Bidyadhar Das

Anthelmintic efficacy of Flemingia vestita (Fabaceae): Effect of genistein on glycogen metabolism in the cestode, Raillietina echinobothrida.

The edible root-tuber peel of Flemingia vestita and its major active component, genistein, have been earlier shown to have a vermifugal/vermicidal effect on cestodes in vitro by causing a flaccid paralysis and alterations in the tegumental architecture and activity of several enzymes associated with the tegumental interface of the parasite. Pursuing further investigation on the mode of action of this putative anthelmintic, the crude peel extract and pure genistein were further tested in respect of glycogen metabolism in the fowl tapeworm, Raillietina echinobothrida. On exposure to the plant root peel crude extract (5 mg/ml) and genistein (0.2 mg/ml), the glycogen concentration was found to decrease by 15-44%, accompanied by an increase of activity of the active form of glycogen phosphorylase (GPase a) by 29-39% and decrease of activity of the active form of glycogen synthase (GSase a) by 36-59% in treated parasites as compared to untreated controls, but without affecting the total activity (a+b) of both the enzymes. Praziquantel (1 microg/ml), the reference drug, also caused quantitative reduction in glycogen level and alterations in enzyme activities somewhat at par with the genistein treatment. These results suggest that this plant-derived component may influence the glycogen metabolism of the parasite by directing it towards utilization of glycogen.

Anthelmintic efficacy of Flemingia vestita (Fabaceae): alteration in the activities of some glycolytic enzymes in the cestode,Raillietina echinobothrida

AbstractThe crude root-peel extract of Flemingia vestita, genistein and praziquantel were tested against some selected glycolytic enzymes—hexokinase (HK), phosphofructokinase (PFK), phosphoenolpyruvate carboxykinase (PEPCK), pyruvate kinase (PK), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and malic enzyme (ME)—of the fowl tape worm, Raillietina echinobothrida. Following exposure to the various treatments, the activities of HK, PFK, PEPCK and LDH increased by 33–39%, 41–125%, 44–49% and 55–67%, respectively, and that of PK decreased by 14–26% in the parasite at the time of paralysis. The MDH and ME activities of the tissue homogenate were also found to be higher by 22–43% and 28–59%, respectively, in the treatments. However, whereas the activity of both cytosolic and mitochondrial MDH increased by 33–58% and 43–73%, respectively, the cytosolic ME activity showed an increase of 33–39%, and there was no significant enhancement in the mitochondrial ME activity. Histochemically, the enhancement in the activities of HK, LDH and MDH was clearly discernible. The enhanced glycolytic activity seems to be a function of anthelmintic stress caused by the phytochemicals.

Genistein from Flemingia vestita (Fabaceae) enhances NO and its mediator (cGMP) production in a cestode parasite, Raillietina echinobothrida.

Cyclic GMP (cGMP) is responsible for various cellular functions including signal pathways and it acts as a mediator for nitric oxide (NO). In order to evaluate the anthelmintic efficacy of the plant-derived isoflavones, the crude peel extract of Flemingia vestita and pure genistein were tested with respect to the activity of nitric oxide synthase (NOS), NO efflux and the cGMP concentration in Rallietina echinobothrida, the cestode parasite of domestic fowl. For comparison, the parasites were also treated with genistein (the major isoflavone present in the crude peel extract), sodium nitroprusside (SNP), a known NO donor, and praziquantel (PZQ), the reference drug. At the time of onset of paralysis in the parasite, the activity of NOS showed a significant increase (35-46%) and a 2-fold increase of NO efflux into the incubation medium in the treated worms in comparison to the respective controls. The cGMP concentration in the parasite tissue increased by 46-84% in the treated test worms in comparison to the controls. The results show that the isoflavones, genistein in particular, from the crude peel extract of F. vestita influence the cGMP concentration in the parasite tissue, which plays a major role in the downstream signal pathways.

Context Dependent Regulation of Human Phosphoenolpyruvate Carboxykinase Isoforms by DNA Promoter Methylation and RNA Stability

