Impact of metal nanoparticles on the structure and function of metabolic enzymes.

The widespread use of nanoparticles raises many serious concerns about the safety and environmental impact of nanoparticles. Therefore, risk assessments of specific nanoparticles in occupational and environmental exposure are essential before their large-scale production and applications, especially in medicine and for usage in household items. In this study, the effects of five different metal nanoparticles on the structure, stability, and function of four metabolic enzymes were evaluated using various biophysical techniques. Our results show that Cu nanoparticles exhibited the most significant adverse effects on the structures, stability, and activities of all the metabolic enzymes. Zn nanoparticles caused moderate adverse effects on these enzymes. The rest of the metal (Al, Fe, and Ni) nanoparticles had a relatively lower impact on the metabolic enzymes. Our data indicated that Cu nanoparticles promote metal-catalyzed disulfide bond formation in these proteins. In summary, some metal nanoparticles can cause adverse effects on the structure, function, and stability of metabolic enzymes. In addition, metal nanoparticles may affect protein homeostasis in the cytosol or extracellular fluids.

Molecular mechanisms of anti-hyperglycemic effects of Costus speciosus extract in streptozotocin-induced diabetic rats.

OBJECTIVES: To investigate the mechanisms of the anti-hyperglycemic effect of Costus speciosus (C. speciosus) root ethanolic extracts (CSREt) by assessing its action on insulin synthesis and glucose catabolic enzyme gene expression and activities in streptozotocin (STZ) diabetic rats. METHODS: This study was carried out at the Biochemical Laboratory, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt between July and August 2013. Sixty male albino rats (120 +/- 20 g weight, and 6 months old) were used and divided into 6 groups (n=10). Two groups served as diabetic and nondiabetic controls. Four groups of STZ diabetic animals were given oral C. speciosus (CSREt) in doses of 200, 400, and 600 mg/kg body weight, and 600 µg/kg body weight of the standard drug glibenclamide for 4 weeks. RESULTS: The CSREt 400 and 600 mg/kg body weight induced a decrease in blood glucose and an increase in serum insulin level, glucokinase (GK), aldolase, pyruvate kinase (PK), succinate dehydrogenase (SDH), and glycogen synthase activities  in addition to a higher expression level of insulin, insulin receptor A (IRA), GK, PK, SDH, and glucose transporting protein. CONCLUSION: The C. speciosus has anti-hyperglycemic activity. It induces insulin secretion and release from cells, as well as stimulates the tissue's insulin sensitivity leading to an increase of the tissues' glucose uptake, storage, and oxidation.

Changes in endoplasmic reticulum stress proteins and aldolase A in cells exposed to dopamine.

In Parkinson's disease, oxidative stress is implicated in protein misfolding and aggregation, which may activate the unfolded protein response by the endoplasmic reticulum (ER). Dopamine (DA) can initiate oxidative stress via H(2)O(2) formation by DA metabolism and by oxidation into DA quinone. We have previously shown that DA quinone induces oxidative protein modification, mitochondrial dysfunction in vitro, and dopaminergic cell toxicity in vivo and in vitro. In this study, we used cysteine- and lysine-reactive fluorescent dyes with 2D difference in-gel electrophoresis, mass spectrometry, and peptide mass fingerprint analysis to identify proteins in PC12 cell mitochondrial-enriched fractions that were altered in abundance following DA exposure (150 muM, 16 h). Quantitative changes in proteins labeled with fluorescent dyes indicated increases in a subset of proteins after DA exposure: calreticulin, ERp29, ERp99, Grp58, Grp78, Grp94 and Orp150 (149-260%), and decreased levels of aldolase A (39-42%). Changes in levels of several proteins detected by 2D difference in-gel electrophoresis were confirmed by western blot. Using this unbiased proteomics approach, our findings demonstrated that in PC12 cells, DA exposure leads to a cellular response indicative of ER stress prior to the onset of cell death, providing a potential link between DA and the unfolded protein response in the pathogenesis of Parkinson's disease.

