Studies onMadhuca butyraceae seed proteins

DefattedMadhuca butyraceae seeds contain 24% of crude protein and 10.4% of saponins. The solubility ofMadhuca seed proteins was determined in water and NaCl as a function of pH and minimum solubility occurred at pH 4.0. The proteins consist of three components with S_20,w values of 2.2, 9.8 and 15.4. On gel filtration the proteins gave three peaks and on diethylaminoethyl cellulose chromatography they resolved into two components. Thein vitro digestibility ofMadhuca seed protein was found to be 69% when assayed with a pepsin-pancreatin system.     

Construction and genetic characterization of lambda specialized transducing particles carrying therglB gene ofEscherichia coli K-12

TherglB gene ofEscherichia coli codes for a restriction activity that cleaves the hydroxymethylated DNA of T2 and T4 phages. Earlier mapping data placed the gene at 98.39 min counterclockwise to thehsd operon. Genetic analysis of the in vivo gene fusions with fusion-transducing phages established the location of therglB gene next to thehsdS gene of thehsdRMS cluster. The methodology used in this study could be extended to similar in vivo physical mapping of closely linked genes.     

Ferric acetate-uranium acetate reagent in cholesterol estimations in plasma and high density lipoprotein fractions: comparison with existing methods

The use of ferric acetate-uranium acetate colour reaction for the estimation of cholesterol in the supernatants of plasma samples after precipitation of low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol by heparin-MnCl2 was assessed and compared with the conventional method using the FeCl3 colour reaction and also with the method using o-phthalaldehyde as the colouring reagent. All three methods gave comparable values when total cholesterol in plasma samples was determined and also when high density lipoprotein (HDL) fractions were separated by ultracentrifugation and the cholesterol contents determined. But when heparin-MnCl2 precipitation was used for HDL separation, and the cholesterol content determined, the FeCl3 method gave significantly lower values. This could be due to interference of the cholesterol colour reaction with FeCl3, due to Mn2+ ions present in the supernatant. Addition of Mn2+ to cholesterol standards and subsequent colour development with ferric acetate-uranium acetate and FeCl3 reagents showed that Mn2+ decreased the absorbancy of the coloured complex at 560 nm only when FeCl3 was used. Percentage recovery of added cholesterol was also lower when the heparin-MnCl2 supernatant was treated with FeCl3 reagent for colour development. Use of ferric acetate-uranium acetate reagent provides a simpler and quicker method. It does not suffer from interference due to the presence of Mn2+ ions and gives results comparable to the o-phthalaldehyde method and those using ultracentrifugation as the separation procedure.     

Studies on cellulase of the cotton wilt pathogen fusarium vasinfectum ATK

Cellulase of the cotton wilt pathogenFusarium vasinfectum was partially purified 19fold. The enzyme preparation had three pH optima at 5.4, 6.0 and between 8.0 and 8.4 and optimum temperature between 35 and 40°C. Irreversible denaturation of the enzyme resulted in 5 min below pH 1.5 and above 70°C. Thiol group inhibitors and EDTA inhibited the cellulase activity completely whereas reducing agents and metal ions Fe⁺⁺, Ca⁺⁺ and Cu⁺⁺ activated. The results are discussed in elation to pathogenicity towards the cotton plant.This research has been financed in part by a grant made by the United States Department of Agriculture, under P.L. 480.     

In vitro effect of prostaglandins,prostaglandin endoperoxides and thromboxane on rat intestinal alkaline phosphatase and (Ca2+-Mg2+) adenosine triphosphatase

The activity of alkaline phosphate and²⁺-Mg²⁺ adenosine triphosphatase, two of the enzymes involved in limpid and calcium uptake across the intestinal membrane, were increased in experimental atherosclerosis. Administration ofAnnapavala sindhooram, an antiatherosclerotic drug, lowers these enzyme levels to near normal values. Prostaglandin E2 stimulated the enzyme activitiesin vitro, while prostaglandin endoperoxide inhibited the activity. Thromboxane and other prostaglandins had no effect on the enzyme activities. Addition of the antiatherosclerotic drug to thein vitro assay system reversed the effect of both prostaglandin E₂ and prostaglandin endoperoxide.     

P. falciparum RH5‐Basigin interaction induces changes in the cytoskeleton of the host RBC

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte‐binding protein homologues (RHs) and erythrocyte‐binding like proteins are two families involved in the invasion leading to merozoite‐red blood cell (RBC) junction formation. Ca²⁺ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca²⁺ signaling, which triggers the erythrocyte‐binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5‐basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5‐Basigin interaction on its own triggers a Ca²⁺ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca²⁺ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.     

A processing product of the Plasmodium falciparum reticulocyte binding protein RH1 shows a close association with AMA1 during junction formation

Plasmodium falciparum responsible for the most virulent form of malaria invades human erythrocytes through multiple ligand‐receptor interactions. The P. falciparum reticulocyte binding protein homologues (PfRHs) are expressed at the apical end of merozoites and form interactions with distinct erythrocyte surface receptors that are important for invasion. Here using a range of monoclonal antibodies (mAbs) against different regions of PfRH1 we have investigated the role of PfRH processing during merozoite invasion. We show that PfRH1 gets differentially processed during merozoite maturation and invasion and provide evidence that the different PfRH1 processing products have distinct functions during invasion. Using in‐situ Proximity Ligation and FRET assays that allow probing of interactions at the nanometre level we show that a subset of PfRH1 products form close association with micronemal proteins Apical Membrane Antigen 1 (AMA1) in the moving junction suggesting a critical role in facilitating junction formation and active invasion. Our data provides evidence that time dependent processing of PfRH proteins is a mechanism by which the parasite is able to regulate distinct functional activities of these large processes. The identification of a specific close association with AMA1 in the junction now may also provide new avenues to target these interactions to prevent merozoite invasion.     

Constructing a collagen hydrogel for the delivery of stem cell-loaded chitosan microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.     

Highly regioselective methylation of inosine nucleotide: an efficient synthesis of 7-methylinosine nucleotide

Abstract

A facile, straightforward, reliable, and an efficient chemical synthesis of inosine nucleotides such as 7-methylinosine 5′-O-monophosphate, 7-methylinosine 5′-O-diphosphate, and 7-methylinosine 5′-O-triphosphate, starting from the corresponding inosine nucleotide is delineated. The present methylation reaction of inosine nucleotide utilizes dimethyl sulfate as a methylating agent and water as a solvent at room temperature. It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylinosine nucleotide in good yields with high purity (>99.5%).