Biomedical subjects

David L Smith

Publications and source records attributed to David L Smith.

At least 55 records · Page 3Linked to original sources

PubMed2004

Co-carriage rates of vancomycin-resistant Enterococcus and extended-spectrum beta-lactamase-producing bacteria among a cohort of intensive care unit patients: implications for an active surveillance program.

OBJECTIVE: To assess the co-colonization rates of extended-spectrum beta-lactamase (ESBL)-producing bacteria and vancomycin-resistant Enterococcus (VRE) obtained on active surveillance cultures. DESIGN: Prospective cohort study. SETTING: Medical and surgical intensive care units (ICUs) of a tertiary-care hospital. PATIENTS: Patients admitted between September 2001 and November 2002 to the medical and surgical ICUs at the University of Maryland Medical System had active surveillance perirectal cultures performed. Samples were concurrently processed for VRE and ESBL-producing bacteria. RESULTS: Of 1,362 patients who had active surveillance cultures on admission, 136 (10%) were colonized with VRE. Among these, 15 (positive predictive value, 11%) were co-colonized with ESBL. Among the 1,226 who were VRE negative, 1,209 were also ESBL negative (negative predictive value, 99%). Among the 1,362 who had active surveillance cultures on admission, 32 (2%) were colonized with ESBL. Among these, 15 (47%) were co-colonized with VRE. Of the 32 patients colonized with ESBL, 10 (31%) had positive clinical cultures for ESBL on the same hospital admission. For these 10 patients, the surveillance cultures were positive an average of 2.7 days earlier than the clinical cultures. CONCLUSIONS: Patients who are colonized with VRE can also be co-colonized with other antibiotic-resistant bacteria such as ESBL-producing bacteria. Our study is the first to measure co-colonization rates of VRE and ESBL-producing bacteria. Isolating VRE-colonized patients would isolate 47% of the ESBL-colonized patients without the need for further testing. Hence, active surveillance for VRE should also theoretically diminish the amount of patient-to-patient transmission of ESBL-producing bacteria.

Carrier State↗

PubMed2004

Thermal stability of human alpha-crystallins sensed by amide hydrogen exchange.

The alpha-crystallins, alphaA and alphaB, are major lens structural proteins with chaperone-like activity and sequence homology to small heat-shock proteins. As yet, their crystal structures have not been determined because of the large size and heterogeneity of the assemblies they form in solution. Because alpha-crystallin chaperone activity increases with temperature, understanding structural changes of alpha-crystallin as it is heated may help elucidate the mechanism of chaperone activity. Although a variety of techniques have been used to probe changes in heat-stressed alpha-crystallin, the results have not yet yielded a clear understanding of chaperone activity. We report examination of native assemblies of human lens alpha-crystallin using hydrogen/deuterium exchange in conjunction with enzymatic digestion and analysis by mass spectrometry. This technique has the advantage of sensing structural changes along much of the protein backbone and being able to detect changes specific to alphaA and alphaB in the native assembly. The reactivity of the amide linkages to hydrogen/deuterium exchange was determined for 92% of the sequence of alphaA and 99% of alphaB. The behavior of alphaA and alphaB is remarkably similar. At low temperatures, there are regions at the beginning of the alpha-crystallin domains in both alphaA and alphaB that have high protection to isotope exchange, whereas the C termini offer little protection. The N terminus of alphaA also has low protection. With increasing temperatures, both proteins show gradual unfolding. The maximum percent change in exposure with increasing temperatures was found in alphaA 72-75 and alphaB 76-79, two regions considered critical for chaperone activity.

Amides↗

PubMed2004

The reaction of alpha-crystallin with the cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) demonstrates close proximity of the C termini of alphaA and alphaB in the native assembly.

