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Background: Glycerol kinase (GK; EC 2.7.1.30) facilitates the entry of glycerol into pathways of glucose and triglyceride metabolism and may play a potential role in Type 2 diabetes mellitus (T2DM). However, the detailed regulatory mechanisms and structure of the human GK are unknown. Methods: The human GK gene was cloned into the pET-24a(+) vector and over-expressed in Escherichia coli BL21 (DE3). Since the protein was expressed as inclusion bodies (IBs), various culture parameters and solubilising agents were used but they did not produce bioactive His-GK; however, co-expression of His-GK with molecular chaperones, specifically pKJE7, achieved expression of bioactive His-GK. The overexpressed bioactive His-GK was purified using coloumn chromatography and characterised using enzyme kinetics. Results: The overexpressed bioactive His-GK was purified apparently to homogeneity (∼295-fold) and characterised. The native His-GK was a dimer with a monomeric molecular weight of ∼55 kDa. Optimal enzyme activity was observed in TEA buffer (50 mM) at 7.5 pH. K+ (40 mM) and Mg2+ (2.0 mM) emerged as prefered metal ions for His-GK activity with specific activity 0.780 U/mg protein. The purified His-GK obeyed standard Michaelis–Menten kinetics with Km value of 5.022 µM (R2=0.927) for its substrate glycerol; whereas, that for ATP and PEP was 0.767 mM (R2=0.928) and 0.223 mM (R2=0.967), respectively. Other optimal parameters for the substrate and co-factors were also determined. Conclusion: The present study demonstrates that co-expression of molecular chaperones assists with the expression of bioactive human GK for its characterisation.

XAS can be a very reliable tool for studying phase evolution and dynamic diffusion in intermetallic Ni/Bi system.

Manipulating light at the sub-wavelength level is a crucial feature of surface plasmon resonance (SPR) properties for a wide range of nanostructures. Noble metals like Au and Ag are most commonly used as SPR materials. Significant attention is being devoted to identify and develop non-noble metal plasmonic materials whose optical properties can be reconfigured for plasmonic response by structural phase changes. Chromium (Cr) which supports plasmon resonance, is a transition metal with shiny finished, highly non-corrosive, and bio-compatible alloys, making it an alternative plasmonic material. We have synthesized Cr micro-rods from a bi-layer of Cr/Au thin films, which evolves from face centered cubic to hexagonal close packed (HCP) phase by thermal activation in a forming gas ambient. We employed optical absorption spectroscopy and cathodoluminescence (CL) imaging spectroscopy to observe the plasmonic modes from the Cr micro-rod. The origin of three emission bands that spread over the UV–Vis-IR energy range is established theoretically by considering the critical points of the second-order derivative of the macroscopic dielectric function obtained from density functional theory (DFT) matches with interband/intraband transition of electrons observed in density of states versus energy graph. The experimentally observed CL emission peaks closely match the s–d and d–d band transition obtained from DFT calculations. Our findings on plasmonic modes in Cr(HCP) phase can expand the range of plasmonic material beyond noble metal with tunable plasmonic emissions for plasmonic-based optical technology.

Glucosinolates, synthesized by the glucosinolate biosynthesis pathway, are the secondary metabolites used as a defence mechanism in the Brassicaceae plants, including Arabidopsis thaliana. The first committed step in the pathway, catalysed by methylthioalkylmalate (MAM) synthase (EC: 2.3.3.17), is to produce different variants of glucosinolates. Phylogenetic analyses suggest that possibly MAM synthases have been evolved from isopropylmalate synthase (IPMS) by the substitutions of five amino acid residues (L143I, H167L, S216G, N250G and P252G) in the active site of IPMS due to point mutations. Considering the importance of MAM synthase in Brassicaceae plants, Petersen et al. (2019) made an effort to characterise the MAM synthase (15 MAM1 variants) in vitro by single substitution or double substitutions. In their study, the authors have expressed the variants in Escherichia coli and analysed the amino acids in the cultures of E. coli in vivo. Since modifying the MAM synthases by transgenic approaches could increase the resistance of Brassicaceae plants for enhancing the defence effect of glucosinolates and their degraded products; hence, MAM synthases should be characterized in detail in vivo in A. thaliana along with the structural analysis of the enzyme for meaningful impact and for its imminent use in vivo.

