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J G Mohanty

Publications and source records attributed to J G Mohanty.

13 recordsLinked to original sources
PubMed2001

Effects of purine and pyrimidine nucleotides on intracellular Ca2+ in human eosinophils: activation of purinergic P2Y receptors.

BACKGROUND: Extracellular adenosine 5'-triphosphate (ATP) increases human eosinophil intracellular Ca(2+) concentration; the mechanism of action is not fully known. ATP, a physiologic regulator, acts through 2 purinergic receptor types: cation channels (P2X) and G protein-coupled receptors (P2Y). OBJECTIVE: This study is aimed at identifying the functional purinergic receptors in human eosinophils. METHODS: The relative potency of ATP, uridine (UTP), cytidine (CTP), and inosine (ITP) 5'-triphosphates (P2Y agonists); 2-methylthio-ATP (P2Y(1) agonist); and 2 P2X agonists, alpha,beta-methylene-ATP and beta,gamma-methylene-ATP on intracellular Ca(2+) concentration was examined in Ca(2+)-sensitive Fura-2-labeled human eosinophils. For comparison, ATP effects were similarly studied in human neutrophils. P2X/P2Y mRNA expression in cells was examined by reverse transcription and PCR. RESULTS: The nucleotide potency order was UTP > or = ATP > ITP >>> 2-methylthio-ATP > alpha,beta-methylene-ATP = beta,gamma-methylene-ATP = CTP = 0 in eosinophils. Pertussis toxin (500 ng/mL) pretreatment abolished the effect of lower (10(-6) mol/L) but not higher (10(-5) mol/L) concentrations of ATP in eosinophils, whereas it attenuated the effects of 10(-4) mol/L ATP in neutrophils. The phospholipase C inhibitor U73122 (2 micromol/L) partially inhibited the effect of ATP in eosinophils but totally blocked it in neutrophils. Both cells constitutively express mRNA for P2X(1), P2X(4), P2X(5), P2Y(1), and P2Y(2), but not P2X(7), with much weaker expressions of P2X(4) and P2X(5) in neutrophils. Eosinophils cultured with the T(H)1 cytokine, IFN-gamma, expressed mRNA for P2X(7), a receptor linked to apoptosis. CONCLUSIONS: These results suggest that the P2 purinergic receptor signal transduction pathways in eosinophils and neutrophils are different and are mediated by more than 1 subtype of functional P2Y receptors.

Adenosine Triphosphate↗
PubMed2000

Hydrogen peroxide release from human eosinophils on fibronectin: scopoletin enhances eosinophil activation.

We have examined the release of H(2)O(2) from PAF or TNFalpha-stimulated human eosinophils on fibronectin (FN)-coated polystyrene plates. H(2)O(2) release was measured by the standard scopoletin-horseradish peroxidase (SCOP-HRP) method and compared with that measured by a new microplate fluorescent assay for H(2)O(2) using a novel HRP substrate A6550. We observed that the SCOP-HRP method gave a 25-fold higher estimate of H(2)O(2) release from eosinophils than did the A6550-HRP method. Microscopic examination of PAF or TNFalpha-stimulated eosinophils in buffer alone or A6550-HRP reaction mixture showed that the cells remained generally round, while eosinophils in SCOP-HRP reaction mixture were spread on the fibronectin-coated surface. Measurement of the cellular ATP content after PAF-stimulation showed that only eosinophils activated in SCOP-HRP had a 50% fall in ATP content. This supported our conclusion that measurement of H(2)O(2) release from eosinophils in SCOP-HRP reaction mixture is problematic since the SCOP-HRP system activates eosinophils. However, we also found that A6550-HRP, when present throughout the incubation, resulted in a lower estimate of H(2)O(2) release than expected. The method used to detect eosinophil H(2)O(2) release greatly influences the absolute amount of H(2)O(2) detected.

Adenosine Triphosphate↗
PubMed1997

A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative.

This study describes a simple, reliable, highly sensitive and quantitative fluorescence microplate-assay of H2O2 from activated leukocytes using a novel horse radish peroxidase (HRP) substrate N-acetyl-3,7-dihydroxyphenoxazine (A6550). Unlike the widely used fluorescent HRP substrate scopoletin, A6550 is non-fluorescent and becomes highly fluorescent upon HRP-catalyzed H2O2 oxidation. Using 50 microM A6550, the change in fluorescence due to H2O2 generated from phorbol 12-myristate 13-acetate-activated human eosinophils and neutrophils is found to have a linear cell dose response up to 1.5 x 10(4) and 5 x 10(4) cells, respectively. The increase in fluorescence from A6550 is specifically due to H2O2 generation since it is inhibitable by catalase. Oxidized A6550 is found to be highly stable and the H2O2 dose response is linear as long as the ratio of A6550:H2O2 in the reaction mixture is higher than five. Unlike scopoletin, A6550 has a very low background, which changes little with time. In addition, the high fluorescent yield of oxidized A6550 results in an increased sensitivity for the detection of H2O2. When the concentrations of A6550 and HRP were 10 microM and 0.2 U/ml, respectively, as low as 2 pmol of H2O2 could be reliably measured. The sensitivity of A6550/H2O2 assay is found to be at least 10-fold higher than with scopoletin as the HRP substrate. The protocol described in this study using A6550 to measure H2O2 release from activated granulocytes can be easily adapted to other cell types which generate H2O2.

