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Biomedical subjects

R S Gilmour

Publications and source records attributed to R S Gilmour.

At least 19 recordsLinked to original sources
PubMed1996

Inositol lipid cycle and autonomous nuclear signalling.

The involvement of phospholipids and in particular polyphosphoinositides in cellular signalling has been documented in detail in the last 20 years. In addition to the plasma membrane localization also the nucleus is shown to be a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. Previous observation have established that the nucleus possesses a specific PLC for inositol lipids, i.e., the PLC beta 1 isoform, which undergoes rapid and transient activation after IGF-I stimulation of quiescent Swiss 3T3 cells and is down-regulated after treatment of Friend erythroleukemia cells with DMSO. Here we have reviewed: (i) the potential of nuclear PLC beta 1 to be a target for anti-cancer drug, (ii) the capability of this PLC isoform, when activated by IGF-I, to be a key signalling molecule in the onset of DNA synthesis, via DAG generation and PKC alpha translocation to the nucleus, (iii) the chromosome mapping of PLC beta 1 gene. The differentiation program of Friend cells can be activated by other agents besides DMSO including tiazofurin, an anti-tumor drug, also capable of affecting the nuclear inositol lipid cycle. Tiazofurin induces a lowering of the activity of PLC beta 1 due to down regulation of this isoform as revealed by both Western blotting and Northern blotting analyses. Using Swiss 3T3 cells stably transformed with an antisense PLC beta 1 construct, the knock-out of the PLC beta 1 gene induces both a loss of PLC beta 1 expression, as determined by Western blots, and a loss of the mitogenic responsiveness to IGF-I. These events show a direct relationship between nuclear PLC beta 1 evoked signals and IGF-I induced cell proliferation. Finally, the assignment of the PLC beta 1 gene to the band q35-36 of rat chromosome 3 paves the way for further genetic studies given the fact that the region where PLC beta 1 gene maps is a hot spot for genetic alterations in a number of experimentally induced rat tumors. Taken as a whole, these results assign a key role to the regulation of nuclear PLC activity and expression both in growth-factor activated mitogenesis and in in vitro erythroid differentiation.

Antineoplastic Agents↗
PubMed1996

Expression and immunohistochemical localization of eight phospholipase C isoforms in adult male mouse cerebellar cortex.

By means of specific polyclonal or monoclonal antibodies we have investigated the expression and the localization of phospholipase C isoforms in the adult mice cerebellar cortex. Western-blot analysis revealed that mouse cerebellum expressed eight phospholipase C isozymes: -beta 1, -beta 2, -beta 3, -beta 4, -gamma 1, -gamma 2, -delta 1, -delta 2. Immunohistochemical analysis carried out on cryosections showed a distinct pattern of expression for each of the isoforms. Purkinje cells had high levels of -beta 1, -beta 3, -gamma 2 and -delta 2 isotypes. The -gamma 2 isozyme was the only one that was identified also in the dendrites of Purkinje cells. In the molecular layer we detected mostly -beta 1 and -gamma 1 isozymes whereas in the granular layer -gamma 1 and -gamma 2 isoforms prodominated. These results indicate a heterogeneity of the phospholipase C isoforms expressed in the layers of mouse cerebellar cortex conceivably due to the fact that these enzymes are coupled to different receptors and perform selective tasks in regulating cell signalling events taking place in the cerebellar cortex of mice.

Amino Acid Sequence↗
PubMed1996

Intravenous infusion of insulin-like growth factor I in fetal sheep reduces hepatic IGF-I and IGF-II mRNAs.

Liver contains the highest concentrations of insulin-like growth factor (IGF) I mRNA in adult rats and sheep and is a major source of circulating IGF-I. In rats, inhibition of hepatic IGF-I production by exogenous IGF-I has been reported. In fetal sheep, skeletal muscle and liver are major sites of IGF-I synthesis and potential sources of circulating IGF-I. To determine whether feedback inhibition of IGF gene expression in fetal liver or muscle by IGF-I occurs, IGF-I and IGF-II mRNAs were measured in these tissues after intravenous infusion of recombinant human IGF-I into fetal sheep. Infusion of IGF-I (26 +/- 4 micrograms.h-1.kg-1; n = 6) or saline (n = 6) commenced on day 120 of pregnancy (term = 150 days) and continued for 10 days. Plasma concentrations of IGF-I were threefold higher in infused fetuses at 130 days of gestation (P < 0.0003), whereas those of IGF-II were unchanged. IGF-I infusion reduced the relative abundance of IGF-I mRNA (P < 0.0002) and IGF-II mRNA (P < 0.01) in fetal liver by approximately 50% but did not alter IGF-I or IGF-II mRNA in skeletal muscle. These results indicate that IGF-I inhibits the expression of both IGF-I and IGF-II genes in fetal liver and that IGF gene expression in fetal liver and muscle is differentially regulated by IGF-I.

