Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies

Monoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87–219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182–204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.

Antigenic determinants at the carboxy terminus of chicken egg white riboflavin carrier protein (RCP): Epitope mapping and antibody-mediated pregnancy curtailment in rodents

The epitopic core sequences recognized by three monoclonal antibodies raised to chicken riboflavin carrier protein (RCP) were mapped to the C-terminal tail-end of the protein using the pepscan method A 21-residue synthetic peptide corresponding to residues 200-219 of the protein and comprising the regions corresponding to the antibodies was synthesized. Administration of polyclonal antibodies specific to this peptide led to termination of early pregnancy in mice. Also, active immunization of rats with the peptide-purified protein derivative conjugate inhibited establishment of pregnancy. These results demonstrate the functional importance of the C-terminal 200-219 region of chicken RCP. Copyright (C) 1996 Elsevier Science Ltd.

Simultaneous purification of biotin-binding proteins-I and -II from chicken egg yolk and their characterization

Chicken egg yolk biotin-binding protein-I (BBP-I) has been purified to homogeneity along with the tetrameric BBP-II by a common protocol. The purification includes delipidation of egg yolk by butanol extraction, DEAE-Sephacel chromatography, treatment with guanidinium chloride and biotin-aminohexyl-Sepharose affinity chromatography. The identity of purified BBP-I was ascertained by its physicochemical properties as well as by its immunological cross-reactivity and precursor-product relationship with BBP-II.

Immunomodulation using agonists and antagonists: potential clinical applications

Introduction: Extensive studies have gone into understanding the differential role of the innate and adaptive arms of the immune system in the context of various diseases. Receptor-ligand interactions are responsible for mediating cross-talk between the innate and adaptive arms of the immune system, so as to effectively counter the pathogenic challenge. While TLRs remain the best studied innate immune receptor, many other receptor families are now coming to the fore for their role in various pathologies. Research has focused on the discovery of novel agonists and antagonists for these receptors as potential therapeutics. Areas covered: In this review, we present an overview of the recent advances in the discovery of drugs targeting important receptors such as G-protein coupled receptors, TRAIL-R, IL-1 beta receptor, PPARs, etc. All these receptors play a critical role in the modulation of the immune response. We focus on the recent paradigms applied for the generation of specific and effective therapeutics for these receptors and their status in clinical trials. Expert opinion: Non-specific activation by antagonist/agonist is a difficult problem to dodge. This demands innovation in ligand designing with the use of strategies such as allosterism and dual-specific ligands. Rigorous preclinical and clinical studies are required in transforming a compound to a therapeutic.

Studying the resistivity imaging of chicken tissue phantoms with different current patterns in Electrical Impedance Tomography (EIT)

A current injection pattern in Electrical Impedance Tomography (EIT) has its own current distribution profile within the domain under test. Hence, different current patterns have different sensitivity, spatial resolution and distinguishability. Image reconstruction studies with practical phantoms are essential to assess the performance of EIT systems for their validation, calibration and comparison purposes. Impedance imaging of real tissue phantoms with different current injection methods is also essential for better assessment of the biomedical EIT systems. Chicken tissue paste phantoms and chicken tissue block phantoms are developed and the resistivity image reconstruction is studied with different current injection methods. A 16-electrode array is placed inside the phantom tank and the tank is filled with chicken muscle tissue paste or chicken tissue blocks as the background mediums. Chicken fat tissue, chicken bone, air hole and nylon cylinders are used as the inhomogeneity to obtained different phantom configurations. A low magnitude low frequency constant sinusoidal current is injected at the phantom boundary with opposite and neighboring current patterns and the boundary potentials are measured. Resistivity images are reconstructed from the boundary data using EIDORS and the reconstructed images are analyzed with the contrast parameters calculated from their elemental resistivity profiles. Results show that the resistivity profiles of all the phantom domains are successfully reconstructed with a proper background resistivity and high inhomogeneity resistivity for both the current injection methods. Reconstructed images show that, for all the chicken tissue phantoms, the inhomogeneities are suitably reconstructed with both the current injection protocols though the chicken tissue block phantom and opposite method are found more suitable. It is observed that the boundary potentials of the chicken tissue block phantoms are higher than the chicken tissue paste phantom. SNR of the chicken tissue block phantoms are found comparatively more and hence the chicken tissue block phantom is found more suitable for its lower noise performance. The background noise is found less in opposite method for all the phantom configurations which yields the better resistivity images with high PCR and COC and proper IRMean and IRMax neighboring method showed higher noise level for both the chicken tissue paste phantoms and chicken tissue block phantoms with all the inhomogeneities. Opposite method is found more suitable for both the chicken tissue phantoms, and also, chicken tissue block phantoms are found more suitable compared to the chicken tissue paste phantom. (C) 2012 Elsevier Ltd. All rights reserved.

