Single Channel Kinetics of a Glutamate Receptor

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the presence of 10^-4 M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.     

Effects of phosphorylation on binding of catecholamines to tyrosine hydroxylase: specificity and thermodynamics

As the catalyst for the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters, the activity of tyrosine hydroxylase is tightly regulated. A principle means of posttranslational regulation is reversible phosphorylation of serine residues in an N-terminal regulatory domain. Phosphorylation of serine 40 has been shown to have a large effect on the rate constant for dissociation of dopamine and a much smaller effect on that for DOPA [Ramsey, A. J., and Fitzpatrick, P. F. (1998) Biochemistry 37, 8980-8986]. To determine the structural basis for the differences in affinity and to further test the validity of the previously proposed model for regulation, the effects of phosphorylation of serine 40 on the affinities for a series of catechols have been determined. The affinities of the unphosphorylated enzyme vary by 3 orders of magnitude due to differences in the rates of dissociation. The highest affinities are found with catecholamines which lack a carboxylate. The affinities of the phosphorylated enzyme show a much smaller range. In the case of binding of dihydroxyphenylalanine, the decrease in affinity upon phosphorylation is due primarily to a decrease in the enthalpy of the interaction. Based upon these results, a structural model for the effect of phosphorylation is proposed.     

Serum dehydroepiandrosterone sulfate concentrations across the life span of laboratory-housed rhesus monkeys

Cross-sectional studies of humans have shown that dehydroepiandrosterone sulfate (DHEAS) peaks shortly after sexual maturation and declines thereafter, suggesting that the progressive reduction in DHEAS may play a role in the aging process and in the development of age-related morbidity. The present study examines changes in DHEAS concentrations across the life span of rhesus monkeys as part of the development of this primate model for studies of aging. Serum concentrations of DHEAS were measured in 792 laboratory-housed rhesus monkeys (Macaca mulatta) aged 0.5-36 years (527 females, 265 males). DHEAS concentrations in all monkeys were used to formulate an equation that describes two levels of decline of DHEAS with age. The most rapid decline occurs from infancy until approximately 5 years of age. The decline then occurs gradually with increasing age. There were no signs of an andrenarche just prior to sexual maturation, as is seen in humans or the great apes. This equation can be used to predict the expected mean serum DHEAS concentration and normal ranges of male or female rhesus monkeys at any age greater than 5 months.     

The Shc locus regulates insulin signaling and adiposity in mammals

Longevity of a p66Shc knockout strain (ShcP) was previously attributed to increased stress resistance and altered mitochondria. Microarrays of ShcP tissues indicated alterations in insulin signaling. Consistent with this observation, ShcP mice were more insulin sensitive and glucose tolerant at organismal and tissue levels, as was a novel p66Shc knockout (ShcL). Increasing and decreasing Shc expression in cell lines decreased and increased insulin sensitivity, respectively - consistent with p66Shc's function as a repressor of insulin signaling. However, differences between the two p66Shc knockout strains were also observed. ShcL mice were fatter and susceptible to fatty diets, and their fat was more insulin sensitive than controls. On the other hand, ShcP mice were leaner and resisted fatty diets, and their adipose was less insulin sensitive than controls. ShcL and ShcP strains are both highly inbred on the C57Bl/6 background, so we investigated gene expression at the Shc locus, which encodes three isoforms, p66, p52, and p46. Isoform p66 is absent in both strains; thus, the remaining difference to which to attribute the 'lean' phenotype is expression of the other two isoforms. ShcL mice have a precise deletion of p66Shc and normal expression of p52 and p46Shc isoforms in all tissues; thus, a simple deletion of p66Shc results in a 'fat' phenotype. However, ShcP mice in addition to p66Shc deletion have a fourfold increase in p46Shc expression in white fat. Thus, p46Shc overexpression in fat, rather than p66Shc deletion, is the likely cause of decreased adiposity and reduced insulin sensitivity in the fat of ShcP mice, which has implications for the longevity of the strain.     

Applications of fluorophore-containing microbial growth media.

Media containing the fluorogenic compound 8-anilino-1-naphthalene sulfonic acid may be used to discriminate between gram-positive and gram-negative bacteria and to differentiate between various species of bacteria. Fluorescent light emitted from colonies of gram-negative bacteria on 8-anilino-1-naphthalene sulfonic acid-containing agar was visually more intense than that on gram-positive bacteria. The emitted light from the gram-negative bacteria differed in wave-lengths from that of light emitted by colonies of gram-positive bacteria. The fluorescent intensity of colonies on complete 8-anilino-1-napthalene sulfonic acid agar supplemented with 1% of single substrates varied depending on the bacterial species, thus allowing the development of profiles used to identify 12 different species.     

