Malonyl-CoA mediates leptin hypothalamic control of feeding independent of inhibition of CPT-1a

Hypothalamic fatty acid metabolism is involved in central nervous system controls of feeding and energy balance. Malonyl-CoA, an intermediate of fatty acid biosynthesis, is emerging as a significant player in these processes. Notably, hypothalamic malonyl-CoA has been implicated in leptin's feeding effect. Leptin treatment increases malonyl-CoA level in the hypothalamic arcuate nucleus (Arc), and this increase is required for leptin-induced decrease in food intake. However, the intracellular downstream mediators of malonyl-CoA's feeding effect have not been identified. A primary biochemical action of malonyl-CoA is the inhibition of the acyltransferase activity of carnitine palmitoyltransferase-1 (CPT-1). In the hypothalamus, the predominant isoform of CPT-1 that possesses the acyltransferase activity is CPT-1 liver type (CPT-1a). To address the role of CPT-1a in malonyl-CoA's anorectic action, we used a recombinant adenovirus expressing a mutant CPT-1a that is insensitive to malonyl-CoA inhibition. We show that Arc overexpression of the mutant CPT-1a blocked the malonyl-CoA-mediated inhibition of CPT-1 activity. However, the overexpression of this mutant did not affect the anorectic actions of leptin or central cerulenin for which an increase in Arc malonyl-CoA level is also required. Thus, CPT-1a does not appear to be involved in the malonyl-CoA's anorectic actions induced by leptin. Furthermore, long-chain fatty acyl-CoAs, substrates of CPT-1a, dissociate from malonyl-CoA's actions in the Arc under different feeding states. Together, our results suggest that Arc intracellular mechanisms of malonyl-CoA's anorectic actions induced by leptin are independent of CPT-1a. The data suggest that target(s), rather than CPT-1a, mediates malonyl-CoA action on feeding.     

The transforming growth factor-beta (TGF-β) mediates acquisition of a mesenchymal stem cell-like phenotype in human liver cells

Transforming growth factor-beta (TGF-β) mediates several and sometime opposite effects in epithelial cells, inducing growth inhibition, and apoptosis but also promoting an epithelial to mesenchymal transition (EMT) process, which enhances cell migration and invasion. TGF-β plays relevant roles in different liver pathologies; however, very few is known about its specific signaling and cellular effects in human primary hepatocytes. Here we show that TGF-β inhibits proliferation and induces pro-apoptotic genes (such as BMF or BIM) in primary cultures of human fetal hepatocytes (HFH), but also up-regulates anti-apoptotic genes, such as BCL-XL and XIAP. Inhibition of the epidermal growth factor receptor (EGFR), using gefitinib, abrogates the increase in the expression of the anti-apoptotic genes and significantly enhances cell death. Simultaneously, TGF-β is able to induce an EMT process in HFH, coincident with Snail up-regulation and a decrease in E-cadherin levels, cells showing mesenchymal proteins and reorganization of the actin cytoskeleton in stress fibers. Interestingly, these cells show loss of expression of specific hepatic genes and increased expression of stem cell markers. Chronic treatment with TGF-β allows selection of a population of mesenchymal cells with a de-differentiated phenotype, reminiscent of progenitor-like cells. Process is reversible and the mesenchymal stem-like cells re-differentiate to hepatocytes under controlled experimental conditions. In summary, we show for the first time that human hepatocytes may respond to TGF-β inducing different signals, some of them might contribute to tumor suppression (growth inhibition and apoptosis), but others should mediate liver tumor progression and invasion (EMT and acquisition of a stem-like phenotype).     

