A smart bioconjugate of alginate and pectinase with unusual biological activity toward chitosan

The commercial preparation of pectinase (Pectinex Ultra SP-L) was conjugated to alginate by noncovalent interactions by employing 1% alginate during the conjugation protocol. The optimum "immobilization efficiency" was 0.76. The pH optimum and the thermal stability of the enzyme remained unchanged upon conjugation with alginate. The soluble bioconjugate showed a 3-fold increase in V(max)/K(m) as compared to the free enzyme when the smart biocatalyst was used for chitosan hydrolysis. Time course hydrolysis of chitosan thus showed higher conversion of chitosan into reducing oligosaccharides/sugars. The smart bioconjugate could be reused five times without any detectable loss of chitosanase activity.     

Construction of an amperometric TG biosensor based on AuPPy nanocomposite and poly (indole-5-carboxylic acid) modified Au electrode

A method is described for construction of an amperometric triglyceride (TG) biosensor based on covalent co-immobilization of lipase, glycerol kinase and glycerol-3-phosphate oxidase onto gold polypyrrole nanocomposite decorated poly indole-5-carboxylic acid electrodeposited on the surface of a gold electrode. The enzyme electrode was characterized by transmission electron microscopy, scanning electron microscopy, electrochemical impedance studies, Fourier transform infrared spectroscopy and cyclic voltammetry. Biosensor showed optimum response within 4 s at pH 6.5 and 35 °C, when polarized at +0.1 V against Ag/AgCl. There was a linear relationship between sensor response and triolein concentration in the range 50–700 mg/dl. Biosensor was employed for determination of TG in serum. Detection limit of the biosensor was 20 mg/dl. Biosensor was evaluated with 91–95 % recovery of added triolein in sera and 4.14 and 5.85 % within and between batch coefficients of variation, respectively. There was a good correlation (r = 0.99) between sera TG values by standard method (Enzymic colorimetric) and the present method. The biosensor was unaffected by a number of serum substances at their physiological concentration. Biosensor lost 50 % of its initial activity after its 100 uses over 7 months, when stored at 4 °C.     

Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-Terminal Residues 23-88 Restores Susceptibility to 22L Prions,but Not to RML Prions in PrP-Knockout Mice

Prion infection induces conformational conversion of the normal prion protein PrP^C, into the pathogenic isoform PrP^Sc, in prion diseases. It has been shown that PrP-knockout (Prnp^0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp^0/0 mice, neither developed the disease nor accumulated MHM2^ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice developed the disease with abundant accumulation of MHM2^ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2^ScΔ23-88, and that the co-expressing wild-type PrP^C can stimulate the conversion of MHM2Δ23-88 to MHM2^ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp^0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp^0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2^ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2^ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice. However, wild-type PrP^Sc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice, compared with RML- and 22L-inoculated Prnp^0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2^ScΔ23-88 without the help of the trans-acting PrP^C, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrP^C stimulates the conversion of MHM2Δ23-88 into MHM2^ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrP^C into PrP^Sc.     

Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3

Sorting nexin 27 (SNX27) contains a PDZ domain that is phylogenetically related to the PDZ domains of the NHERF proteins. Studies on nonepithelial cells have shown that this protein is located in endosomes, where it regulates trafficking of cargo proteins in a PDZ domain–dependent manner. However, the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) primarily through the SNX27 PDZ domain. A combination of knockdown and reconstitution experiments with wild type and a PDZ domain mutant (GYGF → GAGA) of SNX27 demonstrate that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation-based recycling and degradation studies in intestinal epithelial cells show that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ-mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells.     

A new high performance liquid chromatographic method for quantification of atomoxetine in human plasma and its application for pharmacokinetic study

Atomoxetine is the first, non-stimulant alternative to other stimulant medications used for the treatment of Attention-Deficit/Hyperactivity Disorder (ADHD). Reported methods for the determination of atomoxetine include expensive liquid chromatography tandem mass spectrometry (LCMS) and high performance liquid chromatography (HPLC) with liquid scintillation counting (LSC) detection. Till date, no method has been reported in literature to determine atomoxetine using HPLC with UV detection. In this paper, we describe a new HPLC method for the determination of atomoxetine using liquid-liquid extraction with tertiary butyl methyl ether and UV detector. This method was found to be linear over the concentration range of 0.05-3.0 microg/ml. The limit of quantification was 0.05 microg/ml. Intra- and inter-day precision was <15% and accuracy was in the range of 95.67-108.80%. Stability studies showed that atomoxetine was stable in human plasma for short- and long-term period for sample preparation and analysis. This method was used for sample analysis in a pharmacokinetic study of atomoxetine (25mg) in five healthy adult female volunteers. The observed mean+/-S.D. pharmacokinetic parameters Cmax, Tmax and AUC(0-t) were 0.40+/-0.06 microg/ml, 3.40+/-0.42 h and 1.34+/-0.52 microg h/ml, respectively.     

