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D L Smith

Publications and source records attributed to D L Smith.

At least 127 records · Page 7Linked to original sources
PubMed1998

Age-related changes in human lens crystallins identified by HPLC and mass spectrometry.

Analysis of water-soluble crystallins from human lenses, ages 32 week fetal to 55 years has led to identification of the major modifications of the proteins comprising the lens. These modifications were identified by the masses of the proteins determined by electrospray ionization mass spectrometry after the proteins were separated by gel filtration and reversed phase high performance liquid chromatography. Examination of all the proteins isolated from the water soluble portion demonstrated that the major age-related modifications causing significant alteration in the molecular weights of the lens crystallins include truncation of the N-termini of beta B1, beta A3 and beta A1, and partial phosphorylation and C-terminal degradation of alpha-crystallins. N-terminal degradation of beta B1, beta A3 and beta A1 was evident in human lenses less than one year old, and the proportion of these truncated proteins became greater with age. Phosphorylation of alpha A- and alpha B-crystallins increased from the fetal to the 3 year old lens, but did not change with further aging. Minor components indicating truncation of the C-termini of alpha-crystallins were found in older lenses. In contrast to beta B1, beta A3 and beta A1, the masses of the major species of alpha A, alpha B, beta B2, beta A4, gamma S, gamma C, and gamma D did not change with aging. This suggested that the major modifications to these crystallins are limited to deamidation and possibly intra-molecular disulfide bonds. These data, in conjunction with the data in the accompanying manuscript, established deamidation as a common modification, since deamidation, which causes only a one dalton change in mass, is the only modification that is consistent with the absence of a detectable change in molecular weight and the observed increased acidity demonstrated in the two-dimensional gels of the accompanying paper. Other age related changes included a decrease in beta B3 (M(r) 24224), a major component of the fetal lens, which was not detected in lenses older than 3 years, and increases in the ratios of alpha B:alpha A and gamma S:gamma C.

Adolescent↗
PubMed1998

Deamidation and disulfide bonding in human lens gamma-crystallins.

Detailed analysis of the three gamma-crystallins present in the water-soluble portion of human lenses, gammaS, gammaD and gammaC, has identified disulfide bonding and deamidation as the major post-translational modifications of these crystallins. Chromatographic and mass spectrometric techniques were used to isolate and identify water-soluble gamma-crystallins from normal lenses, ages 32 week gestation, 0 day old, 4 day old, 19, 31, 45 and 55 year old. The amino acid sequences of the gamma-crystallins were confirmed and/or corrected by mass spectrometric analysis of peptides produced by enzymatic digestion or chemical fragmentation of the isolated crystallins. The molecular weight of peptides were also used to identify, locate and quantify modifications. Each of the gamma-crystallins had two disulfide bonds as well as several deamidated glutamine and asparagine residues. The extent of disulfide bond formation and deamidation appeared to increase with the age of the lens. This examination of normal human lens gamma-crystallins, the first detailed characterization of the gamma-crystallins, will provide a basis for comparison with modifications found in the water-insoluble portion and in cataractous lenses.

Adult↗
PubMed1998

Double-blind comparison of single-dose alatrofloxacin and cefotetan as prophylaxis of infection following elective colorectal surgery. Trovafloxacin Surgical Group.

BACKGROUND: Alatrofloxacin, the prodrug of trovafloxacin, is a novel fluoroquinolone antimicrobial agent with a broad spectrum, including activity against gram-positive and gram-negative aerobes and anaerobes. Its pharmacokinetic properties (long half-life, excellent tissue distribution, and good safety profile) suggest a role in surgical prophylaxis. This prospective, multicenter, double-blind trial compared alatrofloxacin with cefotetan, an approved drug for surgical prophylaxis, in reducing postoperative infections. METHODS: The efficacy and safety of a single 200-mg intravenous dose of alatrofloxacin were compared to a single 2-g intravenous dose of cefotetan in 492 patients undergoing elective colorectal surgery. The efficacy of alatrofloxacin as a prophylaxis for wound, intra-abdominal, or remote-site postoperative infectious complications was compared with cefotetan in 317 clinically evaluable patients; 161 received alatrofloxacin and 156 received cefotetan. The patients were monitored for infections and safety for 30 days postoperatively. RESULTS: No statistically significant between-treatment difference was detected in successful clinical response rates at the end of the study (72% for each group). The incidence of primary wound infections at the time of hospital discharge was also similar: 21% in patients treated with alatrofloxacin and 18% in those treated with cefotetan. Safety, established by the incidence of adverse events, did not differ statistically between the groups. CONCLUSIONS: A single intravenous dose of alatrofloxacin given within 4 hours prior to surgery was as effective as an intravenous dose of cefotetan in the prevention of postoperative infectious complications in patients undergoing elective colorectal surgery. The safety profiles of the two medications were similar.

