Biomedical subjects

C J Handley

Publications and source records attributed to C J Handley.

At least 73 records · Page 4Linked to original sources

PubMed1984

Turnover of proteoglycans in cultures of bovine articular cartilage.

Proteoglycans in cultures of adult bovine articular cartilage labeled with [35S]sulfate after 5 days in culture and maintained in medium containing 20% fetal calf X serum had longer half-lives (average 11 days) compared with those of the same tissue maintained in medium alone (average 6 days). The half-lives of proteoglycans in cultures of calf cartilage labeled after 5 days in culture and maintained in medium with serum were considerably longer (average 21 days) compared to adult cartilage. If 0.5 mM cycloheximide was added to the medium of cultures of adult cartilage, or the tissue was maintained at 4 degrees C after labeling, the half-lives of the proteoglycans were greater, 24 and greater than 300 days, respectively. Analyses of the radiolabeled proteoglycans remaining in the matrix of the tissue immediately after labeling the tissue and at various times in culture revealed two main populations of proteoglycans; a large species eluting with Kav of 0.21-0.24 on Sepharose CL-2B, of high bouyant density and able to form aggregates with hyaluronate, and a small species eluting with a Kav of 0.63-0.70 on Sepharose CL-2B, of low buoyant density, containing only chondroitin sulfate chains, and unable to form aggregates with hyaluronate. The larger proteoglycan had shorter half-lives than the smaller proteoglycan; in cartilage maintained with serum, the half-lives were 9.8 and 14.5 days, respectively. Labeling cartilage with both [3H]leucine and [35S]sulfate showed the small proteoglycan to be a separate synthetic product. The size distribution of 35S-labeled proteoglycans lost into the medium was shown to be polydisperse on Sepharose CL-2B, the majority eluting with a Kav of 0.27 to 0.35, of high buoyant density, and unable to aggregate with hyaluronate. The size distribution of glycosaminoglycans from 35S-labeled proteoglycans appearing in the medium did not differ from that associated with labeled proteoglycans remaining in the matrix.

Aging↗

PubMed1984

Pregnancy-related changes in the connective tissue of the ovine cervix.

During the course of gestation, changes in the metabolism and chemical composition of the cervical connective tissue occur. The concentrations of collagen, proteoglycan and hyaluronate in the cervix decrease with time of pregnancy, while there is a small but significant increase in tissue hydration. The collagen network loses its dense, ordered appearance and assumes a loose, frayed, disoriented structure. A small molecular weight dermatan sulphate proteoglycan with Kav = 0.48 on Sepharose CL-4B has been isolated from pregnant and nonpregnant ovine cervix. Proteoglycans isolated from dilated cervix yield a second peak at the excluded volume of a Sepharose CL-4B column which may represent a new proteoglycan species, or represent a proteoglycan monomer with larger glycosaminoglycan constituents. The rate of synthesis of proteoglycan is enhanced during pregnancy, and doubles in the time between 140 days and term (dilated). The increased rate of proteoglycan synthesis in the presence of a diminishing hexuronate concentration in the pregnant cervix is indicative of enhanced proteoglycan turnover. This may provide some insight into the mechanism whereby a new matrix with altered mechanical properties is produced, since a rapid turnover of matrix components enables a remodeling of the connective tissue framework. This reorganization of the matrix is discussed with respect to parturition.

Animals↗

PubMed1984

Oxygen free-radicals mediate an inhibition of proteoglycan synthesis in cultured articular cartilage.

Superoxide radical, generated enzymatically by the action of xanthine oxidase on hypoxanthine, significantly inhibited proteoglycan synthesis by cultured bovine articular cartilage. This inhibition was not due to the generation of uric acid or to the generation of superoxide per se. It was immediate in onset and still evident after six days in culture. The inhibition was similar for both 35S-sulphate and 3H-acetate incorporation into glycosaminoglycans and could not be reversed by addition of beta-D-benzyl xyloside. Protein synthesis was also inhibited.

Animals↗

PubMed1984

Treponema pallidum does not synthesise in vitro a capsule containing glycosaminoglycans or proteoglycans.

