Localization of sulfonylurea receptor subunits, SUR2A and SUR2B, in rat heart.

To understand the possible functions and subcellular localizations of sulfonylurea receptors (SURs) in cardiac muscle, polyclonal anti-SUR2A and anti-SUR2B antisera were raised. Immunoblots revealed both SUR2A and SUR2B expression in mitochondrial fractions of rat heart and other cellular fractions such as microsomes and cell membranes. Immunostaining detected ubiquitous expression of both SUR2A and SUR2B in rat heart in the atria, ventricles, interatrial and interventricular septa, and smooth muscles and endothelia of the coronary arteries. Electron microscopy revealed SUR2A immunoreactivity in the cell membrane, endoplasmic reticulum (ER), and mitochondria. SUR2B immunoreactivity was mainly localized in the mitochondria as well as in the ER and cell membrane. Thus, SUR2A and SUR2B are not only the regulatory subunits of sarcolemmal K(ATP) channels but may also function as regulatory subunits in mitochondrial K(ATP) channels and play important roles in cardioprotection.     

Succination of protein thiols during adipocyte maturation: a biomarker of mitochondrial stress

Although obesity is a risk factor for development of type 2 diabetes and chemical modification of proteins by advanced glycoxidation and lipoxidation end products is implicated in the development of diabetic complications, little is known about the chemical modification of proteins in adipocytes or adipose tissue. In this study we show that S-(2-succinyl)cysteine (2SC), the product of chemical modification of proteins by the Krebs cycle intermediate, fumarate, is significantly increased during maturation of 3T3-L1 fibroblasts to adipocytes. Fumarate concentration increased > or =5-fold during adipogenesis in medium containing 30 mm glucose, producing a > or =10-fold increase in 2SC-proteins in adipocytes compared with undifferentiated fibroblasts grown in the same high glucose medium. The elevated glucose concentration in the medium during adipocyte maturation correlated with the increase in 2SC, whereas the concentration of the advanced glycoxidation and lipoxidation end products, N(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one- and two-dimensional electrophoresis and approximately 60 2SC-proteins were detected using an anti-2SC polyclonal antibody. Several of the prominent and well resolved proteins were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. These include cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein. We propose that the increase in fumarate and 2SC is the result of mitochondrial stress in the adipocyte during adipogenesis and that 2SC may be a useful biomarker of mitochondrial stress in obesity, insulin resistance, and diabetes.     

Role of distal upstream sequence in vitamin D-induced expression of human CYP24 gene

The level of CYP24 mRNA in cultured human fibroblasts increases up to 20,000-fold in response to 1,25-dihydroxyvitamin D(3). Two vitamin D-responsive elements (VDREs) located immediately upstream of the CYP24 gene are primarily responsible for the induction. We studied roles of other regions in the 5'-flanking sequence of this gene. A series of deletion constructs between nucleotides -1918 and +209 of the gene were examined for their promoter activities employing the luciferase reporter assay. We found that the VDREs were not sufficient to account for the extent of induction. The sequence between nucleotides -548 and -294, which is located immediately upstream of the VDREs and includes three potential Sp1 sites, acted synergistically with the VDREs for the induction. Further upstream sequence and the 5'-untranslated region did not appear to play a major role in the vitamin D response.     

Sex Attraction and Mating in Bursaphelenchus okinawaensis and B. xylophilus

The fungal feeding, hermaphroditic Bursaphelenchus okinawaensis is a laboratory model to understand the biology of Bursaphelenchus. The extent to which B. okinawaensis can be used to model Bursaphelenchus xylophilus mating was investigated. A chemotaxis assay was conducted to examine whether B. xylophilus and B. okinawaensis produce and respond to volatile sex attractants. Unmated B. xylophilus females were found to attract B. xylophilus males. Similarly, old (sperm depleted) but not young (sperm repleted) B. okinawaensis hermaphrodites attract B. okinawaensis males. Thus, in both species, sperm status corresponds to its ability to attract males. B. xylophilus males also produce a volatile pheromone that attracts both mated and unmated females. A second assay, in which the behavior of males on petri plates in the presence of different females or hermaphrodites of Bursaphelenchus was observed, revealed that B. xylophilus unmated females attract B. okinawaensis males, and B. okinawaensis old hermaphrodites attract B. xylophilus males. These observations suggested that the pheromones of Bursaphelenchus work to some extent across species. Mating behavior through spicule insertion occurs across species, suggesting that postcopulatory mechanisms prevent production of interspecific progeny. The hermaphroditic B. okinawaensis will be a useful model to conduct genetic studies for the understanding of the molecular mechanisms underlying mating behavior in Bursaphelenchus nematodes.     

PGC-1α-mediated changes in phospholipid profiles of exercise-trained skeletal muscle

Exercise training influences phospholipid fatty acid composition in skeletal muscle and these changes are associated with physiological phenotypes; however, the molecular mechanism of this influence on compositional changes is poorly understood. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, the fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training induces these adaptations, together with increased PGC-1α, PGC-1α may contribute to the exercise-mediated change in phospholipid fatty acid composition. To determine the role of PGC-1α, we performed lipidomic analyses of skeletal muscle from genetically modified mice that overexpress PGC-1α in skeletal muscle or that carry KO alleles of PGC-1α. We found that PGC-1α affected lipid profiles in skeletal muscle and increased several phospholipid species in glycolytic muscle, namely phosphatidylcholine (PC) (18:0/22:6) and phosphatidylethanolamine (PE) (18:0/22:6). We also found that exercise training increased PC (18:0/22:6) and PE (18:0/22:6) in glycolytic muscle and that PGC-1α was required for these alterations. Because phospholipid fatty acid composition influences cell permeability and receptor stability at the cell membrane, these phospholipids may contribute to exercise training-mediated functional changes in the skeletal muscle.     

Discovery of selective indole-based prostaglandin D₂ receptor antagonist

A series of N-benzoyl-2-methylindole-3-acetic acids were synthesized and biologically evaluated as prostaglandin (PG) D? receptor antagonists. Some of the selected compounds significantly inhibited OVA-induced vascular permeability in guinea pig conjunctiva after oral dosing. Structure-activity relationship study is presented.     

Autotrophic picoplankton in southern Lake Baikal: abundance, growth and grazing mortality during summer

Autotrophic picoplankton were highly abundant during the thermalstratification period in late July in the pelagic area (waterdepth 500–1300 m) of southern Lake Baikal; maximum numberswere 2 x 10⁶ cells ml^–1 in the euphotic zone ({small tilde}15m). Unicellular cyanobacteria generally dominated the picoplanktoncommunity, although unidentified picoplankton that fluorescedred under blue excitation were also abundant (maximum numbers4 x 10⁵ cells ml^–1) and contributed up to {small tilde}40%of the total autotrophic picoplankton on occasions. Carbon andnitrogen biomasses of autotrophic picoplankton estimated byconversion from biovolumes were 14–84 µg C l^–1and 3.6–21 µg N l^–1. These were comparableto or exceeded the biomass of heterotrophic bacteria. Autotropicpicoplankton and bacteria accounted for as much as 33% of paniculateorganic carbon and 81% of nitrogen in the euphotic zone. Measurementsof the photosynthetic uptake of [^l4C]bicarbonate and the growthof picoplankton in diluted or size-fractionated waters revealedthat 80% of total primary production was due to picoplankton,and that much of this production was consumed by grazers inthe <20 µ.m cell-size category. These results suggestthat picoplankton-protozoan trophic coupling is important inthe pelagic food web and biogeochemical cycling of Lake Baikalduring summer.