Adolescent↗
PubMed1998

Trovafloxacin in the treatment of intra-abdominal infections: results of a double-blind, multicenter comparison with imipenem/cilastatin. Trovafloxacin Surgical Group.

BACKGROUND: Trovafloxacin, a new broad-spectrum fourth-generation quinolone, has in vitro activity against most gram-negative and gram-positive anaerobes and aerobes. Trovafloxacin is available as both an intravenous formulation, alatrofloxacin, and a single daily oral tablet. Excellent tissue pharmacokinetics and oral bioavailability suggest usefulness in the treatment of complicated intra-abdominal infections. Thus, the efficacy of alatrofloxacin followed by oral trovafloxacin was compared with the standard regimen of intravenous imipenem/cilastatin followed by oral amoxicillin/clavulanic acid in this prospective, multicenter, double-blind trial. METHODS: Patients were randomized to receive either 300 mg alatrofloxacin daily followed by 200 mg oral trovafloxacin daily or 1 g imipenem/cilastatin intravenously thrice daily followed by 500 mg oral amoxicillin/clavulanic acid thrice daily for up to 14 days following surgical intervention of a documented intra-abdominal infection. Efficacy was assessed at the end of therapy and at follow-up (day 30). RESULTS: At the end of the study, cure or improvement occurred in 83% (129/156) and 84% (127/152) of clinically evaluable patients in the trovafloxacin and comparative groups, respectively. Pathogen eradication rates, adverse-event profiles, and significant laboratory abnormalities were comparable between groups. CONCLUSION: Intravenous alatrofloxacin with or without oral trovafloxacin was as effective as intravenous imipenem/cilastatin followed by oral amoxicillin/clavulanic acid in complicated intra-abdominal infections.

Abdomen↗
PubMed1998

Regional microbiology of the cystic fibrosis lung: a post-mortem study in adults.

Although the majority of patients with cystic fibrosis (CF) become chronically colonized with Pseudomonas aeruginosa, the mode of acquisition of infection remains unclear. Epidemiological studies using genotyping techniques suggest that person-to-person transmission of this organism may occur. All these studies have utilized sputum or throat swab samples. We have studied the regional microbiological flora of the lungs of five CF patients at post-mortem and genotyped P. aeruginosa strains found therein and in the major airway. We have shown that although in most cases major airway secretions accurately reflect the peripheral lung flora, in cases of multiple strain carriage, major airway cultures may not reflect all strains present in the periphery of the lung. This finding has implications for the interpretation of epidemiological studies that use genotyping of strains from sputum and throat swab samples to assess possible routes of transmission.

Adult↗
PubMed1998

A short outpatient pulmonary rehabilitation programme: immediate and longer-term effects on exercise performance and quality of life.

Pulmonary rehabilitation is widely available in North America and parts of Europe for patients with chronic obstructive pulmonary disease (COPD). Here, we describe the feasibility and benefits of providing a comprehensive but cost effective pulmonary rehabilitation programme in a U.K. district general hospital. Two hundred and sixty-seven patients with respiratory disability were referred for pulmonary rehabilitation. Patients were assessed and recruited onto a 7 week outpatient-based pulmonary rehabilitation programme including elements of exercise and education without longer term maintenance. Exercise performance was measured by the shuttle walking test and a treadmill endurance test, and quality of life as measured by two disease specific questionnaires, the Breathing Problems Questionnaire and the Chronic Respiratory Disease Questionnaire. Of 267 patients referred between 1993 and 1996, 132 (110 with COPD) have completed rehabilitation and could be evaluated. For all graduates, shuttle distance increased by 58 m (27%) and treadmill endurance time increased by 15.9 min (294%). The Breathing Problems Questionnaire proved sensitive to changes in quality of life in some domains. The Chronic Respiratory Disease Questionnaire showed significant improvements in all domains in a sub-set of 57 patients who completed it. Longer term follow-up of 49 patients at a mean of 10.3 months following pulmonary rehabilitation revealed that previous gains in exercise performance and quality of life were maintained with improvements in shuttle walking distance of 33 m (14% over baseline) and endurance time of 16 min (280% over baseline). The Breathing Problems Questionnaire showed no overall change but the Chronic Respiratory Disease Questionnaire showed continued improvement in a small number of patients. We therefore concluded that a short outpatient based pulmonary rehabilitation programme without a maintenance element has produced significant gains in exercise performance and quality of life for 132 patients at a district general hospital in the U.K.