Genomic and proteomic analysis of the effects of cannabinoids on normal human astrocytes.

Delta-9-tetrahydrocannabinol (Delta(9)-THC), the main psychoactive component of marijuana, is known to dysregulate various immune responses. Cannabinoid (CB)-1 and -2 receptors are expressed mainly on cells of the central nervous system (CNS) and the immune system. The CNS is the primary target of cannabinoids and astrocytes are known to play a role in various immune responses. Thus we undertook this investigation to determine the global molecular effects of cannabinoids on normal human astrocytes (NHA) using genomic and proteomic analyses. NHA were treated with Delta(9)-THC and assayed using gene microarrays and two-dimensional (2D) difference gel electrophoresis (DIGE) coupled with mass spectrometry (MS) to elucidate their genomic and proteomic profiles respectively. Our results show that the expression of more than 20 translated protein gene products from NHA was differentially dysregulated by treatment with Delta(9)-THC compared to untreated, control NHA.

Icariside II from Epimedium koreanum inhibits hypoxia-inducible factor-1alpha in human osteosarcoma cells.

Hypoxia-inducible factor-1 (HIF-1) is an important tumor-selective therapeutic target for solid tumors. Icariside II was isolated from Epimedium koreanum through successive fractionation with ethyl acetate, n-butanol, chloroform and hexane, followed by gel column chromatography. Icariside II attenuated the protein level of HIF-1alpha induced by hypoxia in human osteosarcoma (HOS) cells in a concentration-dependent manner, probably by enhancing the interaction rate between von Hippel-Lindau (VHL) and HIF-1alpha. Furthermore, Icariside II down-regulated the levels of HIF-inducible genes involved in angiogenesis, metastasis, and glucose metabolism, such as vascular endothelial growth factor (VEGF), urokinase plasminogen activator receptor (uPAR), adrenomedullin (ADM), matrix metalloproteinase 2 (MMP2), aldolase A, and enolase 1 in HOS cells. Icariside II also inhibited the migration rate in HOS cells and tube formation rate in human umbilical vein endothelium cells (HUVECs). Overall, these results suggest the potential use of Icariside II as a therapeutic candidate against various diseases that involve overexpression of HIF-1alpha.

Effects of depleting the essential central metabolic enzyme fructose-1,6-bisphosphate aldolase on the growth and viability of Candida albicans: implications for antifungal drug target discovery.

The central metabolic enzyme fructose-1,6-bisphosphate aldolase (Fba1p) catalyzes a reversible reaction required for both glycolysis and gluconeogenesis. Fba1p is a potential antifungal target because it is essential in yeast and because fungal and human aldolases differ significantly. To test the validity of Fba1p as an antifungal target, we have examined the effects of depleting this enzyme in the major fungal pathogen Candida albicans. Using a methionine/cysteine-conditional mutant (MET3-FBA1/fba1), we have shown that Fba1p is required for the growth of C. albicans. However, Fba1p must be depleted to below 5% of wild-type levels before growth is blocked. Furthermore, Fba1p depletion exerts static rather than cidal effects upon C. albicans. Fba1p is a relatively abundant and stable protein in C. albicans, and hence, Fba1p levels decay relatively slowly following MET3-FBA1 shutoff. Taken together, our observations can account for our observation that the virulence of MET3-FBA1/fba1 cells is only partially attenuated in the mouse model of systemic candidiasis. We conclude that an antifungal drug directed against Fba1p would have to be potent to be effective.

Characterization of a Mn-dependent fructose-1,6-bisphosphate aldolase in Deinococcus radiodurans.

