Structural characterization of the apo form of a calcium binding protein from Entamoeba histolytica by hydrogen exchange and its folding to the holo state

One of the calcium binding proteins from Entamoeba histolytica (EhCaBP) is a 134 amino acid residue long (M(r) approximately 14.9 kDa) double domain EF-hand protein containing four Ca(2+) binding sites. CD and NMR studies reveal that the Ca(2+)-free form (apo-EhCaBP) exists in a partially collapsed form compared to the Ca(2+)-bound (holo) form, which has an ordered structure (PDB ID ). Deuterium exchange studies on the partially structured apo-EhCaBP reveal that the C-terminal domain is better structured than the N-terminal domain. The protein can be reversibly folded and unfolded upon addition of Ca(2+) and EGTA, respectively. Titration shows a slow initial folding of the apo form with increasing Ca(2+) concentration, followed by a highly cooperative folding to its final state at a certain threshold of Ca(2+). Ca(2+) and the EGTA titration taken together show that site II in the N-terminal domain has the highest affinity for Ca(2+) contrary to earlier studies. Further, this study has thrown light on the relative Ca(2+) binding affinity and specificity of each site in the intact protein. A structural model for the partially collapsed form of apo-EhCaBP and its equilibrium folding to its completely folded holo state has been suggested. Large conformational changes seen in transforming from the apo to holo form of EhCaBP suggest that this protein should be functioning as a sensor protein and might have a significant role in host-parasite recognition.     

Essential role of beta-catenin in postnatal bone acquisition

Mutations in the Wnt co-receptor LRP5 alter bone mass in humans, but the mechanisms responsible for Wnts actions in bone are unclear. To investigate the role of the classical Wnt signaling pathway in osteogenesis, we generated mice lacking the beta-catenin or adenomatous polyposis coli (Apc) genes in osteoblasts. Loss of beta-catenin produced severe osteopenia with striking increases in osteoclasts, whereas constitutive activation of beta-catenin in the conditional Apc mutants resulted in dramatically increased bone deposition and a disappearance of osteoclasts. In vitro, osteoblasts lacking the beta-catenin gene exhibited impaired maturation and mineralization with elevated expression of the osteoclast differentiation factor, receptor activated by nuclear factor-kappaB ligand (RANKL), and diminished expression of the RANKL decoy receptor, osteoprotegerin. By contrast, Apc-deficient osteoblasts matured normally but demonstrated decreased expression of RANKL and increased osteoprotegerin. These findings suggest that Wnt/beta-catenin signaling in osteoblasts coordinates postnatal bone acquisition by controlling the differentiation and activity of both osteoblasts and osteoclasts.     

Sanguinarine: an evaluation of in vivo cytogenetic activity

Sanguinarine (SG), a benzophenanthridine alkaloid, has been shown to possess anti-microbial, anti-inflammatory and antioxidant properties. In the UK and USA its salts has been in use in mouthwashes and toothpastes to inhibit dental plaque and improve gingival health. In India and Nepal consumption of mustard oil contaminated with argemone seeds containing sanguinarine, was associated with "dropsy" syndrome. In the present study, SG was evaluated in vivo in mouse bone marrow cells for its ability to induce clastogenicity and DNA damage in terms of increased sister chromatid exchange (SCE) frequencies. Doses of 5, 10, and 15 mg/kg body weight of SG given intra peritoneally induced a positive dose-dependent significant clastogenicity and SCE frequency increases (trend test alpha < or = 0.05). The minimum effective concentration to induce clastogenic and DNA damage was 10mg of SG/kg body weight. In addition to examining SCEs, the BrdUrd-differential technique was utilized to assess the effect of SG on cell replication. The analysis revealed that SG treatment did not significantly affect the distribution of cells among the different phases of the cell cycle. The proliferation rate index and average generation time data were statistically non-significant. This indicated that the alkaloid was not cytotoxic to the bone marrow cells at the doses tested. Based on the results of the present findings, the use of this alkaloid should be restricted.     

Cardiac overexpression of myotrophin triggers myocardial hypertrophy and heart failure in transgenic mice

Cardiac hypertrophy and heart failure remain leading causes of death in the United States. Many studies have suggested that, under stress, myocardium releases factors triggering protein synthesis and stimulating myocyte growth. We identified and cloned myotrophin, a 12-kDa protein from hypertrophied human and rat hearts. Myotrophin (whose gene is localized on human chromosome 7q33) stimulates myocyte growth and participates in cellular interaction that initiates cardiac hypertrophy in vitro. In this report, we present data on the pathophysiological significance of myotrophin in vivo, showing the effects of overexpression of cardio-specific myotrophin in transgenic mice in which cardiac hypertrophy occurred by 4 weeks of age and progressed to heart failure by 9-12 months. This hypertrophy was associated with increased expression of proto-oncogenes, hypertrophy marker genes, growth factors, and cytokines, with symptoms that mimicked those of human cardiomyopathy, functionally and morphologically. This model provided a unique opportunity to analyze gene clusters that are differentially up-regulated during initiation of hypertrophy versus transition of hypertrophy to heart failure. Importantly, changes in gene expression observed during initiation of hypertrophy were significantly different from those seen during its transition to heart failure. Our data show that overexpression of myotrophin results in initiation of cardiac hypertrophy that progresses to heart failure, similar to changes in human heart failure. Knowledge of the changes that take place as a result of overexpression of myotrophin at both the cellular and molecular levels will suggest novel strategies for treatment to prevent hypertrophy and its progression to heart failure.     

