Isozyme Activity Research Articles

SAT-329 Novel Dehydroepiandrosterone Derivatives Inhibiting Metastatic Prostate Cancer Cell Proliferation

: Metastatic prostate tumor cells mainly express type 1 5α-reductase and have been linked to antiandrogenic resistant prostate cancers. However, the isozyme of type 2 5α-reductase is mostly expressed in normal prostate epithelial cells and non-metastatic cancer cells of the prostate. Therefore, specific inhibitors of type 1 isozyme activity, could decrease the proliferation of these metastatic prostate tumors (in cell line LNCaP, for example). To demonstrate this, the antiproliferative activity of 16 new dehydroepiandrosterone derivatives, previously identified by our group as selective inhibitors of type 1 5α-reductase [1], were evaluated in LNCaP cultures in the presence of testosterone (T) or dihydrotestosterone (DHT) and/or finasteride (F). The viability of these cells was demonstrated by the formation of formazan crystals, which were quantified using a spectrophotometric technique. Western Blot analysis demonstrated the presence of type 1 5α-reductase protein in cells treated with T, T and F, or T plus each of the dehydroepiandrosterone derivatives evaluated. In addition, a significant decrease in LNCaP cell proliferation in cells treated with T plus these derivatives was detected, while T and T plus F treatments increased their spread. These data indicated that 5α-reductase activity in metastatic prostate cell line LNCaP was inhibited by all of the dehydroepiandrosterone derivatives analyzed and, as a consequence, cell proliferation decreased significantly. However, F, which is a specific blocker of type 2 5α-reductase, did not show any effect on cell proliferation compared to cells treated only with T. To determine the toxicity of these steroids, several groups of hamsters were treated with 2 mg/kg of each of these derivatives once every 24 hours for six days. The body weight of these animals increased over six days similarly to that of control hamsters. The weights of prostate and seminal vesicles of the control and treated animals showed no significant differences. In conclusion, 16 new dehydroepiandrosterone derivatives showed an antiproliferative effect on LNCaP cells, due to the inhibition of type 1 5α-reductase activity. In addition, these derivatives showed no toxicity in the tested model. Therefore, these derivatives have significant potential for therapeutical use. Reference: [1] Bratoeff et al. GENZ 2013, 28 (6): 1247-1254. Nothing to Disclose: MC, LB, YH.

Protein Kinase C: Targets to Regenerate Brain Injuries?

Acute or chronic injury to the central nervous system (CNS), causes neuronal death and irreversible cognitive deficits or sensory-motor alteration. Despite the capacity of the adult CNS to generate new neurons from neural stem cells (NSC), neuronal replacement following an injury is a restricted process, which does not naturally result in functional regeneration. Therefore, potentiating endogenous neurogenesis is one of the strategies that are currently being under study to regenerate damaged brain tissue. The insignificant neurogenesis that occurs in CNS injuries is a consequence of the gliogenic/non-neurogenic environment that inflammatory signaling molecules create within the injured area. The modification of the extracellular signals to generate a neurogenic environment would facilitate neuronal replacement. However, in order to generate this environment, it is necessary to unearth which molecules promote or impair neurogenesis to introduce the first and/or eliminate the latter. Specific isozymes of the protein kinase C (PKC) family differentially contribute to generate a gliogenic or neurogenic environment in injuries by regulating the ADAM17 mediated release of growth factor receptor ligands. Recent reports describe several non-tumorigenic diterpenes isolated from plants of the Euphorbia genus, which specifically modulate the activity of PKC isozymes promoting neurogenesis. Diterpenes with 12-deoxyphorbol or lathyrane skeleton, increase NPC proliferation in neurogenic niches in the adult mouse brain in a PKCβ dependent manner exerting their effects on transit amplifying cells, whereas PKC inhibition in injuries promotes neurogenesis. Thus, compounds that balance PKC activity in injuries might be of use in the development of new drugs and therapeutic strategies to regenerate brain injuries.

Glutathione S-transferase enzyme activity and protein expression in patients with recurrent tonsillitis and idiopathic tonsillar hypertrophy.

