Mortality from cardiac arrest.
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Cardiopulmonary Resuscitation↗
Biomedical subjects
R L Brown
Publications and source records attributed to R L Brown.
Cyclic GMP contact points within the 63-kDa subunit and a 240-kDa associated protein of retinal rod cGMP-activated channels.
Ion channels from retinal rods and a variety of other cells are directly gated by cyclic nucleotides. The rod channel is known to contain a 63-kDa subunit, and there is molecular genetic evidence for the existence, in human retina, of a second subunit with a deduced molecular mass of about 100 kDa. When purified from bovine rods, the channel consists of the 63-kDa subunit and a 240-kDa associated protein that has been shown recently to contain a version of the cloned second subunit as part of a larger complex. We had previously shown that a photoaffinity analog of cGMP, 8-(p-azidophenacylthio)-[32P]cGMP, specifically labels both the 63- and 240-kDa proteins. Here the analog was used to identify cGMP-binding regions and amino acid contact points within these proteins. The specific labeling of the 63-kDa subunit was localized to a 66 amino acid fragment (Tyr-515-Met-580) that is contained entirely within a 110 amino acid region proposed to be the cGMP-binding site on the basis of homology with other cyclic nucleotide-binding proteins. Within this fragment, amino acid residues Val-524, Val-525, and Ala-526 were found to contain label. These residues are part of a larger hydrophobic cluster that appears to line the binding pocket. The results also indicate that the 240-kDa protein contains a similar cGMP-binding site. Sequencing of a specifically labeled 8-kDa fragment through 16 amino acid residues indicated that the fragment was derived from the portion of the 240-kDa complex that contains the second subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
Affinity Labels↗
Crystallization of sheep liver cytosolic aldehyde dehydrogenase in a form suitable for high resolution X-ray structural analysis.
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Aldehyde Dehydrogenase↗
Diffusion coefficient of the cyclic GMP analog 8-(fluoresceinyl)thioguanosine 3',5' cyclic monophosphate in the salamander rod outer segment.
Cyclic GMP (cGMP) is the intracellular messenger mediating phototransduction in retinal rods, with its longitudinal diffusion in the rod outer segment (ROS) likely to be a factor in determining light sensitivity. From the kinetics of cGMP-activated currents in the truncated ROS of the salamander (Ambystoma tigrinum), the cGMP diffusion coefficient was previously estimated to be approximately 60 x 10(-8) cm2 s-1. On the other hand, fluorescence measurements in intact salamander ROS using 8-(fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate (Fl-cGMP) led to a diffusion coefficient for this compound of 1 x 10(-8) cm2 s-1; after corrections for differences in size and in binding to cellular components between cGMP and Fl-cGMP, this gave an upper limit of 11 x 10(-8) cm2 s-1 for the cGMP diffusion coefficient. To properly compare the two sets of measurements, we have examined the diffusion of Fl-cGMP in the truncated ROS. From the kinetics of Fl-cGMP-activated currents, we have obtained a diffusion coefficient of 3 x 10(-8) cm2 s-1 for this analog; the cGMP diffusion coefficient measured from the same truncated ROSs was approximately 80 x 10(-8) cm2 s-1. Thus, a factor of 27 appears appropriate for correcting differences in size and intracellular binding between cGMP and Fl-cGMP. Application of this correction factor to the Fl-cGMP diffusion coefficient measurements by Olson and Pugh (1993) gives a cGMP diffusion coefficient of approximately 30 x 10(-8) cm2 s-1, in reasonable agreement with the value measured from the truncated ROS.
Ambystoma↗
Conjoint screening questionnaires for alcohol and other drug abuse: criterion validity in a primary care practice.
The US Preventive Services Task Force recommended that physicians use the CAGE questions to screen patients for alcohol abuse. A similarly brief screening instrument for abuse of other drugs is needed. Two conjoint screening questionnaires for alcohol and other drug abuse were adapted from the CAGE questions and the Short Michigan Alcoholism Screening Test (SMAST). For 124 patients of an academic, community family practice, the conjoint questionnaires and their forerunners were compared with DSM-III-R diagnoses of substance use disorders as measured by the Diagnostic Interview Schedule-Revised (DIS-R). The SMAST and its conjoint analog exhibited low sensitivity. The CAGE Adapted to Include Drugs (CAGE-AID) was more sensitive but less specific for substance abuse than the CAGE, especially when a reduced criterion score was employed. The CAGE-AID was more sensitive than the CAGE for subjects of varying sex, income, and level of education, as well as most patterns of substance use disorders. The diminished specificity of the CAGE-AID may have been, at least in part, artifactual. The CAGE-AID holds promise for identifying primary care patients with alcohol and drug disorders.
