Biomedical subjects
R Read
Publications and source records attributed to R Read.
The cDNA for rat selenoprotein P contains 10 TGA codons in the open reading frame.
Selenoprotein P is a plasma protein recently purified and characterized as containing 7.5 +/- 1.0 selenium atoms/molecule as selenocysteine. In rats maintained on a defined diet containing nutritionally adequate amounts of selenate as the sole selenium source, over half the selenium in plasma is accounted for by selenoprotein P. Its cDNA has been cloned from a rat liver library and sequenced. The sequence is highly unusual, containing 10 TGA codons in its open reading frame prior to the TAA termination codon. TGA designates selenocysteine in other selenoproteins, and limited peptide sequencing that included the amino acids encoded by two of the TGA codons verified that they correspond to selenocysteine. The deduced 366-amino acid sequence is histidine- and cysteine-rich and contains 9 of its selenocysteines in the terminal 122 amino acids. Comparison of the deduced amino acid sequence of selenoprotein P with those of other selenoprotein reveals no significant similarities. Selenoprotein P represents a new class of selenoproteins and is the first protein described with more than 1 selenocysteine in a single polypeptide chain. The primary structure of selenoprotein P suggests that it might be responsible for some of the antioxidant properties of selenium.
Effects of Bordetella pertussis infection on human respiratory epithelium in vivo and in vitro.
Bordetella pertussis infection probably involves attachment to and destruction of ciliated epithelial cells, but most previous studies have used animal tissue. During an epidemic, nasal epithelial biopsy specimens of 15 children (aged 1 month to 3 1/2 years) with whooping cough were examined for ciliary beat frequency, percent ciliation of the epithelium, and ciliary and epithelial cell ultrastructure. In addition, the in vitro effects of filtrates from a 24-h broth culture and of tracheal cytotoxin derived from B. pertussis on human nasal tissue organ culture were measured. B. pertussis was cultured from nasal swabs from 12 children. The mean ciliary beat frequency of their nasal biopsy specimens, 11.3 Hz (range, 10.4 to 13.0 Hz) was similar to that found in biopsy specimens from 10 normal children (mean, 12.5 Hz; range, 11.8 to 13.5 Hz). The abnormalities of the epithelium observed in 14 of 15 patients were a reduction in the number of ciliated cells, an increase in the number of cells with sparse ciliation, an increase in the number of dead cells, and extrusion of cells from the epithelial surface. In vitro, neither culture filtrate nor tracheal cytotoxin had any acute effect on ciliary function, but culture filtrate and tracheal cytotoxin (1 and 5 microM, respectively) caused extrusion of cells from the epithelial surface of turbinate tissue, loss of ciliated cells, an increased frequency of sparsely ciliated cells, and toxic changes in some cells. These changes were dose dependent and progressive, and between 36 and 90 h ciliary beating ceased. The observations made with patient tissue confirm that B. pertussis infection damages ciliated epithelium, and the in vitro experiments suggest that tracheal cytotoxin may be responsible for the abnormalities observed in vivo.
Response of rat selenoprotein P to selenium administration and fate of its selenium.
Selenoprotein P is a glycoprotein that contains greater than 60% of the selenium in rat plasma. Physiological experiments were undertaken to gain insight into selenoprotein P function. Selenium-deficient rats were injected with doses of selenium ranging from 25 to 200 micrograms/kg, and the appearance of selenoprotein P was compared with the appearance of glutathione peroxidase activity in plasma and in liver. Selenoprotein P concentration increased to 35% of control by 6 h, whereas glutathione peroxidase activity increased minimally or not at all. Moreover, in rats given 100 and 200 micrograms selenium/kg, selenoprotein P reached 75% of its concentration in control rats at 24 h, whereas glutathione peroxidase activity reached only 6% of control. Cycloheximide pretreatment blocked the appearance of selenoprotein P in response to selenium injection. Male and female rats had similar concentrations of selenoprotein P. Partially purified selenoprotein P and plasma glutathione peroxidase labeled with 75Se were administered intravenously to selenium-deficient and control rats. 75Se given as selenoprotein P disappeared more rapidly from plasma than did 75Se given as glutathione peroxidase. Selenium deficiency did not significantly affect 75Se disappearance from plasma. At 2 h, brain, but not other tissues, took up more 75Se in selenium-deficient rats than in control rats when 75Se was given as selenoprotein P. This suggests that brain has a specific uptake mechanism for selenium given in the form of selenoprotein P. These results demonstrate that several physiological properties distinguish selenoprotein P from glutathione peroxidase. However, they do not clearly indicate its function.