The palatine tonsil is a significant part of the secondary immune system. Tonsillitis and idiopathic tonsillar hypertrophy (ITH) are the most common pathologies of this component. Although there are studies on their pathogenesis, there is insufficient study of the role of antioxidant agents. Glutathione S-transferase (GST) isozymes contribute to the antioxidation reactions in the tissue via the glutathione pathway. The purpose in this study was to reveal the levels of the GST enzyme activity and protein expression of GSTP1 and GSTA1 isozymes in patients with tonsillitis and tonsil hypertrophy, and to investigate their role in the pathogenesis of these diseases. Sixteen patients with recurrent tonsillitis and 5 patients with ITH and were included in the study. Cytosolic extracts were prepared from post-tonsillectomy tissues of both patient groups and GST enzyme activities were measured. The expression of GSTP1 was found to be significantly higher than GSTA1 in tissue samples of patients with ITH and recurrent tonsillitis (P<0.001). Increased GST activity and GSTP1 isozyme expression were shown in patients with recurrent tonsillitis compared to the idiopathic tonsillar hypertrophy study group. There was a positive correlation between the expressions of GSTP1 (P=0.040; r=0.47). Increased GST activity and GSTP1 isozymes were demonstrated histologically in the pathogenesis of ITH and recurrent tonsillitis. We believe that the data of changes in antioxidant capacity, obtained from studies with more extensive and larger samples, would support our findings.

Influence of Flavonoids from Galium verum L. on the activities of cytochrome P450 isozymes and pharmacokinetic and pharmacodynamic of warfarin in rats

Background: Galium verum L., a traditional Chinese medicine, has been widely used in the folk in China. Preparations of G. verum L. were used to treat thromboembolic disease in clinic for many years and often combined with anticoagulants. Flavonoids from G. verum L. (FGVL) are the main active component. Materials and Methods: We assessed the potential influence of FGVL on the activities of five cytochrome P450 (CYP) enzymes and on the pharmacokinetic of warfarin in rats. The pharmacokinetics of five probe drugs and of warfarin was compared between control and FGVL-pretreated groups, which could estimate the effect on the activities of the five isozymes and warfarin metabolism. Moreover, the potential influence of FGVL on the pharmacodynamic of warfarin was investigated. Results: There was no significant difference in the pharmacokinetic parameters of chlorzoxazone and midazolam between control and FGVL-pretreated groups. However, the pharmacokinetic parameters of caffeine in every dose, metoprolol in middle dose, and tolbutamide in high dose were affected significantly (P < 0.05). It indicated that the metabolism of caffeine was markedly faster in FGVL-pretreated group but metoprolol in the middle dose and tolbutamide in the high-dose FGVL-pretreated group were markedly slower. The anticoagulation of combination group is better than warfarin group or FGVL group. This suggested that FGVL showed no effect on the enzyme activities of CYP2E1 and CYP3A2, induced the enzyme activities of CYP1A2, but inhibited the enzyme activities of CYP2D4 in the middle dose and CYP2C11 in high dose. Conclusion: This indicates that FGVL may increase the anticoagulation of warfarin in clinic dose. The dose of warfarin should be adjusted when combined with FGVL or preparations of G. verum L.

Investigation into the relationship among Cd bioaccumulation, nutrient composition, ultrastructural changes and antioxidative metabolism in lettuce genotypes under Cd stress

Lettuce (Lactuca sativa L.) is known to have high cadmium (Cd) concentrations in its shoots, which makes it necessary to protect against Cd toxicity. Understanding Cd-induced physiological responses in lettuce plants can contribute to the definition of useful strategies to decrease Cd uptake. This study aimed to gain new insights into Cd-induced stress by measuring Cd bioaccumulation, nutrient composition, anatomical and ultrastructural changes, and antioxidative metabolism in three lettuce genotypes characterized as having different degrees of Cd tolerance (Vanda = low, Lidia = medium and Stela = high). Plants were grown hydroponically with Cd concentrations of 0.0 and 0.1 or 0.5 μmol L−1, for 30 days. Cadmium uptake in the lettuce genotypes assayed is controlled by the root/shoot ratio, higher root/shoot ratios allowing greater Cd uptake. The Fe and Ni content increased in shoots of the genotype Lidia, which could be associated with a decrease in oxidative stress in chloroplasts due to superoxide dismutase (SOD) isozyme activity. Cadmium-induced oxidative stress is associated with de-structuring of the phloem and xylem in roots, and starch grain and plastoglobule accumulation in chloroplasts. Lettuce genotypes that presented higher SOD and ascorbate peroxidase (APX) activity presented better preserved anatomical structures. These results suggest that genotypes with less efficient antioxidant defence in the roots tend to take up more Cd, increasing root-to-shoot Cd translocation.

Rice Mutants Lacking Starch Synthase I or Branching Enzyme IIb Activity Altered Starch Biosynthetic Protein Complexes.

