Characterization of adrenergic receptors and related transduction pathways in the liver of the rainbow trout

Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α₁-adrenoceptors in EPI action, at least in some fish species. The role of the α₁- and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist ³H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors (K_d0.36 nM, B_max 8.61 fmol · mg⁻¹ protein). We provide the first demonstration of α₁-adrenoceptors in these same membranes; analysis of binding data with the α₁-adrenergic antagonist ³H-prazosin demonstrated a single class of binding sites with a K_dof 15.4 nM and a B_max of 75.2 fmol · mg⁻¹ protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP₃) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP₃ (K_d 6.03 nM, B_max 90.2 fmol · mg⁻¹ protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α₁-adrenergic agonists are able to increase intracellular concentrations of IP₃ and Ca²⁺ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α₁-binding sites affinity and of α₁-adrenoceptor/IP₃/Ca²⁺ transduction systems.     

Expression and secretion of human lipocortin-1 by promoter and signal sequence of STA1 from Saccharomyces diastaticus

Summary For the secretion of human lipocortin-1 (LC-1) in yeast, a expression and secretion vector was constructed by using the promoter and signal sequence of glucoamylase gene (STA1) of Saccharomyces diastaticus. After the cDNA of human LC-1 was ligated with the secretion vector, the resulting hybrid plasmid was transformed into S. diastaticus. When the recombinant S. diastaticus was cultivated in YPD medium, LC-1 was expressed and secreted into the extracellular medium, yielding LC-1 protein at a concentration of 2.5 g/mL.     

Human immunodeficiency virus type 1 Rev is required in vivo for binding of poly(A)-binding protein to Rev-dependent RNAs.

In the absence of Rev or the Rev-responsive element, the Rev-dependent human immunodeficiency virus type 1 (HIV-1) RNAs do not behave as mRNAs; rather, they exhibit nuclear defects in splicing and/or nuclear export and cytoplasmic defects in stability and translation. A translational initiation factor, eIF-5A, has recently been shown to bind specifically to the Rev activation domain. As the binding of poly(A)-binding protein 1 (PAB1) to the poly(A) tail of mRNAs is involved in both the stability and translation of cytoplasmic mRNAs, we investigated whether Rev might influence the association of PAB1 with cytoplasmic HIV-1 RNAs. Antibodies were generated against PAB1. We used these antibodies in an immunoprecipitation assay to detect specific binding of PAB1 to cytoplasmic mRNAs. We found that in the presence of Rev, PAB1 was associated with Rev-dependent and Rev-independent RNAs in the cytoplasm of transfected cells. However, in the absence of functional Rev, we found little or no PAB1 associated with Rev-dependent RNAs. These RNAs were capable of binding PAB1 in vitro. These results demonstrate that HIV-1 RNAs are defective in PAB1 association in the absence of Rev.     

Rapid screening of an SH2-containing gene using PCR amplification method

Summary To isolate a novel gene that contains an SH2 domain, we devised a rapid and nonradioactive cDNA library screening method using polymerase chain reaction (PCR). For PCR amplification, we designed degenerate oligonucleotide primers from the multialigned DNA sequences of SH2 domains. This method offers an inexpensive and efficient approach for the isolation of clones of interest from cDNA libraries.     

Isolation of Two Plasticizers,Bis(2-ethylhexyl) Terephthalate and Bis(2-ethylhexyl) Phthalate,from Capparis spinosa L. Leaves

Many plants have been known to be contaminated and accumulate plasticizers from the environment, including water sources, soil, and atmosphere. Plasticizers are used to confer elasticity and flexibility to various fiber and plastic products. Consumption of plasticizers can lead to many adverse effects on human health, including reproductive and developmental toxicity, endocrine disruption, and cancer. Herein, we report for the first time that two plasticizers, bis(2-ethylhexyl) terephthalate (DEHT) and bis(2-ethylhexyl) phthalate (DEHP), have been isolated from the leaves of Capparis spinosa L. (the caper bush), a plant that is widely used in food seasonings and traditional medicine. 297 mg/kg of DEHT and 48 mg/kg of DEHP were isolated from dried and grounded C. spinosa L. leaves using column chromatography and semi-preparative high-performance liquid chromatography. Our study adds to the increase in the detection of plasticizers in our food and medicinal plants and to the alarming concern about their potential adverse effects on human health.     

REGENERATION AND SEXUAL DIFFERENTIATION OF GRIFFITHSIA JAPONICA (CERAMIACEAE,RHODOPHYTA) THROUGH SOMATIC CELL FUSION1

Hybrid cells were obtained from somatic cell fusion among male, female, and tetrasporangial plants in Griffithsia japonica Okamura by a wound-healing process. Isolated fusion cells regenerated new mature plants with mixed reproductive structures. The plants regenerated from hybrid cells between male and female plants developed into 1) spermatangiate, 2) carpogonial, 3) bisexual with spermatangia and carpogonial branches, 4) mixed-phase with spermatangia and tetrasporangia, or 5) bisexual/mixed-phase plants with spermatangia, carpogonial branches, and tetrasporangia. About 70% of the plants regenerated from hybrid cells between male and female plants produced tetrasporangia that were always formed with spermatangia on a single cell. Some of those tetrasporangia released tetraspores, six of which gave rise to mature plants. The plants regenerated from hybrid cells between male and tetrasporangial plants developed into spermatangiate, tetrasporangiate, or mixed-phase plants with spermatangia and tetrasporangia. The plants regenerated from hybrid cells between female and tetrasporangial plants developed into carpogonial, tetrasporangiate, or mixed-phase plants with carpogonial branches and tetrasporangia. All types of reproductive structures we re functional.     

Effect of nitrogen application on growth and photosynthetic nitrogen use efficiency in two ecotypes of wild strawberry, Fragaria chiloensis

The relationships between increasing nitrogen fertilization and growth, maximum CO₂ assimilation and the initial slope of the CO₂ response curve were studied in 2 ecotypes of wild strawberry, Fragaria chiloensis (L.) Duchn. Nitrogen accumulation of CA11, an ecotype from a low-nutrient dune site, was greater at all nitrogen concentrations than that of RCP37, an ecotype from a higher-nutrient strand site. Maximum CO₂ assimilation, total Rubisco activity, dry weight, and initiation of leaves and crowns were higher in CAI1 than RCP37 as nitrogen treatment was increased from 0 to 200 mg ^l-1, whereas these parameters were lower in CAl1 when fertilized at 300 mg T¹, but not in RCP37. The mean leaf area of CA11 was greater than RCP37 when grown with no supplemental nitrogen, but mean leaf area of the 2 lines was similar under nitrogen fertilization. Maximum CO₂ assimilation and carboxylation efficiency increased with increasing leaf nitrogen in both clones. At equivalent concentrations of leaf nitrogen, RCP37 had higher CO₂ assimilation and carboxylation efficiency than CA11 and the difference between the 2 clones increased as ieaf nitrogen increased. Thus, RCP37 had a higher photosynthetic nitrogen use efficiency than CA11. However, at a given applied nitrogen level, CA11 allocated more nitrogen to a unit of leaf area so that photosynthetic rates were higher than RCP37, except at the highest application of 300 mg ^l-1. The high nitrogen accumulation capacity and resource allocation to fruiting structures (crowns) in CA11 leads us to suggest that this clone may possess genes that could increase fruit yield in cultivated strawberry.