DFT study of alpha- and beta-D-allopyranose at the B3LYP/6-311++G ** level of theory

One hundred and two conformations of alpha- and beta-D-allopyranose, the C-3 substituted epimer of glucopyranose, were geometry optimized using the density functional, B3LYP, and the basis set, 6-311++G **. Full geometry optimization was performed on different ring geometries and on the hydroxymethyl rotamers (gg/gt/tg). Analytically derived Hessians were used to calculate zero point energy, enthalpy, and entropy. The lowest energy and free energy conformation found is the alpha-tg(g-)-4C1-c conformation, which is only slightly higher in electronic (approximately 0.2 kcal/mol) and free energy than the lowest energy alpha-D-glucopyranose. The in vacuo calculations showed a small (approximately 0.3 kcal/mol) energetic preference for the alpha- over the beta-anomer for allopyranose in the 4C1 conformation, whereas in the 1C4 conformation a considerable (approximately 1.6 kcal/mol) energetic preference for the beta- over the alpha-anomer for allopyranose was encountered. The results are compared to previous aldohexose calculations in vacuo. Boat and skew forms were found that remained stable upon gradient optimization although many starting boat conformations moved to other skew forms upon optimization. As found for glucose, mannose, and galactose the orientation and interaction of the hydroxyl groups make the most significant contributions to the conformation/energy relationship in vacuo. A comparison of different basis sets and density functionals is made in the Discussion section, confirming the appropriateness of the level of theory used here.     

Reversible sequestration of active site cysteines in a 2Fe-2S-bridged dimer provides a mechanism for glutaredoxin 2 regulation in human mitochondria

Human mitochondrial glutaredoxin 2 (GLRX2), which controls intracellular redox balance and apoptosis, exists in a dynamic equilibrium of enzymatically active monomers and quiescent dimers. Crystal structures of both monomeric and dimeric forms of human GLRX2 reveal a distinct glutathione binding mode and show a 2Fe-2S-bridged dimer. The iron-sulfur cluster is coordinated through the N-terminal active site cysteine, Cys-37, and reduced glutathione. The structures indicate that the enzyme can be inhibited by a high GSH/GSSG ratio either by forming a 2Fe-2S-bridged dimer that locks away the N-terminal active site cysteine or by binding non-covalently and blocking the active site as seen in the monomer. The properties that permit GLRX2, and not other glutaredoxins, to form an iron-sulfur-containing dimer are likely due to the proline-to-serine substitution in the active site motif, allowing the main chain more flexibility in this area and providing polar interaction with the stabilizing glutathione. This appears to be a novel use of an iron-sulfur cluster in which binding of the cluster inactivates the protein by sequestering active site residues and where loss of the cluster through changes in subcellular redox status creates a catalytically active protein. Under oxidizing conditions, the dimers would readily separate into iron-free active monomers, providing a structural explanation for glutaredoxin activation under oxidative stress.     

A serial lectin approach to the mucin-type O-glycoproteome of Drosophila melanogaster S2 cells

Identification of mucin-type O-glycosylated proteins with known functions in model organisms like Drosophila could provide keys to elucidate functions of the O-glycan moiety and proteomic analyses of O-glycoproteins in higher eukaryotes remain a challenge due to structural heterogeneity and a lack of efficient tools for their specific isolation. Here we report a strategy to evaluate the O-glycosylation potential of the embryonal hemocyte-like Drosophila Schneider 2 (S2) cell line by expression of recombinant glycosylation probes derived from tandem repeats of the human mucin MUC1 or of the Drosophila salivary gland protein Sgs1. We obtained evidence that mucin-type O-glycosylation in S2 cells grown under serum-free conditions is restricted to the Tn-antigen (GalNAcalpha-Ser/Thr) and the T-antigen (Galbeta1-3GalNAcalpha-Ser/Thr) and this structural homogeneity enables unique glycoproteomic strategies. We present a label-free strategy for the isolation, profiling and analysis of O-glycosylated proteins consisting of serial lectin affinity capture, 2-DE-based glycoprotein analysis by O-glycan specific mAbs and protein identification by MALDI-MS. Protein identity and O-glycosylation was confirmed by ESI-MS/MS with detection of diagnostic sugar oxonium-ion fragments. Using this strategy, we established 2-D reference maps and identified 21 secreted and intracellular mucin-type O-glycoproteins. Our results show that Drosophila S2 cells express O-glycoproteins involved in a wide range of biological functions including proteins of the extracellular matrix (Laminin gamma-chain, Peroxidasin and Glutactin), pathogen recognition proteins (Gnbp1), stress response proteins (Glycoprotein 93), secreted proteases (Matrix-metalloprotease 1 and various trypsin-like serine proteases), protease inhibitors (Serpin 27 A) and proteins of unknown function.     

Not just any ICD device in patients with long-QT syndrome

Patients with LQT syndrome are prone to lifethreatening arrhythmias. After surviving such an event, implantation of an ICD is indicated. There are, however, special subtle demands in the treatment of these patients. In this case report we describe our findings in a patient with LQT1 syndrome, and the pitfalls that can and must be avoided. (Neth Heart J 2007;15:418-21.)     

Changes in sleep quality of athletes under normobaric hypoxia equivalent to 2,000-m altitude: a polysomnographic study.

