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R G Richards

Publications and source records attributed to R G Richards.

At least 19 recordsLinked to original sources
PubMed2002

Simultaneously identifying S-phase labelled cells and immunogold-labelling of vinculin in focal adhesions.

A new combination of autoradiography and immunolabelling techniques is presented that allows the simultaneous identification of both S-phase cells and their focal adhesions using scanning electron microscopy. The technique allows both labels to be discerned visually by their unique shapes and location within and on the cell. S-phase cells were radio-labelled with a pulse of tritiated thymidine, selectively incorporated into synthesizing DNA. The cells were then immunogold-labelled for the focal adhesion protein, vinculin, prepared for autoradiography, and embedded in resin. The resin was then polymerized before removing the substrate, to expose the embedded cell undersurface. Electron-energy 'sectioning' of the sample by varying the accelerating voltage of the electron beam allowed separate S-phase cell identification in one electron-energy 'section' and visualization of immunogold label in another 'section', within the same cell. As a result of applying this technique it was possible to positively identify S-phase cells and immunogold-labelled focal adhesions on the same cell simultaneously, which could be used to quantify focal adhesion sites on different substrates.

Animals↗
PubMed2001

Measurement of fibroblast and bacterial detachment from biomaterials using jet impingement.

Fibroblast and Staphylococcus aureus detachment strength from orthopaedic alloys and a tissue culture plastic (Thermanox) have been investigated with jet impingement. For S. aureus, unlike fibroblasts, detachment is caused more by pressure than shear. For these biomaterials, detachment strength is much higher for S. aureus than fibroblasts. Comparing materials under equivalent flow conditions, S. aureus attach to stainless steel and titanium with equal strength and more strongly than to Thermanox. For fibroblasts, detachment strength from all materials was similar. Fibroblast detachment strength from these biomaterials substantially decreases with time at equal flow rates and increases with flow rate at equal exposure times. Detachment strength is very similar for 3T3 and L929 fibroblasts on Thermanox for equivalent flow rate/time combinations, though enhanced adhesion of 3T3 cells was often noted for metals. Time effects are less evident for S. aureus. S. aureus adhesion to metals is more affected by flow rate than fibroblast adhesion.

Alloys↗
PubMed2001

Immunogold labelling of fibroblast focal adhesion sites visualised in fixed material using scanning electron microscopy, and living, using internal reflection microscopy.

A new immunogold labelling method for the visualisation of vinculin, an integral protein in focal adhesions of cells, is reported. Quantification of vinculin is indicative of substrate cytocompatibility (cytocompatibility is one aspect of biocompatibility; it is the cellular response to a biomaterial). For efficient labelling, most of the cell body above the cell-substrate interface was removed with detergent. The antigen blocking procedure, size of label (5 nm) and duration of silver-enhancement (6 min), for visualisation of the labelled sites on the whole cell by scanning electron microscopy (SEM), were determined. Imaging living cells with interference reflection light microscopy, followed by backscattered electron (BSE) imaging of the same fixed and immunolabelled cells confirmed the results. Collecting low voltage BSE images of embedded cells after the substrate had been removed provided 'sectional' views through the cell. This enabled visualisation of vinculin exclusively within the cell-substrate contact zone; the focal adhesions. The method could be of general use in the imaging of protein distribution at biological tissue/substrate interfaces.

Acrylic Resins↗
PubMed2001

Enhancement of immunogold-labelled focal adhesion sites in fibroblasts cultured on metal substrates: problems and solutions.

Visualisation of cell adhesion patterns by scanning electron microscopy requires special preparation and labelling. The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualise the cell undersurface (embedded in resin after staining with osmium tetroxide) and immunogold-labelled adhesion sites. The gold probe, must be large enough (>40 nm) for detection, while viewing the whole cell, but large gold markers increase steric hindrance and decrease labelling efficiency. This problem can be overcome by using small gold probes (1-5 nm) followed by enlargement with silver enhancement, but osmium tetroxide stain etches the silver. We demonstrated that metal substrates increased this etching. Reducing the concentration of osmium tetroxide and incubation time reduced the amount of etching. We have demonstrated that gold enhancement was not etched by osmium tetroxide, irrespective of the substrate. Therefore, comparative studies of cell adhesion to different biomaterial substrates can be performed using immunogold-labelling with gold enhancement.

Acrylic Resins↗
PubMed2001

E2-induced degradation of uterine insulin receptor substrate-2: requirement for an IGF-I-stimulated, proteasome-dependent pathway.

The insulin receptor substrates are docking proteins that bind various receptor tyrosine kinases and signaling proteins. Previous studies have shown that E2 or progesterone can regulate the relative abundance of insulin receptor substrate-1 and -2 in cells and tissues. For instance, uterine insulin receptor substrate-2 was decreased markedly at 24 h after E2 treatment of mice. In the present study we used various in vivo experimental approaches to examine the mechanism by which E2 influences uterine insulin receptor substrate-2 expression. Uterine insulin receptor substrate-2 mRNA levels were diminished after E2 treatment, but this diminution did not account for the total reduction in insulin receptor substrate-2 protein, suggesting that the E2-induced decrease in insulin receptor substrate-2 is not regulated solely at the mRNA level. Cotreatment with progesterone prevented the E2-stimulated reduction in insulin receptor substrate-2 protein at 24 h after hormone exposure. In addition, MG-132 and epoxomicin, inhibitors of proteasomal protease activity, inhibited the E2-induced decrease in uterine insulin receptor substrate-2 protein levels, and this correlated to an increase in uterine protein ubiquitination. Insulin receptor substrate-2 protein was diminished in uteri of E2-treated insulin receptor substrate-1-null mutant mice, but not in E2-treated IGF-I-null mutant mice. Furthermore, E2-induced diminution of uterine insulin receptor substrate-2 protein was only partially inhibited in the presence of wortmannin, a PI3K inhibitor. Collectively, these data suggest that the E2-induced decrease in uterine insulin receptor substrate-2 requires IGF-I signaling, is not dependent solely on insulin receptor substrate-1 and PI3K, and is blocked by progesterone as well as by pharmacological inhibition of proteasomal protease activity. We speculate that the IGF-I-activated IGF-I receptor, in response to E2, directly or indirectly modifies insulin receptor substrate-2, probably through phosphorylation, leading to ubiquitination and subsequent degradation of this docking protein by the proteasome. This degradation could be a regulatory step to inhibit insulin receptor substrate-2-dependent signaling in the uterus.

