Protein purification using a soluble affinity matrix: purification of estrogen receptor with estradiol-polylysine conjugate.

A new strategy for protein purification using a soluble affinity matrix is described. The method was used for purification of estrogen receptor. Cytosols from rat uteri and human fibroid uterine tissue, after fractionation by ammonium sulfate, were treated with estradiol-polylysine conjugate. The highly basic affinity complex was separated from other proteins by DEAE-Sephacel chromatography. After dissociation of the eluted complex with excess estradiol, the receptor was recovered by CM-Sephadex chromatography. A 2000-fold purification of the rat uterine estrogen receptor was obtained with an activity recovery of 35%.     

Binding of estradiol-peroxidase conjugate to estrogen receptor

The binding of estradiol-horseradish peroxidase conjugate to rat uterine cytosolic estrogen receptor was studied. The conjugate having a steroid to enzyme ratio of 2.8:1 was allowed to bind to protamine precipitated receptor in presence or absence of 100-fold excess of free estradiol. The bound enzyme activity was measured and the data subjected to Scatchard analysis to obtain the dissociation constant and the number of binding sites. Although the binding parameter so obtained differed from values obtained using radiolabelled estradiol, the method may be used for comparative studies.     

Effect of near ultraviolet and visible light on amoeba.

The near ultraviolet and visible light (VL) impinging at an intensity of 2-5 x 10(2) J s-1 m-2 for 2-5 h kills the mitotic and the early S-phase (0- to 15-min-old) amoebae. At the mid- and late S-period only a fraction of cells are killed by VL and G2 phase cells are quite resistant. Amoebae of all cell cycle stages show a delay in the first mitotic division. DNA synthesis, as measured by [3H]thymidine incorporation, is depressed in the VL-exposed early-S amoebae. A concurrent but temporary inhibition in [3H]leucine incorporation also occurs in these cells. However, no significant change in [3H]uridine incorporation has been found. To localize the site of lethal damage, nuclear transplantation studies were undertaken between the control amoebae and the amoebae treated with VL. The nucleus of a VL-exposed early S-phase cell recovers when transplanted immediately after VL exposure into an enucleate G2 cytoplasm but dies if grafted into an enucleat S-phase cytoplasm. The therapeutic effect of the G2 cytoplasm, although at a lower level, is also evident even when the treated early S-phase nucleus is implanted 20 h later, but not after 48 h, into the G2 cytoplasm. The amoeba cytoplasm shows resistance to VL-irradiation, can accept a control nucleus from any cell cycle stage, and function normally. The G2 nucleus also remains apparently unaffected to VL exposure and can survive when it is transfered to the control cytoplasm of any cell-cycle phase. All these findings are discussed in the light of the possible existence of a repair system against VL-induced damage in the G2-phase amoeba.     

Dual binding specificities in MOPC 384 and 870 murine myeloma immunoglobulins.

Homogeneous murine myeloma immunoglobulins (IgA, kappa), M 384, and M 870, bind methyl alpha-D-galactopyranoside and phosphorylcholine at different subsites. Heterologous recombinant immunoglobulins of these two immunoglobulins with M 603 (a homogeneous IgA, kappa with known phosphorylcholine specificity) also bind phosphorylcholine.     

Dichotomy in Growth and Invasion from Low- to High-Grade Glioma Cellular Variants

Glial dysfunction outraging CNS plasticity and integrity results in one of the most dangerous cancers, namely glioma, featuring little median survival period and high recurrence. The hallmark properties of proliferation, invasion and angiogenesis with the infiltrated macrophages in glioma are expected to be tightly coupled or cross-linked, but not properly related so far. The present study is aimed to find a relationship between this featured quadrangle from lower to higher grades (HG) of post-operative glioma tissues and their invading subsets. Elevated Ki67-associated proliferation in lower grades (LG) was supported with VEGF dependent angiogenic maintenance which found a decrease unlikely in HG. In contrast, MMP 2 and 9-associated invasions augmented high in HG with the dominant presence of CD204⁺ M2 polarized macrophages and a general increase in global DNMT1-associated methylation. Marked differences found in ECM invading cellular subsets of HG showing high proliferative capacity indicating rationally for recurrence, contrasting the nature of gross tumor tissue of the same grade. Thus in LG, the neoplastic lesion is more inclined to its growth while in higher grade more disposed towards tissue wreckage in support with cellular environmental milieu whereas the cellular variants and subsets of invaded cells showed different trends. Therefore, some operational dichotomy or coupling among cellular variants in glioma is active in determining its low- to high-grade transition and aggressive progression.

     

An Enzyme from Aristolochia indica Destabilizes Fibrin-β Amyloid Co-Aggregate: Implication in Cerebrovascular Diseases

Fibrinogen and β-amyloid (Aβ) peptide independently form ordered aggregates but in combination, they form disordered structures which are resistant to fibrinolytic enzymes like plasmin and cause severity in cerebral amyloid angiopathy (CAA). A novel enzyme of 31.3 kDa has been isolated from the root of the medicinal plant Aristolochia indica that showed fibrinolytic as well as fibrin-Aβ co-aggregate destabilizing properties. This enzyme is functionally distinct from plasmin. Thrombolytic action of the enzyme was demonstrated in rat model. The potency of the plant enzyme in degrading fibrin and fibrin-plasma protein (Aβ, human serum albumin, lysozyme, transthyretin and fibronectin) co-aggregates was demonstrated by atomic force microscopy, scanning electron microscopy and confocal microscopy that showed better potency of the plant enzyme as compared to plasmin. Moreover, the plant enzyme inhibited localization of the co-aggregate inside SH-SY5Y human neuroblastoma cells and also co-aggregate induced cytotoxicity. Plasmin was inefficient in this respect. In the background of limited options for fragmentation of these co-aggregates, the plant enzyme may appear as a potential proteolytic enzyme.