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Biomedical subjects

L J Murphy

Publications and source records attributed to L J Murphy.

At least 109 records · Page 6Linked to original sources
PubMed1991

The effect of GABA on serum and hepatic polyamine concentrations after partial hepatectomy in rats.

Serum and hepatic polyamine concentrations including putrescine, spermidine and spermine were documented at various time intervals after partial hepatectomy in rats treated with GABA (500 micrograms/gm body wt) or isotonic saline. Aside from a transient decrease in spermidine levels, GABA treatment had no effect on serum polyamine concentrations. In the liver, however, GABA treatment markedly attenuated the increase in hepatic putrescine concentrations that occurs after partial hepatectomy such that levels were 64%, 74% and 100% lower than in saline-treated controls on days 1, 2 and 3 after partial hepatectomy (p less than 0.005, p less than 0.01 and p less than 0.05, respectively). Spermidine and spermine concentrations in the liver were not affected by GABA treatment. To determine the mechanism whereby GABA lowers putrescine concentrations in regenerating liver, ornithine decarboxylase messenger RNA and enzyme activity were documented after GABA treatment. Although ornithine decarboxylase messenger RNA levels were similar, ornithine decarboxylase enzyme activity was significantly inhibited 12 hr after partial hepatectomy in GABA-treated rats compared with saline-treated controls. The results of this study indicate that GABA inhibits hepatic putrescine synthesis at a posttranscriptional level in rats after partial hepatectomy. These results could help to explain the impairment in hepatic regenerative activity that occurs in patients with elevated serum GABA concentrations and fulminant hepatic failure.

Animals↗
PubMed1991

Estrogen induction of insulin-like growth factors and myc proto-oncogene expression in the uterus.

In contrast to the effects observed in vivo, isolated uterine cells cultured in vitro demonstrate little proliferative response to estrogens. Estrogen induced uterine proliferation involves a carefully orchestrated, sequential activation of genes which encode a variety of biologically active molecules. These include nuclear transcription factors, growth factors and growth factor receptors. Expression of these proteins serve to amplify the effect of estrogen through cellular, autocrine and paracrine mechanisms. In this review the effects of estrogen on uterine expression of the myc family of oncogenes and the insulin-like growth factors are discussed.

Animals↗
PubMed1991

Enhanced expression of insulin-like growth factor-binding protein-I in the fasted rat: the effects of insulin and growth hormone administration.

The effect of fasting on insulin-like growth factor-binding protein-1 (IGFBP-1) expression was examined in the rat. Food deprivation for a period of 24 h resulted in a 9.5 +/- 2.0-fold increase in hepatic IGFBP-1 mRNA abundance (P less than 0.001). An increase in circulating IGFBP-1 in sera from fasted rats was demonstrated by immunoblotting, and an increased abundance of a 30-kDa IGFBP in sera from fasted rats was apparent when [125I]IGF-I was used in ligand blotting experiments. Refeeding resulted in a prompt decline in hepatic IGFBP-1 mRNA. Administration of insulin (0.5-4 U, ip) to fasted rats resulted in profound hypoglycemia, but either increased or had no significant effect on hepatic IGFBP-1 mRNA abundance. In contrast, administration of human GH (hGH; 100 micrograms, ip) resulted in a prompt decline in hepatic IGFBP-1 mRNA, followed by a late rebound in IGFBP-1 mRNA to levels greater than those in fasted controls. Furthermore, hepatic IGFBP-1 mRNA levels were significantly lower in hGH-treated (100 micrograms every 8 h) food-deprived rats than in saline-treated food-deprived rats (2.25 +/- 1.55- vs. 8.99 +/- 3.80-fold increase; P less than 0.005). Similar changes were observed when serum IGFBP-1 was quantitated by immunoblotting. The effects of GH could not be explained by secondary hyperinsulinism, since no significant increase in insulin levels was observed in GH-treated rats. From these observations we conclude the enhanced expression of IGFBP-1 in the food-deprived rat may be a consequence of GH deficiency rather than insulin deficiency.

