Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high‐protein feeding to regulate glucose homeostasis in vivo

High‐protein feeding acutely lowers postprandial glucose concentration compared to low‐protein feeding, despite a dichotomous rise of circulating glucagon levels. The physiological role of this glucagon rise has been largely overlooked. We here first report that glucagon signalling in the dorsal vagal complex (DVC) of the brain is sufficient to lower glucose production by activating a Gcgr–PKA–ERK–K_ATP channel signalling cascade in the DVC of rats in vivo. We further demonstrate that direct blockade of DVC Gcgr signalling negates the acute ability of high‐ vs. low‐protein feeding to reduce plasma glucose concentration, indicating that the elevated circulating glucagon during high‐protein feeding acts in the brain to lower plasma glucose levels. These data revise the physiological role of glucagon and argue that brain glucagon signalling contributes to glucose homeostasis during dietary protein intake.     

The unexpected formation of biologically active Cu(II) Schiff mono-base complexes with 2-thiophene-carboxaldehyde and dipropylenetriamine: crystal and molecular structure of CudptaSCl2

Two novel mononuclear Cu(II) coordination compounds of the type [Cu(dptaS)Cl(2)] and [Cu(dptaS)Br(2)] (dptaS=1,3-propanediamine, N(1)-[3-aminopropyl]-N(3)-[2-thienylmethylidene] or Schiff mono-base of dipropylenetriamine with 2-thiophene-carboxaldehyde) were prepared by the hydrolysis of the di-bases, [Cu(dptaSS)Cl(2)] and [Cu(dptaSS)Br(2)] (dptaSS=1,3-propanediamine, N(1)-[2-thienylmethylidene]-N(3)-[[2-thienylmethylidene]aminopropyl] or Schiff di-base of dipropylenetriamine with 2-thiophenecarboxaldehyde) to mono-bases with the release of one aldehyde molecule and freeing of the -NH(2) group of the coordinated dpta ligand. The X-ray determined structure of the compound [Cu(dptaS)Cl(2)] was confirmed by spectroscopic methods, magnetic and molar conductivity measurements. The Cu(II) atom is a five-coordinated CuN(3)Cl(2) chromophore with three nitrogen atoms coming up from the (dptaS) ligand and two chlorine atoms completing the square pyramidal geometry. Antiproliferative activity of both novel compounds was examined against a panel of different normal and cancer cell lines (MRC5, HeLa, MCF7, HT-29 and T47D) and showed that the Cu(II) Schiff mono-bases exhibit increased activity as compared to the starting materials. In vitro studies of plasmid DNA (pDNA) and double stranded DNA (dsDNA) interaction with the compounds under study support this difference. Some of the important factors contributing to the antiproliferative activity of the compounds under study, such as ionic character and dipole moment were also discussed in terms of the density functional theory calculated electronic structures of the ligands and their Cu(II) compounds.     

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.     

Eicosapentaenoic acid and docosahexaenoic acid reduce interleukin-1β-mediated cartilage degradation

Introduction  

In inflammatory joint disease, such as osteoarthritis (OA), there is an increased level of proinflammatory cytokines, such as interleukin (IL)-1β. These cytokines stimulate the production of matrix metalloproteinases (MMPs), which leads to the degradation of the cartilage extracellular matrix and the loss of key structural components such as sulphated glycosaminoglycan (sGAG) and collagen II. The aim of this study was to examine the therapeutic potential of n-3 polyunsaturated fatty acids (PUFAs) in an in vitro model of cartilage inflammation.     

Diorganotin(IV) complexes of dipeptides containing the alpha-aminoisobutyryl residue (Aib): preparation, structural characterization, antibacterial and antiproliferative activities of [(n-Bu)2 Sn(H(-1)L)] (LH=H-Aib-L-Leu-OH, H-Aib-L-Ala-OH)

