Enzyme activity, body measures and heart mass of Sepia officinalis sampled in the English Channel and Adriatic Sea

In the eurythermal cuttlefish Sepia officinalis, performance depends on hearts that ensure systemic oxygen supply over a broad range of temperatures. We therefore aimed to identify adjustments in energetic cardiac capacity and underlying mitochondrial function supporting thermal acclimation and adaptation that could be crucial for the cuttlefish's competitive success in variable environments. Two genetically distinct cuttlefish populations were acclimated to 11, 16 and 21°C. Subsequently, skinned and permeabilised heart fibres were used to assess mitochondrial functioning by means of high-resolution respirometry and a substrate-inhibitor protocol, followed by measurements of cardiac citrate synthase and cytosolic enzyme activities. Temperate English Channel cuttlefish had lower mitochondrial capacities but larger hearts than subtropical Adriatic cuttlefish. Warm acclimation to 21°C decreased mitochondrial complex I activity in Adriatic cuttlefish and increased complex IV activity in English Channel cuttlefish. However, compensation of mitochondrial capacities did not occur during cold acclimation to 11°C. In systemic hearts, the thermal sensitivity of mitochondrial substrate oxidation was high for proline and pyruvate but low for succinate. Oxygen efficiency of catabolism rose as temperature changed from 11 to 21°C via shifts to oxygen-conserving oxidation of proline and pyruvate and via reduced relative proton leak. The changes observed for substrate oxidation, mitochondrial complexes, relative proton leak and heart mass improve energetic efficiency and essentially seem to extend tolerance to high temperatures and reduce associated tissue hypoxia. We conclude that cuttlefish sustain cardiac performance and, thus, systemic oxygen delivery over short- and long-term changes of temperature and environmental conditions by multiple adjustments in cellular and mitochondrial energetics.

The identification of a 9-cis retinol dehydrogenase in the mouse embryo reveals a pathway for synthesis of 9-cis retinoic acid

The ligand-controlled retinoic acid (RA) receptors and retinoid X receptors are important for several physiological processes, including normal embryonic development, but little is known about how their ligands, all-trans and 9-cis RA, are generated. Here we report the identification of a stereo-specific 9-cis retinol dehydrogenase, which is abundantly expressed in embryonic tissues known to be targets in the retinoid signaling pathway. The membrane-bound enzyme is a member of the short-chain alcohol dehydrogenase/reductase superfamily, able to oxidize 9-cis retinol into 9-cis retinaldehyde, an intermediate in 9-cis RA biosynthesis. Analysis by nonradioactive in situ hybridization in mouse embryos shows that expression of the enzyme is temporally and spatially well controlled during embryogenesis with prominent expression in parts of the developing central nervous system, sensory organs, somites and myotomes, and several tissues of endodermal origin. The identification of this enzyme reveals a pathway in RA biosynthesis, where 9-cis retinol is generated for subsequent oxidation to 9-cis RA.

Mitochondrial localization of a NADR-dependent isocitrate dehydrogenase isoenzyme by using the green fluorescent protein as a marker

In this work, we describe the isolation of a new cDNA encoding an NADP-dependent isocitrate dehydrogenase (ICDH). The nucleotide sequence in its 5′ region gives a deduced amino acid sequence indicative of a targeting peptide. However, even if this cDNA clearly encodes a noncytosolic ICDH, it is not possible to say from the targeting peptide sequence to which subcellular compartment the protein is addressed. To respond to this question, we have transformed tobacco plants with a construct containing the entire targeting signal-encoding sequence in front of a modified green fluorescent protein (GFP) gene. This construct was placed under the control of the cauliflower mosaic virus 35S promoter, and transgenic tobacco plants were regenerated. At the same time, and as a control, we also have transformed tobacco plants with the same construct but lacking the nucleotide sequence corresponding to the ICDH-targeting peptide, in which the GFP is retained in the cytoplasm. By optical and confocal microscopy of leaf epiderm and Western blot analyses, we show that the putative-targeting sequence encoded by the cDNA addresses the GFP exclusively into the mitochondria of plant cells. Therefore, we conclude that this cDNA encodes a mitochondrial ICDH.

Glyceraldehyde-3-phosphate dehydrogenase: Nuclear translocation participates in neuronal and nonneuronal cell death

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels increase in particulate fractions in association with cell death in HEK293 cells, S49 cells, primary thymocytes, PC12 cells, and primary cerebral cortical neuronal cultures. Subcellular fractionation and immunocytochemistry reveal that this increase primarily reflects nuclear translocation. Nuclear GAPDH is tightly bound, resisting extraction by DNase or salt treatment. Treating primary thymocytes, PC12 cells, and primary cortical neurons with antisense but not sense oligonucleotides to GAPDH prevents cell death. Because cell-death-associated nuclear translocation of GAPDH and antisense protection occur in multiple neuronal and nonneuronal systems, we propose that GAPDH is a general mediator of cell death and uses nuclear translocation as a signaling mechanism.

