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.

Evaluation of quantity and quality of chitosan produce from Rhizopus oryzae by utilizing food product processing waste whey and molasses.

Low cost whey salt medium (WSM) and molasses salt medium (MSM) have been constructed utilizing food processing byproduct whey and molasses for the production of bio-polysaccharide chitosan from Rhizopus oryzae and subsequently comprehensive physico-chemical characterization of the fungal chitosan has been carried out using various analytical tools to apprehend its biochemical utility. Same has been repeated with chitosan from conventional potato dextrose broth (PDB) for comparison purpose. The yields of chitosan in three different media were 0.62 (WSM), 0.39 (MSM) and 0.63 (PDB) g/L respectively. Molecular weights of the chitosans were in the range of 100-300 kDa. WSM-chitosan and MSM-chitosan were less polydispersed, possessed more hydrated polymorph and loose crystal packing than PDB-chitosan. This indicate that WSM-chitosan and MSM-chitosan are highly exposed to the external reagent hence more reactive to the external reagents with compare to PDB-chitosan. Literature suggest isolated chitosans are useful for specific drug delivery applications.

Targeted apoptosis in ovarian cancer cells through mitochondrial dysfunction in response to Sambucus nigra agglutinin.

Ovarian carcinoma (OC) patients encounter the severe challenge of clinical management owing to lack of screening measures, chemoresistance and finally dearth of non-toxic therapeutics. Cancer cells deploy various defense strategies to sustain the tumor microenvironment, among which deregulated apoptosis remains a versatile promoter of cancer progression. Although recent research has focused on identifying agents capable of inducing apoptosis in cancer cells, yet molecules efficiently breaching their survival advantage are yet to be classified. Here we identify lectin, Sambucus nigra agglutinin (SNA) to exhibit selectivity towards identifying OC by virtue of its specific recognition of α-2, 6-linked sialic acids. Superficial binding of SNA to the OC cells confirm the hyper-sialylated status of the disease. Further, SNA activates the signaling pathways of AKT and ERK1/2, which eventually promotes de-phosphorylation of dynamin-related protein-1 (Drp-1). Upon its translocation to the mitochondrial fission loci Drp-1 mediates the central role of switch in the mitochondrial phenotype to attain fragmented morphology. We confirmed mitochondrial outer membrane permeabilization resulting in ROS generation and cytochrome-c release into the cytosol. SNA response resulted in an allied shift of the bioenergetics profile from Warburg phenotype to elevated mitochondrial oxidative phosphorylation, altogether highlighting the involvement of mitochondrial dysfunction in restraining cancer progression. Inability to replenish the SNA-induced energy crunch of the proliferating cancer cells on the event of perturbed respiratory outcome resulted in cell cycle arrest before G2/M phase. Our findings position SNA at a crucial juncture where it proves to be a promising candidate for impeding progression of OC. Altogether we unveil the novel aspect of identifying natural molecules harboring the inherent capability of targeting mitochondrial structural dynamics, to hold the future for developing non-toxic therapeutics for treating OC.

Use of Spectroscopic Techniques to Reveal the Nature of the Interactions of Two Sialic Acid Specific Lectins with Gold Nanoparticles.

From UV-vis absorption, steady state and time resolved fluorescence measurements coupled with circular dichroism (CD) spectral studies, it was revealed that among the two lectins: Sambucus nigra agglutinin (SNA) and Saraca indica (saracin II), SNA forms stronger binding complex in the ground state with gold nanoparticles (GNPs). From the measurements of Stern-Volmer (SV) constants Ksv, and binding constants K(A) and number of binding sites two important inferences could be drawn. Firstly, the fluorescence quenching is primarily due to static quenching and secondly SNA forms stronger binding with GNPs relative to the other lectin saracin II. Synchronous fluorescence spectral measurements further substantiate this proposition of exhibiting the fully exposed tryptophan residue in case of SNA. It appears that the lectin SNA adopted a relatively looser conformation with the extended polypeptide structures leading to the exposure of the hydrophobic cavities which favoured stronger binding with GNPs. CD measurements demonstrate that gold nanoparticles when interact with the lectins (glycoproteins), no significant distortion in the structural pattern of the later occurs. The unaltered identity in the secondary structural pattern of both SNA and saracin II in presence of gold nanoparticles hints that GNPs may be used as useful drug or drug delivery systems.

A glucose/mannose binding lectin from litchi (Litchi chinensis) seeds: Biochemical and biophysical characterizations.

