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Biomedical subjects

R F Murphy

Publications and source records attributed to R F Murphy.

At least 163 records · Page 9Linked to original sources
PubMed1985

Analysis and isolation of endocytic vesicles by flow cytometry and sorting: demonstration of three kinetically distinct compartments involved in fluid-phase endocytosis.

The existence of three distinct classes of endocytic vesicles that are part of the pathway of fluid-phase endocytosis has been demonstrated by flow cytometry. Amounts of fluorescent and scattered light were measured on a particle-by-particle basis for unfractionated whole cell lysates from cells incubated with fluorescein isothiocyanate-dextran. After 20 min two different fluorescent populations were observed, and after a 180-min incubation a third highly fluorescent population was found. Since the fluorescein isothiocyanate-dextran per fluid-phase vesicle should be a function solely of the external fluorescein isothiocyanate-dextran concentration, the existence of endocytic compartments with widely different amounts of fluorescence could result from a wide range of sizes of initial endocytic vesicles. However, the kinetics of appearance of the intermediate and highly fluorescent vesicles suggest that these compartments become labeled through fusion with the smaller primary endocytic vesicles.

Animals↗
PubMed1985

Release of secretin immunoreactivity from the ileum.

In the rat, about 80% of the intestinal content of secretin immunoreactivity is found distal to the duodenum with a peak in the ileum. This work therefore studied whether the secretin immunoreactivity in distal ileum could be released by substances normally found in the intestinal lumen. An isolated loop of ileum or duodenum was perfused with 100 mM HCl, 3 mM HCl, 50 mM taurocholate, 50 mM oleate, 308 mM glucose, 273 mM amino acids, or 308 mM NaCl in anesthetized rats. Release of secretin immunoreactivity was estimated by the integrated response above basal in portal blood. Substantial release of secretin immunoreactivity was seen after both ileal and duodenal perfusion with concentrated HCl and taurocholate, whereas perfusion with oleate was followed by a lesser response. The response to taurocholate was slower than that to HCl, but more likely represents a physiologic mechanism. The results show that the distal small intestine is capable of contributing to secretin immunoreactivity in the circulation and it is speculated that bile salts and secretin constitute parts of a negative feedback loop.

Animals↗
PubMed1985

Glucagon, secretin and vasoactive intestinal polypeptide immunoreactivities in rat gut after jejuno-ileal bypass and resection.

Jejuno-ileal bypass (JIB) and resection are associated with hyperplastic and other changes in residual functional gut. In studies on endocrine changes, circulating hormone levels are usually measured. In this study with Sprague-Dawley rats, glucagon, secretin and vasoactive intestinal polypeptide (VIP), immunoreactivities were measured in extracts of gut tissues including formerly adjacent segments of functional and bypassed jejunum and ileum. Young adult (200-220 g) and mature (404-440 g) rats received greater than 90% JIB. Though slight differences were observed in the response of the young and old rats to the operations, and between the effects of JIB and resection, the results do not indicate major involvement of secretin, VIP or gut glucagon-like immunoreactivity in the effects of surgery. Changes include increased immunoreactivity of secretin in the upper and of VIP in the lower functional gut following JIB. N-terminal-to central glucagon immunoreactivity in the functional ileum did not change substantially after operations, but was significantly lower in the non-functional than functional ileum after bypass. Plasma levels of this immunoreactivity were higher following operations and highest following resection.

Age Factors↗
PubMed1984

Specificity of cholecystokinin antibody may influence choice of tracer for radioimmunoassay.

When CCK33 was iodinated at His 20 with lactoperoxidase, the labelled hormone was less immunoreactive under radioimmunoassay conditions than CCK39 which is readily iodinated at Tyr 1. Since the difference in immunoreactivities could not be due to different degrees of oxidative damage, the regional specificity of the assay antibody (UT122, from the laboratory of J.C. Thompson, University of Texas) was re-examined. A synthetic parital sequence, CCK33 6-16, which had similar conformation to the same sequence in the intact peptide, as shown by CD analysis, was devoid of immunoreactivity. Results with sulphated and non-sulphated C-terminal octapeptides of the hormone, CCK8 (CCK33 27-33), indicate limited dependence of the C-terminal region for binding to antibody. Thus, the lower binding with 125I-CCK33 than with 125I-CCK39 indicates that His 20 is an important feature of the immunogenic site and that the iodine atom is large enough to cause steric hindrance. This explains why the method of iodination might have to be varied depending on the regional specificity of antibodies.

