Deposition, exhumation, and paleoclimate of an ancient lake deposit, Gale crater, Mars.

The landforms of northern Gale crater on Mars expose thick sequences of sedimentary rocks. Based on images obtained by the Curiosity rover, we interpret these outcrops as evidence for past fluvial, deltaic, and lacustrine environments. Degradation of the crater wall and rim probably supplied these sediments, which advanced inward from the wall, infilling both the crater and an internal lake basin to a thickness of at least 75 meters. This intracrater lake system probably existed intermittently for thousands to millions of years, implying a relatively wet climate that supplied moisture to the crater rim and transported sediment via streams into the lake basin. The deposits in Gale crater were then exhumed, probably by wind-driven erosion, creating Aeolis Mons (Mount Sharp).

Efficacy of eprinomectin and doramectin against Amblyomma americanum (Acari: Ixodidae) on cattle.

Steers were treated with doramectin or eprinomectin by daily oral capsule for 28 consecutive days. The level of doramectin in the serum of steers treated at 200 microg/kg/d reached a maximum of 104.0 +/- 22.1 ppb at day 21 and declined from 93.3 +/- 20.5 ppb on the final day of treatment to below detectable by day 56. Steers treated at 50 microg/kg/d reached a maximum level of doramectin in the serum of 24.7 +/- 1.2 ppb on day 21 and declined from 24.7 +/- 0.6 ppb on the final day of treatment to less than detectable on day 42. Both doramectin dosages provided 100% control of estimated larvae (EL) of Amblyomma americanum (L.) (Acari: Ixodidae) throughout the 28-d treatment period. Daily oral treatment with eprinomectin at a dosage of 200 microg/kg for 28 consecutive days produced a maximum concentration in the serum of 41.6 +/- 11.0 ppb at day 14. On the final day of eprinomectin treatment, the serum concentration was 38.3 +/- 5.9 ppb. Seven days later at day 35, eprinomectin was not detectable in the serum. For steers treated at 50 microg/kg/d for 28 consecutive days, the serum level of eprinomectin reached a maximum of 10.0 +/- 3.0 ppb on day 28 and was undetectable on day 35. Both eprinomectin dosages provided complete control of EL of A. americanum during the 28-d treatment period. Because eprinomectin is efficacious against A. americanum at lower serum levels in cattle and is eliminated from the serum at a more rapid rate than either doramectin or ivermectin, it provides advantages for use in applications such as the medicated bait for control of ticks on white-tailed deer and could have potential for use in the Cattle Fever Tick Eradication Program.

Pharmacokinetics of ivermectin in llamas (Lama glama).

The pharmacokinetic behaviour of ivermectin was investigated in adult llamas (Lama glama) by using high performance liquid chromatography with a lower limit of quantification of 2 ng/ml to measure its concentration in serum. Llamas were treated with one of three commercial formulations (injectable, pour-on or oral paste) at dosages recommended by the manufacturer, or with an experimental injectable sustained-release formulation. In five llamas given 1 per cent ivermectin subcutaneously at 200 microg/kg, the median peak serum concentration (Cmax) was 3 ng/ml and the area under the serum concentration-time curve (AUC) was 13.5 ng x day/ml. In six llamas treated topically with 0.5 per cent ivermedin pour-on at 500 microg/kg, Cmax was 2.5 ng/ml or less and the AUC was 7.75 ng x day/ml or less. In seven llamas with measurable concentrations of ivermedin, the median times to peak serum concentration (tmax) were six days after subcutaneous injection and seven days after treatment with the pour-on formulation. In six llamas, the serum concentration of ivermectin remained less than 2 ng/ml for 124 hours after treatment with a 1.87 per cent oral paste at 200 microg/kg. In five llamas treated subcutaneously with 25 per cent ivermectin sustained-release microspheres at 1500 microg/kg, the median Cmax was 5 ng/ml and the median AUC was 224 ng x day/ml.

The Ivomec SR Bolus for control of Boophilus annulatus (Acari: Ixodidae) on cattle in South Texas.