Amylopectin, the major component of starch, is synthesized by synergistic activity of multiple isozymes of starch synthases (SSs) and branching enzymes (BEs). The frequency and length of amylopectin branches determine the functionality of starch. In the rice endosperm, BEIIb generates short side chains of amylopectin and SSI elongates those branches, which can be further elongated by SSIIa. Absence of these enzymes greatly affects amylopectin structure. SSI, SSIIa, and BEIIb associate with each other and with other starch biosynthetic enzymes although SSIIa is low activity in japonica rice. The aim of the current study was to understand how the activity of starch biosynthetic enzyme complexes is compensated in the absence of SSI or BEIIb, and whether the compensatory effects are different in the absence of BEIIb or in the presence of inactive BEIIb. Interactions between starch biosynthetic enzymes were analyzed using one ss1 null mutant and two be2b japonica rice mutants (a mutant producing inactive BEIIb and a mutant that did not produce BEIIb). Soluble proteins extracted from the developing rice seeds were separated by gel filtration chromatography. In the absence of BEIIb activity, BEIIa was eluted in a broad molecular weight range (60–700 kDa). BEIIa in the wild-type was eluted with a mass below 300 kDa. Further, majority of inactive BEIIb co-eluted with SSI, SSIIa, and BEI, in a mass fraction over 700 kDa, whereas only small amounts of these isozymes were found in the wild-type. Compared with the be2b lines, the ss1 mutant showed subtle differences in protein profiles, but the amounts of SSIIa, SSIVb, and BEI in the over-700–kDa fraction were elevated. Immunoprecipitation revealed reduced association of SSIIa and BEIIb in the ss1 mutant, while the association of BEIIb with SSI, SSIIa, SSIVb, BEI, and BEIIa were more pronounced in the be2b mutant that produced inactive BEIIb enzyme. Mass spectrometry and western blotting revealed that SSI, SSIIa, SSIIIa, BEI, BEIIa, starch phosphorylase 1, and pullulanase were bound to the starch granules in the be2b mutants, but not in the wild-type and ss1 mutant. These results will aid the understanding of the mechanism of amylopectin biosynthesis.

Reframing gene essentiality in terms of adaptive flexibility

BackgroundEssentiality assays are important tools commonly utilized for the discovery of gene functions. Growth/no growth screens of single gene knockout strain collections are also often utilized to test the predictive power of genome-scale models. False positive predictions occur when computational analysis predicts a gene to be non-essential, however experimental screens deem the gene to be essential. One explanation for this inconsistency is that the model contains the wrong information, possibly an incorrectly annotated alternative pathway or isozyme reaction. Inconsistencies could also be attributed to experimental limitations, such as growth tests with arbitrary time cut-offs. The focus of this study was to resolve such inconsistencies to better understand isozyme activities and gene essentiality.ResultsIn this study, we explored the definition of conditional essentiality from a phenotypic and genomic perspective. Gene-deletion strains associated with false positive predictions of gene essentiality on defined minimal medium for Escherichia coli were targeted for extended growth tests followed by population sequencing and transcriptome analysis. Of the twenty false positive strains available and confirmed from the Keio single gene knock-out collection, 11 strains were shown to grow with longer incubation periods making these actual true positives. These strains grew reproducibly with a diverse range of growth phenotypes. The lag phase observed for these strains ranged from less than one day to more than 7 days. It was found that 9 out of 11 of the false positive strains that grew acquired mutations in at least one replicate experiment and the types of mutations ranged from SNPs and small indels associated with regulatory or metabolic elements to large regions of genome duplication. Comparison of the detected adaptive mutations, modeling predictions of alternate pathways and isozymes, and transcriptome analysis of KO strains suggested agreement for the observed growth phenotype for 6 out of the 9 cases where mutations were observed.ConclusionsLonger-term growth experiments followed by whole genome sequencing and transcriptome analysis can provide a better understanding of conditional gene essentiality and mechanisms of adaptation to such perturbations. Compensatory mutations are largely reproducible mechanisms and are in agreement with genome-scale modeling predictions to loss of function gene deletion events.

