Maintenance of a high photosynthetic performance is linked to flooding tolerance in citrus

Citrus trees have been considered as flooding-sensitive although important differences in tolerance among species have been reported. The tolerance to flooding has been linked to optimal photosynthetic performance in other woody plants. To test whether there was a relationship between photosynthetic performance and flooding tolerance, leaf damage, chlorophyll content, net photosynthetic rate, stomatal conductance, the ratio of internal to ambient CO₂ concentration (C_i/C_a), water use efficiency and chlorophyll fluorescence parameters were studied in leaves of three citrus genotypes differing in their tolerance to flooding during continuous substrate flooding and alternate cycles of flooding and recovery. In Cleopatra and Citrumelo genotypes, marked reductions in net photosynthetic rate and stomatal conductance as well as increases in C_i/C_a in response to flooding stress were observed although with differences in the magnitude of the variation. In contrast, in Carrizo, a relatively flooding-tolerant genotype, there were no changes in net photosynthetic rate or in C_i/C_a and only a slight decrease in stomatal conductance occurred in response to flooding. Significant correlation between net photosynthetic rate and chlorophyll fluorescence parameters during flooding indicated a biochemical impairment of photosynthetic activity. This effect was apparently linked to damage in the PSII light-harvesting complexes induced by flooding and a subsequent effect on PSII to PSI electron flow that may alter the redox status in cells. Such biochemical impairment could lead to an increase in oxidative damage in Cleopatra and Citrumelo. The maintenance of good photosynthetic performance together with mechanisms to adjust electron flow in the photosynthetic apparatus could be linked to flooding tolerance in these woody plants.     

Identification of Novel Type 2 Diabetes Candidate Genes Involved in the Crosstalk between the Mitochondrial and the Insulin Signaling Systems

Type 2 Diabetes (T2D) is a highly prevalent chronic metabolic disease with strong co-morbidity with obesity and cardiovascular diseases. There is growing evidence supporting the notion that a crosstalk between mitochondria and the insulin signaling cascade could be involved in the etiology of T2D and insulin resistance. In this study we investigated the molecular basis of this crosstalk by using systems biology approaches. We combined, filtered, and interrogated different types of functional interaction data, such as direct protein–protein interactions, co-expression analyses, and metabolic and signaling dependencies. As a result, we constructed the mitochondria-insulin (MITIN) network, which highlights 286 genes as candidate functional linkers between these two systems. The results of internal gene expression analysis of three independent experimental models of mitochondria and insulin signaling perturbations further support the connecting roles of these genes. In addition, we further assessed whether these genes are involved in the etiology of T2D using the genome-wide association study meta-analysis from the DIAGRAM consortium, involving 8,130 T2D cases and 38,987 controls. We found modest enrichment of genes associated with T2D amongst our linker genes (p = 0.0549), including three already validated T2D SNPs and 15 additional SNPs, which, when combined, were collectively associated to increased fasting glucose levels according to MAGIC genome wide meta-analysis (p = 8.12×10⁻⁵). This study highlights the potential of combining systems biology, experimental, and genome-wide association data mining for identifying novel genes and related variants that increase vulnerability to complex diseases.     

Cannabinoid receptors CB1 and CB2 form functional heteromers in brain

Exploring the role of cannabinoid CB(2) receptors in the brain, we present evidence of CB(2) receptor molecular and functional interaction with cannabinoid CB(1) receptors. Using biophysical and biochemical approaches, we discovered that CB(2) receptors can form heteromers with CB(1) receptors in transfected neuronal cells and in rat brain pineal gland, nucleus accumbens, and globus pallidus. Within CB(1)-CB(2) receptor heteromers expressed in a neuronal cell model, agonist co-activation of CB(1) and CB(2) receptors resulted in a negative cross-talk in Akt phosphorylation and neurite outgrowth. Moreover, one specific characteristic of CB(1)-CB(2) receptor heteromers consists of both the ability of CB(1) receptor antagonists to block the effect of CB(2) receptor agonists and, conversely, the ability of CB(2) receptor antagonists to block the effect of CB(1) receptor agonists, showing a bidirectional cross-antagonism phenomenon. Taken together, these data illuminate the mechanism by which CB(2) receptors can negatively modulate CB(1) receptor function.     

Mechanisms of HDL deficiency in mice overexpressing human apoA-II

To ascertain the mechanisms underlying the hypoalphalipoproteinemia present in mice overexpressing human apolipoprotein A-II (apoA-II) (line 11.1), radiolabeled HDL or apoA-I were injected into mice. Fractional catabolic rate of [(3)H]cholesteryl oleoyl ether HDL ([(3)H]HDL) was 2-fold increased in 11.1 transgenic mice compared with control mice and this was concomitant with increased radioactivity in liver, gonads, and adrenals. However, scavenger receptor class B, type I (SR-BI) was increased only in adrenals. [(3)H]HDL of 11.1 transgenic mice presented greater binding but decreased uptake compared with control mice when Chinese hamster ovary cells transfected with SR-BI were used, thereby pointing to unknown but SR-BI-independent mechanisms as being responsible for the increased (3)H-radioactivity seen in liver and gonads. Synthesis rate (SR) of plasma [(3)H]HDL was 2-fold decreased in 11.1 transgenic mice. Mouse (125)I-apoA-I was 2-fold more rapidly catabolized (mainly by the kidney) in transgenic mice. Mouse apoA-I displacement from HDL by the addition of isolated human apoA-II was reproduced ex vivo; thus, this mechanism may be involved in the increased renal catabolism of apoA-I. ApoA-I SR was 2-fold decreased in 11.1 transgenic mice and this was concomitant with a 2.3-fold decrease in hepatic apoA-I mRNA abundance. Our findings show that multiple mechanisms are involved in the HDL deficiency presented by mice overexpressing human apoA-II.     