The key enzyme of the glycolytic pathway of Deinococcus radiodurans, fructose-1,6-bisphosphate aldolase, could be induced independently by glucose and Mn. The enzyme exhibited the characteristics of the metal-dependent Class II aldolases. Unlike most Class II aldolases, the deinococcal aldolase preferred Mn, not Zn, as a cofactor. The fbaA gene encoding the deinococcal aldolase was cloned and the protein overproduced in various Escherichia coli expression hosts. However, the overexpressed deinococcal enzyme aggregated and formed inclusion bodies. Dissolving these inclusion bodies by urea and subsequent purification by nickel affinity chromatography, resulted in a protein fraction that exhibited aldolase activity only in the presence of Mn. This active aldolase fraction exhibited masses of about 70 kDa and 35 kDa by gel filtration and by SDS gel electrophoresis, respectively, suggesting that the active aldolase was a dimer.

Clotrimazole decreases human breast cancer cells viability through alterations in cytoskeleton-associated glycolytic enzymes.

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Clotrimazole is an anti-fungal azole derivative recently recognized as a calmodulin antagonist with promising anti-cancer effect. Here, we show that clotrimazole induced morphological and functional alterations on human breast cancer derived cell line, MCF-7. The drug decreased cell viability in a dose- and time-dependent manner, exhibiting an IC50 of 88.6+/-5.3 microM and a t0.5 of 89.7+/-7.2 min, with 50 microM clotrimazole. Morphological changes were evident as observed by scanning electron microscopy, which revealed the completely loss of protrusion responsible for cell adhesion after a 180 min of treatment with 50 microM clotrimazole. Giemsa stained cells observed by optical microscopy show morphological alterations and a marked nuclear condensation. These changes occurred in parallel to the detachment of the glycolytic enzymes, 6-phosphofructo-1-kinase and aldolase, from cytoskeleton. After a 45 min treatment with 50 microM clotrimazole, the remaining activities in a cytoskeleton enriched fraction was 16.4+/-3.6% and 41.0+/-15.6% of control for 6-phosphofructo-1-kinase and aldolase, respectively. Immunocytochemistry experiments revealed a decrease in the co-localization of 6-phosphofructo-1-kinase and F-actin after clotrimazole treatment, suggesting the site of detachment of the enzymes. Altogether, our results support evidence for apoptotic events that might be started by clotrimazole involving inhibition of glycolytic flux in MCF-7 cells and makes this drug a promising agent in the fight against human breast cancer.

Flow cytometric screening of aldolase catalytic antibodies.

High-throughput screening of cells expressing active catalytic antibody clones by flow cytometry is described. A fluorogenic retro-aldol retro-Michael substrate was designed and synthesized with incorporation of a chloromethyl moiety for intracellular retention. Hybridoma or transfected mammalian cells expressing catalytic antibody molecules could be rapidly isolated.

Fluorescence analysis of aldolase dissociation from the N-terminal of the cytoplasmic domain of band 3 induced by lanthanide.

The cytoplasmic domain of band 3 (CDB3) offers binding sites for several glycolytic enzymes and regulates the glycolysis of erythrocyte. The interaction between recombinant (His)(6)-tagged CDB3 and aldolase, one of the key enzymes that participated in erythrocyte glycolysis, was investigated in the presence of lanthanide. The results indicate that trace lanthanide blocks the inhibition of CDB3-(His)(6) to aldolase and leads to enhancement of aldolase activity. In agreement with activity studies, fluorescence spectra reveal that 4 microM lanthanum ions induce the complete dissociation of aldolase from the N-terminal of CDB3-(His)(6). Interestingly, the synchronous scanning fluorescence spectra of proteins in the presence of various concentrations of lanthanum ions suggest that the conformational change of CDB3-(His)(6) is significantly attributed to the alteration of tryptophan cluster microenvironment, while the aldolase conformation change is mainly derived from tyrosine microenvironment changes. Based on the observation that lanthanide ions induce the dissociation of aldolase from CDB3-(His)(6), it is suggested that the existence of trace lanthanide may affect the glycolysis of erythrocyte.

Recuperative effect of Semecarpus anacardium linn. nut milk extract on carbohydrate metabolizing enzymes in experimental mammary carcinoma-bearing rats.

