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Shiitake (Lentinula edodes) contains various beneficial compounds and possesses several notable properties. However, there are few reports on its molecular breeding due to delay in development of its gene-modifying technology. Therefore, here, strain UV30 (pyrG -) was bred from the UV-irradiated protoplasts of strain M2. Strain UV30 was uracil-auxotrophic, and the phenylalanine residue in the active centre of orotidine-5-phosphate decarboxylase encoded by pyrG in the strain was substituted with a serine residue. Next, a recycling marker consisting of the upstream sequence of ku80, a repeat sequence (a portion of the downstream sequence of ku80), pyrG, and the downstream sequence of ku80 was introduced into the strain UV30. Consequently, the prototrophic strain ckp2-1, in which ku80 was replaced with the recycling marker, was obtained. After cultivation in complete medium, mycelia from the edges of ckp2-1 colonies were inoculated into a complete medium containing 5-Fluoroorotic acid (5-FOA). A 5-FOA-resistant strain KaM2, in which pyrG sequence was spliced from the recycling marker sequence via homologous recombination, was obtained. In this study, we developed the first marker recycling system for multigene targeting in L. edodes. Moreover, the resulting ∆ku80 strain may serve as a non-homologous end-joining deficient strain for further genetic manipulations., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

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., Competing Interests: Declaration of Competing Interest The authors declare they have no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Alzheimer's disease (AD) is characterized by excessive formation of beta-amyloid peptides (Aβ), 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-APP 695 cells-a model of early AD. Thus, SH-SY5Y-APP 695 cells were incubated with SC and the activity of the mitochondrial respiration chain complexes were measured, as well as levels of ATP, Aβ, ROS, lactate and pyruvate. Incubation of SH-SY5Y-APP 695 cells with SC significantly increased the endogenous respiration of mitochondria and ATP levels, while Aβ 1-40 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.

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 β-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.

Impairment of pancreatic β cells is a principal driver of the development of diabetes. Restoring normal insulin release from the β 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 β cells and subsequently reduced β cell death in the db/db mouse. Orotic acid intake helped to maintain the islet size, number of β cells, and protected insulin secretion in the db/db mouse. In conclusion, orotic acid treatment maintained β cell function and reduced cell death, and may therefore, be a future therapeutic strategy for the prevention and treatment of diabetes., Competing Interests: Declaration of competing interest All authors have no conflict of interest about the manuscript., (Copyright © 2021. Published by Elsevier Inc.)

Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications. The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable. In this study, we developed a screening system for targeted gene knockout using a uracil auxotrophic host (Δ pyrF ) resistant to the highly toxic uracil analog of 5-fluoroorotic acid (5-FOA) converted by PyrF, and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase. The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications. Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutant Δ pyrF at the targeted locus. Double-crossover recombinants were generated, from which the pyrF gene, plasmid backbone, and targeted gene were excised through homologous recombination exchange. These recombinants were rapidly screened by the counterselection agent, 5-FOA. We demonstrated the feasibility and advantage of using this pyrF- based screening system through deleting the otcR gene, which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018. This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species.

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.

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.

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 (KO 2 ), 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.

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeastlike fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 (rap/FK506) and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37° compared to 30° in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNA interference (RNAi) pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30° to the level observed at 37°. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host., Competing Interests: Competing interest statement: A.C. and J.H. are coauthors on a forthcoming position paper. They have not collaborated directly.

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.

Genome sequencing of pathogenic fungi has revealed the presence of various effectors that aid pathogen invasion by the manipulation of plant immunity. Effectors are often individually dispensable because of duplication and functional redundancy as a result of the arms race between host plants and pathogens. To study effectors that have functional redundancy, multiple gene disruption is often required. However, the number of selection markers that can be used for gene targeting is limited. Here, we established a marker recycling system that allows the use of the same selection marker in successive transformations in the model fungal pathogen Colletotrichum orbiculare, a causal agent of anthracnose disease in plants belonging to the Cucurbitaceae. We identified two C. orbiculare homologues of yeast URA3/pyrG, designated as URA3A and URA3B, which can be used as selection markers on medium with no uridine. The gene can then be removed from the genome via homologous recombination when the fungus is grown in the presence of 5-fluoroorotic acid (5-FOA), a chemical that is converted into a toxin by URA3 activity. The ura3a/b double mutants showed auxotrophy for uridine and insensitivity to 5-FOA. Using the ura3a/b mutants, transformation with the URA3B marker and its removal were successfully applied to disrupt the virulence-related gene, PKS1. The pks1 mutants showed a reduction in virulence, demonstrating that the method can be used to study virulence-related genes in C. orbiculare. The establishment of a URA3-based marker recycling system in plant-pathogenic fungi enables the genetic analysis of multiple genes that have redundant functions, including effector genes., (© 2018 The Authors. Molecular Plant Pathology published by BSPP and John Wiley & Sons Ltd.)