Treponema pallidum was investigated for its ability to synthesise glycosaminoglycans or proteoglycans in vitro. Isolated viable T pallidum organisms were incubated with radiolabelled precursors of glycosaminoglycans, sodium 35S-sulphate and 3H-glucosamine (tritiated glucosamine). T pallidum failed to incorporate sodium 35S-sulphate but did incorporate 3H-glucosamine into a macromolecule which may be associated with the surface of the treponeme. This macromolecule was resistant to degradation by specific glycosaminoglycanases. We conclude that T pallidum does not synthesise a capsule containing glycosaminoglycans in vitro.

Chromatography, Gel↗

PubMed1984

Polyanions in syphilis: evidence that glycoproteins and macromolecules resembling glycosaminoglycans are synthesised by host tissues in response to infection with Treponema pallidum.

We investigated by means of radiolabelled precursors the source and nature of the polyanionic macromolecules present in rabbit tissues during active syphilis infection. Previous studies indicated that Treponema pallidum itself does not synthesise glycosaminoglycans, at least in vitro. In replicate experiments on unilaterally infected rabbits, tissue from the orchitic testis incorporated two to three times more 35S-sulphate and 3H-glucosamine (on a wet weight basis) than tissue from the non-orchitic contralateral testis. Incorporation of 35S-sulphate was independent of the number of viable T pallidum organisms present in the infested tissue, which suggested that incorporation represented biosynthesis by the host and not the treponeme. Testes from syphilitic rabbits two days after treatment with high doses (100 mg/kg) of penicillin incorporated less 35S-sulphate than untreated infected testes, but more than normal uninfected rabbit testes. This suggests that active syphilitic infection was necessary for maximum biosynthesis of the macromolecule(s) by host tissue. Hydrodynamic profiles showed incorporation of radiolabelled precursors into two distinct fractions of different sizes, which may represent a proteoglycan and a sulphated glycoprotein. Alcian blue staining of syphilitic testes at or after peak orchitis showed focal deposition of newly synthesised polyanionic components during peak orchitis and a more generalised fibrosis in testes after peak orchitis.

Alcian Blue↗

PubMed1984

In vitro response of chondrocytes to mechanical loading. The effect of short term mechanical tension.

Chick epiphyseal chondrocytes were grown in high density cultures for 14 days, after which the cell layers were placed in a cyclical stretching apparatus and subjected to a strain of 5.5% at a frequency of 0.2 Hz. There was a 1.4- and 1.7-fold increase in the incorporation of 35SO4 and 14C-glucosamine, respectively, into glycosaminoglycans in cultures subjected to mechanical loading for 24 h. No significant change was observed in the hydrodynamic size of the proteoglycans synthesized by chondrocytes subjected to mechanical loading. In this time period there was no increase in 3H-glycine incorporation into acid-insoluble protein, but there was a 2.4-fold increase of 3H-thymidine into DNA in cultures subjected to tensional strain. Concomitant with these changes, the cellular levels of cyclic AMP increased 2.2 times in the mechanically loaded cultures. This is discussed as a possible mechanism whereby chondrocytes respond to mechanical stimuli.

Animals↗

PubMed1983

The effect of serum on biosynthesis of proteoglycans by bovine articular cartilage in culture.

Proteoglycan synthesis by slices of adult bovine articular cartilage is stimulated two-to threefold when tissue is cultured in the presence of fetal calf serum for 5-6 days. After this, essentially steady-state conditions are achieved for up to 14 days in which the high synthetic rates are maintained and the amount of proteoglycan in the tissue remains nearly constant. In the absence of fetal calf serum, synthesis declines to a lower level and there is a gradual, net loss of proteoglycan from the tissue. Tissue maintained without serum for several days rapidly increases synthetic rates to the higher levels over 2-3 days after transferring into medium with serum, and vice versa, indicating that the response of the chondrocytes to serum factors is reversible. The structures of the proteoglycans synthesized under all medium conditions were typical for cartilage. Only small differences in glycosaminoglycan chain sizes and a consistent decrease in the relative amount of keratan sulfate to chondroitin sulfate during the first days in the culture were observed. The net capacity of the cells for chondroitin sulfate synthesis, as estimated by incubation in the presence of exogenous beta-xyloside acceptor, increased (or decreased) in parallel with the changes in endogenous proteoglycan synthesis when cultures were transferred from medium without to medium with serum (or vice versa), suggesting that changes in the net amounts of the enzymes for chondroitin sulfate synthesis are closely coordinated with changes in the amount of core protein being processed to proteoglycans. The responses of calf articular cartilage in the same system were somewhat different. Serum in the medium was required to maintain initial high levels of synthesis. The proteoglycans synthesized contained a lower proportion of keratan sulfate than those initially synthesized in the adult tissue, and there was no change in this proportion with time in culture. The maintenance of steady-state conditions for proteoglycan metabolism by either adult or calf tissue in the presence of serum in these cultures should provide a useful model for studying the regulation of synthesis and catabolism of proteoglycans by chondrocytes residing in a nearly normal extracellular matrix for long periods of time.

