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PubMed2001

TGF-beta1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice.

Abnormal accumulation of the amyloid-beta peptide (Abeta) in the brain appears crucial to pathogenesis in all forms of Alzheimer disease (AD), but the underlying mechanisms in the sporadic forms of AD remain unknown. Transforming growth factor beta1 (TGF-beta1), a key regulator of the brain's responses to injury and inflammation, has been implicated in Abeta deposition in vivo. Here we demonstrate that a modest increase in astroglial TGF-beta1 production in aged transgenic mice expressing the human beta-amyloid precursor protein (hAPP) results in a three-fold reduction in the number of parenchymal amyloid plaques, a 50% reduction in the overall Abeta load in the hippocampus and neocortex, and a decrease in the number of dystrophic neurites. In mice expressing hAPP and TGF-beta1, Abeta accumulated substantially in cerebral blood vessels, but not in parenchymal plaques. In human cases of AD, Abeta immunoreactivity associated with parenchymal plaques was inversely correlated with Abeta in blood vessels and cortical TGF-beta1 mRNA levels. The reduction of parenchymal plaques in hAPP/TGF-beta1 mice was associated with a strong activation of microglia and an increase in inflammatory mediators. Recombinant TGF-beta1 stimulated Abeta clearance in microglial cell cultures. These results demonstrate that TGF-beta1 is an important modifier of amyloid deposition in vivo and indicate that TGF-beta1 might promote microglial processes that inhibit the accumulation of Abeta in the brain parenchyma.

Aged↗

PubMed2002

Primary human cells differ in their susceptibility to rAAV-2-mediated gene transfer and duration of reporter gene expression.

The susceptibility of a variety of different primary tissues was examined to long-term transduction with recombinant adeno-associated virus type 2 (rAAV-2) and factors influencing the transduction efficiency. In contrast to others using cell lines and animal models, emphasis was placed on the use of primary human cells. Enhanced green fluorescent protein (EGFP) marker gene expression was examined using fluorescence-activated cell sorting analysis. The most effective target cells for rAAV-2-mediated gene transfer were bronchial epithelial, artery endothelial as well as smooth and skeletal muscle cells with mean transduction rates ranging from 34.3 to 81.6%. Lower transduction rates between 4.3 and 19.5% were found in chondrocytes, dermal papilla follicle epithelial cells and fibroblasts. No transduction was observed in melanocytes, granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) cells or malignant CD19(+) cells from patients with chronic lymphocytic leukemia. A proportion of EGFP-expressing skeletal muscle and smooth muscle cells was maintained over a period of 6 weeks after transduction (42.7+/-5.4 and 67.1+/-0.9%, respectively). Interestingly, among hair follicle epithelial cells the proportion of transduced cells increased from 8+/-0.5 to 36+/-7.7% in the course of 6 weeks. In contrast, for endothelial cells, bronchial epithelial cells and fibroblasts, a rapid decline in the number of EGFP expressing cells were noted. An inverse relationship between the proportion of cells in G2/M phase of cell cycle and long-term gene expression was observed. All rAAV-2 susceptible primary cells expressed FGFR-1 and the alphaV integrin consistent with their role as co-receptors for AAV-2. In conclusion, AAV-2 is a suitable vector system for transduction and evaluation of functional effects of long-term gene expression in primary human muscle and hair follicle cells.

Cell Line↗

PubMedDate not supplied

Low-molecular-mass endothelial cell-stimulating angiogenic factor in relation to capillary growth induced in rat skeletal muscle by low-frequency electrical stimulation.

