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Biomedical subjects

G M Hodges

Publications and source records attributed to G M Hodges.

At least 37 records · Page 2Linked to original sources
PubMed1985

A methodological basis for SEM autoradiography: biosynthesis and radioligand binding.

A method is described for scanning electron microscope (SEM) autoradiography whereby preservation of high resolution cell surface details is retained together with degelatination of the emulsion without gross loss or redistribution of silver grains. This method should provide a convenient medium-sized marker for SEM (using secondary, backscattered electron and X-ray imaging) topographic studies of biosynthesized molecules, and of cell surface receptors and antigens, using indirect or direct labelling procedures with radio-labelled ligands.

Autoradiography↗
PubMed1985

Changes in surface structure and concanavalin A-binding capacity of urothelium in the mouse bladder after whole-body neutron irradiation.

A broad overview has been compiled of the literature on the effects of radiation on urinary bladder and on selected cell surface markers that may give information on the pathobiological status of the urinary bladder urothelium. Scanning electron microscopy and immunogold labelling have been used in this study which examines the early (6h to 12 day) radiation response of the mouse urinary bladder following whole-body neutron irradiation. Experimentally, after 5 Gy neutron irradiation, changes in the urothelium include surface morphological abnormalities and enhanced concanavalin A surface binding. These changes were most obvious 1 to 5 days post-irradiation, but lessened in their extent from 5 to 12 days after treatment.

Animals↗
PubMed1985

An ultrastructural analysis of the physical organization of collagenous (type I) matrices: one determinant of urothelium maintenance in vitro.

Collagenous matrices, used as cell culture substrata, can be prepared from different collagen types in a variety of forms using a range of polymerization procedures. Type I collagen has been most frequently used either as dried collagen films or hydrated collagen gels. Sheets of isolated bladder urothelium, when plated onto such matrices prepared from type I collagen by different polymerization methods (eg. air-drying; NaOH; NaCl; NH3; or NH3 followed by glutaraldehyde crosslinking) demonstrate the capability of urothelial cells to attach to a variety of differently prepared matrices irrespective of polymerization procedure. In contrast, both cell proliferation and maintenance of the urothelium are markedly influenced by the polymerized form of the collagen matrix. Comparative ultrastructural (scanning and transmission electron microscopy) analysis of these matrices demonstrates dissimilarities in their physical organization. The level of filamentous, fibrillar or fibrous reaggregation of solubilized collagen molecules varies in relation to the polymerization procedure used viz, a) air dried matrices form a dense meshwork of many forms of collagen fibrils and associated filaments with an irregular surface array of coarser collagen fibres; b) matrices prepared by NaOH, NaCl and NH3 polymerization present no major differences and form a felt of interlocking collagen fibres with discrete filamentous networks associated with these fibres; and c) matrices polymerized by NH3 and crosslinked with glutaraldehyde form a dense meshwork of filaments with a more occasional distribution of fibrils associated with filaments or dense "amorphous" aggregates. The level of supramolecular reassemblage of solubilized collagen may be, therefore, a significant factor in determining urothelial cell growth and differentiation on collagen matrices.

Animals↗
PubMed1984

Species cross-reactive membrane-associated urothelial differentiation antigen.

An antigen, termed "urothelium membrane antigen" (UMA), confined to urothelium and most abundantly associated with the asymmetric unit membrane of the terminally differentiated luminal cells was identified with a monospecific (absorbed or affinity purified) rabbit antiserum obtained by immunization with membranes from normal bovine urothelium. A cross-species immunofluorescence-positive reaction was observed in normal bladder: Luminal urothelial membrane reacted most intensely. Underlying normal urothelial cells also showed a weaker cytoplasmic reaction. Specific luminal membrane labeling was confirmed by transmission and scanning electron microscopy. Specificity of the anti-UMA differed from the general epithelial reactivity obtained with antibodies to bovine urothelium cytosol. Anti-UMA did not cross-react with keratin, although there was some evidence of a filamentous localization in the cytoplasm of permeabilized cells. UMA was variably expressed by urothelial carcinoma-derived cell lines to a degree apparently related to the degree of cell differentiation, showing the highest positivity on the well-differentiated RT4 and RT112 lines and only a very weak reaction with the anaplastic MGHU-1 (EJ) and T24 lines. On immunoblots, anti-UMA reacted with a peptide of approximately 54,000 daltons.

