Glucose infusion causes insulin resistance in skeletal muscle of rats without changes in Akt and AS160 phosphorylation

Hyperglycemia is a defining feature of Type 1 and 2 diabetes. Hyperglycemia also causes insulin resistance, and our group (Kraegen EW, Saha AK, Preston E, Wilks D, Hoy AJ, Cooney GJ, Ruderman NB. Am J Physiol Endocrinol Metab Endocrinol Metab 290: E471-E479, 2006) has recently demonstrated that hyperglycemia generated by glucose infusion results in insulin resistance after 5 h but not after 3 h. The aim of this study was to investigate possible mechanism(s) by which glucose infusion causes insulin resistance in skeletal muscle and in particular to examine whether this was associated with changes in insulin signaling. Hyperglycemia (~10 mM) was produced in cannulated male Wistar rats for up to 5 h. The glucose infusion rate required to maintain this hyperglycemia progressively lessened over 5 h (by 25%, P < 0.0001 at 5 h) without any alteration in plasma insulin levels consistent with the development of insulin resistance. Muscle glucose uptake in vivo (44%; P < 0.05) and glycogen synthesis rate (52%; P < 0.001) were reduced after 5 h compared with after 3 h of infusion. Despite these changes, there was no decrease in the phosphorylation state of multiple insulin signaling intermediates [insulin receptor, Akt, AS160 (Akt substrate of 160 kDa), glycogen synthase kinase-3beta] over the same time course. In isolated soleus strips taken from control or 1- or 5-h glucose-infused animals, insulin-stimulated 2-deoxyglucose transport was similar, but glycogen synthesis was significantly reduced in the 5-h muscle sample (68% vs. 1-h sample; P < 0.001). These results suggest that the reduced muscle glucose uptake in rats after 5 h of acute hyperglycemia is due more to the metabolic effects of excess glycogen storage than to a defect in insulin signaling or glucose transport.     

Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation

Synechocystis hemoglobin contains an unprecedented covalent bond between a nonaxial histidine side chain (H117) and the heme 2-vinyl. This bond has been previously shown to stabilize the ferric protein against denaturation, and also to affect the kinetics of cyanide association. However, it is unclear why Synechocystis hemoglobin would require the additional degree of stabilization accompanying the His117-heme 2-vinyl bond because it also displays endogenous bis-histidyl axial heme coordination, which should greatly assist heme retention. Furthermore, the mechanism by which the His117-heme 2-vinyl bond affects ligand binding has not been reported, nor has any investigation of the role of this bond on the structure and function of the protein in the ferrous oxidation state. Here we report an investigation of the role of the Synechocystis hemoglobin His117-heme 2-vinyl bond on structure, heme coordination, exogenous ligand binding, and stability in both the ferrous and ferric oxidation states. Our results reveal that hexacoordinate Synechocystis hemoglobin lacking this bond is less stable in the ferrous oxidation state than the ferric, which is surprising in light of our understanding of pentacoordinate Hb stability, in which the ferric protein is always less stable. It is also demonstrated that removal of the His117-heme 2-vinyl bond increases the affinity constant for intramolecular histidine coordination in the ferric oxidation state, thus presenting greater competition for the ligand binding site and lowering the observed rate and affinity constants for exogenous ligands.     

Evaluation of the predacious mite Hemicheyletia wellsina (Acari: Cheyletidae) as a predator of arthropod pests of orchids

The cheyletid predator Hemicheyletia wellsina was found in association with orchid pests in a Florida greenhouse. The life history of H. wellsina was determined using the two-spotted spider mite, Tetranychus urticae, as prey in the laboratory. Hemicheyletia wellsina is arrhenotokous, with mated females producing a female-biased sex ratio of 3.4:1. Adult longevity averaged 30.2 days, with females producing an average of 18.5 eggs. Adult females live up to 17 days without food, surviving an average of 9.7 days. Hemicheyletia wellsina will readily feed on the phytoseiid Metaseiulus occidentalis, suggesting intraguild predation by H. wellsina on phytoseiids could be important in pest management programs. The average prey consumption of H. wellsina is 1.5 T. urticae females per day. The results of this study provide information on an under-studied group of mite predators. Although H. wellsina does not have the qualities that would be necessary for its use in an augmentative or classical biological control program, its use in naturally occurring biological control could be beneficial.     

