Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments.

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.

Evidence that major 78-44-kD concanavalin A-binding glycopolypeptides in pig epidermis arise from the degradation of desmosomal glycoproteins during terminal differentiation.

The major concanavalin A (Con A)-binding component in urea/deoxycholate/mercaptoethanol extracts from pig ear epidermis had an apparent Mr of 78 kD. In indirect immunofluorescence affinity-purified polyclonal antibodies against this glycopolypeptide strongly stained the surface of suprabasal cells in the epidermis of pig and human skin. Immunocytochemical labeling with gold-labeled second antibody localized this staining to externally disposed, trypsin-sensitive components of desmosomes. Western blotting showed that the 78-kD glycopolypeptide was immunologically related to several other Con A-binding components in pig epidermis. Immunoreactive components with Mr of 115 and 100 kD were membrane-bound, appeared to be susceptible to trypsin in intact epidermis, and were absent from the stratum corneum. Immunoreactive components of lower Mr (78-44 kD) were not membrane-bound, were resistant to trypsin in intact tissue, and were present predominantly in the keratinized layers of pig epidermis. The 115-44-kD glycopolypeptides were also recognized by antisera raised against desmoglein II/desmocollin glycoproteins isolated from bovine spinous layer desmosomes. In addition, these antisera reacted with 120- and 105-kD bands that were apparently not recognized by the anti-78-kD glycopolypeptide antiserum in immunoblotting. In immune precipitation the anti-78-kD glycopolypeptide and antidesmoglein II/desmocollin antisera precipitated comparable amounts of the radioiodinated 78-44-kD components. Both antisera also precipitated the 120- and 105-kD components although the anti-78-kD glycopolypeptide serum was less effective. Little reaction with the 115- and 105-kD components was observed in immune precipitation with either serum. Proteolytic peptide mapping confirmed that the various immunoreactive glycopolypeptides were biochemically as well as immunologically related. The results suggest that terminal differentiation in pig epidermis is accompanied by the orderly degradation of desmoglein II/desmocollin glycoproteins resulting in the accumulation of 78-44-kD glycopolypeptides in the stratum corneum. These glycopolypeptides may represent functionally important nonmembranous domains of cell-adhesion molecules in desmosomes.

The intracellular localisation of proteoglycans and their accumulation in chondrocytes treated with monensin.

Pig laryngeal chondrocytes incubated in the presence of monensin showed inhibition of [35S]sulphate incorporation and decreased secretion of proteoglycan into the culture medium, but no large decrease in protein synthesis. This lead to the intracellular accumulation of proteoglycan protein core, which was detected in immunoprecipitates of cell extracts. Using the same antiserum protein core was localised by electron microscopy with protein A-coated gold. In control chondrocytes, it was detected only in elements of the Golgi and in secretory vesicles, but following monensin treatment labelling was more intense in the Golgi and extended into the distended cisternae of the rough endoplasmic reticulum. The results suggest that monensin blocks proteoglycan protein core translocation between different elements of the Golgi and that this occurs prior to the major site of chondroitin sulphate synthesis on proteoglycan.

Desmoglein II-derived glycopeptides in human epidermis.