The chaperone-like activity of human lens alpha-crystallin in inhibiting the aggregation of denatured proteins suggests a role for alpha-crystallin in cataract prevention. Although a variety of techniques have generated structural information relevant to its chaperone-like activity, the size and heterogeneity of alpha-crystallin have prevented determination of its crystal structure. Even though synthetic cross-linkers have provided considerable information about protein structures, they have not previously been used to study the proximity and orientation of subunits within human alpha-crystallin. Cross-linkers provide structural insight into proteins by binding the side chains of amino acids within close proximity. To identify the cross-linked residues, the modified protein is digested and the resulting peptides are analyzed by mass spectrometry. Analysis of products from the reaction of alpha-crystallin with 3,3'dithiobis(sulfosuccinimidyl propionate), DTSSP, identified several modifications to both alphaA and alphaB. The most structurally informative of these modifications was a cross-link between lysine 166 of alphaA and lysine 175 of alphaB. This cross-link provides experimental evidence supporting theoretical structural models that place the C termini of alphaA and alphaB within close proximity in the native aggregate.

Amino Acid Sequence↗

PubMed2004

Long-term survival after an aggressive surgical approach in patients with breast cancer hepatic metastases.

BACKGROUND: Metastatic breast cancer is generally believed to be associated with a poor prognosis. Therapeutic advances over the past two decades, however, have resulted in improved outcomes for selected patients with limited metastatic disease. METHODS: Between March 1991 and October 2002, 31 patients had hepatic resection for breast cancer metastases limited to the liver. Clinical and pathologic data were collected prospectively from breast and hepatobiliary databases. RESULTS: Median age of patients was 46 years (range, 31 to 70). Liver metastases were solitary in 20 patients and multiple in 11 patients. Median size of the largest liver metastasis was 2.9 cm (range, 1 to 8). Major liver resections (three or more segments resected) were performed in 14 patients, whereas minor resections (fewer than three segments resected) with or without radiofrequency ablation (RFA) were performed in 17 patients. No postoperative mortality occurred. Of the 31 patients, 27 (87%) received either preoperative or postoperative systemic therapy as treatment for metastatic disease. The median survival was 63 months; a single patient died within 12 months of hepatic resection. The overall 2- and 5-year survival rates were 86% and 61%, respectively, whereas the 2- and 5-year disease-free survival rates were 39% and 31%, respectively. No treatment- or patient-specific variables were found to correlate with survival rates. CONCLUSIONS: In selected patients with liver metastases from breast cancer, an aggressive surgical approach is associated with favorable long-term survival. Hepatic resection should be considered a component of multimodality treatment of breast cancer in these patients.

Adult↗

PubMed2003

Solution structure and dynamics of a heat shock protein assembly probed by hydrogen exchange and mass spectrometry.

The solution conformation and dynamics of the 16.9 kDa small heat shock protein from wheat have been studied using a combination of hydrogen/deuterium exchange, proteolytic digestion, and mass spectrometry. At room temperature, HSP16.9 exists as a dodecameric assembly. Regions of HSP16.9 that form extensive and essential intersubunit contacts in the assembly, including residues 1-40 and 131-151, show little or no protection against hydrogen/deuterium exchange after incubation in D(2)O for 5 s. The high levels of hydrogen/deuterium exchange indicate that these regions have experienced large conformational fluctuations in solution, breaking intersubunit contacts and exposing buried amide hydrogens to solvent. When HSP16.9 is pulse labeled for 10 ms, residues 1-40 and 131-151 are substantially more protected than they are after 5 s. Thus, the breaking of intersubunit contacts occurs on a time scale between 10 milliseconds and 5 s. At 42 degrees C, HSP16.9 exists in a suboligomeric form. When the intrinsic temperature dependence of hydrogen/deuterium exchange is taken into account, exchange patterns at 25 and 42 degrees C are identical within experimental error, suggesting that the conformation of individual HSP16.9 subunits is the same in both the dodecameric and subdodecameric forms. Significant protection is seen in regions that form the dimeric interface, suggesting that the stable suboligomeric form is a dimer. Taken together, these results suggest that heat activation of HSP16.9 occurs by shifting the dodecamer <--> dimer equilibrium in favor of free dimers. The conformation of the dimers themselves does not appear to be altered with an increase in temperature.

Amino Acid Sequence↗

PubMed2003

Antisense suppression of a beta-galactosidase gene (TB G6) in tomato increases fruit cracking.