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

Conformation of coenzyme pyrroloquinoline quinone and role of Ca2+ in the catalytic mechanism of quinoprotein methanol dehydrogenase

The ab initio structures of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), semiquinone (PQQH), and dihydroquinone (PQQH2) have been determined and compared with ab initio structures of the (PQQ)Ca2+, (PQQH)Ca2+, and (PQQH2)Ca2+ complexes as well as the x-ray structure of (PQQ)Ca2+ bound at the active site of the methanol dehydrogenase (MDH) of methyltropic bacteria. Plausible mechanisms for the MDH oxidation of methanol involving the (PQQ)Ca2+ complex are explored via ab initio computations and discussed. Considering the reaction of methanol with PQQ in the absence of Ca2+, nucleophilic addition of methanol to the PQQ C-5 carbonyl followed by a retro-ene elimination is deemed unlikely due to large energy barrier. A much more favorable disposition of the methanol C-5 adduct to provide formaldehyde involves proton ionization of the intermediate followed by elimination of methoxide concerted with hydride transfer to the oxygen of the C-4 carbonyl. Much the same transition state is reached if one searches for the transition state beginning with Asp-303–CO2−general-base removal of the methanol proton of the (PQQ)Ca2+O(H)CH3 complex concerted with hydride transfer to the oxygen at C-4. For such a mechanism the role of the Ca2+ moiety would be to (i) contribute to the formation of the ES complex (ii) provide a modest decrease in the pKa of methanol substrate,; and (iii) polarize the oxygen at C-5.

Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: A dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia

Severe jaundice leading to kernicterus or death in the newborn is the most devastating consequence of glucose-6-phosphate dehydrogenase (EC 1.1.1.49; G-6-PD) deficiency. We asked whether the TA repeat promoter polymorphism in the gene for uridinediphosphoglucuronate glucuronosyltransferase 1 (EC 2.4.1.17; UDPGT1), associated with benign jaundice in adults (Gilbert syndrome), increases the incidence of neonatal hyperbilirubinemia in G-6-PD deficiency. DNA from term neonates was analyzed for UDPGT1 polymorphism (normal homozygotes, heterozygotes, variant homozygotes), and for G-6-PD Mediterranean deficiency. The variant UDPGT1 promoter allele frequency was similar in G-6-PD-deficient and normal neonates. Thirty (22.9%) G-6-PD deficient neonates developed serum total bilirubin ≥ 257 μmol/liter, vs. 22 (9.2%) normals (P = 0.0005). Of those with the normal homozygous UDPGT1 genotype, the incidence of hyperbilirubinemia was similar in G-6-PD-deficients and controls (9.7% and 9.9%). In contrast, in the G-6-PD-deficient neonates, those with the heterozygous or homozygous variant UDPGT1 genotype had a higher incidence of hyperbilirubinemia than corresponding controls (heterozygotes: 31.6% vs. 6.7%, P < 0.0001; variant homozygotes: 50% vs. 14.7%, P = 0.02). Among G-6-PD-deficient infants the incidence of hyperbilirubinemia was greater in those with the heterozygous (31.6%, P = 0.006) or variant homozygous (50%, P = 0.003) UDPGT1 genotype than in normal homozygotes. In contrast, among those normal for G-6-PD, the UDPGT1 polymorphism had no significant effect (heterozygotes: 6.7%; variant homozygotes: 14.7%). Thus, neither G-6-PD deficiency nor the variant UDPGT1 promoter, alone, increased the incidence of hyperbilirubinemia, but both in combination did. This gene interaction may serve as a paradigm of the interaction of benign genetic polymorphisms in the causation of disease.

Pinus banksiana has at least seven expressed alcohol dehydrogenase genes in two linked groups

The alcohol dehydrogenase (Adh) gene family is much more complex in Pinus banksiana than in angiosperms, with at least seven expressed genes organized as two tightly linked clusters. Intron number and position are highly conserved between P. banksiana and angiosperms. Unlike angiosperm Adh genes, numerous duplications, as large as 217 bp, were observed within the noncoding regions of P. banksiana Adh genes and may be a common feature of conifer genes. A high frequency of duplication over a wide range of scales may contribute to the large genome size of conifers.

The maturity-onset diabetes of the young (MODY1) transcription factor HNF4α regulates expression of genes required for glucose transport and metabolism

Hepatocyte nuclear factor 4α (HNF4α) plays a critical role in regulating the expression of many genes essential for normal functioning of liver, gut, kidney, and pancreatic islets. A nonsense mutation (Q268X) in exon 7 of the HNF4α gene is responsible for an autosomal dominant, early-onset form of non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young; gene named MODY1). Although this mutation is predicted to delete 187 C-terminal amino acids of the HNF4α protein the molecular mechanism by which it causes diabetes is unknown. To address this, we first studied the functional properties of the MODY1 mutant protein. We show that it has lost its transcriptional transactivation activity, fails to dimerize and bind DNA, implying that the MODY1 phenotype is because of a loss of HNF4α function. The effect of loss of function on HNF4α target gene expression was investigated further in embryonic stem cells, which are amenable to genetic manipulation and can be induced to form visceral endoderm. Because the visceral endoderm shares many properties with the liver and pancreatic β-cells, including expression of genes for glucose transport and metabolism, it offers an ideal system to investigate HNF4-dependent gene regulation in glucose homeostasis. By exploiting this system we have identified several genes encoding components of the glucose-dependent insulin secretion pathway whose expression is dependent upon HNF4α. These include glucose transporter 2, and the glycolytic enzymes aldolase B and glyceraldehyde-3-phosphate dehydrogenase, and liver pyruvate kinase. In addition we have found that expression of the fatty acid binding proteins and cellular retinol binding protein also are down-regulated in the absence of HNF4α. These data provide direct evidence that HNF4α is critical for regulating glucose transport and glycolysis and in doing so is crucial for maintaining glucose homeostasis.