mm-wave Rydberg-Rydberg transitions gauge intermolecular coupling in a molecular ultracold plasma.

Out-of-equilibrium, strong correlation in a many-body system can trigger emergent properties that act to constrain the natural dissipation of energy and matter. Signs of such self-organization appear in the avalanche, bifurcation, and quench of a state-selected Rydberg gas of nitric oxide to form an ultracold, strongly correlated ultracold plasma. Work reported here focuses on the initial stages of avalanche and quench and uses the mm-wave spectroscopy of an embedded quantum probe to characterize the intermolecular interaction dynamics associated with the evolution to plasma. Double-resonance excitation prepares a Rydberg gas of nitric oxide composed of a single selected state of principal quantum number, n0. Penning ionization, followed by an avalanche of electron-Rydberg collisions, forms a plasma of NO+ ions and weakly bound electrons, in which a residual population of n0 Rydberg molecules evolves to a state of high orbital angular momentum, ℓ. Predissociation depletes the plasma of low-ℓ molecules. Relaxation ceases and n0ℓ(2) molecules with ℓ ≥ 4 persist for very long times. At short times, varying excitation spectra of mm-wave Rydberg-Rydberg transitions mark the rate of electron-collisional ℓ-mixing. Deep depletion resonances that persist for long times signal energy redistribution in the basis of central-field Rydberg states. The widths and asymmetries of Fano line shapes witness the degree to which coupling in the arrested bath (i) broadens the allowed transition and (ii) mixes the local network of levels in the ensemble.

Template-oriented genetic algorithm feature selection of analyte wavelets in the Raman spectrum of a complex mixture.

We introduce a fast computational method for feature selection that facilitates the accurate spectral analysis of a chemical species of interest in the presence of overlapping uncorrelated variance. Using a genetic algorithm in a data-driven approach, our method assigns predictors according to a template determined to minimize prediction variance in a calibration space. This template-oriented genetic algorithm (TOGA) efficiently establishes features of greatest significance and determines their optimal combination. We demonstrate the efficacy of TOGA using an elementary model system in which we seek to quantify a target monosaccharide in mixtures containing other sugars added in random amounts. The results establish TOGA as an effective and reliable technique for isolating signature spectra of targeted substances in complex mixtures.

Dissociation and the development of spatial correlation in a molecular ultracold plasma.

Penning ionization initiates the evolution of a dense molecular Rydberg gas to plasma. This process selects for pairs of excited molecules separated by a distance of two Rydberg orbital diameters or less. The deactivated Penning partners predissociate, depleting the leading edge of the distribution of nearest-neighbor distances. For certain density and orbital radii, this sequence of events can form a plasma in which large distances separate a disproportionate fraction of the ions. Experimental results and model calculations suggest that the reduced potential energy of this Penning lattice significantly affects the development of strong coupling in an ultracold plasma.

Evolution from a molecular Rydberg gas to an ultracold plasma in a seeded supersonic expansion of NO.

We report the spontaneous formation of a plasma from a gas of cold Rydberg molecules. Double-resonant laser excitation promotes nitric oxide, cooled to 1 K in a seeded supersonic molecular beam, to single Rydberg states extending as deep as 80 cm;{-1} below the lowest ionization threshold. The density of excited molecules in the illuminated volume approaches 1x10;{13} cm;{-3}. This population evolves to produce free electrons and a durable cold plasma of electrons and intact NO+ ions.

Mode dependent vibrational autoionization of Rydberg states of NO2. II. Comparing the symmetric stretching and bending vibrations.

Triple-resonance excitation and high-resolution photoelectron spectroscopy are combined to characterize the mode selectivity of vibrational autoionization of the high Rydberg states of NO2. Photoelectron spectra and vibrational branching fractions are reported for autoionizing Rydberg states converging to the NO2+ X 1Sigmag +(110) state, that is, with one quantum in the symmetric stretch, nu1, and one quantum in the bending vibration, nu2. These results indicate that autoionization proceeds most efficiently through the loss of one quantum from the symmetric stretch rather than from the bending vibration. The implications of this result are discussed in terms of the autoionization mechanism.

