Characterization of NADP-dependent L-arginine dehydrogenase as a novel amino acid dehydrogenase and its application to an L-arginine assay

L-Arginine dehydrogenase (L-ArgDH, EC 1.4.1.25) is an amino acid dehydrogenase which catalyzes the reversible oxidative deamination of L-arginine to the oxo analog in the presence of NADP. Although the enzyme activity is detected in the cell extract of Pseudomonas aruginosa , the purification and characterization of the enzyme have not been achieved to date. We here found the gene homolog of L-ArgDH in genome data of Pseudomonas veronii and succeeded in expression of P. veronii JCM11942 gene in E. coli. The gene product exhibited strong NADP-dependent L-ArgDH activity. The crude enzyme was unstable under neutral pH conditions, but was markedly stabilized by the addition of 10% glycerol. The enzyme was purified to homogeneity through a single Ni-chelate affinity ch romatography step and consisted of a homodimeric protein with a molecular mass of about 65 kDa. The enzyme selectively catalyzed l-arginine oxidation in the presence of NADP with maximal activity at pH 9.5. The apparent K m values for l-arginine and NADP were 2.5 and 0.21 mM, respectively. The nucleotide sequence coding the enzyme gene ( was determined and the amino acid sequence was deduced from the nucleotide sequence. As an application of the enzyme, simple colorimetric microassay for L-arginine using the enzyme was achieved.

Triple Isozyme Lactic Acid Dehydrogenase Inhibition in Fully Viable MDA-MB-231 Cells Induces Cytostatic Effects That Are Not Reversed by Exogenous Lactic Acid

A number of aggressive human malignant tumors are characterized by an intensified glycolytic rate, over-expression of lactic acid dehydrogenase A (LDHA), and subsequent lactate accumulation, all of which contribute toward an acidic peri-cellular immunosuppressive tumor microenvironment (TME). While recent focus has been directed at how to inhibit LDHA, it is now becoming clear that multiple isozymes of LDH must be simultaneously inhibited in order to fully suppress lactic acid and halt glycolysis. In this work we explore the biochemical and genomic consequences of an applied triple LDH isozyme inhibitor (A, B, and C) (GNE-140) in MDA-MB-231 triple-negative breast cancer cells (TNBC) cells. The findings confirm that GNE-140 does in fact, fully block the production of lactic acid, which also results in a block of glucose utilization and severe impedance of the glycolytic pathway. Without a fully functional glycolytic pathway, breast cancer cells continue to thrive, sustain viability, produce ample energy, and maintain mitochondrial potential (ΔΨM). The only observable negative consequence of GNE-140 in this work, was the attenuation of cell division, evident in both 2D and 3D cultures and occurring in fully viable cells. Of important note, the cytostatic effects were not reversed by the addition of exogenous (+) lactic acid. While the effects of GNE-140 on the whole transcriptome were mild (12 up-regulated differential expressed genes (DEGs); 77 down-regulated DEGs) out of the 48,226 evaluated, the down-regulated DEGS collectively centered around a loss of genes related to mitosis, cell cycle, GO/G1–G1/S transition, and DNA replication. These data were also observed with digital florescence cytometry and flow cytometry, both corroborating a G0/G1 phase blockage. In conclusion, the findings in this work suggest there is an unknown element linking LDH enzyme activity to cell cycle progression, and this factor is completely independent of lactic acid. The data also establish that complete inhibition of LDH in cancer cells is not a detriment to cell viability or basic production of energy.

DIAGNOSTIC UTILITY OF SERUM LACTATE DEHYDROGENASE LEVELS (LDL) IN DIFFERENTIATING MEGALOBLASTIC ANEMIA FROM MYELODYSPLASTIC SYNDROMES IN PAKISTAN

Objective: To study the diagnostic utility of lactate dehydrogenase levels in differentiating megaloblastic anemia from myelodysplastic anemia in Pakistan. Study Design: Comparative cross-sectional study. Place and Duration of Study: Department of Hematology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan from Feb, 2019 to Aug, 2019. Methodology: In this study, total 240 patients (18-75 years of age) males and females were selected by consecutive sampling technique and were equally divided into 3 groups; patients with megaloblastic anemia, patients with myelodysplastic syndromes and healthy control group. The clinical history and duration of anemia were recorded on special designed proforma. The laboratory investigations including lactate dehydrogenase levels were also noted. Both types of anemia were compared on basis of Lactate Dehydrogenase Levels. Results: The lactate dehydrogenase levels in megaloblastic group were more than 3000 IU/L in 58 out of 80 patients (72.5%). On other hand, myelodysplastic group had 79 out of 80 patients with lactic acid dehydrogenase levels below 450 IU/L (98.75%). The difference in lactic acid dehydrogenase levels between both groups was found to be statistically significant. Conclusion: Serum lactate dehydrogenase levels can be used to differentiate megaloblastic anemia from other anemia especially myelodysplastic syndromes before doing a bone marrow examination. High lactate dehydrogenase levels above 3000 IU/L in megaloblastic anemia can differentiate it from other anemia.

