Biomedical subjects
Publications and source records attributed to C V Smith.
The use of 24-h monitoring devices in healthy gravidas confirmed the suspicion that temporary blood pressure elevations relate to ambulation and to emotional upheaval, while the lowest recordings are associated with periods of rest.
The effects of hepatotoxic doses of acetaminophen on tissue concentrations of CoA in the thiol form (CoASH) and as the corresponding mixed disulfide with GSH (CoASSG) were determined to test the hypotheses that early oxidant effects of acetaminophen are expressed principally in the mitochondrial compartment and that oxidative shifts in this redox couple could be employed as biomarkers of mitochondrially compartmentalized oxidant stresses. Administration of 400 mg of acetaminophen/kg to male ICR mice did not change CoASSG concentrations at 2, 4, or 6 h, but CoASH levels were lower than in saline-treated control animals at 2 and 4 h (77 +/- 8 vs 124 +/- 14 nmol/g of liver and 66 +/- 9 vs 142 +/- 7 nmol/g of liver, respectively). HPLC analyses of acid supernatants from livers of mice treated with acetaminophen in vivo showed a peak that coeluted with an adduct generated in vitro by reaction of CoASH with N-acetyl-p-benzoquinone imine, but extensive efforts to characterize further the putative product formed in vivo have been unsuccessful. Decreases in CoASH levels were not observed in mice given comparably hepatotoxic doses of furosemide, which diminishes the concern that the decreases in CoASH levels observed in the acetaminophen-treated mice were simply secondary to injury. Hepatic CoASSG concentrations were elevated 10-20-fold 2 h after administration of 400 or 500 mg of furosemide/kg, but were not different than in saline-treated control mice at 4 or 6 h. Increases in hepatic concentrations of GSSG were observed after 6 h in both the acetaminophen-treated and the furosemide-treated mice, suggesting that these changes may be more reflective of oxidant responses to hepatic necrosis than of thiol oxidation mechanisms involved in mediating the injury. The results presented here are not consistent with oxidant stress mechanisms in initiation of hepatic necrosis by acetaminophen in vivo, but the data suggest possible roles for mitochondrially compartmentalized oxidant effects of furosemide.
Redox cycling metabolism of diquat catalyzes generation of reactive oxygen species, and diquat-induced acute hepatic necrosis in male Fischer 344 (F344) rats has been studied as a model of oxidant mechanisms of cell killing in vivo. At equal doses of diquat, female F344 rats sustained less hepatic damage than did male rats, as estimated by plasma alanine aminotransferase (ALT) activities after 6 h. Biliary efflux of glutathione disulfide (GSSG) was greater in male than in female rats at each dose of diquat, but even comparable rates of GSSG excretion were associated with less hepatic injury in female rats. Hepatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were similar in the two genders, and activities of glutathione reductase (GR) and glutathione S-transferase-alpha (GST-alpha) activities were higher in the male rats. Previous studies in male rats have implicated formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive "protein carbonyls" and related iron chelate-catalyzed redox reactions as mechanisms critical to diquat-induced acute cell death in vivo. However, diquat-treated female rats showed higher levels of DNPH-reactive proteins in livers and in bile than did males, both at identical doses of diquat and at doses that produced similar elevations in plasma ALT activities. In female rats, fragmentation of hepatic deoxyribonucleic acids (DNA) was increased by doses of diquat that did not increase plasma ALT activities, and increased fragmentation was observed prior to elevation of plasma ALT activities. In the present studies, hepatic necrosis was most closely associated with DNA fragmentation, although additional studies are needed to determine the mechanisms responsible for and the pathophysiological consequences of the fragmentation.
