Expression of adrenocorticotropin-releasing hormone precursor gene in placenta and other nonhypothalamic tissues in man

Adrenocorticotropin-releasing hormone (CRH) is a peptide originally isolated from the hypothalamus. Immunocytochemical and RIA studies have revealed that CRH-like peptide is also localized in human nonhypothalamic tissues and some tumors. To see if CRH is synthesized in these nonhypothalamic tissues and tumors, we examined preproCRH mRNA in these tissues by Northern blot analysis using a cloned human preproCRH gene as a probe. PreproCRH mRNA was detected in human hypothalamus, cerebral cortex, adrenal gland, placenta, pheochromocytoma, and thymic carcinoid. The content of preproCRH mRNA in placenta was apparently greater than that in the whole hypothalamus.     

Human corticotropin-releasing hormone test in normal subjects and patients with hypothalamic, pituitary or adrenocortical disorders

Human corticotropin-releasing hormone (hCRH) test was performed in 57 normal volunteers and 102 patients with hypothalamic, pituitary and adrenocortical diseases. Intravenous bolus injection of synthetic hCRH, 100 micrograms for adults or 1.5 micrograms/kg for children, increased plasma ACTH and cortisol levels in about 90% of normal subjects. In 47 patients with Cushing's disease, plasma ACTH tended to show an exaggerated response to hCRH and peak ACTH was the most frequent abnormal component among the several reaction parameters. Poor responders among normal subjects and patients with Cushing's disease had significantly higher plasma cortisol levels before CRH administration. Patients with hypothalamic hypopituitarism showed exaggerated response, whereas patients with primary pituitary lesion, isolated ACTH deficiency or adrenal Cushing's syndrome showed no ACTH response. These differences in the response of patients suggest the value of the hCRH test in their differential diagnosis.     

Immunochemical study on PHI/PHM with use of synthetic peptides

We have synthesized PHI and PHM (human PHI) as well as their fragments, PHI (1-6), PHI (1-15), PHI (14-19), PHI (14-27), PHI (20-27), PHM (1-15) and PHM (13-27), by the solution or solid-phase method for peptide synthesis. Using the highly purified synthetic peptides as immunogens or haptenic immunogens, five kinds of PHI/PHM specific antisera were produced. The major antibody-recognition sites of the five antisera were located respectively in the PHI C-terminal (R8201), in the PHI N-terminal (R8403), in the PHM C-terminal (R8502), and in the PHM whole molecule (R8702 and R8703). Radioimmunoassays (RIAs) with antisera R8201, R8403 and R8502, respectively, showed a wide distribution of immunoreactive (IR) PHI/PHM in porcine and human gastrointestinal and brain tissues. The concentrations of IR-PHI in the porcine gastrointestinal tissues, however, differed between the R8201 and R8403 RIAs employed for measurement. By using these two different PHI RIAs, the IR-PHI in the porcine brain tissue extract was shown to be almost a single component coeluting with synthetic PHI in gel filtration. The IR-PHI in the extract of porcine lower intestine on the other hand, contained, besides a PHI-like component, unidentified component(s) eluting immediately after synthetic PHI in gel filtration; this crossreacted with the PHI C-terminal specific R8201 antiserum but not with the N-terminal specific R8403 antiserum, suggesting the presence of the C-terminal-related fragment(s) of PHI in the tissues.     

Presence of immunoreactive alpha-endorphin in rat pituitary gland.

Utilizing radioimmunoassay for α-endorphin, we attempted to demonstrate immunoreactive α-endorphin in acid extracts of pars distalis and combined pars intermedia and pars nervosa of the rat pituitary gland after chromatography on Sephadex G-25. β-Lipotropin, β-endorphin and γ-endorphin were not converted into α-endorphin during the extraction and gel chromatographic procedures. Concentrations of immunoreactive α-endorphin determined after gel chromatography of extracts from pars distalis and combined pars intermedia and pars nervosa were 1.1±0.6 and 130±17 ng/mg wet tissue (mean±SE), respectively. Serial dilution of these extracts gave parallel lines to the standard curve of synthetic α-endorphin, but not to that of γ-endorphin or δ-endorphin. These results suggest the existence of immunoreactive α-endorphin indistinguishable in molecular size from synthetic α-endorphin in the rat pituitary gland.     

Chloroplast DNA topoisomerase I from cauliflower

An ATP-independent DNA topoisomerase has been isolated from chloroplasts of cauliflower leaves (Brassica oleracea var. botrytis) through DEAE-cellulose, AF-blue Toyopearl, and hydroxyapatite column chromatography. The sedimentation coefficient and Stokes radius of this enzyme are 3.6S and 3.6 nm, respectively, and the molecular weight of native enzyme is estimated to be 54,000. This enzyme changes the linking number in steps of one. The enzyme activity is stimulated by MgCl2, and this enzyme shows optimum activity at 30 degrees C in the range of 3 mM MgCl2 + 100 mM KCl-10 mM MgCl2 + 50 mM KCl. The enzyme activity was reduced remarkably by N-ethylmaleimide, indicating that a free sulfhydryl group is important for the activity; heparin and ellipticine also reduced the activity. Both cauliflower chloroplast topoisomerase and spinach chloroplast topoisomerase can relax positive supercoils as well as negative supercoils. From these properties, cauliflower chloroplast topoisomerase can be classified as a eukaryotic type I DNA topoisomerase.     

Penetration of Eimeria tenella sporozoites under different oxygen concentrations in vitro.

