Key Regulatory Enzyme Research Articles

Fifty years of cycling.

Fifty years ago, the first isolation of conditional budding yeast mutants that were defective in cell division was reported. Looking back, we now know that the analysis of these mutants revealed the molecular mechanisms and logic of the cell cycle, identified key regulatory enzymes that drive the cell cycle, elucidated structural components that underly essential cell cycle processes, and influenced our thinking about cancer and other diseases. Here, we briefly summarize what was concluded about the coordination of the cell cycle 50 years ago and how that relates to our current understanding of the molecular events that have since been elucidated.

Changes in starch quality of mid-season indica rice varieties in the lower reaches of the Yangtze River in last 80 years

Rice (Oryza sativa L.) quality depends mainly on the characteristics of starch stored in kernels. Understanding the changes in starch characteristics in kernels during variety improvement would have great significance to improve rice quality. This study was designed to investigate the starch characteristics in the kernels and associated physiological traits of indica rice varieties in the lower reaches of the Yangtze River in China in last 80 years. Eight representative mid-season indica rice varieties were grown in the field. The results showed that the grain yield was significantly increased with the improvement of varieties and such an increase was mainly attributed to the increase in total number of spikelets. The tall varieties applied in the 1940s–1950s had higher protein content, relative crystallinity and infrared (IR) ratio of 1 045/1 022 cm−1. The semi-dwarf varieties applied in the 1980s–1990s had higher gel consistency, amylopectin content, IR ratio of 1 022/995 cm−1, and breakdown value. With the improvement of varieties, the amylose content, large-sized starch granule number and volume distribution, onset and peak of gelatinization temperature, gelatinization and retrogradation enthalpy, setback value, pasting temperature, viscosity of peak, hot and final, and 1-aminocycopropane-1-carboxylic acid (ACC) concentrations in panicles and root bleeding were gradually decreased, whereas the medium-sized starch granule number and volume distribution, activities of key enzymes in grains, and zeatin (Z)+zeatin riboside (ZR) contents in panicles and root bleeding at grain filling stage were gradually increased. Correlation analysis showed that starch thermodynamic characteristics were closely related to starch structure and components, key enzymes and hormones. The results suggest that starch quality was enhanced through the optimization of starch components, structure, thermodynamics, and the regulation of key enzymes in grains and hormones in panicles and root bleedings at grain filling stage during the improvement of mid-season indica rice.

Correlation of PRL3 expression with colorectal cancer progression.

To evaluate the relationship between phosphatase of regenerating liver 3 (PRL3) expression and clinical outcome in colorectal cancer (CRC). PRL3, a protein tyrosine phosphatase functions as one of the key regulatory enzymes of various signal transduction pathways. PRL3 is highly expressed in a majority of cancers and is a novel potential therapeutic target. PRL3 expression was evaluated by immunohistochemistry in 167 patients with CRC, 37 patients with no disease, and 26 patients with metastatic CRC (mCRC). Phosphorylated Akt at serine 473 (p-Akt S473) expression was also evaluated by immunohistochemistry in mCRC patients. High expression of PRL3 was correlated with CRC progression, and every one unit increase in PRL3 level contributed to an increase in the rate of death by 1%-1.7%. PRL3 expression was significantly higher in liver metastases compared with primary tumors and showed a significant positive correlation with the expression level of p-Akt S473. PRL3 expression levels associated with CRC progression and metastasis, and positively correlated with activated Akt level in mCRC. Together, these findings indicated that PRL3 might be a potential marker for increased risk of CRC-specific tumor burden and identify PRL3 as an attractive therapeutic target for mCRC treatment.

Functional Consequences of Metabolic Zonation in Murine Livers: Insights for an Old Story.

Zone-dependent differences in expression of metabolic enzymes along the portocentral axis of the acinus are a long-known feature of liver metabolism. A prominent example is the preferential localization of the enzyme, glutamine synthetase, in pericentral hepatocytes, where it converts potentially toxic ammonia to the valuable amino acid, glutamine. However, with the exception of a few key regulatory enzymes, a comprehensive and quantitative assessment of zonal differences in the abundance of metabolic enzymes and, much more important, an estimation of the associated functional differences between portal and central hepatocytes is missing thus far. We addressed this problem by establishing a method for the separation of periportal and pericentral hepatocytes that yields sufficiently pure fractions of both cell populations. Quantitative shotgun proteomics identified hundreds of differentially expressed enzymes in the two cell populations. We used zone-specific proteomics data for scaling of the maximal activities to generate portal and central instantiations of a comprehensive kinetic model of central hepatic metabolism (Hepatokin1). The model simulations revealed significant portal-to-central differences in almost all metabolic pathways involving carbohydrates, fatty acids, amino acids, and detoxification.