Redox reactions and fluorescence spectroscopic behaviour of trifluoperazine at the surface of colloidal silica

Fluorescence spectroscopic behaviour and redox reactions of trifluoperazine (TFP) were studied in aqueous solutions in the presence of silica (SiO2) particles. The effect of surface heterogeneity on the secondary reactions of the transients was determined using optical absorption and fluorescence techniques. It appears that electrostatic interaction is the driving force for adsorption of TFP over SiO2 particle. Contrary to the change in fluorescence intensity with time observed in an aqueous solution, fluorescence intensity of TFP did not change significantly with time over the surface of SiO2. Fluorescence microscopic observations of spleen cells treated with TFP over SiO2 showed that the drug got distributed in cells similar to that observed in homogeneous aqueous solution.     

Uteroglobin suppresses SCCA gene expression associated with allergic asthma

Uteroglobin (UG), the founding member of the Secretoglobin superfamily, is a potent anti-inflammatory protein constitutively expressed at a high level in the airway epithelia of all mammals. We previously reported that the lungs of UG-knock-out (UG-KO) mice express elevated levels of Th2 cytokines (e.g. interleukin (IL)-4 and IL-13), which are augmented by allergen sensitization and challenge leading to exaggerated airway inflammation. Notably, these responses are suppressed by recombinant UG treatment (Mandal, A. K., Zhang, Z., Ray, R., Choi, M. S., Chowdhury, B., Pattabiraman, N., and Mukherjee, A. B. (2004) J. Exp. Med. 199, 1317-1330). Recent reports indicate that human orthologs of murine squamous cell carcinoma antigen-2 (SCCA-2/serpinb3a), a serine protease-inhibitor, are overexpressed in the airways of asthmatic patients. We report here that compared with wild type littermates, UG-KO mouse lungs express markedly elevated levels of SCCA-2 mRNA and protein, which are augmented by allergen-challenge. Most importantly, these effects are abrogated by recombinant UG treatment. We further demonstrate that treatment of cultured human bronchial epithelial cells with IL-4 or IL-13 stimulates phosphorylation of STAT-1 and STAT-6 leading to SCCA-1 (SERPINB3) and SCCA-2 (SERPINB4) gene expression. We propose that: (i) IL-4- and IL-13-stimulated SCCA gene expression is mediated via STAT-1 and STAT-6 activation, and (ii) by suppressing the production, and most likely by interfering with the signaling of these cytokines, UG inhibits SCCA gene expression associated with airway inflammation in asthma.     

Fsr1, a striatin homologue,forms an endomembrane‐associated complex that regulates virulence in the maize pathogen Fusarium verticillioides

Fsr1, a homologue of mammalian striatin, containing multiple protein‐binding domains and a coiled‐coil (CC) domain, is critical for Fusarium verticillioides virulence. In mammals, striatin interacts with multiple proteins to form a STRIPAK (striatin‐interacting phosphatase and kinase) complex that regulates a variety of developmental processes and cellular mechanisms. In this study, we identified the homologue of a key mammalian STRIPAK component STRIP1/2 (striatin‐interacting proteins 1 and 2) in F. verticillioides, FvStp1, which interacts with Fsr1 in vivo. Gene deletion analysis indicates that FvStp1 is critical for F. verticillioides stalk rot virulence. In addition, we identified three proteins, designated FvCyp1, FvScp1 and FvSel1, which interact with the Fsr1 CC domain via a yeast two‐hybrid screen. Importantly, FvCyp1, FvScp1 and FvSel1 co‐localize to endomembrane structures, each having a preferred localization in the cell, and they are all required for F. verticillioides stalk rot virulence. Moreover, these proteins are necessary for the correct localization of Fsr1 to the endoplasmic reticulum (ER) and nuclear envelope. Thus, we identified several novel components in the STRIPAK complex that regulates F. verticillioides virulence, and propose that the correct organization and localization of Fsr1 are critical for STRIPAK complex function.     

Freeze-etch appearance of the tight junctions in the epithelium of small and large intestine of mice

Summary The ultrastructure of the central layer and the contributing plasma membranes of tight junctions has been studied in epithelia of the jejunum and colon of mice.Examination of freeze-etched plasma membranes of epithelial cells has revealed that they consist of a central layer, with fracturing characteristics similar to bimolecular lipid leaflets, which is covered on both sides with a layer of particles.The fusion of the outer membrane surfaces of adjacent cells in the region of the tight junction leads to the formation of a new common structure consisting of a meshwork of fibrils embedded in a matrix substance. The fibrils probably contain protein. They have a diameter of 65 ± 10 Å and are linked together so that they form around the distal end of each cell a continuous belt-like meshwork which is extended proximally at the joints where three cells meet. As the fibrillar mesh appears to be strongly attached to the central lipid layer of the two adjoining membranes, in contrast to the weakly bound surrounding matrix, it is believed that the fibrils forming the continuous meshwork could be the mechanical coupling and the sealing elements of the tight junction. Their arrangement in the form of a concertinalike mesh would make the whole structure very flexible. In the region of the junction the membranes are constricted along the lines of attachment to the fibrils and bulge outwards,i.e. towards the cytoplasm, in the areas of the matrix material. In the resulting grooves on the cytoplasmic side of the plasma membranes regularly spaced particles with a diameter of 90 ± 10 Å can be detected. Various observations suggest that these particles could be connected through the central layer of the membranes to the fibrils on the other side. This would offer a possible explanation for the known abhesion properties of tight junctions. The described structures are also evaluated in terms of current theories of cell communication.