Adult↗
Crystallization and preliminary X-ray diffraction studies on cytosolic (class 1) aldehyde dehydrogenase from sheep liver.
The cytosolic (Class 1) aldehyde dehydrogenase (AlDH) from sheep liver has been crystallized in a form suitable for X-ray diffraction studies. The crystals, grown by vapour diffusion using 6.5 to 7.5% methoxypolyethylene glycol 5000 as precipitant, at pH 6.5, are orthorhombic with cell dimensions a = 80.7, b = 92.5, c = 151.6 A, space-group P2(1)2(1)2(1), and one dimer in the asymmetric unit. The crystals diffract to at least 2.8 A resolution. Although unmodified AlDH crystallized readily, a key factor in obtaining diffraction-quality crystals was the covalent attachment of an active site reporter group, provided by 3,4-dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one.
Aldehyde Dehydrogenase↗
PDGF and TGF-alpha act synergistically to improve wound healing in the genetically diabetic mouse.
Impaired wound healing results in significant morbidity for the surgical patient. The genetically diabetic (C57BL/KsJ-db/db) mouse is obese, hyperglycemic, insulin-resistant, and exhibits markedly impaired wound healing. Previous studies have demonstrated that the fibroblast mitogens, BB homodimer of platelet-derived growth factor (PDGF-BB) or basic fibroblast growth factor, plus insulin-like growth factor, act synergistically to enhance wound closure in the genetically diabetic mouse. The purpose of this study was to determine whether the keratinocyte mitogens, epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha), in combination with the fibroblast mitogen, PDGF-BB, would produce a similar synergistic enhancement in tissue repair. Full-thickness skin wounds created on the backs of diabetic mice received topical applications of vehicle (5% polyethylene glycol), PDGF-BB (10 micrograms), EGF (1 microgram), TGF-alpha (1 microgram), or the combination of PDGF (10 micrograms) and EGF (1 microgram) or TGF-alpha (1 microgram) for 5 consecutive days starting at wounding. Application of PDGF-BB or TGF-alpha alone to wounds in diabetic animals improved wound closure when compared to vehicle treatment. EGF did not affect healing and did not have any additive effects when combined with PDGF-BB. Significant improvements in wound closure were observed with the combination of PDGF-BB and TGF-alpha when compared to treatment with the individual growth factors. The PDGF-BB/TGF-alpha combination accelerated healing in the diabetic animals to a rate that was closer to that seen in nondiabetic mice.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Phosphorus magnetic resonance spectra and changes in body composition during weight loss.
OBJECTIVE: The use of magnetic resonance spectroscopy (MRS) to monitor cellular bioenergetics during weight loss may provide novel insights regarding metabolic functioning. METHODS: Changes were noted in 18 sedentary, moderately overweight women following a 7-week program consisting of a low-fat, 422-428 MJ/day (1010-1025 kcal/day) diet, a progressive walking program, a vitamin-mineral-fortified, isolated-protein meal supplement drink and a weekly group meeting. Mitochondrial energy production and utilization were assessed by measuring the concentrations of inorganic phosphate (Pi) and phosphocreatine (PCr) through magnetic resonance spectroscopy. Anthropometric changes were also monitored. RESULTS: Dietary analysis of subjects' pre-intervention food records showed intakes of numerous vitamins and minerals below 100% of the Recommended Dietary Allowances (RDA), most commonly for folic acid, vitamin E, magnesium, iron and zinc. Relative to pre-intervention levels, final measurements showed that all women lost body weight, ranging from 2.6 to 10.0 kg. Body mass index declined in all subjects, from 0.55 to 3.86 kg/m2. All subjects lost fat mass, ranging from 0.9 to 10.4 kg. Seventeen of 18 women showed a decline in their percentage of body fat, ranging from 2.3 to 10.1%. Twelve of 18 subjects showed an increase of 0.1-3.5 kg of fat-free mass, with half of these values increasing by > 1.0 kg. No changes from baseline levels were found in the Pi/PCr ratio over 7 weeks under resting, exercise or recovery conditions, suggesting a preservation in muscle energy function over the course of the study while the subjects were losing fat mass and total body weight. CONCLUSIONS: The possible metabolic advantage associated with a program that employs moderate energy restriction, repletion of vitamin and mineral intakes to RDA levels, and a regular aerobic exercise program is proposed.
Adipose Tissue↗
The adequacy of limb escharotomies-fasciotomies after referral to a major burn center.
