Domain motions and quaternary packing of phosphofructokinase-2 from Escherichia coli studied by small angle x-ray scattering and homology modeling

The binding of MgATP and fructose-6-phosphate to phosphofructokinase-2 from Escherichia coli induces conformational changes that result in significant differences in the x-ray-scattering profiles compared with the unligated form of the enzyme. When fructose- 6-phosphate binds to the active site of the enzyme, the pair distribution function exhibits lower values at higher distances, indicating a more compact structure. Upon binding of MgATP to the allosteric site of the enzyme, the intensity at lower angles increases as a consequence of tetramer formation, but differences along higher angles also suggest changes at the tertiary structure level. We have used homology modeling to build the native dimeric form of phosphofructokinase-2 and fitted the experimental scattering curves by using rigid body movements of the domains in the model, similar to those observed in known homologous structures. The best fit with the experimental data of the unbound protein was achieved with open conformations of the domains in the model, whereas domain closure improves the agreement with the scattering of the enzyme-fructose-6-phosphate complex. Using the same approach, we utilized the scattering curve of the phosphofructokinase-2-MgATP complex to model the arrangement and conformation of dimers in the tetramer. We observed that, along with tetramerization, binding of MgATP to the allosteric site induces domain closure. Additionally, we used the scattering data to restore the low resolution structure of phosphofructokinase-2 (free and bound forms) by an ab initio procedure. Based on these findings, a proposal is made to account for the inhibitory effect of MgATP on the enzymatic activity.     

Distinct thyroid hormone‐dependent expression of trkB and p75NGFR in nonneuronal cells during the critical TH‐dependent period of the cochlea

Analyzing the thyroid hromone (TH)‐dependent period of the inner ear, we observed that the presence of triiodothyronine (T3) between postnatal day 3 (P3) and P12 is sufficient for functional maturation of the auditory system. Within this short time period, an unusual transient TH‐dependent expression of nonneuronal neurotrophin receptors (NT‐R) trkB and p75^NGFR was observed in correlation with neuronal and morphogenetic processes. The availability of thyroid hormone was revealed to be invariably correlated with (a) a transient expression of full‐length trkB in TRα1‐, TRα2‐ and TRβ1‐expressing hair cells concomitant to the segregation of afferent fibers and the synaptogenesis of efferent fibers; and (b) a transient expression of p75^NGFR in TRα1‐ and TRβ1‐expressing great epithelia ridge cells in direct spatiotemporal correlation with the appearance of apoptotic cells and morphogenetic maturation of the organ. For the first time, these data suggest a TH dependency of the expression of neurotrophin receptors in nonneuronal cells. A potential role of these peculiar neurotrophin receptor expression for the conversion of the biological function of TH on innervation patterning and morphogenesis during the critical TH‐dependent period of the inner ear may be considered. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 338–356, 1999     

A network of stimulatory and inhibitory Galpha-subunits regulates olfaction in Caenorhabditis elegans

The two pairs of sensory neurons of C. elegans, AWA and AWC, that mediate odorant attraction, express six Galpha-subunits, suggesting that olfaction is regulated by a complex signaling network. Here, we describe the cellular localization and functions of the six olfactory Galpha-subunits: GPA-2, GPA-3, GPA-5, GPA-6, GPA-13, and ODR-3. All except GPA-6 localize to sensory cilia, suggesting a direct role in sensory transduction. GPA-2, GPA-3, GPA-5, and GPA-6 are also present in cell bodies and axons and GPA-5 specifically localizes to synaptic sites. Analysis of animals with single- to sixfold loss-of-function mutations shows that olfaction involves a balance between multiple stimulatory and inhibitory signals. ODR-3 constitutes the main stimulatory signal and is sufficient for the detection of odorants. GPA-3 forms a second stimulatory signal in the AWA and AWC neurons, also sufficient for odorant detection. In AWA, signaling is suppressed by GPA-5. In AWC, GPA-2 and GPA-13 negatively and positively regulate signaling, respectively. Finally, we show that only ODR-3 plays a role in cilia morphogenesis. Defects in this process are, however, independent of olfactory behavior. Our findings reveal the existence of a complex signaling network that controls odorant detection by C. elegans.     

Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.     