Effects of human neutrophil elastase and Pseudomonas aeruginosa proteinases on human respiratory epithelium.
It has been suggested that proteinase enzymes could play an important role in the pathogenesis of chronic bronchial infections including bronchiectasis and cystic fibrosis (CF). Because Pseudomonas aeruginosa frequently colonizes the respiratory tract in bronchiectasis and CF, we examined the in vitro effects of human neutrophil elastase (HNE) and proteinase enzymes produced by P. aeruginosa (elastase: PE; alkaline proteinase: PAP) on the ciliary beat frequency (CBF) and ultrastructure of human nasal ciliated respiratory epithelium. HNE (500 micrograms/ml) progressively reduced CBF and caused marked epithelial disruption; lower concentrations (100 and 20 micrograms/ml) also caused epithelial disruption but without slowing CBF. The effects of HNE (500 micrograms/ml) were completely abolished by adding alpha 1-antitrypsin (5 mg/ml). There was no synergy between HNE and pyocyanin, a product of P. aeruginosa which slows CBF. PE in phosphate-buffered saline also caused epithelial disruption without slowing CBF; however, PE in medium containing divalent metal ions caused CBF slowing as well as epithelial disruption at 100 micrograms/ml. PAP (500 micrograms/ml) had almost no effect on ciliated epithelium. The effects of HNE and PE on nasal and bronchial epithelium obtained from the same patient were similar. Light and transmission electron microscopy revealed that HNE and PE were cytotoxic and caused detachment of epithelial cells from neighboring cells and the basement membrane. There was cytoplasmic blebbing of the cell surface and mitochondrial damage; however, no increase of abnormalities in the ultrastructure of cilia on living cells was seen. These results support the hypothesis that HNE and PE contribute to the delayed mucociliary clearance and epithelial damage that is observed in patients with chronic bronchial infection.
Recent advances in animal models for evaluating chondroprotective drugs.
While a variety of animal models have been described to evaluate the effects of antiarthritic and chondroprotective drugs on cartilage metabolism, most cannot be said to truly reproduce the temporal changes that occur in human osteoarthritis (OA). Total meniscectomy is a common orthopedic procedure in humans and frequently leads to OA in later years. We investigated the effects of unilateral medial meniscectomy in sheep knee joints subjected to moderate regular exercise. Morphological and biochemical studies of joint cartilage after 6 months revealed slowly progressive changes similar to those seen in early OA, characterized by cartilage fibrillation, chondrocyte hypertrophy, matrix proliferation and marginal osteophyte formation. The proteoglycans synthesized contained more chondroitin-4-sulphate than chondroitin-6-sulfate.
Selenium and amino acid composition of selenoprotein P, the major selenoprotein in rat serum.
Selenoprotein P is the second plasma selenoprotein to be purified. It is a glycoprotein and has been shown to be distinct from plasma glutathione peroxidase. This study characterizes selenoprotein P further. Deglycosylation of the protein shifts its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from Mr 57,000 to Mr 43,000, indicating it has a substantial carbohydrate component. Measurement of selenium indicates a selenium content of 7.5 +/- 1.0 atoms/molecule based on a polypeptide weight of 43,000. Amino acid analysis accounts for all the selenium as selenocysteine. The protein is also rich in cysteine (17 residues) and histidine (23 residues). Fragmentation of selenoprotein P by trypsin and by cyanogen bromide produces peptides with varying selenium content. This indicates that selenium-rich regions of the protein exist. The concentration of selenoprotein P determined by radioimmunoassay in serum from control rats is 26.3 +/- 4.5 micrograms/ml and in serum from selenium-deficient rats it is 2.7 +/- 0.8 micrograms/ml. Depletion of selenoprotein P from control serum using an immunoaffinity column indicates that over 60% of serum selenium in the rat is contained in this protein. These results demonstrate that selenoprotein P is the major form of selenium in rat serum. It is the first selenoprotein described which has more than one selenium atom/polypeptide chain.