PKA and ERK, but not PKC, in the amygdala contribute to pain-related synaptic plasticity and behavior

The laterocapsular division of the central nucleus of the amygdala (CeLC) has emerged as an important site of pain-related plasticity and pain modulation. Glutamate and neuropeptide receptors in the CeLC contribute to synaptic and behavioral changes in the arthritis pain model, but the intracellular signaling pathways remain to be determined. This study addressed the role of PKA, PKC, and ERK in the CeLC. Adult male Sprague-Dawley rats were used in all experiments. Whole-cell patch-clamp recordings of CeLC neurons were made in brain slices from normal rats and from rats with a kaolin/carrageenan-induced monoarthritis in the knee (6 h postinduction). Membrane-permeable inhibitors of PKA (KT5720, 1 μM; cAMPS-Rp, 10 μM) and ERK (U0126, 1 μM) activation inhibited synaptic plasticity in slices from arthritic rats but had no effect on normal transmission in control slices. A PKC inhibitor (GF109203x, 1 μM) and an inactive structural analogue of U0126 (U0124, 1 μM) had no effect. The NMDA receptor-mediated synaptic component was inhibited by KT5720 or U0126; their combined application had additive effects. U0126 did not inhibit synaptic facilitation by forskolin-induced PKA-activation. Administration of KT5720 (100 μM, concentration in microdialysis probe) or U0126 (100 μM) into the CeLC, but not striatum (placement control), inhibited audible and ultrasonic vocalizations and spinal reflexes of arthritic rats but had no effect in normal animals. GF109203x (100 μM) and U0124 (100 μM) did not affect pain behavior. The data suggest that in the amygdala PKA and ERK, but not PKC, contribute to pain-related synaptic facilitation and behavior by increasing NMDA receptor function through independent signaling pathways.     

Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium

K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area.     

Inferring the Distribution and Demography of an Invasive Species from Sighting Data: The Red Fox Incursion into Tasmania

A recent study has inferred that the red fox (Vulpes vulpes) is now widespread in Tasmania as of 2010, based on the extraction of fox DNA from predator scats. Heuristically, this inference appears at first glance to be at odds with the lack of recent confirmed discoveries of either road-killed foxes—the last of which occurred in 2006, or hunter killed foxes—the most recent in 2001. This paper demonstrates a method to codify this heuristic analysis and produce inferences consistent with assumptions and data. It does this by formalising the analysis in a transparent and repeatable manner to make inference on the past, present and future distribution of an invasive species. It utilizes Approximate Bayesian Computation to make inferences. Importantly, the method is able to inform management of invasive species within realistic time frames, and can be applied widely. We illustrate the technique using the Tasmanian fox data. Based on the pattern of carcass discoveries of foxes in Tasmania, we infer that the population of foxes in Tasmania is most likely extinct, or restricted in distribution and demographically weak as of 2013. It is possible, though unlikely, that that population is widespread and/or demographically robust. This inference is largely at odds with the inference from the predator scat survey data. Our results suggest the chances of successfully eradicating the introduced red fox population in Tasmania may be significantly higher than previously thought.     

Rapid vesicular translocation and insertion of TRP channels

The broadly expressed transient receptor potential (TRP) family of ion channels are permeant to cations, most resulting in increased intracellular calcium. However, their regulation and gating is not well understood. Here, we report that growth factor stimulation initiates the rapid translocation of the transient receptor potential ion channel, TRPC5, from vesicles held in reserve just under the plasma membrane. This process, which we term 'rapid vesicular insertion of TRP' (RiVIT), dramatically increases membrane-associated TRPC5 channels and functional TRPC5 current, resulting in tight spatial-temporal control of these Ca(2+)-permeant nonselective channels. Epidermal growth factor (EGF)-induced incorporation of functional TRP channels requires phosphatidylinositide 3-kinase (PI(3)K), the Rho GTPase Rac1 and phosphatidylinositol 4-phosphate 5-kinase (PIP(5)K alpha). The increase in TRPC5 availability affects neurite extension rates in cultured hippocampal neurons, and may be a general mechanism for initiating Ca(2+) influx and cell morphological changes in response to stimuli.