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.     

Redox metabolic and molecular parameters for screening drought tolerant indigenous aromatic rice cultivars

The present work makes an effort to assess and standardize some redox metabolic and molecular parameters for screening drought tolerant indigenous aromatic rice cultivars of West Bengal, India. PEG-induced dehydration stress during early germination caused disruption of redox-homeostasis and oxidative damage in four IARVs (Jamainadu, Tulaipanji, Sitabhog and Badshabhog) by enhancing the accumulation of pro-oxidants [assessed in terms of oxidation of 2′,7′-dichlorofluorescindiacetate (DCFDA), accumulation of \({\text{O}}_{2}^{ \cdot - }\) and H₂O₂ and in situ staining of reactive oxygen species (ROS) in germinating tissue], significant reduction of antioxidative defence (total antioxidant and radical scavenging capacity, total thiol content and activities of antioxidative defence enzymes) and aggravating protein oxidation and lipid peroxidation (assessed in terms of free carbonyl content and accumulation of thiobarbituric acid reactive substances). When compared between the indigenous aromatic rice cultivars, a clear trend in differential redox regulatory properties in which ROS-antioxidant interaction acts at metabolic interface for redox homeostasis was observed in the order Badshabhog > Tulaipanji > Sitabhog > Jamainadu. Moreover, when the efficacy of ascorbate–glutathione cycle for scavenging H₂O₂ generated during dehydration stress was assessed and compared between the landraces exposed to PEG-induced dehydration stress in germinating tissue, it also exhibited almost the same trend with the landrace Tulaipanji and Badsabhog exhibiting maximum and Jamainadu the minimum efficiencies of the redox cycle. The indigenous aromatic rice cultivars Tulaipanji and Badsabhog resist dehydration stress better than the other two landraces due to its early preparedness to combat oxidative stress by up-regulating expression of genes of some enzymes of ascorbate–glutathione cycle along with some other antioxidative enzymes. A model of redox homeostasis in which ROS-antioxidant (ascorbate–glutathione system) acts at metabolic interface for up-regulation of antioxidative gene expression necessary for differential drought stress tolerance among the indigenous aromatic rice varieties is suggested.     

Binding affinity and in vitro cytotoxicity of harmaline targeting different motifs of nucleic acids: An ultimate drug designing approach

The work focuses towards interaction of harmaline, with nucleic acids of different motifs by multispectroscopic and calorimetric techniques. Findings of this study suggest that binding constant varied in the order single‐stranded (ss) poly(A) > double‐stranded calf thymus (CT) DNA > double‐stranded poly(G)·poly(C) > clover leaf tRNA^Phe. Prominent structural changes of ss poly(A), CT DNA, and poly(G)· poly(C) with concomitant induction of optical activity in the bound achiral alkaloid molecule was observed, while with tRNA^Phe, very weak induced circular dichroism perturbation was seen. The interaction was predominantly exothermic, enthalpy driven, and entropy favored with CT DNA and poly(G)·poly(C), while it was entropy driven with poly(A) and tRNA^Phe. Intercalated state of harmaline inside poly(A), CT DNA, and poly(G)·poly(C) was shown by viscometry, ferrocyanide quenching, and molecular docking. All these findings unequivocally pointed out preference of harmaline towards ss poly(A) inducing self‐structure formation. Furthermore, harmaline administration caused a significant decrease in proliferation of HeLa and HepG₂ cells with GI₅₀ of 28μM and 11.2μM, respectively. Nucleic acid fragmentation, cellular ultramorphological changes, decreased mitochondrial membrane potential, upregulation of p53 and caspase 3, generation of reactive oxygen species, and a significant increase in the G₂/M population made HepG₂ more prone to apoptosis than are HeLa cells.