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The aim of this work is to analyze trends in youth transportation fatalities and injuries in North Carolina (NC), assess the implementation of ignition interlock devices (IIDs) in the United States and abroad, discuss policy implications for IIDs, and highlight health equity considerations related to motor vehicle collisions (MVCs). MVCs cause the highest number of unintentional injury-related deaths for children and teenagers in NC, and policymakers should pay special attention to MVCs related to alcohol consumption. IIDs are effective in reducing collision rates and recidivism for driving under the influence of alcohol (DUI). Ignition interlock device requirements have been increasingly implemented globally over the past three decades. However, the adoption of stricter IID policies after first-time DUI offenses in NC and across the U.S. is a prudent public health measure to enhance transportation safety for both adults and children. Evidence-based interventions such as IIDs must also strive to address inequities in transportation safety, and the framing of proposed policies should reflect the tenets of cultural humility.
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Internet , Degeneración Macular , Humanos , Degeneración Macular/diagnóstico , Agudeza VisualRESUMEN
Light touch of a stable reference reduces sway during standing. However, unexpected displacement of a light touch reference leads to short-latency reactions in ankle muscles consistent with a balance reaction, that are replaced by responses in arm muscles on subsequent trials. We anticipated that the excitability of sensorimotor pathways arising from finger cutaneous afferents would reflect these changes in behavior. We hypothesized that (1) interlimb cutaneous reflexes in muscles of the ipsilateral leg, derived from median nerve (MED) stimulation would be facilitated when touch was stable, but reduced when touch was unreliable, (2) intralimb MED reflexes in muscles of the homonymous arm would be facilitated when touch was unreliable and participants tracked the touch reference with arm movements, and (3) radial nerve (RAD) evoked reflexes would be unaffected, given that the RAD innervation territory is not involved in the light touch task. Cutaneous reflexes were evoked using a transcutaneous train of pulses (5 × 1.0 ms square-wave pulses; 300 Hz) and recorded using electromyography of muscles of the ipsilateral arm and leg. As hypothesized, interlimb MED reflexes recorded in soleus (SOL) were larger when touching the stable reference (mean ± SD % MVC; 4.78 ± 1.57) than when not touching a reference (1.00 ± 1.05) or when touching an unstable reference (1.07 ± 1.16). In addition, intralimb MED reflexes in anterior deltoid (AD) were larger when touching an unstable reference (4.50 ± 1.31), compared to touching a stable reference (1.34 ± 1.01) or not touching (1.50 ± 1.00). In contrast, interlimb RAD reflexes in SOL were larger when not touching (4.29 ± 4.34), compared with touching a stable (1.14 ± 1.84) or unstable reference (3.11 ± 4.15). These findings indicate that cutaneous reflexes from the hand are scaled with a rapid change in motor behavior when a touch reference becomes unstable, suggesting that spinal sensorimotor pathways are functionally reweighted based in part upon the reliability of tactile inputs.
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Mano , Reflejo , Estimulación Eléctrica , Electromiografía , Humanos , Músculo Esquelético , Reproducibilidad de los Resultados , Posición de PieRESUMEN
BACKGROUND: Fentanyl is a potent, synthetic opioid at the centre of an international health crisis that has seen thousands of fatal overdoses. Most analytical methods focus on the detection of fentanyl in blood and/or urine (i.e., post-drug use). Harm reduction (including pre-screening before drug use) may be an effective strategy against fentanyl overdoses. METHOD: Paper spray-mass spectrometry (PS-MS) is an inexpensive, direct sampling strategy where a small volume of sample (<10⯵L) is spotted onto a piece of paper that is then wetted and connected to high voltage. Ions are emitted from the paper and enter a mass spectrometer for sensitive and selective semi-quantitation using labeled internal standards. RESULTS: We present the use of PS-MS for the direct measurement of fentanyl and norfentanyl using a custom PS interface, demonstrating that paper tip position and quality can significantly affect quantitative results. Furthermore, we observe comparable calibrations for fentanyl and norfentanyl (0.5 to 600â¯ng/mL) across a variety of complex matrices (methanol, diluted urine, analgesic slurry). Detection limits for fentanyl are as low as 0.049â¯ng/mL (0.4â¯pg total material) in methanol, and 0.66â¯ng/mL (5.3â¯pg total material) spiked in an analgesic slurry (illicit substance simulation). PS-MS was compared with liquid chromatography-MS for the analyses of real urine samples, with satisfactory results. CONCLUSION: PS-MS shows potential as a sensitive and selective direct measurement strategy for use in fentanyl harm reduction strategies, and may also be used for pre-screening in advance of or in combination with more conventional (i.e., chromatographic) analyses.
