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Biomedical subjects

B Rafferty

Publications and source records attributed to B Rafferty.

At least 37 records · Page 2Linked to original sources
PubMed1997

Human meningiomas possess muscarinic acetylcholine receptors: stimulation of phosphatidylinositol turnover by carbachol.

The effect of carbachol, an acetylcholine receptor agonist, on rate of phosphatidylinositol (PI) turnover in cultured human meningioma cells was investigated. Exposure of meningioma cells for 2 h to carbachol (3.12-200 mumol/L) resulted in a dose-dependent stimulation of PI turnover to a maximum of 5.5-fold over basal controls. A time course study showed stimulation of IP3 formation after 30 s followed by increases in IP1 and IP2. The stimulatory effect of carbachol on PI turnover was completely abolished by the muscarinic receptor antagonist, atropine, but was unaltered by the nicotinic antagonist, hexamethonium. Reverse-transcription of meningioma-derived RNA into cDNA followed by amplification by the polymerase chain reaction using specific primers revealed presence of ml type muscarinic receptor mRNA. These results provide evidence that human meningioma cells possess muscarinic acetylcholine receptors the activation of which leads to PI hydrolysis.

Atropine↗
PubMed1996

Lectin-binding assays for the isoforms of human erythropoietin: comparison of urinary and four recombinant erythropoietins.

Assays have been developed for the isoforms of erythropoietin (EPO) based on their binding to eight different lectins. These assays were used to compare the isoform compositions of two preparations of human urinary EPO (uEPO) and four preparations of recombinant DNA-derived human EPO (rEPO), which had been shown to differ in their biological and immunological properties and in their isoform composition as judged by isoelectric focusing and electrophoresis. Agarose-bound Ricinus communis agglutinin I (RCA), Erythrina cristagalli agglutinin (ECA), Maackia amurensis leukoagglutinin (MAL), Sambucus nigra agglutinin (SNA), Lycopersicon esculentum agglutinin (LEA), concanavalin A (Con A), Phaseolus vulgaris agglutinin-L4 (L-PHA) and Agaricus bisporus agglutinin (ABA) were used to bind EPO isoforms possessing: N-glycans containing non-sialylated outer Gal beta 1-4GlcNAc (RCA and ECA), NeuAc alpha 2-3Gal beta 1-4GlcNAc (MAL), NeuAc alpha 2-6Gal (SNA), or repeating Gal beta 1-4GlcNAc sequences (LEA); biantennary N-glycans (Con A); tetraantennary and 2,6-branched triantennary N-glycans (L-PHA); and O-glycans containing NeuAc alpha 2-6GalNAc (SNA) and Gal beta 1-3GalNAc (ABA). Free EPO was measured by mouse spleen cell bioassay or immunoassay. Estimates from most lectin-binding assays were reproducible between assays and batches of lectin-agarose, although batches of MAL- and ABA-agarose, and to a lesser extent LEA-agarose, differed in their EPO-binding. Lectin-binding assays showed differences between the isoform compositions of all EPOs, including the two Chinese hamster ovary cell-derived rEPOs, with RCA- and ECA-binding assays being the most discriminating. Lectin-binding estimates provided evidence that uEPO differs from these rEPOs in its lower content of isoforms with biantennary N-glycans and higher content of those with multiantennary N-glycans, and in its lower content of isoforms with N-glycans possessing repeating Gal beta 1-4GlcNAc sequences and of those with O-glycans containing Gal beta 1-3GalNAc. Lectin-binding estimates also indicated that, contrary to some reports, uEPO possesses Gal beta 1-3GalNAc-containing O-glycans but not NeuAc alpha 2-6GalNAc-containing O-glycans or NeuAc alpha 2-6Gal-containing N-glycans. Most groups of lectin-bound EPO isoforms did not differ in their relative bioactivities and immunoreactivities. However, estimates for ABA-bound EPO isoforms suggested that O-glycans might influence the bioactivity of EPO differently to its immunoreactivity. Furthermore, the bioactivities of some ECA-bound EPO isoforms were higher, and those of some of the MAL-bound EPO isoforms lower, than their immunoreactivities, consistent with the reported enhancement of EPO in vitro bioactivity by desialylation.