Growth and Physiological Responses of Adenophora triphylla (Thunb.) A.DC. Plug Seedlings to Day and Night Temperature Regimes

Adenophora triphylla (Thunb.) A.DC., three-leaf lady bell, is an important medicinal plant used against cancers and obesity. It has been well-established that the temperature regime affects plant growth and development in many ways. However, there is no study available correlating the growth of A. triphylla seedlings with different day and night temperature regimes. In order to find an optimal temperature regime, growth and physiology were investigated in A. triphylla plug seedlings grown in environment-controlled chambers at different day and night temperatures: 20/20 °C (day/night) (TA), 25/15 °C (TB), and 20/15 °C (TC). The seedlings in plug trays were grown under a light intensity of 150 μmol·m−2·s−1 PPFD (photosynthetic photon flux density) provided by white LEDs, a 70% relative humidity, and a 16 h (day)/8 h (night) photoperiod for six weeks. The results showed that the stem diameter, number of roots, and biomass were significantly larger for seedlings in TB than those in TA or TC. Moreover, the contents of total flavonoid, total phenol, and soluble sugar in seedlings grown in TB were markedly higher than those in seedlings in the other two treatments. Soluble protein content was the lowest in seedlings in TC, while starch content was the lowest in seedlings grown in TA. Furthermore, seedlings grown in TB showed significantly lower activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase, and guaiacol peroxidase. Native PAGE (polyacrylamide gel electrophoresis) analysis further proved low activities of antioxidant isozymes in TB treatment. Meanwhile, the lowest content of hydrogen peroxide was observed in seedlings grown in TB. In conclusion, the results suggested that the 25/15 °C (day/night) temperature regime is the most suitable for the growth and physiological development of A. triphylla seedlings.

Inhibitory potential of three Yin-tonification herbal formulas on the activities of human major cytochrome P450 and UDP- glucuronosyltransferases isozymes in vitro

OBJECTIVETo investigate the influence of Yin-tonification herbal formulas Jaeumganghwa-tang (Ziyin Jianghuo Tang, JEGHT), Ssanghwa-tang (Shuanghe Tang, SHT) and Yukmijihwang-tang (Liuwei Di huang Tang, YMJHT) on the activities of human major cytochrome P450 (CYP450s) and UDP-glucuronosyltransferases isozymes (UGTs) in vitro. METHODSThe activities of CYP450s (CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) and UGTs (UGT1A1, UGT1A4 and UGT2B7) were assessed using in vitro fluorescence- and luminescence-based enzyme assays, respectively. The effects of herbal formulas on the activities of CYP450s and UGTs were presented as IC50 values. RESULTSJEGHT showed the potent inhibition of the CYP2D6 activity, with weak inhibition on the activities of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2E1, CYP3A4, UGT1A1, UGT1A4 and UGT2B7. SHT inhibited the activities of CYP1A2 and CYP2E1, whereas the negligible inhibition of the activities of CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A4 and UGT2B7 through SHT was observed. YMJHT inhibited CYP2E1 activity, with a negligible inhibition on the activities of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A4 and UGT2B7. CONCLUSIONThese findings provide information about the potential interactions between three Yin-tonification herbal formulas and conventional drugs.

Effects of Baoyuan decoction, a traditional Chinese medicine formula, on the activities and mRNA expression of seven CYP isozymes in rats

Ethnopharmacological relevanceBaoyuan decoction (BYD), a traditional Chinese medicine (TCM) formula, is composed of four herbs and widely used with western drugs to treat coronary heart disease, aplastic anemia and chronic renal failure in clinic. However, no study of the effect of BYD on the cytochrome P450 (CYP) activities has been reported. Aim of the studyThe purpose of the present study was to evaluate the potential influences of BYD on the activities of seven CYP isozymes (CYP1A2, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) in rats. Materials and methodsA sensitive and selective UPLC-MS/MS method for simultaneous determination of seven probe drugs and internal standard (IS) in rat plasma was developed and validated. The influence of BYD on the activities of CYP isozymes and mRNA expression levels were carried out by comparing plasma pharmacokinetics and real-time reverse transcription-polymerase chain reaction (RT-PCR) of probe drugs between control and BYD treatment groups respectively. ResultsThe calibration curve were linear, with correlation coefficient (r) > 0.99 for seven probe drugs. The intra and inter-assay accuracy and precision of the method were within ± 14.9% and the recoveries ranged from 83.2% to 106.1%. Compared with control group, BYD at low (1.46 g/kg) and high (7.30 g/kg) dosages could significantly increase Cmax and AUC0-t of chlorzoxazone and testosterone, while decrease AUC0-t of phenacetin at high dosage and increase AUC0-t of tolbutamide and metoprolol. Additionally, BYD had increased AUC0-t of bupropion at low dosage and decreased it at high dosage. The mRNA expression results were in accordance with those of pharmacokinetic. ConclusionBYD exhibited inhibitory effects on CYP2C9, CYP2E1, and CYP3A4. Moreover, BYD had induction effects on CYP1A2, and CYP2D6 activities. However, no significant change in CYP2C19 activity was observed. It would be useful for the safe and effective usage of BYD in clinic.

