Altered localization of amyloid precursor protein under endoplasmic reticulum stress

Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.     

Essential role of macrophages in the initiation of allergic rhinitis in mice sensitized intranasally once with cedar pollen: regulation of class switching of immunoglobulin in B cells by controlling interleukin-4 production in T cells of submandibular lymph nodes

The production of allergen-specific IgE antibodies (Abs) in allergen-sensitized patients or animals has a mutual relationship with the immunologic response leading to allergic rhinitis. We recently reported that, after an intranasal injection of cedar pollen into mice, an interleukin-4 (IL-4)-dependent increase in serum nonspecific IgE Abs was a prerequisite for the production of serum allergen-specific IgE Abs. Here, we explored which lymphoid organs were responsive to the intranasally injected allergen and how IL-4 and IgE Abs were produced in the lymphocytes. Time-dependent changes in the total cell numbers and in in vitro IgE Ab production in various lymphoid organs revealed that the submandibular lymph nodes were the main responsible organ. After treatment with allergen (for IgE production) or allergen and complete Freund's adjuvant (for IgG production), we separated submandibular lymph node cells into macrophage-, lymphocyte-, and granulocyte-rich populations by discontinuous Percoll density-gradient centrifugation. Unexpectedly, bulk cells, but not the lymphocyte- or macrophage-rich populations, produced significant amounts of IL-4, IgE, and IgG; whereas production was restored by addition of Mac-1(+) cells from the macrophage-rich to the lymphocyte-rich fraction. Furthermore, a combination of the lymphocyte-rich population (for IgG [or IgE]) production) and the macrophage-rich population (for IgE [or IgG]) production) produced a large amount of IgE (or IgG). These results indicate that, in the initiation of allergic rhinitis, macrophages in the submandibular lymph nodes are essential not only for IL-4 or immunoglobulin production, but also for class switching of immunoglobulin in lymphocytes.     

Exercise-induced muscle damage impairs insulin signaling pathway associated with IRS-1 oxidative modification

Strenuous exercise induces delayed-onset muscle damage including oxidative damage of cellular components. Oxidative stress to muscle cells impairs glucose uptake via disturbance of insulin signaling pathway. We investigated glucose uptake and insulin signaling in relation to oxidative protein modification in muscle after acute strenuous exercise. ICR mice were divided into sedentary and exercise groups. Mice in the exercise group performed downhill running exercise at 30 m/min for 30 min. At 24 hr after exercise, metabolic performance and insulin-signaling proteins in muscle tissues were examined. In whole body indirect calorimetry, carbohydrate utilization was decreased in the exercised mice along with reduction of the respiratory exchange ratio compared to the rested control mice. Insulin-stimulated uptake of 2-deoxy-[(3)H]glucose in damaged muscle was decreased after acute exercise. Tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and phosphatidyl-3-kinase/Akt signaling were impaired by exercise, leading to inhibition of the membrane translocation of glucose transporter 4. We also found that acute exercise caused 4-hydroxy-nonenal modification of IRS-1 along with elevation of oxidative stress in muscle tissue. Impairment of insulin-induced glucose uptake into damaged muscle after strenuous exercise would be related to disturbance of insulin signal transduction by oxidative modification of IRS-1.     

Structural studies of intermediates along the cyclization pathway of Aplysia ADP-ribosyl cyclase

Cyclic ADP-ribose (cADPR) is a calcium messenger that can mobilize intracellular Ca²⁺ stores and activate Ca²⁺ influx to regulate a wide range of physiological processes. Aplysia cyclase is the first member of the ADP-ribosyl cyclases identified to catalyze the cyclization of NAD⁺ into cADPR. The catalysis involves a two-step reaction, the elimination of the nicotinamide ring and the cyclization of the intermediate resulting in the covalent attachment of the purine ring to the terminal ribose. Aplysia cyclase exhibits a high degree of leniency towards the purine base of its substrate, and the cyclization reaction takes place at either the N1- or the N7-position of the purine ring. To decipher the mechanism of cyclization in Aplysia cyclase, we used a crystallization setup with multiple Aplysia cyclase molecules present in the asymmetric unit. With the use of natural substrates and analogs, not only were we able to capture multiple snapshots during enzyme catalysis resulting in either N1 or N7 linkage of the purine ring to the terminal ribose, we were also able to observe, for the first time, the cyclized products of both N1 and N7 cyclization bound in the active site of Aplysia cyclase.     

The structural analysis and the role of calcium binding site for thermal stability in mannanase

Mannanase is an important enzyme involved in the degradation of mannan, production of bioactive oligosaccharides, and biobleaching of kraft pulp. Mannanase must be thermostable for use in industrial applications. In a previous study, we found that the thermal stability of mannanase from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) is enhanced by calcium. Here, we investigated the relationship between the three-dimensional structure and primary sequence to identify the putative calcium-binding site. The results of site-directed mutagenesis experiments indicated that Asp-285, Glu-286, and Asp-287 of StMan (StDEDAAAdC) and Asp-264, Glu-265, and Asp-266 of TfMan (TfDEDAAAdC) were the key residues for calcium binding affinity. Isothermal titration calorimetry revealed that the catalytic domain of StMan and TfMan (StMandC and TfMandC, respectively) bound calcium with a K_a of 3.02 × 10⁴ M⁻¹ and 1.52 × 10⁴ M⁻¹, respectively, both with stoichiometry consistent with one calcium-binding site per molecule of enzyme. Non-calcium-binding mutants (StDEDAAAdC and TfDEDAAAdC) did not show any calorimetric change. From the primary structure alignment of several mannanases, the calcium-binding site was found to be highly conserved in GH5 bacterial mannanases. This is the first study indicating enhanced thermal stability of GH5 bacterial mannanases by calcium binding.     

