Search PubMed⌕ Search

Biomedical subjects

J Melrose

Publications and source records attributed to J Melrose.

At least 55 records · Page 3Linked to original sources
PubMed1994

A solid phase enzyme linked immunofiltration assay for secretory leucocyte proteinase inhibitor.

A solid phase enzyme linked immunosorbent filtration assay (ELIFA) has been developed for secretory leucocyte proteinase inhibitor (SLPI) utilising polyclonal anti-recombinant SLPI (anti-rSLPI) and polyclonal anti-bronchial mucus proteinase inhibitor (anti-BLPI) IgG samples. Millipore HATF nitrocellulose 96-well plates were used as receptacles for the assay and a commercial goat anti-rabbit IgG alkaline phosphatase conjugate was used as a secondary antibody for quantitation of levels of primary antibodies bound to rSLPI in the plate wells. Antigen bound to the HATF plates efficiently and the washing/blocking steps were simplified by vacuum filtration of samples resulting in a rapid and convenient assay system. The ELIFA was also sensitive and a detection limit of 0.1 ng SLPI/well was achieved using either anti-SLPI or anti-BLPI as primary antibodies. This assay was used to demonstrate the production of SLPI at moderate levels (0.5-3 ng/ml media) by human articular chondrocytes grown in monolayer culture.

Adult↗
PubMed1994

Proteoglycan heterogeneity in the normal adult ovine intervertebral disc.

Proteoglycans (PGs) were isolated from 4 M GuHCl extracts of young adult ovine Intervertebral disc (IVD) tissues using sequential CsCl density gradient centrifugation, and a combination of gel-permeation and hydrophobic chromatography. A total of six PG sub-populations were identified in both the Annulus fibrosus (AF) and Nucleus pulposus (NP), i.e. two high buoyant density aggregatable PGs, two high buoyant density non-aggregatable PGs and two small, low-intermediate buoyant density, non-aggregatable, DS-rich PG species. These latter PGs were identified as biglycan and decorin on the basis of analyses of their core protein native size, and glycosaminoglycan composition. Additional low-intermediate buoyant density PG species were also evident in the non-aggregatable PG pool, particularly in extracts of NP tissues. These PGs did not bind to the octyl affinity matrix under the experimental conditions employed and thus were readily separated from the DS-PGs by hydrophobic chromatography, their constituent glycosaminoglycans (CS and KS) also differed and were of a smaller size to the CS and KS chains isolated from the large high buoyant density PGs, the small PGs which did not bind to octyl-sepharose may therefore represent distinct PG species in their own right. Differences were evident in the absolute size, and in the distribution of individual PG species in the respective IVD tissues but the NP always contained a larger proportion of high buoyant density non-aggregatable PGs of somewhat smaller size than those isolated from the AF. Decorin and biglycan, however, were generally more abundant in AF tissues.

Animals↗
PubMed1993

Secretory leucocyte proteinase inhibitor is produced by human articular cartilage chondrocytes and intervertebral disc fibrochondrocytes.

The objective of this study was to examine the expression of the secretory leucocyte proteinase inhibitor (SLPI) gene by human articular cartilage chondrocytes and intervertebral disc fibrochondrocytes. RNA was extracted from human articular cartilage chondrocytes, synovial fibroblasts and fibrochondrocytes of the annulus fibrosus. Expression was analysed by Northern blotting and hybridisation to a SLPI RNA probe. SLPI was isolated from chondrocyte culture supernatant by gel-permeation and cation-exchange chromatography and examined by a solid-phase enzyme-linked immunofiltration assay for SLPI and by SDS/PAGE using Western blotting with polyclonal IgG samples against bronchial mucus leucocyte proteinase inhibitor and SLPI. This identified biochemically and immunologically the major serine proteinase inhibitory protein synthesised by chondrocytes as SLPI. Secretory leucocyte proteinase inhibitor mRNA was detected in RNA preparations from articular cartilage chondrocytes and annulus fibrosus fibrochondrocytes. The results presented here allowed us to deduce that human articular cartilage chondrocytes and intervertebral disc fibrochondrocytes synthesized SLPI whereas human synovial fibroblasts did not.

Adult↗
PubMed1993

Induction of alloantigen-specific hyporesponsiveness in human T lymphocytes by blocking interaction of CD28 with its natural ligand B7/BB1.