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Fentanilo/análogos & derivados , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Fentanilo/análisisRESUMEN
Coking leads to the deactivation of solid acid catalyst. This phenomenon is a ubiquitous problem in the modern petrochemical and energy transformation industries. Here, we show a method based on microwave cavity perturbation analysis for an effective examination of both the amount and the chemical composition of cokes formed over acid zeolite catalysts. The employed microwave cavity can rapidly and non-intrusively measure the catalytically coked zeolites with sample full body penetration. The overall coke amount is reflected by the obtained dielectric loss (εâ³) value, where different coke compositions lead to dramatically different absorption efficiencies (εâ³/cokes' wt%). The deeper-dehydrogenated coke compounds (e.g., polyaromatics) lead to an apparently higher εâ³/wt% value thus can be effectively separated from lightly coked compounds. The measurement is based on the nature of coke formation during catalytic reactions, from saturated status (e.g., aliphatic) to graphitized status (e.g., polyaromatics), with more delocalized electrons obtained for enhanced Maxwell-Wagner polarization.Catalyst deactivation by coke deposition is a major drawback in industrial processes. Here, the authors show a non-intrusive microwave cavity perturbation technique as a powerful tool to determine the nature and extent of coke accumulation in industrially-relevant zeolite catalysts.
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Most studies examine the effects of stress on memory for visual information test memory for entire scenes. However, arousal levels may differentially influence memory for backgrounds as opposed to items. Participants encoded scenes that included a negative-high-arousal, negative-moderate-arousal, or neutral item on a neutral background. After a 30-minute (Experiment 1) or 48-hour delay (Experiment 2), participants underwent a stressor or control task while heart rate was recorded. A recognition memory task was then given with items and backgrounds presented separately. High-arousal images had a greater detriment in background memory than moderate-arousal images. Further, though there was evidence that change in cortisol level at retrieval was associated with impaired memory for items, it was not associated with detriments in background memory. Increased heart rate was associated with impaired memory for both items and backgrounds. This suggests that the level of sympathetic and cortisol reactivity differentially affects memory for items and backgrounds.
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Nivel de Alerta , Hidrocortisona/metabolismo , Memoria , Reconocimiento en Psicología , Adolescente , Femenino , Frecuencia Cardíaca/fisiología , Humanos , Masculino , Saliva/metabolismo , Estrés Fisiológico/fisiología , Estrés Psicológico/psicología , Factores de Tiempo , Adulto JovenRESUMEN
We report a 39-year-old female patient known to have multiple sclerosis (MS), who later developed cerebral glioblastoma. The tumor was documented on the brain-magnetic resonance imaging (MRI) during the work-up for an apparent relapsing MS, and was subsequently confirmed pathologically by stereotactic biopsy and the postmortem brain examination. Our case, as well as others, re-emphasizes the need to evaluate the symptoms and brain MRI carefully, even in well-documented MS subjects. The concurrence of MS and intracranial glioma is uncommon. The possible relationship between the 2 diseases was discussed, and related literature reviewed.
