Direct synthesis of anilines and nitrosobenzenes from phenols.

A one-pot synthesis of anilines and nitrosobenzenes from phenols has been developed using an ipso-oxidative aromatic substitution ((i)SOAr) process. The products are obtained in good yields under mild and metal-free conditions. The leaving group effect on reactions that proceed through mixed quionone monoketals has also been investigated and a predictive model has been established.

The synthesis and characterization of a nuclear membrane affinity chromatography column for the study of human breast cancer resistant protein (BCRP) using nuclear membranes obtained from the LN-229 cells.

BCRP expression has been reported in glioblastoma cell lines and clinical specimens and has been shown to be expressed both in purified nuclei and in the soluble cytoplasmic fraction. To date, the nuclear BCRP has not been characterized. Our laboratory has previously developed an online chromatographic approach for the study of binding interactions between ligands and protein, cellular membrane affinity chromatography. To this end, we have immobilized the nuclear membrane fragments onto an immobilized artificial membrane stationary phase (IAM), resulting in the nuclear membrane affinity chromatography (NMAC) column. Initial characterization was carried out on the radio flow detector, as well as the LC-MSD, using frontal displacement chromatography techniques. Etoposide, a substrate for BCRP, was initially tested, to determine the functional immobilization of BCRP. Frontal displacement experiments with multiple concentrations of etoposide were run and the binding affinity was determined to be 4.54 µM, which is in close agreement with literature. The BCRP was fully characterized on the NMAC column and this demonstrates that for the first time the nuclear membranes have been successfully immobilized.

Development and characterization of immobilized cannabinoid receptor (CB1/CB2) open tubular column for on-line screening.

Cannabinoid receptors, CB1 and CB2, are therapeutic targets in the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. We have developed an on-line screening method for CB1 and CB2 ligands, where cellular membrane fragments of a chronic myelogenous leukemia cell line, KU-812, were immobilized onto the surface of an open tubular (OT) capillary to create a CB1/CB2-OT column. The binding activities of the immobilized CB1/CB2 receptors were established using frontal affinity chromatographic techniques. This is the first report that confirms the presence of functional CB1 and CB2 receptors on KU-812 cells. The data from this study confirm that the CB1/CB2-OT column can be used to determine the binding affinities (K(i) values) for a single compound and to screen individual compounds or a mixture of multiple compounds. The CB1/CB2-OT column was also used to screen a botanical matrix, Zanthoxylum clava-herculis, where preliminary results suggest the presence of a high-affinity phytocannabinoid.

Synthesis and characterization of liquid chromatographic columns containing the immobilized ligand binding domain of the estrogen related receptor alpha and estrogen related receptor gamma.

The ligand binding domains of the estrogen related receptors, ERRalpha and ERRgamma were covalently immobilized onto the surface of an aminopropyl silica liquid chromatography stationary phase to create the ERRalpha-silica and ERRgamma-silica columns and onto the surface of open tubular capillaries to create the ERRalpha-OT and ERRgamma-OT columns. The ERR-silica and ERR-OT columns were characterized using frontal chromatographic techniques with diethylstibesterol and the binding affinities, K(d) values, to the immobilized receptors were consistent with the values obtained by a radioligand binding assay. The ERRgamma-silica column was also characterized using non-linear chromatographic techniques using a series of tamoxifen derivatives. The relative K(d) values obtained for the derivatives were consistent with the relative ability of the compounds to inhibit the cellular proliferation of the human-derived T98G glioma cell line, expressed as IC(50) values. The results indicate that the columns containing immobilized ERRalpha and ERRgamma can be created and used to characterize the binding of compounds to the immobilized receptors and that the relative retention of compounds on these columns reflects the magnitude of their inhibitory activity.

Adenosine A2B receptors are highly expressed on murine type II alveolar epithelial cells.