Two new organotin(IV) complexes with dianionic dipeptides containing the α-aminoisobutyryl residue (Aib) as ligands are described. The solid complexes [(n-Bu)₂Sn(H₋₁L_A)] · 2MeOH (1 · 2MeOH) (L_AH = H-Aib-L-Leu-OH) and [(n-Bu)₂Sn(H₋₁L_B)] · MeOH (2 · MeOH) (L_BH = H-Aib-L-Ala-OH) have been isolated and characterized by single-crystal X-ray crystallography and spectroscopic techniques (H₋₁L²⁻ is the dianionic form of the corresponding dipeptide). Complexes 1 · 2MeOH and 2 · MeOH are monomeric with similar molecular structures. The doubly deprotonated dipeptide behaves as a N(amino), N(peptide), O(carboxylate) ligand and binds to the Sn^IV atom. The five-coordinate metal ion has a distorted trigonal bipyramidal geometry. A different network of intermolecular hydrogen bonds in each compound results in very dissimilar supramolecular features. The IR, far-IR, Raman and ¹¹⁹Sn NMR data are discussed in terms of the nature of bonding and known structures. The antibacterial and antiproliferative activities as well as the effect of the new compounds on pDNA were examined. Complexes 1 and 2 are active against the gram-positive bacteria Bacillus subtilis and Bacillus cereus. The IC₅₀ values reveal that the two compounds express promising cytotoxic activity in vitro against a series of cell lines.     

Antiproliferative activity of mixed-ligand dien-Cu(II) complexes with thiazole, thiazoline and imidazole derivatives.

The reaction of [Cu(dien)NO(3)]NO(3) with 2-amino-5-methylthiazole (2A5MT), 2-amino-2-thiazoline (2A-2Tzn), imidazole (im), N,N'-thiocarbonyldiimidazole (Tcdim), 2-aminothiazole (2AT) and 2-ethylimidazole (2Etim), gave a new series of mixed-ligand compounds of the general formula [Cu(dien)(B)NO(3))]NO(3); (dien, diethylenetriamine; B, 2A5MT, 2A-2Tzn, im, Tcdim, 2AT and 2Etim). The complexes have been characterised by elemental analysis, molar conductivity and magnetic measurements, as well as by electronic and IR spectral studies. According to the above measurements the possible structure of the compounds is the square pyramidal in the solid state and the square planar in aqueous solution. We tested all complexes for antiproliferative (cytostatic and cytotoxic) activity against a panel of cell lines (HeLa, L929, HT-29 and T47D). All [(dien)Cu(B)NO(3))](NO(3)) complexes had an activity against colon cancer cells (HT-29), inducing G2/M cell cycle arrest, an effect that for most of the complexes could be attributed to p34cdc2 inhibition by tyrosine-phosphorylation and/or to induction of (cyclin-dependent kinase inhibitor) p21(WAF1). Other cell lines were resistant to the majority of the complexes, except [Cu(dien)(2A5MT)NO(3))](NO(3)), that had showed the highest anti-proliferative activity against HT-29 cells also. The predilection for colon cancer cells and the relatively low toxicity against normal (L929) cells justify further investigation of this group of compounds.     

Domains of receptor mobility and endocytosis in the membranes of neonatal human erythrocytes in the membranes of neonatal human erythrocytes and reticulocytes are deficient in spectrin

It has previously shown (Schekman, R., and S.J. Singer, Proc. Natl. Acad. Sci. U.S.A. 73:4075-4079) that receptors in the membranes of neonatal human erythrocytes show a restricted degree of lateral mobility, whereas in adult human erythrocytes the receptors are essentially immobile. This restricted mobility is exhibited, for example, when concanavalin A (Con A) induces a limited clustering of its receptors in the neonatal erythrocyte membrane, resulting in the formation of invaginations and endocytic vesicles. This does not happen with adult cells. By the use of indirect immunoferritin labeling of ultrathin frozen sections of Con A-treated neonatal blood cells, we now show that the invaginations and endocytotic vesicles do not stain for spectrin, whereas the adjacent unperturbed membrane is heavily stained. The reticulocytes in the neonatal cell population undergo substantially more Con A-induced invagination and endocytosis than do the erythrocytes. These results lend strong support to the hypothesis that specialized discrete domains exist, or are induced, in the membranes of these neonatal cells, in which receptors are laterally mobile, whereas in the remaining (and predominant) part of the membrane the receptors are immobile. Such mobile domains are characterized by an absence of spectrin. During the maturation of the neonatal reticulocyte to erythrocyte, it is proposed that these domains are in large part, but not completely, eliminated.     

