Molecular cloning and expression of leptin in gray and harbor seal blubber, bone marrow, and lung and its potential role in marine mammal respiratory physiology

Leptin is a multifunctional hormone, produced predominantly in adipocytes. It regulates energy balance through its impact on appetite and fat metabolism, and its concentration indicates the size of body fat reserves. Leptin also plays a vital role in stretch-induced surfactant production during alveolar development in the fetus. The structure, expression pattern, and role of leptin have not previously been explored in marine mammals. Phocid seals undergo cyclical changes in body composition as a result of prolonged fasting and intensive foraging bouts and experience rapid, dramatic, and repeated changes in lung volume during diving. Here, we report the tissue-specific expression pattern of leptin in these animals. This is the first demonstration of leptin expression in the lung tissue of a mature mammal, in addition to its expression in the blubber and bone marrow, in common with other animals. We propose a role for leptin in seal pulmonary surfactant production, in addition to its likely role in long-term energy balance. We identify substitutions in the phocine leptin sequence in regions normally highly conserved between widely distinct vertebrate groups, and, using a purified seal leptin antiserum, we confirm the presence of the leptin protein in gray seal lung and serum fractions. Finally, we report the substantial inadequacies of using heterologous antibodies to measure leptin in unextracted gray seal serum.

Protemic characterization of peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) play quite diverse and important roles in monitoring immune homeostasis. Thus, these subset of blood cells may provide access to potential physiological relevant biomolecules, namely proteins. For this reason, PBMCs represent a promising biological sample in scientific research, particularly as a source of potential biological markers discovery of the most diverse diseases. Prior studies of proteomic characterization of PBMCs from healthy individuals lack either the identification of a large number of proteins or its quantification in a way that is compatible with the search of potential biomarker candidates. Therefore, this study aimed to provide a comprehensive PBMCs proteome characterisation as well as to create a SWATH library. It was also evaluated if by using the BD Vacutainer® CPT™ tubes for PBMCs isolation, it would be possible to identify a larger number of immunologically relevant proteins in comparison to plasma samples. The enrichment test assay revealed that it is possible to identify more immune-related proteins from isolated PBMCs than from plasma. Moreover, the majority of the quantified proteins with an “immune system” GO term assigned is present in higher amounts in PBMCs samples. 2D LC-MS/MS proved to be the best approach to use in qualitative analysis of PBMCs and in the construction of a SWATH library, since it resulted in an increase of both identified and quantified proteins (66.3% and 16.9%, respectively) in comparison to 1D LC-MS/MS. A total of 2071 proteins were identified and it was possible to quantify 922 different proteins among six distinct samples. From these proteins, 445 were commom between all individuals. In conclusion, this work provides a comprehensive PBMCs proteome dataset that will be useful in further studies that focus on the search for potential biological markers of various pathologies in these cells. Additionally, SWATH-MS proved to be a reproducible and effective acquisition method to quantify PBMCs proteins.

Low dose gemcitabine-loaded lipid nanocapsules target monocytic myeloid-derived suppressor cells and potentiate cancer immunotherapy

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Enhanced engraftment and repairing ability of human adipose-derived stem cells, conveyed by pharmacologically active microcarriers continuously releasing HGF and IGF-1, in healing myocardial infarction in rats

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PPARα regulates endothelial progenitor cell maturation and myeloid lineage differentiation through a NADPH oxidase-dependent mechanism in mice

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INVESTIGATION BY MASS SPECTROMETRY OF THE UBIQUITOME AND PROTEIN CARGO OF EXOSOMES DERIVED FROM MYELOID-DERIVED SUPPRESSOR CELLS

Exosomes released by myeloid-derived suppressor cells (MDSC) are 30 nm in diameter extracellular vesicles that have been shown to carry biologically active proteins as well as ubiquitin molecules. Ubiquitin is known to have many functions, including involvement in the formation of exosomes, although the exact role is highly contested. In the study reported here, the proteome and ubiquitome of MDSC exosomes has been investigated by bottom-up proteomics techniques. This report identifies more than 1000 proteins contained in the MDSC exosome cargo and 489 sites of ubiquitination in more than 300 ubiquitinated proteins based on recognition of glycinylglycine tagged peptides without antibody enrichment. This has allowed extensive chemical and biological characterization of the ubiquitinated cohort compared to that of the entire protein cargo to support hypotheses on the role of ubiquitin in exosomes.

