Characterization of human genomic yeast artificial chromosome inserts containing hexokinase 1 coding information on chromosome 10.

Hexokinase 1 (HK1) is one of four mammalian HK isoenzymes and maps to human chromosome 10. Two yeast artificial chromosomes (YACs) were identified in the Washington University human YAC library using polymerase chain reaction (PCR) primers designed with knowledge of the human HK1 cDNA sequence. YAC B129B12 is 120 kb in length and maps entirely to chromosome 10. YAC A159D5 is 400 kb in length and appears to have resulted from a recombination of chromosome 10 with non-chromosome 10 material. We report these YACs as potential resources for those interested in HK1 gene organization and mapping, as well as those desiring additional genomic information and markers on chromosome 10.     

Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site

Aggregation of neurotransmitter receptors at pre- and postsynaptic structures is crucial for efficient neuronal communication. In contrast to the wealth of information about postsynaptic specializations, little is known about the molecular organization of presynaptic membrane proteins. We show here that the metabotropic glutamate receptor mGluR7a, which localizes specifically to presynaptic active zones, interacts in vitro and in vivo with PICK1. Coexpression in heterologous systems induces coclustering dependent upon the extreme C terminus of mGluR7a and the PDZ domain of PICK1. mGluR7a and PICK1 localize to excitatory synapses in hippocampal neurons. Furthermore, whereas transfected mGluR7a clusters at presynaptic sites, mGluR7adelta3 lacking the PICK1 binding site targets to axons but does not cluster. These results suggest that PICK1 is a component of the presynaptic machinery involved in mGluR7a aggregation and in modulation of glutamate neurotransmission.     

Structure and cooperativity of a T-state mutant of histidine decarboxylase from Lactobacillus 30a

Histidine decarboxylase (HDC) from Lactobacillus 30a converts histidine to histamine, a process that enables the bacteria to maintain the optimum pH range for cell growth. HDC is regulated by pH; it is active at low pH and inactive at neutral to alkaline pH. The X-ray structure of HDC at pH 8 revealed that a helix was disordered, resulting in the disruption of the substrate-binding site. The HDC trimer has also been shown to exhibit cooperative kinetics at neutral pH, that is, histidine can trigger a T-state to R-state transition. The D53,54N mutant of HDC has an elevated Km, even at low pH, indicating that the enzyme assumes the low activity T-state. We have solved the structures of the D53,54N mutant at low pH, with and without the substrate analog histidine methyl ester (HME) bound. Structural analysis shows that the apo-D53,54N mutant is in the inactive or T-state and that binding of the substrate analog induces the enzyme to adopt the active or R-state. A mechanism for the cooperative transition is proposed.     

Generation and Characterization of Antibodies against Asian Elephant (Elephas maximus) IgG,IgM, and IgA

Asian elephant (Elephas maximus) immunity is poorly characterized and understood. This gap in knowledge is particularly concerning as Asian elephants are an endangered species threatened by a newly discovered herpesvirus known as elephant endotheliotropic herpesvirus (EEHV), which is the leading cause of death for captive Asian elephants born after 1980 in North America. While reliable diagnostic assays have been developed to detect EEHV DNA, serological assays to evaluate elephant anti-EEHV antibody responses are lacking and will be needed for surveillance and epidemiological studies and also for evaluating potential treatments or vaccines against lethal EEHV infection. Previous studies have shown that Asian elephants produce IgG in serum, but they failed to detect IgM and IgA, further hampering development of informative serological assays for this species. To begin to address this issue, we determined the constant region genomic sequence of Asian elephant IgM and obtained some limited protein sequence information for putative serum IgA. The information was used to generate or identify specific commercial antisera reactive against IgM and IgA isotypes. In addition, we generated a monoclonal antibody against Asian elephant IgG. These three reagents were used to demonstrate that all three immunoglobulin isotypes are found in Asian elephant serum and milk and to detect antibody responses following tetanus toxoid booster vaccination or antibodies against a putative EEHV structural protein. The results indicate that these new reagents will be useful for developing sensitive and specific assays to detect and characterize elephant antibody responses for any pathogen or vaccine, including EEHV.     

Life history trade‐offs and evidence for hierarchical resource allocation in two monocarpic perennials

The evolution of floral display is thought to be constrained by trade‐offs between the size and number of flowers; however, empirical evidence for the trade‐off is inconsistent. We examined evidence for trade‐offs and hierarchical allocation of resources within and between two populations each of the monocarpic perennials, Cardiocrinum cordatum and C. giganteum. Within all populations, flower size–number trade‐offs were evident after accounting for variation in plant size. In addition, variation in flower size explained much variation in flower‐level allocation to attraction, and female and male function, a pattern consistent with hierarchical allocation. However, between population differences in flower size (C. cordatum) and number (C. giganteum) were not consistent with size–number trade‐offs or hierarchical allocation. The population‐level difference in C. cordatum likely reflects the combined influence of a time lag between initiation and maturation of flowers, and higher light levels in one population. Thus, our study highlights one mechanism that may account for the apparent independence of flower size and number in many studies. A prediction of sex allocation theory was also supported. In C. giganteum: plants from one population invested more mass in pistils and less in stamens than did plants from the other population. Detection of floral trade‐offs in Cardiocrinum may be facilitated by monocarpic reproduction, production of a single inflorescence and ease of measuring plant size.     