Semecarpus anacardium Linn. of the family Anacardiaceae has many applications in the Ayurvedic and Siddha systems of medicine. We have tested the antitumour activity of Semecarpus anacardium nut extract against experimental mammary carcinoma in animals. As there is a direct relationship between the proliferation of tumour cells and the activities of the glycolytic and gluconeogenic enzymes, we studied changes in the activities of enzymes involved in this metabolic pathway in the liver and kidney. The enzymes investigated were glycolytic enzymes, namely hexokinase, phosphoglucoisomerase, aldolase and the gluconeogenic enzymes, namely glucose-6-phosphatase and fructose-1,6-biphosphatase in experimental rats. A significant rise in glycolytic enzyme activities and a simultaneous fall in gluconeogenic enzyme activities were found in mammary carcinoma bearing rats. Drug administration returned these enzyme activities to their respective control activities.

Lithium regulation of aldolase A expression in the rat frontal cortex: identification by differential display.

BACKGROUND: Substantial evidence indicates that lithium may exert its therapeutic effects through progressive adaptive changes at the level of gene expression; however, the study of lithium-regulated genes has been primarily undertaken with the "candidate gene" approach based on a specific testable hypothesis. The aim of our study was to identify lithium-regulated genes that would not be predicted a priori by the candidate gene approach. METHODS: Differential display polymerase chain reaction was used to isolate and identify messenger RNAs (mRNAs) that are differentially expressed in the frontal cortex of rats given lithium for 5 weeks to achieve plasma lithium concentrations of 0.6 to 0.9 mmol/L. RESULTS: A putative lithium-regulated complementary DNA fragment (LRG1) was identified. Northern blot analysis revealed that 5 weeks of lithium treatment, but not 1 week, significantly reduced LRG1 mRNA levels. LRG1 mRNA levels were similarly reduced by 5 weeks of carbamazepine, but not valproate administration. Sequence analysis and search of the GenBank database revealed that LRG1 is analogous to the sequence of the gene for rat aldolase A. CONCLUSIONS: These results demonstrate that chronic administration of lithium, but not short-term administration, down regulates the levels of aldolase A mRNA, suggesting this effect may play a role in mediating the therapeutic action of this agent.

Hydrogen exchange demonstrates three domains in aldolase unfold sequentially.

Rabbit muscle aldolase is a homotetramer in which the subunits have a classical alpha/beta-barrel structure and Mr 39,212 Da. We have previously reported that aldolase incubated in 3 M urea has three unfolding domains distinguished by their different unfolding rates. The unfolding rates of these domains were determined from isotope patterns in the mass spectra of peptic fragments derived from aldolase incubated in 3 M urea and pulse labeled in (2)H2O. The present study extends this investigation to more thoroughly characterize the structures of these unfolding intermediates. Mass spectra of intact monomers had four envelopes of isotope peaks corresponding to four structural forms of aldolase. Analysis of the present results suggests that these structural forms consist of native aldolase and three forms that have one to three domains unfolded. The molecular masses of these four structural forms indicate that there are 107 residues in each of the three unfolding domains of aldolase. Present results also show that aldolase remains a tetramer in 4 M urea, even though hydrogen exchange and circular dichroism indicate that it has lost most of its secondary and tertiary structure. The abundances of unfolded domains, which were determined from mass spectra of either intact aldolase or its peptic fragments, were used to determine the abundances of specific, partially unfolded forms of aldolase. Kinetic modeling of the abundances of these structures suggests that these structures are formed sequentially as aldolase unfolds in urea.

The vulnerability of nigral neurons to Parkinson's disease is unrelated to their intrinsic capacity for dopamine synthesis: an in situ hybridization study.

