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Biomedical subjects

J E Scott

Publications and source records attributed to J E Scott.

At least 109 records · Page 6Linked to original sources
PubMed1992

Methyl green and its analogues bind selectively to AT-rich regions of native DNA.

Methyl green has long been used as a DNA stain in histochemistry. The sequence selective binding of the cationic triphenylmethane dyes methyl green, crystal violet and Malachite green to DNA was investigated by DNAase 1 and micrococcal nuclease footprinting. At low concentrations the ligands showed similar footprinting patterns which centred around AT-rich regions with a mild preference for hompolymeric A and T. At higher concentrations the dyes bound to almost all available DNA sites. Models, with and without intercalation are discussed to account for the specific binding.

Base Sequence↗
PubMed1992

Human intervertebral disc acid glycosaminoglycans.

Alcian blue critical electrolyte concentration (CEC) staining of intervertebral discs (annulus and nucleus of cervical, thoracic and lumbar discs) distinguished 3 groups where CEC staining correlated with age (less than 3 months; from 3 months to 5 years; over 10 years). The CEC increased markedly (implying increased sulphation of the glycosaminoglycans) in the period of maturation (fetal life to 10 years) and then remained constant throughout adult life. This is at variance with accepted views which attribute such changes to degenerative changes in old age. The major part of the CEC increase occurred after the disappearance of blood vessels from the disc, which is almost complete by 4 years. Our results are compatible with the hypothesis that keratan sulphate replaces chondroitin sulphate, functionally, in conditions of oxygen lack.

Adult↗
PubMed1992

Morphometry of cupromeronic blue-stained proteoglycan molecules in animal corneas, versus that of purified proteoglycans stained in vitro, implies that tertiary structures contribute to corneal ultrastructure.

Isolated, purified small chondroitin (dermatan) sulphate proteoglycans from corneas of cow and rabbit and cow sclera were stained with Cupromeronic blue in 'model' experiments. The lengths and thicknesses of the images were compared with those of the same proteoglycans stained in the tissue, using the critical electrolyte concentration principle to give specificity for sulphated proteoglycans, and keratanase 1 or chondroitinase ABC digestion to distinguish between chondroitin and keratan sulphate. Corrections for orientation of the stained glycan filaments within the section plane were made to convert the observed lengths to true average lengths. Observed lengths of stained chondroitin (dermatan) sulphate were greater than those of keratan sulphate, both in models and tissues, in agreement with published data from biochemical and rotary-shadowing studies, in both species. Corrected (true) average lengths of stained isolated chondroitin (dermatan) sulphate proteoglycans were slightly, but not significantly, longer than expected from rotary shadowing or biochemical measurements. Keratan sulphate lengths were similarly somewhat longer. The data support the idea that Cupromeronic blue acts as a scaffold that helps maintain polyanion shape against distortion on staining. Stained filaments in tissues were sometimes over twice the length of isolated stained proteoglycans, suggesting that 2 glycan chains were aligned end-to-end. Thicknesses of proteoglycan filaments suggested that at least 2 glycan chains were aligned side-by-side, both in models and in tissues. A scheme for proteoglycan tertiary structure in cornea is proposed, in which glycan chains may bridge collagen fibrils in duplexed forms similar to those observed in rotary shadowed preparations.

Animals↗
PubMed1992

Supramolecular organization of extracellular matrix glycosaminoglycans, in vitro and in the tissues.

Connective tissues maintain shape against external and internal stress. They are molecular hierarchies in which fundamental building units come together in tiers of increasing complexity and mutual interactions, based on information carried in the precursor molecules secreted by cells. The collagen fibril is the end product of well-understood self-aggregation controlled by its amino acid sequences, but the interfibrillar amorphous ground substance has not hitherto been seen as structured by analogous aggregations prescribed by the primary structures of the characteristic glycosaminoglycans dissolved therein. Transmission electron microscopy with morphometry and stereology has demonstrated their existence in tissues. Nuclear magnetic resonance defined their secondary structures, rotary shadowing electron microscopy delineated their aggregates in vitro, and molecular dynamics stimulations showed how the latter can spring from the former. The driving forces to aggregation are hydrophobic and hydrogen bonding, offset by electrostatic repulsion between polyanionic charges. The relative stabilities of the aggregates are determined by this balance, and hence by the position and number of their charges, particularly the sulfate ester groups. Corneal stroma is a system of collagen fibrils, highly ordered to ensure transparency, in which glycosaminoglycan aggregates are suggested to determine the ordered spacing as yardsticks in a way that has parallels in all connective tissues.

