Dehydrogenase Isozymes Research Articles

Evaluation of quercetin as a potential cytoprotector against acetaldehyde using the cultured hepatocyte model with aldehyde dehydrogenase isozyme deficiency.

Protective effect of quercetin against acetaldehyde was evaluated using the cultured hepatocyte models with aldehyde dehydrogenase (ALDH) isozyme deficiency (aldh2-kd and aldh1a1-kd). The quercetin-induced cytoprotection against acetaldehyde in the ALDH1A1-deficient mutant (aldh1a1-kd) was weaker than that in wild type. Furthermore, quercetin did not enhance the ALDH activity in aldh1a1-kd cells, suggesting that ALDH1A1 is involved in the quercetin-induced cytoprotection.

Artemia salina Biological Responses to Zinc Oxide Nanoparticles

The Zinc oxide nanoparticles could be entered and separated in various water bodies through the runoff. The aim of this investigation is to evaluate the influences of different concentrations of zinc oxide nanoparticles on Artemia salina (used as live feed in aquaculture) at developmental, biochemical and molecular levels. The homogeneity among the estimated. Artemia salina was estimated using Inter simple sequence repeats and some isozyme polymorphism. The results showed that samples were highly homogeneous. The hatching percentages decreased as the concentration of Zinc Oxide nanoparticles increased, compared to the control group. The maximum hatching percentage (75.88%) was detected at the control followed by ZT1(65.52%), ZT2 (55.87%) and ZT3(55.22%). The percentage of each developmental stage of A. salina under the experimental conditions, including egg, breakage egg, umbrella stage, Instar 1, Instar 2 and Instar 3, was calculated. Also, the impacts of ZnO NPs on A. salina isozyme variations were evaluated. The results showed some different A. salina tissue responses among the various ZnO NPs concentrations. The total number of bands was 15, 12, 10 and 5 for a-Esterase, b-Esterase, Superoxide dismutase and Lactate dehydrogenase isozymes respectively. The electrophoretic pattern of the A. salina Esterases shows remarkable biochemical markers. Understanding such influences could be helpful for the industry to make informed decisions on whether to use such Zinc oxide nanoparticles or find safer alternatives, ultimately affecting production costs and profitability. The industry should pay attention on developing safer nanoparticles and optimizing their concentrations to reduce long-term costs associated with species depletion and environmental damage.

Fungal glyceraldehyde 3-phosphate dehydrogenase GpdC maintains glycolytic mechanism against reactive nitrogen stress-induced damage.

Highly reactive nitrogen species (RNS) damage proteins, lipids, and nucleotides, and induce disordered intracellular metabolism. Microorganisms that respond to and defend against RNS include fungal pathogens that invade host tissues. However, the full picture of their mechanisms remains unknown. We identified a novel glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme (GpdC) in the fungus Aspergillus nidulans. This isozyme preferred NADP+, which was unlike glycolytic GpdA that uses NAD+ as a cofactor. Exogenous RNS induced expression of the encoding gpdC gene, which when disrupted, decreased intracellular GAPDH activity, mycelial proliferation, and ethanol fermentation under RNS stress. Under these conditions, fungal growth requires glucose instead of non-fermentable carbon sources, and intact pyruvate decarboxylase (pdcA) and alcohol dehydrogenase (alcC) genes indicated that fungal metabolism shifts from respiratory to glycolytic and ethanolic fermentation. These results indicated that GpdC is an alternative GAPDH isozyme that facilitates NADP+-dependent glycolysis and energy conservation, which constitutes a fungal mechanism of stress tolerance via metabolic adaptation.

In Vivo Phosphorylation of the Cytosolic Glucose-6-Phosphate Dehydrogenase Isozyme G6PD6 in Phosphate-Resupplied Arabidopsis thaliana Suspension Cells and Seedlings.

