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Biomedical subjects

P O'Brien

Publications and source records attributed to P O'Brien.

At least 145 records · Page 8Linked to original sources
PubMed1987

Reaction of cytochromes c and c2 with the Rhodobacter sphaeroides reaction center involves the heme crevice domain.

In order to define the interaction domain on Rhodobacter sphaeroides cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged (carboxydinitrophenyl)- (CDNP-) lysines. The reaction mixture was separated into several different fractions by ion-exchange chromatography on (carboxymethyl)cellulose. Tryptic digests of these fractions were analyzed by reverse-phase peptide mapping to determine the lysines that had been modified. Fraction A was found to consist of a mixture of singly labeled derivatives modified at lysine-35, -88, -95, -97, and -105 and several other unidentified lysines comprising 32% of the total. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The second-order rate constant for the reaction of native cytochrome c2 with reaction centers was 2.0 X 10(8) M-1 s-1, while that for fraction A was 20-fold less, 1.0 X 10(7) M-1 s-1. This suggests that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice significantly lowered the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.

Amino Acid Sequence↗
PubMed1987

Genetic analysis of UV mutagenesis of the Escherichia coli glyU gene.

By genetic analysis we examined UV mutagenesis of the Escherichia coli glyU gene. When carried by M13 phage mp9, glyU is subject to induced UV mutagenesis which is dependent on the umuC+ and recF+ genes. When carried by M13 phage mp8, glyU is not subject to induced UV mutagenesis. This difference is correlated with the nature of the target nucleotides: CTC in the mp9 derivative and GAG in the mp8 derivative. Thus, we conclude that the induced (umuC and recF dependent) mutagenesis is locally targeted on pyrimidine cyclobutane or 6-4 dimers. glyU carried by M13 is equally subject to uninduced UV mutagenesis whether carried by mp8 or mp9. This uninduced mutagenesis is independent of the umuC+, recF+ and recA+ genes and we hypothesize that it is regionally targeted on pyrimidine cyclobutane or 6-4 dimers in the vicinity of the target CTC and GAG nucleotides. The role of recF in UV mutagenesis was tested in two ways. First, mutagenesis of glyU carried by M13 mp9 in a recA730 genetic background was found to be recF dependent. Because recA730 renders induced UV mutagenesis partially constitutive, we conclude that the RecF product plays a direct role in UV mutagenesis rather than, or in addition to, any indirect regulatory role it may play. Second, UV mutagenesis of E. coli chromosomal glyU was found to be recF independent while UV mutagenesis of M13-bourne glyU was recF dependent. We conclude that the mechanism of induced UV mutagenesis of the E. coli chromosome is at least partly different from that of M13 phage and we discuss the biochemical basis for such a difference.

Bacterial Proteins↗
PubMed1987

A randomized prospective controlled study of the metabolism and hepatotoxicity of halothane in humans.

In a randomized prospective controlled study in humans, the metabolism and hepatic effects of a single administration of halothane were compared with enflurane and meperidine. Pre- and postoperative antipyrine pharmacokinetics, intraoperative indocyanine green clearance, liver histology, and postoperative liver function tests were determined in 24 patients undergoing abdominal surgery who were randomly allocated to receive either halothane (0.5%, group I), enflurane (0.8%, group II), or meperidine (group III) as a supplement to a common basal anesthetic regimen consisting of thiopental, nitrous oxide/oxygen/muscle relaxant. In addition, end-tidal concentrations of the volatile reductive metabolites of halothane, chlorodifluoroethylene (CDF), and chlorotrifluoroethane (CTF) were determined in group I patients and serum and urinary inorganic fluoride were determined in both group I and II patients. Indocyanine green clearance was measured before anesthesia (stage I), during basal anesthesia (stage II), in the presence of surgical stimuli (stage III), and after introduction of the selected anesthetic agent (stage IV). CDF and CTF were detectable within 20 min of the start of halothane anesthesia in every patient receiving halothane. Peak serum fluoride concentrations occurred at 2 and 24 hr in the enflurane and halothane groups, respectively, whereas urinary fluoride excretion was elevated postanesthesia in the enflurane group only. There was no difference between the pre- and postoperative disposition of antipyrine in group II or III, but after anesthesia, antipyrine clearance was significantly decreased (P less than 0.02) and plasma half-life increased (P less than 0.05) in group I patients (halothane). Concentrations of serum alanine aminotransferase (ALT) and bilirubin were significantly elevated (P less than 0.5) postoperatively in groups I and II but unchanged from preoperative values in group III patients. Three of the 24 liver biopsies taken at the end of stage IV showed several foci of acute liver cell necrosis; of these, two patients were from group I and one from group II. There were no significant differences in liver cell morphology (P greater than 0.5) in biopsies taken at the end of stage IV compared with biopsies at the end of stage III, from groups I and II. The results of this study show that reductive metabolism of halothane occurs routinely in patients undergoing halothane anesthesia under conditions of normoxia. This may be the cause of the changes in antipyrine clearance after halothane anesthesia.