Heavy metals show a wide range of effect on fishes, out of which arsenic (As) and lead (Pb) are among the leading heavy metal toxicants. These heavy metals are known to alter different biochemical parameters, including glycogen level, in different tissues of fishes. Glycogen level in fish serves as the main source of energy; hence, in this study, the acute toxicity test of As and Pb and their effect on the glycogen content and the enzymes involved therein (glycogen phosphorylase, glycogen synthase, hexokinase, phosphofructokinase and pyruvate kinase) were studied in the liver and muscle tissues of Heteropneustes fossilis. The 96 h LC50 values of As2O3 and PbCl2 on H. fossilis were found to be 35.09 ppm and 66.20 ppm, respectively. On acute exposure to 96 h LC50 values of As2O3 and PbCl2, the glycogen concentration showed a gradual decrease in both liver and muscle tissues of H. fossilis. However, on chronic exposure (LC50/20th ppm), the glycogen content in liver and muscle of H. fossilis was depleted till 20 days; whereas after 30 days, the glycogen level was recovered in both the tissues. The activities of glycogen metabolic enzymes (glycogen phosphorylase and glycogen synthase) and few selected glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) were also altered in H. fossilis when exposed to acute and chronic concentration of As2O3 and PbCl2. Our present results showed that As and Pb induced toxicity stress on the catfish, H. fossilis, which might have caused to alter the carbohydrate metabolism in the fish.

Artemisinin (ART) has been in use against different cancer cells and its derivatives and conjugates are more cytotoxic to iron-rich cancer cells. It is desirable to develop easily achievable synthetic 1,2,4-trioxanes having the same pharmacophore as that of ART. To explore more efficient compounds, a 1,2,3-triazole tethered 1,2,4‑trioxane trimer (4T) was synthesized and the anti-cancer effects of ART and 4T on MDA-MB-435 and MDA-MB-231 cells were investigated concerning regulation of osteopontin (OPN) expression, which is associated with cancer progression and malignancy. 1H NMR and 13C NMR, oxidative stress analysis, flow cytometry, western blot, Real-Time PCR, transfections, luciferase assay, cell viability, proliferation, migration and chemotactic invasion assays were used in this study. It was observed that the 4T induced apoptosis by inhibiting Bcl-2 (~0.6-fold) and cleavage of caspase-3 (intrinsic pathway) in these metastatic cancer cells, and also reduced colony formation, migration and invasion of these cancer cells. The treatment of 4T decreased the reduced glutathione level and increased the activities of glucose-6-phosphate dehydrogenase and glutathione reductase in the 4T treated cancer cells as compared to their respective controls. Further, the expression of OPN was diminished (~0.5-fold) by the 4T in these cell lines. It was also observed that the key mitogen-activated protein kinase pathway proteins, mitogen-activated protein kinase kinase1/2 (~1.8-fold) and extracellular signal-regulated kinase1/2 (~16-fold), were also activated following the treatment of the 4T. However, the phosphorylated c-Jun level, a component of activator protein-1, was significantly reduced in these cancer cells upon 4T treatment. Taken together, we hypothesize that 4T may be useful for controlling cancer progression and malignancy.

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.

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 the 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 diferrent concentration of genistein in presence or absence of modulators, and the expression of cytosolic PEPCK (PEPCK-C) and the signalling 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 signalling 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 signalling pathways, which may allow a novel approach to modulate dysfunction in hepatic gluconeogenesis in T2DM. This article is protected by copyright. All rights reserved

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. © 2016 Wiley Periodicals, Inc.

SUMMARYPhosphoenolpyruvate carboxykinase (PEPCK) involved in gluconeogenesis in higher vertebrates opposedly plays a significant role in glucose oxidation of the cestode parasite,Raillietina echinobothrida. Considering the importance of the enzyme in the parasite and lack of its structural details, there exists an urgent need for understanding the molecular details and development of possible modulators. Hence, in this study, PEPCK gene was obtained using rapid amplification of cDNA ends, and various biocomputational analyses were performed. Homology model of the enzyme was generated, and docking simulations were executed with its substrate, co-factor, and modulators. Computer hits were generated after structure- and ligand-based screening using Discovery Studio 4.1 software; the predicted interactions were compared with those of the existing structural information of PEPCK. In order to evaluate the docking simulation results of the modulators, PEPCK gene was cloned and the overexpressed protein was purified for kinetic studies. Enzyme kinetics andin vitrostudies revealed that out of the modulators tested, tetrahydropalmatine (THP) inhibited the enzyme with lowest inhibition constant value of 93 nm. Taking the results together, we conclude that THP could be a potential inhibitor for PEPCK in the parasite.

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.

SUMMARYPhosphoenolpyruvate 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 fromR. 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 withKmappof 46.9 and 22.9µm, respectively, for phosphoenolpyruvate andKmappof 15.4µmfor 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.

