ABSTRACT
This data article is related to the research article entitled "Assessment of structurally modified plant virus as a novel adjuvant in toxicity studies" (Nikitin et al., 2018), devoted to the safety study of structurally modified plant virus - spherical particles (SPs). SPs are generated by thermally denatured tobacco mosaic virus (TMV) coat protein and act as effective adjuvant for development of new vaccine candidates. This article reports the additional results on the toxicity studies of TMV SPs. The weight coefficients of laboratory animals internal organs complements the data of the subchronic toxicity studies. Also plaque-forming cell assay, delayed-type hypersensitivity test and peritoneal macrophage assay as a part of immunotoxicity studies of TMV SPs are presented.
ABSTRACT
In oocytes, nontranslated maternal mRNAs are packaged by protein into messenger ribonucleoprotein particles (mRNPs) that are masked from translation by protein-RNA interactions. Proteins associated with such masked states of mRNAs are particularly abundant in amphibian oocytes. One of these mRNP proteins from Xenopus oocytes, mRNP3+4 (also called FRG Y2a/b or p54/p56), binds to diverse mRNAs independent of their sequence and is the germ line member of the evolutionarily conserved Y box protein multigene family. Xenopus oocytes contain soluble pools of mRNP3+4 6 S oligomers, probably dimers, and larger approximately 15 S particles containing mRNP3+4 and additional proteins. Here we report the purification of this larger form as an approximately 320-kDa particle that contains mRNP3+4 and nine additional polypeptides, including mRNA-binding polypeptides of 34 and 36 kDa and a doublet of 110/105 kDa that proved to be nucleolin. The particle has a protein kinase activity that phosphorylates its own mRNP3+4, nucleolin, and a 31-kDa polypeptide component and exhibits translational inhibition in both the wheat germ extract and rabbit reticulocyte lysate systems. The presence of mRNP3+4 and nucleolin in this large translation regulatory particle suggests that it participates in an early step of mRNP assembly and masking.
Subject(s)
Oocytes/physiology , Protein Biosynthesis , RNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Xenopus Proteins , Animals , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Female , Gene Expression Regulation , Molecular Weight , Multigene Family , Nuclear Proteins/isolation & purification , Nuclear Proteins/metabolism , Phosphoproteins/isolation & purification , Phosphoproteins/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/isolation & purification , Rabbits , Reticulocytes/metabolism , Transcription Factors/isolation & purification , Xenopus laevis , NucleolinABSTRACT
Preincubation of bovine caudate nucleus membranes with the 2',3'-dialdehyde of GTP (oGTP) reduces adenylate cyclase activation by guanylyl imidodiphosphate (GppNHp) in a time-dependent fashion. A slower rate of inhibition is observed if membranes are treated with both GTP and oGTP. The efficacy of oGTP action is enhanced by raising the Mg2+ concentration. Reduction of adenylate cyclase sensitivity to GppNHp is followed by an irreversible decrease of enzyme stimulation by forskolin. Addition of a Lubrol soluble preparation from guinea pig lung membranes to oGTP-treated caudate nucleus membranes causes restoration of the adenylate cyclase sensitivity to GppNHp. These data suggest that oGTP blocks the GTP-binding site of the adenylate cyclase system localized on the Ns protein. Such modification leads to the elimination of the Ns-mediated regulation of the enzyme.
Subject(s)
Caudate Nucleus/enzymology , Guanosine Triphosphate/analogs & derivatives , Adenylyl Cyclase Inhibitors , Animals , Cattle , Colforsin , Diterpenes/pharmacology , Enzyme Activation/drug effects , Guanosine Triphosphate/pharmacology , Guanylyl Imidodiphosphate/pharmacology , Guinea Pigs , Kinetics , Lung/analysis , Magnesium/pharmacology , Polyethylene Glycols/pharmacologyABSTRACT
Guanine nucleotides are successfully used in the studies of regulatory N-proteins coupled with adenylate cyclase. In the present work N-chloroacetylhydrazones of oxo-GTP and oxo-GDP are described. After 4 hr preincubation of these nucleotides with plasma membranes from bovine brain caudate nucleus, the ability of adenylate cyclase to be activated by guanylyl-5'-methylene-diphosphonate is blocked. The degree of inhibition depends on preincubation time and increases in the presence of Mg2+. Guanylyl-5'-methylenediphosphonate protects adenylate cyclase from the action of N-chloroacetylhydrazone of oxo-GTP. These findings suggest that adenylate cyclase activation is diminished as a result of covalent modification of the Ns. N-chloroacetyl-hydrazone of oxo-GDP also causes a loss of the adenylate cyclase sensitivity to the fluoride ion and cholera toxin.
Subject(s)
Adenylyl Cyclase Inhibitors , GTP-Binding Proteins/antagonists & inhibitors , Guanine Nucleotides , Guanosine Diphosphate/analogs & derivatives , Guanosine Triphosphate/analogs & derivatives , Animals , Cattle , Caudate Nucleus/enzymology , Diphosphonates/pharmacology , Enzyme Activation/drug effects , Guanosine Diphosphate/chemical synthesis , Guanosine Diphosphate/pharmacology , Guanosine Monophosphate/analogs & derivatives , Guanosine Monophosphate/pharmacology , Guanosine Triphosphate/chemical synthesis , Guanosine Triphosphate/pharmacology , In Vitro Techniques , Magnesium/pharmacologyABSTRACT
The effect of 5'-(p-bromomethylbenzoyl) adenosine (pBMBA) on adenylate cyclase from bovine caudate nucleus membranes was studied. Adenylyl-5'-methylenediphosphonate (but not adenosine) protected adenylate cyclase against inactivation by this compound. The degree of pBMBA-induced inhibition of adenylate cyclase increased in the presence of Mg2+. 5'-(p-fluorosulfonylbenzoyl) adenosine (pFSBA) was also a specific irreversible inhibitor of adenylate cyclase. It was demonstrated that the enzyme inactivated by pFSBA completely restored its activity under the action of dithiothreitol. The results obtained are indicative of the presence of the -SH group in the enzyme active site.
Subject(s)
Adenosine/analogs & derivatives , Adenylyl Cyclase Inhibitors , Caudate Nucleus/enzymology , Adenosine/pharmacology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , Binding Sites/drug effects , Cattle , Cell Membrane/enzymology , Dithiothreitol/pharmacology , KineticsABSTRACT
6-Cloro-9-beta-d-ribofuranosylpurine 5'-triphosphate (CIRTP) and 6-mercapto-9-beta-d-ribofuranosylpurine 5'-triphosphate (SRTP) irreversibly inhibit adenylate cyclase from rat brain. Adenosine 5'-[beta, gamma -imido] triphosphate protects the enzyme against inactivation by CIRTP and SRTP and acts as a competitive inhibitor with respect to ATP with the Ki value 2 X 10(-4) M. Study of the pH-dependence of the rate of the enzyme inactivation by CIRTP showed that pK for the group modified by this compound is equal to 7.45. Inactivation is first order with respect to the enzyme; the saturation effect is observed at the increased concentration of CIRTP. The k2 and KI values for irreversible inhibition of brain adenylate cyclase by CIRTP were 0.25 min-1 and 1.9 X 10(-4) M, respectively. Adenylate cyclase inhibition by SRTP is also time-dependent. Partial protection against the enzyme inactivation was observed. Dithiothreitol restores the activity of SRTP-inactivated adenylate cyclase. The results obtained indicate the presence of an -SH group in the purine amino group binding area of the enzyme active site.