Chiral cyclopalladated complexes derived from N,N-dimethyl-1-phenethylamine with bridging bis(diphenylphosphine)ferrocene ligand as inhibitors of the cathepsin B activity and as antitumoral agents

Chiral cyclopalladated complexes derived from N,N-dimethyl-1-phenethylamine and the coordinating ligand 1,1'-bis(diphenylphosphine)ferrocene were synthesized and studied as Cathepsin B inhibitors and antitumoral agents against solid tumors. Our results revealed that the palladium compound [Pd2(C2,N-S(-)dmpa)2(mu-dppf)Cl2] (2) was able to inhibit Cathepsin B activity in a reversible fashion. This palladacycle compound binds to free cathepsin B (E) as well as to the enzyme-substrate complex (ES) with dissociation constants of KH=12+/-1 microM and alphaKH=2.4+/-0.3 microM, respectively. The application of this complex, in Walker tumor-bearing rats, resulted in 90% inhibition of the tumor growth. Subcutaneous inoculations of 10(6) tumoral cells produced solid tumors with a mass of 4.0+/-1.0 g in 12 days Walker tumor-bearing rats. However, when these animals were treated with one dose of the palladacycle compound (2.0 mg/kg), the tumoral mass was reduced to 0.3+/-0.1 g. On the other hand, the same complex (2) did not afford any protection to mice bearing the non-metastatic Ehrlich Ascites tumor treated with doses of 0.5, 5.0, and 30 mg/kg for a period of four, three and one day, respectively, beginning 72 h after tumor inoculation. Toxicological studies using mice treated with one high dose of the complex (2) (100 mg/kg) did not show any alterations in red and white blood cell morphology 14 days after the drug administration. Similar results were obtained with hepatic, kidney, and spleen tissues. The results presented in this work introduce the title cyclopalladated complexes as promising antitumoral drugs with reduced toxicity in experimental studies.     

Outlier SNP markers reveal fine‐scale genetic structuring across European hake populations (Merluccius merluccius)

Shallow population structure is generally reported for most marine fish and explained as a consequence of high dispersal, connectivity and large population size. Targeted gene analyses and more recently genome‐wide studies have challenged such view, suggesting that adaptive divergence might occur even when neutral markers provide genetic homogeneity across populations. Here, 381 SNPs located in transcribed regions were used to assess large‐ and fine‐scale population structure in the European hake (Merluccius merluccius), a widely distributed demersal species of high priority for the European fishery. Analysis of 850 individuals from 19 locations across the entire distribution range showed evidence for several outlier loci, with significantly higher resolving power. While 299 putatively neutral SNPs confirmed the genetic break between basins (F_CT = 0.016) and weak differentiation within basins, outlier loci revealed a dramatic divergence between Atlantic and Mediterranean populations (F_CT range 0.275–0.705) and fine‐scale significant population structure. Outlier loci separated North Sea and Northern Portugal populations from all other Atlantic samples and revealed a strong differentiation among Western, Central and Eastern Mediterranean geographical samples. Significant correlation of allele frequencies at outlier loci with seawater surface temperature and salinity supported the hypothesis that populations might be adapted to local conditions. Such evidence highlights the importance of integrating information from neutral and adaptive evolutionary patterns towards a better assessment of genetic diversity. Accordingly, the generated outlier SNP data could be used for tackling illegal practices in hake fishing and commercialization as well as to develop explicit spatial models for defining management units and stock boundaries.     

Structural impact of cations on lipid bilayer models: Nanomechanical properties by AFM-force spectroscopy

Atomic Force Microscopy (AFM) has become an invaluable tool for studying the micro- and nanoworlds. As a stand-alone, high-resolution imaging technique and force transducer, it defies most other surface instrumentation in ease of use, sensitivity and versatility. The main strength of AFM relies on the possibility to operate in an aqueous environment on a wide variety of biological samples, from single molecules – DNA or proteins – to macromolecular assemblies like biological membranes. Understanding the effect of mechanical stress on membranes is of primary importance in biophysics, since cells are known to perform their function under a complex combination of forces. In the later years, AFM-based Force-Spectroscopy (AFM-FS) has provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Lipid membranes are electrostatically charged entities that physiologically coexist with electrolyte solutions. Thus, specific interactions with ions are a matter of considerable interest. The distribution of ions in the solution and their interaction with the membranes are factors that substantially modify the structure and dynamics of the cell membranes. Furthermore, signaling processes are modified by the membrane capability of retaining ions. Supported Lipid Bilayers (SLBs) are a versatile tool to investigate phospholipid membranes mimicking biological surfaces. In the present contribution, we review selected experiments on the mechanical stability of SLBs as models of lipid membranes by means of AFM-FS, with special focus on the effect of cations and ionic strength in the overall nanomechanical stability.     