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first committed step of the oxidative pentose phosphate pathway (OPPP). Our recent phosphoproteomics study revealed that the cytosolic G6PD6 isozyme became hyperphosphorylated at Ser12, Thr13 and Ser18, 48 h following phosphate (Pi) resupply to Pi-starved (-Pi) Arabidopsis thaliana cell cultures. The aim of the present study was to assess whether G6PD6 phosphorylation also occurs in shoots or roots following Pi resupply to -Pi Arabidopsis seedlings, and to investigate its relationship with G6PD activity. Interrogation of phosphoproteomic databases indicated that N-terminal, multi-site phosphorylation of G6PD6 and its orthologs is quite prevalent. However, the functions of these phosphorylation events remain unknown. Immunoblotting with an anti-(pSer18 phosphosite-specific G6PD6) antibody confirmed that G6PD6 from Pi-resupplied, but not -Pi, Arabidopsis cell cultures or seedlings (i.e., roots) was phosphorylated at Ser18; this correlated with a significant increase in extractable G6PD activity, and biomass accumulation. Peptide kinase assays of Pi-resupplied cell culture extracts indicated that G6PD6 phosphorylation at Ser18 is catalyzed by a Ca2+-dependent protein kinase (CDPK), which correlates with the 'CDPK-like' targeting motif that flanks Ser18. Our results support the hypothesis that N-terminal phosphorylation activates G6PD6 to enhance OPPP flux and thus the production of reducing power (i.e., NADPH) and C-skeletons needed to establish the rapid resumption of growth that ensures Pi-resupply to -Pi Arabidopsis.

Acclimation to warm temperatures modulates lactate and malate dehydrogenase isozymes in juvenile Horabagrus brachysoma (Günther)

Differential expression of isozymes enables fish to tolerate temperature fluctuations in their environment. The present study explores the modulation of lactate dehydrogenase (LDH) and cytoplasmic malate dehydrogenase (sMDH) isozyme expression in the heart, muscle, brain, liver, gill, and kidney of juvenile Horabagrus brachysoma after 30 days of acclimation at 26, 31, 33, and 36°C. LDH and sMDH zymography were performed using native polyacrylamide gel electrophoresis. The zymography revealed five distinct bands of LDH isoenzymes (labelled from cathode to anode as LDH-A4, LDH-A3B1, LDH-A2B2, LDH-A1B3, and LDH-B4) and three distinct bands of sMDH isoenzymes (labelled from cathode to anode as sMDH-A2, sMDH-AB, and sMDH-B2), with considerable variation in their expression in the tissues. Acclimation to the test temperatures did not influence the expression patterns of LDH or sMDH isozymes. Densitometric analysis of individual isozyme bands revealed a reduction in the densities of bands containing the LDH-B and sMDH-B molecules, while the densities of bands containing the LDH-A and sMDH-A molecules increased in the gills and muscle, indicating the role of these organs in adaptive responses to thermal acclimation. However, the total densities of the LDH and sMDH isozymes increased with higher acclimation temperatures, indicating that adaptation to increased temperatures in H. brachysoma is primarily characterised by quantitative changes in isozyme expression.

Promotion of NAD+ recycling by the hypoxia-induced shift in the lactate dehydrogenase isozyme profile reduces the senescence of human bone marrow-derived endothelial progenitor cells

Expansion of bone marrow-derived endothelial progenitor cells (EPCs) in vitro to obtain required cell numbers for therapeutic applications faces the challenge of growing cell senescence under the traditional normoxic culture condition. We previously found that 1% O2 hypoxic culture condition is favorable for reducing senescence of EPCs, but the mechanisms underlying the favorability are still unclear. Here, we found that, compared with normoxia, hypoxia induced a shift in lactate dehydrogenase (LDH) isozyme profile, which manifested as decreased LDH2 and LDH1 and increased LDH5, LDH4 and total LDHs. Moreover, under hypoxia, EPCs presented higher LDH activity, which could promote the conversion of pyruvate to lactate, as well as a higher level of NAD+, Bcl2 interacting protein 3 (BNIP3) expression and mitophagy. Additionally, under hypoxia, knock-down of the LDHA subunit increased the LDH2 and LDH1 levels and knock-down of the LDHB subunit increased the LDH5 level, while the simultaneous knock-down of LDHA and LDHB reduced total LDHs and NAD+ level. Inhibition of NAD+ recycling reduced BNIP3 expression and mitophagy and promoted cell senescence. Taken together, these data demonstrated that 1% O2 hypoxia induces a shift in the LDH isozyme profile, promotes NAD+ recycling, increases BNIP3 expression and mitophagy, and reduces EPC senescence. Our findings contribute to a better understanding of the connection between hypoxic culture conditions and the senescence of bone marrow-derived EPCs and provide a novel strategy to improve in vitro expansion of EPCs.