Antisera raised against a major 78 kD glycopeptide from pig epidermis were used to identify desmoglein II-derived glycopeptides in the conA-binding material isolated from human epidermis. In whole CaCl2-separated epidermis the antiserum recognized conA-binding components with apparent Mr of 115, 100, 82, 68, 50, 48, and 46 kD. The 82, 68, 48, and 46 kD immunoreactive bands were present in normal stratum corneum and plantar callus. Psoriatic scales contained significantly more of the 82 kD components and less of the 48 and 46 kD bands. Psoriatic scales also contained a major 50 kD conA-binding component unrelated to keratins or desmoglein II. Proteolytic peptide mapping showed that the major immunoreactive bands in normal stratum corneum and plantar callus were also chemically related. The 82 to 46 kD immunoreactive glycopeptides in plantar callus coincided with the major coomassie blue stained bands and were homogeneous on two-dimensional gels suggesting that this tissue may be a valuable source of human desmoglein II-derived glycopeptides. An antiserum directed against the electrophoretically co-purified 48/46 kD glycopeptides from plantar callus recognized the 82 to 46 kD bands in immunoblotting. In indirect immunofluorescence of frozen skin sections this antiserum stained the surface of epidermal cells in the spinous and granular layers of the tissue. In immunogold labeling of paraformaldehyde-fixed skin sections affinity-purified antibodies stained intact desmosomes in spinous and granular cells and desmosomal remnants in the stratum corneum. The results are consistent with our hypothesis that desmoglein II undergoes limited cleavage to stable fragments during terminal differentiation. Proteolytic degradation appears to be incomplete in psoriatic epidermis.

Basal cell glycoprotein in pig epidermis closely resembles the beta 1 subunit of the integrin family of cell adhesion molecules.

A 135-kD conA-binding glycoprotein isolated from pig epidermis was previously localized to the surface of basal cells in stratified epithelia using affinity-purified antibodies. Preembedding immunoperoxidase electron microscopy has now shown that this glycoprotein is concentrated on the lateral surfaces of basal cells but is not detectable on those surfaces adjacent to the basement membrane indicating a role in cell-cell rather than cell-substrate interactions. The basal cell glycoprotein was shown to resemble the beta 1 subunit of the integrin family following the generation of a specific monoclonal antibody (M5.25). The epidermal glycoprotein recognized by M5.25 and by antibodies against the beta 1 fibronectin receptor from human placenta co-migrated on SDS gels under both reducing and non-reducing conditions. Its response to disulphide reducing agents was characteristic of beta 1 integrin subunits. In addition, the basal cell glycoprotein was shown to bind to the 120-kD cell-binding fragment of fibronectin in a RGD-dependent manner. It was readily detected by immunoblotting whole cell lysates of cultured pig keratinocytes suggesting increased expression in cultured cells compared to fresh epithelial tissue. The results suggest that beta 1 integrin subunits may be involved in cell-cell interactions between basal keratinocytes in pig epidermis and that these receptors are lost from the cell surface during terminal differentiation. Thus modulation of beta 1 integrin subunit expression may play an important role in regulating differentiation in pig epidermis.

Type IX collagen: a possible function in articular cartilage.

The effect of type IX on in vitro fibrillogenesis of type II collagen indicated that, while not preventing fibrillogenesis, the presence of type IX collagen reduced the size of the type II fibre aggregates. This observation is consistent with the in vivo localisation studies of type IX collagen. Using the immunogold labelling technique, type IX collagen was shown to be located evenly on small fibrils which occur at higher concentration closer to the cell. Therefore type IX collagen may function as a regulator of fibre diameter in articular cartilage.

Ultrastructural changes and lipid peroxidation in rat adipomusculocutaneous flap isotransplants after normothermic storage and reperfusion.

Parallel in vivo, histological, and ultrastructural studies were carried out and markers of lipid peroxidation (Schiff's bases [SB] and thiobarbituric-acid-reactive material [TBAR]) were measured in rat adipomusculocutaneous flap isotransplants that had been stored for 0, 2, 4, 6, and 8 hr under normothermic (37 degrees C) conditions and reperfused for specific periods. Flaps stored for 4 hr and treated with intravenous desferrioxamine (DFX) or hypertonic citrate flush (HCA) were also evaluated. In vivo assessment was made after 7 days of reperfusion. Flaps stored for 4 hr eventually exhibited partial necrosis in vivo, and neither DFX or HCA flush increased the area of surviving skin. Electron microscopy revealed extensive storage damage in epidermal, follicle, fat, and smooth muscle cells and in endothelium. HCA significantly preserved fat cells (P = 0.0035) and DFX diminished smooth muscle damage. Reperfusion injury was seen in endothelial cells in the form of swelling that was not prevented by HCA or DFX. Ultrastructural alterations correlated with changes in susceptibility to lipid peroxidation in fat but not in skin. The results of these parallel studies indicate that both free radical-dependent and independent mechanisms operate in ischemia and reperfusion injury in flap tissue and that fat has a greater predisposition to free radical damage than skin.

