Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites.

Epidermal growth factor (EGF)-receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH-3T3 cells lacking endogenous EGF-receptors. Kinetic parameters of the kinase of wild-type and mutant receptors were compared. Both wild-type and mutant EGF-receptors had a Km(ATP) 1-3 microM for the autophosphorylation reaction, and a Km(ATP) of 3-7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF-receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF-receptor (KGSTAENAEYLRV) was phosphorylated by wild-type receptor with a Km of 110-130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF-receptors phosphorylated the peptide substrate with a Km of 70-100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c-src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild-type receptor to 890 microM for mutant receptor and for c-src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650-750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factors I and II in fetal and adult bovine serum. Purification, primary structures, and immunological cross-reactivities

Insulin-like growth factors I and II (IGF-I and IGF-II) were prepared from fetal calf serum and adult bovine serum by gel filtration, immunoaffinity chromatography, chromatofocusing, and reverse-phase high pressure liquid chromatography, and their complete amino acid sequences determined. IGF-I and -II are found in both adult and fetal serum. The sequence of bovine IGF-I is found to be identical to that of human IGF-I, whereas 3 out of 67 amino acid residues are found to be different between bovine and human IGF-II. The differences are located in the C-peptide region of the molecule. Bovine IGF-II shows less than 10% immunological cross-reactivity with antisera against human and rat IGF-II, but is equipotent to human IGF-II in displacing human 125I-labeled IGF-II from human placental receptor. Bovine IGF-I was equipotent to human IGF-I in both radioimmunoassays and radioreceptor assays within the limits of the assay.     

The Impact of Different Long-Term Storage Conditions on the Viability of Lichen-Forming Ascomycetes and their Green Algal Photobiont, Trebouxia spp.

Abstract: Lichen-forming ascomycetes and their green algal photobionts completely die off within approximately 3 years of storage at room temperature. Macroscopically this is recognizable as a colour change, the green shades of the chlorophylls being lost. In fluorescent light microscopy preparations an increase in fungal autofluorescence and a significant decrease in chlorophyll autofluorescence in the Trebouxia cells was observed. In transmission electron microscopy preparations of Xanthoria parietina and its green algal photobiont, Trebouxia arboricola, the fungal membrane systems were found to be largely broken down whereas the shrivelled algal protoplast failed to rehydrate after storage at room temperature. When stored in the desiccated state at - 20 °C, both partners of the symbiosis stayed fully viable for up to 13 years, their colouration and chlorophyll fluorescence being unchanged. Viability was measured as ascospore ejection and germination rates in Xanthoria parietina, soredium germination rates in Xanthoria fallax, Hypogymnia physodes and Parmelia sulcata, and autospore formation rate in Trebouxia cells (green algal photobiont), which had been isolated from the thalli after rehydration. Thallus fragments of Xanthoria parietina were shown to grow normally after one week of storage in LN₂ without any cryoprotectant. In the desiccated state deep-frozen samples can be repeatedly brought to room temperature and back to - 20 °C without any loss of viability. Cryopreservation is therefore a suitable mode of long-term storage of viable lichen thalli for experimental studies or transplant experiments.     

The ultrastructure of campaniform sensilla on the eye of the cricket,Gryllus campestris

Summary The structure of the campaniform sensilla of the cricket eye was investigated by light and electron microscopy. Each sensillum is innervated by a single bipolar neuron. Its axon extends through the retina into a side-branch of the nervus tegumentarius. The dendrite extends through a cuticular channel to the surface of the cornea. The distal part of the dendrite, the sensory process, contains a tubular body and is attached to a cuticular cap which is obliquely inserted into the exocuticle between the corneal lenslets. Some particular structural features as well as the function of the campaniform sensillum of the cricket eye are discussed.Supported by the Deutsche Forschungsgemeinschaft, grant Ho 463/10The authors are indebted to Prof. H. Altner, University of Regensburg, and Mrs. Evelyn Thury, Contron GmbH, München for use of the scanning electron microscope facilities     

Chemical modification of peptides by hydrazine.

High pressure ('performance') liquid chromatography on reverse-phase supports has been used to characterize the products arising from the hydrazine treatment of peptides. In addition to converting arginine residues into ornithine, the reaction was found to cleave predominately Gly-Xaa, Xaa-Gly, Asn-Xaa and Xaa-Ser peptide bonds. Peptide-bond cleavage and deguanidation was studied as a function of time of exposure to hydrazine, hydrazine concentration and temperature. The convenience of this method of chromatography for the rapid low-cost separation and isolation of peptides, as well as their reaction products, is illustrated at the level of material required for solid-phase microsequencing.     