Syncytialization and prolonged exposure to palmitate impacts BeWo respiration.

Placental villous trophoblast mitochondrial respiratory function is critical for a successful pregnancy and environmental influences such as maternal obesity have been associated with respiratory impairment at term. More recently, a gestational high fat diet independent of maternal body composition, has been highlighted as a potential independent regulator of placental mitochondrial metabolism. The current study aimed to characterize the direct impact of a prolonged and isolated exposure to the dietary fatty acids Palmitate (PA) and Oleate (OA) upon placental cell mitochondrial respiratory function. BeWo cytotrophoblast (CT) and syncytiotrophoblast (SCT) cells were treated for 72 h with 100 µM PA, OA or PA+OA (P/O). Live-cell metabolic function was analyzed via the Seahorse XF Mito and Glycolysis Stress tests. Immunoblots and spectrophotometric activity assays were utilized to examine the protein expression and function of electron transport chain (ETC) complexes and key mitochondrial regulatory enzymes. Syncytialization of BeWo cells resulted reduced respiratory activity in conjunction with altered complex I and II activity and decreased pyruvate dehydrogenase (PDH) protein expression and activity. PA and P/O treatments were associated with increased basal and maximal respiratory activities in BeWo CT cells without alterations in protein expression or activity of individual ETC complexes and mitochondrial substrate regulators. The metabolic suppression in BeWo SCTs was consistent with that previously observed in primary human trophoblast cell cultures, while the observed increases in respiratory activity in PA-treated BeWo CTs may be indicative of an early timepoint of specific dietary saturated fat-mediated placental cell mitochondrial dysfunction.

Correlation between expression and activity of ADP glucose pyrophosphorylase and starch synthase and their role in starch accumulation during grain filling under drought stress in rice

ADP glucose pyrophosphorylase (AGPase, EC 2.7.7.27) and starch synthase (SS, EC 2.4.1.21) are key regulatory enzymes involved in the starch biosynthesis. Comprehensive analysis of transcription levels of ADP-glucose pyrophosphorylase and starch synthase genes was performed in leaves, roots, and developing grains of drought susceptible (IR64) and drought-tolerant (N22) cultivars under applied water deficit stress (WDS). AGPase and SS genes are differentially regulated in leaves, roots, and grains under the drought stress. The expression pattern of SS and AGPase genes was correlated with the activity of both AGPase, SS, and starch content of developing grains under the drought. Drought stress reduced transitory starch in leaves and enhanced storage starch in developing grains. An increase in the activity of AGPase in developing grains was due to induced expression of ADP glucose pyrophosphorylase large subunit 3 (AGPL3) in N22 and both ADP glucose pyrophosphorylase small subunit 2 (AGPS2) & ADP glucose pyrophosphorylase large subunit 3 (AGPL3) in IR64 and a positive correlation was established with starch content. Similarly, an increase in the SS activity in developing grains was due to induced expression of soluble starch synthase (SSIIB, SSIVA, and SSIVB) in N22 and SSIVB in IR64.

In silico analysis of SNPs in human phosphofructokinase, muscle (PFKM) gene: An apparent therapeutic target of aerobic glycolysis and cancer

