Biomedical subjects

R A Harrison

Publications and source records attributed to R A Harrison.

At least 127 records · Page 7Linked to original sources

PubMed1989

CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells.

A novel cell surface antigen has been identified on a wide range of lymphoid cells and erythrocytes. A mAb YTH 53.1 (CD59) against this antigen enhanced the lysis of human red cells and lymphocytes by homologous complement. Studies of reactive lysis using different species of C56, and of whole serum used as a source of C7-9, indicated that the inhibitory activity of the CD59 antigen is directed towards the homologous membrane attack complex. CD59 antigen was purified from human urine and erythrocyte stroma by affinity chromatography using the mAb YTH 53.1 immobilized on Sepharose, and, following transient expression of a human T cell cDNA library in COS cells, the corresponding cDNA also identified using the antibody. It was found that the CD59 antigen is a small protein (approximately 20 kD as judged by SDS-PAGE, 11.5 kD predicted from the isolated cDNA) sometimes associated with larger components (45 and 80 kD) in urine. The sequence of CD59 antigen is unlike that of other complement components or regulatory proteins, but shows 26% identity with that of the murine LY-6 antigen. CD59 antigen was released from the surface of transfected COS cells by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor; it is therefore likely to be absent from the surface of affected erythrocytes in the disease paroxysmal nocturnal hemoglobinuria.

Antibodies, Monoclonal↗

PubMed1989

Polyphosphoinositide breakdown and subsequent exocytosis in the Ca2+/ionophore-induced acrosome reaction of mammalian spermatozoa.

An investigation was made of the modifications in phospholipids that occur during the exocytotic event known as the 'sperm acrosome reaction'. Phospholipids were prelabelled with 32P, and exocytosis was induced with Ca2+ and the ionophore A23187. When incubated with [32P]Pi in various media suitable for supporting sperm survival or fertilization in vitro, spermatozoa from all five species examined (ram, boar, guinea pig, mouse and human) incorporated 32P rapidly into the components of the phosphoinositide cycle. There were differences both between species and between media with respect to the actual rate of incorporation of label, and also between species with respect to other phospholipids labelled. Treatment of spermatozoa with Ca2+ and A23187 to induce the acrosome reaction resulted in a rapid breakdown of phosphatidylinositol 4, 5-bisphosphate and phosphatidylinositol 4-phosphate, which was complete within 3 min; there was also a great increase in labelling of phosphatidate. Occurrence of acrosome reactions in the sperm population was only observed after 5-10 min and reached a maximum response of greater than 90% after more than 30 min. The phosphoinositide breakdown was related to subsequent exocytosis: after EGTA/ionophore treatment, neither inositide breakdown nor exocytosis took place; however, later addition of Ca2+ resulted in immediate inositide breakdown, and exocytosis followed, with a delay relative to Ca2+ addition exactly similar to that following standard Ca2+/ionophore treatment. Neomycin inhibited both inositide breakdown and subsequent exocytosis provided it was added together with Ca2+ and ionophore; however, if the drug was added 3 min after Ca2+ and ionophore (by which time inositide breakdown was already complete), exocytosis was not inhibited. Ca2+ seemed to have several consecutive roles in the acrosome reaction. Low (micromolar) levels of free Ca2+ were needed both for phosphoinositide breakdown and for an event downstream of this breakdown; no other bivalent cation could substitute for Ca2+ in either event, and inositide breakdown was actually inhibited by Mg2+. In addition, millimolar levels of Ca2+ were needed for later stages of exocytosis, although this requirement could be satisfied by Sr2+. We conclude that breakdown of polyphosphoinositides is an essential early process after Ca2+ entry in the chain of events that lead to exocytosis in the mammalian sperm acrosome reaction.

Acrosome↗

PubMed1989

The structural basis for neutrophil inactivation of C1 inhibitor.

Limited proteolysis of C1 inhibitor (C1-INH) by neutrophil elastase, Pseudomonas elastase and snake venoms resulted in initial cleavage within the molecule's N-terminus followed by further cleavage within the molecule's C-terminally placed reactive centre. N-Terminal proteolysis occurred at peptide bonds 14-15, 36-37 and 40-41. This had no effect on either the inhibitory activity or the heat-stability of C1-INH. Proteolysis within the reactive centre occurred at peptide bonds 439-440, 440-441, 441-442 and 442-443. Cleavage at any one of these sites inactivated C1-INH and conferred enhanced heat-stability upon a previously heat-labile molecule. Released neutrophil proteinases also cleaved and inactivated C1-INH, suggesting that they may physiologically regulate C1-INH during inflammatory episodes.

