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IntroductionTracking the progress of universal health coverage (UHC) is typically at a country level. However, country-averages may mask significant small-scale variation in indicators of access and use, which would have important implications for policy choice to achieve UHC.MethodsWe conducted a retrospective cross-sectional household and individual-level survey in seven slum sites across Nigeria, Kenya, Bangladesh and Pakistan. We estimated the adjusted association between household capacity to pay and report healthcare need, use and spending. Catastrophic health expenditure was estimated by five different methods.ResultsWe surveyed 7002 households and 6856 adults. Gini coefficients were wide, ranging from 0.32 to 0.48 across the seven sites. The total spend of the top 10% of households was 4–47 times more per month than the bottom 10%. Households with the highest budgets were: more likely to report needing care (highest vs lowest third of distribution of budgets: +1 to +31 percentage points (pp) across sites), to spend more on healthcare (2.0 to 6.4 times higher), have more inpatient and outpatient visits per year in five sites (1.0 to 3.0 times more frequently), spend more on drugs per visit (1.1 to 2.2 times higher) and were more likely to consult with a doctor (1.0 to 2.4 times higher odds). Better-off households were generally more likely to experience catastrophic health expenditure when calculated according to four methods (−1 to +12 pp), but much less likely using a normative method (−60 to −80 pp).ConclusionsSlums have a very high degree of inequality of household budget that translates into inequities in the access to and use of healthcare. Evaluation of UHC and healthcare access interventions targeting these areas should consider distributional effects, although the standard measures may be unreliable.

International audience; Oceania includes sovereign states as well as overseas territories of metropolitan powers. In both cases, contemporary geopolitical borders are legacies of colonialism. As in many (de)colonised places, materialisations of spatially anchored social imaginaries and practices of self and otherness, play a role in the everyday politics of Oceania's communities and states. Notably, cultural intimacies (Herzfeld, 2016) in this region are also shaped by tensions between islands in an archipelagic unit. Characterised by plural identities, Oceania's communities must navigate solidarities within the colonial borders of unitary sovereign states or non-sovereign island territories. Given this context, we ask whether spatially anchored identities within archipelagic contexts are politically engaged, playing a role in the politics of state (dis)cohesion across the region. This paper presents findings from 73 interviews across four pairs of Pacific islands - Wallis-Futuna, Tahiti-Mangareva, Rarotonga-Manihiki, Pohnpei-Chuuk - exploring how communities define their own identities and the identity of those on 'the other island'. We find both sides in agreement on six complementary and rather respectful identities. We therefore suggest that while political tensions and calls for secession in archipelagos are real, it is unlikely that identity politics at this point in time inflames political break ups.

Within the United Kingdom (UK), assisted living technologies are mostly provided through statutory health and social care services following assessment of individual need and application of eligibility criteria. This paper describes the first UK study to explore and develop business approaches and innovations required to make electronic assisted living technologies more accessible to consumers in their fifties and sixties. A robust mixed-method approach was used including a large sample size for a consumer survey, triangulation of methods and confirmation of research findings through validation workshops. This three-year study makes significant and original contributions to understanding consumer needs in this rapidly changing market and offers unique insights into the needs and wants of people aged 50–70. Analysis shows significant differences between consumer and business perceptions, indicating that marketing is not closely aligned to consumers' needs and is affecting the development of the market. New approaches to consumer-led business models are presented to improve information and marketing aimed at 50–70-year-old consumers. A ‘Broker/Independent Advisor’ business model showed most potential for meeting the needs of both consumer and business stakeholders. Findings support future development of an assisted living consumer market to meet growing levels of need and demand, and to offer greater consumer choice of mainstream technologies to enable people to age in place.

The existence of an association between child pedestrian accidents and socio-economic deprivation in Great Britain is well established. The factors driving this association are complex and difficult to isolate. This study uses accident prediction models to investigate the links between child pedestrian casualties and a range of environmental and socio-economic factors commonly linked to deprived areas and people. Separate models are constructed relating to the areas in which the children become casualties, and the areas in which the children reside. Significant socio-economic factors include: single-parenthood, reliance on income support, and crime; and environmental factors include domestic garden area, junction density and pedestrian and vehicular flow density. The study found that factors pertaining to the local environment were more prevalent in the models considering accident locations, whilst socio-economic factors were of greater influence in the residency model.