The specificity of T lymphocyte activation is determined by engagement of the T cell receptor (TCR) by peptide/major histocompatibility complexes expressed on the antigen-presenting cell (APC). Lacking costimulation by accessory molecules on the APC, T cell proliferation does not occur and unresponsiveness to subsequent antigenic stimulus is induced. The B7/BB1 receptor on APCs binds CD28 and CTLA-4 on T cells, and provides a costimulus for T cell proliferation. Here, we show that prolonged, specific T cell hyporesponsiveness to antigenic restimulation is achieved by blocking the interaction between CD28 and B7/BB1 in human mixed leukocyte culture (MLC). Secondary T cell proliferative responses to specific alloantigen were inhibited by addition to the primary culture of monovalent Fab fragments of anti-CD28 monoclonal antibody (mAb) 9.3, which block interaction of CD28 with B7/BB1 without activating T cells. Hypo-responsiveness was also induced in MLC by CTLA4Ig, a chimeric immunoglobulin fusion protein incorporating the extracellular domain of CTLA-4 with high binding avidity for B7/BB1. Cells previously primed could also be made hyporesponsive, if exposed to alloantigen in the presence of CTLA4Ig. Maximal hyporesponsiveness was achieved in MLC after 2 d of incubation with CTLA4Ig, and was maintained for at least 27 d after removal of CTLA4Ig. Accumulation of interleukin 2 (IL-2) and interferon gamma but not IL-4 mRNA was blocked by CTLA4Ig in T cells stimulated by alloantigen. Antigen-specific responses could be restored by addition of exogenous IL-2 at the time of the secondary stimulation. Addition to primary cultures of the intact bivalent anti-CD28 mAb 9.3, or B7/BB1+ transfected CHO cells or exogenous IL-2, abrogated induction of hyporesponsiveness by CTLA4Ig. These data indicate that interaction of CD28 with B7/BB1 during TCR engagement with antigen is required to maintain T cell competence and that blocking such interaction can result in a state of T cell hyporesponsiveness.

Animals↗
PubMed1992

Development of an avidin-biotin competitive inhibition assay and validation of its use for the quantitation of human intervertebral disc serine proteinase inhibitory proteins.

A simple convenient method has been developed for the quantitation of serine proteinase inhibitors (SPIs) in tissue extracts. The method is based on the competitive binding to trypsin and chymotrypsin immobilized using glutaraldehyde on 96-well microtiter plate wells of native SPIs and a biotinylated secretory proteinase inhibitor (SLPI) standard. The bound SLPI standard was visualized using an avidin-alkaline phosphatase conjugate and inhibition curves were determined using absorbancy measurements at 405 nm. The standard assay had a range between 0.02 and 1 microgram SLPI/well and a lower detection limit of 20 ng SLPI/well; an improved microassay had a detection limit of 2 ng SLPI/well. Only active free inhibitor was detected in the assay since denatured and/or enzyme-inhibitor complexes did not bind to the plates. A range of SPI species was demonstrable in human bronchial mucus and intervertebral disc SPI samples using this technique. Quantitation of SPI levels in a number of intervertebral disc samples indicated that the SPIs were depleted in degenerate discs compared to nondegenerate discs (P less than 0.05, n = 12). Since the immobilized trypsin and chymotrypsin microplates used in this assay may be prepared in advance (and are stable at 4 degrees C for at least 1 month) the remaining two steps of the assay (the inhibition step and visualization) may be completed in 2-3 h; thus the assay is simple, convenient, and fast. All reagents (other than the biotinylated SLPI standard) are readily available commercially, and in principle the assay could be adapted to other systems provided defined biotinylated standards were available.

Avidin↗
PubMed1992

A longitudinal study of the matrix changes induced in the intervertebral disc by surgical damage to the annulus fibrosus.