Biological Assay↗
PubMed1995

Characterization of a rat anterior pituitary cell bioassay.

We have described the protocols and characterization of a pituicyte culture, which became established as a reliable and reproducible bioassay for the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The bioassay was used to measure the bioactivity of factors that inhibit and stimulate gonadotrophin secretion. The protocol that was used involved the culling of female Wistar rats (200 to 250 g weight), at random stages of their cycle, and dispersal of their pituicytes in a concentration of 0.4 x 10(6) cells.ml-1.well-1 in serum-free medium (Dulbecco's modified Eagle's medium/Ham's F12 mixture, supplemented with insulin and transferrin) in Falcon 3047 24-well culture plates. After 24 h of pre-culture, the medium was changed and the cells cultured for a further 48 h. The supernatant was removed and assayed for basal secretion of FSH and LH. The cells were then stimulated with 10(-8) M GnRH for 4 h and the supernatant assayed for gonadotrophin-releasing hormone (GnRH)-stimulated FSH and LH secretion. All samples were assayed as pairs of duplicates (i.e. quadruplicate samples) which were randomly added to the plates to minimize plate effects. Random number tables were used to achieve this randomization.

Animals↗
PubMed1995

Ophthalmologic examination of patients with seasonal affective disorder, before and after bright light therapy.

PURPOSE: We assessed the potential ocular hazards of bright light therapy for patients with seasonal affective disorder, after both short- and long-term treatment, and identified prospective patients with pre-existing ocular abnormalities. METHODS: Fifty patients with seasonal affective disorder received daily exposure to artificial light in the morning or evening for 30 minutes at an illuminance level of 10,000 lux (irradiant dose, 0.016 J/cm2). Ophthalmologic examinations were performed before and after short-term treatment (two to eight weeks) and after three to six years of use during the fall and winter months. Over the four years of patient intake, the eye examination included subsets of the following tests: visual acuity, intraocular pressure, slit-lamp biomicroscopy, direct and indirect ophthalmoscopy, color vision, visual field, fundus photography, Amsler grid, ocular motility, pupillary reactions, contrast sensitivity, stereopsis, and the macular stress test. RESULTS: No ocular changes were detected after short-term treatment. Long-term treatment (three to six years) of 17 patients, with cumulative exposure durations of 60 to 1,250 hours, also resulted in no ocular abnormalities. CONCLUSIONS: Light therapy yields about 75% clinical remissions. It is effective as an antidepressant and appears safe for the eyes. Current knowledge is insufficient to specify any definite ocular contraindications for bright light therapy, although we recommend that patients with preexisting ocular abnormalities and those using photosensitizing drugs undergo treatment only with periodic ophthalmologic examination.

Adolescent↗
PubMed1995

In vitro bioactivity of gonadotrophin surge attenuating factor is not affected by an antibody to human inhibin.

The effect of anti-inhibin antibodies on gonadotrophin surge attenuating/inhibiting factor (GnSAF/GnSIF) and its effect on gonadotrophin secretion in a pituitary cell bioassay were determined by culturing rat pituitary cells in a serum-free medium to which inhibin and a partially purified preparation of gonadotrophin surge inhibiting factor (GnSIF) were added. The samples were treated with an anti-inhibin antiserum and basal and GnRH-stimulated FSH and LH secretion were measured by radioimmunoassay of the culture supernatant. Inhibin had a dose-dependent inhibitory effect on both basal and GnRH-stimulated FSH secretion and also on GnRH-stimulated LH secretion. Anti-inhibin antibody blocked the inhibition of FSH secretion, except at the highest two doses of human inhibin (1.25 ng ml-1, P = 0.04; 2.5 ng ml-1, P = 0.0004). The GnSIF preparation inhibited basal and GnRH-stimulated FSH and GnRH-stimulated LH secretion and was not affected by the anti-inhibin antibody. The novel hormone, GnSIF, is different from inhibin.

Animals↗
PubMed1995

Differences in carbohydrate composition of FSH preparations detected with lectin-ELISA systems.