The involvement of endothelial cell-stimulating angiogenic factor (ESAF) in capillary growth was studied in adult rat skeletal muscles (tibialis anterior, TA, and extensor digitorum longus, EDL) in which capillary growth was induced by chronic unilateral electrical stimulation (10 Hz, 8 h/day for 7 days), and in sham-operated and unoperated control muscles. ESAF was assayed by its ability to activate latent collagenase in units per hour per milligram protein. Anatomical capillary density (CD, number of capillaries per mm2) and capillary per fibre ratio (C/F) were estimated in frozen sections from the same muscles after staining for endothelial alkaline phosphatase. In control muscles, ESAF levels were inversely related to capillary supply, being highest (1.82 +/- 0.25 units) in the glycolytic cortex of TA (CD 273 +/- 18/mm2, C/F 1.26 +/- 0.07), lowest (1.04 +/- 0.02 units) in its oxidative highly capillarized core (CD 862 +/- 60/mm2, C/F 2.05 +/- 0.04), and intermediate in EDL (ESAF 1.59 +/- 0.37 units, CD 527 +/- 26/mm2, C/F 1.44 +/- 0.06). Neither capillary supply nor ESAF levels were affected by sham operation. However, chronic electrical stimulation increased capillary supply significantly in EDL (CD 61% greater than in controls, C/F 45% greater) and ESAF levels were elevated 3-fold to 4.77 +/- 0.74 units. In TA muscles, stimulation increased capillary supply specifically in the glycolytic cortex (C/F 2.51 +/- 0.09, p < 0.0001 vs. control) and ESAF levels were increased significantly in this region to 3.19 +/- 0.55 units (p < 0.05, vs. control). C/F ratio and ESAF in the oxidative core of TA (2.31 +/- 0.05 and 1.48 +/- 0.23 units, respectively) were not significantly different from control values. Thus, chronic electrical stimulation, which is known to increase both shear stress and wall tension in capillaries and induce angiogenesis, also increased ESAF activity.

Animals↗

PubMed1995

Transforming growth factor-beta 1 serves as an autocrine inhibitor of human endothelial cell/lymphocyte adhesion.

Other investigators have shown that exogenously administered transforming growth factor-beta (TGF-beta) inhibits lymphocyte adherence to vascular endothelial cells (VEC). We examined the role of TGF-beta 1 as an autocrine mediator of lymphocyte adhesion to adult human VECs. VECs were harvested from eight saphenous or cadaveric iliac veins using 0.2% collagenase. Low-passage VECs in MCDB + 0.1% BSA were pretreated for 24 hr with monoclonal anti-TGF-beta 1 antibody (5 micrograms/ml), LPS (5 micrograms/ml), or IL-1 (10 U/ml). Adherence of fluorescently labeled lymphocytes to pretreated VECs was quantitated and results were expressed as relative adhesion compared to untreated control. Total mRNA from LPS- or IL-1-treated VECs was subjected to Northern analysis to determine relative TGF-beta 1 expression. Total TGF-beta 1 protein concentration in supernatants from LPS- or IL-1-treated VECs was determined by ELISA. Data (means +/- SEM) were analyzed by ANOVA with a Newman-Keuls posttest. Neutralizing endogenous TGF-beta 1 with anti-TGF-beta 1 antibody significantly increased adhesion of lymphocytes to VEC monolayers compared to control (125 +/- 3 vs 101 +/- 2%, P < 0.01, n = 8). The level of adhesion was equivalent to that seen with IL-1 stimulation (131 +/- 6%). Spearman correlation of lymphocyte adherence to IL-1- or LPS-treated VECs vs TGF-beta 1 mRNA expression or vs relative TGF-beta 1 protein concentration showed significant inverse relationships (r = -0.82, P < 0.001, and r = -0.87, P < 0.001, respectively). Endogenous TGF-beta 1's inhibitory effect on lymphocyte adhesion was blocked by a specific neutralizing antibody. VEC TGF-beta 1 mRNA expression and TGF-beta 1 production were inversely proportional to lymphocyte adhesion, suggesting down-regulation of TGF-beta 1 in response to proinflammatory cytokines. Together, these observations support the hypothesis that TGF-beta 1 has an autocrine inhibitory role in regulation of lymphocyte adhesion to VECs.

