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Biomedical subjects

J E Scott

Publications and source records attributed to J E Scott.

At least 73 records · Page 4Linked to original sources
PubMed1995

Extracellular matrix, supramolecular organisation and shape.

Connective tissue function is defined as the formation and maintenance of shape, without which centralised physiologies (circulatory, digestive or nervous) could not have evolved. Two elements, inextensible (collagenous) fibrils and compression-resistant interfibrillar soluble polymers (proteoglycans), cope with all usual stresses. Relationships between the two are highly specific, as demonstrated by electron histochemistry based on Cupromeronic blue and critical electrolyte concentration (CEC) methodologies. Recent ideas on (1) the protofibrillar or modular structure of collagen fibrils, (2) the nature of specific binding sites for proteoglycans on fibrils, and (3) fundamental similarities in secondary and tertiary structures of the glycosaminoglycans (hyaluronan, chondroitin, keratan and dermatan sulphates) are described. They have greatly illuminated the study of extracellular matrix structure and function in normal, pathological (osteogenesis imperfecta) and ageing tissues. The small proteoglycans are proposed to be tissue organisers, orienting and ordering the collagen fibrils--thus shaping the tissue at a molecular and ultimately macro level. These interfibrillar structures are based on their bifunctional character, the protein parts binding to collagen fibrils at specific sites and the glycosaminoglycans duplexing and aggregating to hold the proteins and hence the collagen fibrils at defined distances from each other, rather like yardsticks. Examples of the way these functions work in specific tissues are drawn from the cornea and vitreous humour of the eye and developing tendon.

Animals↗
PubMed1995

Tendon response to tensile stress: an ultrastructural investigation of collagen:proteoglycan interactions in stressed tendon.

Tendons are parallel arrays of collagenous fibres which are specialised to resist and transmit tensile stresses. The response of tendon fibres to tensile stress is age-dependent and complex. Elastic elongation at low stress is accompanied by the disappearance of alternate light and dark bands seen in transmitted polarised light. This region of the stress/strain curve is associated with straightening of fibre 'crimps'. At higher stress, elongation is still elastic and reversible until break point is reached. This behaviour may be associated with straightening of a helical arrangement of collagen fibrils. In addition to the collagen fibrils, there are transverse and longitudinal proteoglycan filaments, many of which bridge and link between the fibrils. We have investigated the effect of various levels of stress from very low up to breaking point on the appearance of the proteoglycan filaments and their relationships with the collagen fibrils. Proteoglycan-collagen fibril interactions in rat and mouse tail and flexor digitorum tendons were visualised by Cupromeronic blue staining, applied to dissected fibres in the resting state and at stresses up to breaking. Proteoglycan filaments were seen to be orthogonally arranged in every D period, probably at the d band in mature tendons. In immature tendons proteoglycan filaments took up more varied orientations, but were mainly orthogonal or axially arranged with respect to the collagen fibrils. Both pictures appeared unchanged after application of stress of any level up to breaking point. Young tendons ruptured at lower stresses than mature tendons. It is suggested that PG bridges between collagen fibrils play a part in transmitting and resisting tensile stresses in tendons, contributing to the strength of the tissue.

Animals↗
PubMed1994

Influence of protein kinase C activation by 4 beta-phorbol ester or 1-oleoyl-2-acetylglycerol on disaturated phosphatidylcholine synthesis and secretion, and protein phosphorylation in differentiating fetal rabbit type II alveolar cells.