BACKGROUND: Lectins are highly important biomolecules to study several biological processes. A novel α-D-glucose/mannose specific lectin was isolated from the seeds of litchi fruits (Litchi chinensis) and its various biophysical and biochemical properties were studied. METHODS: Purification was done by successive Sephadex G 100 and Con A-Sepharose 4B affinity chromatography. SDS-PAGE, Surface Plasmon Resonance (SPR), steady state absorbance, fluorescence, time-correlated single-photon counting, circular dichroism and antibiofilm activity by measuring total protein estimation and azocasein degradation assay have been performed. RESULTS: The purified lectin is a homodimer of molecular mass ~ 54 kDa. The amount of lectin required for hemagglutination of normal human O erythrocytes was 6.72 µg/ml. Among the saccharides tested, Man-α-(1,6)-Man was found to be the most potent inhibitor (0.01 mM) determined by hemagglutination inhibition assay. Steady state and time resolved fluorescence measurements revealed that litchi lectin formed ground state complex with maltose (Ka=4.9 (±0.2)×104 M-1), which indicated static quenching (Stern-Volmer (SV) constant Ksv=4.6 (±0.2)×104 M-1). CD measurements demonstrated that litchi lectin showed no overall conformational change during the binding process with maltose. The lectin showed antibiofilm activity against Pseudomonus aeruginosa. CONCLUSIONS: A novel homodimeric lectin has been purified from the seeds of litchi fruits (Litchi chinensis) having specificity for α-d-glucose/mannose. The thermodynamics and conformational aspects of its interaction with maltose have been studied in detail. The antibiofilm activity of this lectin towards Pseudomonus aeruginosa has been explored. GENERAL SIGNIFICANCE: The newly identified litchi lectin is highly specific for α-d-glucose/mannose with an important antibiofilm activity towards Pseudomonus aeruginosa.

A study on antifungal activity of water-soluble chitosan against Macrophomina phaseolina.

The objective of this study was to evaluate antifungal effect of water-soluble chitosan (s-chitosan) on Macrophomina phaseolina (M. phaseolina) causing jute seedling infection and monitor the change in activity of released enzymes during infection. The minimum inhibitory concentration (MIC) of s-chitosan for M. phaseolina was found at 12.5g/l and s-chitosan exhibited fungistatic mode of action against this pathogen. The application of s-chitosan (12.5g/l) during infection of jute seedlings by M. phaseolina inhibited fungal infection and length of the seedlings was found almost similar to seedlings without infection. M. phaseolina infected jute seedlings showed length of 22mm over 10 days of incubation and it increased to 58mm in presence of s-chitosan (12.5g/l) during incubation for 10 days. TEM study indicated presence of hyphae in the cortical and epidermal cells of fungus infected jute seedlings indicating colonization by the fungus and it disappeared after treatment with s-chitosan. The changes in enzyme profiles of jute seedling during prevention of fungal infection using s-chitosan helped in proper understanding of mode of action of s-chitosan as antifungal agent. The activity of defense related enzymes like chitosanase and peroxidase in infected seedlings was observed to be enhanced after treatment with s-chitosan.

Jacalin bound plasma O-glycoproteome and reduced sialylation of alpha 2-HS glycoprotein (A2HSG) in rheumatoid arthritis patients.

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 (p<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.

Altered glycosylation and expression of plasma alpha-1-acid glycoprotein and haptoglobin in rheumatoid arthritis.

Altered glycosylation patterns in plasma proteins are found to be associated with the pathogenesis of various malignancies and autoimmune disorders. Our previous studies demonstrated the occurrence of some differentially glycosylated plasma proteins in rheumatoid arthritis (RA) patients. The current study was conducted to evaluate the alterations in expression and glycosylation of major acute phase proteins from wheat germ agglutinin enriched RA patients' plasma. Immunoblotting studies revealed a significant enhancement in the plasma levels of alpha-1 acid glycoprotein (AGP) and haptoglobin (Hp) in RA patients with respect to healthy controls. Monosaccharide analysis by high performance anion exchange-chromatography with pulse amperometric detection showed significant variations in the relative percentage of galactose, glucosamine and mannose in AGP and of mannose in Hp in RA patients. Altered patterns of mannosylation in AGP and Hp were also established by enzyme linked immunosorbent assay and Western blotting using Concanavalin-A lectin. These results could give information for understanding the disease pathogenesis and may provide an insight into the development and progression of the disease.

Macoma birmanica agglutinin recognizes glycoside clusters of beta-GlcNAc/Glc and alpha-Man.

