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Biomedical subjects

G Milne

Publications and source records attributed to G Milne.

At least 19 recordsLinked to original sources
PubMed1996

Collagen type X: a component of the surface of normal human, pig, and rat articular cartilage.

Collagen type X, a protein generally associated with hypertrophic chondrocytes of avian and mammalian growth plate during endochondral growth of long bones, has been previously shown to be present during disruption of normal metabolic status of articular cartilage during osteoarthritis. We have demonstrated that collagen type X is present as a component of normal articular cartilage in adult human, growing pig and new-born rat. The protein was immunolocalized in these tissues at the surface of the articular cartilage and for human tissue there was some staining of chondrocytes adjacent to the "tidemark' zone. The presence of collagen type X was confirmed by isolating the protein from these tissues and its identification by SDS-PAGE and immunodetection with antibodies specific for collagen type X.

Adult↗
PubMed1994

Secretory and absorptive activity of oesophageal epithelium: evidence of circulating mucosubstances.

The space between the oesophageal basal and prickle epithelial cells appears empty by standard ultrastructural preparative techniques. Fixation of human oesophageal biopsies with a variety of agents, including tannic acid, glutaraldehyde-lysine, cetylpyridinium chloride and Ruthenium Red shows that this space is filled with mucosubstances, some free, some attached to the cells as a glycocalyx. There is evidence that this material is secreted constitutively by the basal and prickle cells. This secretion may be changed or blocked by incubating oesophageal biopsies in the presence of colchicine or dinitrophenol. Incubation at 16 degrees C has the same effect. Absorption from the intercellular space may be followed using the fluid phase marker, horseradish peroxidase. Early endosomes may also be shown by their acid phosphatase activity. Incubation of biopsies at 20-22 degrees C allows early endosomes to accumulate material, but not pass it on the late endosomes.

Absorption↗
PubMed1992

Turnover rates of different collagen types measured by isotope ratio mass spectrometry.

The rates of collagen turnover in different tissues have been estimated in growing rats previously exposed to gaseous 18O2. The abundance of the stable isotope was measured using isotope ratio mass spectrometry following combustion of isolated collagen-derived hydroxyproline. Using this method, problems of label reutilization associated with radiolabelling methods are avoided. In general the results confirm the slow turnover rates with half-lives of total collagen in skin, muscle and gut of 74, 45 and 244 d, respectively. The use of cyanogen bromide digests of whole tissues followed by isolation of collagen type-specific peptides has allowed the comparison of turnover rates of collagen types I and III, indicating that collagen type III is turned over more rapidly than type I.

Collagen↗
PubMed1992

Flow cytometry of oesophageal mucosal biopsies; epidermal growth factor receptor, and CD15.

Flow cytometry may be used to examine the properties of single or isolated cells. We have shown that it is possible to disaggregate and label oesophageal epithelial cells for two surface markers, CD15 and epidermal growth factor receptor. We have previously demonstrated these markers in oesophageal squamous cells using immunoperoxidase techniques. These labelled disaggregated cells could then be measured by flow cytometry.

Adult↗
PubMed1991

Leakiness of gastric superficial and foveolar cells. A quantitative electron microscopic study using tannic acid.

Two adjacent antral biopsies were taken endoscopically from 41 patients attending a clinic for the investigation of upper gastrointestinal tract symptoms. One was processed for histopathological grading and assessment of the Helicobacter pylori by light microscopy. The other was fixed in tannic acid, which demonstrates cell leakiness, and processed for electron microscopy. On each grid, the light (electron lucent) and dark (electron dense-leaky) gastric superficial and foveolar cells away from mechanical trauma of biopsy were counted, along with the number of H. pylori which were in contact with the epithelial cells. There was a significant relationship between histological grade of gastritis and the extent of electron microscopic damage, i.e., the degradation of normal to leaky cells and the number of H. pylori per leaky cell. If the patients were divided into two groups with minimal and maximal gastritis and compared against each other, then there was a significant difference between the two groups with respect to surface cell damage, polymorphs per unit area, the number of glands invaded by polymorphs, light/dark cells, Helicobacter grades assessed light microscopically or totals counted in contact with epithelial cells by electron microscopy, the number of Helicobacter per cell, and the age of the patients. There was no difference between the two groups on their endoscopic appearances or treatment with H2 blockers or NSAIDS.

Adolescent↗
PubMed1991

Uptake of horseradish peroxidase by human oesophageal explants over 24 h.

Human oesophageal biopsies, endoscopically and histologically normal, were incubated in Ham's F10 for periods up to 24 hours in the presence of horseradish peroxidase. The fluid phase marker was taken up most avidly by the prickle cells but to a lesser extent in the functional layers and by basal cells. Endocytosed markers proceeded to multivesicular bodies (segrosomes). Horseradish peroxidase was later deposited in lysosome-like structures and also in the Golgi apparatus. The lesser uptake by the functional cells may represent reduced access of the marker to the cells due to the intercellular barrier.