The contribution of the dopamine-synthetic capacity of nigral neuronal subregions to their vulnerability to degeneration in idiopathic Parkinson's disease (IPD) was explored using semiquantitative in situ hybridization to study expression of mRNA encoding the rate-limiting dopamine synthetic enzyme, tyrosine hydroxylase (TH). Expression of mRNA, the structural protein, beta-tubulin, and the glycolytic enzyme, fructose-1,6, biphosphate aldolase (aldolase C) was studied in parallel in individual neurons of the substantia nigra pars compacta (SNc) in matched groups of IPD and control subjects. TH mRNA expression was found to be heterogeneously expressed in nigral neurons in control and IPD subjects. There was no significant difference in mean values for TH mRNA expression between control and IPD cases and none between nigral subregions, either in control subjects or in established IPD subjects in this study, but there was evidence for a selective upregulation of TH mRNA expression in non-melanized neurons in IPD. There was no relationship between TH mRNA expression disease duration or L-dopa dosage in the IPD group. Mean TH mRNA values for two additional 40-year-old control subjects fell within the range of values of the aged-control group. Aldolase C and beta-tubulin expression did not differ between control and IPD groups or between nigral subregions. These findings suggest that regulation of dopamine synthesis at the level of the cell body does not play a part in determining the pattern of nigral cell vulnerability in IPD. The heterogeneous pattern of TH synthesis was not age-dependent and may be of physiological significance in nigral function. There was no evidence for compensatory upregulation of TH synthesis in surviving melanized neurons in IPD but non-melanized neurons may be involved in this process. Surviving nigral neurons in IPD appear to retain the capacity for normal aldolase C and beta-tubulin peptide synthesis. Long-term L-dopa treatment does not appear to compromise normal function of nigral dopaminergic neurons.

Catalytic and physico-chemical characteristics of goat spleen cathepsin B.

To improve the level of purity of cathepsin B, we have modified the published procedure [Agarwal, S.K. and Khan, M.Y. (1987) Biochem. Int. 15,785-792] by incorporating CM-Sephadex ion exchange chromatography and chromatofocusing. The enzyme thus isolated could be resolved into one 26 kDa major and a minor 27 kDa protein bands on SDS-PAGE. The two components, however, could not be separated by gel filtration and they eluted, in a single peak corresponding to a molecular mass of 28.1 kDa. Among the various substrates tested, Z-Phe-Arg-MCA with a Km of 0.058 mM and hemoglobin with a Km of 1.449 microM were the most preferred synthetic and protein substrates respectively. It was found to be a glycoprotein with an acidic pI of 4.8. The enzyme was activated by various thiol-reducing reagents and inhibited by cysteine proteinase inhibitors, divalent cations, lysyl group modifiers, anti-inflammatory drug and denaturing agents. The hydrodynamic behaviour of cathepsin B suggested a compact and globular conformation. Immunodiffusion studies with anti-goat cathepsin B indicated a tissue/ species dependence.

Redox-regulated expression of glycolytic enzymes in resting and proliferating rat thymocytes.

Resting rat thymocytes partially degrade glucose aerobically to CO2 and H2O and produce reactive peroxide anions. In contrast proliferating cells, due to enhanced induction of glycolytic enzymes, degrade glucose almost completely to lactate thus minimizing the production of reactive oxygen species. In this paper we show that under conditions of oxidative stress the induction of the glycolytic enzymes in cultured rat thymocytes is markedly reduced. Furthermore, transfection assays with a rat hepatoma cell line and Drosophila Schneider cells revealed that reactive oxygen intermediates dramatically decrease the transcriptional activities of the Sp1-dependent aldolase A and pyruvate kinase M2 promoters leading to reduced reporter gene expression. These results indicate that cellular redox changes can regulate gene expression by reversible oxidative inactivation of Sp1 binding.

Significant suppression of rat liver aldolase B by a toxic coplanar polychlorinated biphenyl, 3,3',4,4',5-pentachlorobiphenyl.