Adult↗

PubMed1997

Effect of obesity on total and free insulin-like growth factor (IGF)-1, and their relationship to IGF-binding protein (BP)-1, IGFBP-2, IGFBP-3, insulin, and growth hormone.

OBJECTIVES: We investigated the effect of obesity on the serum levels of total and free IGF-1 and their relationship to the circulating levels of insulin and IGF binding proteins (IGFBPs) in age and sex-matched groups. SUBJECTS: The study included 43 obese subjects (ideal body weight; IBW > 120%) and 45 controls (IBW < 100%). All of the subjects were male. MEASUREMENT: Total IGF-1, free IGF-1, IGFBP-1, IGFBP-2, IGFBP-3, and insulin were measured in obese subjects and normal control subjects. RESULTS: No significant differences in the circulating levels of total and IGFBP-3 were observed between the obese and control groups. In contrast to total IGF-1, free IGF-1 in obese subjects was significantly increased compared to normal controls (P < 0.05). Serum total and free IGF-1 were inversely correlated with age (r = -0.42, P = 0.001, and -0.44, P = 0.001). Fasting serum insulin concentrations were elevated in all the obese subjects (P < 0.05) and positively correlated with IBW (r = 0.57, P = 0.001). The levels of serum GH and IGFBP-1 were suppressed in all the obese subjects (P < 0.05). IGFBP-1 was inversely correlated with IBW (r = -0.51, P = 0.001) and serum insulin concentrations (r = -0.48, P = 0.001). The IGFBP-2 concentrations were also suppressed in obese subjects and inversely related to free IGF-1 (r = -0.48, P = 0.001). Using multiple linear regression analysis, total IGF-1 and insulin concentrations were positively correlated (r = 0.58, P = 0.001) and free IGF-1 and IGFBP-1 concentrations were negatively correlated (r = -0.57, P = 0.001). CONCLUSION: We confirmed that total IGF-1 and IGFBP-3 concentrations were not significantly different between the obese and control groups, despite GH hyposecretion in obesity. We also found that free IGF-1 concentrations were higher in obese subjects than in normal controls. It seems likely that overnutrition and chronic hyperinsulinaemia in obesity may alter this regulated growth response by insulin stimulation of IGF-1 production and suppression of hepatic IGFBP-1 and IGFBP-2 production, which may inhibit IGF-1 bioactivity.

Adult↗

PubMed2005

Differentiation of pluripotent C3H10T1/2 cells rapidly elevates CYP1B1 through a novel process that overcomes a loss of Ah Receptor.

Stimulation of C3H10T1/2 cells by an adipogenic hormonal mixture (IDM) consisting of insulin (I), dexamethasone (D), and methylisobutylxanthine (M) substantially induces cytochrome P450 (CYP) 1B1 expression. This stimulation represents up to 40% of the level produced by maximum activation of the arylhydrocarbon receptor (AhR) with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dexamethasone and methylisobutylxanthine in combination produced near maximum elevation of CYP1B1 along with a subsequent decline in AhR that paralleled the rise in peroxisome proliferator-activated receptorgamma1 (PPARgamma1). Inhibitors of AhR activity, which block TCDD induction, did not affect this increase of CYP1B1 expression, which was, therefore, independent of AhR activity. These responses were unaffected by inhibition of DNA synthesis, which was required for PPARgamma1 induction and terminal differentiation. Induction of CYP1B1 mRNA was paralleled by increased CYP1B1 promoter-luciferase reporter activity. The initial 0.8kb of promoter region, which was sufficient for 24h near maximum stimulation, did not contain either the key AhR-responsive elements that mediate the TCDD response or CREB and SF1 elements that mediate cAMP stimulation of rat CYP1B1 in steroidogenic cells. This reporter response to IDM stimulation, but not to TCDD, was maintained in AhR-null fibroblasts. CYP1B1 expression, unlike TCDD induction, was stimulated by IDM in only about half the cells. CYP1B1 expression partially overlapped with PPARgamma expression, which was also inversely related in clonal sub-lines. CYP1B1 expression may, therefore, represent an early stage of differentiation that requires factors associated with DNA synthesis to subsequently generate PPARgamma1.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Control of nitric oxide production by endogenous TGF-beta1 and systemic nitric oxide in retinal pigment epithelial cells and peritoneal macrophages.