There is renewed interest in Europe in the potential role of new automated technologies for urban transport. One such system is personal rapid transit (PRT), a system of automated demand-responsive vehicles designed to transport individuals directly to their destinations. Assessing the contribution of such a system when applied extensively in an urban area is challenging. Methods used to assess the potential viability of traditional systems have to be updated to incorporate the new technologies. Several interrelated methods are examined to assess the feasibility and potential benefits of a citywide PRT system by using a case study region. Microsimulation analysis is used to obtain relationships between network characteristics, level of service, demand, and system performances. Outputs from this analysis feed into the strategic model that is used to test the contribution that such a system will make. Outputs from both the microsimulation and strategic modeling are used in the creation and running of a business case tool that provides the basis of the economic justification for such a scheme. Finally, barriers to the introduction of a PRT system are described, along with means of overcoming them.

Assessment practice at tertiary education has become a topic of intensive review and discussion internationally. It is widely acknowledged that assessment is crucial for learning and is a main source for directing students’ learning practices. With increasing demand to nurture graduate capabilities and generic skills alongside students’ major studies, there is a call for improvement in assessment practice to align with these new demands. This paper reports on a trial project conducted at a tertiary institution language department, which was designed to improve assessment practice. The trial took a participatory action research approach with a distributive leadership model. Through a series of analyses, presentations, workshops and round table discussions the whole of the teaching staff was engaged in a process of renewal of assessment practice for the department. The creation of a Statement of Assessment Principles emerged from this process. Based on these principles the staff took on the challenge of reworking assessment tasks for all units to be offered in the coming semester to ensure they aligned with this newly developed statement of principles. Data was collected from the participating staff members through individual and group interviews. This paper discusses the transition experienced of teaching staff members. It examines changes that took place in beliefs and understandings about assessment, their expectations and concerns about the implementation stage and their responses to the concentrated nature of the renewal process that was used in this trial. A future project will follow the implementation and will utilize the experience of the participants to develop a set of guidelines, in an endeavour to ensure this renewed approach to assessment can be sustained through staff and syllabus changes. Future papers may emerge from this stage.

SUMMARY A case of mucopolysaccharidosis is described, in which the principal glycosaminoglycan stored in the liver and excreted in the urine was chondroitin sulphate. Both isomers were present in equal amounts. The clinical features were similar to those of the Hurler syndrome or mucopolysaccharidoses type I (McKusick 1966). RESUME Une forme de mucopolysaccharidose avec surcharge viscerale et elimination urinaire excessive de chondroitine sulfate Les auteurs decrivent un cas de mucopolysaccharidose dans lequel le principal glycosaminoglycan stocke dans le foie et elimine dans les urines etait la chondroitine sulfate. Les deux isomeres etaient presents en quantiteegaie. Les symptomes cliniques etaient semblables e ceux du syndrome de Hurler autrement dit du type I des mucopolysaccharidoses selon la classification de McKusick en 1966. ZUSAMMENFASSUNG Eine Form von Mukopolysaccharidose, bei der Chondroitinsulfat in Organen gespeichert und in hohem Maβe in Urin ausgeschieden wird Es wird ein Fall von Mukopolysaccharidose beschrieben, bei dem von Glykoaminogly-kanen hauptsachlich das Chondroitinsulfat in der Leber gespeichert und im Urin ausgeschieden wurde. Beide Isomeren waren in gleichem Mase vertreten. Das klinische Bild ahnelte dem beim Hurler Syndrom oder bei der Mukopolysaccharidose Typ I, wie auch von McKusick 1966 berichtet wurde. RESUMEN Una forma de mucopolisacaridosis con almacenamiento visceral y excesiva excrecion urinaria de condroitin sulfato Se describe un caso de mucopolisacaridosis en que el principal acumulo de glicosamino-glican en el higado y excretado en orina era el condroitin sulfato. Ambos isomeros estaban presentes en cantidades iguales. Los caracteres clinicos eran similares a los del sindrome de Hurler o mucopolisacaridosis tipo I descrita por McKusick en 1966.

