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 in 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 immuno-precipitated 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.Abbreviations CerKin protein kinase catalytic domain of PhyCer - EC-R chimeric receptor consisting of the extracellular, the membrane spanning and the juxtamembrane domains of the human epidermal growth factor receptor (EGF-R) linked to the protein kinase catalytic domain of PhyCer - EGF-R epidermal growth factor receptor - mAb monoclonal antibody - PhyCer phytochrome gene in Ceratodon encoding a phytochrome type which has a C-terminal domain homologous to the catalytic domain of eucaryotic protein kinases - PK protein kinase - PVDF polyvinyl difluoride - Ser serine - Thr threonine - Tyr tyrosine Dr. Patricia Algarra was supported by the Alexander von Humboldt Foundation, Germany. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany.     

Increased chilling tolerance and altered carbon metabolism in tomato leaves following application of mechanical stress

We investigated the effects of brushing on the chilling tolerance and metabolism of nonstructural carbohydrates (soluble sugars and starch) in tomato leaves before, during and after a chilling stress. Tomato plants ( Lycopersicon esculentum Mill. cv. Caruso) were cultivated either without mechanical stress application (control plants) or with daily brushing treatments for 15 days (brushed plants), prior to a 7-day chilling treatment (8/5°C day/night). Brushing resulted in shorter plants with a 34% reduction in leaf dry weight per area and a 59% reduction of soluble sugars and starch, on a dry weight basis. The sugar to starch ratio was not affected by brushing. A greater chilling tolerance in the brushed plants was demonstrated by the maintenance of a significantly higher PSII efficiency in brushed plants (42%) compared to that of the control plants (30%) after 7 days of chilling treatment, less visible damage to the leaf tissue, and a more rapid resumption of growth during 3 days of recovery as compared to control plants. During the chilling treatment levels of soluble sugars per leaf dry weight increased 15-fold in the brushed plants and 5-fold in control plants. In the present study we have demonstrated that brushing can increase chilling tolerance in tomato plants. The observed differences in chilling tolerance and concentration of soluble sugars in the leaves may indicate an involvement of soluble sugar levels in acclimation to chilling.     

Differential accumulation of the transcripts of 22 novel protein kinase genes in Arabidopsis thaliana

22 novel members of the Arabidopsis thaliana protein kinase family (AKs) were identified by using degenerate oligonucleotide primers directed to highly conserved amino acid sequences of the protein kinase (PK) catalytic domain. Of these 22 genes, 16 turned out to carry intron sequences. Homologies of AK sequences were detected to S-locus receptor protein kinases (SRKs) from Brassica spp., to SRK-like PKs from maize and A. thaliana and to several other receptor PKs from A. thaliana. Sequence similarity was also detected to Ca²⁺-dependent PKs (CDPKs) from rape and soybean, to SNF1 and to CDC2 homologues. The genomic organization and the accumulation of the mRNAs from these 22 AK genes were investigated.     

BBReader: A computer program for the combined use of the BioMagResBank and PDB databases

A computer program (BBReader) was developed which performs an inverse search in theBioMagResBank database. Given (cross) peak positions of a protein, the program searchesfor atoms with matching chemical shifts and suggests possible assignments for user-specifiedhomo- and heteronuclear one- to three-dimensional COSY- and NOESY-type experiments.It can handle 1H, 13C and 15N spectra. Distance information from PDB files can be utilizedfor filtering possible NOESY cross peak assignments.     

Stable carbon isotopes in tree rings of beech: climatic versus site-related influences

 Stable carbon isotopes in tree rings are a promising tool in palaeoclimate research, provided attempts are made to disentangle climatic from local effects (e.g. soil properties, competition, light). The ¹³C/¹²C variations in cellulose of tree rings of beech (Fagus sylvatica) were determined at several sites in the Swiss Central Plateau covering the last 50 years. We chose sites which differ in moisture conditions and sampled cores from four to six trees per site. The mean ¹³C/¹²C series from the different dry sites (distant by up to 40 km) are closely interrelated suggesting a common external cause. Correlation analysis with climate data proved the total precipitation in the months May, June and July to have the strongest effect on the carbon isotopes (r =  – 0.73). This result is in agreement with the commonly used model which relates the isotope discrimination to the water use efficiency. On the other hand, the isotope series of the wet sites are not as well correlated to the climate. At two of the sites (a dry and a humid) tree ring width suddenly increased. We used this effect as a test-case to study the influence of local growth conditions on the climate-isotope relationship. Received: 17 April 1996 / Accepted: 2 September 1996     

Cholinergic Control of Nerve Growth Factor in Adult Rats: Evidence from Cortical Cholinergic Deafferentation and Chronic Drug Treatment