Both in vivo and in vitro experiments demonstrate that transforming growth factor-beta1 (TGF-beta1) suppresses expression of the inducible form of nitric oxide synthase (iNOS). In this study, we examined the effects of exogenous and endogenous TGF-beta1 on retinal pigment epithelial (RPE) cells and resident peritoneal macrophages ex vivo using cells from TGF-beta1 null (TGF-beta1-/-) mice or age-matched wild-type (TGF-beta1+/+) or heterozygous (TGF-beta1+/-) littermates. RPE cells from both TGF-beta1-/- mice and TGF-beta1+/+ littermates produced NO and were immunocytochemically positive for iNOS protein only following treatment with interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS); however, RPE cells from TGF-beta1-/- mice produced 40% more NO than cells from TGF-beta1+/+ mice. In contrast, resident peritoneal macrophages from both TGF-beta1+/+ and TGF-beta1-/- mice expressed iNOS protein without stimulation and in the absence of detectable production of NO. The expression of iNOS was increased by treatment with IFN-gamma, resulting in detectable levels of NO. Macrophages from TGF-beta1+/+ mice appeared to produce NO in a manner inversely proportional to the serum content of NO2- and NO3- of the mice from which the cells were obtained; no such correlation existed in TGF-beta1+/- or TGF-beta1-/- mice. Treatment of RPE cells or macrophages from both TGF-beta1+/+ and TGF-beta1-/- mice with exogenous TGF-beta1 decreased both iNOS protein and NO production. These findings demonstrate a novel role of endogenous TGF-beta1 in coupling systemic NO production to the production of NO by macrophages, and demonstrate that endogenous and exogenous TGF-beta1 can act differently to suppress NO production.

Animals↗

PubMed2006

Endoglin regulates cyclooxygenase-2 expression and activity.

The endoglin heterozygous (Eng(+/-)) mouse, which serves as a model of hereditary hemorrhagic telangiectasia (HHT), was shown to express reduced levels of endothelial NO synthase (eNOS) with impaired activity. Because of intricate changes in vasomotor function in the Eng(+/-) mice and the potential interactions between the NO- and prostaglandin-producing pathways, we assessed the expression and function of cyclooxygenase (COX) isoforms. A specific upregulation of COX-2 in the vascular endothelium and increased urinary excretion of prostaglandin E(2) were observed in the Eng(+/-) mice. Specific COX-2 inhibition with parecoxib transiently increased arterial pressure in Eng(+/-) but not in Eng(+/+) mice. Transfection of endoglin in L6E9 myoblasts, shown previously to stimulate eNOS expression, led to downregulation of COX-2 with no change in COX-1. In addition, COX-2 promoter activity and protein levels were inversely correlated with endoglin levels, in doxycyclin-inducible endothelial cells. Chronic NO synthesis inhibition with N(omega)-nitro-l-arginine methyl ester induced a marked increase in COX-2 only in the normal Eng(+/+) mice. N(omega)-nitro-l-arginine methyl ester also increased COX-2 expression and promoter activity in doxycyclin-inducible endoglin expressing endothelial cells, but not in control cells. The level of COX-2 expression following transforming growth factor-beta1 treatment was less in endoglin than in mock transfected L6E9 myoblasts and was higher in human endothelial cells silenced for endoglin expression. Our results indicate that endoglin is involved in the regulation of COX-2 activity. Furthermore, reduced endoglin levels and associated impaired NO production may be responsible, at least in part, for augmented COX-2 expression and activity in the Eng(+/-) mice.