Chondrocytes are specialised cells which produce and maintain the extracellular matrix of cartilage, a tissue that is resilient and pliant. In vivo, it has to withstand very high compressive loads, and that is explicable in terms of the physico-chemical properties of cartilage-specific macromolecules and with the movement of water and ions within the matrix. The functions of the cartilage-specific collagens, aggrecan (a hydrophilic proteoglycan) and hyaluronan are discussed within this context. The structures of cartilage collagens and proteoglycans and their genes are known and a number of informative mutations have been identified. In particular, collagen fibrillogenesis is a complex process which can be altered by mutations whose effects fit what is known about collagen molecular structural functions. In other instances, mutations have indicated new functions for particular molecular domains. As cartilage provides the template for the developing skeleton, mutations in genes for cartilage-specific proteins often produce developmental abnormalities. The search for mutations amongst such genes in heritable disorders is being actively pursued by many groups, although mutation and phenotype are not always well correlated, probably because of compensatory mechanisms. The special nature of the chondrocyte is stressed in connection with its cell involvement in osteoarthritis, the most widespread disease of diarthrodial joints.

The release rates of specific components of the proteoglycan aggregates (G1 domain, the chondroitin sulfate and keratan sulfate containing portion of the protein core, and link protein) of the articular cartilage of mature beagles were studied at early stages of canine experimental osteoarthritis (OA), generated by transection of the anterior cruciate ligament. Analysis of cartilage explants and synovial fluids indicates that at early stages of experimental OA, there is increased release of the proteoglycan aggregates of the articular cartilage. This involves a release from the tissue of the components of the proteoglycan that are specifically involved with aggregation together with the glycosaminoglycans of the proteoglycan. These components were detected at elevated levels in the media of explants of cartilage from the operated joint, and in the synovial fluids of the operated joints.

Objective To investigate the effects of culture conditions, serum and specific cytokines such as insulin-like growth factor (IGF) 1 and interleukin (IL) 1alpha on phenotype and cell survival in cultures of Syrian hamster embryonic chondrocyte-like cells (DES4(+).2). Methods Proteins and RNA extracted from subconfluent and confluent early- and late-passage DES4(+).2 cells cultured in the presence or absence of serum and IL-1alpha or IGF-1 or both cytokines together were analysed for the expression of chondrocyte-specific genes and for the chondrogenic transcription factor Sox-9 by Western and Northern blotting. Apoptosis was assessed by agarose gel electrophoresis of labelled low-molecular weight DNA extracted from DES4(+).2 cells and another Syrian hamster embryonic chondrocyte-like cell line, 10W(+).1, cultured under the different conditions and treatments. Results Early passage DES4(+).2 cells expressed chondrocyte-specific molecules such as collagen types alpha1(II) and alpha1(IX), aggrecan, biglycan and link protein and collagen types alpha1(I) and alpha1(X) mRNAs, suggesting a prehypertrophic chondrocyte-like phenotype. The expression of all genes investigated was cell density- and serum-dependent and was low to undetectable in cell populations from later passages. Early-passage DES4(+).2 and 10W(+).1 cells survived when cultured at low cell density, but died by apoptosis when cultured at high cell density in the absence of serum or IGF-1. IGF-1 and IL-1alpha had opposite and antagonistic effects on the chondrocyte phenotype and survival. Whereas IL-1alpha acting alone suppressed cartilage-specific gene expression without significantly affecting cell survival, IGF-1 increased the steady-state mRNA levels and relieved the IL-1alpha-induced suppression of all the chondrocyte-specific genes investigated; it also enhanced chondrocyte survival. Suppression of the chondrocyte phenotype by the inflammatory cytokine IL-1alpha correlated with marked down-regulation of the transcription factor Sox-9, which was relieved by IGF-1. The expression of the Sox9 gene was closely correlated with the expression of the chondrocyte-specific genes under all conditions and treatments. Conclusions The results suggest that the effects of cartilage anabolic and catabolic cytokines IGF-1 and IL-1alpha on the expression of the chondrocyte phenotype are mediated by Sox-9. As Sox-9 appears to be essential for matrix production, the potent effect of IL-1alpha in suppressing Sox-9 expression may limit the ability of cartilage to repair during inflammatory joint diseases.