Undifferentiated type II alveolar cells were isolated from the fetal rabbit lung on the 24th gestational day, grown in vitro for 2-3 days, and used to test the hypothesis that activation of protein kinase C by 4 beta-phorbol ester (TPA) or the diacylglycerol analogue, sn-1-oleoyl-2-acetylglycerol (OAG), stimulates disaturated phosphatidylcholine (DSPC) synthesis and secretion. To measure secretion, cells were prelabelled with [3H]choline in serum-free medium or medium with 10% carbon-stripped fetal bovine serum for 24 h. The radiolabel was removed and TPA (10(-6)-10(-9) M) or OAG (125, 250 or 500 microM) was incubated with the cells for 2 h. The medium was removed and filtered. Fresh medium with the same compound was added for an additional 16 h. To measure synthesis, cells were incubated with [3H]choline and concurrently TPA or OAG was added. Cells were removed at 2 or 18 h. After 2 h at concentrations of 10(-8) M, TPA augmented the release of 3H-labelled DSPC, the major component of the surfactant, by cells incubated in serum-free medium. In the presence of carbon-stripped fetal bovine serum, TPA (10(-7) and 10(-6) M) induced release of DSPC. The incorporation of [3H]choline into intracellular DSPC was increased after 2 or 18 h in fetal alveolar cells exposed to TPA at 10(-9) M or higher. OAG also significantly significantly stimulated the release of labelled DSPC after 2 h at all concentrations tested. In contrast, OAG-exposed cells displayed a reduction of [3H]choline incorporation into cellular DSPC. Characterization of radioactive material released by prelabelled fetal type II cells showed that phorbol ester stimulation increased the release of material which co-migrated with adult rabbit lung lamellar bodies on a sucrose gradient. Electrophoretic examination of [gamma-32P]ATP phosphorylation sites in fetal type II cells cells showed that TPA and OAG induced an increase in phosphorylation of a group of proteins with apparent molecular masses of 45, 50 and 55 kDa. Addition of phosphatidylserine to the incubations produced substantial increase in the phosphorylation of these proteins, particularly in the presence of TPA. Fetal type II cells also displayed a phosphorylation product with an apparent molecular mass of 97 kDa. This protein as well as two high-molecular-mass products appeared to be particular to cells incubated with TPA plus phosphatidylserine and may in part account for the different action of TPA compared to OAG with regard to synthesis and secretion of DSPC by the fetal type II cells.

Animals↗
PubMed1994

On the polylactose nature of chondroitin and keratan sulphates.

The recognition that keratan and chondroitin sulphates are based on the same polylactose backbone simplifies the study of heteroduplex formation between them, suggests insights into structure-function relationships in tissues, and permits new definitions of the linkage regions.

Animals↗
PubMed1994

Hyaluronan-mediated protective effect against cell damage caused by enzymatically produced hydroxyl (OH.) radicals is dependent on hyaluronan molecular mass.

Hyaluronan (HA) protected tendon fibroblasts against cell damage mediated by hydroxyl radicals (OH.) as demonstrated by release of 51Cr from labelled cells. Protection afforded by high molecular mass (M(r)) HA (1218 kDa) was much more effective than that provided by lower (176 kDa and 668 kDa) M(r) HA. OH. was generated by coupling H2O2 produced by glucose oxidase:glucose to [Fe(2+)-EDTA] chelate in a Fenton-type system. The flux of OH. was measured by a spectrofluorimetric assay of salicylate produced by the reaction of benzoate with OH.. Cell damage caused by the OH. generating system was prevented in the presence of catalase, which destroyed H2O2. Damage caused in a standard incubation time increased with increased amounts of glucose oxidase. Protection against OH.-mediated cell damage increased with increasing concentration of HA. The presence of HA did not interfere with the enzyme-Fenton system, as monitored by production of gluconate. On the other hand, HA scavenged OH. produced by the enzyme-Fenton system, as shown by competition with benzoate, which produced less salicylate in the spectrofluorimetric assay in the presence of HA. The reaction of OH. with HA was measured directly by a pulse radiolysis technique in which a hydrated electron (eaq-) produced OH. by the reaction with nitrous oxide. Second order rate constants obtained in distilled H2O or in phosphate buffer showed no dependence on HA M(r). Similarly, fluorimetric assay of the flux of in the enzyme-Fenton system confirmed that HA competed with benzoate, thus lowering salicylate production, and the flux was also independent of the molecular mass of HA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Stimulation of signal transduction pathways in osteoblasts by mechanical strain potentiated by parathyroid hormone.