Animals↗
PubMed1991

Multihormonal regulation of insulin-like growth factor-binding protein-1 in rat H4IIE hepatoma cells: the dominant role of insulin.

Circulating levels of insulin-like growth factor-binding protein-1 (IGFBP-1) and the abundance of hepatic IGFBP-1 mRNA are increased in streptozotocin-diabetic rats and are regulated in accordance with insulin and metabolic status. We recently purified rat IGFBP-1 from medium conditioned by well differentiated rat H4IIE hepatoma cells. Since this cell line provides a useful model for examining the effects of hormones on hepatocellular function, we used H4IIE cells to examine the relative role that insulin and other factors may play in the regulation of IGFBP-1 production. H4IIE cells were stabilized in serum-free medium, then treated with specific hormones. The availability of IGFBPs in conditioned medium was estimated by [125I]IGF-I binding assay, and specific BPs were assessed by Western ligand and immunoblot analyses. The abundance of IGFBP-1 mRNA was determined by Northern and slot blot analysis. Initial studies revealed that [125I]IGF-I-binding activity in conditioned medium was reduced after 24-h incubation with 100 nM insulin (52 +/- 4% of control; P less than 0.001). In contrast, binding activity was increased after only 4 h of incubation with 75 microM 8-(4-chlorophenylthio)cAMP (8-CPT-cAMP) or 1 microM dexamethasone (P less than 0.001 vs. control for each), but these effects were prevented by insulin. Ligand and immunoblotting demonstrated that insulin decreased the production of 32K and 34K forms of IGFBP-1, while both 8-CPT-cAMP and dexamethasone increased the production of IGFBP-1; again, insulin prevented the effects of 8-CPT-cAMP and dexamethasone. Of note, 1 microM rat GH, testosterone, progesterone, or 17 beta-estradiol had no effect on either IGF-binding activity or IGFBP-1 production. Northern and slot blot analyses revealed that 100 nM insulin profoundly lowered the abundance of IGFBP-1 mRNA in H4IIE cells (4 +/- 0.6% of control at 4 h; P less than 0.001), while IGFBP-1 mRNA was increased 2-fold during incubation with 75 microM 8-CPT-cAMP (P less than 0.001) and 9-fold with 1 microM dexamethasone (P less than 0.001). Once again, the effect of insulin was dominant; insulin both prevented and reversed the effects of maximally effective concentrations of 8-CPT-cAMP and dexamethasone. To determine whether this effect of insulin reflected altered generation or stability of IGFBP-1 mRNA, H4IIE cells were incubated with 2.5 micrograms/ml actinomycin-D with or without insulin, and mRNA was quantitated by Northern blot.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Maternal insulin-like growth factor-binding protein messenger ribonucleic acid during rat pregnancy.

Pregnancy is associated with rapid growth of maternal and fetal tissues. Insulin-like growth factors (IGF-I and -II) have roles in mediating both fetal and placental growth. In this study serum IGFs and IGF-binding proteins (IGFBPs) were characterized, IGFBP protease activity was quantified, and hepatic IGFBP-1, -2, -3, and -4 mRNA were investigated throughout rat pregnancy. IGF-I in maternal serum was elevated (P less than or equal to 0.001) on days 5 and 10 (d5 and d10) of gestation compared to levels in nonpregnant controls (NP), but was significantly decreased below NP levels (P less than or equal to 0.001) after d10 of pregnancy. Serum IGF-II levels were unaffected by pregnancy. Using Western ligand blotting (WLB), six IGFBP bands were visualized in NP, d5, and d10 pregnancy rat sera. At 15 and 20 days gestation, the IGFBP-3 bands were no longer detectable by WLB. Using an in vitro IGFBP protease assay, sera from rats at 15 and 20 days gestation proteolyzed 63 +/- 4% and 81 +/- 5% of recombinant human IGFBP-3, respectively. Regression analyses demonstrated that serum IGF-I was positively correlated with serum IGFBP-3 (r2 = 0.73; P = 0.001), whereas serum IGFBP-3 (r2 = -0.85; P = 0.001) and serum IGF-I (r2 = -0.78; P = 0.001) were negatively correlated with serum protease activity. In addition, no change was observed in liver IGFBP-3 mRNA during pregnancy, further suggesting that protease activity is primarily responsible for the decrease in serum IGFBP-3. However, IGFBP-1 and -4 mRNA levels were increased 3- to 11-fold after d5 of gestation. The hormonal and/or metabolic regulators of hepatic IGFBP-1 and -4 expression during rat pregnancy remain to be determined.

