Orotic acid induces apoptotic death in ovarian adult granulosa tumour cells and increases mitochondrial activity in normal ovarian granulosa cells.

Orotic acid (OA) is a natural product that acts as a precursor in the pyrimidine nucleotide biosynthesis pathway. Most studies concerning administration of OA focus on its therapeutic effects; however, its effect on tumours is unclear. We aimed to determine whether treatment with OA influences the viability and apoptosis of normal (HGrC1) and tumour-derived (KGN) human ovarian granulosa cells. The effects of OA (10-250 µM) on viability and apoptosis of both cell lines were determined by using alamarBlue and assessing caspase-3/7 activity, respectively. Annexin V binding and loss of membrane integrity were evaluated in KGN cells. The cell cycle and proliferation of HGrC1 cells were assessed by performing flow cytometric and DNA content analyses, respectively. The influence of OA (10 and 100 µM) on cell cycle- and apoptosis-related gene expression was assessed by RT-qPCR in both cell lines. Mitochondrial activity was analysed by JC-1 staining in HGrC1 cells. In KGN cells, OA reduced viability and increased caspase-3/7 activity, but did not affect mRNA expression of Caspase 3, BAX, and BCL2. OA enhanced proliferation and mitochondrial activity in HGrC1 cells without activating apoptosis. This study demonstrates that the anti-cancer properties of OA in ovarian granulosa tumour cells are not related to changes in apoptosis-associated gene expression, but to increased caspase-3/7 activity. Thus, OA is a promising therapeutic agent for ovarian granulosa tumours. Further, our results suggest that differences in basal expression of cell cycle- and apoptosis-related genes between the two cell lines are responsible for their different responses to OA.

Combination of Secondary Plant Metabolites and Micronutrients Improves Mitochondrial Function in a Cell Model of Early Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by excessive formation of beta-amyloid peptides (Aß), mitochondrial dysfunction, enhanced production of reactive oxygen species (ROS), and altered glycolysis. Since the disease is currently not curable, preventive and supportive approaches are in the focus of science. Based on studies of promising single substances, the present study used a mixture (cocktail, SC) of compounds consisting of hesperetin (HstP), magnesium-orotate (MgOr), and folic acid (Fol), as well as the combination (KCC) of caffeine (Cof), kahweol (KW) and cafestol (CF). For all compounds, we showed positive results in SH-SY5Y-APP695 cells-a model of early AD. Thus, SH-SY5Y-APP695 cells were incubated with SC and the activity of the mitochondrial respiration chain complexes were measured, as well as levels of ATP, Aß, ROS, lactate and pyruvate. Incubation of SH-SY5Y-APP695 cells with SC significantly increased the endogenous respiration of mitochondria and ATP levels, while Aß1-40 levels were significantly decreased. Incubation with SC showed no significant effects on oxidative stress and glycolysis. In summary, this combination of compounds with proven effects on mitochondrial parameters has the potential to improve mitochondrial dysfunction in a cellular model of AD.

Sea Cucumber Saponins Derivatives Alleviate Hepatic Lipid Accumulation Effectively in Fatty Acids-Induced HepG2 Cells and Orotic Acid-Induced Rats.

The sulfated echinoside A (EA) and holothurin A (HA) are two prominent saponins in sea cucumber with high hemolytic activity but also superior lipid-lowering activity. Deglycosylated derivatives EA2 and HA2 exhibit low hemolysis compared to EA and HA, but their efficacies on lipid metabolism regulation remains unknown. In this study, fatty acids-treated HepG2 cells and orotic acid-treated rats were used to investigate the lipid-lowering effects of sea cucumber saponin derivatives. Both the saponin and derivatives could effectively alleviate lipid accumulation in HepG2 model, especially EA and EA2. Moreover, though the lipid-lowering effect of EA2 was not equal with EA at the same dosage of 0.05% in diet, 0.15% dosage of EA2 significantly reduced hepatic steatosis rate, liver TC and TG contents by 76%, 41.5%, and 63.7%, respectively, compared to control and reversed liver histopathological features to normal degree according to H&E stained sections. Possible mechanisms mainly included enhancement of fatty acids ß-oxidation and cholesterol catabolism through bile acids synthesis and excretion, suppression of lipogenesis and cholesterol uptake. It revealed that the efficacy of EA2 on lipid metabolism regulation was dose-dependent, and 0.15% dosage of EA2 possessed better efficacy with lower toxicity compared to 0.05% dosage of EA.