Phosphofructokinase, muscle (PFKM), a key glycolytic regulatory enzyme, is a potential target for cancer therapeutic studies accredited to the employed inefficient phenomenon known as Warburg effect. PFKM is encoded by PFKM gene located at chromosome 12q13.11. Single nucleotide polymorphisms (SNPs) are known to profoundly affect gene expression and protein function. Therefore, the first attempt was made to computationally identify putative functional PFKM variants. These SNPs were further explored to find their probable association with different cancer types. A total of 9694 SNPs were retrieved from dbSNP database. Of which, only 85 validated SNPs with ≥10% minor allele frequency (MAF) were subjected to analysis by softwares including Ensembl Genome browser, FuncPred (SNPinfo), regulomeDB (v 2.0), SIFT and PolyPhen-2. The relative analysis of output obtained classified the selected-SNPs into 11 highly prioritized (HP), 20 moderately prioritized and 54 not/poorly prioritized SNPs. The 11 HP-SNPs were found to have the highest likelihood of being functionally important, evidenced by previous association of rs2269935, rs11168417, rs11609399 and rs2228500 HP-SNPs with cachexia, lung and breast cancer. The study warrants further experiments to confirm the predictive role of prioritized SNPs in cancer etiology and also provides directions to fellow researchers.

Okra Modulates Regulatory Enzymes and Metabolites of Glucose-Utilizing Pathways in Diabetic Rats

Objective Using a rat diabetes model, the authors examined how substrates and products of glycolysis and key regulatory enzymes for glycolysis, gluconeogenesis, Kreb’s cycle, and glycogen metabolism react to treatment with okra diet therapy, relative to glibenclamide treatment. Method The animal grouping involved normoglycemic rats, untreated diabetic rats, and diabetic rats treated with glibenclamide, 50% w/w okra sauce, exclusive okra sauce diet, or sauce without okra. Alloxan monohydrate was the diabetogenic agent. Insulin and adiponectin were assayed with enzyme-linked immunosorbent assay (ELISA) while the metabolites and enzymes were assed using standard spectrophotometric methods. Results The exclusive diet therapy significantly (p < 0.05) improved insulin activities after 60 days and reversed the altered adiponectin activities. Glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-bisphosphate levels were depleted during diabetes, but phosphoenolpyruvate and pyruvate accumulated during the first short phase of diabetes. Rats in the glibenclamide and 100% okra diet groups showed comparable hexokinase, phosphofructokinase, and pyruvate kinase activities relative to the normoglycemic rats, while the gluconeogenic enzymes, glucose-6-phosphatase, and fructose-1,6-bisphosphatase remained altered. The authors observed that extended treatment with glibenclamide restored the activities of all the Kreb’s cycle enzymes, while succinate dehydrogenase and α-ketoglutarate dehydrogenase were nonresponsive to the okra diet therapy relative to their control levels. The glycogen stores were normalized by the exclusive diet therapy, but glycogen synthase and phosphorylase activities were unresponsive. Conclusions Okra diet has shown insulin-sensitizing potentials with prolonged intake during diabetes as well as the potential to reverse alterations in the major carbohydrate-metabolizing enzyme.

Identification and characterization of CTP:phosphocholine cytidylyltransferase CpCCT1 in the resurrection plant Craterostigma plantagineum

Phosphatidylcholine is a major phospholipid which is shown to be involved in stress adaptation. Phosphatidylcholine increased during dehydration in Craterostigma plantagineum, and therefore we characterized CTP:phosphocholine cytidylyltransferase (CpCCT1), a key regulatory enzyme for phosphatidylcholine synthesis in plants. The CpCCT1 gene from the resurrection plant C. plantagineum was cloned and the amino acid sequence was compared with homologs from other species including yeast and rat. CCT proteins have conserved catalytic and membrane-binding domains while the N-terminal and C-terminal domains have diverged. The tissue specific expression analysis indicated that CpCCT1 is expressed in all tested tissues and it is induced by dehydration and in response to 0.5 M NaCl solutions. In plants exposed to low temperature in the dark, the CpCCT1 transcript increased after 4 h at 4 °C. CpCCT1 expression also increased during mannitol and sorbitol treatments in a concentration dependent manner. Phytohormones such as abscisic acid and indole-3-acetic acid also trigged transcript accumulation. Comparisons of transcript and protein accumulations for different treatments (except for dehydration) suggest transcriptional and translational control mechanisms. Analysis of promoter activity and polysome occupancy suggest that CpCCT1 gene expression is mainly under translational regulation during dehydration.

Protein sumoylation with SUMO1 promoted by Pin1 in glioma stem cells augments glioblastoma malignancy.