Rhizopus delemar causes devastating mucormycosis in immunodeficient individuals. Despite its medical importance, R. delemar remains understudied largely due to the lack of available genetic markers, the presence of multiple gene copies due to genome duplication, and mitotically unstable transformants resulting from conventional and limited genetic approaches. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system induces efficient homologous and non-homologous break points and generates individual and multiple mutant alleles without requiring selective marker genes in a wide variety of organisms including fungi. Here, we have successfully adapted this technology for inducing gene-specific single nucleotide (nt) deletions in two clinical strains of R. delemar: FGSC-9543 and CDC-8219. For comparative reasons, we first screened for spontaneous uracil auxotrophic mutants resistant to 5-fluoroorotic acid (5-FOA) and obtained one substitution (f1) mutationin the FGSC-9543 strain and one deletion (f2) mutation in the CDC-8219 strain. The f2 mutant was then successfully complemented with a pyrF-dpl200 marker gene. We then introduced a vector pmCas9:tRNA-gRNA that expresses both Cas9 endonuclease and pyrF-specific gRNA into FGSC-9543 and CDC-8219 strains and obtained 34 and 42 5-FOA resistant isolates, respectively. Candidate transformants were successively transferred eight times by propagating hyphal tips prior to genotype characterization. Sequencing of the amplified pyrF allele in all transformants tested revealed a single nucleotide (nt) deletion at the 4th nucleotide before the protospacer adjacent motif (PAM) sequence, which is consistent with CRISPR-Cas9 induced gene mutation through non-homologous end joining (NHEJ). Our study provides a new research tool for investigating molecular pathogenesis mechanisms of R. delemar while also highlighting the utilization of CRISPR-Cas9 technology for generating specific mutants of Mucorales fungi., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mucormycosis-an emergent, deadly fungal infection-is difficult to treat, in part because the causative species demonstrate broad clinical antifungal resistance. However, the mechanisms underlying drug resistance in these infections remain poorly understood. Our previous work demonstrated that one major agent of mucormycosis, Mucor circinelloides, can develop resistance to the antifungal agents FK506 and rapamycin through a novel, transient RNA interference-dependent mechanism known as epimutation. Epimutations silence the drug target gene and are selected by drug exposure; the target gene is re-expressed and sensitivity is restored following passage without drug. This silencing process involves generation of small RNA (sRNA) against the target gene via core RNAi pathway proteins. To further elucidate the role of epimutation in the broad antifungal resistance of Mucor, epimutants were isolated that confer resistance to another antifungal agent, 5-fluoroorotic acid (5-FOA). We identified epimutant strains that exhibit resistance to 5-FOA without mutations in PyrF or PyrG, enzymes which convert 5-FOA into the active toxic form. Using sRNA hybridization as well as sRNA library analysis, we demonstrate that these epimutants harbor sRNA against either pyrF or pyrG, and further show that this sRNA is lost after reversion to drug sensitivity. We conclude that epimutation is a mechanism capable of targeting multiple genes, enabling Mucor to develop resistance to a variety of antifungal agents. Elucidation of the role of RNAi in epimutation affords a fuller understanding of mucormycosis. Furthermore, it improves our understanding of fungal pathogenesis and adaptation to stresses, including the evolution of drug resistance., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Preliminary study results have shown that rats with non-alcoholic fatty liver disease (NAFLD) induced by 1% orotic acid-containing diet have decreased hepatic CYP2D activity. This study aims to evaluate the possible pharmacokinetic changes in NAFLD as a result of reduced metabolic activity of CYP2D., Methods: The pharmacokinetics of metoprolol and its metabolites, O-desmethyl metoprolol (DMM) and α-hydroxy metoprolol (HM), was investigated in NAFLD and control rats following intravenous (1 mg/kg) and oral (2 mg/kg) administration of metoprolol. The hepatic CYP2D expression was also investigated., Results: NAFLD rats had lower CYP2D expression (by 36.6%) and slower intrinsic clearance (CLint) of metoprolol and formation of HM (by 40.1% and 37.2%, respectively). There were no significant changes in the pharmacokinetics of metoprolol and its metabolites following intravenous administration. In contrast, oral administration of metoprolol resulted in significantly increased total area under plasma concentration-time curve (AUC) of metoprolol (by 127%) and decreased metabolite formation ratios (AUCDMM/AUCMetoprolol [by 42.8%], AUCHM/AUCMetoprolol [by 35.0%]) in NAFLD rats. Moreover, these changes were well correlated with severity of steatosis as quantified by hepatic triglyceride contents., Conclusions: NALFD can lead to a reduction in the hepatic CLint of a drug if it is a substrate of the CYP2D subfamily. The decreased clearance may result in elevated drug concentrations and increased exposure.

