Potassium ion accumulation at the external surface of the nodal membrane in frog myelinated fibers.

Potassium accumulation associated with outward membrane potassium current was investigated experimentally in myelinated fibers and analyzed in terms of two models-three-compartment and diffusion in an unstirred layer. In the myelinated fibers, as in squid giant axons, the three-compartment model satisfactorily describes potassium accumulation. Within this framework the average space thickness, theta, in frog was 5,900 +/- 700 A, while the permeability coefficient of the external barrier, PK, was (1.5 +/- 0.1) X 10(-2) cm/s. The model of ionic diffusion in an unstirred aqueous layer adjacent to the axolemma, as an alternative explanation for ion accumulation, was also consistent with the experimental data, provided that D, the diffusion constant, was (1.8 +/- 0.2) X 10(-6) cm/s and l, the unstirred layer thickness, was 1.4 +/- 0.1 micron, i.e., similar to the depth of the nodal gap. An empirical equation relating the extent of potassium accumulation to the amplitude and duration of depolarization is given.     

Two hybrid plasmids with D. melanogaster DNA sequences complementary to mRNA coding for the major heat shock protein.

The isolation and partial characterization of two cloned segments of Drosophila melanogaster DNA containing "heat shock" gene sequences is described. We have inserted sheared embryonic D. melanogaster DNA by the poly(dA-dt) connector method (Lobban and Kaiser, 1973) into the R1 restriction site of the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975). A collection of independent hybrid plasmids was screened by colony hybridization (Grunstein and Hogness, 1975) for sequences complementary to in vitro labeled polysomal poly(A)+ heat shock RNA. Two clones were identified which contain sequences complementary to a heat shock mRNA species that directs the in vitro synthesis of the 70,000 dalton heat-induced polypeptide. Both cloned segments hybridize in situ to the heat-induced puff sites located at 87A and 87C of the salivary gland polytene chromosomes.     

C1 proteins: a class of high-mobility-group non-histone chromosomal proteins from the fruit fly Ceratitis capitata

1. CM-cellulose chromatography of a fraction soluble in 5% perchloric acid from Ceratitis capitata chromatin yields three proteins, C1a1, C1a2 and C1b, which have been purified to electrophoretical homogeneity. 2. C1a1, C1a2 and C1b analyse like high mobility group (HMG) non-histone chromosomal proteins, although they do not exactly correspond with those from vertebrates. It is proposed that C1 proteins, as well as Drosophila D1 [Rodríguez Alfageme et al. (1980) Chromosoma, 78, 1-31] are representative of a class of insect-specific HMG proteins. Tryptic fingerprints show that C1a1 and C1a2 are very similar, but C1b is a somewhat distinct protein. Circular dichroism studies have shown that these preparations do not appreciably fold on increasing ionic strength. 3. The interactions between DNA and C1 proteins have been studied. These proteins precipitate DNA in 0.15 M NaCl, 0.015 M sodium citrate and the precipitation curves are cooperative. Soluble complexes between C1 proteins and DNA could be prepared in low ionic strength media and their thermal denaturation profiles obtained. C1 proteins do not destabilize DNA under the conditions used to prepare the complexes but the three proteins stabilize DNA to a different degree. From these studies it has been concluded that the association constant of C1b to DNA is smaller than that of C1a1 and C1a2.     

Evolution of mouse B1 repeats: 7SL RNA folding pattern conserved

Summary In a recent report mouse B1 genomic repeats were divided into six families representing different waves of fixation of B1 variants, consistent with the retroposition model of human Alu elements. These data are used to examine the distribution of nucleotide substitutions in individual genomic repeats with respect to family consensus sequences and to compare the minimal energy structures of the corresponding B1 RNAs. By an enzymatic approach the predicted structure of B1 RNAs is experimentally confirmed using as a model sequence an RNA of a young B1 family member transcribed in vitro by T7 RNA polymerase. B1 RNA preserves folding domains of the Alu fragment of 7SL RNA, its progenitor molecule. Our results reveal similarities among 7SL-like retroposons, human Alu, and rodent B1 repeats, and relate the evolutionary conservation of B1 family consensus sequences to selection at the RNA level.     

Compositional analysis of Helicobacter pylori rough-form lipopolysaccharides.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the macromolecular heterogeneity of lipopolysaccharides (LPS) from seven fresh clinical isolates and three culture collection strains of the human pathogen Helicobacter pylori. All the clinical isolates produced smooth-form LPS with O side chains of relatively homogeneous chain length, whereas the culture collection strains yielded rough-form LPS. A better yield of the latter LPS was obtained when combined protease pretreatment and hot phenol-water extraction were used than when the conventional phenol-water technique alone was used for extraction. The LPS of the three culture collection strains (S-24, C-5437, and NCTC 11637) were chemically characterized. Constituents common to all the LPS were fucose, D-mannose, D-glucose, D-galactose, D-glycero-D-manno-heptose, L-glycero-D-manno-heptose, and 3-deoxy-D-manno-2-octulosonic acid. The molar ratios of the hexoses differed between different strains, thereby reflecting structural differences. Phosphate, phosphorylethanolamine, and pyrophosphorylethanolamine were present also. Free lipid A contained D-glucosamine and fatty acids, with phosphate and a minor amount of ethanolamine. The major fatty acids were ester- and amide-bound 3-hydroxyoctadecanoic acid and ester-bound octadecanioc and 3-hydroxyhexadecanoic acids, with minor amounts of ester-bound tetradecanoic and hexadecanoic acids. In addition to the uncommonly long 3-hydroxy fatty acids, an unusual phosphorylation pattern was deduced to be present in the lipid A.     