Structural and functional interaction between polyamine related molecules and biological membranes

Changes induced by PA on nucleic acid (NA) conformation and synthesis is proven to be a major reason for PA essentiality (1-3). However, PA interactions with other polyanions, for instance polyanionic membrane lipid bilayers and glyosaminoglycans have received less attention (3-4). The functional importance of these interactions still is an obscure but interesting area of cell and molecular biology, especially in mammalian cells for which specific PA transport systems are not fully characterized (5). In mammals, activity and turnover of the polyamine (PA) synthesis key enzyme is controlled by a set of proteins: Antizymes (OAZ1-3) and antizyme inhibitors (AZIN1 and 2). It is demonstrated that AOZ modulate polyamine uptake (6), and that PA transport to mitochondria is linked to the respiratory chain state and modulates mitochondrial permeability transition (7). Antizyme expression variants have been located in mitochondria, being proposed as a proapoptotic factor (7-8). AZIN 2 is only expressed in a reduced set of tissues that includes mast cells, where it is associated to mast cell granules membrane (9). This fact, together to the abnormalities observed in bone marrow derived mast cell granules when they are differentiated under restricted PA synthesis conditions (10 and unpublished results), point out to important roles of PA and their related proteins in structure and function of mast cell granules. We will also present novel biophysical results on tripartite interactions of PA that remark the interest of the characterization of PA interactions with lipid bilayers for biomedicine and biotechnology. Thus, the information reported in this paper integrates previously reported information with our still unpublished results, all indicating that PA and their related proteins also are important factors for structure and dynamics of biological membranes and their associated functions essential in human physiology; for instance, solute interchange with the environment (uptake and secretion), oxidative metabolism and apoptosis. The importance of these involved processes for human homeostasis claim for further research efforts. 1. Ruiz-Chica J, Medina MA, Sánchez-Jiménez F and Ramírez FJ (2001) Fourier Transform Raman study of the structural specificities on the interaction between DNA and biogenic polyamines. Biophysical J. 80:443-454. 2. Lightfoot HL, Hall J (2014) Endogenous polyamine function--the RNA perspective. Nucleic Acids Res. 42:11275-11290. 3. Igarashi K, Kashiwagi K (2010) Modulation of cellular function by polyamines. Int J Biochem Cell Biol. 42:39-51. 4. Finger S, Schwieger C, Arouri A, Kerth A, Blume A (2014) Interaction of linear polyamines with negatively charged phospholipids: the effect of polyamine charge distance. Biol Chem. 395:769-778. 5. Poulin R, Casero RA, Soulet D. (2012) Recent advances in the molecular biology of metazoan polyamine transport. Amino Acids. 42:711-723. 6. Kahana C (2009) Regulation of cellular polyamine levels and cellular proliferation by antizyme and antizyme inhibitor. Essays Biochem. 4:47-61. 7. Agostinelli E, Marques MP, Calheiros R, Gil FP, Tempera G, Viceconte N, Battaglia V, Grancara S, Toninello A (2010) Polyamines: fundamental characters in chemistry and biology. Amino Acids 38:393-403. 8. Liu GY, Liao YF, Hsu PC, Chang WH, Hsieh MC, Lin CY, Hour TC, Kao MC, Tsay GJ, Hung HC (2006) Antizyme, a natural ornithine decarboxylase inhibitor, induces apoptosis of haematopoietic cells through mitochondrial membrane depolarization and caspases' cascade. Apoptosis 11:1773-1788. 9. Kanerva K, Lappalainen J, Mäkitie LT, Virolainen S, Kovanen PT, Andersson LC (2009). Expression of antizyme inhibitor 2 in mast cells and role of polyamines as selective regulators of serotonin secretion. PLoS One 31:e6858. 10. García-Faroldi G, Rodríguez CE, Urdiales JL, Pérez-Pomares JM, Dávila JC, Pejler G, Sánchez-Jiménez F, Fajardo I (2010) Polyamines are present in mast cell secretory granules and are important for granule homeostasis. PLoS One 30:e15071.