Aged↗
PubMed1998

Characterization of 5-HT1A receptor functional coupling in cells expressing the human 5-HT1A receptor as assessed with the cytosensor microphysiometer.

The functional activity of a series of 5-HT1A receptor ligands has been evaluated in a cell line expressing the human 5-HT1A receptor (h5-HT1A x CHO) using the agonist-stimulated increase in extracellular acidification rate, measured with the microphysiometer, as a functional assay. Both 5-CT and 8-OH-DPAT were potent agonists in stimulating an increase in extracellular acidification rate in h5-HT1A x CHO cells with estimated EC50 values of 1.2 and 7.8 nM, respectively. Additionally, these two 5-HT1A receptor agonists elicited a similar maximum response. Concentration-dependent agonist activity was also observed in the presence of buspirone, ipsapirone, BMY7378, NAN-190 and WAY100135, and each of these compounds behaved as partial 5-HT1A receptor agonists. The selective 5-HT1A receptor antagonist WAY100635 produced a potent (IC50, 2.3 nM) and complete block of the 8-OH-DPAT-stimulated response. An evaluation of the inhibitory activity of a series of 5-HT1A receptor antagonists produced the following rank order of potency; WAY100635 > LY206130 (IC50, 7.1 nM) > WAY100135 (30.8 nM) > pindolol (76.2 nM) > (-)UH-301 (92.8 nM). Parallel studies on the inhibition of forskolin-stimulated adenylyl cyclase activity in hS-HT1A x CHO cells revealed that agonist potencies were generally similar between the two functional assays and were in good agreement with the estimated 5-HT1A receptor binding affinities. However, the relative efficacies determined for the partial agonists in the cAMP assay were substantially greater than those observed with the microphysiometer. Finally, antagonists were considerably weaker in the cAMP assay compared with the microphysiometer. The evaluation of 5-HT1A ligands using the microphysiometer, which represents a very distinct indice of 5-HT1A receptor function compared with the cAMP assay, results in a different profile of functional activity.

Animals↗
PubMed1998

Design, synthesis, and biological evaluation of spider toxin (argiotoxin-636) analogs as NMDA receptor antagonists.

PURPOSE: Twelve synthetic spider toxin analogs were prepared in an effort to better understand the structure-activity relationships of the polyamine portion of argiotoxin-636 (Arg-636), a noncompetitive NMDA receptor (NMDAR) antagonist. METHODS: The 1,13-diamino-4,8-diazatridecane portion of the side chain of Arg-636 was systematically modified in an effort to further our knowledge of the structural requirements for the alkyl linker spacing between the amine nitrogens. Systematic isosteric replacement of each of the amine nitrogens in the polyamine moiety with either oxygen or carbon provided a series of compounds which were evaluated in vitro for NMDAR antagonist activity. RESULTS: One-half of the heteroatoms found in Arg-636 were removed to provide analogs which maintained in vitro potency below 1 microM. However, these simplified analogs produced similar or more pronounced effects on the cardiovascular system than Arg-636 in vivo. CONCLUSIONS: In this set of analogs, a minimum of three basic nitrogens in the side chain was required for maximum potency as NMDAR antagonists. Isosteric nitrogen substitutions in the polyamine chain reduced the in vitro potency of these analogs. An analog binding-conformation model was proposed to rationalize the inactivity of these isosterically substituted analogs.