The adenosine A(2B) receptor (A(2B)R) has a wide tissue distribution that includes fibroblasts and endothelial and epithelial cells. The recent generation of an A(2B)R(-/-) mouse constructed with a beta-galactosidase (beta-gal) reporter gene under control of the endogenous promoter has provided a valuable tool to quantify A(2B)R promoter activity (29). To determine the sites of expression of the A(2B) receptor in the mouse lung, histological and flow cytometric analysis of beta-gal reporter gene expression in various lung cell populations was performed. The major site of A(2B)R promoter activity was found to be the type II alveolar epithelial cells (AECs), identified by coexpression of prosurfactant protein C, with relatively less expression in alveolar macrophages, bronchial epithelial cells, and cells of the vasculature. Highly purified type II AECs were prepared by fluorescence-activated sorting of enhanced green fluorescent protein (eGFP)-positive cells from transgenic mice expressing eGFP under control of the surfactant protein C promoter (21). The type II cells expressed 89-fold higher A(2B)R mRNA than pulmonary leukocytes, and the A(2B)R was shown to be functional, as treatment of purified type II AECs with the nonspecific adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) induced an increase in intracellular cAMP greater that the beta-adrenergic agonist isoproterenol that was inhibited completely following treatment by ATL-802, a novel, highly potent (K(i) = 8.6 nM), and selective (>900 fold over other adenosine receptor subtypes) antagonist of the mouse A(2B)R.

Characterization of a multiple ligand-gated ion channel cellular membrane affinity chromatography column and identification of endogenously expressed receptors in astrocytoma cell lines.

Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [(3)H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) and heteromeric nicotinic acetylcholine receptors (alpha(x)beta(y) nAChRs), which was confirmed by the addition of subtype-specific inhibitors, alpha-bungarotoxin (alpha7 nAChR) and kappa-bungarotoxin (alpha(x)beta(y) nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), gamma-aminobutyric acid (GABA(A)) and N-methyl-D-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABA(A) receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors.

Gating kinetics of the alpha1I T-type calcium channel.

The alpha1I T-type calcium channel inactivates almost 10-fold more slowly than the other family members (alpha1G and alpha1H) or most native T-channels. We have examined the underlying mechanisms using whole-cell recordings from rat alpha1I stably expressed in HEK293 cells. We found several kinetic differences between alpha1G and alpha1I, including some properties that at first appear qualitatively different. Notably, alpha1I tail currents require two or even three exponentials, whereas alpha1G tails were well described by a single exponential over a wide voltage range. Also, closed-state inactivation is more significant for alpha1I, even for relatively strong depolarizations. Despite these differences, gating of alpha1I can be described by the same kinetic scheme used for alpha1G, where voltage sensor movement is allosterically coupled to inactivation. Nearly all of the rate constants in the model are 5-12-fold slower for alpha1I, but the microscopic rate for channel closing is fourfold faster. This suggests that T-channels share a common gating mechanism, but with considerable quantitative variability.

The treatment of eruptive syringomas in an African American patient with a combination of trichloroacetic acid and CO2 laser destruction.

BACKGROUND: Eruptive syringomas are uncommon benign adnexal neoplasms. They are numerous and disseminated and often have a predilection for the neck, face, chest, and axillary fossae. Because they are persistent, usually numerous, and often on exposed sites, the lesions may be disfiguring and often pose significant cosmetic concerns for patients. Many treatment modalities such as dermabrasion, electrodesiccation with curettage, and scissors excision have been tried with some success, but more recently lasers have provided good to excellent results. OBJECTIVE: To describe an approach to the treatment of eruptive syringomas in an African American patient with a combination of trichloroacetic acid (TCA) and CO2 laser resurfacing, providing acceptable cosmetic results without significant side effects. METHODS: We describe an African American patient with eruptive syringomas of the face treated with a combination of TCA and CO2 laser resurfacing with good results. RESULTS: While the syringomas were not completely ablated, the combination of TCA and CO2 laser resurfacing provided acceptable cosmetic results without significant side effects. CONCLUSION: The TCA pretreatment probably removed some of the bulk of the surface of the lesions, thereby reducing the number of laser passes required to flatten the remainder of the lesions and thus lessening the potential for thermal damage at the treated sites and of surrounding normal skin.

Acoustic imaging of objects buried in soil.

In this study, we demonstrate an acoustic system for high-resolution imaging of objects buried in soil. Our goal is to image cultural artifacts in order to assess in a rapid manner the historical significance of a potential construction site. We describe the imaging system and present preliminary images produced from data collected from a soil phantom. A mathematical model and associated computer software are developed in order to simulate the signals acquired by the system. We have built the imaging system, which incorporates a single element source transducer and a receiver array. The source and receiver array are moved together along a linear path to collect data. Using this system, we have obtained B-mode images of several targets by using delay-and-sum beamforming, and we have also applied synthetic aperture theory to this problem.

