Hormonal regulation of the synthesis and mRNA content of the regulatory subunit of cyclic AMP-dependent protein kinase type II in cultured rat ovarian granulosa cells

These studies were undertaken to determine the molecular events by which estradiol and follicle-stimulating hormone (FSH) stimulate in ovarian granulosa cells the increase in the content of one of the regulatory subunits of cAMP-dependent protein kinase type II, RII51 (Mr = 51,000), and its electrophoretic variants RII51.5 (Mr = 51,500) and RII52 (Mr = 52,000). To analyze the de novo synthesis of RII51/52, granulosa cells were cultured (10(6) cells/ml) for 0, 12, 24, or 48 h with estradiol (10 nM) +/- FSH (12.5, 25, and 50 ng/ml), 8-bromo-cAMP (0.25-3 mM), or forskolin (0.5-100 microM) and then pulse-labeled with [35S]methionine (300 microCi/ml; 4 h). Labeled RII51, present either in urea extracts of total cellular protein or after partial purification from a soluble cell extract by cAMP-Sepharose chromatography, was quantitated by autoradiography of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and by excision of the silver-stained spots of the RII51 variants from the gels and counting. Synthesis of RII51 and its electrophoretic variants was low in cells cultured with estradiol alone for 48 h, whereas it was increased 4-5-fold in cells cultured with estradiol and FSH. Changes in the synthesis of actin were minor throughout the culture period regardless of hormone treatment. Pulse-chase experiments using [35S]methionine provided evidence that the isoelectric variants RII51.5 and RII52 may be derived from RII51 by post-translation modification, such as phosphorylation. Labelling with [32P]orthophosphate showed that RII52 contained more radioactivity than RII51.5 and RII51. Northern and filter hybridization assays demonstrated a 6-10-fold dose- and time-dependent increase in the amount of RII51 mRNA in granulosa cells exposed to estradiol and FSH or estradiol and forskolin compared to those cultured with estradiol alone. In vitro translation of poly(A)+ mRNA of granulosa cells from estradiol- and FSH-treated hypophysectomized rats also demonstrated an increase in the content of translatable RII51 mRNA. These studies indicate that in cultured rat granulosa cells the synthesis of RII51 and the content of its mRNA are selectively increased by estradiol and cAMP in a time- and dose-dependent manner. Based on these observations, RII51 appears to be a useful marker to determine the molecular (genomic?) sites of estradiol and FSH action in differentiating rat granulosa cells.     

Volatile constituents of male and female boll weevils and their frass

The volatile constituents of male and female boll weevils, Anthonomus grandis, and their frass were analysed by GLC-MS. The 4 previously identified components of the male pheromone were present only in the male volatile oil (3·9%) and in the male frass volatile oil (38·9%). The compounds found in one or both sexes and their frass include 26 carbonyls, 23 hydrocarbons, 12 alcohols, 6 phenols, 4 esters, 3 furans, 1 ether, and 1 lactone. Also found were 2 compounds containing nitrogen, 1 halogen, and 1 sulphur. There were 33 terpenes and 24 aromatic compounds. 3,7-Dimethyl-1-octanol comprised 15·6 per cent of the male frass oil. Carvone was found only in females (5·4%) and in female frass (6·8%). A series of monoterpene aldehydes (M⁺ 152) were found only in the female frass oil. A pheromone rôle for these components was suggested.     

Pecan weevil sex attractant: Bioassay and chemical studies

When the distillable oil from adult pecan weevils, Curculio caryae, of both sexes was investigated separately with an integrated gas chromatographymass spectrometry system, evidence was obtained for a number of di- and trisubstituted aromatic hydrocarbons, seven phenolic and carbonyl aromatic compounds, and at least three alcohols. The major compounds were a series of alkanes and alkenes, four esters and four heterocyclic compounds. Among these, eleven compounds were unique to males and four were unique to females. The volatiles were also bioassayed in the field and each attracted the opposite sex. This investigation was part of a study made to identify the possible rôle of these compounds as a pheromone produced by the pecan weevil.     

Arteriosclerosis in rat aortic allografts: Dynamics of cell growth,apoptosis and expression of extracellular matrix proteins

Transplant vasculopathy is a key factor behind the late loss of transplanted organs. Since effective treatment is still lacking, a further understanding of the pathology of this process is important. Here, a rat model of aortic allografts was used and analyzed by immunohistochemistry and biochemical tests. Infrarenal aortic segments were transplanted from F344 to Lewis rats and analysed after 1–12 weeks using isografts as controls. After 1 week, endothelial cells gradually disappeared at the graft lumen as shown by von Willebrand factor staining and cellular activation was detected in the adventitia and intima using cellular retinol-binding protein-1 as a marker. Subsequently, proliferating smooth muscle cells, lymphocytes and macrophages accumulated in the intima as indicated by the appearance of staining for cell- and proliferation-specific antigens (smooth muscle -actin, CD45_RC, ED1, cyclin D1 and proliferating cell nuclear antigen). After 4–8 weeks, TUNEL- and Fas-positive cells were observed in the media, denoting progressive apoptosis. In parallel, the developing neointima contained increased immunoreactivity for fibronectin and osteopontin. At the end of the observation period, an accumulation of macrophages and calcification was observed in the media and endothelial cells reappeared at the graft surface. The findings demonstrate major cellular and structural changes in the transplanted artery, including activation, proliferation and apoptosis of SMCs, and an altered composition of the extracellular matrix. Possibly, the observed changes in SMC phenotype, cell cycle and apoptosis during development of transplant arteriosclerosis are related to the expression of extracellular matrix proteins.     

Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells

Plasma fibronectin promotes modulation of rat arterial smooth muscle cells from a contractile to a synthetic phenotype during the first few days in primary culture. This process includes cell adhesion and spreading, loss of myofilaments, and formation of a widespread rough endoplasmic reticulum and a prominent Golgi complex. The structural reorganization is accompanied by activation of overall RNA and protein synthesis. Moreover, the cells gain the ability to replicate their DNA and divide in response to platelet-derived growth factor. Here, it is demonstrated that the power of fibronectin to bring about this change in the differentiated properties of the smooth muscle cells resides in a 105-kD cell-binding fragment, whereas a 70-kD collagen-binding fragment and a 31-kD heparin-binding fragment are inactive in this respect. Laminin, another adhesive glycoprotein and a component of the basement membrane that normally surrounds arterial smooth muscle, was contrarily found to maintain the cells in a contractile phenotype. However, with increasing time more and more cells went through the modulation into a synthetic phenotype. This "catch-up" was counteracted by a peptide that contained the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser). Hence, it is possible that the delayed modulation on laminin was due to production of fibronectin by the cells themselves. In support of this notion, fibronectin isolated from smooth muscle cultures was found to be as effective as plasma fibronectin in stimulating the phenotypic modulation. Moreover, using a combination of chemical, immunochemical, and immunocytochemical methods, it was demonstrated that the cells secreted fibronectin as well as laminin at an increasing rate during the first 4 d in primary culture and, notably, cells cultured on laminin produced more fibronectin than cells cultured on fibronectin. Newly synthesized fibronectin was incorporated into a network of pericellular and intercellular fibrils, whereas laminin formed a more diffuse layer covering the cells in a basement membrane-like manner. Taken together, the findings suggest diverse roles for fibronectin and laminin in the control of the differentiated properties of arterial smooth muscle cells. They further indicate that the ability of arterial smooth muscle cells to produce fibronectin and laminin early in primary culture is not directly related to the phenotypic state as determined morphologically and by measurement of overall rates of RNA and protein synthesis. This may be due to the cells being able to sense the macromolecular composition of the pericellular matrix and to modify their secretory activity accordingly.(ABSTRACT TRUNCATED AT 400 WORDS)     

TrackBeam, a simple tool for estimating potential exposure to the solar beam underneath tree canopies

A simple tool, TrackBeam, for rapid in situ estimates of potential exposure to the solar beam of objects underneath tree canopies is presented, tested and demonstrated to be useful in insect conservation studies. The tool can be used whenever accurate data on sun exposure is of use e.g., in ecological studies of saproxylic insects. TrackBeam draws upon the principles behind the analysis of hemispherical photographs but its use is much less dependent on the weather at the time of sampling. It may be used for detecting canopy openings in directions corresponding to the solar path. Based on this, periods of time with potential beam exposure and/or the proportion of sky that is not obscured by canopy for objects such as dead wood may be estimated. The results of using TrackBeam compares well between operators and with the corresponding results of analysed hemispherical photographs. Results are presented which show that TrackBeam was successfully used to characterise the habitat light requirements of the saproxylic beetle Melanotus castanipes (Coleoptera: Elateridae).     

Survival estimation of a cryptic antelope via photographic capture–recapture

Adult survival is a primary determinant of abundance and dynamics of large herbivore populations. For species that are inconspicuous, however, accurate survival estimation depends on accommodating low detection probability. For species with individually recognizable markings, photographic capture–recapture (CR) provides an approach to estimate population parameters while accounting for imperfect detection. I investigated the use of photographic CR for a cryptic large herbivore, the nyala, in a region of Hluhluwe–iMfolozi Park, South Africa. I conducted photographic sampling based on the closed robust design, with 5–6 daily sampling occasions nested within three week‐long sampling periods, which delineated one dry and one wet season. Detection differed between sexes: encounter probability of female adults depended on whether individuals fell into high‐encounter (seasonal range: 0.61–0.71) or low‐encounter (seasonal range: 0.29–0.40) groups, whereas male adults had a constant encounter probability of 0.39 per day. For both sexes, monthly survival probability was ≥0.93 and did not differ appreciably between seasons or sexes. Given the role of survival in population dynamics, photographic CR has the potential to provide survival estimates for cryptic large herbivores that lack such information.     

In silico identification and screening of CYP24A1 inhibitors: 3D QSAR pharmacophore mapping and molecular dynamics analysis

Vitamin D is a key signalling molecule that plays a vital role in the regulation of calcium phosphate homeostasis and bone remodelling. The circulating biologically active form of vitamin D is regulated by the catabolic mechanism of cytochrome P450 24-hydroxylase (CYP24A1) enzyme. The over-expression of CYP24A1 negatively regulates the vitamin D level, which is the causative agent of chronic kidney disease, osteoporosis and several types of cancers. In this study, we found three potential lead molecules adverse to CYP24A1 through structure-based, atom-based pharmacophore and e-pharmacophore-based screening methods. Analysis was done by bioinformatics methods and tools like binding affinity (binding free energy), chemical reactivity (DFT studies) and molecular dynamics simulation (protein–ligand stability). Combined computational investigation showed that the compounds NCI_95001, NCI_382818 and UNPD_141613 may have inhibitory effects against the CYP24A1 protein.