Androstadienes↗
PubMed2000

Activator protein-2 regulates human placental lactogen gene expression.

DNase I footprint analysis of the human placental lactogen-A (hPL-A) promoter using nuclear extracts from purified human trophoblast cells and BeWo choriocarcinoma cells revealed five protected regions within the proximal 325 bp. Two of the protected regions, FP4 (-289--267) and FP5 (-167--154), are homologous to regions on the human growth hormone (hGH) promoter that bind transcription factors AP-2 and/or NFI. Competitive gel shift assays and supershift assays indicated that FP4 forms complexes with activator protein-2 (AP-2) and nuclear factor I (NFI), while FP5 forms a complex with AP-2 alone. In transient transfection studies in human trophoblast cells, hPL promoter constructs containing point mutations in the AP-2 binding sites of FP4 and/or FP5 were 60-80% less active than plasmids containing the wild-type promoter. A mutation in the NFI binding site of FP4, however, had little effect on promoter activity in these cells. Overexpression of AP-2 in HepG2 cells co-transfected with the wild-type hPL promoter resulted in a significant increase in promoter activity. Taken together, these findings suggest a critical role for AP-2 in the regulation of hPL gene expression.

Base Sequence↗
PubMed2000

Freeze-substitution of rabbit tibial articular cartilage reveals that radial zone collagen fibres are tubules.

Investigations of the micromorphology of rabbit tibial articular cartilage using scanning and transmission electron microscopy revealed that the collagenous elements in the tissue form fluid-containing tubular structures. The commonly described radial or deep zone longitudinal fibres were found to be tubular structures with internal diameters of 1-2 microm. The walls of the tubules were composed of tightly packed fibrils of collagen. The tangential zone, close to the tibial plateau, was composed mainly of a spongy arrangement of collagen fibrils, containing bunches of tangentially lying small (< 1 microm) diameter tubules. The application of conventional chemical fixation techniques resulted in the fine detail of this tissue being obscured. When the tissue was frozen, followed by cryo-scanning electron microscopy or freeze-drying, prior to observation in the scanning electron microscope the tubule structures were not obviously present. It was only by applying freeze-substitution techniques, followed by critical point drying or resin embedding, that the structure was revealed clearly. Segregation of water into ice crystals did occur during the freezing process, but the formation of those crystals played no part in creating the tubular morphology observed. A similar structure was still revealed following pre-treatment with glycerol, methanol or Triton X-100, provided that concentration of these additives was not too high. The walls of the tubules in the radial region were composed of straight, longitudinally arranged as well as helically arranged, 30 nm diameter fibrils. The lumen of the tubules appears to be lined by a circumferentially arranged array of approximately 10 nm diameter fibres, spaced at regular intervals of 50-70 nm.

Animals↗
PubMed2000

Mitogen-activated protein kinase activates human placental lactogen-B enhancer by an NF-IL6-dependent pathway.

Computer analysis of the human placental lactogen-B (hPL-B) enhancer reveals two putative binding sites for the transcription factor NF-IL6, but the role of NF-IL6 in the regulation of the enhancer is unknown. Using gel mobility shift and supershift assays, we demonstrated that NF-IL6 binds to both enhancer sites. Transient transfection studies indicated that the transcription factor NF-IL6 stimulates hPL-B enhancer activity by 4.4-fold in primary cultures of human trophoblast cells and by 32.0- and 8.4-fold in JAR and BeWo choriocarcinoma cells, respectively. Overexpression of MEK (mitogen-activated protein [MAP] kinase kinase), which is known to stimulate phosphorylation of NF-IL6, induced a 3.6-fold increase in hPL-B enhancer activity. The induction by MEK was completely inhibited by an expression plasmid for a dominant/negative mutant of NF-IL6 or by mutation of the NF-IL6 binding sites on the enhancer. PD98059, an inhibitor of MEK, inhibited hPL release from cultured trophoblast cells by about 50%. Taken together, these results indicate that MAP kinase stimulates the hPL-B enhancer by an NF-IL-6-dependent pathway.

CCAAT-Enhancer-Binding Proteins↗
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

EGF-related peptides and their receptors in mammary gland development.

The discovery of multiple EGF-like ligands and erbB receptors offers the potential for a highly diverse signaling system allowing specific ligand/receptor complexes to be created in response to a certain hormone(s) or stage of mammary development. The known erbB receptors and several of the erbB-related ligands are synthesized by the normal mammary gland and have different temporal and spatial expression patterns. For instance, cumulative findings support the concepts that the EGF receptor has an essential role in morphogenesis of the mammary gland and that activation of this receptor occurs in response to estradiol-stimulated synthesis of an EGF receptor ligand in mammary stromal cells. The importance of both epithelial and stromal mammary cells in the hormonal activation of erbB-related pathways is underscored in this review. Current experimental protocols that utilize erbB mutant mice or enable detection of phosphorylated erbB members and their proximal substrates should permit more precise identification of the pathways operative in the mammary gland.

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↗