Coupling of electron orbital motion with rotation in the high Rydberg states of BH.

We have applied optical-optical-optical triple resonance spectroscopy to resolve a system of high Rydberg states in BH that serves quantitatively to characterize a fundamental example of electron-orbital-cation core rotational coupling. The third-color ionization-detected absorption spectrum originating from the photoselected 3s B1Sigma+ Rydberg state with vibrational and total angular momentum quantum numbers, v'=1 and N'=0 consists entirely of vibrationally autoionizing resonances for which final N=1 that converge in series to the BH+v+=1 rotational limits, N+=0, 1, and 2. For series with l=1 converging to N+=0 and 2, Rydberg orbital and rotational angular momenta couple to systematically perturb level energies and distribute lifetime in a well-isolated two-channel rotronic interaction that spans hundreds of wave numbers.

A new instrument adapted to in situ Raman analysis of objects of art.

The analysis of precious artefacts and antiquities demands care in order to minimise the risk of accidental damage during measurement. Mobile fibre-optic-based Raman instruments offer a means to avoid destructive sampling and eliminate the need to transport artefacts for spectrochemical analysis. In this work we present a new mobile instrument developed and optimised for the in situ Raman investigation of objects of art and antiquities. The instrument is controlled by a portable computer. Selected mounts cover many types of artefacts. Newly written software routines organise spectra together with measurement parameters and facilitate calibration of the instrument. The present paper describes this new Raman instrument and discusses some challenges in the transition from a laboratory environment to in situ investigations in museums.

Acute respiratory infection in patients with cystic fibrosis with mild pulmonary impairment: comparison of two physiotherapy regimens.

Chest physiotherapy is an essential part of the management of cystic fibrosis, yet comparatively few studies have investigated the commonly used forms of chest physiotherapy during acute respiratory exacerbations. Fifteen subjects with cystic fibrosis and predominantly mild pulmonary impairment completed a randomised cross-over trial with 24 hours between treatments. The active cycle of breathing techniques (ACBT) assisted by a physiotherapist was compared with the ACBT performed independently by the patient. Measurement outcomes included pulmonary function tests, indirect calorimetry and oximetry parameters. Energy expenditure was not significantly different between the two treatment regimens, though significant improvements in pulmonary function were apparent 24 hours following the therapist-assisted ACBT. In this group of subjects, neither form of treatment proved superior in terms of energy consumption, but a reduction in airways obstruction was observed as a carry-over effect following the therapist-assisted ACBT.

Ultrafast time-resolved soft x-ray photoelectron spectroscopy of dissociating Br2.

The dissociation of excited state Br2 is probed with the novel technique of ultrafast soft x-ray photoelectron spectroscopy. Excited Br2 molecules are prepared in the dissociative (1)Pi(u) state with 80 fs, 400 nm pulses, and a series of photoelectron spectra are obtained during dissociation with pulses of soft x-ray light (47 nm, 26.4 eV, 250 fs). The formation of Br atoms is readily detected and the data support an extremely fast dissociation time for Br2 on the order of 40 fs. Amplitudes of the pump-probe features reveal that the ionization cross section of atomic Br at 47 nm is approximately 40 times larger than that of Br2.

A region of the rat N-methyl-D-aspartate receptor 2A subunit that is sufficient for potentiation by phorbol esters.

N-methyl-D-aspartate (NMDA) receptors are modulated by protein kinase C (PKC) in vivo and in heterologous expression systems. In heterologous expression systems, PKC-mediated modulation is subunit specific with NR2A-containing receptors being potentiated by phorbol 12-myristate 13-acetate (PMA), while NR2C-containing receptors are inhibited or unaffected. In the present study we have produced chimeric receptors containing NR2A and NR2C to define the components of NR2A which are sufficient for potentiation by PMA. Amino acids 1105-1400 of NR2A placed onto the C-terminus of NR2C at amino acid 1102 was the minimum region sufficient for producing a PMA-stimulated receptor. This suggests that this region contains structural determinants for PKC-mediated potentiation of NR2A receptors.