Orotic acid protects pancreatic ß cell by p53 inactivation in diabetic mouse model.

Impairment of pancreatic ß cells is a principal driver of the development of diabetes. Restoring normal insulin release from the ß cells depends on the ATP produced by the intracellular mitochondria. In maintaining mitochondrial function, the tumor suppressor p53 has emerged as a novel regulator of metabolic homeostasis and participates in adaptations to nutritional changes. In this study, we used orotic acid, an intermediate in the pathway for de novo synthesis of the pyrimidine nucleotide, to reduce genotoxicity. Administration of orotic acid reduced p53 activation of MIN6 ß cells and subsequently reduced ß cell death in the db/db mouse. Orotic acid intake helped to maintain the islet size, number of ß cells, and protected insulin secretion in the db/db mouse. In conclusion, orotic acid treatment maintained ß cell function and reduced cell death, and may therefore, be a future therapeutic strategy for the prevention and treatment of diabetes.

Hypoxia Active Platinum(IV) Prodrugs of Orotic Acid Selective to Liver Cancer Cells.

Platinum(IV) complexes of orotic acid selectively target liver cancer cells displaying enhanced activity and higher uptake in Hep G2. The comparatively higher expression of Organic Anion Transporter 2 (OAT2) in Hep G2 and decrease in toxicity in the presence of OAT2 inhibitor suggest its involvement in the uptake of the complexes. They are resistant to sequestration by the copper transporter ATP7B, unlike cisplatin and oxaliplatin.

In Vitro Interaction of 5-Aminoorotic Acid and Its Gallium(III) Complex with Superoxide Radical, Generated by Two Model Systems.

Increased levels of the superoxide radical are associated with oxidative damage to healthy tissues and with elimination of malignant cells in a living body. It is desirable that a chemotherapeutic combines pro-oxidant behavior around and inside tumors with antioxidant action near healthy cells. A complex consisting of a pro-oxidant cation and antioxidant ligands could be a potential anticancer agent. Ga(III) salts are known anticancer substances, and 5-aminoorotic acid (HAOA) is a ligand with antioxidant properties. The in vitro effects of HAOA and its complex with Ga(III) (gallium(III) 5-aminoorotate (GaAOA)) on the in vitro accumulation of superoxide and other free radicals were estimated. Model systems such as potassium superoxide (KO2), xanthine/xanthine oxidase (X/XO), and rat blood serum were utilized. Data suggested better antioxidant effect of GaAOA compared to HAOA. Evidently, all three ligands of GaAOA participated in the scavenging of superoxide. The effects in rat blood serum were more nuanced, considering the chemical and biochemical complexity of this model system. It was observed that the free-radical-scavenging action of both compounds investigated may be manifested via both hydrogen donation and electron transfer pathways. It was proposed that the radical-scavenging activities (RSAs) of HAOA and its complex with Ga(III) may be due to a complex process, depending on the concentration, and on the environment, nature, and size of the free radical. The electron transfer pathway was considered as more probable in comparison to hydrogen donation in the scavenging of superoxide by 5-aminoorotic acid and its gallium(III) complex.

Metabolic engineering generates a transgene-free safety switch for cell therapy.

Safeguard mechanisms can ameliorate the potential risks associated with cell therapies but currently rely on the introduction of transgenes. This limits their application owing to immunogenicity or transgene silencing. We aimed to create a control mechanism for human cells that is not mediated by a transgene. Using genome editing methods, we disrupt uridine monophosphate synthetase (UMPS) in the pyrimidine de novo synthesis pathway in cell lines, pluripotent cells and primary human T cells. We show that this makes proliferation dependent on external uridine and enables us to control cell growth by modulating the uridine supply, both in vitro and in vivo after transplantation in xenograft models. Additionally, disrupting this pathway creates resistance to 5-fluoroorotic acid, which enables positive selection of UMPS-knockout cells. We envision that this approach will add an additional level of safety to cell therapies and therefore enable the development of approaches with higher risks, especially those that are intended for limited treatment durations.

