Genetic Determinants for Gestational Diabetes Mellitus and Related Metabolic Traits in Mexican Women

Epidemiological and physiological similarities among Gestational Diabetes Mellitus (GDM) and Type 2 Diabetes (T2D) suggest that both diseases, share a common genetic background. T2D risk variants have been associated to GDM susceptibility. However, the genetic architecture of GDM is not yet completely understood. We analyzed 176 SNPs for 115 loci previously associated to T2D, GDM and body mass index (BMI), as well as a set of 118 Ancestry Informative Markers (AIMs), in 750 pregnant Mexican women. Association with GDM was found for two of the most frequently replicated T2D loci: a TCF7L2 haplotype (CTTC: rs7901695, rs4506565, rs7903146, rs12243326; P=2.16x10^-06; OR=2.95) and a KCNQ1 haplotype (TTT: rs2237892, rs163184, rs2237897; P=1.98x10^-05; OR=0.55). In addition, we found two loci associated to glycemic traits: CENTD2 (60’ OGTT glycemia: rs1552224, P=0.03727) and MTNR1B (HOMA B: rs1387153, P=0.05358). Remarkably, a major susceptibility SLC16A11 locus for T2D in Mexicans was not shown to play a role in GDM risk. The fact that two of the main T2D associated loci also contribute to the risk of developing GDM in Mexicans, confirm that both diseases share a common genetic background. However, lack of association with a Native American contribution T2D risk haplotype, SLC16A11, suggests that other genetic mechanisms may be in play for GDM.     

Mesenchymal stem cell conditioned medium increases glial reactivity and decreases neuronal survival in spinal cord slice cultures

Ex vivo spinal cord slice cultures (SCSC) allow study of spinal cord circuitry, maintaining stimuli responses comparable to live animals. Previously, we have shown that mesenchymal stem/stromal cell (MSC) transplantation in vivo reduced inflammation and increased nerve regeneration but MSC survival was short-lived, highlighting that beneficial action may derive from the secretome. Previous in vitro studies of MSC conditioned medium (CM) have also shown increased neuronal growth. In this study, murine SCSC were cultured in canine MSC CM (harvested from the adipose tissue of excised inguinal fat) and cell phenotypes analysed via immunohistochemistry and confocal microscopy. SCSC in MSC CM displayed enhanced viability after propidium iodide staining. GFAP immunoreactivity was significantly increased in SCSC in MSC CM compared to controls, but with no change in proteoglycan (NG2) immunoreactivity. In contrast, culture in MSC CM significantly decreased the prevalence of βIII-tubulin immunoreactive neurites, whilst Ca²⁺ transients per cell were significantly increased. These ex vivo results contradict previous in vitro and in vivo reports of how MSC and their secretome may affect the microenvironment of the spinal cord after injury and highlight the importance of a careful comparison of the different experimental conditions used to assess the potential of cell therapies for the treatment of spinal cord injury.     

Investigation of charged polymer influence on green fluorescent protein thermal stability

Methods of stabilization and formulation of proteins are important in both biopharmaceutical and biocatalysis industries. Polymers are often used as modifiers of characteristics of biological macromolecules to improve the biochemical activity and stability of proteins or drug bioavailability. Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Relative thermal stability was undertaken by incubation of GFP at varying temperatures and GFP fluorescence was used as a reporter for unfolding. At 80°C, DEAE-dextran did not have any effect on GFP fluorescence, indicating that it does not confer stability.     

LPS–protein aggregation influences protein partitioning in aqueous two-phase micellar systems

Lipopolysaccharide endotoxins (LPS) are the most common pyrogenic substances in recombinant peptides and proteins purified from Gram-negative bacteria, such as Escherichia coli. In this respect, aqueous two-phase micellar systems (ATPMS) have already proven to be a good strategy to purify recombinant proteins of pharmaceutical interest and remove high LPS concentrations. In this paper, we review our recent experimental work in protein partitioning in Triton X-114 ATPMS altogether with some new results and show that LPS–protein aggregation can influence both protein and LPS partitioning. Green fluorescent protein (GFPuv) was employed as a model protein. The ATPMS technology proved to be effective for high loads of LPS removal into the micelle-rich phase (%REM_LPS?>?98 %) while GFPuv partitioned preferentially to the micelle-poor phase (K _GFPuv?<?1.00) due to the excluded-volume interactions. However, theoretically predicted protein partition coefficient values were compared with experimentally obtained ones, and good agreement was found only in the absence of LPS. Dynamic light scattering measurements showed that protein–LPS interactions were taking place and influenced the partitioning process. We believe that this phenomenon should be considered in LPS removal employing any kind of aqueous two-phase system. Nonetheless, ATPMS can still be considered as an efficient strategy for high loads of LPS removal, but being aware that the excluded-volume partitioning theory available might overestimate partition coefficient values due to the presence of protein–LPS aggregation.     

Immunohistological localization of serotonin in the CNS and feeding system of the stable fly Stomoxys calcitrans L. (Diptera: Muscidae)

Serotonin, or 5-hydroxytryptamine (5-HT), plays critical roles as a neurotransmitter and neuromodulator that control or modulate many behaviors in insects, such as feeding. Neurons immunoreactive (IR) to 5-HT were detected in the central nervous system (CNS) of the larval and adult stages of the stable fly, Stomoxys calcitrans, using an immunohistological technique. The location and pattern of the 5-HT IR neurons are described and compared for these two different developmental stages. Anatomical features of the fly feeding system were analyzed in third instar larvae and adult flies using a combination of histological and immunohistological techniques. In third instar larvae, the cibarial dilator muscles were observed within the cibarial pump skeleton and innervated by 5-HT IR neurons in nerves arising from the brain. There were four pairs of nerves arising from the frontal surface of the larval brain that innervate the cibarial pump muscles, pharynx, and muscles controlling the mouth hooks. A strong serotoninergic innervation of the anterior stomatogastric system was observed, which suggests 5-HT may play a role in the coordination of different phases of food ingestion by larvae. Similarly, many 5-HT IR neurons were found in both the brain and the thoracico-abdominal ganglia in the adult, some of which innervate the cibarial pump dilator muscles and the stomatogastric muscles. This is tnhe first report describing neuromuscular structures of the stable fly feeding system. The results reported here suggest 5-HT may play a critical role in feeding behaviors of stable fly larvae and adults.     