The tumorigenic potential of glioma stem cells (GSCs) is associated with multiple reversible molecular alternations, but the role of posttranslational protein sumoylation in GSCs has not been elucidated. The development of GSC-targeting drugs relies on the discovery of GSC-preferential molecular modifications and the relevant signaling pathways. In this work, we investigated the protein sumoylation status, the major sumoylated substrate, and the key regulatory enzyme in GSCs to explore the therapeutic potential of disrupting protein sumoylation for glioblastoma (GBM) treatment. Patient-derived GSCs, primary GBM sections, and intracranial GBM xenografts were used to determine protein sumoylation and the related molecular mechanisms by immunoblot, quantitative PCR, immunoprecipitation, immunofluorescence, and immunohistochemistry. Orthotopic GBM xenograft models were applied to investigate the inhibition of tumor growth by disrupting protein sumoylation with short hairpin (sh)RNAs or molecular inhibitors. We show that high levels of small ubiquitin-related modifier 1 (SUMO1)-but not SUMO2/3-modified sumoylation are preferentially present in GSCs. The promyelocytic leukemia (PML) protein is a major SUMO1-sumoylated substrate in GSCs, whose sumoylation facilitates its interaction with c-Myc to stabilize c-Myc proteins. The prolyl-isomerase Pin1 is preferentially expressed in GSCs and functions as the key enzyme to promote SUMO1 sumoylation. Disruption of SUMO1 sumoylation by Pin1 silencing with shRNAs or inhibition with its inhibitor Juglone markedly abrogated GSC maintenance and mitigated GSC-driven tumor growth. Our findings indicate that high SUMO1-modified protein sumoylation as a feature of GSCs is critical for GSC maintenance, suggesting that targeting SUMO1 sumoylation may effectively improve GBM treatment.

Regulation of glucose and lipid metabolism by insulin and glucagon in vivo and in vitro in common carp Cyprinus carpio L.

Our objective is to study the low metabolic utilization of dietary carbohydrates in fish. The regulation of key enzymes involved in glucose and lipid metabolism in the common carp (Cyprinus carpio L.), known to be relatively tolerant to dietary carbohydrates, were assessed. We performed intraperitoneal (i.p.) administration of insulin and glucagon to common carp and also subjected common carp primary hepatocytes to insulin or glucagon. The results showed that insulin administration induced hypoglycaemia through up-regulation of the expression of the glycolysis genes glucokinase (GK) and hexose kinase (HK), coupled with increased gene expression of fatty acid synthase (FAS), acetyl-CoA carboxylase-1 (ACC-1), sterol-regulator element-binding protein-1 (SREBP-1). Insulin also limited the expression of the gluconeogenesis genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), coupled with decreased gene expression of lipoprotein lipase (LPL) and carnitine palmitoyl transferase I (CPT-1). However, glucagon had the opposite effect. The results showed that glucagon induced hyperglycemia with increased expression of PEPCK and G6Pase and increased lipolytic capacity as indicated by lower mRNA levels of SREBP-1. Glucagon (i.p.) significantly increased the activity of PEPCK and LPL in hepatopancreas. In addition, the mRNA expression of HK showed an up-regulated trend and the mRNA expression of PEPCK and G6Pase showed a down-regulated trend in the group of glucose (i.p.). This study demonstrates that insulin and glucagon possess the intrinsic ability to regulate hepatic energy metabolism in common carp, the regulation of which seems to be similar to that of the mammals at least at the molecular level.

Upregulated PKM2 in Macrophages Exacerbates Experimental Arthritis via STAT1 Signaling.

Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis. We hypothesize that Pkm2, as a key regulatory enzyme of glycolysis pathway, triggers the activation of macrophages (Mφ), which results in proinflammatory cytokine production during the arthritis progress. In this study, Pkm2 was found to be overexpressed in ED1-positive Mφ in spleens and synovial tissues from arthritic rats via immunofluorescence, Western blotting, and quantitative RT-PCR. To reveal the role of Pkm2, Dark Agouti rats were treated with either Pkm2 enzyme inhibitor shikonin or the RNA interference plasmids of Pkm2 and negative control plasmids, respectively, via i.p. injection. Pkm2 intervention could alleviate the severity of pristane-induced arthritis in aspects of the macroscopic arthritis score, perimeter changes of midpaw, and the synovitis and destruction of the bone and cartilage as well as reduce the ED1 and p-Stat1-positive cell population in rat synovial tissues. Silencing Pkm2 by RNA interference in classical activated rat and mouse Mφ resulted in less Tnf-α, Il-1β production via Stat1 signaling. Collectively, Pkm2 is highly expressed in ED1-positive Mφ of spleens and synovial tissues from arthritic rats and promotes Mφ activation via Stat1 signaling. Pkm2 might be a promising selective metabolic target molecule for rheumatoid arthritis treatment.