New insight on obesity and adipose-derived stem cells by comprehensive metabolomics

Obesity affects the functional capability of adipose-derived stem cells (ASCs) and their effective use in regenerative medicine through mechanisms still poorly understood. Here we employed a multiplatform (LC/MS, CE/MS, GC/MS) metabolomics untargeted approach to investigate the metabolic alteration underlying the inequalities observed in obese-derived ASCs. The metabolic fingerprint (metabolites within the cells) and footprint (metabolites secreted in the culture medium) from humans or mice, obese and non-obese derived ASCs, were characterized by providing valuable information. Metabolites associated to glycolysis, TCA, pentose phosphate pathway and polyol pathway were increased in the footprint of obese-derived human ASCs indicating alterations in the carbohydrate metabolism; whereas from the murine model, deep differences in lipid and amino acid catabolism were highlighted. Therefore, new insights on the ASCs metabolome were provided that enhance our understanding of the processes underlying the ASCs stemness capacity and its relationship with obesity, in different cell models.

Exploring the role of miR-34a in regulating adipose inflammation during obesity

Background: Obesity is not a new disease, with roots that can be traced back to 400 BC. However, with the staggering increase in individuals that are overweight and obese since the 1980s, now over a quarter of individuals in Europe and the Americas are classed as obese. This presents a global health problem that needs to be addressed with novel therapies. It is now well accepted that obesity is a chronic, low-grade inflammatory condition that could predispose individuals to a number of comorbidities. Obesity is associated with cardiovascular diseases (CVDs) and type 2 diabetes (T2D) as part of “the metabolic syndrome,” and as first identified by Dr Vauge, central distribution of white adipose tissue (WAT) is an important risk factor in the development of these diseases. Subsequently, visceral WAT (vWAT) was shown to be an important factor in this association with CVDs and T2D, and increasing inflammation. As the obese WAT expands, mainly through hypertrophy, there is an increase in inflammation that recruits numerous immune cells to the tissue that further exacerbate this inflammation, causing local and systemic inflammatory and metabolic effects. One of the main types of immune cell involved in this pathogenic process is pro-inflammatory M1 adipose tissue macrophages (ATMs). MicroRNAs (miRNAs) are a species of small RNAs that post-transcriptionally regulate gene expression by targeting gene mRNA, causing its degradation or translational repression. These miRNAs are promiscuous, regulating numerous genes and pathways involved in a disease, making them useful therapeutic targets, but also difficult to study. miR-34a has been shown to increase in the serum, liver, pancreas, and subcutaneous (sc)WAT of patients with obesity, non- alcoholic fatty liver disease (NAFLD) and T2D. Additionally, miR-34a has been shown to regulate a number of metabolic and inflammatory genes in numerous cell types, including those in macrophages. However, the role of miR-34a in regulating vWAT metabolism and inflammation is poorly understood. Hypothesis: miR-34a is dysregulated in the adipose tissue during obesity, causing dysregulation of metabolic and inflammatory pathways in adipocytes and ATMs that contribute to adipose inflammation and obesity’s comorbidities, particularly T2D. Method/Results: The role of miR-34a in adipose inflammation was investigated using a murine miR-34a-/- diet-induced obesity model, and primary in vitro models of adipocyte differentiation and inflammatory bone marrow-derived macrophages (BMDMs). miR-34a was shown to be ubiquitously expressed throughout the murine epididymal (e)WAT of obese high-fat diet (HFD)-fed WT mice and ob/ob mice, as well as omental WAT from patients with obesity. Additionally, miR-34a transcripts were increased in the liver and brown adipose tissue (BAT) of ob/ob and HFD-fed WT mice, compared to WT controls. When miR-34a-/- mice were fed HFD ad libitum for 24 weeks they were significantly heavier than their WT counterparts by the end of the study. Ex vivo examinations showed that miR-34a-/- eWAT had a smaller adipocyte area on chow, which significantly increased to WT levels during HFD-feeding. Additionally, miR-34a-/- eWAT showed basal increases in cholesterol and fatty acid metabolism genes Cd36, Hmgcr, Lxrα, Pgc1α, and Fasn. miR-34a-/- iBAT showed basal reductions in Cebpα and Cebpβ, with increased Pgc1α expression during HFD- feeding. The miR-34a-/- liver additionally showed increased basal transcript expression of Pgc1α, suggesting miR-34a may broadly regulate PGC1α. Accompanying the ex vivo changes in cholesterol and fatty acid metabolism genes, in vitro miR-34a-/- white adipocytes showed increased lipid content. An F4/80high macrophage population was identified in HFD-fed miR-34a-/- eWAT, with increased Il-10 transcripts and serum IL-5 protein. Following these ex vivo observations, BMDMs from WT mice upregulated miR-34a expression in response to TNFα stimulation. Additionally, miR-34a-/- BMDMs showed an ablated CXCL1 response to TNFα. Conclusion: These findings suggest miR-34a has a multi-factorial role in controlling a susceptibility to obesity, by regulating inflammatory and metabolic pathways, potentially through regulation of PGC1α.

