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Biomedical subjects

S Ayad

Publications and source records attributed to S Ayad.

At least 19 recordsLinked to original sources
PubMed2001

Interscalene brachial plexus anesthesia for shoulder surgery: report of a complicated intraoperative course.

We present a case of abrupt hemodynamic and mental status changes that occurred during shoulder surgery. During interscalene anesthesia for rotator cuff repair, there was abrupt onset of altered mental status and hemodynamic changes, which had a variety of possible contributing causes. Complete recovery occurred during care in the post-anesthesia care unit. A variety of physiologic changes can occurred during interscalene anaesthesia for shoulder surgery, which require prompt identification and management.

Brachial Plexus↗
PubMed2001

A novel cell culture model of chondrocyte differentiation during mammalian endochondral ossification.

Endochondral ossification (EO) occurs in the growth plate where chondrocytes pass through discrete stages of proliferation, maturation, hypertrophy, and calcification. We have developed and characterized a novel bovine cell culture model of EO that mirrors these events and will facilitate in vitro studies on factors controlling chondrocyte differentiation. Chondrocytes derived from the epiphyses of long bones of fetal calves were treated with 5-azacytidine (aza-C) for 48 h. Cultures were maintained subsequently without aza-C and harvested at selected time points for analyses of growth and differentiation status. A chondrocytic phenotype associated with an extensive extracellular matrix rich in proteoglycans and collagen types II and VI was observed in aza-C-treated and -untreated cultures. aza-C-treated cultures were characterized by studying the expression of several markers of chondrocyte differentiation. Parathyroid hormone-related protein (PTHrP) and its receptor, both markers of maturation, were expressed at days 5-9. Type X collagen, which is restricted to the stage of hypertrophy, was expressed from day 11 onward. Hypertrophy was confirmed by a 14-fold increase in cell size by day 15 and an increased synthesis of alkaline phosphatase during the hypertrophic period (days 14-28). The addition of PTHrP to aza-C-treated cultures at day 14 led to the down-regulation of type X collagen by 6-fold, showing type X collagen expression is under the control of PTHrP as in vivo. These findings show that aza-C can induce fetal bovine epiphyseal chondrocytes to differentiate in culture in a manner consistent with that which occurs during the EO process in vivo.

Animals↗
PubMed2000

Re-expression of procollagen type IIA in oral squamous cell carcinoma.

There are two forms of procollagen type II (IIA and IIB), both of which are expressed during chondrogenesis. Procollagen type IIA also is present at sites of developmental epithelial-mesenchymal interaction. Malignant transformation is associated with disturbed epithelial-mesenchymal interaction and the reappearance of fetal characteristics. This study aims to determine whether or not procollagen type IIA is re-expressed in oral squamous cell carcinoma (OSCC). Immunoperoxidase techniques were applied to frozen and paraffin sections of OSCC (n = 30) and normal oral mucosa (n = 5). In the carcinoma group, strong cytoplasmic staining for collagen type II was present (25/30). Staining was weak or absent in the stroma, and absent from the normal oral mucosa. Frozen sections from 10 of the carcinoma cases which showed positive staining were incubated with antibodies specific for procollagen type IIA and visualised using immunofluorescence. Staining was evident in each case and was particularly strong in the region of the basement membrane. Slot-blot analysis of collagen extracts from OSCC supported the immunohistochemical findings. We conclude, therefore, that procollagen type IIA is re-expressed in OSCC.

Carcinoma, Squamous Cell↗
PubMed1999

The distribution of type VI collagen in the developing tissues of the bovine femoral head.

Type VI collagen appears central to the maintenance of tissue integrity. In adult articular cartilage, type VI collagen is preferentially localised in the chondron where it may be involved in cell attachment. In actively remodelling developing cartilage, the distribution is less certain. We have used confocal immunohistochemistry and in situ hybridisation to investigate type VI collagen distribution in third trimester bovine proximal femoral epiphyses. In general, type VI collagen immunofluorescence was concentrated in the chondrocyte pericellular matrix, with staining intensity strongest in regions which persist to maturity and weakest in regions that remodel during development. Type VI collagen was also present in cartilage canals. In the growth plate and around the secondary centre of ossification, the intensity of type VI collagen stain rapidly decreased with chondrocyte maturation and was absent at hypertrophy, except where canal branches penetrated the growth plate and stain was retained around the adjacent chondrocytes. In situ hybridisation confirmed the presence of type VI collagen mRNA in cartilage canal mesenchymal cells but the signal was low in chondrocytes, suggesting minimal levels of synthesis and turnover. The results are consistent with a role for type VI collagen in stabilising the extracellular matrix during development.

