Crystal structure of glutamate-1-semialdehyde aminomutase: An α2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity

The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC 5.4.3.8), an α2-dimeric enzyme from Synechococcus, has been determined by x-ray crystallography using heavy atom derivative phasing. The structure, refined at 2.4-Å resolution to an R-factor of 18.7% and good stereochemistry, explains many of the enzyme’s unusual specificity and functional properties. The overall fold is that of aspartate aminotransferase and related B6 enzymes, but it also has specific features. The structure of the complex with gabaculine, a substrate analogue, shows unexpectedly that the substrate binding site involves residues from the N-terminal domain of the molecule, notably Arg-32. Glu-406 is suitably positioned to repel α-carboxylic acids, thereby suggesting a basis for the enzyme’s reaction specificity. The subunits show asymmetry in cofactor binding and in the mobilities of the residues 153–181. In the unliganded enzyme, one subunit has the cofactor bound as an aldimine of pyridoxal phosphate with Lys-273 and, in this subunit, residues 153–181 are disordered. In the other subunit in which the cofactor is not covalently bound, residues 153–181 are well defined. Consistent with the crystallographically demonstrated asymmetry, a form of the enzyme in which both subunits have pyridoxal phosphate bound to Lys-273 through a Schiff base showed biphasic reduction by borohydride in solution. Analysis of absorption spectra during reduction provided evidence of communication between the subunits. The crystal structure of the reduced form of the enzyme shows that, despite identical cofactor binding in each monomer, the structural asymmetry at residues 153–181 remains.

Exercise-induced changes in expression and activity of proteins involved in insulin signal transduction in skeletal muscle: Differential effects on insulin-receptor substrates 1 and 2

Level of physical activity is linked to improved glucose homeostasis. We determined whether exercise alters the expression and/or activity of proteins involved in insulin-signal transduction in skeletal muscle. Wistar rats swam 6 h per day for 1 or 5 days. Epitrochlearis muscles were excised 16 h after the last exercise bout, and were incubated with or without insulin (120 nM). Insulin-stimulated glucose transport increased 30% and 50% after 1 and 5 days of exercise, respectively. Glycogen content increased 2- and 4-fold after 1 and 5 days of exercise, with no change in glycogen synthase expression. Protein expression of the glucose transporter GLUT4 and the insulin receptor increased 2-fold after 1 day, with no further change after 5 days of exercise. Insulin-stimulated receptor tyrosine phosphorylation increased 2-fold after 5 days of exercise. Insulin-stimulated tyrosine phosphorylation of insulin-receptor substrate (IRS) 1 and associated phosphatidylinositol (PI) 3-kinase activity increased 2.5- and 3.5-fold after 1 and 5 days of exercise, despite reduced (50%) IRS-1 protein content after 5 days of exercise. After 1 day of exercise, IRS-2 protein expression increased 2.6-fold and basal and insulin-stimulated IRS-2 associated PI 3-kinase activity increased 2.8-fold and 9-fold, respectively. In contrast to IRS-1, IRS-2 expression and associated PI 3-kinase activity normalized to sedentary levels after 5 days of exercise. Insulin-stimulated Akt phosphorylation increased 5-fold after 5 days of exercise. In conclusion, increased insulin-stimulated glucose transport after exercise is not limited to increased GLUT4 expression. Exercise leads to increased expression and function of several proteins involved in insulin-signal transduction. Furthermore, the differential response of IRS-1 and IRS-2 to exercise suggests that these molecules have specialized, rather than redundant, roles in insulin signaling in skeletal muscle.

Human MTH1 protein hydrolyzes the oxidized ribonucleotide, 2-hydroxy-ATP

The human nucleotide pool sanitization enzyme, MTH1, hydrolyzes 2-hydroxy-dATP and 8-hydroxy-dATP in addition to 8-hydroxy-dGTP. We report here that human MTH1 is highly specific for 2-hydroxy-ATP, among the cognate ribonucleoside triphosphates. The pyrophosphatase activities for 8-hydroxy-GTP, 2-hydroxy-ATP and 8-hydroxy-ATP were measured by high-performance liquid chromatography. The kinetic parameters thus obtained indicate that the catalytic efficiencies of MTH1 are in the order of 2-hydroxy-dATP > 2-hydroxy-ATP > 8-hydroxy-dGTP > 8-hydroxy-dATP >> dGTP > 8-hydroxy-GTP > 8-hydroxy-ATP. Notably, MTH1 had the highest affinity for 2-hydroxy-ATP among the known substrates. ATP is involved in energy metabolism and signal transduction, and is a precursor in RNA synthesis. We suggest that the 2-hydroxy-ATP hydrolyzing activity of MTH1 might prevent the perturbation of these ATP-related pathways by the oxidized ATP.