Apparent change in ion selectivity caused by changes in intracellular K(+) during whole-cell recording.

In whole-cell recordings from HEK293 cells stably transfected with the delayed rectifier K(+) channel Kv2.1, long depolarizations produce current-dependent changes in [K(+)](i) that mimic inactivation and changes in ion selectivity. With 10 mM K(o)(+) or K(i)(+), and 140-160 mM Na(i,o)(+), long depolarizations shifted the reversal potential (V(R)) toward E(Na). However, similar shifts in V(R) were observed when Na(i,o)(+) was replaced with N-methyl-D-glucamine (NMG(+))(i, o). In that condition, [K(+)](o) did not change significantly, but the results could be quantitatively explained by changes in [K(+)](i). For example, a mean outward K(+) current of 1 nA for 2 s could decrease [K(+)](i) from 10 mM to 3 mM in a 10 pF cell. Dialysis by the recording pipette reduced but did not fully prevent changes in [K(+)](i). With 10 mM K(i,o)(+), 150 mM Na(i)(+), and 140 mM NMG(o)(+), steps to +20 mV produced a positive shift in V(R), as expected from depletion of K(i)(+), but opposite to the shift expected from a decreased K(+)/Na(+) selectivity. Long steps to V(R) caused inactivation, but no change in V(R). We conclude that current-dependent changes in [K(+)](i) need to be carefully evaluated when studying large K(+) currents in small cells.

High contrast ultrasound images of defects in food package seals.

Previous work to detect defects in food packaging seals using pulse-echo ultrasound inspired the backscattered amplitude integral (BAI) imaging technique, which could reliably identify channels with diameters 38 microm or larger at a center frequency of 17.3 MHz (lambda=86 microm). The current study presents two new processing techniques that more reliably reveal smaller channels ( approximately 6 microm in diameter). The RF sampling technique (RFS) displays a single, time-gated, pressure value from the received (not envelope-detected) RF waveform at each transducer position. The RF correlation technique (RFC) calculates the correlation coefficients of the RF signals with a reference signal that does not pass through a channel. The correlation coefficient can be calculated for the entire RF signal (RFCE) or over a short segment of the RF signal (RFCS). The performance of these imaging methods for detecting channel defects is investigated for plastic and aluminum foil trilaminate films with 6, 10, 15, 38, and 50 microm channels filled with water or air. Data are collected with a focused ultrasound transducer (17.3 MHz, 6.35 mm in diameter, f/2, 173 microm -6 dB pulse-echo lateral beamwidth at the focus) scanned over a rectangular grid, keeping the package in the focus. Performance is measured using detection rates, image contrast, and contrast-to-noise ratio (CNR). Both RFS and RFCS have improved detection rates relative to BAI for channels 15 microm or smaller. The RFCS technique is the most effective at smoothing the background, leading to the greatest CNR improvements.

A 5-year residency curriculum in obstetrics, gynecology, and family medicine.

OBJECTIVE: To develop a curriculum for residents in obstetrics and gynecology that also provides training in family medicine. METHODS: We designed a 5-year curriculum with 36 months of obstetrics and gynecology, 12 of which are as chief resident, with a 4-month rotation through family medicine to meet the primary care requirements, and rotations of 1 month each in geriatrics and emergency medicine. The curriculum includes the 30 months of required rotations mandated by family medicine (three of which are in obstetrics and gynecology), with the 6 months' available elective time allocated to obstetrics and gynecology. RESULTS: The Residency Review Committee for Obstetrics and Gynecology accredited the curriculum, which meets the Accreditation Council for Graduate Medical Education Special Requirements for Family Medicine, in August 1996. CONCLUSION: This 5-year residency curriculum educates physicians in both obstetrics and gynecology, and family medicine, and graduates are eligible to pursue board certification in both specialties.

The development of a nurse-directed computerized functional electrical stimulation program.

