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Biomedical subjects

D L Smith

Publications and source records attributed to D L Smith.

At least 163 records · Page 9Linked to original sources
PubMed1997

Dose-dependent pain-facilitatory and -inhibitory actions of neurotensin are revealed by SR 48692, a nonpeptide neurotensin antagonist: influence on the antinociceptive effect of morphine.

Neurotensin has bipolar (facilitatory and inhibitory) effects on pain modulation that may physiologically exist in homeostasis. Facilitation predominates at low (picomolar) doses of neurotensin injected into the rostroventral medial medulla (RVM), whereas higher doses (nanomolar) produce antinociception. SR 48692, a neurotensin receptor antagonist, discriminates between receptors mediating these responses. Consistent with its promotion of pain facilitation, the minimal antinociceptive responses to a 30-pmol dose of neurotensin microinjected into the RVM were markedly enhanced by prior injection of SR 48692 into the site (detected using the tail-flick test in awake rats). SR 48692 had a triphasic effect on the antinociception from a 10-nmol dose of neurotensin. Antinociception was attenuated by femtomolar doses, attenuation was reversed by low picomolar doses (corresponded to those blocking the pain-facilitatory effect of neurotensin) and the response was again blocked, but incompletely, by higher doses. The existence of multiple neurotensin receptor subtypes may explain these data. Physiologically, pain facilitation appears to be a prominent role for neurotensin because the microinjection of SR 48692 alone causes some antinociception. Furthermore, pain-facilitatory (i.e., antianalgesic) neurotensin mechanisms dominate in the pharmacology of opioids; the response to morphine administered either into the PAG or systemically was potentiated only by the RVM or systemic injection of SR 48692. On the other hand, reversal of the enhancement of antinociception occurred under certain circumstances with SR 48692, particularly after its systemic administration.

Analgesics, Opioid↗
PubMed1996

Mass spectrometric determination of isotopic exchange rates of amide hydrogens located on the surfaces of proteins.

The rates at which peptide amide hydrogens in folded proteins undergo isotopic exchange are reduced by factors of 10(0)-10(-8) relative to exchange rates at the same peptide linkages in unfolded proteins. To measure the isotopic exchange rates of the most rapidly exchanging peptide amide hydrogens in proteins, a flow-quench deuterium exchange-in step has been added to the protein fragmentation/mass spectrometry method (Zhang, Z.; Smith, D. L. Protein Sci. 1993, 2, 522-531). Isotopic exchange rates in eight short segments spanning the entire backbone of cytochrome c have been determined for exchange-in times of 0.2-120 s. These results show that the isotopic exchange rates of 10 of the peptide amide hydrogens in cytochrome c are similar to those expected for unfolded cyt c, while the exchange rates for 33 other non-hydrogen-bonded amide hydrogens are much less than expected for unfolded cyt c. Since the isotopic exchange rates of the most rapidly exchanging amide hydrogens in folded proteins are a direct measure of their access to the aqueous solvent, the ability to determine these isotopic exchange rates points to the possibility of using quenched-flow amide hydrogen exchange and mass spectrometry as a tool for identifying protein surfaces involved with binding.

Amides↗
PubMed1996

Amide hydrogen exchange determined by mass spectrometry: application to rabbit muscle aldolase.

