Role of pepsin in modifying the allergenicity of Bhetki (Lates calcarifer) and Mackerel (Rastrelliger kanagurta) fish

94-100The effect of pepsin digestion on the allergenicity of raw and thermally processed (boiled and fried) fish muscle extracts of two widely consumed fishes bhetki (Lates calcarifer) and mackerel (Rastrelliger kanagurta) was studied. Sere were collected from 110 patients who were hypersensitive to fish, as evidenced by their clinical history, symptoms and positive skin-prick test results. The various extracts after digestion with pepsin at different times of incubation were tested for specific IgE-binding activity by ELISA and immunoblotting with patients’ sera. All the extracts of both the fishes retained their allergenicity as evidenced by ELISA and immunoblotting. In bhetki, maximum allergenicity was found in the pepsin-digested fried extract, whereas similar treatment decreased the allergenicity in fried mackerel. Results showed that raw as well as thermally processed allergens of both the fishes maintained strong allergenicity, even after digestion with pepsin for different time periods. The study revealed that the fish proteins played an important role in manifestation of allergy, due to their stable structure, which was retained even after pepsin and heat treatment

Identification of allergens in Indian fishes: Hilsa and Pomfret exemplified by ELISA and immunoblotting

1170-1175Enzymed-linked immunosorbant assay of hilsa and pomfret muscle extracts showed specific IgE binding to ten allergic patients' sera, the results corroborated to that of skin prick test. Comparison of allergen profiles of the two fish extracts by immunoblotting revealed a common antigenic protein of 50 kDa and some high molecular weight fish allergens instead of low molecular weight parvalbumin found in several fishes. Purified and well characterized fish allergens are always considered better than crude fish extracts for diagnostic use

Phosphorylated Proteins from Serum: A Promising Potential Diagnostic Biomarker of Cancer

Cancer is a fatal disease worldwide. Each year ten million people are diagnosed around the world, and more than half of patients eventually die from it in many countries. A majority of cancer remains asymptomatic in the earlier stages, with specific symptoms appearing in the advanced stages when the chances of adequate treatment are low. Cancer screening is generally executed by different imaging techniques like ultrasonography (USG), mammography, CT-scan, and magnetic resonance imaging (MRI). Imaging techniques, however, fail to distinguish between cancerous and non-cancerous cells for early diagnosis. To confirm the imaging result, solid and liquid biopsies are done which have certain limitations such as invasive (in case of solid biopsy) or missed early diagnosis due to extremely low concentrations of circulating tumor DNA (in case of liquid biopsy). Therefore, it is essential to detect certain biomarkers by a noninvasive approach. One approach is a proteomic or glycoproteomic study which mostly identifies proteins and glycoproteins present in tissues and serum. Some of these studies are approved by the Food and Drug Administration (FDA). Another non-expensive and comparatively easier method to detect glycoprotein biomarkers is by ELISA, which uses lectins of diverse specificities. Several of the FDA approved proteins used as cancer biomarkers do not show optimal sensitivities for precise diagnosis of the diseases. In this regard, expression of phosphoproteins is associated with a more specific stage of a particular disease with high sensitivity and specificity. In this review, we discuss the expression of different serum phosphoproteins in various cancers. These phosphoproteins are detected either by phosphoprotein enrichment by immunoprecipitation using phosphospecific antibody and metal oxide affinity chromatography followed by LC-MS/MS or by 2D gel electrophoresis followed by MALDI-ToF/MS analysis. The updated knowledge on phosphorylated proteins in clinical samples from various cancer patients would help to develop these serum phophoproteins as potential diagnostic/prognostic biomarkers of cancer

Antiproliferative effect of T/Tn specific Artocarpus lakoocha agglutinin (ALA) on human leukemic cells (Jurkat, U937, K562) and their imaging by QD-ALA nanoconjugate