Global gene expression profile in myelodysplastic syndromes using SAGE

The molecular pathogenesis of myelodysplastic syndromes (MDS) is poorly understood. In order to expand our knowledge of genetic defects in MDS, we determined the overall profile of genes expressed in bone marrow from patients with refractory anemia with excess blasts ( RAEB) by serial analysis of gene expression ( SAGE). The present report describes a partial transcriptome of RAEB bone marrow derived from 56,694 sequenced tags that provides information about expressed gene products. This is the first attempt to determine an overall profile of gene expression specifically in RAEB at diagnosis using SAGE, which should be useful in the understanding of the physiopathology of MDS and in identifying the genes involved.

SCFAs Induce Mouse Neutrophil Chemotaxis through the GPR43 Receptor

Short chain fatty acids (SCFAs) have recently attracted attention as potential mediators of the effects of gut microbiota on intestinal inflammation. Some of these effects have been suggested to occur through the direct actions of SCFAs on the GPR43 receptor in neutrophils, though the precise role of this receptor in neutrophil activation is still unclear. We show that mouse bone marrow derived neutrophils (BMNs) can chemotax effectively through polycarbonate filters towards a source of acetate, propionate or butyrate. Moreover, we show that BMNs move with good speed and directionality towards a source of propionate in an EZ-Taxiscan chamber coated with fibrinogen. These effects of SCFAs were mimicked by low concentrations of the synthetic GPR43 agonist phenylacetamide-1 and were abolished in GPR43(-/-) BMNs. SCFAs and phenylacetamide-1 also elicited GPR43-dependent activation of PKB, p38 and ERK and these responses were sensitive to pertussis toxin, indicating a role for Gi proteins. Phenylacetamide-1 also elicited rapid and transient activation of Rac1/2 GTPases and phosphorylation of ribosomal protein S6. Genetic and pharmacological intervention identified important roles for PI3K gamma, Rac2, p38 and ERK, but not mTOR, in GPR43-dependent chemotaxis. These results identify GPR43 as a bona fide chemotactic receptor for neutrophils in vitro and start to define important elements in its signal transduction pathways.

Humoral responses and immune protection in mice immunized with irradiated T. gondii tachyzoites and challenged with three genetically distinct strains of T. gondii

Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds. T. gondii also causes human toxoplasmosis; although toxoplasmosis is generally a benign disease, ocular, congenital or reactivated disease is associated with high numbers of disabled people. Infection occurs orally through the ingestion of meat containing cysts or by the intake of food or water contaminated with oocysts. Although the immune system responds to acute infection and mediates the clearance of tachyzoites, parasite cysts persist for the lifetime of the host in tissues such as the eye, muscle, and CNS. However, T. gondii RH strain tachyzoites irradiated with 255 Gy do not cause residual infection and induce the same immunity as a natural infection. To assess the humoral response in BALB/c and C57BL/6J mice immunized with irradiated tachyzoites either by oral gavage (p.o.) or intraperitoneal (i.p.) injection, we analyzed total and high-affinity IgG and IgA antibodies in the serum. High levels of antigen-specific IgG were detected in the serum of parenterally immunized mice, with lower levels in mice immunized via the oral route. However, most serum antibodies exhibited low affinity for antigen in both mice strain. We also found antigen specific IgA antibodies in the stools of the mice, especially in orally immunized BALB/c mice. Examination of bone marrow and spleen cells demonstrated that both groups of immunized mice clearly produced specific lgG, at levels comparable to chronic infection, suggesting the generation of IgG specific memory. Next, we challenged i.p. or p.o. immunized mice with cysts from ME49. VEG or P strains of T. gondii. Oral immunization resulted in partial protection as compared to challenged naive mice: these findings were more evident in highly pathogenic ME49 strain challenge. Additionally, we found that while mucosal IgA was important for protection against infection, antigen-specific IgG antibodies were involved with protection against disease and disease pathogenesis. Most antigen responsive cells in culture produced specific high-affinity IgG after immunization, diverse of the findings in serum IgG or from cells after infection, which produced low proportion of high-avidity IgG. (C) 2011 Elsevier B.V. All rights reserved.