Second-messenger systems have been implicated to transmit mechanical stimulation into cellular signals; however, there is no information on how mechanical stimulation is affected by such systemic factors as parathyroid hormone (PTH). Regulation of adenylyl cyclase and phosphatidylinositol pathways in rat dentoalveolar bone cells by mechanical strain and PTH was investigated. Two different cell populations were isolated after sequential enzyme digestions from dentoalveolar bone (group I and group II) to study potential differences in response. Mechanical strain was applied with 20 kPa of vacuum intermittently at 0.05 Hz for periods of 0.5, 1, 5, 10, and 30 minutes and 1, 3, and 7 days using the Flexercell system. Levels of cAMP, measured by RIA, and levels of inositol 1,4,5-triphosphate (IP3) and protein kinase C activity (PKC), measured by assay systems, increased with mechanical strain. When PTH was added to the cells, there was a significant increase in levels of all the intracellular signals, which appeared to potentiate the response to mechanical strain. IP3 levels (0.5 minute) peaked before those of PKC activity (5 minutes), which in turn peaked before those of cAMP (10 minutes). Group II cells showed higher levels of cAMP and IP3 than the group I cells. This suggests that the former may ultimately play the predominant roles in skeletal remodeling in response to strain. Immunolocalization of the cytoskeleton proteins vimentin and alpha-actinin, focal contact protein vinculin, and PKC showed a marked difference between strained and nonstrained cells. However, the addition of PTH did not cause any significant effect in cytoskeleton reorganization. Staining of PKC and vimentin, alpha-actinin, and vinculin suggests that PKC participates actively in the transduction of mechanical signals to the cell through focal adhesions and the cytoskeleton, although only PKC seemed to change with short time periods of strain. In conclusion, dentoalveolar osteoblasts responded to mechanical strain initially through increases in levels of IP3, PKC activity, and later cAMP, and this response was potentiated when PTH was applied together with mechanical strain.

Adenylyl Cyclases↗
PubMed1994

Perinatal lung development following maternal exposure to methylmercuric chloride.

Mercury ingested from dietary sources has potent neurotoxic and teratogenic effects. Initial studies have shown that mercury may also affect fetal lung development. Since these pulmonary effects may play a role in subsequent neonatal morbidity and mortality due to compromising of the development of the lung, mercury effects in fetal and neonatal lung were investigated. Methylmercuric chloride (MMC), 1,000 ppm (15 mg/kg of body weight); was administered via an intragastric tube to timed-pregnant Swiss/Webster mice on day 9 of gestation. Lungs from fetuses on gestational day 18 and from neonates on days 1, 5, or 10 after birth were studied. Significant changes in MMC-exposed lungs compared to controls occurred at postnatal day 1. At this time, lung weight per gram body weight increased, phospholipid content per gram of lung or per microgram of DNA decreased, while DNA per gram of lung increased. Methylmercury appears to have delayed lung maturation. Cuboidal epithelial cells in alveolar tubules contained conspicuous glycogen deposits, and differentiation of alveolar type II cells was adversely affected. These results suggest that prenatal exposure to methylmercury may be detrimental to lung development, specifically to the initiation of surfactant synthesis, by delaying the normal pattern of maturation of the alveolar type II cells within the lungs.

Animals↗
PubMed1994

A specific fluorometric assay for hexosamines in glycosaminoglycans, based on deaminative cleavage with nitrous acid.

Glycosaminoglycan (GAG) hexosamines were measured after deacetylation (2 h acid hydrolysis), deaminative cleavage by nitrous acid, and coupling of the 2,5-anhydro sugars thus produced with 3,5-diaminobenzoic acid (DABA) to give a fluorescent product. Analyses were performed after the addition of an aliquot of potassium acetate solution to the acid hydrolysates, to adjust the pH. Results on a series of GAGs were compared with an Elson-Morgan (E-M) procedure. Our method is faster, more convenient, 10-20 times more sensitive, and always gave higher figures for hexosamine content. In particular it avoids long, destructive hydrolysis in concentrated strong acid and the complications of the Moggridge-Neuberger effect. Results agreed with calculations and titrimetric data. Using internal standards, our method is tolerant of cetylpyridinium chloride, peptides, and salts. A simple fluorometer easily handled less than 1 microgram GAG per sample in 1.0 ml volume. Results are available in 4-5 h. The method is suitable for automation. A strongly polycationic environment, as in chitosan, markedly slowed nitrous acid deamination, possibly because of Donnan-type exclusion from the domain of the polycation of the cationic nitrosonium species. Whereas all other alpha- and beta-hexosaminides gave yield-hydrolysis time profiles that peaked in approximately 1 h in the DABA method, the E-M profiles, particularly for 2-sulfamato-alpha-hexosaminides, took longer and/or achieved lower optimal yields. All the usually encountered forms of the 2-amino-2-deoxy group (free, acetylated, sulfamato) are readily assayed using the same reagents, with appropriate prefluorometric stages.