Animals↗
PubMed1996

The ontogeny of hepatic growth hormone receptor and insulin-like growth factor I gene expression in the sheep fetus during late gestation: developmental regulation by cortisol.

The effects of cortisol on hepatic GH receptor and insulin-like growth factor-I (IGF-I) gene expression were investigated in sheep fetuses during late gestation and after experimental manipulation of plasma cortisol levels by fetal adrenalectomy and exogenous infusion of cortisol. Hepatic GH receptor and IGF-I messenger RNA (mRNA) levels increased with increasing gestational age in parallel with the normal rise in fetal cortisol levels toward term (145 +/- 2 days). These increases in mRNA abundance toward term were prevented when the prepartum cortisol surge was abolished by fetal adrenalectomy and were stimulated prematurely in fetuses younger than 130 days by exogenous infusion of cortisol. Both the class 1 and class 2 transcripts of the IGF-I gene were increased when cortisol levels were elevated either endogenously or exogenously. However, there were no significant changes in fetal plasma IGF-I levels either with increasing gestational age or in response to experimental manipulation of the fetal cortisol level. When the data from all the fetuses were combined irrespective of treatment or gestational age, there were significant positive correlations between the log plasma cortisol concentration in utero and the abundance of GH receptor and IGF-I mRNA in the fetal liver. There was also a significant inverse relationship between log plasma cortisol and the ratio of class 1 to class 2 transcript abundance in the fetal liver. These findings show that cortisol is a physiological regulator of hepatic GH receptor and IGF-I gene expression in fetal sheep during late gestation and indicate that it preferentially increases the class 2 transcript of the IGF-I gene. The prepartum cortisol surge therefore appears to have an important maturational role in initiating the perinatal switch from the fetal to adult modes of somatotrophic regulation.

Adrenalectomy↗
PubMed1996

The effects of cortisol on the growth rate of the sheep fetus during late gestation.

Using indwelling crown-rump length (CRL)-measuring devices, the growth rate of sheep fetuses was monitored during late gestation and after experimental manipulation of fetal plasma cortisol by exogenous infusion and fetal adrenalectomy. In intact control fetuses, the increment in CRL declined progressively during the last 20-25 days of gestation: mean +/- S.E.M. values fell from 5.5 +/- 0.4 mm/day (n = 12) at 21-25 days before delivery to 2.5 +/- 0.3 mm/day (n = 12) in the last 5 days before birth (P < 0.01). These changes closely parallelled the normal prepartum increase in fetal plasma cortisol which rose from 19.3 +/- 3.3 nmol/l (n = 10) at 21-25 days before birth to 177.4 +/- 19.0 nmol/l (n = 10) in the final 5 days before delivery (P < 0.01). When this cortisol surge was prevented by fetal adrenalectomy, there was no decrease in CRL increment towards normal term: mean CRL increment in the 5 days before normal term (4.8 +/- 0.6 mm/day, n = 5) was similar to that observed at 21-25 days before term (4.7 +/- 0.4 mm/day, n = 5). At delivery at term, the body weight (4.116 +/- 0.280 kg, n = 5) and CRL (51.9 +/- 1.7 cm, n = 5) of the adrenalectomized fetuses were significantly greater than the corresponding values in their sham-operated controls (2.877 +/- 0.070 kg and 47.1 +/- 1.6 cm, n = 6, respectively). In contrast with the sham-operated controls, plasma glucose and insulin levels in the adrenalectomized fetuses decreased towards term. Infusion of cortisol into the preterm fetus for 5 days increased fetal plasma cortisol to term levels and decreased the CRL increment to a value (1.8 +/- 0.5 mm/day, n = 8) which was similar to that observed in untreated controls during the last 5 days before spontaneous delivery at term (2.1 +/- 0.3 mm/day, n = 6). There were no significant alterations in the fetal arterial concentrations of plasma glucose or insulin in response to fetal cortisol infusion. When all the data were combined irrespective of treatment or proximity to delivery, the fetal plasma concentrations of cortisol (P < 0.001) and glucose (P < 0.04), but not insulin (P > 0.05), had a significant effect on the fetal CRL increment measured over 5-day periods during the last 25-30 days of gestation. These findings show that cortisol inhibits growth of the axial skeleton in the sheep fetus during late gestation. They also indicate that the prepartum cortisol surge may be responsible for the normal decline in fetal growth rate observed towards term in this species.