Animals↗

PubMed1983

Biosynthesis of proteoglycans by isolated rabbit glomeruli.

Isolated rabbit glomeruli were incubated in vitro with 35SO4 in order to analyze the proteoglycans synthesized. Proteoglycans extracted with 4 M guanidine HCl from whole isolated glomeruli and from purified glomerular basement membrane (GBM) were analyzed by gel filtration chromatography. Two types of sulfated proteoglycans were found to be synthesized by rabbit glomeruli and these contained either heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycan chains. These glycosaminoglycans were characterized by their sensitivity to selective degradation by nitrous acid or chondroitinase ABC, respectively. The major proteoglycan extracted from the whole glomeruli was a chondroitin/dermatan sulfate species (75%), while purified GBM contained mostly heparan sulfate (70%). The glycosaminoglycan chains were estimated to be about 12,000 molecular weight which is consistent with previous estimates for similar molecules extracted from the rat GBM.

Animals↗

PubMed1983

An ovarian tumour specific mucin antigen--immunohistological and biochemical studies.

A new mucin antigen was detected in a mucinous cystadenoma of human ovary. An antiserum produced in rabbits against the crude cyst fluid, following appropriate absorptions, showed immunoreactivity exclusively with tumour epithelium, particularly endocervical type epithelium. The antigen responsible for the immunoreactivity was isolated in the fraction with density 1.45g/ml following density gradient ultracentrifugation in cesium chloride, which indicated that it was indeed a glycoprotein. The native glycoprotein was very large since it was excluded from Sepharose CL-2B. The complex could be dissociated following reduction with dithiothreitol. The subunits were included in Sepharose CL-2B and the position of elution would indicate a molecular weight in the order of 1-5 x 10(6). This suggests the native antigen was a complex made up of subunits held together by disulphide bonds. Amino acid analysis of the new antigen showed a resemblance with intestinal mucins, as two-thirds of the protein consists of threonine, serine, proline, alanine and glycine. This similarity in peptide core may explain the potential of the epithelium in ovarian mucinous cystadenoma to produce inappropriate intestinal mucins during the process of malignant transformation.

Adenocarcinoma↗

PubMed1983

Transport of neutral amino acids by adult articular cartilage.

The transport of three neutral amino acids: alanine, serine and leucine and the non-metabolizable analogue aminoisobutyric acid (AIB) was characterized in bovine articular cartilage slices. Alanine and serine were strongly concentrated intracellularly by a factor of 4.5 and 5 respectively and were transported in a sodium dependent and pH sensitive manner. AIB was concentrated by a factor of 1.8 with respect to the extracellular medium and was also transported in a sodium dependent and pH sensitive manner. Leucine was concentrated weakly across the chondrocyte membrane (X1.1) and its transport was independent of sodium ion concentration and the lowering of the extracellular pH. The apparent Km of transport of alanine, serine, leucine and AIB was determined to be 0.46 mM, 0.69 mM, 0.93 mM and 0.66 mM respectively and the values of Vmax were 35.8, 52.1, 14.8 and 6.8 pmol/min per mg respectively. It was shown that both alanine and serine were transported predominantly by system ASC (63.8% and 67.4% respectively) with a small percentage of transport occurring via system A. AIB was transported mainly by system A (76.0%) and leucine was transported equally via systems L (48.4%) and ASC (45.0%).

Alanine↗

PubMed1983

Biosynthesis and turnover of proteoglycans in organ culture of bovine articular cartilage.