A toxic coplanar polychlorinated biphenyl, 3,3',4,4',5-pentachlorobiphenyl (PenCB), significantly suppresses the expression of liver aldolase B in rats. Hepatic aldolase activity in PenCB-treated rats was significantly reduced to about 50% of that in free- and pair-fed control groups. The reduced aldolase activity following PenCB-treatment was due to the marked suppression of the expression of aldolase B shown by immunoblot analysis after SDS-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. The suppression of rat liver aldolase B could be a key biochemical lesion caused by PenCB.

Studies on the effect of fenbendazole and mebendazole on some enzymes of swine kidney worm Stephanurus dentatus.

The effect of fenbendazole and mebendazole on the activity of some enzymes of the homogenates of swine kidney worm Stephanurus dentatus was investigated. Fenbendazole at 10(-5) M inhibited malate oxidation by 49% and 51% and oxaloacetate reduction by 33% and 40% whereas, mebendazole at 10(-5) M diminished malate oxidation by 25% and 35% and oxaloacetate reduction by 12% and 14% in male and female S. dentatus, respectively. Lactate dehydrogenase activity was inhibited by 45% and 50% in male and female worm respectively by 10(-5) M fenbendazole. Aldolase activity in both male and female S. dentatus was inhibited by 10(-5) M fenbendazole and mebendazole. Fenbendazole at 10(-5) M caused moderate inhibition of acid and alkaline phosphomonoesterases but mebendazole did not show a significant effect on these enzymes. Cholinesterase activity was not affected significantly with either compound. The possible mode of action of the two compounds is compared.

Drosophila melanogaster aldolase: characterization of the isozymes alpha, beta, and gamma generated from a single gene.

Three isozymic forms, alpha, beta, and gamma, of Drosophila melanogaster aldolase are produced from a single gene by alternative usage of the triple exons 4 (4 alpha, 4 beta, and 4 gamma) [Shaw-Lee et al. (1992) J. Biol. Chem. 267, 3959-3967; Kim et al. (1992) Mol. Cell. Biol. 12, 773-783; Kai et al. (1992) J. Biochem. 112,677-688]. The expression plasmids for the respective isozymes were transfected into Escherichia coli cells, and the isozymes alpha and beta were purified to homogeneity by a simple procedure, though isozyme gamma was only partially purified. These isozymes are active towards two substrates, fructose-1,6-bisphosphate (Fru-1,6-P2) and fructose-1-phosphate (Fru-1-P), with a preference for Fru-1,6-P2 over Fru-1-P, but they have different kcat/Km values towards these two substrates; isozyme alpha shows the highest value for Fru-1,6-P2. These isozymes show similarity in optimal pHs, thermal stability, and Km values for both Fru-1,6-P2 and Fru-1-P. They are composed of four identical subunits of 40 kDa, forming a tetramer with a molecular weight of approximately 160 kDa. The three isozymes are different in primary structure only at the carboxyl-terminal region encoded by the respective exon 4. Therefore, this region should be primarily responsible for the distinct characteristics of these isozymes.

Effect of Plumbagin on some glucose metabolising enzymes studied in rats in experimental hepatoma.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) isolated from Plumbago zeylanica Linn, when administered orally, at a dosage of 4 mg/kg body weight induces tumour regression in 3-methyl-4-dimethyl aminoazobenzene (3MeDAB) induced hepatoma in Wistar male rats. The purpose of this investigation was to identify the changes in the rate of glycolysis and gluconeogenesis in tumour-bearing rats and the effects of treatment with Plumbagin. The levels of certain glycolytic enzymes, namely, hexokinase; phosphoglucoisomerase; and aldolase levels increased (p < 0.001) in hepatoma bearing rats, whereas they decreased in Plumbagin administered rats to near normal levels. Certain gluconeogenic enzymes, namely, glucose-6-phosphatase and fructose-1,6-diphosphatase decreased (p < 0.001) in tumour hosts, whereas Plumbagin administration increased the gluconeogenic enzyme levels in the treated animals. These investigations indicate the molecular basis of the different biological behaviour of 3MeDAB induced hepatoma and the anticarcinogenic property of Plumbagin against hepatoma studied in rats.