Carbohydrate Sequence↗
PubMed1991

Chondroitin sulphate and keratan sulphate are almost isosteric.

Keratan sulphate and chondroitin sulphate (KS and CS) in the 2-fold helical configurations that are prevalent in solution are of very similar tacticity. The chiral centres, anionic sites and hydrophobic patches are in identical conformations. Only the position of the acetamido group varies from CS to KS, but part of its intramolecular H-bonding potential in CS is retained in KS. The formation of tertiary aggregates, observed in vitro and in tissues, is explicable on these bases. The proposal that KS may be a functional substitute for CS [Scott & Haigh (1988) J. Anat. 158, 95-108] under low-O2 conditions is relevant.

Acetamides↗
PubMed1991

Secondary and tertiary structures of hyaluronan in aqueous solution, investigated by rotary shadowing-electron microscopy and computer simulation. Hyaluronan is a very efficient network-forming polymer.

1. Hyaluronan from mesothelioma fluid, rooster comb and streptococci was examined by rotary shadowing and electron microscopy. All preparations showed extensive branched networks, but high-viscosity hyaluronan networks were essentially infinite, with no individual 'molecules' that were not integrated via multiple branched points into the meshwork. Low-viscosity hyaluronan, recovered after papain digestion of mesothelioma fluid, showed occasional single filaments that were independent of the main aggregates, some of which were themselves independent of other aggregates. 2. Hyaluronan is a polymer with a very marked capability to form meshworks at very low dilution (less than 1 microgram/ml). The longer the hyaluronan molecule, the more branching is potentially possible, and the more extensive and coherent is the network, with every hyaluronan molecule in contact with every other in the solution, via the network. This behaviour accounts for the mechanical properties of the soft tissues (e.g. vitreous humour) and fluids (e.g. synovial fluid) of which hyaluronan is a major component. 3. The hyaluronan twofold helix, previously demonstrated to be present in solution [Heatley & Scott (1988) Biochem. J. 254, 489-493] was shown by computer simulation and energy calculations to be sterically capable of extensive duplex formation, probably driven by interactions between the large hydrophobic patches on alternate sides of the tape-like polymer, forming stable aggregates at biological temperatures in water. This 'stickiness' is postulated to be the basis of the network-forming and laterally aggregating behaviour of hyaluronan. 4. The tertiary structures formed by hyaluronan may not be possible in the case of chondroitin 4-sulphate.

Animals↗
PubMed1991

Proteoglycan: collagen interactions in connective tissues. Ultrastructural, biochemical, functional and evolutionary aspects.

Electron histochemical investigations of mammalian and echinoderm tissues, using cupromeronic blue to stain proteoglycans (PGs) specifically in critical electrolyte concentration methods, showed that collagen fibrils are associated with keratan sulphate and chondroitin (dermatan) sulphate ('tadpole') PGs at the a, c, d and e bands on the fibril surface, giving rise to the 'one proteoglycan: one binding site' hypothesis. Intra-fibrillar PGs have been observed, distributed in a regular way which suggests that collagen fibrils are aggregates of 'protofibrils', some of which carry PGs at their surfaces. A scheme for remodelling of collagen fibrils, based on recycling of these protofibrils, is outlined. The choice of which tadpole PG to use to carry out a given function is decided to a considerable extent by the availability of oxygen to the relevant tissue element.

Animals↗
PubMed1991

Autoradiographic localization and effects of exogenous radioactively labelled surfactant in the lung of the prematurely delivered fetal rabbit.