Antipyrine↗
PubMed1986

The involvement of carboxylate groups of putidaredoxin in the reaction with putidaredoxin reductase.

Modification of carboxyl groups on putidaredoxin with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) resulted in loss of putidaredoxin reductase activity. The modification did not affect the visible absorption spectrum of putidaredoxin, indicating that the iron-sulfur center was not perturbed. In order to identify the carboxyl groups labeled by EDC, native and EDC-treated putidaredoxin were digested with a combination of trypsin and Staphylococcus aureus protease, and the resulting peptides were separated by high pressure liquid chromatography. The most heavily modified carboxyl groups were found to be those at residues 58, 65, 67, 72, and 77. These carboxyl groups are located in the same general region of the protein as those on adrenodoxin that have been shown to be involved in binding to both adrenodoxin reductase and cytochrome P-450scc. Chemical modification was also used to compare the role of lysine, arginine, and histidine residues on putidaredoxin and adrenodoxin. Modification of lysine and arginine residues had no effect on the reductase activity of either protein. The reductase activity of adrenodoxin was unaffected by labeling with 1 eq of diethyl pyrocarbonate/histidine residue, but labeling with a second equivalent completely abolished both activity and the iron-sulfur center spectrum. In contrast, modification of the 2 histidines in putidaredoxin with 1 eq each resulted in nearly complete loss of reductase activity. There was no significant activity for adrenodoxin in the putidaredoxin reductase assay or for putidaredoxin in the adrenodoxin reductase assay, demonstrating that, in spite of the structural similarity between the two proteins, they are not interchangeable functionally.

Adrenodoxin↗
PubMed1986

Protective effects of the synthetic prostaglandin enprostil on the gastric microvasculature after ethanol injury in the rat.

The effect of pretreatment with the synthetic prostaglandin E2 analogue enprostil on ethanol damage to the rat gastric mucosa was studied. Microvascular casts were prepared and studied by scanning electron microscopy. The permeability of mucosal capillaries to fluorescein isothiocyanate-labeled albumin (FITC-albumin) given intravenously was examined by fluorescence microscopy. After administration of ethanol (1 ml absolute ethanol intragastrically) alone, casts showed gross disruption of the normal structure, with large foci of loss of the patency of the capillary network, frequently extending to the level of the submucosal vessels. There was exudation of casting material into the mucosal interstitium and onto the surface of the cast. After administration of FITC-albumin, there was a marked increase in interstitial fluorescence throughout the full thickness of the mucosa. Pretreatment with enprostil (1 microgram/kg intragastrically) prevented most of the damaging effects of ethanol. Increased microvascular permeability to FITC-albumin was noted only in the most superficial layers of the mucosa. These studies characterize the effect of ethanol on the gastric microvasculature and indicate that pretreatment with enprostil restricts this damaging effect to the superficial mucosal microvessels. These studies further suggest that microvascular damage is an early event in ethanol injury, apparently preceding epithelial erosion.