Animals↗

PubMed1999

Insulin-like growth factor binding protein-1 as a predictor of glucose-stimulated hyperinsulinemia in prepubertal obese children.

OBJECTIVE: The present study was undertaken to examine the association of a glucose-stimulated insulin response with the fasting insulin-like growth factor-binding protein (IGFBP)-1 concentration in prepubertal obese children. SUBJECTS AND METHODS: The fasting levels of serum insulin and IGFBP-1 were measured in 17 obese and 16 control children. Furthermore, we performed an oral glucose tolerance test in obese children and examined the association of the area under the curve (AUC) for insulin with the fasting IGFBP-1 level. RESULTS: The mean serum level of IGFBP-1 was significantly lower in obese children (41.0 +/- 4.8 micrograms/l. P < 0.005) than in controls (91.2 +/- 9.9 micrograms/l). Although there was an inverse relationship between the fasting levels of serum insulin and IGFBP-1 in all subjects (r = -0.42, P < 0.05), no significant correlation between these two parameters was observed in the obese group alone. In obese children, the fasting IGFBP-1 level correlated inversely with AUC-insulin (r = -0.70, P < 0.005), whereas there was no significant relationship between the fasting insulin level and AUC-insulin. CONCLUSION: The present study suggests that the serum level of IGFBP-1 may be an early predictor of insulin resistance in prepubertal obesity.

Area Under Curve↗

PubMed1987

Different types of modification in c-fos and its associated protein p39: modulation of DNA binding by phosphorylation.

We have studied the biosynthesis and biochemical properties of the c-fos gene product and its associated protein (p39) in growth factor-stimulated fibroblasts. c-fos is a markedly acidic protein that is extensively post-translationally modified by phosphorylation and another type of modification not changing its relative molecular mass (Mr). More than 10 different forms of c-fos protein can be identified by two-dimensional gel analysis. In c-fos-transformed cells, however, most of the highly modified forms are missing. The affinity for DNA of less phosphorylated c-fos-protein complexes is higher than that of the highly modified ones. The transforming potential of c-fos protein and its affinity for DNA thus seems to be inversely correlated with the extent of its phosphorylation. In contrast to c-fos, p39 is a basic protein that is rendered even more basic by post-translational modification. Two other forms of p39 differing in specific domains of the protein (p41 and p43) were also found to be complexed with c-fos.

Animals↗

PubMed2005

Propofol dose-dependently reduces tumor necrosis factor-alpha-Induced human umbilical vein endothelial cell apoptosis: effects on Bcl-2 and Bax expression and nitric oxide generation.

We investigated whether propofol can inhibit tumor necrosis factor (TNF)-alpha-induced apoptosis in cultured human umbilical vein endothelial cells (HUVECs). Isolated HUVECs were cultured in Dulbecco's modified Eagle medium supplemented with 20% bovine calf serum. HUVECs in untreated and propofol control groups were cultured at 37 degrees C for 24.5 h. HUVECs in the TNF treatment groups were initially cultured for 30 min in the presence of TNF or various concentrations of propofol, respectively, which were then cultured for 24 h with the addition of TNF at 40 ng/mL in the medium. Apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and confirmed by electron microscopy. The antiapoptotic Bcl-2 and proapoptotic Bax protein expressions were measured by immunocytochemical analysis. TNF stimulation resulted in a reduced Bcl-2/Bax ratio and increased apoptotic index (AI: percentage of apoptotic cells) in HUVECs. Propofol, at concentrations >/=12 muM, significantly (P < 0.001) and dose-dependently attenuated TNF-induced increase in AI and decrease in Bcl-2/Bax ratio. This was accompanied by increases in nitric oxide production. There is an inverse correlation between the ratio of Bcl-2/Bax expression and AI (P = 0.0009). These results suggest that propofol, at clinical relevant concentrations, can reduce TNF-induced HUVEC apoptosis.