Methyltetrahydrofolate-homocysteine methyltransferase reductase gene and congenital heart defects in Down syndrome

Congenital heart defects (CHD) are the most common abnormalities occurring in 40% -60% of Down syndrome (DS) patients. The 5-methyltetrahydrofolate homocysteine methyl transferase reductase (MTRR) is one of the key regulatory enzymes involved in folate pathway. Disrupted folate pathway due to MTRR polymorphism could be a risk factor for CHD in DS. The aim of the study was to determine the association between polymorphism MTRR 66A&gt; G and CHD in DS. Additionally, the impact of maternal endogenous factors on CHD was analyzed, intake of folate through diet, periconceptional folic acid supplementation, smoking and alcohol drinking. A total of 155 children with DS and 148 their mothers have been enrolled in this study. Genotyping was performed by PCR-RFLP. The frequency of alleles and genotypes of MTRR 66A&gt; G polymorphisms was not significantly different between a group with CHD compared to a group without CHD among DS subjects as well as in their mothers. The mothers with mutated homozygous genotypes who have taken folic acid preparations from the fourth week before pregnancy to eight weeks of pregnancy were more likely to have DS-CHD+ child. The study results suggested that maternal MTRR 66A&gt; G polymorphisms associated with their lifestyle habits such as folic acid intake could altered individual risk for CHD in DS child.

Characterizing the regulation of pyruvate kinase in response to hibernation in ground squirrel liver (Urocitellus richardsonii).

The Richardson's ground squirrel (Urocitellus richardsonii) undergoes numerous changes to its core physiological and metabolic processes over the months it spends hibernating during the winter. Winter torpor is characterized by an overall reduction in metabolic rate, a lowering of core body temperature, and a switch to preferential consumption of lipids instead of carbohydrates. The alterations in central metabolic pathways are often accomplished by the regulation of key enzymes within the glycolytic pathway. The regulation of one such enzyme, pyruvate kinase (PK), was characterized in the present study in the liver of torpid ground squirrels. PK was purified from liver tissue of euthermic and hibernating U. richardsonii and subsequently assayed to determine the kinetic parameters of the enzyme at 22° and 5°C. Additional studies assessed the relative degree of post-translational modifications in PK from control and hibernating ground squirrels. The results from this study demonstrated significantly lowered maximal activity in the hibernating form of the enzyme and decreased sensitivity to the activator FBP when compared to the control. Immunoblotting demonstrated increased relative serine and threonine phosphorylation (~3 fold) in the hibernating PK. Taken together these results suggest that phosphorylation of liver PK is an important step in inhibiting glycolytic activity in the liver of the Richardson's ground squirrel during torpor.

Glyphosate-based herbicide formulations and reproductive toxicity in animals

The adoption of genetically engineered (GE) crops in agriculture has increased dramatically over the last few decades. Among the transgenic plants, those tolerant to the herbicide glyphosate are among the most common. Weed resistance to glyphosate-based herbicides (GBHs) has been on the rise, leading to increased herbicide applications. This, in turn, has led to increased glyphosate residues in feed. Although glyphosate has been considered to be generally safe to animal health, recent studies have shown that GBHs have potential to cause adverse effects in animal reproduction, including disruption of key regulatory enzymes in androgen synthesis, alteration of serum levels of estrogen and testosterone, damage to reproductive tissues and impairment of gametogenesis. This review emphasizes known effects of GBHs on reproductive health as well as the potential risk GBH residues pose to animal agriculture.