Animals↗
PubMed1986

Plasma exchange in chronic inflammatory demyelinating polyradiculoneuropathy.

Plasma exchange has been reported to be efficacious in chronic inflammatory demyelinating polyradiculoneuropathy. We performed a prospective double-blind trial in which patients with static or worsening disease were randomly assigned to plasma exchange (n = 15) or to sham exchange (n = 14) for three weeks. After three weeks, we observed statistically significant differences in combined measurements of nerve conduction (total, motor, proximal, velocity, and amplitude) favoring patients who had received plasma exchange. Improvement to a greater degree than for any patient receiving sham exchange was detected in the neurologic-disability score in five patients (P = 0.025) and in subset scores for weakness and reflex in four patients (P less than 0.057). We conclude that for some patients with chronic inflammatory demyelinating polyradiculoneuropathy, plasma exchange has an ameliorating effect on neurologic dysfunction and nerve conduction, but in others no improvement is observed. Because plasma was replaced with normal serum albumin, a humoral factor or factors may have a role in the neurologic deficit of this disorder.

Chronic Disease↗
PubMed1986

Fiber loss is primary and multifocal in sural nerves in diabetic polyneuropathy.

Pathological, morphometric, and teased fiber studies of sural nerve from 36 diabetic patients with (n = 32) and without (n = 4) neuropathy and from 47 healthy subjects provide evidence that in diabetic polyneuropathy: (1) fiber loss is primary; (2) demyelination and remyelination with or without onion bulb formation are secondary; (3) remaining fibers, on average, have the same ratio of small to large fibers as in healthy individuals, but with a greatly increased variability; and (4) the spatial distribution of fiber loss is both diffuse and multifocal. Criteria developed during the study of experimental models of ischemic neuropathy were employed to assess whether ischemic nerve damage had occurred in diabetic polyneuropathy. We conclude that there is increasing evidence that microvascular pathological abnormality and ischemia may be involved in the pathogenesis of human diabetic polyneuropathy. Cases with selective loss of small or large afferent fibers are probably extremes of a normal distribution and not different disorders.

Adult↗
PubMed1986

The spatial distribution of fiber loss in diabetic polyneuropathy suggests ischemia.

Characterization and quantitation of the spatial distribution of pathological abnormalities along the length of nerves may be helpful in understanding the underlying mechanisms of diabetic polyneuropathy. To this end, by examining transverse sections of nerve roots and proximal-to-distal levels of lower limb nerves in 9 controls and 15 diabetic patients with polyneuropathy, we have determined the myelinated fiber (MF) number, size distribution, median diameter, and variability of density (MFs/mm2) among frames and among fascicles. Even in cases with mild polyneuropathy, fiber loss, a decrease in the median diameter, and an increase in the variability of density among frames and among fascicles began in proximal nerve and extended to distal levels. Multifocal fiber loss along the length of nerves and sprouting provide the best explanation for these findings. The pattern is dissimilar from that observed in diffuse metabolic disease of Schwann cells, neuronal degeneration, and dying-back neuropathy, but like that found in experimental ischemic neuropathy induced by embolization of nerve capillaries.

Adult↗
PubMed1986

Effort angina with adequate beta-receptor blockade: comparison with diltiazem alone and in combination.

