Phosphate Enzyme Research Articles

Automated System for Attomolar-Level Detection of MiRNA as a Biomarker for Influenza A Virus.

We have developed an automated sensing system for the repeated detection of a specific microRNA (miRNA) of the influenza A (H1N1) virus. In this work, magnetic particles functionalized with DNAs, target miRNAs, and alkaline phosphate (ALP) enzymes formed sandwich structures. These particles were trapped on nickel (Ni) patterns of our sensor chip by an external magnetic field. Then, additional electrical signals from electrochemical markers generated by ALP enzymes were measured using the sensor, enabling the highly sensitive detection of target miRNA. The magnetic particles used on the sensor were easily removed by applying the opposite direction of external magnetic fields, which allowed us to repeat sensing measurements. As a proof of concept, we demonstrated the detection of miRNA-1254, one of the biomarkers for the H1N1 virus, with a high sensitivity down to 1 aM in real time. Moreover, our sensor could selectively detect the target from other miRNA samples. Importantly, our sensor chip showed reliable electrical signals even after six repeated miRNA sensing measurements. Furthermore, we achieved technical advances to utilize our sensor platform as part of an automated sensing system. In this regard, our reusable sensing platform could be utilized for versatile applications in the field of miRNA detection and basic research.

Design of charge converting lipid nanoparticles via a microfluidic coating technique.

It was the aim of this study to design charge converting lipid nanoparticles (LNP) via a microfluidic mixing technique used for the preparation and coating of LNP. LNP consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, N-(carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG-2000-DSPE), and various cationic surfactants were prepared at diverging flow rate ratios (FRR) via microfluidic mixing. Utilizing a second chip in the microfluidic set-up, LNP were coated with polyoxyethylene (9) nonylphenol monophosphate ester (PNPP). LNP were examined for their stability in different physiologically relevant media as well as for hemolytic and cytotoxic effects. Finally, phosphate release and charge conversion of PNPP-coated LNP were evaluated after incubation with alkaline phosphatase and on Caco2-cells. LNP produced at an FRR of 5:1 exhibited a size between 80 and 150nm and a positive zeta potential. Coating with PNPP within the second chip led to LNP exhibiting a negative zeta potential. After incubation with 1 U/ml alkaline phosphatase for 4h, zeta potential of the LNP containing 1,2-dioleoyloxy-3-trimethylammonium-propane chloride (DOTAP) as cationic component shifted from - 35mV to approximately + 5mV. LNP prepared with other cationic surfactants remained slightly negative after enzymatic phosphate cleavage. Manufacturing of LNP containing PNPP and DOTAP via connection of two chips in a microfluidic instrument proves to show efficient change in zeta potential from negative to positive after incubation with alkaline phosphatase.

Dietary β-glucan ameliorates metabolic stress caused by a high dietary carbohydrate level in Nile tilapia