Anesthetics, Intravenous↗

PubMed2005

Epidermal growth factor mediates detachment from and invasion through collagen I and Matrigel in Capan-1 pancreatic cancer cells.

BACKGROUND: Pancreatic adenocarcinoma is a highly invasive neoplasm. Epidermal growth factor (EGF) and its receptor are over expressed in pancreatic cancer, and expression correlates with invasion and metastasis. We hypothesized that EGF receptor and integrin signalling pathways interact in mediating cellular adhesion and invasion in pancreatic cancer, and that invasiveness correlates temporally with detachment from extracellular matrix. METHODS: We tested this hypothesis by investigating the role of EGF in mediating adhesion to and invasion through collagen I and Matrigel in the metastatic pancreatic adenocarcinoma cell line Capan-1. Adhesion and invasion were measured using in vitro assays of fluorescently-labeled cells. Adhesion and invasion assays were also performed in the primary pancreatic adenocarcinoma cell line MIA PaCa-2. RESULTS: EGF inhibited adhesion to collagen I and Matrigel in Capan-1 cells. The loss of adhesion was reversed by AG825, an inhibitor of erbB2 receptor signalling and by wortmannin, a PI3K inhibitor, but not by the protein synthesis inhibitor cycloheximide. EGF stimulated invasion through collagen I and Matrigel at concentrations and time courses similar to those mediating detachment from these extracellular matrix components. Adhesion to collagen I was different in MIA PaCa-2 cells, with no significant change elicited following EGF treatment, whereas treatment with the EGF family member heregulin-alpha elicited a marked increase in adhesion. Invasion through Matrigel in response to EGF, however, was similar to that observed in Capan-1 cells. CONCLUSION: An inverse relationship exists between adhesion and invasion capabilities in Capan-1 cells but not in MIA PaCa-2 cells. EGF receptor signalling involving the erbB2 and PI3K pathways plays a role in mediating these events in Capan-1 cells.

Biocompatible Materials↗

PubMed1995

The Bacillus subtilis histone-like protein Hbsu is required for DNA resolution and DNA inversion mediated by the beta recombinase of plasmid pSM19035.

The beta recombinase, encoded by the Gram-positive bacterial plasmid pSM19035, is unable to mediate DNA recombination in vitro unless a host factor is provided. The factor has now been identified as the Bacillus subtilis Hbsu protein. Hbsu is a nonspecific DNA-binding and DNA-bending protein. The beta recombinase, in the presence of highly purified Hbsu protein, is able to catalyze in vitro intramolecular recombination between two specific recombination sites on a supercoiled DNA molecule. DNA resolution was obtained when the two crossing over sites (six sites) were directly oriented, whereas DNA inversion was the product when the six sites were in inverse orientation. The ability of the Escherichia coli chromatin-associated proteins HU, IHF, Fis, and H-NS to substitute for Hbsu was investigated. HU efficiently stimulated beta-mediated recombination, while the effect of IHF was partial and that of Fis and H-NS was undetectable. In addition, the beta protein was able to mediate DNA recombination in both wild-type and IHF-deficient E. coli cells, but failed to do so in an HU-deficient strain. The data presented provide direct evidence that a chromatin-associated protein is strictly required for beta-mediated recombination.

Bacillus subtilis↗

PubMed1991

[Interaction between interleukin-1 and tumor necrosis factor productions by peripheral blood monocytes and nutritional disturbance in active pulmonary tuberculosis].

