Regeneration of ethyl parathion antibodies for repeated use in immunosensor: a study on dissociation of antigens from antibodies

Reliable analysis using an immunosensor strongly depends on the specificity, activity, and sensitivity of the antibody. Immobilization of antibody on the solid matrix enables its repeated use, for which it is required to dissociate the antigens and antigen-enzyme conjugate from the immobilized antibody matrix after each use and while doing so, a maximum retention of activity and specificity are crucial requirements. In the present investigation, on the development of an immunosensor for the organophosphorus pesticide ethyl parathion (EP) using EP antibodies, different dissociating agents such as organic solvents, detergents and acidic buffers, that is, dimethyl sulphoxide (DMSO), Tween-20, cetyl trimethylammonium bromide (CTAB), methanol, chloroform, guanidium chloride (GdmCl), glycine-HCl (Gly-HCl) buffer in the pH range of 1.5-3.0, pierce buffer and combination of DMSO and methanol in phosphate buffer and Gly-HCl buffer and salts like NaCl and MgCl2 were used. Generally about 50-60% dissociation was obtained with some degree of denaturation of the antibody immobilized on the sepharose matrix. However, 1% DMSO in combination with 0.2 M Gly-HCl buffer at a pH of 2.3 showed 97% dissociation and the immobilized antibody retained sufficient activity to carry out 14 reproducible assays for EP.     

High-throughput fluorescent tagging of full-length Arabidopsis gene products in planta

We developed a high-throughput methodology, termed fluorescent tagging of full-length proteins (FTFLP), to analyze expression patterns and subcellular localization of Arabidopsis gene products in planta. Determination of these parameters is a logical first step in functional characterization of the approximately one-third of all known Arabidopsis genes that encode novel proteins of unknown function. Our FTFLP-based approach offers two significant advantages: first, it produces internally-tagged full-length proteins that are likely to exhibit native intracellular localization, and second, it yields information about the tissue specificity of gene expression by the use of native promoters. To demonstrate how FTFLP may be used for characterization of the Arabidopsis proteome, we tagged a series of known proteins with diverse subcellular targeting patterns as well as several proteins with unknown function and unassigned subcellular localization.     

Biosensor based on Langmuir–Blodgett films of poly(3-hexyl thiophene) for detection of galactose in human blood

An amperometric biosensor was developed to estimate galactose in human blood serum. Monolayers of poly(3-hexyl thiophene) were placed on glass plates coated with indium tin oxide formed by dispensing a mixed solution of stearic acid in chloroform on to a water sub-phase. Galactose oxidase was mixed with poly(3-hexyl thiophene)/stearic acid in chloroform and dispensed on to the air-water interface of Langmuir-Blodgett trough. These monolayers were transferred on to glass plates which were used as working electrodes with platinum as a reference electrode. The amperometric galactose biosensor thus fabricated had a linear response from 0.05 to 0.5 g galactose l(-1) in blood serum. The normal level in blood is < 0.05 g galactose l(-1) in adults and 0-0.2 g galactose l(-1) in infants. In case of galactosemia, this increases to above 0.2 g galactose l(-1) in infants.     

Diaryl naphthyl methanes a novel class of anti-implantation agents

Diaryl naphthyl methanes and the corresponding 1, 2, 3, 4- and 5, 6, 7, 8-tetrahydro naphthyl methane derivatives have been synthesized as novel estrogen receptor binding ligands. The secondary and tertiary amino alkoxy derivatives of diaryl naphthyl and tetrahydro naphthyl methane interact with the estrogen receptor to elicit promising estrogenic, antiestrogenic and implantation inhibition activities in rats. The most active compounds in this series are 7, 9 and 20, cent percent active in preventing implantation in rats at 2.5 mgkg(-1) dose.     