Carbohydrates are an excellent cost-friendly energy source for fish, and they have been used to improve growth performance and reduce dietary protein levels. However, the aquaculture industry has frequently overused high dietary carbohydrate levels, which has caused metabolic disturbances in fish. This deleterious effect is often diagnosed at the end of the production cycle, affects fish health and causes significant losses to farmers. In this context, β-glucan, which is broadly known as an immunomodulator, may ameliorate the deleterious effects caused by high levels of dietary carbohydrates. Therefore, we investigated the effect of dietary β-glucan on the reduction of metabolic disturbances in Nile tilapia fed a high carbohydrate diet. A total of 352 juvenile Nile tilapias (25.39 ± 0.83 g) were fed isoproteic (280 g kg−1 PD) and isoenergetic diets (3130 kcal kg−1) containing low amounts of carbohydrates (CHO-L 270 g kg−1) or high amounts of carbohydrates (CHO-H 620 g kg−1) with four levels of β-glucan (βG; 0, 1, 3 and 6 g kg−1) for 10 weeks. We evaluated the productive performance, plasma glucose, serum triglycerides, hepatosomatic index, total liver and muscle lipid, hepatic glycogen, hepatic activity of hexokinase, glucokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, malic enzyme, liver histopathology and innate immune responses. Fish fed a high carbohydrate diet had significantly increased productive performance and lipogenesis, presenting higher hepatosomatic indexes, triglycerides, hepatic glycogen, muscular lipid, hepatic activity of glucose-6-dehydrogenase phosphate, and malic enzyme. Additionally, histological analyses indicated expressive cytoplasm vacuolization. The innate immune responses such as leukocyte respiratory activity, complement and lysozyme were also significantly decreased for the high carbohydrate diet, indicating the occurrence of immunosuppression in fish. These results also indicate impaired metabolic functioning. However, β-glucan ameliorated metabolic stress. Specifically, we found a significant decrease in the serum levels of triglycerides, hepatosomatic index, muscular lipid, and hepatic activities of malic and glucose-6-dehydrogenase phosphate enzymes in fish fed high amounts of carbohydrates containing glucan. There was also a significant decrease in hepatic activity of the glycolytic pathway, hexokinase and pyruvate kinase. Moreover, β-glucan modulated the respiratory activity of leukocytes and complement, confirming its immunomodulating effect on fish. In conclusion, β-glucan mitigated the metabolic disturbances caused by the high levels of carbohydrates in the diet of Nile tilapia. This finding indicates that besides having an immunomodulating effect, β-glucan also has metabolic functions/benefits such as reducing glycolysis and lipogenesis.

Biochemical and Bioinformatic Studies of Mutations of Residues at the Monomer-Monomer Interface of Human Ornithine Aminotransferase Leading to Gyrate Atrophy of Choroid and Retina.

Deficit of human ornithine aminotransferase (hOAT), a mitochondrial tetrameric pyridoxal-5'-phosphate (PLP) enzyme, leads to gyrate atrophy of the choroid and retina (GA). Although 70 pathogenic mutations have been identified, only few enzymatic phenotypes are known. Here, we report biochemical and bioinformatic analyses of the G51D, G121D, R154L, Y158S, T181M, and P199Q pathogenic variants involving residues located at the monomer-monomer interface. All mutations cause a shift toward a dimeric structure, and changes in tertiary structure, thermal stability, and PLP microenvironment. The impact on these features is less pronounced for the mutations of Gly51 and Gly121 mapping to the N-terminal segment of the enzyme than those of Arg154, Tyr158, Thr181, and Pro199 belonging to the large domain. These data, together with the predicted ΔΔG values of monomer-monomer binding for the variants, suggest that the proper monomer-monomer interactions seem to be correlated with the thermal stability, the PLP binding site and the tetrameric structure of hOAT. The different impact of these mutations on the catalytic activity was also reported and discussed on the basis of the computational information. Together, these results allow the identification of the molecular defects of these variants, thus extending the knowledge of enzymatic phenotypes of GA patients.

Information-Rich, Dual-Function 13C/2H-Isotopic Crosstalk NMR Assay for Human Serine Racemase (hSR) Provides a PLP-Enzyme "Partitioning Fingerprint" and Reveals Disparate Chemotypes for hSR Inhibition.

The first dual-function assay for human serine racemase (hSR), the only bona fide racemase in human biology, is reported. The hSR racemization function is essential for neuronal signaling, as the product, d-serine (d-Ser), is a potent N-methyl d-aspartate (NMDA) coagonist, important for learning and memory, with dysfunctional d-Ser-signaling being observed in some neuronal disorders. The second hSR function is β-elimination and gives pyruvate; this activity is elevated in colorectal cancer. This new NMR-based assay allows one to monitor both α-proton-exchange chemistry and β-elimination using only the native l-Ser substrate and hSR and is the most sensitive such assay. The assay judiciously employs segregated dual 13C-labeling and 13C/2H crosstalk, exploiting both the splitting and shielding effects of deuterium. The assay is deployed to screen a 1020-compound library and identifies an indolo-chroman-2,4-dione inhibitor family that displays allosteric site binding behavior (noncompetitive inhibition vs l-Ser substrate; competitive inhibition vs adenosine 5'-triphosphate (ATP)). This assay also reveals important mechanistic information for hSR; namely, that H/D exchange is ∼13-fold faster than racemization, implying that K56 protonates the carbanionic intermediate on the si-face much faster than does S84 on the re-face. Moreover, the 13C NMR peak pattern seen is suggestive of internal return, pointing to K56 as the likely enamine-protonating residue for β-elimination. The 13C/2H-isotopic crosstalk assay has also been applied to the enzyme tryptophan synthase and reveals a dramatically different partition ratio in this active site (β-replacement: si-face protonation ∼6:1 vs β-elimination: si-face protonation ∼1:3.6 for hSR), highlighting the value of this approach for fingerprinting the pyridoxal phosphate (PLP) enzyme mechanism.