Cytochrome P450 induction in rat hepatocytes assessed by quantitative real-time reverse-transcription polymerase chain reaction and the RNA invasive cleavage assay.

The acceleration of drug discovery due to combinatorial chemistry and high-throughput screening methods has increased the numbers of candidate pharmaceuticals entering the drug development phase, and the capability to accurately predict whether drug candidates will induce various members of the drug-metabolizing cytochrome P450 (CYP) enzyme superfamily is currently of great interest to the pharmaceutical industry. In the present study, we describe the rapid and reliable analysis of CYP induction in a readily obtained model system (cultured rat hepatocytes) using both real-time quantitative reverse transcription-polymerase chain reaction (real-time RT-PCR) and the RNA invasive cleavage assay. The levels of members in the three primary inducible rat CYP subfamilies (CYP1A1, CYP2B1/2, and CYP3A1) were analyzed in untreated and induced (beta-naphthoflavone, phenobarbital, and hydrocortisone) hepatocyte cultures under various media conditions to screen for optimal CYP induction profiles. The fold inductions measured by real-time RT-PCR and the RNA invasive cleavage assay were also compared with enzyme activity measurements in parallel cultures using liquid chromatography/double mass spectrometry-based assays, and the sensitivity and the specificity of the two RNA analysis methods were compared. Using these techniques, various culture conditions were examined for optimizing induction of the three CYP subfamily members. Both real-time RT-PCR and the RNA invasive cleavage assay prove to be effective methods for determining the effects of drugs on specific CYPs in primary rat hepatocytes.

Protection against glutamate toxicity through inhibition of the p44/42 mitogen-activated protein kinase pathway in neuronally differentiated P19 cells.

Excessive levels of the neurotransmitter glutamate trigger excitotoxic processes in neurons that lead to cell death. N-Methyl-D-aspartate (NMDA) receptor over-activation is a key excitotoxic stimulus that leads to increases in intracellular calcium and activation of downstream signaling pathways, including the p44/42 mitogen-activated protein (MAP) kinase pathway. In the present study, we have demonstrated that 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a potent and selective inhibitor of the p44/42 MAP kinase signaling pathway, prevents glutamate-induced death in neuronally differentiated P19 cells. In addition, we show that differentiated, but not undifferentiated, P19 cells expressed zeta1, epsilon1, and epsilon2 subunits of the NMDA receptor. Differentiated P19 cells exhibited specific NMDA receptor binding and intracellular calcium responses to glutamate that were blocked by the selective NMDA receptor antagonist [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not U0126. Glutamate treatment of differentiated P19 cells triggered a rapid and sustained induction in p42 MAP kinase phosphorylation that was blocked by U0126. Pretreatment of differentiated P19 cells with U0126, but not other classes of protein kinase inhibitors, protected against glutamate-induced cell death. Post-treatment with U0126, even as late as 6 hr after glutamate application, also protected against glutamate toxicity. These results suggest that the p44/42 MAP kinase pathway may be a critical downstream signaling pathway in glutamate receptor-activated toxicity.

Nile Red binding to HepG2 cells: an improved assay for in vitro studies of hepatosteatosis.

Nile Red is a fluorescent dye used extensively to study fat accumulation in many types of cells; unfortunately protocols that work well for most cells are not effective for studying drug-induced lipid accumulation in cultured liver cells and hepatocyte-derived cell lines. Using human hepatoma (HepG2) cells, we have developed a simple Nile Red binding assay as a screen for steatosis-inducing compounds. Increases in Nile Red binding in response to known hepatotoxic compounds were observed after incubating treated cells with 1 microM Nile Red for several hours, washing away free Nile Red, and then allowing redistribution, and/or clearance of the lipid-indicator dye. Several compounds known to cause hepatic fat accumulation in vivo were examined and most robustly increased Nile Red binding in HepG2 cells. These include estrogen and other steroids, ethionine, cyclosporin A, and valproic acid. Required concentrations for increased Nile Red binding were generally three-fold or more lower than the cytotoxic concentration determined by a resazurin reduction assay in the same cells. Qualitatively similar Nile Red binding results were obtained when primary canine or rat hepatocytes were used. Morphological differences in Nile Red staining were observed by confocal fluorescence microscopy in HepG2 cells after treatment with different compounds and likely reflect distinct toxicological mechanisms.