Orotic acid-treated hepatocellular carcinoma cells resist steatosis by modification of fatty acid metabolism.

BACKGROUND: Orotic acid (OA) has been intensively utilized to induce fatty liver in rats. Although the capacity of OA to cause steatosis is species-specific, previous in vitro studies indicate that humans could also be susceptible to OA-induced fatty liver. The aim of the present study was to re-elucidate the potential of OA exposure to modulate the cellular mechanisms involved in both non-alcoholic fatty liver disease pathogenesis and cellular protection from lipid accumulation. In addition, alterations in detailed fatty acid (FA) profiles of cells and culture media were analyzed to assess the significance of lipid metabolism in these phenomena. METHODS: In our experiments, human hepatocellular carcinoma HepG2 cells were exposed to OA. Bacterial endotoxin, lipopolysaccharide (LPS), was used to mimic hepatic inflammation. The lipogenic and inflammatory effects of OA and/or LPS on cells were assessed by labeling cellular lipids with Nile red stain and by performing image quantifications. The expression levels of key enzymes involved in de novo lipogenesis (DNL) and of inflammatory markers related to the disease development were studied by qRT-PCR. FA profiles of cells and culture media were determined from total lipids with gas chromatography-mass spectrometry. RESULTS: Our data indicate that although OA possibly promotes the first stage of DNL, it does not cause a definite lipogenic transformation in HepG2 cells. Reduced proportions of 16:0, increased stearoyl-Coenzyme A desaturase 1 mRNA expression and relatively high proportions of 16:1n-7 suggest that active delta9-desaturation may limit lipogenesis and the accumulation of toxic 16:0. Inflammatory signaling could be reduced by the increased production of long-chain n-3 polyunsaturated FA (PUFA) and the active incorporation of certain FA, including 18:1n-9, into cells. In addition, increased proportions of 20:4n-6 and 22:6n-3, total PUFA and dimethyl acetal 18:0 suggest that OA exposure may cause increased secretion of lipoproteins and extracellular vesicles. CONCLUSIONS: The present data suggest that, apart from the transcription-level events reported by previous studies, modifications of FA metabolism may also be involved in the prevention of OA-mediated steatosis. Increased delta9-desaturation and secretion of lipoproteins and extracellular vesicles could offer potential mechanisms for further studies to unravel how OA-treated cells alleviate lipidosis.

In vitro Studies of Antitumor Activity of Vanadium Complexes with Orotic and Glutamic Acids / Estudos in vitro da Atividade Antitumoral de Complexos de Vanádio com Ácidos Órotico e Glutâmico / Estudios in vitro de la Actividad Antitumoral de Complejos de Vanadio con Ácidos Orótico y Glutámico

Introduction: Three vanadium complexes with orotic and glutamic acids, in their anion forms, were prepared and their in vitro cytotoxicity toward human lung fibroblasts (MRC-5), human hepatocellular carcinoma (HepG2) and human colorectal adenocarcinoma (Caco-2) are reported. Objective: Describe the synthesis and characterization of new vanadium complexes with orotic and glutamic acids, and test its antitumor activity against HepG2 and Caco-2. Method: The complexes were formulated as VO (oro), VO (α-glu) and VO (γ-glu) based on chemical, thermogravimetric analyses and infrared spectra. Results: Resazurin assay demonstrates its cytotoxicity against the HepG2 and Caco-2 cell lines with the IC50 ranging from 7.90 to 44.56 µmol.L-1. The cytotoxicity profiles indicate that the tumoral lines show more activity than the cells MRC-5, with selectivity indexes ranging from 1.58 to 8.96. Conclusion: The three complexes had better in vitro activity than cisplatin for both normal and cancer cell lines. The IC50 values are two to six times better for the cancer cell ines and five to seven times better for the normal cell lines. This study indicates that the complexes obtained are promising candidates for antitumor drugs.