Malnutrition is frequently observed in patients with pulmonary tuberculosis. We have already reported the nutritional disturbance in those patients by comprehensive nutritional assessment. But the mechanism of this nutritional disturbance remains unclear. We anticipated that cytokines contributed to the nutritional disturbance. To elucidate this mechanism we measured the productions of interleukin-1 (IL-1) and tumor necrosis factor (TNF) by peripheral blood monocytes, and correlated them with nutritional parameters in those patients. These cytokines had been reported to mediate metabolic alterations in inflammatory process. Subjects were 45 patients with bacteriologically confirmed pulmonary tuberculosis and their controls matched by age and sex. Adherent monocyte at 0.5 x 10(6)/ml were stimulated by lipopolysaccharide (LPS), and the culture supernatant was measured by ELISA for IL-1 and TNF. In order to assess nutritional status we measured serum albumin, transferrin, prealbumin, retinol binding protein, branched chain amino acid (BCAA)/aromatic amino acid (AAA) ratio as amino acid imbalance index, % ideal body weight (%IBW), % arm muscle circumference (% AMC) as muscle mass index, % triceps skin fold thickness (% TSF), as fat store index. The results were as follows: (1) Patients with active pulmonary tuberculosis were confirmed to be malnourished in visceral proteins, plasma amino acid, and anthropometric indices. (2) In patients with moderate or mild nutritional depletion the production of IL-1 and TNF was higher than that in healthy controls, and significantly correlated inversely with the nutritional parameters. (3) In patients with severe nutritional depletion the production of IL-1 and TNF was lower than that in healthy controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1989

Epidermal growth factor: modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport.

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose- and time-dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4-6 hours. EGF also stimulated the initial rate of putrescine transport in a dose- and time-dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 microM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 microM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF-treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 micrograms/ml) and actinomycin D (50 micrograms/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca++ while alpha-aminoisobutyric acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO-treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.

Animals↗

PubMed2004

Basal and stimulated levels of growth hormone, insulin-like growth factor-I (IGF-I), IGF-I binding and IGF-binding proteins in beta-thalassemia major.

A significant percentage of children with beta-thalassemia major shows retardation in longitudinal growth as they progress towards puberty due to skeletal dysplasia, endocrine gland hypofunction or trace element deficiencies. The aim of this study was to evaluate GH/IGF-I secretion and action in prepubertal patients with beta-thalas-semia major. Eight prepubertal patients with short stature (group A) and seven prepubertal patients with normal stature (group B) were studied. Basal and stimulated (after administration of the hexapeptide Hexarelin) GH levels were measured with IRMA (Nichols); IGF-I and IGFBP-3 levels were measured with RIA (Nichols). IGF-I binding proteins (IGFBPs) were analyzed qualitatively with Western ligand blot. IGF-I binding to B-lymphocytes of the patients was also measured with competitive binding studies using human recombinant IGF-I and 125I-IGF-I (Amersham). Basal GH levels did not differ statistically between the groups. Peak GH levels after Hexarelin stimulation test were higher in group A (A: 27.9 +/- 15.6 ng/ml vs B: 9.1 +/- 4.7 ng/ml) (Wilcoxon test, p < 0.05). IGF-I levels in the two groups were low-normal and comparable (A: 168.0 +/- 81.6 ng/ml vs B: 126.6 +/- 25.5 ng/ml). IGFBP-3 levels were low in both groups (A: 1.21 +/- 0.27 microg/ml vs B: 1.08 +/- 0.20 microg/ml). Western ligand blot did not reveal any discernible difference in IGFBPs. However, IGF-I binding on B-lymphocytes was at least 20% lower in group A compared to group B (t-test, p < 0.01). IGF-I binding inversely correlated with peak GH levels (r = -0.54, p < 0.05). Patients in group A were older and chronological age correlated with IGF-I levels (r = 0.53, p < 0.05) whereas it inversely correlated with IGF-I binding (r = -0.63, p < 0.05). Moreover, patients in group A had higher ferritin levels. No correlation was found between ferritin levels, desferrioxamine dose/compliance or liver enzyme levels and the parameters of the GH axis studied. However, desferrioxamine dose x years correlated with IGFBP-3 (r = 0.56, p < 0.05) and correlated inversely with IGF-I binding (r = -0.74, p < 0.01). In conclusion, we have shown adequate GH secretion, higher secretive capacity after the administration of Hexarelin and lower IGF-I binding in prepubertal beta-thalassemic patients with short stature. Whatever the cause, reduced IGF-I action has to be considered when treating beta-thalassemic patients with short stature.

