The ratio of morning cortisol to CRP prospectively predicts first-onset depression in at-risk adolescents.

RATIONALE: Early-onset adolescent depression is related to poor prognosis and a range of psychiatric and medical comorbidities later in life, making the identification of a priori risk factors for depression highly important. Increasingly, dysregulated levels of immune and neuroendocrine markers, such as C-reactive protein (CRP) and cortisol, have been demonstrated as both precursors to and consequences of depression. However, longitudinal research with adolescent populations is limited and demonstrates mixed immuno-endocrine-depression links. OBJECTIVE: This study explored the putative bidirectional relationship between salivary measures of cortisol (Cort) and CRP, including the novel Cort:CRP ratio and depression. METHODS: Participants from the randomized control trial 'Sleep and Education: learning New Skills Early' (SENSE) Study were 122 adolescents at risk for depression (73 females) aged 12-16 years (M = 12.71 years, SD = 1.01 years) assessed at baseline (T1), post-intervention (T2), and a two-year follow-up (T3). RESULTS: Logistic regression results demonstrated that adolescents with higher T1 Cort:CRPmorn ratio levels were two-fold more likely to develop a first-onset depressive disorder from T2 to T3 as compared to adolescents with lower Cort:CRPmorn ratio levels, ß = 0.73, t (36) = 2.15, p = .04, OR = 2.08. This effect was not moderated by treatment condition (ß = -1.38, t (13) = -1.33, p = .20) and did not change when controlling for known risk factors for depression, including sex, age, body-mass index, socio-economic status, T1 anxiety disorder, nor T1 sleep disturbance, anxiety, or depressive symptoms (ß = 0.91, t (31) = 2.14, p = .04). CONCLUSION: Results highlight potential immuno-endocrine dysregulation as an underlying risk factor for adolescent first-onset depression, and may inform the development of targeted, preventative biobehavioral treatment strategies for youth depression.

Acetylcholine noise: analysis after chemical modification of receptor.

The elementary voltage pulses ("shot effects") produced by the action of acetylcholine molecules on the receptor were studied by analyzing the membrane voltage fluctuations ("noise") after acetylcholine application at the frog neuromuscular junction. The amplitude of these pulses was decreased after treatment with a disulfide-bond reducing agent. The shot effect may thus depend on the structure or conformation of the receptor molecule.

X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases.

Lipidic cubic phases provide a continuous three-dimensional bilayer matrix that facilitates nucleation and growth of bacteriorhodopsin microcrystals. The crystals diffract x-rays isotropically to 2.0 angstroms. The structure of this light-driven proton pump was solved at a resolution of 2.5 angstroms by molecular replacement, using previous results from electron crystallographic studies as a model. The earlier structure was generally confirmed, but several differences were found, including loop conformations and side chain residues. Eight water molecules are now identified experimentally in the proton pathway. These findings reveal the constituents of the proton translocation pathway in the ground state.

A Synchrotron X-ray Study of a Solid-Solid Phase Transition in a Two-Dimensional Crystal.

A measurement and interpretation on a molecular level of a phase transition in an ordered Langmuir monolayer is reported. The diagram of surface pressure (pi) versus molecular area of a monolayer of chiral (S)-[CF(3)-(CF(2))(9)-(CH(2))(2)-OCO-CH(2)-CH (NH(3)(+))CO(2)(-)] over water shows a change in slope at about pi(s)= 25 millinewtons per meter. Grazing-incidence x-ray diffraction and specular reflectivity measurements indicate a solid-solid phase transition at pi(s). The diffraction pattren at low pressures reveals two diffraction peaks of equal intensities, with lattice spacings d of 5.11 and 5.00 angstroms; these coalesce for pi >/=pi(s). Structural models that fit the diffraction data show that at pi> pi(s) the molecules pack in a two-dimensional crystal with the molecules aligned vertically. At pi < pi(s) there is a molecular tilt of 16 degrees +/- 7 degrees . Independent x-ray reflectivity data yield a tilt of 26 degrees +/- 7 degrees . Concomitant with the tilt, the diffraction data indicate a transition from a hexagonal to a distorted-hexagonal lattice. The hexagonal arrangement is favored because the -(CF(2))(9)CF(3) moiety adopts a helical conformation. Compression to 70 millinewtons per meter yields a unit cell with increased crystallinity and a coherence length exceeding 1000 angstroms.

Gating of CaMKII by cAMP-regulated protein phosphatase activity during LTP.

