Carbohydrate induced modulation of cell membrane VII. Binding of exogenous lectin increases osmofragility of erythrocytes

AbstractDue to their multivalent binding character, lectins when added exogenously will cross-link membrane surface receptors leading to lateral molecular reorganizations in the plane of the bilayer. This study reports for the first time that agglutination of rabbit erythrocytes by lentil lectin and concanavalin A increases their osmofragility. Increase in osmofragility was detected by measuring the hemolysis of erythrocytes in hypotonic as well as in isotonic solutions. It was also found that agglutination per se does not increase osmofragility but the binding of legume lectin is essential since human Rh+ cells agglutinated by a monoclonal antibody do not exhibit hemolysis

Immune Responses to Defined Plasmodium Falciparum Antigens and Disease Susceptibility in Two Subpopulations of Northern India

The aim of this study was to investigate the prevalence of naturally acquired immune response to malaria in individuals of different age groups belonging to areas of northern India, Loni PHC (LN) and Dhaulana PHC (SD) of district Ghaziabad. Plasmodium falciparum-infected erythrocyte lysate and six synthetic peptides from different stages of P. falciparum (CSP, MSP1, AMA1, RAP1, EBA175 and PfG27) were used to determine both humoral and cellular immune responses. Plasma of individual subject was also analyzed for IL-4, IL-10, IFN-γ and TNF-α level. We observed an age-wise increasing trend of immunity in these two populations. There was a significant association between the number of antibody responders and recognition of stage-specific epitopes by antibodies. Peripheral blood mononuclear cells of more than 75% of individuals proliferated in response to stimulation by all the antigens in LN area. IL-4 and IL-10 responses were significantly higher in individuals of LN Area; whereas IFN-g and   TNF-a responses were higher in individuals of SD Area. It was also noticed that the frequency of responders to stage-specific antigens was higher in individuals from the LN area where the frequency of malaria was lower. The naturally acquired immune responses to P. falciparum antigens reflected the reduced risk of malaria in the study groups. The results demonstrated immunogenicity of the epitopes to P. falciparum in population of this endemic zone

Advances in Adoptive Cellular Therapy (ACT)

Adoptive T cell therapy (ACT) is getting acknowledged as the Advanced Therapy Medicinal Products (ATMPs) in many countries and it has evolved as one of the newest regimens to treat cancer. Developed gradually by the basic understanding of cells, involved in innate and adaptive immunity, ACT has emerged as one of the successful immunotherapies in recent times. It broadly includes various cell types such as stem cells, T cells, dendritic cells and Natural Killer cells. By the applications of genetic engineering and advanced cell culture techniques, these cells from patients’ blood, can be manipulated to train them for better efficacy against specific tumor cells. However, only some cells’ subsets have shown promising regression for certain cancer cells types. To understand the reason behind this, technical knowledge about the tumor antigens presentation, tumor microenvironment (TME), hosts’ immune responses and possible issues in the manufacturing of adoptive cellular material for infusion in patients are being explored further. This chapter brings together development of immune cells from basic research to clinical use, newer approaches which have been taken to address the resistance of ACT and future promises of this therapy

Cleavage of Kininogen and Subsequent Bradykinin Release by the Complement Component: Mannose-Binding Lectin-Associated Serine Protease (MASP)-1

Bradykinin (BK), generated from high-molecular-weight kininogen (HK) is the major mediator of swelling attacks in hereditary angioedema (HAE), a disease associated with C1-inhibitor deficiency. Plasma kallikrein, activated by factor XIIa, is responsible for most of HK cleavage. However other proteases, which activate during episodes of angioedema, might also contribute to BK production. The lectin pathway of the complement system activates after infection and oxidative stress on endothelial cells generating active serine proteases: MASP-1 and MASP-2. Our aim was to study whether activated MASPs are able to digest HK to release BK. Initially we were trying to find potential new substrates of MASP-1 in human plasma by differential gel electrophoresis, and we identified kininogen cleavage products by this proteomic approach. As a control, MASP-2 was included in the study in addition to MASP-1 and kallikrein. The proteolytic cleavage of HK by MASPs was followed by SDS-PAGE, and BK release was detected by HPLC. We showed that MASP-1 was able to cleave HK resulting in BK production. MASP-2 could also cleave HK but could not release BK. The cleavage pattern of MASPs is similar but not strictly identical to that of kallikrein. The catalytic efficiency of HK cleavage by a recombinant version of MASP-1 and MASP-2 was about 4.0×102 and 2.7×102 M−1s−1, respectively. C1-inhibitor, the major inhibitor of factor XIIa and kallikrein, also prevented the cleavage of HK by MASPs. In all, a new factor XII- and kallikrein-independent mechanism of bradykinin production by MASP-1 was demonstrated, which may contribute to the pro-inflammatory effect of the lectin pathway of complement and to the elevated bradykinin levels in HAE patients

Isolation, purification and characterization of a protease from the seeds of Artocarpus heterophyllus