Aminobenzoates↗
PubMed1994

The chemical morphology of extracellular matrix in experimental rat liver fibrosis resembles that of normal developing connective tissue.

The time course of development of extracellular matrix (ECM) in experimentally induced fibrosis (thioacetamide administration followed for 12 weeks or bile duct ligation for 8 weeks) in adult rats was examined by light and electron microscopy, using Alcian blue or Cupromeronic blue staining for sulphated proteoglycans (PGs) in critical electrolyte concentration techniques. Proteodermatan sulphate (PDS) was regularly observed at the gap zone of the collagen fibrils. Morphometry of uranyl acetate-stained collagen fibrils, polarity of their banding patterns (a-e), statistics of d/e band occupancies by PDS, and lengths and thicknesses of PG filaments were quantified. Biochemical analyses showed that the ECM components collagen, hyaluronan, chondroitin and dermatan sulphates increased by 5-10 fold, roughly in parallel, as did heparan sulphate and DNA. Water and lipid contents also increased sharply. Thioacetamide treatment was much slower than bile duct ligation in producing fibrotic changes of equal severity. Sulphation of anionic glycosaminoglycans (AGAGs) decreased with increasing severity of fibrosis. Biochemical and ultrastructural methods correlated well. The large increase in dermatan sulphate was quantitatively as expected, given that it is collagen fibril surface-associated, and there was an increase of collagen content together with a decrease in fibril thicknesses. The increase in DNA reflected the marked increase in cell numbers in fibrotic livers. The chemical morphology of the new connective tissue closely resembled that in e.g. developing young tendon, in that fibrils were thinner, and AGAG levels were higher. The collagen fibrils were often disarranged, rather than ordered and parallel as in normal ECM. No other indication of abnormality in the new ECM was obtained.

Alcian Blue↗
PubMed1994

Molecular modelling of secondary and tertiary structures of hyaluronan, compared with electron microscopy and NMR data. Possible sheets and tubular structures in aqueous solution.

Electron microscopy shows that hyaluronan (HA) forms sheets and tube-like structures in solution. Molecular modelling by Tartu plastic space-filling atomic models revealed that hydroxymethyl and carboxylate groups of HA anti-parallel chains can be joined by H-bonds. Using these bonds, HA molecules can be modelled as sheets and tubules. These tertiary structures have three kinds of lateral contact: (1) antiparallel chains stacked by hydrophobic patches; (2) parallel chains joined by both stacking interactions and H-bonds; and (3) crossing chains joined by H-bonds and stacking interactions. Sheet and tubular structures may explain some viscoelastic and biological properties of HA.

Carbohydrate Conformation↗
PubMed1994

Distribution and characteristics of Ca+2-phosphatidylserine-dependent protein kinase C in subcellular fractions and lamellar bodies of adult rabbit lung.

Pulmonary surfactant prevents lung collapse at minimal alveolar diameter. Since surfactant acts extracellularly, secretion is vitally important in regulating the alveolar surfactant levels. Studies with phorbol esters which stimulate protein kinase C (PKC) activity suggest PKC is involved in regulating surfactant secretion. This study was done to characterize PKC activity in adult rabbit lung fractions. Lungs were removed, homogenized and subcellular fractions prepared by centrifugation on a discontinuous sucrose gradient. Calcium-phosphatidylserine-dependent PKC activity was assayed in fractions in the presence of 4 microM phorbol 12-myristate 13-acetate, 1mM EDTA, 8 mole% phosphatidylserine and 1mM Ca2+ by measuring the transfer of 32P from [gamma-32P]ATP to protein. Concurrent assays were done without Ca+2 or PS. Ca+2-PS dependent PKC activity was defined as the difference between the two. Select fractions were incubated with PKC inhibitors sangivamycin, acridine orange or 9-aminoacridine and activity measured. The results showed the majority of the PKC activity was in the cytosolic fraction (87%, specific activity, 142 pmoles/min/mg) but the lamellar bodies also appeared to contain a small amount of PKC activity (approximately 4.0%, 151 pmoles/min/mg). PKC inhibitors were used to examine the characteristics of the enzyme in the microsomal and lamellar body fractions. Sangivamycin was the most potent inhibitor. Some differences in the inhibition characteristics between the lamellar body and microsomal fractions were observed. However using an add-back approach with the lamellar body fraction, indicated that the small quantity of activity in this fraction be attributed to contamination by microsomes. These results indicate that PKC is active in adult rabbit lung subcellular compartments but is probably not associated with the intracellular surfactant storage organelles.