Virtual screening of triazoles inhibitors of 11β-hydroxysteroid dehydrogenaseenzymes using -ADME-moleculardocking, and molecular dynamics simulation studies

Hypertension, or elevated arterial blood pressure, is a substantial public health problem. The two 11β hydroxysteroid dehydrogenase (11β HSD) isozymes catalyze the interconversion of cortisol and cortisone. Our research consists in studying the inhibition of the enzymes with some derivatives of 1,2,4 triazoles by means of molecular docking and dynamics approaches. The interactions between the studied inhibitors and our target were further explored through molecular docking and molecular dynamics simulations, in the presence of water molecules. The molecular dynamics study was done for the best der ivatives of 1,2,4 triazoles inhibitors (deducted from the docking best scores for L2 and L1, and lowest score for Lref) A f e w k e y r e s i du e s N 14 with oxygen receptor interaction H donor) at the binding site of (11β HSD1) and (11β HSD2) were identified. Obtained Docking and molecular dynamics result, both leads to the same conclusion and predict that L2 subsisted derivatives of 1, 2, 4 triazoles is the best inhibitor candidate.

Association of serum lactate dehydrogenase (LDH) isozyme with prognosis of patients with metastatic clear cell renal cell carcinoma (mCRCC).

709 Background: LDH isozyme is a tetramer of two subunits, H chain and M chain, and is present in all living tissue. Five types of molecular forms characterize the LDH pattern, and tumor tissues relatively consist LDH-4 and LDH-5, composed with a high ratio of the M chain, compared to normal tissues. This study analyzed the association between LDH isozyme and prognosis of mCRCC after nephrectomy. Methods: Clinical records of mCRCC patients those who were initially diagnosed M0 disease at Nippon Medical School between 2012 and 2016 were retrospectively reviewed. LDH isozyme values before operation and at time of metastasis were checked. Isozyme patterns were classified into 6 types, LDH 1-5 dominant and common type, according to the most composed molecular form. Results: Out of 38 patients, 33 patients (87%) were male, 5 patients (13%) were female. Median age was 65 years old (36-87). pT1 was seen in 3 cases, pT2 in 4, pT3 in 27, and pT4 in 4. Pathological grade 2 were 9 cases, G3 in 21, and G4 in 8. As for the IMDC risk, favourable was 2 cases (5%), intermediate in 26 (68%) and poor in 10 (26%). Median LDH was 163 IU/L (113-317), and isozyme dominant pattern were as follows: LDH-2 in 9 cases (24%), LDH-3 in 6 (16%), LDH-4 in 4, LDH-5 in 11. 8 cases were common type, and no cases showed LDH-1 dominant. Median time from surgery to recurrence was 10 months (1-104), median follow-up period after recurrence was 18 months (4-72). 16 deaths occurred. No significant correlation was seen between pre-operative LDH isozyme pattern and pathological grade or pT stage. Pre-operative LDH isozyme did not correlate with the time to recurrence (p=0.7420). The median OS for LDH-4 dominant at the time of metastasis was 10.9 months, significantly shorter than other isozyme types (P=0.0134). Conclusions: LDH-4 dominant isozyme pattern at the time of recurrence has a short OS, proposing as a prognostic predictor in mRCC.

Effect of B-NIPOx in Experimental Trypanosoma cruzi Infection in Mice.

Chagas disease is caused by Trypanosoma cruzi and represents a major public health problem, which is endemic in Latin America and emerging in the rest of the world. The two drugs that are currently available for its treatment, Benznidazole and Nifurtimox, are partially effective in the chronic phase of the disease. In this study, we designed and synthesized the benzyl ester of N-isopropyl oxamic acid (B-NIPOx), which is a non-polar molecule that crosses cell membranes. B-NIPOx is cleaved inside the parasite by carboxylesterases, releasing benzyl alcohol (a molecule with antimicrobial activity), and NIPOx, which is an inhibitor of α-hydroxy acid dehydrogenase isozyme II (HADH-II), a key enzyme in T. cruzi metabolism. We evaluated B-NIPOx cytotoxicity, its toxicity in mice, and its inhibitory activity on purified HADH-II and on T. cruzi homogenates. We then evaluated the trypanocidal activity of B-NIPOx in vitro and in vivo and its effect in the intestine of T. cruzi-infected mice. We found that B-NIPOx had higher trypanocidal activity on epimastigotes and trypomastigotes than Benznidazole and Nifurtimox, that it was more effective to reduce blood parasitemia and amastigote nests in infected mice, and that, in contrast to the reference drugs, it prevented the development of Chagasic enteropathy.