The effect of colloid formulation on colloid osmotic pressure in horses with naturally occurring gastrointestinal disease

Background

Naturally occurring gastrointestinal disease is an important cause of acute hypoproteinemia in adult horses and hydroxyethyl starch colloid fluid treatment is a component of supportive care in these cases to improve plasma volume and maintain colloid osmotic pressure (COP). The objectives of the present study were to compare 2 formulations of high molecular weight hydroxyethyl starch and their relative effect on COP, acid-base status, and survival of horses with acute hypoproteinemia secondary to gastrointestinal disease.

Methods

Twenty adult horses, ≥ 1 year of age, were prospectively enrolled, with informed client consent, if they developed acute hypoproteinemia, defined as a plasma total protein <5.0 g/dL or albumin <2.2 g/dL during hospitalization while undergoing treatment for gastrointestinal disease. Horses were randomly assigned to receive a rapid infusion of either 6% hydroxyethyl starch in 0.9% saline or 6% hydroxyethyl starch in lactated ringers solution at a dose of 10ml/kg. Venous blood gas analysis, COP, and PCV were evaluated before and after colloid administration.

Results

For both groups, average COP prior to treatment was 11.0 mmHg (9.7 – 12.2 mmHg) and post colloid treatment was 13.2 mmHg (12.0 -14.7 mmHg) [Normal range 18 – 22 mmHg]. COP was significantly increased with colloid treatment (p<0.001) but this increase was not significantly different between treatment groups. Venous pH did not change significantly with treatment. Twelve horses survived to hospital discharge and survival did not differ significantly between treatment groups.

Conclusions

Post-treatment COP improved approximately 20% regardless of the formulation used, however, values did not reach the normal range of COP observed in healthy horses. Acid-base parameters were not significantly impacted by either treatment. Further study is needed to determine how these two products compare with regards to other outcome measures. Evaluation of the relative effects of colloid formulation in horses with clinical disease is a future area of interest.

     

Effect of Geographic Origin and Ex Situ Growing Site on Phenology,Morphology, and Seed Yield of Yarrow (Achillea millefolium L.) Germplasm from the Rhaetian Alps,Italy

Species adapted to prevailing soil and climatic conditions and native to the same geographic context are increasingly recommended for ecological restoration at high altitude. Better knowledge is required on the level of variation within these species for morpho‐physiological traits and seed yield. Adequate and affordable seed production is a prerequisite for native species to be widely adopted for restoration interventions. This study evaluated the variation of yarrow (Achillea millefolium L.) germplasm collected in the Rhaetian Alps, Italy. The worth of yarrow for restoration at high altitude has been repeatedly noted. The main goal of the study was to identify promising materials for selection purposes. The explored mountain section harbored valuable variation for traits of interest. Altitude, climate, and soil should frame an ecological correspondence between the collection site and the target restoration site, to secure the value of the native germplasm outside the boundaries of its collection area. The work also assessed any interaction between the germplasm and the ex situ growing environments, represented by a mountain and a lowland site, and verified the feasibility of the latter for seed multiplication. The growing site remarkably affected several morpho‐physiological characters. The lack of difference in mean seed yield between the two altitude‐contrasting sites suggested that seed multiplication of yarrow could also be conveniently carried out in lowland environments, with advantages in terms of production costs. Nonetheless, different yield responses in the two sites were observed, emphasizing the need of choosing the material to multiply based on the environment adopted for seed production.     

Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection

Background

Paracoccin is a dual-function protein of the yeast Paracoccidioides brasiliensis that has lectin properties and N-acetylglucosaminidase activities. Proteomic analysis of a paracoccin preparation from P. brasiliensis revealed that the sequence matched that of the hypothetical protein encoded by PADG-3347 of isolate Pb-18, with a polypeptide sequence similar to the family 18 endochitinases. These endochitinases are multi-functional proteins, with distinct lectin and enzymatic domains.