The protein fragmentation/mass spectrometry method described by Zhang and Smith [(1993) Protein Sci. 2, 522-531] has been extended to measure amide hydrogen exchange rates in rabbit muscle aldolase, a homotetramer with M(r) = 157,000. Following a period of deuterium exchange, the partially deuterated protein was proteolytically fragmented into peptides whose deuterium contents were determined by directly coupled HPLC fast atom bombardment mass spectrometry. Hydrogen exchange rates were determined for amide hydrogens located in short segments derived from 85% of the aldolase backbone. Isotopic exchange rate constants spanning the range from 100 to 0.001 h-1 were determined for the exchange-in times used in this study (2.5 min to 44 h). The exchange rates for amide hydrogens located within short segments differed by as much as 10(4), demonstrating that local structural features dramatically affect the isotopic exchange rates in large proteins. A high level of correlation between the slowing of hydrogen exchange and intramolecular hydrogen bonding in aldolase was found. An exception to this correlation occurs at the subunit interface, where the amide hydrogens in one peptide segment with few amide hydrogen bonds have slower exchange rates than expected, suggesting that the amide hydrogens in this region are effectively shielded from the deuterated solvent. Isotope patterns observed for most peptides were binomial, indicating that hydrogen exchange proceeds through the EX2 mechanism (uncorrelated exchange). However, bimodal isotope patterns were found for peptides derived from three short segments of aldolase (including residues 58-64, 279-283, and 326-337), suggesting structural differences in these regions. A high level of correlation was found between crystallographic B-factors and amide hydrogen exchange rates, suggesting an isotopic exchange mechanism involving localized low-amplitude, high-frequency motions that do not require collective motion of many residues. From a methodology viewpoint, these results demonstrate that the combination of protein fragmentation with mass spectrometry is a useful method for determining the rates at which amide hydrogens located over major portions of large proteins undergo isotopic exchange.

Amides↗
PubMed1996

Thermal-induced unfolding domains in aldolase identified by amide hydrogen exchange and mass spectrometry.

Amide hydrogen exchange has been measured in short segments of intact rabbit muscle aldolase at temperatures of 14-50 degrees C by the protein fragmentation/mass spectrometry method (Zhang Z, Smith DL, 1993, Protein Sci 2:522-531). Deuterium levels in some segments did not change over the temperature range of the measurements, whereas deuterium levels in other segments increased rapidly with temperature. These results demonstrate that the equilibrium constant for local unfolding, Kunf, of some segments increases with temperature in the low temperature range (14-30 degrees C) of this study. Aldolase begins to lose activity at temperatures above 40 degrees C. In the 40-50 degrees C temperature range, Kunf is greater than 10(-4) in some regions and less than 10(-6) in other regions. This wide range of regional stability in the temperature range where aldolase begins to denature is interpreted in terms of cooperative unfolding/folding domains. Regions of highest stability were located along the hydrophobic subunit binding surface. It is proposed that hydrogen exchange might be used to identify unfolding domains in multidomain proteins whose thermodynamic properties have been determined by differential scanning calorimetry.

Amides↗
PubMed1996

Modifications of the water-insoluble human lens alpha-crystallins.

Since the water-insoluble crystallins of the lens may be the precursors of cataract, identifying the modifications that differentiate the water-insoluble from the water-soluble crystallins may provide the basis for understanding the chemistry leading to cataract. This investigation of the alpha-crystallins of the water-insoluble urea-soluble portion of 45-year-old normal clear lenses, isolated using gel filtration, ion exchange and reversed phase chromatography, has employed state-of-the-art mass spectrometric techniques to identify and locate the modifications of the water-insoluble alpha-crystallins. Modifications present in the isolated alpha-crystallins were identified by the molecular weights of the modified proteins, by the molecular weights of peptides produced by enzymatic digestion of the proteins, and by the fragmentation patterns produced by collisional activation of the peptides. Modifications that are either unique to the water-insoluble alpha-crystallins or are more prevalent in the water-soluble portion than in the water-soluble part include complete oxidation of the two Cys residues of alpha A-crystallin to form an intra-molecular disulfide bond, partial truncation at both the C-termini and N-termini of alpha A- and alpha B-crystallins, partial oxidation of Met residues to methionine sulfoxide, partial deamidation of several Asn and Gln residues, and evidence of peptide bond cleavage at some of the deamidated residues. Although many reactions have been proposed to contribute to the insolubility of crystallins, this compilation of in vivo post-translational modifications of water-insoluble alpha-crystallins delineates products that are actually present at levels of 5% or more. From these results, it is hypothesized that alpha-crystallin becomes water-insoluble following deamidation of various Asn and Gln residues which cause conformational changes leading to formation of an intra-molecular disulfide bond between the Cys residues of alpha A-crystallin.

