Sleep-wake sensitive mechanisms of adenosine release in the basal forebrain of rodents : an in vitro study

Adenosine acting in the basal forebrain is a key mediator of sleep homeostasis. Extracellular adenosine concentrations increase during wakefulness, especially during prolonged wakefulness and lead to increased sleep pressure and subsequent rebound sleep. The release of endogenous adenosine during the sleep-wake cycle has mainly been studied in vivo with microdialysis techniques. The biochemical changes that accompany sleep-wake status may be preserved in vitro. We have therefore used adenosine-sensitive biosensors in slices of the basal forebrain (BFB) to study both depolarization-evoked adenosine release and the steady state adenosine tone in rats, mice and hamsters. Adenosine release was evoked by high K+, AMPA, NMDA and mGlu receptor agonists, but not by other transmitters associated with wakefulness such as orexin, histamine or neurotensin. Evoked and basal adenosine release in the BFB in vitro exhibited three key features: the magnitude of each varied systematically with the diurnal time at which the animal was sacrificed; sleep deprivation prior to sacrifice greatly increased both evoked adenosine release and the basal tone; and the enhancement of evoked adenosine release and basal tone resulting from sleep deprivation was reversed by the inducible nitric oxide synthase (iNOS) inhibitor, 1400 W. These data indicate that characteristics of adenosine release recorded in the BFB in vitro reflect those that have been linked in vivo to the homeostatic control of sleep. Our results provide methodologically independent support for a key role for induction of iNOS as a trigger for enhanced adenosine release following sleep deprivation and suggest that this induction may constitute a biochemical memory of this state

The deuteron: structure and form factors

A brief review of the history of the discovery of the deuteron in provided. The current status of both experiment and theory for the elastic electron scattering is then presented.Comment: 80 pages, 33 figures, submited to Advances in Nuclear Physic

Dysmorphometrics: the modelling of morphological abnormalities

<p>Abstract</p> <p>Background</p> <p>The study of typical morphological variations using quantitative, morphometric descriptors has always interested biologists in general. However, unusual examples of form, such as abnormalities are often encountered in biomedical sciences. Despite the long history of morphometrics, the means to identify and quantify such unusual form differences remains limited.</p> <p>Methods</p> <p>A theoretical concept, called dysmorphometrics, is introduced augmenting current geometric morphometrics with a focus on identifying and modelling form abnormalities. Dysmorphometrics applies the paradigm of detecting form differences as outliers compared to an appropriate norm. To achieve this, the likelihood formulation of landmark superimpositions is extended with outlier processes explicitly introducing a latent variable coding for abnormalities. A tractable solution to this augmented superimposition problem is obtained using Expectation-Maximization. The topography of detected abnormalities is encoded in a dysmorphogram.</p> <p>Results</p> <p>We demonstrate the use of dysmorphometrics to measure abrupt changes in time, asymmetry and discordancy in a set of human faces presenting with facial abnormalities.</p> <p>Conclusion</p> <p>The results clearly illustrate the unique power to reveal unusual form differences given only normative data with clear applications in both biomedical practice & research.</p

Genetic polymorphism of the iron-regulatory protein-1 and -2 genes in age-related macular degeneration

Iron can be involved in the pathogenesis of AMD through the oxidative stress because it may catalyze the Haber–Weiss and Fenton reactions converting hydrogen peroxide to free radicals, which can induce cellular damage. We hypothesized that genetic polymorphism in genes related to iron metabolism may predispose individuals to the development of AMD and therefore we checked for an association between the g.32373708 G>A polymorphism (rs867469) of the IRP1 gene and the g.49520870 G>A (rs17483548) polymorphism of the IRP2 gene and AMD risk as well as the modulation of this association by some environmental and life-style factors. Genotypes were determined in DNA from blood of 269 AMD patients and 116 controls by the allele-specific oligonucleotide-restriction fragment length polymorphism and the polymerase chain reaction-restriction fragment length polymorphism. An association between AMD, dry and wet forms of AMD and the G/G genotype of the g.32373708 G>A-IRP1 polymorphism was found (OR 3.40, 4.15, and 2.75). On the other hand, the G/A genotype reduced the risk of AMD as well as its dry or wet form (OR 0.23, 0.21, 0.26). Moreover, the G allele of the g.49520870 G>A-IRP2 polymorphism increased the risk of the dry form of the disease (OR 1.51) and the A/A genotype and the A allele decreased such risk (OR 0.43 and 0.66). Our data suggest that the g.32373708 G>A-IRP1 and g.49520870 G>A-IRP2 polymorphisms may be associated with increased risk for AMD

Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase

This work was supported in part by funds from an Oklahoma State Regent Grant for Higher Education (021606), P20RR016478 grant from the National Center for Research Resources (NCRR) a component of National Institute of Health (NIH), and a grant from the University of Central Oklahoma office of Research and Grants to L.C.The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification.Yeshttp://www.plosone.org/static/editorial#pee

Wolbachia infections that reduce immature insect survival: Predicted impacts on population replacement

