Biomedical subjects
C J Campbell
Publications and source records attributed to C J Campbell.
Purification and partial characterization of rat factor D.
Rat factor D has been purified to homogeneity (10,559-fold) from serum by chromatography on CM-Sepharose Fast Flow, phenyl-Sepharose CL-4B and Mono S and has been shown to resemble its human and mouse counterparts both in substrate specificity and in its susceptibility to inhibition by the organophosphorous inhibitor di-isopropylfluorophosphate. The rat enzyme, however, is heavily glycosylated and binds to wheat-germ lectin-Sepharose 6MB and 5-hydroxytryptamine-agarose, but not to concanavalin A-Sepharose 4B. All of the carbohydrate chains are N-linked. Enzymic removal of this carbohydrate decreased the Mr by approx. 15,000. The deglycosylated rat enzyme had the same mobility as native human factor D on SDS/PAGE, corresponding to an Mr of 24,500. N-Terminal sequence analysis of the first 30 amino acids of rat factor D highlighted the sequence similarity with human factor D (greater than 76%) and, in particular, with mouse adipsin (greater than 93%).
Reduction of non-steroidal anti-inflammatory drug induced gastric damage in the rat by soluble pepstatin derivatives.
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Peripheral and intracardiac levels of atrial natriuretic factor during cardiothoracic surgery.
Immunoreactive atrial natriuretic factor (ANF) levels were measured in blood taken from superior vena cava, right atrium, pulmonary artery, pulmonary vein, left atrium, and radial arteries in patients undergoing cardiac surgery. Significant (p less than 0.05) differences were seen between sites. Levels of atrial natriuretic factor were seen to rise from superior vena cava (27.5 pmol/L) to right atrium (54.3 pmol/L) and then fall in pulmonary artery (38.5 pmol/L). A further significant fall was seen in pulmonary vein (32.7 pmol/L) samples. There was no significant change in levels in left atrium (32.1 pmol/L) or radial artery (30 pmol/L). The fall between radial artery levels and superior vena caval levels was also significant. The rise from superior vena cava to right atrium is to be expected as this represents the major site of addition of atrial natriuretic factor to the circulation. We propose that the fall in levels from right atrium to pulmonary artery and from pulmonary artery to pulmonary vein suggests uptake and therefore possible local hormonal action on both right ventricle and pulmonary vasculature. The fall from radial artery to vena cava would be in keeping with atrial natriuretic factor's known systemic uptake and action.
Substrate and inhibitor studies with human gastric aspartic proteinases.
The separation of pepsin isoenzymes 1, 2, 3 and 5 (gastricsin) in human gastric juice was effected by chromatography on Mono Q ion-exchanger, and slow-moving proteinase was purified to homogeneity by using a modified procedure incorporating a novel affinity-chromatography step. The pH-activity profiles of these enzymes with mucus glycoprotein and basement-membrane substrates were determined; the profiles for pepsin 2 were noticeably different, and, in general, the pH optima for the hydrolysis of basement membrane were more acidic. Pepsin 1 expressed larger specificity constants (kcat./Km) than pepsin 3 with a series of synthetic peptide substrates, reflecting greater binding (smaller Km) by pepsin 1. Inhibitor studies at pH 1.7 and 4.5 with a series of P2-substituted lactoyl-pepstatins implied that valine at position P2 was optimal for inhibiting pepsins 1, 2 and 3 but detrimental for pepsin 5, whereas lysine at position P2 was tolerated well by pepsin 5 but not by pepsins 1, 2 and 3. The potency of lactoyl-pepstatin with lysine at position P2 did not increase as a function of pH. P2-substituted lactoyl-pepstatins failed to show any inhibitory selectivity among pepsins 1, 2 and 3.
Atrial natriuretic factor release in postoperative cardiothoracic surgical patients.
Immunoreactive atrial natriuretic factor (ANF) levels were measured preoperatively and for 5 days postoperatively in 22 patients undergoing cardiothoracic surgery. They were studied in 4 groups according to surgical procedure (mitral valve replacement, aortic valve replacement, coronary artery bypass grafting, and lung resection). The highest preoperative ANF levels were observed in the aortic valve group (mean 40.9 pmol/L) which were 2.5 (95% CI: 0.7 to 8.6) to 3.5 (95% CI: 0.9 to 13.9) times higher than the other groups. Values tended to peak on the 3rd and 4th postoperative days in all groups, although significantly elevated postoperative ANF concentrations occurred only in the coronary artery bypass group where the levels increased by a factor of 3.2 (95% CI: 1.3 to 7.5). As a separate part of the study, measurement of ANF release before, during, and after cardiopulmonary bypass in 6 patients tended to show a fall in ANF levels when on bypass, with a return to baseline levels on cessation of bypass.
The barrier function in extracapsular cataract surgery.
