Identification and partial characterization of a latent ATP,Mg-dependent protein phosphatase in rabbit skeletal muscle cytosol

Summary Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase F_A-dependent form upon incubation at 30°C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent M _r in sucrose density-gradient centrifugation (70 000) and in gel filtration (110 000).Abbreviations PMSF Phenylmethanesulphonyl Fluoride - TLCK L-l-chloro-3-(4-tosylamido)-7-amino2-heptanone-hydrochloride - TPCK L-l-chloro-3-(4-tosvlamido)-4-phenyl-2-butanone     

Rapid Test for Urease and Phenylalanine Deaminase Production

A rapid urea-phenylalanine medium was effective for the identification of Proteus and, with one exception, Providencia. Most Klebsiella and a few Enterobacter were urease-positive with this method.     

Interleukin-8 activates microtubule-associated protein 2 kinase (ERK1) in human neutrophils

The signal transduction initiated by the human cytokine interleukin-8 (IL-8), the main chemotactic cytokine for neutrophils, was investigated and found to encompass the stimulation of protein kinases. More specifically, IL-8 caused a transient, dose and time dependent activation of a Ser/Thr kinase activity towards myelin basic protein (MBP) and the MBP-derived peptide APRTPGGRR patterned after the specific concensus sequence in MBP for ERK enzymes. The activated MBP kinase was furthermore identified as an extracellular signal regulated kinase (ERK1) based on several criteria such as substrate specificity, molecular weight, activation-dependent mobility shift, and recognition by anti-ERK antibodies. For comparison, the chemotactic response of neutrophils to a stimulus of bacterial origin (fMet-Leu-Phe or fMLP) was also examined and found to involve the activation of a similar ERK enzyme. The present data clearly indicate that in terminally differentiated, non-proliferating human cells, the MBP kinase/ERK activity can serve other purposes than mitogenic signaling, and that processes such as chemotaxis, induced by bacterial peptides as well as by human cytokines like IL-8, involve the regulation of ERK enzyme.Abbreviations IL-8 interleukin-8 - fMLP fMet-Leu-Phe - MBP myelin basic protein - ERK extracellular signal regulated kinase - MAP2 microtubule-associated protein 2 - PK-A cAMP dependent protein kinase - PKI protein kinase inhibitor - PMSF phenyl-methanesulfonyl fluoride - PVDF poly-vinylidene difluoride - HBSF Hank's buffered salt solution - DAB 3,3-diaminobenzidine tetrahydrochloride - PNPP p-nitrophenyl-phosphate - HSA human serum albumin - EGTA [ethylenebis (oxyethylenenitrilo)]tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Intragenic Suppressors of Folding Defects in the P22 Tailspike Protein

Within the amino acid sequences of polypeptide chains little is known of the distribution of sites and sequences critical for directing chain folding and assembly. Temperature-sensitive folding (tsf) mutations identifying such sites have been previously isolated and characterized in gene 9 of phage P22 encoding the tailspike endorhamnosidase. We report here the isolation of a set of second-site conformational suppressors which alleviate the defect in such folding mutants. The suppressors were selected for their ability to correct the defects of missense tailspike polypeptide chains, generated by growth of gene 9 amber mutants on Salmonella host strains inserting either tyrosine, serine, glutamine or leucine at the nonsense codons. Second-site suppressors were recovered for 13 of 22 starting sites. The suppressors of defects at six sites mapped within gene 9. (Suppressors for seven other sites were extragenic and distant from gene 9.) The missense polypeptide chains generated from all six suppressible sites displayed ts phenotypes. Temperature-sensitive alleles were isolated at these amber sites by pseudoreversion. The intragenic suppressors restored growth at the restrictive temperature of these presumptive tsf alleles. Characterization of protein maturation in cells infected with mutant phages carrying the intragenic suppressors indicates that the suppression is acting at the level of polypeptide chain folding and assembly.     

Bacterial composition of soils of the Lake Wellman area, Darwin Mountains, Antarctica

The aim of this study was to examine the bacterial composition of high latitude soils from the Darwin–Hatherton glacier region of Antarctica. Four soil pits on each of four glacial drift sheets were sampled for chemical and microbial analyses. The four drifts—Hatherton, Britannia, Danum, and Isca—ranged, respectively, from early Holocene (10 ky) to mid-Quaternary (ca 900 ky). Numbers of culturable bacteria were low, with highest levels detected in soils from the younger Hatherton drift. DNA was extracted and 16S rRNA gene clone libraries prepared from samples below the desert pavement for each of the four drift sheets. Between 31 and 262 clones were analysed from each of the Hatherton, Britannia, and Danum drifts. Bacterial sequences were dominated by members of the phyla Deinococcus-Thermus, Actinobacteria, and Bacteroidetes. Culturable bacteria, including some that clustered with soil clones (e.g., members of the genera Arthrobacter, Adhaeribacter, and Pontibacter), belonged to Actinobacteria and Bacteroidetes. The isolated bacteria are ideal model organisms for genomic and phenotypic investigations of those attributes that allow bacteria to survive and/or grow in Antarctic soils because they have close relatives that are not tolerant of these conditions.     

Ordinary ethics: lay people's deliberations on social sex selection

Abstract

This article summarises the results of a research project that used a scenario about sex selection of embryos for social reasons as a basis for discussion groups with lay people. The aim of the research was to examine the processes by which non-professionals make ethical evaluations in relation to a contested area in medical genetics. We note in particular the role played in the discussions by expressions of instinct; making distinctions; rational argument; reference to principles; use of personal experience; analogies and examples; slippery slope arguments and meta-reflections. The implications for developing processes of public consultation and debate are also considered.