The biosynthesis and turnover of proteoglycans (PG) in organ cultures of bovine metacarpalphalangeal cartilage are described. After a few days in the presence of fetal calf serum, the amount of PG in the tissue is maintained at a constant level and the rate of PG biosynthesis is maintained at a high rate. Under these "steady state" conditions PG biosynthesis and turnover must be in balance. Exposure of tissue to lipopolysaccharides reduces the rate of synthesis while increasing the rate of turnover, thereby yielding net loss of PG from the matrix. After an initial mild trypsin digestion to remove PG from the matrix, turnover of PG is reduced in the cultures and a net increase of PG occurs with time. It is suggested that viable chondrocytes can regulate local concentrations of PG in their surrounding matrix by altering both biosynthetic and turnover parameters.

Animals↗

PubMed1982

Aggregation of human platelets by amniotic fluid.

Twenty-four amniotic fluid samples were examined for their effects on human platelets. All samples caused irreversible platelet aggregation. The active material precipitated with high-speed ultracentrifugation and was completely inhibited by prior incubation with purified collagenase. The presence of free collagen in amniotic fluid was further confirmed by polyacrylamide-gel electrophoresis and hydroxyproline assays. Beside platelet-aggregating activity, amniotic fluid samples were also shown to significantly shorten the recalcification time of normal plasma. This procoagulant activity appears to be related to the presence of thromboplastin, collagen and other as yet unidentified procoagulant material in amniotic fluid. The presence of activators of platelets and clotting factors in amniotic fluid would account for the strong clot-promoting activity of this fluid. These studies suggest that the ideal management of the coagulopathy of pregnancy should include a combination of anticoagulant and antiplatelet drugs.

Amniotic Fluid↗

PubMed1982

The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study.

Primary cultures of neonatal rat aortic smooth muscle cells inoculated at high densities (1 X 10(6) cells/25 cm2 Falcon flask) with adequate nutrient media and pH control grow rapidly and form multilayers of cells with typical "hill and valley" organization. After 10 days growth insoluble elastin formation could be visualized by phase contrast microscopy as small particles which grew rapidly to become larger irregular refractile aggregates and later coalesced to form larger aggregates and small fibres. With light and electronmicroscopy, elastin was the predominant matrix protein formed, with the "hill regions" of cultures containing abundant elastin aggregates and some collagen. In 2-week-old cultures differentiation could be observed within the cell multilayer. The older deeper cells contained more protein synthesis organelles and myofilaments and were in close association with large often coalescing elastin aggregates; compared to younger more superficial cells which contained more free polyribosomes less myofilaments, and were associated with fewer and small elastin aggregates. In older cultures this differentiation was not apparent; the cells contained many myofilaments, dense bodies, and lysosomes. Elastin aggregates and newly formed elastic fibres were abundant in the matrix. Quantitative analysis of insoluble elastin formation in the cell layer during the 4-week culture period indicated continuous biosynthesis and deposition which paralleled that of desmosine formation. Amino-acid analysis of a hot alkali insoluble residue (regarded as elastin) from 30-day-old cultures gave a profile identical with neonatal rat aortic elastin in vivo. Insoluble collagen formation in the cell layer tended to plateau after the log phase of growth was completed (10 days). Proteoglycans were found predominantly in the supernatant media. Glycosaminoglycan analysis revealed a profile of dermatan sulphate (32%), chondroitin 4-sulphate (43%), keratan and heparan sulphate (30%), with only a trace of hyaluronic acid. This study indicates that primary cultures of neonatal rat aortic smooth muscle cells remain differentiated in culture and have the unique capacity to continue to synthesize and deposit large amounts (mg) of insoluble elastin which aggregate and from elastic fibres in vitro.

Animals↗

PubMed1981

Effect of dibutyryl cyclic AMP on the sulphation of proteoglycans by chondrocytes.