Cell Membrane↗
PubMed1990

Isolation and characterization of a metalloproteinase secreted by rat glioma cells in serum-free culture.

The isolation of a metalloproteinase secreted by a rat glioma cell line (BT5C) in serum-free media is described. After affinity purification, the activity was present as a double band with Mr 86000 and 76000 both of which required CaCl2 for activity. The enzyme was able to degrade gelatin but not casein. It was unable to degrade native types I, III, IV and V collagens but their denatured counterparts were degraded. Using a radiolabel release assay the enzyme was inhibited by EDTA, 1:10 phenanthroline and TIMP confirming that it belongs to the family of metalloproteinases. Its activity was not affected by either serine or cysteine protease inhibitors. The proteinase was activated by APMA but was unaffected by trypsin treatment.

Animals↗
PubMed1990

A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media.

A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed. Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates. The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay. The assay is sensitive, reproducible, and convenient for small sample volumes. The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme. It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue.

Animals↗
PubMed1989

Endocytotic pathways across the human gall-bladder mucosa: permeability studies using horseradish peroxidase.

Human gall-bladder epithelium obtained straight from the operating theatre was incubated in an Ussing chamber with the fluid phase marker, horseradish peroxidase (HRP), for up to 60 min. When the marker was presented on the apical surface, within 30 min it had moved readily across the apical cytoplasm in transport vesicles to receptosomes and into the lateral intercellular space, extending across the basement membrane into the lamina propria. When HRP was presented at the basal aspect, within 30 min it had moved through the lamina propria, across the basement membrane and into the lateral intercellular space. By 60 min, only small amounts had been taken up by the epithelial cells and transported to receptosomes. These data indicate a rapid transmucosal endocytotic pathway for blood- or bile-borne macromolecules.

Cell Membrane Permeability↗
PubMed1988

The fine structure and histochemistry of human bile duct in obstruction and choledocholithiasis.

Biopsies from the common bile ducts from seven patients undergoing surgery for biliary obstruction due to stones or malignancy were studied histochemically and electron microscopically. The surface of the bile duct is lined by a tall epithelium which extends into diverticula. Apically, they contain some neutral and sialated mucosubstances. Fucosyl residues were found in the Golgi apparatus and along the apical cell membrane. The latter is lined by microvilli. There was a well-developed rough endoplasmic reticulum and Golgi apparatus and a small number of apical secretory droplets. Large numbers of lipid droplets were present basally in some cells. Lipid-containing macrophages were also seen intra-epithelially and in the lamina propria. This suggests a possible pathway for lipid transport. The glands were lined by cuboidal cells, some containing much mucus--sulphated, sialated, and neutral with a basal nucleus. A well-developed endoplasmic reticulum and Golgi apparatus were found with abundant secretory droplets. The glandular epithelium contained lysozyme, alpha-1-antitrypsin, and alpha-1-antichymotrypsin. These may play a protective role. The lamina propria contained scattered smooth muscle cells amongst the fibroblasts and inflammatory cells.

Cholestasis, Extrahepatic↗
PubMed1988

Glycosaminoglycans in human gallbladder basement membrane: nature and quantitative changes in chronic cholecystitis.

Using cuprolinic blue as a stain along with enzymic digestion, heparan sulphate has been identified as the main glycosaminoglycan in the basement membrane of human gallbladder epithelium. The amount of glycosaminoglycans was quantified by counting the number of molecular profiles cm-2 in electron micrographs of mildly, moderately and severely inflamed gallbladders. There is a significant increase (P = 0.009) in the amount of glycosaminoglycans in the basement membranes of severely inflamed gallbladders compared with cases of mild chronic cholecystitis. Differences, although present, are less significant when mild and moderate or moderate and severe cholecystitis are compared. The findings suggest that there is a continuous accumulation of heparan sulphate in the basement membrane in chronic cholecystitis which increases in amount with the severity of inflammation.

Basement Membrane↗
PubMed1987

Permeability of the mouse gallbladder to blood-borne horseradish peroxidase.

Horseradish peroxidase (HRP) was administered intravenously to mice by bolus injection. The subsequent uptake and fate of the HRP by the lateral and basal cell surfaces of resting and stimulated gallbladder epithelial cells was followed by light and electron microscopy. At 10 min after injection, HRP was visible in the lamina propria of the gallbladder and within 20 min of injection, HRP had permeated the basement membrane and had entered the lateral intercellular space, extending as far as the apical tight junction. Over the following 30 min, there was evidence of vesicular epithelial HRP uptake and 1 h after injection, HRP was visible in epithelial secretory granules within the lumen of the gallbladder and apical transport vesicles. These data provide evidence of a blood-to-bile transport pathway which could represent an important route of entry to bile by various blood-borne macromolecules.

