Characterization and expression of the phytochrome gene family in the moss Ceratodon purpureus.

In the moss Ceratodon purpureus, phytochrome is encoded by two different genes, CpPHY1 and CpPHY2. CpPHY2 represents a conventional type phytochrome characterized by a C-terminus homologous to the catalytic domain of bacterial sensor histidine kinases, whereas CpPHY1 represents an unique phytochrome, which carries a C-terminus homologous to the catalytic domain of eukaryotic serine/threonine/tyrosine kinases. Southern blot analysis revealed that CpPHY1 is present in different Ceratodon cultivars which were collected in Germany and in Finland, implying that CpPHY1 represents a functional and active gene in Ceratodon, but CpPHY1 homologous genes could not be detected in another moss, Physcomitrella patens, or in Arabidopsis thaliana. cDNA analysis of CpPHY1 revealed the presence of a hitherto unnoticed intron within the 3' region. This results in a change of the sequence of the 11 C-terminal amino acids from KLSSHSYLTSK to FSSYQDSYPSTEELS. CpPHY1 and CpPHY2 mRNAs appear to accumulate in a light-independent manner, with CpPHY2 being much more strongly expressed than CpPHY1. Accordingly, in crude protein extracts, CpPHY2 is clearly detectable by Western blot analysis, whereas CpPHY1 is not. Light-dependent expression of CpPHY2 can be detected at the post-transcriptional level; during a 7-day period of dark adaptation, pronounced CpPHY2 accumulation occurs. Upon transfer to white light, dark-accumulated CpPHY2 is depleted within 24 h. That depletion can be completely inhibited by the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), implying that photosynthesis is strongly involved in the adjustment of phytochrome steady-state concentrations in Ceratodon. The presence of an ORF within the 5' UTR region of CpPHY2 (uORF) encoding peptide MKEFSSTSRSLMIVGIY suggests regulation at the translational level. The uORF resides on a short intron which is excised from the 5' leader in a light-dependent manner, resulting in the formation of an alternative uORF encoding peptide MEEEEDCVP.

[Which variables predict the appearance of right bundle-branch block and its course after heart transplantation?]. / Qué variables predicen la aparición de un bloqueo de rama derecha del haz de His y su evolución tras el trasplante cardíaco?

AIM: The purpose of this study was to analyze a series of variables in donors as well as of the preoperative and early and late postoperative of patients developing a right branch block of the bundle of His (RBBBH) in the first week after heart transplantation (HT), and to evaluate factors predicting the disappearance or progression of this conduction disorder. MATERIAL AND METHODS: 58 consecutive patients having undergone an orthotopic HT were studied. 15 of them showed a RBBBH after the HT (age: 43 +/- 13, 12 male, 3 female) and 43 did not (age: 54 +/- 17, 40 male and 3 female). ECGs and echocardiographic studies were performed after 1 week, 1, 3 and 6 months and 1 year. The following factors were analyzed: age and sex of the donor baseline cardiopathy, donor's weight related to recipient's weight, time of ischaemia and cardiopulmonary by-pass, number of rejections per patient/year, previous pulmonary vascular resistance. These parameters were compared among the patients who showed RBBBH and those who did not, and between those whose blocks disappeared in the follow-up and those whose blocks persisted. RESULTS: We found differences is the sex of donors, age of recipients, baseline etiology and time of cardiopulmonary by-pass (with block: 43 +/- 13 years old, dilated cardiomyopathy 73%, 106 +/- 25 minutes, whereas without block: 54 +/- 17 years old, dilated cardiomyopathy 42%, 92 +/- 18). The different parameters between the patients whose block underwent a regression or a progression were sex, lung resistances and right ventricle diameter (progression of the block: men 100%, 3.43% +/- 1.05 UW, progressively growing ventricular diameters. No progression or regression of the block: men 67%, 1.63 +/- 0.74 UW, ventricular diameters with progressive decrease). CONCLUSION: Younger recipients, with a diagnosis of dilated cardiomyopathy, to whom a woman's heart is implanted and who show a longer extracorporal circulation time are those who show a higher incidence of RBBBH: Male patients with high pulmonary resistances undergo a progressive increase in the diameter of the right ventricle and a progressive increase in the RBBBH degree.

Sequence similarities of phytochrome to protein kinases: implication for the structure, function and evolution of the phytochrome gene family.