Introdução: Foram preparados três complexos de vanádio com ácidos orótico e glutâmico, em suas formas aniônicas, e foi testada sua citotoxicidade in vitro para fibroblastos pulmonares humanos (MRC-5), carcinoma hepatocelular humano (HepG2) e adenocarcinoma colorretal humano (Caco-2). Objetivo: Descrever a síntese e caracterização de novos complexos de vanádio com ácidos orótico e glutâmico e testar sua atividade antitumoral contra HepG2 e Caco-2. Método: Os complexos foram formulados como VO (oro), VO (α-glu) e VO (γ-glu) com base em análises químicas, termogravimétricas e espectros no infravermelho. Resultados: O ensaio de resazurina demonstrou sua citotoxicidade contra as linhagens celulares HepG2 e Caco-2 com o IC50 variando de 7,90 a 44,56 µmol.L-1. Os perfis de citotoxicidade indicam que as linhas tumorais apresentam maior atividade que as células MRC-5, com índices de seletividade variando de 1,58 a 8,96. Conclusão: Os três complexos tiveram melhor atividade in vitro do que a cisplatina, tanto para linhagens celulares normais como cancerosas. Os valores de IC50 são de duas a seis vezes melhores para as linhagens celulares cancerosas e de cinco a sete vezes melhores para as linhagens celulares normais. Este estudo indica que os complexos obtidos são promissores candidatos a fármacos antitumorais.

Introducción: Tres complejos de vanadio con ácidos orótico y glutámico, en sus formas aniónicas, fueram preparados. Su citotoxicidad in vitro hacia los fibroblastos pulmonares humanos (MRC-5), el carcinoma hepatocelular humano (HepG2) y el adenocarcinoma colorrectal humano (Caco-2) son reportados. Objetivo: Los principales objetivos de este trabajo son describir la síntesis y caracterización de nuevos complejos de vanadio con ácidos orótico y glutámico y probar su actividad antitumoral contra el HepG2 y el Caco-2. Método: Los complejos fueron formulados como VO (oro), VO (α-glu) y VO (γ-glu) basados en análisis químicos, termogravimétricos y espectros infrarrojos. El ensayo de resazurina demuestra su citotoxicidad contra las líneas celulares HepG2 y Caco-2 con el IC50 que van de 7,90 a 44,56 µmol.L-1. Los perfiles de citotoxicidad indican que las líneas tumorales presentan mayor actividad que los MRC-5, con índices de selectividad que van de 1,58 a 8,96. Conclusión: Los tres complejos tuvieron mejor actividad in vitro que el cisplatino, tanto para líneas celulares normales como para líneas celulares cancerosas. Los valores del IC50 son de dos a seis veces mejores para las líneas celulares de cáncer y de cinco a siete veces mejores para las líneas celulares normales. Este estudio indica que los complejos obtenidos son candidatos prometedores para fármacos antitumorales.

Effects of extracellular orotic acid on acute contraction-induced adaptation patterns in C2C12 cells.

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.

[The influence of 'potassium orotate' on neocollagenogenesis in implantation of polypropylene endoprosthesis and polypropylene combined with polylactic acid endoprosthesis]. / Vliyanie orotata kaliya na neokollagenogenez pri implantatsii polipropilenovogo endoproteza i endoproteza iz polipropilena s polimolochnoi kislotoi v eksperimente.

AIM: To study neocollagenogenesis after implantation of polypropylene endoprosthesis and polypropylene combined with polylactic acid endoprosthesis on background of «potassium orotate¼ administration. MATERIAL AND METHODS: We used two different types of endoprosthesis in the experiment. The first type was made of just polypropylene, the second type was made of polypropylene combined with polylactic acid. Histological examination was performed using polarizing microscopy. Collagen types I and III ratio in connective tissue around the prosthesis was analyzed according to the color that was individual for each type. RESULTS: The results were significantly better in case of collagenogenesis stimulation with Potassium orotate within 30 days and later for one type of endoprosthesis. Also we revealed that collagenogenesis and paraprosthesis capsule formation were more active in case of combined endoprosthesis. We revealed stimulating action of «Potassium Orotate¼ for collegenogenesis process, this fact was proved by increased collagen I/III ratio. CONCLUSION: Optimization of collagenogenesis was based on persistent 1,37-fold increase of collagen I/III ratio in case of combined endoprosthesis after 90 days. It was manifested by accelerated formation of connective tissue capsule and facilitated early isolation of the implant from surrounding tissues.