Animals↗
PubMed1991

Effect of insulin and statil on aldose reductase expression in diabetic rats.

Tissue accumulation of sorbitol secondary to enhanced polyol-pathway activity is believed to play an important role in the development of diabetic complications. We previously demonstrated sorbitol accumulation, due in part to enhanced expression of aldose reductase (AR) in the diabetic kidney. In this study, we quantitated AR enzyme activity, immunoreactivity, and mRNA in various tissues from nondiabetic and diabetic BB/Wor rats 3 mo after onset of diabetes. In addition, the effects of intensive insulin treatment (3-6 U/day) and the effects of the AR inhibitor Statil (25 mg.kg-1.day-1) on AR expression were determined. Of 13 tissues examined, AR activity was significantly increased in the lens, kidney, sciatic nerve, skeletal muscle, retina, and spinal cord from diabetic rats compared with age-matched nondiabetic control rats. In most tissues, AR immunoreactivity and AR mRNA were proportionately elevated. Intensive insulin treatment, which normalized blood glucose and glycosylated hemoglobin, significantly reduced AR activity and immunoreactivity. AR mRNA abundance was also reduced in tissues from insulin-treated diabetic rats. Statil treatment had no significant effect on AR immunoreactivity or AR mRNA abundance, although AR activity in tissues from Statil-treated diabetic rats was significantly reduced compared with untreated diabetic rats. These studies demonstrate that the expression of the AR gene is upregulated in most tissues of the diabetic rat, that insulin treatment reverses this phenomenon, and that AR inhibition has no effect on AR gene expression.

Aldehyde Reductase↗
PubMed1991

Nutrition and somatomedin. XXV. Regulation of insulinlike growth factor binding protein 1 in primary cultures of normal rat hepatocytes.

Although mRNAs encoding insulinlike growth factor (IGF) binding proteins (BPs) are present in adult rat liver and IGF BP-1 circulates at elevated levels in diabetic animals, there is little knowledge of the metabolic regulation of IGF BPs in normal tissues. We examined the release of IGF BPs by adult rat hepatocytes maintained in primary culture. When cultured for 2 days in the absence of added insulin, hepatocytes released a BP identified as BP-1 on the basis of approximately 30,000-Mr on ligand blotting and reactivity with antiserum to human BP-1 in immunoblotting and immunoprecipitation studies. Release of BP-1 was sensitive to insulin with suppression of 24 +/- 4, 73 +/- 5, and 64 +/- 14% at 10(-10), 10(-8), and 10(-6) M insulin, respectively; ED50 was approximately 1.7 x 10(-9) M, which is within the physiological range. Suppression by insulin was reversible and began within 3 h. Because normal hepatocytes in primary culture exhibit insulin-responsive release of both BP-1 and IGF-1, this system may be an ideal model for studies of molecular mechanisms of metabolic regulation.

Animals↗
PubMed1990

Regulation of insulin-like growth factor binding protein-3 expression by dexamethasone.

Dexamethasone (DXM), a potent long acting glucocorticoid results in growth retardation when administered to children and experimental animals. We have used ligand blotting and RNA blotting techniques to examine the effects of DXM on serum insulin-like growth factor binding protein 3 (IGFBP-3) levels and hepatic IGFBP-3 mRNA abundance. A time- and dose-dependent increase in the 39-42 kDa serum IGF binding proteins and in IGFBP-3 mRNA abundance was observed in DXM-treated rats. A significant increase in serum IGF binding capacity was seen with as little as 0.1 microgram/100 g body weight. A significant increase in IGFBP-3 mRNA abundance was apparent as early as 1 h following DXM administration. IGFBP-3 mRNA levels reached a peak at 3 h (1.9 +/- 0.2 fold, p less than 0.005) and declined to normal levels in 6-12 h after DXM administration. Since the IGF binding proteins may be able to inhibit the action of the IGFs, enhanced expression of IGFBP-3 may be one of the mechanisms involved in DXM-induced growth retardation.