Macoma birmanica agglutinin (MBA) that seems to play crucial roles in the innate immunity of marine bivalve, M. birmanica has been earlier defined as GlcNAc/Man specific. However, most complementary carbohydrate structures to its binding domain and ligand clustering in its recognition profile have not been established. In this study, the complete recognition profile of MBA was examined by enzyme-linked lectin-sorbent assay and inhibition assay. Among the monosaccharides tested, GlcNAc was more reactive followed by Man and Glc, others were non-reactive; revealing the importance of equatorial -NAc group at C-2, -OH group at C-4 and C-6, and pyranose conformation of hexose. Moreover, beta-glycosides of GlcNAc and Glc were more potent whereas for Man it was alpha-glycoside. MBA recognized both exposed and internal alpha-Man and beta-GlcNAc/Glc residues well with most linkages except (beta1-4). This binding pattern was further extended and confirmed by polyvalent glycoside clusters of GlcNAc(beta1-2)Man(alpha1-, which was a better inhibitor than Man(alpha1-2/3/6)Man(alpha1- or Man(alpha1-3/6)Man(beta1- present in well-defined naturally occurring glycoproteins. This broad range specificity explains the importance of MBA as an important pattern recognition molecule that provides more realistic picture of carbohydrate-based immune response triggering.

Enhanced expression of alpha1-acid glycoprotein and fucosylation in hepatitis B patients provides an insight into pathogenesis.

Altered glycosylation and concentration of alpha1-acid glycoprotein has been known to be related to the pathogenesis of the hepatic diseases. The present study investigated enhanced fucosylation of AGP in the sera of chronic hepatitis B (HBV-CH) and hepatitis B cirrhosis (HBV-LC) patients by high performance anion exchange chromatography and by ELISA using fucose binding Aleuria aurantia lectin. The concentration of AGP determined by ELISA using monoclonal anti-human AGP (mAb-AGP) showed high level of AGP in HBV-CH and HBV-LC patients. This was further judged by association constant (K (A)) measured by surface plasmon resonance analysis. There was no apparent linkage variation of sialic acid among different patient groups when tested with two sialic acid binding lectins viz., Maackia amurensis agglutinin (MAA, NeuAc alpha2-3-) and Sambucus nigra agglutinin (SNA, NeuAc alpha2-6-) respectively. There was no change of oligosaccharide branching in HBV-CH in comparison to controls whereas a slight change was observed in HBV-LC using ConA. The above results suggest that the changes in concentration of AGP and fucosylation have a prognostic value of hepatitis diseases and it could be possible to use AGP as diagnostic marker besides clinical examination and routine laboratory investigation.

Influence of plant growth hormones on the growth of Mucor rouxii and chitosan production.

Supplementation of molasses-salt medium with plant growth hormones, viz., indoleacetic acid, indolebutyric acid, kinetin and gibberellic acid, increased chitosan production by Mucor rouxii as well as its growth at different optimum concentrations. The increase in yield of chitosan was found to range from 34% to 69% and mycelial growth from 12% to 17.4%. Gibberellic acid was the most potent in this respect. Sixty-nine percent more chitosan over the control could be obtained from 1l of the medium supplemented with 3mg gibberellic acid. Degree of acetylation of chitosan ( approximately 13%) was not changed due to addition of hormone in the medium but weight average molecular weight of chitosan increased by more than 50%. Thus, the plant growth hormones add a value to chitosan by increasing its molecular weight.

Purification and characterization of an N-acetylglucosamine specific lectin from marine bivalve Macoma birmanica.

A calcium independent lectin of molecular mass 47kDa was isolated from the foot muscle of marine bivalve Macoma birmanica by ammonium sulphate precipitation followed by affinity chromatography on immobilized GlcNAc column and designated as M. birmanica agglutinin (MBA). The lectin agglutinated rabbit erythrocytes strongly compared to human erythrocytes over a wide pH range from 5 to 9 and up to 50 degrees C. MBA is a glycoprotein and consists of 7.63% sugar. Among the tested sugars for analysis of carbohydrate recognition properties, Me-betaGlcNAc was the most potent inhibitor followed by Me-alphaMan. Enzyme linked solid phase assay revealed that MBA interacted well with complex type N-linked glycans and moderately to high mannose type N-linked glycans. Fluorescence study of MBA indicated that tryptophan was present in a non-hydrophobic region and its binding to GlcNAc was neither quenched nor altered lambda(max) position. The denaturation of MBA induced by urea was a reversible process and urea could not significantly change the Trp environment. MBA interacted with both Gram-positive and Gram-negative bacteria by recognizing their surface exposed GlcNAc containing antigens.

Catfish (Clarias batrachus) serum lectin recognizes polyvalent Tn [alpha-D-GalpNAc1-Ser/Thr], Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc1-Ser/Thr], and II [beta-D-Galp(1-->4)-beta-D-GlcpNAc1-] mammalian glycotopes.