To determine the frequency of inadequate decompression and its complications, the medical records of 108 pediatric burn patients requiring escharotomies or fasciotomies were reviewed. Of 108 patients, 100 (93%) had escharotomies or fasciotomies performed at an outlying facility before transfer. Of these 100 patients, 44 (117 limbs) were inadequately decompressed and required further decompression after admission to our facility. Initial mean compartment pressures were 50.3 +/- 1.3 mm Hg, which were reduced to 16.3 +/- 0.5 mm Hg after decompression. Pulses were present in 74% of limbs requiring decompression. Twenty limbs required decompression despite noncircumferential burns. Complications of inadequate or delayed decompression included foot drop in 20 patients (35 limbs) and muscle necrosis in 13 patients (23 limbs). Four patients (seven limbs) required amputations because of progressive muscle necrosis and infection. Complications of the procedure itself were limited to bleeding in three patients. In conclusion, compartment pressures should be followed in patients with significant burns since pressures may increase over time and pulses are not predictive of ischemia. Failure to decompress extremities with elevated pressures leads to significant but preventable complications.
Burns↗
Nutrition considerations in the neonatal extracorporeal life support patient.
Extracorporeal life support (ECLS) is a type of heart-lung bypass commonly used to support neonates with life-threatening respiratory or cardiac failure. These patients are among the most critically ill patients in the neonatal intensive care unit and are at significant nutritional risk. Unfortunately, there is a paucity of literature regarding guidelines for nutrition support of the neonatal ECLS patient. The purpose of this article is to provide an overview of the indications, techniques, and complications of ECLS and to address the approach to nutrition support of these patients. Emphasis will be placed on issues unique to the neonatal ECLS patient.
Enteral Nutrition↗
Activation of retinal rod cGMP-gated channels: what makes for an effective 8-substituted derivative of cGMP?
Analogs of cGMP bearing diverse substituents at the C8 position of the guanine ring system have been shown to activate the cGMP-activated channel of retinal rods at concentrations lower than cGMP itself. In an effort to understand this behavior, we synthesized eight novel C8-substituted derivatives and tested their ability to activate channels in excised patches from salamander rod outer segments. We express the effectiveness of each analog as a ratio (in brackets) of the concentration required to open half of the channels in a patch to that required of 8-Br-cGMP, previously shown to be about 10 times more effective than cGMP. Five of the derivatives contained a thio substitution at C8: n-propylthio-cGMP [0.61], sulfoethylthio-cGMP [0.90], carboxyethylthio-cGMP [0.97], aminoethylthio-cGMP [2.8], and (trimethylamino)ethylthio-cGMP [8.5]. Three of the derivatives contained an amino substitution at C8: carboxyethylamino-cGMP [22], n-propylamino-cGMP [25], and aminoethylamino-cGMP [230]. The results indicate that thio-substitution at C8 produces more effective analogs than does amino-substitution, regardless of the chemical nature of the terminal functional group. Derivatives containing neutral and apolar tails opened channels at much lower concentrations than their positively-charged counterparts with the same C8 substituent. Analogs having negatively-charged tails were also more effective than those with positive charge but not quite as effective as those with neutral tails.(ABSTRACT TRUNCATED AT 250 WORDS)
Ambystoma↗
Frequency and determinants of screening for breast cancer in primary care group practice.
BACKGROUND: Many studies reporting the frequency of breast cancer screening have been based only on physician and patient surveys or on data from quality assurance studies and do not assess the reliability of information obtained from these various sources. METHODS: To obtain more complete data we studied mammography performed in a 3-year period, 1988 through 1991, in 24 nonacademic primary care group practices by both auditing the medical records and obtaining questionnaire responses from 1819 women aged 53 to 62 years and from their 98 physicians in the nonmetropolitan Midwest. RESULTS: Medical record data indicated that mammography was performed in all 3 years in 16.7%, in at least two of 3 years in 49.8%, and in at least one of 3 years in 81.7% of women. While patient reports of a family history of breast cancer, health insurance coverage for mammography, and greater annual household income were each significant predictors, a patient report that a clinic staff member had discussed mammography was the strongest predictor of greater frequency of mammography. CONCLUSIONS: In this study of self-selected physicians and their patients, record-documented mammographic examinations were considerably more frequent than has been reported in some studies, but occurred at rates consistent with quality assurance data for the region. These data suggest that clinic staff initiatives with screening mammography have a large impact.
Breast Neoplasms↗
Specific labeling and permanent activation of the retinal rod cGMP-activated channel by the photoaffinity analog 8-p-azidophenacylthio-cGMP.
