Superoxide radical production by sponges Sycon sp.

AbstractUsing the catechol Tiron as an O−⋅2 scavenger, we showed that sea sponges (Sycon sp.) produce superoxide radicals in sea water at a high rate without any stimuli added. The rate of O−⋅2 outflow from sponges to their water surroundings reaches a value of 0.5 nmol/min per sponge at pH 6.5. The generation of O−⋅2 was inhibited by Cu,Zn-superoxide dismutase, and restored by the addition of KCN. We also confirmed the abiotic production of O−⋅2 in sea water, detected earlier with a different method by Petasne and Zika [Nature 325 (1987) 516–518]

Single fluorescent protein-based Ca2+ sensors with increased dynamic range

<p>Abstract</p> <p>Background</p> <p>Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca²⁺sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).</p> <p>Results</p> <p>Here we report significant progress on the development of the latter type of Ca²⁺sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca²⁺concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca²⁺-free and Ca²⁺-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca²⁺response to a prolonged glutamate treatment in cortical neurons.</p> <p>Conclusion</p> <p>We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.</p

A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants.

We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu(222)-->Gly (E222G) substitution, which removed the evolutionarily invariant Glu(222). Re-introduction of Glu(222) into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of 'humanized' aceGFP and beta-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells

Single fluorescent protein-based Casensors with increased dynamic range-0

<p><b>Copyright information:</b></p><p>Taken from "Single fluorescent protein-based Casensors with increased dynamic range"</p><p>http://www.biomedcentral.com/1472-6750/7/37</p><p>BMC Biotechnology 2007;7():37-37.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1931437.</p><p></p>amino acid residues (145–148) and sensitive domains (calmodulin and M13) within Casensors

Single fluorescent protein-based Casensors with increased dynamic range-3

<p><b>Copyright information:</b></p><p>Taken from "Single fluorescent protein-based Casensors with increased dynamic range"</p><p>http://www.biomedcentral.com/1472-6750/7/37</p><p>BMC Biotechnology 2007;7():37-37.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1931437.</p><p></p>5–565 nm emission filter) and fura-2FF (black dashed line, ratio of 340 nm and 380 nm excited green fluorescence, 505–530 nm emission filter) are shown

Single fluorescent protein-based Casensors with increased dynamic range-1

<p><b>Copyright information:</b></p><p>Taken from "Single fluorescent protein-based Casensors with increased dynamic range"</p><p>http://www.biomedcentral.com/1472-6750/7/37</p><p>BMC Biotechnology 2007;7():37-37.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1931437.</p><p></p>presence of 0.5 mM EGTA (dashed lines) or 1 mM Ca(solid lines) at pH 7.4. c,d. Absorbance in the presence of 0.5 mM EGTA (dashed lines) or 0.4 mM Ca(solid lines) at pH 7.4. e,f. Dependence of sensors fluorescence on pH in the presence of 0.5 mM EGTA (dashed lines) or of 0.2 mM Ca(solid lines). g,h. Catitration curves, at pH 7.4

Single fluorescent protein-based Casensors with increased dynamic range-2

<p><b>Copyright information:</b></p><p>Taken from "Single fluorescent protein-based Casensors with increased dynamic range"</p><p>http://www.biomedcentral.com/1472-6750/7/37</p><p>BMC Biotechnology 2007;7():37-37.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1931437.</p><p></p>) to Caionophore A23187. c,d. HeLa cells expressing Case12 are shown before (b) and after (c) ionophore addition. e-h. Fluorescence changes of M21 (human Melanoma-derived) cells expressing Case12 in response to 100 μM ATP. Images were captured every 0.294 sec on the confocal microscope. e,f. Individual responses of two selected cells within 400 s after ATP addition. g,h. The same cells, first 60 s of response. i. PC12 cells response to 500 uM carbachol (CCH). j. PC12 cells response to 30 mM KCl. For i and j first and second arrows indicate the moments of a compound addition and of washing start, respectively

Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina

Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like chromoproteins with red-shifted absorption, we performed a broad search in blue-coloured Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disc. A novel blue chromoprotein, aeCP597, with an absorption maximum at 597 nm determining the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chemically identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is determined by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm respectively. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins