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Responsiveness to 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] may be diminished in osteoporosis and inflammatory arthritis. The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is produced in excess in these disorders and has been shown to decrease osteoblast transcriptional responsiveness to vitamin D and to inhibit the binding of the vitamin D receptor (VDR) and its nuclear partner the retinoid X receptor (RXR) to DNA. Previous studies have shown that a vitamin D (VDRE) or retinoid X DNA response element (RXRE) is sufficient to confer TNF-alpha inhibition of vitamin D or retinoid-stimulated transcription in the absence of known TNF-alpha-responsive DNA sequences. We tested the hypothesis that the TNF-alpha-stimulated transcription factor nuclear factor (NF)-kappaB could, in part, mediate TNF-alpha action by inhibiting the transcriptional potency of the VDR and RXR at their cognate cis regulatory sites. Osteoblastic ROS 17/2.8 cells transfected with a dose of NF-kappaB comparable to that stimulated by TNF-alpha decreased 1,25(OH)(2)D(3)-stimulated transcription. This inhibitory effect of NF-kappaB was not observed on basal transcription of a heterologous reporter in the absence of the VDRE. The effects of NF-kappaB and TNF-alpha were comparable but not additive. COS-7 cells were cotransfected with reporters under the regulation of VDRE or RXRE along with vectors expressing VDR, RXR, and NF-kappaB nuclear proteins. Reconstituted NF-kappaB and the NF-kappaB subunit p65 alone, but not p50, dose dependently suppressed basal and ligand-stimulated transcription. p65 overexpression completely abrogated enhanced VDRE-mediated transcriptional activity in response to 1,25(OH)(2)D(3). Electrophoretic mobility shift experiments did not reveal a direct effect of recombinant NF-kappaB or its individual subunits on the binding of heterodimeric VDR-RXR to DNA. These results suggest that TNF-alpha inhibition of hormone-stimulated transcriptional activation may be mediated by activation of NF-kappaB. In contrast, the inhibitory effect of TNF-alpha on binding of receptors to DNA is unlikely to be mediated by NF-kappaB and is not necessary for inhibition of transcription.

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A calmodulin-like domain protein kinase (DcCPK1, previously designated CDPK431) cloned from carrot (Daucus carota L.) was expressed at high levels in Escherichia coli and partially purified. Ca(2+)-induced gel mobility shift and (45)Ca(2+) ligand binding assays confirmed that recombinant DcCPK1 binds Ca(2+) through its calmodulin-like domain and undergoes a significant conformational change. Ca(2+) activated the kinase activity of recombinant DcCPK1 (K(0.5)=1.7 microM) up to 20-fold. Ca(2+) combined with certain lipids, including phosphatidic acid, phosphatidylserine and phosphatidylinositol, but not diolein or lysophosphatidylcholine, provided even greater Ca(2+)-dependent protein kinase activity. DcCPK1 phosphorylated casein and histone III-S, and a variety of peptide substrates containing a hydrophobic and a basic residue situated P-5 and P-3 amino acids N-terminal to a Ser or Thr residue. The calmodulin and protein kinase inhibitors, W-7 and staurosporine, inhibited CDPK activity. The similarities between DcCPK1 and mammalian protein kinase C (PKC) in substrate specificity, sensitivity to inhibitors, and activation by Ca(2+) and phospholipid suggest that various CDPK isoforms may be responsible for some PKC-like activities in plant cells.

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Although the liver is the major source of circulating insulin-like growth factor-I (IGF-I), relatively little is known about the regulation of IGF-I gene transcription in this tissue. Since transcripts are initiated largely in exon 1, we established an in vitro transcription system to evaluate activation of transcription via the major exon 1 initiation site. Transcription of a G-free cassette reporter was directed by rat IGF-I genomic fragments, and the adenovirus major late promoter was used as an internal control. Tissue specificity was demonstrated by a 60-90% decrease in transcripts with spleen extracts as compared with liver. 54 base pairs (bp) of upstream sequence were sufficient to direct IGF-I gene transcription, and activity increased 5-fold with 300 bp of upstream sequence. DNase I footprinting revealed four protected regions between -300 and -60 bp; binding was confirmed by gel shift analysis, and tissue specificity was demonstrated by reduced shifts with spleen extracts. The necessity of transcription factor binding to such sites was established by competition analysis, which revealed a specific decrease in IGF-I transcription in the presence of a competing fragment. Use of this in vitro transcription system should permit analysis of the function of individual transcription factors involved in regulation of IGF-I gene expression.

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Insulin-like growth factor-I (IGF-I) gene transcription is mediated largely via exon 1. In an initial search for regulatory regions, rat hepatocytes were transfected with IGF-I constructs. Since omission of downstream sequences led to reduced expression, we then used in vitro transcription to evaluate potential metabolic regulation via downstream regions. With templates including 219 base pairs of downstream sequence, transcriptional activity was reduced 70-90% with hepatic nuclear extracts from diabetic versus normal rats. However, activity was comparable with templates lacking downstream sequences. The downstream region contained six DNase I footprints, and templates with deletion of either region III or V no longer provided reduced transcriptional activity with nuclear extracts from diabetic rats. Nuclear protein binding to regions III and V appeared to be metabolically regulated, as shown by reduced DNase I protection and activity in gel mobility shift assays with nuclear extracts from diabetic rats. Southwestern blotting probes corresponding to regions III and V recognized a approximately 65-kDa nuclear factor present at reduced levels in diabetic rats. These findings indicate that a downstream region in exon 1 may be important for both IGF-I expression and metabolic regulation. Altered concentration or activity of a transcription factor(s) binding to this region may contribute to reduced IGF-I gene transcription associated with diabetes mellitus.

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Synthesis of insulin-like growth factor-I (IGF-I) and IGF binding protein-1 (IGFBP-1) is altered in diabetes and malnutrition, but underlying processes are poorly understood. To study molecular mechanisms, we examined regulation of IGF-I and IGFBP-1 gene transcription in primary cultures of rat hepatocytes. Transcription of the IGF-I and IGFBP-1 genes was measured as incorporation of [alpha-32P]UTP into preinitiated message in isolated nuclei. IGFBP-1 gene transcription was not sensitive to reduction in amino acid concentration from 5x to 0.5x rat arterial plasma levels. However, IGF-I gene transcription fell 60-70% in response to reduced provision of amino acids. Culture with 10(-9) M insulin lowered IGFBP-1 gene transcription 50% below control levels (10-11 M) but did not affect IGF-I gene transcription; 10(-6) M insulin raised IGF-I gene transcription 2-fold. After an acute reduction in insulin concentration, IGFBP-1 transcription began to rise within 30 min, but IGF-I gene transcription was unchanged over 120 min. Similarly, 3-6 h were required for stimulation of IGF-I gene transcription by insulin, but a 40% decrease in IGFBP-1 gene transcription could be detected within 15 min after adding 10(-6) M insulin, and suppression of IGFBP-1 transcription by insulin was unaffected by the presence of cycloheximide. Effects of insulin on IGFBP-1 gene transcription were not mimicked or antagonized by phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)

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Although it is known that circulating levels of the insulin-like growth factors (IGFs) and the IGF-binding proteins (IGFBPs) fluctuate in response to changes in nutritional status, there is little information regarding either relative contributions from different dietary components or regulation by insulin in nondiabetic subjects. To define dietary contributions to IGF regulation, the authors examined the effects of fasting and hypocaloric diets of differing nutritional composition on serum IGF-1 and a IGFBP-1 in 16 healthy, obese adult women. Subjects received an isocaloric diet for 6 days, followed by 14 days of calorie restriction (fasting or a hypocaloric diet enriched in either protein, fat, or carbohydrate), and by 4 days refeeding. All diets produced 6-8% weight loss over 14 days with little difference between groups. The "protein-sparing" diet sustained nitrogen balance (+1.2 g/d, versus -4.5 g/d for the other three groups; p < 0.05). Serum IGF-1 levels decreased during calorie restriction with fasting or with diets high in fat or carbohydrates (CHO; combined mean 40 +/- 7%) but showed little change with the high protein regimen (3 +/- 16%; p < 0.05 compared to the other diets). In contrast, IGFBP-1 increased during calorie restriction in all four groups but significantly less with the high CHO diet (43 +/- 17% above baseline) than with the other diets (168 +/- 31%; p < 0.05). Levels of IGF-1 were correlated with nitrogen balance (r = 0.51; p < 0.05) but levels of IGFBP-1 were not. Although IGFBP-1 levels inversely correlated with measures of insulin secretion, IGF-1 levels did not.(ABSTRACT TRUNCATED AT 250 WORDS)

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Insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (BP-1) are critical cell regulators, with regulation and action in endocrine, paracrine, and autocrine modes. Although IGF-I and BP-1 are thought to be modulated mainly at the level of synthesis, underlying molecular mechanisms are poorly understood. To examine regulation by insulin, we used run-on assays to measure IGF-I and BP-1 gene transcription rates in nuclei isolated from the livers of normal and diabetic rats. Streptozotocin (STZ)-treated rats exhibited 20-25% weight loss, a 2.5- to 3-fold increase in serum glucose, and a 50-60% fall in circulating IGF-I levels (all P less than 0.001). Diabetic animals also had a 45% reduction in hepatic IGF-I mRNA and over 400% increases in BP-1 mRNA (both P less than 0.005); all parameters were restored toward normal after treatment with insulin. Metabolically responsive IGF-I gene transcription was evaluated effectively with a 3.2-kilobase BglII/EcoRI genomic probe located down-stream from all initiation sites in exon 1, while BP-1 gene transcription was studied with a cDNA probe. Animals treated with 144 mg/kg STZ exhibited 50-97% decreases in IGF-I gene transcription (P less than 0.05), while insulin treatment raised IGF-I gene transcription to control levels (P less than 0.02). IGF-I gene transcription appeared to be more sensitive to metabolic status than IGF-I mRNA levels, resulting in a modest correlation between transcription rates and mRNA levels (r = 0.68; P less than 0.001). In contrast, changes in BP-1 mRNA and gene transcription appeared to be exquisitely sensitive to metabolic status.(ABSTRACT TRUNCATED AT 250 WORDS)

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When previous data suggested a growth hormone-releasing factor (GRF)-sensitive branch in intracellular hormone processing, the monensin-sensitive Golgi apparatus seemed a likely candidate. We examined monensin's effect on basal and GRF-stimulated release of newly synthesized and stored rat growth hormone (rGH) and rat prolactin (rPRL). 14C-Pre-labeled, perifused rat pituitary fragments were exposed to [3H]leucine in 0-10 microM monensin; a pulse of 3 nM GRF assessed subsequent secretory responsivity. Monensin dose-dependently reduced basal release of stored [14C]rGH and [14C]rPRL. GRF-stimulated release of stored [14C]hormone was doubled after 0.03 microM and 0.1 microM monensin; higher concentrations diminished stored hormone release. Low concentrations of monensin accelerated basal (0.03 microM and 0.1 microM) and GRF-stimulated (0.03 microM) [3H]rGH and [3H]rPRL release without altering recovery; higher monensin concentrations (greater than or equal to 1 microM) reduced basal, and abolished GRF-stimulated, new hormone release and reduced total [3H]rGH and [3H]rPRL recovery. These data are consistent with a GRF-sensitive and monensin-influenced branch in intracellular hormone processing that regulates the fraction of new hormone exiting the cell without prior immersion in storage compartments.

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Growth hormone (GH) pulses in vivo are associated with increased hypothalamic portal growth hormone releasing hormone (GH-RH) concentration and can be prevented by GH-RH antisera. GH pulses are also associated with prior reduction of portal somatostatin (SRIF) concentrations, although SRIF antisera do not abolish GH pulses. In vitro, pulses of GH-RH as well as SRIF withdrawal are followed by pulses of GH release; the presence of GH-RH enhances post-SRIF GH release. We asked four questions: (1) During combined GHRH-SRIF exposure in vitro, must SRIF withdrawal be complete to produce a pulse of GH release, or is there a threshold diminution of SRIF which permits it? (2) When pulsatile GH release does occur, is it an all-or-none phenomenon, or is it titratable by fractional reduction of SRIF? (3) Does varying the GH-RH concentration while administering SRIF systematically alter GH release in response to fractional SRIF reduction? (4) Given a small but distinct effect of GH-RH on release of stored prolactin (PRL) in this system, does fractional SRIF reduction alter PRL release in parallel? Rat pituitary tissue whose hormone stores had been prelabeled with tritium was perifused for 120 min in combined 25 nM SRIF and 3 or 10 nM rat GH-RH (rGH-RH). Then, while maintaining rGH-RH concentrations, the SRIF concentration was left unchanged (control) or was reduced to 20, 15, 10, 5, or 0 nM for 60 min. Release of stored rGH and rPRL was assessed by immunoprecipitation.(ABSTRACT TRUNCATED AT 250 WORDS)

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Somatostatin (SRIF) and GRFs play key roles in regulating GH secretion. We previously presented a model of SRIF-cAMP interaction; SRIF blocks rat (r) GH release without preventing its accumulation in a potentially releasable pool. This phenomenon may represent a mechanism whereby tonic SRIF inhibition and its subsequent reduction or withdrawal can modulate the magnitude if not the initiation of rGH pulses. Herein we test that model using human GRF-44 (hGRF-44). Tritium-prelabeled rat anterior pituitary fragments were perifused until stored [3H]rGH and [3H]rPRL release rates were stable. SRIF (10 or 25 nM), with and without hGRF-44 (3 or 10 nM), was added in short (1-h hGRF-44) and long (3-h hGRF-44) protocols; SRIF was then withdrawn while hGRF-44 was continued. Release of stored prelabeled [3H]rGH and [3H]rPRL was assessed by immunoprecipitation. Effects on PRL release were followed for comparison. SRIF-induced inhibition of release was only partially reversed by hGRF-44. At these concentrations and so long as SRIF was present, hGRF-44 could not stimulate the rate of hormone release to values above pre-SRIF basal rates. On the other hand, the amplitude of post-SRIF rebound release was increased by prolonging exposure to SRIF alone, by including hGRF-44 with SRIF, by increasing the amount of hGRF-44 included with SRIF, by prolonging exposure to hGRF-44 plus SRIF, and by using a smaller concentration of SRIF during exposure to hGRF-44. Interaction of hGRF-44-SRIF effects generated peak rates of hormone release after SRIF withdrawal which exceeded the maximum rates achieved using hGRF-44 alone in this system. Lactotroph responses were much smaller, but qualitatively resembled somatotroph responses. We conclude that the interplay of simultaneous hGRF-44 and SRIF effects can regulate the amplitude of rGH pulses. Although GRF can initiate physiological GH release, and GRF antisera can block GH pulses, we suggest that the surge of release that follows reduction of SRIF-induced inhibitory tone in vitro represents a potential mechanism that could contribute to the initiation of some pulses of release. Finally, we also present a theoretical model of secretagogue interactions at the cellular level to explain our results. The model is compatible with either a homogeneous cell population in which each secretory cell has multiple capabilities or a heterogeneous cell population composed of cell subgroups with complementary secretory abilities.

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UNLABELLED: Human pancreatic growth hormone-releasing factor-44 (hpGRF-44) differentially stimulates release of stored and newly synthesized rGH without altering rGH synthesis over 3 h in static in vitro incubation; hpGRF-44 also stimulates release of stored, but not newly synthesized, rPRL. To study the time course of pre-labeled, stored hormone release without pharmacologically interrupting synthesis, the current experiments were performed in perifusion. Fifteen minute pulses of 0.1 to 10 nM hpGRF-44 stimulated stored [3H]rGH release (to 890% of base); 1.0 to 10 nM hpGRF-44 stimulated stored [3H]rPRL release (to 440% of base). Pulses of 0.1 to 1.0 mM (Bu) 2cAMP also stimulated release of [3H]rGH (to 570% of base) and [3H]rPRL (to 410% of base). However, peak [3H]rGH and [3H]rPRL responses to hpGRF-44 required 10 min, while peak responses to (Bu) 2cAMP required 25 min. Continuous hpGRF-44 stimulated an initial surge of stored [3H]rGH release which was not sustained; the diminishing release was not explained by hpGRF-44 degradation. Total radioimmunoassayable (RIA) hormone release roughly paralleled release of stored immunoprecipitable (IPn) hormone. CONCLUSIONS: in pituitary perifusion, hpGRF-44 stimulates release of both stored rGH and rPRL as shown in static incubation, but the response is biphasic: initial rapid release is followed by a progressively lesser response; and the response is both more acute and less well sustained than that resulting from exposure to (Bu) 2cAMP.

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Effects of synthetic human pancreatic GH-releasing factor-44 (hpGRF-44) on synthesis and release of rat pituitary GH and PRL were examined in vitro in a static incubation system. A double label, specific immunoprecipitation protocol permitted simultaneous study of hormone synthesis as well as release of both stored and newly synthesized hormone. Synthetic hpGRF-44 (0.3 and 3.0 nM) stimulated the release of stored GH 240% beyond the basal level, while simultaneously stimulating the release of newly synthesized GH by 610%. Despite the stimulation of release, hpGRF-44 did not alter GH synthesis (102% of control value). A small but statistically significant increase in release of stored PRL occurred in response to hpGRF-44, while release of newly synthesized PRL and PRL synthesis were unaffected. In contrast, 1 mM (Bu)2cAMP stimulated the release of both newly synthesized and stored GH and PRL. We conclude that hpGRF-44 differentially stimulates GH release from separate intracellular compartments and that the lactotroph may also, under certain conditions, respond to this secretagogue.

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Conventional methods for dialyzing numerous samples are either expensive or tedious and inefficient. These disadvantages were overcome through the construction and use of a Plexiglas dialysis sample holder (DSH). Large numbers of dialysis samples having 0.5 to 2.0-ml volumes may be attached to numbered positions on the DSH. Sample identification is greatly simplified and considerable savings in time and material are achieved. Furthermore, the risk of sample spill or mixing during filling or emptying of dialysis sacks, and the risk of leaks in dialysis tubing, are minimized.

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