Cytoplasmic and mitochondrial isoforms of phosphoenolpyruvate carboxykinase (PEPCK‐C and PEPCK‐M) regulate hepatic gluconeogenesis to control systemic glucose homeostasis. Transcriptional and post‐transcriptional mechanisms may govern synthesis, maintenance and cooperative function of compartmentalized PEPCK enzymes. In a comparative analysis, we show that tumor cells consistently transcribe and translate higher levels of enzymatically active PEPCK‐C than PEPCK‐M and both the isoforms were present at lower levels in normal fibroblasts. Unlike in PEPCK‐M, absence of glucose reduced the PEPCK‐C mRNA and protein levels only in HepG2 cells. Interestingly, isoflavone genistein significantly increased PEPCK‐C mRNA and protein levels in normal fibroblasts indicating cell type specific control mechanisms. Genistein also significantly affected RNA stability of PEPCK‐C but not PEPCK‐M in HepG2 cells. This was due to the conserved and functional mRNA destabilizing AU rich sequences at the 3′‐UTR region of PEPCK‐C gene and was confirmed by luciferase reporter assays suggesting that glucose deprivation and genistein targets these sequences for mRNA degradation in HepG2 cells but not in fibroblasts. Analysis of promoter methylation by luciferase reporter assays and bisulfite DNA sequencing suggested that PEPCK‐C but not PEPCK‐M promoter was activated by 5‐aza‐2‐deoxycytidine by inducing cytosine demethylation at the specific CpG dinucleotides of 5′‐UTR region. Taken together, our data suggests stable PEPCK‐M activity and identifies intricate relationship between (1) mRNA stability and (2) promoter DNA methylation as two mechanisms of gene expression that distinguishes PEPCK‐C and PEPCK‐M enzyme activities in a context and cell type dependent manner during gluconeogenesis. J. Cell. Biochem. 117: 2506–2520, 2016.

Genistein represses PEPCK-C expression in an insulin-independent manner in HepG2 cells and in alloxan-induced diabetic mice

Genistein has been reported to exert beneficial effects on type 2 diabetes mellitus (T2DM); however, the underlying molecular mechanisms involved therein have not been clearly elucidated. To address this question, the effect of genistein on the expression of phosphoenolpyruvate carboxykinase (PEPCK), and glucose production in HepG2 cells and in alloxan‐induced diabetic mice was investigated. HepG2 cells were exposed to different concentration of genistein in presence or absence of modulators, and the expression of cytosolic PEPCK (PEPCK‐C) and the signaling pathways was studied. Further, the biological relevance of the in vitro study was tested in alloxan‐induced diabetic mice. Genistein lowered PEPCK‐C expression and glucose production in HepG2 cells accompanied with increased in phosphorylation states of AMPK, MEK½, ERK½, and CRTC2. Treatment with the AMPK inhibitor (compound C) enhanced genistein‐induced MEK½ and ERK½ activity indicating a potential cross‐talk between the two signaling pathways. In vivo, genistein also reduced fasting glucose levels accompanied with reduced PEPCK‐C expression and increased in AMPK and ERK½ phosphorylation states in the liver of genistein‐treated alloxan‐induced diabetic mice. Genistein fulfills the criteria of a suitable anti‐diabetic agent by reducing glucose production and inhibiting PEPCK‐C expression in HepG2 cells and also in alloxan‐induced diabetic mice. These results indicate that genistein is an effective candidate for preventing T2DM through the modulation of AMPK‐CRTC2 and MEK/ERK signaling pathways, which may allow a novel approach to modulate dysfunction in hepatic gluconeogenesis in T2DM.

Differential kinetics at PK/PEPCK branch point in the cestode, Raillietina echinobothrida.

Pyruvate kinase (PK; EC 2.7.1.40) and phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) are essential regulatory enzymes of glucose oxidation in helminths, the PK/PEPCK branch point being the first divergent step between carbohydrate catabolism of the parasites and their hosts. Recently, PEPCK from the cestode parasite, Raillietina echinobothrida, has been purified and characterized. In order to find out the differential kinetics, if any, at PK/PEPCK branch point in the parasite, in this study, we purified and characterized the parasite PK and compared it with the parasite PEPCK. The purified PK displayed standard Michaelis-Menten kinetics with Kmapp of 77.8 µM for its substrate PEP, whereas the Kmapp was 46.9 µM for PEPCK. PEP exhibited differential kinetics at PK/PEPCK branch point of the parasite and behaved as a homotropic effector for PEPCK, but not for PK. The inhibitory constant (Ki) for genistein and daidzein (phytochemicals from Flemingia vestita) was determined and discussed. From these results, we hypothesize that PK/PEPCK branch point is a probable site for anthelmintic action.