When Hereford heifers infested with Boophilus annulatus (Say) were treated with a single Ivomec SR Bolus, the concentration of ivermectin in the serum of the treated cattle reached a maximum of 8.8 +/- 0.9 ppb at 2 wk posttreatment. The single bolus treatment resulted in 84.4% control of standard engorging B. annulatus females on treated cattle over the 20-wk trial. Although fewer engorged ticks were collected from the sentinel heifers exposed in the treated pasture than those in the control pasture at weeks 4, 10, and 16 posttreatment, none of the differences was statistically significant. Each exposure of sentinel cattle found free-living ticks in both the treated and control pastures, indicating the infestation was not eliminated by the treatment. When the trial was repeated using two Ivomec SR Boluses/heifer, the concentration of ivermectin in the serum of the treated cattle reached a maximum level of 31.2 +/- 3.9 ppb at week 13 posttreatment. The use of two boluses/heifer resulted in 99.6% control of standard engorging B. annulatus females over the 20-wk trial. No ticks were found on sentinels placed in the treated pasture after week 9 posttreatment, an indication that the treatment had eliminated the free-living population in the treated pasture. From these studies, we conclude that a single Ivomec SR Bolus is incapable of sufficient control of B. annulatus to meet the rigid requirements of the Cattle Fever Tick Eradication Program in South Texas. Although two boluses per animal did eliminate the ticks from treated heifers and the pasture they were in, the treatment would not be sufficiently efficacious for mature cattle (>400 kg) for it to be useful in the program.

Evaluation of horn flies (Diptera: Muscidae) from a pyrethroid susceptible colony for general and permethrin esterase activities.

In this study we describe a nonradioactive single-fly microassay for permethrin hydrolysis. We used this assay with a microplate assay for general esterase activity to evaluate the permethrin hydrolyzing and general esterase activities of aging pyrethroid-susceptible male and female horn flies, Haematobia irritans (L.). We found substantial gender- and age-related differences regarding general esterase activity, permethrin sensitivity, and permethrin hydrolyzing activity within the colony. Extracts of female flies collected 48 h after receiving their first blood meal yielded significantly greater esterase activity than male extracts. Aging female flies were more tolerant of permethrin than were male flies. In addition, a positive correlation was found to exist between the general esterase activity of aging females and their ability to hydrolyze permethrin.

Control of Boophilus annulatus (Acari: Ixodidae) on cattle using injectable microspheres containing ivermectin.

The efficacy of an injectable microsphere formulation of ivermectin for control of the cattle tick, Boophilus annulatus (Say), was tested on 2 groups of 6 Hereford heifers held on separate 7-ha, tick-infested, buffel grass pastures. Cattle in one pasture were injected subcutaneously in the neck with a controlled-release microsphere formulation of ivermectin at the rate of 2.4 mg AI/kg body weight; the other group was injected with carrier only. Beginning 4 wk after injection and continuing throughout the remainder of the test (16 wk), no engorged ticks (> or = 5.5 mm) were found on any of the treated cattle, whereas large numbers of engorged ticks were found on the untreated controls. During this period, a few ticks were recovered from untreated sentinel animals placed in the treatment pasture during 7-8 wk after treatment, but none were recovered from animals exposed from 11-12 wk or 14-15 wk. Large numbers of B. annulatus ticks were found on untreated sentinel cattle placed in the control pasture during these same periods. Although the cattle, pastures, and tick habitat were approximately equal, the treated cattle gained an average of 77 kg compared with an average of 42 kg for the control group. This technology offers a possible alternative to the current official program of dipping and vacating pastures for eradication of Boophilus sp. infestations from the quarantine zone in southern Texas. Larger scale testing is needed to determine the potential of the injectable microsphere formulation and to optimize its use in eradication or control strategies.

Delivery of ivermectin by injectable microspheres.

