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R G Richards

Publications and source records attributed to R G Richards.

At least 19 recordsLinked to original sources
PubMed1998

Apolipoprotein A-I stimulates human placental lactogen release by activation of MAP kinase.

Apolipoprotein A-I (apo A-I) stimulates human placental lactogen (hPL) release via protein kinase C (PKC)-dependent pathways. Since PKC has been shown to activate the MAP kinase cascade in other cell types, we examined the effect of two inhibitors of the MAP kinase cascade on apo A-I-induced hPL secretion and the effect of apo A-I on MAP kinase activity in human trophoblast cells. Apigenin (10 microM) and PD98059 (100 microM) inhibited apo A-I-induced hPL release by 94 and 73%, respectively. Moreover, apo A-I activated MAP kinase in a time- and dose-dependent manner. Activation of PKC by phorbol myristate acetate (PMA) stimulated MAP kinase activity, and down-regulation of PKC completely prevented apo A-I-stimulation of MAP kinase activity. Taken together, these results strongly suggest that activation of MAP kinase is involved in the intracellular mechanism of apo A-I-induced hPL release.

Apolipoprotein A-I↗
PubMed1998

Insulin-like growth factor-1 (IGF-1) receptor-insulin receptor substrate complexes in the uterus. Altered signaling response to estradiol in the IGF-1(m/m) mouse.

Some of the actions of estradiol occur through stimulation of growth factor pathways in target organs. Tyrosine-phosphorylated (Tyr(P)) insulin-like growth factor-1 receptor (IGF-1R) and the insulin receptor substrate (IRS)-1 are found in the uterus of mice treated with estradiol. Immunoprecipitates of uterine Tyr(P) IRS-1 contained both p85, the regulatory subunit of phosphatidylinositol (PI) 3-kinase, and PI 3-kinase catalytic activity. Estradiol also stimulated binding of IRS-1 and PI 3-kinase to the IGF-1R. Depletion of IRS-1 from uterine extracts reduced PI 3-kinase associated with the receptor, which suggests that binding of the enzyme to IGF-1R occurs primarily in a complex that also contains IRS-1. Following treatment with estradiol, formation of Tyr(P) IGF-1R, Tyr(P) IRS-1, and the p85.IRS-1 complex was very weak in the uterus of IGF-1(m/m) mice, which are severely deficient in IGF-1. This indicated that most, if not all, of the estradiol-stimulated Tyr phosphorylation of uterine IRS-1 originates from ligand activation of IGF-1R kinase. IRS-2 was also Tyr-phosphorylated in the normal uterus and bound more IGF-1R and p85 in response to estradiol; however, a marked decrease in levels of uterine IRS-2 occurred 12-24 h after treatment with estradiol. Since IRS-2 was present in IGF-1R precipitates and a recombinant form of IGF-1 (long R3 IGF-1) stimulated formation of Tyr(P) IRS-2, hormonal activation of this docking protein probably occurs through the IGF-1R. In summary, our findings show that estrogen activation of uterine IGF-1R kinase results in enhanced binding of p85 (PI 3-kinase) to IRS-1 and IRS-2. The formation of one or both of these complexes may be important for the potent mitogenic action of this steroid. That estradiol stimulated a decrease of IRS-2, but not of IRS-1, suggests that these docking proteins have different roles in hormone-induced signaling in the uterus.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Activation and function of the epidermal growth factor receptor and erbB-2 during mammary gland morphogenesis.

The hormonal stimulation of mammary gland morphogenesis is believed to occur through growth factor receptor signaling pathways. To determine the importance of the epidermal growth factor receptor (EGFR) pathway, we examined extracts of inguinal mammary glands from prepubertal and pubertal mice for tyrosine-phosphorylated EGFR and other erbB receptors. Tyrosine phosphorylation of both EGFR and erbB-2 was detected in normal female BALB/c mice at 5-6 weeks of age, but not during the prepubertal stage, e.g., 24 days of age. Treatment of mice with estradiol or epidermal growth factor also stimulated the formation of mammary EGFR/erbB-2 phosphotyrosine. Waved-2 mice, which have impaired EGFR kinase activity, exhibited less mammary development than did wild-type (wt) mice when both were evaluated at 36 days of age. Because EGFR knockout (KO) mice die shortly after birth, glands from the newborns were implanted under the renal capsules of female nude mice. Under these conditions, extensive ductal growth was observed in mammary glands from wt animals; in contrast, glands from EGFR KO mice failed to grow beyond rudimentary structures. Tissue recombinants revealed that the wt fat pad supported the morphogenesis of EGFR KO epithelium, whereas the EGFR KO fat pad did not. Taken together, these data suggest that EGFR is essential for morphogenesis of the mammary ducts and functions during this period of mammary development as a heterodimer with erbB-2 in the mammary stroma.