Cloning and expression of phosphoenolpyruvate carboxykinase from a cestode parasite and its solubilization from inclusion bodies using l-arginine

Phosphoenolpyruvate carboxykinase is an essential regulatory enzyme of glycolysis in the cestode parasite, Raillietina echinobothrida, and is considered a potential target for anthelmintic action because of its differential activity from that of its avian host. However, due to the unavailability of its structure, the mechanism of regulation of PEPCK from R. echinobothrida (rePEPCK) and its interaction with possible modulators remain unclear. Hence, in this study, the rePEPCK gene was cloned into pGEX-4T-3 and overexpressed for its characterization. On being induced by IPTG, the recombinant rePEPCK was expressed as inclusion bodies (IBs); hence, various agents, like different inducer concentrations, temperature, time, host cell types, culture media, pH, and additives, were used to bring the protein to soluble form. Finally, a significant amount (∼46%) of rePEPCK was solubilized from IBs by adding 2M l-arginine. Near-UV circular dichroism spectra analysis indicated that l-arginine (2M) had no effect on the conformation of the protein. In this study, we have reported a yield of ∼73mg of purified rePEPCK per 1L of culture. The purified rePEPCK retained its biological activity, and Km of the enzyme for its substrate was determined and discussed. The availability of recombinant rePEPCK may help in biochemical- and biophysical-studies to explore its molecular mechanisms and regulations.

Biological significance of phosphoenolpyruvate carboxykinase in a cestode parasite, Raillietina echinobothrida and effect of phytoestrogens on the enzyme from the parasite and its host, Gallus domesticus

Phosphoenolpyruvate carboxykinase (PEPCK) is involved in glycolysis in the cestode parasite, Raillietina echinobothrida; whereas, it executes a gluconeogenic role in its host, Gallus domesticus. Because of its differing primary function in the cestode parasite and its host, this enzyme is regarded as a plausible anthelmintic target. Hence, the biological significance of PEPCK in the parasite was analysed using siRNA against PEPCK from R. echinobothrida (RePEPCK). In order to find out the functional differences between RePEPCK and GdPEPCK (PEPCK from its host, G. domesticus), PEPCK genes from both sources were cloned, over-expressed, characterized, and some properties of the purified enzymes were compared. RePEPCK and GdPEPCK showed a standard Michaelis-Menten kinetics with K mapp of 46.9 and 22.9 µ m, respectively, for phosphoenolpyruvate and K mapp of 15.4 µ m for oxaloacetate in GdPEPCK decarboxylation reaction. Here, we report antagonist behaviours of recombinant PEPCKs derived from the parasite and its host. In search of possible modulators for PEPCK, few phytoestrogens were examined on the purified enzymes and their inhibitory constants were determined and discussed. This study stresses the potential of these findings to validate PEPCK as the anthelmintic drug target for parasitism management.

Genistein: is the multifarious botanical a natural anthelmintic too?

Genistein (4′,5,7-trihydroxyisoflavone) is naturally present in plants of the soy family and is known to have various pharmacological activities, such as anti-cancer, anti-diabetic, anti-oxidant, etc. The phytoestrogen is one of the major isoflavones found in some medicinal plants having anthelmintic properties. This review describes the putative role of genistein as an anthelmintic, which has been tested on some helminth parasites in vitro. Genistein has been shown to cause paralysis and alterations in the tegument and tegumental enzymes (acid phosphatase, alkaline phosphatase, adenosine triphosphatase, and 5′-nucleotidase) of helminth parasites. Alterations in the activities of several enzymes associated with the coordination system (specifically non-specific esterases, acetylcholine esterase, and nitric oxide synthase), and changes in the concentration of nitric oxide, cGMP, free amino acid pool, and tissue ammonia are observed in helminth parasites treated with genistein. The phytoestrogen also affects the carbohydrate metabolism by altering the activities of key enzymes involved in glycogen- and glucose-metabolism of a cestode parasite. Considering the significance of phosphoenolpyruvate carboxykinase (PEPCK) in glycolysis of the cestode parasite, Ki of the phytoestrogen for PEPCK in the parasite has been determined, and molecular docking of genistein into the active site of the enzyme has also been described. The potential beneficial role of genistein as a natural alternative in management of helminth parasites needs to be further explored, particularly considering its in vivo efficacy and pharmacokinetics.

Proteases of Parasitic Helminths: Their Metabolic Role in Establishment of Infection in the Host

Proteases catalyze hydrolysis of peptide bonds in proteins and play an important role in the survival of living organisms, encoded by about 2% of the whole genome in all kind of organisms. Mostly they are nonspecific, while some are highly specific toward a peptide bond. Generally, proteases are grouped into different clan, family, and type, depending on kinds of reaction they catalyze, mechanism of catalysis, and their molecular structure and homology. Proteases control many biological processes in living organisms including helminths. There are about 1828 sequences that pertain to 25 genera of helminth parasites. In this chapter, we have discussed various types of proteases found in helminth parasites, like aspartic-, cysteine-, metallo-, and serine proteases, and their possible role in these parasites and their hosts.