Dipeptidyl peptidase IV is involved in the cellulose-responsive induction of cellulose biomass-degrading enzyme genes in Aspergillus aculeatus.

We screened for factors involved in the cellulose-responsive induction of cellulose biomass-degrading enzyme genes from approximately 12,000 Aspergillus aculeatus T-DNA insertion mutants harboring a transcriptional fusion between the FIII-avicelase gene (cbhI) promoter and the orotidine 5'-monophosphate decarboxylase gene. Analysis of 5-fluoroorodic acid (5-FOA) sensitivity, cellulose utilization, and cbhI expression of the mutants revealed that a mutant harboring T-DNA at the dipeptidyl peptidase IV (dppIV) locus had acquired 5-FOA resistance and was deficient in cellulose utilization and cbhI expression. The deletion of dppIV resulted in a significant reduction in the cellulose-responsive expression of both cbhI as well as genes controlled by XlnR-independent and XlnR-dependent signaling pathways at an early phase in A. aculeatus. In contrast, the dppIV deletion did not affect the xylose-responsive expression of genes under the control of XlnR. These results demonstrate that DppIV participates in cellulose-responsive induction in A. aculeatus.

A yeast-based high-throughput screen for modulators of phosphodiesterase activity.

Cell-based high-throughput screens (HTSs) targeting heterologously expressed proteins in yeast identify compounds that often display relevant biological activity when tested in cell culture. We developed a fission yeast-based HTS to detect small-molecule inhibitors of mammalian cyclic nucleotide phosphodiesterases (PDEs). These screens are carried out in Schizosaccharomyces pombe using a PKA-repressed fbp1-ura4 reporter whose expression due to low PKA activity prevents cells from growing in medium containing the pyrimidine analog 5-fluoro orotic acid (5FOA). We describe here the steps required to construct strains for screening and to optimize conditions for successful screens.

Carboxyamidotriazole-orotate inhibits the growth of imatinib-resistant chronic myeloid leukaemia cells and modulates exosomes-stimulated angiogenesis.

The Bcr/Abl kinase has been targeted for the treatment of chronic myelogenous leukaemia (CML) by imatinib mesylate. While imatinib has been extremely effective for chronic phase CML, blast crisis CML are often resistant. New therapeutic options are therefore needed for this fatal disease. Although more common in solid tumors, increased microvessel density was also reported in chronic myelogenous leukaemia and was associated with a significant increase of angiogenic factors, suggesting that vascularity in hematologic malignancies is a controlled process and may play a role in the leukaemogenic process thus representing an alternative therapeutic target. Carboxyamidotriazole-orotate (CTO) is the orotate salt form of carboxyamidotriazole (CAI), an orally bioavailable signal transduction inhibitor that in vitro has been shown to possess antileukaemic activities. CTO, which has a reduced toxicity, increased oral bioavailability and stronger efficacy when compared to the parental compound, was tested in this study for its ability to affect imatinib-resistant CML tumor growth in a xenograft model. The active cross talk between endothelial cells and leukemic cells in the bone marrow involving exosomes plays an important role in modulating the process of neovascularization in CML. We have thus investigated the effects of CTO on exosome-stimulated angiogenesis. Our results indicate that CTO may be effective in targeting both cancer cell growth and the tumor microenvironment, thus suggesting a potential therapeutic utility for CTO in leukaemia patients.

Role of the AMPK/SREBP-1 pathway in the development of orotic acid-induced fatty liver.

