Rhythmic,reciprocal ghrelin and leptin signaling: new insight in the development of obesity

The hypothalamus integrates metabolic, neural and hormonal signals to evoke an intermittent appetitive drive in the daily management of energy homeostasis. Three major players identified recently in the feedback communication between the periphery and hypothalamus are leptin, ghrelin and neuropeptide Y (NPY). We propose that reciprocal circadian and ultradian rhythmicities in the afferent humoral signals, anorexigenic leptin from adipocytes and orexigenic ghrelin from stomach, encode a corresponding discharge pattern in the appetite-stimulating neuropeptide Y network in the hypothalamus. An exquisitely intricate temporal relationship among these signaling modalities with varied sites of origin is paramount in sustenance of weight control on a daily basis. Our model envisages that subtle and progressive derangements in temporal communication, imposed by environmental shifts in energy intake, impel a positive energy balance culminating in excessive weight gain and obesity. This conceptual advance provides a new target for designing pharmacologic or gene transfer therapies that would normalize the rhythmic patterns of afferent hormonal and efferent neurochemical messages.     

Regulated delivery of AMPA receptor subunits to the presynaptic membrane

In recent years, a role for AMPA receptors as modulators of presynaptic functions has emerged. We have investigated the presence of AMPA receptor subunits and the possible dynamic control of their surface exposure at the presynaptic membrane. We demonstrate that the AMPA receptor subunits GluR1 and GluR2 are expressed and organized in functional receptors in axonal growth cones of hippocampal neurons. AMPA receptors are actively internalized upon activation and recruited to the surface upon depolarization. Pretreatment of cultures with botulinum toxin E or tetanus toxin prevents the receptor insertion into the plasma membrane, whereas treatment with alpha-latrotoxin enhances the surface exposure of GluR2, both in growth cones of cultured neurons and in brain synaptosomes. Purification of small synaptic vesicles through controlled-pore glass chromatography, revealed that both GluR2 and GluR1, but not the GluR2 interacting protein GRIP, copurify with synaptic vesicles. These data indicate that, at steady state, a major pool of AMPA receptor subunits reside in synaptic vesicle membranes and can be recruited to the presynaptic membrane as functional receptors in response to depolarization.     

Oligonucleotides containing an acridine group covalently bonded to the nucleotide flanking the 3'-3' phosphodiester junction for alternate strand triple helix formation

Oligonucleotides with a 3'-3' inversion of polarity and containing an acridine group attached to nucleotide base flanking the 3'-3' phosphodiester bon have been synthesized, characterized and used as third strand in alternate triple helix formation. CD melting studies and molecular mechanics calculations have been carried out to investigate these triplex structures.     

Differential effects of ebselen on neutrophil recruitment,chemokine, and inflammatory mediator expression in a rat model of lipopolysaccharide-induced pulmonary inflammation

We postulated that the seleno-organic compound ebselen would attenuate neutrophil recruitment and activation after aerosolized challenge with endotoxin (LPS) through its effect as an antioxidant and inhibitor of gene activation. Rats were given ebselen (1-100 mg/kg i.p.) followed by aerosolized LPS exposure (0.3 mg/ml for 30 min). Airway inflammatory indices were measured 4 h postchallenge. Bronchoalveolar lavage (BAL) fluid cellularity and myeloperoxidase activity were used as a measure of neutrophil recruitment and activation. RT-PCR analysis was performed in lung tissue to assess gene expression of TNF-alpha, cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage-inflammatory protein-2 (MIP-2), ICAM-1, IL-10, and inducible NO synthase. Protein levels in lung and BAL were also determined by ELISA. Ebselen pretreatment inhibited neutrophil influx and activation as assessed by BAL fluid cellularity and myeloperoxidase activity in cell-free BAL and BAL cell homogenates. This protective effect was accompanied by a significant reduction in lung and BAL fluid TNF-alpha and IL-1 beta protein and/or mRNA levels. Ebselen pretreatment also prevented lung ICAM-1 mRNA up-regulation in response to airway challenge with LPS. This was not a global effect of ebselen on LPS-induced gene expression, because the rise in lung and BAL CINC-1 and MIP-2 protein levels were unaffected as were lung mRNA expressions for CINC-1, MIP-2, IL-10, and inducible NO synthase. These data suggest that the anti-inflammatory properties of ebselen are achieved through an inhibition of lung ICAM-1 expression possibly through an inhibition of TNF-alpha and IL-1 beta, which are potent neutrophil recruiting mediators and effective inducers of ICAM-1 expression.     