Animals↗
PubMed1994

Trichloroethylene-induced pneumotoxicity in fetal and neonatal mice.

Trichloroethylene (TCE) has previously been shown, in adult mouse lung, to alter the distribution of phospholipid associated with the pulmonary surfactant and to affect the activity of phospholipase A2, an enzyme involved in synthesis of the surfactant. However there are no data available on the effects of maternal exposure to TCE on fetal lung development. To determine if maternal TCE exposure may affect fetal pulmonary development, pregnant mice were treated with TCE (3000 mg/kg body wt.) administered intraperitoneally on day 17 of pregnancy. Fetuses were examined on the 18th and 19th gestational days and the 1st, 5th and 10th postnatal days. TCE caused increased mortality among 18- and 19-day-old fetuses and 1-day-old newborn mice. In addition mean body weight was reduced on the 1st postnatal day in animals exposed to TCE. Specific lung weights were also significantly reduced on both the 18th and 19th gestational days. More importantly, total lung phospholipid content was depressed immediately before birth; total DNA was not affected. These results suggest that prenatal maternal exposure to TCE may delay pulmonary maturation during the critical period when surfactant synthesis is initiated.

Analysis of Variance↗
PubMed1994

Case report: the antenatal ultrasound diagnosis of megalourethra.

The antenatal ultrasound findings in a fetus which postnatally had fusiform megalourethra, rectal agenesis, recto-vesical fistula and unilateral renal hypoplasia are described. At 16 weeks gestation a cystic area was seen arising from the perineum. It later became evident that this was the urethra. Initially, an echogenic mass was noted in the pelvis, but by the second trimester, fluid-filled loops of bowel were evident. This combination of developmental abnormalities though rare is well defined. Only one previously recorded case has been suspected antenatally.

Anus, Imperforate↗
PubMed1994

Effects of smoke inhalation on alveolar surfactant subtypes in mice.

The effects of smoke inhalation on alveolar surfactant subtypes were examined in mice exposed for 30 minutes to smoke generated from the burning of a flexible polyurethane foam. At 4 or 12 hours after the exposure, three surfactant pellets, P10, P60, and P100, and a supernatant, S100, were prepared by sequential centrifugation of lavage fluids at 10,000 g for 30 minutes (P10), 60,000 g for 60 minutes (P60), and 100,000 g for 15 hours (P100 and S100). Phospholipid analysis and electron microscopy were performed on each fraction. Smoke exposure dramatically altered the normal distributions of these fractions: it significantly increased the phospholipid content of the heavier subtype, P10, which is thought to represent newly secreted surfactant; had no effect on the intermediate form, P60; and dramatically increased the phospholipid content (approximately fivefold) of the lighter subtypes, P100 and S100, which are believed to represent catabolic end-products of alveolar surfactant. Only P100 was structurally altered by the smoke. These results represent alterations of the normal metabolic processing of alveolar surfactant. Whereas the mechanism is yet to be defined, it seems to involve a small but significant increase in the newly secreted surfactant, as well as an excessively high accumulation of the structurally altered catabolic forms of the secreted surfactant.

Animals↗
PubMed1994

Keratan sulphate--a 'reserve' polysaccharide?