Long-term potentiation (LTP) at the Schaffer collateral-CA1 synapse involves interacting signaling components, including calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclic adenosine monophosphate (cAMP) pathways. Postsynaptic injection of thiophosphorylated inhibitor-1 protein, a specific inhibitor of protein phosphatase-1 (PP1), substituted for cAMP pathway activation in LTP. Stimulation that induced LTP triggered cAMP-dependent phosphorylation of endogenous inhibitor-1 and a decrease in PP1 activity. This stimulation also increased phosphorylation of CaMKII at Thr286 and Ca2+-independent CaMKII activity in a cAMP-dependent manner. The blockade of LTP by a CaMKII inhibitor was not overcome by thiophosphorylated inhibitor-1. Thus, the cAMP pathway uses PP1 to gate CaMKII signaling in LTP.

Mental Health screening in cystic fibrosis centres across Europe.

OBJECTIVES: The Cystic Fibrosis (CF) International Mental Health Guidelines Committee published consensus statements for screening and treating depression and anxiety in individuals with CF and their caregivers. This work aimed to evaluate the dissemination and implementation of the guidelines in Europe two years following their publication. METHODS: A 28-item survey was developed by the multidisciplinary ECFS Mental Health Working Group and emailed to approximately 300 CF centres across Europe. The survey evaluated (a) who should be responsible for mental health (MH) care, (b) the current awareness and agreement of the guidelines, (c) the provision of recommended MH screening and follow-up care, and (d) successes, barriers and required resources/training needs. RESULTS: Responses were received from 187 centres (28 countries represented). There was consensus that a psychologist should be responsible for MH care, although members of the multidisciplinary team (MDT) believed they should also share this responsibility. Sixty-two percent of respondents were aware of the guidelines; 82% percent fully, and 12% partially, agreed with them. Fifty percent (94 centres) had implemented screening. In the past year approximately 6000 patients and 2000 caregivers had been screened, with 80% of respondents using the recommended screening tools. Respondents reported 551 referrals for moderate/severe psychopathology and 84 urgent suicide ideation referrals. CONCLUSIONS: The challenges of different healthcare systems and language barriers are being overcome with a greater awareness of the importance of mental health among the MDT. MH screening is feasible and gaining momentum in both Western and Eastern Europe.

Salivary C-reactive protein among at-risk adolescents: A methods investigation of out of range immunoassay data.

Inflammatory markers including C-Reactive Protein (CRP) are increasingly used within research and clinical settings. Yet, varying methodologies for cleaning immunoassay data with out of range (OOR) samples may alter characteristic levels of CRP, thereby obscuring interpretation and reliability. This study investigated the influence of eight immunoassay OOR data treatment techniques on salivary CRP (sCRP) samples from at-risk adolescents. Participants from the 'Sleep and Education: learning New Skills Early' (SENSE) Study were 86 adolescents at-risk for depression (50 female), aged 14.29 years (SD = 1.04). ANOVA results showed no statistically significant differences in average morning (F(7, 590) = 1.24, p = .28) and evening (F(7, 599)=1.29, p = .25) values produced by each OOR data cleaning technique. However, varying techniques produced differences in the magnitude of Pearson's correlations between consecutive saliva samples (r's between 0.27-0.78), and influenced the significance of a sCRP diurnal pattern; two techniques produced statistically higher morning than evening sCRP levels (t(85) = 2.70, p = .01 and t(85) = 2.67, p = .01), whereas six techniques failed to find statistical differences between morning and evening sCRP levels (p's >.05). Varying techniques also produced statistically divergent associations between sCRP and age and depressive symptoms. Results from this study provide evidence for the temporal stability of sCRP among adolescents, show winsorization as an effective OOR data management technique, and highlight the influence of methodological decisions in cleaning salivary biomarker data and the need for consistency within the field.

Postsynaptic cAMP pathway gates early LTP in hippocampal CA1 region.

The role of the cAMP pathway in LTP was studied in the CA1 region of hippocampus. Widely spaced trains of high frequency stimulation generated cAMP postsynaptically via NMDA receptors and calmodulin, consistent with the Ca2+/calmodulin-mediated stimulation of postsynaptic adenylyl cyclase. The early phase of LTP produced by the same pattern of high frequency stimulation was dependent on postsynaptic cAMP. However, synaptic transmission was not increased by postsynaptic application of cAMP. Early LTP became cAMP-independent when protein phosphatase inhibitors were injected postsynaptically. These observations indicate that in early LTP the cAMP signaling pathway, instead of transmitting signals for the generation of LTP, gates LTP through postsynaptic protein phosphatases.

Protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple membrane at 1.9 A resolution.