Effect of methyl jasmonate on phenolic acids accumulation and the expression profile of their biosynthesis-related genes in Mentha spicata hairy root cultures

Mentha spicata L., as a rich source of phenolic acids, is known for its therapeutic importance. Elicitors play a crucial role in biosynthetic pathways to improve plant secondary metabolites production. The purpose of this study was to investigate the effect of methyl jasmonate (MeJA) as a potent elicitor, on phenolic acids production and the relative changes in expression of their biosynthesis-related key genes (PAL, TAT, C4H, HPPR, and 4CL) in M. spicata hairy root cultures, to elucidate the interrelation between phenolic acids accumulation and the regulation mechanism of gene expression. The results showed that the relative expression levels of phenylpropanoid pathway genes, i.e., PAL, C4H, 4CL, and HPPR in the tyrosine-derived pathway increased 4.04-fold, 3.62-fold, 1.75-fold, and 1.45-fold in comparison to untreated controls, respectively. High-performance liquid chromatography analysis indicated that MeJA dramatically increased rosmarinic acid content (55.44 µg g−1 dry wt, about 11.84-fold) after 6 h exposure to elicitor. Moreover, caffeic acid, chlorogenic acid, and cinnamic acid contents were also enhanced significantly (p < 0.05) in response to MeJA treatment. On the other hand, the application of MeJA had a negative effect on both TAT expression level and lithospermic acid B accumulation. Our results implied that MeJA has a significant impact on RA, CA, CGA, and CIA accumulation, which might be the consequence of gene activation from the phenylpropanoid pathway (PAL, C4H, and 4CL) and HPPR, a key regulatory enzyme of the tyrosine-derived pathway in the elicitor-treated hairy root cultures of M. spicata. MeJA is an effective elicitor for stimulating valuable phenolic acids production in M. spicata hairy root cultures.

De novo transcriptome analysis unravels tissue-specific expression of candidate genes involved in major secondary metabolite biosynthetic pathways of Plumbago zeylanica: implication for pharmacological potential.

The present study provides comparative transcriptome analysis, besides identifying functional secondary metabolite genes of Plumbago zeylanica with pharmacological potential for future functional genomics, and metabolomic engineering of secondary metabolites from this plant towards diversified biomedical applications. Plumbago zeylanica is a widely used medicinal plant of the traditional Indian system of medicine with wide pharmacological potential to treat several disorders. The present study aimed to carry out comparative transcriptome analysis in leaf and root tissue of P. zeylanica using Illumina paired end sequencing to identify tissue-specific functional genes involved in the biosynthesis of secondary metabolites, contributing to its therapeutic efficacy. De novo sequencing assembly resulted in the identification of 62,321 "Unigenes" transcripts with an average size of 1325bp. Functional annotation using BLAST2GO resulted in the identification of 50,301 annotated transcripts (80.71%) and GO assigned to 18,814 transcripts. KEGG pathway annotation of the "Unigenes" revealed that 2465 transcripts could be assigned to 242 KEGG pathway maps wherein the number of transcripts involved in secondary metabolism was distinct in root and leaf transcriptome. Among the secondary metabolite biosynthesis pathways, the cluster of "Unigenes" encoding enzymes of 'Phenylpropanoid biosynthesis pathway' represents the largest group (84 transcripts) followed by 'Terpenoid Backbone biosynthesis' (48 transcripts). The transcript levels of the candidate unigenes encoding key enzymes of phenylpropanoid (PAL, TAL) and flavanoid biosynthesis (CHS, ANS, FLS) pathways were up-regulated in root, while the expression levels of candidate "Unigenes" transcript for monoterpenoid (DXS, ISPF), diterpenoid biosynthesis (SPS, SDS) and indole alkaloid pathways (STR) were significantly higher in leaf of P. zeylanica. Interestingly, validation of differential gene expression profile by qRT-PCR also confirmed that candidate "Unigenes" enzymes of phenylpropanoid and flavonoid biosynthesis were highly expressed in the root, while the key regulatory enzymes of terpenoid and indole alkaloid compounds were up-regulated in the leaf, suggesting that (differences in) the levels of these functional genes could be attributed to the (differential) pharmacological activity (between root and leaf) in tissues of P. zeylanica.

Changes in arginase isoforms in a murine model of neonatal brain hypoxia-ischemia.