Proteins containing an uncleaved signal for glycophosphatidylinositol membrane anchor attachment are retained in a post-ER compartment

Glycophosphatidylinositol (GPI)-anchored membrane proteins are initially synthesized with a cleavable COOH-terminal extension that signals anchor attachment. Overexpression in COS cells of hGH-DAF fusion proteins containing the GPI signal of decay accelerating factor (DAF) fused to the COOH-terminus of human growth hormone (hGH), produces both GPI-anchored hGH-DAF and uncleaved precursors that retain the GPI signal. Using hGH-DAF fusion proteins containing a mutated, noncleavable GPI signal, we show that uncleaved polypeptides are retained inside the cell and accumulate in a brefeldin A-sensitive, Golgi-like juxtanuclear structure. Retention requires the presence of either a functional or a noncleavable GPI signal; hGH-DAF fusion proteins containing only the COOH-terminal hydrophobic domain (a component of the GPI signal) are secreted. Immunofluorescence analysis shows colocalization of the retained, uncleaved fusion proteins with both a Golgi marker and with p53, a marker of the ER-Golgi intermediate compartment. Since N-linked glycosylation is postulated to facilitate the transport of proteins to the cell surface, we engineered a glycosylation site into hGH-DAF. Glycosylation failed to completely override the transport block, but allowed some uncleaved hGH-DAF to pass through the secretory pathway and acquire endoglycosidase H resistance. The retained molecules remained endoglycosidase H sensitive. We suggest that the uncleaved fusion protein is retained in a sorting compartment between the ER and the medial Golgi complex. We speculate that a mechanism exists to retain proteins containing an uncleaved GPI signal as part of a system for quality control.     

Salmonella serotype typhimurium phage typing. A critical evaluation

Of 12,930 Salmonella serotype typhimurium strains, phage typed during 1985-1988, 45.68% were "nontypable" by Anderson's set; the percent of typable strains decreased from 54.17 in 1986 to 30.54 in 1988. Of 90 phage patterns of sensitivity, 22 were currently encountered. Phage types 1, 18 and 104 were most frequent to strain of both human and non-human origin. In food generating S. typhimurium outbreaks, phage types 1 and 36 were prevalent. Except lysotypes 198 and 95, isolated from "single cases" in man only, all other phage types were common in man and animals, too. Introducing other typing methods to serotype typhimurium "nontypable" strains (by Anderson's set) was considered necessary for epidemiological purposes.     

Characterization of a fibrinogenase from northern copperhead (Agkistrodon contortrix mokasen) venom

One of the fractions obtained by the carboxymethylcellulose ion-exchange chromatography of northern copperhead (Agkistrodon contortrix mokasen) venom prevented the thrombin-induced clotting of fibrinogen by proteolytically degrading the fibrinogen. The active component has been further purified to apparent electrophoretic homogeneity by molecular sieve chromatography. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated a molecular weight of 22 900 +/- 600 for the purified enzyme. In addition to its fibrinogenase activity, it catalyzed the hydrolysis of hide power azure and had an intraperitoneal LD50 value in mice of less than 5.1 microgram/g body weight. The enzyme rapidly destroyed fibrinogen's ability to form clots. Electrophoresis of fibrinogen which had been incubated only a few minutes with the fibrinogenase revealed the rapid disappearance of the alpha-chain and the appearance of lower molecular weight fragments. The neutral pH optimum and ethylenediamine-tetraacetic acid (EDTA) and dithiothreitol sensitivity indicated that this enzyme belonged to the class metalloproteinases. Atomic absorption studies have revealed one zinc atom per molecule of protein. The apoenzyme's activity was restored by incubation with ZnCl2.     

Stability of the Plasma Membrane in Saccharomyces rouxii and Its Relationship to Glucose Tolerance

The stability of spheroplasts from the osmotrophic yeast Saccharomyces rouxii was studied in buffered solutions of mannitol and glucose. The plasma membranes from cells grown in high glucose concentrations were more stable to osmotic lysis than were membranes from cells grown in lower glucose concentrations. Mannitol was a better osmotic stabilizer than glucose, except when the cells were grown in a high glucose concentration. Spheroplasts from a glucose tolerant-deficient mutant were much less stable than the corresponding spheroplasts from the parent strain, especially when suspended in glucose solutions. These results suggest an involvement of the plasma membrane in the glucose-tolerant mechanism of S. rouxii.