It has been proposed that ageing of human dermal fibroblasts occurs as a multi-stage process during which cells progress from a mitotic to a post-mitotic state. We describe the development of a simple and novel cell-cloning model for identifying and quantifying the different fibroblast morphotypes associated with the induction of post mitotic behaviour. We have found that under atmospheric (20%) oxygen tension a significant proportion of human dermal fibroblasts are rapidly induced to switch from a mitotic to a post-mitotic phenotype. In contrast, under more physiological (4%) oxygen conditions, the induction of a post-mitotic phenotype is largely prevented. Increasing oxidative stress by addition of hydrogen peroxide or depletion of glutathione also induced a switch from a mitotic to a post-mitotic phenotype in these cells, whereas addition of the anti-oxidant N-acetylcysteine under atmospheric (20%) oxygen tension potently inhibited this process. In addition, a statistically significant correlation was observed between the magnitude of intracellular glutathione depletion and the reduction in the population of mitotic cells in this model. We propose that the switch from a mitotic to a post-mitotic phenotype represents a process of cellular ageing and that standard atmospheric oxygen tension imposes a substantial oxidative stress on dermal fibroblasts which accelerates this process in culture. The data also suggest that intracellular glutathione levels strongly influence the induction of a post-mitotic phenotype and that, by implication, depletion of glutathione may play a significant role in the progression of cellular ageing in human skin.

In UMR-106 osteosarcoma cells we found that PTH activated both the cAMP/protein kinase A and the Ca2+-dependent phosphoinositide/protein kinase C (PKC) pathways, but prostaglandin E2 (PGE2) activated only the cAMP pathway. Activation of PKC by the phorbol ester PMA had no effect on cAMP production but enhanced PTH-stimulated cAMP production by 50% or more; the effect on PGE2-induced cAMP was negligible. Inhibition of the α-subunit of the inhibitory guanine nucleotide binding protein (Gi) by pertussis toxin pretreatment also enhanced PTH-mediated cAMP production but had no effect on PGE2-induced cAMP production. These results suggest that although PTH-mediated adenylate cyclase activity is regulated via both the stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, only Gs regulates PGE2-mediated adenylate cyclase activity in UMR-106 cells. Costimulation with pertussis toxin and PMA did not increase PTH-stimulated cAMP production above that obtained with PMA alone. This implies a similar target of action for pertussis toxin and PMA, that is, the α-subunit of Gi. The α-subunit of Gi was found to be a substrate for in vitro PKC phosphorylation of membrane fractions from UMR-106 cells, seen as a ±40 kD band on SDS-PAGE. Stimulation of in situ 32P-labeled cells with either PMA or PTH also enhanced incorporation of 32P into the 40 kD band. Using the peptide antisera AS/7 and EC/2, we showed that pertussis toxin-labeled subunits of both Gi1α/Gi2α and Gi3α could be immunoprecipitated, respectively, but immuinoprecipitation of membrane proteins after in situ phosphorylation and stimulation with PMA precipitated only Gi2α. We therefore conclude that modulation of adenylate cyclase activity by phorbol esters in UMR-106 osteosarcoma cells can be ascribed, at least in part, to PKC-mediated phosphorylation of the α-subunit of the Gi2 component of the adenylate cyclase regulatory complex.