Curcumin-Arteether Combination Therapy of Plasmodium berghei-Infected Mice Prevents Recrudescence Through Immunomodulation

Earlier studies in this laboratory have shown the potential of artemisinin-curcumin combination therapy in experimental malaria. In a parasite recrudescence model in mice infected with Plasmodium berghei (ANKA), a single dose of alpha, beta-arteether (ART) with three oral doses of curcumin prevented recrudescence, providing almost 95% protection. The parasites were completely cleared in blood with ART-alone (AE) or ART+curcumin (AC) treatments in the short-term, although the clearance was faster in the latter case involving increased ROS generation. But, parasites in liver and spleen were not cleared in AE or AC treatments, perhaps, serving as a reservoir for recrudescence. Parasitemia in blood reached up to 60% in AE-treated mice during the recrudescence phase, leading to death of animals. A transient increase of up to 2-3% parasitemia was observed in AC-treatment, leading to protection and reversal of splenomegaly. A striking increase in spleen mRNA levels for TLR2, IL-10 and IgG-subclass antibodies but a decrease in those for INF gamma and IL-12 was observed in AC-treatment. There was a striking increase in IL-10 and IgG subclass antibody levels but a decrease in INF gamma levels in sera leading to protection against recrudescence. AC-treatment failed to protect against recrudescence in TLR2(-/-) and IL-10(-/-) animals. IL-10 injection to AE-treated wild type mice and AC-treated TLR22/2 mice was able to prolong survival. Blood from the recrudescence phase in AE-treatment, but not from AC-treatment, was able to reinfect and kill naive animals. Sera from the recrudescence phase of AC-treated animals reacted with several parasite proteins compared to that from AE-treated animals. It is proposed that activation of TLR2-mediated innate immune response leading to enhanced IL-10 production and generation of anti-parasite antibodies contribute to protective immunity in AC-treated mice. These results indicate a potential for curcumin-based combination therapy to be tested for prevention of recrudescence in falciparum and relapse in vivax malaria.

Chemical unfolding of chicken villin headpiece in aqueous dimethyl sulfoxide solution: cosolvent concentration dependence, pathway, and microscopic mechanism

Unfolding of a protein often proceeds through partial unfolded intermediate states (PUIS). PUIS have been detected in several experimental and simulation studies. However, complete analyses of transitions between different PUIS and the unfolding trajectory are sparse. To understand such dynamical processes, we study chemical unfolding of a small protein, chicken villin head piece (HP-36), in aqueous dimethyl sulfoxide (DMSO) solution. We carry out molecular dynamics simulations at various solution compositions under ambient conditions. In each concentration, the initial step of unfolding involves separation of two adjacent native contacts, between phenyl alanine residues (11-18 and 7-18). This first step induces, under appropriate conditions, subsequent separation among other hydrophobic contacts, signifying a high degree of cooperativity in the unfolding process. The observed sequence of structural changes in HP-36 on increasing DMSO concentration and the observed sequence of PUIS, are in approximate agreement with earlier simulation results (in pure water) and experimental observations on unfolding of HP-36. Peculiar to water-DMSO mixture, an intervening structural transformation (around 15% of DMSO) in the binary mixture solvent retards the progression of unfolding as composition is increased. This is reflected in a remarkable nonmonotonic composition dependence of RMSD, radius of gyration and the fraction of native contacts. At 30% mole fraction of DMSO, we find the extended randomly coiled structure of the unfolded protein. The molecular mechanism of DMSO induced unfolding process is attributed to the initial preferential solvation of the hydrophobic side chain atoms through the methyl groups of DMSO, followed by the hydrogen bonding of the oxygen atom of DMSO to the exposed backbone NH groups of HP-36.