Abstract: It is well documented that nerve growth factor (NGF) plays an important role in maintaining functions of cholinergic basal forebrain neurons. In the present study, we tested the hypothesis that cholinergic activity controls NGF levels in cholinoceptive neurons of the cerebral cortex and hippocampus. To address that question, we used both cholinergic deafferentation of cerebral cortex and hippocampus by cholinergic immunolesion with 192IgG-saporin and chronic pharmacological treatment of sham-treated and immunolesioned rats with the cholinergic agonist pilocarpine and the cholinergic antagonist scopolamine. We observed an increase in NGF protein levels in the cortex and hippocampus after cholinergic immunolesions and also after muscarinic receptor blockade by chronic intracerebroventricular scopolamine infusion in sham-treated rats after 2 weeks. There was no further increase in the accumulation of NGF after scopolamine treatment of immunolesioned rats. Chronic infusion of pilocarpine had no effect on cortical and hippocampal NGF protein levels in sham-treated rats. In rats with cholinergic immunolesions, however, pilocarpine did prevent the lesion-induced accumulation of NGF. There was no effect of cholinergic lesion and drug treatment on cortical or hippocampal NGF mRNA levels, consistent with the importance of NGF retrograde transport as opposed to its de novo synthesis. This study provides strong evidence for the hypothesis that there is cholinergic control of cortical and hippocampal NGF protein but not mRNA levels in adult rats.     

Crystallization of the complex of human IFN-γ and the extracellular domain of the IFN-γ receptor

A complex of human interferon-γ (IFN- γ) with the soluble extracellular domain of the IFN- γ receptor α-chain (IFN-γ-R) has been crystallised. Crystals of the complex were grown using PEG 4000 as the precipitating agent in the presence of β-octyl glucoside. The receptor-ligand complex crystallizes in a monoclinic space group and diffracts to about 3.0 Å resolution. Isomorphous crystals have been obtained with complex containing selenomethionine and cysteine mutants of IFN-γ, which may facilitate the ongoing X-ray structure determination. © 1995 Wiley-Liss, Inc.     

Immunohistochemical localization of cytochrome P450 3A in human pulmonary carcinomas and normal bronchial tissue

The cytochrome P450 (CYP) enzymes metabolize drugs and other xenobiotics in liver and also in some extrahepatic tissues. We have studied the expression and localization of CYP3A in primary lung tumours and normal lung tissue from the same patients. Thirtytwo patients undergoing partial or total lung resection for therapy of primary pulmonary carcinoma were included in this study. Immunohistochemical staining for CYP3A was performed with a modification of the ABC technique. Eight of the 32 cases of primary pulmonary carcinoma showed expression of CYP3A. In 12 of the 32 cases of normal tissue, the seromucous glands were positive for CYP3A. The bronchial epithelium was positive for CYP3A in 11 cases. We observed no correlation between CYP3A expression in tumour tissue and that in seromucous glands or bronchial epithelium. We conclude that CYP3A is present in both normal and cancerous lung tissue. Our findings suggest, however, no co-expression of CYP3A in lung cancer.     

Developmental regulation of presence of binding sites for neoglycoproteins and endogenous lectins in various embryonic stages of human lung,liver and heart

Protein-carbohydrate interactions are supposed to play key roles in the mechanisms of cell adhesion, biosignalling and intracellular routing, warranting the analysis of the developmental course of expression of epitopes of this system. Thus, a panel of carrier-immobilized carbohydrate ligands was used as probes, namely lactose,N-acetylgalactosamine,N-acetylglucosamine, mannose, fucose and maltose. Additionally, an antibody to an endogenous -galactoside-binding lectin (anti-galectin-1), the biotinylated lectin and two further human lectins, namely the macrophage migration inhibitory factor-binding sarcolectin and serum amyloid P component (SAP) that displays selectivity for sulphated sugars and mannose-6-phosphate, were included. They enabled us to assess the extent of the presence of respective binding sites in fixed sections from human lungs (pulmonary epithelial cells), livers (hepatocytes) and hearts (myocard cells) of 10–50 weeks gestation. Invariably, specific binding was detected in the three organ types, at least in certain stages. In most of the cases, the intensity of staining exhibited developmental regulation. The apparent patterns reveal similarities between the different cell types, as seen with immobilizedN-acetylglucosamine as well as with labelled galectin-1 and sarcolectin. However, drastic differences among such patterns with nearly opposite developmental courses do also occur, as detected for carrier-attached mannose and maltose residues. These results point to a potential importance for the detected glycohistochemical features in human development and substantiate the possibility of differential regulation of the presence of binding sites for distinct sugars within a certain organ and between the individual cell types of the monitored organs.