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Biomedical subjects

Z Nevo

Publications and source records attributed to Z Nevo.

At least 55 records · Page 3Linked to original sources
PubMed1990

Decrease in the basal levels of cytosolic free calcium in chondrocytes during aging in culture: possible role as differentiation-signal.

Cell- and matrix-related parameters, which characterize the aging and differentiation process of cartilage in vivo, were measured in cultured chick epiphyseal chondrocytes during maintenance in a suspension culture for 34 days. A gradual decrease in the rates of proliferation and an increase in the size of the cells were observed. Ultrastructural examination revealed increased vacuolization and appearance of glycogen-storing pools. The rate of proteoglycan synthesis gradually increased. Age-related changes in the composition of the proteoglycan consisted of an increase in the ratio of keratan sulfate/chondroitin sulfate. The results indicate that the process of aging in culture resembles maturation and differentiation of cartilage tissue in vivo. The levels of cytosolic free calcium ions ([Ca2+]i) were measured in fura-2-loaded cells during the course of aging in culture. A gradual decrease in [Ca2+]i was observed. In 5-day cultures, a value of 184 nM [Ca2+]i was measured; this value decreased to 61 nM in 34-day cultures. On the basis of the present data and the previous results, which showed that cartilage-derived growth factors caused a decrease in [Ca2+]i, concomitantly with enhancing differentiation, whereas factors which elevated [Ca2+]i caused an increase in proliferation and a decrease in proteoglycan synthesis, we suggest a model for control of chondrocyte differentiation and aging. The model suggests that the rate of differentiation may be paced by changes in steady-state levels of [Ca2+]i.

Animals↗
PubMed1990

Regenerating hyaline cartilage in articular defects of old chickens using implants of embryonal chick chondrocytes embedded in a new natural delivery substance.

Partial and full thickness defects were created mechanically in articular cartilage and subchondral bone of the tibiotarsal joint condyles of 3-year-old chickens. The wounds were then repaired using embryonal chick chondrocytes embedded in a new biocompatible, hyaluronic acid-based delivery substance. Controls were similarly operated on but received either no treatment or implants of the delivery substance only. Animals were killed from 1 week to 6 months postoperatively. Sections from the two groups were examined and compared macroscopically, histologically, and histochemically. Results of 6-month follow-up showed that only the defects of the experimental chickens were completely filled with reparative hyaline cartilage tissue, with no signs of inflammation or immunologic rejection. Initially the entire defect cavity, whether partial thickness or full thickness up to the deep regions in the subchondral bone, was filled with cartilaginous reparative tissue. Relatively rapid maturation occurred under the tidemark; chondrocytes hypertrophied, were invaded with vascular elements and ossified. In the superficial areas, the reparative tissue remained cartilaginous and matured as typical hyaline cartilage tissue. These results indicate that aged chicken cartilage and its accompanying thin and spongy osteoporotic bone offer a favorable host environment for embryonal cell implants.

Animals↗
PubMed1989

Fate of allogeneic embryonal chick chondrocytes implanted orthotopically, as determined by the host's age.

Chondrocytes derived from chick embryos can be successfully implanted in defects of adult chick articular cartilage surfaces. Such implants thrive in their implantation site and create a new articular surface. The chondrocytes mature and hypertrophy in the orthotopic site without invoking an immune response. Eventually a steady state is reached in which mature chondrocytes resurface the defect while in the deeper areas spongy bone replaces the hypertrophic chondrocytes. Time schedules of these repair events have been studied in hosts of different ages. We compared 4-month-old chicks with 3-year-old chickens. The embryonal chondrocytes implanted in the latter group underwent an accelerated aging process. The defects were completely filled-up after 1 month as compared with 2-3 months in the younger age group. Endochondral ossification in the older group was evident as early as 2 months post implantation and was completed after 6 months. This contrasts with the situation in the younger group where the chondrocytes only began to hypertrophy after 6 months. At this stage endochondral ossification was hardly seen at all. A unique response to the cartilaginous implants is seen in the old group only in the vicinity of the reparative tissue, accumulation of hematopoietic centers. This study seem to indicate that the host's environment affects the "biological clock", i.e. rate and degree of aging of the implanted cells, as well as their matrices.

Age Factors↗
PubMed1988

Slowing down aging of cultured embryonal chick chondrocytes by maintenance under lowered oxygen tension.