Catalase↗
PubMed1995

Receptor proteins on newborn Balb/c mouse brain cells for coxsackievirus B3 are immunologically distinct from those on HeLa cells.

Newborn Balb/c mice are highly susceptible to infection by the six coxsackievirus serotypes of group B (CVB) and it is known that receptor for these viruses are in highest concentration in the brain as compared to other tissues. Therefore, proteins from the brain tissues of these animals were solubilized (Brain-Ext) and characterized for the identification of mouse brain receptor (MBR) proteins. Virus-blot analyses of Brain-Ext suggested that each of three virus variants of CVB3-(N, W and RD) recognized four receptor proteins designated p46, p44, p36 and p33 according to their molecular size. Similar analyses of cultured neurons from newborn Balb/c mice revealed the presence of the same four receptor proteins, while astrocytes appeared to possess only p46 and/or p44. Isoelectric focusing of Brain-Ext, focused MBR proteins in the pH range 4.0-8.5, with a peak around pH 5.7. P46 was found to be neuraminidase sensitive. A polyclonal rat antiserum (anti-MBR) protected cultured neurons and astrocytes against infection by CVB3, inhibited virus binding to these cells and recognized the same four receptor proteins on western-blots as detected on virus-blots by CVB3. However, a rabbit polyclonal anti-HeLa cell antiserum, which strongly binds to HeLa cells and protects them from CVB3 infection, neither recognized any of the receptor proteins in western-blot analyses of Brain-Ext nor inhibited CVB3 infection on cultured neurons and astrocytes. Conversely, anti-MBR did not recognize any of the receptor proteins by western-blot analysis of HeLa cell extracts nor did it inhibit CVB3 infection of HeLa cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Coxsackievirus B3 adapted to growth in RD cells binds to decay-accelerating factor (CD55).

A coxsackievirus B3 (CB3) isolate adapted to growth in RD cells shows an alteration in cell tropism as a result of its capacity to bind a 70-kDa cell surface molecule expressed on these cells. We now show that this molecule is the complement regulatory protein, decay-accelerating factor (DAF) (CD55). Anti-DAF antibodies prevented CB3 attachment to the cell surface. Radiolabeled CB3 adapted to growth in RD cells bound to CHO cells transfected with human DAF, whereas CB3 (strain Nancy), the parental strain, did not bind to DAF transfectants. These results indicate that growth of CB3 in RD cells selected for a virus strain that uses DAF for cell surface attachment.

Antibodies, Monoclonal↗
PubMed1993

Attempts to purify a second cellular receptor for a coxsackievirus B3 variant, CB3-RD from HeLa cells.

A coxsackievirus B3 variant, CB3-RD, isolated on rhabdomyosarcoma (RD) cells is known to bind HeLa cells at two different receptor protein sites, HR1 and HR2. Since HR2 occurs in almost 50 fold excess of HR1 in HeLa cells, purification of HR2 was attempted, to obtain its partial N-terminal amino acid sequence and its further characterization. This study describes the purification of HR2 from octylthioglucoside solubilized HeLa cell membranes (HeLa-OTG) by preparative isoelectric focusing (IEF) followed by either preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or affinity chromatography on immobilized receptor monoclonal antibody, RmcA (RmcA-agarose). IEF of HeLA-OTG showed that both HR2 and HR1 could be well separated by this technique and focused with peak maxima around pH 3.7 and 6.7, respectively. Both RmcA and CB3-RD recognized HR2 as doublet bands (60 kD major polypeptide and a minor 55 kD polypeptide) on electroblots under non-reducing conditions. Preparative SDS-PAGE of the pool of IEF fractions containing HR2 (IEF pool) and simultaneous elution of polypeptides from the bottom of the gel during electrophoresis, is shown to be a useful technique in purifying HR2 with only one contaminating polypeptide (65 kD). However, affinity chromatography of the IEF pool on RmcA-agarose yielded HR2 without any detectable contaminating polypeptide. A quantitative chemiluminescence assay was developed to estimate the amount of HR2 on HeLa cells and in solution, when dot blotted on polyvinylidene difluoride (PVDF) membranes and probed with RmcA. Assays revealed that about 1.2% of the total HR2 present on HeLa cells could be obtained by IEF followed by affinity chromatography. Efforts are continuing to obtain sufficient quantities of purified HR2 for partial N-terminal amino acid sequencing.