The distribution of fluorescein between the anterior chamber and the anterior vitreous was measured in two groups of patients after oral administration: group I, extracapsular cataract extraction (ECCE) patients with intact capsule and posterior chamber intraocular lens (PC IOL) (n = 12); group II, intracapsular cataract extraction (ICCE) patients with anterior chamber IOL (AC IOL) (n = 13). The fluorescein concentrations were measured by fluorophotometry and the penetration ratios were calculated. The penetration of fluorescein into the anterior vitreous was significantly less in the ECCE group (group I, penetration ratio = 2.03 +/- 1.00 X 10(-3) min-1; group II, penetration ratio = 5.99 +/- 4.89, X 10(-3) min-1, P less than 0.01). The authors concluded that in ECCE versus ICCE a significantly smaller proportion of fluorescein is found in the anterior vitreous relative to the aqueous after passage through the blood-aqueous barrier. This suggests a barrier to posterior movement of other molecules that may initially gain access to the eye in the anterior segment (e.g., prostaglandins).
A short N-proximal region of prochymosin inhibits the secretion of hybrid proteins from Escherichia coli.
A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.
Proteolytic activities of human Campylobacter pylori and ferret gastric Campylobacter-like organism.
The levels of proteolytic activity in cell washes, lysates and pellets of C. pylori and gastric Campylobacter-like organisms isolated from humans and ferrets, respectively, have been studied using porcine mucus glycoprotein and bovine haemoglobin substrates. The total haemoglobin degrading activity, expressed by 10(12)-10(13) cfu of either organism, was no greater than 3 micrograms chymotrypsin equivalents. The mucolytic specific activity (rate of mucus peptide bond hydrolysis by bacterial protein) of the fractions tested from both organisms did not exceed 2nmol/min/mg protein. This value is 1000-fold lower than expected from published data. Electrophoretic profiles suggested that the mucolytic activity assessed by fluorimetry was insufficient to alter the quaternary structure of mucus and hence may not significantly contribute to the undermining of gastric mucus integrity.
Translational coupling in the threonine operon of Escherichia coli K-12.
In an attempt to express the two distal genes of the Escherichia coli threonine operon, the majority of the first gene in the operon, thrA, was removed and a series of transcriptional fusions were constructed placing the thrB and thrC genes downstream of either the trp or hybrid tac promoter. Analysis of the proteins produced by cells containing these fusions revealed that although the distal gene, thrC, was efficiently expressed, the proximal gene, thrB, was not expressed at a detectable level. A translational fusion was constructed which fused the cat gene in phase to the last 800 base pairs of thrA followed by thrB and thrC. Cells containing this fusion produced high levels of both the thrB and thrC gene products, showing that translation of thrB requires translation through thrA; thus, thrA and thrB are translationally coupled. In addition, it was found that a sequence between 220 and 57 base pairs before the start of thrB was necessary to allow translational coupling to occur.
Purification and partial characterization of rat intestinal cefuroxime axetil esterase.
An esterase which hydrolyses the cephalosporin antibiotic, cefuroxime axetil has been isolated from rat intestinal washings and purified. Closely related cefuroxime esters were extremely poor substrates, but p-nitrophenyl acetate and alpha-naphthyl acetate were slowly hydrolysed by the purified enzyme. Analysis by gel filtration gave an Mr = 51,000 and on SDS-polyacrylamide gel electrophoresis the esterase resolved into two main bands of Mr = 31,500 and 26,800. Analytical isoelectric focusing resolved purified esterase into multiple forms active toward alpha-naphthyl acetate, the isoelectric points of which ranged from pH 4.5 to 6.3. The esterase bound specifically to Con A-Sepharose suggesting it could be a glycoprotein. Esterase activity was unaffected by the presence of dihydroxy bile salts (1-8 mM) and inhibition studies using organophosphates and eserine salicylate have classified the enzyme as a carboxylesterase.
The rapid purification and partial characterization of human serum angiotensinogen.
Human angiotensinogen has been purified 390-fold from serum by a rapid high-yielding procedure that involved chromatography on Blue Sepharose, phenyl-Sepharose, hydroxyapatite and immobilized 5-hydroxytryptamine (5-HT). Angiotensinogen was specifically bound to immobilized 5-HT, which effected a partial resolution into multiple forms, which were also evident when analysed by SDS/polyacrylamide-gel electrophoresis (Mr 59,400, 60,600, 62,600 and 63,800). This heterogeneity was confirmed by resolution into six main bands on isoelectric focusing, ranging from pI 4.40 to 4.82. N-terminal analysis, digestion with human renal renin and deglycosylation studies implied that the preparation comprised several forms of angiotensinogen, varying in their degree of glycosylation. The presence of sialic acid was shown to be a major factor in determining the heterogeneity.
The role of the available sulphydryl group in liver butyryl-coenzyme A synthetase.
Butyryl-CoA synthetase from acetone-dried ox liver mitochondria can be activated up to 80% by low concentrations of iodoacetate with the concomitant modification of one available enzyme thiol group. The relative molecular mass of the carboxymethylated enzyme was unchanged, and there were no major changes in the complex kinetic properties of the enzyme. Extraction experiments on liver mitochondria indicate that in vivo the enzyme is linked to a mitochondrial membrane by a bond cleavable by disulphide bond reducing agents.