<p>Abstract</p> <p>Background</p> <p>The evolutionary success of <it>Wolbachia </it>bacteria, infections of which are widespread in invertebrates, is largely attributed to an ability to manipulate host reproduction without imposing substantial fitness costs. Here, we describe a stage-structured model with deterministic immature lifestages and a stochastic adult female lifestage. Simulations were conducted to better understand <it>Wolbachia </it>invasions into uninfected host populations. The model includes conventional <it>Wolbachia </it>parameters (the level of cytoplasmic incompatibility, maternal inheritance, the relative fecundity of infected females, and the initial <it>Wolbachia </it>infection frequency) and a new parameter termed relative larval viability (<it>RLV</it>), which is the survival of infected larvae relative to uninfected larvae.</p> <p>Results</p> <p>The results predict the <it>RLV </it>parameter to be the most important determinant for <it>Wolbachia </it>invasion and establishment. Specifically, the fitness of infected immature hosts must be close to equal to that of uninfected hosts before population replacement can occur. Furthermore, minute decreases in <it>RLV </it>inhibit the invasion of <it>Wolbachia </it>despite high levels of cytoplasmic incompatibility, maternal inheritance, and low adult fitness costs.</p> <p>Conclusions</p> <p>The model described here takes a novel approach to understanding the spread of <it>Wolbachia </it>through a population with explicit dynamics. By combining a stochastic female adult lifestage and deterministic immature/adult male lifestages, the model predicts that even those <it>Wolbachia </it>infections that cause minor decreases in immature survival are unlikely to invade and spread within the host population. The results are discussed in relation to recent theoretical and empirical studies of natural population replacement events and proposed applied research, which would use <it>Wolbachia </it>as a tool to manipulate insect populations.</p

Both Positive and Negative Selection Pressures Contribute to the Polymorphism Pattern of the Duplicated Human CYP21A2 Gene.

The human steroid 21-hydroxylase gene (CYP21A2) participates in cortisol and aldosterone biosynthesis, and resides together with its paralogous (duplicated) pseudogene in a multiallelic copy number variation (CNV), called RCCX CNV. Concerted evolution caused by non-allelic gene conversion has been described in great ape CYP21 genes, and the same conversion activity is responsible for a serious genetic disorder of CYP21A2, congenital adrenal hyperplasia (CAH). In the current study, 33 CYP21A2 haplotype variants encoding 6 protein variants were determined from a European population. CYP21A2 was shown to be one of the most diverse human genes (HHe=0.949), but the diversity of intron 2 was greater still. Contrary to previous findings, the evolution of intron 2 did not follow concerted evolution, although the remaining part of the gene did. Fixed sites (different fixed alleles of sites in human CYP21 paralogues) significantly accumulated in intron 2, indicating that the excess of fixed sites was connected to the lack of effective non-allelic conversion and concerted evolution. Furthermore, positive selection was presumably focused on intron 2, and possibly associated with the previous genetic features. However, the positive selection detected by several neutrality tests was discerned along the whole gene. In addition, the clear signature of negative selection was observed in the coding sequence. The maintenance of the CYP21 enzyme function is critical, and could lead to negative selection, whereas the presumed gene regulation altering steroid hormone levels via intron 2 might help fast adaptation, which broadly characterizes the genes of human CNVs responding to the environment

Chronic Hypoxia Impairs Muscle Function in the Drosophila Model of Duchenne's Muscular Dystrophy (DMD)

Duchenne's muscular dystrophy (DMD) is a severe progressive myopathy caused by mutations in the DMD gene leading to a deficiency of the dystrophin protein. Due to ongoing muscle necrosis in respiratory muscles late-stage DMD is associated with respiratory insufficiency and chronic hypoxia (CH). To understand the effects of CH on dystrophin-deficient muscle in vivo, we exposed the Drosophila model for DMD (dmDys) to CH during a 16-day ascent to the summit of Mount Denali/McKinley (6194 meters above sea level). Additionally, dmDys and wild type (WT) flies were also exposed to CH in laboratory simulations of high altitude hypoxia. Expression profiling was performed using Affymetrix GeneChips® and validated using qPCR. Hypoxic dmDys differentially expressed 1281 genes, whereas the hypoxic WT flies differentially expressed 56 genes. Interestingly, a number of genes (e.g. heat shock proteins) were discordantly regulated in response to CH between dmDys and WT. We tested the possibility that the disparate molecular responses of dystrophin-deficient tissues to CH could adversely affect muscle by performing functional assays in vivo. Normoxic and CH WT and dmDys flies were challenged with acute hypoxia and time-to-recover determined as well as subjected to climbing tests. Impaired performance was noted for CH-dmDys compared to normoxic dmDys or WT flies (rank order: Normoxic-WT ≈ CH-WT> Normoxic-dmDys> CH-dmDys). These data suggest that dystrophin-deficiency is associated with a disparate, pathological hypoxic stress response(s) and is more sensitive to hypoxia induced muscle dysfunction in vivo. We hypothesize that targeting/correcting the disparate molecular response(s) to hypoxia may offer a novel therapeutic strategy in DMD