FSH is a glycoprotein containing N-linked carbohydrates which exhibit a variety of forms ranging from mono- to multibranched structures. Variation in glycosylation, particularly the degree of terminal sialylation, determines the half-life of the hormone and hence its in vivo bioactivity. The glycoform content of FSH preparations can differ according to the source (e.g. pituitary, urine), cell line (for rDNA-derived material) and selectivity of purification procedures, and may create difficulties in the preparation and characterization of standards and therapeutic products. In order to develop a simple method to detect changes in glycocomposition, an FSH ELISA was modified by the incorporation of lectins of recognized sugar specificity, and used to examine the terminal sugar composition of ampouled preparations of pituitary, urinary and rDNA-derived FSH. FSH was captured with a specific monoclonal antibody (MAb) and detected with either biotinylated anti-FSH MAb (ELISA) or the sugar-specific lectins (L-ELISA) from Triticum vulgaris (sialic acid, SA), Sambucus nigra (alpha 2,6-linked SA), Maackia amurensis (alpha 2,3-linked SA) or Ricinus communis (free terminal galactose; GAL). Relative estimates of the amounts of terminal SA, its different forms and GAL were derived from the L-ELISA/ELISA data compared with the highly sialylated 1st International Standard for pituitary FSH (IS) 83/575. All the FSH preparations had less SA than the IS with the ratio of alpha 2,3- and alpha 2,6-linked SA varying between preparations. The amounts of alpha 2,6-linked SA relative to the IS were not significantly different in the urinary and pituitary preparations whereas alpha 2,3-linked SA in all preparations was generally less than that of the standard.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Conformation↗
PubMed1994

Fungal spores in faeces as evidence of fungus ingestion by sheep.

In Ireland, upland areas are becoming extensively planted with coniferous forest to which sheep are allowed access. Such forest produces many more fruiting bodies of basidiomycetes than the blanket bog on which it was planted. Faecal samples taken from hill and adjacent forest (Picea abies) grazing areas showed an autumnal increase in radiocaesium in the forest samples compared with the hill samples, and there was an indication of higher in vivo radiocaesium activity in the autumn, particularly in individual animals which are known to graze the forest. The peak faecal and in vivo radiocaesium levels coincided with the fungal growing season. A new method is presented using fungal spores, which proves the ingestion of fungi by sheep and permits the identification and quantification of the fungi consumed.

Animal Feed↗
PubMed1994

Seasonal variations in the transfer of 137Cs and 40K to pasture grass and its ingestion by grazing animals.

Samples of grass and sheep faeces were taken monthly over a 12-month period from three pasture sites. Biomass, 137Cs and 40K were measured. The activity concentration of these two radionuclides in grass was influenced by season and soil type. The seasonal patterns of 137Cs and 40K transfer to grass were inversely related, but evidence is presented which suggests that soil contamination of grass samples contributed to the 137Cs and 40K activity concentrations of the grass and augmented the difference between their patterns of transfer. This was influenced by season, climate and farm management. A parallel seasonality was identified in the 137Cs activity concentration of the faeces of sheep grazing these pastures. This was not observed for 40K in sheep faeces.

Animals↗
PubMed1994

Assessment of the role of soil adhesion in the transfer of 137Cs and 40K to pasture grass.

137Cs and 40K were analyzed in pasture grass and soils throughout a 12-month period. The titanium content of the samples was determined as a marker of soil contamination of the grass samples. The 137Cs activity concentration in the grass was closely correlated with titanium and both increase in the winter months suggesting that increased soil contamination of grass is responsible for the increased 137Cs activity concentration observed at this time. The method greatly overestimates the contribution made by soil to the 137Cs activity concentrations of the grass samples and the results are highly variable. Some problems associated with the method are discussed and it is proposed that this method is unreliable.

Adhesiveness↗
PubMed1994

Human acoustic neuromas secrete interleukin-6 in cell culture: possible autocrine regulation of cell proliferation.

Interleukin-6 (IL-6) secretion by cell cultures of human acoustic neuromas was examined. Secretory rates varied from 0.02 to 5.4 ng/10(5) cells per 4 days, depending on the tumor. The IL-6 immunoreactivity eluted from a Sephadex G-100 column in a major peak corresponding to an M(r) of 30,000 and a lesser peak corresponding to an M(r) of 50,000. Western blot analysis revealed three IL-6 immunoreactive bands with M(r)s corresponding to 53,000, 29,000, and 24,000. Tumor necrosis factor-alpha, interleukin-1-beta, and cholera toxin all stimulated IL-6 secretion. An antisense phosphorothioate oligonucleotide against IL-6 messenger RNA inhibited both [3H]thymidine uptake and IL-6 secretion by acoustic neuroma cells in culture. In addition, [3H]thymidine uptake was inhibited by a specific polyclonal antibody against IL-6. We conclude that human acoustic neuroma cells produce and secrete IL-6, which may act in an autocrine manner to stimulate cellular proliferation.