Cultured epiphyseal-chondrocytes from embryonic chick may serve as a useful in vitro model to study aging processes in cartilage. The accelerated aging process in cultured chondrocytes is completed within a month and is manifested by typical changes in both cellular and extracellular compartments. Under common maintenance conditions, cells show a gradual loss of replicative capacity, increase in the rate of proteoglycan synthesis and age-dependent changes in the structure and composition of proteoglycan. An environmental factor--reduced oxygen tension--was found to slow down aging processes and preserve the young features of chondrocytes for a longer duration in culture. Cultures maintained under lower oxygen tension had higher proliferation rate, smaller cell size, lower rate of proteoglycan synthesis, and lower content of keratan sulfate side chains in the proteoglycan. In addition higher concentrations of free cytosolic calcium [Ca2+]in as compared to control cultures, was found. It is suggested that the increased proliferation rate and the decrease in proteoglycan synthesis caused by low oxygen tension may be signalled by the higher [Ca2+]in in these cells.

Animals↗
PubMed1987

Murine macrophage heparanase: inhibition and comparison with metastatic tumor cells.

Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells (B16-BL6 melanoma; ESb T-lymphoma) attach, invade, and penetrate confluent vascular endothelial cell monlayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the [35S]O4 = -labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The macrophages do not store the heparanase intracellularly but it is instead found pericellularly and requires a continuous cell-matrix contact at the optimal pH for maintaining cell growth. The degradation of [35S]O4 = -labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10 micrograms/ml), arteparon (10 micrograms/ml), and heparin at a concentration of 3 micrograms/ml. In contrast, other glycosaminoglycans such as hyaluronic acid, dermatan sulfate, and chondroitin sulfate as well as the specific inhibitor of exo-beta-glucuronidase D-saccharic acid 1,4-lactone failed to inhibit the degradation of sulfated proteoglycans in the subendothelial extracellular matrix. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. However, the following antiproteases--alpha 2-macroglobulin, antithrombin III, leupeptin, and phenylmethylsulfony fluoride (PMSF)--failed to inhibit this degradation process, and only alpha 1-antitrypsin inhibited the heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associated enzyme, was inhibited by heparin to the same extent as the macrophage heparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase, inhibited at concentrations of 1 and 3 micrograms/ml, respectively. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood-borne tumor metastasis.

Animals↗
PubMed1987

Use of cultured embryonal chick epiphyseal chondrocytes as grafts for defects in chick articular cartilage.

Full-thickness defects in articular cartilage were repaired with cultured homologous embryonic chick epiphyseal chondrocytes embedded in a biological resorbable immobilization vehicle (BRIV). This graft was successfully transplanted in mechanically induced defects in the surface of condylar articular cartilage of the tibiotarsal joint of four-month-old roosters. Healing of the defects was observed macroscopically, histologically, and histochemically and with the use of biochemical analyses for six months. Chondrocyte proliferation was seen 48 hours after implantation, and a hyaline cartilage matrix surrounding the cells was present two weeks later. Within eight weeks, the defects were completely filled with hyaline cartilage, which integrated smoothly with the neighboring cartilage without the formation of fibrous tissue at the interface. The cell content and rate of proteoglycan synthesis remained high for four months, then declined slowly to the level of the surrounding cartilage. Six months after transplantation, the cartilaginous tissue in the wound at levels below the ossification front showed penetration by vascular elements and young bone trabeculae at the margins of the reparative tissue. No signs of immunogenic rejection of the implants were observed. These results may be related to the employment of a capable source of cells, i.e., cultured chondrocytes characterized by a high mitotic rate and an early stage of development. The transplanted cells grew well and maintained their initial rate of proliferation, with definite maturation and transformation. The resulting cartilage was structurally reorganized according to the host pattern and under the influence of multitudinous environmental conditions. The articular zone preserved its cartilaginous phenotype, whereas the subchondral regions were transformed into bone.

Animals↗
PubMed1985

Decrease in cytosolic free Ca2+ and enhanced proteoglycan synthesis induced by cartilage derived growth factors in cultured chondrocytes.

Cartilage-derived growth factors, enhance proteoglycan synthesis in cultured chick-embryo chondrocytes, and have almost no effect on cell proliferation. Addition of cartilage derived growth factors to cartilage cells loaded with the fluorescent Ca2+ indicator quin 2, caused a rapid, concentration dependent decrease in cytoplasmic free Ca2+. This decrease persisted also in Ca2+-free medium, indicating that it is not mediated by a decrease in the passive permeability of cell membrane to Ca2+. Addition of the Ca2+ ionophore A23187, with or without cartilage derived factors, caused an increase in cytoplasmic free Ca2+ together with inhibition of proteoglycan synthesis and enhanced cell proliferation. The results may indicate that whereas cell proliferation in chondrocytes is signaled by an increase in cytoplasmic Ca2+ ([Ca2+]in), proteoglycan synthesis is signaled by a decrease in [Ca2+]in. The data lead to suggesting a mechanism for antagonistic regulation of cell proliferation and the expression of the differentiated state.