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Astrocitoma/complicaciones , Neoplasias Encefálicas/complicaciones , Esclerosis Múltiple/complicaciones , Adyuvantes Inmunológicos/uso terapéutico , Adulto , Astrocitoma/patología , Astrocitoma/cirugía , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/cirugía , Resultado Fatal , Femenino , Humanos , Interferón beta-1a , Interferón beta/uso terapéutico , Imagen por Resonancia Magnética , Esclerosis Múltiple/tratamiento farmacológico , Resultado del TratamientoRESUMEN
On October 9, 2001, a letter containing anthrax spores was mailed from New Jersey to Washington, DC. The letter was processed at a major postal facility in Washington, DC, and opened in the Senate's Hart Office Building on October 15. Between October 19 and October 26, there were 5 cases of inhalational anthrax among postal workers who were employed at that major facility or who handled bulk mail originating from that facility. The cases of 2 postal workers who died of inhalational anthrax are reported here. Both patients had nonspecific prodromal illnesses. One patient developed predominantly gastrointestinal symptoms, including nausea, vomiting, and abdominal pain. The other patient had a "flulike" illness associated with myalgias and malaise. Both patients ultimately developed dyspnea, retrosternal chest pressure, and respiratory failure requiring mechanical ventilation. Leukocytosis and hemoconcentration were noted in both cases prior to death. Both patients had evidence of mediastinitis and extensive pulmonary infiltrates late in their course of illness. The durations of illness were 7 days and 5 days from onset of symptoms to death; both patients died within 24 hours of hospitalization. Without a clinician's high index of suspicion, the diagnosis of inhalational anthrax is difficult during nonspecific prodromal illness. Clinicians have an urgent need for prompt communication of vital epidemiologic information that could focus their diagnostic evaluation. Rapid diagnostic assays to distinguish more common infectious processes from agents of bioterrorism also could improve management strategies.
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Carbunco/diagnóstico , Bacillus anthracis/aislamiento & purificación , Bioterrorismo , Infecciones del Sistema Respiratorio/microbiología , Esporas Bacterianas/aislamiento & purificación , Dolor Abdominal/complicaciones , Carbunco/sangre , Carbunco/fisiopatología , Carbunco/terapia , Antibacterianos/uso terapéutico , Sangre/microbiología , Bradicardia/etiología , District of Columbia , Disnea/complicaciones , Resultado Fatal , Fiebre/complicaciones , Paro Cardíaco/etiología , Homicidio , Humanos , Leucocitosis , Masculino , Mediastinitis/diagnóstico por imagen , Persona de Mediana Edad , Náusea/complicaciones , Exposición Profesional , Derrame Pleural/diagnóstico por imagen , Servicios Postales , Radiografía Torácica , Respiración Artificial , Infecciones del Sistema Respiratorio/sangre , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/fisiopatología , Infecciones del Sistema Respiratorio/terapia , Taquicardia/etiología , Tomografía Computarizada por Rayos XRESUMEN
Taking advantage of a PCR technique that allows amplification of all variable region genes with equal efficiency, we defined three novel waves of TCR delta-chain transcription during thymic ontogeny. The canonical DV101-D2-J2 rearrangement was confined to a narrow window from days 14 to 18 of gestation, indicating that the postulated two consecutive gamma delta precursor waves bearing this canonical DV101 rearrangement will coincide on day 16. Neonatal delta-chain transcripts used a second wave of diverse V alpha gene segments that are exclusively located in the delta locus-proximal gene cluster of intermingled single members of different V alpha subfamilies. In the adult, only expression of a clan of three homologous subfamilies, ADV7, DV104, and ADV17, persists. The members of the ADV7 subfamily are also scattered across the alpha locus, but their usage does not show the position-dependent bias of the other V alpha-to-delta rearrangements.