Amino Acid Sequence↗
PubMed1984

Acidification of internalized class I major histocompatibility complex antigen by T lymphoblasts.

It has previously been shown that activated murine T lymphocytes express intracellular vesicles containing the class I major histocompatibility complex (MHC) antigen H-2K. Evidence has also been provided that such vesicles may be part of a cellular pathway of spontaneous H-2K antigen internalization and recycling, which is specific to T-lymphoid cells. Dual fluorescence flow cytometry has now been used to establish that H-2K antigen is acidified upon internalization in concanavalin A-stimulated but not lipopolysaccharide-stimulated murine splenocytes, thus providing further support that in T lymphoblasts this class I MHC antigen may travel intracellular routes similar to those reported for other cell surface receptors.

Animals↗
PubMed1984

Reduced insulin endocytosis in serum-transformed fibroblasts demonstrated by flow cytometry.

Neoplastic transformation often results in the loss of growth control and concomitant changes in cell surface properties. The changes in endocytosis of a variety of probes after serum or anchorage transformation were measured for mouse fibroblasts by flow cytofluorometry. No major differences in dextran (fluid phase) or histone (nonspecific-adsorptive) endocytosis were observed among four cell lines with different growth properties. However, decreased receptor-mediated internalization of alpha 2-macroglobulin was observed for cell lines transformed to either serum or anchorage independence. Furthermore, increased wheat germ agglutinin and decreased insulin endocytosis were observed, but only in serum transformants. The changes specific to serum transformants were not accounted for by changes in binding of wheat germ agglutinin or insulin. The possible implications of these observations regarding serum transformation and the insulin requirement for growth in serum-free medium are discussed.

Animals↗
PubMed1984

A proposal for a flow cytometric data file standard.

The increasing complexity of multiparameter data collection and analysis in flow cytometry and the development of relatively inexpensive arc-lamp-based flow cytometers, which increases the probability that laboratories or institutions may have more than one type of instrument, creates a need for shareable analysis programs and for the transport of flow cytometric data files within an installation or from one institution to another. To address this need, we propose a standard file format to be used for all flow cytometric data. The general principles of this proposal are: (1) The data file will contain a minimum of three segments, TEXT, DATA, and ANALYSIS; (2) The TEXT and ANALYSIS segments consist of KEYWORDS, which are the names of data fields, and their values; (3) All TEXT is encoded in ASCII; (4) KEYWORDS and their values may be of any length; (5) Certain KEYWORDS will be standard, i.e., having specified formats to be recognized by all programs. The structure of the DATA segment will be uniquely defined by the values of KEYWORDS in the TEXT area. It may be in any bit resolution, facilitating compatibility between machines with different word length and/or allowing bit compression of the data. The structured nature of the TEXT area should facilitate management of flow cytometric data using existing data base management systems. The proposed file format has been implemented on VAX, PDP-11, and HP9920 based flow cytometry data acquisition systems.

Computers↗
PubMed1984

Internalization and acidification of insulin by activated human lymphocytes.

The binding and internalization of fluorescein isothiocyanate-conjugated insulin by nonactivated and phytohemagglutinin-activated circulating human lymphocytes was measured by flow cytometry. In confirmation of previous results, negligible binding or internalization was observed for unstimulated cells, while activated lymphocytes showed significant insulin binding. The majority of this insulin was demonstrated to be internalized via receptor-mediated endocytosis and acidified within 60 min after addition of insulin. Dual-fluorescence flow cytometry, using antibodies specific for human T cell subsets, was used to show that the expression of insulin binding sites occurs for at least some cells from both the helper/inducer and cytotoxic/suppressor T cell subsets. Insulin internalization is not an artifact of in vitro stimulation, since more than 90% of the unstimulated lymphocytes from a patient with a helper T cell leukemia are positive for insulin internalization. The usefulness of flow cytometric analysis for measuring lymphocyte activation in unstimulated populations and the therapeutic potential of the reported findings for control of lymphocyte proliferation are discussed.