Diabetes is an increasingly common metabolic disorder with high comorbidity and societal and personal costs. Insulin replacement therapy is limited by a lack of oral bioavailability. Recent studies suggest vanadium has therapeutic potential. A newly synthesized complex between oxidovanadium (IV) and orotic acid (OAH 3 ), [(OAH 1 )(VO)(NH 3 ) 2 ].3H 2 O, was characterized using spectroscopic and thermogravimetric techniques. In vivo potential was assessed in a streptozocin-induced rat model of diabetes. OAH 3 acts as a bidentate ligand in the formation of the dark green, crystalline oxidovanadium (IV) complex in a square pyramidal configuration. Treatment with oxidovanadium (IV)-orotate in vivo significantly improved many biochemical parameters with minimal toxicity and restored pancreatic and hepatic histology. The results of the present work describe a safe, new compound for the treatment of diabetes.

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.

Thermoanaerobacter kivui is one of the very few thermophilic acetogenic microorganisms. It grows optimally at 66°C on sugars but also lithotrophically with H 2 + CO 2 or with CO, producing acetate as the major product. While a genome-derived model of acetogenesis has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in carbon and energy metabolism have been carried out. To address this issue, we developed a method for targeted markerless gene deletions and for integration of genes into the genome of T. kivui The strain naturally took up plasmid DNA in the exponential growth phase, with a transformation frequency of up to 3.9 × 10 -6 A nonreplicating plasmid and selection with 5-fluoroorotate was used to delete the gene encoding the orotate phosphoribosyltransferase ( pyrE ), resulting in a Δ pyrE uracil-auxotrophic strain, TKV002. Reintroduction of pyrE on a plasmid or insertion of pyrE into different loci within the genome restored growth without uracil. We subsequently studied fructose metabolism in T. kivui The gene fruK (TKV_c23150) encoding 1-phosphofructosekinase (1-PFK) was deleted, using pyrE as a selective marker via two single homologous recombination events. The resulting Δ fruK strain, TKV003, did not grow on fructose; however, growth on glucose (or on mannose) was unaffected. The combination of pyrE as a selective marker and the natural competence of the strain for DNA uptake will be the basis for future studies on CO 2 reduction and energy conservation and their regulation in this thermophilic acetogenic bacterium. IMPORTANCE Acetogenic bacteria are currently the focus of research toward biotechnological applications due to their potential for de novo synthesis of carbon compounds such as acetate, butyrate, or ethanol from H 2 + CO 2 or from synthesis gas. Based on available genome sequences and on biochemical experiments, acetogens differ in their energy metabolism. Thus, there is an urgent need to understand the carbon and electron flows through the Wood-Ljungdahl pathway and their links to energy conservation, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Unfortunately, genetic systems have been reported for only a few acetogenic bacteria. Here, we demonstrate proof of concept for the genetic modification of the thermophilic acetogenic species Thermoanaerobacter kivui The genetic system will be used to study genes involved in biosynthesis and energy metabolism, and may further be applied to metabolically engineer T. kivui to produce fuels and chemicals., (Copyright © 2018 American Society for Microbiology.)

The effects of six nucleating agents (NAs), i.e., orotic acid (OA), potassium salt of 3,5-bis(methoxycarbonyl)benzenesulfonate (LAK-301), substituted-aryl phosphate salts (TMP-5), talc (TALC), N' 1 ,N' 6 -dibenzoyladipohydrazide (TMC-306) and N 1 ,N 1' -(ethane-1,2-diyl)bis(N 2 -phenyloxalamide) (OXA), on the crystallization behavior of poly(lactic acid) (PLA) were compared by DSC. Under the same dosing of 0.5wt%, the nucleation effect of the NAs for PLA declines in the order of TMC-306≈OXA>TALC≫TMP-5≈LAK-301≈OA. The nucleation efficiency (NE) of TMC-306 and OXA is around 50%, which is almost 2 times of the NE of TALC. In the best case of the PLA/TMC-0.5% sample, the half-time of crystallization decreases from 30s to 9s with decreasing the crystallization temperature from 120°C to 100°C, which is of great significance to the fast production of highly crystallized PLA materials. As high-efficient NAs, TMC-306 and OXA are able to accelerate the crystallization rate of PLA even upon fast cooling at 50°C/min, while make no difference on PLA crystal form, as identified by WAXD. DMA analysis shows that the storage modulus of PLA is significantly improved by TMC-306 and OXA., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single-stranded carrier DNA and polyethylene glycol (lithium acetate treatment), generally used for ascomycetous yeast transformation. In the rice-derived P. antarctica strain GB-4(0), PaURA3, a homologue of the Saccharomyces cerevisiae orotidine-5'-phosphate decarboxylase gene (URA3), was selected as the target locus. A disruption cassette was constructed by linking the nouseothricine resistance gene (natMX4) to homologous DNA fragments of PaURA3, then electroporated into the strain GB-4(0). We obtained strain PGB015 as one of the PaURA3 disruptants (Paura3Δ::natMX4). Then the PCR-amplified PaURA3 fragment was introduced into PGB015, and growth of transformant colonies but not background colonies was observed on selective media lacking uracil. The complementation of uracil-auxotrophy in PGB015 by introduction of PaURA3 was also performed using lithium acetate treatment, which resulted in a transformation efficiency of 985 CFU/6.8 μg DNA and a gene-targeting ratio of two among 30 transformants. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