T/Tn specificity of Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha (Moraceae) fruit and a heterodimer (16 kD and 12 kD) of molecular mass 28 kD, was further confirmed by SPR analysis using T/Tn glycan containing mammalian glycoproteins. N-terminal amino acid sequence analysis of ALA showed homology at 15, 19-21, 24-27, and 29 residues with other lectin members of Moraceae family viz., Artocarpus integrifolia (jacalin) lectin, Artocarpus hirsuta lectin, and Maclura pomifera agglutinin. It is mitogenic to human PBMC and the maximum proliferation was observed at 1 ng/ml. It showed an antiproliferative effect on leukemic cells, with the highest effect toward Jurkat cells (IC50 13.15 ng/ml). Synthesized CdS quantum dot-ALA nanoconjugate was employed to detect the expression of T/Tn glycans on Jurkat, U937, and K562 leukemic cells surfaces as well as normal lymphocytes by fluorescence microscopy. No green fluorescence was observed with normal lymphocytes indicating that T/Tn determinants, which are recognized as human tumor associated structures were cryptic on normal lymphocyte surfaces, whereas intense green fluorescent dots appeared during imaging of leukemic cells, where such determinants were present in unmasked form. The above results indicated that QD-ALA nanoconjugate is an efficient fluorescent marker for identification of leukemic cell lines that gives rise to high quality images

Binding mechanism of methyl-α-N-acetyl-D-galactopyranosyl amine to <i>Artocarpus lakoocha </i>lectin, artocarpin: A proton nuclear magnetic resonance study

299-306The dynamics of the binding mechanism between Artocarpus lakoocha lectin and Me-α-D-GalNAc has been studied using 1H NMR spectroscopy. Various thermodynamic parameters have been calculated with the help of temperature dependence of line broadening of the methoxy group resonance of Me-α-D-GalNAc. No change in the chemical shift has been observed while full line width at half height of the sugar protons was found to increase with increasing temperature indicating that the binding ligand is in fast exchange. No chemical shift between bound and free ligands has been observed.The activation parameters obtained from the association and dissociation rate constants suggest that the association process is controlled by high activation entropy which is due to the specific orientation of both lectin and sugar whereas the contribution of activation enthalpy is small. On the other hand, the dissociation reaction is controlled by high activation enthalpy due to the break in the interaction between the sugar and the lectin. From NMR data a two-step binding mechanism has been proposed. The associated complex is stabilized mainly by hydrogen bonding and vander Waals attractions while hydrophobic interaction is not significant as indicated by the negative entropy and enthalpy values

Jacalin Bound Plasma O-Glycoproteome and Reduced Sialylation of Alpha 2-HS Glycoprotein (A2HSG) in Rheumatoid Arthritis Patients

<div><p>Glycosylation studies of plasma proteins can reveal information about the onset and progression of diseases, where in the glycan biosynthetic pathways are disturbed as in rheumatoid arthritis (RA). The present study was focused on analysis of O-linked glycoproteins of plasma in RA patients. Two dimensional gel electrophoresis of jacalin bound plasma of RA patients revealed a number of differentially expressed protein spots as compared to healthy controls. Eighteen protein spots were found to have statistically significant (<em>p</em><0.05) difference in their expression level from four sets of gels and were identified by MALDI-TOF MS. Most of the identified proteins were predicted to be O-glycosylated proteins by Net–O-Gly 3.1 algorithm. Among these the alpha 2HS glycoprotein (A2HSG) was found to be down regulated whereas inter alpha trypsin inhibitor H4 (ITIH4) was up regulated and this was validated by Western blotting. The glycosylation studies showed the reduced N-linked sialylation of A2HSG in RA patients. Altered glycoprotein expression and functional as well as structural studies of glycans might help in the diagnosis of RA and understanding the disease pathogenesis.</p> </div

Prediction of O-glycosylation sites using Net-O-Glyc3.1 program.

<p>Prediction of O-glycosylation sites using Net-O-Glyc3.1 program.</p

Glycosylation studies in A2HSG.

<p>2-DE Western blot of A2HSG in untreated plasma showed the higher p<i>I</i> of A2HSG in RA patients compared to control. The treatment with sialidase shifted the p<i>I</i> of control towards higher p<i>I</i> in comparison to that of RA, while the shift in p<i>I</i> was found to be equal in both the samples after PNGase F treatment. The experiment was repeated thrice with different pooled samples.</p