Reactive oxygen species (ROS) are implicated as agents of cellular damage in pulmonary oxygen toxicity. Glutathione (GSH) and GSH-dependent antioxidant enzymes protect against damage by ROS, and recycling of glutathione disulfide (GSSG) to GSH by glutathione reductase (GR) is essential for the optimum functioning of this system. Exposure to hyperoxia inhibits lung development in newborn animals and humans, and attenuates cell growth in proliferating cell cultures. Considerable evidence supports a role for ROS as growth-altering molecules. Previously, we have observed that gene transfer of GR to mitochondria in H441 cells, using a vector containing a mitochondrial leader sequence (LGR), protected these cells against t-BuOOH-induced cytotoxicity. The present studies tested the hypothesis that gene transfer of LGR would attenuate the cytostatic effects of hyperoxia exposure in H441 cells. H441 cells (0.9 x 10(6) cells/plate) transfected with adenovirus containing LGR or the complementary DNA (cDNA) for manganese superoxide dismutase in reverse orientation (DOS) as a control construct, and untransfected cells (CON) were maintained in 21% oxygen (normoxia) or 95% oxygen (hyperoxia) for 48 h, and cell growth was assessed by cell counts and by reduction of the tetrazolium dye 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan. Cells maintained in normoxia achieved normal growth (CON, 1.98; DOS, 1.91; LGR, 2.0 x 10(6) cells/plate). Hyperoxia inhibited cell growth and the reduction of MTT; however, cells transfected with LGR had greater mitochondrial GR activities (CON, 16+/-2; DOS, 19+/-3; LGR, 322+/-18 mU/mg of protein), sustained more normal growth patterns (CON, 1.25+/-0.12; DOS, 1.24 +/-0.21, LGR, 1.8+/-0.25 x 10(6) cells/plate), and had less inhibition of MTT reduction (CON, 29; DOS, 27; LGR, 16% inhibition, P<0.01) after exposure to hyperoxia for 48 h than was observed in cells transfected with DOS or in control cells not infected with virus. In addition, resistant cells had higher mitochondrial GSH levels and maintained mitochondrial GSH/GSSG ratios in hyperoxia, suggesting that maintaining mitochondrial GSH homeostasis determined critical aspects of cell division in these studies. The mechanisms for sustaining cell growth during hyperoxia in H441 cells with enhanced mitochondrial GR activities are unknown, but similar effects in infants exposed to supplemental oxygen could be highly beneficial.
In this investigation, we tested the hypothesis that the cytochrome P-450 (CYP) inhibitor 1-aminobenzotriazole (ABT) alters the susceptibility of rats to hyperoxic lung injury. Male Sprague-Dawley rats were treated i.p. with ABT (66 mg/kg), i.v. with N-benzyl-1-aminobenzotriazole (1 micromol/kg), or the respective vehicles, followed by exposure to >95% oxygen for 24, 48, or 60 h. Pleural effusion volumes were measured as estimates of hyperoxic lung injury, and lung microsomal ethoxyresorufin O-deethylation (EROD) (CYP1A1) activities and CYP1A1 apoprotein levels were determined by Western blotting. ABT-pretreated animals exposed to hyperoxia died between 48 and 60 h, whereas no deaths were observed with up to 60 h of hyperoxia in vehicle-treated animals. In addition, three of four ABT-treated rats exposed to hyperoxia for 48 h showed marked pleural effusions. Exposure of vehicle-treated rats to hyperoxia led to 6.3-fold greater lung EROD activities and greater CYP1A1 apoprotein levels than in air-breathing controls after 48 h, but both declined to control levels by 60 h. Liver CYP1A1/1A2 enzymes displayed responses to hyperoxia and ABT similar to the effects on lung CYP1A1. N-Benzyl-1-aminobenzotriazole markedly inhibited lung microsomal pentoxyresorufin O-depentylation (principally CYP2B1) activities in air-breathing and hyperoxic animals but did not affect lung EROD or liver CYP activities. In conclusion, the results suggest that induction of CYP1A enzymes may serve as an adaptive response to hyperoxia, and that CYP2B1, the major pulmonary CYP isoform, does not contribute significantly to hyperoxic lung injury.