A 5 x 5-mm anterolateral incision was made in the annulus fibrosus (AF) of lumbar discs of 16 sheep; four animals of similar age not operated on were used as controls. The experimental animals were sacrificed 2, 4, 6, 8, 12, and 18 months postoperatively (PO), and the incised and adjacent lumbar discs were collected. Discs were dissected into four zones: AF (zones 1 and 2) and nucleus pulposus (NP) (zones 3 and 4) corresponding to the half of the AF in which the cut was made and its opposite half, and the complementary halves of the NP. Each zone was analyzed for moisture, proteoglycan (PG), collagen, and noncollagenous protein (NCP) content. The PG extractability, aggregation, and hydrodynamic size were also examined. The NP of injured discs showed a significant loss of PGs and collagen 8 months PO, but NCP levels increased. In the incised discs, PG aggregation initially declined but recovered to within control values 6-8 months PO. The NP of discs adjacent to the incised disc also showed time-dependent changes in matrix components that included loss of collagen and PG; however, the AF matrix remained essentially uneffected. Double immunodiffusion studies indicated that a sizeable proportion of the NCPs present in the injured discs (but not the adjacent lumbar discs) were derived from serum.

Animals↗
PubMed1992

A comparison of the high buoyant density proteoglycans isolated from the intervertebral discs of chondrodystrophoid and non-chondrodystrophoid dogs.

Groups of purebred beagles and greyhounds of similar ages (1.5-2.5 years) were used for the study. Intervertebral disc proteoglycans (PGs) were radiolabelled in vivo (with [35SO4(2-)], 24 hours and 60 days prior to euthanasia, when lumbar discs were dissected into nucleus pulposus (NP) and annulus fibrosus (AF). Aliquots of each disc region were separately analysed for total PG content as hexuronate. The remaining tissue was subjected to extraction with 4.0 M GuHCl. High buoyant density PGs were isolated from these extracts by CsCl density gradient ultracentrifugation. The hydrodynamic size and aggregatability of the 24-hour, 60-day-old, and resident PG populations were determined by Sepharose CL2B chromatography in the presence or absence of excess hyaluronic acid. While the hydrodynamic sizes of the newly synthesized (24-hour) disc PG preparations appeared to be similar, the 60-day-old greyhound disc PGs were found to be larger than the corresponding beagle disc PG populations. However, the keratan sulphate-core protein complexes prepared by chondroitinase ABC digestion of the newly synthesized (24 hour) disc PGs showed that the greyhound disc preparations were also larger than those from beagle discs. Approximately 80% of the newly synthesized PGs from beagle and greyhound discs were capable of aggregating with hyaluronic acid, however, this was reduced to 55% for the 60 day-old PGs in NP and AF and even less for the resident PG populations (as determined by hexuronate analysis). Significantly, PG aggregation was lower in the greyhound NP and AF preparations than in the corresponding PGs isolated from the beagle disc.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The serine proteinase inhibitory proteins of the human intervertebral disc: their isolation, characterization and variation with ageing and degeneration.

Serine proteinase inhibitory proteins (SPIs) were extracted from human disc tissues using 2 M GuHCl and subjected to CsCl density gradient ultracentrifugation. The SPIs recovered in the low buoyant density fractions (rho < or = 1.35 g/ml) were purified by a combination of gel-permeation, ion-exchange, trypsin affinity, and reverse-phase high performance chromatographies. Characterisation of the major disc SPI by polyacrylamide gel electrophoresis, isoelectric focussing, enzyme inhibition and pH stability studies indicated that this small molecular weight (12-14 kDa), highly basic (pI > 9.5), acid-stable but alkaline-labile protein possessed potent inhibitory activity against bovine pancreatic trypsin and chymotrypsin, and human leukocyte elastase and cathepsin G. Two-major and two-minor low molecular weight cationic SPI species were identified by reverse-phase HPLC. The predominant species was identical to a human articular cartilage SPI sharing amino terminal sequence homology with the mucus proteinase inhibitors (MPIs). It also cross-reacted with an antiserum to the MPIs and behaved identically to secretory leucocyte proteinase inhibitor (SLPI) when examined by reverse phase HPLC, and SDS PAGE. A higher molecular weight (54 kDa), anionic (pI approximately 4.6) SPI was also purified and identified as alpha 1-proteinase inhibitor (alpha 1-PI). Quantification of alpha 1-PI and the small molecular weight cationic disc inhibitors indicated that the latter were depleted in morphologically degenerate disc tissues while levels of alpha 1-PI were somewhat higher although a large proportion of the alpha 1-PI was inactive. A depletion of total SPI levels was evident overall in degenerate discs suggesting a functional role for these inhibitory proteins in the maintenance of IVD matrix homeostasis.