Animals↗
PubMed1997

Community immunization programme in response to an outbreak of invasive Neisseria meningitidis serogroup C infection in the Trent region of England 1995-1996.

Between 8 December 1995 and 16 January 1996 seven laboratory confirmed cases of septicaemia owing to infection with Neisseria meningitidis serogroup C strains and one highly probable case of meningococcal septicaemia occurred in three electoral wards in south Rotherham and the Retford area of north Nottinghamshire. All cases occurred among children aged 1-17 years. One patient died. The public health response to this outbreak was the largest community prophylactic antibiotic and immunization programme against meningococcal infection, to date, in the United Kingdom. The target group for each Health Authority was 8900 for Rotherham Health Authorities and 8000 for North Nottinghamshire Health. Local logistical factors led to differences in the implementation of the programme by each Health Authority. At the completion of each programme, 8320 doses of vaccine had been administered (92.5 per cent coverage) during the Rotherham Health Authorities programme and 7660 (95.7 per cent coverage) during the North Nottinghamshire Health programme. The additional financial cost of the exercise amounted to approximately Pounds 125000 for each Health Authority. This paper describes the evolution of the outbreak, the decision-making process resulting in the immunization programme in each Health Authority, the implementation of each programme, problems identified and lessons learned.

Adolescent↗
PubMed1997

Effects of acute endotoxemia on serum somatotropin and insulin-like growth factor I concentrations in prepubertal gilts.

OBJECTIVE: To evaluate effects of endotoxemia on serum somatotropin (ST) and insulin-like growth factor I (IGF-I) concentrations in finishing pigs. ANIMALS: Eight female pigs (98 +/- 2 kg) randomly assigned to IV administration (time 0) of saline solution (n = 4) or Escherichia coli lipopolysaccharide (LPS; 5 micrograms/kg of body weight; n = 4). PROCEDURE: Serum ST concentration was determined in serum samples obtained at 20-minute intervals for 6 hours after treatment. Serum IGF-I concentration was determined in samples collected at 1-hour intervals for 6 hours and at 12, 15, 18, 24, 48, 72, and 96 hours after treatment. RESULTS: One distinct pulse of ST (peak 10.5 +/- 0.5 ng/ml) was observed at 40 minutes in each pig after administration of LPS. Control pigs had 2.25 +/- 0.48 ST pulses during the 6 hours of frequent sample collection; however, magnitude of the ST pulses was similar between gilts given LPS and control gilts. A temporal association between ST pulses and saline administration was not evident. Serum IGF-I concentration was similar between gilts of the LPS and control groups prior to treatment. The IGF-I concentration was similar between gilts of the LPS and control groups prior to treatment. The IGF-I concentration was lower (P < 0.01) in gilts of the LPS group (44 +/- 5 ng/ml) than in gilts of the control group (157 +/- 4 ng/ml) at 24 hours. The difference in IGF-I concentrations between groups was evident for 96 hours. CONCLUSIONS: Immediate release of ST was attributed to stress associated with acute endotoxemia and stimulation of the pituitary gland; immune stimulation by LPS may have contributed to the changes in IGF-I concentration. Because feed consumption was similar between the 2 groups of pigs, suppression of IGF-I concentration for 96 hours after administration of LPS was attributable to factors in addition to transient feed restriction. Thus, acute endotoxemia altered the positive association between ST and IGF-I, and provided evidence for a potential mechanism of impaired growth in endotoxemic animals.

Animals↗
PubMed1996

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.

Animals↗
PubMed1996

Human placental tissue expresses a novel 22.7 kDa apolipoprotein A-I-like protein.

