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The G protein-coupled delta opioid receptor (DOR) plays a critical role in pain control. Emerging evidence shows that DOR also plays a role in neuronal differentiation and survival. Nerve growth factor (NGF) is known to be critical for the development and maintenance of the central and peripheral nervous systems. Our previous studies have shown that sustained activation of NGF/PI3K/Akt/NF-kappaB signaling is essential for NGF-induced dor gene expression during neuronal differentiation and that the epigenetic modifications at histone 3 lysine 9 temporally correlate with the dor gene transcription. In this study, we cloned the rat dor gene promoter and identified an NGF-responsive region similar to that from the mouse dor gene promoter. We further identified p300, a known NF-kappaB binding partner with intrinsic histone acetyltransferase activity, to be dynamically associated with the dor gene. We also found that assembling of RNA polymerase II (Pol II) at the promoter took place before NGF stimulation, indicating that p300 could only interact with preassembled Pol II at the promoter after NGF stimulation. Taken together, these results implicate that preassembly of the Pol II preinitiation complex, sustained activation of PI3K/Akt/NF-kappaB signaling, and dynamic p300 association at the promoters sequentially is one of the mechanisms of induction of the late phase genes during NGF-induced neuronal differentiation.

Reactions of [cyclopentadienyl)(1,3-dichlorobenzene)M] + PF 6 - complexes (M= Fe, Ru) with phenoxide nucleophiles were found to proceed with excellent selectivity under mild conditions to give products of monosubstitution. Using aminophenoxides, a preference for O-arylation was observed, while amino alcohols such as prolinol react selectively on nitrogen. Methodology for sequential selective displacement of both chlorides by different nucleophiles is reported

A systematic method is described for the optimization of a mobile phase for the simultaneous determination of 24 neurochemicals consisting of catecholamine, serotonin, their precursors and metabolites and related materials. This mobile phase contained sodium acetate (0.04 M), citric acid (0.01 M), sodium chloride (0.0126 M), sodium octyl sulfate (91 mg/l), tetrasodium EDTA (50 mg/l) and 10% (v/v) methanol. When this optimum mobile phase was applied to the analysis of brain tissues of the Swiss male mouse, twelve neurochemicals were quantified in the free state: tyrosine, L-beta-3,4-dihydroxyphenylanine, dopamine, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylacetic acid, norepinephrine, 3-methoxy-4-hydroxyphenylglycol, DL-3,4-dihydroxymandelic acid, DL-4-hydroxy-3-methoxymandelic acid, serotonin, L-tryptophan, 5-hydroxyindole-3-acetic acid and DL-synephrine and normetanephrine, appearing as a fused peak. This fused peak was present on the chromatogram tracings of all the mouse brain tissues. The separable neurochemicals not found by this procedure in the Swiss male mouse tissues were DL-3,4-dihydroxyphenylglycol,5-hydroxytryptophan, epinephrine, DL-octopamine, metanephrine, deoxyepinephrine, homovanillyl alcohol, N-acetylserotonin, tyramine and 3-methyltyramine.

International audience; no abstract

OBJECTIVE. The goal of this study was to evaluate the role of factors that may determine the efficacy of treatment with delayed head cooling and mild systemic hypothermia for neonatal encephalopathy.METHODS. A total of 218 term infants with moderate to severe neonatal encephalopathy plus abnormal amplitude-integrated electroencephalographic recordings, assigned randomly to head cooling for 72 hours, starting within 6 hours after birth (with the rectal temperature maintained at 34.5 ± 0.5°C), or conventional care, were studied. Death or severe disability at 18 months of age was assessed in a multicenter, randomized, controlled study (the CoolCap trial).RESULTS. Treatment, lower encephalopathy grade, lower birth weight, greater amplitude-integrated electroencephalographic amplitude, absence of seizures, and higher Apgar score, but not gender or gestational age, were associated significantly with better outcomes. In a multivariate analysis, each of the individually predictive factors except for Apgar score remained predictive. There was a significant interaction between treatment and birth weight, categorized as ≥25th or CONCLUSIONS. Outcomes after hypothermic treatment were strongly influenced by the severity of neonatal encephalopathy. The protective effect of hypothermia was greater in larger infants.

Adaptive optical-coherence-domain reflectometry (OCDR) is performed by use of an adaptive interferometer and homodyne detection. The adaptive element of the interferometer is a photorefractive quantum-well device in a two-wave mixing geometry. The mixing self-adaptively maintains constant relative phase between the signal and reference waves and dynamically compensates gross movements of the sample or optical components as well as image speckle. The application described here is used for laser ranging into and through turbid media. Adaptive OCDR is a bridge between conventional optical coherence tomography and adaptive holographic optical coherence imaging. The insertion loss for the adaptive performance is -15 dB, but adaptive OCDR has potential applications for coherence tomography under conditions of large target motion and low background. We also demonstrate its potential application for optoacoustics and laser-based ultrasound detection. (C) 2004 Optical Society of America.