Microbial (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate reductase (IspH) and its biotechnological potential: A mini review.

(E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) reductase (IspH) is a [4Fe-4S] cluster-containing enzyme, involved in isoprenoid biosynthesis as the final enzyme of the methylerythritol phosphate (MEP) pathway found in many bacteria and malaria parasites. In recent years, many studies have revealed that isoprenoid compounds are an alternative to petroleum-derived fuels. Thus, ecofriendly methods harnessing the methylerythritol phosphate pathway in microbes to synthesize isoprenoid compounds and IspH itself have received notable attention from researchers. In addition to its applications in the field of biosynthesis, IspH is considered to be an attractive drug target for infectious diseases such as malaria and tuberculosis due to its survivability in most pathogenic bacterium and its absence in humans. In this mini-review, we summarize previous reports that have systematically illuminated the fundamental and structural properties, substrate binding and catalysis, proposed catalytic mechanism, and novel catalytic activities of IspH. Potential bioengineering and biotechnological applications of IspH are also discussed.

Purple urine bag syndrome: a perplexing finding in a urethral catheter related urinary tract infection

The appearance of purple coloured urine within the Urethral Catheter-Urine Bag system is often frightening to patients and their relatives and gets health care givers perplexed. The purple colour is said to be a by-product of chemical transformation of indole to indigo or indirubin, or a mixture of the two. Both Indigo and Indirubin are derivatives of colonic bacterial metabolism of dietary Tryptophan using either the Sulphatase or Phosphate enzymes or both. It is seen in chronically ill patients with chronic constipation and on long standing urethral catheterisation. The organisms commonly cultured from such purplish urine are Escherichia coli, proteus mirabilis, klebsiella pneumoniae and Enterobacter species. We reported such a case in a 98-year old paraplegic male patient with a histologically proven metastatic prostate adenocarcinoma and chronic constipation. The urine and catheter tip culture yielded klebsiella specie. We wrote to draw attention to its occurrence and highlight a simple method of its treatment.

Study of lipid profile levels and liver's enzymes in serum patients infected with diabetes mellitus type 2 After 3 years from infected

This research aims to the relation between long period of infection with diabetes mellitus type 2 and some drugs that cause decrease lipids in serum blood and measure liver's enzymes and study the relationship between formation stone in gallbladder and high level of cholesterol and triglyceride. the studied measurer levels of some Biochemical profile such as : (blood glucose , Glycosylate hemoglobin HbA1c , cholesterol, Triglyceride ,High density lipioprotein – cholesterol(HDL-C) ,low density lipioprotein- cholesterol(LDL-C), glutamic pyruvate transaminase enzyme(GPT),glutamic oxaloacetate transaminase enzyme(GOT), Alkaline phosphates enzyme )and measure of serum total protein ,Albumin , Globulin in patients infected with diabetes type2 before and after treatment for three years. Blood samples collected before and after three years infected and treated and the above mention parameters were measured as scheduled. This study contains (100) samples Diabetes patients un controlling glucose divided in to (50males , 50females ) and 50 healthy person divided in (25 males ,25 females )from the same area no infected with diabetes mellitus depended as a control group . The patients infected with diabetic treated by daonil and Metra famine and lipids drugs( Gemfibrozl (lopid 600). High levels in lipids profile for three years caused to stone in gall bladder in female group more than male group due to may be to relation between the infection with diabetes mellitus and length period infection with high level of cholesterol and triglyceride . It is clear that treatment of diabetes by lipids drugs caused decreasing in blood level profile, also liver founded in good function and high enzyme level induce to high level of cholesterol but no dangerous or risk about it due to infected with diabetes after three years. Blood glucose continuous with high level but don’t increase above before treatment that refers to patients not engagement with diet and sport exercises and the patients did not care about diabetes and complications.

