Cloning, genomic organization and expression pattern of a novel Drosophila gene, the disco-interacting protein 2 (dip2), and its murine homolog.

We report the cloning and initial characterization of a novel gene encoding the Disco interacting protein 2 (Dip2). dip2 DNA complementary to RNA (cDNA) showed a high degree of sequence similarity to cDNAs of unknown function previously identified in humans and Caenorhabditis elegans. We have cloned the mouse homolog of the dip2 cDNA and characterized the expression of this gene by Northern blotting analysis and in situ hybridization to whole mount embryos. Our observations demonstrate that there is a remarkable degree of sequence conservation at the dip2 locus that is reflected in the nervous system-specific expression of both the Drosophila and mouse homologs.

Enhanced osteoblast development after continuous infusion of hPTH(1-84) in the rat.

Rats and humans respond to intermittent treatment with parathyroid hormone (PTH) with increased bone density and cancellous bone volume. In the rat, osteoblast expression of insulin-like growth factor-I (IGF-I) is elevated by intermittent PTH. We examined the effect of continuous infusion of rhPTH(1-84), a bone catabolic regime, on the IGF system in rat pelvis. Female Sprague-Dawley rats (12 weeks, 250 g) were randomly assigned to receive 0, 0.1, 1, or 5 microg/100 g body weight (b.w.) rhPTH(1-84) (0, 0.106, 1.06, or 5.305 nmol/kg) in vehicle (1% normal rat serum in saline) delivered by subcutaneous Alzet minipump. After 7 days, blood was taken for serum chemistry and pelvises were processed for immunocytochemistry. Sections of pelvis from rats continuously infused with 0.1 or 1 microg/100 g b.w. rhPTH(1-84) for 7 days did not differ significantly from those of the vehicle-treated controls. However, continuous infusion of 5 microg/100 g b.w. rhPTH(1-84) resulted in a dramatic increase in cellular development, with trabeculae surrounded by many layers of large, plump osteoblasts. All pelvis osteoblasts expressed osteocalcin, but only those from rats that received 0, 0.1, or 1 microg/100 g b.w. rhPTH(1-84) showed positive staining for IGF-I. The extra-abundant osteoblasts from rats that received 5 microg/100 g b.w. rhPTH(1-84) did not stain for IGF-I. However, although all osteoblasts stained positively for IGF binding proteins (IGFBPs)-3, -4, and -5, staining for these IGFBPs increased as the dose of rhPTH(1-84) (and osteoblast number) increased. These results suggest that continuous infusion of PTH has a direct effect on osteoblast development (either recruitment or proliferation), decreases the expression of IGF-I, and enhances the expression of IGFBPs in pelvis, factors which may interact to bring about negative bone balance.

Parenting and potency: alternative routes to reproductive success in male Mongolian gerbils

Adult male Mongolian gerbils, Meriones unguiculatusgestated in intrauterine positions between two female fetuses (2F males) are less likely than are adult males gestated between two male fetuses (2M males) to impregnate strange female gerbils with whom they are paired. The reduced copulatory success of 2F males is correlated with both lower circulating levels of and reduced sensitivity to testosterone. We asked whether 2F male gerbils compensated for their reduced copulatory success by increasing their parental effort. 2F male gerbils engaged in less sexual activity with their mates, but were more frequently in contact with pups than were 2M males, huddling over the young when their mates were absent from the nest. Although there were no differences in rates of survival or growth of pups reared by pairs consisting of a female and either a 2M or 2F male, mates of 2F males delivered significantly more pups as a consequence of copulations occurring during postpartum oestrus than did either mates of 2M males or females rearing young alone. We interpreted these results as consistent with Ketterson & Nolan's (1992, Am. Nat. (Supplement)140, 533-562) hypothesis of a testosterone-mediated trade-off between investment in sexual and parental behaviours.1997The Association for the Study of Animal Behaviour

Increase of basic fibroblast growth factor (FGF) and FGF receptor messenger RNA during rat thyroid hyperplasia: temporal changes and cellular distribution.

Goitre was induced in adult rats by acute (1 or 2 weeks) or chronic (4 or 10 weeks) administration of methimazole together with a low iodine diet. Involution of thyroid growth was then observed at 16 weeks, 4 weeks after withdrawal of goitrogens and reversion to a normal diet. Experimental animals quickly became hypothyroid compared with controls and exhibited thyroid hyperplasia (control (n = 10): total serum thyroxine (T4) 66 +/- 4 nmol/l, thyroid weight 5 +/- 1 mg/100 g body weight, means +/- S.D.; experimental (n = 10): T4 undetectable, thyroid weight 27 +/- 4 mg/100 g body weight after 2 weeks of treatment). Thyroid growth rate subsequently slowed between 2 and 10 weeks. Messenger RNA for basic fibroblast growth factor (basic FGF) and for the high-affinity FGF receptor, was compared in the thyroids and livers of control and goitrous rats by ribonuclease protection assay. Low levels of mRNA for basic FGF and its receptor were detectable in thyroids from control rats at all times, while none was detected in the livers from any animal. Basic FGF and receptor mRNAs increased, and were detected at greatest abundance in hyperplastic thyroids at 1 and 2 weeks respectively, during goitre formation, but subsequently declined in parallel with thyroid growth rate at 4 and 10 weeks. When quantified by radioimmunoassay, basic FGF extracted from thyroids was fivefold greater than in controls after 1 week of goitrogen treatment (control (n = 4): 24 +/- 9 pmol/micrograms DNA; goitre (n = 4): 100 +/- 16 pmol/micrograms DNA; P < 0.05). Basic FGF and FGF receptor mRNAs localized by in situ hybridization predominantly to the epithelial cell population within follicles. Localization by immunohistochemistry demonstrated that basic FGF was present in the thyroids of control rats, and was largely associated with the basement membrane of follicles. During thyroid hyperplasia, increased basic FGF immunoreactivity appeared over the cytoplasm of follicular epithelial cells and was lost from the extracellular matrix. Thyroid involution following removal of goitrogen/low iodine treatment was associated with a decrease in mRNA for basic FGF or its receptor, and a loss of immunoreactive basic FGF from the cytoplasm of follicular cells. These results suggest that autocrine expression of basic FGF and FGF receptor could contribute to thyroid hyperplasia in rats.