Evidence for anaerobic metabolism in the larval tiger beetle, Phaeoxantha klugii (Col. Cicindelidae) from a Central Amazonian floodplain (Brazil)

Abstract. The tiger beetle, Phaeoxantha klugii Chaudoir, survives the annual inundation period in Central Amazonian floodplains as a third‐instar larvae submerged in the soil at approximately 29 °C for up to 3.5 months. Previous studies showed an exceptional anoxia resistance in these larvae and this study investigates whether they perform anaerobiosis. Larvae collected in the field were exposed to a pure nitrogen atmosphere for 0–9 days in the laboratory. The content of lactate, alanine, free sugars and glycogen is analysed in surviving larvae. Lactate and alanine contents rise during anoxia from around 1.5 and 7 to 6–14 and 15–22 µmol g^?1 fresh mass, respectively, providing evidence for anaerobic metabolism. Both compounds show a steep increase during the first 12 h and a tendency to rise further with increasing duration of anoxic conditions, indicating a significant metabolic depression within the first day. Content of free sugars and glycogen varies greatly between individuals and ranges from 0.08–2.5 and 0.05–2.9 mg g^?1 fresh mass, respectively. Whether glycogen is used as metabolic substrate for anaerobiosis could not be verified. The findings for free sugars indicate that larvae apparently maintain the ability to regulate the level of glucose and/or trehalose even after 9 days of anoxia.     

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.     

Ceramide is a potent activator of plasma membrane Ca2+-ATPase from kidney-promixal tubule cells with protein kinase A as an intermediate

The kidney-proximal tubules are involved in reabsorbing two-thirds of the glomerular ultrafiltrate, a key Ca(2+)-modulated process that is essential for maintaining homeostasis in body fluid compartments. The basolateral membranes of these cells have a Ca(2+)-ATPase, which is thought to be responsible for the fine regulation of intracellular Ca(2+) levels. In this paper we show that nanomolar concentrations of ceramide (Cer(50) = 3.5 nm), a natural product derived from sphingomyelinase activity in biological membranes, promotes a 50% increase of Ca(2+)-ATPase activity in purified basolateral membranes. The stimulatory effect of ceramide occurs through specific and direct (cAMP-independent) activation of a protein kinase A (blocked by 10 nm of the specific inhibitor of protein kinase A (PKA), the 5-22 peptide). The activation of PKA by ceramide results in phosphorylation of the Ca(2+)-ATPase, as detected by an anti-Ser/Thr specific PKA substrate antibody. It is observed a straight correlation between increase of Ca(2+)-ATPase activity and PKA-mediated phosphorylation of the Ca(2+) pump molecule. Ceramide also stimulates phosphorylation of renal Ca(2+)-ATPase via protein kinase C, but stimulation of this pathway, which inhibits the Ca(2+) pump in kidney cells, is counteracted by the ceramide-triggered PKA-mediated phosphorylation. The potent effect of ceramide reveals a new physiological activator of the plasma membrane Ca(2+)-ATPase, which integrates the regulatory network of glycerolipids and sphingolipids present in the basolateral membranes of kidney cells.     

Rapid regulation of Na+ fluxes and ammonia excretion in response to acute environmental hypoxia in the Amazonian oscar, Astronotus ocellatus

The Amazonian oscar is extremely resistant to hypoxia, and tolerance scales with size. Overall, ionoregulatory responses of small ( approximately 15 g) and large oscars ( approximately 200 g) to hypoxia were qualitatively similar, but the latter were more effective. Large oscars exhibited a rapid reduction in unidirectional Na(+) uptake rate at the gills during acute hypoxia (Po(2) approximately 10 mmHg), which intensified with time (7 or 8 h); Na(+) efflux rates were also reduced, so net balance was little affected. The inhibitions were virtually immediate (1st h) and preceded a later 60% reduction (at 3 h) in gill Na(+)-K(+)-ATPase activity, reflected in a 60% reduction in maximum Na(+) uptake capacity without change in affinity (Km) for Na(+). Upon acute restoration of normoxia, recovery of Na(+) uptake was delayed for 1 h. These data suggest that dual mechanisms may be involved (e.g., immediate effects of O(2) availability on transporters, channels, or permeability, slower effects of Na(+)-K(+)-ATPase regulation). Ammonia excretion appeared to be linked indirectly to Na(+) uptake, exhibiting a Michaelis-Menten relationship with external [Na(+)], but the Km was less than for Na(+) uptake. During hypoxia, ammonia excretion fell in a similar manner to Na(+) fluxes, with a delayed recovery upon normoxia restoration, but the relationship with [Na(+)] was blocked. Reductions in ammonia excretion were greater than in urea excretion. Plasma ammonia rose moderately over 3 h hypoxia, suggesting that inhibition of excretion was greater than inhibition of ammonia production. Overall, the oscar maintains excellent homeostasis of ionoregulation and N-balance during severe hypoxia.