Antisense suppression of a tomato beta-galactosidase gene (TBG6) was used to study its role in fruit development, cell wall-modification, and fruit firmness. TBG6 mRNA is highly abundant during the early stages of fruit development, but the levels decline sharply after the breaker stage with the start of the respiratory climacteric and a concomitant increase in ethylene production. Two antisense lines were obtained with significantly reduced levels of TBG6 mRNA at all stages of fruit development. At 30 d after pollination (dap), TBG6 mRNA levels were reduced by up to 98% and 88% in lines 6-2 and 6-10, respectively. Morphological phenotypes observed in the antisense lines included increased fruit cracking, reduced locular space, and a doubling in the thickness of the fruit cuticle. Two biochemical changes in antisense lines, compared with wild-type lines, were a reduction of exo-galactanase activity at the breaker +3 d stage and a reduction in the cell wall galactosyl content at the 20 dap stage. In addition, transgenic lines exhibited a 35-39% reduction in fruit firmness at the 20 dap stage, but their texture was equivalent to the wild type at 30 dap and beyond. Although the exact function of the TBG6 product is still unknown, these results implicate an important role for this enzyme in early fruit growth and development in tomato.

Cell Wall↗

PubMed2003

Quaternary structure of aldolase leads to differences in its folding and unfolding intermediates.

Pulsed hydrogen exchange mass spectrometry has been used to investigate folding of rabbit muscle aldolase, an alpha/beta-barrel protein exhibiting the classic TIM structure. Aldolase unfolded in GdHCl refolded as the denaturant concentration was reduced by dialysis. Samples withdrawn during dialysis were pulse-labeled with deuterium to identify unfolded regions in structural forms highly populated during the folding process. Intact, labeled aldolase was digested into fragments, which were analyzed by HPLC electrospray ionization mass spectrometry to detect the H/D exchange along the aldolase backbone. For some concentrations of GdHCl, bimodal distributions of deuterium were found for most peptic fragments, indicating that regions represented by these fragments were either unfolded or folded in the intact polypeptide prior to labeling. The extent of folding was determined from these mass spectra, as well as by CD (220 nm) and enzymatic activity. These results show that folding to the active form involves three domains and two intermediates. Approximately 110 residues fold to highly compact forms in each step. These results also show that each folding domain includes widely separated regions of the backbone. When compared with the results of a previous study of aldolase unfolding, these results show that the folding and unfolding domains include most of the same residues. However, three short segments change domains depending on whether the process is folding or unfolding. These changes are attributed to the very stable quaternary structure of rabbit muscle aldolase.

Animals↗

PubMed2003

Mass spectrometric quantification of acetylation at specific lysines within the amino-terminal tail of histone H4.

Electrospray ionization mass spectrometry, a leading method for the quantification of biomolecules, is useful for the analysis of posttranslational modifications of proteins. Here we describe a mass spectrometric approach for determining levels of acetylation at individual lysine residues within the amino-terminal tail of histone H4. Because of the high density of acetylatable lysine residues within this short span of amino acids, collision-induced dissociation tandem mass spectrometry was required. In addition, it was necessary to develop an algorithm to determine the fraction of acetylation at specific lysine residues from fragment ions containing more than one lysine residue. This is the first report of direct measurement of endogeneous levels of acetylation at individual lysine residues within the amino-terminal tail of yeast histone H4 and is the first use of tandem mass spectrometry for quantification of peptides containing multiple sites of modification.

Acetylation↗

PubMed2003

Downsizing improves sensitivity 100-fold for hydrogen exchange-mass spectrometry.

This paper describes a method that substantially improves the sensitivity of high-performance liquid chromatography hydrogen exchange-mass spectrometry (HPLC HX MS). The success of this method relies on using a capillary HPLC column (0.1mm IDx5cmL) to increase the sensitivity of electrospray ionization, while keeping analysis times short to minimize hydrogen/deuterium (H/D) exchange. A small, immobilized pepsin column and a capillary C18 trap were included in the capillary HPLC MS system to provide rapid digestion, peptide concentration, and desalting while maintaining slow H/D exchange conditions. To minimize the analysis time, dead volumes and capacities of all components were optimized. Fully deuterated cytochrome c and its fully deuterated peptic peptides were used to evaluate deuterium recovery at amide linkages. The deuterium recovery measured at low flow rates using this system spanned a range of 66-77% (average of 71%), which was similar to the range measured for a much larger system (67-80%, average 75%). Signal levels of most peptides for the downsized system increased by about 100-fold compared with the signal for the larger system. These results greatly strengthen the HPLC HX MS technique for studies where the quantity of protein is small.