BACKGROUND: Bacteriorhodopsin (bR) from Halobacterium salinarum is a proton pump that converts the energy of light into a proton gradient that drives ATP synthesis. The protein comprises seven transmembrane helices and in vivo is organized into purple patches, in which bR and lipids form a crystalline two-dimensional array. Upon absorption of a photon, retinal, which is covalently bound to Lys216 via a Schiff base, is isomerized to a 13-cis,15-anti configuration. This initiates a sequence of events - the photocycle - during which a proton is transferred from the Schiff base to Asp85, followed by proton release into the extracellular medium and reprotonation from the cytoplasmic side. RESULTS: The structure of bR in the ground state was solved to 1.9 A resolution from non-twinned crystals grown in a lipidic cubic phase. The structure reveals eight well-ordered water molecules in the extracellular half of the putative proton translocation pathway. The water molecules form a continuous hydrogen-bond network from the Schiff-base nitrogen (Lys216) to Glu194 and Glu204 and includes residues Asp85, Asp212 and Arg82. This network is involved both in proton translocation occurring during the photocycle, as well as in stabilizing the structure of the ground state. Nine lipid phytanyl moieties could be modeled into the electron-density maps. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of single crystals demonstrated the presence of four different charged lipid species. CONCLUSIONS: The structure of protein, lipid and water molecules in the crystals represents the functional entity of bR in the purple membrane of the bacteria at atomic resolution. Proton translocation from the Schiff base to the extracellular medium is mediated by a hydrogen-bond network that involves charged residues and water molecules.

Mitogen-activated protein kinase regulates early phosphorylation and delayed expression of Ca2+/calmodulin-dependent protein kinase II in long-term potentiation.

Activation of mitogen-activated protein kinase (MAPK) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are required for numerous forms of neuronal plasticity, including long-term potentiation (LTP). We induced LTP in rat hippocampal area CA1 using theta-pulse stimulation (TPS) paired with beta-adrenergic receptor activation [isoproterenol (ISO)], a protocol that may be particularly relevant to normal patterns of hippocampal activity during learning. This stimulation resulted in a transient phosphorylation of p42 MAPK, and the resulting LTP was MAPK dependent. In addition, CaMKII was regulated in two, temporally distinct ways after TPS-ISO: a transient rise in the fraction of phosphorylated CaMKII and a subsequent persistent increase in CaMKII expression. The increases in MAPK and CaMKII phosphorylation were strongly colocalized in the dendrites and cell bodies of CA1 pyramidal cells, and both the transient phosphorylation and delayed expression of CaMKII were prevented by inhibiting p42/p44 MAPK. These results establish a novel bimodal regulation of CaMKII by MAPK, which may contribute to both post-translational modification and increased gene expression.

Long-term potentiation induced by theta frequency stimulation is regulated by a protein phosphatase-1-operated gate.

Long-term potentiation (LTP) can be induced in the Schaffer collateral-->CA1 synapse of hippocampus by stimulation in the theta frequency range (5-12 Hz), an effect that depends on activation of the cAMP pathway. We investigated the mechanisms of the cAMP contribution to this form of LTP in the rat hippocampal slice preparation. theta pulse stimulation (TPS; 150 stimuli at 10 Hz) by itself did not induce LTP, but the addition of either the beta-adrenergic agonist isoproterenol or the cAMP analog 8-bromo-cAMP (8-Br-cAMP) enabled TPS-induced LTP. The isoproterenol effect was blocked by postsynaptic inhibition of cAMP-dependent protein kinase. Several lines of evidence indicated that cAMP enabled LTP by blocking postsynaptic protein phosphatase-1 (PP1). Activators of the cAMP pathway reduced PP1 activity in the CA1 region and increased the active form of inhibitor-1, an endogenous inhibitor of PP1. Postsynaptic injection of activated inhibitor-1 mimicked the LTP-enabling effect of cAMP pathway stimulation. TPS evoked complex spiking when isoproterenol was present. However, complex spiking was not sufficient to enable TPS-induced LTP, which additionally required the inhibition of postsynaptic PP1. PP1 inhibition seems to promote the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII), because (1) a CaMKII inhibitor blocked the induction of LTP by TPS paired with either isoproterenol or activated inhibitor-1 and (2) CaMKII in area CA1 was activated by the combination of TPS and 8-Br-cAMP but not by either stimulus alone. These results indicate that the cAMP pathway enables TPS-induced LTP by inhibiting PP1, thereby enhancing Ca(2+)-independent CaMKII activity.

Lipidic cubic phase crystallization of bacteriorhodopsin and cryotrapping of intermediates: towards resolving a revolving photocycle.