Phytochrome, the best characterised plant photoreceptor, is encoded by a small multigene family within the plant kingdom. The different phytochrome types are composed of a conserved light-sensing chromophore domain of about 80 kDa and a less-conserved C-terminal domain of about 50 kDa. The C-terminus of phytochrome of the moss Ceratodon purpureus is homologous to the catalytic domain of eukaryotic serine/threonine or tyrosine protein kinases; in contrast, for all other phytochromes (conventional phytochromes) sequence similarities within the C-terminal domain to the catalytic domain of bacterial histidine kinases have been reported. We performed careful sequence comparisons of the putative catalytic domains of phytochrome with each other, with authentic serine/threonine, tyrosine and with histidine kinases. We report that conventional phytochromes exhibit structural elements of the catalytic domains of both histidine and, to a lesser extent, of serine/threonine and tyrosine kinases. The significance of these observations is discussed in the framework of the structure, function and evolution of phytochrome.

Analysis of the protein kinase activity of moss phytochrome expressed in fibroblast cell culture.

In the moss Ceratodon purpureus a phytochrome gene encodes a phytochrome type (PhyCer) which has a C-terminal domain homologous to the catalytic domain of eukaryotic protein kinases (PKs). PhyCer exhibits sequence conservation to serine/threonine as well to tyrosine kinases. Since PhyCer is expressed very weakly to moss cells, to investigate the proposed PK activity of PhyCer, we overexpressed PhyCer transiently in fibroblast cells. For this purpose we made a chimeric receptor, EC-R, which consists of the extracellular, the membrane-spanning and the juxtamembrane domains of the human epidermal growth-factor receptor (EGF-R) linked to the PK catalytic domain of PhyCer (CerKin). The expression of EC-R in transiently transfected cells was confirmed with antibodies directed against the extracellular domain of EGF-R or against CerKin. Both EGF-R and EC-R were immunoprecipitated from lysates of overexpressing cells with antibodies against the extracellular domain of EGF-R. Phosphorylation experiments were performed with the immunoprecipitates and the phosphorylation products were subjected to phosphoamino acid analysis. Phosphorylation products specifically obtained with EC-R-transfected cells exhibit phosphorylation on serine and threonine residues. In EC-R transfected cells the endogenous EGF-R showed enhanced phosphorylation of serine and threonine residues compared to EGF-R immunoprecipitated from control cells. Although CerKin is closest to the catalytic domain of a protein tyrosine kinase from Dictyostelium discoideum, EC-R does not appear to phosphorylate tyrosine residues in vitro. From our data we conclude that PhyCer carries an active PK domain capable of phosphorylating serine and threonine residues.

Biochemical evidence that phytochrome of the moss Ceratodon purpureus is a light-regulated protein kinase.

The phytochrome gene of the moss Ceratodon purpureus (phyCer) codes for a novel phytochrome polypeptide with a predicted molecular mass of 145 kDa that has a COOH-terminal domain which is homologous to the catalytic domain of eukaryotic protein kinases. In this paper we report the first biochemical evidence that in fact, as predicted from the gene sequence, PhyCer represents an active, light-regulated protein kinase. In vitro phosphorylation experiments with protonemata extracts revealed the existence of a 140 kDa protein, phosphorylated in a red/far-red light dependent manner. The binding of a polyclonal antibody directed to the protein kinase catalytic domain of PhyCer enhanced the phosphorylation of a 140 kDa band when assayed in a renaturation-auto-phosphorylation experiment with nitrocellulose bound protein. These findings strongly implicate that the phyCer gene product has protein kinase activity and is capable of auto-phosphorylation. The results of the renaturation-phosphorylation experiments were essentially the same, no matter whether protein extracts from light grown or dark adapted moss protonemata were used. Thus, phyCer expression most likely is not light regulated.

Phycobilisome Heterogeneity in the Red Alga Porphyra umbilicalis.

Phycobilisomes were isolated from Rhodophyceae brought from the field (Porphyra umbilicalis) or grown in culture under laboratory conditions (Antithamnion glanduliferum). In P. umbilicalis two kinds of well-coupled (ellipsoidal and hemidiscoidal) phycobilisomes were detected, in contrast to A. glanduliferum cultured algae in which only one kind of well-coupled, ellipsoidaltype phycobilisome appeared. The new phycobilisome-type particle detected in P. umbilicalis is characterized by an impoverishment in R-phycoerythrin and by sedimentation at lower density. The comparison between both phycobilisomes of P. umbilicalis allows determination of the presence of one colorless linker polypeptide (30 kilodaltons) associated with R-phycocyanin and allophycocyanin and two (40 and 38 kilodaltons) associated to R-phycoerythrin. The percentage of linker polypeptides associated with this pigment is low in the new phycobilisome-like particle detected. This suggests that part of the R-phycoerythrin is less strongly bound to the phycobilisome than the other pigments. This feature could probably explain the existence of two kinds of phycobilisomes as intermediary steps of phycobilisome organization in algae exposed to rapid changes in environmental factors. In contrast, algae growing in culture and adapted to specific conditions do not present intermediary organization steps. Polypeptide composition and identification are given for this phycobilisome-like particle.