Since apolipoprotein A-I (apo A-I) and HDL stimulate the expression of the placental hormone human placental lactogen (hPL), experiments were performed to determine whether the human placenta synthesizes apo A-I. Western blot analysis of a partially purified extract of human term placenta with an antiserum to human apo A-I yielded an immunoreactive band with an apparent mass of approximately 23.5 kDa, which is smaller than human plasma apo A-I (28 kDa). HPLC chromatography of the partially purified placental extract on a preparative reverse-phase C-18 column yielded two fractions that reacted to the apo A-I antiserum. The mass of both fractions by mass spectral analysis was 22 721 daltons, and N-terminal amino acid sequences were identical to the first four amino acids of apo A-I (Asp, Glu, Pro, Pro). The apo A-I-like protein was not a proteolytic product of apo A-I since Northern analysis of placental RNA with a 641 bp apo A-I cDNA fragment encoding most of the 5' region of the apo A-I mRNA detected a single band of 850 nt, which is smaller than the size of apo A-I mRNA (1100 nt). Placental mRNA, however, did not hybridize with a 3' apo A-I riboprobe, indicating that the 3' region of the apo A-I-like mRNA is different from that of apo A-I mRNA. Differences in the mRNAs were confirmed by S1 nuclease analysis of placental RNA with a cDNA probe that included the 3' end of the apo A-I cDNA and by RT-PCR analysis with a series of oligonucleotide primers that span the entire cDNA for apo A-I. Since there is only a single apo A-I gene in the human genome, these findings strongly suggest that human placental tissue expresses a novel 22.7 kDa apo A-I-like protein (ALP) that results from alternative splicing of the apo A-I primary transcript.

Apolipoprotein A-I↗
PubMed1996

The effect of surface roughness on fibroblast adhesion in vitro.

Adhesion of tissue cells to biomaterial implants is a major factor of their biocompatibility. Quantitative or qualitative adhesion measurements would therefore be useful in the screening of new implant materials. Results from a quantitative method of measuring the total cell adhesion area of cultured cells is presented. It is postulated that the more compatible the surface, the greater the amount of cell adhesion. Fibroblastic cells were cultured on discs of plastic (Thermanox), commercially pure titanium (ISO 5832/2) or steel (ISO 5832/1), as used in AO fixation plates. The cells were fixed, stained, embedded in resin and their discs removed. Backscattered electron (BSE) imaging in a scanning electron microscope displayed the stained cells within the unstained resin. Imaging at high beam energy allowed visualization of the entire cell. Low beam energy displayed the regions of cell contact with the substrate, i.e. the focal adhesion sites. Images were analyzed with an image analysis and measurement system which allowed the percentage of the cell area involved with adhesion to be calculated. Results show that the material roughness, with the materials and cells tested, does not affect the total amount of cell adhesion. Implant surface design to encourage cell adhesion and discourage bacterial adhesion is discussed.

Analysis of Variance↗
PubMed1996

Microwave-enhanced fixation of rabbit articular cartilage.

Cartilage, being highly aqueous, is difficult to preserve for electron microscopy without artefacts. Microwave-enhanced fixation is suggested as a standard method for block samples of this material, with dimensions of up to 12 x 7 x 3 mm. Cartilage samples from the tibial plateau of adult rabbits were fixed by conventional, cryo- or microwave-enhanced fixation. Constant or cyclical microwave irradiation of samples, immersed in fixatives, was carried out to varying final solution temperatures. Microwave-enhanced fixation and staining is shown to be both rapid and reproducible, giving fine structural preservation. Below 323 K microwave fixation always gave excellent preservation of the fine structure within seconds. At higher temperatures thermal artefacts were introduced. In this study the microwave-enhanced fixation is equal in quality to the test conventional immersion fixation and is nearly as fast as cryo-preservation. It provides a standardized, reproducible fixation for morphological studies on cartilage with good process control.

Animals↗
PubMed1996

Human dermal fibroblast cells express prolactin in vitro.