Women Physiologists , edited by Lynn Bindman, Alison Bruding and Tilli Tansey. Portland Press, 1993. £16.95. ISBN 1-85578-021-6. The Physiological Society originated as a scientific dining club in 1879 for men interested in animal physiology. It was not until 1915 that the first woman was elected as a member, and it is the 75th anniversary of this event that is marked by this book, which consists of brief biographies of 18 women physiologists whose scientific careers are outlined. As an introduction there is a short history of women and the Physiological Society. The next section is devoted to those - 8 out of the 18 - who received public recognition in the form of F.R.S. and/or D.B.E.: they are considered at greater length. All the biographies are written by women, themselves physiologists of high standing, who knew their subjects personally or through close associates. (The final section has details of each contributor.)

Monomer and aggregated proteoglycans were prepared from pig laryngeal cartilage. Vascoelastic flow properties, comprising linear complex dynamic shear modulus, nonlinear steady-state shear-rate dependent viscosity, and primary normal stress difference, were measured in proteoglycan solutions containing varying proportions of aggregate (0-80%) and at different concentrations (10-50 mg/ml). Results were analyzed using the simple Oldroyd four-parameter nonlinear rate-type rheological equation. All solution properties were strongly dependent on proteoglycan concentration and on the proportion of aggregates present. Aggregation was found to have a great effect on the zero shear-rate viscosity at 50 mg/ml, which increased fivefold from 0-100% aggregate. The results showed that network formation in proteoglycan solutions increased with concentration from 10-50 mg/ml and also increased with aggregation. All proteoglycan solutions showed shear thinning, which was most marked with aggregated proteoglycan at high concentration (50 mg/ml), where the viscosity decreased tenfold from the zero shear-rate limit to the infinite shear-rate limit. The intermolecular interactions in the network were therefore increasingly disrupted by increasing shear rate, but repeated measurements showed that these were reversible changes and that testing did not induce disaggregation or degradation of proteoglycan. These rheological properties show that aggregation is likely to immobilize proteoglycan at high concentration within cartilage and to contribute to the material properties of the porous solid matrix of articular cartilage that are important for its load-bearing function.

The chondrocyte is a specialized cell that synthesizes proteoglycans of a type found only in cartilage and nucleus pulposus. These proteoglycans are distinct in forming multiple aggregates of unique structure in which hyaluronic acid provides a central chain to which many proteoglycan molecules are bound at one end only. Chondrocytes were isolated from adult cartilage and used in suspension culture to test the effect of compounds in the medium on the synthesis of proteoglycans. Hyaluronic acid alone, among a number of compounds extracted from or analogous to those in cartilage, reduced the incorporation of [$^{35}$S]sulphate into macromolecular material. Oligosaccharides of hyaluronic acid of the size of decasaccharides and above also had this effect but hyaluronic acid already bound to proteoglycan did not. The proportion of total labelled material associated with the cells increased at the expense of that in the medium. Treatment of the cells with trypsin abolished the effect of hyaluronic acid but treatment with chondroitinase did not. It is suggested that hyaluronic acid interacts with proteoglycans at the cell surface by a specific mechanism similar to that involved in proteoglycan aggregation, as a result of which the secretion and synthesis of proteoglycans is reduced.

