are cationic molecules synthesized via a highly regulated pathway, obtained from the diet or produced by the gut microbiota. They are involved in general molecular and cellular phenomena that play a role also in vascular disease. Bicuspid aortic valve (BAV) is a congenital malformation associated to a greater risk of thoracic ascending aorta (TAA) aneurysm, whose pathogenesis is not yet well understood. We focused on differential analysis of key members of polyamine pathway and on polyamine concentration in non-dilated TAA samples from patients with either stenotic tricuspid aortic valve (TAV) or BAV (diameter ≤ 45 mm), vs. normal aortas from organ donors, with the aim of revealing a potential involvement of polyamines in early aortopathy. Changes of gene expression in TAA samples were evaluated by RT-PCR. Changes of ornithine decarboxylase 1 (ODC1), a key enzyme in polyamine formation, and cationic amino acid transporter 1 (SLC7A1/CAT-1) expression were analyzed also by Western blot. ODC1 subcellular localization was assessed by immunohistochemistry. Polyamine concentration in TAA samples was evaluated by HPLC. BAV TAA samples showed an increased concentration of putrescine and spermidine vs. TAV and donor samples, together with a decreased mRNA level of polyamine anabolic enzymes and of the putative polyamine transporter SLC7A1/CAT-1. The catabolic enzyme spermidine/spermine N1-acetyltransferase 1 showed a significant mRNA increase in TAV samples only, together with a decreased concentration of spermine. The decreased expression of SLC7A1/CAT-1 and ODC1 mRNAs in BAV corresponded to increased or unchanged expression of the respective proteins. ODC was located mainly in smooth muscle cell (SMC) nucleus in TAV and donor samples, while it was present also in SMC cytoplasm in BAV samples, suggesting its activation. In conclusion, BAV, but not TAV non-dilated samples show increased polyamine concentration, accompanied by the activation of a regulatory negative feedback mechanism.

Purpose: Ornithine decarboxylase 1 (ODC1)-an oncogene involved in the biosynthesis of polyamines-is commonly upregulated and associated with poor prognosis in numerous cancers. However, the role and mechanism of ODC1 in hepatocellular carcinoma (HCC) remains unclear. The aim of the present study was to investigate the role of ODC1 in HCC and clarify the latent molecular mechanisms. Material and methods: We used samples obtained from The Cancer Genome Atlas. The expression of ODC1 was also assessed in our additional HCC samples and HCC cell lines. The roles of ODC1 in HCC cell proliferation, migration and invasion in vitro were investigated using the cell-counting kit-8 assay, 5-ethynyl-2´-deoxyuridine assay, colony formation assay, flow cytometry, wound healing assay and transwell assay, respectively. The effect of ODC1 on HCC cell proliferation in vivo was investigated by constructing a xenotransplanted tumor model in nude mice. Quantitative real-time polymerase chain and western blotting were used to detect the expression levels of ODC1 in mimetic hypoxia, nutrient depleted, and acidotic microenvironment. The relationships between ODC1, the AKT/GSK3β/β-catenin pathway, and acidotic microenvironment were further investigated through western blotting, immunohistochemical staining, and immunofluorescence. Results: ODC1 was upregulated in HCC tissues and cell lines, and co-expressed with KI67 and PCNA (P<0.05). A decrease in the expression of ODC1 inhibits proliferation, migration, invasion, and induces cell cycle arrest in HCC cell lines in vitro, while suppressing HCC cell proliferation in vivo (P<0.05). Furthermore, the expression of ODC1 was increased in the mimetic acidotic microenvironment, while the interference with the expression of ODC1 reversed the effect of the acidotic microenvironment through regulation of AKT/GSK3β/β-catenin and related downstream proteins. Conclusion: ODC1 is an unfavorable gene in HCC patients,promoting HCC cell proliferation, migration and invasion via the AKT/GSK3β/β-catenin pathway and modulation of the acidotic microenvironment.

