Biomedical subjects

G A Tennent

Publications and source records attributed to G A Tennent.

34 records · Page 2Linked to original sources

PubMed1993

Molecular characterization of Limulus polyphemus C-reactive protein. II. Asparagine-linked oligosaccharides.

The N-linked oligosaccharides of C-reactive protein (CRP) from the arachnid Limulus polyphemus, the horseshoe crab, were characterized after their release by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. High-voltage paper electrophoresis of the reduced oligosaccharides revealed only neutral species. Gel-permeation chromatography on Bio-Gel P4 yielded five fractions. The oligosaccharide fractions were further fractionated using high-voltage borate paper electrophoresis and Dionex BioLC ion-exchange chromatography. The oligosaccharides were structurally characterized by sequential exoglycosidase digestion, fragmentation by acetolysis and methylation analysis. Three major structures were found, of which two were the biantennary oligomannose type with compositions Man5GlcNAc2 (B-1), Man4GlcNAc2 (C-3) and one was the monoantennary structure Man3GlcNAc2 (D-1). The biantennary oligomannose structures B-1 and C-3 contained the structural unit Man alpha 6Man alpha 6R. This unusual arrangement of mannose linkages suggests a biosynthetic pathway in Limulus which differs from that reported in mammals, plants and the parasitic protozoa.

Animals↗

PubMed1993

Human lysozyme gene mutations cause hereditary systemic amyloidosis.

Hereditary non-neuropathic systemic amyloidosis (Ostertag-type) is a rare autosomal dominant disease in which amyloid deposition in the viscera is usually fatal by the fifth decade. In some families it is caused by mutations in the apolipoprotein AI gene but in two unrelated English families under our care the amyloid deposits did not contain apoAI, despite a report that this may have been the case in one of them. Lysozyme is a ubiquitous bacteriolytic enzyme present in external secretions and in polymorphs and macrophages, but its physiological role is not always clear. Here we report that in these two families, lysozyme is the amyloid fibril protein. Affected individuals are heterozygous for point mutations in the lysozyme gene that cause substitution of highly conserved residues, namely threonine for isoleucine at position 56 in one family, and histidine for aspartic acid at residue 67 in the other. Amyloid fibrils from one individual were composed of the full-length Thr-56 variant lysozyme molecule. To our knowledge, this is the first report of naturally occurring variants of human lysozyme and of lysozyme-associated disease. As the structures of human and hen egg-white lysozyme are known to atomic resolution and their folding and structure-function relationships have been exhaustively analysed, our observations should provide a powerful model for understanding amyloidogenesis.

Amino Acid Sequence↗

PubMed1993

The International Standard for Thyroxine Binding Globulin.

OBJECTIVE: Thyroxine binding globulin is the major thyroid hormone binding and transport protein of the plasma, and its quantitative estimation is therefore of clinical importance. The objectives of the present study were to prepare and ampoule a preparation of thyroxine binding globulin suitable to serve as an international standard, to confirm the suitability of the preparation in a multi-centre collaborative study, and to assign an ampoule content. DESIGN: The collaborative study was designed with the following aims: (1) to compare the candidate preparation with local standards; (2) to calibrate the preparation with local standards and to confirm the assigned ampoule content; (3) to assess the stability of the ampouled preparation. MEASUREMENTS AND RESULTS: Thyroxine binding globulin was purified by a combination of affinity and conventional chromatography. Analysis by gel electrophoresis, N-terminal sequencing and electrospray mass spectroscopy showed the preparation to be > 99% pure and to have a structure consistent with the known structure of thyroxine binding globulin. Thyroxine binding globulin assays performed in the multi-centre collaborative study indicated that the preparation behaved as thyroxine binding globulin when compared to local standards and that the ampoule content was consistent with that assigned on the basis of physicochemical measurements of protein concentration. CONCLUSIONS: The thyroxine binding globulin preparation coded 88/638 was established by the Expert Committee on Biological Standardization of the World Health Organization as the International Standard for Thyroxine Binding Globulin, 30.0 International Units per ampoule, with the additional information that for the preparation in 88/638, 1 IU is equivalent to 1 microgram. The preparation is available from the National Institute for Biological Standards and Control.

Chromatography, Affinity↗

PubMed1993

Studies of the structure and binding properties of hamster female protein.