Expression of aldehyde dehydrogenase 1A1 in oral squamous cell carcinoma and its correlation with clinicopathological parameters.

Background:Aldehyde dehydrogenase 1A1 (ALDH1A1) is a key aldehyde dehydrogenase (ALDH) isozyme, related to the cancer stem cells which are responsible for initiating tumor growth, progression, and recurrence. High expression of ALDH1A1 has been reported in several tumor types in humans and its expression is associated with poor prognosis. The aim of this study was to assess the expression of the ALDH1A1 in oral squamous cell carcinoma (SCC) and its correlation with various clinicopathological parameters.Materials and Methods:ALDH1A1 expression was analyzed by using immunohistochemistry on paraffin blocks of 112 cases of primary oral SCC and their corresponding 68 lymph nodes with metastatic deposits. ALDH1A1 expression was also correlated with various clinicopathological parameters. Statistical analysis was done with statistical analysis software, the Statistical Package for the Social Sciences version 21.0.Results:High ALDH1A1 expression was observed in 31.2% of cases of primary oral SCC as compared to 73.5% in lymph node metastasis. A statistically significant difference (P = 0.04) was observed in high TNM stages (68.6%) of the tumor as compared to low TNM stages (31.4%). However, histopathological grades of tumor showed nonsignificant correlation with ALDH1A1 expression (P = 0.093). 40.2% of patients were expired at the end of the study, and the rate of mortality was significantly higher (P = 0.01) in patients with high ALDH1A1 expression as compared to low expression (60.0% vs. 31.2%).Conclusion:High ALDH1A1 expression was associated with higher TNM tumor stage and high nodal stage. It was also associated with high mortality rate which validates it as a marker of invasiveness and poor prognosis in oral SCC.

Glucocorticoid and mineralocorticoid receptor expression in critical illness: A narrative review.

The glucocorticoid receptor (GCR) and the mineralocorticoid receptor (MR) are members of the steroid receptor superfamily of hormone-dependent transcription factors. The receptors are structurally and functionally related. They are localized in the cytosol and translocate into the nucleus after ligand binding. GCRs and MRs can be co-expressed within the same cell, and it is believed that the balance in GCR and MR expression is crucial for homeostasis and plays a key role in normal adaptation. In critical illness, the hypothalamic-pituitary-adrenal axis is activated, and as a consequence, serum cortisol concentrations are high. However, a number of patients exhibit relatively low cortisol levels for the degree of illness severity. Glucocorticoid (GC) actions are facilitated by GCR, whose dysfunction leads to GC tissue resistance. The MR is unique in this family in that it binds to both aldosterone and cortisol. Endogenous GCs play a critical role in controlling inflammatory responses in critical illness. Intracellular GC concentrations can differ greatly from blood levels due to the action of the two 11β-hydroxysteroid dehydrogenase isozymes, type 1 and type 2. 11β-hydroxysteroid dehydrogenases interconvert endogenous active cortisol and intrinsically inert cortisone. The degree of expression of the two isozymes has the potential to dramatically influence local GC availability within cells and tissues. In this review, we will explore the clinical studies that aimed to elucidate the role of MR and GCR expression in the inflammatory response seen in critical illness.

NADP+ -dependent isocitrate dehydrogenase isozymes from a psychrotrophic bacterium, Psychrobacter sp. strain 13A.

The genes encoding dimeric and monomeric isocitrate dehydrogenase (IDH) isozymes from a psychrotrophic bacterium, strain 13A (13AIDH-D and 13AIDH-M, respectively), were cloned and sequenced. The deduced amino acid sequences of these two IDHs showed high degrees of identity with those of bacteria of genus Psychrobacter. Analysis of the 16S ribosomal RNA gene of the strain 13A revealed that this bacterium is classified to genus Psychrobacter. The optimum temperatures for activities of 13AIDH-D and 13AIDH-M were 55°C and 45°C, respectively, indicating that they are mesophilic. On the contrary, 13AIDH-D maintained 90% of its maximum activity after incubation for 10 min at 50°C, while the 13AIDH-M activity was completely lost under the same condition. In addition, 13AIDH-D showed much higher specific activity than 13AIDH-M. From northern and western blot analyses, the 13AIDH-D gene was found to be not transcribed under the growth conditions tested in this study. However, the catalytic ability of the mesophilic 13AIDH-M was concluded to be enough to sustain the growth of strain 13A at low temperatures. Therefore, a novel pattern of the contribution of IDH isozymes in cold-living bacteria to their growth at low temperatures was confirmed in strain 13A.