Inorganic salts influence IAA ionization and adventitious rhizogenesis in Pongamia pinnata

The basal cut end of coppice shoot cuttings of Pongamia pinnata was treated for 24 h with 0 (water treated control) or 5.0 mmol/L of KMnO4, KCI, and KH2PO4 or 2.5 mmol/L of K2HPO4 and K2SO4. Inorganic salts of P, S, Cl and Mn significantly influenced IAA ionization and adventitious rhizogenesis. P and S salts had lower IAA ionization potential, but more pronounced effect on adventitious rhizogenesis than Cl and Mn salts. The linear regression analysis also established negative correlations between salt induced IAA ionization with various characteristics of adventitious rhizogenesis such as sprouting (r = -0.83, p < 0.05), rooting (r = -0.82, p < 0.05), root number (r = -0.95, p < 0.01), and root length (r = -0.80, p < 0.1). The implication of IAA ionization in adventitious rhizogenesis has been discussed and the possible role of inorganic salts therein suggested.     

Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives

A new series of betulinic acid derivatives have been synthesized by introducing heterocyclic ring between C-2 and C-3 positions of betulinic acid. Further modifications were also carried out by reduction of C-20(29) unsaturated bond and substitution of C-28 carboxyl group by ester and amide linkage to enhance the selectivity. Compound 11 resulted in IC(50) of 2.44, 2.5, and 2.7 microg/ml on MIAPaCa, PA-1, and SW620 cancer cell lines, respectively. Compound 38 resulted in IC(50) of 0.67 microg/ml on MIAPaCa cell line.     

Spinigerin induces apoptotic like cell death in a caspase independent manner in Leishmania donovani

Antimicrobial peptides (AMPs) are multifunctional components of the innate immune system. Chemotherapeutic agents used for treatment of visceral leishmaniasis (VL) are now threatened due to the emergence of acquired drug resistance and toxicity. AMPs are attractive alternative to conventional pharmaceuticals. In this study, first time we explored the antileishmanial activity of spinigerin originally derived from Pseudacanthotermes spiniger. Leishmania donovani promastigotes present apoptosis-like cell death upon exposure to spinigerin (IC₅₀, 150 μM). The infection rate was reduced by 20% upon exposure to 150 μM spinigerin but no cytotoxicity on host macrophages was observed. Elevation of intracellular ROS level and down-regulation of two ROS detoxifying enzymes, ascorbate peroxidase (APx) and trypanothione reductase (TR) suggested essential role of ROS machinery during spinigerin mediated cell death. About 97% cell population was found to be Annexin-V positive; 44% cells being highly Annexin-V positive. Moreover, we observed morphological changes like cell rounding, nuclear condensation, oligonucleosomal DNA degradation and TUNEL positive cells without loss of membrane integrity upon spinigerin exposure, suggests apoptosis-like death. Interestingly, collapse in mitochondrial membrane potential and increased level of intracellular ROS and calcium were not associated with caspase like activity. Computational analysis suggests spiningerin interacts with trypanothione reductase and thus probably interferes its function to detoxify the toxic ROS level. Therefore, spinigerin induces apoptosis-like cell death in L. donovani in a caspase-independent manner. The study elucidates the antileishmanial property of spinigerin that may be considered for future chemotherapeutic option alone or adjunct with other drug regimens for improved treatment of visceral leishmaniasis.     

Hydrogen peroxide detoxifying enzymes show different activity patterns in host and non-host plant interactions with Magnaporthe oryzae Triticum pathotype

Wheat blast caused by the hemibiotroph fungal pathogen Magnaporthe oryzae Triticum (MoT) pathotype is a destructive disease of wheat in South America, Bangladesh and Zambia. This study aimed to determine and compare the activities of antioxidant enzymes in susceptible (wheat, maize, barley and swamp rice grass) and resistant (rice) plants when interacting with MoT. The activities of reactive oxygen species-detoxifying enzymes; catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione S-transferase (GST), peroxidase (POX) were increased in all plants in response to MoT inoculation with a few exceptions. Interestingly, an early and very high activity of CAT was observed within 24 h after inoculation in wheat, barley, maize and swamp rice grass with lower H₂O₂ concentration. In contrast, an early and high accumulation of H₂O₂ was observed in rice at 48 hai with little CAT activity only at a later stage of MoT inoculation. The activities of APX, GST and POD were also high at an early stage of infection in rice. However, these enzymes activities were very high at a later stage in wheat, barley, maize and swamp rice grass. The activity of GPX gradually decreased with the increase of time in rice. Taken together, our results suggest that late and early inductions of most of the antioxidant enzyme activities occurs in susceptible and resistant plants, respectively. This study demonstrates some insights into physiological responses of host and non-host plants when interacting with the devastating wheat blast fungus MoT, which could be useful for developing blast resistant wheat.