Today's spinal-cord-injured (SCI) person is discharged from the inpatient clinical setting very early in his or her recovery process. Faced with the tremendous challenges of relearning the skills of daily living and psychologically adjusting to a catastrophic injury, the newly injured person is thrust into an overwhelming environment. As early as 1994, when inpatient stays were longer, concern was expressed about the impact of early discharge on the health and well-being of persons with SCI (Ditunno & Formal, 1994). For over 10 years, the Medical Illness Counseling Center (MICC) has offered a community-based, nurse-directed program of Computerized Functional Electrical Stimulation (CFES) for persons with SCI. The program is founded on the belief that when multi-system deterioration associated with paralysis is avoided and a behavioral approach is used, the person with SCI will have a renewed sense of well-being that enables him or her to overcome the challenges of daily living. Over time, the need for expansion of the program became apparent; it evolved into a comprehensive package of medical, nursing, and psychological care. This article describes the essential elements that comprised a successful program design, the benefits of participation in CFES, and the significance of this technology in a nurse-managed setting.

Middle- and Neighborhood-Scale Variations of PM10 Source Contributions in Las Vegas, Nevada.

The Las Vegas Valley PM10 Study was conducted during 1995 to determine the contributions to PM10 aerosol from fugitive dust, motor vehicle exhaust, residential wood combustion, and secondary aerosol sources. Twenty-four-hr PM10 samples were collected at two neighborhood-scale sites every sixth day for 13 months. Five week-long intensive studies were conducted over a middle-scale sub-region at 29 locations that contained many construction projects emitting fugitive dust. The study found that the zone of influence around individual emitters was less than 1 km. Most of the sampling sites in residential and commercial areas yielded equivalent PM10 concentrations in the neighborhood region, even though they were more distant from each other than they were from the nearby construction sources. Based on chemical mass balance (CMB) receptor modeling, fugitive dust accounted for 80-90% of the PM10, and motor vehicle exhaust accounted for 3-9% of the PM10 in the Las Vegas Valley.

Synaptic potentials mediated via alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in rat hippocampal interneurons.

Exogenous application of acetylcholine elicits inward currents in hippocampal interneurons that are mediated via alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors, but synaptic responses mediated via such receptors have never been reported in mammalian brain. In the present study, EPSCs were evoked in hippocampal interneurons in rat brain slices by electrical stimulation and were recorded by using whole-cell voltage-clamp techniques. Nicotinic EPSCs were isolated pharmacologically, using antagonists to block other known types of ligand-gated ion channels, and then were tested with either alpha-bungarotoxin or methyllycaconitine, which are selective antagonists for nicotinic acetylcholine receptors that contain the alpha7 receptor subunit. Each antagonist proved highly effective at reducing the remaining synaptic current. Evoked alpha7-mediated nicotinic EPSCs also were desensitized by superfusion with 1 microM nicotine, had extrapolated reversal potentials near 0 mV, and showed strong inward rectification at positive potentials. In several interneurons, methyllycaconitine-sensitive spontaneous EPSCs also were observed that exhibited a biphasic decay rate very similar to that of the alpha7-mediated evoked response. These studies provide the first demonstration of a functional cholinergic synapse in the mammalian brain, in which the primary postsynaptic receptors are alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors.

Acetylcholine activates an alpha-bungarotoxin-sensitive nicotinic current in rat hippocampal interneurons, but not pyramidal cells.

The effects of acetylcholine on both pyramidal neurons and interneurons in the area CA1 of the rat hippocampus were examined, using intracellular recording techniques in an in vitro slice preparation. In current-clamp mode, fast local application of acetylcholine (ACh) to the soma of inhibitory interneurons in stratum radiatum resulted in depolarization and rapid firing of action potentials. Under voltage-clamp, ACh produced fast, rapidly desensitizing inward currents that were insensitive to atropine but that were blocked by nanomolar concentrations of the nicotinic alpha7 receptor-selective antagonists alpha-bungarotoxin (alphaBgTx) and methyllycaconitine. Nicotinic receptor antagonists that are not selective for alpha7-containing receptors had little (mecamylamine) or no effect (dihydro-beta-erythroidine) on the ACh-induced currents. Glutamate receptor antagonists had no effect on the ACh-evoked response, indicating that the current was not mediated by presynaptic facilitation of glutamate release. However, the current could be desensitized almost completely by bath superfusion with 100 nM nicotine. In contrast to those actions on interneurons, application of ACh to the soma of CA1 pyramidal cells did not produce a detectable current. Radioligand-binding experiments with [125I]-alphaBgTx demonstrated that stratum radiatum interneurons express alpha7-containing nAChRs, and in situ hybridization revealed significant amounts of alpha7 mRNA. CA1 pyramidal cells did not show specific binding of [125I]-alphaBgTx and only low levels of alpha7 mRNA. These results suggest that, in addition to their proposed presynaptic role in modulating transmitter release, alpha7-containing nAChRs also may play a postsynaptic role in the excitation of hippocampal interneurons. By desensitizing these receptors, nicotine may disrupt this action and indirectly excite pyramidal neurons by reducing GABAergic inhibition.