B-Lymphocytes↗

PubMed1997

Abdominal fat determines growth hormone-binding protein levels in healthy nonobese adults.

The circulating high affinity GH-binding protein (GHBP), which derives from the extracellular domain of the hepatic GH receptor, correlates inversely to GH levels and directly to body mass index (BMI) in healthy adults. As GH secretion and adiposity are also interrelated, we tested the hypothesis that body composition more than GH, determines GHBP levels in healthy adults. Forty-two healthy adults [21 females and 21 males; mean age, 39.4 yr range, 27-59 yr); mean BMI, 23.9 kg/m2 (range, 18.9-34.7 kg/m2)], underwent anthropometric measurements (BMI, W/H ratio, computed tomography scan, dual energy x-ray absortiometry (DEXA) scan, and bioimpedance) in addition to two GH stimulation tests (arginine and clonidine) and a 24-h GH profile. By simple linear regression, serum GHBP correlated positively to several indices of adiposity: intraabdominal fat (r = 0.537; P = 0.001), sc abdominal fat (r = 0.680; P < 0.001), BMI (r = 0.483; P = 0.001), W/H ratio (r = 0.452; P = 0.003), total body fat (DEXA scanning; r = 0.503; P = 0.002), and body fat (bioimpedance; r = 0.354; P = 0.023). Lean body mass estimated by DEXA scan was negatively associated with GHBP (r = 0.541; P < 0.001). GHBP was inversely proportional to arginine-stimulated GH release (r = -0.346; P = 0.027) and negatively associated with several measures of spontaneous GH release as estimated by deconvolution analysis (GH mass, GH production rate, and mean GH; r = -0.371; P = 0.017, r = -0.393; P = 0.011, and r = -0.343; P = 0.028, respectively)). With multiple linear regression analyses, indices of adiposity were significant determinants of GHBP levels, whereas GH status did not contribute independently to the prediction of GHBP. Neither insulin-like growth factor I nor fasting insulin levels correlated to GHBP levels. In conclusion, GHBP levels in normal adults seem to be determined by abdominal fat mass rather than GH secretion.

Abdomen↗

PubMed1989

Insulin-like growth factor (IGF)-binding proteins inhibit the biological activities of IGF-1 and IGF-2 but not des-(1-3)-IGF-1.

(1) Many cell types secrete insulin-like growth factor (IGF)-binding proteins that can be expected to sequester free IGF and modify the biological activities of the growth factors. (2) A binding protein purified from bovine kidney (MDBK) cells potently inhibited the ability of IGF-2 to stimulate DNA synthesis or protein accumulation as well as to reduce rates of protein breakdown in chick embryo fibroblasts. The binding protein did not influence the biological activities of des-(1-3)-IGF-1, while effects on IGF-1 were intermediate. Since the chick embryo fibroblasts contain only the type 1 IGF receptor, the MDBK-cell binding protein must have reduced the accessibility of IGF-2 and IGF-1 to that receptor. Binding to the type 2 receptor on L6 myoblasts was also inhibited. (3) Inhibiting effects on both protein breakdown responsiveness to IGF and IGF binding to cell receptors were also observed with human amniotic fluid binding protein, although here IGF-1 and IGF-2 were equipotent. These results contrast with stimulatory responses on different IGF-1 actions of the same binding protein reported previously [Elgin, Busby & Clemmons (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3254-3258]. (4) The biological potencies of IGF-1, IGF-2 and des-(1-3)-IGF-1 correlate inversely with their binding to proteins released into the medium by cells, so that the enhanced potency of des-(1-3)-IGF-1 is a consequence of it not binding to purified binding proteins or those released by cultured cells.

Animals↗