This study demonstrates the synthesis and release of prolactin (PRL) from dermal fibroblasts (>98%) in vitro, suggesting a potential local source of PRL in skin. PRL release was first detected in confluent cultures (0.25 x 10(6) plated cells) on or before day 18 and increased to a maximal level of 2 ng/72 h by day 30. Medroxyprogesterone acetate and estradiol (E2) had no effect on PRL release, but prostaglandin E2 (PGE2) reduced the time required for PRL induction to 6-9 days. The steroids and PGE2 together were synergistic, reaching maximal values of approximately 10 ng/72 h after 2 or more weeks of treatment. Dibutyryl-cyclic AMP, a second messenger in prostaglandin signal transduction, was also synergistic with medroxyprogesterone acetate and E2, but induced significant PRL expression in the absence of the steroids (28 and 12 ng/72 h, respectively). The increase in PRL release was not a result of increased cell proliferation, because the PRL-secreting cultures had 32.2 +/- 8.8% less DNA (N = 3 individuals, 93% confidence limit) than control cultures after 3 weeks of treatment with dibutyryl-cyclic AMP, medroxyprogesterone acetate, and E2. Dermal fibroblast PRL was immunologically and electrophoretically identical to decidual and pituitary PR-Ls, and Northern blot analysis demonstrated a PRL mRNA size of 1.15 kb. Maximal PRL release from fibroblast cells was 32.0 +/- 6.1 ng/72 h (mean +/- SD at 95% confidence limit) for a donor population representing both males (n = 15) and females (n = 7) between the ages of 20-week gestation to 52 years. In contrast to term decidual fibroblast cells that also express PRL, dermal fibroblasts did not co-express insulin-like growth factor-binding protein-1.

Blotting, Western↗
PubMed1996

A novel method for viewing heavy metal stained and embedded biological tissue by field emission scanning electron microscopy.

Backscattered electron (BSE) imaging was used to display heavy metal stained biological structures of various embedded specimens. Samples were fixed, stained and embedded in resin blocks as with preparation for the transmission electron microscope (TEM). Blocks were trimmed to center the specimens in a trapezoidal face of up to 5 mm2 and their sides painted with conductive silver paint leaving the face uncovered. Blocks were sputter coated with 6-8 nm of silver, chromium or aluminum, with aluminum providing the best specimen contrast in BSE mode. Samples were examined in a field emission scanning electron microscope operated at a high emission current of 50 microA. Both the fixation protocol and microscope operating parameters were optimized to maximize the number of BSE available from the smallest probe. An accelerating voltage of 10 keV was found optimal for resolution and contrast. The technique allowed the direct visualization of embedded samples at resolutions beyond light microscopy with good contrast, without cutting sections, and avoiding grid bars obscuring areas of interest. The two dimensional images provided averaged information on the internal structures of the specimens in relation to the predicted emission depth of the BSE. The technique could be used for rapid diagnostics in pathological examinations, or for routine preselection of areas of interest within a sample face before final trimming for ultrathin sectioning for higher resolution TEM study.

Animals↗
PubMed1995

Laminin decreases PRL and IGFBP-1 expression during in vitro decidualization of human endometrial stromal cells.

The expression of laminin, a major constituent of endometrial cell basement membranes, is increased during differentiation of human endometrial stromal cells (decidualization). To determine whether laminin plays a role in decidualization, we studied the effects of laminin substrate on the synthesis and release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two major secretory proteins of decidualized stromal cells. Endometrial stromal cells were plated on laminin as well as several other extracellular matrix (ECM) proteins (types 1 and IV collagen or fibronectin) and on plastic, and cultured in media containing medroxyprogesterone acetate (MPA) and estradiol. Cells cultured on plastic or ECM proteins displayed similar morphological changes indicative of decidualization. However, the release of PRL and IGFBP-1 from cells cultured on plastic and ECM proteins (types 1 and IV collagen and fibronection) was approximately 2.1-fold and 2.8-fold greater respectively, than from cells cultured on laminin. The decrease in PRL and IGFBP-1 expression in cells cultured on laminin was not due to differences in initial cell attachment efficiency or final DNA content. In addition, laminin had no effect on the content of laminin protein or fibronectin mRNA levels, indicating that the effects of laminin on PRL and IGFBP-1 were specific. PGE2 stimulated the release of PRL and IGFBP-1 from cells cultured on laminin to levels comparable to those from cells cultured on plastic or other ECM proteins. This indicates that the decrease in PRL and IGFBP-1 release by laminin was not due to a generalized unresponsiveness. In contrast to the effects of laminin during decidualization, PRL expression was not altered by laminin in terminally differentiated decidual cells isolated at term. Our results support a role for laminin in selectively regulating PRL and IGFBP-1 gene expression during in vitro decidualization of human endometrial stromal cells.