Amino Acid Sequence↗
PubMed1996

Etiology and outcome of pediatric burns.

A 6-year retrospective review of burn victims hospitalized at a major burn center was conducted to determine the etiology and outcome of pediatric burns. Four hundred forty-nine patients under age 16 years were identified and stratified by age, sex, burn size, presence or absence of inhalation injury, cause of burn, and county of residence. The mean patient age was 4.3 +/- 0.2 years, and the male:female ratio was 1.9:1. There were 21 deaths overall (4.7%), the majority of which (18) were among children under 4 years of age. With respect to large burns, defined as > and = 30% total body surface area (TBSA), the mortality rate for children under age 4 was significantly higher than that for older children (46.9% v 12.5%; P < .01), despite the nearly identical mean burn size of the two groups. Except for burn incidence, there were no significant differences between males and females. The mean burn size was 15.1% +/- 0.7%, and was significantly larger for nonsurvivors than survivors (55.3% +/- 5.7 v 13.1% +/- 0.5%; P < .01). Inhalation injuries were strongly associated with large burns and were present in all 15 flame-burn fatalities. Scalds were the most common type of burn among children under 4 years of age; flame burns predominated in older children. There were 6 deaths related to scalds, all of which occurred in children under 4. Burn type, size, and mortality rate did not differ between children from urban and rural counties. Large burn size was the strongest predictor of mortality, followed by (in order) age less than 4 and the presence of inhalation injury. Infants and young children have the highest risk of death from burn injury. Burns smaller than 30% TBSA without an inhalation injury (such as small scald injuries) occasionally are lethal in infants and small children, despite modern therapy.

Adolescent↗
PubMed1996

Calcium bioavailability from bovine milk and dairy products in premenopausal women using intrinsic and extrinsic labeling techniques.

Stable isotopes were used to compare calcium fractional absorption from intrinsically and extrinsically labeled bovine milk as well as intrinsically labeled dairy product and cheese analogue. Healthy Caucasian women were fed a controlled diet for 4 d during the follicular phase of their menstrual cycle. With breakfast on the third day, participants ingested milk containing 44Ca (intrinsic) and 42CaCl2 (extrinsic) or dairy products containing 44Ca. Total feces were collected for 2 d prior to and 10 d after isotope ingestion. Polyethylene glycol was administered to monitor completeness of fecal collections. Total calcium was determined by atomic absorption spectrophotometry, and isotopic abundance was determined by high resolution fast atom bombardment mass spectrometry. Fractional absorption was determined as the difference between the administered isotopic dose and the quantity of 44Ca or 42Ca excreted in feces. The fractional absorption of calcium from milk was not affected by the method of labeling, lactose content, fermentation or the chemical form of calcium in dairy products or cheese analogue.

Absorption↗
PubMed1996

Outcome for patients colonised with Burkholderia cepacia in a Birmingham adult cystic fibrosis clinic and the end of an epidemic.