Adult rabbit lung surfactant was radioactively labelled with [3H]palmitate and isolated by centrifugation. This material was instilled into the trachea of fetal rabbits prematurely delivered on the 27th gestational day. A similar preparation of unlabelled surfactant was used to measure the effects on pressure-volume characteristics in lungs of 27th day fetuses. Tissue sections were prepared from the lungs of all animals and morphometric and autoradiographic determinations made. Surfactant instillation improved pressure-volume relationships in fetal rabbit lungs. Histologically, although only the middle right lobe seemed to show significant qualitative improvement in expansion after surfactant treatment, quantitative assessment indicated that the surfactant preparation had significantly increased the mean alveolar cross-sectional areas in all three lobes of right lungs. In addition, distribution of autoradiographic grains indicated that 8-25% were located over the alveolar spaces while approximately half this percentage was present over tissue at the level of the alveolus. These results indicate that intratracheal instillation of surfactant supplements the endogenous surfactant at the level of the alveolus.

Animals↗
PubMed1991

Copper tetrapyridino 'phthalocyanin' (Cuprolinic blue) differs in shape from the palladium and platinum analogues, and this affects staining of polynucleotides.

Palladium and platinum homologues of the copper-containing tetrapyridinotetraazaporphin cationic dye Cuprolinic blue were prepared. The affinities of all three dyes for a series of polyanions, with and without nucleotide bases, were assessed using the CEC (critical electrolyte concentration) approach, and expressed as molarities of MgCl2. Whereas Cuprolinic blue bound to RNA with much greater avidity than to DNA, the Pt and Pd analogues showed very high affinity for both. Patterns of affinities for non-nucleotide polyanions showed no differences. 1H and 13C NMR spectra showed that, although the Cu compound was planar, the Pt and Pd homologues must be non-planar. Taken together with previous findings, on e.g. methyl green and pyronin, these results suggest that DNA finds it easier to accommodate pairs of out-of-plane aromatic rings than RNA in stable complexes. CEC analyses can, as demonstrated here, uncover quite subtle differences between molecular recognition patterns of very closely related reagents.

Coloring Agents↗
PubMed1991

Effects of smoke inhalation on surfactant phospholipids and phospholipase A2 activity in the mouse lung.

The effects of smoke inhalation on the pulmonary surfactant system were examined in mice exposed for 30 minutes to smoke generated from the burning of polyurethane foam. At 8 or 12 hours after exposure, surfactants were isolated separately from lung lavage (extracellular surfactant) and residual lung tissue (intracellular surfactant) for phospholipid analysis. Calcium-dependent phospholipase A2 (PLA2) was measured on a microsomal fraction prepared from the tissue homogenate. Smoke inhalation produced a twofold increase in extracellular surfactant total phospholipid. While there was no change in the total phospholipid or phosphatidylcholine (PC) content of the intracellular surfactant, smoke inhalation significantly decreased the disaturated species of PC (DSPC). The specific activity of PLA2 was reduced by more than 50% in both groups of exposed mice. Smoke inhalation appears to result in selective depletion of the DSPC of intracellular surfactant and PLA2 involved in its synthesis. This depletion may be compensated for by increased secretion or slower breakdown of the material present in the extracellular compartment.

Animals↗
PubMed1990

A comparative biochemical and ultrastructural study of proteoglycan-collagen interactions in corneal stroma. Functional and metabolic implications.