Opioid peptides exert their regulatory effects on both central and peripheral nervous systems via multiple opioid receptors that are linked to seemingly identical sets of guanine nucleotide-binding regulatory proteins (G proteins). In contrast to the mu-opioid receptor, the delta-opioid receptor can efficiently stimulate phospholipase C via G16. We used a series of mu/delta-opioid receptor chimeras to examine the involvement of intracellular receptor domains in the recognition of G16. After ascertaining that the chimeras can bind opioid ligands with high affinity and elicit inhibition of adenylyl cyclase, COS-7 cells were cotransfected with cDNAs encoding Galpha16 and a mu/delta-opioid receptor chimera and assayed for [D-Ala2,D-Leu5]enkephalin-induced stimulation of phospholipase C. Our results indicate that (i) the carboxy terminal tail of the delta-opioid receptor is necessary but insufficient for conferring coupling to Galpha16, (ii) the third inner loop together with the carboxy terminal tail of the delta-opioid receptor can provide sufficient contact domains for Galpha16, and (iii) the first inner loop of the delta-opioid receptor, in particular Leu80, as well as the fifth transmembrane domain and/or the third extracellular loop may also contribute in defining the fidelity of interaction between the delta-opioid receptor and Galpha16. These results indicate that efficient coupling of the delta-opioid receptor to Galpha16 requires the participation of most of the intracellular regions, including the first intracellular loop.

Morphine is used to treat pain in several medical conditions including cancer. Here we show that morphine, in a concentration typical of that observed in patients' blood, stimulates human microvascular endothelial cell proliferation and angiogenesis in vitro and in vivo. It does so by activating mitogen-activated protein kinase/extracellular signal-regulated kinase phosphorylation via Gi/Go-coupled G protein receptors and nitric oxide in these microvascular endothelial cells. Other contributing effects of morphine include activation of the survival signal PKB/Akt, inhibition of apoptosis, and promotion of cell cycle progression by increasing cyclin D1. Consistent with these effects, morphine in clinically relevant doses promotes tumor neovascularization in a human breast tumor xenograft model in mice leading to increased tumor progression. These results indicate that clinical use of morphine could potentially be harmful in patients with angiogenesis-dependent cancers.

We report on laboratory experiments dealing with the interaction of clay-sized sediments with oil slicks on Class 1–2 rivers. We find that given enough time, clay-sized sediments can sink nearly all surface oil. In addition, the greater the viscosity of the oil, the less the degree of interaction. We present plots for estimating the rate at which sediments attach to and bring down oil slicks – at the maximum rate observed, about 15% of a slick of intermediate fuel oil (IFO) could be sunk in 1 km of rapids with 0.5 m waves; half of a slick could be sunk in about 4500 m of river length.

An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.

Electro-optical properties of thin film are of great interest owing to the perpetual demand for miniaturization and higher speed devices for communication, electronic, and biomedical applications. The thickness of polymer films developed for these applications has decreased dramatically making interfacial effects significant. It is well documented that, in submicron thickness range, both film/substrate & film/air interface are critical. In this study, we probe the dynamics of electro-optical polymer thin films in the sub-micron thickness regime to understand interfacial effects. The polymer chain dynamics of Polypropylene oxide (PPO) under electric field are investigated in this study. The effects of electric field strength, frequency, and polymer molecular weight on the polymer chain dynamics under electric field are studied. Experimental results show that PPO exhibits both piezoelectric and electrorestrictive effects at significantly high frequencies (101kHz range). Conventional organic materials are responsive only at frequencies in

To date, the visualization of delta-opioid receptor (DOR) internalization has been largely focused on the events of short-term agonist treatment in transfected non-neuronal cells. In this study, we followed DOR trafficking upon prolonged agonist exposure in the neuronally derived neuro2a cells, stably transfected with the fusion DOR (HA-DOR) cDNA. Internalization of surface DOR was clearly visualized in 5 min of exposure to agonist (100 nM DADLE), and the cell surface DOR remained low throughout the entire 24 h agonist exposure. Significant intracellular accumulation was visible at 20 min exposure, and increased to a maximum at 4 h, after which intracellular DOR staining gradually diminished. DOR intracellular staining was enhanced in the presence of agonist and chloroquine, a lysosomotropic agent, suggesting that internalized receptors were targeted to lysosomes and degraded upon prolonged treatment. Time-dependent colocalization of DOR with transferrin and LAMP-2 following short-term and prolonged agonist exposure further confirmed that receptor was distributed to early endosomes (sequestration) and subjected to lysosomes for degradation (down-regulation), respectively.