Ultrastructural and experimental investigations of sperm-egg interactions in fertilization ofHydra carnea

Summary Fertilization in the freshwater hydrozoanHydra carnea has been examined by light, scanning and transmission electron microscopy. Sperm penetrate the jelly coat which covers the entire egg surface only at the site of the emission of the polar bodies. The egg surface exhibits a small depression, the so called fertilization pit at this site. Sperm-egg fusion takes place only at the bottom of the fertilization pit.Hydra sperm lack a structurally distinct acrosome and in most of the observed cases, fusion was initiated by contact between the membrane of the lateral part of the sperm head and the egg surfacce. Neither microvilli nor a fertilization cone are formed at the site of gamete fusion. The process of membrane fusion takes only a few seconds and within 1 to 2 min sperm head and midpiece are incorporated in the egg.Electron dense material is released by the egg upon insemination but cortical granule exocytosis does not occur and a fertilization envelope is not formed. The possible polyspermy-preventing mechanisms in hydrozoans are discussed. Hydra eggs can be cut into halves whereupon the egg membranes reseal at the cut edges and the fragments assume a spherical shape. Fragments containing the female pronucleus can be inseminated and exhibit normal cleavage and development. The observation that in such isolated parts the jelly coat will not fuse along the cut edges was used to determine its role in site-specific gamete fusion. These experiments indicate that site-specificity of gamete fusion can be attributed to special membrane properties at the fertilization pit.     

Evidence for subclasses of SH groups in (Na++K+)-ATPase.

Changes in the chemical reactivity of the sulfhydryl groups of (Na⁺ + K⁺)-dependent ATPase can be indicative of conformational changes induced by activating ions. Cyanylation of these groups by 5 mM 2-nitro-5-thiobenzoic acid caused a partial inhibition of enzymatic activity. Both this loss and the incorporation of radioactive cyanide from the ¹⁴C-labeled reagent were reduced by inclusion of 50 mM ATP and 150 mM Na⁺ in the incubation. When 10 mM Mg⁺⁺ was added in addition, the inactivation was not different from that produced by cyanylation reagent alone, but the radioactive labeling of protein increased significantly. The data indicate that the sulfhydryl groups of this enzyme exist in two populations, one of which must be free if the enzyme is to function. The other, not essential for enzymatic activity, becomes accessible only when the Na⁺ and Mg⁺⁺-dependent phosphorylation of the enzyme alters its conformation. Inactivation of the enzyme by freezing and thawing increases the incorporation of radioactivity but destroys the responsiveness of labeling to cations and ATP.     

Contributions to the diversity in cryptogamic covers in the mid‐Palaeozoic: Nematothallus revisited

Compression fossils from the Silurian and Devonian of southern Britain, composed of cuticles and tubes, were described by W. H. Lang as the genus Nematothallus and placed, with Prototaxites, in Nematophytales, related neither to algae nor tracheophytes. Dispersed cuticles of Nematothallus and perforated forms assigned to Cosmochlaina were frequently recovered in macerates, their affinities being unresolved. New collections from a Lochkovian locality in the Welsh Borderland permitted the reconstruction of the stratified thalli of these nematophytes; they comprise a superficial cortex (which produced the cuticles) overlying a palisade zone composed of septate, parallel tubes, presumed to be hyphae, and a basal zone comprising wefts of randomly interwoven hyphae. Excellent three dimensional preservation allows the erection of a new species of Nematothallus, N. williamii. A similar anatomy is seen in a new group of fossils with either circular incisions in the cortex or complete separation of thickened cortical cells, presumably comprising a developmental sequence. By their stratified organization the nematophytes differ from extant and extinct algae and bryophytes and the enigmatic Spongiophyton. A complex anatomy and septate tubes suggest affinity with lichenized fungi. Limited data support a fungal rather than embryophyte chemistry, but a photobiont is missing. Nematophytes, globally widespread in cryptogamic covers from mid‐Ordovician times, added to the biodiversity in early terrestrial ecosystems and enhanced chemical weathering. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 505–534.     

Structural Basis for Eliciting a Cytotoxic Effect in HER2-Overexpressing Cancer Cells via Binding to the Extracellular Domain of HER2

1. Download : Download high-res image (208KB)
2. Download : Download full-size image