Roles of Escherichia coli heat shock proteins DnaK, DnaJ and GrpE in mini-F plasmid replication

Summary A subset of Escherichia coli heat shock proteins, DnaK, DnaJ and GrpE were shown to be required for replication of mini-F plasmid. Strains of E. coli K12 carrying a missense mutation or deletion in the dnaK, dnaJ, or grpE gene were virtually unable to be transformed by mini-F DNA at the temperature (30° C) that permits cell growth. When excess amounts of the replication initiator protein (repE gene product) of mini-F were provided by means of a multicopy plasmid carrying repE, these mutant bacteria became capable of supporting mini-F replication under the same conditions. However, the copy number of a high copy number mini-F plasmid was reduced in these mutant bacteria as compared with the wild type in the presence of excess RepE protein. Furthermore, mini-F plasmid mutants that produce altered initiator protein and exhibit a very high copy number were able to replicate in strains deficient in any of the above heat shock proteins. These results indicate that the subset of heat shock proteins (DnaK, DnaJ and GrpE) play essential roles that help the functioning of the RepE initiator protein in mini-F DNA replication.     

Effects of temperature on cytomorphogenesis and ultrastructure ofMicrasterias denticulata Bréb

Summary Exposure of growingMicrasterias cells to high (32°–36°C) and low (3°–10°C) temperatures produces changes in morphology that are accompanied by several ultrastructural alterations. Whereas low temperatures essentially cause simplification of cell ornamentation, a variety of cell malformations result from high temperature treatment. These are the loss of cell symmetry leading to markedly aberrant cell shapes and an increase of main lobes with reduced degree of differentiation. Preliminary studies indicate that a shift in the distribution of membrane-associated Ca²⁺ by elevated temperatures probably underlies these abnormal cytomorphogenetic events. Both, low and high temperature cause a reduction in size of the young half cell and affect cytoplasmic streaming. Moreover, nuclear migration is retarded and chloroplast arrangement is influenced by temperature treatment at both ranges. Growth velocity of primary wall responsible for cell shaping is increased at high and slowed down at low temperatures compared to cells grown at 20°C.The main ultrastructural alterations induced by high temperatures are an increase in amount and length of ER cisternae, the appearance of heat shock granule aggregations localized in the cytoplasm, a reduced number of ribosomes and polysomes, the presence of oil bodies in growing cells and a varying thickness of the primary wall. Influences of low temperatures on ultrastructure are less pronounced. They are manifested in the formation of large aggregations of ER cisternae slightly differing from those found in untreated cells, a disturbed arrangement of the microtubule system surrounding the nucleus and a decrease of the number of cell wall forming cytoplasmic vesicles.It is thought that most of the temperature effects are due to an influence on membranes probably an alteration of ionic currents and, in addition, a modulation of normal protein synthesis.Dedicated to my teacher Professor Oswald Kiermayer in deep gratitude     

Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m₁ muscarinic acetylcholine receptor. Stimulation of m₁ receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule-associated protein.     

Thiol-induced oligomerization of α-lactalbumin at high pressure

Denaturation and aggregation of-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, gel-permeation chromatography, sodium dodecyl sulfate and gel electrophoresis. It was found that the unfolding of-lactalbumin at high pressure is reversible even in basic pH and at a protein concentration as large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregation of-lactalbumin at high pressure increases significantly in the presence of low-molecular reducing agents such as cysteine, 2-mercaptoethanol, and dithiothreitol. Maximal yield of-lactalbumin oligomerization (over 90%) was achieved in the presence of cysteine at the molar cysteine/protein ratioq=2 and atpH 8.5. Apparent molecular weight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varyingpH and reducing agent. The size distribution shifts in the direction of very large, poorly soluble particles whenpH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH 5.5 when cysteine (q=2) is used as reducing agent. The oligomers of-lactalbumin are stabilized mainly by nonnative interchain disulfide bridges. Circular dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermolecular-sheets.     

Localization of α1-adrenoceptors in rat and human hearts by immunocytochemistry

The localization of the ai adrenoceptors (₁-AR) in the heart tissues from rat and human and in the cultured heart cells from neonatal rats was studied by indirect immunofluorescence and postembedding electronmicroscopical immuno-gold technique. With antipeptide antibodies directed against the second extracellular loop of the human ₁-AR (AS sequence 192–218), this receptor was found to be localized along the sarcolemma in both human and rat hearts. Similar localization sites were detected in cultivated rat neonatal cardiomyocytes. Beside the localization in cardiomyocytes, ₁-AR were identified in endothelial cells of capillaries and smooth muscle cells of coronary vessels, in neuronal endings, in mast cells of cultivated heart cells but not, or in less amount in fibroblasts. Interestingly, in the right atrium of rat heart the localization of ₁-AR was found to be near or on atrial natriuretic factor (ANF) granules, providing the basis for the -adrenergic influence on ANF release. The immunocytochemical studies further confirm and complete the findings known by using autoradiographic binding studies with specific ligands.     

The mitochondrial inner membrane anion channel is inhibited by DIDS

The mitochondrial inner membrane anion channel (IMAC) is a channel, identified by flux studies in intact mitochondria, which has a broad anion selectivity and is maintained closed or inactive by matrix Mg²⁺ and H⁺. We now present evidence that this channel, like many other chloride/anion channels, is reversibly blocked/inhibited by stilbene-2,2-disulfonates. Inhibition of malonate transport approaches 100% with IC₅₀ values of 26, 44, and 88 M for DIDS, H₂-DIDS, and SITS respectively and Hill coefficients 1. In contrast, inhibition of Cl^– transport is incomplete, reaching a maximum of about 30% at pH 7.4 and 65% at pH 8.4 with an IC₅₀ which is severalfold higher than that for malonate. The IC₅₀ for malonate transport is decreased about 50% by pretreatment of the mitochondria withN-ethylmaleimide. Raising the assay pH from 7.4 to 8.4 increases the IC₅₀ by about 50%, but under conditions where only the matrix pH is made alkaline the IC₅₀ is decreased slightly. These properties and competition studies suggest that DIDS inhibits by binding to the same site as Cibacron blue 3GA. In contrast, DIDS does not appear to compete with the fluorescein derivative Erythrosin B for inhibition. These findings not only provide further evidence that IMAC may be more closely related to other Cl^– channels than previously thought, but also suggest that other Cl^– channels may be sensitive to some of the many regulators of IMAC which have been identified.     

Biochemical and immunocytochemical identification of conglutin γ, a lupin seed lectin,in the roots of young germinating seedlings

Summary Polyclonal antibodies directed against polypeptide epitopes of conglutin , a glycosylated lectin in lupin seed, have been used to identify and quantify this protein in root extracts of germinating lupins. The highest conglutin content was found in protein extracts of root elongation zones after 5 to 7 days germination. Root conglutin showed the same subunit composition, glycosylation pattern, isoelectric point, and lectin activity as the cotyledonary one. Immunolocalization experiments on root thin sections demonstrated that conglutin is chiefly present in the intercellular spaces of the cortical parenchyma, where it forms large aggregates. Labelling of the Golgi complexes and the area between the plasmalemma and cell wall revealed the conglutin pathway from post-synthetic processing to excretion via the secretory system.Abbreviations EDTA ethylene diamino tetraacetic acid - IEF isoelectric focusing - LRW London resin white - NC nitrocellulose - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - P_i inorganic phosphate - SDS sodium dodecylsulphate - TEM transmission electron microscopy