Animals↗
PubMed1987

The identification of the N tau-methyl histidine-containing dipeptide, balenine, in muscle extracts from various mammals and the chicken.

The occurrence of the dipeptide, balenine (beta-alanyl-N tau-methyl histidine), has been identified by comparative chromatography in extracts of muscles from 9 species of mammal, including man, and the chicken. The identity of the dipeptide from 6 mammalian species and chicken was confirmed by preparative isolation prior to determination of the amino acid composition and N-terminal residue. In all cases, the dipeptide contained equimolar amounts of beta-alanine and N tau-methyl histidine with beta-alanine as the N-terminus. The concentration of the dipeptide varied dramatically between species, being just detectable in the muscle of man and rat while present at concentrations of up to 14 mumol/g wet muscle in adult pigs. It is proposed that balenine, like carnosine and anserine, should be regarded as a normal constituent of muscle.

Animals↗
PubMed1987

The retention and metabolism of N tau-methylhistidine by cockerels: implications for the measurement of muscle protein breakdown determined from the excretion of N tau-methylhistidine in excreta.

Excreta were collected for four consecutive days from 4- to 18-week-old cockerels following subcutaneous injection of N tau-[14CH3]methylhistidine. The recoveries of radioactivity in excreta were incomplete and progressively decreased with increasing age. Most of the radioactivity not recovered in excreta after 4 d was found in skeletal muscle where greater than 55% of the radioactivity present was in the N tau-methylhistidine-containing dipeptide, balenine. This peptide appeared to be relatively stable so that most of the labelled N tau-methylhistidine incorporated was not released during the period of the recovery measurements. The total pool of non-protein bound N tau-methylhistidine (free (free N tau-methylhistidine + balenine) in pectoral and mixed thigh muscles increased with age and relative to the daily excretion of N tau-methylhistidine. At 18 weeks the pool was 3.3 times the daily excretion of N tau-methylhistidine. These observations account for the decreasing recoveries of radioactivity in excreta described previously, due to progressive dilution of labelled N tau-methylhistidine in an expanding pool of non-protein-bound N tau-methylhistidine, part of which was relatively stable. It is concluded that excretion of N tau-methylhistidine by 4- to 18-week-old cockerels cannot be used as a reliable index of muscle protein breakdown in vivo.

Age Factors↗
PubMed1987

Cell death in granulomata: the role of apoptosis.

Unequivocal apoptosis were seen by light microscopy in examples of leprosy, sarcoidosis, tuberculosis, Crohn's disease and foreign body granulomata. A limited electron microscopic investigation showed typical apoptotic bodies in both sarcoid and leprosy granulomata. The number of apoptosis and mitoses in granulomata were counted and their densities calculated. The wide variation in the results between individual lesions may reflect differences in disease activity.

Cell Survival↗
PubMed1986

Sex- and phenotype-dependent metabolism of N tau-methylhistidine by mice.

The metabolism of N tau-[Me-14C]methylhistidine by mice was influenced by both the sex and the phenotype of the animal. After subcutaneous injection of labelled N tau-methylhistidine, recoveries of radioactivity in excreta were incomplete, and the distributions of radioactivity showed that different mouse phenotypes degraded N tau-methylhistidine in vivo to different extents. Hence the excretion of N tau-methylhistidine by mice cannot be used as an index of protein breakdown in vivo.

Animals↗
PubMed1986

Evidence for renal tubular resorption of collagen fragments from immunostaining of rat kidney with antibodies specific for denatured type I collagen.

Antibodies to rat collagens I and III were raised in sheep. The antisera to collagen I were separated by affinity chromatography into components specific for either native or denatured forms. Immunolabelling of rat kidney sections with antibodies to native collagen I showed staining only of the interstitial matrix. By contrast, antibodies to denatured collagen I revealed the presence of immunoreactive material primarily in the upper part of the proximal tubules, detected in both fixed and cryostat sections. In fixed material, the granular appearance of staining in the region of the brush border was shown to be distinct from the protein droplets counterstained by toluidine blue. Collagen III antibodies stained the interstitial matrix in a similar pattern to that for native collagen I, but no proximal tubule staining was observed despite the fact that antibodies to denatured collagen III were shown to be present. No material reactive with denatured collagen I antibodies was detected in urine or serum by an inhibition ELISA technique. The results are discussed in terms of renal tubular resorption of collagen degradation products.

Absorption↗