Improved cannulation method for extracorporeal membrane oxygenation.
Extracorporeal membrane oxygenation has been shown to be useful for patients in reversible cardiogenic shock. Effective arterial cannulation techniques for infants have been developed that are simple to use and require minimal subsequent vascular repair or reconstruction after removal. Groin cannulation in adults frequently requires bidirectional arterial cannulation to ensure adequate distal perfusion as well as frequent complex arterial repairs after discontinuation. We describe a simple arterial cannulation technique using a single right-angle, high-flow arterial cannula. With this technique adequate bidirectional arterial perfusion is maintained with a single arterial cannula while the need for vascular repairs or reconstruction is minimized.
Bronchial anomaly of the right upper lobe.
This case report presents a rare anomaly of right upper lobe bronchial anatomy. During routine right upper lobe resection for carcinoma, a common right upper and middle lobe bronchus was found. The resection was completed as a right upper and middle bilobectomy. Knowledge of this uncommon variant was beneficial in performing the pulmonary resection. A review of the literature is presented.
Clinical and laboratory findings in the Paul-Bunnell negative glandular fever-fatigue syndrome.
Forty-one patients with recurrent fatigue were studied for evidence of symptom clustering, abnormal laboratory findings and infection with novel viruses. Symptom enquiry and investigations were repeated 4 months later. Four patients were found to have diseases compatible with their symptoms. In those remaining, an initial acute onset of symptoms was associated with an intermittent course, tender glands and a raised number of T suppressor lymphocytes. Raised numbers of T suppressor lymphocytes at follow-up correlated with resolution of symptoms. Antibodies to human herpesvirus 6 (HHV-6) were found in 75% of the patients as compared to 53% of a control group and more patients than controls were strongly seropositive. Some patients with chronic fatigue have a pattern of illness which suggests glandular fever, although acute infection with Epstein-Barr virus (EBV) is not demonstrated. Primary or reactivation infection with HHV-6 may have a role in this syndrome.
The influence of weight-bearing exercise on articular cartilage of meniscectomized joints. An experimental study in sheep.
Unilateral medial meniscectomy was undertaken in 29 purebred adult merino sheep. Arthrotomy (without meniscectomy) was conducted in 11 animals, and six were used as nonoperated controls. All animals were followed with either three- or six-month active or passive postoperative management. Active animals traversed a total of 360 km after three months and 1040 km after six months. The passive group was housed in pens allowing limited weight-bearing exercise. After death, morphologic changes were recorded and cartilages from the medial and lateral joint regions were examined for water, collagen, and proteoglycan (as hexuronate) content. Proteoglycan aggregation and extractability under nondissociative (0.4 molar GuHCl) and dissociative (4.0 molar GuHCl) conditions were also determined. The collagen content of the medial cartilage of the passively maintained meniscectomized animals was reduced, relative to external controls. Although proteoglycan content was elevated in medial cartilage of the passive group three months postoperatively, these levels returned to the control range after six months. However, low-salt (0.4 molar GuHCl) extractability of proteoglycans still remained high. All cartilages of control and meniscectomized joints showed an elevation of proteoglycan content in the active groups three months postoperatively. The cartilages in the medial region of meniscectomized animals showed the largest increase, but these levels declined after six months. Proteoglycan aggregation and water content were still elevated relative to controls six months postoperatively. The collagen levels in the three-month or six-month actively maintained meniscectomized group were not distinguishable from control values. Morphologically, joints of the passively and actively maintained animals showed focal surface fibrillation and erosions. However, in the active group, osteophytes were common and well developed six months postoperatively. These studies indicate that, while weight-bearing exercise after meniscectomy appears to be beneficial to the quality of the cartilaginous matrix, it is also accompanied by osteophytosis and cartilage hyperplasia.
Active Epstein-Barr virus infection in post-viral fatigue syndrome.
Serological evidence of active infection with Epstein-Barr virus (EBV) was found in 25 of 124 patients (20%) with the post-viral fatigue syndrome (PVFS). In another study on the same group of patients around 50% were found to have evidence of chronic enterovirus infection. No overlap was found between those patients with enterovirus infection and those with active EBV infection. We suggest that there are multiple causes of PVFS and that, in the absence of coexisting immunosuppressive disease which may itself reactivate the virus, EBV may be the aetiological agent in a predominantly female subgroup of patients with PVFS. Furthermore, the disease process in this subgroup may be immunopathological in nature.