1. Corneas of mouse, rat, guinea pig, rabbit, sheep, cat, dog, pig and cow were quantitatively analysed for water, hydroxyproline, nucleic acid, total sulphated polyanion, chondroitin sulphate/dermatan sulphate and keratan sulphate, several samples or pools of tissue from each species being used. Ferret cornea was similarly analysed for water and hydroxyproline on one pool of eight corneas. Pooled frog (38) and ferret (eight) corneas and a single sample of human cornea were qualitatively examined for keratan sulphate and chondroitin sulphate/dermatan sulphate by electrophoresis on cellulose acetate membranes. Nine species (mouse, frog, rat, guinea pig, rabbit, sheep, cat, pig and cow) were examined by light microscopy and six (mouse, frog, rat, guinea pig, rabbit and cow) by electron microscopy, with the use of Alcian Blue or Cupromeronic Blue in critical-electrolyte-concentration (CEC) methods to stain proteoglycans. 2. Water (% of wet weight), hydroxyproline (mg/g dry wt.) and chondroitin sulphate (mg/g of hydroxyproline) contents were approximately constant across the species, except for mouse. 3. Keratan sulphate contents (mg/g of hydroxyproline) increased with corneal thickness, whereas dermatan sulphate contents decreased. The oversulphated domain of keratan sulphate was absent from mouse and frog corneas, increasing as percentage of total keratan sulphate with increasing corneal thickness. Sulphation of dermatan sulphate was essentially complete (i.e. one sulphate group per disaccharide unit). 4. Chondroitin sulphate/dermatan sulphate proteoglycans were present at the d bands of the collagen fibrils of all species examined, orthogonally arrayed, with high frequency, and occasionally at the e bands. Keratan sulphate proteoglycans were present at the a and c bands of all species examined, but with far higher frequency in the thicker corneas, where keratan sulphate contents were high. 5. Alcian Blue CEC staining showed much higher sulphation of keratan sulphate in thick corneas, e.g. that of cow, than in thin corneas, e.g. that of mouse, in keeping with biochemical analyses. 6. It is suggested that the constancy of interfibrillar volumes is regulated via the swelling and osmotic pressure of the interfibrillar polyanions, by adjustment of the extent of sulphation in two independent proteoglycan populations, to achieve an 'average sulphation' of the total polyanion similar to that of fully sulphated chondroitin sulphate/dermatan sulphate. 7. The balance of synthesis of the two kinds of proteoglycans may be determined by the O2 supply to the avascular cornea. O2 supply may also determine the conversion of chondroitin sulphate into dermatan sulphate.

Animals↗
PubMed1990

Northern Region Fetal Abnormality Survey results 1987.

The UK Northern Region Fetal Abnormality Survey was notified of 572 suspected fetal abnormalities in 1987. Of these, 265 (46%) had pediatric surgical implications. Because the registry traces the postnatal outcome of each notification and records congenital abnormalities diagnosed postnatally, it is possible to measure both sensitivity and selectivity of antenatal diagnosis of fetal abnormalities. Cases were divided into five groups: diaphragmatic hernia, abdominal wall defects, intestinal anomalies, neural tube defects, and urological anomalies. Each group comprised cases that were unsuspected, suspected but not confirmed, and suspected and confirmed. Sensitivity was highest in the urological group, but 30% of the notified cases were unconfirmed so selectivity was higher in the abdominal wall and neural tube groups. None of the diaphragmatic hernias and only 14.3% of the intestinal anomalies were correctly suspected. Only 13.5% of all neural tube defects were treated surgically. The Survey has improved cooperation and communication between specialists concerned with the management of fetal abnormalities and this in turn has improved patient care.

Abdominal Muscles↗
PubMed1990

Examination of corneal proteoglycans and glycosaminoglycans by rotary shadowing and electron microscopy.

Proteoglycans (PGs) from cornea and their relevant glycosaminoglycan (GAG) chains, dermatan sulphate (DS) and keratin sulphate (KS), were examined by electron microscopy following rotary shadowing, and compared with hyaluronan (HA), chondroitin sulphate (CS), alginate, heparin, heparan sulphate (HS) and methyl cellulose. Corneal DS PG had the tadpole shape previously seen in scleral DS FG, and the images from corneal KS PG could be interpreted similarly, although the GAG (KS) chains were very much fainter than those of DS PG GAG. Isolated GAG (KS, DS, CS, HA, etc.) examined in the same way showed images that decreased very significantly in clarity and contrast, in the sequence HA greater than DS greater than CS greater than KS. The presence of secondary and tertiary structures in the GAGs may be at least partly responsible for these variations. HA appeared to be double stranded, and DS frequently self-aggregated, KS and HS showed tendencies to coil into globular shapes. It is concluded that it is unsafe to assume the absence of GAGs, based on these techniques, and quantitative measurements of length may be subject to error. The results on corneal DS PG confirm and extend the hypothesis that PGs specifically associated with collagen fibrils are tadpole shaped.

Alginates↗