Adult↗
PubMed1994

Secretion of interleukin-6 by human meningioma cells: possible autocrine inhibitory regulation of neoplastic cell growth.

Using cell culture techniques, the authors have previously shown that human meningioma cells secrete an autocrine growth stimulator related to platelet-derived growth factor. Here, they further demonstrate potential autocrine inhibitory regulation of meningioma cell growth by interleukin (IL)-6. Constitutive IL-6 production was detected in all meningiomas studied, in the form of protein as well as IL-6-specific messenger ribonucleic acid. The IL-6 immunoreactivity in conditioned medium from three different meningioma cultures eluted from a Sephadex G-100 column was evidenced by a single peak corresponding to a molecular weight of about 32 kD. Interleukin-6 secretion was remarkably stimulated by tumor necrosis factor-alpha, IL-1 beta, and IL-4, and was also influenced by a combination of epidermal growth factor and bromocriptine. Recombinant IL-6 exhibited a significant dose-dependent inhibitory effect on meningioma cell proliferation. The maximum effect was observed at concentrations of 10 to 100 pg/ml, with the decrease in thymidine incorporation ranging from 21% to 35% versus control. Addition of an anti-IL-6 antibody enhanced the growth-stimulating effect of meningioma-derived conditioned medium. The rate of IL-6 secretion tended to show an inverse correlation with meningioma growth rate. The results presented here and the previous results suggest that the regulation of meningioma cell proliferation is defined by a complex network of autocrine stimulation, autocrine inhibition, and influences from multiple exogenous factors.

Blotting, Northern↗
PubMed1993

Interactive effects of interleukin-6, 17 beta-estradiol and progesterone on growth and 17 beta-hydroxysteroid dehydrogenase activity in human breast carcinoma cells.

It has been demonstrated that reductive 17 beta-hydroxysteroid dehydrogenase activity (17-HSD) in the human breast cancer cell line MCF-7 can be stimulated by 17 beta-estradiol (E2), progesterone (P) and interleukin-6 (IL-6). We have examined the interactive effects of these factors on growth and reductive 17-HSD activity of MCF-7 cells cultured under defined conditions in phenol red-free medium. E2 stimulated growth of MCF-7 cells in a dose-dependent manner, while IL-6 had a growth inhibitory effect and in combination with E2, it reduced or abolished the stimulatory effects of the steroid. Both E2 and IL-6 stimulated 17-HSD activity by a maximum of 2- to 5-fold, but, in combination, the stimulatory effects ranged from 7- to 10-fold, indicating a strong synergism between the 2 factors. P had growth stimulatory effects on MCF-7, but when combined with IL-6 had no further positive or negative growth effects. Both factors stimulated reductive 17-HSD activity and simultaneous treatment with P and IL-6 indicated a synergy between the 2 factors. These results provide evidence of powerful interactive effects between steroidal and paracrine control of human breast epithelial cells in vitro.

17-Hydroxysteroid Dehydrogenases↗
PubMed1992

Effects of topography on caesium-137 in montane peat soils and vegetation.

This study investigated the relationship between altitude and 137Cs fallout in soils and plants. The soils and plants, Calluna vulgaris, Erica cinerea and Molinia caerulea, were sampled across a transect of two valleys in north-western Ireland. The results provided evidence that greater 137Cs deposition to soils occurred at higher altitudes, and the data supported the findings of a previous investigation in the same area. A valley effect, whereby greater concentrations of 137Cs accumulated in plants on the lower altitudes of the valley floors, was shown to be significant. Deposition values for total 137Cs in soils were not correlated with plant concentrations, although a relationship between concentrations in C. vulgaris and M. caerulea was found to be statistically significant. No relationship was observed between the 137Cs from weapons fallout with that of Chernobyl origin in the 0-10 cm depth soils, although a significant correlation emerged between the same two components measured in C. vulgaris. The results suggest that existing definitions of concentration ratios and transfer factors are, for many reasons, inappropriate for studies of soil to plant transfer of 137Cs in peatland ecosystems.

