Structural studies leading to the discovery of a cannabinoid binding site.
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Analgesics↗
Biomedical subjects
G Milne
Publications and source records attributed to G Milne.
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↗
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↗
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↗
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↗
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↗
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↗
The uptake of lead by small intestine, colon and gallbladder of the guinea pig in vivo.
The binding and uptake of lead by the jejunal, colonic and gallbladder epithelium of guinea pig has been investigated by electron microscopy. Binding occurred rapidly, the most marked by the microvilli was by the jejunum, followed by colon and gallbladder. The tracer was subsequently internalised in small membrane bound vesicles and smooth endoplasmic reticulum. By 30 min, it appeared in multivesicular bodies at all three sites.
Animals↗
Microwave fixation: its potential for routine techniques, histochemistry, immunocytochemistry and electron microscopy.
Human tissues, both biopsy and postmortem, and tissues from rodents were fixed by microwaves at various temperatures and compared against formaldehyde-fixed material. Conventional stains, including trichromes, worked well. Red cells were lysed, but white cells were fixed, thus permitting diagnoses of various inflammatory states. Malignant cells were equally well-preserved by the two methods. Histochemical investigations of mucosubstances, lipids and various hydrolases showed no significant difference between the two techniques. Some neurological stains, however, were not as good following microwave treatment. Immunocytochemical localization of IgA, IgM and IgG showed no significant difference after microwave fixation compared to that in tissues fixed with formaldehyde. Microwave fixation did not lead to a greater tissue shrinkage than that obtained with formaldehyde fixation. Both were significantly less than that following treatment with phosphate-buffered saline alone. Electron microscopy gave results which were interpretable, but with damage resembling early postmortem change. Microwave fixation is complete in approximately 1-2 min. The mechanism of fixation appears to be due to denaturation associated with disulphide bond formation and a decrease in solubility of proteins.
Animals↗
The uptake of cationized ferritin by guinea-pig gall bladder in vitro.
Tissue pieces of guinea-pig gall bladder were grown in vitro for up to ten days. Over this period at different intervals, specimens were exposed to cationized ferritin in culture medium for 1 h and then grown in ferritin free medium for up to 24 h. Other specimens were grown in culture medium containing cationized ferritin for up to 24 h. Both treatments produced a similar morphological sequence. Electron microscopy at all intervals studied showed the cationized ferritin was first bound by the apical cell membrane, clumped and internalized in large 400 nm vesicles. It was then carried to lysosomes in the region of the Golgi apparatus. Within 1 h, the marker was exocytosed in clumps into the lateral intercellular space, accumulating against the basement membrane in a roughly regular approximately 60 nm array. This pathway of cationized ferritin through the gall bladder epithelium is the same as that followed in vivo although the time taken was shorter in vitro.
Animals↗
The uptake of horseradish peroxidase and its subsequent redistribution by guinea pig gallbladder in vivo.
Horseradish peroxidase (HRP) was injected into the emptied gallbladder of guinea pigs at laparotomy. The fate of the HRP was observed by light and electron microscopy over the following 2 hr. Within 5 min. HRP appeared in pits and apical vesicles either as a rim of increased electron density or more evenly distributed throughout the pit or vesicle. The pits and vesicles were more frequently seen at the edge of the cell apex. From 15 min. HRP was identified in the basolateral cell space in increasing quantities with spill over through the basement membrane into the lamina propria: by 30 min. and 2 hr, little further change was observed. The quantity of nonspecific electron density in epithelial cell multivesicular bodies and residual bodies made the assessment of intracellular handling of the HRP impossible. Dilution of the HRP in the lamina propria made the identification of the eventual fate of the HRP difficult to determine.
Animals↗
Gallbladder epithelial acid hydrolases in human cholecystitis.
The lysosomal enzymes beta-glucuronidase and acid phosphatase were studied in 112 patients with cholecystitis. Acid phosphatase activity was generally lower in patients with cholesterol stones compared with cases with pigment stones. beta-glucuronidase activity was higher in acalculous cholecystitis than in any other group, a fact compatible with the concept that in lithiasis the enzyme is secreted into the bile and therefore may participate in nidus formation. Histochemistry at light microscopical level clearly demonstrates the lysosomal distribution of these enzymes and their presence in the macrophages infiltrating lamina propria in cholesterolosis. Electron histochemistry in 45 patients showed acid phosphatase activity in lysosomes and some in mucous droplets. Thiamine pyrophosphatase activity, a marker for the Golgi system, showed a close association with these mucous droplets. The secretion of mucus will be accompanied by a secretion of acid phosphatase, and by implication other acid hydrolases, into the bile.
Acid Phosphatase↗
Tissue culture of guinea-pig gall-bladder epithelium.
Tissue culture of gall-bladder was attempted in the following media: Dulbecco, Eagle's minimum essential medium, NCTC 135, medium 199 and Ham's F12. Growth occurred in all of them for up to 2 weeks assessed by light microscopy. No enhancement of growth was induced by collagenase trypsin insulin or hydrocortisone. Scanning electron microscopy confirmed that new cells colonised the free surface of the explant. Transmission electron microscopy showed good preservation of the original tall epithelial cells for the period of study. The new migrating cells were flatter, but retained the morphological features of the columnar cells. Secretory granules were absent after 1 day in culture but increased amounts of glycogen and lipid began to appear in the epithelium.