Enterovirus B, Human↗
PubMed1989

Detergent solubilised bovine viral diarrhea virus elicits a similar immune response as the inactivated virus.

The immune response to two detergent solubilised preparations of Bovine Viral Diarrhea Virus (BVDV) proteins were compared with the response to a chemically inactivated BVDV preparation. The antibody titres were measured with the standard immunofluorescence assay and with a newly developed enzyme linked immunosorbent assay (ELISA). The range of viral protein specificity of the antibodies was compared using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. The results clearly demonstrate that the detergent solubilised viral proteins are equally as effective immunogens as the killed BVDV vaccine both in terms of antibody titers and specificity.

Animals↗
PubMed1989

Purification of IgG from serum with caprylic acid and ammonium sulphate precipitation is not superior to ammonium sulphate precipitation alone.

Immunoglobulin G (IgG) from bovine serum raised against Aeromonas Salmonicida was purified by ammonium sulphate precipitation (ASP) or caprylic acid treatment followed by ammonium sulphate precipitation (CAAS). Purity of IgG samples prepared by both methods were examined by High Performance Gel Permeation Chromatography, electrophoresis and antibody activity assay. Results suggest that IgG prepared by ASP is better than that obtained by CAAS method in terms of the yield of the IgG monomers and the recovery of the antibody activity.

Aeromonas↗
PubMed1988

High-performance liquid chromatographic separation and immunological characterization of soluble bovine viral diarrhea virus antigen.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis of the protein extracts from bovine viral diarrhea virus (BVDV, Singer strain) infected primary calf testicle cells (soluble antigen) showed the presence of four virus specific polypeptides of 105, 90, 84 and 67 kiloDaltons (kD) the 84-kD being the most abundant. Anion-exchange high-performance liquid chromatography (HPLC) of soluble antigen separated the virus specific polypeptides in individual peaks while the gel permeation HPLC collected all of them in a single protein aggregate peak of 290 kD. Except for the 84-kD polypeptide peak in anion-exchange HPLC, all peak fractions were found to be heterogeneous in nature having more than one polypeptide. Analysis of the antisera raised against the peaks having antigen activity showed that antisera against the 84-kD polypeptide peak did not neutralise BVDV while those against the fractions containing the 90- and 105-kD polypeptides neutralised the virus.

Animals↗
PubMed1986

2-deoxy-D-glucose inhibition of herpes simplex virus type-1 receptor expression.

Growth of HEp-2 cells in 2-deoxy-D-glucose (2-DOG) supplemented media decreased the cells' binding capacity for herpes simplex virus type-1 KOS(HSV-1) but not vesicular stomatitis virus. HEp-2 cells tolerated up to 30 mM 2-DOG, but 2-DOG was toxic for Vero cells over 2 mM. The reduction in binding was maintained for at least 24 h even after careful removal of the inhibitor and growth in normal media. Complete regeneration of the receptor sites on HEp-2 cells was observed 8 h after mild trypsinization of cells grown in either normal or the 2-DOG supplemented media. Specific glycoprotein characteristics of the HSV-1 binding site were indicated by its inactivation upon trypsinization (0.1 mg per 5 X 10(5) cells for 30 s) and blocking by wheat germ agglutinin but not limulin. These results suggest that 2-DOG inhibits the proper expression of cell surface glycoprotein HSV-1 receptor sites on HEp-2 cells.

Animals↗
PubMed1985

A micropreparation of fluorescein conjugates of immunoglobulin G and Fab from serum.

Protein A-Sepharose CL-4B (PAS) was used to isolate rabbit immunoglobulin G from crude anti-herpes simplex virus-1 serum. Papain treatment of the PAS-bound immunoglobulin G released Fab fragments from the solid support, while Fc-containing fragments remained bound to PAS. PAS-immobilized immunoglobulin G was fluoresceinated by reaction with fluorescein isothiocyanate followed by papain cleavage to yield fluorescein-conjugated Fab fragments in solution. These fragments retained activity toward herpes simplex virus-1 infected Vero cells as evaluated by immunofluorescence. This novel procedure represents the fastest and simplest method for preparing Fab or fluoresceinated Fab fragments directly from any volume of immune serum.

Animals↗