Adrenalectomy↗
PubMed1996

Effects of growth hormone administration and dietary protein intake on insulin-like growth factor I and growth hormone receptor mRNA Expression in porcine liver, skeletal muscle, and adipose tissue.

The effects of growth hormone (GH) and dietary protein on expression of IGF-I and GH receptor (GHR) genes in liver, muscle, and fat of pigs were investigated. Forty-eight intact male Large White x Landrace pigs were allotted to eight treatment groups (four diets with or without GH). The pigs were restriction-fed one of four diets, which differed only in their protein content (9.9, 13.1, 16.2, and 19.4%, as-fed basis), for a total of 3 wk. The pigs were then injected intramuscularly with either porcine GH (50 micrograms.kg-1.d-1 of rpST) or vehicle for the last 7 d. Pigs were slaughtered 4 h after the final injection. Total RNA was extracted from all tissues and then RNase protection assays were performed to measure expression of IGF-I and GHR genes. Expression of IGF-I mRNA was found to be GH responsive in the liver, semitendinosus (ST), and adipose tissue (P < .01) but not in longissimus muscle (LD). Dietary protein increased IGF-I expression only in the adipose tissue (P < .01). Expression of class 2 transcripts of IGF-I were observed only in the livers of GH-treated pigs, with no effect of dietary protein. Expression of GHR mRNA was found to increase with GH administration in liver and skeletal muscle (LD and ST, P < .05) but not in adipose tissue. There were diet x GH interactions on GHR expression in liver, ST, and adipose tissue, resulting in the highest GHR expression being in the high protein-fed, GH-treated group for liver, but in the low protein-fed, GH-treated group for muscle and adipose tissue. This study demonstrates tissue-specific control of expression of the two genes and also tissue-specific promoter usage (IGF-I exon 2 in liver) in response to GH administration.

Adipose Tissue↗
PubMed1995

Phosphoinositide signaling in nuclei of Friend cells: tiazofurin down-regulates phospholipase C beta 1.

Previous investigations have demonstrated the presence of conventional lipid kinases and phospholipase C (PLC) activities in nuclei of Friend erythroleukemia cells. Moreover, when Friend erythroleukemia cells are treated for 96 h with the antitumor drug tiazofurin, the induction of erythroid differentiation is accompanied by changes in amounts of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate due to the inhibition of an uncharacterized nuclear PLC activity. Here, we show that the nuclear PLC beta 1 isoform is down-regulated by tiazofurin (5 microM) treatment of Friend erythroleukemia cells as shown by both Western blot and Northern blot analyses for PLC beta 1 message. This indicates that PLC beta 1 down-regulation is tightly linked with erythroid differentiation of Friend erythroleukemia cells and that the autonomous nuclear signaling via inositol lipid cycle can be controlled by the antitumor drug tiazofurin.

Animals↗
PubMed1995

Nuclear inositol lipid cycle and differentiation.

Previous investigations from our laboratory and others have shown the existence of an autonomous intranuclear inositide cycle endowed with conventional lipid kinases and PLC which in PC12 pheochromocytoma cells, human osteosarcoma SaOS-2 cells, rat liver and Swiss 3T3 cells is the isoform beta 1, which in the latter cells is activated upon IGF-I stimulation. The behavior of the nuclear inositol lipid cycle has been investigated in nuclei of Friend erythroleukemia cells. These nuclei possess both lipid kinases and PLC. The cycle upon treatment with differentiating agents (i.e., DMSO and tiazofurin) is characterized by an accumulation of polyphosphoinositides and a decrease of DAG due to down-regulation of a specific PLC. Indeed, even if both beta 1 and gamma 1 isoforms are present in these nuclei, when Friend cells undergo terminal erythroid differentiation only the PLC beta 1 isoform is down-regulated as shown by immunochemical and immunocytochemical analysis, by direct determination of enzymatic activity and in the presence of neutralizing monoclonal antibodies as well as by Northern blot for PLC beta 1 message, whilst the amount of PLC gamma 1 and its activity are unaffected by erythroid differentiation. In conclusion, the presence of a specific nuclear PLC whose activity and expression are down-regulated during differentiation of erythroleukemia cells points out a role for nuclear phosphoinositide signalling in the processes of cell differentiation and hints at the nuclear PLC beta 1 as an important step of the cycle in relation to the erythroid differentiative commitment of murine erythroleukemia cells.

Animals↗
PubMed1995

Inositides in nuclei of Friend cells: changes of polyphosphoinositide and diacylglycerol levels accompany cell differentiation.

