Expression of uncoupling proteins-1,-2 and-3 mRNA is induced by an adenocarcinoma-derived lipid-mobilizing factor

The abnormalities of lipid metabolism observed in cancer cachexia may be induced by a lipid-mobilizing factor produced by adenocarcinomas. The specific molecules and metabolic pathways that mediate the actions of lipid-mobilizing factor are not known. The mitochondrial uncoupling proteins-1, -2 and -3 are suggested to play essential roles in energy dissipation and disposal of excess lipid. Here, we studied the effects of lipid-mobilizing factor on the expression of uncoupling proteins-1, -2 and -3 in normal mice. Lipid-mobilizing factor isolated from the urine of cancer patients was injected intravenously into mice over a 52-h period, while vehicle was similarly given to controls. Lipid-mobilizing factor caused significant reductions in body weight (-10%, P=0.03) and fat mass (-20%, P<0.01) accompanied by a marked decrease in plasma leptin (-59%, P<0.01) and heavy lipid deposition in the liver. In brown adipose tissue, uncoupling protein-1 mRNA levels were elevated in lipid-mobilizing factor-treated mice (+96%, P<0.01), as were uncoupling proteins-2 and -3 (+57% and +37%, both P<0.05). Lipid-mobilizing factor increased uncoupling protein-2 mRNA in both skeletal muscle (+146%, P<0.05) and liver (+142%, P=0.03). The protein levels of uncoupling protein-1 in brown adipose tissue and uncoupling protein-2 in liver were also increased with lipid-mobilizing factor administration (+49% and +67%, both P=0.02). Upregulation by lipid-mobilizing factor of uncoupling proteins-1, -2 and -3 in brown adipose tissue, and of uncoupling protein-2 in skeletal muscle and liver, suggests that these uncoupling proteins may serve to utilize excess lipid mobilized during fat catabolism in cancer cachexia.

Conformational dynamics associated with ligand binding to vertebrate hexa-coordinate hemoglobins

Neuroglobin (Ngb) and cytoglobin (Cygb) are two new additions to the globin family, exhibiting heme iron hexa-coordination, a disulfide bond and large internal cavities. These proteins are implicated in cytoprotection under hypoxic-ischemic conditions, but the molecular basis of their cytoprotective function is unclear. Herein, a photothermal and spectroscopic study of the interactions of diatomic ligands with Ngb, Cygb, myoglobin and hemoglobin is presented. The impact of the disulfide bond in Ngb and Cygb and role of conserved residues in Ngb His64, Val68, Cys55, Cys120 and Tyr44 on conformational dynamics associated with ligand binding/dissociation were investigated. Transient absorption and photoacoustic calorimetry studies indicate that CO photo-dissociation from Ngb leads to a volume expansion (13.4±0.9 mL mol-1), whereas a smaller volume change was determined for Ngb with reduced Cys (ΔV=4.6±0.3 mL mol-1). Furthermore, Val68 side chain regulates ligand migration between the distal pocket and internal hydrophobic cavities since Val68Phe geminate quantum yield is ∼2.7 times larger than that of WT Ngb. His64Gln and Tyr44Phe mutations alter the thermodynamic parameters associated with CO photo-release indicating that electrostatic/hydrogen binding network that includes heme propionate groups, Lys 67, His64, and Tyr 44 in Ngb modulates the energetics of CO photo-dissociation. In Cygb, CO escape from the protein matrix is fast (< 40 ns) with a ΔH of 18±2 kcal mol-1 in Cygbred, whereas disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4 kcal mol-1. Therefore, the disulfide bond modulates conformational dynamics in Ngb and Cygb. I propose that in Cygb with reduced Cys the photo-dissociated ligand escapes through the hydrophobic tunnel as occurs in Ngb, whereas the CO preferentially migrates through the His64 gate in Cygbox. To characterize Cygb surface 1,8-ANS interactions with Cygb were investigated employing fluorescence spectroscopy, ITC and docking simulations. Two 1,8-ANS binding sites were identified. One binding site is located close to the extended N-terminus of Cygb and was also identified as a binding site for oleate. Furthermore, guanidinium hydrochloride-induced unfolding studies of Cygb reveal that the disulfide bond does not impact Cygb stability, whereas binding of cyanide slightly increases the protein stability.