It is well known that 5-fluoroorotic acid (5-FOA)-resistant mutants isolated from wild-type Cryptococcus neoformans are exclusively either ura3 or ura5 mutants. Unexpectedly, many of the 5-FOA-resistant mutants isolated in our selective regime were Ura + . We identified CNM00460 as the gene responsible for these mutations. Cnm00460 belongs to the nucleobase cation symporter 1/purine-related transporter (NCS1/PRT) super family of fungal transporters, representative members of which are uracil transporter, uridine transporter and allantoin transporter of Saccharomyces cerevisiae. Since the CNM00460 gene turned out to be involved in utilization of orotic acid, most probably as transporter, we designated this gene Orotic Acid Transporter 1 (OAT1). This is the first report of orotic acid transporter in this family. C. neoformans has four members of the NCS1/PRT family, including Cnm00460, Cnm02550, Cnj00690, and Cnn02280. Since the cnm02550∆ strain showed resistance to 5-fluorouridine, we concluded that CNM02550 encodes uridine permease and designated it URidine Permease 1 (URP1). We found that oat1 mutants were sensitive to 5-FOA in the medium containing proline as nitrogen source. A mutation in the GAT1 gene, a positive transcriptional regulator of genes under the control of nitrogen metabolite repression, in the genetic background of oat1 conferred the phenotype of weak resistance to 5-FOA even in the medium using proline as nitrogen source. Thus, we proposed the existence of another orotic acid utilization system (tentatively designated OAT2) whose expression is under the control of nitrogen metabolite repression at least in part. We found that the OAT1 gene is necessary for full pathogenic activity of C. neoformans var. neoformans.

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.

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, is the leading cause of heart disease in Latin America. T. cruzi dihydroorotate dehydrogenase (DHODH), which catalyzes the production of orotate, was demonstrated to be essential for T. cruzi survival, and thus has been considered as a potential drug target to combat Chagas disease. Here we report the design and synthesis of 75 compounds based on the orotate structure. A comprehensive structure-activity relationship (SAR) study revealed two 5-substituted orotate analogues (5u and 5v) that exhibit K i app values of several ten nanomolar level and a selectivity of more than 30,000-fold over human DHODH. The information presented here will be invaluable in the search for next-generation drug leads for Chagas disease., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Aim: To study neocollagenogenesis after implantation of polypropylene endoprosthesis and polypropylene combined with polylactic acid endoprosthesis on background of «potassium orotate» 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 Orotate» 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.

Orotate (OA) is well-known as a precursor in biosynthesis of pyrimidines; in mammals it is released from the mitochondrial dihydroorotate dehydrogenase (DHODH) for conversion to UMP by the cytoplasmic UMP synthase enzyme. OA is also a normal part of the diet, being found in milk and dairy products, and it is converted to uridine for use in the pyrimidine salvage pathway predominantly in liver, kidney and erythrocytes. Early research into nutrition identified orotate as "vitamin B13," and its use as a complex with organic cations or metal ions was promulgated in body-building, and in assisting therapies of metabolic syndromes. It has recently been established that the amelioration of gout by dairy products arises from the competition of orotate and urate at the hURAT1 transporter. The orotic aciduria that arises in children with defective UMP synthase can be rescued by oral uridine therapy, since UMP is the end-product and also a feedback inhibitor of the de novo pathway. In contrast, Miller (dysmorphology) syndrome is connected with defects in DHODH, and hence in the supply of OA, and cannot be helped by uridine. Other models of dysmorphisms are connected with enzymes early in the pyrimidine de novo pathway. We conclude that the OA molecule is itself required for the regulation of genes that are important in the development of cells, tissues and organisms.

Predictable integration of foreign biological signals and parts remains a key challenge in the systematic engineering of synthetic cellular actuations, and general methods to improve signal transduction and sensitivity are needed. To address this problem we modeled and built a molecular signal buffer network in Saccharomyces cerevisiae inspired by chemical pH buffer systems. The molecular buffer system context-insulates a riboswitch enabling synthetic control of colony formation and modular signal manipulations. The riboswitch signal is relayed to a transcriptional activation domain of a split transcription factor, while interacting DNA-binding domains mediate the transduction of signal and form an interacting molecular buffer. The molecular buffer system enables modular signal inversion through integration with repressor modules. Further, tuning of input sensitivity was achieved through perturbation of the buffer pair ratio guided by a mathematical model. Such buffered signal tuning networks will be useful for domestication of RNA-based sensors enabling tunable outputs and library-wide selections for drug discovery and metabolic engineering.

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.