Cancer microcell initiation and determination

BackgroundCancer remains one of the leading causes of death worldwide, despite the possibilities to detect early onset of the most common cancer types. The search for the optimal therapy is complicated by the cancer diversity within tumors and the unsynchronized development of cancerous cells. Therefore, it is necessary to characterize cancer cell populations after treatment has been applied, because cancer recurrence is not rare. In our research, we concentrated on small cancer cell subpopulation (microcells) that has a potential to be cancer resistance source. Previously made experiments has shown that these cells in small numbers form in specific circumstances after anticancer treatment.MethodsIn experiments described in this research, the anticancer agents’ paclitaxel and doxorubicin were used to stimulate the induction of microcells in fibroblast, cervix adenocarcinoma, and melanoma cell lines. Mainly for the formation of microcells in melanoma cells. The drug-stimulated cells were then characterized in terms of their formation efficiency, morphology, and metabolic activity.ResultsWe observed the development of cancer microcells and green fluorescent protein (GFP) transfection efficiency after stress. In the time-lapse experiment, we observed microcell formation through a renewal process and GFP expression in the microcells. Additionally, the microcells were viable after anticancer treatment, as indicated by the nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) enzyme activity assay results. Taken together, these findings indicate that cancer microcells are viable and capable of resisting the stress induced by anticancer drugs, and these cells are prone to chemical substance uptake from the environment.ConclusionMicrocells are not only common to a specific cancer type, but can be found in any tumor type. This study could help to understand cancer emergence and recurrence. The appearance of microcells in the studied cancer cell population could be an indicator of the individual anticancer therapy effectiveness and patient survival.

Evi1 upregulates Fbp1 and supports progression of acute myeloid leukemia through pentose phosphate pathway activation

Evi1 is a transcription factor essential for the development as well as progression of acute myeloid leukemia (AML) and high Evi1 AML is associated with extremely poor clinical outcome. Since targeting metabolic vulnerability is the emerging therapeutic strategy of cancer, we herein investigated a novel therapeutic target of Evi1 by analyzing transcriptomic, epigenetic, and metabolomic profiling of mouse high Evi1 leukemia cells. We revealed that Evi1 overexpression and Evi1‐driven leukemic transformation upregulate transcription of gluconeogenesis enzyme Fbp1 and other pentose phosphate enzymes with interaction between Evi1 and the enhancer region of these genes. Metabolome analysis using Evi1‐overexpressing leukemia cells uncovered pentose phosphate pathway upregulation by Evi1 overexpression. Suppression of Fbp1 as well as pentose phosphate pathway enzymes by shRNA‐mediated knockdown selectively decreased Evi1‐driven leukemogenesis in vitro. Moreover, pharmacological or shRNA‐mediated Fbp1 inhibition in secondarily transplanted Evi1‐overexpressing leukemia mouse significantly decreased leukemia cell burden. Collectively, targeting FBP1 is a promising therapeutic strategy of high Evi1 AML.

PPARγ-Induced Global H3K27 Acetylation Maintains Osteo/Cementogenic Abilities of Periodontal Ligament Fibroblasts.

