Mechanisms of Human Erythrocytic Bioactivation of Nitrite

Nitrite signaling likely occurs through its reduction to nitric oxide (NO). Several reports support a role of erythrocytes and hemoglobin in nitrite reduction, but this remains controversial, and alternative reductive pathways have been proposed. In this work we determined whether the primary human erythrocytic nitrite reductase is hemoglobin as opposed to other erythrocytic proteins that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. Our studies show that NO is formed by red blood cells and inhibits platelet activation. Nitric oxide formation and signaling can be recapitulated with isolated deoxyhemoglobin. Importantly, there is limited NO production from erythrocytic xanthine oxidoreductase and nitric-oxide synthase. Under certain conditions we find dorzolamide (an inhibitor of carbonic anhydrase) results in diminished nitrite bioactivation, but the role of carbonic anhydrase is abrogated when physiological concentrations of CO₂ are present. Importantly, carbon monoxide, which inhibits hemoglobin function as a nitrite reductase, abolishes nitrite bioactivation. Overall our data suggest that deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions and accounts for nitrite-mediated NO signaling in blood.     

Identification of a common risk haplotype for canine idiopathic epilepsy in the ADAM23 gene

Background

Idiopathic epilepsy is a common neurological disease in human and domestic dogs but relatively few risk genes have been identified to date. The seizure characteristics, including focal and generalised seizures, are similar between the two species, with gene discovery facilitated by the reduced genetic heterogeneity of purebred dogs. We have recently identified a risk locus for idiopathic epilepsy in the Belgian Shepherd breed on a 4.4 megabase region on CFA37.

Results

We have expanded a previous study replicating the association with a combined analysis of 157 cases and 179 controls in three additional breeds: Schipperke, Finnish Spitz and Beagle (p_c = 2.9e–07, p_GWAS = 1.74E-02). A targeted resequencing of the 4.4 megabase region in twelve Belgian Shepherd cases and twelve controls with opposite haplotypes identified 37 case-specific variants within the ADAM23 gene. Twenty-seven variants were validated in 285 cases and 355 controls from four breeds, resulting in a strong replication of the ADAM23 locus (p_raw = 2.76e–15) and the identification of a common 28 kb-risk haplotype in all four breeds. Risk haplotype was present in frequencies of 0.49–0.7 in the breeds, suggesting that ADAM23 is a low penetrance risk gene for canine epilepsy.

Conclusions

These results implicate ADAM23 in common canine idiopathic epilepsy, although the causative variant remains yet to be identified. ADAM23 plays a role in synaptic transmission and interacts with known epilepsy genes, LGI1 and LGI2, and should be considered as a candidate gene for human epilepsies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1651-9) contains supplementary material, which is available to authorized users.     

The karyopherin Kap95 and the C-termini of Rfa1, Rfa2, and Rfa3 are necessary for efficient nuclear import of functional RPA complex proteins in Saccharomyces cerevisiae

Nuclear protein import in eukaryotic cells is mediated by karyopherin proteins, which bind to specific nuclear localization signals on substrate proteins and transport them across the nuclear envelope and into the nucleus. Replication protein A (RPA) is a nuclear protein comprised of three subunits (termed Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae) that binds single-stranded DNA and is essential for DNA replication, recombination, and repair. RPA associates with two different karyopherins in yeast, Kap95, and Msn5/Kap142. However, it is unclear which of these karyopherins is responsible for RPA nuclear import. We have generated GFP fusion proteins with each of the RPA subunits and demonstrate that these Rfa-GFP chimeras are functional in yeast cells. The intracellular localization of the RPA proteins in live cells is similar in wild-type and msn5Δ deletion strains but becomes primarily cytoplasmic in cells lacking functional Kap95. Truncating the C-terminus of any of the RPA subunits results in mislocalization of the proteins to the cytoplasm and a loss of protein-protein interactions between the subunits. Our data indicate that Kap95 is likely the primary karyopherin responsible for RPA nuclear import in yeast and that the C-terminal regions of Rfa1, Rfa2, and Rfa3 are essential for efficient nucleocytoplasmic transport of each RPA subunit.     