Accidents↗
PubMed1991

Measurement of cytokine production by the monocytic cell line Mono Mac 6 using novel immunoradiometric assays for interleukin-1 beta and interleukin-6.

Immunoradiometric assays for interleukin-1 beta and interleukin-6 were developed using affinity-purified IgG fractions from antisera initially raised for radioimmunoassay. Capture and detector functions were carried out by the same antibody preparation within each assay. The assays were precise, rapid and 6-8-fold more sensitive than the RIA systems previously employed. IRMAs were used for the initial characterisation of a candidate cell line (Mono Mac 6), under investigation in a 'monocyte test' for pyrogen detection, and permitted rapid and specific determination of the cytokines under stimulatory and inhibitory conditions.

Animals↗
PubMed1991

Interleukin 6 is secreted by breast fibroblasts and stimulates 17 beta-oestradiol oxidoreductase activity of MCF-7 cells: possible paracrine regulation of breast 17 beta-oestradiol levels.

Previous studies have demonstrated that cultured human breast fibroblasts secrete a high-molecular-weight polypeptide which stimulates the ability of human breast-cancer MCF-7 cells to convert oestrone (E1) to the biologically more active 17 beta-oestradiol (E2). This effect is mediated by an increase in reductive E2 oxidoreductase (EOR) activity. We have identified the fibroblast-derived stimulatory factor as interleukin 6 (IL6) or an immunologically related peptide. Human breast fibroblasts in culture secreted up to 10 ng IL6/ml medium during 24 hr of incubation. The effects of IL6 and breast fibroblast conditioned medium (CM) on reductive EOR activity of MCF-7 cells were similar; both CM and IL6 potently stimulated enzyme activity in a dose-dependent manner, and both exerted synergistic stimulatory effects in combination with E2. A polyclonal neutralizing antibody to IL6 completely abolished the reductive EOR-stimulating activity of CM. These results indicate that breast stromal fibroblasts may have a paracrine role in regulation of breast-cancer-tissue levels of E2, and that this effect is mediated by IL6 or a closely related peptide.

Breast↗
PubMed1991

Assay of pyrogens by interleukin-6 release from monocytic cell lines.

A novel in-vitro system has been developed for the detection and quantification of pyrogen in pharmaceutical products. The measured variable was evoked secretion of the pyrogenic cytokine interleukin-6 from MONO MAC 6 monocytic cells incubated with the product. The interleukin-6 was detected using a specific and sensitive ELISA developed for this purpose. The test system detected pyrogenic contamination in 3 batches of therapeutic human serum albumin which had caused adverse reactions in recipients. The contamination was not detected in conventional tests: the rabbit pyrogen test and the limulus amoebocyte lysate test.

Animals↗
PubMed1990

The relationship between the circulating concentrations of interleukin 6 (IL-6), tumor necrosis factor (TNF) and the acute phase response to elective surgery and accidental injury.

The possible role of Interleukin 6 (IL-6) and tumour necrosis factor (TNF) in the regulation of the acute phase response to injury was studied in thirteen subjects undergoing elective surgery or suffering from multiple accidental injuries. The temporal changes in the circulating concentrations of these cytokines were related to the circulating acute phase protein concentrations (C-reactive protein and alpha 1 antichymotrypsin), white cell count, blood loss and duration of surgery. Circulating acute phase protein concentrations rose in all subjects during a thirty hour period following injury but none of the subjects showed a detectable rise in circulating concentrations of TNF. Peak circulating concentrations of IL-6 were detected between 4-6 hours after surgery/trauma but these showed a poor correlation with blood loss, fever, white cell count or duration of surgery. The peak concentrations of IL-6 typically occurred before the rise in circulating acute phase protein concentration. The data do not suggest a role for circulating TNF in the regulation of the acute phase response to injury. In contrast, IL-6 appears to be variably involved in the acute phase response but its precise role in mediating fever, leucocytosis and synthesis of acute phase proteins is as yet uncertain.

Acute-Phase Proteins↗