Animals↗
PubMed1982

Urothelium-specific antibody and lectin surface mapping of bladder urothelium.

Coupled ligand-colloidal gold complexes were found to provide a convenient approach for the localization of scanning electron microscopy of cell surface membrane antigens and lectin-binding sites on bladder urothelium and for the immunocytochemical identification of urothelial cell populations at different stages of differentiation. The ligands used to prove the membrane were a urothelium-specific rabbit antibody raised to a urothelial membrane-associated antigen (UMA), and two lectins: Concanavalin A (Con A) and peanut agglutinin (PNA). A complex luminal surface distribution pattern was demonstrated by the UMA antigen related to the stage of urothelial cell maturation and differentiation. UMA could be detected on the surface of immature and early differentiating intermediate cells, but was absent from the late differentiation stage, becoming re-expressed as the cells matured and was found in greatest abundance on the terminally differentiated superficial cells. It was absent on cells in benign hyperplasia of the urothelium. Cellular and regional differences in lectin binding to the urothelial cell surface was suggested with Con A receptors localized uniformly over the superficial cells, and PNA receptors confined to linear arrays or occasional clusters over the apical surface but evenly dispersed over the lateral surface of these cells.

Animals↗
PubMed1981

Cytochemical markers of bladder carcinogenesis.

Enzyme cytochemical and immunocytochemical techniques at the light and electron microscope levels were used to study the distribution of potential markers of chemical transformation in rodent bladders. In rat tumours induced by in vivo treatment with methylnitrosourea, alkaline phosphatase localization was normal on the external surface of the plasma membranes of some cells but abnormal in others where reaction product was seen only on intracellular membranes. 5'-Nucleotidase localization was abnormal in all cells, being seen on endoplasmic reticulum and nuclear membranes only, while in normal bladders only ectoenzyme localization was seen. Heterogeneity of alkaline phosphatase amd 5'-nucleotidase localization was seen on the plasma membranes of these tumours after 15 days in organ culture. Some cells produced enzyme and others did not; in other cells only parts of the membrane reacted heavily, while other regions were negative. In transformed cell cultures and tumours of mouse bladder derived by in vitro treatment of explants with dimethylbenz (a) anthracene, a bimodal pattern of alkaline phosphatase localization was seen. Cells had either normal ectoenzyme reaction product or abnormal intracellular membrane reaction product. 5'-Nucleotidase and ADPase were lost after transformation while cAMP-phosphodiesterase was retained as an ectoenzyme. Mg.ATPase and a cAMP-independent, calcium-insensitive 'protein phosphatase' were induced in transformed cell cultures. An epithelial antigen was detected in the cytoplasm of both normal and transformed cells associated with reticular cytoplasmic ground substance, plasma membrane vesicles and cytoskeletal elements.

5'-Nucleotidase↗
PubMed1981

Colloidal gold markers and probes for routine application in microscopy.

Colloidal gold can be used as an electron-dense cytochemical probe for direct or indirect labelling techniques. Gold markers can be prepared in a size range of 5-150 nm, and show size-dependent shape characteristics and absorption spectra. Size and shape distribution increases with mean particle diameter, with appreciable overlapping between populations of different mean size. Using radio isotope-binding assays, spectrophotometric analysis and an innovative rapid microtitration technique, the effect of pH, ionic strength and protein concentration on gold-protein interaction has been studied. Efficient adsorption of protein to gold occurs at, or near, the pI of the protein. The amount of protein needed to effect stabilization is both a function of pH and of ionic strength, but does not reflect the amount of protein binding for all proteins. There is evidence for multilamellar adsorption of proteins to gold, which is discussed in context of the bioactivity, and stability of the probe. A working protocol for the routine reproducible manufacture of protein-gold probes is given, making use of the microtitration assay.