Direct evidence in vivo of impaired macrophage-specific reverse cholesterol transport in ATP-binding cassette transporter A1-deficient mice

The ATP-binding cassette transporter A1 (ABCA1) is a key regulator of high-density lipoprotein (HDL) metabolism. There is strong evidence that ABCA1 is a key regulator of reverse cholesterol transport (RCT). However, this could not be proved in vivo since hepatobiliary cholesterol transport was unchanged in ABCA1-deficient mice (ABCA1-/-). We used ABCA1-/- mice to test the hypothesis that ABCA1 is a critical determinant of macrophage-specific RCT. Although this cell-specific RCT only accounts for a tiny part of total RCT, it is widely accepted that it may have a major impact on atherosclerosis susceptibility. [(3)H]cholesterol-labeled endogenous macrophages were injected intraperitoneally into wild-type ABCA1+/+, ABCA1+/- and ABCA1-/- mice maintained on a chow diet. A direct relationship was observed between ABCA1 gene dose and plasma [(3)H]cholesterol at 24 and 48 h after the injection of tracer into the mice. Forty-eight hours after this injection, ABCA1-/- mice had significantly reduced [(3)H]cholesterol in liver (2.8-fold), small intestine enterocytes (1.7-fold) and feces (2-fold). To our knowledge, this is the first direct in vivo quantitative evidence that ABCA1 is a critical determinant of macrophage-specific RCT.     

Differences in nuclear retention characteristics of agonist-activated glucocorticoid receptor may determine specific responses

We studied the glucocorticoid response to the synthetic steroid pregna-1,4-diene-11beta-ol-3,20-dione (DeltaHOP) in several cell types and correlated its biological effect with the ability of the glucocorticoid receptor (GR) to be retained in the nuclear compartment. We observed that the DeltaHOP-transformed GR was diffusely distributed in the nucleus compared to the discrete structures observed for the dexamethasone (DEX)-transformed GR. Despite the fact that the receptor was entirely nuclear upon binding of each steroid and exhibited identical nuclear export rates, a greater amount of DeltaHOP-transformed GR was recovered in the cytoplasmic fraction after hypotonic cell lysis. Furthermore, accelerated nuclear export of GR was evidenced in digitonin-permeabilized cells treated with ATP and molybdate. Inasmuch as limited trypsinization of DEX-GR and DeltaHOP-GR complexes yielded different proteolytic products, we conclude that GR undergoes a differential conformational change upon binding of each ligand. We propose that these conformational differences may consequently lead to changes of stability in the interaction of the GR with chromatin. Therefore, the dynamic exchange of liganded GR with chromatin is likely to have significant consequences for the observed pleiotropic physiological responses triggered by glucocorticoid ligands, not only in different tissues but also in the same cell type.     

Vibrational coupling as diagnostic for intramolecular hydrogen bonds of carbohydrates in aqueous solution

The coupling of nuC-O and deltaO-D vibrations in the 1200-900 cm(-1) IR range leads to band shifting in opposite directions, which provides information on intramolecular hydrogen bonding of carbohydrates in aqueous solution. The aqueous solution IR spectra of 2-acetamide-1,6-anhydro-2-deoxy-D-glucopyranose and cis-1,2-cyclopentanediol and tetrahydrofuran-ethanol mixtures are reported. Frequency upshifting upon deuteration is observed for the nuC-O bands of both a hydrogen bond acceptor and donor in ether-hydroxyl and hydroxyl-hydroxyl contacts. The 1200-900 cm(-1) range can be used to reveal the presence of intramolecular hydrogen bonds in aqueous carbohydrates, unlike the OH stretching region, which cannot be used in this sense due to carbohydrate band masking by the strong nuOH IR absorption of water.     

Isolation and characterization of bovine brain myelin distribution of 5′-nucleotidase

Myelin was isolated from bovine brain by several published procedures and modifications of these procedures. High activity of the myelin marker (2,3-cyclic nucleotide 3-phosphohydrolase) and low activity of contaminants markers in white matter homogenates in respect to cerebral cortex showed the white matter to be better than the cerebral cortex or the whole brain for myelin isolation. A procedure is described for the preparation of purified myelin from bovine white matter which yielded a content of protein (40%), myelin marker (51%), and 5-nucleotidase (25%) in purified myelin higher than by any used method. Acetylcholinesterase or succinate dehydrogenase was lower than 7% of its activity in the white matter homogenate, and monoamine oxidase and NADPH: cytochrome c reductase were not recovered in myelin fraction. Morphologically, myelin fraction was shown to mainly consist of multilamellar membranes of different sizes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of myelin fraction showed a characteristic protein pattern of myelin. When our procedure was applied to frozen white matter, lower protein (32%) and myelin marker (34%) and similar 5-nucleotidase activity (24%) were recovered in myelin, increasing its recovery in denser fractions of white matter.