Animals↗

PubMed1989

Leucine kinetics in surgical patients. I: A study of the effect of surgical 'stress'.

At present there is no consensus about the effect of surgical and accidental trauma on protein metabolism. Leucine kinetics, an index of protein turnover, have been studied in ten patients before intra-abdominal surgery and on either the third (n = 5) or seventh (n = 5) postoperative day. Only small changes in leucine kinetics were noted in the early postoperative study but, by the seventh day, there was evidence of increased synthesis as part of an overall elevation of turnover rates. It is suggested that the previously expressed opinion that protein synthesis is reduced after surgery must be re-examined.

Aged↗

PubMed1989

Leucine kinetics in surgical patients. II: A study of the effect of malignant disease and tumour burden.

Isotope studies of protein turnover have given conflicting evidence about the effects of a malignant tumour on the protein metabolism of the host. Leucine kinetics have been studied, as an index of protein turnover, using a well established methodology. Twenty patients were studied, five with benign disease of the colon, ten with localized malignant tumours of the large bowel, and five in whom there was evidence of metastatic disease from a colonic primary. No effect was observed on the leucine turnover of the patients that might be attributable to the presence or size of the tumour burden. It is suggested that some of the previously conflicting results may be attributable to variability in the experimental design, the heterogeneous patient groups studied and the methods used to express the results obtained.

Aged↗

PubMed1989

Sensitivity of anthropometric techniques for clinical trials in ankylosing spondylitis.

To determine whether anthropometric techniques widely used for assessment of ankylosing spondylitis (AS) would be useful outcome measures in long-term clinical trials, 52 AS patients at the Royal National Hospital for Rheumatic Diseases were studied. All patients had well documented AS with a mean age of 38.1 years, and had been diagnosed for an average of 7.6 years. Measurements were taken before (B) and after (A) 3-week intensive inpatient physical therapy (PT). Short-term therapeutic effects (over 3 weeks) were significant (adjusted p less than 0.0001) for all five measures tested (chest expansion, finger-to-floor distance, height, lumbar flexion, and cervical rotation). Height (adjusted p less than 0.0001), finger-to-floor distance (adjusted p = 0.047) and cervical rotation (adjusted p = 0.012) diminished over the course of follow-up. Therefore, 3 weeks of hospitalization with intensive physical therapy produces measurable short-term change; minute but measurable change with treatment occurs even in long-standing AS; and detectable changes in physical measurements occur over a 5-year period even in long-standing AS. Anthropometric measurements are useful outcome variables for long-term clinical trials in AS, but the potential for improvement in clinical measurements in long-standing AS is predictably small.

Adult↗

PubMed1989

Vertical banded gastroplasty in the treatment of morbid obesity: results of three year follow up.

Vertical banded gastroplasty has replaced jejunoileal bypass in the surgical treatment of morbid obesity. Although this procedure carries a low incidence of early postoperative complications there is little information on either longer term follow up or the impact on nutritional status. The present study describes the outcome in 42 patients with morbid obesity who underwent vertical banded gastroplasty and were followed up in a special nutrition clinic between nine and 36 months postoperatively. Body mass index (BMI) fell from a preoperative value of 47.4 (6.7) kg/m2 (mean (SD] to 37.5 (5.8), 35 (6.2), 33.9 (6.1), and 33.1 (5.7) kg/m2 at 6, 12, 24, and 36 months respectively. Immediate postoperative complications were minimal but during follow up four patients developed stenosis of the gastroplasty stoma and required reoperation. Plasma albumen concentrations were maintained but three patients developed iron deficiency anaemia. Most patients were able to eat a normal diet in reduced quantities and noted a marked improvement in the quality of life. The benefits and safety of vertical banded gastroplasty suggest an important role for this operation in the management of morbid obesity.

Adult↗

PubMed1989

Pathology resulting from the administration of a live attenuated anti-Schistosoma haematobium vaccine in baboons.