Animals↗
Anionic groups in basement membranes.
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Animals↗
The uptake of cationised ferritin and its subsequent redistribution by gallbladder epithelium in vivo.
Cationised ferritin was injected into the gallbladders of guinea pigs at operation and its subsequent fate studied with an electron microscope over a 24-hr period. The apical cell membrane of the epithelium was tagged and showed patching. The cationised ferritin was internalised into small vesicles, some derived from coated pits, within 5 min. Other vesicles were found at the same time near the lateral cellular membrane. Cationised ferritin was found in lysosomes and in residual bodies in large quantities and occasionally in multivesicular bodies and vesicles near the Golgi apparatus. Clumps of (cationised) ferritin appeared in the lateral intercellular space, 2 hr after exposure, increasing in number with time up to 5 hr. Similar clumps also were found between the basal cell membrane and the lamina rara interna of the basement membrane in a fairly regular pattern. By 5 hr the marker was beginning to pass through the basement membrane. The process was largely complete by 10 hr. Twenty-four hours after injection of cationised ferritin, virtually no marker was found in either the epithelium or underlying connective tissue.
Animals↗
Changes in the collagen of rat heart in copper-deficiency-induced cardiac hypertrophy.
Groups of rats were maintained from weaning on copper-supplemented or copper-deficient diets. After 2, 4, 6, 8 or 12 weeks animals were killed, their hearts were removed and the solubility and phenotype composition of the collagen of the hearts was studied. In the hearts from the copper-supplemented animals 35 to 45% of the collagen could be extracted after two treatments with pepsin but up to 74% could be extracted from the hypertrophied copper-deficient hearts. Almost all of the increase in the soluble material was found in the first pepsin extract. Electrophoretic analysis of the extracts showed that in the copper-supplemented hearts the ratio of Type III/Type I collagen increased about threefold between 2 and 6 weeks after weaning but by 8 weeks it had returned to about the level found at 2 weeks and was maintained at this level to the end of the experimental period. In the copper-deficient hearts the ratio of Type III/Type I collagen had increased about fivefold by 6 weeks after weaning and from 8 weeks to the end of the experimental period it remained at two to three times the value found in the copper-supplemented hearts. There was an indication that the relative proportion of Type V collagen was reduced in the copper-deficient hearts. The possible significance of these results in relation to the known pathological effects of copper deficiency on the cardiovascular system is discussed.
Animals↗
Oesophageal epithelial ultrastructure after incubation with gastrointestinal fluids and their components.
Oesophageal biopsies from endoscopically normal patients were incubated in the following for up to 30 min-Ham's F10 medium:isotonic saline:0.1 N HCl:20, 2 and 0.2 mM bile acids:trypsin:pepsin:lipase; autologous gastric and duodenal juices. The biopsies were then examined by electron microscopy. No morphological change was produced by Ham's F10 or saline. HCl caused little damage. Gastric juice produced widespread severe damage, as did pepsin. Duodenal juice and the enzymes tested caused lysis and internalisation of desmosomes and peripheral cytoplasmic vacuolation. Bile acids split desmosomes and induced microvesiculation of cell membranes. Similar microvesiculation was also induced by duodenal juice. All media except hydrochloric acid eventually produced organelle damage and leaky and disrupted cells. The functional and superficial cells appeared to be able to withstand attack from these experimental media better then did prickle and basal cells. Membrane coating granules were secreted in the presence of hydrochloric acid, but not with enzymes.
Bile Acids and Salts↗
The urinary excretion of N tau-methyl histidine by cattle: validation as an index of muscle protein breakdown.
1. The recoveries of radioactivity in cattle urine following the intravenous administration of N tau-[14CH3]methyl histidine were essentially quantitative in 5--7 d in non-lactating cows, bulls and steers and did not change with age. 2. The N tau-methyl histidine was excreted unchanged in urine. 3. N tau-methyl histidine occurred in muscle extracts both in the free form and as a perchloric acid-soluble, acid-labile form which accounted for approximately 85% of the total non-bound N tau-methyl histidine in muscle and appeared identical to a similar component identified in muscle extracts of sheep and pigs. 4. There was probably an age-related decrease in the concentration of the acid-labile component in muscle but which did not produce a measurable change in recovery of radioactivity in urine. 5. The daily excretion of N tau-methyl histidine (E, mumol) by male cattle was highly correlated with live weight (W, kg) by the equation: E = 50 . 4 + 3 . 536 (+/- 0 . 044)W (r 0. 997). The excretions progressively decreased from 4 . 04 mumol/d per kg at 100 kg weight to 3 . 62 mumol/d per kg at 600 kg. 6. By the criterion of the rate of clearance of labelled N tau-methyl histidine from the body, the excretion of N tau-methyl histidine in urine appears to be a valid index of muscle protein breakdown in cattle.
Aging↗