Saphenous vein graft patency 1 year after coronary artery bypass surgery and effects of antiplatelet therapy. Results of a Veterans Administration Cooperative Study.
To determine whether antiplatelet therapies improve saphenous vein graft patency after coronary artery bypass grafting, we compared 1) aspirin (325 mg once daily), 2) aspirin (325 mg three times daily), 3) aspirin and dipyridamole (325 mg and 75 mg, respectively, three times daily), 4) sulfinpyrazone (267 mg three times daily), and 5) placebo (three times daily). Therapy with dipyridamole and sulfinpyrazone was started 48 hours before bypass graft surgery, and aspirin treatment was begun 12 hours before surgery as a single 325-mg dose. Postoperative treatment was started 6 hours after surgery and continued for 1 year. Graft patency data were obtained early (median, 9 days) and late (median, 367 days) after surgery. The early graft occlusion rate was decreased with all aspirin treatment regimens compared with that of the placebo regimen. At 1 year, in 406 patients with 1,315 grafts, the graft occlusion rate in all of the aspirin groups combined was 15.8% compared with 22.6% for the placebo group (p = 0.029). The patients taking aspirin once daily had a lower occlusion rate (13.2%) compared with the patients receiving placebo (p = 0.050). At 1 year, in the vein grafts placed to vessels less than or equal to 2.0 mm in diameter (804 distal sites), the graft occlusion rate in all of the aspirin groups was 20.1% compared with 32.3% for the placebo group (p = 0.008). In the vein grafts placed to vessels greater than 2.0 mm in diameter (511 distal sites), there was no difference in the occlusion rates between aspirin and the placebo group at 1 year (8.7% vs. 9.0%, p = 0.918).(ABSTRACT TRUNCATED AT 250 WORDS)
IgG1 subclass deficiency in patients with chronic fatigue syndrome.
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The statistical analysis of graft patency data in a clinical trial of antiplatelet agents following coronary artery bypass grafting.
Because most coronary artery bypass patients receive more than one graft at surgery, it is most important to determine whether statistical analysis of graft patency should be performed on the premise that the multiple grafts within patients are dependent or independent experimental units. Veterans Administration Cooperative Study No. 207 was a multicenter clinical trial comparing four different antiplatelet regimens to placebo in the prevention of graft occlusion following coronary artery bypass grafting. Using the results from the 1-week postoperative angiograms from the Veterans Administration Cooperative Study No. 207, in which there were 3.2 distal anastomoses per patient, we have tested the hypothesis that grafts within patients tend to act dependently with respect to patency or occlusion by comparing the graft patency data to a binomial distribution (i.e., that distribution that would have been manifest if grafts were independent). Because the graft patency results in Study No. 207 significantly deviated from the binomial distribution (p = 0.0003), a more appropriate analysis for graft patency data was applied using a ratio estimate as applied to cluster sampling. The statistical methods used in 11 previous clinical trials of antithrombotic therapy after coronary artery bypass grafting were examined. Only one of the previous studies used such an analysis, and three additional reports attempted to correct for dependency of grafts within patients in their analyses using other statistical methods. In seven of the studies the investigators did not address the potential problem of a dependent relationship between multiple grafts within patients. We conclude that grafts within patients act as dependent experimental units and that the ratio estimate as applied to cluster sampling may be appropriately applied to these data.
Improvement in early saphenous vein graft patency after coronary artery bypass surgery with antiplatelet therapy: results of a Veterans Administration Cooperative Study.