Search for very high energy gamma radiation from the radio bright region DR4 of the SNR G78.2+2.1

Data from the HEGRA air shower array are used to set an upper limit on the emission of gamma-radiation above 25 (18) TeV from the direction of the radio bright region DR4 within the SNR G78.2 + 2.1 of 2.5 (7.1). 10^-13 cm^-2 sec^-1. The shock front of SNR G78.2 + 2.1 probably recently overtook the molecular cloud Gong 8 which then acts as a target for the cosmic rays produced within the SNR, thus leading to the expectation of enhanced gamma-radiation. Using a model of Drury, Aharonian and Völk which assumes that SNRs are the sources of galactic cosmic rays via first order Fermi acceleration, we calculated a theoretical prediction for the gamma-ray flux from the DR4 region and compared it with our experimental flux limit. Our 'best estimate' value for the predicted flux lies a factor of about 18 above the upper limit for gamma-ray energies above 25 TeV. Possible reasons for this discrepancy are discussed.

Enantioselective aldol reactions with aqueous 2,2-dimethoxyacetaldehyde organocatalyzed by binam-prolinamides under solvent-free conditions

Aqueous 2,2-dimethoxyacetaldehyde (60% wt solution) is used as an acceptor in aldol reactions, with cyclic and acyclic ketones and aldehydes as donors, organocatalyzed by 10 mol % of N-tosyl-(Sa)-binam-l-prolinamide [(Sa)-binam-sulfo-l-Pro] at rt under solvent-free conditions. The corresponding monoprotected 2-hydroxy-1,4-dicarbonyl compounds are obtained in good yields and with high levels of diastereo- and enantioselectivity mainly as anti-aldols. In the case of 4-substituted cyclohexanones a desymmetrization process takes place to mainly afford the anti,anti-aldols. 2,2-Dimethyl-1,3-dioxan-5-one allows the synthesis of a useful intermediate for the preparation of carbohydrates in higher yield, de and ee than with l-Pro as the organocatalyst.

Organocatalytic Asymmetric α-Chlorination of 1,3-Dicarbonyl Compounds Catalyzed by 2-Aminobenzimidazole Derivatives

Bifunctional chiral 2-aminobenzimidazole derivatives 1 and 2 catalyze the enantioselective stereodivergent α-chlorination of β-ketoesters and 1,3-diketone derivatives with up to 50% ee using N-chlorosuccinimide (NCS) or 2,3,4,4,5,6-hexachloro-2,5-cyclohexadien-1-one as electrophilic chlorine sources.

Coast, North Africa, Tunisia and Algeria, 1829 (Image 1 of 2) (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Carte comparée des régences d'Alger et de Tunis, dressée par le Chev. Lapie, Géographe du Roi ; les noms anciens ont été revus par ... Hase ; les noms arabes par ... Taubert ; gravé par Flahaut ; écrit par Lallemand. It was published by Chez Ch. Picquet, Quai Conti, no. 17 in 1829. Scale [ca. 1:1,632,653], 16 km=9.8 cm. This layer is image 1 of 2 total images of the two sheet source map, representing the western portion of the map. Covers the coast of North Africa including Tunisia, Algeria, and portions of Morocco and Libya. Map in French. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Europe Lambert Conformal Conic coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, roads, cities and other human settlements, territorial boundaries, shoreline features, and more. Relief shown by hachures. This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Dinoflagellate cysts in surface sediment samples of METEOR cruises M20/2, M23/1, M34/1, M34/2, M34/3 and M34/4