The early history of keratan sulphate and its proteoglycans is briefly described. Studies were overlooked that could have had a profound influence on later work. Early methods of writing the structures of keratan and chondroitin sulphates obscured the fundamental relationships between them. Both are now seen to be based on the same polymer backbone poly(Gal beta 1:4 Glc beta 1-3). Confusion over the complicated sulphation patterns in keratan sulphate was clarified by the domain structure idea by the group of Helmut Greiling. Keratan sulphate is characteristic of avascular tissues (cartilages, intervertebral discs, corneal stromas) that get their oxygen supplies by diffusion. Stockwell's early idea that the distribution of keratan sulphate in cartilages was a response to the poor supply of oxygen has been generalised, to the hypothesis that keratan sulphate is a functional substitute for chondroitin sulphate under conditions of oxygen lack. The keratan:chondroitin sulphate ratios in discs, corneas of different species, and changes therein with age can be explained on this basis. The biochemical controlling step is probably the NAD:NADH ratio. Keratan sulphate may thus be a 'reserve' polysaccharide, able to do the job of chondroitin sulphate in adverse conditions of oxygen supply. Keratan and chondroitin/dermatan sulphates have similar functions in corneal stroma, and probably in the other connective tissues in which they are found. They swell the collagenous matrix, keeping the fibrils apart. Even more importantly, they probably act as tissue organisers, orienting the fibrils vis-a-vis each other via specific interactions of their proteoglycan protein cores with the fibrils.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

The chemical morphology of age-related changes in human intervertebral disc glycosaminoglycans from cervical, thoracic and lumbar nucleus pulposus and annulus fibrosus.

Hyaluronan (HA), chondroitin and keratan sulphates (CS, KS), collagen and dry weights were measured in the annulus fibrosus and nucleus pulposus of human cervical, thoracic and lumbar intervertebral discs aged 36-79 y. Alcian blue-critical electrolyte concentration (CEC) staining of sections extended the results. The collagen, total polyanion, HA, CS and KS contents of the nucleus pulposus and annulus fibrosus were plotted for all 3 regions against age. Regional differences and age-related trends were found. For regional differences, the collagen content of the nucleus pulposus was highest in cervical discs and lowest in lumbar discs. In contrast, the total polyanion content of the nucleus pulposus was highest in lumbar discs and lowest in cervical discs. These differences were seen in fetal and adult discs. With respect to age-related trends, the collagen content of the annulus fibrosus was higher in adults and children than in neonates and infants. The collagen content of the nucleus pulposus increased with age in thoracic and lumbar discs, but it was consistently high in cervical discs. There was generally a downward trend of total polyanion and CS with increase in age. This was quite consistent for the annulus fibrosus in all regions and there were dramatic decreases in the lumbar nucleus pulposus in all adults compared with infants and children. These trends were least evident in the cervical nucleus pulposus where infant values were low. CS changes correlated with water content. HA and KS increased in all discs with increasing maturity. Oversulphated KS, absent from fetal discs, reached mature levels by 10 y. Many of the changes occurred before maturity. Glycosaminoglycan (GAG) levels correlated with increasing compressive loads. Higher collagen levels in the cervical nucleus pulposus correlated with greater ranges of torsional and shearing strains in cervical discs. High GAG levels in cervical annulus fibrosus probably facilitate lamellar movements during torsional and flexional movements by lubrication and increase of tissue compressibility. Increased KS/CS ratios before maturity correlated with decreased disc blood supply. Ambient O2 tensions may determine KS/CS balance, the former consuming little O2 during biosynthesis.

Adult↗
PubMed1993

The nomenclature of glycosaminoglycans and proteoglycans.

1. The proposed terms and codes provide concise, quantitative information on the polymer backbone, state of oxidation, patterns of sulphation and epimerization, and proportions of monomeric units. Oversulphated domains and special units are easily recognized. 2. They are intermediate between general statements (e.g. 'chondroitin sulphate') and detailed primary structures. 3. They bring terminology into line with current analytical techniques, which depend largely on enzymes developed by S. Suzuki and co-workers. Current enzyme nomenclature (chondroitinase ABC, AC) can still be used. 4. They are not primarily intended to be spoken, although some codes are easily articulated. 5. They have capacity and flexibility to accommodate future developments.

Carbohydrate Sequence↗