Calcium channel blockers and beta-receptor blockers improve symptoms of myocardial ischemia by potentially different mechanisms. Accordingly, combination therapy may entail additive benefits. Twenty-four patients with symptomatic stable effort angina despite full beta-blockade were randomized to a double-blind Latin square protocol in which they received propranolol in a dose producing full beta-receptor blockade, diltiazem, 240 mg/day, in divided doses and a combination of propranolol and diltiazem, 240 or 360 mg/day. Treadmill testing (Bruce protocol) was utilized to assess exercise tolerance, radionuclide ventriculography to assess left ventricular function and clinical follow-up to assess adverse effects and overall clinical response. Comparable treadmill exercise times were observed with monotherapy (344 +/- 83 seconds with propranolol and 341 +/- 87 seconds with diltiazem) and the lower dose combination (361 +/- 87 seconds). With propranolol and diltiazem, 360 mg/day, however, there was a significant increase in treadmill time (393 +/- 106 seconds; p less than 0.05). In five patients whose treadmill exercise was limited by angina on all therapies, there was a significant improvement in the time to onset of chest pain with both low dose and high dose combinations (311 +/- 71 seconds, p less than 0.05 and 336 +/- 76 seconds, p less than 0.01, respectively). Improved treadmill performance was supported by the clinical response, while an increase in adverse effects was not observed. Thirteen of 24 patients blindly selected the higher dose diltiazem combination as their optimal therapy. Left ventricular dilation was observed (by radionuclide ventriculography) in response to exercise in each phase of therapy; this was related to stress-induced ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗
PubMed1986

An approach to analyzing UV mutagenesis in E. coli.

UV mutagenesis of single-strand DNA phage can be divided into three types: induced untargeted; induced targeted; and uninduced targeted. We report the development of new tools to determine the number of processes which contribute to these types of mutagenesis. An E. coli tRNA gene, glyU, has been cloned using M13 derivatives mp8 and mp9 as vectors. The nucleotide sequence of glyU and its flanking regions is presented. In this paper, phage glyU anticodon mutants are detected by their ability to suppress GAA and GAT missense mutations in trpA. We used phage carrying GAG and CTC at the anticodon position and found results consistent with the hypothesis that two processes act to produce the transition to GAA suppression: an uninduced regionally targeted process; and an induced locally targeted process with some untargeted activity. The transversion frequency to GAT suppression on the other hand responded as if only an uninduced locally targeted process was involved. Thus, we hypothesize that the new tools have discriminated three different processes of mutagenesis and we discuss further work designed to test this hypothesis.

Anticodon↗
PubMed1986

Morphological and biochemical studies of canine progressive rod-cone degeneration. 3H-fucose autoradiography.

Visual cell pathology and rod outer segment renewal were investigated in normal and PRCD-affected miniature poodles using 3H-fucose autoradiography. Twenty-four hours following the intravitreal injection of 3H-fucose, label accumulated diffusely over cone OS and in a banded pattern at the rod OS base. In normal rods, the band of 3H-label was displaced sclerad with time. PRCD-affected rods in the early stages of the disease (stages 0-1) also showed a similar 3H-label pattern but a significantly (P less than 0.001) reduced renewal rate (control = 2.35 +/- 0.43 mu/24 hr; affected = 0.99 +/- mu/24 hr). This abnormal renewal rate was present in central, equatorial, and peripheral visual cells and was not associated with the presence or density of pigment in the RPE cell layer. Biochemical studies indicated that the 3H-label was present as an integral membrane component in the rod OS and confirmed that canine rhodopsin is a fucosylated glycoprotein. The 3H-band in the rod OS layer disappeared in stage 2 of the disease; diffuse label now was present over rod OS that had decreased length and were reduced in number. At this stage of the disease, interphotoreceptor space was invaded by phagocytic cells, and photoreceptor nuclei were lost from the outer nuclear layer. These late degenerative changes were more extensive in the superior and inferior retinal meridians.

Animals↗
PubMed1986

Effects of in vivo treatment with ibuprofen on macrophage function in breast and colon cancer patients.