Six new chalcones from Angelica keiskei inducing adiponectin production in 3T3-L1 adipocytes

Angelica keiskei (Ashitaba in Japanese), a traditional herb in Japan, contains abundant prenylated chalcones. It has been reported that the chalcones from A. keiskei showed such bioactivities as anti-bacterial, anti-cancer and anti-diabetic effects. Xanthoangelol, 4-hydroxyderricin and six new chalcones were isolated in this study from an ethanol extract of A. keiskei by octadecyl silyl (ODS) and silica gel chromatography, and identified by 1D- and 2D-nuclear magnetic resonance (NMR) and high-resolution mass spectrometric analyses. The chalcones from A. keiskei markedly increased the expression of the adiponectin gene and the production of adiponectin in 3T3-L1 adipocytes. These results suggest that the chalcones from A. keiskei might be useful for preventing the metabolic syndrome.     

Decreased Serum Levels of Mature Brain-Derived Neurotrophic Factor (BDNF), but Not Its Precursor proBDNF, in Patients with Major Depressive Disorder

Background

Meta-analyses have identified serum levels of brain-derived neurotrophic factor (BDNF) as a potential biomarker for major depressive disorder (MDD). However, at the time, commercially available human ELISA kits are unable to distinguish between proBDNF (precursor of BDNF) and mature BDNF because of limited BDNF antibody specificity. In this study, we examined whether serum levels of proBDNF, mature BDNF, and matrix metalloproteinase-9 (MMP-9), which converts proBDNF to mature BDNF, are altered in patients with MDD.

Methodology/Principal Findings

Sixty-nine patients with MDD and 78 age- and gender-matched healthy subjects were enrolled. Patients were evaluated using 17 items on the Structured Interview Guide for the Hamilton Depression Rating Scale. Cognitive impairment was evaluated using the CogState battery. Serum levels of proBDNF, mature BDNF, and MMP-9 were measured using ELISA kits. Serum levels of mature BDNF in patients with MDD were significantly lower than those of normal controls. In contrast, there was no difference in the serum levels of proBDNF and MMP-9 between patients and normal controls. While neither proBDNF nor mature BDNF serum levels was associated with clinical variables, there were significant correlations between MMP-9 serum levels and the severity of depression, quality of life scores, and social function scores in patients.

Conclusions/Significance

These findings suggest that mature BDNF may serve as a biomarker for MDD, and that MMP-9 may play a role in the pathophysiology of MDD. Further studies using larger sample sizes will be needed to investigate these results.     

Reducing salt intake for prevention of cardiovascular diseases in high-risk patients by advanced health education intervention (RESIP-CVD study), Northern Thailand: study protocol for a cluster randomized trial

ABSTRACT: BACKGROUND: Decreasing salt consumption can prevent cardiovascular diseases (CVD). Practically, it is difficult to promote people's awareness of daily salt intake and to change their eating habits in terms of reducing salt intake for better cardiovascular health. Health education programs visualizing daily dietary salt content and intake may promote lifestyle changes in patients at high risk of cardiovascular disease. METHODS: This is a cluster randomized trial. A total of 800 high-CVD-risk patients attending diabetes and hypertension clinics at health centers in Muang District, Chiang Rai province, Thailand, will be studied with informed consent. A health center recruiting 100 participants is a cluster, the unit of randomization. Eight clusters will be randomized into intervention and control arms and followed up for 1 year. Within the intervention clusters the following will be undertaken: (1) salt content in the daily diet will be measured and shown to study participants; (2) 24-hour salt intake will be estimated in overnight-collected urine and the results shown to the participants; (3) a dietician will assist small group health education classes in cooking meals with less salt. The primary outcome is blood pressure change at the 1-year follow-up. Secondary outcomes at the 1-year follow-up are estimated 24-hoursalt intake, incidence of CVD events and CVD death. The intention-to-treat analysis will be followed.Blood pressure and estimated 24-hour salt intake will be compared between intervention and control groups at the cluster and individual level at the 1-year follow-up. Clinical CVD events and deaths will be analyzed by time-event analysis. Retinal blood vessel calibers of CVD-risk patients will be assessed cross-sectionally. Behavioral change to reduce salt intake and the influencing factors will be determined by structured equation model (SEM). Multilevel regression analyses will be applied. Finally, the cost effectiveness of the intervention will be analyzed. DISCUSSION: This study is unique as it will recruit the individuals most vulnerable to CVD morbidity and mortality by applying the general Framingham CVD risk scoring system. Dietary salt reduction will be applied as a prioritized, community level intervention for the prevention of CVD in a developing country.Trial registrationISRCTN39416277.