Ordering the Cytochrome c–initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9–dependent Manner

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1–mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c–inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.     

Application study of 1,2-α-l-fucosynthase: introduction of Fucα1-2Gal disaccharide structures on N-glycan,ganglioside, and xyloglucan oligosaccharide

We have recently generated a highly efficient 1,2-α-l-fucosynthase (BbAfcA N423H mutant) by protein engineering of 1,2-α-l-fucosidase from Bifidobacterium bifidum JCM 1254. This synthase could specifically introduce H-antigens (Fucα1-2Gal) into the non-reducing ends of oligosaccharides and in O-linked glycans in mucin glycoprotein. In the present study, we show an extended application of the engineered 1,2-α-l-fucosynthase by demonstrating its ability to insert Fuc residues into N- and O-glycans in fetuin glycoproteins, GM1 ganglioside, and a plant-derived xyloglucan nonasaccharide. This application study broadens the feasibility of this novel H-antigen synthesis technique in functional glycomics.     

Biopolymer - Surfactant Interaction: 4 Kinetics of Binding of Cetyltrimethyl Ammonium Bromide with Gelatin,Hemoglobin, β-lactoglobulin and Lysozyme

Abstract

The binding of CTAB with the proteins, gelatin, hemoglobin, β-lactoglobulin and lysozyme follow first order kinetics and occurs either in two or three distinct stages. The number of stages depends on the overall configuration of the biopolymers. The denatured protein, gelatin has shown three-stage kinetics under all conditions, whereas the native proteins, hemoglobinn, β-lactoglobulin and lysozyme have exhibited two stage kinetics. Heat treated lysozyme in 8 mol dm-³ urea medium has also shown a two-stage kinetics. On the basis of non interacting binding sites on the proteins and independent sequential binding, the rates of reaction have been observed to increase with temperature and follow the trend k₁ >> k₂ > k₃. The interaction of CTA⁺ with the proteins is both electrostatic and hydrophobic. Hemoglobin has shown maximum reaction rate whereas, β-lactoglobulin has shown a minimum. The activation parameters for the kinetic process have exhibited almost non-variant Δ G? and Δ H? < T Δ S? The formation of activation complex in the Eyring model is entropy controlled so also the overall kinetics. An isokinetic entropy-enthalpy compensation phenomenon has been observed for the respective kinetic stages.     

14-3-3 proteins,red light and photoperiodic flowering: A point of connection?

The 14-3-3 family of proteins is well known for participating in signal transduction by binding specifically phosphorylated proteins, thereby completing their kinase-induced transition in activity or localization. This interaction-based modulation of signal flux through metabolic pathways is a critical feature of many important eukaryotic signal transduction cascades. Only recently, however, have studies in Arabidopsis thaliana described that some of the most fundamental plant signal transduction pathways, including the photoperiodic flowering pathway, are functionally affected by 14-3-3s. There are pivotal points in the photoperiod pathway that are characterized by the accumulation, localization and stability of critical protein factors, all of which are strongly affected by light quality and photoperiod duration. These mechanisms (localization, phosphorylation, regulated proteolysis) are the same as those regulated by 14-3-3 proteins in other systems. Yet it is only recently that well characterized 14-3-3 genetic tools have become available in sufficient diversity to make it possible to truly tie 14-3-3 interactions to light signaling and flowering. This review presents an overview of photoperiodic flowering signaling and direct 14-3-3 participation in the process, coupled with a discussion of the overlapping and specific roles of 14-3-3s which present confounding issues in the functional dissection of this family of signaling proteins.Key words: isoform specificity, protein interaction, phosphorylation, signaling     

Molecular weights of wheat γ2-, β6-, α7-, α8- and α9-gliadins

The molecular weights of wheat γ₂-, β6-, α7-, α8- and α9-gliadins were calculated with the aid of a computer technique from sedimentation equilibrium data obtained in an ultracentrifuge equipped with photoelectric scanner. The dissociative solvents, all at pH 3.1 by addition of HCl, included 3 M urea, 0.15 M KCl; 8 M urea, 0.15 M KCl and 6 M guanidine-HCl. The minimum molecular weights for γ₂-, α7- and α9-gliadins, obtained in 6 M guanidine-HCl, were 34 600, 30 400 and 30 900, respectively. The β6- and α8-gliadins gave minimum molecular weights of 33 000 and 36 900, respectively, in 3 M urea, 0.15 M KCl.     

Synthesis and properties of alkylglucosides with mild detergent action: improved synthesis and purification of β-1-octyl-, -nonyl-, and -decyl-glucose. Synthesis of β-1-undecylglucose and β-1-dodecylmaltose

Medium chain β-1-alkylglycosides show interesting mild detergent properties. Therefore, their synthesis and purification have been investigated and improved so as to permit preparation of 50–100 g amounts. Preparatory methods are presented for the already known compounds β-1-octyl-, β-1-nonyl and β-1-decyl-glucose and for the new compounds β-1-undecylglucose and β-1-dodecylmaltose. Some relevant properties such as melting point, optical rotation, critical micelle concentration and NMR-spectra have been determined. They illustrate the suitability of this class of detergents for membrane research.     

Characterization of caveolins from human knee joint cartilage: expression of caveolin-1, -2, and -3 in chondrocytes and association with integrin β1

Interactions between the extracellular matrix (ECM) and chondrocytes are of great importance for structure and function of cartilage. The present study was undertaken to answer the question whether caveolins take part in integrin-mediated cell–ECM interactions in the human cartilage. In samples of human knee joint cartilage, we detected the caveolin subtypes -1, -2, and -3 by immunohistochemical methods. Double-label experiments revealed a colocalization of caveolin with β1-integrin. Results of immunoprecipitation and immunoblotting assays show that β1-integrins associate with all three caveolin subtypes in human chondrocytes and indicate that they are part of the same complexes. Furthermore, immunoelectron microscopy shows the localization of β1-integrin in caveolae-like structures of the cell membrane. The data stimulate further investigations on the role of the caveolin–integrin complex for integrin-mediated signaling pathways in chondrocytes. Accepted: 17 December 1999