We report here the characterization of hamster female protein (FP), a member of the pentraxin family of plasma proteins, as a molecule composed of glycosylated subunits of 25,655 MW containing a single intrachain disulphide bridge. In the presence of EDTA the subunits are non-covalently associated as pentamers of mass approximately 128,000 MW, and in the presence of calcium they aggregate further, probably to form decamers. This pentamer-decamer transition at physiological ionic strength has not been described in other pentraxins. As previously reported, FP shares the capacity of C-reactive protein (CRP) in other species to bind phosphocholine and we show here that it also resembles human CRP in binding only weakly to agarose, to human AA amyloid fibrils in vitro, and to mouse AA amyloid deposits in vivo. It thus differs markedly from human and mouse serum amyloid P component (SAP) but it is nevertheless deposited in hamster AA amyloid in vivo and clearly is the hamster counterpart of SAP in other species. These results illustrate the subtle diversity among members of the otherwise conserved pentraxin family of vertebrate plasma proteins.

Alpha-Globulins↗

PubMed1992

Apolipoprotein AI mutation Arg-60 causes autosomal dominant amyloidosis.

A mutation in the gene for apolipoprotein AI (apoAI) was identified in an English family with autosomal dominant non-neuropathic systemic amyloidosis. The plasma of all affected individuals contained a variant apoAI with one additional charge, as well as normal apoAI. The propositus was heterozygous; the coding region of his apoAI gene contained both the normal sequence and a single-base substitution changing the codon for residue 60 of the mature protein from CTG (leucine) to CGG (arginine). Allele-specific oligonucleotide hybridization showed that the other affected individuals were also heterozygotes and that there was concordance of the mutant allele with the presence of variant plasma apoAI. Amyloid fibrils isolated from the spleen of the propositus consisted of proteins that ran as a doublet with an apparent mass of approximately 10 kDa in SDS/PAGE and a trace band at 28 kDa. Electrospray mass spectrometry of the purified 10-kDa material revealed components with mass corresponding to the N-terminal 88, 92, 93, and 94 residues of apoAI each with substitution of arginine for leucine. These observations were confirmed by direct protein sequencing and laser desorption time-of-flight mass analysis. No material with the normal apoAI sequence was detected. The trace band at 28 kDa yielded the N-terminal sequence of mature apoAI, indicating that intact or minimally degraded apoAI was also present in the fibril preparation. Discovery of this mutation and the detailed characterization of the apoAI fragments that form the amyloid fibrils open additional avenues for investigation of amyloidogenesis.

Amino Acid Sequence↗

PubMed1991

Serum amyloid P component in chronic renal failure and dialysis.

A normal reference interval for serum amyloid P component (SAP) concentration in the serum was established in 500 healthy adult individuals (274 women, 226 men), by electroimmunoassay calibrated with standards of highly purified, isolated SAP. The mass of SAP in these was determined from the extinction coefficient of SAP at 280 nm measured here precisely for the first time by spectrophotometry and cryogenic drying. The mean (SD, range) SAP concentration was significantly lower in women: 24 mg/l (8, 8-55), compared to 32 mg/l (7, 12-50) in men (P less than 0.001). In renal insufficiency patients, 38 with chronic renal failure, 79 on hemodialysis and 66 on continuous ambulatory peritoneal dialysis, the mean values for SAP concentration were all significantly higher than normal (range of means, 39-59 mg/l in men and 35-42 mg/l in women), but did not correlate with serum creatinine, duration of dialysis or the presence of an acute phase response. The metabolism of SAP is thus altered in renal failure and is not normalized by dialysis, but it is not clear whether this is relevant to the pathogenesis of dialysis related arthropathy and amyloidosis.

Adult↗

PubMed1991

Studies in vivo and in vitro of serum amyloid P component in normals and in a patient with AA amyloidosis.

Pure serum amyloid P component (SAP) was isolated from a normal donor pool, from individuals with the different genotypes of an MspI restriction fragment length polymorphism (RFLP) linked to the SAP gene, and from a patient with AA amyloidosis. The SAP preparations were all identical and all behaved as a single homogeneous species in polyacrylamide gel electrophoresis, isoelectric focussing, reverse-phase chromatography, binding in vitro to phosphoethanolamine-Sepharose (binding constant 2.4 x 10(7) l/mol) and AL amyloid fibrils (1.6 x 10(8) l/mol), and binding to amyloid deposits in vivo in mice with casein-induced amyloidosis. The in vivo metabolism of 125I-SAP from a single donor was normal and identical in three healthy individuals representing the three different MspI RFLP genotypes. There is thus no frequent polymorphism of SAP in normal subjects, and SAP altered with respect to the characteristics studied here is not a necessary condition for pathogenesis of systemic AA amyloidosis.

Amyloidosis↗

PubMed1991

Plasma leucocyte elastase concentrations in smokers.