The periodontal ligament is a soft connective tissue embedded between the alveolar bone and cementum, the surface hard tissue of teeth. Periodontal ligament fibroblasts (PDLF) actively express osteo/cementogenic genes, which contribute to periodontal tissue homeostasis. However, the key factors maintaining the osteo/cementogenic abilities of PDLF remain unclear. We herein demonstrated that PPARγ was expressed by in vivo periodontal ligament tissue and its distribution pattern correlated with alkaline phosphate enzyme activity. The knockdown of PPARγ markedly reduced the osteo/cementogenic abilities of PDLF in vitro, whereas PPARγ agonists exerted the opposite effects. PPARγ was required to maintain the acetylation status of H3K9 and H3K27, active chromatin markers, and the supplementation of acetyl-CoA, a donor of histone acetylation, restored PPARγ knockdown-induced decreases in the osteo/cementogenic abilities of PDLF. An RNA-seq/ChIP-seq combined analysis identified four osteogenic transcripts, RUNX2, SULF2, RCAN2, and RGMA, in the PPARγ-dependent active chromatin region marked by H3K27ac. Furthermore, RUNX2-binding sites were selectively enriched in the PPARγ-dependent active chromatin region. Collectively, these results identified PPARγ as the key transcriptional factor maintaining the osteo/cementogenic abilities of PDLF and revealed that global H3K27ac modifications play a role in the comprehensive osteo/cementogenic transcriptional alterations mediated by PPARγ.

Effects of different fertilizers on some soil enzymes activity and chlorophyll contents of two cotton (G. hirsutum L.) varieties grown in a saline and non-saline soil

Cotton plant has two physiological development periods which are vegetative and generative periods. The activities of urease, dehydrogenase, glucosidase and alkaline phosphatase enzymes in the soil continue during the growth of plants. In this study, three different fertilizer sources (diammonium phosphate (DAP), vermicompost and manure) and two cotton varieties (Candia and Lima) were used in non-saline and saline soil conditions. The experimental layout was randomized plots with 3 replications. Total chlorophyll content of Candia and Lima cotton (Gossypium hirsutum L.) cultivars and urease, dehydrogenase, glucosidase and alkaline phosphate enzyme activities of the soil were determined. The chlorophyll content of cotton plants was significantly (p < 0.01) different in fertilizer treatments applied in non-saline and saline soils. The lowest chlorophyll value (17.83) in non-saline soil was obtained in the control, while the highest chlorophyll value (32.64) was recorded in the DAP fertilizer treatments. The lowest chlorophyll value (20.33) in saline soil was obtained from DAP fertilizer, while the highest chlorophyll value (24.11) was obtained in the vermicompost application. The fertilizer treatment significantly affected the urease activity in non-saline (p < 0.01) and saline (p < 0.05) soils. The highest mean urease enzyme activity in non-saline (102.74 µg N g−1 dry soil h−1) and saline (120.23 µg N g−1 dry soil h−1) soils was obtained in the DAP treatments. The highest mean dehydrogenase enzyme activity in non-saline (35.99 µg TPF g−1 dry soil 24 h−1) and saline (48.63 µg TPF g−1 dry soil 24 h−1) soils was obtained in cattle manure application. The difference between fertilizer applications was significant at p < 0.01 level in both soils.

Screening of compound library identifies novel inhibitors against the MurA enzyme of Escherichia coli.

Bacterial cell has always been an attractive target for anti-infective drug discovery. MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) enzyme of Escherichia coli (E.coli) is crucial for peptidoglycan biosynthetic pathway, as it is involved in the early stages of bacterial cell wall biosynthesis. In the present study we aim to identify novel chemical structures targeting the MurA enzyme. For screening purpose, we used in silico approach (pharmacophore based strategy) for 52,026 library compounds (Chembridge, Chemdiv and in house synthetics) which resulted in identification of 50 compounds. These compounds were screened in vitro against MurA enzyme and release of inorganic phosphate (Pi) was estimated. Two compounds (IN00152 and IN00156) were found to inhibit MurA enzyme > 70% in primary screening and IC50 of 14.03 to 32.30 μM respectively. These two hits were further evaluated for their mode of inhibition studies and whole-cell activity where we observed 2-4 folds increase in activity in presence of Permeabilizer EDTA (Ethylenediaminetetraacetic acid). Combination studies were also performed with known antibiotics in presence of EDTA. Hits are reported for the first time against this target and our report also support the use of OM permeabilizer in combination with antibacterial compounds to address the permeability and efficacy issue. These lead hits can be further optimized for drug discovery. KEY POINTS: • Emerging Gram negative resistant strains is a matter of concern. • Need for new screening strategies to cope with drying up antibiotics pipeline. • Outer membrane permeabilizers could be useful to improve potency of molecules to reach its target.