Background:Arginases (ARG-isoforms, ARG-1/ARG-2) are key regulatory enzymes of inflammation and tissue repair, however their role after neonatal brain hypoxia (H) and hypoxia-ischemia (HI) remains unknown.Methods:C57BL/6-mice subjected to the Vannucci procedure on postnatal day 9 were sacrificed at different timepoints. The degree of brain damage was assessed histologically. ARG spatiotemporal localization was determined via immunohistochemistry. ARG expression was measured by Western blot and activity spectrophotometrically.Results:ARG-isoforms expression increased during neurodevelopment (P9-P17) in the cortex and hippocampus. This was suppressed with H and HI only in the hippocampus. In the cortex, both isoforms increased with H alone and only ARG-2 increased with HI at 3 d. ARG activity during neurodevelopment remained unchanged but increased at 1 d with H and not HI. ARG-1 localized with microglia at the injury site as early as 4 h after injury, while ARG-2 localized with neurons.Conclusions:ARG-isoforms expression increases with age from P9-P17 but is suppressed by injury specifically in the hippocampus and not in the cortex. Both, levels and activity of ARG-isoforms increase with H while ARG-1 immunolabelling is upregulated in the HI cortex. Evidently, ARG-isoforms in brain differ in spatiotemporal localization, expression and activity during neurodevelopment and after injury.

Progress on epigenetic regulation of iron homeostasis

Iron homeostasis plays an important role for the maintenance of human health. It is known that iron metabolism is tightly regulated by several key genes, including divalent metal transport-1(DMT1), transferrin receptor 1(TFR1), transferrin receptor 2(TFR2), ferroportin(FPN), hepcidin(HAMP), hemojuvelin(HJV) and Ferritin H. Recently, it is reported that DNA methylation, histone acetylation, and microRNA (miRNA) epigenetically regulated iron homeostasis. Among these epigenetic regulators, DNA hypermethylation of the promoter region of FPN, TFR2, HAMP, HJV and bone morphogenetic protein 6 (BMP6) genes result in inhibitory effect on the expression of these iron-related gene. In addition, histone deacetylase (HADC) suppresses HAMP gene expression. On the contrary, HADC inhibitor upregulates HAMP gene expression. Additional reports showed that miRNA can also modulate iron absorption, transport, storage and utilization via downregulation of DMT1, FPN, TFR1, TFR2, Ferritin H and other genes. It is noteworthy that some key epigenetic regulatory enzymes, such as DNA demethylase TET2 and histone lysine demethylase JmjC KDMs, require iron for the enzymatic activities. In this review, we summarize the recent progress of DNA methylation, histone acetylation and miRNA in regulating iron metabolism and also discuss the future research directions.

Functional and correlation analyses of dihydroflavonol-4-reductase genes indicate their roles in regulating anthocyanin changes in Ginkgo biloba

Anthocyanins, a class of flavonoids, are synthesized in a branch of the flavonoid pathway. Dihydroflavonol-4-reductase (DFR) catalyzes the first reaction leading to anthocyanins, and it is considered a key regulatory enzyme of anthocyanin biosynthesis in plants. In this research, we isolated three new DFR genes by searching the whole-genomic sequence data of Ginkgo biloba. The genes have similar structures and are clustered in the same scaffold. Sequence analyses of GbDFR proteins revealed that these GbDFRs have a closer relationship to Arabidopsis thaliana DFRs than previously identified GbDFRs. Expression analyses revealed that GbDFR4 and GbDFR6 were preferentially expressed in leaves and fruit, respectively. Young and mature leaves shared similar gene expression patterns, while fruit harvested in May had significantly higher expression levels of GbDFRs than fruit harvested in June. Transgenic tobacco plants ectopically overexpressing GbDFR genes showed higher DFR enzymatic activity levels, higher anthocyanin contents and darker flowers than the control. Ginkgo seedlings treated with NaCl showed increased total flavonoid and anthocyanin contents, and the expression levels of GbDFR genes were also induced. In contrast, UV-B irradiation increased the total flavonoid content but decreased both the anthocyanin content and GbDFR expression levels. A salicylic acid treatment induced and inhibited the total flavonoid content with and without light, respectively. In contrast, both the GbDFR expression levels and the anthocyanin content were inhibited by the salicylic acid treatment regardless of the light conditions. This research validated the functions of GbDFRs and expands our understanding of the environmental stress-response mechanisms of ginkgo trees.