Tescalcin/c-Src/IGF1R\u03b2-mediated STAT3 activation enhances cancer stemness and radioresistant properties through ALDH1

Tescalcin (TESC; also known as calcineurin B homologous protein 3, CHP3) has recently reported as a regulator of cancer progression. Here, we showed that the elevation of TESC in non-small cell lung cancer (NSCLC) intensifies epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties, consequently enhancing the cellular resistance to γ-radiation. TESC expression and the phosphorylation (consequent activation) of signal transducer and activator of transcription 3 (STAT3) were upregulated in CSC-like ALDH1high cells than in ALDH1low cells sorted from A549 NSCLC cells. Knockdown of TESC suppressed CSC-like properties as well as STAT3 activation through inhibition of insulin-like growth factor 1 receptor (IGF1R), a major signaling pathway of lung cancer stem cells. TESC activated IGF1R by the direct recruitment of proto-oncogene tyrosine kinase c-Src (c-Src) to IGF1Rβ complex. Treatment of IGF1R inhibitor, AG1024, also suppressed c-Src activation, implicating that TESC mediates the mutual activation of c-Src and IGF1R. STAT3 activation by TESC/c-Src/IGF1R signaling pathway subsequently upregulated ALDH1 expression, which enhanced EMT-associated CSC-like properties. Chromatin immunoprecipitation and luciferase assay demonstrated that STAT3 is a potential transcription activator of ALDH1 isozymes. Ultimately, targeting TESC can be a potential strategy to overcome therapeutic resistance in NSCLC caused by augmented EMT and self-renewal capacity.

Cloning and characterisation of four catA genes located on the chromosome and large plasmid of Pseudomonas putida ND6

Although the functional redundancy of catechol 1,2-dioxygenase (C12O) genes has been reported in several microorganisms, limited enzymes were characterised, let alone the advantage of the coexistence of the multiple copies of C12O genes. In this study, four novel C12O genes, designated catA, catAI, catAII and catAIII, in the naphthalene-degrading strain Pseudomonas putida ND6, were cloned and characterised. Phylogenetic analysis of their deduced amino acid sequences revealed that the four C12O isozymes each formed independent subtrees, together with homologues from other organisms. All four enzymes exhibited maximum activity at pH 7.4 and higher activity in alkaline than in acidic conditions. Furthermore, CatA, CatAI and CatAIII were maximally active at a temperature of 45°C, whereas a higher optimum temperature was observed for CatAII at a temperature of 50°C. CatAI exhibited superior temperature stability compared with the other three C12O isozymes, and kinetic analysis indicated similar enzyme activities for CatA, CatAI and CatAII, whereas that of CatAIII was lower. Significantly, among metal ions tested, only Cu2 + substantially inhibited the activity of these C12O isozymes, thus indicating that they have potential to facilitate bioremediation in environments polluted with aromatics in the presence of metals. Moreover, gene expression analysis at the mRNA level and determination of enzyme activity clearly indicated that the redundancy of the catA genes has increased the levels of C12O. The results clearly imply that the redundancy of catA genes increases the available amount of C12O in P. putida ND6, which would be beneficial for survival in challenging environments. How to cite: Li S, Qin K, Li H, et al. Cloning and characterisation of four catA genes located on the chromosome and large plasmid of Pseudomonas putida ND6. Electron J Biotechnol 2018;34.

Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase Activity in the Serum of Patients with Non-alcoholic Fatty Liver Disease.

Non-alcoholic liver disease (NAFLD) is one of the most common causes of chronic liver disease, and its prevalence and medical importance is increasing worldwide. Changes in enzyme activity in liver cells in various liver diseases are reflected by an increase in serum enzymatic activity. For example, alcohol dehydrogenase activity (ADH) and aldehyde dehydrogenase (ALDH), that occur in the liver in large quantities, correlate with disease severity during cirrhosis. In the current study, the activity of ADH isoenzymes and ALDH in the serum of patients with NAFLD was investigated. Serum samples were collected for routine biochemical studies from 55 patients with NAFLD patients and from 50 healthy individuals. Class I and II ADH and ALDH activity were measured by spectrofluorometric method. Photometric methods were used to measure ADH class III, IV and total ADH activity. Total ADH activity was significantly higher in non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) than in healthy individuals (44 and 48.5% activity, respectively). The median total activity of ADH was 1,164 mU/l in patients with NAFLD, 1,258 mU/l in NASH and 648 mU/l in the control group. The increase in ADH class I and II isoenzyme in serum of patients with NAFL and NASH was statistically significant. The activity of ADH I, ADH II, and total ADH significantly increased with increasing disease progression. The activity of isozymes of class I and II alcohol dehydrogenase in patients with NAFLD is enhanced and appears to be due to the release of these isoenzymes from damaged hepatocytes.

