Evidence for the concentration of F-actin and myosin in synapses and in the plasmalemmal zone of axons

Fluorescent staining with phalloidin, a specific probe for F-actin, and antibodies to non-muscle myosin from thymus was used to localize actin and myosin in brain neurons of the rat. Phalloidin and anti-myosin displayed a preferential affinity for synaptic formations in the cerebellum, the brain stem, the spinal cord and the retina. The conclusion that F-actin and myosin are concentrated in synaptic terminals was further established by simultaneous staining of isolated rat brain synaptosomes with phalloidin and anti-thymus myosin as well as by the demonstration of a selective affinity of anti-thymus myosin for a 200 000-Mr protein band in gel electrophoretograms of synaptic fractions. Apart from synaptic areas, phalloidin and anti-thymus myosin reacted also, albeit rather weakly, with a narrow circumferential layer located in the area of the plasma membrane of virtually all axons in the white matter and the spinal roots. The spatial coexistence of myosin and actin in brain synapses and axons is of particular interest in view of various dynamic functions that have been proposed for axonal and synaptic actin.     

A kinetic study of interactions of (Rp)- and (Sp)-adenosine cyclic 3',5'-phosphorothioates with type II bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase

The stereoselectivity of the adenosine cyclic 3',5'-phosphate (cAMP) binding sites on the regulatory subunit of the type II bovine cardiac muscle cAMP-dependent protein kinase was investigated by examining the interactions of (Rp)- and (Sp)-adenosine cyclic 3',5'-phosphorothioates (cAMPS) with these sites. While activation of the holoenzyme and binding to the regulatory subunit of the type II kinase were observed for both of these diastereomers, there were significant differences between the interactions of the cAMPS isomers with the enzyme. In particular, the Sp isomer is more potent than the Rp species not only in the activation of reconstituted, as well as directly isolated, holoenzyme but also in the inhibition of [3H]cAMP binding to the regulatory subunit. A marked preference for the binding of the Sp isomer to site 2 in the regulatory subunit exists. Hydrogen bonding of a functional group on the regulatory subunit with preferential orientation toward the exocyclic oxygen rather than the sulfur of the thiophosphoryl residue may be involved in the observed selectivity of cAMPS binding and activation. In addition to our findings on the stereoselectivity of the binding of cAMPS to cAMP-dependent protein kinase, we have established a method for the reconstitution of holoenzyme from the purified subunits without subjecting the regulatory protein to denaturing conditions.     

Loss of tRNA 5-methyluridine methyltransferase and pseudouridine synthetase activities in 5-fluorouracil and 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur)-treated Escherichia coli

The degradation of human erythrocyte membrane proteins in relation to the identification of the monosaccharide transporter has been investigated in whole membrane preparations and membrane protein extracts by polyacrylamide gel electrophoresis in sodium n-dodecyl sulphate and iodine-125 labelling. Evidence is presented for the degradation of band 3 polypeptide to lower molecular weight material some of which appears in region 4.5 of the polyacrylamide gel electrophoresis profile. It is found that the degradation process is inhibited by phenylmethylsulphonyl fluoride and is only significant in membrane extracts in the absence of detergent (Triton X-100) and on prolonged incubation at 37 degrees C, conditions which do not prevail during the isolation of membrane protein extracts for reconstitution studies. Extracts of band 3 and band 4.5 have been prepared and reconstituted in bilayer lipid membranes. The permeabilities of the reconstituted systems to D-glucose have been investigated and it is found that only bilayers incorporating band 4.5 exhibited enhanced monosaccharide transport. A linear relationship between D-glucose transport and the concentration of protein in the aqueous phase bathing the bilayers suggests a partitioning of the protein into the bilayer. Reconstitution is stereospecific and inhibited by cytochalasin B.     

Pulsation of nuclei in insect oocytes is reversibly inhibited by cytochalasin B

Oocyte nuclei of the dipteran insect Heteropeza pygmaea display swift pulsating movements during in vitro follicle formation in the ovaries. Low doses of cytochalasin B (CB) completely inhibit the nuclear movements within a few minutes and cause the nuclei to assume spherical shapes. If the drug is removed, nuclear pulsation is resumed within 5–10 min. Phalloidin and colchicine do not affect the nuclear movements. Actin is shown by indirect immunofluorescence microscopy to be present in considerable amounts all over the cytoplasm of the oocytes. It is concluded that microfilaments are responsible for pulsation of the oocyte nuclei, whereas microtubules are not involved.     

Effect of pH and bicarbonate on phosphoenolpyruvate carboxykinase and glutaminase mRNA levels in cultured renal epithelial cells.