Lipid peroxidation and ultrastructural changes in rat lung isografts after single-passage organ flush and 48-hour cold storage with and without one-hour reperfusion in vivo.

Rat lung isografts were preserved for 48 hr at 0 degrees C using a simple organ flush technique. After storage alone, isotonic saline flush resulted in significantly raised indices of lipid peroxidation in vitro (Schiff bases and thiobarbituric-acid-reactive material [TBAR]). Lungs flushed with hypertonic citrate (HCA) had significantly less oxidative damage than saline-flushed lungs. The addition to the HCA flush of verapamil, a calcium channel blocker, or desferrioxamine, an iron chelator, significantly reduced TBA reactivity in stored lungs compared with HCA alone. After 1-hr reperfusion in vivo, lipid peroxidation was reduced in HCA-flushed lungs compared with saline flush (TBAR alone), but no additional protection from the use of desferrioxamine or verapamil was demonstrated. Electron microscopy after saline flush and storage alone showed gross endothelial swelling and fragmentation. Reperfusion with blood for 1 hr resolved cell swelling, but alveolar/capillary wall rupture occurred. HCA protected against cell swelling, but endothelial vesiculation and widening of the basement membrane were observed. After reperfusion, HCA-flushed lungs developed much endothelial loss that was considerably reduced by the use of desferrioxamine and verapamil. The lipid peroxidation results suggest that iron- and calcium-mediated free radical production may be important mechanisms in oxidative damage to stored rat lungs. Electron microscopy findings correlated with biochemical evidence of free-radical-mediated injury. Reduction of endothelial loss on reperfusion by the use of verapamil and desferrioxamine provides circumstantial evidence that ischemia and reperfusion damage of organs stored for transplantation is partly due to Fe++(+)- and Ca+(+)-dependent mechanisms that probably involve increased free radical production.

The distribution of aggregating proteoglycans in articular cartilage: comparison of quantitative immunoelectron microscopy with radioimmunoassay and biochemical analysis.

Electron microscopic immunolocalization and radioimmunoassay have been used to determine the variation with depth of the hyaluronate-binding region of proteoglycan in articular cartilage. The cartilage was cut into serial sections from the articular surface to the bony margin, the proteoglycans were extracted from each section and determined by radioimmunoassay using antibodies raised against proteoglycan binding region. Proteoglycans were found to be most abundant in the middle zone and least abundant near the articular surface. Biochemical analysis for hexuronate in the same extracts showed a distribution of proteoglycan in agreement with these and other published results. The binding region antiserum was used for electron microscopic immunolocalization of proteoglycan with ultrathin sections of cartilage embedded in Lowicryl K4M resin. After digestion of the sections with chondroitinase ABC, the proteoglycans were localized using the antiserum and protein A-coated gold particles as immunolabel. The density of labeling was quantified using a Magiscan image analysis system. Throughout the depth of the cartilage matrix labeling was higher in the pericellular regions compared to the intercellular regions, and variation of the amount of immunolabel with depth was found to show a good correlation with the results from radioimmunoassay. Intracellular labeling of proteoglycans was mainly found over the Golgi region and in membrane-bound (secretory) vesicles.

A study of the distribution of 7-ethoxycoumarin O-de-ethylase activity in human placental subcellular fractions.