Characterization of NADP-dependent L-arginine dehydrogenase as a novel amino acid dehydrogenase and its application to an L-arginine assay

Purpose: The primary aim of this study was the purification and characterization of an NADP-dependent L arginine dehydrogenase (L-ArgDH, EC 1.4.1.25) as a novel amino acid dehydrogenase from Pseudomonas veronii. We then applied the enzyme to an L-arginine assay. Methods: An L-ArgDH gene from P. veronii JCM11942 was amplified by PCR using primers based on the N and C-terminal sequences inferred from a putative L-ArgDH gene (PverR02_12350) found in the P. veronii genome. The L-ArgDH activity of the product expressed in Escherichia coli was confirmed, after which the enzyme was purified, characterized, and applied to an L-Arg microassay. Results: The P. veroniiJCM11942 gene was expressed in E. coli, and the gene product exhibited strong NADP dependent L-ArgDH activity. The crude enzyme was unstable but was stabilized by the presence of 10% glycerol under neutral pH conditions. The enzyme was purified to homogeneity through a single Ni-chelate affinity chromatography step and consisted of a homodimeric protein with a molecular mass of about 65 kDa. The enzyme selectively catalyzed L-arginine oxidation in the presence of NADP, with maximal activity at pH 9.5. The apparent Km values for L-arginine and NADP were 2.5 and 0.21 mM, respectively. A simple colorimetric microassay for L-arginine was achieved using the enzyme. Conclusions: The L-ArgDH gene from P. veronii JCM 11942 was successively expressed in E. coli. The product exhibited NADP-dependent L-ArgDH dehydrogenase activity, and the enzyme was purified and characterized as a novel amino acid dehydrogenase. Furthermore, a simple colorimetric assay for L-arginine using L-ArgDH was achieved. Conflict of interest: The authors declare that they have no competing interests.

Toward an Understanding Of the Structural and Mechanistic Aspects of Protein-Protein Interactions in 2-Oxo Acid Dehydrogenase Complexes

The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the TCA cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging. DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxo acid dehydrogenase complexes, their function and regulation.

Incidence of intravascular hemolysis after transcatheter mitral valve repair

Background Chronic subclinical intravascular hemolysis is a common complication after valve replacement associated with worse prognosis, occurring in up to 80% of patients after mitral valve surgery. While serious intravascular hemolysis after MitraClip implantation has been reported anecdotally, data on the impact of transcatheter mitral valve repair on the prevalence of subclinical hemolysis are lacking. Methods and results From August 2017 to November 2019, 77 patients with high perioperative risk and moderate-to-severe or severe mitral regurgitation were prospectively enrolled in a single-center trial. All participants were treated with transcatheter edge-to-edge mitral valve repair using the MitraClip NT, NTR or XTR system. Before and three months after the procedure, all patients underwent comprehensive clinical assessment including laboratory measurement of hemoglobin, haptoglobin and lactic acid dehydrogenase in venous blood samples. Presence of subclinical intravascular hemolysis was defined as hemoglobin <13.8 g/dl for males or <12.4 g/dl for females, haptoglobin <65 mg/dl and lactic acid dehydrogenase >250 U/l. Levels of the hemolysis marker haptoglobin significantly decreased three months after the intervention (127±71 mg/dl at three months vs. 158±73 mg/dl at baseline, p<0.001), accompanied by an increase in lactic acid dehydrogenase (251±88 U/l vs. 222±55 U/l, p<0.01), implying the induction of intravascular hemolysis by transcatheter mitral valve repair. Higher residual mitral regurgitation was associated with lower haptoglobin levels three months after mitral valve repair (p<0.05), hinting that shear stress caused by regurgitation flow is the primary mechanism for hemolysis after MitraClip implantation. Concurrently, we observed a trend towards an increase in the presence of subclinical intravascular hemolysis (9.1% at three months vs. 3.9% at baseline, p=0.289). Hemoglobin levels remained unchanged (12.1±1.5 g/dl at three months vs 12.3±1.8 g/dl at baseline, p=0.107). No patient needed treatment for intravascular hemolysis. Conclusion Transcatheter edge-to-edge mitral valve repair in a high-risk collective is associated with the induction of hemolysis. Yet, prevalence of subclinical intravascular hemolysis is low when compared to mitral valve surgery, emphasizing the good safety profile of minimal-invasive mitral valve therapy. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): ReForM-B research grant, University of Regensburg