Cells of the ciliated protozoan Tetrahymena thermophila, grown in proteose peptone medium up to late logarithmic phase, harvested by centrifugation, and resuspended in fresh medium to almost the same cell density, underwent one more division cycle within 5 h after inoculation, thereafter being definitely in full stationary phase. This growth cycle proved to be a useful tool to investigate the activation and deactivation of ornithine decarboxylase ODC1 in Tetrahymena: In late logarithmic phase the cells contained a very low specific activity of ODC of about 3 nmol CO2.h-1.mg-1 in the soluble protein fraction. After growth stimulation the activity was increased up to 100-fold within 1 h. This high activity was maintained for about 5 h-about as long as division activity-then rapidly declined with a half life time (t1/2) of about 15 min to the original low level. Inhibition assays with cycloheximide and actinomycin D revealed that: i. the rapid increase of ODC activity was biphasic with one component of translation of preexisting mRNA and one component of translation of newly transcribed mRNA; ii. the t1/2 of the mRNA of ODC was estimated to be about 2 h; iii. inhibition of protein biosynthesis before ODC inactivation at 5 h caused a decrease of ODC with a t1/2 of 55 min instead of 15 min. These findings suggest that ODC activity in Tetrahymena is regulated on both levels: transcription and translation and by an inactivating protein factor which is regulated at the level of biosynthesis.

Polyamines (PAs) are ubiquitous polycations derived from basic l-amino acids whose physiological roles are still being defined. Their biosynthesis and functions in nitrogen-fixing rhizobia such as Sinorhizobium meliloti have not been extensively investigated. Thin layer chromatographic and mass spectrometric analyses showed that S. meliloti Rm8530 produces the PAs, putrescine (Put), spermidine (Spd) and homospermidine (HSpd), in their free forms and norspermidine (NSpd) in a form bound to macromolecules. The S. meliloti genome encodes two putative ornithine decarboxylases (ODC) for Put synthesis. Activity assays with the purified enzymes showed that ODC2 (SMc02983) decarboxylates both ornithine and lysine. ODC1 (SMa0680) decarboxylates only ornithine. An odc1 mutant was similar to the wild-type in ODC activity, PA production and growth. In comparison to the wild-type, an odc2 mutant had 45 % as much ODC activity and its growth rates were reduced by 42, 14 and 44 % under non-stress, salt stress or acid stress conditions, respectively. The odc2 mutant produced only trace levels of Put, Spd and HSpd. Wild-type phenotypes were restored when the mutant was grown in cultures supplemented with 1 mM Put or Spd or when the odc2 gene was introduced in trans. odc2 gene expression was increased under acid stress and reduced under salt stress and with exogenous Put or Spd. An odc1 odc2 double mutant had phenotypes similar to the odc2 mutant. These results indicate that ODC2 is the major enzyme for Put synthesis in S. meliloti and that PAs are required for normal growth in vitro.

The urea cycle is an endogenous source of arginine that also supports removal of nitrogenous waste following protein metabolism. This cycle is considered inefficient in salmonids, where only 10-15% of nitrogenous waste is excreted as urea. In rainbow trout, arginine is an essential amino acid that has attracted attention due to its many functional roles. These roles include the regulation of protein deposition, immune responses and polyamine synthesis; the latter is directly linked to the urea cycle and involved in tissue repair. The key enzymes used in the urea cycle, namely arginase, ornithine transcarbamylase, argininosuccinate synthase and argininosuccinate lyase, in addition to two rate limiting enzymes required for polyamine synthesis (ornithine decarboxylase and s-adenosylmethionine decarboxylase) are poorly studied in fishes, and their responses to inflammation remain unknown. To address this knowledge gap, we characterised these gene families using phylogenetics and comparative genomics, investigated their mRNA distribution among a panel of tissues and established their transcriptional responses to an acute inflammatory response caused by bacterial infection in liver and muscle. Gene duplicates (paralogues) were identified for arginase (ARG1a, 1b, 2a and 2b), ornithine decarboxylase (ODC1 and 2) and s-adenosylmethionine decarboxylase (SAMdc1 and 2), including paralogues retained from an ancestral salmonid-specific whole genome duplication. ARG2a and 2b were highly upregulated following bacterial infection in liver, whereas ARG1b was downregulated, while both paralogues of SAMdc and ODC were upregulated in liver and unchanged in muscle. Overall, these findings improve our understanding of the molecules supporting the urea cycle and polyamine synthesis in fish, highlighting major changes in the regulation of these systems during inflammation.

BACKGROUND

Chronic hypoxia-induced pulmonary hypertension and vascular remodeling have been shown to be associated with ornithine decarboxylase 1 (ODC1). However, few animal studies have investigated the role of ODC1 in acute hypoxia.

OBJECTIVE

We investigated ODC1 gene expression, morphologic and functional changes, and the effect of L-arginine as an attenuator in lung tissues of rats exposed to acute hypobaric hypoxia at a simulated altitude of 6000 m.