Sporozoites of Eimeria tenella (Wisconsin strain) were inoculated onto monolayers of normal chicken kidney fibroblasts and cultured in RPMI-1640 supplemented with 5% fetal bovine serum, sodium bicarbonate, and gentamicin under either aerobic, 5% CO2/95% air, or anaerobic conditions. Penetration of fibroblasts by sporozoites under CO2 or anaerobic conditions at 2 and 24 hr postinoculation was 3-4 times greater than that in the aerobic atmosphere. Effect of reduced oxygen concentrations, i.e., 20.0, 12.5, and 5.0% oxygen, was also investigated in an N2-O2-CO2 incubator. Under 5.0 and 12.5% oxygen at 2 and 24 hr postinoculation, the number of sporozoites that penetrated was about 4 and 2 times greater, respectively, than under 20.0% O2. These results indicate that lower oxygen concentrations provide for greater penetration by E. tenella sporozoites in cultured cells.     

Association of the Myosin-binding Subunit of Myosin Phosphatase and Moesin: Dual Regulation of Moesin Phosphorylation by Rho-associated Kinase and Myosin Phosphatase

To examine the role of bone morphogenetic protein (BMP) signaling in chondrocytes during endochondral ossification, the dominant negative (DN) forms of BMP receptors were introduced into immature and mature chondrocytes isolated from lower and upper portions of chick embryo sternum, respectively. We found that control sternal chondrocyte populations expressed type IA, IB, and II BMP receptors as well as BMP-4 and -7. Expression of a DN-type II BMP receptor (termed DN-BMPR-II) in immature lower sternal (LS) chondrocytes led to a loss of differentiated functions; compared with control cells, the DN-BMPR- II–expressing LS chondrocytes proliferated more rapidly, acquired a fibroblastic morphology, showed little expression of type II collagen and aggrecan genes, and upregulated type I collagen gene expression. Expression of DN-BMPR-II in mature hypertrophic upper sternal (US) chondrocytes caused similar effects. In addition, the DN-BMPR-II–expressing US cells exhibited little alkaline phosphatase activity and type X collagen gene expression, while the control US cells produced both alkaline phosphatase and type X collagen. Both DN-BMPR-II–expressing US and LS chondrocytes failed to respond to treatment with BMP-2 . When we examined the effects of DN forms of types IA and IB BMP receptors, we found that DN-BMPR-IA had little effect, while DN-BMPR-IB had similar but weaker effects compared with those of DN-BMPR-II. We conclude that BMP signaling, particularly that mediated by the type II BMP receptor, is required for maintenance of the differentiated phenotype, control of cell proliferation, and expression of hypertrophic phenotype.     

Bleomycin-induced β-lactamase overexpression in Escherichia coli carrying a bleomycin-resistance gene from Streptomyces verticillus and its application to screen bleomycin analogues

Abstract A bleomycin-resistance gene, designated blmA , has been cloned from bleomycin-producing Streptomyces verticillus by Sugiyama et al. (Gene 151 (1994) 11–16). The present study shows that Escherichia coli harboring the blmA -carrying pUC plasmid overproduced β-lactamase, encoded by an ampicillin-resistance gene on the plasmid, when cultured in the presence of bleomycin, which suggests that bleomycin may act as an inducer (or an activator) for the expression of the specific gene in the presence of blmA . We constructed a vector, designated pMAB50, which senses bleomycin and produces a pigment, using blmA and a Streptomyces tyrosinase gene located under the control of β-lactamase promoter: E. coli harboring pMAB50 produced the melanin pigment in the presence of bleomycin-type antibiotics, suggesting that the transformed E. coli can be employed as a reporter organism to screen bleomycin analogues.     

Variation in life-history traits of male Japanese fluvial sculpin Cottus pollux in relation to nest abundance along a stream course

Life-history theory predicts the occurrence of variation in the life-history traits of fish populations under different environmental conditions; however, most studies have focused on such variation between geographically separated populations. We compared breeding characteristics and life-history traits of the Japanese fluvial sculpin (Cottus pollux), a bottom-dwelling nest-holding fish, between two adjacent sites sub-divided by a weir along a stream course in central Japan. Males in the area with a lower abundance of nest sites reached sexual maturity at an earlier age and had a shorter life span than males in the area with sufficient nest abundance. Size-dependent male reproduction was found only in areas with a shortage of nest sites, supporting the assumption of competitive exclusion among males for nests. Females matured at the same age in both sites with no differences in age-specific growth rates and mortality. Our results provide evidence for life-history variation in age and size at maturity and age-specific mortality schedule of males in nest-holding fishes in a single stream population via different sexual selection regimes related to differences in nest abundance between sites.     

Body size and temperature dependence of routine metabolic rate and critical oxygen concentration in larvae and juveniles of the round crucian carp Carassius auratus grandoculis Temminck & Schlegel 1846

Routine metabolic rate (RMR, mgO₂ g^?1 h^?1) and critical oxygen concentration (Pc, a hypoxia tolerance indicator, mgO₂ L^?1) were determined in larvae and juveniles of round crucian carp, Carassius auratus grandoculis Temminck & Schlegel 1846, by measuring oxygen consumption at 15°C, 20°C, and 30°C. In addition, the dependence of RMR and Pc on fish body weight (W, g) and temperature (T, °C) was examined to construct minimal mathematical models. RMR depended on W and showed smaller values in larger individuals. RMR was different among the three temperature conditions and showed higher values at higher temperatures. Pc was significantly related to W and was low in larger individuals; that is, larger individuals had a higher hypoxia tolerance. In contrast, Pc was independent of temperature, implying that seasonal temperature fluctuations do not cause seasonal disequilibrium of hypoxia tolerance in round crucian carp. The RMR and Pc models were RMR = 0.0674W^?0.193e^0.0562T and Pc = 1.35W^?0.107, respectively. The metabolic information clarified in this study is essential for habitat quality assessments and aquaculture management of this species.