Εύρεση βιοδεικτών που σχετίζονται με την ενδομήτρια υπολειπόμενη ανάπτυξη με χρήση μεταβολωμικής

Η ενδομήτρια υπολειπόμενη ανάπτυξη (IUGR) αναφέρεται στην αδυναμία του εμβρύου να επιτύχει το γενετικά καθορισμένο ενδογενές δυναμικό αύξησης. Η IUGR παθολογία χαρακτηρίζεται από την περιορισμένη μεταφορά οξυγόνου και θρεπτικών συστατικών από τη μητέρα στο έμβρυο και σχετίζεται με αυξημένη περιγεννητική νοσηρότητα και θνησιμότητα, καθώς και απώτερες επιπλοκές κατά την παιδική ηλικία και την ενήλικο ζωή. Η παρούσα διατριβή εστίασε στην εφαρμογή της μεταβολωμικής για τον εντοπισμό δευτερογενών μεταβολιτών που χαρακτηρίζουν την IUGR παθολογία. Η μεταβολωμική πραγματοποιήθηκε με χρήση με φασματοσκοπίας πυρηνικού μαγνητικού συντονισμού (NMR) σε δείγματα ομφαλικού και μητρικού αίματος. Η μελέτη περιλάμβανε 84 κυήσεις εκ των οποίων οι 48 αφορούσαν IUGR περιπτώσεις και οι 36 κυήσεις νεογνών με φυσιολογική ανάπτυξη (AGA). Με εφαρμογή τεχνικών επιβλεπόμενης και μη επιβλεπόμενης πολυμεταβλητής στατιστικής ανάλυσης επιτεύχθηκε ο διαχωρισμός των ομφαλικών και των μητρικών δειγμάτων μεταξύ των IUGR και AGA υποσυνόλων. Το μεταβολικό προφίλ των μητρικών προσομοιάζει αυτό των ομφαλικών δειγμάτων στις IUGR κυήσεις. Στο φάσμα NMR ταυτοποιήθηκαν 56 μεταβολίτες, εκ των οποίων 7 φάνηκε ότι συμμετέχουν παρόμοια στη διάκριση μεταξύ IUGR και AGA περιπτώσεων. Αυτή η συσχέτιση πιθανώς αναδεικνύει ότι μεταξύ των εμβρύων με IUGR παθολογία και των μητέρων τους επιδρούν κοινοί μηχανισμοί που διαφοροποιούν το μεταβολικό τους αποτύπωμα. Αυτό το συμπέρασμα, εφόσον επικυρωθεί με επιπρόσθετες μελέτες, είναι δυνατόν να οδηγήσει στην χρήση βιοδεικτών στο αίμα των εγκύων με σκοπό την ανάπτυξη νέων διαγνωστικών εργαλείων για την IUGR. Η ανάδειξη υποψήφιων βιοδεικτών σε πρώιμο στάδιο της κύησης θα μπορούσε να συμβάλλει στην ανάσχεση των αρνητικών επιπτώσεων της IUGR είτε μέσω κάποιας φαρμακευτικής ή διατροφικής παρέμβασης. Συγκεκριμένα, η ενίσχυση της γλυκονεογένεσης και η καταστολή της γλυκερονεογένεσης στην ομάδα των IUGR νεογνών φαίνεται να αποδίδεται στη σχέση της IUGR παθολογίας με την αντίσταση στην ινσουλίνη. Η αύξηση των BCAAs και κατ’ επέκταση του 3 μέθυλο-2-οξοβαλερικού οξέος στις IUGR ομάδες, πιθανόν να σχετίζεται με την έλλειψη ή δυσλειτουργία του πολυ-ενζυμικού συμπλέγματος BCKD (Branched chain α-keto acid dehydrogenase). Τέλος, η μείωση της θρυπτοφάνης στα IUGR νεογνά φαίνεται να σχετίζεται με την ανάπτυξη μηχανισμών προσαρμογής για την κάλυψη των αυξημένων αναγκών τους σε σεροτονίνη. Τα αποτελέσματα της παρούσας διατριβής επιβεβαιώνουν προηγούμενα διφορούμενα αποτελέσματα που σχετίζονται με την περίπλοκη παθολογία της ενδομήτριας υπολειπόμενης ανάπτυξης, και προσφέρουν νέα δεδομένα με έμφαση σε δυνητικούς βιοδείκτες για μελλοντική εφαρμογή στην έγκαιρη διάγνωση.