A bioabsorbable, injectable microsphere formulation containing ivermectin in poly-(lactide-co-glycolide) copolymer (PLA/PGA) was developed to provide long-lasting delivery of the drug for control of livestock pests. A solvent-evaporation technique was used to produce the spherical beads containing approximately 30% ivermectin and ranging in size from 25-250 microns. The pattern of delivery of the drug into the blood stream of Spanish goats was characterized for a 50:50 PLA/PGA, a 90:10 PLA/PGA copolymer formulation, and a PLA monomer formulation. When the 50:50 PLA/PGA formulation was used in cattle at the rate of 2 mg (AI)/kg body weight, 2 peaks of 45-50 ppb of ivermectin in serum were observed. The 1st peak was at approximately 1 wk after injection and the 2nd peak, which was broader than the 1st, occurred at approximately 6-7 wk after injection. Percentage of inhibition of estimated larvae for the lone star tick, Amblyomma americanum (L.), placed on treated cattle was 100% for the first 8 wk after injection and was 75, 57, 46 and 44% for wk 9, 10, 11, and 12, respectively. The treatment provided 98-100% control of larval horn flies, Haematobia irritans (L.), in the manure of treated cattle for 10 wk. The bioassay results against lone star ticks and larval horn flies were in agreement with the serum concentration data. The injectable microsphere formulation of ivermectin should be useful in a variety of other applications ranging from the control of Boophilus spp. ticks in south Texas to heartworms in pets.

Protective effect of glutamine from glucocorticoid-induced muscle atrophy occurs without alterations in circulating insulin-like growth factor (IGF)-I and IGF-binding protein levels.

We investigated whether the prevention of glucocorticoid-induced muscle atrophy by glutamine infusion is associated with alterations in serum levels of insulin-like growth factor (IGF)-I and its binding proteins (IGFBPs). Hormone (cortisol acetate [CA], 100 mg/kg body wt/day) and vehicle (carboxymethyl cellulose [CMC])-treated female rats were infused with either saline or glutamine (240 mM, 0.75 ml/hr) for a 7-day period. Glutamine infusion prevented over 70% of the skeletal muscle mass loss due to the glucocorticoid injections. Serum IGF-I concentrations, which were measured by radioimmunoassay (RIA) after acid solid-phase extraction of IGFBPs, were not significantly different among groups (range of means: 373-395 ng/ml). Saline/CA treatment resulted in a 2-fold increase in circulating levels of IGFBP-3 (38- to 50-kDa bands from ligand blotting measurements) versus the saline/CMC group. Levels of 30- to 32-kDa bands were increased by approximately 3-fold in the CA-treated rats. Immunoprecipitation studies suggested that the increase in the 30- to 32-kDa binding proteins were not due to elevated levels of IGFBP-1, -2, or -5. None of the treatments significantly modified circulating levels of IGFBP-4 (24 kDa). Glutamine infusion did not reverse the effects of glucocorticoids on circulating levels of 38- to 50- and 30- to 32-kDa IGFBPs. We conclude that the attenuation of glucocorticoid-induced muscle atrophy by glutamine infusion is not associated with changes in circulating levels of IGF-I or IGFBPs.

Microdissection and DOP-PCR-based reverse chromosome painting as a fast and reliable strategy in the analysis of various structural chromosome abnormalities.

Reverse chromosome painting has become a powerful tool in clinical genetics for the characterization of cytogenetically unclassifiable aberrations. In this report, the application of a sensitive and rapid procedure for the complete and precise identification of four different de novo structural chromosome abnormalities is presented. These chromosome rearrangements include a marker derived from chromosome 3(cen-q11), an interstitial deletion of chromosome 13 [del(13)(q14q22)], an unbalanced translocation [46,XY, -4, +der(4)t(4;8)(p 15.2;p21.1)] leading to Wolf-Hirschhorn syndrome, and a partial inverted duplication in conjunction with a partial deletion of chromosome 5p [46,XX, -5, +der(5)(:p13-p15.1::p15.1-qter)] which is responsible for the manifestation of the cri-du-chat syndrome. The importance of a fast and reliable evaluation of complex chromosome aberrations in pre- and postnatal diagnosis with regard to comprehensive genetic counselling is emphasized.

Systemic treatment of white-tailed deer with ivermectin-medicated bait to control free-living populations of lone star ticks (Acari:Ixodidae).

Whole-kernel corn was treated with 10 mg ivermectin per 0.45 kg corn and fed at rate of approximately .45 kg/deer per day to white-tailed deer confined in the treatment pasture, whereas deer in an adjacent control pasture received a similar ration of untreated corn. Treatments were dispensed from February through September of 1992 and 1993, and free-living populations of lone star ticks. Amblyomma americanum (L.), were monitored in both pastures using dry-ice traps to quantify nymphs and adults and flip-cloths to assay the relative abundance of larval masses. Control values that were calculated for all ticks collected in both pastures during 1993 showed 83.4% fewer adults, 92.4% fewer nymphs and 100.0% fewer larval masses in the treatment versus control pasture. Serum ivermectin concentrations in treated deer averaged 21.7 and 28.3 ppb during 1992 and 1993, respectively. These values compared favorably with the goal concentration of 30.0 ppb which was anticipated under ideal conditions. This study demonstrates that a freely consumed, systemically active acaricidal bait ingested by white-tailed deer under nearly wild conditions can significantly reduce the abundance of all stages of free-living long star ticks.

Regional distribution and characterization of nitric oxide synthase activity in the brain of the common marmoset.

The distribution of nitric oxide synthase (NOS) within the brain of the common marmoset, a non-human primate species, was investigated using the [3H]L-citrulline formation assay and Western blot analysis. No hemispheric asymmetry of specific NOS activity was shown. The highest levels of NOS were found in the putamen and caudate nucleus--more than twice those in the cortex and the cerebellum, the brain regions with the lowest activities. The regional distribution pattern was similar to that in the ferret brain and contrasted to that in the mouse and bovine brain. Analysis of NOS catalytic activities in subcellular fractions revealed marked differences in the subcellular localization. Neuronal NOS accounted mainly for the measured catalytic activity in the brain. Differences in the regional distribution pattern of brain NOS activity among species may be indicative of diversities in the functional role of nitric oxide and NOS in mammals.

Nutritional insult and recovery in the neonatal rat cerebellum: insulin-like growth factors (IGFs) and their binding proteins (IGFBPs).

Alterations in growth caused by neonatal malnutrition may be mediated in part by changes in insulin-like growth factor (IGF) and IGF binding protein (IGFBP) expression. Since the neonatal rat cerebellum undergoes a transient, proliferative growth phase in the first two weeks of life, this structure was used to determine whether alterations in circulating and tissue IGFs and IGFBPs may mediate effects of impaired nutrition on the developing central nervous system. Gravid rats were placed on a 4% (protein-calorie deprived, D) or 20% (control, C) protein diets one day prior to delivery and allowed to nurse their pups postpartum. Pups nursing from D mothers received a limited volume of milk and were calorically deprived. Some litters of D pups were foster fed by C mothers from day 8 to day 13 to constitute a recovery group (R). Cerebellar weight, protein, and DNA content in D pups were less than C, p < 0.001. In R pups, DNA and protein returned to C levels by day 13. Between days 6 and 13, serum IGF-I levels rose from 158 +/- 18 to 210 +/- 18 ng/ml in C but remained low in D (47 +/- 6 ng/ml and 25 +/- 3 ng/ml), respectively. In R pups, serum IGF-I partially recovered during this time, and increased from 49 +/- 5 to 110 +/- 7 ng/ml. In cerebellar extracts, IGF-I levels in both C and D were lower at 13 days than at 6 days, p < 0.05 and p < 0.005, respectively. IGF-I levels in C were similar at day 9 and day 11 and were consistently higher than D (11.84 +/- 0.83 vs 8.56 +/- 0.92 ng/g, p < 0.02 C vs D). In R, IGF-I was reduced on day 11, but was similar to C on day 13. Serum IGF-II in D was lower than C, p < 0.01, and did not increase in the R group. Cerebellar IGF-II was virtually undetectable in either group. Immunoprecipitation and ligand blotting studies of serum demonstrated that circulating levels of 32-34 K IGFBPs were increased 3-4 fold in D vs C, reflecting high levels of IGFBP-1 and/or -2, while levels of 24 K IGFBP-4 were lower in D vs C. By contrast, immunoprecipitation and ligand blotting of cerebellar extracts detected IGFBP-2 and -4, but did not detect IGFBP-1. Further, tissue levels of IGFBP-2 were not increased in D vs C, and levels of IGFBP-4 also were not markedly affected by nutritional deprivation. These results suggest that alterations in tissue content and the availability of IGF-I only modestly contributed to the effects of impaired nutrition in the developing central nervous system.