Humoral defence factors in Indian river prawn, Macrobrachium malcolmsonii

A preliminary study on humoral defence factors of Indian river prawn, Macrobrachium malcolmsonii was carried out. The serum of animals collected from intermoult stage had an average total protein content of 9.65 g dl(-1) and lysozyme-like activity of 0.04 unit ml(-1). The phenoloxidase activity in haemocyte lysate supernatant was triggered significantly (P < 0.05) by chitosan, Gram-positive and Gram-negative bacterial cells in comparison to other elicitors viz., levamisole, trypsin and glucan. The serum contained an agglutinin against Gram-negative bacteria and a haemagglutinin against a wide range of vertebrate erythrocytes. The haemagglutinin was stable over a wide range of pH (3.0-7.0) and temperatures (-30 degrees C to 60 degrees C). A 413 kDa N-acetyl galactosamine-specific haemagglutinin was purified by single step affinity chromatography and the protein was found to be calcium ion-dependent in nature and made up of five different subunits of varied molecular weights on denaturing SDS-PAGE.     

Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases

Modular polyketide synthases (PKSs) are large multi-enzymatic, multi-domain megasynthases, which are involved in the biosynthesis of a class of pharmaceutically important natural products, namely polyketides. These enzymes harbor a set of repetitive active sites termed modules and the domains present in each module dictate the chemical moiety that would add to a growing polyketide chain. This modular logic of biosynthesis has been exploited with reasonable success to produce several novel compounds by genetic manipulation. However, for harnessing their vast potential of combinatorial biosynthesis, it is essential to develop knowledge based in silico approaches for correlating the sequence and domain organization of PKSs to their polyketide products. In this work, we have carried out extensive sequence analysis of experimentally characterized PKS clusters to develop an automated computational protocol for unambiguous identification of various PKS domains in a polypeptide sequence. A structure based approach has been used to identify the putative active site residues of acyltransferase (AT) domains, which control the specificities for various starter and extender units during polyketide biosynthesis. On the basis of the analysis of the active site residues and molecular modelling of substrates in the active site of representative AT domains, we have identified a crucial residue that is likely to play a major role in discriminating between malonate and methylmalonate during selection of extender groups by this domain. Structural modelling has also explained the experimentally observed chiral preference of AT domain in substrate selection. This computational protocol has been used to predict the domain organization and substrate specificity for PKS clusters from various microbial genomes. The results of our analysis as well as the computational tools for prediction of domain organization and substrate specificity have been organized in the form of a searchable computerized database (PKSDB). PKSDB would serve as a valuable tool for identification of polyketide products biosynthesized by uncharacterized PKS clusters. This database can also provide guidelines for rational design of experiments to engineer novel polyketides.     

Enzymatic E-colicins bind to their target receptor BtuB by presentation of a small binding epitope on a coiled-coil scaffold

Toxins and viruses often initiate their attacks by binding to specific proteins on the surfaces of target cells. Bacterial toxins (e.g. bacteriocins) and viruses (bacteriophages) targeting Gram-negative bacteria typically bind to outer membrane proteins. Bacterial E-colicins target Escherichia coli by binding to the outer membrane cobalamin transporter BtuB. Colicins are tripartite molecules possessing receptor-binding, translocation, and toxin domains connected by long coiled-coil alpha-helices. Surprisingly, the crystal structure of colicin E3 does not possess a recognizable globular fold in its receptor-binding domain. We hypothesized that the binding epitope of enzymatic E-colicins is a short loop connecting the two alpha-helices that comprise the coiled-coil region and that this flanking coiled-coil region serves to present the loop in a binding-capable conformation. To test this hypothesis, we designed and synthesized a 34-residue peptide (E-peptide-1) corresponding to residues Ala366-Arg399 of the helix-loop-helix region of colicin E3. Cysteines placed near the ends of the peptide (I372C and A393C) enabled crosslinking for reduction of conformational entropy and formation of a peptide structure that would present the loop epitope. A fluorescent analog was also made for characterization of binding by measurement of fluorescence polarization. Our analysis shows the following. (i). E-peptide-1 is predominantly random coil in aqueous solution, but disulfide bond formation increases its alpha-helical content in both aqueous buffer and solvents that promote helix formation. (ii). Fluorescein-labeled E-peptide-1 binds to purified BtuB in a calcium-dependent manner with a Kd of 43.6 +/- 4.9 nm or 2370 +/- 670 nm in the presence or absence of calcium, respectively. (iii). In the presence of calcium, cyanocobalamin (CN-Cbl) displaces E-peptide-1 with a nanomolar inhibition constant (Ki = 78.9 +/- 5.6 nm). We conclude that the BtuB binding sites for cobalamins and enzymatic E-colicins are overlapping but inequivalent and that the distal loop and (possibly) the short alpha-helical flanking regions are sufficient for high affinity binding.     