Functional genomic and molecular analyses of transcripts related to glycerol synthesis and trafficking in rainbow smelt (Osmerus mordax) using cold temperature-induced models of glycerol production

The rainbow smelt (Osmerus mordax) is an anadromous teleost that produces type II antifreeze protein (AFP) and accumulates modest urea and high glycerol levels in plasma and tissues as adaptive cryoprotectant mechanisms in sub-zero temperatures. It is known that glyceroneogenesis occurs in liver via a branch in glycolysis and gluconeogenesis and is activated by low temperature; however, the precise mechanisms of glycerol synthesis and trafficking in smelt remain to be elucidated. The objective of this thesis was to provide further insight using functional genomic techniques [e.g. suppression subtractive hybridization (SSH) cDNA library construction, microarray analyses] and molecular analyses [e.g. cloning, quantitative reverse transcription - polymerase chain reaction (QPCR)]. Novel molecular mechanisms related to glyceroneogenesis were deciphered by comparing the transcript expression profiles of glycerol (cold temperature) and non-glycerol (warm temperature) accumulating hepatocytes (Chapter 2) and livers from intact smelt (Chapter 3). Briefly, glycerol synthesis can be initiated from both amino acids and carbohydrate; however carbohydrate appears to be the preferred source when it is readily available. In glycerol accumulating hepatocytes, levels of the hepatic glucose transporter (GLUT2) plummeted and transcript levels of a suite of genes (PEPCK, MDH2, AAT2, GDH and AQP9) associated with the mobilization of amino acids to fuel glycerol synthesis were all transiently higher. In contrast, in glycerol accumulating livers from intact smelt, glycerol synthesis was primarily fuelled by glycogen degradation with higher PGM and PFK (glycolysis) transcript levels. Whether initiated from amino acids or carbohydrate, there were common metabolic underpinnings. Increased PDK2 (an inhibitor of PDH) transcript levels would direct pyruvate derived from amino acids and / or DHAP derived from G6P to glycerol as opposed to oxidation via the citric acid cycle. Robust LIPL (triglyceride catabolism) transcript levels would provide free fatty acids that could be oxidized to fuel ATP synthesis. Increased cGPDH (glyceroneogenesis) transcript levels were not required for increased glycerol production, suggesting that regulation is more likely by post-translational modification. Finally, levels of a transcript potentially encoding glycerol-3-phosphatase, an enzyme not yet characterized in any vertebrate species, were transiently higher. These comparisons also led to the novel discoveries that increased G6Pase (glucose synthesis) and increased GS (glutamine synthesis) transcript levels were part of the low temperature response in smelt. Glucose may provide increased colligative protection against freezing; whereas glutamine could serve to store nitrogen released from amino acid catabolism in a non-toxic form and / or be used to synthesize urea via purine synthesis-uricolysis. Novel key aspects of cryoprotectant osmolyte (glycerol and urea) trafficking were elucidated by cloning and characterizing three aquaglyceroporin (GLP)-encoding genes from smelt at the gene and cDNA levels in Chapter 4. GLPs are integral membrane proteins that facilitate passive movement of water, glycerol and urea across cellular membranes. The highlight was the discovery that AQP10ba transcript levels always increase in posterior kidney only at low temperature. This AQP10b gene paralogue may have evolved to aid in the reabsorption of urea from the proximal tubule. This research has contributed significantly to a general understanding of the cold adaptation response in smelt, and more specifically to the development of a working scenario for the mechanisms involved in glycerol synthesis and trafficking in this species.

Expression of the retinoic acid catabolic enzyme CYP26B1 in the human brain to maintain signaling homeostasis

Funding was provided by the Wellcome Trust grant WT081633MA-NCE and Biological Sciences Research Council Grant BB/K001043/1. Prof Fragoso is the recipient of a Post Doctoral Science without Borders grant from the Brazilian National Council for Scientific and Technological Development (CNPq, 237450/2012-7).