A novel DNA/protein complex interacts with the insulin-like growth factor binding protein-1 (IGFBP-1) insulin response sequence and is required for maximal effects of insulin and glucocorticoids on promoter function.

Glucocorticoids stimulate and insulin inhibits hepatic production of IGFBP-1 at the level of gene transcription. We previously identified contiguous insulin and glucocorticoid response sequences in the proximal rat IGFBP-1 promoter. This insulin response sequence (IRS) is palindromic (CAAAACAAACTTATTTTG) and each half resembles an IRS in the phosphoenolpyruvate carboxykinase (PEPCK) gene. We have reported that both the IGFBP-1 and PEPCK IRSs bind hepatocyte nuclear factor-3 (HNF-3) proteins [1]. We now report that IRSs from the IGFBP-1 and PEPCK, as well as an IRS which also binds HNF-3 in the rat tyrosine aminotransferase (TAT) gene, also interact with another DNA/protein complex in gel shift studies. Further, methylation interferences studies, gel shift and transient transfection studies with site-specific mutations identified a single base in the first half of the IRS that is critical both for interactions with proteins in this complex, and for maximal effects of insulin and glucocorticoids, on promoter function. Of note, a 250-fold excess of an oligo containing a C/EBP binding site (but not other AT-rich sequences) inhibits the formation of this complex in gel shift assays. Nevertheless, interactions with this C/EBP site are negligible at lower titers (< or = 100-fold excess), and antibodies against known C/EBP proteins do not react with this complex. Similarly, preincubation with CHOP, a truncated member of the C/EBP family which contains a beta-leucine zipper domain, does not prevent or alter the mobility of this novel DNA/protein complex, indicating that components of this complex do not form heterodimers with beta-ZIP proteins. We conclude that HNF-3 proteins and this novel C/EBP-related DNA/protein complex may play an important role in mediating interactions between glucocorticoids and insulin in the regulation of IGFBP-1 and perhaps multiple hepatic genes.

Moxidectin: pharmacokinetics and activity against horn flies (Diptera: Muscidae) and trichostrongyle nematode egg production.

The concentration of moxidectin, a macrocyclic lactone endectocide, in the blood serum of cattle resulting from single and daily subcutaneous injections and oral dosing was determined as a function of time. When given as a single subcutaneous (SC) injection, the drug peaked between 4 and 6 h post-treatment. As a single oral dose, the peak serum level occurred at 1 day post-treatment. Daily SC injections and oral doses resulted in a gradual increase in blood serum level over the 21 days of treatment but did not reach a plateau during this time. Horn flies, Haematobia irritans (Linnaeus), feeding on the blood of treated cattle drawn on Day 21 of daily treatment showed a decline in survival and egg production, but a negligible effect on egg hatching. Dose-mortality data on adult horn flies showed an LC-50 and LC-90 value of 10 ppb and 19 ppb in the blood, respectively. Moxidectin was also found to have larvicidal activity against the immature stages of the horn fly in the manure of treated cattle. Moxidectin administered at 100, 50 and 25 micrograms kg-1 as a daily oral medication was 100% effective in eliminating trichostrongyle egg counts by Day 3 of the treatment. Counts remained negative to the end of the trial.

Dideoxy sequencing and structural analysis of the rat insulin-like growth factor binding protein-1 gene.

Insulin-like growth factor binding protein-1 (IGFBP-1) is an important modulator of IGF bioavailability. To facilitate studies of IGFBP-1 regulation and function in rodent models, we cloned the rat IGFBP-1 gene and analyzed its structure by dideoxy sequencing. The rat IGFBP-1 gene is relatively small (approximately 5 kb) and contains 4 exons and 3 introns, similar to the human IGFBP-1 gene.