ADP regulates SNF1, the Saccharomyces cerevisiae homolog of AMP-activated protein kinase

The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.     

Air trapping on chest CT is associated with worse ventilation distribution in infants with cystic fibrosis diagnosed following newborn screening

Background

In school-aged children with cystic fibrosis (CF) structural lung damage assessed using chest CT is associated with abnormal ventilation distribution. The primary objective of this analysis was to determine the relationships between ventilation distribution outcomes and the presence and extent of structural damage as assessed by chest CT in infants and young children with CF.

Methods

Data of infants and young children with CF diagnosed following newborn screening consecutively reviewed between August 2005 and December 2009 were analysed. Ventilation distribution (lung clearance index and the first and second moment ratios [LCI, M₁/M₀ and M₂/M₀, respectively]), chest CT and airway pathology from bronchoalveolar lavage were determined at diagnosis and then annually. The chest CT scans were evaluated for the presence or absence of bronchiectasis and air trapping.

Results

Matched lung function, chest CT and pathology outcomes were available in 49 infants (31 male) with bronchiectasis and air trapping present in 13 (27%) and 24 (49%) infants, respectively. The presence of bronchiectasis or air trapping was associated with increased M₂/M₀ but not LCI or M₁/M₀. There was a weak, but statistically significant association between the extent of air trapping and all ventilation distribution outcomes.

Conclusion

These findings suggest that in early CF lung disease there are weak associations between ventilation distribution and lung damage from chest CT. These finding are in contrast to those reported in older children. These findings suggest that assessments of LCI could not be used to replace a chest CT scan for the assessment of structural lung disease in the first two years of life. Further research in which both MBW and chest CT outcomes are obtained is required to assess the role of ventilation distribution in tracking the progression of lung damage in infants with CF.     

Degree of landscape fragmentation influences genetic isolation among populations of a gliding mammal

Forests and woodlands are under continuing pressure from urban and agricultural development. Tree-dependent mammals that rarely venture to the ground are likely to be highly sensitive to forest fragmentation. The Australian squirrel glider (Petaurus norfolcensis) provides an excellent case study to examine genetic (functional) connectivity among populations. It has an extensive range that occurs in a wide band along the east coast. However, its forest and woodland habitat has become greatly reduced in area and is severely fragmented within the southern inland part of the species' range, where it is recognised as threatened. Within central and northern coastal regions, habitat is much more intact and we thus hypothesise that genetic connectivity will be greater in this region than in the south. To test this we employed microsatellite analysis in a molecular population biology approach. Most sampling locations in the highly modified south showed signatures of genetic isolation. In contrast, a high level of genetic connectivity was inferred among most sampled populations in the more intact habitat of the coastal region, with samples collected 1400 km apart having similar genetic cluster membership. Nonetheless, some coastal populations associated with urbanisation and agriculture are genetically isolated, suggesting the historic pattern observed in the south is emerging on the coast. Our study demonstrates that massive landscape changes following European settlement have had substantial impacts on levels of connectivity among squirrel glider populations, as predicted on the basis of the species' ecology. This suggests that landscape planning and management in the south should be focused on restoring habitat connectivity where feasible, while along the coast, existing habitat connectivity must be maintained and recent losses restored. Molecular population biology approaches provide a ready means for identifying fragmentation effects on a species at multiple scales. Such studies are required to examine the generality of our findings for other tree-dependent species.     

Acetylation of nuclear hormone receptor superfamily members: thyroid hormone causes acetylation of its own receptor by a mitogen-activated protein kinase-dependent mechanism