Development of enzyme-linked immunosorbent assays for measurement of leukotrienes and prostaglandins.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the leukotrienes LTC4 and LTB4 and the prostaglandins 6-keto PGF1 alpha and thromboxane (TxB2). In an indirect assay procedure for all 4 eicosanoids a BSA conjugate of the leukotrienes or an ovalbumin conjugate of the prostaglandins was absorbed to polystyrene microtiter plates. Samples containing the respective eicosanoids were incubated in the coated wells with specific rabbit antisera. The wells were then incubated successively with a goat anti-rabbit antibody linked to fluorescein and a rabbit anti-fluorescein antibody linked to alkaline phosphatase. The resultant assays for LTC4, LTB4, 6-keto PGF1 alpha, and TxB2, gave steep, sensitive inhibition curves; IC50s were 0.2, 10, 1, and 0.4 pmol respectively with minimal cross-reactivity to other eicosanoids. The sensitivities and specificities were comparable to those found in the RIA, and the levels determined in this assay correlate well with those determined in non-immunological assays.

Statistical consequences of reducing the number of rabbits utilized in eye irritation testing: data on 67 petrochemicals.

Data from 67 six-rabbit eye irritation tests were used to generate two-, three-, and four-rabbit Draize scores. The 15 two-rabbit, 20 three-rabbit, and 15 four-rabbit "subsample" scores for each of the 67 petrochemicals tested were used to establish prediction intervals for the original six-rabbit scores. Prediction interval length shortens with increasing sample size, is widest in the middle portion of the Draize scale, and can be used to select the minimum number of rabbits necessary to satisfy a required level of precision. Also, the ability of each subsample size to correctly classify the test materials according to an in-house irritation classification system was evaluated. Results indicate that subsamples of size 2, 3, 4, and 5 were 88, 93, 95%, and 96% accurate, respectively (compared to six rabbits), at correctly classifying the irritation potential of the materials tested.

Subchronic inhalation toxicology of ethylene glycol monoethyl ether in the rat and rabbit.

The subchronic inhalation toxicology of ethylene glycol monoethyl ether (EGEE) was evaluated in rats and rabbits using a 13-week exposure regimen. Groups of 20 rabbits (10 M, 10 F) and 30 rats (15 M, 15 F) were exposed to a vapor of 25 ppm, 100 ppm, or 400 ppm, 6 hr/day, 5 days/week. The control groups received air only. Physical examinations and body weight measurements were conducted on all animals pretest and weekly throughout the study. Ophthalmoscopic examination was performed pretest and at termination. Evaluation of hematology and clinical chemistry was conducted on 10 animals per sex per group from each species after 13 weeks of study. Histopathological changes were assessed for all animals from the high-dose and control groups. In addition, selected tissues were examined from all animals from the mid- and low-dose groups. Both species exhibited an increased incidence of lacrimation and mucoid nasal discharge, but the response was not consistently dose-related. Rats exposed to EGEE showed no compound-related effects except for a decrease in pituitary to body weight ratio for high-dose males and a decrease in absolute spleen weight for all female animals. The spleen to body weight ratio was also less than controls for the females in the low- and high-dose groups. Pathological changes supportive of these organ weight changes were not observed. The rabbit is the more sensitive species to the subchronic toxicological effects from EGEE. Mean body weight values for low- and high-dose animals were decreased; the mid-dose animals, however, showed no change.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukotriene A4: preparation and enzymatic conversion in a cell-free system to leukotriene B4.

Treatment of leukotriene A4 (LTA4) methyl ester with sodium hydroxide in aqueous methanol at 4 degrees C afforded LTA4, the presence of which was inferred from the UV spectrum of the compound, its rate of reaction with water, and the identity of the hydration products obtained. The half-life of LTA4 in water (pH 7.4, room temperature) was increased from 14 to 500 s by 1 mg/ml of bovine serum albumin. This stabilized (chiral) LTA4 was converted to LTB4 by an epoxide hydrolase activity in the 100,000 x g supernatant fraction from sonified rat basophilic leukemia cells. Neither the ester of LTA4 nor the biologically incorrect enantiomer of LTA4 was metabolized to LTB4 under these conditions.