Validation of Housekeeping Genes for Gene Expression Studies in <Emphasis Type="Italic">Symbiodinium</Emphasis> Exposed to Thermal and Light Stress

Unicellular photosynthetic algae (dinoflagellate) from the genus Symbiodinium live in mutualistic symbiosis with reef-building corals. Cultured Symbiodinium sp. (clade C) were exposed to a range of environmental stresses that included elevated temperatures (29°C and 32°C) under high (100 μmol quanta m⁻² s⁻¹ Photosynthetic Active Radiation) and low (10 μmol quanta m⁻² s⁻¹) irradiances. Using real-time RT-PCR the stability of expression for the nine selected putative housekeeping genes (HKGs) was tested. The most stable expression pattern was identified for cyclophilin and S-adenosyl methionine synthetase (SAM) followed by S4 ribosomal protein (Rp-S4), Calmodulin (Cal), and Cytochrome oxidase subunit 1 (Cox), respectively. Thermal stress alone resulted in the highest expression stability for Rp-S4 and SAM, with a minimum of two reference genes required for data normalization. For Symbiodinium exposed to both, light and thermal stresses, at least five reference genes were recommended by geNorm analysis. In parallel, the expression of Hsp90 for Symbiodinium in culture and in symbiosis within coral host (Acropora millepora) was evaluated using the most stable HKGs. Our results revealed a drop in Hsp90 expression after an 18 h-period and a 24 h-period of exposure to elevated temperatures indicating the similar Hsp90 expression profile in symbiotic and non-symbiotic environments. This study provides the first list of the HKGs and will provide a useful reference in future gene expression studies in symbiotic dinoflagellates.     

Secoiridoid content of Blackstonia perfoliata in vivo and in vitro

Summary This study reports the analysis of secondary metabolites of gentiopicrin, swertiamarin, and sweroside in shoot and root cultures of yellow wort (Blackstonia perfoliata), which were initiated from seeds, grown on Murashige and Skoog (MS) medium. Shoot cultures of B. perfoliata were inoculated with suspension of Agrobacterium rhizogenes strain A4M70GUS and hairy roots appeared at the infected sites after 3 wk of inoculation. Tips of adventitious roots of B. perfoliata were grown on hormone-free MS medium and three clones of the transformed roots regenerated shoots spontaneously. Gentiopicrin, swertiamarin, and sweroside were detected in both roots and shoots of B. perfoliata in vitro and in vivo, but gentiopicrin was found to be the major compound. The concentration of growth regulator in the medium affected the production of secoiridoids in B. perfoliata in vitro, where the level of gentiopicrin was higher in plants grown in the presence of indole-3-butyric acid, but the presence of 6-benzylaminopurine was inhibitory to secoiridoid production.     

The mathematical analysis of the heart rate and blood lactate curves during incremental exercise testing

This paper describes a new mathematical approach for the analysis of HR (heart rate) and BL (blood lactate) curves during incremental exercise testing using a HR/BL curve and its derivatives, taking into account the native shape of all curves, without any linear approximation. Using this approach the results indicate the appearance of three characteristic points (A, B and C) on the HR/BL curve. The point A on the HR/BL curve which is the value that corresponds to the load (12.73 ± 0.46 km h-1) at which BL starts to increase above the resting levels (0.9 ± 0.06 mM), and is analogous to Lactate Turn Point 1 (LTP1). The point C on the HR/BL curve which corresponds to a BL of approximately 4mM, and is analogous to LTP2. The point B on the HR/BL curve, which corresponds to the load (16.32 ± 0.49 km h-1) at which the moderate increase turns into a more pronounced increase in BL. This point has not been previously recognized in literature. We speculate this point represents attenuation of left ventricular ejection fraction (LVEF) increase, accompanied by the decrease in diastolic time duration during incremental exercise testing. Proposed mathematical approach allows precise determination of lactate turnpoints during incremental exercise testing.     

Versatile capacity of shuffled cytochrome P450s for dye production

DNA family shuffling is a relatively new method of directed evolution used to create novel enzymes in order to improve their existing properties or to develop new features. This method of evolution in vitro has one basic requirement: a high similarity of initial parental sequences. Cytochrome P450 enzymes are relatively well conserved in their amino acid sequences. Members of the same family can have more than 40% of sequence identity at the protein level and are therefore good candidates for DNA family shuffling. These xenobiotic-metabolising enzymes have an ability to metabolise a wide range of chemicals and produce a variety of products including blue pigments such as indigo. By applying the specifically designed DNA family shuffling approach, catalytic properties of cytochrome P450 enzymes were further extended in the chimeric progeny to include a new range of blue colour formations. This mini-review evokes the possibility of exploiting directed evolution of cytochrome P450s and the novel enzymes created by DNA family shuffling for the production of new dyes.     

Dual activation of the inositol-triphosphate-calcium and cyclic nucleotide intracellular signaling systems by adrenocorticotropin in rat adrenal cells

Hormones which primarily utilize cAMP as their intracellular "second messenger" are generally not thought to activate the IP3-Ca++ signalling system. Presently, we show that ACTH, at certain concentrations, can activate both the cAMP and IP3-Ca++ intracellular signalling systems.