Because the androgen and estrogen nuclear hormone receptors are subject to acetylation, we speculated that the nuclear thyroid hormone receptor-beta1 (TRbeta1), another superfamily member, was also subject to this posttranslational modification. Treatment of 293T cells that contain TRbeta1(wt) with l-thyroxine (T4)(10(-7)M, total concentration) resulted in the accumulation of acetylated TR in nuclear fractions at 30-45 min and a decrease in signal by 60 min. A similar time course characterized recruitment by TR of p300, a coactivator protein with intrinsic transacetylase activity. Recruitment by the receptor of SRC-1, a TR coactivator that also acetylates nucleoproteins, was also demonstrated. Inhibition of the MAPK (ERK1/2) signal transduction cascade by PD 98059 blocked the acetylation of TR caused by T4. Tetraiodothyroacetic acid (tetrac) decreased T4-induced acetylation of TR. At 10(-7)M, 3,5,3'-triiodo-l-thyronine (T3) was comparably effective to T4 in causing acetylation of TR. We studied acetylation in TR that contained mutations in the DNA-binding domain (DBD) (residues 128-142) that are known to be relevant to recruitment of coactivators and to include the MAPK docking site. In response to T4 treatment, the K128A TR mutant transfected into CV-1 cells recruited p300, but not SRC-1, and was subject to acetylation. R132A complexed with SRC-1, but not p300; it was acetylated equally well in both the absence and presence of T4. S142E was acetylated in the absence and presence of T4 and bound SRC-1 under both conditions; this mutant was also capable of binding p300 in the presence of T4. There was no serine phosphorylation of TR in any of these mutants. We conclude that (1) TRbeta1, like AR and ER, is subject to acetylation; (2) the process of acetylation of TR requires thyroid hormone-directed MAPK activity, but not serine phosphorylation of TR by MAPK, suggesting that the contribution of MAPK is upstream in the activation of the acetylase; (3) the amino acid residue 128-142 region of the DBD of TR is important to thyroid hormone-associated recruitment of p300 and SRC-1; (4) acetylation of TR DBD mutants that is directed by T4 appears to be associated with recruitment of p300.     

Brain, spine, and muscle cytochrome Cu-A redox patterns of change during hypothermic circulatory arrest in swine

Past near infrared spectroscopy (NIRS) studies have reported different changes in cytochrome C oxidase (Cyt) redox status during similar interventions that cause tissue ischaemia. We investigated whether there were distinctive differences when NIRS signals were obtained simultaneously from different tissues during total circulatory arrest. Forty-two healthy 10 kg commercial swine (Sus scrofa) on cardiopulmonary bypass, each underwent 2 to 8 sequential periods of hypothermic circulatory arrest for 7.5 min. Prior to each arrest, key physiologic variables were adjusted to 1 of 81 combinations of high, normal, or low levels of core temperature, hematocrit, pH, and serum glucose. Each combination was repeated at least twice. Simultaneous NIRS monitoring yielded 202 brain, 191 spine, and 199 muscle Cyt data sets, which were then classified into 13 distinctive patterns of change. The data sets always differed between tissues in the same arrest trial and subject. Typically, brain Cyt rapidly became more reduced at the start of arrest and changed little thereafter, muscle Cyt behaved comparably to brain Cyt but continued to become reduced throughout the arrest, and spine Cyt either did not change status or gradually became more reduced over the course of arrest. The spine pattern's mean rate of change was 12 times slower than those of the brain or muscle. The Cyt patterns of change were classified into 13 groups which were significantly related to core temperature in the brain and spine, and hematocrit in muscle. The respiratory response in mitochondria during systemic circulatory arrest differs between brain, spine and muscle tissues in the same subject.     

Human dorsal and ventral auditory streams subserve rehearsal-based and echoic processes during verbal working memory

To hear a sequence of words and repeat them requires sensory-motor processing and something more-temporary storage. We investigated neural mechanisms of verbal memory by using fMRI and a task designed to tease apart perceptually based ("echoic") memory from phonological-articulatory memory. Sets of two- or three-word pairs were presented bimodally, followed by a cue indicating from which modality (auditory or visual) items were to be retrieved and rehearsed over a delay. Although delay-period activation in the planum temporale (PT) was insensible to the source modality and showed sustained delay-period activity, the superior temporal gyrus (STG) activated more vigorously when the retrieved items had arrived to the auditory modality and showed transient delay-period activity. Functional connectivity analysis revealed two topographically distinct fronto-temporal circuits, with STG co-activating more strongly with ventrolateral prefrontal cortex and PT co-activating more strongly with dorsolateral prefrontal cortex. These argue for separate contributions of ventral and dorsal auditory streams in verbal working memory.