Animals↗
PubMed1998

Glycoproteins of trabecular meshwork, cornea and sclera.

PURPOSE: To analyse high-molecular-weight matrix glycoproteins in trabecular meshwork, cornea and sclera using SDS/PAGE and immuno- and lectin blotting. METHOD: Extracts of normal trabecular meshwork (TM), cornea and sclera were analysed under reducing conditions on SDS/ PAGE. Western blots were stained for total protein, and major high-molecular-weight components were identified by immunoblotting with antibodies to fibronectin (FN) and type VI collagen. Lectin blotting with PSA, MPA and DSA identified some of the glycoprotein glycans. RESULTS: FN antibody bound to the 240 kDa band in TM, cornea and sclera. Type VI collagen antibody bound more strongly to one band and less so to two other bands at approximately 200 kDA in normal TM and to a ladder of bands in cornea and sclera. PSA and DSA bound at 240, 200 and 140 kDa in TM, cornea and sclera. MPA bound at 240, 200 and 140 kDa in TM and at 240, 200 and approximately 120 kDA in cornea and sclera. CONCLUSIONS: FN is a component of the band at 240 kDA in TM, cornea and sclera. Normal TM was found to contain relatively more of one of the isoforms of the alpha 3 (VI) chain whilst cornea and sclera contained all the alpha 3 (VI) isoforms. Complex N-linked bi/tri-antennary glycans were localised in FN and the alpha 1, alpha 2 and alpha 3 (VI) chains in TM, cornea and sclera. O-linked glycans (identified by MPA binding) were located in FN and alpha 3 (VI) chains of TM, cornea and sclera.

Aged↗
PubMed1998

Characterization of two corneal epithelium-derived antigens associated with vasculitis.

PURPOSE: In a previous investigation into corneal autoimmunity, it was demonstrated that a putative autoantigen, a protein of 66 kDa, present in bovine corneal epithelium, binds circulating autoantibodies in approximately 60% of patients with Wegener's granulomatosis (WG). The aim of the present study was to characterize and identify the 66-kDa protein. METHODS: A purification protocol was established for the 66-kDa protein using standard chromatography techniques. During the purification procedure it became clear that the 66-kDa protein detected in patients' sera was in fact two proteins, both running at 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, that eluted in different fractions on DE-52 chromatography columns. These two proteins have been labeled bovine corneal epithelial antigen-A and -B (BCEA-A and BCEA-B). Further investigations of antibody binding have demonstrated that patients' sera bind to either one or the other of these proteins with no cross-reactivity between them. Separated BCEA-A and BCEA-B protein extracts were immunoblotted with 27 WG patients' sera, 10 Churg-Strauss syndrome (CSS) patients' sera, 31 rheumatoid arthritis (RA) patients' sera, and 40 healthy control subjects' sera from the blood bank. RESULTS: Forty-six percent of WG patients' sera had antibodies to one of the 66-kDa antigens, whereas none of the healthy control subjects' sera had 66-kDa antibodies (P < 10(-5)). In the WG group, 31% were positive to BCEA-A (versus controls, P = 0.0023), and 15% were positive to BCEA-B. WG patients with peripheral ulcerative keratitis (PUK) had a significant association with anti-BCEA-A antibodies when compared with healthy control subjects (50%, P < 10(-6)). However, in the RA group with no eye disease there was an association with BCEA-A (25%, P = 0.011) but not in the RA group with PUK. The frequency of anti-BCEA-B antibodies was significantly increased in patients with CSS (60%, P < 10(-7)). CONCLUSIONS: In summary, it has been shown that vasculitis patients have antibodies to two 66-kDa corneal antigens and that autoantibodies to these antigens are mutually exclusive. It has also been shown that antibodies to BCEA-B are associated with CSS, whereas BCEA-A antibodies are associated with WG and RA.

Adult↗
PubMed1997

Trabecular bone formation in the healing of the rodent molar tooth extraction socket.