Vitreous fluorophotometer data analysis by deconvolution.
The measurement process in fluorophotometry inherently involves a loss of information due to the finite sampling volume of the instrument. Mathematically, the effect is expressed as a convolution of the actual fluorescein distribution with the spread function of the instrument. Scattering by the ocular media can increase the spread function over that due to the instrument alone. A method is proposed for deconvolution of vitreous fluorophotometry data. Simulation studies and analyses of patient data demonstrate recovery of information with this method, including enhancement of retinal peaks and resolution of detail in the posterior vitreous which was not apparent from the original scans.
Widespread absence of outer dynein arms in the spermatozoids of lower plants.
Transverse sections of flagellar axonemes from a variety of lower plant spermatozoids were examined by electron microscopy. Motile sperm of four ferns (Marsilea, Pteridium, Lygodium and Aneimia), a horsetail (Equisetum) and a liverwort (Marchantia) were fixed in the presence of tannic acid to visualise the dynein arms. In all cases the inner dynein arms were clearly resolved but the outer arms were absent. Absence of outer arms therefore appears to be a common feature of the archegoniate plants. The implications of these findings to our understanding of the evolution of the land plants and the role of the dynein arms in flagellar beating is discussed.
Measurement of rat-intestinal cefuroxime axetil esterase activity: comparison of an h.p.l.c. and coupled-enzyme assay.
An alternative method to h.p.l.c. for determining cefuroxime axetil esterase (CAE) activity has been developed which involves coupling acetaldehyde, produced in the esterase reaction, with alcohol dehydrogenase (ADH) to provide a direct reading spectrophotometric assay. The optimum temperature and concn. of NADH, cefuroxime axetil and ADH for the assay are 37 degrees C, 160 microM, 2.9 mM and 160 U/ml, respectively. The coupled assay was more reproducible but less sensitive than the h.p.l.c. assay, and the two methods gave results that were not significantly different (P greater than 0.05). Both assays responded linearly when CAE activity was measured as a function of protein concn., however, the coupled assay was impaired at ionic strengths greater than 0.2 M NaCl, whereas no adverse effects were seen with the h.p.l.c. assay up to 0.5 M NaCl.
Desmoplastic fibroma of bone. A report of eight cases and review of the literature.
Desmoplastic fibroma of bone is a rare benign tumor consisting of thin, wavy fibroblasts set in an abundant matrix of collagen fibers. At times it is difficult to distinguish desmoplastic fibroma from other fibrous lesions, especially low-grade fibrosarcomas. Fewer than eight cases have been previously reported. We have reviewed the diagnostic and therapeutic findings of eighty additional cases. Six patients had the lesions located in an extremity and two had an axial lesion. The average age of the patients was twenty-five years (range, twelve to fifty-six years) and all of the patients had more than two years of follow-up (range, two to seventeen years). The radiographic findings in all but one patient were of a purely lytic, honeycombed lesion that often widened the bone, and was metaphyseal in long bones. The tumor replaced the medullary cavity with a grayish-white, rubbery to firm tissue that was often, but not always, contained by a rim of periosteal reactive bone. Histologically, the features were: (1) prominent loose bundles of fibrous tissue composed of slim, spindle-shaped fibroblasts with wavy, elongated nuclei; (2) variable amounts of bands of collagen fibers; and (3) absence of mitoses or atypical cells. Areas of metaplastic bone were found only around sites, of pathological fractures. The biology of desmoplastic fibroma is different from that of other benign fibrous lesions in that the lesion is very destructive locally and often recurs after incomplete excision. It is also distinguished from low-grade malignant lesions (for example, fibrosarcoma) in that metastases have never been reported. In our series an intralesional excision was initially performed in six of the eight patients and a marginal resection, in two. There were four recurrences, treated by a marginal resection in two patients and repeat curettage in one. The recurrence in the fourth patient required an amputation above the knee after two additional intralesional procedures had been unsuccessful. Wide or marginal resection appears to be the treatment of choice when the lesion is located in a site that can be resected without significant loss of function. In other areas, an attempt at curettage, instillation of phenol, and bone-grafting seems to be warranted, resorting to more radical procedures only if this fails to control local disease.
Ox liver butyryl-coenzyme A synthetase--a subunit enzyme?
Butyryl-CoA synthetase extracted from untreated or freeze-dried liver mitochondria consisted of two fractions of Mr 40,000 and 46,000 as determined by gel permeation chromatography, the higher value being confirmed by sedimentation equilibrium. A red pigment having the characteristic of haem was associated with the enzyme. This appeared to be an artifact of isolation. A number of active bands were obtained on polyacrylamide gel electrophoresis and isoelectric focusing, and on treatment with sodium dodecylsulphate or 6 M guanidine hydrochloride both dissociation and aggregation of the enzyme fractions occurred. As no evidence of protease contamination or proteolytic action could be detected, it is suggested that the active enzymes contain more than one polypeptide chain.