Animals↗
PubMed1998

Solution structure of a zinc domain conserved in yeast copper-regulated transcription factors.

The three dimensional structure of the N-terminal domain (residues 1-42) of the copper-responsive transcription factor Amtl from Candida glabrata has been determined by two-dimensional 1H-correlated nuclear magnetic resonance (NMR) methods. The domain contains an array of zinc-binding residues (Cys-X2-Cys-X8-Cys-X-His) that is conserved among a family of Cu-responsive transcription factors. The structure is unlike those of previously characterized zinc finger motifs, and consists of a three-stranded antiparallel beta-sheet with two short helical segments that project from one end of the beta-sheet. Conserved residues at positions 16, 18 and 19 form a basic patch that may be important for DNA binding.

Amino Acid Sequence↗
PubMed1998

Selected physiological and psychological responses to live-fire drills in different configurations of firefighting gear.

The purpose of this study was to examine selected physiological and psychological responses to strenuous live-fire drills in different configurations of protective firefighting gear. Career firefighters (n = 10) performed three sets of firefighting drills in a training structure that contained live fires in two different configurations of firefighting gear. On separate days subjects wore: (a) the NFPA 1500 (1987) standard configuration, and (b) a hip-boot configuration of the firefighting gear. Physiological and psychological measurements were recorded pre-activity and at the end of each trial. Repeated measures ANOVA revealed a strong trend for performance time to be greater in the 1500 gear than in the hip-boot gear. There was a significant Time x Gear interaction for tympanic membrane temperature, with temperature being greater in the 1500 gear. Perceptions of effort and thermal sensations were also greater in the 1500 gear than in the hip-boot configuration of the gear. There was little difference in mean performance on cognitive function measures between the two gear configurations, but there was greater variability in performance in the 1500 gear. These data suggest that performing strenuous firefighting drills in the current NFPA 1500 standard configuration results in longer performance time, greater thermal strain, and greater perception of effort and thermal sensation.

Adult↗
PubMed1998

Emerging treatments for epidemic (AIDS-related) Kaposi's sarcoma.

Kaposi's sarcoma (KS) is an opportunistic tumor that develops with increased frequency (100,000-fold) after HIV infection. KS causes significant morbidity from mucocutaneous involvement and mortality from complications of visceral sites of disease such as the lungs, gastrointestinal tract, and the liver. Progressive unraveling of the KS pathogenesis has lead to the development of novel therapeutic approaches. Newest therapies are first evaluated in patients with limited tumor burden. These include: 1) inhibitors of angiogenesis such as vascular endothelial growth factor signaling inhibitor (SU 5416), and several other inhibitors of angiogenesis such as the dipeptide IM 862, TNP-470, Col-3, and thalidomide; 2) topical and systemic retinoids; 3) antiviral agents specific for Kaposi's sarcoma herpesvirus and human herpesvirus-8, or HIV; and 4) pregnancy-related factors. Patients with advanced disease such as widespread mucocutaneous disease, lymphedema, and visceral disease are treated most effectively with cytotoxic agents. The most active agents include liposomal anthracyclines, paclitaxel, vinca alkaloids, and bleomycin. The combination of liposomal anthracyclines and paclitaxel, with and without the most promising biologicals, should now be studied to further reduce the toxicity, and enhance the antitumor effects. Furthermore, identification of risk factors for KS should serve to explore prophylactic therapies.

AIDS-Related Opportunistic Infections↗
PubMed1998

A gene coding for tomato fruit beta-galactosidase II is expressed during fruit ripening. Cloning, characterization, and expression pattern.