Animals↗
PubMed1985

Arthritis induced by a T-lymphocyte clone that responds to Mycobacterium tuberculosis and to cartilage proteoglycans.

Adjuvant arthritis characterized by chronic inflammation of the joints of rats is induced by immunization to Mycobacterium tuberculosis. To learn how autoimmune arthritis may be caused by a microbial antigen, we isolated a T-lymphocyte clone specific for M. tuberculosis antigens that was strongly arthritogenic. We now report that the clone recognized, in addition to M. tuberculosis antigens, antigens present in human synovial fluid, medium of chondrocyte cultures, and proteoglycans purified from cartilage. These observations indicate that the target antigen for the arthritogenic clone resides in the proteoglycan component of cartilage. As this arthritogenic clone shows specificity for both a M. tuberculosis antigen and a cartilage constituent we conclude that disease is probably caused by antigenic cross-reactivity. Thus, an autoimmune disease may be triggered by structural mimicry between antigens in the environment and self-antigens in the individual.

Animals↗
PubMed1984

The effect of serum growth factors and xyloside on molecular aging of proteoglycan in embryonal chick cartilage.

The effects of normal human serum, insulin-like growth factor and beta-D-xyloside on the synthesis of proteoglycan, as well as their differential effect on the synthesis of chondroitin sulfate and keratan sulfate side-chains, were studied in chick embryonal cartilage. The glycosaminoglycans found in the incubation medium were mainly intact carbohydrate moieties of partially degraded proteoglycan molecules, whereas the tissue-bound glycosaminoglycans were of intact proteoglycan molecules. In incubations with normal human serum, the synthesis of the chondroitin sulfate side-chains of the tissue-bound glycosaminoglycans was preferentially stimulated, while the percentage of medium glycosaminoglycan (out of the total glycosaminoglycan in tissue and medium) was reduced, compared to control incubations. In incubations with insulin-like growth factor, the synthesis of the keratan sulfate side-chains of the tissue-bound glycosaminoglycan was preferentially stimulated, whereas the percentage of the medium glycosaminoglycan resembled that of control incubations. In incubations with xyloside, a marked reduction of tissue-bound glycosaminoglycan was noticed, mainly of chondroitin sulfate chains, and only a slight decrease in keratan sulfate chains. Human serum of various age groups stimulated proteoglycan synthesis in embryonal chick cartilage to almost the same extent. However, sera from babies and adults were found to stimulate chondroitin sulfate chains preferentially, whereas serum of aged subjects preferentially enhanced the synthesis of keratan sulfate chains. These findings suggest that the synthesis and/or degradation of the various types of glycosaminoglycan chains (chondroitin sulfate and keratan sulfate) of cartilage proteoglycan can be regulated differentially by serum growth factors. Secondly, the growth hormone-mediated serum factor (insulin-like growth factor) seems to play a role in molecular aging of proteoglycans.

Adolescent↗
PubMed1984

Extracellular matrix (ECM) proteoglycans produced by cultured bovine corneal endothelial cells.

The nature of the proteoglycan(s) (PG) found in the extracellular matrix (ECM) layer produced by cultured bovine corneal endothelial (BCE) cells is analyzed. The PG(s) account for approximately 5 to 6% of the dry weight of the ECM, regardless of the amount of extracellular soluble PG available in the medium. A 4 M guanidinium chloride (GuCl) extract of ECM was separated on a dissociative cesium chloride (CsCl) gradient (1.25 g/cm3 starting density). Results showed one main peak of PG substance(s) comprising 91% of the total labelled substance and uronic acid, banding at a specific buoyant density of 1.29 g/cm3. The molecular weight of this major PG(s) as estimated by gel filtration on Sepharose CL-4B ranged from 0.5 to 0.7 X 10(6). Further chemical analysis of the main PG(s) band revealed a protein moiety accounting for 45% of the weight and carbohydrates-glycosaminoglycans (GAG) accounting for the remaining 55%. Analysis of the GAG chains (over the entire gradient) showed a composition, based on the susceptibility of the PG substance(s) to degrading enzymes, of 50% heparan sulfate, 43.5% dermatan sulfate, and 6.5% chondroitin 4- and 6-sulfate chains. BCE cell cultures grown in the presence of beta-D-xyloside produced an ECM lacking more than 90% of the GAG content found in the control ECM. The medium-soluble GAG chains, produced in vast excess in cultures grown in the presence of beta-D-xyloside, are composed mainly of chondroitin 4- and 6-sulfates.

Animals↗
PubMed1983

Fresh and cryopreserved fetal bones replacing massive bone loss in rats.