Baboons (Papio anubis) were injected in the leg muscle with 18,000 20 Krad irradiated schistosomula of Schistosoma haematobium. Four protocols were followed: single, primary injection; single injection into animals primed by patent S. haematobium infection; secondary vaccine injection following an earlier injection; and single injection following praziquantel treatment of infected animals. Injection of the putative vaccine elicited localized mixed inflammatory infiltration at the site of injection which was both intense and prolonged. Three grades of tissue reaction were seen: the relatively mild primary response; the response in infected animals which had enhanced tissue eosinophilia; and the response in animals primed by prior injection and drug-treated prior infection. The latter 2 showed intensification of eosinophilia, stellate abscesses in the lesion centers, and perischistosomular Hoeppli precipitates. Intramuscular lesions peaked at 14 days for the primary response and at 7 days for all secondary responses. Traces of the milder lesions persisted beyond 4 weeks; more severe reactions healed more rapidly. Some schistosomula survived for 14 days in the milder reactions. A few larvae were deposited in the skin by backflushing of the injectate which produced local inflammation. Compared to mice, live schistosome vaccines injected into baboons elicited greater local inflammation; however, while evidence suggested that sporadic vaccine schistosomula did reach the lymphatic nodes draining the injection sites, no systemic lesions were found and the injection sites healed in approximately 5-6 weeks without permanent damage.

Animals↗

PubMed1988

Absence of active protein kinase C in ram spermatozoa.

At fertilization, mammalian spermatozoa undergo a Ca2+-dependent exocytotic event known as the acrosome reaction. As protein kinase C (PKc) has been implicated in exocytosis in some other cell systems, we have searched for PKc in ram spermatozoa. We have found that: (a) no changes in protein phosphorylation pattern could be induced in the intact cells by phorbol dibutyrate (PDBu), a compound which binds to and stimulates PKc; (b) no changes in protein phosphorylation pattern could be detected during the course of the Ca2+/ionophore-induced acrosome reaction (when greater than 95% of the cells underwent exocytosis); (c) there was no effect of PDBu on the exocytotic response to various Ca2+ and ionophore levels; (d) no specific PDBu binding could be detected in the cells (this binding is considered to be indicative of the presence of active PKc). We conclude that potentially active PKc is not present in ram spermatozoa.

Animals↗

PubMed1988

Analysis of the interaction between properdin and factor B, components of the alternative-pathway C3 convertase of complement.

The interactions between Factor B (B), its activation products Ba and Bb, properdin (P) and C3i or C3b, components that together form the alternative-pathway C3 convertase enzyme of human complement, have been analysed. Fluid-phase complexes of the purified components C3i, B and P were probed with the homobifunctional cross-linking reagent disuccinimidyl tartarate, and efficient cross-linking of B to P was observed. The 140 kDa B-P conjugate formed was cleaved by Factor D to yield a single product of 85 kDa. This is consistent with a Ba-P heterodimer, and suggests that the initial interaction of B and P includes an interaction of P with the Ba domain of intact B. (The Ba fragment is not retained in the active P-stabilized complex, C3bBbP). By contrast, no cross-linking of P to the Bb domain of B could be demonstrated. Binding studies on cellular intermediates also provided evidence for a site of interaction between B and P, with high concentrations of B inhibiting P binding to EAC3b (sheep erythrocytes coated with antibody and C3b). Neither isolated Ba nor Bb had any effect on the P-EAC3b interaction. High concentrations of B also accelerated the decay of the functional EAC3bBbP complex. These data indicate that the positive co-operativity of binding to C3i or to C3b between B and P is mediated, at least in part, through a direct interaction between B and P.

Complement Activating Enzymes↗

PubMed1988

Dysfunctional C1-inhibitor(At), isolated from a type II hereditary-angio-oedema plasma, contains a P1 'reactive centre' (Arg444----His) mutation.

Simple rapid procedures for identification and analysis of dysfunctional C1-inhibitor proteins mutated at the reactive-centre P1 residue have been developed and used to define structurally a C1-inhibitor protein, C1-inhibitor(At), isolated from an individual with hereditary angio-oedema. The observed mutation, Arg444----His, is compatible with a single base change in the codon used for Arg444 in the native protein.

Amino Acid Sequence↗

PubMed1988

Hyaluronidase in ram semen. Quantitative determination, and isolation of multiple forms.