N-Methyl-D-aspartate receptor mediated toxicity in nonneuronal cell lines: characterization using fluorescent measures of cell viability and reactive oxygen species production.

Cells transfected with specific N-methyl-D-aspartate (NMDA) receptor subtypes undergo cell death that mimics glutamate-induced excitotoxicity pharmacologically. We have further characterized the mechanisms of cell death resulting from NMDA receptor activation in such cells through development of cell counting methods based on co-transfection with green fluorescent protein. When co-transfected with NMDA receptors, GFP expression was limited to live cells as indicated by the observation that GFP was only detected in cells which were positive for markers of live cells, and was found in no cells which were trypan blue or propidium iodide positive. Using co-transfection with green fluorescent protein and cell counting of viable cells with a fluorescence activated cells sorter, we confirmed the subunit-specific profile of NMDA receptor-mediated cell death in cells transfected with NMDA receptors. Toxicity was greatest in the NR1A/2A receptor, less in the NR1A/2B receptor, and least in NR1A/2C receptors. Cell death also differed pharmacologically between subunit combinations. Cell death in cells transfected with NR 1A/2A was blocked by amino-phosphonovaleric acid at lower concentrations than in cells transfected with NR 1A/2B. In cells transfected with the NR1A/2A or NR1A/2B combinations but not NR1A/2C, cell death was also associated with production of reactive oxygen species. In addition, removal of the final 400 amino acids of the C-terminal region of NR2A decreased cell death. The use of GFP based cell counting provides a sensitive mechanism for assessing the mechanism of excitotoxicity in transfected cell models.

Actions of butyrophenones and other antipsychotic agents at NMDA receptors: relationship with clinical effects and structural considerations.

Haloperidol inhibits NMDA receptors with higher affinity for NMDA receptors composed of NR1/2B compared with NR1/2A. To assess whether the clinical effects of haloperidol and other antipsychotic agents are mediated through this site on NMDA receptors and to examine structure activity relationships at this site, we examined the ability of a variety of drugs with neuroleptic actions to inhibit NMDA receptor function. Many antipsychotic agents inhibit 125I-MK 801 binding to the NMDA receptor with IC50 values in the micromolar range. The rank order of potency for inhibition of binding to adult rat forebrain was trifluperidol (TFP) > clozapine = fluphenazine = reduced haloperidol = spiperone = trifluoperazine = butaclamol >> pimozide = risperidone = sulpiride. These findings match the molecular biological specificity of the agents, with trifluperidol having a marked preference for NR1/2B (epsilon2) receptors. Mutations at epsilon2E201, which alter the effects of haloperidol, also decrease the affinity of TFP but not other modulators, showing that the effect of TFP but not other modulators is mediated by this residue of the NMDA receptor. The present results demonstrate that while TFP acts on NMDA receptors in a manner similar to haloperidol, other antipsychotic agents do not share the specific pharmacological properties of this action, suggesting that their clinical mechanism is not mediated by this receptor.

Opposing contributions of NR1 and NR2 to protein kinase C modulation of NMDA receptors.

N-Methyl-D-aspartate (NMDA) receptors mediate increases in intracellular calcium that can be modulated by protein kinase C (PKC). As PKC modulation of NMDA receptors in neurons is complex, we studied the effects of PKC activation on recombinant NMDA receptor-mediated calcium rises in a nonneuronal mammalian cell line, human embryonic kidney 293 (HEK-293). Phorbol 12-myristate 13-acetate (PMA) pretreatment of HEK-293 cells enhanced or suppressed NMDA receptor-mediated calcium rises based on the NMDA receptor subunit composition. NR2A or NR2B, in combination with NR1(011), conveyed enhancement whereas NR2C and NR2D conveyed suppression. The PKC inhibitor bisindolylmaleimide blocked each of these effects. The region on NR2A that conveyed enhancement localized to a discrete segment of the C terminus distal to the portion of NR2C that is homologous to NR2A. Calcium-45 accumulation, but not intracellular calcium store depletion, matched PMA effects on NMDA receptor-mediated calcium changes, suggesting that these effects were not due to effects on intracellular calcium stores. The suppression of intracellular calcium transients seen with NR2C was eliminated when combined with NR1 splice variants lacking C-terminal cassette 1. Thus, the intracellular calcium effects of PMA were distinguishable based on both the NR1 splice variant and the NR2 subunit type that were expressed. Such differential effects resemble the diversity of PKC effects on NMDA receptors in neurons.