Methodology/principal findings

The multi-exon assembly and the largest exon of the predicted ORF (PADG-3347), was cloned and expressed in Escherichia coli cells, and the features of the recombinant proteins were compared to those of the native paracoccin. The multi-exon protein was also used for protection assays in a mouse model of paracoccidioidomycosis.

Conclusions/Significance

Our results showed that the recombinant protein reproduced the biological properties described for the native protein—including binding to laminin in a manner that is dependent on carbohydrate recognition—showed N-acetylglucosaminidase activity, and stimulated murine peritoneal macrophages to produce high levels of TNF-α and nitric oxide. Considering the immunomodulatory potential of glycan-binding proteins, we also investigated whether prophylactic administration of recombinant paracoccin affected the course of experimental paracoccidioidomycosis in mice. In comparison to animals injected with vehicle (controls), mice treated with recombinant paracoccin displayed lower pulmonary fungal burdens and reduced pulmonary granulomas. These protective effects were associated with augmented pulmonary levels of IL-12 and IFN-γ. We also observed that injection of paracoccin three days before challenge was the most efficient administration protocol, as the induced Th1 immunity was balanced by high levels of pulmonary IL-10, which may prevent the tissue damage caused by exacerbated inflammation. The results indicated that paracoccin is the protein encoded by PADG-3347, and we propose that this gene and homologous proteins in other P. brasiliensis strains be called paracoccin. We also concluded that recombinant paracoccin confers resistance to murine P. brasiliensis infection by exerting immunomodulatory effects.     

Meiotic self-pairing of the Psalidodon (Characiformes,Characidae) iso-B chromosome: A successful perpetuation mechanism

B chromosomes are non-essential additional genomic elements present in several animal and plant species. In fishes, species of the genus Psalidodon (Characiformes, Characidae) harbor great karyotype diversity, and multiple populations carry different types of non-essential B chromosomes. This study analyzed how the dispensable supernumerary B chromosome of Psalidodon paranae behaves during meiosis to overcome checkpoints and express its own meiosis-specific genes. We visualized the synaptonemal complexes of P. paranae individuals with zero, one, or two B chromosomes using immunodetection with anti-medaka SYCP3 antibody and fluorescence in situ hybridization with a (CA)₁₅ microsatellite probe. Our results showed that B chromosomes self-pair in cells containing only one B chromosome. In cells with two identical B chromosomes, these elements remain as separate synaptonemal complexes or close self-paired elements in the nucleus territory. Overall, we reveal that B chromosomes can escape meiotic silencing of unsynapsed chromatin through a self-pairing process, allowing expression of their own genes to facilitate regular meiosis resulting in fertile individuals. This behavior, also seen in other congeneric species, might be related to their maintenance throughout the evolutionary history of Psalidodon.     

The FTO gene is associated with adulthood obesity in the Mexican population

Common polymorphisms in the fat mass and obesity-associated gene (FTO) have shown strong association with obesity in several populations. In the present study, we explored the association of FTO gene polymorphisms with obesity and other biochemical parameters in the Mexican population. We also assessed FTO gene expression levels in adipose tissue of obese and nonobese individuals. The study comprised 788 unrelated Mexican-Mestizo individuals and 31 subcutaneous fat tissue biopsies from lean and obese women. FTO single-nucleotide polymorphisms (SNPs) rs9939609, rs1421085, and rs17817449 were associated with obesity, particularly with class III obesity, under both additive and dominant models (P = 0.0000004 and 0.000008, respectively). These associations remained significant after adjusting for admixture (P = 0.000003 and 0.00009, respectively). Moreover, risk alleles showed a nominal association with lower insulin levels and homeostasis model assessment of B-cell function (HOMA-B), and with higher homeostasis model assessment of insulin sensitivity (HOMA-S) only in nonobese individuals (P (dom) = 0.031, 0.023, and 0.049, respectively). FTO mRNA levels were significantly higher in subcutaneous fat tissue of class III obese individuals than in lean individuals (P = 0.043). Risk alleles were significantly associated with higher FTO expression in the class III obesity group (P = 0.047). In conclusion, FTO is a major risk factor for obesity (particularly class III) in the Mexican-Mestizo population, and is upregulated in subcutaneous fat tissue of obese individuals.