Oxidative modifications of low-density lipoproteins (LDL) may contribute to the pathogenesis of atherosclerosis. Although the oxidation products of the lipid components of LDL have been studied extensively, less is known about the oxidation products of the apoprotein, apolipoprotein B-100. To identify the specific oxidative modifications, we oxidized LDL in the presence of Cu(2+), treated with DNPH, precipitated and delipidated the protein, digested the protein with trypsin, and analyzed the peptides by high-performance liquid chromatography. We isolated nine peptides that exhibited measurable absorbance at 365 nm, which is characteristic of hydrazones derived from DNPH and is not observed in peptides derived from unoxidized LDL. Unexpectedly, we obtained the same peptides with absorbance at 365 nm in Cu(2+)-oxidized LDL not treated with DNPH. N-terminal sequence analyses and mass spectrometry indicated that the peptides isolated from the Cu(2+)-oxidized LDL all contained kynurenine residues in place of Trp residues found in the native apoprotein. The product profile we observed in Cu(2+)-oxidized LDL was remarkably different from the profiles observed in LDL oxidized by HOCl or myeloperoxidase in vitro, and the preferential oxidation of Trp to kynurenine in Cu(2+)-catalyzed oxidation of LDL contrasts with the products observed following oxidation of LDL with HOCl or myeloperoxidase. Our studies to date support the working hypothesis that the specific products of protein oxidation are sufficiently distinct to be developed as biomarkers of proposed mechanisms of oxidation of LDL and biological molecules in other toxicities and diseases.
BACKGROUND: Collagenase infusion into the pancreatic duct is an essential step in human islet isolation. We developed a new method for ductal canulation and collagenese infusion. METHODS: A total of 53 pancreata were divided into two groups: group 1 (n=23), the new tube method, and group 2 (n=30), the standard angiocatheter method. In group 1, a polyethylene tube was inserted into the duct and pushed to the tail. The tail was first expanded, followed by expansion of the body and then the head, by pulling out the tube. RESULTS: Total islet number and number/g pancreas (mean+/-SE) were significantly higher in group 1 (481,123+/-43,218 and 8,010+/-722) (mean+/-SE) than in group 2 (300,974+/-35,122 and 5,090+/-515, P<0.01). Total islet equivalent number and islet equivalent number per gram pancreas were also significantly higher in group 1 (319,176+/-39,354 and 5,455+/-652) than in group 2 (202,022+/-23,331 and 3,722+/-468, P<0.04). Islet purity and fragmentation showed no differences between the groups. CONCLUSIONS: The tube method improved islet yields. We recommended this method for human islet isolation.
OBJECTIVE: To determine if treatment of a positive amniotic fluid culture for mycoplasmal colonization obtained at genetic amniocentesis is associated with improved pregnancy outcome. STUDY DESIGN: A retrospective analysis of 2718 genetic amniocentesis specimens cultured for Ureaplasma/Mycoplasma was undertaken. Specimens were obtained between March 1993 and January 1997. The Irvine culture kit was used to culture all specimens. Data collected included indication for amniocentesis, gestational age at amniocentesis, karyotype, gestational age at delivery, pregnancy outcome, and any antimicrobial treatment. RESULTS: During this time period 44 patients were found to be culture-positive for Ureaplasma/Mycoplasma. Thirty-five were treated with oral erythromycin. Mid-trimester loss was 11.4% and 44.4% (p = 0.04) in the treated and untreated groups, respectively. Preterm delivery was similar in the two groups, 19.4% and 20% (p = NS). CONCLUSION: Treatment of an amniotic mycoplasmal colonization with erythromycin was associated with fewer mid-trimester losses after genetic amniocentesis. Preterm delivery rates between the two groups were similar, which may indicate recolonization.