Flow Cytometry↗
PubMed1984

Plasma and brain cholecystokinin levels in cancer anorexia.

The syndrome of cancer anorexia includes early satiety in man and a reduction in the duration of feeding in experimental animals. These aberrations suggest dysfunction of peripheral and/or central nervous system satiety mechanisms in tumor-bearing individuals. Since the gut peptide, cholecystokinin (CCK), has been implicated as a potent satiety cue in man and animals, plasma and brain concentrations of CCK were measured by radioimmunoassay in anorectic tumor-bearing rats. Plasma concentrations of immunoreactive CCK were not significantly altered in either an acute Walker 256 carcinosarcoma or more chronic methylcholanthrene-induced sarcoma animal model of cancer anorexia. However, levels of immunoreactive CCK were significantly reduced in the hypothalamus and cerebral cortex of animals bearing the methylcholanthrene sarcoma during both mild and severe anorexia. These data demonstrate that elevations in immunoreactive CCK are not a major factor in the etiology of cancer anorexia. If brain CCK is involved in satiety, tumor-bearing rats may be attempting to compensate for their anorexia by down-regulating CCK production.

Animals↗
PubMed1984

Convention on nomenclature for DNA cytometry. Committee on Nomenclature, Society for Analytical Cytology.

Analysis of cellular DNA content by cytometry is important in clinical and biological research. Measurements are used widely to assess the relative DNA content of tumor stemlines and to assist in the detection and evaluation of malignant diseases. A review of the literature on DNA measurements in solid tumor and leukemias reveals a confusing variety of terms applied for the description of similar results. In order to facilitate the understanding of data and to standardize the terminology for DNA analyses, a questionnaire was distributed to more than 500 investigators. Subsequently, a workshop on terminology was held at the Combined Conference on Analytical Cytology and Cytometry IX and VIth International Symposium on Flow Cytometry, Schloss Elmau, West Germany, 18-23 October, 1982. The workshop nominated a nine-member committee to develop guidelines for nomenclature to be used in reporting results from analyses by DNA cytometry. The committee was charged by the Council of the Society for Analytical Cytology to complete this task and to publish its recommendations in Cytometry and in Cancer Genetics and Cytogenetics. The following guidelines are based on the questionnaires returned and the discussion at the workshop; they represent the unanimous recommendations of the committee. The five guidelines given herein apply to measurements of relative DNA content of cells that have been stained appropriately and analyzed by cytometry.

Animals↗
PubMed1984

A method for incorporating macromolecules into adherent cells.

We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture. We refer to the method as "scrape-loading." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture. About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G. On average, 10(7) dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape-loading in 10 mg/ml dextran. The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, 1774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules.

Animals↗
PubMed1984

Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6.

The acidification of various ligands was measured on a cell by cell basis for cell suspensions by correlated dual fluorescence flow cytometry. Mouse 3T3 cells were incubated with a mixture of fluorescein- and rhodamine-conjugated ligands, and the ratio of fluorescein and rhodamine fluorescence was used as a measure of endosome pH. The calibration of this ratio by both fluorometry and flow cytometry is described. Dual parameter histograms of average endosome pH per cell versus amount of internalization were calculated from this data, for samples in the absence and presence of chloroquine added to neutralize acidic cellular vesicles. The kinetics of acidification of insulin were measured and compared with previous results obtained with the chloroquine ratio technique. Rapid acidification of internalized ligand was observed both for insulin, which was mostly internalized via nonspecific pathways, and for alpha 2-macroglobulin, which was mainly internalized by specific receptor-mediated endocytosis. The average pH observed for internalized insulin was less than pH 6 within 10 min after addition of insulin. At 30 min, the average pH began to decrease to approximately pH 5, presumably because of fusion of endosomes with lysosomes.

Animals↗
PubMed1984

Effect of extragastric vagotomy on pancreatic polypeptide in dogs.