Influence of alkyl chain length on sulfated zirconia catalysed batch and continuous esterification of carboxylic acids by light alcohols

The impact of alkyl chain length on the esterification of C2–C16 organic acids with C1–C4 alcohols has been systematically investigated over bulk and SBA-15 supported sulfated zirconias (SZs). Rates of catalytic esterification for methanol with acetic acid are directly proportional to the sulfur content for both SZ and SZ/SBA-15, with the high dispersion of SZ achievable in conformal coatings over mesoporous SBA-15 confering significant rate-enhancements. Esterification over the most active 0.24 mmol gcat−1 bulk SZ and 0.29 mmol gcat−1 SZ/SBA-15 materials was inversely proportional to the alkyl chain length of alcohol and acid reactants; being most sensitive to changes from methanol to ethanol and acetic to hexanoic acids respectively. Kinetic analyses reveal that these alkyl chain dependencies are in excellent accord with the Taft relationship for polar and steric effects in aliphatic systems and the enthalpy of alcohol adsorption, implicating a Langmuir–Hinshelwood mechanism. The first continuous production of methyl propionate over a SZ fixed-bed is also demonstrated.

AltitudeOmics: Red Blood Cell metabolic adaptation to high altitude hypoxia

Red blood cells (RBCs) are key players in systemic oxygen transport. RBCs respond to in vitro hypoxia  through  the so-called  oxygen-dependent  metabolic  regulation,  which  involves  the competitive  binding  of  deoxyhemoglobin  and  glycolytic  enzymes  to  the  N-terminal  cytosolic domain  of  band  3.  This  mechanism  promotes  the  accumulation  of  2,3-DPG,  stabilizing  the deoxygenated state of hemoglobin, and cytosol acidification, triggering oxygen off-loading through the  Bohr  effect.  Despite  in  vitro  studies,  in  vivo adaptations  to  hypoxia  have  not  yet  been completely elucidated. Within  the  framework  of  the AltitudeOmics  study,  erythrocytes  were  collected  from  21 healthy volunteers at sea level, after exposure to high altitude (5260m) for 1, 7 and 16days, and following  reascent  after  7days  at 1525m.  UHPLC-MS  metabolomics  results  were  correlated  to physiological and athletic performance parameters. Immediate  metabolic  adaptations  were  noted as early as a few hours from ascending  to >5000m, and maintained for 16 days at high altitude.  Consistent with the mechanisms elucidated in vitro, hypoxia promoted glycolysis and deregulated the pentose phosphate pathway, as well purine catabolism, glutathione homeostasis, arginine/nitric oxide and sulphur/H2S metabolism. Metabolic adaptations were preserved one week after descent, consistently with improved physical performances in comparison to the first ascendance, suggesting a mechanism of metabolic memory.

Immunity and Arginine Deprivation in Alzheimer's Disease

The pathogenesis of Alzheimer’s disease (AD) is a critical unsolved question, and while recent studies have demonstrated a strong association between altered brain immune responses and disease progression, the mechanistic cause of neuronal dysfunction and death is unknown. We have previously described the unique CVN-AD mouse model of AD, in which immune-mediated nitric oxide is lowered to mimic human levels, resulting in a mouse model that demonstrates the cardinal features of AD, including amyloid deposition, hyperphosphorylated and aggregated tau, behavioral changes and age-dependent hippocampal neuronal loss. Using this mouse model, we studied longitudinal changes in brain immunity in relation to neuronal loss and, contrary to the predominant view that AD pathology is driven by pro-inflammatory factors, we find that the pathology in CVN-AD mice is driven by local immune suppression. Areas of hippocampal neuronal death are associated with the presence of immunosuppressive CD11c+ microglia and extracellular arginase, resulting in arginine catabolism and reduced levels of total brain arginine. Pharmacologic disruption of the arginine utilization pathway by an inhibitor of arginase and ornithine decarboxylase protected the mice from AD-like pathology and significantly decreased CD11c expression. Our findings strongly implicate local immune-mediated amino acid catabolism as a novel and potentially critical mechanism mediating the age-dependent and regional loss of neurons in humans with AD.