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Cryopreservation of pancreatic islets provides many advantages for clinical transplantation. Unfortunately, the freezing and thawing processes lead to a significant loss of islets. In this study, an attempt was made to increase the yield and viability of islets after cryopreservation and thawing. By using canine islets, we evaluated whether beraprost sodium (BPS), a stable prostacyclin analog, protects islets during the freeze-thaw processes and improves the recovery of frozen-thawed islets. Canine islets were frozen and thawed by the procedures used routinely for storage of human islets. In this study, we deliberately used islets of lower purity (60+/-3.6%), which is undesirable for cryopreservation. The recovery of viable islets after thawing is poorer with islets of lower purity than with highly purified islets. BPS was added to both the cryopreservation solutions containing dimethyl sulfoxide (DMSO) and the thawing solution containing sucrose. After thawing, the islet recovery (islet number after thawing divided by islet number before freezing) was 71.1+/-12.7% with 1 nM BPS, 77.8+/-5.6% with 10 nM BPS, 79.3+/-6.7% with 100 nM BPS, and 69.2+/-7.2% in control preparations without BPS. Islet viability assessed by supravital staining was 57.5+/-5.6%, 64.7+/-7.0%, 67.5+/-6.5%, and 57.7+/-4.9% with 1 nM, 10 nM, and 100 nM BPS and controls, respectively. Both islet recovery and viability were significantly better with 10 nM and 100 nM BPS than with the controls (p<0.03). After 3 days in culture, islet numbers in the 10 nM and 100 nM BPS groups were significantly higher and showed better insulin-release responses than those from the 1 nM BPS and control groups. Histologically, islet structure was well preserved in the 10 nM and 100 nM BPS groups, whereas many islets of the control group were smaller and fragmented. Electron microscopic examination revealed that 10 nM and 100 nM BPS preserved the microstructure of islet cells, and signs of apoptosis or necrosis were rare. It was concluded that BPS improved the recovery and viability of canine islets after cryopreservation and thawing. BPS would be a useful agent for improving the recovery of cryopreserved human islets for clinical transplantation.
Increased generation of reactive oxygen species (ROS) and low levels of antioxidants may cause morbidity in premature infants on supplemental oxygen. Glutathione (GSH)-dependent antioxidant systems protect against ROS, and regenerating GSH from GSH disulfide (GSSG) by the flavoenzyme GSH reductase (GR) is essential for the optimal function of this system. Previously, we have observed enhanced resistance to t-butyl hydroperoxide (t-BuOOH) in Chinese hamster ovary cells stably transfected with a vector (leader sequence GR [LGR]) for human GR cDNA that contained a functional synthetic mitochondrial targeting signal. The present studies were designed to investigate adenovirus-mediated gene transfer of LGR to H441 cells and resistance of such cells to t-BuOOH. Adenovirus-mediated transfection of H441 cells with LGR increased total GR activities more than 11-fold (mitochondria more than 10-fold and cytosolic more than 7-fold) and protected against t-BuOOH cytotoxicity, as indicated by lower fractional release of cellular lactate dehydrogenase (LDH) than was observed in wild-type untransfected cells (CON) or in cells transfected with a control gene (human manganese superoxide dismutase in the antisense orientation [DOS]) (*LGR 6.6 +/- 1.7; DOS 16 +/- 1.8; CON 16.6 +/- 0.7% LDH release). In addition, cells transfected with LGR retained higher GSH/GSSG ratios (*LGR 66 +/- 0.4; DOS 47 +/- 1; CON 52.6 +/- 2.3) and released less GSH + GSSG to the media in response to challenge with t-BuOOH (*LGR 0.05 +/- 0.01; DOS 0.08 +/- 0.01; CON 0.07 +/- 0.01 nmol/mg of protein) than did wild-type cells or cells transfected with a control vector, indicating an enhanced ability of the LGR cells to reduce GSSG formed in response to exposure to t-BuOOH. In conclusion, adenovirus-mediated gene transfer of LGR enhanced cellular GR activities and protected H441 cells from oxidant stresses.