Human placental homogenates from maternal smokers and non-smokers were fractionated using differential centrifugation techniques. Yields of the various subfractions were determined and their homogeneity assessed using electron microscopic procedures. The distribution and response of 7-ethoxycoumarin O-de-ethylase activity towards inhibition by dimethylsulphoxide, alpha-naphthoflavone and 9-hydroxyellipticine inhibitors in the placental subfractions were investigated. The low yield of microsomal protein obtained following differential centrifugation of placental homogenates (2.5 +/- 0.2 mg protein per g placenta) highlights the extremely refractory nature of human placental tissue towards homogenization. Enzymic studies showed that the majority (75 per cent) of the original O-de-ethylase activity in homogenates from smokers and non-smokers was to be found in the crude nuclear fraction. The 7-ethoxycoumarin O-de-ethylase activity present in both homogenate and crude nuclear preparations from a maternal smoker was found to be inhibited by both alpha-naphthoflavone and 9-hydroxyellipticine to a lesser extent than the O-de-ethylase activity which was present in both mitochondrial and microsomal fractions. While this observation suggests the existence of more than one induced O-de-ethylase activity in the human placenta, the possibility that such differences in inhibitory response may be due to other factors (e.g. inhibitor solubility effects) cannot be excluded. Studies using the above inhibitors also confirmed the results of earlier work by demonstrating that the O-de-ethylase activity in placental homogenates and subfractions from non-smokers is qualitatively different from the O-de-ethylase activities induced as a result of maternal smoking.

Effects of hypothermic storage on the vascular endothelium: a scanning electron microscope study of morphological change in human vein.

OBJECTIVE: The aims of this study were: i) to identify morphological changes occurring in the endothelium of human umbilical veins subjected to the typical storage procedures used in transplantation and ii) to determine the relative efficacy of preservation solutions containing intra and extracellular levels of sodium and potassium. EXPERIMENTAL DESIGN: Prospective. PROCEDURE: Scanning electron micrographs were taken pre and post cold hypoxic storage of human umbilical veins for 3 or 16 hours. RESULTS: Cold preservation resulted in severe cell detachment with subsequent loss of monolayer continuity and exposure of thrombogenic basal membrane components (highly significant after only 3 hours of cold storage, Kruskal-Wallis, p < 0.01). The morphological alterations culminated in EC with spherical shapes. Cytoplasmic membranes presented an increased number of microvilli and intercellular processes, followed by microvillous swelling and surface blebbing as damage increased. Bleb detachment was seen in severely damaged specimens. However, morphological preservation was not significantly affected by the duration of hypoxia or the ionic balance of the solution tested. CONCLUSIONS: The present results demonstrate that even short periods (3 hours) of cold storage without revascularization cause significant morphological damage to the endothelium. The ionic composition of the preservation solution did not significantly affect the process. The morphological changes seen in this study could explain storage-related problems such as loss of normal vascular permeability and increased thrombogenicity, problems often associated with transplantation procedures.

Accumulation of membrane-bound melanosomes occurs in Langerhans cells of patients with the Leopard syndrome.

The Langerhans cells in the lentigines of four patients with the Leopard syndrome contained large membrane bound accumulations of melanin granules. Giant melanosomes were only seen in two patients. The patients had no immune-based symptoms relating to their lentigines. The Leopard Syndrome, also known as multiple lentigines syndrome, progressive cardiomyopathic lentiginosis, lentiginosis profusa syndrome and the cardiocutaneous syndrome, refers to an inherited abnormality of the skin, often associated with cardiomyopathy. The aetiology of the condition is so far unknown and the penetrance is variable. Here we describe electron microscopical findings of large accumulations of melanin within Langerhans cells.

Technical difficulties overcome in the use of Lowicryl 4KM EM embedding resin.

Several technical difficulties have been overcome in the use of Lowicryl 4KM resin. In order to embed and section tissue satisfactorily in the resin, it has been found necessary to thoroughly degass the resin before infiltration and polymerisation. After irradiation with UV light, the blocks are further polymerised by exposure to daylight for 2-3 weeks and then stored under partial vacuum over dessicant.

Reperfusion injury in rat adipomusculocutaneous flaps: an ultrastructural and microangiographic study.