Amides↗

PubMed2003

Expression of betaA2-crystallin in human lenses.

Evidence of betaA2-crystallin expression has been detected in human lenses. The protein, which co-elutes with betaA1/A3 from reversed phase HPLC separation of beta(L)-crystallins, accounts for 1-2% of the lens crystallins. Its molecular mass, M(r) 22 006, is consistent with the cDNA deduced sequence with addition of acetylation at the N-terminal serine residue. Approximately 20% of the protein is phosphorylated at Ser30.

Acetylation↗

PubMed2003

Human beta-crystallins modified by backbone cleavage, deamidation and oxidation are prone to associate.

Information about beta-crystallins and their post-translational modifications has been scarce because of difficulties in isolating the individual beta-crystallins. These difficulties arise because the beta-crystallin sequences are highly homologous and because beta-crystallins undergo many age-related modifications that lead to a variety of molecular masses and a range of acidities for each crystallin. In this study, human beta-crystallins were isolated using several steps of chromatography both before and after two-dimensional gel electrophoresis. Many previously unidentified in vivo modifications, including deamidations among all beta-crystallins except betaB3, truncation of betaA3, betaB1 and betaA4, and oxidation of some methionines and tryptophans were located among the isolated beta-crystallins. Many modifications occurred before age 20 with modest increases in modification for beta-crystallins from lenses 20-87 years old. The tendency of the modified beta-crystallins to form non-covalent complexes was evident from their chromatographic behaviour. The presence in these complexes of betaB2-crystallin, the least modified and most soluble of the beta-crystallins, points to a possible role for betaB2 in solubilizing the more heavily modified beta-crystallins. The greater solubility of beta-crystallins compared with alpha- and gamma-crystallins in aging lenses may be due to beta-crystallin modifications and their non-covalent associations.

Adult↗

PubMed2003

Low-volume resuscitation with a polymerized bovine hemoglobin-based oxygen-carrying solution (HBOC-201) provides adequate tissue oxygenation for survival in a porcine model of controlled hemorrhage.

BACKGROUND: We have shown in a previous work that HBOC-201 is able to reverse anaerobic metabolism at low volumes in a porcine model of controlled hemorrhage. On the basis of these results, we hypothesize that low-volume resuscitation with HBOC-201 in a porcine model of controlled hemorrhage provides adequate tissue oxygenation to limit end-organ damage and allow for survival of the animal. METHODS: Twenty-four Yorkshire swine (55-65 kg) were rapidly hemorrhaged to a mean arterial pressure (MAP) of 30 mm Hg, maintained hypotensive for 45 minutes, and then divided into four groups. The first group, Shed Blood (BL), was resuscitated with shed blood to baseline MAP. A second group, Shed Blood (60), underwent resuscitation for four hours at an MAP of 60 mm Hg with shed blood. The third group, LR + Blood, was resuscitated with lactated Ringer's (maximum, 40 mL/kg) followed by shed blood to baseline MAP. The final group, HBOC (60), underwent resuscitation for 4 hours at an MAP of 60 mm Hg with HBOC-201. Hemodynamic variables, urine output, blood gas analyses, lactate levels, and jejunal oximetry were followed throughout the experiment. Animals were allowed to survive and underwent necropsy on postinjury day 3. Histologic comparisons were made. Data were analyzed using analysis of variance/Duncan's multiple range test. RESULTS: All animals survived the hemorrhage/resuscitation. One animal in the LR + Blood group died on postinjury day 1. Heart rate, MAP, and arterial pH were similar between groups. Cardiac output was significantly lower throughout resuscitation in the HBOC (60) group. Jejunal oximetry was similar throughout the experiment in all groups, revealing a decline in Po2 during hemorrhage and return to baseline or near baseline during resuscitation. There was no evidence of renal dysfunction. Histologically, one animal in the LR + Blood group and four of six animals in the HBOC (60) group demonstrated mild hepatocellular damage. All other tissues examined were found to have no significant abnormalities. Elevations in serum aspartate aminotransferase levels were noted when comparing the HBOC (60) group to the Shed Blood (BL) and Shed Blood (60) groups on day 2. Significant decreases in hemoglobin levels were noted in the HBOC (60) group compared with all other groups beginning on day 2. CONCLUSION: Low-volume resuscitation with HBOC-201 provides adequate tissue oxygenation for survival in a porcine model of controlled hemorrhagic shock with no long-term organ dysfunction identified. Although some animals did show mild hepatocellular damage with elevations of aspartate aminotransferase at day 2, these findings did not appear to have clinical relevance, and the enzyme elevations were trending toward normal by the third postoperative day. Decreases in hemoglobin levels at the later time points were expected, given the half-life of the product.