In this study, the capacity of macrophages from breast and colon cancer patients to become cytotoxic for tumor cells after in vivo administration of the prostaglandin inhibitor, Ibuprofen, has been investigated. Prior to the administration of the drug, each patient's macrophages were tested for their ability to kill tumor cells in the presence and absence of 10(-7) M Ibuprofen. The patients were given 400 mg Ibuprofen 4 times daily for 28 days. At days 14 and 28 after commencing the study, blood samples were drawn and the macrophages were tested for their ability to kill tumor cells in vitro. The patients were divided into 3 groups: Category I, patients possessing non-cytotoxic macrophages in the presence and absence of Ibuprofen; Category II, patients possessing non-cytotoxic macrophages in the absence of the drug and cytotoxic macrophages in the presence of the drug; and Category III, patients possessing cytotoxic macrophages in the absence of the drug and non-cytotoxic macrophages in the presence of the drug. Category I cancer patients generally responded the most effectively to in vivo administration of Ibuprofen at day 28 whereas Category II patients responded the most effectively at day 14. Category II patients became non-responsive when tested again at day 28 and Category I patients became non-responsive when the study was extended to day 42. Category III patients were generally non-responsive at all time points studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed1985

Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate.

Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.

Animals↗
PubMed1985

Identification of the binding site on cytochrome c1 for cytochrome c.

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.

Amino Acid Sequence↗
PubMed1985

Recurrent aneurysms and late vascular complications following repair of abdominal aortic aneurysms.

Between 1970 and 1976, 1,112 patients underwent abdominal aortic aneurysm repair. Follow-up, ranging from six to 12 years, was complete in 1,087 patients (97.7%). The most frequent cause of late deaths was coronary artery disease (45.6%), but significant morbidity related to the peripheral vascular system had developed in 94 patients, and led to 8.4% (48 patients) of all late deaths. Forty-nine true, 14 anastomotic, and five dissecting aneurysms were detected in 59 patients (5.4%) a mean (+/- SD) of 5.2 +/- 3.1 years after the initial aneurysm repair. These aneurysms were located in the thoracic (24), thoracoabdominal (five), or abdominal aorta (11), and in the iliac (six), femoral (17), popliteal (four), and renal arteries (one). Only one of 26 patients presenting with a rupture of one of these secondary aneurysms survived. There was a significant association between preoperative hypertension and recurrent aneurysm. These findings suggest that subsequent vascular disease, including recurrent aneurysms and graft complications, cause significant late morbidity and mortality after repair of abdominal aortic aneurysm. Careful follow-up and adequate control of hypertension may allow reduction in morbidity and an improvement in late survival.

Adult↗
PubMed1985

Homozygosity in a population of variable size and mutation rate.

A formula is obtained for the probability that two genes at a single locus, sampled at random from a population at time t, are of particular types. The model assumed is a diffusion approximation to a neutral Wright-Fisher model in which mutation is not necessarily symmetric and the population size is a function of time. It is shown that for symmetric mutation in a population undergoing a step-function type bottleneck, homozygosity increases with decreasing population size. A formula is given for the distribution of the number of segregating sites occurring in two randomly sampled sequences of completely linked sites, with general mutation at a site and identical mutation structure between sites. We give similar results for a population of fixed size but for which the mutation rate is a function of time, and not necessarily symmetric. We confirm the intuitively clear effect that increasing the mutation rate decreases homozygosity.

Alleles↗
PubMed1985

Pathology of colposcopic findings in 2635 diethylstilbestrol-exposed young women.

An analysis of 6055 colposcopically directed biopsy specimens from 2635 diethylstilbestrol (DES)-exposed women and 445 biopsy specimens from 277 nonexposed women was undertaken to correlate microscopic findings with colposcopic patterns. All examinations were performed using a standardized protocol which required that each participant have colposcopy, cytologic smears, and biopsy of abnormal colposcopic lesions. The findings of colposcopic "columnar epithelium, gland openings, and Nabothian cysts" correlated most often with glandular epithelium in the biopsy specimen. "White epithelium," which includes three related colposcopic patterns, mosaicism, punctation, and white epithelium, was associated most frequently (82-93% of cases) with squamous metaplasia, but occasionally with dysplasia and carcinoma in situ (CIS)(0-6%). The presence of dysplasia or CIS in any individual biopsy specimen occurred most frequently with the observation of higher graded lesions by colposcopy or a prior diagnosis of dysplasia.

Carcinoma in Situ↗