Continuous cell lines (used in the manufacture of monoclonal antibodies and other therapeutic proteins) may harbour retroviral particles; nevertheless, they are deemed acceptable if the production system is validated for clearance of these contaminants. As part of a campaign to evaluate and validate appropriate virus removal methodologies for monoclonal immunoglobulin G (IgG) products, studies were undertaken with a hydrophilic polyvinylidene fluoride (PVDF) microporous membrane—the Ultipor® VF grade DV50 virus removal membrane. Testing conducted with murine leukaemia virus, a specific model virus used in viral validation studies to document removal of retroviruses, demonstrated a greater that 7 log clearance by the Ulipor® VR grade DV50 membrane. Filterability testing established the feasability of positioning the filter at either of two different stages during manufacture. The results presented in this study demonstrate that membrane filtration through the Ultipor VF grade DV50 virus removal filter can constitute part of the purification regimen to effect removal of endogenous retrovirus-like contaminants from monoclonal lgG products.

Expression of the structural proteins of human immunodeficiency virus type 1 (HIV-1) requires the direct interaction of multiple copies of the viral protein Rev with its target RNA, the Rev response element (RRE). RRE is a complex 351-nt RNA that is highly structured and located within the viral env gene. During initial Rev-RRE recognition, Rev binds with high affinity to a bubble structure located within the RRE RNA stem-loop II. We have used a site-specific photocrosslinking method based on 6-thioguanosine (6-thioG) photochemistry to probe the conformation of the high-affinity binding site of RRE RNA and its interactions with Rev protein under physiological conditions. A minimal duplex RNA containing the bubble region of RRE and 12 flanking base pairs was synthesized chemically. Two different RRE constructs with a single photoactive nucleoside (6-thio-dG or 6-thioG) at position 47 or 48 were synthesized. Upon UV irradiation, 6-thioG at both positions formed interstrand covalent crosslinks in RRE RNA. Mapping of crosslink sites by RNA sequencing revealed that 6-thioG at position 47 or 48 crosslinked to A73. In the presence of Rev, both RNA-RNA and RNA-protein crosslinks were observed, however, the RNA-RNA crosslink site was unchanged. Our results provide direct evidence that, during RNA-protein recognition, Rev is in close proximity to O6 of G47 and G48 in the major groove of RRE RNA. Our results also show that the bubble region of RRE RNA has a biologically relevant structure where G47 and G48 are in close proximity to A73 and this RNA structure is not changed significantly upon Rev binding. We propose that Rev protein recognizes and binds to specific structural elements of RRE RNA containing non-Watson-Crick base pairs and such structures could be a determinant for recognition by other RNA-binding proteins. Our site-specific crosslinking methods provide a general approach to capture dynamic states of biologically relevant RNA structures that are otherwise missed by NMR and X-ray crystallographic studies.

Bezoars are food or fiber concretions of the alimentary tract, which infrequently cause intestinal obstruction. The authors describe a case of a 7-year-old child with pica in whom a synthetic fiber bezoar obstruction of the small intestine developed at the site of a previously stapled intestinal anastomosis.

The chromatographic purification of vWF (von Willebrand Factor) from human plasma represents a challenge because it consists of multimers with molecular weights ranging from 0.5 to 10 million Daltons. Phage peptide library screening yielded a lead peptide (RLRSFY) that interacts with vWF. Conservative substitutions of terminal residues of the lead peptide led to a second peptide, RVRSFY, which was more efficient in the affinity chromatographic purification of vWF from protein mixtures. Adsorption isotherm measurements indicated multiple interactions between vWF and the immobilized peptide RVRSFY. Increases in peptide density on the chromatographic supports resulted in stronger association constants and higher maximum protein binding capacities. When the peptide density was lower than 32 mg/mL, there was no measurable interaction between vWF and immobilized peptide RVRSFY in HEPES buffer containing 0.5 M NaCl at pH 7. An increase in peptide density from 32 to 60 mg/mL increased the association constants from 0.9 x 10(6) to 2 x 10(6) (M-1). Divalent salts (calcium and magnesium chloride) were used to elute the retained vWF with 82.5% of the activity recovered. The interactions between vWF and the immobilized peptide RVRSFY are dominated by ionic attractions and also involve hydrophobic interactions at close contact. Finally, the purification of vWF from crude material PEG filtrate of a cryoprecipitate of human plasma is demonstrated using affinity chromatography with immobilized N-acetyl-RVRSFYK.