There is a large interest in identifying, lineage tracing, and determining the physiologic roles of monophagocytes in Alzheimer’s disease. While Cx3cr1 knock-in fluorescent reporting and Cre expressing mice have been critical for studying neuroimmunology, mice that are homozygous null or hemizygous for CX3CR1 have perturbed neural development and immune responses. There is, therefore, a need for similar tools in which mice are CX3CR1+/+. Here, we describe a mouse where Cre is driven by the Cx3cr1 promoter on a bacterial artificial chromosome (BAC) transgene (Cx3cr1-CreBT) and the Cx3cr1 locus is unperturbed. Similarly to Cx3cr1-Cre knock-in mice, these mice express Cre in Ly6C-, but not Ly6C+, monocytes and tissue macrophages, including microglia. These mice represent a novel tool that maintains the Cx3cr1 locus while allowing for selective gene targeting in monocytes and tissue macrophages.

The study of immunity in Alzheimer’s requires the ability to identify and quantify specific immune cell subsets by flow cytometry. While it is possible to identify lymphocyte subsets based on cell lineage-specific markers, the lack of such markers in brain myeloid cell subsets has prevented the study of monocytes, macrophages and dendritic cells. By improving on tissue homogenization, we present a comprehensive protocol for flow cytometric analysis, that allows for the identification of several cell types that have not been previously identified by flow cytometry. These cell types include F4/80hi macrophages, which may be meningeal macrophages, IA/IE+ macrophages, which may represent perivascular macrophages, and dendritic cells. The identification of these cell types now allows for their study by flow cytometry in homeostasis and disease.

(Table 1) Organic acid analyses from Leg 135 interstitial waters

For the first time, short-chain organic acids are described in interstitial waters from sediments and lithified materials in a backarc setting. Organic acids in interstitial waters from the Tonga forearc region were also analyzed and compared with previous organic acid analyses from the Mariana and Bonin forearc interstitial waters. In the Tonga backarc setting, propionate typically dominates the organic acid assemblage, and organic acids are a consistent feature of these interstitial waters. The persistent presence of ammonia and the dominance of propionate over formate in the backarc interstitial waters suggest that the organic acids in this setting have their origin in reductive deamination of amino acids derived from sedimentary proteinaceous material. The organic acid assemblage revealed in the samples from Hole 841B in the Tonga forearc are similar to the organic acid assemblage detected in the Mariana forearc, that is, formate dominates the assemblage over acetate or propionate. These forearc organic acid assemblages may both have formed by a similar mechanism.

(Table 1) Organic acid analyses from ODP Leg 125 interstitial waters

For the first time, short-chain organic acids are described from serpentine-associated interstitial waters. In this geologic setting, formate typically dominates the organic acid assemblage. Within the forearc setting, the organic acids are associated only with unconsolidated serpentine. Their existence may be the result of alkaline hydrolysis of ester linkages in organic matter that has been entrained in the serpentine diapir.

Substituição de fonte de amido por fibra solúvel em detergente neutro na dieta de vacas

v. 17, n .2, p. 164-174, abr./jun. 2016.

Characterization of the glial scar tissue in a murine model of spinal cord compression, with focus on hyaluronan