The direct vagal innervation of the pancreas in dogs was interrupted by extragastric vagotomy (EGV). The response of pancreatic polypeptide (PP) to a protein meal and to hypoglycemia was compared preoperatively, after EGV and after truncal vagotomy (TV). EGV had no detectable effect on PP secretion under basal or stimulated conditions. After TV, the PP response to a protein meal was reduced and totally abolished in response to insulin hypoglycemia when compared to preoperative results. This indicates that direct innervation of the pancreas is of little importance for the release of PP but that vagal innervation of the stomach is important provided that the vagal fibers to PP cells all pass through these extragastric vagal branches.

Animals↗
PubMed1984

Appraisal of endocrine function of segmental autotransplanted pancreas in dogs.

There is increasing interest in pancreatic transplantation for patients with diabetes. In experimental models, endocrine function is usually monitored by determination of insulin and glucose levels in plasma. In this study following a segmental pancreatic autotransplant to the iliac fossa in dogs, a combined analysis of three pancreatic islet hormones, insulin, pancreatic polypeptide (PP) and glucagon was undertaken by radioimmunoassay of plasma. These were measured under basal conditions and following provocation with a standard meal, arginine, secretin and bombesin infusions. Immunohistochemical and electron microscopic examination of transplanted tissue was also performed. Circulating insulin and glucose levels in the surviving dogs with transplants reflected normoglycaemia with a normal tolerance to iv glucose and immunohistochemical detection of endocrine cells producing insulin, PP and glucagon. Secretory granules were found in A and B cells by electron microscopy. The normal circulating glucagon immunoreactivity could have originated in gastric antral A cells as well as in pancreatic tissue. It was not possible, however, to stimulate the autotransplanted pancreas to release detectable PP into the circulation.

Animals↗
PubMed1984

Sensitive radioimmunoassay for measurement of circulating peptide YY.

A radioimmunoassay with sufficient sensitivity to detect the recently discovered, circulating peptide YY in the majority of healthy adults has been developed. The antiserum to synthetic peptide YY raised in rabbits essentially showed no cross-reactivity with bovine or porcine synthetic pancreatic polypeptide, neurotensin, or neuropeptide Y, but cross-reacted to the extent of 0.03% with synthetic peptide HI. The minimum detectable limit of the assay for plasma peptide YY was 20 pg/ml with coefficients of variation less than 16%. The immunoreactivity of peptide YY isolated from healthy adult plasma had a gel-filtration chromatographic profile similar to that of synthetic peptide YY. Fasting levels of peptide YY in plasma from 127 of 137 (92.7%) ostensibly healthy adults were measurable and showed a log-normal distribution. Using the maximum-likelihood estimation method, we calculated the normal range to be 13-178 pg/ml with a geometric mean value of 49 pg/ml. The fasting plasma peptide YY levels showed no significant age or sex dependency.

Adult↗
PubMed1984

Decrease in alkaline secretion during duodenal ulceration induced by mepirizole in rats.

The mechanisms by which the potent antiinflammatory agent, mepirizole, causes duodenal ulceration were investigated in the rat. After subcutaneous administration of 200 mg/kg of mepirizole, basal gastric acid secretion remained unchanged for 5 h but duodenal alkaline output, reliably measured, decreased significantly (p less than 0.05) within 2 h. The decrease was maximal (-45%) at 3 h and persisted for a total of 6 h. The duodenal alkaline secretion returned to near normal by 24 h. A dose-response study showed that the threshold ulcerogenic dose of mepirizole (30 mg/kg) did not significantly reduce alkaline secretion, whereas higher doses did. Plasma levels of immunoreactivity of gastrin, pancreatic polypeptide, vasoactive intestinal polypeptide, and secretin were not changed at either 6 or 24 h after oral mepirizole. Vasoactive intestinal peptide levels in the duodenal mucosa were increased by 158% at 24 h after administration. Secretin levels in the duodenal mucosa were decreased by greater than 60% at both 6 and 24 h after drug treatment. Intravenous secretin (1 CU/kg X h) had no effect on duodenal alkaline secretion in either saline- (154 mM NaCl) or mepirizole-treated animals. Exogenous 16,16-dimethyl prostaglandin E2 (10 micrograms/kg X h, i.v.) reversed the action of mepirizole on duodenal alkaline secretion. These findings suggest that mepirizole causes a reduction in duodenal alkaline secretion that can be reversed by administration of an exogenous prostaglandin.

16,16-Dimethylprostaglandin E2↗