Synergism of Toll-like receptor-induced interleukin-12p70 secretion by monocyte-derived dendritic cells is mediated through p38 MAPK and lowers the threshold of T-helper cell type 1 responses

Toll-like receptors (TLRs) recognise specific molecular signatures of pathogens and trigger antimicrobial defence responses. Thereby, two independent signalling pathways can be distinguished: The inflammatory signalling pathway acting via the adapter molecule MyD88, leading to the activation of nuclear factor-κB (NF-κB) and mitogen activated protein kinases (MAPK) such as SAPK/JNK and p38 MAPK and the interferon (IFN) dependent pathway that signals via TRIF and results in the production of IFN-α/β. Several evolutionarily conserved molecular patterns are expressed by pathogens, leading to the question if concerted targeting of different TLRs may induce exaggerated immune responses by signalling via both TLR pathways. Here we report that monocyte-derived dendritic cells (MoDCs) combine and integrate signals received via the IFN-dependent pathway by engagement of TLR3 (poly I:C) and activation of TRIF with the MyD88-dependent pathway by ligation of TLR2 (PGN), TLR2/TLR6 (zymosan) and TLR5 (flagellin). The generally low IL-12p70 inducers resulted in combination of both pathways in cytokine levels similar to LPS, which acts via TLR4 and induces recruitment of MyD88/Tirap and TRIF/TRAM adapter proteins. The combination of TLR3 (poly I:C) or TLR4 (LPS) engagement with TLR8 (R848) ligation induced synergistic effects on cytokine production with a boost especially in IL-12p70 secretion. SB203580, a specific p38 MAPK inhibitor, completely blocked TLR ligand mediated IL-12p70 secretion, whereby specific inhibitors for SAPK/JNK (SP600125) and NF-κB (PDTC) only repressed partially the IL-12p70 secretion. Enhanced phosphorylation in poly I:C and R848 activated MoDCs revealed the critical contribution of p38 MAPK in synergistically induced IL-12p70 induction. Further investigation of primary and recall CD8⁺ T cell responses to the MUC_12-20 M1.2 peptide LLLLTVLTV and the influenza A virus matrix_58-66 peptide GILGFVFTL proved that synergistically activated MoDCs were superior compared with LPS or R848 alone. The results indicate that dendritic cells process, combine and integrate signals delivered by pathogens to launch effective adaptive immune responses.     

Copper(II) Schiff base coordination compounds of dien with heterocyclic aldehydes and 2-amino-5-methyl-thiazole: synthesis, characterization, antiproliferative and antibacterial studies. Crystal structure of CudienOOCl2

A new series of coordination compounds of the starting materials [Cu(dienX(2)Y(2))] and their adducts [Cu(dienXXY(2))(2a-5mt)] (where dien=diethylenetriamine, dienXX=Schiff bases of diethylenetriamine with 2-furaldehyde or 2-thiophene-carboxaldehyde, X=O, S, Y=Cl, Br, NO(3) and 2a-5mt=2-amino-5-methylthiazole) were synthesized by stepwise reactions and their structures were established by C, H, N, Cu analysis, spectroscopic, magnetic and molar conductivity measurements. The isolated compounds are monomers, paramagnetic and electrolytic compounds of the type 1:1. In all cases, the pentadentate Schiff base (dienXX) is bonded in a tridentate fashion through the 3 N atoms. In the CudienXXY(2) compounds the coordination sphere is completed by two Cl or Br or NO(3) groups in a square pyramidal arrangement. The proposed structure for this type of compound was further supported by X-ray diffraction analysis of the compound [Cu(dienOO)Cl(2)]. Its basal plane consists of three Cu-N contacts [2.017(2), 2.025(2) and 2.012(2) A] from dienOO, and the Cl(1) atom, while the Cl(2) atom possesses the apical position, the relevant distances being 2.2732(7) A for Cu-Cl(1) and 2.6051(7) A Cu-Cl(2). In the CudienX(2)Y(2).2a-5mt adducts the coordination sphere of copper is further completed by the nitrogen ring atom of the 2a-5mt, forming an octahedral configuration. The study of the biological activity of the compounds synthesized against a panel of different normal and cancer cell lines (MRC5, HeLa, MCF7, HT-29, OAW42, T47D) and bacteria (E. coli, B. cereus, B. subtilis) showed that the adducts of the type [Cu(dienXXY(2))(2a-5mt)] exhibit increased activity both in cancer cells and in bacteria, compared to the starting material of type [Cu(dienXXY(2))].