Size shifting of solid lipid nanoparticle system triggered by alkaline phosphatase for site specific mucosal drug delivery

We aim to prepare a size-shifting nanocarrier for site-targeting mucosal drug delivery that can penetrate through mucus gel layer and remain close to the absorption membrane. As nanocarriers can be engineered to penetrate mucus but they can also back diffuse into outer mucus regions, a size shifting to micron range once they have reached the absorption membrane would prevent back-diffusion effect and extend drug release over a long period of time. For this purpose, we loaded solid lipid nanoparticles (SLN) with a phosphate ester surfactant and octadecylamine. Alkaline phosphatase (AP), a membrane bound enzyme was for the first time utilized as an in situ partner for triggering the size conversion at epithelial cell surface. Having the size of ~120 nm, SLN with hydrophilic and phosphate-decorated shells were shown to penetrate through mucus gel and form aggregates above cell layer surface. Aggregates of 5–8 µm were formed due to interparticle interactions induced by enzymatic phosphate removal after ~30 min in contact with isolated AP. The developed SLN system could be a potential tool for mucosal drug delivery to AP-expressing tissues like colon, lung, cervix, vagina and some mucus-secreting tumors.

Promiscuous enzymes generating d-amino acids in mammals: Why they may still surprise us?

Promiscuous catalysis is a common property of enzymes, particularly those using pyridoxal 5'-phosphate as a cofactor. In a recent issue of this journal, Katane et al. Biochem. J. 477, 4221-4241 demonstrate the synthesis and accumulation of d-glutamate in mammalian cells by promiscuous catalysis mediated by a pyridoxal 5'-phosphate enzyme, the serine/threonine dehydratase-like (SDHL). The mechanism of SDHL resembles that of serine racemase, which synthesizes d-serine, a well-established signaling molecule in the mammalian brain. d-Glutamate is present in body fluids and is degraded by the d-glutamate cyclase at the mitochondria. This study demonstrates a biochemical pathway for d-glutamate synthesis in mammalian cells and advances our knowledge on this little-studied d-amino acid in mammals. d-Amino acids may still surprise us by their unique roles in biochemistry, intercellular signaling, and as potential biomarkers of disease.

Abdominal Positron Emission Tomography Combined With Magnetic Resonance Imaging in Chronic Granulomatous Disease

Abdominal Positron Emission Tomography Combined With Magnetic Resonance Imaging in Chronic Granulomatous Disease

Assessing calcium availability from limestone sources through bone and blood status of chickens

Calcium (Cu) availability from limestone (procured from different cement factories) was assessed through bone and blood status of chickens using the completely randomized design. Five hundred and twenty five (525) straight run broiler chicks that were 28 days Of age (trial l), four hundred and twenty (420) broiler finishers that were 56 days of age (trial 2), and 210 layers that had been laying for six months (trial 3) were used in the study. Six limestone dietary treatments and a control accounted for the seven diets that were assessed Twelve birds per treatment and 4 per replicate that had their weights close to the mean of the pen were selected in the broiler trials (trials I and 2) and three birds per pen and nine per diet were selected in the layer trial (trial 3) for bone and blood samples analysis. Bone samples were analyzed for bone weight, bone length, ash weight, percentage ash, Ca and phosphorus contents of ash and blood samples were analyzed for plasma Ca and alkaline phosphates activity. Results showed similar influence of sources on bone weight, bone length, ash weight and percentage ash. Ca and P contents of ash were significantly (P&lt;0.05) affected by limestone sources but generally. Limestone sources produced mean values that were equal to or than the control diet in the starter phase (trail 1). In the finisher phase (trial 2), only Ca content of ashy varied significantly where Jakura source produced the least mean value. The layer trial (trial 3) also produced mean values that were significant (P &lt;0.05) for Ca content of ash with the Sokoto source producing the least mean value. Plasma Ca and alkaline phosphates enzyme activity attained published values for chickens. It was concluded based on the information from this study that the (h from tested limestone sources was generally available for chicken production.