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Regulación del Desarrollo de la Expresión Génica/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/biosíntesis , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Timo/embriología , Timo/crecimiento & desarrollo , Envejecimiento/genética , Envejecimiento/inmunología , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos/genética , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/inmunología , Secuencia de Bases , Feto/inmunología , Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T/genética , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T/genética , Edad Gestacional , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Familia de Multigenes/inmunología , Timo/inmunología , Timo/metabolismo , Transcripción Genética/inmunologíaRESUMEN
The N and C abundances and isotopic compositions of acid-insoluble carbonaceous material in thirteen primitive chondrites (five unequilibrated ordinary chondrites, three CM chondrites, three enstatite chondrites, a CI chondrite and a CR chondrite) have been measured by stepped combustion. While the range of C isotopic compositions observed is only delta 13C = 30%, the N isotopes range from delta 15N approximately -40 to 260%. After correction for metamorphism, presolar nanodiamonds appear to have made up a fairly constant 3-4 wt% of the insoluble C in all the chondrites studied. The apparently similar initial presolar nanodiamond to organic C ratios, and the correlations of elemental and isotopic compositions with metamorphic indicators in the ordinary and enstatite chondrites, suggest that the chondrites all accreted similar organic material. This original material probably most closely resembles that now found in Renazzo and Semarkona. These two meteorites have almost M-shaped N isotope release profiles that can be explained most simply by the super-position of two components, one with a composition between delta 15N = -20 and -40% and a narrow combustion interval, the other having a broader release profile and a composition of delta 15N approximately 260%. Although isotopically more subdued, the CI and the three CM chondrites all appear to show vestiges of this M-shaped profile. How and where the components in the acid-insoluble organics formed remains poorly constrained. The small variation in nanodiamond to organic C ratio between the chondrite groups limits the local synthesis of organic matter in the various chondrite formation regions to at most 30%. The most 15N-rich material probably formed in the interstellar medium, and the fraction of organic N in Renazzo in this material ranges from 40 to 70%. The isotopically light component may have formed in the solar system, but the limited range in nanodiamond to total organic C ratios in the chondrite groups is consistent with most of the organic material being, presolar.
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Carbono/química , Diamante/análisis , Meteoroides , Nitrógeno/química , Isótopos de Carbono , Exobiología , Medio Ambiente Extraterrestre , Calor , Isótopos de Nitrógeno , Tamaño de la Partícula , Sistema SolarRESUMEN
TCR delta-chain gene rearrangements were analyzed at different stages of thymic ontogeny. The VDJ delta junctional sequences of the dominantly expressed TCR delta-chain V7 subfamily are highly conserved in fetal and neonatal thymocytes. They are equally conserved in C delta-targeted mice lacking cell surface expression of gamma delta receptors, indicating evolutionary selection acting at the level of DNA rearrangement. Yet, in C delta-mutant mice, the frequency of in-frame transcripts is reduced to 61%, from 88% in wild-type mice, suggesting cellular selection of this DV7-overexpressing gamma delta thymocyte subset. In contrast, in genomic DNA rearrangements, no difference was found in the frequency of productive rearrangements between mutant (26%) and wild-type mice (31%). This in-frame rate, characteristic of random rearrangements, indicates that positive selection is not required for thymic maturation of gamma delta T cells. The results are discussed with regard to models of alpha beta/gamma delta lineage commitment.
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Desarrollo Embrionario y Fetal/genética , Desarrollo Embrionario y Fetal/inmunología , Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T/fisiología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Linfocitos T/citología , Linfocitos T/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Evolución Molecular , Genes Dominantes/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Familia de Multigenes/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/química , Linfocitos T/químicaRESUMEN
The technique of inverse PCR permits the rapid amplification and identification of unknown DNA segments adjacent to well characterized core regions. In the field of immunology anchored PCR and inverse PCR are useful methods for examining junctional diversity and unknown variable gene segments of rearranged T cell receptor genes. We have applied an improved inverse PCR protocol to study the repertoire of gamma delta T cell receptor genes in the developing thymus of the mouse.
