Correlation between persistent forms of zeaxanthin-dependent energy dissipation and thylakoid protein phosphorylation

High light stress induced not only a sustained form of xanthophyll cycle-dependent energy dissipation but also sustained thylakoid protein phosphorylation. The effect of protein phosphatase inhibitors (fluoride and molybdate ions) on recovery from a 1-h exposure to a high PFD was examined in leaf discs of Parthenocissus quinquefolia (Virginia creeper). Inhibition of protein dephosphorylation induced zeaxanthin retention and sustained energy dissipation (NPQ) upon return to low PFD for recovery, but had no significant effects on pigment and Chl fluorescence characteristics under high light exposure. In addition, whole plants of Monstera deliciosa and spinach grown at low to moderate PFDs were transferred to high PFDs, and thylakoid protein phosphorylation pattern (assessed with anti-phosphothreonine antibody) as well as pigment and Chl fluorescence characteristics were examined over several days. A correlation was obtained between dark-sustained D1/D2 phosphorylation and dark-sustained zeaxanthin retention and maintenance of PS II in a state primed for energy dissipation in both species. The degree of these dark-sustained phenomena was more pronounced in M. deliciosa compared with spinach. Moreover, M. deliciosa but not spinach plants showed unusual phosphorylation patterns of Lhcb proteins with pronounced dark-sustained Lhcb phosphorylation even under low PFD growth conditions. Subsequent to the transfer to a high PFD, dark-sustained Lhcb protein phosphorylation was further enhanced. Thus, phosphorylation patterns of D1/D2 and Lhcb proteins differed from each other as well as among plant species. The results presented here suggest an association between dark-sustained D1/D2 phosphorylation and sustained retention of zeaxanthin and energy dissipation (NPQ) in light-stressed, and particularly photoinhibited, leaves. Functional implications of these observations are discussed.This revised version was published online in October 2005 with corrections to the Cover Date.     

Ameliorated hepatic insulin resistance is associated with normalization of microsomal triglyceride transfer protein expression and reduction in very low density lipoprotein assembly and secretion in the fructose-fed hamster

To determine whether reduction of insulin resistance could ameliorate fructose-induced very low density lipoprotein (VLDL) oversecretion and to explore the mechanism of this effect, fructose-fed hamsters received placebo or rosiglitazone for 3 weeks. Rosiglitazone treatment led to normalization of the blunted insulin-mediated suppression of the glucose production rate and to a approximately 2-fold increase in whole body insulin-mediated glucose disappearance rate (p < 0.001). Rosiglitazone ameliorated the defect in hepatocyte insulin-stimulated tyrosine phosphorylation of the insulin receptor, IRS-1, and IRS-2 and the reduced protein mass of IRS-1 and IRS-2 induced by fructose feeding. Protein-tyrosine phosphatase 1B levels were increased with fructose feeding and were markedly reduced by rosiglitazone. Rosiglitazone treatment led to a approximately 50% reduction of VLDL secretion rates (p < 0.05) in vivo and ex vivo. VLDL clearance assessed directly in vivo was not significantly different in the FR (fructose-fed + rosiglitazone-treated) versus F (fructose-fed + placebo-treated) hamsters, although there was a trend toward a lower clearance with rosiglitazone. Enhanced stability of nascent apolipoprotein B (apoB) in fructose-fed hepatocytes was evident, and rosiglitazone treatment resulted in a significant reduction in apoB stability. The increase in intracellular mass of microsomal triglyceride transfer protein seen with fructose feeding was reduced by treatment with rosiglitazone. In conclusion, improvement of hepatic insulin signaling with rosiglitazone, a peroxisome proliferator-activated receptor gamma agonist, is associated with reduced hepatic VLDL assembly and secretion due to reduced intracellular apoB stability.     

A neuronal inhibitory domain in the N-terminal half of agrin.

Agrin is required for appropriate pre- and postsynaptic differentiation of neuromuscular junctions. While agrin's ability to orchestrate postsynaptic differentiation is well documented, more recent experiments have suggested that agrin is also a "stop signal" for the presynaptic neuron, and that agrin has actions on neurons in the CNS. To elucidate the neuronal activities of agrin and to define the receptor(s) responsible for these functions, we have examined adhesions of neurons and their neurite-outgrowth responses to purified agrin in vitro. We find that both full-length agrin and the C-terminal 95 kDa of agrin (agrin c95), which is sufficient to induce postsynaptic differentiation, are adhesive for chick ciliary ganglion (CG) and forebrain neurons. Consistent with previous findings, our results show that N-CAM binds to full-length agrin, and suggest that alpha-dystroglycan is a neuronal receptor for agrin c95. In neurite outgrowth assays, full-length agrin inhibited both laminin- and N-cadherin-induced neurite growth from CG neurons. The N-terminal 150 kDa fragment of agrin, but not agrin c95, inhibited neurite outgrowth, indicating that domains in the N-terminal portion of agrin are sufficient for this function. Adhesion assays using protein-coated beads and agrin-expressing cells revealed differential interactions of agrin with members of the immunoglobulin superfamily of cell adhesion molecules. However, none of these, including N-CAM, appeared to be critical for neuronal adhesion. In summary, our results suggest that the N-terminal half of agrin is involved in agrin's ability to inhibit neurite outgrowth. Our results further suggest that neither alpha-dystroglycan nor N-CAM, two known binding proteins for agrin, mediate this effect.     