Bacteriorhodopsin is a small retinal protein found in the membrane of the halophilic bacterium Halobacterium salinarum, whose function is to pump protons across the cell membrane against an electrostatic potential, thus converting light into a proton-motive potential needed for the synthesis of ATP. Because of its relative simplicity, exceptional stability and the fundamental importance of vectorial proton pumping, bacteriorhodopsin has become one of the most important model systems in the field of bioenergetics. Recently, a novel methodology to obtain well-diffracting crystals of membrane proteins, utilizing membrane-like bicontinuous lipidic cubic phases, has been introduced, providing X-ray structures of bacteriorhodopsin and its photocycle intermediates at ever higher resolution. We describe this methodology, the new insights provided by the higher resolution ground state structures, and review the mechanistic implications of the structural intermediates reported to date. A detailed understanding of the mechanism of vectorial proton transport across the membrane is thus emerging, helping to elucidate a number of fundamental issues in bioenergetics.

Molecular weight determination of membrane proteins by sedimentation equilibrium at the sucrose or nycodenz-adjusted density of the hydrated detergent micelle.

The determination of the molecular weight of a membrane protein by sedimentation equilibrium is complicated by the fact that these proteins interact with detergents and form complexes of unknown density. These effects become marginal when running sedimentation equilibrium at gravitational transparency, i.e., at the density corresponding to that of the hydrated detergent micelles. Dodecyl-maltoside and octyl-glucoside are commonly used for dissolving membrane proteins. The density of micelles thereof was measured in sucrose or Nycodenz. Both proved to be about 50% lower than those of the corresponding non-hydrated micelles. Several membrane proteins were centrifuged at sedimentation equilibrium in sucrose- and in Nycodenz-enriched solutions of various densities. Their molecular weights were then calculated by using the resulting slope value at the density of the hydrated detergent micelles, i.e. at gravitational transparency, and the partial specific volume corrected for a 50% hydration of the membrane protein. The molecular weights of all measured membrane proteins, i.e. of photosystem II complex, reaction center of Rhodobacter sphaeroides R26, spinach photosystem II reaction center (core complex), bacteriorhodopsin, OmpF-porin and rhodopsin from Bovine retina corresponded within +/-15% to those reported previously, indicating a general applicability of this approach.

pH dependence of the acetylcholine receptor channel: a species variation.

The effects of pH changes on the miniature endplate current (mepc) and on endplate current fluctuations (acetylcholine [ACh] noise) were examined at the neuromuscular junction in vitro in two species of frogs. In Rana pipiens the relationship between the decay time constant of the mepc (tau') and pH had a symmetrical bell shape; the value of tau' being largest at pH 7 and decreasing at more acid or more alkaline pH. In acid pH the mepc amplitude (A) decreased relative to its value at pH 7, and in alkaline pH A increased. In Rana ridibunda a narrower and asymmetric bell-shaped dependence of tau' on pH, having a maximum of pH 5.5, was found. The mepc amplitude was again reduced in acid pH but had a peak at pH 5.5. Also, its value at pH 9 was larger than at pH 7. These results were obtained with a number of different buffers and were not found to be sensitive to the nature of the buffer chosen. By performing ACh-noise analysis we found that in Rana pipiens at acid pH (5.5-5.0), the single channel conductance (gamma) and the single channel open time (tau) were significantly reduced relative to their value at pH 7. However, in Rana ridibunda at acid pH (5.4) gamma was unchanged and tau was markedly increased relative to their values at pH 7. The results can be explained quantitatively by electrostatic interaction between two fixed and titratable ionic groups and a mobile charge in the receptor molecule. The model fits the data for groups having pKs approximately 4.8 and approximately 9.8 for Rana pipiens and approximately 4.6 and approximately 6.3 for Rana ridibunda. The groups can be tentatively identified as amino acid residues; glutamic or aspartic and lysine or tyrosine for Rana pipiens; glutamic or aspartic and histidine for Rana ridibunda. The difference in the fitted values of the other model parameters for these two species can be attributed to differences in the spatial configuration of the charged groups.

Assessing the functionality of a membrane protein in a three-dimensional crystal.

Hexagonal microcrystals of bacteriorhodopsin embedded in a lipidic cubic phase have been investigated by time-resolved FT-IR and resonance Raman spectroscopy. Retinal isomerization, conformational changes in the protein backbone and proton translocation are virtually indistinguishable from those in the native membrane. The protein is thus fully active in three-dimensional crystals.

Gonadotropin-releasing hormone receptor expression in Xenopus oocytes.