The aim of this study was to investigate the nature of the template structure on which trabecular bone formation occurs during healing of the rodent tooth extraction socket, a well studied bone healing system. The presence of collagen type II mRNA has previously been described in the healing socket, although the formation of the protein or cartilage has not been observed. However, recent evidence from developmental and other bone healing studies indicates that collagen type III may be important in forming the preliminary scaffold on which bone trabeculae are formed. The maxillary right molar teeth were removed from rats under general anaesthesia and the animals killed at various times afterward. The tissues were examined using histological, in situ hybridization, and immunohistochemical staining techniques. It was concluded that collagen type IIA mRNA was produced by osteoblast cells of the socket, but that collagen type II, if present, would account for less than 0.01% of the total proteins extracted. During bone formation, Sharpey's fibers were seen radiating from the peripheral bone toward the center of the socket. These optically active collagen fibers were inserted into the forming bone trabeculae and were recognized by antibodies raised against collagen type III. The arrangement and composition of these fibers therefore suggest that they form a preliminary framework on which deposition of woven bone trabeculae occurs.

Animals↗
PubMed1996

Type VI collagen is present in human and bovine vitreous.

BACKGROUND: Heterotypic (mixed) collagen fibrils that contain collagen types II, IX and V/XI have previously been identified in vitreous gel. The purpose of this study was to determine whether vitreous gel also contains type VI collagen microfibrils, as they are widely distributed in other tissues where they may play an important structural role. METHODS: Bovine and human vitreous gel was analysed by rotary shadowing electron microscopy after bacterial collagenase digestion and gel filtration chromatography. Bovine vitreous was extracted in 4 M guanidine hydrochloride, and following isopycnic centrifugation fractions were analysed by western blotting. RESULTS: Type VI collagen microfibrils were identified by electron microscopy in human and bovine vitreous. Type VI collagen was identified by western blot analysis. CONCLUSION: Type VI collagen is present in vitreous and may participate in the structural assembly of the vitreous gel.

Animals↗
PubMed1996

Increased metabolism of collagen VI in canine osteoarthritis.

The integrity of joint function depends on both the extracellular matrix and the chondrocytes of articular cartilage. It has been suggested that there is a reciprocal relationship between these two components, which is altered in osteoarthritis. The immunohistochemical distribution of type VI collagen in normal and osteoarthritic canine cartilage was investigated by immunofluorescence and immunoelectron microscopy. Immunogold labelling showed that in normal cartilage type VI collagen was concentrated in the capsule adjacent to the chondrocyte complex. However, in osteoarthritic cartilage, type VI collagen was also observed throughout the cartilage matrix and was greatly increased in the territorial matrix and pericellular capsule surrounding the chondrocytes. Naturally occurring canine osteoarthritis provides a useful model for the study of the human disease, particularly the early stages. The changes in type VI collagen observed in osteoarthritis suggest an attempt at cartilage repair, resulting in the reorganization of the matrix.

Animals↗
PubMed1996

The expression of types X and VI collagen and fibrillin in rat mandibular condylar cartilage. Response to mastication forces.

Types X and VI collagen and fibrillin were localized by in situ hybridization and immunohistochemical methods in the mandibular condyles of rats, and the response of these molecules to post-weaning diets of soft food, ordinary pellets, or hardened pellets was studied. Type X collagen was synthesized, particularly in conditions of soft food consistency, by cells in the perichondrium-periosteum and in the bone and by cells at the erosion front between cartilage and bone. Type X collagen synthesis diminished under higher compression forces due to mastication and with increasing age. Type VI collagen and fibrillin were synthesized by cells in the perichondrium-periosteum and by chondrocytes and by stromal osteoblasts and were not modified by higher mechanical forces. In contrast to previous findings in the growth plate of long bones, type X collagen in the mandibular condyle was not synthesized by hypertrophic chondrocytes but was associated with cells of the osteoblastic rather than the chondroblastic phenotype.

Animals↗
PubMed1994

Identification of alternatively spliced variants of type II procollagen in vitreous.

Adult and foetal bovine vitreous were analyzed for the presence of unprocessed and partially processed forms of type II procollagen. Type II procollagen can exist in two alternatively spliced forms: a short form which lacks a large part of the N-propeptide due to the splicing out of exon 2 and a long form in which exon 2 is expressed and which has a full-sized N-propeptide. Both splice variants were demonstrated and analyses of type II pN-collagen demonstrated that the long form predominated, the ratio of the long form to the short form being 5:1 in foetal vitreous and 1.5:1 in adult vitreous. In foetal vitreous 35% of the type II collagen extracted was in the pN form, but there was very little unprocessed type II procollagen or type II pC-collagen. In contrast, adult vitreous extracts contained relatively less type II pN-collagen, but contained larger amounts of type II pC-collagen and unprocessed type II procollagen.