Friend erythroleukemia cells were labelled with high levels of [3H]myo-inositol and the radioactivity in PI, PIP and PIP2, extracted from isolated nuclei, was measured. A parallel analysis employing a picomole sensitive assay for both PIP and DAG has been carried out. The results indicate that the differentiation process is characterised by an accumulation of nuclear PIP and PIP2 and by a decrease of DAG mass. We suggest that as differentiation proceeds toward erythrocytes in Friend cells, this is accompanied by a reduction in the amount of these messengers in the nucleus.

Animals↗
PubMed1995

Localization of messenger ribonucleic acids for insulin-like growth factor I (IGF-I), IGF-II, and the type 1 IGF receptor in the ovine ovary throughout the estrous cycle.

The distribution pattern of messenger RNAs (mRNAs) for insulin-like growth factor I (IGF-I), IGF-II, and the type 1 IGF receptor and the detection of IGF-binding sites in sections of ovine ovary were demonstrated using in situ hybridization and autoradiography. Ovaries were collected from 30 ewes at time points throughout the estrous cycle. Luteal IGF-II mRNA and IGF-binding site concentrations altered significantly during the cycle, peaking on day 8 (midluteal phase; P < 0.01) and day 15 (late luteal phase; P < 0.001), respectively. In contrast, mRNA expression for IGF-I and the type 1 IGF receptor in the corpus luteum was low and did not vary. IGF-binding sites and mRNAs for IGF-II and the type 1 IGF receptor were also present at low and constant concentrations in ovarian stroma. There was no detectable follicular expression of IGF-I mRNA, although there were high concentrations of IGF-II and the type 1 IGF receptor mRNAs, which both varied significantly with follicular size (P < 0.001 and 0.01, respectively), with the highest concentrations in small follicles (< 2 mm in diameter). Follicular IGF-II expression was confined to the theca, whereas the type 1 IGF receptor was present in both theca and granulosa. IGF-binding site concentrations were significantly higher (P < 0.01) in atretic than healthy follicles, but were uninfluenced by follicular size. These results suggest that IGF-II, in contrast to IGF-I, appears to be the most significant IGF in luteal and, particularly, follicular development in the ewe.

Animals↗
PubMed1995

Effect of restriction of placental growth on expression of IGFs in fetal sheep: relationship to fetal growth, circulating IGFs and binding proteins.

To determine whether tissue production of the IGFs is altered when fetal growth is retarded, IGF-I and -II mRNAs were measured in tissues of fetal sheep subjected to placental restriction and the relationships between IGF gene expression, circulating IGF protein and fetal growth were examined. The majority of potential placental attachment sites were surgically removed from the uterus of 12 non-pregnant ewes to restrict placental size in a subsequent pregnancy. Blood and tissues were collected at 121 days of gestation (term = 150) in 12 fetuses with restricted placental size and eight normal fetuses. IGF-I and IGF-II mRNA was detected by solution hybridization/ribonuclease protection assay in placenta and all fetal tissues studied. IGF-I mRNA was most abundant in skeletal muscle and liver and IGF-II mRNA was highest in kidney and lung. Restriction of placental size reduced fetal weight by 17% and reduced the pO2 (18%) and glucose concentration (23%) of fetal blood. Placental restriction also reduced IGF-I mRNA in fetal muscle (P < 0.002), lung (P < 0.05) and kidney (P < 0.01) but had no significant effect on IGF-II mRNA in any tissue. IGF-I mRNA in fetal liver, kidney and skeletal muscle correlated positively with the concentration of IGF-I protein in fetal blood (P < 0.01). There was no relationship between the concentration of IGF-II protein in fetal blood and IGF-II mRNA in any fetal tissue examined. The concentration of IGF-binding protein-3 (IGFBP-3) in fetal arterial blood plasma measured by RIA correlated positively with fetal weight and with plasma IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Hormonal control of insulin-like growth factor-I and growth hormone receptor mRNA expression by porcine hepatocytes in culture.