Artemisinin and its analogs have shown potent anticancer activity in primary cancer cultures and cell lines by inhibiting cancer proliferation, metastasis, and angiogenesis. Despite its apparent compatibility to normal cells and low IC50 values in comparison to the commonly used anticancer drugs, the underlying mechanisms behind their cytotoxic effects are not yet fully understood. Surprisingly, the efficacy of synthetic 1,2,4-trioxanes against cancer has not been explored yet. Given the high antitumor activity of artemisinin dimers in comparison to their monomers, we report here the synthesis of simple 1,2,3-triazole conjugated 1,2,4-trioxanes and their potential antitumor activity by studying their inhibitory effect on osteopontin (OPN) expression in MDA-MB-435 breast cancer cells. It may be noted that despite being a strong marker to identify human tumor metastasis, no study on effect of artemisinin and its synthetic and semisynthetic derivatives on OPN expression has ever been studied. Although our initial studies did not notice any straight-line relationship between the number of trioxane units in a molecule to the extent of inhibition of OPN protein expression, we could observe better results in some cases in comparison to artemisinin. We have observed that artemisinin did not show appreciable OPN downregulation in MDA-MB-435 cancer cells, but dihydroartemisinin (DHA) and some synthetic 1,2,4-trioxane monomers and dimers showed downregulation of OPN expression. Therefore, these compounds may act as an anti-metastatic agent in controlling breast cancer cells metastasis.

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.

SUMMARYPhosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) is an essential regulatory enzyme of glycolysis in helminths in contrast to its role in gluconeogenesis in their host. Previously we have reported that phytochemicals fromFlemingia vestita(Family: Fabaceae), genistein in particular, have vermifugal action and are known to affect carbohydrate metabolism in the cestode,Raillietina echinobothrida. In order to determine the functional differences of PEPCK from the parasite and its avian host (Gallus domesticus), we purified the parasite enzyme apparently to homogeneity, and characterized it. The native PEPCK is a monomer with a subunit molecular weight of 65 kDa. The purified enzyme displayed standard Michaelis-Menten kinetics withKm value of 42·52μM for its substrate PEP. TheKi for the competitive inhibitors GTP, GMP, ITP and IMP for the carboxylation reaction were determined and discussed. In order to identify putative modulators from plant sources, phytochemicals fromF. vestitaandStephania glabrawere tested on the purified PEPCK, which resulted in alteration of its activity. From our results, we hypothesize that PEPCK may be a potential target site for anthelmintic action.

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. © 2016 Wiley Periodicals, Inc.

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.

The alcoholic crude root-peel extract of Flemingia vestita and its major isoflavone, genistein, have been shown to have a vermifugal/vermicidal effect by causing a flaccid paralysis accompanied by alterations in the structural architecture of the tegumental interface and metabolic activity in Raillietina echinobothrida, the cestode of domestic fowl. In the present study, the crude root-peel extract and pure genistein were tested in vitro with respect to Ca2+ homeostasis and the occurrence of some metal ions was detected in the parasite. Live cestodes were incubated in pre-defined concentrations of the crude root-peel extract, genistein and praziquantel (as reference drug), till the paralysis time with simultaneous maintenance of respective controls. In the parasite tissue, a significant amount of Ca2+ (approximately 400 microg/g dry tissue wt) was found to be present besides magnesium, iron, zinc, lead and chromium, whilst manganese, cadmium and nickel were below the level of detection. The Ca2+ concentration was decreased significantly by 39%-49%, in the parasite tissue exposed to the test materials in comparison to the respective controls. There was also an increase in Ca2+ efflux by 91%-160% into the culture medium under similar treatments. The changes in Ca2+ homeostasis may be related to the rapid muscular contraction and consequent paralysis in the parasite due to the anthelmintic stress caused by the phytochemicals of F. vestita.

The root-tuber peel of Flemingia vestita and its active component, genistein, were tested in respect of glucose metabolism in the cestode, Raillietina echinobothrida. Live R. echinobothrida, collected from the intestine of freshly slaughtered domestic fowl, were incubated at 39+/-1 degrees C in defined concentrations of the root-peel crude extract (5 mg/ml), genistein (0.2 mg/ml) and praziquantel (1 microg/ml) in phosphate buffered saline with 1% of dimethyl sulphoxide with simultaneous maintenance of controls. In the treated worms, there was a significant decrease in the glycogen concentration accompanied with the decrease of glucose by 14-32%, whereas the malate concentration increased by 49-134% as compared to controls. Both in controls and treated parasites, however, the pyruvate content was not measurable. While alanine and lactate contents showed a decline by 7-25% in the parasites exposed to all test materials, the lactate efflux into the incubation medium showed 37-71% increase in treatments indicating an overall increase of lactate production in comparison to controls. The results showing a decline in the glycogen and glucose contents and a significant rise in the malate content and lactate efflux under treatment conditions suggest that the energy demand in the parasites possibly got enhanced under stress, though it did not influence a switch over towards aerobic degradation of glucose in the parasites.