Analysis of Variance↗

PubMed2003

Methylation and carbamylation of human gamma-crystallins.

Accessible sulfhydryls of cysteine residues are likely sites of reaction in long-lived proteins such as human lens crystallins. Disulfide bonding between cysteines is a major contributor to intermolecular cross-linking and aggregation of crystallins. A recently reported modification of gammaS-crystallins, S-methylation of cysteine residues, can prevent disulfide formation. The aim of this study was to determine whether cysteines in gammaC-, gammaD-, and gammaB-crystallins are also S-methylated. Our data show that all the gamma-crystallins are S-methylated, but only at specific cysteines. In gammaD-crystallin, methylation is exclusively at Cys 110, whereas in gammaC- and gammaB-crystallins, the principal methylation site is Cys 22 with minor methylation at Cys 79. gammaD-crystallin is the most heavily methylated gamma-crystallin. gammaD-Crystallins from adult lenses are 37%-70% methylated, whereas gammaC and gammaB are approximately 12% methylated. The specificity of gamma-crystallin methylation and its occurrence in young clear lenses supports the idea that inhibition of disulfide bonding by S-methylation may play a protective role against cataract. Another modification, not reported previously, is carbamylation of the N termini of gammaB-, gammaC-, gammaD-crystallins. N-terminal carbamylation is likely a developmentally related modification that does not negatively impact crystallin function.

Adolescent↗

PubMed2003

Microbial diversity of biofilms in dental unit water systems.

We investigated the microbial diversity of biofilms found in dental unit water systems (DUWS) by three methods. The first was microscopic examination by scanning electron microscopy (SEM), acridine orange staining, and fluorescent in situ hybridization (FISH). Most bacteria present in the biofilm were viable. FISH detected the beta and gamma, but not the alpha, subclasses of Proteobacteria: In the second method, 55 cultivated biofilm isolates were identified with the Biolog system, fatty acid analysis, and 16S ribosomal DNA (rDNA) sequencing. Only 16S identified all 55 isolates, which represented 13 genera. The most common organisms, as shown by analyses of 16S rDNA, belonged to the genera Afipia (28%) and Sphingomonas (16%). The third method was a culture-independent direct amplification and sequencing of 165 subclones from community biofilm 16S rDNA. This method revealed 40 genera: the most common ones included Leptospira (20%), Sphingomonas (14%), Bacillus (7%), Escherichia (6%), Geobacter (5%), and Pseudomonas (5%). Some of these organisms may be opportunistic pathogens. Our results have demonstrated that a biofilm in a health care setting may harbor a vast diversity of organisms. The results also reflect the limitations of culture-based techniques to detect and identify bacteria. Although this is the greatest diversity reported in DUWS biofilms, other genera may have been missed. Using a technique based on jackknife subsampling, we projected that a 25-fold increase in the number of subclones sequenced would approximately double the number of genera observed, reflecting the richness and high diversity of microbial communities in these biofilms.

Acridine Orange↗

PubMed2002

Alpha-crystallin regions affected by adenosine 5'-triphosphate identified by hydrogen-deuterium exchange.