A new calcium dependent GalNAc/Gal specific lectin was isolated from the serum of Indian catfish, Clarias batrachus and designated as C. batrachus lectin (CBL). It is a disulfide-linked homodecameric lectin of 74.65kDa subunits and the oligomeric form is essential for its activity. Binding specificity of CBL was investigated by enzyme-linked lectin-sorbent assay using a series of simple sugars, polysaccharides, and glycoproteins. GalNAc was more potent inhibitor than Gal; and alpha glycosides of both were more inhibitory than their beta counterparts. CBL showed maximum affinity for human tumor-associated Tn-antigens (GalNAcalpha1-Ser/Thr) at the molecular level and was 3.5 times higher than GalNAc. CBL interacted strongly with polyvalent Tn and Talpha (Galbeta1,3GalNAcalpha1-) as well as multivalent-II (Galbeta1,4GlcNAcbeta1-) antigens containing glycoproteins and intensity of inhibition was 10(3)-10(5) times more than monovalent ones. The overall specificity of CBL lies in the order of polyvalent Tn, Talpha and II>>>>monovalent Tn > or = Me-alphaGalNAc>monovalent Talpha> Me-betaGalNAc>Me-alphaGal>monovalent T>GalNAc>monovalent F>monovalent II>Me-betaGal>Gal.

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 (IC(50) 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.

Enhancement of growth and chitosan production by Rhizopus oryzae in whey medium by plant growth hormones.

The effect of some plant growth hormones, viz., gibberellic acid, indole-3-acetic acid, indole-3-butyric acid, and kinetin on chitosan production by Rhizopus oryzae in deproteinized whey was studied. Hormones, at different concentrations, increase the mycelial growth by 19-32%. However, increase in chitosan content of the mycelia was relatively small (1.7-14.3%) over the control. Maximum enhancement was observed with gibberellic acid. Fifty percent more chitosan could be obtained from 1L of whey containing 0.1mg/L gibberellic acid. Hormones, at higher dose, instead of stimulation inhibited both growth and mycelial chitosan content. This study showed that hormones have no influence on degree of deacetylation of chitosan but increase the quality of the chitosan by increasing weight average molecular weight and decreasing polydispersity. All the hormones had been found to enhance chitin deacetylase activity of R. oryzae by 1.067-1.267-fold and may be one of the reasons for increased chitosan production.

Role of pepsin in modifying the allergenicity of bhetki (Lates calcarifer) and mackerel (Rastrelliger kanagurta) fish.

The 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.

Multivalent II [beta-D-Galp-(1-->4)-beta-D-GlcpNAc] and Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc] specific Moraceae family plant lectin from the seeds of Ficus bengalensis fruits.

A galactose specific lectin was isolated from the seeds of Ficus bengalensis (Moraceae) fruits and designated as F. bengalensis agglutinin (FBA). The lectin was purified by affinity repulsion chromatography on fetuin-agarose and was a monomer of molecular mass 33kDa. Like other Moraceae family lectins, carbohydrate-binding activity of FBA was independent of any divalent cation. FBA did not bind with simple saccharides, however sugar ligands with aromatic aglycons showed pronounced binding. The combining site of FBA recognized preferably Galbeta1,4GlcNAcbeta1-(II) followed by Galbeta1,3GalNAcalpha1-(Talpha) containing glycotopes. Interaction with saccharides revealed that the combining site of FBA could well accommodate a tetrasaccharide, asialo GM1 glycan (Galbeta1,3GalNAcbeta1,4Galbeta1,4Glcbeta1-), whereas polyvalent Tn (GalNAcalpha1-Ser/Thr), one of the well-recognized ligands of Moraceae family lectin, did not interact well with FBA.

Identification of allergens in Indian fishes: hilsa and pomfret exemplified by ELISA and immunoblotting.

Enzymed-linked immunosorbent 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.

Adsorption of a model anionic dye, eosin Y, from aqueous solution by chitosan hydrobeads.

The process of sorption is being increasingly used for ecofriendly and economic remediation of textile dye effluents. The present model study deals with the adsorption of a model anionic dye, eosin Y, from wastewater using conditioned chitosan hydrobeads. Conditioning reduced the pH sensitivity and maintained the maximum sorption capacity of the beads near pH 8. To understand the chemicophysical characteristics of the adsorption process we studied, the kinetics and isotherm behavior of the system. It was observed that temperature played a significant role in the process. The Langmuir model was found to be most appropriate for the description of the adsorption process. The kinetic results followed a second-order equation. It was observed that 1 g of chitosan adsorbed approximately 76 mg of eosin Y. The dye was desorbed from the beads by changing the pH of the solution, and the conditioned chitosan beads were reused five times without any loss of mechanical and chemical efficacy.