The associations between cigarette smoking, plasma leucocyte elastase concentration, peripheral leucocyte count and FEV1 were examined in 148 men, 72 of whom were current cigarette smokers, 40 of whom were ex-smokers, and 36 who had never smoked. All men were part of a long-term survey. Smokers had significantly higher plasma leucocyte elastase concentrations than ex-smokers or those who had never smoked. Mean current FEV1 was lower, and the annual decline in FEV1 in the preceding 10 years was faster in smokers than the other two groups. A few smokers had slight increases in serum C-reactive protein concentrations. Although peripheral blood leucocyte counts were higher in smokers than in non-smokers or ex-smokers, no association was found in any of the three groups of men between plasma elastase concentration and peripheral leucocyte count, nor between either of these two variables and annual decline in FEV1 or current level of FEV1. There was also no relation between plasma elastase concentration and reported daily cigarette consumption or mixed expired carbon monoxide in smokers. The results indicate that some male smokers have increased in vivo release of elastase from peripheral blood neutrophils at a time when there is no evidence of acute infection. Because leucocyte elastase is a strong candidate for pulmonary tissue damage, further studies of the mechanisms that increase plasma concentrations are indicated.

Adult↗

PubMed1990

Pentraxin-chromatin interactions: serum amyloid P component specifically displaces H1-type histones and solubilizes native long chromatin.

Pure serum amyloid P component (SAP) and native long chromatin, mixed together at wt/wt ratios between 1:1 and 1:2 in the presence of physiological concentrations of NaCl and calcium, both remained in solution, whereas each alone precipitates rapidly under these conditions. This solubilization accompanies the binding of SAP to chromatin and the displacement of H1-type histones, which are essential for condensation and higher order folding of chromatin. Such binding of SAP to chromatin is remarkable since displacement of H1 and H5 by salt alone requires approximately 0.5 M NaCl. SAP also bound to nucleosome core particles forming soluble complexes with an apparent stoichiometry of 1:2, a result that is compatible with attachment of SAP at the nucleosome dyad, the site of H1 in intact chromatin. SAP thus undergoes a specific, avid interaction with chromatin that promotes its solubilization and may thereby contribute to the physiological handling of chromatin released from cells in vivo. In contrast, C-reactive protein (CRP) did not bind significantly to either chromatin or to core particles at physiological ionic strength. Incubation of chromatin with either normal serum, or acute phase human serum containing raised levels of CRP, did not induce complement activation regardless of the presence of added SAP or CRP, nor was any cleavage of DNA observed.

Animals↗

PubMed1989

Serum amyloid A protein (SAA) in horses: objective measurement of the acute phase response.

A sensitive and precise immunoassay for equine serum amyloid A protein (SAA) was established and used to determine, for the first time, the circulating concentration of this protein in health and disease. As in other species, equine SAA was present only at trace levels in healthy animals but behaved as an extremely sensitive and rapidly responding acute phase reactant following most forms of tissue injury, infection and inflammation, objectively reflecting the extent and activity of disease. Measurements of SAA should make a significant contribution to diagnosis and management of viral and bacterial infection in horses, and routine serial assays could provide an objective criterion for monitoring prospectively the general health of horses in training and racing.

Acute-Phase Reaction↗

PubMed1983

Demonstration of amyloid A (AA) protein and amyloid P component (AP) in deposits of systemic amyloidosis associated with renal adenocarcinoma.

The fibril protein in three cases of systemic amyloidosis associated with renal adenocarcinoma was identified as amyloid A protein (AA) using immunohistochemical staining techniques. Amyloid P component was also present in all deposits. In one case unfixed snap frozen tissues were studied directly whilst in the other two, which were investigated retrospectively, fixed paraffin sections were stained by immunofluorescence and immunoperoxidase methods after treatment with trypsin. Enzymatic digestion was necessary to restore reactivity with anti-AP but anti-AA reacted well without any such treatment. These findings establish that renal carcinoma, the carcinoma most frequently associated with systemic amyloidosis, causes the same type of secondary or reactive systemic amyloid as chronic infections and inflammatory disorders.

Adenocarcinoma↗

PubMed1982

The acute-phase response in (NZB X NZW)F1 and MRL/l MICE.

The acute-phase plasma protein response to disease activity in murine models of autoimmune lupus-like disease was investigated by measurement of the concentration of serum amyloid P component (SAP) in NZB X W and MRL/l mice. The levels of SAP, which is a major acute-phase protein in mice, did not rise at all in response to progression of disease in NZB X W mice between the ages of 1 and 9 mo. This resembles the behavior of acute-phase proteins such as C-reactive protein and serum amyloid A protein in human systemic lupus erythematosus, and just as in human lupus, where the occurrence of intercurrent microbial infection can stimulate an acute-phase response, so injection of bacterial lipopolysaccharide or casein into the NZB X W mice stimulated "normal" acute-phase SAP production. In marked contrast, MRL/l mice developed greatly increased levels of SAP, which correlated closely with progression of their pathology as they aged. The disease profile of the MRL/l strain includes rheumatoid factors and spontaneous polyarthritis and their SAP response resembles the behavior of acute phase proteins in human rheumatoid arthritis. Different patterns of acute-phase response in different autoimmune disorders may thus be a reflection of the genetic predisposition to particular diseases and/or contribute to their pathogenesis. The existence of animal counterparts for the various clinical patterns of human acute-phase protein production will assist in experimental investigation of the underlying mechanisms and of the biological role of the acute-phase response.