The effects of various hormones commonly added to hepatocyte culture media upon the expression of the GH receptor (GHR) and insulin-like growth factor-I (IGF-I) genes in cultured porcine hepatocytes were investigated. Preliminary investigations indicated that there was an absolute requirement only for insulin, with high losses of cell viability upon long term exclusion of insulin from the culture medium. The decline in GHR expression with time in culture was found to be less when high levels of glucose were included in the medium. Therefore the basal culture medium used in these studies was Williams' medium E supplemented with 0.2% (w/v) BSA, 5000 mg glucose/l and 100 nmol porcine insulin/l. The addition of dexamethasone (10 nmol/l) increased the expression of both GHR and IGF-I (class 1 transcripts only) mRNA (p < 0.001 and P < 0.05 respectively), and resulted in an increased responsiveness of IGF-I mRNA expression to GH (1 microgram/ml), when the two were added in combination (although only class 1 transcripts were shown to be statistically significant, P < 0.01). The addition of either thyroid hormone (1 nmol/l T3 or T4) alone also increased the expression of GHR mRNA (P < 0.01) in addition to the dexamethasone stimulated expression, with T4 appearing to decrease IGF-I expression slightly (P < 0.05) (either on its own or with T3). As with dexamethasone, the thyroid hormones increased the response of IGF-I mRNA expression to GH (1 microgram/ml) when added in combination with GH (P < 0.001). These observations demonstrate one possible mechanism for the interactions of glucocorticoids and thyroid hormones with the GH-IGF axis.

Animals↗
PubMed1994

Selective nuclear translocation of protein kinase C alpha in Swiss 3T3 cells treated with IGF-I, PDGF and EGF.

To determine the subcellular distribution of PKC after GFs treatment we have employed a combined immunochemical and in situ confocal microscopy analysis. In quiescent Swiss 3T3 cells only a faint PKC positivity was observable in the nucleus while a strong reaction was seen in the cytoplasm. IGF-I and to a lesser extent PDGF and EGF induced, after 45 min of treatment, a nuclear translocation of PKC detected by a pan-anti-PKC antibody and nuclear fluorescence was distributed in the nuclear interior except for the nucleolar regions. Bombesin and FGF did not affect the sub-cellular distribution of the enzyme. To further the understanding of which PKC isoform was involved in the translocation process, we have tested nine isozyme-specific anti-PKC antibodies. Immunoblotting analysis revealed the presence in Swiss 3T3 fibroblasts of alpha, beta I, epsilon and zeta isoforms. In isolated nuclei from GF-exposed cells only the alpha isozyme was detected: immunostaining was very intense after IGF-I treatment and clearly observable after PDGF and EGF stimulation. This result was strongly supported by the in situ confocal microscopy which parallels the Western blot analysis. These data demonstrate that several, but not all, GFs acting through tyrosine kinase receptor induce the intranuclear translocation of PKC alpha and, because of the dramatic effect of IGF-I, strengthen the case for a link between the activation of nuclear inositol lipid cycle and PKC translocation induced by this GF.

3T3 Cells↗
PubMed1994

Phosphoinositide signaling in nuclei of Friend cells: phospholipase C beta down-regulation is related to cell differentiation.

Previous investigations have demonstrated the existence of an autonomous intranuclear inositide cycle endowed with conventional lipid kinases and phospholipase C (PLC) which is the isoform beta in Swiss 3T3 cells, PC12 pheochromocytoma cells, human osteosarcoma SaOS-2 cells, and rat liver. The presence of PLC has been investigated in nuclei of Friend erythroleukemia cells. Both beta and gamma isoforms are present in these nuclei. When Friend cells undergo terminal erythroid differentiation in the presence of dimethyl sulfoxide the PLC beta isoform is down-regulated as shown by immunochemical and immunocytochemical analysis, by determination of enzymatic activity directly and in the presence of neutralizing monoclonal antibodies and also by Northern blot for PLC beta message. By contrast, the amount of PLC gamma and its activity are unaffected by erythroid differentiation. Thus, the presence of a nuclear PLC beta, the activity and expression of which are modulated during differentiation of erythroleukemia cells, implicates a role for nuclear phosphoinositide signaling in the processes of cell determination and indicates the nuclear PLC beta as a key enzyme of the cycle in relation to the erythroid differentiative commitment of murine erythroleukemia cells.

Animals↗
PubMed1994

Partial characterization of a cDNA for human stearoyl-CoA desaturase and changes in its mRNA expression in some normal and malignant tissues.

A segment of 712 bases coding for part of the human stearoyl-CoA desaturase gene was made by polymerase chain reaction (PCR) using primers based on published rat cDNA sequences. The human PCR product was confirmed by DNA sequencing. It was next cloned into a vector from which anti-sense, highly radioactive RNA transcripts were made in vitro using T7 polymerase. The transcripts were used to probe desaturase mRNA in a number of human tumour and control tissues, using a very sensitive solution hybridization/RNase protection assay. Increased desaturase mRNA levels were found in colonic and oesophageal carcinomas and in hepatocellular adenoma; however, no consistent trend was seen in hepatocellular carcinoma. It is suggested that certain classes of tumour may exhibit increased levels of desaturase mRNA.

Amino Acid Sequence↗