Alternative Splicing↗
PubMed1994

Extraction and characterization of the tissue forms of collagen types II and IX from bovine vitreous.

We report for the first time that, after centrifugation of adult bovine vitreous, the hyaluronan-rich supernatant contains collagens which can be isolated in their intact forms by precipitation with 4.5 M NaCl. This precipitate constituted approx. 4% of the total vitreous collagen and comprised collagen types IX and II (in the approximate ratio of 4:1) with negligible amounts of type-V/XI collagen. Type-II collagen was present partly in a pro-alpha 1(II) form, suggesting that there is active synthesis of type-II collagen into the matrix of adult bovine vitreous. Type-IX collagen was purified (2-2.5 mg/l of vitreous) and its glycosaminoglycan chain composition was analysed. Bovine vitreous type-IX collagen always possessed a glycosaminoglycan chain of comparatively low M(r) that was predominantly 4-sulphated, with chondroitin 6-sulphate representing a more minor component. By contrast, chick vitreous has been shown to contain type-IX collagen which always possesses a high-M(r) chondroitin sulphate chain that is predominantly 6-sulphated. The functional significance of these different glycosaminoglycan chain lengths and sulphation patterns is discussed.

Animals↗
PubMed1994

Rat fibrosarcoma as a source of stable co-polymer of type I and type III collagens.

During conventional procedures for the extraction of collagen from rat fibrosarcoma it was observed that a significant fraction of the acid-soluble collagen remained insoluble when subsequently fractionated at neutral pH. Analysis of this collagenous component, using amino acid analysis, cyanogen bromide peptide mapping, proteinase susceptibility and the action of denaturing and reducing agents, revealed different properties when compared to other collagen species. The results indicate that the tumour collagen fraction is probably a stable complex of type I and III collagens. This native collagen heteropolymer represented approximately 18% of the total collagen from rat fibrosarcoma and as such provides a unique natural source of this collagen complex for further analytical studies.

Amino Acids↗
PubMed1994

Conformation dependence of integrin-type II collagen binding. Inability of collagen peptides to support alpha 2 beta 1 binding, and mediation of adhesion to denatured collagen by a novel alpha 5 beta 1-fibronectin bridge.

The mechanism of interaction of chondrocytic cells with cartilage-specific type II collagen has been examined using HCS-2/8 human chondrosarcoma cells as a model system. By the criteria of specific collagen secretion and integrin expression profile, HCS-2/8 have a similar differentiated phenotype to normal chondrocytes and are therefore a good model system. HCS-2/8 cells were able to attach and spread on both native and heat-denatured pepsinised type II collagen, and assays using denatured cyanogen bromide fragments apparently localised the major cell binding site to the CB10 fragment. However, when they were used as soluble inhibitors, cyanogen bromide fragments were found to block adhesion to denatured collagen, but had no effect on either attachment or spreading on the native molecule. The inability of cyanogen bromide fragments to reproduce the cell binding site of native collagen demonstrated a strict dependence on collagen conformation. This was also reflected in the receptors that were employed by HCS-2/8 cells for binding to type II collagen: binding to native collagen was mediated by the integrin alpha 2 beta 1 while binding to denatured collagen was mediated by a novel alpha 5 beta 1-fibronectin bridge. The identification of this bridge adds to the mechanisms by which cells can bind to denatured collagens. The previously characterised KDGEA active site peptide from type I collagen was found to be inactive as an inhibitor of type II collagen-mediated adhesion. The implications of these findings for the strategies used to identify adhesive active sites within collagens are discussed. In particular, these data suggest that, unlike other integrin ligands, a synthetic peptide-based approach is not suitable for the identification of collagen active sites.

Amino Acid Sequence↗
PubMed1992

Extraction and characterisation of the intact form of bovine vitreous type IX collagen.

We provide the first biochemical characterisation of intact type IX collagen extracted from bovine vitreous. It possesses a shortened alpha 1(IX) chain (M(r) 64K) compared to its cartilage counterpart (M(r) 84K). All the vitreous type IX collagen is in a proteoglycan form, its glycosaminoglycan constituent being a chondroitin/dermatan sulphate component of M(r) 15-60K attached to the alpha 2(IX) chain. This contrasts with previous findings in chick vitreous where a very long glycosaminoglycan chain of M(r) approximately 350K was demonstrated.

Animals↗