Identification of the allosteric site for neutral amino acids in the maize C4 isozyme of phosphoenolpyruvate carboxylase: The critical role of Ser-100

The isozymes of photosynthetic phosphoenolpyruvate carboxylase from C4 plants (PEPC-C4) play a critical role in their atmospheric CO2 assimilation and productivity. They are allosterically activated by phosphorylated trioses or hexoses, such as d-glucose 6-phosphate, and inhibited by l-malate or l-aspartate. Additionally, PEPC-C4 isozymes from grasses are activated by glycine, serine, or alanine, but the allosteric site for these compounds remains unknown. Here, we report a new crystal structure of the isozyme from Zea mays (ZmPEPC-C4) with glycine bound at the monomer-monomer interfaces of the two dimers of the tetramer, making interactions with residues of both monomers. This binding site is close to, but different from, the one proposed to bind glucose 6-phosphate. Docking experiments indicated that d/l-serine or d/l-alanine could also bind to this site, which does not exist in the PEPC-C4 isozyme from the eudicot plant Flaveria, mainly because of a lysyl residue at the equivalent position of Ser-100 in ZmPEPC-C4 Accordingly, the ZmPEPC-C4 S100K mutant is not activated by glycine, serine, or alanine. Amino acid sequence alignments showed that PEPC-C4 isozymes from the monocot family Poaceae have either serine or glycine at this position, whereas those from Cyperaceae and eudicot families have lysine. The size and charge of the residue equivalent to Ser-100 are not only crucial for the activation of PEPC-C4 isozymes by neutral amino acids but also affect their affinity for the substrate phosphoenolpyruvate and their allosteric regulation by glucose 6-phosphate and malate, accounting for the reported kinetic differences between PEPC-C4 isozymes from monocot and eudicot plants.

Inhibitory properties of some heavy metals on carbonic anhydrase I and II isozymes activities purified from Van Lake fish (Chalcalburnus Tarichi) gill.

In this study, CA I and II isoenzymes were purified from Van Lake fish gills by using Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography and to determine the effects of some metals on the enzyme activities. For purified CA I isoenzyme, yield, specific activity, and purification fold were obtained as 42.07%, 4948.12 EU/mg protein, and 116.61 and for CA II isoenzyme, 7%, 1798.56 EU/mg protein, and 42.38 respectively. Activity of CA was determined by measuring "CO2-hydratase activity". Purity control was checked by SDS-PAGE. In vitro inhibitory effect of Cu2+, Ag+, Cd2+, Ni2+ metal ions, and arsenic (V) oxide were also examined for both isozymes activities. Whereas Cu2+, Ag+, Cd2+, and Ni2+ ions showed inhibitory effects on both isozymes, arsenic (V) oxide showed activation effect. IC50 values were calculated by drawing activity %-[I] graphs for metal ions exhibiting inhibitory effects. IC50 values were determined as 3.39, 6.38, 13.52, and 206μM for CA I isozyme and 6.16, 20.29, 46, and 223μM for CA II isozyme respectively.

Modulation of Arachidonic Acid Metabolism in the Rat Kidney by Quercetin: Implications for Regulation of Blood Pressure

BackgroundWe investigated the effects of quercetin (QR), a plant polyphenol from the flavonoid group, on Arachidonic acid (AA) metabolism in the kidney and its effect on arterial blood pressure, using spontaneously hypertensive rats (SHR) as our animal model system.MethodsRats were treated for 7 weeks with QR (10, 30, or 60 mg/L) added to their drinking water; drinking water alone was used as our control. Mean arterial pressure (MAP) was measured at 7‐day intervals throughout the study. At the end of the treatment, rats were euthanized, followed by preparation of kidney microsomes and cytosols to measure the activity (using LC/MS/MS) and expression (using immunoblotting) of enzymes involved in regulation of vasoactive metabolites: CYP4A, the key enzyme in the formation of 20‐hydroxyeicosatetraenoic acid, and the soluble epoxide hydrolase (sEH), which is responsible for the degradation of the vasodilator metabolites such as epoxyeicosatetraenoic acids.ResultsWe found that treatment with QR lead to significant reductions in both the expression and activity of renal CYP4A isozyme. Only the high dose QR resulted in a significant reduction in the expression, but not the activity, of renal sEH. Consistent with these data, we have found that treatment with QR resisted the progressive rise in MAP in the developing SHR in a dose‐dependent manner.ConclusionThis is the first demonstration that QR modulates the metabolism of AA by both P450 enzymes and sEH in SHR rats. This may represent a novel mechanism by which QR protects SHR rats against the progressive rise in blood pressure.Support or Funding InformationPacific University Research Incentive GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulation of Arachidonic Acid Metabolism in the Rat Kidney by Thymoquinone: Implications for Regulation of Blood Pressure

