Photochromic biliproteins can be switched by light between two states, initiated by Z/E photoisomerization of the linear tetrapyrrole chromophore. The cyanobacterium Anabaena sp. PCC 7120 contains three genes coding for such biliproteins, two coding for phytochromes (aphA/B) and one for the alpha subunit of phycoerythrocyanin (pecA). (a) aphA was overexpressed in Escherichia coli with N-terminal His and S tags, and the protein was reconstituted by an optimized protocol with phycocyanobilin (PCB), to yield the photochromic chromoprotein, PCB-AphA, carrying the PCB chromophore. (b) AphA chromophorylation is autocatalytic such as in other phytochromes. (c) AphA chromophorylation is also possible by chromophore transfer from the PCB-carrying biliprotein, phycocyanin (CPC). The autocatalytic transfer is very slow, and it is enhanced more than 100-fold by catalysis of PCB:CpcA lyase and alpha-CPC as donor. (d) Through deletion mutations of aphA, a short sequence IQPHGV [amino acids (aa) 26-31] was found essential for the lyase activity of AphA, indicating an interaction of the N terminus with the chromophore-binding domain around cysteine 259. (e) A motif of at least 23 aa, starting with this sequence and located approximately 250 aa N terminal of the chromophore-binding cysteine, is proposed to relate to the lyase function in plant and most prokaryotic phytochromes. (f) Long-range interactions in AphA are further supported by blue-shifted absorptions (

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In the central nervous system, L-type voltage-gated calcium channels (LTCCs) come in two isoforms, namely Cav1.2 and Cav1.3 channels. It has been shown previously that these channels differ in biophysical properties, in subcellular localization, and in the coupling to the gene transcription machinery. In previous work on rat hippocampal neurons we have identified an excitatory cation conductance and an inhibitory potassium conductance as important LTCC coupling partners. Notably, a stimulus-dependent interplay of LTCC-mediated Ca(2+) influx and activation of these Ca(2+)-dependent conductances was found to give rise to characteristic voltage responses. However, the contribution of Cav1.2 and Cav1.3 to these voltage responses remained unknown. Hence, the relative contribution of the LTCC isoforms therein was the focus of the current study on hippocampal neurons derived from genetically modified mice, which either lack a LTCC isoform (Cav1.3 knockout mice) or express a dihydropyridine-insensitive LTCC isoform (Cav1.2DHP(-)-knockin mice). We identified common and alternate ion channel couplings of Cav1.2 and Cav1.3 channels. Whereas hyperpolarizing Ca(2+)-dependent conductances were coupled to both Cav1.2 and Cav1.3 channels, an afterdepolarizing potential was only induced by the activity of Cav1.2 channels. Unexpectedly, the activity of Cav1.2 channels was found at relatively hyperpolarized membrane voltages. Our data add important information about the differences between Cav1.2 and Cav1.3 channels that furthers our understanding of the physiological and pathophysiological neuronal roles of these calcium channels. Moreover, our findings suggest that Cav1.3 knockout mice together with Cav1.2DHP(-)-knockin mice provide valuable models for future investigation of hippocampal LTCC-dependent afterdepolarizations.

We found nil prevalence of OBI in CHD patients and a very low prevalence (<1%) in KTxR suggesting that routine screening for HBV DNA is not required in CHD population in our region. However, in KTxR, pretransplant screening with HBV DNA should be considered. Testing for HBV DNA in PBMCs does not seem to be of additional value.