Colloids↗
PubMed1981

Cell surface distribution of fibronectin in cultures of fibroblasts and bladder derived epithelium: SEM-immunogold localization compared to immunoperoxidase and immunofluorescence..

Expression of cell surface fibronectin in cultures of untransformed fibroblasts is well documented, but little is known of its presence and distribution in cultured epithelial cells. Using species monospecific anti-fibronectin antibodies, the distribution of fibronectin in untransformed fibroblasts and in normal and neoplastic bladder epithelial cells was characterized by indirect labelling experiments using immunogold scanning electron microscopy (SEM). The surface matrix of fibronectin expressed in rodent and human fibroblast cell lines was demonstrated with ease by SEM of gold-tagged second antibodies. However, no fibronectin could be detected on any of the mouse and human bladder epithelium-derived cells studied in single or in mixed epithelial-fibroblast cultures. These SEM-immunogold observations were compared to and confirmed by immunofluorescence and immunoperoxidase microscopy. Immunofluorescence and SEM localization of the fibronectin in the extracellular matrix presented similar distribution patterns but the higher resolution of the SEM provided a more detailed analysis.

Animals↗
PubMed1980

A fine fibrous silica contaminant of flour in the high oesophageal cancer area of north-east Iran.

We report here the discovery and characterization of a fibrous mineral contaminant of the diet in that area of north-east Iran where oesophageal cancer has a very high incidence. This contaminant has a smoothly tapering shape and is between 50 and 150 micrometers long. The greatest diameter is between 1 and 10 micrometers and this decreases to a sharply pointed tip with a radius of curvature of between 0.25 and 0.60 micrometers. Electron microscope X-ray analysis shows that this fibre consists almost entirely of silica. It is free from alkali metals, aluminium and iron, and therefore differs from other known natural or manmade mineral fibres. Examination of the seeds of more than sixty different species of weed know to contaminate the wheat in this area of the Middle East shows that the fibre originates from the seeds of the common Mediterranean grass Phalaris minor. This seed bears fibres of the same dimensions, composition and birefringence, borne upon the inflorescence bracts which envelop the pericarp of the seeds of this and other members of the phalaris genus. They are broken off from the seed when the wheat is milled but persist in the flour, where up to 3,000 are found in each gram. Similar fibres can be isolated in quantity from the seeds of related species which are grown commercially, and they have a similar size and composition. When cells of the 3T3 mouse fibroblast line are exposed to these fibres in semi-solid suspension culture, their proliferation is stimulated more than 100-fold. We present an hypothesis for the involvement of these plant mineral fibres in the aetiology of oesophageal cancer in Iran and in other areas of high incidence.

Animals↗
PubMed1980

A review of the colloidal gold marker system.

Colloidal gold can be used as a particulate marker for the detection and localization of target molecules by various modes of microscopy (light and fluorescent microscopy, scanning and transmission electron microscopy) using both direct and indirect labeling approaches. Several techniques are available for the preparation of gold markers in a size range of 5nm to 150nm, their mean size and shape characteristics and absorption spectra varying with particle size. Under appropriate conditions, colloidal gold will bind macromolecules by non-covalent electrostatic adsorption with little change in the specific activity of the bound macromolecule. This interaction is influenced by a number of factors including ionic concentration, pH conditions (in correlation with the protein pI values) and protein stabilizing levels. Presence of reactive protein on probes can be demonstrated and quantitated by direct and indirect radioactive binding assays and agglutination assays. These assays provide convenient procedures for characterizing stability, and behaviour in storage, of gold probes. Stability of gold probes under conditons where competing proteins are present, under freeze-thaw cycles and under SEM preparation conditions have been evaluated in this paper. Some of the basic procedures in the application of gold probes to cell labeling are briefly discussed together with certain limitations of the colloidal gold marker system. A bibliography of gold probe cell labeling studies is included.