Solvent Sensitivity of Protein Unfolding: Dynamical Study of Chicken Villin Headpiece Subdomain in Water Ethanol Binary Mixture

We carry out a series of long atomistic molecular dynamics simulations to study the unfolding of a small protein, chicken villin headpiece (HP-36), in water-ethanol (EtOH) binary mixture. The prime objective of this work is to explore the sensitivity of protein unfolding dynamics toward increasing concentration of the cosolvent and unravel essential features of intermediates formed in search of a dynamical pathway toward unfolding. In water ethanol binary mixtures, HP-36 is found to unfold partially, under ambient conditions, that otherwise requires temperature as high as similar to 600 K to denature in pure aqueous solvent. However, an interesting course of pathway is observed to be followed in the process, guided by the formation of unique intermediates. The first step of unfolding is essentially the separation of the cluster formed by three hydrophobic (phenylalanine) residues, namely, Phe-7, Phe-11, and Phe-18, which constitute the hydrophobic core, thereby initiating melting of helix-2 of the protein. The initial steps are similar to temperature-induced unfolding as well as chemical unfolding using DMSO as cosolvent. Subsequent unfolding steps follow a unique path. As water-ethanol shows composition-dependent anomalies, so do the details of unfolding dynamics. With an increase in cosolvent concentration, different partially unfolded intermediates are found to be formed. This is reflected in a remarkable nonmonotonic composition dependence of several order parameters, including fraction of native contacts and protein-solvent interaction energy. The emergence of such partially unfolded states can be attributed to the preferential solvation of the hydrophobic residues by the ethyl groups of ethanol. We further quantify the local dynamics of unfolding by using a Marcus-type theory.

Study on the biomass yield of Duckweed (Lemna minor) (L.) in hydroponic cultures containing different concentrations of aquaeous extract of chicken manure

The biomass yields of duck week (Lemna minor(L) was monitored in hydroponic media prepared by variously extracting 0.50, 1.00 and 2.00g of dried chicken manure per liter of city water (tap water) supply. The culture media consisting of aqueous extract of the various manure treatments were made up to 12 liters in all cases with tap water as control. Plastic baths of 25 liters capacity with 0.71 super(m2) surface area were used as culture facility. Each bath was stocked at a density of 30g super(m-2) with fresh weed samples (i.e 21.30g/bath). Maximum yields were obtained at all treatment levels and control on day 3 and based on the highest yield of 0.37gm super(-2)d super(-1) (dry matter) obtained at 1.00gL manure treatment which was however not significantly higher (P>0.05) than the 0.36gm super(-2)d super(-1) (dry matter) at 0.05gl super(-1) media manure content, an average manure level of 0.75l super(-1) was selected and used to determine the operational plant density. Thus fresh weights of 30 to 300gm super(-2) was grown in triplicate at 30g intervals for a period of 3 days. A regression equation of Y=2.6720+0.0021x with a corresponding maximum density or operational plant density of 266gm super(-2) and yield of 0.98gm super(-2), d super(-1) (dry matter) were obtained. Further growth trials were carried out at the operational density and manure levels of 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2.00gl super(-1) media manure concentration giving a significantly higher yield (P<0.05) of 17gm super(-2), d super(-1) (dry matter). This yield was however doubled to between 2.21 and 2.24gm super(-2) d super(-1) (equivalent to 7.96 to 8.06mt.ha-1, Yr-1 dry matter on extrapolation) if 25% and 75% respectively of the total weed cover were harvested daily within the experimental period. The role of some dissolved plant nutrients (DPN) were also discussed