Carrier Proteins↗
PubMed1995

Microjet impingement followed by scanning electron microscopy as a qualitative technique to compare cellular adhesion to various biomaterials.

Adhesion of cells to biomaterial surfaces is one of the major factors which mediates their biocompatibility. Quantitative or qualitative cell adhesion measurements would be useful for screening new implant materials. Microjet impingement has been evaluated by scanning electron microscopy, to determine to what extent it measures cell adhesion. The shear forces of the impingement, on the materials tested here, are seen to be greater than the cohesive strength of the cells in the impinged area, causing their rupture. The cell bodies are removed during impingement, leaving the sites of adhesion and other cellular material behind. Thus the method is shown not to provide quantification of cell adhesion forces for the metals and culture plastic tested. It is suggested that with highly adherent biomaterials, the distribution and patterns of these adhesion sites could be used for qualitative comparisons for screening of implant surfaces.

3T3 Cells↗
PubMed1995

Fibroblast cells from term human decidua closely resemble endometrial stromal cells: induction of prolactin and insulin-like growth factor binding protein-1 expression.

Term decidual tissue consists of terminally differentiated decidual cells, bone marrow-derived cells, and fibroblast cells. Since undifferentiated decidual cells are fibroblast-like cells of the endometrial stroma, the possibility exists that the fibroblast cells in term decidua are undifferentiated decidual cells. To test this hypothesis, a purified population of fibroblast cells was isolated from term decidua and treated under conditions that are known to induce differentiation of endometrial stromal cells. By flow cytometry and immunocytochemistry, the fibroblast cells from term decidua were shown to be free of cells expressing bone marrow-derived cell-surface antigens and the epithelial cell marker cytokeratin. In addition, they tested positive for the cytoskeletal protein vimentin, thus establishing that they were mesenchymal cells. As with endometrial stromal cells, continuous treatment of the decidual fibroblast cells with the progesterone analog medroxyprogesterone acetate and estradiol in combination with either dibutyryl-cAMP or prostaglandin E2 induced cell aggregation and the expression of prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1). When cells were plated at an initial cell density of 0.25 x 10(6) cells/well in a 24-well culture dish with medium changes every three days, PRL was first detected on Days 4-6, and the peak of averaged 24 h-PRL release (30 ng/well) occurred on Days 26-28. The mRNA for decidual fibroblast PRL followed a temporal pattern corresponding to that of the released hormone. The size of the PRL mRNA was 1.15 kb, corresponding to the alternately spliced PRL mRNA reported for decidualized endometrial stromal cells and other extrapituitary sources of PRL.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Surface↗
PubMed1995

Backscattered electron imaging of the undersurface of resin-embedded cells by field-emission scanning electron microscopy.

In this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c 3T3 fibroblasts were cultured on either 13-mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field-emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 microm, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values to give 'sections' of information from increasing depths within the sample. At 3-4 keV only structures a very short distance into the material were observed, essentially the areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metals were intensely bound became more evident. The technique allowed stepwise 'sectional' information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light-microscope-based system.

3T3 Cells↗
PubMed1994

Lipopolysaccharide-induced increases in porcine serum cortisol and progesterone concentrations are not mediated solely by prostaglandin F2 alpha.

The increase in steroid hormone blood levels in response to bacterial lipopolysaccharide (LPS) appears to be an important mechanism by which mammalian species regulate inflammation. This study examined changes in serum concentrations of cortisol, progesterone, and 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) in diestrous pigs following the intravenous injection of LPS and determined whether indomethacin would attenuate these changes. Serum cortisol and progesterone concentrations increased (P < 0.05) within 30 min after the administration of LPS, and the increases in steroid hormones were accompanied by a sharp, transient increase (P < 0.05) in PGFM levels. In the presence of indomethacin, serum PGFM levels did not change (P > 0.05); however, LPS enhanced (P < 0.05) cortisol and progesterone concentrations, although the increases were delayed. Serum concentrations of cortisol acutely increased (P < 0.05) immediately following both infusions of indomethacin. In summary, cortisol and progesterone concentrations increased irrespective of serum PGFM concentrations, thereby indicating that prostaglandin F2 alpha was not the sole mediator of LPS-induced changes in cortisol and progesterone concentrations.

Animals↗