Adhesion of flowing monocytes to hypoxia-reoxygenation-exposed endothelial cells: role of Rac1, ROS,and VCAM-1

Production ofreactive oxygen species (ROS) by ischemic tissue afterischemia-reperfusion (I/RP) is an important factor that contributes to tissue injury. The small GTPase Rac1 mediates the oxidative burst, and ROS act on signaling pathways involved in expression of inflammatory genes. Because there is evidence implicating monocytes in the pathogenesis of I/RP injury, our objective was todetermine the molecular mechanisms that regulate adhesive interactions between monocytes and hypoxia-reoxygenation (H/RO)-exposed cultured endothelial cells (ECs). When U937 cells were perfused over human umbilical vein ECs at 1 dyn/cm², H (1 h at 1%O₂)/RO (13 h) significantly increased the fluxes of rollingand stably adherent U937 cells. Either EC treatment with theantioxidant pyrrolidine dithiocarbamate (PDTC) or infection withAdRac1N17, which results in expression of the dominant-negative form ofRac1, abolished H/RO-induced ROS production, attenuated rolling, andabolished stable adhesion of U937 cells to H/RO-exposed ECs. Infectionwith AdRac1N17 also abolished H/RO-induced upregulation of vascularcell adhesion molecule (VCAM)-1. In turn, blocking VCAM-1 abolishedU937 cell stable adhesion and slightly increased rolling. We concludedthat the Rac1-dependent ROS partially regulate rolling and exclusivelyregulate stable adhesion of monocytic cells to ECs after H/RO and thatstable adhesion, but not rolling, is mediated by ROS-induced expressionof VCAM-1.

     

Herpetic stromal keratitis in the absence of viral antigen recognition

Herpetic stromal keratitis (HSK), resulting from ocular infection with herpes simplex virus (HSV), is thought to represent a T cell mediated immunopathologic lesion. Antigens recognized by the inflammatory T cells remain unresolved and non-TCR mediated activation of T cells (bystander activation) is considered as also involved. This report documents further evidence for the bystander activation mechanisms using three T cell transgenic RAG-/- mouse strains. Accordingly HSK occurred in PCC RAG-/-, P14 RAG-/-, and OT-1 RAG-/- mice. In none of the models could HSV specific T cell reactivity be demonstrated and animals were unprotected from lesion development by immunization prior to HSV ocular infection. The results support the role of bystander activation as a mechanism of T cell mediated immunopathology and show that CD8(+) as well as CD4(+) T cells can participate in HSK lesion development.     

Alpha-crystallin binds to the aggregation-prone molten-globule state of alkaline protease: implications for preventing irreversible thermal denaturation

Alpha-crystallin, the major eye-lens protein with sequence homology with heat-shock proteins (HSPs), acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain more insight into its chaperoning ability, we used a protease as the model system that is known to require a propeptide (intramolecular chaperone) for its proper folding. The protease ("N" state) from Conidiobolus macrosporus (NCIM 1298) unfolds at pH 2.0 ("U" state) through a partially unfolded "I" state at pH 3.5 that undergoes transition to a molten globule-(MG) like "I(A)" state in the presence of 0.5 M sodium sulfate. The thermally-stressed I(A) state showed complete loss of structure and was prone to aggregation. Alpha-crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The alpha-crystallin-bound I(A) state exhibited native-like secondary and tertiary structure showing the interaction of alpha-crystallin with the MG state of the protease. 8-Anilinonaphthalene sulphonate (ANS) binding studies revealed the involvement of hydrophobic interactions in the formation of the complex of alpha-crystallin and protease. Refolding of acid-denatured protease by dilution to pH 7.5 resulted in aggregation of the protein. Unfolding of the protease in the presence of alpha-crystallin and its subsequent refolding resulted in the generation of a near-native intermediate with partial secondary and tertiary structure. Our studies represent the first report of involvement of a molecular chaperone-like alpha-crystallin in the unfolding and refolding of a protease. Alpha-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of the alkaline protease and keeps it in a near-native, folding-competent intermediate state.     

Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds

Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two-dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28-6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman-Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman-Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera, a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase-like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique.     

Hyperexpression and purification of biologically active human luteinizing hormone and human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

The glycoprotein hormones, luteinizing hormone (LH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), and follicle stimulating hormone (FSH), play important roles in overall physiology and reproduction. These hormones are heterodimeric molecules consisting of an identical alpha subunit non-covalently associated with the hormone-specific beta subunit. The inherent structural intricacies possessed by these hormones make them very interesting model systems for structure-function relationship studies of complex dimeric glycoproteins. The structural studies, as well as, the therapeutic applications require large quantities of biologically active hormones free of any contaminants. In this study, we report hyperexpression and purification of biologically active recombinant hLH and hCG expressed using Pichia pastoris expression system. A combination of hydrophobic interaction chromatography and ion exchange chromatography has been used to purify these recombinant hormones to homogeneity. Using a number of biochemical and immunological criteria, the recombinant hormones have been shown to be similar to the natural hormones and were equally biologically active. The preliminary data also suggested that P. pastoris cells express a low molecular weight isoform of hCG that appeared to be less glycosylated. This isoform exhibited lesser affinity for the receptor as compared to hCG, but was found to be fully biologically active.     

A conserved chromatin architecture marks and maintains the restricted germ cell lineage in worms and flies

In C. elegans, mRNA production is initially repressed in the embryonic germline by a protein unique to C. elegans germ cells, PIE-1. PIE-1 is degraded upon the birth of the germ cell precursors, Z2 and Z3. We have identified a chromatin-based mechanism that succeeds PIE-1 repression in these cells. A subset of nucleosomal histone modifications, methylated lysine 4 on histone H3 (H3meK4) and acetylated lysine 8 on histone H4 (H4acetylK8), are globally lost and the DNA appears more condensed. This coincides with PIE-1 degradation and requires that germline identity is not disrupted. Drosophila pole cell chromatin also lacks H3meK4, indicating that a unique chromatin architecture is a conserved feature of embryonic germ cells. Regulation of the germline-specific chromatin architecture requires functional nanos activity in both organisms. These results indicate that genome-wide repression via a nanos-regulated, germ cell-specific chromatin organization is a conserved feature of germline maintenance during embryogenesis.     

Inhibitors of Abeta production: solid-phase synthesis and SAR of alpha-hydroxycarbonyl derivatives

Inhibitors of amyloid-beta (Abeta) protein production have been widely pursued as a potential treatment for Alzheimer's disease. Following the identification of a 5 microM screening hit, SAR was initiated using solid-phase synthetic techniques. Two series of alpha-hydroxy esters and ketones which are sub-micromolar inhibitors of Abeta production were identified. The most potent alpha-hydroxyketone identified is approximately 30-fold more potent than the initial lead.