Highlights of strategies adapted by two Pennisetum glaucum (L.) R. Br. races in a simulated drought stress experiment

Pearl millet (Pennisetum glaucum) is an important staple food crop in Asia and Africa. The most severe abiotic stress that hinders growth and development of crop is Drought. Four genotypes of P. glaucum were grown for 4 weeks in a greenhouse facility with the aim to identify several physiological responses observed after 12 days of drought stress and transcriptional profiling related to drought tolerance. Distinct parameters like relative water content (RWC), water use efficiency (WUE), stomata quantification (SI%), electron transport rate (ETR), photochemical (qP) and non-photochemical quenching (qNP) were measured under simulated drought condition for the selected four genotypes. The genotypes were grouped into two pearl millet races viz., nigritarum and globosum. Genotype 1P35451 of the nigritarum race showed augmentation in dry weight (DW) and tolerance index (TI) under the condition of decreased soil water due to transpiration. It exhibited a remarkable ability for recovery of leaf after an imposed drought condition in RWC, WUE and SI. Also, the transcriptional expression of CBF, RubSc and PIP 2;3 genes indicated that they were quick to respond to drought. CBF gene has proven to be useful for evaluating the ability to withstand drought in both races. RubSc and PIP2:3 displayed a pronounced increase in their expression level in nigritarum race. However, the drought tolerant genotype 1P13013 of globosum functioned in stomatal movement which is well linked with the expression of the Nicotinamide Adenine Dinucleotide Phosphate (NADP) enzyme. Therefore, the results indicate that mRNA expression of genes in both races under drought stress provides information on their ability to resist drought and could supply valuable molecular information for drought-tolerant elite breeding programs in the future.

Mechanism of eukaryotic serine racemase-catalyzed serine dehydration

Eukaryotic serine racemase (SR) is a pyridoxal 5′-phosphate enzyme belonging to the Fold-type II group, which catalyzes serine racemization and is responsible for the synthesis of D-Ser, a co-agonist of the N-methyl-d-aspartate receptor. In addition to racemization, SR catalyzes the dehydration of D- and L-Ser to pyruvate and ammonia. The bifuctionality of SR is thought to be important for D-Ser homeostasis. SR catalyzes the racemization of D- and L-Ser with almost the same efficiency. In contrast, the rate of L-Ser dehydration catalyzed by SR is much higher than that of D-Ser dehydration. This has caused the argument that SR does not catalyze the direct D-Ser dehydration and that D-Ser is first converted to L-Ser, then dehydrated. In this study, we investigated the substrate and solvent isotope effect of dehydration of D- and L-Ser catalyzed by SR from Dictyostelium discoideum (DdSR) and demonstrated that the enzyme catalyzes direct D-Ser dehydration. Kinetic studies of dehydration of four Thr isomers catalyzed by D. discoideum and mouse SRs suggest that SR discriminates the substrate configuration at C3 but not at C2. This is probably the reason for the difference in efficiency between L- and D-Ser dehydration catalyzed by SR.

MP57-17 PROGNOSTIC IMPACT OF PHOSPHATIDYLINOSITOL PHOSPHATE AND ITS ASSOCIATED ENZYME LEVELS IN PATIENTS WITH RENAL CELL CARCINOMA TREATED SURGICALLY

