A SIGNAL GLYCOPROTEIN WITH α-d-MANNOSYL RESIDUES IS INVOLVED IN THE WOUND-HEALING RESPONSE OF ANTITHAMNION SPARSUM (CERAMIALES,RHODOPHYTA)1

A variety of fluorescein isothiocyanate-labeled lectins specific for different sugar moieties were examined as probes for the wound-healing response in the filamentous red alga Antithamnion sparsum Tokida. Among them, only concanavalin A (ConA) and Lens culrinaris agglutinin (LCA), which have specificity to α-D-mannosyl residues, bound specifically to repair cells during the wound-healing process. When ConA or LCA was added at various time intervals after wounding, it first bound (3 h post-wounding) as a thin layer at the tips of the adjacent cells. Later (4–5 h post-wounding) labeling also appeared at the tips of the repair cells. Intense labeling at these sites continued throughout the healing process until repair cell fusion, at which time the lectin labeling was reduced to a narrow ring around the area of fusion. When added to plants prior to wounding and continually monitored, these same lectins acted as inhibitors to the wound-healing response. Other control lectins showed no inhibitory effects. A crude extract solution obtained from decapitated filaments stimulated the wound-healing response, and a lectin-binding component bound strongly to a protein-binding transfer membrane. These results suggest that the labeled compound is a glycoprotein that has α-D-mannosyl residues and is similar to the repair hormone rhodomorphin found in Griffithsia pacifica Kylin.     

An SAR sequence containing 395 bp DNA fragment mediates enhanced,gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants

A 395 bp fragment located downstream from the soybean heat shock geneGmhsp 17.6-L exhibits several characteristics of scaffold attachment region (SAR) sequences. It contains matrix consensus elements, a topoisomerase II binding sequence and it associates with the isolated nuclear scaffold of soybeanin vitro. Chimaeric genes containing the SAR_L fragment either at one side (5 or 3) or at both sides of a heat shock promoter-regulated -glucuronidase reporter gene were constructed. A five-to nine-fold increase of heat-inducible -glucuronidase activity was observed in transgenic tobacco plants containing constructs with SAR_L fragments either at both sides or with at least one SAR_L copy located upstream from the reporter gene. The gene copy number is positively correlated with the level of heat-inducible reporter gene activity in these. plants but positional effects are not entirely eliminated. Thus, SAR sequences may potentially be used to increase gene expression, via as yet unknown mechanisms, and to reduce adverse effects on the expression of multiple gene copies in transgenic plants.     

ULTRASTRUCTURAL ASPECTS OF SEXUAL REPRODUCTION IN THE RED TIDE DINOFLAGELLATE GONYAULAX TAMARENSIS1

The marine dinoflagellate Gonyaulax tamarensis Lebour is best known for its propensity to form blooms known as red tides in coastal waters worldwide. This paper examines the sexual cycle of this organism using light and electron microscopy. Sexual reproduction begins with contact between thecate gametes which subsequently shed their thecae to fuse along their pellicular layers. Nuclear fusion occurs well after cytoplasmic fusion and is characterized by several distinctive features: a highly vesiculate nucleoplasm without microtubules; nucleoli and V-shaped chromosomes abut the nuclear envelope distal to the region of nuclear contact; and each chromosome possesses a longitudinal line, the central chromosomal axis. Fusion results in a planozygote with numerous cytoplasmic storage products and a slightly thickened layer beneath the pellicle. Subsequent loss of thecal plates and a thickening of the sub-pellicular layer results in a non-motile hypnozygote. A newly-formed hypnozygote possesses numerous minute papillae along its outer surface, formed by the up-folding of the accumulating wall layer. Maturation of the hypnozygote wall results in a smooth three-layered wall, the outermost layer of which is the pellicular layer. Hypnozygote germination produces a large quadriflagellate plan-omeiocyte with a single nucleus and thecal plates identical to vegetative cells. Two subsequent divisions, presumably meiotic, result in Jour cells morphologically identical to vegetative cells.     

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.     

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.     

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     

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.