Spinal cord injury (SCI) is a devastating neurological disorder that affects thousands of people each year. Although in recent decades significant progress has been made in relation to understanding the molecular and cellular events underlying the nervous damage, spinal cord injury is still a highly disabling condition for which there is no curative therapy. People affected by spinal cord injuries manifested dysfunction or loss, temporary or permanent, of motor, sensory and / or autonomic functions depending on the spinal lesion damaged. Currently, the incidence rate of this type of injury is approximately 15-40 cases per million people worldwide. At the origin of these lesions are: road accidents, falls, interpersonal violence and the practice of sports. In this work we placed the hypothesis that HA is one of the component of the scar tissue formed after a compressive SCI, that it is likely synthetised by the perilesional glial cells and that it might support the permeation of the glial scar during the late phase of SCI. Nowadays, much focus is drawn on the recovery of CNS function, made impossible after SCI due to the high content of sulfated proteoglycans in the extracellular matrix. Counterbalancing the ratio between these proteoglycans and hyaluronic acid could be one of the experimental therapy to re-permeate the glial scar tissue formed after SCI, making possible axonal regrowth and functional recovery. Therefore, we established a model of spinal cord compression in mice and studied the glial scar tissue, particularly through the characterization of the expression of enzymes related to the metabolism of HA and the subsequent concentration thereof at different distances of the lesion epicenter. Our results show that the lesion induced in mice shows results similar to those produced in human lesions, in terms of histologic similarities and behavioral results. but these animals demonstrate an impressive spontaneous reorganization mechanism of the spinal cord tissue that occurs after injury and allows for partial recovery of the functions of the CNS. As regards the study of the glial scar, changes were recorded at the level of mRNA expression of enzymes metabolizing HA i.e., after injury there was a decreased expression of HA synthases 1-2 (HAS 1-2) and an increase of the expression HAS3 synthase mRNA, as well as the enzymes responsible for the HA catabolism, HYAL 1-2. But the amount of HA measured through the ELISA test was found unchanged after injury, it is not possible to explain this fact only with the change of expression of enzymes. At two weeks and in response to SCI, we found synthesized HA by reactive astrocytes and probably by others like microglial cells as it was advanced by the HA/GFAP+ and HA/IBA1+ cells co-location.

The role of glucocorticoids in naturally fasting grey seal (Halichoerus grypus) pups: dexamethasone stimulates mass loss and protein utilisation, but not departure from the colony

Seals must manage their energy reserves carefully while they fast on land to ensure that they go to sea with sufficient fuel to sustain them until they find food. Glucocorticoids (GCs) have been implicated in the control of fuel metabolism and termination of fasting in pinnipeds. Here we tested the hypothesis that dexamethasone, an artificial GC, increases fat and protein catabolism, and induces departure from the breeding colony in wild, fasting grey seal pups. A single intramuscular dose of dexamethasone completely suppressed cortisol production for 24–72 h, demonstrating activation of GC receptors. In experiment 1, we compared the effects of a single dose of dexamethasone or saline administered 10 days after weaning on fasting mass and body composition changes, cortisol, blood urea nitrogen (BUN) and glucose levels, and timing of departure from the colony. In experiment 2, we investigated the effects of dexamethasone on short-term (5 days) changes in mass loss, body composition and BUN levels. In experiment 1, dexamethasone induced a short-lived increase in mass loss, but there was no difference in timing of departure between dexamethasone- and saline-treated pups (N=10). In experiment 2, dexamethasone increased protein and water loss and prevented a decrease in BUN levels (N=11). Our data suggest changes in cortisol contribute to regulation of protein catabolism in fasting seal pups, irrespective of the sex of the animal, but do not terminate fasting. By affecting the rate of protein depletion, lasting changes in cortisol levels could influence the amount of time seal pups have to find food, and thus may have important consequences for their survival.

Developmental programming of polycystic ovary syndrome (PCOS): prenatal androgens establish pancreatic islet α/β cell ratio and subsequent insulin secretion

Exogenous androgenic steroids applied to pregnant sheep programmes a PCOS-like phenotype in female offspring. Via ultrasound guidance we applied steroids directly to ovine fetuses at d62 and d82 of gestation, and examined fetal (day 90 gestation) and postnatal (11 months old) pancreatic structure and function. Of three classes of steroid agonists applied (androgen - Testosterone propionate (TP), estrogen - Diethystilbesterol (DES) and glucocorticoid - Dexamethasone (DEX)), only androgens (TP) caused altered pancreatic development. Beta cell numbers were significantly elevated in prenatally androgenised female fetuses (P=0.03) (to approximately the higher numbers found in male fetuses), whereas alpha cell counts were unaffected, precipitating decreased alpha:beta cell ratios in the developing fetal pancreas (P=0.001), sustained into adolescence (P=0.0004). In adolescence basal insulin secretion was significantly higher in female offspring from androgen-excess pregnancies (P=0.045), and an exaggerated, hyperinsulinaemic response to glucose challenge (P=0.0007) observed, whereas prenatal DES or DEX treatment had no effects upon insulin secretion. Postnatal insulin secretion correlated with beta cell numbers (P=0.03). We conclude that the pancreas is a primary locus of androgenic stimulation during development, giving rise to postnatal offspring whose pancreas secreted excess insulin due to excess beta cells in the presence of a normal number of alpha cells.