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Reacción en Cadena de la Polimerasa/métodos , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Tampones (Química) , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genéticaRESUMEN
The mu opioid receptor was shown to be phosphorylated at a basal rate in the absence of agonist, measured in permeabilized HEK293 cells transfected with an epitope tagged mu receptor (EE-mu) [Arden, J., Segredo, V., Wang, Z., Lameh, J. and Sadee, W. (1995) J. Neurochem. 65, 1636-1645]. In the present study, basal phosphorylation was found to be Ca2+ dependent; however, several inhibitors of protein kinase C and Ca2+-calmodulin dependent kinases failed to affect basal mu receptor phosphorylation. Thus, the basal mu receptor phosphorylating activity differed from the main kinases involved in receptor regulation. The general kinase inhibitor H7 (100 microM) suppressed basal mu receptor phosphorylation. Pretreatment with the agonist morphine, followed by drug removal, resulted in a sustained increase of basal mu receptor phosphorylation. The gradual agonist dependent modulation of basal mu receptor phosphorylation suggests a novel regulatory mechanism which may play a role in narcotic tolerance and dependence.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Calcio/metabolismo , Morfina/farmacología , Receptores Opioides mu/metabolismo , Animales , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Línea Celular , Inhibidores Enzimáticos/farmacología , Humanos , Isoquinolinas/farmacología , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidoresAsunto(s)
Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Secuencia de Bases , Carbacol/farmacología , Línea Celular , Cartilla de ADN , Epítopos , Humanos , Riñón/citología , Riñón/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Fosfatidilinositoles/metabolismo , Mutación Puntual , Receptor Muscarínico M1 , Receptores Muscarínicos/químicaRESUMEN
We expressed the cloned mu-opioid receptor (muR) in high abundance (5.5 x 10(6) sites/cell) with an amino-terminal epitope tag (EYMPME) in human embryonic kidney 293 cells. The epitope-tagged receptor (EE-muR) was similar to the untagged mu R ligand binding and agonist-dependent inhibition of cyclic AMP accumulation. By confocal microscopy, the labeled receptor was shown to be largely confined to the plasma membrane. Pretreatment with morphine failed to affect the cellular distribution of the receptor as judged by immunofluorescence and tracer binding studies. In contrast, exposure to the mu-specific peptide agonist [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist-induced cellular redistribution of EE-muR. Tracer binding studies suggested partial net internalization and a small degree of down-regulation caused by DAMGO. EE-muR-containing membranes were solubilized in detergent [3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate] and immunoprecipitated by an anti-epitope monoclonal antibody. Immunoblotting revealed a prominent band at approximately 70 kDa with weaker bands at approximately 65 kDa. EE-muR was labeled with [gamma-32P]ATP in permeabilized cells, immunoprecipitated, and analyzed by polyacrylamide gel electrophoresis autoradiography. A prominent band at 65-70 kDa indicated the presence of basal receptor phosphorylation occurring in the absence of agonist, which was enhanced approximately 1.8-fold with the addition of morphine. In conclusion, intracellular trafficking of the muR appears to depend on the agonist, with morphine and DAMGO having markedly different effects. Unlike other G protein-coupled receptors, basal phosphorylation is substantial, even in the absence of agonist.
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Epítopos , Membranas Intracelulares/metabolismo , Receptores Opioides mu/inmunología , Receptores Opioides mu/metabolismo , Lugares Marcados de Secuencia , Animales , Secuencia de Bases , Línea Celular , Humanos , Microscopía Confocal , Sondas Moleculares/genética , Datos de Secuencia Molecular , Fosforilación , Ratas , Receptores Opioides mu/genéticaRESUMEN
We have determined the precise chromosomal location, the exon structure, and the expression pattern of CGM2, a member of the carcinoembryonic antigen (CEA) gene family. CGM2 cDNA was amplified by reverse transcription-polymerase chain reaction (RT/PCR) from the colon adenocarcinoma cell line, LS174T. A defective exon is missing from this cDNA clone, leading to a novel domain organization for the human CEA family with two immunoglobulin-like domains. The derived C-terminal domain predicts that the CGM2 protein is membrane-bound through a glycosyl phosphatidylinositol anchor. RT/PCR analyses identified CGM2 transcripts in mucinous ovarian and colonic adenocarcinomas as well as in adjacent colonic tissue, but not in other tumors including leukocytes from six chronic myeloid leukemia patients. Thus, unlike several other family members, CGM2 is not expressed in granulocytes but reveals a more CEA-like expression pattern. Northern blot analyses identified a 2.5-kilobase CGM2 mRNA that is strongly down-regulated in colonic adenocarcinomas compared with adjacent colonic mucosa, suggesting a possible tumor suppressor function. In addition, a 3.2-kilobase transcript was observed in a number of colon tumors that is not detectable in normal colonic tissue. This mRNA species could represent a tumor-specific CGM2 splice variant.