To enhance the limited information available about the palaeo-ecological significance of calcareous dinoflagellates, we have studied their lateral distribution in surface sediments of the equatorial and south Atlantic between 13°N and 36°S. Calcareous dinoflagellate cysts appear to be widely distributed throughout the studied area. In the surface sediments, concentrations (cyst per gram dry sediment) of the vegetative stage Thoracosphaera heimii are generally higher than that of the (presumably) calcareous resting cysts. Distribution patterns in surface sediments of Orthopithonella granifera (Fütterer) Keupp and Versteegh, Rhabdothorax spp. Kamptner., Sphaerodinella albatrosiana (Kamptner) Keupp and Versteegh S. albatrosiana praratabulated, Sphaerodinella tuberosa var. 1 (Kamptner) Keupp and Versteegh and S. tuberosa var. 2 and the ratios between these species have been compared with temperature, salinity, density and stratification gradients in the upper water column. Rhabdothorax spp. is characteristically present in sediments of more temperate regions characterized by high seasonality. Dinoflagellates producing these cysts are able to tolerate high nutrient concentrations, and mixing of the water column. S. albatrosiana is abundant in regions characterized by high sea surface temperatures and oligotrophic surface water conditions. In contrast, the distribution of S. tuberosa var. 2 is negatively related to temperature. The other cyst species did not show a characteristic pattern in relation to the studied environmental gradients. The ratio of Sphaerodinella tuberosa var. 2 to Orthopithonella granifera can be used for reconstructing the presence of stratification in the upper 50 m of the water column, whereas the ratios of S. tuberosa var. 2 to Sphaerodinella albatrosiana and of O. granifera to Rhabdothorax spp. might be used for palaeotemperature reconstructions. Calcareous dinoflagellate cysts are abundant in oligotrophic areas and may be useful for the reconstruction of palaeoenvironmental conditions.

(Table 2) Dinoflagellate cysts in surface sediment samples of METEOR cruises M20/2, M23/1, M34/2 and M34/2

The organic walled cyst content of 41 surface sediment samples from the south-eastern South Atlantic Ocean have been studied to create a dataset that can be used for palaeoceanographic reconstructions. In order to obtain insight into which environmental factors influence the distribution of individual cyst species, the cyst associations have been compared with oceanographic characteristics of the overlying water masses, i.e. temperature, salinity, density and stratification gradients. The associations and relationships have been established by visual examination of the dataset and the multivariate ordination techniques, Detrended Correspondence Analysis and Canonical Correspondence Analysis. Special attention has been given to the factors of transport and preservation of the cysts. Five associations have been recognised as being characteristic of (1) areas influenced by coastal upwelling and/or river outflow, (2) open ocean, (3) Agulhas Current and southern Benguela Current, (4) Benguela Current and (5) Walvis Bay, shelf break area. The factors dominant in influencing either directly or indirectly the cyst distributions appear to be the stratification in the upper 50 m of the water column, nutrient concentration and seasonality. Variations in sea surface temperatures and salinities have only minor effect on cyst distribution.

Performance of commercial enzyme-linked immunosorbent assays (ELISA) for diagnosis of HSV-1 and HSV-2 infection in a clinical setting

Thesis (Master's)--University of Washington, 2016-06

Conformational analysis of the natural iron chelator myo-inositol 1,2,3-trisphosphate using a pyrene-based fluorescent mimic

myo-Inositol phosphates possessing the 1,2,3-trisphosphate motif share the remarkable ability to completely inhibit iron-catalysed hydroxyl radical formation. The simplest derivative, myo-inositol 1,2,3-trisphosphate [Ins(1,2,3)P3], has been proposed as an intracellular iron chelator involved in iron transport. The binding conformation of Ins(1,2,3)P3 is considered to be important to complex Fe3+ in a 'safe' manner. Here, a pyrene-based fluorescent probe, 4,6-bispyrenoyl-myo-inositol 1,2,3,5-tetrakisphosphate [4,6-bispyrenoyl Ins(1,2,3,5)P4], has been synthesised and used to monitor the conformation of the 1,2,3-trisphosphate motif using excimer fluorescence emission. Ring-flip of the cyclohexane chair to the penta-axial conformation occurs upon association with Fe3+, evident from excimer fluorescence induced by π-π stacking of the pyrene reporter groups, accompanied by excimer formation by excitation at 351 nm. This effect is unique amongst biologically relevant metal cations, except for Ca 2+ cations exceeding a 1:1 molar ratio. In addition, the thermodynamic constants for the interaction of the fluorescent probe with Fe3+ have been determined. The complexes formed between Fe 3+ and 4,6-bispyrenoyl Ins(1,2,3,5)P4 display similar stability to those formed with Ins(1,2,3)P3, indicating that the fluorescent probe acts as a good model for the 1,2,3-trisphosphate motif. This is further supported by the antioxidant properties of 4,6-bispyrenoyl Ins(1,2,3,5)P4, which closely resemble those obtained for Ins(1,2,3)P3. The data presented confirms that Fe3+ binds tightly to the unstable penta-axial conformation of myo-inositol phosphates possessing the 1,2,3-trisphosphate motif. © 2010 The Royal Society of Chemistry.