Effects of glucocorticoids on circulating levels and hepatic expression of insulin-like growth factor (IGF)-binding proteins and IGF-I in the adrenalectomized streptozotocin-diabetic rat.

Circulating levels and hepatic expression of insulin-like growth factor-binding protein-1 (IGFBP-1) are increased in insulin-deficient streptozotocin (STZ)-diabetic rats. Glucocorticoids stimulate and insulin suppresses hepatocellular expression of IGFBP-1 in vitro. We asked whether increased IGFBP-1 expression in STZ-diabetic animals is due to an effect of insulin deficiency per se or whether insulin deficiency represents a permissive state where glucocorticoids may play an important role in the regulation of IGFBP-1 and other circulating peptides involved in the modulation of IGF bioactivity. Intact female Sprague-Dawley-derived rats and rats undergoing bilateral adrenalectomy (ADNX) were injected with STZ (140 mg/kg) or buffer. Corticosterone acetate (50 mg/kg) or vehicle was administered to diabetic and nondiabetic animals immediately after ADNX and 24 h later. All rats were killed 48 h after surgery and/or STZ administration. Serum [125I]IGF-I-binding activity was increased 4-fold (P < 0.01), and Western ligand and immunoblotting demonstrated that levels of IGFBP-1 were high in intact STZ-diabetic animals. ADNX prevented these effects of STZ-diabetes, and corticosterone treatment restored serum IGF-binding activity and IGFBP-1 to intact diabetic levels. Similarly, Northern analysis demonstrated that the abundance of hepatic IGFBP-1 mRNA was increased 6-fold in intact STZ-diabetic animals (P < 0.01), but not in adrenalectomized diabetic animals. Corticosterone treatment restored hepatic IGFBP-1 mRNA to intact diabetic levels, and serum concentrations of corticosterone correlated with the abundance of IGFBP-1 mRNA (r = 0.475; P < 0.01), indicating that glucocorticoids play an important role in the regulation of expression of IGFBP-1 in insulin-deficient animals. In contrast, neither ADNX nor corticosterone altered the abundance of hepatic IGFBP-1 mRNA levels in nondiabetic animals. This pattern of regulation appeared to be specific; serum levels of immunoreactive IGFBP-2 and -4 tended to rise in adrenalectomized animals, and levels of IGFBP-3 were not affected by either ADNX or corticosterone treatment. Of note, serum levels of IGF-I by RIA were reduced in STZ-diabetic animals compared to control values (168 +/- 16 vs. 587 +/- 55 ng/ml, respectively; P < 0.01), were partially restored toward control values with ADNX (320 +/- 22 ng/ml), and were reduced again by corticosterone treatment (195 +/- 26 ng/ml), indicating that glucocorticoids also contribute to the regulation of IGF-I levels in insulin-deficient animals. The abundance of IGF-I mRNA was reduced in STZ-diabetic animals, and ADNX also partially prevented this effect of diabetes.(ABSTRACT TRUNCATED AT 400 WORDS)

Divergent effects of phorbol esters and insulin on insulin-like growth factor binding protein-1 (IGFBP-1) production and mRNA in rat H4IIE hepatoma cells.

125I-IGF-I binding assay, western ligand and immunoblotting, and northern analysis of total RNA reveal that phorbol ester agonists of protein kinase C rapidly enhance IGFBP-1 production and increase the abundance of IGFBP-1 mRNA in rat H4IIE hepatoma cells. In combination with insulin, a potent inhibitor of IGFBP-1 gene transcription, this early effect of phorbol esters is dominant. These results demonstrate divergent regulation of IGFBP-1 by phorbol esters and insulin and indicate that protein kinase C may play a critical role in the regulation of IGFBP-1 and modulation IGF bioactivity in metabolic disease.

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)

Circulating levels of insulin-like growth factor binding protein-1 (IGFBP-1) and hepatic mRNA are increased in the small for gestational age (SGA) fetal rat.

