The formation of ferritin from apoferritin. Inhibition and metal ion-binding studies

Inhibition by Zn(2+) of iron uptake by apoferritin at very low substrate concentrations is shown to be competitive. It is proposed that Zn(2+) competes with Fe(2+) for sites on the protein at which the oxidation of Fe(2+) is catalysed. Interpretation of titration data suggests there are two independent classes of binding site for Zn(2+) and several other cations. Sites in one such class are probably on the external surface of the apoferritin molecule. The catalytic binding sites are presumed to be internal and may involve histidine or possibly cysteine as ligands.     

Predicting Absolute Risk of Type 2 Diabetes Using Age and Waist Circumference Values in an Aboriginal Australian Community

Objectives

To predict in an Australian Aboriginal community, the 10-year absolute risk of type 2 diabetes associated with waist circumference and age on baseline examination.

Method

A sample of 803 diabetes-free adults (82.3% of the age-eligible population) from baseline data of participants collected from 1992 to 1998 were followed-up for up to 20 years till 2012. The Cox-proportional hazard model was used to estimate the effects of waist circumference and other risk factors, including age, smoking and alcohol consumption status, of males and females on prediction of type 2 diabetes, identified through subsequent hospitalisation data during the follow-up period. The Weibull regression model was used to calculate the absolute risk estimates of type 2 diabetes with waist circumference and age as predictors.

Results

Of 803 participants, 110 were recorded as having developed type 2 diabetes, in subsequent hospitalizations over a follow-up of 12633.4 person-years. Waist circumference was strongly associated with subsequent diagnosis of type 2 diabetes with P<0.0001 for both genders and remained statistically significant after adjusting for confounding factors. Hazard ratios of type 2 diabetes associated with 1 standard deviation increase in waist circumference were 1.7 (95%CI 1.3 to 2.2) for males and 2.1 (95%CI 1.7 to 2.6) for females. At 45 years of age with baseline waist circumference of 100 cm, a male had an absolute diabetic risk of 10.9%, while a female had a 14.3% risk of the disease.

Conclusions

The constructed model predicts the 10-year absolute diabetes risk in an Aboriginal Australian community. It is simple and easily understood and will help identify individuals at risk of diabetes in relation to waist circumference values. Our findings on the relationship between waist circumference and diabetes on gender will be useful for clinical consultation, public health education and establishing WC cut-off points for Aboriginal Australians.     

Human 2'-phosphodiesterase localizes to the mitochondrial matrix with a putative function in mitochondrial RNA turnover

The vertebrate 2-5A system is part of the innate immune system and central to cellular antiviral defense. Upon activation by viral double-stranded RNA, 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2'-5'-oligoadenylate synthetases. These unusual oligonucleotides activate RNase L, an unspecific endoribonuclease that mediates viral and cellular RNA breakdown. Subsequently, the 2-5As are removed by a 2'-phosphodiesterase (2'-PDE), an enzyme that apart from breaking 2'-5' bonds also degrades regular, 3'-5'-linked oligoadenylates. Interestingly, 2'-PDE shares both functionally and structurally characteristics with the CCR4-type exonuclease-endonuclease-phosphatase family of deadenylases. Here we show that 2'-PDE locates to the mitochondrial matrix of human cells, and comprise an active 3'-5' exoribonuclease exhibiting a preference for oligo-adenosine RNA like canonical cytoplasmic deadenylases. Furthermore, we document a marked negative association between 2'-PDE and mitochondrial mRNA levels following siRNA-directed knockdown and plasmid-mediated overexpression, respectively. The results indicate that 2'-PDE, apart from playing a role in the cellular immune system, may also function in mitochondrial RNA turnover.     

Indirect selection for increased susceptibility to permethrin in diamondback moth (Lepidoptera: Plutellidae)

We have been exploring the behavioral response ot insect pests to heterogeneous distribution of toxins (low dose with refugia), and its genetic correlation with physiological tolerance to these toxins. A field-collected population of diamondback moth, Plutella xylostellu (L.) (Lepidoptera Plutellidae), from Celeryville, OH, was selected with permethrin to determine whether low heterogeneous doses could lead to increased susceptibility to permethrin by selecting indirectly on behavior. Two replicates of each of three selection regimes: uniform high concentration hypothesized to result in increased physiological tolerance, heterogeneous low concentration hypothesized to result in increased susceptibility through indirect selection on behavior, and a control with no exposure to permethrin, were maintained in 1-in3 cages in a greenhouse, for 33 generations. All life stages of the diamondback moth were exposed to the selection regimes, and new generations were started with a random selection of pupae from the previous generation. Lines selected with uniform high concentrations developed 76-fold levels of resistance to permethrin by the 17th generation, with little changes thereafter. For generations 1-20, lines selected with heterogeneous low concentrations remained slightly lower in LC50 but not significantly different from the unselected control lines. Based on confidence intervals from probit analyses, the LC50 of the lines selected with heterogeneous low concentration, however, were significantly lower than those of the control lines in generations 21-33. Our results demonstrate that selection on behavioral responses can result in greater susceptibility than no selection at all, despite exposure to the toxin and ample genetic variation and potential for increased physiological tolerance. The implications of our findings, which are based on selection scenarios that could take place in field situations, are that behavioral responses can prevent and even decrease the levels of resistance in insect populations, an important result with respect to resistance and resistance management.     

Morphological and molecular characterization of a Hirsutella species infecting the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), in Florida

The Asian citrus psyllid, Diaphorina citri Kuwayama, is an invasive pest that vectors citrus greening disease, which recently was detected in Florida. Mycosed adult D. citri were collected at four sites in central Florida between September 2005 and February 2006. Observation of the cadavers using scanning electron microscopy revealed that the pathogen had branched synnemata supporting monophiladic conidiogenous cells. A high-fidelity polymerase chain reaction (PCR) assay was used to amplify the 18S rRNA, 28S rRNA and beta-tubulin genes of the pathogen for phylogenetic analysis. The morphological and genetic data indicated that the pathogen was a novel isolate related to Hirsutella citriformis Speare. PCR assays using isolate-specific primers designed from the unique putative intron region of the beta-tubulin sequence distinguished the psyllid pathogen from five related Hirsutella species. The pathogen was maintained in vivo by exposing healthy D. citri to the synnemata borne on field-collected cadavers. Infected psyllids had an abundance of septate hyphal bodies in their hemolymph and exhibited behavioral symptoms of disease. In vitro cultures of the pathogen were slow-growing and produced synnemata similar to those found on mycosed D. citri. In laboratory bioassays, high levels of mortality were observed in D. citri that were exposed to the conidia-bearing synnemata produced in vivo and in vitro.     

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.