The addition of a 1 mM N6, O2'-dibutyryl cyclic AMP to incubations of foetal calf articular cartilage for a 4 h period resulted in a 13 and a 15% decrease, respectively, in the rate of incorporation of [3H]glucosamine and [14C]glucose into glycosaminoglycans. Under the same conditions, a 41% increase in the rate of incorporation of [35S]sulphate into glycosaminoglycans was observed. Dibutyryl cyclic AMP had no effect on protein synthesis over a 4 h period or on the hydrodynamic size of the proteoglycan subunits or glycosaminoglycans synthesized by the cartilage. When the glycosaminoglycans were digested with chondroitin ABC lyase and the resulting disaccharides analysed on Dowex 1 (formate form), it was observed that dibutyryl cyclic AMP increased the degree of sulphation of the disaccharides. Furthermore, an over-sulphated disaccharide corresponding to chondroitin 4,6-disulphate was identified. It was concluded that dibutyryl cyclic AMP was stimulating the process of sulphation of glycosaminoglycans by cartilage.

Animals↗

PubMed1980

Molecular size distribution of proteoglycan subunits and glycosaminoglycans synthesized by chondrocytes under conditions of reduced proteoglycan synthesis.

The rate of proteoglycan synthesis by chondrocytes in vitro was depressed by either omitting L-glutamine from the incubation medium or by addition of proteoglycan subunit to the medium. The molecular size distribution on Sepharose 2B of the proteoglycan subunits synthesized by the chondrocytes under these conditions of reduced proteoglycan synthesis was found to be the same as those synthesized by the control cells. Likewise, the molecular size distribution on Sepharose 6B CL of the glycosaminoglycan chains synthesized by the depressed cells was found to be similar to that observed in untreated chondrocytes. This work demonstrates that, under conditions of reduced proteoglycan synthesis, fewer proteoglycan subunits are synthesized by chondrocytes and that the molecular size distribution of these macromolecules is similar to those synthesized by untreated cells.

Animals↗

PubMed1980

Extracellular matrix metabolism by chondrocytes. 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells.

The incorporation of [3H]glycine into acid-insoluble protein and of [3H]acetate into glycosaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gradients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of polyribosomes that was revered by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence of L-glutamine was also demonstrated for other avain connective tissues.

Amino Acids, Essential↗

PubMed1979

Permeability of composite chondrocyte-culture-millipore membranes to solutes of varying size and shape.

A model connective-tissue system was developed that is amenable to the determination of permeability coefficients of diffusing solutes. The system involves the culture of 13-day chick-embryo chondrocytes on a Millipore filter (HA:0.45 micron pore size) to form what is, in effect, a confluent, extremely thin cartilage slice of uniform thickness. These cultured chondrocyte membranes were used to measure the steady-state flux of radioactively labelled low-molecular-weight solutes and micro-ions. Similarly, the permeability coefficients of either radioactively labelled or enzymically active proteins across the membranes were determined. The membrane was found to have no marked effects on the diffusional behaviour of low-molecular-weight non-electrolytes (water, proline, ribose, glucose, sorbitol, raffinose). For micro-ions (Na+, SO42-, Cl-, glutamate, glucuronate,), the diffusive behaviour was found to be markedly affected by the ionic strength of the solvent used in a manner which was consistent with a Donnan distribution resulting from the immobilized proteoglycans. Globular proteins permeated the membrane at rates which decreased as the molecular size of the diffusing solute increased. The apparent diffusion rates of fibrinogen and of collagen through the membranes were greater than would be expected on the basis of their diffusion coefficients in free solution. Reasons for this behaviour are discussed.

Animals↗

PubMed1979

Extracellular matrix metabolism by chondrocytes. 5. The proteoglycans and glycosaminoglycans synthesized by chondrocytes in high density cultures.

Proteoglycans were extracted from the extracellular matrix of cultures of embryonic chick chondrocytes grown at high density and were purified by CsC1 density gradient centrifugation. The chemical, physical and hyaluronate binding properties of the proteoglycans were similar to those observed in proteoglycans from other hyaline cartilages. Proteoglycans in the media were also purified and on analysis showed three populations of proteoglycans to be present. One population had the physical characteristics of a typical proteoglycan subunit and bound hyaluronate, the other two populations were unable to complex with hyaluronate but one had the physical characteristics of the proteoglycan subunit and the other was of smaller molecular weight. The small molecular weight appears to be a product of the enzymatic degradation of the larger molecular weight species.

Cartilage↗