Insulin-like growth factors (IGFs) circulate in association with a family of specific binding proteins (BPs). Recently, we reported that circulating levels of IGFBP-1 and IGFBP-2 are increased in streptozotocin-diabetic adult rats and are differentially regulated in accordance with insulin and metabolic status. Since IGF BPs appear to be important modulators of IGF bioactivity in post-natal life, we asked whether serum levels of IGF BPs also might be altered in utero when the delivery of maternal nutrients is restricted and fetal growth is impaired. Bilateral uterine artery ligation or sham surgery was performed on maternal rats on d 19 of gestation (term 21.5 d). One day after ligation (d 20), fetuses were (SGA) compared to shams (3.1 +/- 1 vs 3.7 +/- 0.2 g, p less than .02) and serum levels of glucose (70 +/- 5 vs 96 +/- 6 mg/dL, p less than .01) and insulin (62 +/- 4 vs 138 +/- 14 microU/mL) also were reduced. In contrast, serum [125I]IGF-I binding activity was markedly increased in SGA litters compared to sham (65 +/- 5% maximum binding with 2.5 microL/mL SGA serum vs 14 +/- 3% for sham serum, p less than .001), and correlated with fetal weight (r = -0.539, p less than .05) and insulin (r = -0.622, p less than .05). Ligand blotting with [125I]IGF-I revealed that serum levels of IGFBP-1 (32 K) were greater in SGA than shams, while immunoblotting with specific antiserum demonstrated that levels of IGFBP-2 (34 K), the major fetal rat IGF BP, were similar in serum from SGA and shams litters. Affinity labeling and immunoprecipitation confirmed that IGF binding activity is increased in SGA, due largely to increased availability of IGFBP-1. In addition, Northern analysis of hepatic RNA revealed that the abundance of IGFBP-1 mRNA is increased in the SGA fetal rat, while hepatic mRNA for IGFBP-2 is similar in SGA and sham-operated litters. We conclude that circulating levels of IGFBP-1 and the abundance of hepatic mRNA are increased in the SGA fetal rat. IGFBP-1 may be an important modulator of IGF bioactivity and somatic growth in utero.

Production of the rat type 1 insulin-like growth factor-binding protein by well differentiated H4EIIC3 hepatoma cells: identification, purification, and N-terminal amino acid analysis.

We recently identified a 32 K mol wt insulin-like growth factor (IGF)-binding protein (BP) which is markedly increased in the serum of streptozotocin-diabetic rats and recognized by antiserum against the human amniotic fluid IGFBP (hIGFBP-1). In the present study we sought to confirm that this protein represents the rat homolog of IGFBP-1 (rIGFBP-1), and that rIGFBP-1 may, therefore, play an important role in the regulation of IGF bioactivity in experimental diabetes. Since the abundance of related hepatic mRNA is high in diabetic rats, we asked whether well differentiated H4EIIC3 rat hepatoma cells produce rIGFBP-1 and provide sufficient amounts of this protein for purification and further characterization. Specific IGF-binding activity in hepatoma conditioned medium was detected initially by incubation with 125I-labeled recombinant human IGF-II and precipitation with polyethylene glycol. Ligand blotting demonstrated a 32 K BP, identical in size to the major low mol wt IGFBP found in diabetic rat serum. Affinity labeling and immunoprecipitation confirmed that this BP is related to human IGFBP-1 and is distinct from the fetal rat IGFBP, rIGFBP-2. Incorporation of [35S]methionine into 32 K BPs confirmed synthesis by hepatoma cells. For purification of BPs, conditioned medium was collected in roller culture, and BPs were purified by ammonium sulfate precipitation, Sephadex G-75 chromatography, and reverse phase HPLC. Partial amino acid sequencing of purified protein demonstrated 68% identity with the human IGFBP-1 and distinguished this BP from previously characterized rat IGFBPs. Purified protein bound both IGF-I and IGF-II with high affinity. We conclude that the 32 K IGFBP produced by H4EIIC3 hepatoma cells in culture represents the rat form of IGFBP-1 (rIGFBP-1). Regulation of rIGFBP-1 may play an important role in the modulation of IGF bioactivity in experimental animals with metabolic disease. The availability of purified rIGFBP-1 and identification of a cell line that produces this BP will greatly facilitate future studies of IGFBP-1 in the rat model.