BACKGROUND: There has been increasing concern since 1979 about the emergence of Pseudomonas cepacia (Burkholderia cepacia) in patients with cystic fibrosis in the UK and elsewhere. Colonisation of the sputum has been shown to be associated with increased morbidity and mortality. Evidence suggests person to person transmission and some centres have segregated those colonised with B cepacia from other patients with cystic fibrosis. The outcome of patients colonised by B cepacia has been studied, together with the effects of strict segregation. METHODS: The outcome in 18 patients with sputum colonised by B cepacia was compared with that in age, sex, and severity matched controls with no evidence of B cepacia colonisation by a retrospective case note study. RESULTS: No difference between cases or controls were found in the 24 month period prior to colonisation by B cepacia in lung function, number of days in hospital, or outpatient visits. Colonisation led to an increased rate of loss of lung function and utilisation of hospital services. There was an increase in the numbers of transplants and deaths amongst the cases. Since 1992 there have been only three new cases of B cepacia colonisation and the incidence and prevalence of the organism has fallen dramatically since segregation commenced. CONCLUSIONS: B cepacia appears to be linked to the decline in colonised individuals. There was no evidence that colonisation occurred in patients declining for other reasons. B cepacia colonisation confers a worse prognosis than Pseudomonas aeruginosa alone. Segregation appears to limit the spread of the organism from infected individuals to other patients with cystic fibrosis.

Adult↗
PubMed1996

Biochemical and pharmacological characterization of multiple beta-endorphinergic antinociceptive systems in the rat periaqueductal gray.

Based on the differential abilities of the opioid antagonists naltrexone and D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP) to antagonize the antinociceptive action of beta-endorphin and morphine in the rat periaqueducatal gray (PAG), three pharmacologically distinct mechanisms were determined to mediate the antinociceptive effect of beta-endorphin. Two of these mechanisms are unique to beta-endorphin, possess a high affinity for CTP and can be discriminated based on their differential sensitivity to naltrexone. The third mechanism displays characteristics common to that activated by morphine. The results of radioligand binding studies were consistent with these observations. [125I]-beta-Endorphin labeled a population of sites in the PAG which (compared to those labeled by [3H]morphine) displayed a significantly higher affinity for CTP. In addition, a naltrexone-insensitive binding component was identified in the [125I]-beta-endorphin, but not [3H]morphine assays. Furthermore, comparable competitor affinities were determined across assays, suggesting an interaction of the radioligands with common PAG sites. A naltrexone-insensitive component to beta-endorphin antinociception also was identified in studies which evaluated the ability of the antagonist to shift the beta-endorphin dose-response curve. Interestingly, the ability of low doses of CTP and naltrexone to inhibit increasing doses of beta-endorphin was described by a U-shaped dose effect curve. The response to low and high, but not intermediate, doses of beta-endorphin were antagonized by picomole doses of both antagonists. As there was no evidence for allosteric interactions between [125I]-beta-endorphin binding sites in the PAG, it appears that beta-endorphin also may activate pain facilitory mechanisms which counterbalance its overall antinociceptive effect.

Amino Acid Sequence↗
PubMed1996

Reliability of infra-red tympanic thermometry (ITT).

BACKGROUND: The ability to take a patient's temperature quickly, easily, and accurately is desirable in a variety of situations. However, in order to have confidence in the values obtained, it is important to quantify the reliability of the measurement. PURPOSE: The purpose of this study, therefore, was to determine the reliability of temperature measurements obtained by infra-red tympanic membrane thermometers. METHOD: This study examined intra-tester, inter-tester and inter-instrument reliability using two commercially available tympanic membrane thermometers. Forty-four college students (mean age 20.2 +/- 3.6 yr) had their tympanic membrane temperature taken by two investigators, each using two different instruments (ThermoScan Pro-1 and FirstTemp Genius). RESULTS: The results indicated a statistically significant difference between testers for each instrument. Similarly, there was a significant statistical difference between instruments within each tester. Correlations were moderately high (r = 0.66-0.88) between testers for each instrument, whereas correlations within instruments varied considerably between testers (r = 0.35-0.78). The intra-tester reliability was good for both testers using both instruments with all coefficients of variation (CV) less than 2%. Statistically significant differences were found between testers for each instrument and between instruments for each tester. A 2 x 2 ANOVA revealed a significant main effect for Tester and a significant Tester x Instrument interaction. CONCLUSION: We conclude that individual investigators are able to obtain reliable temperature measures when using either the FirstTemp Genius or ThermoScan Pro-1 instruments under resting laboratory conditions. However, inter-tester and inter-instrument reliability should be considered when collecting or comparing data.