In vitro setting of dose-effect relationships of 32 metal compounds in the Balb/3T3 cell line, as a basis for predicting their carcinogenic potential

The results are reported of the second stage in a programme for a systematic in vitro study on the carcinogenic potential of metal compounds with Balb/3T3 clone A31-1-1 mouse fibroblasts. Nineteen metal compounds that exhibited a strong cytotoxic effect during a previous screening run with a 100 microM fixed dose were tested with a 72-hour exposure over a wide range of concentrations (from 0.1 microM to 1000 microM), to produce dose-effect curves to permit extrapolation of the 50% inhibition concentration (IC50) values for each metal compound. This allows the establishment of a suitable range of doses for individual metal species, for use in the subsequent Balb/3T3 assay based on a two-stage concurrent cytotoxicity and morphological transformation protocol. Another 13 metal compounds were also tested, to determine whether the Balb/3T3 cell transformation assay is really a valuable in vitro model in relation to the problem of metal speciation. Of the metal compounds assayed, 26 showed a dose related cytotoxic response with calculated IC50 values ranging from 0.25 microM (CH3HgCl) to 140 microM [(C5H5)2TiCl2], whereas six metal compounds, namely (NH4)6Mo7O24*4H2O, CH3AsO(OH)2, C2H6AsNaO2(3H2O, KBr, CrCl3*6H2O and (NH4)2[TiO(C2O4)2]*H2O, displayed no observable cytotoxicity or low cytotoxicity at all the doses tested. The determination of IC50 values permits a ranking of the cytotoxicity responses of metal compounds with the highest cytotoxicities. Dose-effect curves and IC50 values of different chemical forms of individual metal compounds of As, Br, Cr, Hg, Ir, Pt, Te, Ti and V (cationic/anionic inorganic or organometallic species) showed clearly how the chemical nature of the metal strongly influences the toxic response. This confirms that the Balb/3T3 cell line is a valuable in vitro model with respect to the problem of metal speciation. This is a fundamental aspect to be considered when incorporating the results from in vitro cell transformation assays of the carcinogenic potential of metal compounds into regulatory testing schemes. In this context, the choice of test metal species for the development and validation of such assays cannot disregard the possibility that humans will be exposed to specific chemical forms of individual metal compounds (different oxidation states, and inorganic or organometallic natures) that can profoundly affect their toxicity.     

Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells

The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a approximately 95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).     

Activated Notch4 inhibits angiogenesis: role of beta 1-integrin activation

Notch4 is a member of the Notch family of transmembrane receptors that is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions. The sprouting of endothelial cells from microvessels, or angiogenesis, involves the modulation of the endothelial cell phenotype. Based on the function of other Notch family members and the expression pattern of Notch4, we postulated that Notch4 activation would modulate angiogenesis. Using an in vitro endothelial-sprouting assay, we show that expression of constitutively active Notch4 in human dermal microvascular endothelial cells (HMEC-1) inhibits endothelial sprouting. We also show that activated Notch4 inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane in vivo. Activated Notch4 does not inhibit HMEC-1 proliferation or migration through fibrinogen. However, migration through collagen is inhibited. Our data show that Notch4 cells exhibit increased beta1-integrin-mediated adhesion to collagen. HMEC-1 expressing activated Notch4 do not have increased surface expression of beta 1-integrins. Rather, we demonstrate that Notch4-expressing cells display beta1-integrin in an active, high-affinity conformation. Furthermore, using function-activating beta 1-integrin antibodies, we demonstrate that activation of beta1-integrins is sufficient to inhibit VEGF-induced endothelial sprouting in vitro and angiogenesis in vivo. Our findings suggest that constitutive Notch4 activation in endothelial cells inhibits angiogenesis in part by promoting beta 1-integrin-mediated adhesion to the underlying matrix.