Pore Formation Triggered by Legionella spp. Is an Nlrc4 Inflammasome-Dependent Host Cell Response That Precedes Pyroptosis

Legionella pneumophila, the etiological agent of Legionnaires disease, is known to trigger pore formation in bone marrow-derived macrophages (BMMs) by mechanisms dependent on the type IVB secretion system known as Dot/Icm. Here, we used several mutants of L. pneumophila in combination with knockout mice to assess the host and bacterial factors involved in pore formation in BMMs. We found that regardless of Dot/Icm activity, pore formation does not occur in BMMs deficient in caspase-1 and Nlrc4/Ipaf. Pore formation was temporally associated with interleukin-1 beta secretion and preceded host cell lysis and pyroptosis. Pore-forming ability was dependent on bacterial Dot/Icm but independent of several effector proteins, multiplication, and de novo protein synthesis. Flagellin, which is known to trigger the Nlrc4 inflammasome, was required for pore formation as flaA mutant bacteria failed to induce cell permeabilization. Accordingly, transfection of purified flagellin was sufficient to trigger pore formation independent of infection. By using 11 different Legionella species, we found robust pore formation in response to L. micdadei, L. bozemanii, L. gratiana, L. jordanis, and L. rubrilucens, and this trait correlated with flagellin expression by these species. Together, the results suggest that pore formation is neither L. pneumophila specific nor the result of membrane damage induced by Dot/Icm activity; instead, it is a highly coordinated host cell response dependent on host Nlrc4 and caspase-1 and on bacterial flagellin and type IV secretion system.

Cutting Edge: Nucleotide-Binding Oligomerization Domain 1-Dependent Responses Account for Murine Resistance against Trypanosoma cruzi Infection

An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)like receptor proteins in host response to T cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1(-/-) mice showed an impaired induction of NF-kappa B-dependent products in response to infection and failed to restrict T cruzi infection in presence of IFN-gamma. Despite normal cytokine production in the sera, Nod1(-/-) mice were highly susceptible to T cruzi infection, in a similar manner to MyD88(-/-) and NO synthase 2(-/-) mice. These studies indicate that Nod1-dependent responses account for host resistance against T cruzi infection by mechanisms independent of cytokine production. The Journal of Immunology, 2010, 184: 1148-1152.

Hematopoietic response of rats exposed to the impact of an acute psychophysiological stressor on responsiveness to an in vivo challenge with Listeria monocytogenes: Modulation by Chlorella vulgaris prophylactic treatment

In this study, we investigated the hematopoietic response of rats pretreated with CV and exposed to the impact of acute escapable, inescapable or psychogenical stress on responsiveness to an in vivo challenge with Listeria monocytogenes. No consistent changes were observed after exposure to escapable footshock. Conversely, the impact of uncontrollable stress (inescapable and psychogenical) was manifested by an early onset and increased severity and duration of myrelossuppression produced by the infection. Small size CFU-CM colonies and increased numbers of clusters were observed, concurrently to a greater expansion in the more mature population of bone marrow granulocytes. No differences were observed between the responses of both uncontrollable stress regimens. CV prevented the myelossuppression caused by stress/infection due to increased numbers of CFU-GM in the bone marrow. Colonies of cells tightly packed, with a very condensed nucleus; in association with a greater expansion in the more immature population of bone marrow granulocytes were observed. Investigation of the production of colony-stimulating factors revealed increased colony-stimulating activity (CSA) in the serum of normal and infected/stressed rats treated with the algae. CV treatment restored/enhanced the changes produced by stress/infection in total and differential bone marrow and peripheral cells counts. Further studies demonstrated that INF-gamma is significantly reduced, whereas IL-10 is significantly increased after exposure to Uncontrollable stress. Treatment with CV significantly increased INF-gamma levels and diminished the levels of IL-10. Uncontrollable stress reduced the protection afforded by CV to a lethal dose of L. monocytogenes, with survival rates being reduced from (50%) in infected rats to 20% in infected/stressed rats. All together, our results suggest Chlorella treatment as an effective tool for the prophylaxis of post-stress myelossupression, including the detrimental effect of stress on the course and outcome of infections. (C) 2008 Elsevier Inc. All rights reserved.