B7-H1 (programmed death-1 ligand) on dendritic cells is involved in the induction and maintenance of T cell anergy

In an effort to identify immunoregulatory molecules on dendritic cells (DC), we generated and screened for mAbs capable of modulating the T cell stimulatory function of DC. A particularly interesting mAb was mAb DF272. It recognizes monocyte-derived DC, but not blood monocytes or lymphocytes, and has profound immunomodulatory effects on DC. Treatment of DC with intact IgG or Fab of mAb DF272 enhanced their T cell stimulatory capacity. This effect on DC was accompanied by neither an up-regulation of costimulatory molecules such as B7.1 (CD80), B7.2 (CD86), and MHC class II molecules nor by an induction of cytokine production, including IL-1, TNF-alpha, IL-10, and IL-12. Moreover, the well-established inhibitory function of IL-10-treated DC could be reverted with mAb DF272. Even T cells, anergized because of stimulation with IL-10-treated DC, could be reactivated and induced to proliferate upon stimulation with mAb DF272-treated DC. Furthermore, mAb DF272-treated DC favored the induction of a type-1 cytokine response in T cells and inhibited IL-10 production. By using a retrovirus-based cDNA expression library generated from DC, we cloned and sequenced the mAb DF272-defined cell surface receptor and could demonstrate that it is identical with B7-H1 (programmed death-1 ligand), a recently identified new member of the B7 family of costimulatory molecules. Our results thus demonstrate that the mAb DF272-defined surface molecule B7-H1 represents a unique receptor structure on DC that might play a role in the induction and maintenance of T cell anergy.     

Early Life Experiences and Exercise Associate with Canine Anxieties

Personality and anxiety disorders across species are affected by genetic and environmental factors. Shyness-boldness personality continuum exists across species, including the domestic dog, with a large within- and across-breed variation. Domestic dogs are also diagnosed for several anxiety-related behavioral conditions, such as generalized anxiety disorders, phobias, and separation anxiety. Genetic and environmental factors contributing to personality and anxiety are largely unknown. We collected questionnaire data from a Finnish family dog population (N = 3264) in order to study the associating environmental factors for canine fearfulness, noise sensitivity, and separation anxiety. Early life experiences and exercise were found to associate with anxiety prevalence. We found that fearful dogs had less socialization experiences (p = 0.002) and lower quality of maternal care (p < 0.0001) during puppyhood. Surprisingly, the largest environmental factor associating with noise sensitivity (p < 0.0001) and separation anxiety (p = 0.007) was the amount of daily exercise; dogs with noise sensitivity and separation anxiety had less daily exercise. Our findings suggest that dogs share many of the same environmental factors that contribute to anxiety in other species as well, such as humans and rodents. Our study highlights the importance of early life experiences, especially the quality of maternal care and daily exercise for the welfare and management of the dogs, and reveals important confounding factors to be considered in the genetic characterization of canine anxiety.     

The CATP-8/P5A-type ATPase functions in multiple pathways during neuronal patterning

The assembly of neuronal circuits involves the migrations of neurons from their place of birth to their final location in the nervous system, as well as the coordinated growth and patterning of axons and dendrites. In screens for genes required for patterning of the nervous system, we identified the catp-8/P5A-ATPase as an important regulator of neural patterning. P5A-ATPases are part of the P-type ATPases, a family of proteins known to serve a conserved function as transporters of ions, lipids and polyamines in unicellular eukaryotes, plants, and humans. While the function of many P-type ATPases is relatively well understood, the function of P5A-ATPases in metazoans remained elusive. We show here, that the Caenorhabditis elegans ortholog catp-8/P5A-ATPase is required for defined aspects of nervous system development. Specifically, the catp-8/P5A-ATPase serves functions in shaping the elaborately sculpted dendritic trees of somatosensory PVD neurons. Moreover, catp-8/P5A-ATPase is required for axonal guidance and repulsion at the midline, as well as embryonic and postembryonic neuronal migrations. Interestingly, not all axons at the midline require catp-8/P5A-ATPase, although the axons run in the same fascicles and navigate the same space. Similarly, not all neuronal migrations require catp-8/P5A-ATPase. A CATP-8/P5A-ATPase reporter is localized to the ER in most, if not all, tissues and catp-8/P5A-ATPase can function both cell-autonomously and non-autonomously to regulate neuronal development. Genetic analyses establish that catp-8/P5A-ATPase can function in multiple pathways, including the Menorin pathway, previously shown to control dendritic patterning in PVD, and Wnt signaling, which functions to control neuronal migrations. Lastly, we show that catp-8/P5A-ATPase is required for localizing select transmembrane proteins necessary for dendrite morphogenesis. Collectively, our studies suggest that catp-8/P5A-ATPase serves diverse, yet specific, roles in different genetic pathways and may be involved in the regulation or localization of transmembrane and secreted proteins to specific subcellular compartments.