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) - a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite., (© 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Background: The discovery of novel influenza virus inhibitors remains an important priority in light of the emergence of drug-resistant viruses. Toward this end, a library of over 6,000 compounds was tested for antiviral activity., Methods: Strains of influenza virus were evaluated by cytopathic effect (CPE) inhibition and virus yield reduction assays. Intracellular nucleoside triphosphate pools were analysed by strong anion exchange HPLC. Dihydroorotate dehydrogenase inhibition assays were conducted. Influenza virus-infected mice were treated for 5 days with D282., Results: A non-nucleoside, 4-[(4-butylphenyl)amino]-2-methylene-4-oxo-butanoic acid (D282), was discovered that inhibited influenza A and B virus CPE by 50% at 6-31 μM (giving selectivity indices of >13 to >67, based on cytotoxicity of >400 µM in stationary cell cultures). Ribavirin (positive control) was active at 14-44 µM (yielding selectivity indices of >9 to >29, with >400 µM toxicity). D282 and ribavirin inhibited virus yield by 90% at 9.5 ±3.3 and 10.8 ±3.2 µM, respectively. The antiviral activity of D282 in vitro was reversed by addition of uridine, cytidine and orotic acid. D282 exhibited an uncompetitive inhibition of mouse liver dihydroorotate dehydrogenase (inhibitor constant [Ki] of 2.3 ±0.9 µM, Michaelis constant [Km] of 150 ±16 µM). Because cellular pyrimidine biosynthesis was inhibited, D282-treated cells had decreased uridine triphosphate and cytidine triphosphate levels. D282 (≤100 mg/kg/day) failed to prevent death of mice infected with influenza., Conclusions: D282 was active against influenza A and B viruses by inhibiting de novo pyrimidine biosynthesis. Although effective in vitro, the compound, like others in its class, was devoid of antiviral activity in infected mice.

We have developed a range of vectors for allelic replacements in Staphylococcus aureus to facilitate genetic work in this opportunistic pathogen. The central feature of the vector range is a selection/counterselection system that takes advantage of the 5-fluoroorotic acid (FOA) resistance and pyrimidine prototrophy caused by the loss and gain, respectively, of the pyrF and pyrE genes. This system allows for stringent counterselection of the vectors during the second homologous recombination of a classic allelic replacement. The basic vector pRLY2, which contains the pyrFE genes from Bacillus subtilis, was combined with chloramphenicol, erythromycin, and tetracycline resistance genes and four different versions of nonreplicative or conditionally replicative origins of replication. The choice between these 12 different pRLY vectors allows for high versatility and ensures that the vectors can be used in virtually any genetic background. Finally, as proof of concept, we present six deletions or modifications of components in the S. aureus degradosome as well as the operon containing the cshB DEAD box helicase.

Aims: As cardiac performance is closely related to its energy supply, our study investigated the effect of the orotic acid cardioprotective agent on the pathways of energy supply, in both conditions of normal flow and ischemia., Main Methods: Male Wistar rats were fed during nine days with a balanced diet only or supplemented with 1% orotic acid., Key Findings: Dietary administration of orotic acid increased the cardiac utilization of fatty acids, activity of the lipoprotein lipase, expression of the gene of peroxisome proliferator-activated receptor α and its target enzymes. In addition, orotic acid increased the myocardial uptake and incorporation of glucose, glycogen content and level of GLUT4, concentration of glycolytic metabolites and lactate production in both experimental conditions, baseline and after regional ischemia., Significance: Thus, in orotic acid hearts there was a simultaneous stimulus of fatty acid oxidation and glycolytic pathway, reflected in increased energetic content even in pre-ischemia. The analysis of the cardiac contractility index showed a positive inotropic effect of orotic acid due, at least in part, to the increased availability of energy. The result allows us to suggest that the metabolic changes induced by orotic acid result in appreciable alterations on myocardial contractile function., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

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.

Nucleotides are synthesized from de novo and salvage pathways. To characterize the uridine salvage pathway, two genes, UKL1 and UKL2, that tentatively encode uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) bifunctional enzymes were studied in Arabidopsis thaliana. T-DNA insertions in UKL1 and UKL2 reduced transcript expression and increased plant tolerance to toxic analogs 5-fluorouridine and 5-fluorouracil. Enzyme activity assays using purified recombinant proteins indicated that UKL1 and UKL2 have UK but not UPRT activity. Subcellular localization using a C-terminal enhanced yellow fluorescent protein fusion indicated that UKL1 and UKL2 localize to plastids. The ukl2 mutant shows reduced transient leaf starch during the day. External application of orotate rescued this phenotype in ukl2, indicating pyrimidine pools are limiting for starch synthesis in ukl2. Intermediates for lignin synthesis were upregulated, and there was increased lignin and reduced cellulose content in the ukl2 mutant. Levels of ATP, ADP, ADP-glucose, UTP, UDP, and UDP-glucose were altered in a light-dependent manner. Seed composition of the ukl1 and ukl2 mutants included lower oil and higher protein compared with the wild type. Unlike single gene mutants, the ukl1 ukl2 double mutant has severe developmental defects and reduced biomass accumulation, indicating these enzymes catalyze redundant reactions. These findings point to crucial roles played by uridine salvage for photoassimilate allocation and partitioning.

