AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism

ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca²⁺/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease.     

Enhanced Activation of Memory,but Not Na?ve,B Cells in Chronic Hepatitis C Virus-Infected Patients with Cryoglobulinemia and Advanced Liver Fibrosis

Mixed cryoglobulinemia is the most common extrahepatic disease manifestation of chronic hepatitis C virus (HCV) infection, where immunoglobulins precipitate at low temperatures and cause symptoms such as vasculitis, glomerulonephritis and arthralgia. HCV-associated cryoglobulinemia is also strongly linked with the development of B cell non-Hodgkin lymphoma. Abnormal B cell function in HCV infections can lead to the formation of HCV cryoglobulin complexes that usually comprise monoclonal rheumatoid factor and HCV-specific immune complexes. The aim of this study was to characterize the activation phenotype of B cells from patients with chronic HCV infection in comparison to healthy controls using flow cytometry. In addition, we determined how the activation status varies depending on the presence of cryoglobulinemia and advanced liver fibrosis. We found that only memory B cells, not naïve cells, were significantly activated in chronic HCV infection when compared with healthy controls. We also identified markers of memory B cell activation that were specific for HCV patients with cryoglobulinemia (CD86, CD71, HLA-DR) and advanced liver disease (CD86). Our results demonstrate that HCV infection has differential effects on B cells depending on the severity of hepatic and extrahepatic disease.     

Biocontrol agents promote growth of potato pathogens,depending on environmental conditions

There is a pressing need to understand and optimize biological control so as to avoid over‐reliance on the synthetic chemical pesticides that can damage environmental and human health. This study focused on interactions between a novel biocontrol‐strain, Bacillus sp. JC12GB43, and potato‐pathogenic Phytophthora and Fusarium species. In assays carried out in vitro and on the potato tuber, the bacterium was capable of near‐complete inhibition of pathogens. This Bacillus was sufficiently xerotolerant (water activity limit for growth = 0.928) to out‐perform Phytophthora infestans (~0.960) and challenge Fusarium coeruleum (~0.847) and Fusarium sambucinum (~0.860) towards the lower limits of their growth windows. Under some conditions, however, strain JC12GB43 stimulated proliferation of the pathogens: for instance, Fusarium coeruleum growth‐rate was increased under chaotropic conditions in vitro (132 mM urea) by >100% and on tubers (2‐M glycerol) by up to 570%. Culture‐based assays involving macromolecule‐stabilizing (kosmotropic) compatible solutes provided proof‐of‐principle that the Bacillus may provide kosmotropic metabolites to the plant pathogen under conditions that destabilize macromolecular systems of the fungal cell. Whilst unprecedented, this finding is consistent with earlier reports that fungi can utilize metabolites derived from bacterial cells. Unless the antimicrobial activities of candidate biocontrol strains are assayed over a full range of field‐relevant parameters, biocontrol agents may promote plant pathogen infections and thereby reduce crop yields. These findings indicate that biocontrol activity, therefore, ought to be regarded as a mode‐of‐behaviour (dependent on prevailing conditions) rather than an inherent property of a bacterial strain.     

Validation of an experimental model of fetal limb growth and development: Practical applications for the study of fetal defects associated with therapeutic agents in pregnant women

Summary Numerous musculoskeletal disorders which occur during pregnancy require an effective, nonpharmacological treatment that is known to have no adverse effects on fetal development. The present report describes morphological and histological changes occurring in fetal mouse limbs maintained in a serum-free organ culture system. Limbs maintained in this organ culture system show a progressive rise in limb dimensions including surface area, perimeter, and limb length. Histological analysis of serial cross sections of the limbs revealed a statistically significant increase in histological scores of limb development, thickness of epidermal layer, and amount of collagen deposited in the bone and dermis of limbs by Day 4 of culture. Therefore, this in vitro model combined with contemporary computer image analysis represents an excellent experimental model which can be used readily to examine the effects of therapeutic modalities on the growth and development of many different organ systems.     

The role of homophilic binding in anti-tumor antibody R24 recognition of molecular surfaces. Demonstration of an intermolecular beta-sheet interaction between vh domains

The murine antibody R24 and mouse-human Fv-IgG1(kappa) chimeric antibody chR24 are specific for the cell-surface tumor antigen disialoganglioside GD3. X-ray diffraction and surface plasmon resonance experiments have been employed to study the mechanism of "homophilic binding," in which molecules of R24 recognize and bind to other molecules of R24 though their heavy chain variable domains. R24 exhibits strong binding to liposomes containing disialoganglioside GD3; however, the kinetics are unusual in that saturation of binding is not observed. The binding of chR24 to GD3-bearing liposomes is significantly weaker, suggesting that cooperative interactions involving antibody constant regions contribute to R24 binding of membrane-bound GD3. The crystal structures of the Fabs from R24 and chR24 reveal the mechanism for homophilic binding and confirm that the homophilic and antigen-binding idiotopes are distinct. The homophilic binding idiotope is formed largely by an anti-parallel beta-sheet dimerization between the H2 complementarity determining region (CDR) loops of two Fabs, while the antigen-binding idiotope is a pocket formed by the three CDR loops on the heavy chain. The formation of homophilic dimers requires the presence of a canonical conformation for the H2 CDR in conjunction with participation of side chains. The relative positions of the homophilic and antigen-binding sites allows for a lattice of GD3-specific antibodies to be constructed, which is stabilized by the presence of the cell membrane. This model provides for the selective recognition by R24 of cells that overexpress GD3 on the cell surface.     