Adolescent↗
PubMed1992

A comparative study of the low-molecular mass serine proteinase inhibitors of human connective tissues.

Low molecular mass serine proteinase inhibitors isolated from human articular cartilage, intervertebral disc, meniscus, and costal cartilage were compared chromatographically. Similar charge and size properties were exhibited when these inhibitors were examined by gel permeation and cation exchange chromatography. The individual proteinase inhibitory species separated by these procedures all cross-reacted with a polyclonal antibody raised against the mucous proteinase inhibitors (MPIs) obtained from human bronchial secretions, however the distribution of these MPI-like species varied with the origin of the connective tissue. The major inhibitory species present in human articular cartilage and intervertebral disc were purified to homogeneity using gel filtration, cation exchange, trypsin affinity and high performance reverse phase chromatography. The amino-terminal sequences of the purified cartilage intervertebral disc inhibitors was found to be identical to the published sequence of MPIs isolated from parotid and seminal secretions. These findings indicate that the endogenous small molecular mass cationic serine proteinase inhibitory proteins present in human cartilaginous connective tissues are members of the MPI family of proteinase inhibitors.

Amino Acid Sequence↗
PubMed1991

The influence of scoliosis and ageing on proteoglycan heterogeneity in the human intervertebral disc.

Proteoglycans (PGs) purified from 19-year-old male (19M), 53-year-old female (53F), and 15-16-year-old adolescent idiopathic scoliotic (AIS) disc tissues were characterised by their aggregation properties with hyaluronic acid (HA), glycosaminoglycan (GAG), and amino acid composition, electrophoretic mobilities by composite agarose polyacrylamide gel electrophoresis (CAPGE), and hydrodynamic size by Sepharose CL2B gel permeation chromatography. Proteoglycan levels were similar in the AIS and 53F discs, but considerably higher levels were found in the 19M discs. Proteoglycan populations assessed by the aforementioned criteria demonstrated a similar level of heterogeneity in the AIS and 53F discs but this differed with the 19M sample. Two small Mr, low buoyant density PG species were particularly prominent in AIS and 53F discs: these were similar to two nonaggregating PG species formerly reported by di Fabio et al.; however, the corresponding PG species in 19M discs were of somewhat different composition. Collectively, these data suggest similar processing of PGs in the AIS and 53F discs. Furthermore, the pattern of normal age changes in the PGs from AIS discs appears to be disturbed, most likely arising as a consequence of the abnormal biomechanical forces intrinsic to the deformity.

Adolescent↗
PubMed1989

Isolation and characterisation of a neutral proteinase from the canine intervertebral disc.

A neutral proteinase of 94 kDa capable of degrading gelatin, canine disc proteoglycan, and L-lysine and L-arginine peptide substrates has been isolated from the greyhound intervertebral disc. Strong inhibition of this proteinase with class-specific inhibitors, such as APMSF, TLCK and benzamidine indicated a 'serine'-type specificity. Metallo, aspartyl- and cysteine proteinase inhibitors were devoid of significant action. Degradation of the resident canine disc proteoglycan monomer by the disc proteinase was shown to occur at the hyaluronic acid binding region, thereby diminishing its ability to aggregate with hyaluronic acid. The hydrodynamic size of the proteoglycan degradation products was only slightly less than that of the intact disc proteoglycan subunits.

Animals↗
PubMed1989

Lysozyme, a major low-molecular-weight cationic protein of the intervertebral disc, which increases with ageing and degeneration.

Lysozyme was demonstrated to increase in content in the human intervertebral disc in a significant age-dependent manner. In young discs (less than 20 years) the levels were 440 U/g wet weight, whereas in older discs (greater than 65 years) the concentration was 3,460 U/g wet weight. These findings contrasted with the hexuronate level (as a marker of proteoglycans) and neutral sugar content of the tissues which decreased with age. The level of lysozyme determined in the disc tissue also correlated with the morphological grade of disc degeneration.

Adolescent↗
PubMed1988

The quantitative discrimination of corneal type I, but not skeletal type II, keratan sulfate in glycosaminoglycan mixtures by using a combination of dimethylmethylene blue and endo-beta-D-galactosidase digestion.