Cyclic nucleotide-gated ion channels play a key role in visual excitation and a variety of other signaling pathways. The photoaffinity probe 8-p-azidophenacylthio-cGMP (APT-cGMP) has been developed for structural and functional studies of the cGMP-activated channel of retinal rod outer segments. Using this analog, we have demonstrated both specific labeling of the channel in a partially purified biochemical preparation from bovine rod outer segments and permanent activation of the channel current in excised membrane patches from salamander outer segments. After UV illumination, a 32P-labeled version of APT-cGMP was shown by SDS/PAGE and autoradiography to be covalently attached to the 63-kDa channel subunit. This incorporation was significantly reduced by 8-Br-cGMP but was not reduced by 5'-GMP. In patch-clamp experiments APT-cGMP was a potent activator of the channel; APT-cGMP typically opened half of the channels in a patch at a 10-fold lower concentration than cGMP. Exposure of membrane patches to UV light in the presence of APT-cGMP resulted in a persistent current observed in the absence of bath-applied nucleotide. This current increased with repeated exposure of the patch to both UV light and fresh APT-cGMP, approaching the maximum current originally evoked by saturating (500 microM) cGMP. At this point, addition of 500 microM cGMP caused a negligible increase in current. The persistent current had several other properties expected of current through cGMP-activated channels: it was outwardly rectifying; outward current was blocked > 90% by 2 mM internal Mg2+, whereas inward current was blocked much less efficiently; a low concentration of cGMP caused a larger increase in current atop a half-maximal persistent current than it did originally. We conclude that the persistent current was caused by the covalent tethering of cGMP moieties to channel binding sites, resulting in irreversible channel activation. APT-cGMP should prove useful for further studies of these and similar cGMP-binding sites and in the identification of unknown cGMP-binding proteins.
Affinity Labels↗
Kidney-related Munchausen's syndrome and the Red Baron.
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Anti-Glomerular Basement Membrane Disease↗
Comparison of motor vehicle damage documentation in emergency medical services run reports compared with photographic documentation.
STUDY OBJECTIVE: To determine whether emergency medical services (EMS) run reports adequately document vehicle damage when compared with vehicle photographs by using a traffic accident scoring system. DESIGN: A prospective study consisting of three phases: photographing motor vehicle collisions and collecting their respective EMS run reports, traffic accident damage score development, and comparison of photographs to the run reports by emergency medical technicians using the traffic accident damage score. SETTING: Data were collected in North Carolina and Ohio from motor vehicle crashes to which nine different EMS squads responded during a three-year period. TYPE OF PARTICIPANTS: EMS squads ranged from basic to paramedic levels of training. MEASUREMENTS AND MAIN RESULTS: Three emergency medical technicians were unable to determine the area of vehicle damage in 48% and the severity of damage in 61% of the EMS run reports. In contrast, there were no instances in which all three emergency medical technicians were unable to determine both area and severity of damage from the photographs. CONCLUSION: Most EMS run reports do not document vehicle damage adequately.
Accidents, Traffic↗
Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods.
The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.
Animals↗
Plans for the In-house Construction of Environmental Enrichment Devices.
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Journal Article↗
Functional regions of the inhibitory subunit of retinal rod cGMP phosphodiesterase identified by site-specific mutagenesis and fluorescence spectroscopy.
In the dark, the activity of the cGMP phosphodiesterase (PDE) of retinal rod outer segments is held in check by its two inhibitory gamma subunits. Following illumination, gamma is rapidly removed from its inhibitory site by transducin, the G-protein of the visual system. In order to probe the functional roles of specific regions in the PDE gamma primary sequence, 10 variants of PDE gamma have been produced by site-specific mutagenesis and expression in bacteria and their properties compared to those of protein containing the wild-type bovine PDE gamma amino acid sequence. Three questions were asked about each mutant: What is its affinity for the alpha beta catalytic subunit of PDE? Does it inhibit catalytic activity? If so, can transducin relieve this inhibition? Binding to PDE alpha beta was determined directly using fluorescein-labeled gamma by measuring the increase in emission anisotropy that occurs when gamma binds to alpha beta. Inhibition of PDE alpha beta was measured by reconstitution of the gamma variants with gamma-free PDE generated by limited digestion with trypsin or endoproteinase Arg-C. Unlike trypsin, the latter enzyme did not remove PDE's ability to bind membranes and be activated by transducin, so that transducin activation of PDE containing specific gamma variants could be assayed directly. The results indicate that mutations in many regions of gamma affect its binding to alpha beta. A mutant missing the last five carboxy-terminal residues (83-87) was totally lacking in inhibitory activity. However, it still bound to PDE alpha beta tightly, although with a 100-fold lower dissociation constant (approximately 5 nM) than that of wild-type gamma (approximately 50 pM).(ABSTRACT TRUNCATED AT 250 WORDS)
3',5'-Cyclic-GMP Phosphodiesterases↗