METHODS

Sprague-Dawley rats exposed to simulated hypobaric hypoxia (6000 m) for 24, 48, or 72 hours were treated with L-arginine (L-arginine group, 20 mg/100 g intraperitoneal; n=15) or untreated (non-L-arginine group, n=15). Control rats (n=5) were maintained at 2260 m in a normal environment for the same amount of time but were treated without L-arginine. The mean pulmonary artery pressure was measured by PowerLab system. The morphologic and immunohistochemical changes in lung tissue were observed under a microscope. The mRNA and protein levels of ODC1 were measured by real-time polymerase chain reaction and Western-blot, respectively.

RESULTS

Hypobaric hypoxia induced pulmonary interstitial hyperemia and capillary expansion in the lungs of rats exposed to acute hypoxia at 6000 m. The mean pulmonary artery pressure and the mRNA and protein levels of ODC1 were significantly increased, which could be attenuated by treatment with L-arginine.

CONCLUSIONS

L-arginine attenuates acute hypobaric hypoxia-induced increase in mean pulmonary artery pressure and ODC1 gene expression in lung tissues of rats. ODC1 gene contributes to the development of hypoxic pulmonary hypertension.

Spermidine, a polyamine that induces macroautophagy/autophagy, exhibits anti-aging properties. It is thought that these properties of spermidine are primarily due to its ability to modulate autophagy, but the mechanistic details were hitherto unclear. Studying the effects of spermidine on B lymphocytes, Zhang et al uncover the molecular mechanism that places spermidine at the crossroads of autophagy and immune senescence. Their work highlights the role of spermidine as an anti-aging metabolite that exerts its effects through the translational control of autophagy.Abbreviations: EIF5A, eukaryotic translation initiation factor 5A; HC, hematopoietic cell; ODC1, ornithine decarboxylase 1; PBMCs, peripheral blood mononuclear cells.

To investigate the physiological responses of Oreochromis aureus to salinity fluctuations at the molecular level. We used RNA-seq to explore the differentially expressed genes (DEGs) in the liver and spleen of O. aureus at 0, 3, 7 and 11 ppt (parts per thousand) salinity levels. Herein, De novo assembly generated 71,009 O. aureus unigenes, of which 34,607 were successfully mapped to the four major databases. A total of 120 shared DEGs were identified in liver and spleen transcripts, of which 83 were up-regulated and 37 were down-regulated. GO and KEGG analysis found a total of 26 significant pathways, mainly including energy metabolism, immune response, ion transporters and signal transduction. The trend module category of DEGs showed that the genes (e.g., FASN, ODC1, CD22, MRC, TRAV and SLC7 family) involved in the change-stable-change (1) and the constant-change categories (2) were highly sensitive to salinity fluctuations, which were of great value for further study. Based on these results, it would help provide basic data for fish salinity acclimation, and provide new insights into evolutionary response of fish to various aquatic environments in the long-term stress adaptation mechanism.

Keywords: Energy metabolism; Immune response; Liver and spleen transcript; Oreochromis aureus; Salinity stress.

The urea cycle (UC) removes the excess nitrogen and ammonia generated by nitrogen-containing compound composites or protein breakdown in the human body. Research has shown that changes in UC enzymes are not only related to tumorigenesis and tumor development but also associated with poor survival in hepatocellular, breast, and colorectal cancers (CRC), etc. Cytoplasmic ornithine, the intermediate product of the urea cycle, is a specific substrate for ornithine decarboxylase (ODC, also known as ODC1) for the production of putrescine and is required for tumor growth. Polyamines (spermidine, spermine, and their precursor putrescine) play central roles in more than half of the steps of colorectal tumorigenesis. Given the close connection between polyamines and cancer, the regulation of polyamine metabolic pathways has attracted attention regarding the mechanisms of action of chemical drugs used to prevent CRC, as the drug most widely used for treating type 2 diabetes (T2D), metformin (Met) exhibits antitumor activity against a variety of cancer cells, with a vaguely defined mechanism. In addition, the influence of metformin on the UC and putrescine generation in colorectal cancer has remained unclear. In our study, we investigated the effect of metformin on the UC and putrescine generation of CRC in vivo and in vitro and elucidated the underlying mechanisms. In nude mice bearing HCT116 tumor xenografts, the administration of metformin inhibited tumor growth without affecting body weight. In addition, metformin treatment increased the expression of monophosphate (AMP)-activated protein kinase (AMPK) and p53 in both HCT116 xenografts and colorectal cancer cell lines and decreased the expression of the urea cycle enzymes, including carbamoyl phosphate synthase 1 (CPS1), arginase 1 (ARG1), ornithine trans-carbamylase (OTC), and ODC. The putrescine levels in both HCT116 xenografts and HCT116 cells decreased after metformin treatment. These results demonstrate that metformin inhibited CRC cell proliferation via activating AMPK/p53 and that there was an association between metformin, urea cycle inhibition and a reduction in putrescine generation.