Electron microscopy (EM), microangiography (MA) and histological studies were carried out to demonstrate the vascular basis of ischaemic and reperfusion injury in rat adipomusculocutaneous free flaps which had been stored under normothermic conditions (37 degrees C) for specific periods of time. MA revealed that areas destined to die reperfused for at least 6 h in this model. EM showed progressive endothelial damage as a result of both storage and reperfusion. The use of the iron chelator desferrioxamine (DFX) did not benefit the endothelium or improve salvage of ischaemic flaps.

Scanning electron microscopic changes in morphology of pulmonary endothelium in rat lung isografts following hypothermic ischaemic storage and transplantation.

Endothelial monolayer integrity is a critical factor limiting vascular permeability of solid organs in transplantation. Several in vitro, ex vivo and in vivo studies suggest that damage to endothelial cells (EC) due to hypothermia and ischaemia-reperfusion injury causes morphological and functional damage to the endothelium leading to parenchymal oedema and haemorrhage. Aiming to study morphological changes to arterial pulmonary EC subjected to transplantation procedures, random scanning electron micrographs of vascular endothelium of rat lungs were taken. Forty-eight rat lungs were hypothermically stored for 48 or 72 hours in two different preservation solutions and studied either at the end of the cold storage period, or 5 min, 24 h or 4 weeks following transplantation. After 5 minutes of revascularization, micrographs showed EC shape variations, bleb formation and cell retraction with intercellular gap formation. Twenty-four hours after transplantation loss of monolayer continuity was widely extended. Four weeks of revascularization resulted in either well preserved specimens with nearly normal endothelium, or badly preserved arteries with fibrotic degeneration of the luminal vessel wall. The morphological disruptions found in this study help to explain the alterations in permeability control and vascular dysfunction observed in lung transplantation.

Ultrastructural changes in rat adipomusculocutaneous flaps during warm ischaemic storage and reperfusion.

Electron microscopy (EM) was used to evaluate ultrastructural changes in adipomusculocutaneous rat flaps which had been stored for 0, 2, 4, 6 and 8 hours under normothermic conditions (37 degrees C). The effects of treatment with desferrioxamine (DFX) or hypertonic citrate flush (HCA), prior to 4 hours of storage, were compared to untreated flaps which had been stored for 4 hours. Ultrastructural changes caused by 30 minutes of reperfusion, were also studied. Most ultrastructural alterations occurred between 2 and 4 hours of warm storage and there were further changes in some cells after short periods of reperfusion. DFX decreased smooth muscle damage and HCA protected adipocytes but neither of these agents preserved endothelial cells. These studies indicated that free radical-dependent and independent mechanisms were both involved in events which led to flap necrosis after periods of warm storage and reperfusion.

Morphological changes in rat single lung isografts after long-term survival.

A high priority in organ transplantation research is to increase the number of hours that an organ can be successfully preserved. Transplant programmes rely on hypothermia and flush solutions to maintain organ viability during the storage period. We studied long-term morphology in lungs stored for 24 or 48 hours using two modified versions of University of Wisconsin solution, one mimicking the extracellular medium and the other the intracellular medium. Four weeks after transplantation, X-ray and angiograms were used to assess the proportion of ventilating tissue, and light and electron microscopy to analyse morphology. Pulmonary tissue presented near-normal histological appearance in well preserved areas while fibrosis and chronic inflammation were found in scarring processes. Electron microscopy studies revealed some damage-related changes in tissue which appeared histologically normal. Four weeks after transplantation, quality and quantity of recovery were uniform for both solutions tested after 24 hours of storage. However, without reaching significance, after 48 hours the quantity of successfully preserved pulmonary tissue was greater in the group stored in the intracellular solution.

Immunoelectron microscopic studies reveal differences in distribution of sialo-oligosaccharide receptors for Mycoplasma pneumoniae on the epithelium of human and hamster bronchi.