Effects of sodium hyaluronate and triamcinolone acetonide on proteoglycan metabolism in equine articular chondrocytes treated with interleukin-1

The objective of this study is to determine if the effects of a high molecular weight sodium hyaluronate (HA) alone or in combination with triamcinolone acetate (TA) can mitigate chondrocyte proteoglycan catabolism caused by interleukin-1 (IL-1) administration. Chondrocytes were collected from fetlock joints of ten horses euthanized for reasons unrelated to joint disease. Chondrocyte pellets were treated with media (negative control); media containing IL-1 only (positive control); or media containing IL-1 with HA only (0.5 or 2.0 mg/mL), TA only (0.06 or 0.6 mg/mL), or HA (0.5 or 2.0 mg/mL) and TA (0.06 or 0.6 mg/mL) in combination. Chondrocyte pellets were assayed for newly synthesized GAG, total GAG content, total DNA content, and mRNA levels of collagen type II, aggrecan, and COX-2. The high concentration of HA (2.0 mg/mL) increased GAG synthesis while the high concentration of TA (0.6 mg/mL) decreased loss of GAG into the media. Both the high concentration of HA and TA increased the total GAG content within the pellet. There was no change in pellet DNA content with either treatment. TA reduced COX-2 mRNA levels as well as aggrecan and collagen type II expression. Treatment with HA had no effect on mRNA levels of COX-2, aggrecan or collagen type II. These results indicate that the high concentration of HA or TA alone or in combination will mitigate effects of IL-1 administration on proteoglycan catabolism of equine articular chondrocytes.

Characterizing neural tissues with mass spectrometry imaging via enhanced computational approaches

Neuropeptides affect the activity of the myriad of neuronal circuits in the brain. They are under tight spatial and chemical control and the dynamics of their release and catabolism directly modify neuronal network activity. Understanding neuropeptide functioning requires approaches to determine their chemical and spatial heterogeneity within neural tissue, but most imaging techniques do not provide the complete information desired. To provide chemical information, most imaging techniques used to study the nervous system require preselection and labeling of the peptides of interest; however, mass spectrometry imaging (MSI) detects analytes across a broad mass range without the need to target a specific analyte. When used with matrix-assisted laser desorption/ionization (MALDI), MSI detects analytes in the mass range of neuropeptides. MALDI MSI simultaneously provides spatial and chemical information resulting in images that plot the spatial distributions of neuropeptides over the surface of a thin slice of neural tissue. Here a variety of approaches for neuropeptide characterization are developed. Specifically, several computational approaches are combined with MALDI MSI to create improved approaches that provide spatial distributions and neuropeptide characterizations. After successfully validating these MALDI MSI protocols, the methods are applied to characterize both known and unidentified neuropeptides from neural tissues. The methods are further adapted from tissue analysis to be able to perform tandem MS (MS/MS) imaging on neuronal cultures to enable the study of network formation. In addition, MALDI MSI has been carried out over the timecourse of nervous system regeneration in planarian flatworms resulting in the discovery of two novel neuropeptides that may be involved in planarian regeneration. In addition, several bioinformatic tools are developed to predict final neuropeptide structures and associated masses that can be compared to experimental MSI data in order to make assignments of neuropeptide identities. The integration of computational approaches into the experimental design of MALDI MSI has allowed improved instrument automation and enhanced data acquisition and analysis. These tools also make the methods versatile and adaptable to new sample types.