Cartilaginous fetal bones from rat preserved by deep freezing procedures were compared to comparable fresh bones with regard to the following parameters: chemical composition, water and uronic acid contents; cell viability measured by the rate of proteoglycan synthesis; mineralization-ossification status by calcium binding; matrix integrity by the release of uronic acid containing substances; and biological activity as transplants inducing the formation of bone. The transplanted material was chemically analyzed and checked for its rate of proteoglycan synthesis. The quality of the formed bone was similar whether isogeneic or allogeneic, fresh or cryopreserved bone was employed as transplant material. Evidently those various fetal bones may be of clinical value whenever the need for replacement of massive bone loss arises. Although the viability and the cartilaginous nature of the graft are critical, the isogeneity and freshness are of a quantitative advantage only. These biochemical observations were confirmed by roentgenological and histological evaluations of the grafts. An optimal cryopreserving procedure and tests for examining bone candidates for successful grafting are described.

Animals↗
PubMed1982

Age-related decrease in the activity of UDP-xylose:core protein xylosyltransferase in rat costal cartilage.

The activity of UDP-xylose:core protein xylosyltransferase (EC 2.4.2.26) in costal cartilage of young rats (3 months) and old rats (36 months) was measured. The enzyme activity in cartilage of young rats (mean +/- S.D.) is 3370 +/- 1440 Bq h-1 mg-1 DNA, which is about three times higher than that determined in cartilage of old rats (1090 +/- 520 Bq h-1 mg-1 protein). The amount of galactosamine-containing proteoglycosaminoglycans that are extractable with 4 M guanidinium chloride from cartilage is significantly higher in young rats (29.1 +/- 4.8 nmol GalN per mg cartilage wet weight) than in old animals (5.8 +/- 3.0 nmol GalN per mg cartilage wet weight). Thus, if xylosyltransferase activity is referred to the amount of galactosamine-containing proteoglycans in cartilage, nearly identical values are obtained (young rats, 80 +/- 30 Bq h-1 mumol-1 GalN; old rats, 85 +/- 35 Bq h-1 mumol-1 GalN). The results support the assumption that the synthesis of proteochondroitin sulfate is diminished in costal cartilage of old rats by a mechanism involving a reduced activity of xylosyltransferase.

Aging↗
PubMed1982

The role of sperm-bound hyaluronidase in the dispersal of the cumulus oophorus surrounding rat ova.

Only a very small portion (4-7%) of the hyaluronidase of rat sperm obtained from the caput or cauda epididymis was related during incubation in capacitation medium for up to 24 h at 37 degree C. A portion of the cell-associated hyaluronidase was accessible to external substrates, allowing sperm suspensions to lyse glycosaminoglycans of cumulus clots rapidly, even without capacitation. This process appears to represent the employment of an enzyme bound to the sperm cell, analogous to the use of solid-phase enzymes in current enzyme technology.

Animals↗
PubMed1981

The effect of insulin-like growth factor (IGF) and of human serum on steps in proteoglycan synthesis.

Embryonic chick pelvic cartilages were incubated in the presence of insulin like growth factor (IGF) (1 - 100 microunits/ml), as well as normal human serum (5%), with radiolabelled precursors of proteoglycan (PG) synthesis: L-[3-3H]serine, D-[6-3H]glucosamine and [35S]Na2SO4. IGF alone (1 - 15 microunits/ml), stimulated in a dose-dependent manner D-[6-3H]glucosamine incorporation into tissue-bound and soluble isolated glycosaminoglycan (GAG) chains. L-[3-3H]serine incorporation into PG molecules was not stimulated by IGF (1 - 100 microunits/ml), despite the increase in the uptake of this precursor into intact cartilage. [35S]Na2SO4 incorporation was unaffected by IGF. Serum promoted the uptake of all three precursors into tissue-bound glycosaminoglycans. It was postulated that IGF could stimulate proteoglycan synthesis not only by elongating existing chondroitin sulphate chains but also by increased synthesis of other sugar chains e.g. keratan sulphate and oligosaccharides.

Animals↗
PubMed1980

Hormonal and local agents as extracellular regulators of cartilage growth measured by the rate of proteoglycan synthesis. A hypothesis.

Following the rate of proteoglycan synthesis as the metabolic parameter of the growth of cartilage cells, indicates that hormonal and local agents are two distinct types of extracellular regulators responsible for altering the basic metabolic rates of cells. Such changes in growth rate occur regularly at physiological developmental stages (e.g., during embryonic life and at advancing age), and at pathological events (e.g., wounds and repair).

Animals↗
PubMed1979

Growth factors.

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Animals↗