Animals↗
PubMed1990

Dexamethasone increases hepatic insulin-like growth factor binding protein-1 (IGFBP-1) mRNA and serum IGFBP-1 concentrations in the rat.

Glucocorticoid excess is associated with growth retardation in both man and experimental animals. We have previously reported that dexamethasone (DXM) inhibits growth hormone induction of insulin-like growth factor-I (IGF-I) mRNA in the hypophysectomized rat and reduces steady-state IGF-I mRNA levels in the intact rat. However, in these experiments, DXM had surprisingly little effect on serum IGF-I concentrations. Here, we have examined the effect of DXM on hepatic insulin-like growth factor binding protein-1 (IGFBP-1) mRNA levels and on serum IGFBP-1 concentrations. After a single ip injection of DXM, 6 micrograms/100 g body wt, IGFBP-1 mRNA increased 2.24 +/- 0.25-fold, P less than 0.05 at 1 h and declined to normal levels in 6-12 h. A dose-dependent increase in increased hepatic IGFBP-1 mRNA abundance was seen in rats killed 1 h after an ip injection of DXM, 0.1 to 60 micrograms/100g body wt. As little as 1 microgram/100g body wt, significantly enhanced hepatic IGFBP-1 mRNA levels; 2.02- +/- 0.38-fold, P less than 0.05. This effect appeared to be post-transcriptional, since IGFBP-1 transcription in hepatic nuclei from rats treated with DXM was not significantly different from untreated rats. When DXM, 1 microgram/rat, was administered daily for 6 days a significant increase in IGFBP-1 mRNA was detected; 2.02 +/- 0.39-fold, P less than 0.05. A more marked increase was seen with 6 and 60 micrograms/rat of DXM; 4.47 +/- 1.08- and 10.61 +/- 0.31-fold, respectively. Serum was analyzed by sodium dodecyl sulfate-plyacrylamide gel electrophoresis and immunoblotting using antisera raised against a synthetic peptide derived from the predicted sequence of rat IGFBP-1. The antisera recognized a doublet of approximately 30 kDa. A dose-dependent increase in the abundance of these BPs were seen in the serum from rats treated chronically with DXM. The observations reported here clearly demonstrate that DXM increases hepatic IGFBP-1 mRNA and serum IGFBP-1 concentrations. If IGFBP-1 functions to decrease the bioavailability of IGF-1 in vivo the enhanced expression of IGFBP-1 may be an additional mechanism whereby glucocorticoid excess results in growth retardation.

Animals↗
PubMed1990

Expression of insulin-like growth factor-I and insulin-like growth factor-binding protein-1 in the rat uterus during decidualization.

Decidualization of the uterus involves proliferation and differentiation of uterine cells. The effects of decidualization on uterine expression of insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) have been examined in the hypophysectomized-ovariectomized (hypox-ovx) rat and the pituitary-intact (ovx) rat. Decidualization was induced by uterine stimulation of animals treated with a combination of 17 beta-estradiol and progesterone. The patterns of change in uterine IGF-I mRNA and IGFBP-1 mRNA abundance were similar to hypox-ovx rats, hypox-ovx rats replaced with GH and T4, and ovx rats. The changes in IGF-I mRNA abundance were temporally related to 17 beta-estradiol injections. IGFBP-1 mRNA was undetectable early in the decidualization process and reached maximal levels on day 6. Mechanical separation of the deciduoma tissue from the underlying myometrium revealed that the deciduoma tissue was depleted in IGF-I mRNA, while the majority of the IGFBP-1 was located in the deciduoma tissue. The in situ hybridization technique was used to localize IGF-I and IGFBP-1 mRNA in the decidualized uterus. The majority of the IGF-I expression was localized to the outer stroma and smooth muscle cell layer, whereas IGFBP-1 mRNA was detected in uterine epithelial cells and stromal glands. These experiments demonstrated that uterine IGF-I and IGFBP-1 expression during the process of decidualization are pituitary independent. Furthermore, our observations support the hypothesis that the expression of IGFBP-1, a protein capable of inhibiting the mitogenic activity of IGF-I, in deciduoma tissue may inhibit paracrine IGF-1 actin and allow for the differentiation of stromal tissue.