BackgroundWe investigated the effects of thymoquinone (TQ), a Quinone phytochemical in the plant Nigella Sativa, on Arachidonic acid (AA) metabolism in the kidney and its effect on arterial blood pressure, using spontaneously hypertensive rats (SHR) as our animal model system.MethodsRats were treated for 7 weeks with TQ (10, 20, or 40 mg/kg) added to their drinking water; drinking water alone was used as our control. Mean arterial pressure (MAP) was measured at 7‐day intervals throughout the study. At the end of the treatment, rats were euthanized, followed by preparation of kidney microsomes to measure the activity (using LC/UV and fluorescence assay) and expression (using immunoblotting) of enzymes involved in regulation of vasoactive metabolites: CYP4A, the key enzyme in the formation of 20‐hydroxyeicosatetraenoic acid, and the soluble epoxide hydrolase (sEH), which is responsible for the degradation of the vasodilator metabolites such as epoxyeicosatetraenoic acids.ResultsWe found that treatment with TQ leads to significant reductions in both the expression and activity of renal CYP4A isozyme. Only the high dose TQ resulted in a significant reduction in the expression and activity of renal sEH. Consistent with these data, we have found that treatment with TQ resisted the progressive rise in MAP in the developing SHR in a dosedependent manner.ConclusionThis is the first demonstration that TQ modulates the metabolism of AA by both kidney P450 enzymes and sEH in SHR rats. This may represent a novel mechanism by which TQ protects SHR rats against the progressive rise in blood pressure.Support or Funding InformationPacific University Research Incentive GrantMedical Research Foundation of OregonThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Foliar or subirrigational silicon supply modulates salt stress in strawberry during vegetative propagation

Availability of silicon (Si) to the plant as a silicic acid could be a limiting factor for crop productivity. Although the effect of Si has been shown to vary by plant species, Si has been shown to improve photosynthesis, and to remediate nutrient imbalances and abiotic stresses in plants. Plants of two Korean strawberry cultivars ‘Seolhyang’ and ‘Maehyang’ at a propagation stage was grown for 58 days with a complete nutrient solution (EC of 0.8 dS m−1) and also with supplementation of either 0, 35, or 70 mg L−1 Si from either potassium silicate (K2SiO3), sodium silicate (Na2SiO3), or calcium silicate (CaSiO3). The Si in solution was supplied to the roots through subirrigational nutrient solution or to the leaves by daily foliar applications. The growth and development of the plants were assessed after this period. Then the plants were subjected to one of different salt stress conditions (EC of either 0.0, 0.8, 1.6, or 2.4 dS m−1) for 14 days. The high EC (2.4 dS m−1) resulted in oxidative stress in the form of H2O2 and O 2 −1 in the leaves of the Si-non-treated plants as compared to Si-treated plants, and no or less stress was observed in the 0.0, and 0.8 or 1.6 dS m−1, respectively. However, Si, especially supplied from K2SiO3, relieved the stress level. Interestingly, the isozyme activities of three important antioxidant enzymes, superoxide dismutase, catalase and guaiacol peroxidase, were abundantly increased in the Si-treated plants, particularly with K2SiO3, even under the high EC (2.4 dS m−1) treatment and decreased in the Si-non-treated plants. The observed responses to Si supply in high salt-stressed plants indicate that Si, particularly supplied from K2SiO3, has a significant role in limiting the negative effects of salt stress by maintaining antioxidative enzymes during the vegetative propagation.