Introduction The evolution of multicellular organisms made necessary the development of an extracellular matrix to protect cells and to bind them together in a spatial arrangement required for specialized anatomical and physiological functions. Thus the extent of the matrix and how it is organized in different parts of the body depend on the specialized functions of cells. The matrix in its turn influences the sorting and organization of cells during embryonic development of animals (see Hay, 198 1). Collagen is the principal structural protein of animals, a primitive form of which was evolved by Porifera (Mathews, 1967; Adams, 1978). It has proved so suitable that it is highly conserved and remains the prime structural protein of higher animals, providing the fibrous framework for the body. The extracellular matrix also contains polyanionic macromolecules which fill the interfibrillar space and complement the role of collagen by retaining water in the tissue and controlling its flow (see below). In Nature, polyanions predominate in the pericellular environment, and it has been suggested (Scott, 1975, 1979) that this predominance arose because the Donnan effects of polyanions would exclude from their domains the extremely reactive hydrated electrons produced by ionizing radiations on water. Pericellular polyanions would thus have protected primitive organisms from damage by hydrated electrons and their reactive products generated by the intense radiation that reached the biosphere when there was a thinner protective ozone layer because there was less oxygen in the atmosphere. The acidic polysaccharides of the extracellular matrix of vertebrates are much less diverse than those of invertebrates (Mathews, 1967). In mammals there are a limited number, based on differences in repeating disaccharide units of which they are composed. They are collectively known as glycosaminoglycans and are long unbranched chains with many carboxy and/or sulphate groups made up of disaccharide units of hexosamine and uronic acid or hexosamine and galactose (for reviews, see Muir & Hardingham, 1975: Hardingham, 198 1). However, with the probable exception of hyaluronic acid, all glycosaminoglycans are attached at the reducing end to specific proteins to form large macromolecules known as proteoglycans, a term introduced in 1967 (see Balazs & Gibbs, 1970). Several glycosaminoglycan chains are attached laterally to the protein, from which they extend outwards like a bottle brush owing to electrostatic repulsion of their negatively charged groups. The physical properties of different tissues of the body depend mainly on differences in the proportion of collagen to proteoglycan. which varies widely, and also on the type of proteoglycan and collagen and how these are organized in the matrix. The specific anatomical distribution of different proteoglycans and collagen implies that they have different functions, although these are not yet known precisely. The organizing functions of proteoglycans can be roughly separated into space-filling functions and specific interactions; however, much more is known about the former than the latter. The space-filling role of proteoglycans is very important in cartilage. where the concentration of proteoglycans is higher than in any other tissue. Most progress has therefore been made in elucidating structure and function of proteoglycans of cartilage, which have particular features necessary for this space-filling role, which will be discussed in detail below.

We have assessed the effectiveness of transplanted histocompatible fibroblasts as a long-lived source of lysosomal enzymes for replacement therapy in three patients with Hunter's syndrome, over periods ranging from 2.5 to 3.75 yr. The level of Hunter corrective factor excreted by all three patients increased after transplantation, as did the activity of alpha-L-idurono-2-sulfate sulfatase in serum, when measured directly with a radioactive disulfated disaccharide substrate. Sulfatase activity was also raised in leukocyte homogenates from the two patients that we were able to assess. These increases in enzyme activity were accompanied by corresponding increases in catabolism of heparan and dermatan sulfates, as shown by (a) a decrease in sulfate:uronic ratios of urinary oligosaccharides, (b) an increase in iduronic acid monosaccharide, and (c) a normalization of Bio-Gel P-2 gel filtration profiles. Both the increase in enzyme activity and increased catabolism were maintained during the period of study and were not affected by either a gradual decrease or total withdrawal of immunosuppressive therapy.

A detailed study of the biochemistry of each of the lower lumbar intervertebral discs from 3 spines aged 8, 16, and 44 years has shown progressive changes down the spine in a number of biochemical parameters. These were most apparent in the 44-year-old spine. The chemical composition of proteoglycans of the nucleus pulposus and of its constituent proteoglycans differed from those of the corresponding annulus fibrosus of all three spines. The interaction of proteoglycans with collagen, as assessed by extractability, changed markedly with advancing age, while the molecular size of the proteoglycans from both regions decreased and their keratan sulphate content increased. These changes would be expected to affect the mechanical properties of the disc.