Oxidative modification of LDL may be important in the initiation and/or progression of atherosclerosis, but the precise mechanisms through which low density lipoprotein (LDL) is oxidized are unknown. Recently, evidence for the existence of HOCl-oxidized LDL in human atherosclerotic lesions has been reported, and myeloperoxidase (MPO), which is thought to act through production of HOCl, has been identified in human atherosclerotic lesions. In the present report we describe the formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive modifications in the apolipoprotein (apo) by exposure of LDL to myeloperoxidase in vitro. In contrast with the complex mixture of peptides from oxidation of LDL with reagent HOCl, oxidation with MPO in vitro produced a major tryptic peptide showing absorbance at 365 nm. This peptide was isolated and characterized as VELEVPQL(*C)SFILK..., corresponding to amino acid residues 53-66...on apoB-100. Mass spectrometric analyses of two tryptic peptides from oxidation of LDL by HOCl indicated formation of the corresponding methionine sulfoxide (M=O), cysteinyl azo (*C), RS -N= N-DNP, derivatives of EEL(*C)T(M=O)FIR and LNDLNS VLV(M=O)PTFHVPFTDLQVPS(*C)K, which suggest oxidation to the corresponding sulfinic acids (RSO2H) by HOCl. The present results demonstrate that DNPH-reactive modifications other than aldehydes and ketones can be formed in the oxidation of proteins and illustrate how characterization of specific products of protein oxidation can be useful in assessing the relative contributions of different and unexpected mechanisms to the oxidation of LDL and other target substrates. The data also suggest a direct interaction of the LDL particle with the active site on myeloperoxidase and indicate that effects of the protein microenvironment can greatly influence product formation and stability.
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Infants and adults on oxygen often are treated with glucocorticoids in an attempt to reduce lung inflammatory injury. However, glucocorticoids hasten the development of hyperoxic lung injury in some animal models. The purpose of this study was to test the hypothesis that dexamethasone alters the lung inflammatory responses to hyperoxia exposure. We studied male Sprague-Dawley rats, placing them in >95% oxygen immediately after administration of 0, 0.1, 1, or 10 mg/kg of dexamethasone. At 0, 24, or 48 hr of exposure to hyperoxia, extravascular lung water contents were measured, and lung inflammatory responses were assessed by lung myeloperoxidase activities, lung neutrophil counts, and lung expression of E-Selectin and intercellular adhesions molecule-1 (ICAM-1). Dexamethasone, independent of exposure to hyperoxia, led to marked increases in lung neutrophil counts, without increases in lung myeloperoxidase activities or increases in the expression of the adhesion molecules. Hyperoxia exposure also enhanced lung neutrophil accumulation, and extravascular lung water increased earlier in animals exposed to hyperoxia and dexamethasone than in those exposed to hyperoxia alone. In conclusion, the increase in lung neutrophils in dexamethasone-treated rats without enhanced expression of E-Selectin or intracellular adhesions molecule-1 suggests that dexamethasone leads to lung neutrophil accumulation by its effect on neutrophils. The more rapid development of hyperoxic lung injury associated with earlier lung neutrophil accumulation suggests that dexamethasone-induced lung neutrophil sequestration primes the lung for the development of hyperoxic lung injury and supports further the conclusion that lung inflammation contributes significantly to the development of hyperoxic lung injury.
Examination of the X-ray crystal structure of the 43 kDa N-terminal domain of the DNA gyrase B protein (GyrB) shows that the majority of the interactions with bound ATP are made with subdomain 1 (residues 2-220). However, two residues from subdomain 2, Gln335 and Lys337, interact with the gamma-phosphate of ATP. The proposed roles for these residues include nucleotide binding, transition-state stabilization, and triggering protein conformational changes. We have used site-directed mutagenesis to convert Gln335 to Asn and Ala and Lys337 to Gln and Ala in the N-terminal domain of GyrB. Two of the resultant mutant proteins, GyrB43(Q335A) and GyrB43(K337Q), were shown to be correctly folded, and their interactions with ATP have been analyzed in detail. The Q335A protein is apparently unchanged with regard to nucleotide binding and hydrolysis, whereas the K337Q protein shows a modest decrease in nucleotide binding and a drastic reduction in ATPase activity. This is manifested by a approximately 10(3)-fold decrease in kcat. When the two mutations were moved into full-length GyrB, the Q335A mutation again showed little or no effect on activity, whereas the K337Q mutation had undetectable supercoiling and ATPase activities. We conclude that Gln335 is dispensable for ATP binding and hydrolysis by the gyrase B protein, whereas Lys337 has a critical role in the ATPase reaction and is likely to be a key residue in transition-state stabilization.
