Biomedical subjects
Publications and source records attributed to H Muir.
Hemoglobin Collingwood is a new unstable hemoglobin variant arising from the substitution of the internal valine at position beta 60 (E4) by alanine. The variant retains normal oxygen binding characteristics and is not associated with clinical symptoms, though there is evidence suggesting increased red cell turnover.
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Ultrastructural changes in articular cartilage were studied in joint laxity induced by severing the anterior cruciate ligament of the right knee in sixteen mature dogs. The left knees provided controls; sham operations on six other dogs consisted of stab incision only, leaving the ligament intact. Cartilage from the medial tibial condyles was examined at intervals from two days to eighteen months later. In the superficial zone of the cartilage, collagen fibrils became abnormally widely spaced at four days, and narrower fibrils appeared from seven days after operation. Chondrocytes, particularly in the middle zone, became more active, with hypertrophy of cytoplasmic organelles detectable from four days. Superficial cells were initially healthy and became more numerous while their lipid content increased. The articular surface was fissured from two months and cell degeneration was rarely seen until several months after operation. These findings correlate with previous biochemical studies and are similar to early changes noted in degeneration of human articular cartilage.
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Human fibroblasts totally deficient in beta-glucuronidase acquired high levels of enzyme activity when co-cultured with mouse or rabbit lymphocytes. Direct cell-to-cell contact was obligatory for this process. The enzyme acquired by the fibroblasts was shown to be identical to beta-glucuronidase from donor lymphocytes by its position of elution from DEAE-cellulose, thermal stability, mobility on polyacrylamide gels and by its antigenic determinants. The enzyme extracted from deficient fibroblasts after co-culture with lymphocytes showed no evidence of any hybridisation between human and mouse or rabbit sub-units. It is concluded that during direct cell interaction, enzymically active beta-glucuronidase is transferred directly from donor lymphocytes to deficient fibroblasts by a mechanism, previously shown not to involve normal receptor mediated endocytosis.
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Fibroblasts deficient in beta-glucuronidase acquired high levels of this enzyme when they were co-cultured with concanavalin A-stimulated lymphocytes. Acquired enzyme activity, determined using a single-cell cytochemical assay, was directly proportional to the number of lymphocytes added and persisted for several days in fibroblasts maintained at high density. Lymphocytes did not secret significant levels of beta-glucuronidase into their culture medium, and did not release other substances able to induce synthesis of the enzyme by the deficient fibroblasts. Nor did beta-glucuronidase acquisition result from concanavalin A-mediated uptake of enzyme, since alpha-methylmannoside did not reduce acquired activity. Moreover, lymphocytes from various sources, whether unstimulated or activated by a different mitogen, bacterial lipopolysaccharide, were equally effective in promoting the appearance of beta-glucuronidase. Deficient fibroblasts did not acquire beta-glucuronidase by active endocytosis when co-cultured with lymphocytes, since enzyme extracted from lymphocytes was not itself effective in this respect. Furthermore, mannose 6-phosphate, which did inhibit, endocytosis by deficient fibroblasts of exogenous beta-glucuronidase prepared from 3T3 cells, had no effect on enzyme acquisition by fibroblasts during their co-culture with lymphocytes. Conversely, inhibitors of protein synthesis and energy metabolism, which did not interfere with endocytosis of exogenous enzyme, abolished the acquisition of beta-glucuronidase during co-culture. Deficient fibroblasts did not acquire beta-glucuronidase when they were cultured together with lymphocytes but separated from them by Millipore membranes permeable to exogenous enzyme. Thus, although the mechanism of acquisition is still unclear, the present results suggest that beta-glucuronidase is transferred from lymphocytes to deficient fibroblasts by a process in which direct cell-to-cell contact is obligatory.
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This paper describes a new method of magnifying small images in photographic film by means of a scanning electron microscope (SEM) operated in the backscattered electron detection mode. The study included tests of several types of radiographic film, transmission electron microscopy film, and black and white 35 mm film. The electron optical enlargement method is particularly useful in situations where the film sample is opaque to light and for generating enlarged images at magnifications beyond the reach of light optical enlargement methods, i.e. up to approximately 2000X with ease and rapidity in a single step. The electron optical enlargements compare favorably in contrast and detail with the enlargements made with a light microscope and with a darkroom enlarger.
The semilunar menisci of the knee have an important mechanical function and are commonly involved in joint degeneration. However, previously published analyses of the compositions of normal and degenerate human menisci vary widely. In the present study the glycosaminoglycan content and composition of selected areas of the menisci of eight normal knees of working foxhounds were determined. The menisci contained 10% less water and abut 8-fold less glucosaminoglycan than did the articular cartilage of these animals. Although the glycosaminoglycan composition was the same in different regions of the menisci, the total amounts varied considerably. Of the chondroitinase digestible material, approx. 60% was chondroitin 6-sulphate, 25% chondroitin 4-sulphate, 10% chondroitin and 5% dermatan sulphate. Hyaluronic acid accounted for about 6% of the total uronic acid.
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An animal model of intervertebral disc degeneration produced by surgical ventral disc herniation in the rabbit is described. Histologic studies showed proliferation of cells on the inner third of the annular wound, with metaplasia into fibrocartilage in the first 2 weeks following injury. Progressive fibrocartilaginous change occurred, reproducing the morphologic changes of disc degeneration over the first 6 weeks involving nearly the entire disc. Proteoglycans (total and newly synthesized) were studied qualitatively and quantitatively for periods of 1 to 200 days after herniation. There were two periods of time during the early course of degeneration when the ability of the proteoglycans to aggregate by interaction with hyaluronic acid was recovered, but this decreases progressively after 6-7 weeks. There was an immediate loss of water content from the injured disc which was restored only transiently during the first 2 days after herniation. Thereafter the water content progressively decreased. The uronic acid content of the disc changed in parallel with the changes in water content. The hyaluronic acid content decreased rapidly after herniation. However, the size of the proteoglycan monomers did not change with degeneration. The biochemical and morphologic changes are correlated, and an early repair mechanism is postulated to exist after injury.
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Osteoarthrosis was induced in one knee joint of dogs by an established surgical procedure. Changes in the articular cartilage in the biosynthesis of collagen and other proteins were sought by radiochemical labelling in vivo, with the following findings. (1) Collagen synthesis was stimulated in all cartilage surfaces of the experimental joints at 2, 8 and 24 weeks after surgery. Systemic labelling with [3H]proline showed that over 10 times more collagen was being deposited per dry weight of experimental cartilage compared with control cartilage in the unoperated knee. (2) Type-II collagen was the radiolabelled product in all samples of experimental cartilage ranging in quality from undamaged to overtly fibrillated, and was the only collagen detected chemically in the matrix of osteoarthrotic cartilage from either dog or human joints. (3) Hydroxylysine glycosylation was examined in the newly synthesized cartilage collagen by labelling dog joints in vivo with [3H]lysine. In experimental knees the new collagen was less glycosylated than in controls. However, no difference in glycosylation of the total collagen in the tissues was observed by chemical analysis. (4) Over half the protein-bound tritium was extracted by 4 M-guanidinium chloride from control cartilage labelled with [3H]proline, compared with one-quarter or less from experimental cartilage. Two-thirds of the extracted tritium separated in the upper fraction on density-gradient centrifugation in CsCl under associative conditions. Much of this ran with a single protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions. The identity of this protein was unknown, although it resembled serum albumin in mobility afte disulphide-bond cleavage.
The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 x 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 x 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol..wt. 670 000), an average Kd of 12 x 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.
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.
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