The dynamic, steady-shear and transient shear flow properties of precisely prepared link-stable (s0 136, 66% aggregate) and link-free (s0 93, 59% aggregate) proteoglycan aggregate solutions at concentrations ranging from 10 to 50 mg/ml were determined using a cone-on-plate viscometer in a mechanical spectrometer. All proteoglycan solutions tested possessed: (1) linear viscoelastic properties - as measured by the dynamic complex modulus under small amplitude steady oscillatory conditions (1 less than or equal to omega less than or equal to 100 rad/s) - and (2) nonlinear shear-rate dependent apparent viscosities and primary normal stress difference under steady shearing conditions (0.25 less than or equal to gamma less than or equal to 250 s-1). Our transient flow data show that all proteoglycan aggregate solutions exhibited transient stress overshoot effects in shear stress and normal stress. From these steady and transient flow data, we conclude that link protein stabilized aggregates have significant effects on their dynamic and steady-shear properties as well as transient flow properties. The transient stress overshoot data provide a measure of the energy per unit volume of fluid required to overcome the proteoglycan networks in solution from a resting state. Thus we found that link-stable aggregates form much stronger networks than link-free aggregates. This is corroborated by the fact that link-stable aggregates form more elastic (lower than delta) and stiffer (higher [G*]) networks than link-free aggregates. The complete spectrum of viscometric flow data is entirely compatible with the proposed role of link protein in adding structural stability to the proteoglycan-hyaluronate bond. In cartilage, the enhanced strength of the networks formed by link-stable aggregates may play an important role in determining the material properties of the tissue and thereby contribute to the functional capacity of cartilage in diarthrodial joints.

Although many studies have attempted to elucidate how cytotoxic T (Tc) lymphocytes cause the death of target cells, the mechanism is still controversial. In the present study the effect on the integrity of the lysosomes of the target cell has been investigated. We show here that the specific recognition and attachment of cloned type A influenza-specific Tc cells to A/X31 influenza virus-infected target cells caused rapid change in the amount of lysosomal naphthylamidase activity that was bound within the lysosomes, indicating that the lysosomal membianes in the target cells had been totally labilized. Target cells infected with type B influenza virus served as controls. We therefore suggest that the viral specificity of Tc, lymphocytes allows for recognition and intimate membrane contact with suitably infected targets. This intimate contact induces sufficient perturbation of the target cell plasma membrane so as to cause total labilization of the target cell lysosomes which could account for intracellular lysis.

An electric field causes partial alignment of macromolecules in a dilute solution. The accompanying changes in the solution birefringence offer a sensitive and quick means of monitoring the rates of particle orientation and hence the size of the solute molecules. Such measurements are reported for dilute solutions of proteoglycans in the absence and presence of added hyaluronic acid. The proteoglycan molecules are shown to be some 580 nm long. In the presence of hyaluronic acid they form aggregates that appear to be consistent with the model previously proposed in which the proteoglycans attach radially to the extended hyaluronic acid chain. The electric-birefringence relaxation rates indicate aggregates of similar length to that of the extended hyaluronic acid chain, with the proteoglycans spaced on average at 29nm intervals. A proteoglycan sample the cystine residues of which had been reduced and alkylated showed no evidence of aggregation with hyaluronic acid up to the concentrations of the acid corresponding to 1% of the total uronic acid content. The electric-birefringence method is shown to have a large potential in the study of associating polysaccharide solutions.

Low molecular mass proteoglycans (PG) were isolated from human articular cartilage and from pig laryngeal cartilage, which contained protein cores of similar size (Mr 40-44 kDa). However, the PG from human articular cartilage contained dermatan sulphate (DS) chains (50% chondroitinase AC resistant), whereas chains from pig laryngeal PG were longer and contained only chondroitin sulphate (CS). Disaccharide analysis after chondroitinase ABC digestion showed that the human DS-PG contained more 6-sulphated residues (34%) than the pig CS-PG (6%) and both contained fewer 6-sulphated residues than the corresponding high Mr aggregating CS-PGs from these tissues (86% and 20% from human and pig respectively). Cross-reaction of both proteoglycans with antibodies to bovine bone and skin DS-PG-II and human fibroblasts DS-PG suggested that the isolated proteoglycans were the humans DS-PG-II and pigs CS-PG-II homologues of the cloned and sequenced bovine proteoglycan. Polyclonal antibodies raised against the pig CS-PG-II were shown to cross-react with human DS-PG-II. SDS/polyacrylamide-gel analysis and immunoblotting of pig and human cartilage extracts showed that some free core protein was present in the tissues in addition to the intact proteoglycan. The antibodies were used in a competitive radioimmunoassay to determine the content of this low Mr proteoglycan in human cartilage extracts. Analysis of samples from 5-80 year-old humans showed highest content (approximately 4 mg/g wet wt.) in those from 15-25 year-olds and lower content (approximately 1 mg/g wet wt.) in older tissue (greater than 55 years). These changes in content may be related to the deposition and maintenance of the collagen fibre network with which this class of small proteoglycan has been shown to interact.