Orotic acid (OA), an intermediate in pyrimidine metabolism, has been used for a variety of purposes, such as dietary supplements. Although it is well documented that OA induces fatty liver in a species-specific manner, the precise molecular mechanisms remain unclear. The present study investigated the role of the adenosine monophosphate-activated protein kinase (AMPK)-sterol regulatory element-binding protein-1 (SREBP-1) pathway in the OA-induced fatty liver. Treatment with OA suppressed the phosphorylation of AMPK via proteasomal degradation of upstream kinase LKB1 and induced activation of SREBP-1 in both human hepatoma cell lines and primary rat hepatocytes. OA-induced SREBP-1 transcriptional activity was suppressed by cotreatment with aminoimidazole carboxamide ribonucleotide (AICAR) or metformin, or by overexpression of constitutively active AMPK (CA-AMPK) in the human hepatoma cell line. Importantly, in vivo data corroborated these results. Feeding 1% OA with diet decreased the phosphorylation of AMPK and increased the maturation of SREBP-1 and the expression of SREBP-responsive genes in the rat liver. OA-induced lipid accumulation was also completely inhibited by rapamycin. Mouse hepatocytes and mice were resistant to OA-induced lipogenesis because of little if any response in AMPK and downstream effectors. In conclusion, OA induces hepatic lipogenesis, mediated predominantly by the AMPK/SREBP-1 pathway in rat hepatocytes and human hepatoma cell lines.

New classes of PDE7 inhibitors identified by a fission yeast-based HTS.

Studies of the phosphodiesterase PDE7 family are impeded by there being only one commercially available PDE7 inhibitor, BRL50481. The authors have employed a high-throughput screen of commercial chemical libraries, using a fission yeast-based assay, to identify PDE7 inhibitors that include steroids, podocarpanes, and an unusual heterocyclic compound, BC30. In vitro enzyme assays measuring the potency of BC30 and 2 podocarpanes, in comparison with BRL50481, produce data consistent with those from yeast-based assays. In other enzyme assays, BC30 stimulates the PDE4D catalytic domain but not full-length PDE4D2, suggesting an allosteric site of action. BC30 significantly enhances the anti-inflammatory effect of the PDE4 inhibitor rolipram as measured by release of tumor necrosis factor alpha from activated monocytes. These studies introduce several new PDE7 inhibitors that may be excellent candidates for medicinal chemistry because of the requirements for drug-like characteristics placed on them by the nature of the yeast-based screen.

Experimental evidence that ornithine and homocitrulline disrupt energy metabolism in brain of young rats.

Tissue accumulation of ornithine (Orn), homocitrulline (Hcit), ammonia and orotic acid (Oro) is the biochemical hallmark of patients affected by hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, a disorder clinically characterized by neurological symptoms, whose pathophysiology is practically unknown. In the present study, we investigated the in vitro effect of Orn, Hcit and Oro on important parameters of energy metabolism in brain of 30-day-old Wistar rats since mitochondrial abnormalities have been observed in the affected patients. We first verified that Orn and Hcit significantly inhibited the citric acid cycle (inhibition of CO(2) synthesis from [1-(14)C] acetate, as well as aconitase and alpha-ketoglutarate dehydrogenase activities), the aerobic glycolytic pathway (reduced CO(2) production from [U-(14)C] glucose) and moderately the electron transfer flow (inhibitory effect on complex I-III). Hcit, but not Orn, was also able to significantly inhibit the mitochondrial creatine kinase activity. Furthermore, this inhibition was prevented by GSH, suggesting a possible role of reactive species oxidizing critical thiol groups of the enzyme. In contrast, the other enzyme activities of the citric acid cycle and of the electron transfer chain, as well as synaptic Na(+),K(+)-ATPase were not altered by either Orn or Hcit at concentrations as high as 5.0 mM. Similarly, Oro did not interfere with any of the tested parameters. Taken together, these data strongly indicate that Orn and Hcit compromise brain energy metabolism homeostasis and Hcit also interferes with cellular ATP transfer and buffering. It is therefore suggested that Orn and especially Hcit may be involved in the neurological damage found in patients affected by HHH syndrome.

The role of DNA mismatch repair in generating genetic diversity and drug resistance in malaria parasites.