Cofactor Specificity of Glucose-6-Phosphate Dehydrogenase Isozymes in Pseudomonas putida Reveals a General Principle Underlying Glycolytic Strategies in Bacteria.

ABSTRACTGlucose-6-phosphate dehydrogenase (G6PDH) is widely distributed in nature and catalyzes the first committing step in the oxidative branch of the pentose phosphate (PP) pathway, feeding either the reductive PP or the Entner-Doudoroff pathway. Besides its role in central carbon metabolism, this dehydrogenase provides reduced cofactors, thereby affecting redox balance. Although G6PDH is typically considered to display specificity toward NADP+, some variants accept NAD+ similarly or even preferentially. Furthermore, the number of G6PDH isozymes encoded in bacterial genomes varies from none to more than four orthologues. On this background, we systematically analyzed the interplay of the three G6PDH isoforms of the soil bacterium Pseudomonas putida KT2440 from genomic, genetic, and biochemical perspectives. P. putida represents an ideal model to tackle this endeavor, as its genome harbors gene orthologues for most dehydrogenases in central carbon metabolism. We show that the three G6PDHs of strain KT2440 have different cofactor specificities and that the isoforms encoded by zwfA and zwfB carry most of the activity, acting as metabolic “gatekeepers” for carbon sources that enter at different nodes of the biochemical network. Moreover, we demonstrate how multiplication of G6PDH isoforms is a widespread strategy in bacteria, correlating with the presence of an incomplete Embden-Meyerhof-Parnas pathway. The abundance of G6PDH isoforms in these species goes hand in hand with low NADP+ affinity, at least in one isozyme. We propose that gene duplication and relaxation in cofactor specificity is an evolutionary strategy toward balancing the relative production of NADPH and NADH.IMPORTANCE Protein families have likely arisen during evolution by gene duplication and divergence followed by neofunctionalization. While this phenomenon is well documented for catabolic activities (typical of environmental bacteria that colonize highly polluted niches), the coexistence of multiple isozymes in central carbon catabolism remains relatively unexplored. We have adopted the metabolically versatile soil bacterium Pseudomonas putida KT2440 as a model to interrogate the physiological and evolutionary significance of coexisting glucose-6-phosphate dehydrogenase (G6PDH) isozymes. Our results show that each of the three G6PDHs in this bacterium display distinct biochemical properties, especially at the level of cofactor preference, impacting bacterial physiology in a carbon source-dependent fashion. Furthermore, the presence of multiple G6PDHs differing in NAD+ or NADP+ specificity in bacterial species strongly correlates with their predominant metabolic lifestyle. Our findings support the notion that multiplication of genes encoding cofactor-dependent dehydrogenases is a general evolutionary strategy toward achieving redox balance according to the growth conditions.

Cancer-Testis Antigen Lactate Dehydrogenase C4 in Human Cancers: Old and New Perspectives

Lactate dehydrogenase (LDH) isozymes are abundant in various mammalian tissue types and accountable for the interconversion of pyruvate and lactate during the last step of anaerobic glycolysis...

Reconstitution of oxaloacetate metabolism in the tricarboxylic acid cycle in Synechocystis sp. PCC 6803: discovery of important factors that directly affect the conversion of oxaloacetate.