Animals↗
PubMed1990

Localization of the sites of synthesis and action of insulin-like growth factor-I in the rat uterus.

In this report we have used in situ hybridization to localize insulin-like growth factor-I (IGF-I) expression in the uterus and have examined the effects of exogenous IGF on 3H-methyl thymidine incorporation into DNA, in uterine sections in organ culture. IGF-I mRNA was detected in all layers of the uterus but was particularly abundant in the outer longitudinal smooth muscle layer. Although IGF-I mRNA was detectable in untreated, immature rat uteri, the abundance in each layer of the uterus was increased after 17 beta-estradiol (E2) administration. A similar increase was seen in uteri from ovariectomized, hypophysectomized rats treated with E2. IGF-I when added to uterine sections in organ culture had no significant effect on DNA synthesis in the absence of E2. However, a dose-dependent increase in DNA synthesis was seen in the presence of E2. The half-maximal and maximal responses required 1 and 10 ng IGF-I, respectively. Autoradiographic localization of 3H-methyl thymidine incorporation into DNA demonstrated that the majority of the DNA synthesis occurred in the stromal layer. These findings are consistent with the hypothesis that IGF-I may function as a paracrine growth factor, mediating stromal-myometrial cell interaction.

Animals↗
PubMed1990

Identification and characterization of a rat decidual insulin-like growth factor-binding protein complementary DNA.

A partial cDNA which encodes the rat homolog of human placental protein-12, the low mol wt insulin-like growth factor-binding protein (IGFBP-1), has been isolated from a rat decidual cDNA library using low stringency hybridization with a human IGFBP-1 cDNA probe. The incomplete cDNA obtained from this library was used to screen a rat liver cDNA library from which a full-length cDNA was obtained. The predicted amino acid sequence of rat IGFBP-1 showed 66%, 29%, and 34% sequence identity with the human IGFBP-1, the human GH-dependent binding protein IGFBP-3, and rat IGFBP-2, the BP secreted by buffalo rat liver cells, respectively. The rat IGFBP-1 cDNA hybridized with a 1.6-kilobase transcript which was easily detected in uterine RNA from the pseudopregnant rat and RNA from the liver and kidney of adult rats. A low level of expression was apparent in the brain and the diestrous uterus. Of the tissues examined the order of abundance of IGFBP-1 mRNA was deciduoma tissue greater than or equal to liver much greater than kidney much greater than uterus greater than brain. The 1.6-kb mRNA was more abundant in RNA from neonatal rat liver than in that from maternal liver, but was not detected in total RNA (50 micrograms) from other neonatal rat tissues (kidney, lung, brain, and heart). Under stringent conditions, rat IGFBP-1 did not hybridize with RNA from the BRL-3A rat liver cell line. In food-deprived rats, hepatic IGFBP-1 mRNA was increased 10.0 +/- 2.2-fold compared to that in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Transcriptional regulation of rat insulin-like growth factor-binding protein-1 expression by growth hormone.

The low mol wt insulin-like growth factor-binding protein (IGFBP-1) originally isolated from amniotic fluid has been considered to be GH independent. In this report we have examined the effect of GH on hepatic IGFBP-1 expression in the hypophysectomized rat. Using a rat IGFBP-1 cDNA probe we now demonstrated that hepatic IGFBP-1 expression is up-regulated in the GH-deficient rat. In addition, using ligand blotting, an increase in the abundance of a 30-kDa [125I]IGF-I-BP was detected in both hepatic extracts and serum from hypophysectomized rats. After a single ip injection of GH, the IGFBP-1 transcription rate was reduced within 30 min to the levels seen in the sham-operated control rats. Similarly, hepatic IGFBP-1 mRNA abundance was reduced after both acute GH injection and chronic GH treatment for 8 days. These data demonstrate that IGFBP-1 expression is inversely regulated by GH.