A study was made of hyaluronidase in ram semen. The end-group assay conditions used to determine activity quantitatively were chosen to ensure reliability as well as sensitivity [Gacesa, Savitsky, Dodgson & Olavesen (1981) Anal. Biochem. 118, 76-84]; they led to 1 W.H.O. Standard International Hyaluronidase Unit displaying 0.1263 EC munit (1 EC unit of activity releases 1 mumol equivalent of N-acetylglucosamine end groups/min at 37 degrees C). All the activity in the semen was shown to be sperm-derived, and intact spermatozoa were estimated to contain 1.23 EC units per 10(9) cells. In a low-ionic-strength medium, only some 20% of the hyaluronidase was extractable, although up to 80% of the activity could be extracted as the ionic strength was increased; further addition of detergent extracted the remainder. During purification of the enzyme, it was found that inclusion of poly(vinyl alcohol) in the media stabilized the activity; detergent inclusion also improved the yield, especially during early stages. As a consequence both of reliable quantitative determination and of stabilization, a number of forms of hyaluronidase could be isolated in high yield, by using anion-exchange chromatography, cation-exchange chromatography, affinity chromatography and gel filtration. The existence of all these forms was confirmed by electrophoresis and immunoblotting with the use of a monoclonal anti-(ram hyaluronidase) antibody, and their presence in very freshly prepared sperm extracts was demonstrated. The specific activity of the isolated major hyaluronidase form was 15.0 EC units/mg; this was equivalent to 119,000 W.H.O. units/mg, higher than any other previously reported values.

Animals↗

PubMed1988

Preliminary characterization of the multiple forms of ram sperm hyaluronidase.

An investigation was made of the inter-relationships and characteristics of various hyaluronidase forms isolated from ram spermatozoa. They were shown to be members of an oligomeric series, apparently formed by intermolecular disulphide cross-linking. Two monomer species were detected, alpha (Mr 89,600) and beta (Mr 81,200). Although the alpha species predominated, the two were evenly distributed throughout the oligomer population, and they shared antigenic determinants; the beta species did not arise from the alpha species as a result of catabolism following cell disruption. The oligomeric series was of the form [Hyal]n, where n = 1, 2, 4, 5, 6, 7 etc.; no trimer was detectable. Though essentially cationic, part of the hyaluronidase population also had anionic characteristics, probably due to oxidation of free thiol groups. In the anionic subpopulation tetramers and higher oligomers predominated, whereas the non-anionic subpopulation was composed of monomers, dimers and tetramers. The pH optimum of the monomer was 4.3 in 0.2 M-NaCl/0.1 M-sodium citrate, whereas that of the anionic oligomers was 4.9. Both serum albumin and polylysine stimulated enzyme activity at pH 4.0 in the absence of NaCl; polylysine was particularly effective. NaCl diminished the stimulatory effects, and essentially suppressed them above the pH optimum. The specific activities of different oligomer populations were the same as that of the monomer, and conversion of oligomers into monomer by reduction had likewise no effect upon the specific activity. Low concentrations of poly(vinyl alcohol), poly(ethylene glycol) or polyvinylpyrrolidone stabilized soluble hyaluronidase activity by preventing the enzyme's binding to surfaces; solutions of anionic oligomers were further stabilized by NaCl. Enzyme preparations were stable for several months frozen in the presence of poly(vinyl alcohol) and salt.

Animals↗

PubMed1988

Analysis of the interactions between properdin, the third component of complement (C3), and its physiological activation products.

The interactions of properdin with both surface-bound and fluid-phase C3 (the third component of complement) and its activation products have been investigated by using a purified preparation of the 'native' form. At physiological ionic strength, a weak interaction with cell-bound C3b (the larger activation fragment of C3) could be demonstrated. In the presence of Factor B this interaction was enhanced, and further enhancement was seen when C3bBb sites were formed on the erythrocytes. The avidities of properdin for cell-bound iC3b (the initial product of Factors I and H action on C3b) and C3b were compared at low ionic strength, with that measured for iC3b being less than that for C3b. In contrast, the affinities of properdin for fluid-phase C3b, iC3b and C3c (the larger product of Factors I and H or CR1 (the C3b receptor) action on iC3b) were all very similar, and apparently much weaker than that for cell-bound C3b. No interaction with either native C3 or, more surprisingly, C3i (haemolytically inactive C3) could be detected. Properdin also inhibited Factor I binding to, and action upon, cell-bound C3b, but did not inhibit Factor I action on fluid-phase C3b. These data permit a more detailed description of the roles of properdin in the alternative pathway of complement activation, emphasizing its importance in concentrating activation at the activating surface.