Long-chain sialo-oligosaccharides with poly-N-acetyllactosamine backbones (Ii antigen type) are major host cell receptors for the human pathogen Mycoplasma pneumoniae. Previous immunofluorescence studies of the human bronchial epithelium, using sequence-specific monoclonal antibodies to the branched I-type and linear i-type backbones, have indicated that sialylated and nonsialylated long-chain sequences of both types are richly expressed on the ciliated cells, where they are polarized at the apical aspects. These sequences are lacking in the goblet cells. In the present study, the display of these oligosaccharides has been investigated by electron microscopy (immunogold labelling) in the human bronchial epithelium and in that of the hamster, an animal model commonly used for M. pneumoniae infection. In the human bronchial epithelium, the long-chain branched sequences have been detected along the entire length of the cilia and on microvilli, whereas the linear sequences are confined to the microvilli and the basal aspects of the cilia. On the ciliated epithelial cells of the hamster, by contrast, the branched and linear sequences (sialo- and asialo-) have been detected exclusively on microvilli. A further striking difference is that in the hamster these structures are expressed in abundance on the goblet cells and in the intracellular globules. We suggest that the latter finding may partly explain the relatively large doses of M. pneumoniae required to establish experimental infection in the hamster, as the receptor-bearing secreted mucus may have a protective role in binding to the microorganisms, leading to their clearance by bronchociliary action.

Immunochemical studies on the synthesis and secretion of link protein and aggregating proteoglycan by chondrocytes.

Chondrocytes from pig laryngeal cartilage were maintained in culture, and the biosynthesis and secretion of link protein and proteoglycan were studied using immunochemical, biochemical and immunolocalisation techniques. In the presence of monensin there was a dose-dependent inhibition of link protein secretion which was very similar to that of aggregating proteoglycan, and suggested that they followed the same intracellular pathway during biosynthesis. In the presence of cycloheximide there was a similar dose-dependent inhibition of the secretion of both link protein and proteoglycan. Kinetics of secretion following inhibition of synthesis by cycloheximide showed that both proteins had similar intracellular pool sizes. Analysis of protein core and glycosaminoglycan biosynthesis showed that the time for synthesis and glycosylation of proteoglycan was 22 minutes, and this was quickly followed (within 6 minutes) by secretion. Intracellular electron microscopic immunolocalisation using protein A-gold showed link protein to be present in the Golgi cisternae and vesicles, and double-labelling experiments showed link protein only to be detected in vesicles that also labelled for proteoglycan protein core. When chondrocytes were maintained in monolayer culture for 10 days the rate of biosynthesis and secretion of proteoglycan increased although that of link protein remained constant. The control of their biosynthesis was thus shown to be independent. Within 4 hours of secretion a high proportion of link protein was incorporated into proteoglycan aggregates.

A type VI collagen-related glycopolypeptide is the major concanavalin A-binding component in pig skin.

The major concanavalin A-binding component in urea/deoxycholate/mercaptoethanol extracts of pig skin was a collagenous disulphide-cross-linked glycopolypeptide with an apparent molecular mass of 150 kDa and a pI of 5.5. Antiserum against the electrophoretically purified glycopolypeptide gave strong dermal staining similar to that seen with fluorescent concanavalin A. Immunocytochemical labelling showed prominent labelling of 3-4 nm dermal microfilaments, particularly those associated with dermal blood vessels and mast cells. Immunoblotting with authentic antiserum indicated that the major skin glycopolypeptide was probably identical with collagen-like glycoprotein, the tissue form of the alpha 1/alpha 2 subunits of type VI collagen. This was confirmed by immunoblotting of authentic type VI collagen from pepsin-treated pig skin. Immunoblotting, metabolic labelling with [3H]glucosamine and immune precipitation showed that an immunoreactive collagenous glycopolypeptide was synthesized and secreted by cultured pig skin fibroblasts. The results suggest that type VI collagen is the major concanavalin A-binding component in pig skin.