Leptin activation of mTOR pathway in intestinal epithelial cell triggers lipid droplet formation,cytokine production and increased cell proliferation

Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-β production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-β production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma.     

Physiology and Phylogeny of Green Sulfur Bacteria Forming a Monospecific Phototrophic Assemblage at a Depth of 100 Meters in the Black Sea

The biomass, phylogenetic composition, and photoautotrophic metabolism of green sulfur bacteria in the Black Sea was assessed in situ and in laboratory enrichments. In the center of the western basin, bacteriochlorophyll e (BChl e) was detected between depths of 90 and 120 m and reached maxima of 54 and 68 ng liter⁻¹. High-pressure liquid chromatography analysis revealed a dominance of farnesyl esters and the presence of four unusual geranyl ester homologs of BChl e. Only traces of BChl e (8 ng liter⁻¹) were found at the northwestern slope of the Black Sea basin, where the chemocline was positioned at a significantly greater depth of 140 m. Stable carbon isotope fractionation values of farnesol indicated an autotrophic growth mode of the green sulfur bacteria. For the first time, light intensities in the Black Sea chemocline were determined employing an integrating quantum meter, which yielded maximum values between 0.0022 and 0.00075 μmol quanta m⁻² s⁻¹ at the top of the green sulfur bacterial layer around solar noon in December. These values represent by far the lowest values reported for any habitat of photosynthetic organisms. Only one 16S rRNA gene sequence type was detected in the chemocline using PCR primers specific for green sulfur bacteria. This previously unknown phylotype groups with the marine cluster of the Chlorobiaceae and was successfully enriched in a mineral medium containing sulfide, dithionite, and freshly prepared yeast extract. Under precisely controlled laboratory conditions, the enriched green sulfur bacterium proved to be capable of exploiting light intensities as low as 0.015 μmol quanta m⁻² s⁻¹ for photosynthetic ¹⁴CO₂ fixation. Calculated in situ doubling times of the green sulfur bacterium range between 3.1 and 26 years depending on the season, and anoxygenic photosynthesis contributes only 0.002 to 0.01% to total sulfide oxidation in the chemocline. The stable population of green sulfur bacteria in the Black Sea chemocline thus represents the most extremely low-light-adapted and slowest-growing type of phototroph known to date.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, <Emphasis Type="Italic">in vitro</Emphasis>effects on cartilage metabolism and intra-articular levels after injection

Introduction  

Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration.     

Vertical ground reaction forces diminish in mice after botulinum toxin injection

We examined changes in weight-bearing ability in mice after injection with botulinum toxin type A (BTX) to determine whether BTX can be used to isolate the effects of muscle on bone. As ambulation patterns were previously shown to improve within two weeks post-injection, we hypothesized that BTX injection to the posterior hindlimb would not significantly affect the mouse's ability to bear weight in the affected limb one week post-injection. Female BALB/c mice (N=13, 16-17 week old) were injected with either 20 μL of BTX (1U/100 g) or saline (SAL) in the left posterior hindlimb. Vertical ground reaction forces (GRF), hindlimb muscle cross-sectional area (MCSA), and tibial bone micro-architecture were assessed for 42 d following injection. Peak and average vertical GRF were 11±1% and 23±3% lower, respectively, in the BTX-injected hindlimb within 4d post-injection and remained lower than the SAL-injected hindlimb 14-21 d post-injection (15±4% and 10±2%, respectively). Time between forelimb and hindlimb peaks was 30-40% greater in the BTX-injected hindlimb than SAL-injected hindlimb 4-14 d post-injection. Peak vertical GRF recovered earlier following BTX injection than MCSA or bone volume fraction. These results indicate that weight-bearing ability recovered despite persistent muscle atrophy, and that weight-bearing alone was insufficient to maintain bone in the absence of muscle activity. We suggest that the absence of high-frequency signals typically associated with fast-twitch muscle activity may be contributing to the ongoing degradation of bone after BTX injection.     

Involvement of ERK in BMP-2 induced osteoblastic differentiation of mesenchymal progenitor cell line C3H10T1/2

The signaling mechanisms responsible for bone morphogenetic protein (BMP) induced osteoblast differentiation remains poorly understood. Previous research demonstrated that Smad proteins are the substrates and the mediators of BMP bound serine/threonine receptor kinase. In the present study, we examined the possible involvement of extracellular signal-regulated kinase (Erk) in the BMP induced osteoblast differentiation of mesenchymal progenitor cell C3H10T1/2. Our results indicate that BMP-2 inducement increased MAP kinase activity in mesenchymal progenitor cell line C3H10T1/2. Contrary to previous reports, this increased MAP kinase activity showed a latent but sustained pattern. Elevation of Erk1 and Erk2 protein levels was observed simultaneously. RT-PCR results demonstrated that the elevation of Erk protein level in BMP-2 induced cells was from the upregulation of mRNA expression. Furthermore, upregulated Erk proteins present enhanced phosphorylation. By using a dominant-negative Erk2 cell line, we demonstrated that nonfunctional Erk2 partially eliminated BMP-2 induced cell proliferation and ALP activity in the C3H10T1/2 cell. These results indicate that Erk is involved in BMP-2 induced osteoblast differentiation. The results also demonstrate that a latent and sustained signaling pattern exists in BMP induced signaling cascade.     

ESTIMATION OF AGE-SPECIFIC PROBABILITIES OF FIRST RETURN AND ANNUAL SURVIVAL RATES FOR THE MALE GRAY SEAL (HALICHOERUS GRYPUS) ON SABLE ISLAND FROM CAPTURE-RECAPTURE DATA

We used a longitudinal capture-recapture study to estimate the age-specific probabilities of first return to the breeding colony and annual survival rates for male gray seals ( Halichoerus grypus ), based on resightings of seals branded as young on Sable Island. We estimated that the average age of first returns for seals born in 1969–1970 to be 9.1 (SE 0.4) yr; for seals born in 1973–1974 it is estimated to be 9.8 (SE 0.2) yr. The estimated annual survival rate of these males was estimated to be 0.976 (SE 0.003).     

Cytokine profiles in the joint depend on pathology,but are different between synovial fluid,cartilage tissue and cultured chondrocytes

Introduction

This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.

Methods

Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).

Results

In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.

Conclusions

Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.     

Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.