Nitric oxide and attenuated reflex cutaneous vasodilation in aged skin

Thermoregulatory cutaneous vasodilation is diminished in the elderly. The goal of this study was to test the hypothesis that a reduction in nitric oxide (NO)-dependent mechanisms contributes to the attenuated reflex cutaneous vasodilation in older subjects. Seven young (23 +/- 2 yr) and seven older (71 +/- 6 yr) men were instrumented with two microdialysis fibers in the forearm skin. One site served as control (Ringer infusion), and the second site was perfused with 10 mM N(G)-nitro-l-arginine methyl ester to inhibit NO synthase (NOS) throughout the protocol. Water-perfused suits were used to raise core temperature 1.0 degrees C. Red blood cell (RBC) flux was measured with laser-Doppler flowmetry over each microdialysis fiber. Cutaneous vascular conductance (CVC) was calculated as RBC flux per mean arterial pressure, with values expressed as a percentage of maximal vasodilation (infusion of 28 mM sodium nitroprusside). NOS inhibition reduced CVC from 75 +/- 6% maximal CVC (CVC(max)) to 53 +/- 3% CVC(max) in the young subjects and from 64 +/- 5% CVC(max) to 29 +/- 2% CVC(max) in the older subjects with a 1.0 degrees C rise in core temperature. Thus the relative NO-dependent portion of cutaneous active vasodilation (AVD) accounted for approximately 23% of vasodilation in the young subjects and 60% of the vasodilation in the older subjects at this level of hyperthermia (P < 0.001). In summary, NO-mediated pathways contributed more to the total vasodilatory response of the older subjects at high core temperatures. This suggests that attenuated cutaneous vasodilation with age may be due to a reduction in, or decreased vascular responsiveness to, the unknown neurotransmitter(s) mediating AVD.     

Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor

l-Glutamate is a major excitatory neurotransmitter that binds ionotropic and metabotropic glutamate receptors. Cerebral endothelial cells from many species have been shown to express several forms of glutamate receptors; however, human cerebral endothelial cells have not been shown to express either the N-methyl-D-aspartate (NMDA) receptor message or protein. This study provides evidence that human cerebral endothelial cells express the message and protein for NMDA receptors. Human cerebral endothelial cell monolayer electrical resistance changes in response to glutamate receptor agonists, antagonists, and second message blockers were tested. RT-PCR and Western blot analysis were used to demonstrate the presence of the NMDA receptor. Glutamate and NMDA (1 mM) caused a significant decrease in electrical resistance compared with sham control at 2 h postexposure; this response could be blocked significantly by MK-801 (an NMDA antagonist), 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethyoxybenzoate (an intracellular Ca2+ antagonist), and N-acetyl-L-cystein (an antioxidant). Trans(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid, a metabotropic receptor agonist (1 mM), did not significantly decrease electrical resistance. Our results are consistent with a model where glutamate, at excitotoxic levels, may lead to a breakdown in the blood brain barrier via activation of NMDA receptors.     

Ontogeny of uteroplacental progestagen production in pregnant mares during the second half of gestation

In pregnant mares during late gestation, little, if any, progesterone (P4) is found in the maternal circulation. Hence, quiescence of the equine uterus is believed to be maintained by metabolites of pregnenolone and P4 known as progestagens, which are produced by the uteroplacental tissues. However, little is known about the ontogeny, distribution, or actual rates of uteroplacental progestagen production in pregnant mares and their fetuses during the second half of pregnancy. Therefore, the present study measured the rates of uteroplacental uptake and output of eight specific progestagens in chronically catheterized, pregnant pony mares from 180 days to term. No significant uteroplacental uptake of any of the eight individual progestagens was observed from the uterine circulation. In contrast, significant uteroplacental uptake was observed for five of the eight individual progestagens from the umbilical circulation, and the uptakes increased toward term. The major uteroplacental progestagen outputs were 5 alpha-pregnane-3,20-dione (5 alphaDHP) and 20 alpha-hydroxy-5 alpha-pregnan-3-one (20 alpha 5P). These were released into both the umbilical and uterine circulations at rates that increased toward term. The majority of the total uteroplacental 20 alpha 5P output was distributed into the uterine circulation at all gestational ages studied. In contrast, distribution of the total uteroplacental 5 alphaDHP output switched from preferential delivery into the uterine circulation before 220 days of gestation to release predominantly into the umbilical circulation after 260 days. These findings demonstrate that uteroplacental progestagen production changes during the second half of gestation, which may have important implications for the maintenance of pregnancy and the onset of labor in the mare.