The rodent GnRH receptor was characterized in Xenopus oocytes injected with RNA isolated from rat pituitary and from a gonadotrope cell line, alpha T3, derived from a transgenic mouse. Three to 4 days after 150-200 ng RNA injection, 93% of the oocytes, which were recorded by voltage clamp, responded to 10(-7) M GnRH. The mean inward currents obtained after RNA injection were 620 +/- 88 nA (n = 22) with pituitary RNA and 1415 +/- 598 (n = 4) with alpha T3 RNA. The threshold GnRH concentration able to evoke the dose dependent current after pituitary RNA injection was 3 x 10(-9) M GnRH. The GnRH receptor response of the oocyte was antagonized by [D-Phe2,6,Pro3] GnRH and [N-Ac-D-Na](2)1, D-alpha D-Me, pCl-Phe2, D-Arg6, D-Ala10-NH2]GnRH and could be elicited by D-Ser(But)6,Pro9-N-ethylamide GnRH (buserelin). The reversal potential of the GnRH generated current as determined by voltage-ramp was -22.5 +/- 1.0 mV (n = 7) and -25.6 +/- 3.3 mV (n = 3) in pituitary and cell line RNA-injected oocytes respectively, consistent with the chloride reversal potential. The GnRH receptor response was virtually eliminated by intracellular EGTA injection but was unaffected by ligand application in calcium-free perfusate. The GnRH-evoked response is mimicked by intracellular injection of inositol 1,4,5-trisphosphate. To determine the size of the GnRH receptor mRNA, alpha T3 RNA was size fractionated through a sucrose gradient. The maximal GnRH response was induced by a fraction larger than the 28S ribosomal peak. Thus we find that oocytes injected with RNA from an appropriate source develop an electrophysiological response to GnRH which is dependent on intracellular calcium mobilization, is independent of extracellular calcium, and may be mediated by inositol 1,4,5-trisphosphate.

Detergent-free membrane protein crystallization.

A comprehensive understanding of structure-function relationships of proteins requires their structures to be elucidated to high resolution. With most membrane proteins this has not been accomplished so far, mainly because of their notoriously poor crystallizability. Here we present a completely detergent-free procedure for the incorporation of a native purple membrane into a monoolein-based lipidic cubic phase, and subsequent crystallization of three-dimensional bacteriorhodopsin crystals therein. These crystals exhibit comparable X-ray diffraction quality and mosaicity, and identical crystal habit and space group to those of bacteriorhodopsin crystals that are grown from detergent-solubilized protein in cubic phase.

Molecular mechanism for the crystallization of bacteriorhodopsin in lipidic cubic phases.

Crystals of transmembrane proteins may be grown from detergent solutions or in a matrix of membranous lipid bilayers existing in a liquid crystalline state and forming a cubic phase (in cubo). While crystallization in micellar solutions appears analogous to that for soluble proteins, crystallization in lipidic matrices is poorly understood. As this method was shown to be applicable to several membrane proteins, understanding its mechanism will facilitate a rational design of crystallization, minimizing the laborious screening of a large number of parameters. Using polarization microscopy and low-angle X-ray diffraction, experimental evidence is provided to support a mechanistic model for the in cubo crystallization of bacteriorhodopsin in a lipid matrix. Membrane proteins are thought to reside in curved lipid bilayers, to diffuse into patches of lower curvature and to incorporate into lattices which associate to form highly ordered three-dimensional crystals. Critical testing of this model is necessary to generalize it to other membrane proteins.

Peripheral neuropathy in arsenic smelter workers.

We conducted a double-blind controlled study of individuals exposed to arsenic trioxide in a copper-smelting factory. Subjects fell into three categories of peripheral neuropathy: none, subclinical, and clinical. The subclinical group had no symptoms or signs of numbness or reduced reflexes, but did have reduced nerve conduction velocity and amplitude measurements. Clinical neuropathy groups had signs and symptoms of neuropathy and electrophysiologic abnormalities. The clinical and subclinical groups correlated with increased content of arsenic in urine, hair and nails. The incidence of subclinical and clinical neuropathy was greater in arsenic workers than in unexposed controls.

Differential solubilities in subregions of the membrane: a nonsteric mechanism of drug specificity.

We studied the effect of four volatile anesthetics and convulsants on the GABA- and glutamate-induced conductance change in crab muscle. The intensity of drug action correlated with the solubility parameter (delta) values of the four drugs. Thus, the higher that value was for a given drug, the stronger was its effect on the glutamate response but the weaker was it on the response to gamma-aminobutyric acid (GABA). We suggest that different gating molecules are housed in specific subregions of the membrane, each characterized by a particular value of the solubility parameter. The differential distribution of drugs in these subregions may be a nonsteric mechanism for drug specificity.