Synthetic aperture techniques with a virtual source element.

A new imaging technique has been proposed that combines conventional B-mode and synthetic aperture imaging techniques to overcome the limited depth of field for a highly focused transducer. The new technique improves lateral resolution beyond the focus of the transducer by considering the focus a virtual element and applying synthetic aperture focusing techniques. In this paper, the use of the focus as a virtual element is examined, considering the issues that are of concern when imaging with an array of actual elements: the tradeoff between lateral resolution and sidelobe level, the tradeoff between system complexity (channel count/amount of computation) and the appearance of grating lobes, and the issue of signal to noise ratio (SNR) of the processed image. To examine these issues, pulse-echo RF signals were collected for a tungsten wire in degassed water, monofilament nylon wires in a tissue-mimicking phantom, and cyst targets in the phantom. Results show apodization lowers the sidelobes, but only at the expense of lateral resolution, as is the case for classical synthetic aperture imaging. Grating lobes are not significant until spatial sampling is more than one wavelength, when the beam is not steered. Resolution comparable to the resolution at the transducer focus can be achieved beyond the focal region while obtaining an acceptable SNR. Specifically, for a 15-MHz focused transducer, the 6-dB beamwidth at the focus is 157 mum, and with synthetic aperture processing the 6-dB beamwidths at 3, 5, and 7 mm beyond the focus are 189 mum, 184 mum, and 215 mum, respectively. The image SNR is 38.6 dB when the wire is at the focus, and it is 32.8 dB, 35.3 dB, and 38.1 dB after synthetic aperture processing when the wire is 3, 5, and 7 mm beyond the focus, respectively. With these experiments, the virtual source has been shown to exhibit the same behavior as an actual transducer element in response to synthetic aperture processing techniques.

Age-related changes in effects of insulin-like growth factor I on human osteoblast-like cells.

The role of insulin-like growth factor I (IGF-I) in extracellular matrix metabolism was studied in both proliferating and confluent human osteoblast-like cultures derived from donors of different ages. In proliferating cultures, recombinant human (rh)IGF-I was found to increase the incorporation of [3H]thymidine in a dose- and age-dependent manner. To study cell proliferation dynamically, continuous growth curves with and without rhIGF-I were modelled by a modified logistic function. Increasing doses of rhIGF-I decreased the lag time and maximal growth rates, whereas plateau values decreased only at the highest dose (100 ng/ml). In post-proliferative cell strains, rhIGF-I (0.1-100 ng/ml) increased levels of type I collagen, biglycan and decorin, and to a smaller extent fibronectin and thrombospondin, whereas it decreased the levels of hyaluronan and a versican-like proteoglycan when protein and proteoglycan metabolism were followed by steady-state radiolabelling with [3H]proline, [3H]glucosamine or [35S]sulphate. These responses to rhIGF-I were found to be age-dependent, with osteoblast-like cells derived from younger patients being more responsive to rhIGF-I. When extracellular matrix turnover was analysed by pulse-chase experiments, rhIGF-I had no effect. The steady-state levels of collagen, decorin, hyaluronan and a versican-like proteoglycan for bone cells treated with rhIGF-I on day 7 in culture were equivalent to levels of these matrix components in untreated osteoblasts grown for 14 days. These results are consistent with rhIGF-I's altering cellular proliferative capacity and matrix synthesis, causing a change in the osteoblast differentiated state.