Investigations on the mass and neutron scattering densities of acidic polysaccharides are reported. The neutron scattering length densities of neutral and acidic sugars are sensitive to chemical composition, and are almost always higher than those of proteins. These findings were based on crystal data, and were confirmed by preliminary neutron scattering experiments, which gave matchpoints of 52% 2H2O for hyaluronate and 60 ± 5% for chondroitin sulphate. Densitometric studies gave the partial specific volumes v of 0.58 ml g−1 for hyaluronate and 0.53 ml g−1 for chondroitin sulphate, and these could be completely interpreted in terms of partial specific volumes from crystal data and involving electrostriction. Neutron scattering studies on the hyaluronate binding region of proteoglycan show that it has a scattering density equivalent to 45% 2H2O. Densitometry gave a partial specific volume (v) of 0.66 ml g−1. Both can be accounted for by the above studies of v for sugars and published values of v for amino acid residues. A molecular weight of 81,000 ± 20,000 is found from neutron scattering, in satisfactory agreement with biochemical estimates of 83,000. The amino acid and sugar composition of the binding region is reported. The radius of gyration (RG) of the binding region at infinite contrast is 42 ± 2 A. showing that the binding region in 0.2 m-NaCl is elongated and globular. The dependence of RG on the solvent contrast shows that the carbohydrate content is on the surface of the molecule with a higher scattering density than that of the protein. The Stokes radius of 66 A from quasielastic light-scattering shows that the binding region is strongly hydrated, and is consistent with an oligosaccharide structure that is extended semi-rigidly into the solvent and traps much water. The globular nature of the protein component is further demonstrated by the observation of slowly exchanging NH backbone protons in its nuclear magnetic resonance spectrum, and most of these are exchanged on heating to 50 °C. There was no change in the RG of the binding region in H2O when a ligand of hyaluronate(dodecasaccharide, HA12) was added to it. This suggested that the shape of the binding site for HA12 was preformed.

Experimental intervertebral disc degeneration produced in rabbits by ventral nuclear herniation reliably produces vertebral osteophytes. Osteophytes arise from proliferating inner annular fibers which undergo metaplasia into cartilage, calcify, and proceed through an endochondral ossification sequence. Proteoglycans extracted from the osteophytes reveal that the degree of aggregation, molecular size of the monomer, and the chondroitin sulfate/keratin sulfate ratio are directly related to the cartilage state of the tissue.

The structure of newly synthesized proteoglycans from explant cultures of cartilage from joints subjected to transection of the anterior cruciate ligament (osteoarthritic) and from normal (non- or sham-operated) joints was examined. The structure of the products of proteoglycan turnover was also examined using explants of normal and osteoarthritic cartilage maintained in culture for a 48 h chase period. The findings were as follows: Newly synthesized (/sup 35/S)-proteoglycans extracted from cartilage explants from osteoarthritic joints whether examined 3 weeks, 3 months, or 6 months after surgery were larger than those from corresponding normal cartilage. This can be explained by the synthesis in osteoarthritic cartilage of abnormally long chondroitin sulfate chains on newly synthesised proteoglycans. The extracts also contained a newly formed small proteoglycan species that was unable to interact with hyaluronic acid. The proportion of this species was higher in osteoarthritic cartilage compared with normal, examined 3 weeks after surgery, but was generally absent from cartilage obtained 3 and 6 months after surgery. Compared with controls, a smaller proportion of the (/sup 35/S)-proteoglycans released into the maintenance medium of explant cultures of osteoarthritic cartilage during a 48 h chase period was able to interact with hyaluronic acid. However, although furnished withmore » longer (/sup 35/S)-glycosaminoglycan chains, these proteoglycans were smaller than those from control explants.« less

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.