The NR2B-specific interactions of polyamines and protons with the N-methyl-D-aspartate receptor.

Many compounds exhibit NR2B-specific modulation of the N-methyl-D-aspartate receptor, although their mechanism(s) of action are largely unknown. Using chimeric NR2A/NR2B subunits, we have located a region of NR2B (amino acids 138-238) which regulated glycine-independent polyamine stimulation. Mutation of glutamate 201 in this region affected stimulation by polyamines in the order E201D < E201A < E201N < E201R. The relief of proton inhibition of the N-methyl-D-aspartate-induced currents mediated by these mutant receptors correlated with the reduction in glycine-independent polyamine stimulation. Electrophysiological evidence with a triple mutant of NR2A further supports the hypothesis that polyamine stimulation may be linked to the relief of tonic inhibition by protons and demonstrates the crucial role of amino acids 200 and 201 in polyamine stimulation. Polyamines and protons, therefore, share common NR2B determinants.

N-methyl-D-aspartate receptors expressed in a nonneuronal cell line mediate subunit-specific increases in free intracellular calcium.

N-methyl-D-aspartate (NMDA) receptors can mediate cell death in neurons and in non-neuronal cells that express recombinant NMDA receptors. In neurons, increases in intracellular calcium correlate with NMDA receptor-mediated death, supporting a key role for loss of cellular calcium homeostasis in excitotoxic cell death. In the present study, free intracellular calcium concentrations were examined in response to activation of recombinant NMDA receptors expressed in human embryonic kidney 293 cells. Intracellular calcium was measured in transfected cell populations by cotransfection with the calcium-sensitive, bioluminescent protein aequorin and by single cell imaging with the fluorescent calcium indicator fluo-3. Agonist application to NR1/2A or NR1/2B-transfected cells elicited robust rises in intracellular calcium. NR1/2A responses were inhibited by the noncompetitive antagonists MK-801 and dextromethorphan and were dependent on extracellular calcium but not on intracellular calcium stores. In contrast, no detectable intracellular calcium responses were observed in NR1/2C-transfected cells. These findings indicate that NMDA receptors in the absence of other neuron-specific factors can mediate increases in intracellular calcium with subunit specificity and extracellular calcium dependence.

Biomedical considerations and clinical patterns related to disorders of the glenoid labrum in the predominantly stable glenohumeral joint.

SUMMARY. The roles of the glenoid labrum and long head of biceps are reviewed together with their significance in stability of the glenohumeral joint. Clinical presentations related to disorders of the glenoid labrum and long head of biceps not associated with frank instability but commonly responsible for dysfunction in the athletic shoulder, are reviewed from the perspective of pertinent anatomical, biomechanical and kinematic knowledge. Copyright 1996 Harcourt Publishers Ltd.

Naltrexone reverses ethanol-induced dopamine release in the nucleus accumbens in awake, freely moving rats.

The effect of the opioid receptor antagonist, naltrexone, on ethanol-induced changes in extracellular dopamine and serotonin in the nucleus accumbens was investigated using in vivo microdialysis in awake, freely moving rats. Locally applied ethanol (5% infused transprobe) resulted in substantial increases in dopamine in dialysate. Administration of naltrexone (cumulative dosing with 0.25-1.0 mg/kg i.p.) during ethanol administration dose-dependently reversed ethanol-induced increases in extracellular dopamine and its metabolite homovanillic acid but not serotonin. These data demonstrate an essential role for the endogenous opioid system in stimulation of dopamine release by ethanol in a brain area associated with reward and support the opioid system as a prime target for pharmacological modulation of the rewarding effects and consumption of ethanol.