ATP interaction with lens alpha-crystallins leading to enhanced chaperone activity is not yet well understood. One model for chaperone activity of small heat shock proteins proposes that ATP causes small heat shock proteins to release substrates, which are then renatured by other larger heat shock proteins. A similar role has been proposed for ATP in alpha-crystallin chaperone activity. To evaluate this model, ATP-induced structural changes of native human alpha-crystallin assemblies were determined by hydrogen-deuterium exchange. In these experiments, hydrogen-deuterium exchange, measured by mass spectrometry, gave direct evidence that ATP decreases the accessibility of amide hydrogens in multiple regions of both alphaA and alphaB. The surface encompassed by these regions is much larger than would be shielded by a single ATP, implying that multiple ATP molecules bind to each subunit and/or ATP causes a more compact alpha-crystallin structure. Such a conformational change could release a bound substrate. The regions most affected by ATP are near putative substrate binding regions of alphaA and alphaB and in the C-terminal extension of alphaB. The widespread decrease in hydrogen-deuterium exchange with particularly large decreases near substrate binding regions suggests that ATP releases substrates via both direct displacement and a global conformational change.

Adenosine Triphosphate↗

PubMed2002

S-methylated cysteines in human lens gamma S-crystallins.

The proteins of the eye lens, which do not turn over throughout life, undergo many modifications, some of which lead to senile cataract. We describe a modification, S-methylation of cysteine, that may serve to protect the lens from detrimental modifications. The modification was detected as a +14 Da peak in electrospray ionization mass spectra of human lens gammaS-crystallins. Derivatization of gammaS-crystallin with iodoacetamide showed reaction at only six of the seven cysteines, indicating the modification blocked reaction at one cysteine. Further analysis of the modified gammaS-crystallin as tryptic peptides located the modification primarily at Cys 26, with smaller amounts at Cys 24. Tandem mass spectrometry and exact mass measurements showed that the modification was S-methylation. Methylation of proteins has been documented at several other amino acid residues, but S-methylation of cysteine residues has previously been detected only as part of a methyltransferase DNA repair mechanism or at trace amounts in hemoglobin. The high levels of S-methylated cysteines in lens nuclei and the specificity for Cys 26 and Cys 24 suggest the reaction is enzymatically mediated. This modification is particularly important because it blocks disulfide bonding of gammaS-crystallins and, thereby, inhibits formation of the high-molecular weight assemblies associated with cataract. Evidence of more S-methylation in soluble than in insoluble gammaS-crystallins supports the contention that S-methylation of gammaS-crystallin inhibits protein insolubilization and may offer protection against cataract.

Adult↗

PubMed2002

Deamidation in human gamma S-crystallin from cataractous lenses is influenced by surface exposure.

A major component of human nuclear cataracts is water-insoluble, high molecular weight protein. A significant component of this protein is disulfide bonded gamma S-crystallin that can be reduced to monomers by dithiothreitol. Analysis of this reduced gamma S-crystallin showed that deamidation of glutamine and asparagine residues is a principal modification. Deamidation is one of the modifications of lens crystallins associated with aging and cataractogenesis. One proposed hypothesis of cataractogenesis is that it develops in response to altered surface charges that cause conformational changes, which, in turn, permit formation of disulfide bonds and crystallin insolubility. This report, showing deamidation among the disulfide bonded gamma S-crystallins from cataractous lenses, supports this hypothesis.

Amides↗

PubMed2002

Calcium absorption from three salts and CaSO(4)-fortified bread in premenopausal women.

Two studies were conducted to measure calcium absorption from calcium sulfate fortified bread and three salts (calcium lactate, calcium carbonate, and calcium sulfate) in healthy premenopausal women using a crossover design. In study I, calcium fractional absorption levels from the three salts labeled with a stable isotope, 44Ca, were not significantly different (0.039-0.47) as determined by the fecal recovery method. In a second study, absorption of 45Ca from CaSO(4)-fortified labeled bread or labeled milk was measured in serum exactly 5 h postingestion. Fractional calcium absorption was slightly but significantly greater from fortified bread than from milk (mean within subject difference = 0.0675 +/- 0.016). Calcium sulfate is a bioavailable fortificant for white bread that compares favorably with milk and two other salts.

Adsorption↗