Amyloid↗

PubMed1982

Enumeration of lymphocyte populations defined by surface markers in the whole blood of patients with Crohn's disease.

The proportions and absolute numbers of different lymphocyte populations were determined using alkaline phosphatase-labelled reagents in the whole peripheral blood of 22 patients with Crohn's disease. Monoclonal mouse anti-T cell antibody (OKT3) was used to identify T cells, polyvalent F(ab')2 anti-human immunoglobulin for B cells, C3b for C3b-receptor bearing cells, and soluble IgG antibody-antigen complexes for Fc(gamma)-receptor bearing cells. Endogenous myeloperoxidase served to distinguish monocytes. Application of this methodology to whole blood avoids the inevitable loss of cells which accompanies separation of mononuclear cells from blood and therefore permits precise enumeration of lymphocyte populations in the circulation. No significant difference from healthy adult controls was observed in any of the lymphocyte subsets tested.

Adult↗

PubMed1981

Enumeration of T and B lymphocytes in whole peripheral blood: absence of a null cell population.

Alkaline phosphatase-labelled F(ab')2 polyvalent anti-human immunoglobulin stained a mean of 12% (s.d. 4.2) of lymphocytes in the whole peripheral blood of 15 normal individuals. However, when the sensitivity of detection of the bound anti-immunoglobulin reagent was enhanced by adding complexes of alkaline phosphatase with F(ab')2 anti-alkaline phosphatase, a mean of 22.1% (s.d. 7.8) of lymphocytes were positive. The mean number of T lymphocytes demonstrated in the same blood samples using a monoclonal anti-T lymphocyte antibody (OKT3) was 78% (s.d. 4.1) and was not increased by immunoenzyme enhancement. In five individuals the blood was washed at 37 degrees C to remove passively adsorbed IgG and was then studied using the enhanced method together with monoclonal anti-kappa and anti-lambda antibodies. The mean +/- s.d. number of kappa-positive lymphocytes was 15.5 +/- 4.6% and lambda-positive was 7.9 +/- 1.1%. The sum of these was the same as the number of cells stained either with anti-kappa and anti-lambda together or with the conventional polyvalent anti-immunoglobulin, confirming that the enhancement procedure was detecting integral membrane immunoglobulin and not passively adsorbed IgG. Application to the same blood sample of both the anti-T cell antibody and the enhancement procedure with polyvalent anti-immunoglobulin stained 99-100% of lymphocytes. The present observations confirm that there are two populations among the B-lymphocytes, the B-major cells with readily demonstrable surface immunoglobulin and the B-minor cells on which surface immunoglobulin is demonstrable only by very sensitive techniques (Haegert & Coombs, 1979). The B-major and B-minor cells together account for all the non-T lymphocytes and there are virtually no so-called 'null' cells in normal peripheral blood. These findings have significant implications for the use of surface membrane immunoglobulin as a marker in the typing of normal and abnormal lymphocyte populations.

Adult↗

PubMedDate not supplied

Immunohistochemical demonstration of amyloid P component in cerebro-vascular amyloidosis.

The presence of amyloid P component (AP) in cerebral amyloid deposits was sought using a direct immunoperoxidase technique. AP was detected in the amyloid deposits in the vessel walls but was absent from the intracerebral plaques in tissue from patients with senile cerebral amyloidosis. AP was also present in the amyloid deposits in the vessel walls of patients with the Icelandic form of hereditary cerebral haemorrhage associated with amyloidosis.

Amyloid↗

PubMedDate not supplied

Continuous subcutaneous insulin infusion does not provoke significant acute-phase response.

C-reactive protein (CRP), the classical acute-phase reactant, and serum amyloid A protein (SAA), the putative precursor of AA-type amyloid fibrils, were measured in 62 diabetic patients. They were all attending their regular clinic appointments and had been asymptomatic during the 2 wk preceding sampling. CRP and SAA levels were similar in 18 patients on continuous subcutaneous insulin infusion (CSII), 27 patients treated by conventional insulin therapy (CIT), nine treated by diet only, and eight treated by diet and oral hypoglycemic agents, and were almost entirely within the normal range. It is concluded that CSII does not provoke an acute-phase reaction in diabetic patients and, while caution should always be exercised with a new form of treatment, it does not seem likely that CSII will predispose to the development of reactive systemic amyloidosis.

Adult↗