This study evaluated the sleep quality of athletes in normobaric hypoxia at a simulated altitude of 2,000 m. Eight male athletes slept in normoxic condition (NC) and hypoxic conditions equivalent to those at 2,000-m altitude (HC). Polysomnographic recordings of sleep included the electroencephalogram (EEG), electrooculogram, chin surface electromyogram, and electrocardiogram. Thoracic and abdominal motion, nasal and oral airflow, and arterial blood oxygen saturation (Sa(O(2))) were also recorded. Standard visual sleep stage scoring and fast Fourier transformation analyses of the EEG were performed on 30-s epochs. Subjective sleepiness and urinary catecholamines were also monitored. Mean Sa(O(2)) decreased and respiratory disturbances increased with HC. The increase in respiratory disturbances was significant, but the increase was small and subclinical. The duration of slow-wave sleep (stage 3 and 4) and total delta power (<3 Hz) of the all-night non-rapid eye movement sleep EEG decreased for HC compared with NC. Subjective sleepiness and amounts of urinary catecholamines did not differ between the conditions. These results indicate that acute exposure to normobaric hypoxia equivalent to that at 2,000-m altitude decreased slow-wave sleep in athletes, but it did not change subjective sleepiness or amounts of urinary catecholamines.     

Genome-wide association study of idiopathic hypersomnia in a Japanese population

Sleep and Biological Rhythms - Idiopathic hypersomnia (IH) is a rare sleep disorder characterized by excessive daytime sleepiness, great difficulty upon awakening, and prolonged sleep time. In...     

Effect of gap junction-mediated intercellular communication on TGF-β induced epithelial-to-mesenchymal transition

Epithelial-to-mesenchymal transition (EMT) is the process in which epithelial cells lose cell polarity and cell adhesion with surrounding cells to obtain migratory and invasive abilities. On the other hand, the expression of connexin is decreased or lacked in the many types of tumor cells. This study examined the effect of gap junctional intercellular communication (GJIC) on EMT induced by the transforming growth factor-β1 (TGF-β1). To investigate the effect of GJIC on EMT in U2OS cells, smooth muscle 22-α (sm22α) promoter-driven luciferase reporter gene was introduced into Cx43-expressing cells (U2OS-Luc Cx43) and into the control parental cell line (U2OS-Luc). TGF-β1 induced the expression of EMT markers and the sm22α promoter activity of U2OS-Luc cells. Sm22α promoter activity of U2OS cells was neither dependent on the expression of Cx43 nor on the establishment of GJIC among U2OS cells. Furthermore, we found that the homocellular communication among tumor cells did not affected the tumor cell growth and migration. However, we revealed that tumor cell density was an important factor for tumor cells to acquire metastatic phenotype. Interestingly, the co-culture of U2OS cells with osteoblasts revealed that sm22α promoter activity was inhibited only by the GJIC established between these two cell types. These results suggest that normal osteoblast cells negatively regulate the EMT of tumor cells, at least in part. Thus, Cx43-mediated GJIC may have anti-metastatic activity in tumor cells. Our findings provide a new insight into the role of GJIC in cancer progression and metastasis and identify potential therapeutic targets for the treatment of cancer.     

Multi‐proxy analyses of Late Cretaceous coprolites from Germany

A total of 462 coprolites from three localities exposing Upper Cretaceous deposits in the Münster Basin, northwestern Germany, have been subjected to an array of analytical techniques, with the aim of elucidating ancient trophic structures and predator–prey interactions. The phosphatic composition, frequent bone inclusions, size and morphology collectively suggest that most, if not all, coprolites were produced by carnivorous (predatory or scavenging) vertebrates. The bone inclusions further indicate that the coprolite producers preyed principally upon fish. Putative host animals include bony fish, sharks and marine reptiles – all of which have been previously recorded from the Münster Basin. The presence of borings and other traces on several coprolites implies handling by coprophagous organisms. Remains of epibionts are also common, most of which have been identified as the encrusting bivalve Atreta. Palynological analyses of both the coprolites and host rocks reveal a sparse assemblage dominated by typical Late Cretaceous dinoflagellates, and with sub‐ordinate fern spores, conifer pollen grains and angiosperm pollen grains. The dinoflagellate key taxon Exochosphaeridium cenomaniense corroborates a Cenomanian age for the Plenus Marl, from which most studied coprolites derive. The findings of this study highlight the potential of a multi‐proxy approach when it comes to unravelling the origin, composition and importance of coprolites in palaeoecosystem analyses.     

The retinol-retinoic acid metabolic pathway is impaired in the lumbar spine of a rat model of congenital kyphoscoliosis

Although congenital scoliosis is defined as a genetic disease characterized by a congenital and abnormal curvature of the spinal vertebrae, our knowledge of the genetic underpinnings of the disease is insufficient. We herein show that the downregulation of the retinol-retinoic acid metabolism pathway is involved in the pathogenesis of congenital scoliosis. By analyzing DNA microarray data, we found that the expression levels of genes associated with the retinol metabolism pathway were decreased in the lumbar spine of Ishibashi rats (IS), a rat model of congenital kyphoscoliosis. The expression of Adh1 and Aldh1a2 (alcohol dehydrogenase), two enzymes that convert retinol to retinoic acid in this pathway, were decreased at both the gene and protein levels. Rarα, a receptor of retinoic acid and bone morphogenetic protein 2, which play a central role in bone formation and are located downstream of this pathway, were also downregulated. Interestingly, the serum retinol levels of IS rats were higher than those of wild-type control rats. These results indicate that the adequate conversion from retinol to retinoic acid is extremely important in the regulation of normal bone formation and it may also be a key factor for understanding the pathogenesis of congenital scoliosis.