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathways in nature. Oxygenic photoautotrophic bacteria, cyanobacteria, have an unusual TCA cycle. The TCA cycle in cyanobacteria contains two unique enzymes that are not part of the TCA cycle in other organisms. In recent years, sustainable metabolite production from carbon dioxide using cyanobacteria has been looked at as a means to reduce the environmental burden of this gas. Among cyanobacteria, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) is an optimal host for sustainable metabolite production. Recently, metabolite production using the TCA cycle in Synechocystis 6803 has been carried out. Previous studies revealed that the branch point of the oxidative and reductive TCA cycles, oxaloacetate metabolism, plays a key role in metabolite production. However, the biochemical mechanisms regulating oxaloacetate metabolism in Synechocystis 6803 are poorly understood. Concentrations of oxaloacetate in Synechocystis 6803 are extremely low, such that in vivo analysis of oxaloacetate metabolism does not seem realistic. Therefore, using purified enzymes, we reconstituted oxaloacetate metabolism in Synechocystis 6803 in vitro to reveal the regulatory mechanisms involved. Reconstitution of oxaloacetate metabolism revealed that pH, Mg2+ and phosphoenolpyruvate are important factors affecting the conversion of oxaloacetate in the TCA cycle. Biochemical analyses of the enzymes involved in oxaloacetate metabolism in this and previous studies revealed the biochemical mechanisms underlying the effects of these factors on oxaloacetate conversion. In addition, we clarified the function of two l-malate dehydrogenase isozymes in oxaloacetate metabolism. These findings serve as a basis for various applications of the cyanobacterial TCA cycle.

유산소운동이 당뇨쥐 해마의 신경세포 LDH 동위효소, MCT2 및 GLUT3 단백질 발현에 미치는 영향

In the present study, the effects of aerobic exercise on expression of Lactate dehydrogenase (LDH) isozymes, Monocarboxylate transporter2 (MCT2) and Glucose transporter3 (GLUT3) in hippocampal neuron of Streptozotocin (STZ)-induced diabetic rats. The experimental animals were divided into three groups (n=10 in each group): the normal control group (CON), the diabetes mellitus group (DM), the diabetes and exercise group(DM-Ex). Animals in the exercise group were submitted to daily exercise in the water pool during 4weeks. Native polyacrylamide gel electrophoresis analysis for the purification of LDH isozyme was performed. Western blotting and immunohistochemistry analysis for the expression of MCT2 and GLUT3 was performed. Here in this study, LDH A4 and LDH A3B in DM were predominantly expressed compared to DM-Ex (p<.05), and LDH AB3 and LDH B4 in DM-Ex were predominantly expressed compared to DM (p<.05). MCT2 activity of DM and DM-Ex were significantly lower than that of CON (p<.05). MCT2 activity of DM-Ex was significantly higher than that of DM (p<.05). GLUT3 activity of DM and DM-Ex were significantly lower than that of CON (p<.05). GLUT3 activity of DM-Ex was significantly higher than that of DM (p<.05). The results of the present study indicate that aerobic exercise affects the production of ATP using lactic acid and the activity of neurons by adapting to the energy metabolic demands of diabetes in the hippocampal neurons.

Effect of active-site aromatic residues Tyr or Phe on activity and stability of glucose 6-phosphate dehydrogenase from psychrophilic Arctic bacterium Sphingomonas sp.

Cold-adapted enzymes maintain correct conformation at their active sites despite their intrinsically flexible structures. The psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 has two glucose 6-phosphate dehydrogenase (G6PD) isozymes, SpG6PD1 involved in the Entner-Doudoroff pathway and SpG6PD2 in the oxidative pentose phosphate pathway. Structural modeling of SpG6PD1 showed that the hydroxyl group of Tyr177 participates in substrate binding by forming a hydrogen bond with the phosphate group of glucose 6-phosphate, whereas in SpG6PD2, a Phe residue is present in the corresponding position of Tyr177. In this study, we investigated how subtle differences in aromatic residues in the substrate-binding pocket of SpG6PD1 affect enzymatic activity and stability. Mutations of Tyr177 to Ala, His, Phe, and Trp caused increases in the rigidity of the SpG6PD1 structure. Particularly, mutants Y177F and Y177W showed increased thermal stabilities compared to wild-type (WT) but 3- and 15-fold lower catalytic efficiencies, respectively. However, mutants Y177A and Y177H became heat-labile at moderate temperatures. These results indicate that an aromatic residue (Tyr or Phe) is necessary for the substrate-binding pocket of SpG6PD1; Tyr with its hydroxyl group is preferred for enzymatic activity, whereas the more hydrophobic Phe is preferred for thermal stability. Substitutions of bulky Trp for Tyr or Phe at this position resulted in substantial loss of activity. Our study suggests that delicate adjustment of aromatic residues can regulate the activity and stability of psychrophilic G6PD isozymes involved in different metabolic pathways.