The quantitation of individual glycosaminoglycans in mixtures of polyanions using the dimethylmethylene blue (DMB) method described by R. W. Farndale, D. J. Buttle, and A. J. Barrett (1986, Biochim. Biophys. Acta 883, 173) is dependent on enzymatic hydrolysis by specific polysaccharidases. While using this method to examine the keratan sulfate (KS) of the intervertebral disc we found that digestion with commercially available keratanase decreased binding to DMB by less than 30%, whereas corneal KS was reduced by 85%. However, by preincubating the KS fractions with endo-beta-D-galactosidase prior to keratanase treatment the corneal KS could be completely digested and disc KS digestion increased to 60%. It is suggested that the resistance of the disc KS to these digestive procedures arises from branching and/or sites of multisulfation on the polysaccharide chain. Agarose gel electrophoresis and compositional analyses of the keratan sulfates supported such an interpretation.

Chondroitin Sulfate Proteoglycans↗
PubMed1988

Cysteine proteinase inhibitors of the canine intervertebral disc.

Several species of cysteine proteinase inhibitors have been demonstrated in the greyhound intervertebral disc which were resolved into four species (Mr 15,800, 16,600, 17,200 and 17,800) by gelatin-SDS-polyacrylamide gel electrophoresis. Reductive alkylation did not affect their inhibitory capability nor their electrophoretic migration on gelatin-SDS-polyacrylamide gel electrophoresis. The cysteine proteinase inhibitors from the nucleus pulposus and annulus fibrosus were identical as assessed by the aforementioned criteria, although the level in the nucleus was found to be higher than that in the annulus. Ion-exchange chromatography demonstrated distinct acidic and basic forms of the disc cysteine proteinase inhibitor. The latter species was the most abundant and its Mr was determined to be 16,900 by gelatin-SDS-polyacrylamide gel electrophoresis. Both forms were shown to be strongly inhibitory against the cysteine proteinases, papain and ficin, but were less strongly inhibitory against cathepsin B (EC 3.4.22.1). Presumably these disc cysteine proteinase inhibitors play a regulatory role in the metabolism of proteoglycans and collagen by endogenous cysteine proteinases.

Animals↗
PubMed1987

Neutral proteinases of the human intervertebral disc.

Disc tissue consisting of pooled annuli fibrosus and nuclei pulposus from the cadaver of an adolescent aged 19 years was extracted with 4.0 M Gu-HCl. Proteins of low buoyant density (p less than or equal to 1.38 g/ml) containing the disc enzymes and inhibitors were separated from proteoglycans of high buoyant density (p greater than or equal to 1.50 g/ml) by density gradient ultracentrifugation. Sephadex G-75F gel chromatography followed by trypsin affinity chromatography was then used to resolve disc proteolytic and trypsin inhibitory activities. The results obtained were strongly suggestive of the presence of a high molecular weight zymogen which upon activation generated a population of smaller molecular weight proteinases. The disc proteinases obtained by this process showed similar properties in terms of: their pH optima (7.4-7.6); their inhibition patterns by class-specific proteinase inhibitors; their variation of activity as a function of NaCl and lysine concentrations; and the hydrodynamic size of their proteoglycan degradation products. The activated disc neutral proteinase demonstrated many characteristics in common with plasmin; however, unlike the latter, the disc proteinases also showed some calcium dependence.

Adolescent↗
PubMed1987

Low molecular weight serine proteinase inhibitors of the human intervertebral disc.

Human lumbar disc tissue when extracted with 4M GuHCl and subjected to dissociative CsCl density gradient ultracentrifugation yielded trypsin inhibitor activity in the low bouyant density fractions (rho less than or equal to 1.38 g/ml). Disc proteoglycans sedimented in the high bouyant density fractions (rho greater than or equal to 1.5 g/ml). Sephadex G75F gel filtration of the low bouyant density protein fractions afforded a major low molecular weight (Kav = 0.5) trypsin inhibitor pool which was further purified by trypsin affinity chromatography. This latter step facilitated separation of the trypsin inhibitors from neutral proteinase activity also present. The trypsin inhibitor fraction so isolated was shown to possess potent inhibitory activity against a range of human serine proteinases including leukocyte elastase and cathepsin G, urokinase, kallikrein, plasmin and thrombin. Significantly this serine proteinase inhibitor preparation effectively prevented degradation of proteoglycans by a neutral proteinase also isolated from the human intervertebral disc.

Centrifugation, Density Gradient↗