[Molecular phylogeny of gastrotricha based on 18S rRNA genes comparison: rejection of hypothesis of relatedness with nematodes].

Gastrotrichs are meiobenthic free-living aquatic worms whose phylogenetic and intra-group relationships remain unclear despite some attempts to resolve them on the base of morphology or molecules. In this study we analysed complete sequences of the 18S rRNA gene of 15 taxa (8 new and 7 published) to test numerous hypotheses on gastrotrich phylogeny and to verify whether controversial interrelationships from previous molecular data could be due to the short region available for analysis and the poor taxa sampling. Data were analysed using both maximum likelihood and Bayesian inference. Results obtained suggest that gastrotrichs, together with Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea and Lophotrochozoa, comprise a clade Spiralia. Statistical tests reject phylogenetic hypotheses regarding Gastrotricha as close relatives of Nematoda and other Ecdysozoa or placing them at the base of bilaterian tree close to acoels and nemertodermatides. Within Gastrotricha, Chaetonotida and Macrodasyida comprise two well supported clades. Our analysis confirmed the monophyly of the Chaetonotidae and Xenotrichulidae within Chaetonida as well as Turbanellidae and Thaumastodermatidae within Macrodasyida. Mesodasys is a sister group of the Turbanellidae, and Lepidodasyidae appears to be a polyphyletic group as Cephalodasys forms a separate lineage at the base of macrodasyids, whereas Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To infer a more reliable Gastrotricha phylogeny many species and additional genes should be involved in future analyses.

A major nucleolar protein B23 as a marker of proliferation activity of human peripheral lymphocytes.

A novel monoclonal antibody (Mab) (called 3C9) against a major nucleolar phosphoprotein B23 was used to study B23 qualitative and quantitative alterations in phytohemagglutinin (PHA) -stimulated human peripheral blood lymphocytes in indirect immunofluorescence and Western blots. It was shown that lymphocyte proliferation was accompanied by gradual augmentation of nucleoli and their accumulation of the protein B23 up to 2-fold by 16 h and 40-50 fold by 72 h, as compared with the non-stimulated cells. By parallel immunolabeling with the anti-Ki-67 antibody, it was shown that the early changes of B23 amount and localization occurred before an appearance of Ki-67 protein, a well-known marker of proliferating cells. Our results evidence that antibodies against B23 might be applied for recognition of human peripheral lymphocytes at early stages of their activation for proliferation, preceding the S-phase.

[The role of calmodulin (delta-subunit) in the activation of phosphorylase kinase from rabbit skeletal muscles]. / Rol' kal'modulina (delta-sub''edinitsy) v protsesse aktivatsii kinazy fosforilazy iz skeletnykh myshts krolika.

A comparative study on the structure of nonactivated and activated forms of phosphorylase kinase was carried out. The enzyme was activated by incubation in alkaline medium (pH 8.5), by phosphorylation with cAMP-dependent protein kinase and by limited proteolysis. The comparative analysis was based on the use of hydrophobic chromatography on phenyl-sepharose and electrophoresis in polyacrylamide gel density gradient. Activation of the enzyme was accompanied by separation of a low molecular weight component (Mr about 17 000). Using chromatography on phenyl-sepharose, this low molecular weight protein was obtained in a homogeneous state. It was found that the properties of the protein are close to those of calmodulin. The presence of calmodulin in phosphorylase kinase preparations was judged upon by the activation of the calmodulin-dependent form of phosphodiesterase. The boiled and subtilisin-treated kinase activates phosphodiesterase in the same way as does bovine brain calmodulin. The experimental results suggest that the delta-subunit is a protein inhibitor of the enzyme.

Study of properties of phosphorylase kinase using hydrophobic chromatography.

The structure of nonactivated and activated forms of phosphorylase kinase has been investigated. The enzyme activation was achieved by phosphorylation with cAMP-dependent protein kinase as well as by incubation of the enzyme in an alkaline medium (pH 8.8). For structural comparison of the enzymic forms, hydrophobic chromatography on phenyl-Sepharose and polyacrylamide gel electrophoresis were used. It has been shown that the enzyme activation results in a release of a low molecular weight component (Mr 16 000). The properties of this component resemble those of calmodulin. Evidence for the formation of an unstable nonactivated phosphorylase kinase - calmodulin complex may be important for the correct understanding of the mechanism of enzyme activation.

[Properties of the catalytically active fragment obtained by limited proteolysis of phosphorylase kinase]. / Svoistva kataliticheski aktivnogo fragmenta, poluchennogo putem ogranichennogo proteoliza kinazy fosforilazy.

The activation of phosphorylase kinase by limited proteolysis with subtilisin results in a formation of new enzyme forms differing in their molecular weights. Using gradient electrophoresis in polyacrylamide gel, it was shown that the high molecular weight fraction is made up of active fragments having different molecular weights. The low molecular weight fraction was found to contain only one active fragment with molecular weight of 80 000. Disc electrophoresis in polyacrylamide gel demonstrated that the active fragments of the high and low molecular weight fractions are not homogenous. The kinetic properties of the low molecular weight fragment were investigated. It was found that at pH 8.2 the native non-activated kinase and the catalytically active fragment have identical Km values for the substrates (phosphorylase B and MgATP); however, unlike the non-activated kinase, this fragment possesses a decreased sensitivity to Ca2+ and effectors (glycogen and glucose 6-phosphate) and has no optimum of activity within the pH range of 6.0-9.0.