Extracellular hyaline material in association with other cytologic features in aspirates from collagenous spherulosis and adenoid cystic carcinoma of the breast

OBJECTIVE: To reevaluate breast aspirates showing extracellular hyaline material (EHM) and globules to assess if clinicoradiologic and cytologic features could help in differentiating between benign and malignant lesions, especially collagenous spherulosis (CS) and adenoid cystic carcinoma (ACC). STUDY DESIGN: Fine needle aspiration was performed on 884 patients with breast lumps. The cytomorphologic features of 6 cases showing EHM, including classic hyaline globules (HGs), were analyzed in detail. Three cases also had hemorrhagic nipple discharge. Tissue diagnosis (4) and mammography (6) were available. RESULTS: Aspirate smears revealed high cellularity composed of monolayers: clusters of uniform, small cells; EHM; and HGs surrounded by similar cells. Benign naked nuclei and stromal fragments (4), nuclear pleomorphism (3), apocrine cells (2), foam cells (2), naked HGs (2) and spindle cells in proximity to HGs were also seen (4). Nipple discharge smears showed foam cells, erythrocytes (3) and epithelial cell clusters with hyaline material (1). The cytologic diagnosis was CS (4) and ACC (2). Histopathology confirmed the diagnosis of CS (2) and ACC (1). CONCLUSION: There may be a morphologic overlap between the cytomorphologic features of CS and ACC, leading to diagnostic errors. The presence of EHM and HGs in association with bland cellular features should be interpreted with caution to avoid erroneous diagnoses. Histopathology is mandatory in these cases because of their different prognostic implications.     

Paternal reciprocal translocation t(11;16)(p13;q24.3) in a Silver-Russel syndrome patient

We describe a 7-month-old male child with Silver-Russel syndrome (SRS) phenotype, presented with two major clinical features: low birth weight, short stature, and minor features, such as macrocephaly, clinodactyly, essential for the diagnosis of SRS. Routine cytogenetic studies with GTG-banding showed 46,XY,t(11;16)(p13;q24.3). Fluorescence in situ hybridisation (FISH) with single copy probes BAC (11p13) and PAC (16q24.3), showed a reciprocal translocation. Chromosomal analysis of the mother was normal and the phenotypically normal father had apparently identical translocation t(11;16)(p13;q24.3). The disruption of growth factor genes at 11p and 16q breakpoint regions due to reciprocal translocation in the father might have caused SRS phenotype in the child.     

Enzymatic digestion--a safe and rapid technique for individual separation of Macrobrachium rosenbergii embryos for cryopreservation studies

A new, safe, and rapid technique for the individual separation of the embryos of giant freshwater prawn Macrobrachium rosenbergii de Man is described. Two protease enzymes, e.g., trypsin and collagenase were used. Embryos in the advanced stage of development (gray embryos with eyespot and heart beat) were selected for the study. Treatment with collagenase and trypsin at respective concentrations of 0.05 and 0.25% for 30 min resulted in 100% separation of 35-40 mg of embryonic mass (approximately 180 embryos). A chelating agent, EDTA (ethylenediaminetetraacetic acid disodium salt: dihydrate) at 400 mg l(-1) enhanced the activity of trypsin. Trypsin and collagenase, when used together, were found to act synergistically. The separated embryos revealed no morphological injury when observed under the microscope. Further, in vitro hatching of the separated embryos was successful indicating that the present technique is safe and effective in achieving individual separation of prawn embryos.