Animals↗
PubMed1990

Insulin regulation of IGF-I expression in rat aorta.

Smooth muscle cell proliferation may be important in pathogenesis of atherosclerosis. Because insulinlike growth factor I (IGF-I) is a potent mitogen for arterial smooth muscle cells in culture, we examined the hypothesis that IGF-I functions as an autocrine growth factor in the aorta. We also investigated the role of insulin in regulation of IGF-I expression in the aorta. With immunohistochemical and in situ hybridization techniques, IGF-I immunoreactivity and IGF-I mRNA were localized to the smooth muscle layer of the aorta. In diabetic rats, aortic IGF-I mRNA abundance was significantly reduced to 60.3 +/- 2.9% of controls (P less than 0.01). In nondiabetic rats, administration of insulin as an acute bolus (10 U i.p.) or a chronic infusion (2.4 U/day for 5 days) resulted in an approximately twofold increase in abundance of IGF-I mRNA in the aorta. These observations are consistent with the hypothesis that IGF-I may function as an autocrine growth factor in the aorta and suggest that one of the mechanisms whereby hyperinsulinism may favor atherogenesis is enhanced expression of IGF-I in the vessel wall.

Animals↗
PubMed1990

Characterization of two preparations of antibodies to basic fibroblast growth factor which exhibit distinct patterns of immunolocalization.

Immunoglobulins reactive against basic fibroblast growth factor (bFGF) were obtained from the serum of a single rabbit immunized against residues [1-24] of bFGF conjugated to keyhole limpet hemocyanin (KLH). Pure immunoglobulin preparations no. 1 and no. 2 were prepared using different affinity chromatography columns and preabsorption to KLH-coupled Sepharose for preparation no. 1. Both preparations no. 1 and no. 2 were specific for bFGF in in vitro assays. Competition with synthetic peptides suggests that preparations no. 1 and no. 2 recognize predominantly epitope(s) within residues [16-24]bFGF or residues [1-10]bFGF, respectively, in situ. Furthermore, no. 2 (but not no. 1) antibodies can react with tissue-(heparin-)-bound antigen. When used in indirect immunofluorescence for bFGF in frozen heart sections, preparation no. 1 stained predominantly muscle intercalated discs (IcDs); muscle nuclei were also stained, in an overall punctate fashion. Preparation no. 2 stained muscle nuclei strongly, in association with the nuclear envelope; it also stained basement-membrane associated bFGF. Differences in immunostaining were also observed in uterine smooth muscle and kidney sections but not in skeletal muscle. It is plausible that accessibility of various epitopes within the amino-terminal region depends strongly on the local interactions of bFGF. Our data illustrate the importance of using several different antibodies to localize bFGF in a tissue.

Absorption↗
PubMed1990

Epidermal growth factor: receptor and ligand expression in human breast cancer.

The epidermal growth factor (EGF) gene is expressed by most human breast cancer cell lines as well as 83% of human breast cancers in vivo. Furthermore, EGF mRNA is detectable in normal human breast tissue. These data suggest that EGF may have a functional role in both normal and neoplastic human breast tissue. Expression of EGF was generally highest in steroid receptor positive human breast tumor biopsies and cell lines. EGF expression was increased by progestins in T-47D and ZR 75 human breast cancer cells. Furthermore, progestins specifically increased the level of TGF-alpha and EGF-receptor mRNA in T-47D cells. Under these same conditions progestins inhibit growth of the cells. Regulation of expression of EGF, TGF-alpha and the EGF-receptor is unlikely to be directly related to the mechanism of progestin induced growth inhibition in T47-D cells. T-47D-5 cells are more sensitive than T-47D cells to progestin and antiestrogen induced growth inhibition. T-47D-5 cells do not express EGF and contain very low levels of TGF-alpha mRNA. The higher level of EGF and TGF-alpha expression in T-47D cells may be one mechanism by which these cells decrease their sensitivity to growth inhibition by progestins and antiestrogens.

Breast Neoplasms↗