Uridine-5'-monophosphate synthase (UMPS), the critical step of the de novo pyrimidine biosynthesis pathway, which is a housekeeping plastid process in higher plants, was investigated in a marine diatom, the most crucial primary producer in the marine environment. A mutagenesis using an alkylation agent, N-ethyl-N-nitrosourea, was carried out to the marine diatom Phaeodactylum tricornutum. Cells were treated with 1.0 mg mL(-1) N-ethyl-N-nitrosourea and were screened on agar plates containing 100 to 300 mg L(-1) 5-fluoroorotidic acid (5-FOA). Two clones survived the selection and were designated as Requiring Uracil and Resistant to FOA (RURF) 1 and 2. The 50% effective concentration of 5-FOA on growth of RURF1 was about 5 mm, whereas that in wild-type cells was 30 μm. The ability to grow in the absence of uracil was restored by a P. tricornutum gene that potentially encoded UMPS or the human umps gene, HUMPS. Because the P. tricornutum gene was able to restore growth in the absence of uracil, it was designated as ptumps, encoding a major functional UMPS in P. tricornutum. RNA interference to the ptumps targeting the 5' region of ptumps resulted in the occurrence of a clear RURF phenotype in P. tricornutum. This RNA interference phenotype was reverted to the wild type by the insertion of HUMPS, confirming that the ptumps encodes UMPS. These results showed direct evidence of the occurrence of novel-type UMPS in a marine diatom and also revealed the potential usage of this gene silencing and complementation system for molecular tools for this organism.

The ability to engineer genetic programs governing cell fate will permit new safeguards for engineered organisms and will further the biological understanding of differentiation and aging. Here, we have designed, built and implemented a genetic device in the budding yeast Saccharomyces cerevisiae that controls cell-cycle progression selectively in daughter cells. The synthetic device was built in a modular fashion by combining timing elements that are coupled to the cell cycle, i.e. cell-cycle specific promoters and protein degradation domains, and an enzymatic domain which conditionally confers cell arrest. Thus, in the presence of a drug, the device is designed to arrest growth of only newly-divided daughter cells in the population. Indeed, while the engineered cells grow normally in the absence of drug, with the drug the engineered cells display reduced, linear growth on the population level. Fluorescence microscopy of single cells shows that the device induces cell arrest exclusively in daughter cells and radically shifts the age distribution of the resulting population towards older cells. This device, termed the 'daughter arrester', provides a blueprint for more advanced devices that mimic developmental processes by having control over cell growth and death.

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.

Background: Nonalcoholic fatty liver disease is the most common chronic liver disease in the world, and is becoming increasingly prevalent. Saponins of sea cucumber (SSC) are proven to exhibit various biological activities. Therefore, the present study was undertaken to examine the effect of saponins extracted from sea cucumber (Pearsonothuria graeffei) on the preventive activity of fatty liver in rats., Methods: Male Wistar rats were randomly divided into five groups, including normal control group, fatty liver model group, SSC-treated group with SSC at levels of 0.01%, 0.03% and 0.05%. Model rats were established by administration with 1% orotic acid (OA). After the experiment period, serum total cholesterol (TC), triglyceride (TG), and hepatic lipid concentrations were determined. To search for a possible mechanism, we examined the changes of key enzymes and transcriptional factors involved in hepatic lipids biosynthesis, fatty acid beta-oxidation., Results: Both 0.03% and 0.05% SSC treatment alleviated hepatic steatosis and reduced serum TG and TC concentration significantly in OA fed rats. Hepatic lipogenic enzymes, such as fatty acid synthase (FAS), malic enzyme (ME), and glucose-6-phosphate dehydrogenase (G6PDH) activities were inhibited by SSC treatment. SSC also decreased the gene expression of FAS, ME, G6PDH and sterol-regulatory element binding protein (SREBP-1c). Otherwise, the rats feeding with SSC showed increased carnitine palmitoyl transferase (CPT) activity in the liver. Hepatic peroxisome proliferator-activated receptor (PPARalpha), together with its target gene CPT and acyl-CoA oxidase (ACO) mRNA expression were also upregulated by SSC., Conclusions: According to our study, the lipids-lowering effect of dietary SSC may be partly associated with the enhancement of beta-oxidation via PPARalpha activation. In addition, the inhibited SREBP-1c- mediated lipogenesis caused by SSC may also contribute to alleviating fatty liver.

We aim to create an Aspergillus oryzae mutant with a highly reduced chromosome, but better growth, by eliminating the nonessential regions coding various dispensable functions for its better industrial use. In our previous study, we successfully determined the outline of essential and nonessential regions by constructing a series of large chromosomal deletions in A. oryzae chromosome 7. Based on these results, we here constructed two mutants, designated RkuAF7A and RkuAF7B, lacking 24.7 and 24% (725 and 705 kb) of wild type chromosome 7, respectively, using multiple large-scale chromosomal deletions in a recursive pyrG-mediated transformation system. Both showed higher amylase activity in DPY liquid medium and faster growth rate on malt agar medium relative to the parent strain. The two mutants also displayed soft fluffy hyphal morphology when grown in DPY liquid media. In addition, the gene expression profile obtained by DNA microarray indicated that although the deletion regions were fewer than 2% of the whole genome, the effect on whole gene expression exceeded 20%. Among these, the genes involved in secondary metabolism showed a relatively large change in their gene expression levels. Together, the constructed mutants showing better growth and potential usefulness is possibly suitable for further industrial use.

