5'-AMP-activated protein kinase controls insulin-containing secretory vesicle dynamics

Changes in 5'-AMP-activated protein kinase (AMPK) activity have recently been implicated in the control of insulin secretion by glucose (da Silva Xavier, G., Leclerc, I., Varadi, A., Tsuboi, T., Moule, S. K., and Rutter, G. A. (2003) Biochem. J. 371, 761-774). Here, we examine the possibility that activation of AMPK may regulate distal steps in insulin secretion, including vesicle movement and fusion with the plasma membrane. Vesicle dynamics were imaged in single pancreatic MIN6 beta-cells expressing lumen-targeted pH-insensitive yellow fluorescent protein, neuropeptide Y.Venus, or monomeric red fluorescent protein by total internal reflection fluorescence and Nipkow disc confocal microscopy. Overexpression of a truncated, constitutively active form of AMPK (AMPKalpha1, 1-312, T172D; AMPK CA), inhibited glucose-stimulated (30 versus 3.0 mM) vesicle movements, and decreased the number of vesicles docked or fusing at the plasma membrane, while having no effect on the kinetics of individual secretory events. Expression of the activated form of AMPK also prevented dispersal of the cortical actin network at high glucose concentrations. Monitored in permeabilized cells, where the effects of AMPK CA on glucose metabolism and ATP synthesis were bypassed, AMPK CA inhibited Ca2+ and ATP-induced insulin secretion, and decreased ATP-dependent vesicle movements. These findings suggest that components of the vesicle transport network, including vesicle-associated motor proteins, may be targets of AMPK in beta-cells, dephosphorylation of which is required for vesicle mobilization at elevated glucose concentrations.     

20 S proteasome from Saccharomyces cerevisiae is responsive to redox modifications and is S-glutathionylated

The 20 S proteasome core purified from Saccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor gamma-glutamylcysteine. Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH. Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and gamma-glutamylcysteine) through the oxidized Cys-sulfenic acid form. S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane. Compounds such as N-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity. In vivo experiments revealed that 20 S proteasome extracted from H(2)O(2)-treated cells showed decreased chymotrypsin-like activity accompanied by S-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH(4). Moreover, cells pretreated with H(2)O(2) showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels. The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis.     

RhoA/ROCK regulation of neuritogenesis via profilin IIa-mediated control of actin stability

Neuritogenesis, the first step of neuronal differentiation, takes place as nascent neurites bud from the immediate postmitotic neuronal soma. Little is known about the mechanisms underlying the dramatic morphological changes that characterize this event. Here, we show that RhoA activity plays a decisive role during neuritogenesis of cultured hippocampal neurons by recruiting and activating its specific kinase ROCK, which, in turn, complexes with profilin IIa. We establish that this previously uncharacterized brain-specific actin-binding protein controls neurite sprouting by modifying actin stability, a function regulated by ROCK-mediated phosphorylation. Furthermore, we determine that this novel cascade is switched on or off by physiological stimuli. We propose that RhoA/ROCK/PIIa-mediated regulation of actin stability, shown to be essential for neuritogenesis, may constitute a central mechanism throughout neuronal differentiation.     

Honey bee (Hymenoptera: Apidae) foraging in response to preconditioning with onion flower scent compounds

Onion (Allium cepa L.) seed production has long been plagued with yield problems because of lack of pollination by the honey bee, Apis mellifera L. To attempt to attract more pollinators to the onion seed production field, honey bees were conditioned to associate onion floral odor components with a reward. Isolated nucleus hives of honey bees were fed 30% sucrose solutions scented with a 0.2% solution of onion floral odor compounds. After feeding on these solutions for 6 wk, bees were not found to prefer onion flowers to two competing food sources, carrot and alfalfa flowers, at the 5% significance level. However, there was an overall trend indicating a change in honey bee behavior, with fewer "trained" bees visiting alfalfa and carrot and more visiting onion. Thus, it may be possible to alter honey bee behavior with preconditioning but probably not to a degree that would be economically significant.     

Pathogenesis of renal ischemia/reperfusion injury: lessons from knockout mice

Ischemia/reperfusion-induced acute renal failure is a common clinical problem associated with a high morbidity and mortality. Upon hypoxic injury, the depletion of ATP causes mitochondrial dysfunction, and accumulation of intracellular sodium, calcium and reactive oxygen species. Subsequently, multiple enzyme systems including proteases, nitric oxide synthases, phospholipases and endonuclease are activated and responsible for cytoskeleton disruption, membrane damage, and DNA degradation, and eventually cell death. Ischemia/reperfusion injury also activates complement, cytokines, and chemokines, which are cytotoxic themselves, but also attract leukocytes into the ischemic area to cause further damage. The vascular endothelial cell injury and dysfunction prolong ischemia and induce vascular congestion, edema, and further infiltration of inflammatory cells. Many players in renal ischemia/reperfusion injury and their mechanisms have been investigated using genetically manipulated mouse models. In this review, we focus on the information gathered from these studies. Deficiency of the Na/Ca exchanger, inducible nitric oxide synthase, Caspase-1, A3 adenosine receptor, C3, C5, C6, Factor B, or midkine protects the kidney against I/R injury. Conversely, deficiency of the interleukin-1 receptor, osteopontin, C4, or recombination activation gene-1 is not protective, while the absence of adrenomedullin or endothelin receptor B delays the recovery of ischemia/reperfusion injury. The knowledge obtained from these studies provides new direction for designing potential therapeutic agents for treating ischemia/reperfusion injury.     

Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.     

Climate,body condition and spleen size in birds

Climatic conditions may impact on the body condition of animals and thereby affect their survival prospects. However, climate may also impact directly on the survival prospects of animals by affecting the size of immune defence organs that are used for defence against parasites. We used a large long-term database on body condition and size of the spleen in birds to test for immediate and delayed relationships between climatic conditions as indexed by the North Atlantic Oscillation (NAO) and body condition and spleen mass, respectively. Across 14 species of birds, spleen mass was significantly positively correlated with the NAO index, while the delayed effect of NAO on spleen mass was not significant. Spleen mass was positively related to body condition, but body condition did not depend significantly on NAO or delayed NAO effects. Bird species with a strong positive effect of NAO on spleen mass tended to have small spleens for their body size, while species with a strong negative effect of NAO on spleen mass tended to have relatively large spleens. Since bird species with relatively large spleen have been shown to suffer more from the negative effects of parasites, we can infer that the effects of climate as indexed by NAO on the size of the spleen depends on the importance of parasite-mediated natural selection.Due to an error in the citation line, this revised PDF (published in December 2003) deviates from the printed version, and is the correct and authoritative version of the paper.     

Probing the volume changes during voltage gating of Porin 31BM channel with nonelectrolyte polymers

To probe the volume changes of the voltage-dependent anion-selective channel (VDAC), the nonelectrolyte exclusion technique was taken because it is one of the few existing methods that may define quite accurately the rough geometry of lumen of ion channels (in membranes) for which there is no structural data.Here, we corroborate the data from our previous study [FEBS Lett. 416 (1997) 187] that the gross structural features of VDAC in its highest conductance state are asymmetric with respect to the plane of the membrane, and state that this asymmetry is not dependent on sign of voltage applied. Hence, the plasticity of VDAC does not play a role in the determination of lumen geometry at this state and the asymmetry is an internal property of the channel.We also show that the apparent diameter of the cis segment of the pore decreases slightly from 2 to 1.8 nm when the channel's conductance decreases from its high to low state. However, the trans funnel segment undergoes a more marked change in polymer accessible volume. Specifically, its larger diameter decreases from approximately 4 to 2.4 nm. Supposing the channel's total length is 4.6 nm, the apparent change in channel volume during this transition is estimated to be about 10 nm(3), i.e. about 40% of the channel's volume in the high conductance state.     

Characterization of the membrane-destabilizing properties of different pH-sensitive methacrylic acid copolymers

The intracellular delivery of active biomacromolecules from endosomes into the cytoplasm generally requires a membrane-disrupting agent. Since endosomes have a slightly acidic pH, anionic carboxylated polymers could be potentially useful for this purpose since they can destabilize membrane bilayers by pH-triggered conformational change. In this study, five different pH-sensitive methacrylic acid (MAA) copolymers were characterized with respect to their physicochemical and membrane lytic properties as a function of pH. pH-dependent conformational changes were studied in aqueous solution by turbidimetry and spectrofluorimetry. The hydrophobic domains that formed upon a decrease in pH were found to be dependent on copolymer's composition. Hemolysis and cytotoxicity assays demonstrated that the presence of the hydrophobic ethyl acrylate monomer and/or sufficient protonation of the carboxylic acid groups were important parameters for efficient membrane destabilization. Excessive copolymer hydrophobicity was not associated with membrane destabilization, but resulted in high macrophage cytotoxicity. Overall, this study gave more insights into the structure-activity relationship of MAA copolymers with membrane bilayers. Gaining knowledge of modulation of the physicochemical properties of copolymers and the optimization of copolymer-lipid interactions may lead to the elaboration of much more efficient drug delivery systems.     

Crystal structure of human phosphoglucose isomerase and analysis of the initial catalytic steps

The second enzyme in the glycolytic pathway, phosphoglucose isomerase (PGI), catalyses an intracellular aldose-ketose isomerization. Here we describe the human recombinant PGI structure (hPGI) solved in the absence of active site ligands. Crystals isomorphous to those previously reported were used to collect a 94% complete data set to a limiting resolution of 2.1 A. From the comparison between the free active site hPGI structure and the available human and rabbit PGI (rPGI) structures, a mechanism for protein initial catalytic steps is proposed. Binding of the phosphate moiety of the substrate to two distinct elements of the active site is responsible for driving a series of structural changes resulting in the polarisation of the active site histidine, priming it for the initial ring-opening step of catalysis.