Hyperoxia, used therapeutically in the treatment of respiratory insufficiencies, can cause lung injury, probably through the actions of reactive oxygen species. The present studies were designed to test the hypothesis that oxidation of specific proteins would provide useful biomarkers of the onset of tissue injury, and thereby provide clues as to the mechanisms responsible. We exposed adult male Sprague-Dawley rats to room air or to greater than 95% O2 for 60 h and examined proteins in pleural effusion and broncho-alveolar lavage (BAL) fluids, and in lung tissue homogenates and subfractions. Oxidation of protein thiols was assessed by derivatization with monobromobimane, separation by electrophoresis, and visualization of the fluorescent thioether derivatives. Derivatization with 2,4-dinitrophenylhydrazine (DNPH), electrophoresis, and western analysis was employed to assess a different class of oxidative modifications, frequently termed 'protein carbonyls'. In addition, we investigated the effects of the 21-aminosteroid U-74389G, 10 mg/kg, given intraperitoneally every 12 h, on biomarkers of protein oxidation and on manifestations of lung injury. Hyperoxia caused lung injury evidenced by pleural effusions, increases in BAL protein concentrations, and pulmonary edema; U-74389G attenuated the first two indices of lung injury, but did not alter edema. Protein thiol status of the fractions studied were not affected notably by hyperoxia, or by the aminosteroid. The formation of DNPH-reactive sites on a limited number of proteins by hyperoxia was observed, and some of these effects were attenuated in the animals given U-74389G. Histological examination of lung tissues showed accumulation of intra-alveolar protein exudates in hyperoxic rats, and a significant attenuation of this effect was observed in the animals treated with U-74389G. In conclusion, studies of shifts in protein thiol status that may be caused by hyperoxia will require increasingly specific methods of analysis, and characterization of the specific DNPH-reactive proteins formed in hyperoxia may provide critical insights into the mechanisms of lung injury. Administration of U-74389G offers some degree of protection against hyperoxia and attenuation of these biomarkers of oxidation, but the precise mechanisms by which this protection is effected will require additional study.
We reported previously that Fischer-344 (F344) rats were more susceptible to hyperoxic lung injury than were Sprague Dawley (SD) rats. In the present study we exposed adult male F344 and SD rats to >95% oxygen for up to 48 h, and measured lung wet-to-dry weight ratios and lavage protein concentrations as indices of lung injury. In addition, we measured nonheme iron contents in the lung subcellular fractions and in bronchoalveolar lavages (BAL), and we derivatized samples from the subcellular compartments and lavages with 2,4-dinitrophenylhydrazine (DNPH), separated the proteins electrophoretically, and detected DNPH-derivatized proteins by western blotting. After 48 h of hyperoxia, BAL protein and nonheme iron concentrations were higher in F344 rats than in SD rats (2.17+/-0.77 versus 0.17+/-0.17 mg/ml, and 1.61+/-0.45 versus 0.45+/-0.18 nmol/ml, respectively, both P<0.05). In addition, two DNPH-reactive proteins of about 40 and 120 kDa were identified in the lavage fluids of hyperoxic F-344 rats that were not observed similarly in hyperoxic SD rats or in air-breathing rats of either strain. N-terminal amino acid sequences of the two DNPH-reactive proteins 100% identical over 16 residues to rat beta-casein, which is a potent neutrophil chemotaxin, and has been reported to be a product of cytotoxic T-lymphocytes. There were no significant alterations in iron contents in lung subcellular fractions in either strain of rat as a consequence of hyperoxia-exposure, nor were there any significant alterations in DNPH-reactive carbonyls, as determined by western blotting. These data suggest that increased iron concentrations in the airspaces reflect altered iron homeostasis, which may contribute to the greater susceptibility of F344 rats than SD rats to hyperoxic lung injury. The identification of oxidized beta-casein in the BAL of the hyperoxic F344 rats suggests a role for cytotoxic T-lymphocytes in hyperoxic lung inflammation and injury, although the nature of this possible involvement is not known at this time.
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