(Table 2, Page 287) Chemical composition of manganese nodules from GH76-1 cruise

Chemical analyses of manganese nodules from the Central Pacific Basin show that their chemical composition varies regionally, although that of the associated sediments is markedly uniform throughout the basin. Mn content varies from 16 to 32% in average. Its higher value is generally found in nodules from siliceous clay and a few from deep-sea clay. Fe content tends to enrich in nodules from deep-sea clay area. Most manganese nodules, except those from deep-sea clay, are remarkably depleted in Fe compared with ones from the other Pacific regions. Mostly, Cu and Ni contents exceed 1% in nodules from siliceous clay, and decrease towards the northwest of the basin where deep-sea clay is distributed. The inter-element relationship between manganese nodules and associated sediments suggests that the mechanism of incorporation of major and minor elements in nodules is apparently different from that of the associated sediments. This finding seems to provide a new interpretation on the problem why manganese nodules having low accumulation rate are not buried by the associated sediments with greater sedimentation rate and then occur on sediment-seawater interface.

(Table XIII-2, Page 166) Physical characteristics of manganese nodules collected in the Pacific Ocean during cruise GH77-1 of R/V Hakurei Maru

In the GH77-1 cruise, manganese nodules were obtained from 31 stations among 37 total ones. Here are reported the preliminary results of the observation mainly done on-board. Special attention was paid to confirming the applicability of the nodule type classification tentatively established in the previous GH76-1 cruise and to delineating the pattern of the nodule distribution and clarifying its relation to the geological factors, such as topography, surface sediment types, and substrate stratigraphy. In addition, a short description of the obtained rock samples from a few stations is included in this chapter.

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the developing male Wistar(Han) rat II: chronic dosing causes developmental delay

We have investigated whether fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes defects in the male reproductive system of the rat, using chronically exposed rats to ensure continuous exposure of the fetus. 5-6 week old rats were exposed to control diet, or diet containing TCDD, to attain an average dose of 2.4, 8 and 46 ng TCDD kg-1 day-1 for twelve weeks, whereupon the rats were mated, and allowed to litter; rats were switched to control diet after parturition. Male offspring were allowed to develop until kills on PND70 (25 per group), or PND120 (all remaining animals). Offspring from the high dose group showed an increase in total litter loss, and the number of animals alive on post-natal day (PND) 4 in the high dose group was ~26% less than control. The high and medium dose offspring showed decreased weights at various ages. Balano-preputial separation was significantly delayed in all three dose groups, compared to control. There were no significant effects of maternal treatment when the offspring were subjected to a functional observational battery, or learning tests, with the exception that the high dose group showed a deficit in motor activity. 20 rats per group were mated to females, and there were no significant effects of maternal treatment on the fertility of these rats, nor on the F1 or F2 sex ratio. Sperm parameters at PND70 and 120 showed no significant effect of maternal treatment, with the exception that there was an increase in the proportion of abnormal sperm in the high dose group at PND70; this is associated with the developmental delay in puberty in this dose group. There were no remarkable findings of maternal treatment on organ weights, with the exception that testis weights were reduced by ~10% at PND70 (but not PND120), and although the experiment was sufficiently powered to detect small changes, ventral prostate weight was not reduced. There were no significant effects of maternal treatment upon histopathological comparison of high dose and control group organs. These data confirm that developmental exposure to TCDD shows no potent effect on adult sperm parameters or accessory sexual organs, but show that delay in BPS occurs after exposure to low doses of TCDD, and this is dependent upon whether TCDD is administered acutely or chronically.