A gluconeogenic strain of renal epithelial cells (LLC-PK1-F+) was used to characterize the effect of pH and bicarbonate concentration on the levels of phosphoenolpyruvate carboxykinase (PCK) and glutaminase (GA) mRNAs. The levels of both mRNAs are markedly dependent upon medium glucose concentration. The level of PCK mRNA is increased with increasing glucose concentration from 0 to 40 mM, whereas the level of GA mRNA is maximal between 3 and 5 mM glucose. When LLC-PK1-F+ cells are grown with 5 mM glucose and then subjected to an acute decrease in pH (from 7.4 to 6.9) and bicarbonate concentration (from 25 to 10 mM), the level of PCK mRNA exhibits a biphasic response. The PCK mRNA is initially increased 4-fold within 3 h, then decreases slightly and subsequently increases between 10 and 20 h to a level that is 17-fold greater than normal. Only the initial increase parallels the changes observed in vivo. In contrast, after onset of acidosis, the level of GA mRNA initially remains unchanged, is then increased 8-fold between 10 and 16 h, and then decreases slightly. This response closely mimics the results obtained in vivo. A decrease in media pH at constant bicarbonate causes a marked increase in both mRNAs. However, the levels of the two mRNAs are also elevated by decreasing bicarbonate at a constant pH. Thus, both parameters independently affect the level of the two mRNAs. The use of actinomycin D to measure the half-lives of PCK and GA mRNAs at pH 7.4 and 6.9 indicates that stabilization may fully account for the induction of GA mRNA and contributes to the inductive effects of decreased pH and/or bicarbonate on PCK mRNA. Following recovery from acidic conditions, the two mRNAs exhibit a rapid and coordinate decrease (t1/2 approximately 20 min). Dexamethasone had no effect on the level of either mRNA, whereas cAMP increased only PCK mRNA. The latter effect was additive with the increase caused by decreased pH and/or bicarbonate and was reversed by incubating in alkalotic media. Thus, the induction of PCK and GA mRNAs during acidosis is initiated in direct response to a decrease in extracellular pH and/or bicarbonate.     

Targeted disruption of the Myxococcus xanthus orotidine 5''-monophosphate decarboxylase gene: effects on growth and fruiting-body development.

The Myxococcus xanthus gene coding for orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23) was cloned. The M. xanthus uraA gene efficiently complemented an Escherichia coli OMP decarboxylase mutant, permitting it to grow in the absence of uracil. Electroporation of M. xanthus with a circular plasmid carrying a selectable uraA::kan gene disruption resulted in homologous recombination at the chromosomal uraA locus. Chromosomal integration of the gene disruption plasmid created heterozygous (uraA+/uraA::kan) tandem duplications. These tandem duplications were unstable and segregated auxotrophic uraA::kan daughters at frequencies of 2 x 10(-4) to 8 x 10(-4) per viable cell. Rare uraA::kan segregants were easily obtained by selecting for resistance to the toxic analog 5-fluoroorotic acid. Our experiments suggest that the cloned uraA gene could facilitate the use of gene duplications in the genetic analysis of M. xanthus development. The uraA mutants could utilize uracil, uridine, or uridine 5'-phosphate for growth, indicating that M. xanthus has pyrimidine salvage pathways. During multicellular development, uraA::kan gene disruption mutants sporulated to wild-type levels but formed smaller and more numerous aggregates than did their uraA+ parent, regardless of whether uracil was added to the medium. Pyrimidine deprivation of uraA mutants, under conditions that otherwise supported vegetative growth, failed to induce fruiting-body development or sporulation.     

The yeast SEC17 gene product is functionally equivalent to mammalian alpha-SNAP protein.

The SEC17 gene of Saccharomyces cerevisiae is required for vesicular transport between the endoplasmic reticulum and the Golgi apparatus. Here we report that the product of the SEC17 gene has the exact biochemical properties expected for a yeast homologue of the mammalian transport factor, alpha-SNAP. The DNA sequence of SEC17 codes for a protein of predicted molecular mass of 33 kDa. Immunoblotting indicates that Sec17p fractionates as a peripheral membrane protein and is mostly soluble when overexpressed, suggesting the presence of a saturable membrane receptor for Sec17p. Sec17p was purified from yeast cytosol using a SNAP-dependent in vitro mammalian Golgi transport assay. Kinetic analysis using this assay shows Sec17p acts temporally close to the fusion of transport vesicles with the medial Golgi compartment. In yeast extracts, Sec17p binds to Sec18p with a 1:1 stoichiometry. The interaction between Sec17p and Sec18p requires an activity provided by yeast membranes, and this putative membrane receptor activity is not extracted by high salt treatment of membranes.