In eukaryotes, ribosomes are made from precursor rRNA (pre-rRNA) and ribosomal proteins in a maturation process that requires a large number of snoRNPs and processing factors. A fundamental problem is how the coordinated and productive folding of the pre-rRNA and assembly of successive pre-rRNA-protein complexes is achieved cotranscriptionally. The conserved protein Mrd1p, which contains five RNA binding domains (RBDs), is essential for processing events leading to small ribosomal subunit synthesis. We show that full function of Mrd1p requires all five RBDs and that the RBDs are functionally distinct and needed during different steps in processing. Mrd1p mutations trap U3 snoRNA in pre-rRNP complexes both in base-paired and non-base-paired interactions. A single essential RBD, RBD5, is involved in both types of interactions, but its conserved RNP1 motif is not needed for releasing the base-paired interactions. RBD5 is also required for the late pre-rRNP compaction preceding A(2) cleavage. Our results suggest that Mrd1p modulates successive conformational rearrangements within the pre-rRNP that influence snoRNA-pre-rRNA contacts and couple U3 snoRNA-pre-rRNA remodeling and late steps in pre-rRNP compaction that are essential for cleavage at A(0) to A(2). Mrd1p therefore coordinates key events in biosynthesis of small ribosome subunits.

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.

Purpose: Mildronate and orotic acid act as modulators of energy metabolism and are considered as cardioprotective agents. This study was performed to compare the cardioprotective effects of mildronate, orotic acid and mildronate orotate., Methods: Male Wistar rats received mildronate, orotic acid or mildronate orotate for 14 days. The isolated rat heart infarction and isoproterenol-induced ischemia models were used to test the cardioprotective effects of compounds studied. Experimental arrhythmias were induced by the ligation of the left anterior descending coronary artery for 10 min with subsequent reperfusion or by administration of calcium chloride or aconitine at arrhythmogenic doses., Results: The data obtained showed a statistically significant decrease of necrotic area in 25% of infarcted rat hearts after 14 days of treatment with mildronate and orotic acid, whereas mildronate orotate decreased the infarct size by 50%. Moreover, we found that the administration of mildronate and its orotate salt decreased the duration and incidence of arrhythmias in experimental arrhythmia models., Conclusions: The study provides experimental evidence that the combination of orotic acid and mildronate possesses additive pharmacological effects and that mildronate orotate might be considered as a powerful therapeutic agent facilitating recovery from ischemia-reperfusion injury.

Resistance to antimalarial antifolates necessitates a search for new antimetabolites targeting other enzymes of the folate metabolic pathway. In this study, 5-fluoroorotate (FOA), reported to be an inhibitor of thymidylate synthase, was assayed against Plasmodium berghei NK 65 in mice, with(out) an oral uridine supplement. FOA (2.5 and 5.0 mg/kg bw.) was tested alone, or in a double and triple combination with a fixed oral dose of 1.25 and 2.5 mg/kg of pyrimethamine (PYR); 1.0 and 2.0 mg/kg of dapsone (DAP); 1.0 and 2.0 mg/kg of artesunate (ART). FOA achieved high suppression which ranged from 95.7% to aparasitaemic, activity that was dose-dependent. At the highest dosages used, FOA-PYR and FOA-DAP-ART combinations were synergistic with 100% cure rate, while FOA-PYR-ART was antagonistic. Drugs in a synergistic combination may exert less resistance selection pressure, thus FOA-PYR and FOA-DAP-ART warrant further evaluation with an ultimate object of possible clinical use against drug-resistant malaria.

Bovine serum is an essential factor for the continuous in vitro growth of Babesia bovis parasites. Culture media typically contain 40% (v/v) of bovine serum. In the present study assays with low-serum media were performed. The growth of B. bovis Mo7 was successively lower in media with 30%, 20% and, principally, with 10% of serum. Without serum, the parasites were not able to propagate. In media with 10% of serum, the supplementation with Albumax II improved visibly the growth of B. bovis at the end of each cycle. Regarding the addition of orotic acid, no considerable effect was observed in media with 20% or 10% of serum, with or without Albumax II. B. bovis parasites cultured in vitro in all these media maintain their typical morphology during the intraerythrocytic stages.