Although the mechanisms by which malaria parasites develop resistance to drugs are unclear, current knowledge suggests a main mechanism of resistance is the alteration of target enzymes by point mutation. In other organisms, defects in DNA mismatch repair have been linked to increased mutation rates and drug resistance. We have identified an unusual complement of mismatch repair genes in the Plasmodium genome. An initial functional test of two of these genes (PfMSH2-1 and PfMSH2-2) using a dominant mutator assay showed an elevation in mutation frequency with the PfMSH2-2 homolog, indirectly demonstrating a role for this gene in mismatch repair. We successfully disrupted PbMSH2-2 in the Plasmodium berghei laboratory isolate NK65, and showed that this gene is not essential for parasite growth in either the asexual (rodent) or sexual (mosquito) stages of the lifecycle. Although we observed some differences in levels of drug resistance between wild type and mutant parasites, no uniform trend emerged and preliminary evidence does not support a strong link between PbMSH2-2 disruption and dramatically increased drug resistance. We found microsatellite polymorphism in the PbMSH2-2 disrupted parasites in less than 40 life cycles post-transfection, but not in PbMap2K disrupted controls or mosquito-passaged wild type parasites, which suggests a possible role for PbMSH2-2 in preventing microsatellite slippage, similar to MSH2 in other organisms. Our studies suggest that Plasmodium species may have evolved a unique variation on the highly conserved system of DNA repair compared to the mismatch repair systems in other eukaryotes.

The Escherichia coli protein YjjG is a house-cleaning nucleotidase in vivo.

House-cleaning enzymes protect cells from the adverse effects of noncanonical metabolic chemical compounds. The Escherichia coli nucleotide phosphatase YjjG (B4374, JW4336) functions as a house-cleaning phosphatase in vivo. YjjG protects the cell against noncanonical pyrimidine derivatives such as 5-fluoro-2'-deoxyuridine (5-FdUridine), 5-fluorouridine, 5-fluoroorotic acid (5-FOA), 5-fluorouracil, and 5-aza-2'-deoxycytidine. YjjG prevents the incorporation of potentially mutagenic nucleotides into DNA as shown for 5-bromo-2'-deoxyuridine (BrdU). Its enzymatic activity in vitro towards noncanonical 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP) is higher than towards canonical thymidine monophosphate (dTMP). The closest homolog in humans, HDHD4, does not show a protective effect against noncanonical nucleotides, excluding an involvement of HDHD4 in resistance against noncanonical nucleotides used for cancer chemotherapy. The substrate spectrum of YjjG suggests that its in vivo substrates are noncanonical pyrimidine derivatives, which might also include oxidized nucleobases such as 5-formyluracil and 5-hydroxyuracil.

New lanthanum (III) complex--synthesis, characterization, and cytotoxic activity.

The complex of lanthanum (III) was synthesized reacting the respective inorganic salt with 5-aminoorotic acid in amounts equal to the metal:ligand molar ratio of 1:3. The complex was prepared by adding an aqueous solution of lanthanum (III) nitrate to an aqueous solution of the ligand, subsequently raising the pH of the mixture gradually to approx. 5.0 through addition of a dilute solution of sodium hydroxide. The structure of the final complex was determined by means of spectral data (IR, Raman,( 1)H-NMR) and elemental analysis. Significant differences in the IR spectrum of the complex were observed as compared to the spectrum of the ligand. A comparative analysis of the Raman spectrum of the complex with that of the free 5-aminoorotic acid allowed a straightforward assignment of the vibrations of the ligand groups involved in coordination. The ligand and the complex were tested for the cytotoxic activities on the chronic myeloid leukemia derived K-562, overexpressing the BCR-ABL fusion protein and the non-Hodgkin lymphoma derived DOHH-2, characterized by a re-expression of the anti-apoptotic protein bcl-2 cell lines. The results obtained indicate that the tested compounds exerted a considerable cytotoxic activity upon the evaluated cell lines in a concentration-dependent matter, which enabled the construction of dose-response curves and the calculation of the corresponding IC(50 )values. The inorganic salt exerted a very weak cytotoxic effect on these cells, which is in contrast to the lanthanum (III) complex.