The C-terminal domain of the epsilon subunit of the chloroplast ATP synthase is not required for ATP synthesis

The epsilon subunit of the ATP synthases from chloroplasts and Escherichia coli regulates the activity of the enzyme and is required for ATP synthesis. The epsilon subunit is not required for the binding of the catalytic portion of the chloroplast ATP synthase (CF1) to the membrane-embedded part (CFo). Thylakoid membranes reconstituted with CF1 lacking its epsilon subunit (CF1-epsilon) have high ATPase activity and no ATP synthesis activity, at least in part because the membranes are very leaky to protons. Either native or recombinant epsilon subunit inhibits ATPase activity and restores low proton permeability and ATP synthesis. In this paper we show that recombinant epsilon subunit from which 45 amino acids were deleted from the C-terminus is as active as full-length epsilon subunit in restoring ATP synthesis to membranes containing CF1-epsilon. However, the truncated form of the epsilon subunit was significantly less effective as an inhibitor of the ATPase activity of CF1-epsilon, both in solution and bound to thylakoid membranes. Thus, the C-terminus of the epsilon subunit is more involved in regulation of activity, by inhibiting ATP hydrolysis, than in ATP synthesis.     

Does nociceptin play a role in pain disorders in man?

Nociceptin-immunoreactive cellbodies were detected in the human trigeminal ganglion, while no such fibers were identified in the temporal artery or in dermal tissue from the neck region. In four healthy subjects receiving nociceptin into the temporal muscle in an open labeled design no pain was detected. In 10 healthy subjects who received 200pmol of nociceptin into tender non-dominant trapezius muscles in a placebo-controlled, randomized, balanced, and double-blinded design local tenderness increased (P=0.025) while no pain was noted. Thus, the action of nociceptin should be searched for in the trigeminal ganglion and/or in the central nervous system (CNS).     

Fasting and postprandial overproduction of intestinally derived lipoproteins in an animal model of insulin resistance. Evidence that chronic fructose feeding in the hamster is accompanied by enhanced intestinal de novo lipogenesis and ApoB48-containing lipoprotein overproduction

Insulin-resistant states are characterized by hypertriglyceridemia, predominantly because of overproduction of hepatic very low density lipoprotein particles. The additional contribution of intestinal lipoprotein overproduction to the dyslipidemia of insulin-resistant states has not been previously appreciated. Here, we have investigated intestinal lipoprotein production in a fructose-fed hamster model of insulin resistance previously documented to have whole body and hepatic insulin resistance, and hepatic very low density lipoprotein overproduction. Chronic fructose feeding for 3 weeks induced significant oversecretion of apolipoprotein B48 (apoB48)-containing lipoproteins in the fasting state and during steady state fat feeding, based on (a) in vivo Triton WR1339 studies of apoB48 production as well as (b) ex vivo pulse-chase labeling of intestinal enterocytes from fasted and fed hamsters. ApoB48 particle overproduction was accompanied by increased intracellular apoB48 stability, enhanced lipid synthesis, higher abundance of microsomal triglyceride transfer protein mass, and a significant shift toward the secretion of larger chylomicron-like particles. ApoB48 particle overproduction was not observed with short-term fructose feeding or in vitro incubation of enterocytes with fructose. Secretion of intestinal apoB48 and triglyceride was closely linked to intestinal enterocyte de novo lipogenesis, which was up-regulated in fructose-fed hamsters. Inhibition of fatty acid synthesis by cerulenin, a fatty acid synthase inhibitor, resulted in a dose-dependent decrease in intestinal apoB48 secretion. Overall, these findings further suggest that intestinal overproduction of apoB48 lipoproteins should also be considered as a major contributor to the fasting and postprandial dyslipidemia observed in response to chronic fructose feeding and development of an insulin-resistant state.     

GLP-1 and GIP are colocalized in a subset of endocrine cells in the small intestine

BACKGROUND: The incretin hormones GIP and GLP-1 are thought to be produced in separate endocrine cells located in the proximal and distal ends of the mammalian small intestine, respectively. METHODS AND RESULTS: Using double immunohistochemistry and in situ hybridization, we found that GLP-1 was colocalized with either GIP or PYY in endocrine cells of the porcine, rat, and human small intestines, whereas GIP and PYY were rarely colocalized. Thus, of all the cells staining positively for either GLP-1, GIP, or both, 55-75% were GLP-1 and GIP double-stained in the mid-small intestine. Concentrations of extractable GIP and PYY were highest in the midjejunum [154 (95-167) and 141 (67-158) pmol/g, median and range, respectively], whereas GLP-1 concentrations were highest in the ileum [92 (80-207) pmol/l], but GLP-1, GIP, and PYY immunoreactive cells were found throughout the porcine small intestine. CONCLUSIONS: Our results provide a morphological basis to suggest simultaneous, rather than sequential, secretion of these hormones by postprandial luminal stimulation.     

Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis

Protective immunity against Mycobacterium tuberculosis involves major histocompatibility complex class I (MHC-I)- and CD1-restricted CD8 T cells, but the mechanisms underlying antigen delivery to antigen-presenting molecules remain enigmatic. Macrophages, the primary host cells for mycobacteria, are CD1-negative. Here we show that M. tuberculosis phagosomes are secluded from the cytosolic MHC-I processing pathway and that mycobacteria-infected cells lose their antigen-presenting capacity. We also show that mycobacteria induce apoptosis in macrophages, causing the release of apoptotic vesicles that carry mycobacterial antigens to uninfected antigen-presenting cells (APCs). Inhibition of apoptosis reduced transfer of antigens to bystander cells and activation of CD8 T cells. Uninfected dendritic cells, which engulfed extracellular vesicles, were indispensable for subsequent cross-presentation of antigens, through MHC-I and CD1b, to T cells from mycobacteria-sensitized donors. This new 'detour' pathway for presentation of antigens from a phagosome-contained pathogen shows the functional significance of infection-induced apoptosis in the activation of CD8 T cells specific for both protein and glycolipid antigens in tuberculosis.