Diurnal variation in vascular function: role of sleep

Although vascular function is lower in the morning than afternoon, previous studies have not assessed the influence of prior sleep on this diurnal variation. The authors employed a semiconstant routine protocol to study the contribution of prior nocturnal sleep to the previously observed impairment in vascular function in the morning. Brachial artery vascular function was assessed using the flow-mediated dilation technique (FMD) in 9 healthy, physically active males (mean ± SD: 27 ± 9 yrs of age), at 08:00 and 16:00 h following, respectively, 3.29 ± .37 and 3.24 ± .57 h prior sleep estimated using actimetry. Heart rate and systolic and diastolic blood pressures were also measured. The data of the experimental sleep condition were compared with the data of the "normal" diurnal sleep condition, in which FMD measurements were obtained from 21 healthy individuals who slept only during the night, as usual, before the morning test session. The morning-afternoon difference in FMD was 1 ± 4% in the experimental sleep condition compared with 3 ± 4% in the normal sleep condition (p =?.04). This difference was explained by FMD being 3 ± 3% lower in afternoon following the prior experimental sleep (p =?.01). These data suggest that FMD is more dependent on the influence of supine sleep than the endogenous circadian timekeeper, in agreement with our previous finding that diurnal variation in FMD is influenced by exercise. These findings also raise the possibility of a lower homeostatic "set point" for vascular function following a period of sleep and in the absence of perturbing hemodynamic fluctuation.     

Cutting Edge: Selective role of ubiquitin in MHC class I antigen presentation

The importance of ubiquitination in MHC class I-restricted Ag processing remains unclear. To address this issue, we overexpressed wild-type and dominant-negative lysineless forms of ubiquitin (Ub) in mammalian cells using an inducible vaccinia virus system. Overexpression of the lysineless Ub nearly abrogated polyubiquitination and potently inhibited epitope presentation from a cytosolic N-end rule substrate as well as endoplasmic reticulum (ER)-targeted model Ags. In contrast, there was little impact on Ag presentation from cytosolic proteins. These trends were location dependent; redirecting cytosolic Ag to the ER rendered presentation lysineless Ub-sensitive, whereas retargeting exocytic Ag to the cytosol had the inverse effect. This dichotomy was further underscored by small interfering RNA knockdown of the ER-associated Ub ligase Hrd1. Thus, Ub-dependent degradation appears to play a major role in the MHC class I-restricted processing of ER-targeted proteins and a more restricted role in the processing of cytosolic proteins.     

Retrograde Traffic Out of the Yeast Vacuole to the TGN Occurs via the Prevacuolar/Endosomal Compartment

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid–containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.     

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M, species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation. © 1996 Wiley-Liss, Inc.     

A Highlights from MBoC Selection: Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking

The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.