Abstract B048: Activation of PKC inhibits oncogenic KRAS-driven malignancies

Abstract The extremely high mutation rate of KRAS in three of the top four deadly cancers (lung, colon, and pancreas) makes it a major target in anticancer drug discovery. However, no effective anti-KRAS inhibitors have yet been developed into clinically useful drugs. Therefore, identifying “druggable” signaling components crucial for KRAS can provide a new approach for targeting KRAS-driven malignancies. We have reported that oncogenic KRAS uniquely elicits a tumorigenic phenotype through downregulation of noncanonical Fzd8/Wnt/Ca2+ signaling. Binding of calmodulin (CaM) to KRAS, but not to H- or N-RAS, appears to be responsible for this major difference. The total intracellular concentration of CaM has been reported to be significantly below the total concentration of its targets, making CaM a limiting factor in their regulation. Therefore, the unique binding of KRAS to CaM, which competes for a limiting pool of CaM, can be sufficient to affect CaM-dependent signaling pathways. Serine-181 in the hypervariable region of KRAS, as the essential domain for KRAS to bind to CaM, has been identified as the phosphorylation site of PKC. The KRAS-V12-S181D (pseudo-phosphorylated) variant lost the ability to suppress CaMKii activity and Fzd8 expression, suggesting that the interaction between KRAS and CaM is an important pathway for KRAS to inhibit Fzd8-mediated noncanonical Wnt/Ca2+ signaling. Thus, we hypothesize to target KRAS selectively by blocking this specific interaction between KRAS and CaM through the activation of PKC. The activation of PKC by a non-tumor promoting PKC activator, prostratin, successfully interrupts the KRAS-CaM interaction, rewires the sequential downstream signaling pathways, and, most importantly, represses the tumor initiation and growth in KRAS-driven cancers. The pancreatic cancer cells treated with Gο6984 (targeting conventional and novel PKCs), but not Gο6976 (targeting conventional PKCs), lost the response to prostratin treatment on activation of CaMKii. Next, each individual of PKC isozyme in conventional and novel subfamilies has been knocked down by siRNA, followed by the treatment of prostratin. The cells, where PKCδ has been knocked down, failed to activate CaMKii in the response to prostratin, when compared to the cells with the depletions of other PKC isoforms. The pancreatic cancer cells where PKCδ, but not PKCα, has been knocked down by shRNA lost their sensitivity to prostratin on cell viability and growth in vitro. The treatment of prostratin increased the phosphorylated PKCδ at serine-299 in KRAS transformed cells, suggesting prostratin acts as a PKCδ activator. The cellular thermal shift assay suggested that, when compared to PMA, prostratin preferentially stabilized PKCδ in a dose-dependent manner. To further define the specificity of prostratin-induced PKC activation on phosphorylation of KRAS, we treated the LSL-KRAS-G12D mice carrying the additional mutation at serine-181 (S181S to A, resistant to phosphorylation) with prostratin post-adenoCre infection. The lung tumors from LSL-KRAS-G12D-S181S mice, but not -S181A ones, showed increased phosphorylated CaMKii in the response to prostratin treatment. The results suggest that activation of certain specific PKC isozymes may provide a novel approach to rewire KRAS-mediated signaling pathway and to sequentially suppress its malignant features in human cancers. Citation Format: Man-Tzu Wang, Jacqueline Galeas, Frank McCormick. Activation of PKC inhibits oncogenic KRAS-driven malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B048.

A novel quantification method for serine hydrolases in cellular expression system using fluorophosphonate-biotin probe

In the present study, we established a quantitative western blotting method to measure the expression level of recombinant serine hydrolases based on their catalytic mechanism. Fluorophosphonate (FP)-biotin was selected as a universal probe to quantify their expression levels, since FP moiety irreversibly inhibits serine hydrolases through strong stoichiometric binding to active serine residue. The linearity of detection using FP-biotin was assessed on three serine hydrolases; human carboxylesterase (CES) 1, butyrylcholinesterase and porcine liver esterases (PLE). Similar response signals were obtained from the equimolar concentrations of these enzymes and excellent linearity was observed at the range of 0.4–3.4pmol/lane (r2>0.99). Accuracy and precision of the proposed method were proved using PLE with recovery of 97.1–107.2% and relative standard deviation of 5.56%. PLE was selected as a calibration standard because of its high stability and commercial availability. As an application of the developed method, we measured the expression levels of four recombinant CES isozymes from human and cynomolgus macaque in S9 fraction of HEK293 cell homogenates. The expression levels of human CES1 and CES2, and cynomolgus macaque CES1 and CES2 were 2.51±0.1, 1.63±0.17, 0.79±0.09 and 1.37±0.13pmol/5μg S9 protein, respectively. Based on these determinations, their hydrolytic activities were accurately assessed. Cynomolgus CESs showed lower hydrolysis activities for p-nitrophenyl esters than human CESs. The hydrolase activities of CES2 isozymes were higher than CES1 in both species. Three to five folds faster hydrolysis for p-nitrophenyl butyrate than p-nitrophenyl acetate was observed in all CES isozymes except of cynomolgus CES1 that showed nearly same hydrolysis for both substrates. The provided method could be widely used for universal quantitative analysis of recombinant serine hydrolases.