Adolescent↗
PubMed1996

Management and outcome of splenic injury: the results of a five-year statewide population-based study.

Although splenectomy was the preferred method of treating the injured spleen in the past, the methods of splenorrhaphy and nonoperative management have appeared to gain in popularity. The purpose of this study was to determine whether the management of splenic injuries has changed over the course of time and if there has been any differences in the morbidity and mortality associated with different methods of treatment. We retrospectively examined the discharge records from 2627 patients with splenic injuries from the North Carolina Discharge Database. There were 2258 adults and 369 pediatric patients for evaluation. The rate of nonoperative therapy increased from 33.9 per cent to 46.3 per cent over the 5 years of the study, whereas the rate of splenectomy decreased from 52.9 per cent to 43.4 per cent over the same time period. Splenorrhaphy was used in approximately 10 per cent of the injuries over the course of the entire study period. Adults treated nonoperatively required late operation 6.0 per cent (49/811) of the time. The pediatric late operation rate for nonoperative management was 0.4 per cent(1/231). Reoperation after splenorrhaphy was 2.9 per cent (7/240) for adult patients and 4.3 per cent (2/47) for pediatric patients. The majority of adults (57.2%) with an Injury Severity Score (ISS) < or = 15 were able to be cared for via nonoperative methods, whereas the majority of adults (66.4%) with an ISS > 15 required splenectomy. The majority of pediatric patients were able to be cared for in a nonoperative fashion in both the ISS < or = 15 (83.4%) and ISS > 15 (45.5%).

Adult↗
PubMed1996

Physiological, psychophysical, and psychological responses of firefighters to firefighting training drills.

This study was designed to describe the physiological, psychophysical, and psychological responses of firefighters to firefighting drills in a training structure containing live fires. Fifteen male firefighters, wearing standard turnout gear which resulted in full encapsulation, performed two firefighting tasks (advancing fire hose, chopping wood) while inside the training structure. Measurements of heart rate, tympanic membrane temperature, blood lactate, perceptions of respiration, mood, perceived exertion, and thermal sensation were obtained after 8 min of advancing fire hose, and again after 8 min of chopping. Heart rate and temperature increased significantly from baseline and from advancing hose to wood chopping, whereas blood lactate increased initially after advancing the hose and remained elevated at the end of the chopping task. At the completion of the test (both tasks), mean heart rate (182.3 b.min-1), temperature (40.1 degrees C, [104.1 degrees F]), and blood lactate (3.8 mMol) suggested that the firefighting tasks used in this study impose considerable physiological strain on firefighters. Psychophysical and psychological data mirrored the greater physiological strain following firefighting tasks performed in a hot environment while wearing full turnout gear.

Adult↗
PubMed1995

Inhibition of matrix metalloproteinases by hydroxamates containing heteroatom-based modifications of the P1' group.

In this study, structure-based drug design of matrix metalloproteinase inhibitors [human fibroblast collagenase (HFC), human fibroblast stromelysin (HFS), and human neutrophil collagenase (HNC)] was utilized in the development of potent hydroxamates which contain novel, heteroatom-based modifications of the P1' group. A series containing a P1' butyramide group resulted in a nanomolar potent and selective HNC inhibitor as well as a dual HFS/HNC inhibitor. Benzylic ethers with a four- or five-carbon methylene linker in the P1' position also produced nanomolar potent HFS/HNC inhibition and micromolar potent HFC inhibition as expected. Surprisingly, the phenolic ethers of the same overall length as the benzylic ethers showed nanomolar potencies against HFC, as well as HFS and HNC. The potency profile of the phenolic ethers was optimized by structure-activity relationships of the phenolic group and the C-terminal amide. These inhibitors may help elucidate the in vivo roles of matrix metalloproteinases in normal and disease states.

Crystallography, X-Ray↗