beta-Galactosidases (EC 3.2.1.23) constitute a widespread family of enzymes characterized by their ability to hydrolyze terminal, nonreducing beta-D-galactosyl residues from beta-D-galactosides. Several beta-galactosidases, sometimes referred to as exo-galactanases, have been purified from plants and shown to possess in vitro activity against extracted cell wall material via the release of galactose from wall polymers containing beta(1-->4)-D-galactan. Although beta-galactosidase II, a protein present in tomato (Lycopersicon esculentum Mill.) fruit during ripening and capable of degrading tomato fruit galactan, has been purified, cloning of the corresponding gene has been elusive. We report here the cloning of a cDNA, pTombetagal 4 (accession no. AF020390), corresponding to beta-galactosidase II, and show that its corresponding gene is expressed during fruit ripening. Northern-blot analysis revealed that the beta-galactosidase II gene transcript was detectable at the breaker stage of ripeness, maximum at the turning stage, and present at decreasing levels during the later stages of normal tomato fruit ripening. At the turning stage of ripeness, the transcript was present in all fruit tissues and was highest in the outermost tissues (including the peel). Confirmation that pTombetagal 4 codes for beta-galactosidase II was derived from matching protein and deduced amino acid sequences. Furthermore, analysis of the deduced amino acid sequence of pTombetagal 4 suggested a high probability for secretion based on the presence of a hydrophobic leader sequence, a leader-sequence cleavage site, and three possible N-glycosylation sites. The predicted molecular mass and isoelectric point of the pTombetagal 4-encoded mature protein were similar to those reported for the purified beta-galactosidase II protein from tomato fruit.

Amino Acid Sequence↗
PubMed1998

Oligohistidine tag mutagenesis of the lambda holin gene.

Holins are a diverse group of small integral membrane proteins elaborated by bacteriophages to lyse bacterial hosts and effect release of progeny phages in a precisely timed manner. Recently, the holin S gene of phage lambda was overexpressed and the holin protein was purified to homogeneity by means of an oligohistidine tag procedure and immobilized metal affinity chromatography (D. L. Smith, D. K. Struck, J. M. Scholtz, and R. Young, J. Bacteriol. 180:2531-2540, 1998). Numerous locations within the S gene were tested as sites for an oligohistidine-tag-encoding insertion which preserves holin function. The lysis phenotypes of these alleles, expressed from moderate-copy-number transactivation plasmids, were characterized. A striking class of mutants, previously referred to as early-dominant, have been found to have severe lysis defects but are fully functional in the presence of wild-type protein. Results presented here reveal that the early-dominance phenotype is independent of S107 inhibitor function. The results provide insight into the mechanism of hole formation and indicate that, while oligomerization is required in the pathway to hole formation, a nucleation event may also be required.

Alleles↗
PubMed1998

Purification and biochemical characterization of the lambda holin.

Holins are small phage-encoded cytoplasmic membrane proteins, remarkable for their ability to make membranes permeable in a temporally regulated manner. The purification of S105, the lambda holin, and one of the two products of gene S is described. Because the wild-type S105 holin could be only partially purified from membrane extracts by ion-exchange chromatography, an oligohistidine tag was added internally to the S105 sequence for use in immobilized metal affinity chromatography. An acceptable site for the tag was found between residues 94 and 95 in the highly charged C-terminal domain of S. This allele, designated S105H94, had normal lysis timing under physiological expression conditions. The S105H94 protein was overproduced, purified, and characterized by circular dichroism spectroscopy, which revealed approximately 40% alpha-helix conformation, consistent with the presence of two transmembrane helices. The purified protein was then used to achieve release of fluorescent dye loaded in liposomes in vitro, whereas protein from an isogenic construct carrying an S mutation known to abolish hole formation was inactive in this assay. These results suggest that S is a bitopic membrane protein capable of forming aqueous holes in bilayers.

Amino Acid Sequence↗
PubMed1998

Effects of short-term inactivity on glucose tolerance, energy expenditure, and blood flow in trained subjects.