To determine whether specific antiplatelet therapies improved vein graft patency after coronary artery bypass grafting (CABG) we compared (1) aspirin, 325 mg daily, (2) aspirin, 325 mg three times daily, (3) aspirin plus dipyridamole (325 mg and 75 mg, respectively, three times daily), (4) sulfinpyrazone (267 mg three times daily), and (5) placebo (three times daily). Therapy, except aspirin, was started 48 hr before CABG. When aspirin was a treatment, one 325 mg dose was given 12 hr before surgery and therapy was maintained thereafter according to the assigned regimen. Angiographic graft patency data were obtained within 60 days of surgery. Analysis of early graft patency in 555 patients (1781 grafts), revealed the following graft patency rates: aspirin daily, 93.5%; aspirin three times daily, 92.3%; aspirin and dipyridamole, 91.9%; and sulfinpyrazone, 90.2%. All aspirin-containing therapeutic regimens improved (p less than .05) graft patency compared with placebo (85.2%). Chest tube drainage measured within the first 35 hr after CABG revealed that the median loss with aspirin daily (965 ml), aspirin three times daily (1175 ml), and aspirin plus dipyridamole (1000 ml) exceeded (p less than .02) that with placebo (805 ml), while median loss with sulfinpyrazone (775 ml) did not. The reoperation rate was greater (p less than .01) in all the treatment groups that received aspirin (6.5%) compared with the two nonaspirin groups (1.7%). Overall operative mortality was 2.3%, without significant differences among treatment groups. Transient renal insufficiency occurred in 5.3% of patients taking sulfinpyrazone. Thus, early vein graft patency was improved after CABG with all aspirin-containing drug regimens.(ABSTRACT TRUNCATED AT 250 WORDS)
Identification of spectrin-related peptides associated with the reticulocyte heme-controlled alpha subunit of eukaryotic translational initiation factor 2 kinase and of Mr 95,000 peptide that appears to be the catalytic subunit.
An isolation procedure for the reticulocyte heme-controlled alpha subunit of eukaryotic translational initiation factor 2 (eIF-2 alpha) kinase is described which yields different fractions with kinase activity. Each is associated with a different spectrin-related peptide as identified by anti-spectrin monoclonal antibodies. The most abundant of these peptides is the Mr 90,000 species characterized previously (Kudlicki, W., Fullilove, S., Kramer, G., and Hardesty, B. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5332-5336). Association with the spectrin-related peptides appears to account for the heterogeneity of the enzyme during its isolation and for its highly asymmetric structure. Isolated alpha or beta spectrin subunits as well as the separated homogeneous Mr 90,000 peptide cause an increase in the initial rate of eIF-2 alpha phosphorylation that is related to a decrease in Km with little or no effect on Vmax for the phosphorylation reaction. Fractionation of highly purified eIF-2 alpha kinase preparations using affinity chromatography on monoclonal anti-spectrin antibodies has separated eIF-2 alpha kinase activity from the Mr 100,000 phosphopeptide which copurifies with the kinase during all other purification steps. A Mr 95,000 peptide, detectable only by photoaffinity labeling with 8-azido-[alpha 32P]ATP, is shown to be distinct from the Mr 100,000 phosphopeptide and appears to be the catalytic subunit of the eIF-2 alpha kinase.
The matrix components of the epiphyseal growth plate and articular cartilages from dogs treated with ammonium tetrathiomolybdate, a copper antagonist.
As part of a project to study the effect of copper deficiency (CD) on bone development in young dogs, the composition and metabolism of proteoglycans (PGs) and extractability of collagens in the epiphyseal growth plate cartilage (EGPC) and articular cartilages (AC) were investigated. Copper deficiency was induced by feeding ammonium tetrathiomolybdate (TTM) a copper antagonist. The collagen of cartilages from TTM-treated animals was significantly more soluble in 0.5 saline than control tissues. While no distinction between TTM-treated and control cartilages was evident in terms of PG content or extractability under associative (0.5 M-GuHCl) or dissociative (4.0 M-GuHCl) conditions, the sedimentation behaviour of the PG aggregates following CsCl density gradient ultracentrifugation suggested less polydispersity of PGs in preparations from the TTM-treated animals. Moreover, analysis of the PG monomers from EGPC of TTM animals showed galactosamine/glucosamine ratios higher than control preparations, suggesting a reduced keratan sulphate content in these preparations. Organ culture of EGPC showed a significant reduction in the incorporation of 35S into PGs and of 3H-thymidine into DNA in the tissues of TTM-treated animals relative to controls. From these findings we deduce that the catabolism of PGs and the extent of collagen cross-linking in EGPC of TTM-treated animals may be reduced relative to age-matched control tissues.