The crude root-peel extract of Flemingia vestita, containing genistein as the major isoflavone, has a vermifugal/vermicidal effect. It acts by causing flaccid paralysis accompanied by alterations in the activities of several tegumental enzymes and other metabolic activities in the fowl tapeworm, Raillietina echinobothrida. To elucidate the mode of action of the putative phytochemicals on energy metabolism, crude root-peel extract, pure genistein and praziquantel were tested on glucose 6-phosphate dehydrogenase (G6PDH) and enzymes of gluconeogenesis—pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and fructose 1,6-bisphosphatase (FBPase)—in R. echinobothrida. The activities of G6PDH, PEPCK and FBPase were largely restricted to the cytosolic fraction, while PC was confined to the mitochondrial fraction. Following treatments, the G6PDH activity was decreased by 23–31%, whereas the activities of PC and PEPCK were increased by 32–44% and 44–49%, respectively. There was no significant effect by any of the treatments on FBPase activity. We hypothesize that the phytochemicals from F. vestita, genistein in particular, influence the key enzymes of these pathways, which is perhaps a function of high energy demand of the parasite under anthelmintic stress.

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 μg/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.

The nodular tapeworm, Raillietina echinobothrida is a well studied avian gastrointestinal parasite of family Davaineidae (Cestoda: Cyclophyllidea). It is reported to be the largest in size and second most prevalent species infecting chicken in north-east India. In the present study, morphometrical methods coupled with the molecular analysis of the second internal transcribed spacer (ITS2) region of ribosomal DNA were employed for precise identification of the parasite. The annotated ITS2 region was found to be 446 bp long and further utilized to elucidate the phylogenetic relationships and its species-interrelationships at the molecular level. In phylogenetic analysis similar topology was observed among the trees obtained by distance-based neighbor-joining as well as character-based maximum parsimony tree building methods. The query sequence R. echinobothrida is well aligned and placed within the Davaineidae group, with all Raillietina species well separated from the other cyclophyllidean (taeniid and hymenolepid) cestodes, while Diphyllobothrium latum (Pseudophyllidea: Diphyllobothriidae) was rooted as an out-group. Sequence similarities indeed confirmed our hypothesis that Raillietina spp. are neighboring the position with other studied species of order Cyclophyllidea against the out-group order Pseudophyllidea. The present study strengthens the potential of ITS2 as a reliable marker for phylogenetic reconstructions.

Cyclic GMP (cGMP) mediates various physiological functions of nitric oxide (NO) synthesized by nitric oxide synthase (NOS). A crude peel extract and purified fraction of Flemingia vestita, as well as a crude rhizome extract of Stephania glabra and fractions were tested with respect to the activity of NOS, NO efflux and cGMP concentration in the cestode Raillietina echinobothrida in order to find out the possible mode of anthelmintic action of these plant-derived components. For comparison purposes, the parasites were also treated with pure genistein, sodium nitroprusside (SNP-a known NO donor), and the reference drug, praziquantel (PZQ). At the time of onset of paralysis in the parasites, a significant increase (32%-87%) in the NOS activity and a two to three fold increase of NO efflux into the incubation medium were observed in the treated parasites in comparison to their respective controls. The cGMP concentration in the treated parasites' tissue was also increased by 44%-103%. However, in the presence of NG-nitro-L-arginine methyl ester, a potent inhibitor of NOS, there was no increase in the cGMP concentration in the parasite tissue. This study indicates that the phytochemicals, in particular genistein and tetrahydropalmatine, from F. vestita and S. glabra, respectively, disturb the downstream signalling pathway of NO, as indicated by the change in cGMP concentration in the parasite tissue.

SUMMARYCyclic 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 ofFlemingia vestitaand pure genistein were tested with respect to the activity of nitric oxide synthase (NOS), NO efflux and the cGMP concentration inRallietina 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 ofF. vestitainfluence the cGMP concentration in the parasite tissue, which plays a major role in the downstream signal pathways.

The 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.

A coprological survey was made to assess the spectrum of helminth parasites in Mithun and other co-existing wild livestock in Bhutan and neighbouring Arunachal Pradesh (India). Nematodes with direct life cycle were found to be most predominant helminth infection among these animals. Cestodes and trematodes were poorly represented as their occurrence is primarily determined by the presence of the invertebrate intermediate hosts in the locality of the final host.