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Adenocarcinoma/genética , Antígenos de Neoplasias/genética , Antígeno Carcinoembrionario/genética , Moléculas de Adhesión Celular/genética , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cósmidos , ADN Complementario , Regulación hacia Abajo , Exones , Proteínas Ligadas a GPI , Humanos , Mucosa Intestinal/inmunología , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
A retrospective study was conducted of 14 affectively ill patients who were treated with long-term unilateral maintenance electroconvulsive therapy (MECT). The patients had received an inpatient course of ECT before being referred for MECT. The average age was 57 +/- 16 years (range 30-91). The average interval between inpatient ECT was 2.4 +/- 0.9 days, in contrast to the interval between MECT, which was 12.1 +/- 11.3 days. The average time to start MECT after inpatient ECT was 16.2 +/- 16.2 days. The average duration of MECT was 81 +/- 104 days (maximum 571). Patients' affective symptoms continued to improve during the course of MECT based on Carroll Depression Ratings. Adjustment of the electrical dose and caffeine augmentation were used to keep the seizure durations >30 s by electroencephalograph (EEG) monitoring. Over time, most treatments were administered using the maximal charge provided by the Mecta SR-1. Despite considerable time intervals between MECT treatments, seizure durations did not increase. Additionally, high stimulus charge and frequently administered caffeine were used to maintain seizure length. The apparent anticonvulsant effect of ECT was not lost over the time span of MECT. This has clinical implications if the anticonvulsant effects of ECT contribute to determining the clinical response.
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One hypothesis for the origin of the nanometer-size diamonds found at the Cretaceous-Tertiary (K-T) boundary is that they are relict interstellar diamond grains carried by a postulated asteroid. The (13)C/(12)C and (15)N/(14)N ratios of the diamonds from two sites in North America, however, show that the diamonds are two component mixtures differing in carbon and nitrogen isotopic composition and nitrogen abundance. Samples from a site from Italy show no evidence for either diamond component. All the isotopic signatures obtained from the K-T boundary are material well distinguished from known meteoritic diamonds, particularly the fine-grain interstellar diamonds that are abundant in primitive chondrites. The K-T diamonds were most likely produced during the impact of the asteroid with Earth or in a plasma resulting from the associated fireball.
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We measured dose-response curves for carbachol stimulation of phosphatidyl inositol (PI) turnover with mutants of the Hm1 muscarinic cholinergic receptor having various deletions from amino acids 219 to 358 of the large third intracellular (i3) loop (208 to 366). These deletions had only small or no effects on the ability of Hm1 transfected into HEK 293 cells to stimulate PI turnover. This result indicates that only small regions of 9 to 11 amino acids adjacent to trans-membrane domains (TMDs) 5 and 6 can be directly involved in G protein coupling. Point mutations were constructed to test the role of charged amino acids in these junctions. A triple point mutation of Hm1 (E214 A/ E216K/ E221 K), which mimics the charge distribution in Hm2 (negatively coupled to cAMP) over the first 14 amino acids of i3, and a double point mutation in the N terminal junction, K359A/K361A, both failed to affect carbachol stimulated PI turnover. Therefore, charge distribution in the loop junctions appears to play a minor role in G protein coupling of Hm1 in HEK 293 cells.