Animals↗
PubMed1979

Colloidal gold probes--a further evaluation.

Colloidal gold provides an immuno- and cytochemical marker suitable both for TEM and SEM. Various parameters involved in the preparation and stability of gold markers of various sizes and of probes have been reviewed and modifications in methodology suggested based on a study of factors affecting the performance of the gold marker system. In particular, dependence of protein adsorption on pH conditions correlating with protein pI is further confirmed. Presence of bioactive protein in gold probes is demonstrated and quantitated by radioassay (125I) and agglutination procedures.

Cell Line↗
PubMed1976

Scanning electron microscopy of cell-surface changes in methylnitrosurea (MNU)-treated rat bladders in vivo and in vitro.

Scanning electron microscopy has been used (1) to characterize epithelial cells of bladders from normal rats and from rats treated with a single initiating but non-carcinogenic dose of 2 mg methylnitrosurea (MNU), 24 h and 6 weeks after treatment; and (2) to compare morphological aspects of epithelial differentiation in organ culture of bladder explants taken from untreated and MNU-treated rats at these time intervals. There are marked differences in vivo between the surface organization of normal urothelium and urothelium undergoing reversible hyperplasia following MNU treatment. Maturation of the normal rat bladder epithelium in vivo is shown to be related to a series of well-defined cell-surface changes readily identified by SEM. By contrast the maturation response is perturbed in the hyperplastic epithelium; the cells lose their ability to differentiate normally and form instead an excess of stubby globular microvilli which project from the cell surface. In organ culture, maturation of normal bladder epithelium (both in re-epithelialized areas of the explant and in areas of epithelial outgrowth over cellulose acetate substrates) can be also related to a series of cell surface changes showing close similarities to those in vivo. However, epithelial maturation remains defective in organ cultures of bladders from MNU-treated animals. The closely parallel behaviour of the bladder epithelium in vivo and in vitro in both normal and treated tissues underlines the potential value of the bladder organ culture system for studying the comparative biology of hyperplastic development produced by a single initiating dose of MNU and suggests it will be useful with which to study carcinogenesis following multiple doses of MNU.

Animals↗
PubMed1976

Chemically-defined medium for growth and differentiation of mixed epithelial and connective tissues in organ culture.

The effect on tissue differentiation and growth in vitro of certain of the factors implicated in collagen synthesis (ascorbic acid, alpha-ketoglutarate and oxygen) and the influence of hydrocortisone was studied using organ cultures of fetal mouse mandible as a mixed epithelial and connective tissue system. Using serum-free Waymouth's MB 752/1 chemically-defined medium, addition of high levels of ascorbic acid (300mug per ml), hydrocortisone (1mug per ml) and oxygen (95%) enhanced differentiation in a number of tissues, in particular skin and appendages, tooth germs and bone, while osteoid and dentine production were noticeable promoted. It is suggested that an essential aspect of media design for organ culture involves the incorporaation of collagen-promoting factors to the in vitro enviornment particularly with regard to the controlling role implicated for collagen in a variety of biological processess.

Animals↗
PubMed1975

Quantitative evaluation of autoradiographs by X-ray spectroscopy.

Measurements of mass of developed silver in autoradiograms have been made using X-ray microanalysis. This procedure has been applied to a scanning electron microscopic study of protease-induced cell stimulation in 3H-thymidine-labelled bladder tissues in vitro. Differences in silver concentrations over individual nuclei were demonstrated by X-ray analysis and quantitative data was obtained showing an increased rate of 3H-thymidine incorporation in protease-treated cultures. The initial evidence indicates that X-ray microanalysis could provide a potentially useful quantitative procedure for autoradiography.

Animals↗