Avian biodiversity in the coastal wetlands of the Okahukura Peninsula

The Tapora Landcare Group, operating on the Okahukura Peninsula, has the long-term goal of making this region predator fenced. The aim of this study was to obtain information on the current status of avian biodiversity and the bird community across the band of coastal wetlands on the Okahukura Peninsula. Bird counts were conducted and playback lures used to detect three cryptic wetland species: fernbirds (Bowdleria punctata); spotless crakes (Porzana tabuensis); and banded rails (Gallirallus philippensis). Fernbirds and banded rails were detected at seven of the eight wetland sites sampled whereas spotless crakes were detected at two sites. The native species with the highest relative abundance across the eight sites were silvereyes (Zosterops lateralis) and South Island pied oystercatchers (Haematopus finschi). Changes in avian biodiversity over time in the region can now be monitored, and comprehensive long-term data on the status of avian biodiversity over time obtained.     

Disruption and eradication of P. aeruginosa biofilms using nitric oxide-releasing chitosan oligosaccharides

Biofilm disruption and eradication were investigated as a function of nitric oxide- (NO) releasing chitosan oligosaccharide dose and the results compared with control (ie non-NO-releasing) chitosan oligosaccharides and tobramycin. Quantification of biofilm expansion/contraction and multiple-particle tracking microrheology were used to assess the structural integrity of the biofilm before and after antibacterial treatment. While tobramycin had no effect on the physical properties of the biofilm, NO-releasing chitosan oligosaccharides exhibited dose-dependent behavior with biofilm degradation. Control chitosan oligosaccharides increased biofilm elasticity, indicating that the scaffold may mitigate the biofilm disrupting power of nitric oxide somewhat. The results from this study indicate that nitric oxide-releasing chitosan oligosaccharides act as dual-action therapeutics capable of eradicating and physically disrupting P. aeruginosa biofilms.     

Postnatal Temporal,Spatial and Modality Tuning of Nociceptive Cutaneous Flexion Reflexes in Human Infants

Cutaneous flexion reflexes are amongst the first behavioural responses to develop and are essential for the protection and survival of the newborn organism. Despite this, there has been no detailed, quantitative study of their maturation in human neonates. Here we use surface electromyographic (EMG) recording of biceps femoris activity in preterm (<37 weeks gestation, GA) and term (≥37 weeks GA) human infants, less than 14 days old, in response to tactile, punctate and clinically required skin-breaking lance stimulation of the heel. We show that all infants display a robust and long duration flexion reflex (>4 seconds) to a single noxious skin lance which decreases significantly with gestational age. This reflex is not restricted to the stimulated limb: heel lance evokes equal ipsilateral and contralateral reflexes in preterm and term infants. We further show that infant flexion withdrawal reflexes are not always nociceptive specific: in 29% of preterm infants, tactile stimulation evokes EMG activity that is indistinguishable from noxious stimulation. In 40% of term infants, tactile responses are also present but significantly smaller than nociceptive reflexes. Infant flexion reflexes are also evoked by application of calibrated punctate von Frey hairs (vFh), 0.8–17.2 g, to the heel. Von Frey hair thresholds increase significantly with gestational age and the magnitude of vFh evoked reflexes are significantly greater in preterm than term infants. Furthermore flexion reflexes in both groups are sensitized by repeated vFh stimulation. Thus human infant flexion reflexes differ in temporal, modality and spatial characteristics from those in adults. Reflex magnitude and tactile sensitivity decreases and nociceptive specificity and spatial organisation increases with gestational age. Strong, relatively non-specific, reflex sensitivity in early life may be important for driving postnatal activity dependent maturation of targeted spinal cord sensory circuits.     

Role for mTOR signaling and neuronal activity in morphine-induced adaptations in ventral tegmental area dopamine neurons

While the abuse of opiate drugs continues to rise, the neuroadaptations that occur with long-term drug exposure remain poorly understood. We describe here a series of chronic morphine-induced adaptations in ventral tegmental area (VTA) dopamine neurons, which are mediated via downregulation of AKT-mTORC2 (mammalian target of rapamycin complex-2). Chronic opiates decrease the size of VTA dopamine neurons in rodents, an effect seen in humans as well, and concomitantly increase the excitability of the cells but decrease dopamine output to target regions. Chronic morphine decreases mTORC2 activity, and overexpression of Rictor,?a component of mTORC2, prevents morphine-induced changes in cell morphology and activity. Further, local knockout of Rictor in VTA decreases DA soma size and reduces rewarding responses to morphine, consistent with the hypothesis that these adaptations represent a mechanism of reward tolerance. Together, these findings demonstrate a novel role for AKT-mTORC2 signaling in mediating neuroadaptations to opiate drugs of abuse.