Keywords: ARG1; CPS1; ODC; OTC; colorectal cancer (CRC); human colorectal cancer HCT116 cell line; metformin; p53; proliferation; putrescine; urea cycle.

Quantitative real-time PCR is one of the most widely used techniques for measuring changes in the expression of target transcripts due to its sensitivity, specificity, and cost-effectiveness. However, the essential step that determines appropriate and correct data interpretation is the selection of proper endogenous control genes. Identifying useful reference genes with stable expression is critical for accurate normalization and precise results. Functional divergence of duplicated genes in tetraploid species, like common carp, can complicate the choice for a proper reference gene. In the present study, we determined the expression stability of duplicated genes of 40s, b2m, ef1α, gapdh, g6pd, and odc1 in different tissues of common carp (Cyprinus carpio L.). Gene expression analysis comprised healthy control fish, fish under bacterial and parasitic infections, and across the early stage of common carp development. Obtained data were compared with the actb gene, which is used widely as a reference in RT-qPCR analysis. The application of the three different algorithms - geNorm, NormFinder, BestKeeper, allowed comparative evaluation of the expression stability of the tested genes. Subsequently, the RefFinder, a web-based tool, was used to rank the examined housekeeping genes comprehensively. We demonstrate variable transcription stability levels in the examined mRNAs as well as differences in expression between paralog gene copies. The 40s, b2m, ef1α and actb genes showed the most stable expression across all physiological conditions and tissues. The gapdh, odc1, and g6pd gene variants demonstrated lower stability. Differences in expression patterns between duplicated genes underline the possibility of functional divergence between them. This aspect should be considered in polyploid species before selecting the reference gene(s). Our study also points on the importance of choice for a reference gene (paralog) when expressing newly identified genes and the spatial expression profile is performed.

Keywords: RT-qPCR; common carp; gene expression; reference gene.

Bladder cancer has a high incidence worldwide accompanies by high recurrent rate after treatment. The emergence of primary or acquired chemotherapy resistance leads to poor efficacy in many cases. To explore the underlying mechanisms of drug resistance, we firstly established a drug-resistant cell model T24/THP by repeated exposure of T24 cells to pirarubicin (THP) whose concentration increases gradually. Non-targeted metabolomics was performed to identify metabolic changes and key metabolism pathways variance in T24/THP cells. Pathway enrichment analysis demonstrated that the arginine and proline metabolic pathway was the most significantly changed pathway, where two representative members of polyamine, putrescine and spermidine were remarkably down regulated in T24/THP. Subsequent experiments further confirmed that ornithine decarboxylase (ODC1) and spermidine synthase (SRM), the key enzymes involved in the synthesis of these compounds, also showed a stable low expression in T24/THP. However, knocking down of ODC1 and SRM sensitized cells to chemotherapy treatment while overexpression of these two enzymes enhances chemotherapy resistance. This leaded to the point that ODC1 and SRM themselves are more likely to promote the drug resistance, which appears to contradict their low expression in T24/THP. We hypothesize that their diminished levels were due to the declined activity of genes upstream. According to this line of thought, we found that c-MYC was also down-regulated in T24/THP and its content could be significantly affected by drug administration. In addition, c-MYC could not only regulate the expression levels of ODC1 and SRM but also influence drug resistance in T24/THP. In conclusion, alterations in gene expression of ODC1 and SRM in drug resistance cell line is probably mediated by some upstream regulators rather than antineoplastic agents alone. Exploration of upstream signals and research on detailed regulatory mechanism, thereby understanding the actual role of c-MYC and polyamine in response to chemotherapy, can become a potential field direction to overcome drug resistance in bladder cancer.

Keywords: Bladder cancer; Metabonomics; Multi-drug resistance; Polyamine metabolism.