The purpose of this investigation was to examine the effects of 7-10 days of inactivity (IA) on glucose tolerance (GT), resting metabolic rate (RMR), thermic effect of a meal (TEM), and limb blood flow in endurance-trained men. Eight highly trained (peak O2 consumption 64 +/- 2 ml . kg-1 . min-1) endurance athletes participated in this study involving two identical test days, one approximately 24 h after a normal training bout (Tr) and the second after 7-10 days of IA. The following tests were conducted at each visit: 75-g oral glucose tolerance test (OGTT), RMR, and TEM and measurements of calf and forearm blood flow (BF) by using venous occlusive plethysmography. Body weight remained unchanged during this short period of IA (Tr, 78.5 +/- 1 kg; IA, 78.7 +/- 1 kg). The area under the glucose and insulin curves increased 65% (Tr, 3,375 +/- 877 vs. IA, 5,559.4 +/- 621 mg . dl-1 . 180 min-1) and 73% (Tr, 2,182.5 +/- 270 vs. IA, 3,793.1 +/- 739 microU . ml-1 . 180 min-1) after IA, respectively (P < 0.01). RMR decreased significantly (4%; 1.5 +/- 0. 02 vs. 1.44 +/- 0.02 kcal/min; P < 0.05) and respiratory exchange ratio during the OGTT increased (4%, 0.812 +/- 0.011 vs. 0.842 +/- 0. 009; P < 0.05) after IA, whereas TEM increased similarly in the Tr and IA states. In the Tr state, mean calf BF increased by 22% (3.17 +/- 0.22 vs. 3.87 +/- 0.38 ml . 100 ml-1 . min-1; P < 0.05) during the OGTT but remained unchanged after IA, whereas no differences at rest or during OGTTs existed between the two conditions for forearm BF. Incremental insulin area above fasting during the OGTT was correlated with mean calf BF in the Tr (r = 0.76, P < 0.05) and IA (r = 0.72, P < 0.05) states. In conclusion, 7-10 days of IA results in a deterioration in GT and a reduction in RMR. After glucose ingestion, calf BF was elevated compared with resting levels in the Tr state but was unchanged in the IA state; however, limb BF was not related to GT or RMR. Thus our findings raise questions regarding the relative contribution of BF in modulating glucose tolerance and energy expenditure in endurance athletes in their habitual Tr or IA state.

Adult↗
PubMed1998

Local structure and dynamics in proteins characterized by hydrogen exchange and mass spectrometry.

Amide hydrogen exchange rates, determined by NMR spectroscopy, have become an important tool that is often used to investigate structure and dynamics of small proteins. Recent developments in mass spectrometry and sample handling methods make possible measurement of deuterium levels at peptide amide linkages in polypeptides. The ability to make these measurements has led to development of the protein fragmentation/mass spectrometry approach for determining amide hydrogen exchange rates in short segments of intact proteins following their incubation in D2O. Partially deuterated proteins are proteolytically fragmented into peptides whose molecular weights are determined by on-line liquid chromatography/mass spectrometry. Deuterium levels, which are determined from the molecular weights of the peptic fragments, can be used to determine amide hydrogen exchange rates. Details of the protein fragmentation/mass spectrometry approach, along with a brief review of the theory of amide hydrogen exchange, are described. The ability to detect and locate minor structural differences in proteins by the protein fragmentation/mass spectrometry approach is illustrated using oxidized and reduced cytochrome c. These results show that oxidation of iron has little effect on the N- and C-terminal regions, but significantly destabilizes the interior regions of cytochrome c. The ability to detect localized unfolding in large proteins is illustrated with aldolase that was equilibrated in acid. Despite the success achieved by NMR spectroscopy for determining amide hydrogen exchange rates, mass spectrometry is advantageous because it permits studies of large proteins, requires only picomoles of protein, and provides a direct measure of structural heterogeneity.

Deuterium↗
PubMed1998

The lambda holin accumulates beyond the lethal triggering concentration under hyperexpression conditions.

Most bacteriophages terminate infection by creating lesions in the cytoplasmic membrane, which not only cause immediate cell death but also allow escape of a phage-encoded endolysin. Destruction of the peptidoglycan and cell lysis follows very rapidly, allowing efficient release of the progeny virions. These membrane lesions are formed by a small integral membrane protein called a holin. Holins have highly charged carboxyl-termini that are thought to have two transmembrane alpha-helical domains. Holins are believed to oligomerize and form large holes in the inner membrane. The prototype holin is the S protein from bacteriophage lambda. Scheduling of the lytic event is determined in part by the "structure directed initiation" or sdi translational control region. Inductions of S, cloned under a variety of native and nonnative promoters but with native translational control, resulted in cell lysis at about 1000 molecules of holin per cell, and thus do not produce biochemically useful amounts of S protein. By utilizing a plasmid-based system with the T7 RNA polymerase promoter in tandem with a consensus ribosome binding site, Coomassie blue-detectable quantities of S protein were obtained upon induction, corresponding to an approximately 100-fold increase over the normal lethal level of holin. Characterization of this expression system is presented and discussed with respect to the current model of holin function.

Amino Acid Sequence↗