Nuliglucaemia Lucidae: Extreme Deprivation of Blood Glucose as Organic Context for Cancer Treatment with Antimetabolites

The present report describes our clinical findings regarding the use of high dose intravenous insulin in cancer patients, as a means to deplete the blood compartment of glucose molecules. The purpose of this intervention is to create a favorable physiological state for the competitive inhibition of several rate-limiting enzymes within cancer cells, with structural analogues that behave as antimetabolites. Regardless of their histological origin, virtually all solid tumors reported on to date (February 2020) are found to be hypercaptant in PET-CT scans following the intravenous injection of 2-¹⁸fluoro-deoxy-D-glucose. Most solid tumors display a hypermetabolic phenotype (SUVmax ≥ 3), with marked overexpression of glucose transporters (GLUTs) in the outer membrane of their anaplastic cells subpopulations. The fact that neoplastic cells also overexpress glycolytic, fermentative and glutaminolytic enzymes up to an order of magnitude relative to healthy cells further strengthens the argument for a competitive inhibition with antimetabolites. The rationale for a deep, systemic deprivation of glucose was suggested by classical enzymological work concerning competitive inhibition (the Woods principle), and our group has shown that a metabolic intervention with structural analogues of glucose and pyruvate is strongly enhanced by a systemic suppression of the natural substrates of hexokinase 2 (HK-2) and lactic dehydrogenase isozyme A (LDH-A), followed by the timely introduction of several non- metabolizable analogues. Sustained, deep hypoglycemia (&lt;10mg/dl) under physiological ketosis provides an advantageous context for antitumor treatment with structural analogues of glucose, pyruvate and glutamine. Data provided in this report demonstrates the feasibility and safety of the procedure.

Effects of Maternal Chewing on Prenatal Stress-Induced Cognitive Impairments in the Offspring via Multiple Molecular Pathways.

We aimed to investigate the effects of maternal chewing on prenatal stress-induced cognitive impairments in the offspring and to explore the molecular pathways of maternal chewing in a mice model. Maternal chewing ameliorated spatial learning impairments in the offspring in a Morris water maze test. Immunohistochemistry and Western blot findings revealed that maternal chewing alleviated hippocampal neurogenesis impairment and increased the expression of hippocampal brain-derived neurotrophic factor in the offspring. In addition, maternal chewing increased the expression of glucocorticoid receptor (GR) and 11β-hydroxysteroid dehydrogenase isozyme 2 (11β-HSD2) and decreased the expression of 11β-HSD1 in the placenta, thereby attenuating the increase of glucocorticoid in the offspring. Furthermore, maternal chewing increased the expression of 11β-HSD2, FK506-binding protein 51 (FKBP51) and FKBP52 and decreased the expression of 11β-HSD1, thereby increasing hippocampal nuclear GR level. In addition, maternal chewing attenuated the increase in expression of DNMT1 and DNMT3a and the decrease in expression of histone H3 methylation at lysine 4, 9, 27 and histone H3 acetylation at lysine 9 induced by prenatal stress in the offspring. Our findings suggest that maternal chewing could ameliorate prenatal stress-induced cognitive impairments in the offspring at least in part by protecting placenta barrier function, alleviating hippocampal nuclear GR transport impairment and increasing the hippocampal brain-derived neurotrophic factor (BDNF) level.

Cloning, expression, identification and characterization of borneol dehydrogenase isozymes in Pseudomonas sp. TCU-HL1

Borneol is a bicyclic plant monoterpene. It can be degraded by soil microorganisms through the conversion of borneol dehydrogenase (BDH) and a known camphor degradation pathway. Recombinant BDH from Pseudomonas sp. TCU-HL1 was produced in the form of inclusion body. The refolded BDH1 tends to precipitate. Insoluble recombinant BDH1 was converted into a soluble form by adding glycerol in LB medium. The kcat and kcat/Km values of soluble form BDH1 for (+)-borneol turned out to be about 34-fold and 45-fold higher, respectively, than those of the refolded enzyme. On the other hand, a gene knockout mutant, TCU-HL1Δbdh, was constructed to investigate the possible presence of a second copy of the bdh gene in TCU-HL1 genome. A new gene, bdh2, encoding a BDH isozyme, was identified, and the recombinant BDH2 protein was produced in a soluble form. Both bdh1 and bdh2 genes are expressed in the crude extract of wild type TCU-HL1, as shown by RT-qPCR results. Both BDH isozymes prefer to degrade (+)-borneol, rather than (−)-borneol, probably because (+)-camphor is the main form present in nature.