690-702Proteases are being widely used in various industries like detergent, leather, food and pharmaceuticals.Protease was purified to homogeneity from the seeds of Artocarpus heterophyllus. The enzyme was found to be a tetramer having molecular mass of 74 kDa. Gelatin zymography showed a clear band of proteolysis. The enzyme isolated and purified was a serine protease, as indicated by its inhibition with PMSF. The enzyme was stable at broad pH and temperature ranges with pH and temperature optima at 8.5 and 50°C, respectively. The presence of some divalent ions enhanced the activity. With the addition of calcium, change in absorption and emission spectra was observed in spectrofluorometric analysis. The Km and Vmax for the enzyme was found to be 0.229 μM and 0.014 μM min1, respectively, using BAPNA as a substrate. The enzyme consisted 4.44% alpha helix and 44.17% beta sheets when measured by CD spectra. Dynamic light scattering of the protease for particle size distribution revealed the mono-dispersity of the sample. Easy purification and paramount stability of protease makes it a good candidate for industrial and pharmaceutical applications

Purification and characterization of lipase from a new strain Staphylococcus argenteus MG2 (MTCC 12820)

The lipase enzyme was isolated and purified from Staphylococcus argenteus MG2 (MTCC 12820) to homogeneity using ammonium sulphate precipitation followed by chromatographic techniques. This process resulted in a purification factor of 40.96-fold and a 26.25% recovery with a specific activity of 744.68 U mg-1. The molecular weight of the purified lipase was determined by SDS-PAGE to be 45 kDa. The Km and Vmax values of the purified lipase were calculated to be 4.95 mM and 79.36 µmol/min/mg-1, respectively. The maximum lipase activity was observed at pH 7.0 and 30 ºC with 100% stability, and it was also found to be stable in a broad range of pH (5-12) and temperature (30-90 ºC). The enzymatic activity of this Staphylococcal lipase was increased by Ca2+ to 105.71% at a concentration of 1 mM CaCl2. Additionally, it exhibited marked stability and activity in organic solvents. In the presence of 1% SDS surfactant, it retained 85.16% residual activity, while the metal chelator EDTA (inhibitor) reduced the lipase activity to 83.87% residual activity at a concentration of 1% w/v. This alkali-stable and thermo-stable lipase can be exploited by extending its use in the preparation of detergents and in various industrial and biotechnological applications

ESR studies on the effect of ionic radii on displacement of Mn2+ bound to a soluble β-galactoside binding hepatic lectin

AbstractBinding of divalent metal ions to hepatic soluble β-galactoside binding lectin was studied using electron spin resonance (ESR) spectroscopy. The Mn2+ bound to hepatic lectin could be displaced by Mg2+, Cu2+, Ni2+ and Ca2+ but not by Sr2+. As the ionic radii of Mg2+ (0.65 Å), Cu2+ (0.73 Å) and Ni2+ (0.72 Å) are appreciably smaller than Ca Mn2+ binding site is more accessible to Mg2+, Cu2+, and Ni2+ as compared to Ca2+, the ionic radius of Mn2+ being 0.80 Å. Sr2+ with an ionic radius of 1.13 is thus unable to displace bound Mn2+. Surprisingly, the presence of specific sugars like α-lactose, or α-d-galactose facilitated the displacement of bound Mn2+ by metal ions whereas non-specific sugars, i.e. α-d-glucose, β-d-fructose and α-d-ribose had no effect. It appears that minor perturbations in the saccharide binding site significantly affect the ability of the metal binding site to ligate bivalent metals

EPR studies on Fcϒ receptor-mediated changes in lymphocyte membrane

59-61Biophysical evidence has been presented for the interaction of human lymphocyte membrane Fc receptors with aggregated IgG by severely restricting the rotational mobility of the cell surface proteins, as well as membrane lipids. Decrease in membrane fluidity was more prominent with aggregated IgG since the multivalency of Fc regions in aggregated IgG cross-linked cell surface Fc receptor

The ORF3 Protein of Hepatitis E Virus Delays Degradation of Activated Growth Factor Receptors by Interacting with CIN85 and Blocking Formation of the Cbl-CIN85 Complex▿

Hepatitis E virus (HEV) causes an acute self-limiting disease that is endemic in developing countries. Previous studies suggested that the ORF3 protein (pORF3) of HEV is required for infection in vivo and is likely to modulate the host response. Our previous work showed that pORF3 localizes to early and recycling endosomes and causes a delay in the postinternalization trafficking of epidermal growth factor receptor (EGFR) to late endosomes/lysosomes. Here we report that pORF3 also delays the trafficking and degradation of activated hepatocyte growth factor receptor (c-Met) and delineate the mechanistic details of these effects. A mutant ORF3 protein, which does not localize to endosomes, also showed similar effects on growth factor receptor trafficking, making this effect independent of the endosomal localization of pORF3. The ORF3 protein was found to interact with CIN85, a multidomain adaptor protein implicated in the Cbl-mediated downregulation of receptor tyrosine kinases. This interaction competed with the formation of the growth factor receptor-Cbl-CIN85 complex, resulting in the reduced ubiquitination of CIN85 and trafficking of the growth factor receptor complex toward late endosomes/lysosomes. We propose that through its effects on growth factor receptor trafficking, pORF3 prolongs endomembrane growth factor signaling and promotes cell survival to contribute positively to viral replication and pathogenesis