Complement Activation↗

PubMed1988

The strength, contractile properties and radiological density of skeletal muscle before and 1 year after gastroplasty.

1. Skeletal muscle strength, contractile properties and radiological composition have been studied in seven morbidly obese adults (six female) before and 1 year after gastroplasty operations. The mean body weight fell from 138.3 kg (SD 25.2) to 99.7 kg (SD 23.0) (P less than 0.001). 2. The strength and contractile properties (force/frequency, relaxation rate and fatiguability) of both the adductor pollicis and quadriceps muscles were unaffected by the weight loss. 3. Computerized axial tomography scans obtained 1 year after surgery showed that the quadriceps contained an abnormally high proportion of fat. The mean fat content was 10.8% (range 3.0-30.1%) compared with 1.6% (range 0-5%) for normal muscle. Two individuals were scanned before and after surgery and the fat content of their quadriceps fell from 12.6% and 6.9% to 3.1% and 3.0%, respectively. 4. It is concluded that in obese individuals large amounts of weight can be lost, from both subcutaneous and intramuscular fat stores, without compromising either the strength or contractile properties of skeletal muscles. These results do not support the claim that skeletal muscle contractility is a sensitive indicator of changes in nutritional status.

Adult↗

PubMed1988

Structure of C3f, a small peptide specifically released during inactivation of the third component of complement.

C3f, a peptide presumed to be generated by the combined actions of factors I and H on fluid-phase C3b, has been isolated and sequenced. The peptide is 17 residues long and has a molecular weight of 1,847 daltons. The amino-terminal sequence is, with the exception of a single residue, identical to that deduced for the 46-kilodalton polypeptide seen transiently in the generation of iC3b from C3b, and is in full agreement with the sequence deduced from cDNA analysis. In addition, high-pressure liquid chromatography of the digestion of C3b by factor I has shown that C3f is the sole peptide released during iC3b generation.

Amino Acid Sequence↗

PubMed1988

Multiple forms of ram and bull sperm hyaluronidase revealed by using monoclonal antibodies.

Two monoclonal anti-sperm hyaluronidase-producing cell lines were isolated following inoculation of mice with ram sperm hyaluronidase monomer. Both lines produced antibodies of the IgG1 class; these bound to ram hyaluronidase after 'Western blotting' but did not recognize the native enzyme. Whereas the 1A4 antibody was specific for ram hyaluronidase, and did not react with 'blotted' bull, boar or rabbit hyaluronidase, the 1D6 antibody recognized bull as well as ram hyaluronidase. The antibodies could be used for immunocytochemical localization of hyaluronidase in fixed spermatozoa. However, although some form of denaturation was required to unmask or form the epitopes with which the antibodies reacted, the degree and type of fixation required was critical, for the epitopes were readily destroyed; in particular, they were very sensitive to chemical modification such as glutaraldehyde treatment. It could be demonstrated that, like ram, bull spermatozoa contained an extended oligomeric family of hyaluronidase forms, apparently the result of intermolecular disulphide cross-linking of monomers. In spermatozoa of both species, the enzyme was confined to the anterior acrosomal region of the head.

Acrosome↗

PubMed1987

A major immunogen in Schistosoma mansoni infections is homologous to the heat-shock protein Hsp70.

A 70,000 mol wt protein of Schistosoma mansoni was shown to be a major immunogen that invariably elicited an antibody response in infected humans. The universality of the response to this abundant antigen was confirmed in experimental animals and included the antibody response associated with the protective irradiated cercarial vaccine. We identified the 70,000 mol wt antigen as an S. mansoni homologue of the major eukaryotic heat-shock protein hsp70 by DNA sequence analysis of a cDNA insert from a lambda gt11 clone expressing the antigen and located the immunodominant epitope near the COOH-terminus of the molecule. The antigenic relationship of hsp70 to schistosome infections suggested an important role for this protein in parasite development and pathogenesis.

Amino Acid Sequence↗