Search PubMed⌕ Search

Biomedical subjects

N Fleming

Publications and source records attributed to N Fleming.

At least 55 records · Page 3Linked to original sources
PubMed1983

Radioimmunoassay of rat submandibular salivary gland mucin.

A solid phase radioimmunoassay (RIA) for rat submandibular mucin (RSM) was developed and applied to studies of mucin concentration, antigenicity and secretion. The assay utilizes the affinity of 125I-Protein A for IgG to quantitate antigen-antibody immunocomplexes immobilized in the wells of plastic microtitre plates. The technique was highly reproducible and capable of detecting as little as 3 ng mucin protein. The submandibular glands of rats weighing 150-180 g contained 257 micrograms of mucin (dry wt) per mg protein, which is equivalent to about 3.6 mg of mucin per gland. The antiserum to RSM was cross-reactive with mucins from rat sublingual and pig submandibular glands, and rat, human and pig small intestine. No cross-reactivity was detected with mucins from mouse, canine or bovine submandibular glands, and there was no evidence that ABH blood-group sugars contributed to mucin antigenicity. The RIA was used to estimate secretion from dispersed rat submandibular gland cells and gave a more specific and accurate assay of mucin release than previous assays of precursor-labelled radioactive glycoproteins in the culture medium. The beta-adrenergic agonist, isoproterenol, stimulated immunoreactive mucin secretion from cultured cells to approximately twice the level of unstimulated or propranolol-inhibited controls. The RIA appears to offer promising new approaches for studies on mucin metabolism and secretion in health and disease.

Animals↗
PubMed1982

Purification and immunofluorescent localization of rat submandibular mucin.

Rat submandibular mucin (RSM) was purified by acid precipitation, then alcohol precipitation of the 30000g supernatant of gland homogenate, followed by column chromatography on Sephadex G-200. The mucin, which was eluted in the void volume, had an amino acid profile typical of a salivary mucus glycoprotein with high proportions of threonine, serine and proline (48.8% of total amino acids), and low proportions of aromatic and basic amino acids. It consisted of 63% (w/w) carbohydrate, which was shown by g.l.c. analysis to contain N-acetylglucosamine, N-acetylgalactosamine, galactose, sialic acid and fucose in the proportions 1.0:3.4:2.6:3.1:1.2. After staining of the mucin with periodic acid/Schiff reagent, analytical equilibrium ultracentrifugation in a CsCl density gradient produced a symmetrical peak of buoyant density 1.449g/ml, without evidence of protein contaminants. Sedimentation velocity centrifugation revealed a major periodate/Schiff-positive component (S(0) (20,w) 5.06) with an associated shoulder of slower sedimenting material, suggesting polydispersity in the size of the mucin. Our findings suggest that the RSM purified in these studies has a molecular weight between 200000 and 1x10(6). Antibody to RSM was prepared in a rabbit and produced a single precipitin line on immunoelectro-osmophoresis with the mucin. Immunofluorescence studies showed that the antibody localized only to submandibular acinar cells and confirmed that these cells were the source of RSM. The antibody was not directed towards the blood-group-A determinant (terminal N-acetylgalactosamine) present in the mucin.

Amino Acids↗
PubMed1980

The secretory response in dissociated acini from the rat submandibular gland.

Rat submandibular gland was dissociated by enzymatic digestion with collagenase and hyaluronidase, followed by mild mechanical shearing and filtration through a nylon mesh. The dissociated cell populations contained predominantly groups of acinar cells which maintained their acinar arrangement. The morphological and functional viability of the cells was confirmed by electron microscopic examination and a normal secretory response to beta-adrenergic or cholinergic stimulation was observed. Both isoproterenol (IPR) and carbachol caused the fusion of secretory granules into large vacuoles which were also continuous with the lumen, and into which the secretory product was released. Secretion was assessed quantitatively from the incorporation of 14C-glucosamine into the acinar cells and its subsequent release into the culture medium as labelled glycoprotein. IPR stimulated secretion to 125% of untreated controls in the concentration range t x 10(-5) to 5x 10(-7) M and to 110% of controls at 5 x 10(-8) M, after 40 min incubation. Carbachol stimulated secretion to 131% of controls at 5 x 10(-5) M and to 115% at 5 x 10(-6) M but had no effect at 5 x 10(-7) or 5 x 10(-8) M. The secretory response was blocked by the respective beta-adrenergic and cholinergic antagonists, propranolol and atropine. These findings show that dissociated rat submandibular acinar cells provide a useful in vitro model for the study of mucus synthesis and secretion.

Animals↗
PubMed1978

Inhibition of lactate dehydrogenase in cultured SIRC cells by cigarette smoke.

SIRC cell monolayer cultures were exposed to whole smoke from a mid tar and nicotine level research cigarette (ASFC, 72 puffs), or from a high tar and nicotine level reference cigarette (Kentucky 2RI, 48 puffs) over a period of 65 days. The activity and distribution of lactate dehydrogenase (LDH) in the cells were investigated, and the electrophoretic characteristics of its isozymes studied. Cell morphology was examined by light microscopy and by transmission- and scanning electron microscopy. LDH activity was reduced by exposure to smoke from both cigarette types, the greater inhibitory effect being produced by that of the Kentucky cigarette. In addition, cells exposed to this high tar and nicotine smoke displayed intramitochondrial granules which were larger and more numerous than those found in cells exposed to the mid tar and nicotine smoke, or in the control cells. It is speculated that cation accumulation in the mitochondria may be involved in the observed inhibition of LDH activity.

Cells, Cultured↗
PubMed1977

The activity and distribution of gamma-glutamyl transpeptidase (y-GT) in human lung cancers serially transplanted in nude mice.

Gamma-glutamyl transpeptidase (y-GT) activity and distribution were investigated in different types of human lung cancers (three epidermoid carcinomas, one large cell carcinoma) which were maintained by serial transplantation in nude mice. All transplanted tumour fragments were positive for the enzyme. In the epidermoid carcinomas, y-GT levels were related to the degree of tumour differentiation. Enzyme activity in tumour fragments was always higher than that found in normal adult human lung tissue, and was, in general, maintained throughout the transplantation series.

Adult↗
PubMed1977

The activity and distribution of gamma-glutamyl transpeptidase (y-GT) in human foetal organs.

The activity and distribution of y-GT was investigated in a number of organs from human foetuses aged from 14 to 24 weeks post menstruationem. Over this period, enzyme activity increased in the kidney, pancreas and thymus, but decreased in the small intestine. No trend could be established for the liver, although activity was high. In the lung, spleen, brain and adrenals, y-GT was either detectable at very low levels or could not be demonstrated. The possible relationship between y-GT activity in some human tumours and the enzyme level in the corresponding foetal organs is discussed.

Female↗
PubMed1977

Morphology, histochemistry and isozymes of monkey kidney cells during long-term exposure to cigarette smoke.

Monkey kidney cells (Vero) in monolayer culture were exposed to 260 puffs of whole smoke from a mid tar range cigarette over a period of 240 days. Cell morphology was investigated at the light and electron microscopic level. The histochemical characteristics of several hydrolytic enzymes and dehydrogenases were examined, and the isozymes of LDH, MDH and G6PD studied. For all these parameters smoke exposed cells displayed the same features as sham exposed and negative controls. The cells were assessed as non malignant by their failure to form tumours in the nude mouse.

Acid Phosphatase↗
PubMed1975

Hepatocyte function in long-term organ culture of Amphimuma means liver.

Fragments of liver from the adult urodele Amphiuma means, the Congo eel, were maintained in organ culture for up to 70 days. The normal electrophoretic patterns of several enzymes were retained. The activities of ornithine transcarbamylase, arginase, glutamate oxalacetate transaminase and glutamate pyruvate transaminase, and urea production, glucose uptake and tissue glycogen content remained relatively constant throughout the culture period. Histological organization and hepatocyte ultrastructure were also retained. Liver fragments survived better in media based on MEM or BME than in medium based on Leibovitz L15. Since many aspects of tissue-specific structure and function are retained, long-term amphibian organ culture is well suited to studies on the control of hepatocyte function and on the effects of metabolites, hormones, drugs and toxins.

Alanine Transaminase↗