You have accessJournal of UrologyKidney Cancer: Localized: Surgical Therapy IV (MP57)1 Apr 2020MP57-17 PROGNOSTIC IMPACT OF PHOSPHATIDYLINOSITOL PHOSPHATE AND ITS ASSOCIATED ENZYME LEVELS IN PATIENTS WITH RENAL CELL CARCINOMA TREATED SURGICALLY Shintaro Narita*, Hiroki Nakanishi, Yoshinori Matsuda, Huang Mingguo, Mizuki Kobayashi, Akane Kikuchi, Taketoshi Nara, Sohei Kanda, Kazuyuki Numakura, Atsushi Koizumi, Mitsuru Saito, Takamitsu Inoue, Shigeru Satoh, Takehiko Sasaki, and Tomonori Habuchi Shintaro Narita*Shintaro Narita* More articles by this author , Hiroki NakanishiHiroki Nakanishi More articles by this author , Yoshinori MatsudaYoshinori Matsuda More articles by this author , Huang MingguoHuang Mingguo More articles by this author , Mizuki KobayashiMizuki Kobayashi More articles by this author , Akane KikuchiAkane Kikuchi More articles by this author , Taketoshi NaraTaketoshi Nara More articles by this author , Sohei KandaSohei Kanda More articles by this author , Kazuyuki NumakuraKazuyuki Numakura More articles by this author , Atsushi KoizumiAtsushi Koizumi More articles by this author , Mitsuru SaitoMitsuru Saito More articles by this author , Takamitsu InoueTakamitsu Inoue More articles by this author , Shigeru SatohShigeru Satoh More articles by this author , Takehiko SasakiTakehiko Sasaki More articles by this author , and Tomonori HabuchiTomonori Habuchi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000926.017AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The phosphoinositide (PIP) is associated with the risk of developing renal cell carcinoma (RCC). However, the levels of lipid metabolites, including PIPs phosphate derivatives, in patients with RCC is unknown. Thus, we investigated the PIP profile and its impact on clinical outcomes in patients with RCC who underwent surgery. METHODS: A total of 85 patients with RCC treated with surgery between 2011 and 2014 were included. The level of PIPs (PI, PIP1 and PIP2) in the tumor and its surrounding normal tissues was assessed by LC–MS–MS using a selected reaction monitoring with electrospray ionization. The mRNA expression of 16 phosphatases associated with the PIPs in the tumor was measured by qRT–PCR. RESULTS: No significant difference was observed in the PIP level between the tumors and normal surrounding tissues. The patients with high-grade tumor had significantly lower PIP1 and PIP2 (C38:4) tumor-to-normal (T/N) ratio than those with low-grade tumor. The metastatic-free survival (MFS) of patients with low PIP1 and PIP2 (C38:4) T/N ratios was significantly lower than that of patients with high PIP1 and/or PIP2 (C38:4) T/N ratios (p = 0.007). However, no significant difference was observed in the cancer-specific survival between the two groups (p = 0.590). The 5-year MFS of patients with low PIP1 and PIP2 (C38:4) T/N ratios was 67.0% and that of patients with either high PIP1 or PIP2 T/N ratio was 96.2%. In a multivariable analysis, pT stage and low PIP1 and PIP2 T/N ratios were found to be the independent prognostic factors of poor MFS in patients with RCC who underwent surgery (hazard ratio [HR] = 6.8, p = 0.015 and HR = 9.23, p = 0.041, respectively). Several candidate phosphatases associated with high-grade tumor were assessed using VIP score. Among the different types of phosphatases, the T/N ratios of patients with MTM1 and MTMR6 mRNA significantly correlated with PIP1 and PIP2 (C38:4) T/N ratios. CONCLUSIONS: Low PIP1 and PIP2 (C38:4) T/N ratios are associated with cancer aggressiveness and poor MFS in patients with RCC who underwent surgery. The PIP levels in surgical specimens can be a potential biomarker of oncological outcomes in patients with RCC, and targeting the enzymes associated with PIPs may be a novel treatment strategy for RCC. Source of Funding: None © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e868-e869 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shintaro Narita* More articles by this author Hiroki Nakanishi More articles by this author Yoshinori Matsuda More articles by this author Huang Mingguo More articles by this author Mizuki Kobayashi More articles by this author Akane Kikuchi More articles by this author Taketoshi Nara More articles by this author Sohei Kanda More articles by this author Kazuyuki Numakura More articles by this author Atsushi Koizumi More articles by this author Mitsuru Saito More articles by this author Takamitsu Inoue More articles by this author Shigeru Satoh More articles by this author Takehiko Sasaki More articles by this author Tomonori Habuchi More articles by this author Expand All Advertisement PDF downloadLoading ...