Controls on carbon consumption during Alaskan wildland fires

A method was developed to estimate carbon consumed during wildland fires in interior Alaska based on medium‐spatial scale data (60 m cell size) generated on a daily basis. Carbon consumption estimates were developed for 41 fire events in the large fire year of 2004 and 34 fire events from the small fire years of 2006–2008. Total carbon consumed during the large fire year (2.72 × 10⁶ ha burned) was 64.7 Tg C, and the average carbon consumption during the small fire years (0.09 × 10⁶ ha burned) was 1.3 Tg C. Uncertainties for the annual carbon emissions ranged from 13% to 21%. Carbon consumed from burning of black spruce forests represented 76% of the total during large fire years and 57% during small fire years. This was the result of the widespread distribution of black spruce forests across the landscape and the deep burning of the surface organic layers common to these ecosystems. Average carbon consumed was 3.01 kg m^?2 during the large fire year and 1.69 kg m^?2 during the small fire years. Most of the carbon consumption was from burning of ground layer fuels (85% in the large fire year and 78% in small fire years). Most of the difference in average carbon consumption between large and small fire years was in the consumption of ground layer fuels (2.60 vs. 1.31 kg m^?2 during large and small fire years, respectively). There was great variation in average fuel consumption between individual fire events (0.56–5.06 kg m^?2) controlled by variations in fuel types and topography, timing of the fires during the fire season, and variations in fuel moisture at the time of burning.     

Isolation and characterization of an Isaria fumosorosea isolate infecting the Asian citrus psyllid in Florida

A fungal pathogen that killed adult Diaphorina citri Kuwayama (Asian citrus psyllid) (Hemiptera: Psyllidae) in Florida citrus groves during the fall of 2005 was identified and characterized. Investigation of this pathogen is important because D. citri vectors citrus greening disease (Huanglongbing), which was reported in Florida in 2005. The morphological and genetic data generated herein support identification of the fungus as Isaria fumosorosea Wize (Ifr) (=Paecilomyces fumosoroseus) (Hypocreales: Cordycipitaceae) from the Asian citrus psyllid (Ifr AsCP). Koch’s postulates were fulfilled after the fungus was isolated in vitro and transmitted to healthy psyllids, which then exhibited a diseased-phenotype similar to that observed in the field. Both in vitro growth characteristics and two Ifr AsCP-specific molecular markers discriminated the psyllid pathogen from another local Ifr isolate, Ifr 97 Apopka. These molecular markers will be useful to track the dynamics of this disease in D. citri populations. The potential for utilizing Ifr to complement existing psyllid pest management strategies is discussed.     

Erythrocyte agglutination by wheat germ agglutinin: ionic strength dependence of the contact seam topology

Abstract

In this work, we describe a process for production of a Pichia pastoris strain which overproduces large quantities of the human glycine receptor. Subsequent purification yielded functional, uniform protein with expression yields of up to 5 mg per liter cell culture. As the wild-type protein is prone to proteolytic degradation, the labile sites were removed by mutagenesis resulting in an intracellular loop 2 deletion mutant with N-terminal modifications. This variant of the receptor is both stable during purification and storage on ice for up to a week as a complex with an antagonist. The quality of the protein is suitable for biophysical characterization and structural studies. The interaction of the agonist glycine and the antagonist strychnine with purified protein was analyzed by isothermal titration calorimetry. Strychnine binding is driven enthalpically with a K_D of 138 ± 55 nM, a ΔH of ?9708 ± 1195 cal/mol and a ΔS of ?1.0 ± 4.1 cal/mol/K, whereas glycine binding is driven by entropy with a K_D of 3.2 ± 0.8 μM, a ΔH of ?2228 ± 1012 cal/mol and ΔS of 17.7 ± 2.8 cal/mol/K. Strychnine and glycine binding is competitive with a stoichiometry of one ligand molecule to one pentameric glycine receptor.