The synthesis and in-vitro biological evaluation of the amide series 4 of 5-(4-chlorobenzoyl)aminoorotic acid 2 are presented. The biological properties of a few 5-(4-chlorobenzoyl)amino-2,6-dihydroxy-N-substituted-4-pyrimidinecarboxamide derivatives 4 tested here were compared with those of the isosteric isothiazole derivative MR-2/94 (5-(4-chlorobenzoyl)amino-N-(4-chlorophenyl)-3-methyl-4-isothiazolecarboxamide), which possesses a strong immunosuppressive and anti-inflammatory activity [1, 2], It must be suggested that replacement of the isothiazole by a pyrimidine core ring system resulted in considerable lowering of the anti-inflammatory and immunotropic actions of the obtained amides. Physicochemical properties of 2-(4-chlorophenyl)-6,8-dihydroxy-4H-pyrimido[5,4-d]-1,3-oxazin-4-on 3 are also briefly described.

Dexrazoxane (ICRF-187) is clinically used as a doxorubicin cardioprotective agent and to prevent anthracycline extravasation injury. It may act by preventing iron-based oxygen free radical damage through the iron-chelating ability of its metabolite N,N'-[(1S)-1-methyl-1,2-ethanediyl]bis[(N-(2-amino-2-oxoethyl)]glycine (ADR-925). Dexrazoxane undergoes an initial metabolism to its two one-ring open intermediates [N-(2-amino-2-oxoethyl)-N-[(1S)-2-(3,5-dioxo-1-piperazinyl)-1-methylethyl]glycine (B) and N-(2-amino-2-oxoethyl)-N-[(2S)-2-(3,5-dioxo-1-piperazinyl)propyl]glycine (C)] and is then further metabolized to its presumably active metal-chelating form ADR-925. We previously showed that the first ring opening reaction is catalyzed by dihydropyrimidinase and the second by dihydroorotase (DHOase), but not vice versa. To determine whether DHOase was important in the metabolism of dexrazoxane, its metabolism and that of B and C to ADR-925 were measured in rats that were pretreated with the DHOase inhibitor 5-aminoorotic acid. In rats pretreated with 5-aminoorotic acid the area-under-the-curve concentration of ADR-925 was reduced 5.3-fold. In rats treated with a mixture of B and C, the maximum concentration of ADR-925 in the plasma was significantly decreased in rats pretreated with 5-aminoorotic acid, which indicates that DHOase directly metabolized B and C. Both heart and liver tissue levels of ADR-925 in rats were also greatly reduced by pretreatment with 5-aminoorotic acid. Together these results indicate that the metabolism of dexrazoxane and of B and C is mediated by DHOase. These results provide a mechanistic basis for the antioxidant cardioprotective activity of dexrazoxane.

The cerium (III), lanthanum (III) and neodymium (III) complexes of 5-aminoorotic acid were synthesized and their structures were determined by means of theoretical study, spectral and physicochemical analysis. Significant differences in the spectra of the complexes were observed as compared to the spectra of the ligand. The ligand and the complexes were tested for the antioxidant activity. The results obtained indicate that the tested compounds exerted a considerable antioxidant effect upon the evaluated radicals' formation in the blood plasma of Wistar rat.

The yeast split-ubiquitin system has previously been shown to be suitable to detect protein interactions of membrane proteins and of transcription factors in vivo. Therefore, this technology complements the classical split-transcription factor based yeast two-hybrid system (Y2H). Success or failure of the Y2H depends primarily on the ability to avoid false-negative and false-positive hits that become a limiting factor for the value of the system, especially in large scale proteomic analyses. We provide here a systematic assessment of parameters to help improving the quality of split-ubiquitin cDNA-library screenings. We experimentally defined the optimal 5-fluoroorotic acid (5-FOA) concentration as a key parameter to increase the reproducibility of interactions and, at the same time, to keep non-specific background growth low. Furthermore, we show that the efficacy of the 5-FOA selection is modulated by the plating density of the yeast clones. Moreover, a reporter-specific class of false-positive hits was identified, and a simple phenotypic assay for efficient de-selection was developed. We demonstrate the application of this improved system to identify novel interacting proteins of the human Frizzled 1 receptor. We identified several novel interactors with components of the Wnt-Frizzled signalling pathways and discuss their potential roles as direct mediators of Frizzled receptor signalling. The present work is the first example of a split-ubiquitin interaction screen using an in-situ expressed receptor of the serpentine class, emphasizing the suitability of the described improvements in the screening protocol.

In a URA3/5-FOA-based dosage-suppressor screen, we isolated a plasmid containing the little-characterized ORF YJL055W. Further analysis showed that this gene did not suppress the mutation of interest. Instead, overexpression of Yjl055Wp directly suppressed the non-viability of URA3(+) cells in the presence of 5-FOA. Overexpression of Yjl055Wp also suppressed the lethality induced by 5-FU, but deletion of YJL055W had no detectable effect on resistance to either 5-FOA or 5-FU. Based on these observations and a previous report that a yjl055wDelta mutant has increased sensitivity to purine-analogue mutagens, we suggest that Yjl055Wp may function in one of several pathways for the detoxification of base analogues. However, its precise mechanism of action remains unknown.