E. M. Karger

Mutational analysis of human prothymosin α reveals a bipartite nuclear localization signal

Mutants of human prothymosin α with impaired ability to inhibit yeast Saccharomyces cerevisiae. cerevisiae cell growth were characterized. Two types of prothymosin α‐inactivating mutations were observed. Mutations that belong to the first type compromised the nuclear entry of prothymosin α by affecting its nuclear localization signal. Analysis of subcellular distribution of GFP‐prothymosin α fusions revealed a bipartite nuclear localization signal that is both necessary and sufficient for nuclear import of the protein in human cells. Mutations of the second type abrogated the inhibitory action of prothymosin α through an unknown mechanism, without influencing the nuclear import of the protein.

Age-associated murine cardiac lesions are attenuated by the mitochondria-targeted antioxidant SkQ1

Age-related changes in mammalian hearts often result in cardiac hypertrophy and fibrosis that are preceded by inflammatory infiltration. In this paper, we show that lifelong treatment of BALB/c and C57BL/6 mice with the mitochondria-targeted antioxidant SkQ1 retards senescence-associated myocardial disease (cardiomyopathy), cardiac hypertrophy, and diffuse myocardial fibrosis. To investigate the molecular basis of the action of SkQ1, we have applied DNA microarray analysis. The global gene expression profile in heart tissues was not significantly affected by administration of SkQ1. However, we found some small but statistically significant modifications of the pathways related to cell-to-cell contact, adhesion, and leukocyte infiltration. Probably, SkQ1-induced decrease in leukocyte and mesenchymal cell adhesion and/or infiltration lead to a reduction in age-related inflammation and subsequent fibrosis. The data indicate a causative role of mitochondrial reactive oxygen species in cardiovascular aging and imply that SkQ1 has potential as a drug against age-related cardiac dysfunction.

Binding of monoclonal antibodies to the movement protein (MP) of Tobacco mosaic virus: influence of subcellular MP localization and phosphorylation.

Monoclonal antibodies (mAbs) to recombinant movement protein (MP(REC)) of Tobacco mosaic virus (TMV) were used to reveal the dependence of MP epitope accessibility to mAbs on subcellular MP localization and post-translational MP phosphorylation. Leaves of Nicotiana benthamiana or N. tabacum were inoculated mechanically with TMV or agroinjected with an MP expression vector. At different time post-inoculation, ER membrane- and cell wall-enriched fractions (ER-MP and CW-MP, respectively) were isolated and analysed. The N-terminal region (residues 1-30) as well as regions 186-222 and 223-257 of MP from the CW and ER fractions were accessible for interaction with mAbs. By contrast, the MP regions including residues 76-89 and 98-129 were not accessible. The C-terminal TMV MP region (residues 258-268) was inaccessible to mAbs not only in CW-MP, but also in ER-MP fractions. Evidence is presented that phosphorylation of the majority of TMV MP C-terminal sites occurred on ER membranes at an early stage of virus infection, i.e. not after, but before reaching the cell wall. C-terminal phosphorylation of purified MP(REC) abolished recognition of C-proximal residues 258-268 by specific mAbs, which could be restored by MP dephosphorylation. Likewise, accessibility to mAbs of the C-terminal MP epitope in ER-MP and CW-MP leaf fractions was restored by dephosphorylation. Substitution of three or four C-terminal Ser/Thr residues with non-phosphorylatable Ala also resulted in abolition of interaction of mAbs with MP.

The effect of aerobic exercise on the expression of genes in skeletal muscles of trained and untrained men

It is well recognized that PGC-1α protein is a key regulator of mitochondrial biogenesis. Mechanical and metabolic perturbations in a skeletal muscle during and after aerobic exercise lead to an increased expression of PGC-1α gene. This increased expression of PGC-1α gene after exercise depends on the relative workload intensity and does not depend on the fitness level. The goal of this study was to compare mRNA expression of PGC-1α, TFAM, and TFB2M regulators of mitochondrial biogenesis and FOXO1 and Atrogin-1 proteolysis-related genes in a skeletal muscle of untrained and trained men after aerobic exercise with the same relative workload. This study showed that PGC-1α gene expression after exercise was the same in the two groups, but the expression of TFAM and TFB2M genes was higher in untrained muscles than in trained ones. In contrast, the expression of FOXO1 and Atrogin-1 genes increased only in the muscles of trained men.

Previously unclassified mutation of mtDNA m.3472T>C: Evidence of pathogenicity in Leber’s hereditary optic neuropathy

Leber’s hereditary optic neuropathy (LHON) refers to a group of mitochondrial diseases and is characterized by defects of the mitochondrial electron transport chain and decreased level of oxidative phosphorylation. The list of LHON primary mtDNA mutations is regularly updated. In this study, we describe the homoplasmic nucleotide substitution m.3472T>C in the MT-ND1 (NADH-ubiquinone oxidoreductase chain 1) gene and specific changes in cell metabolism in a patient with LHON and his asymptomatic sister. To confirm the presence of mutation-related mitochondrial dysfunction, respiration of skin fibroblasts and platelets from the patient and his sister was studied, as well as the mitochondrial potential and production of reactive oxygen species in the skin fibroblasts. In addition, based on characteristics of the toxic effect of paraquat, a new approach was developed for detecting the functional activity of complex I of the mitochondrial respiratory chain.

Mapping of Epitopes of the Recombinant Movement Protein of the Tobacco Mosaic Virus with the Use of Monoclonal Antibodies

The movement protein (MP) of the tobacco mosaic virus (TMV) provides the intercellular transport of the viral RNA through plasmodesmata. MP fulfils its function while interacting with host cell factors on the whole way of its intracellular movement from the subcellular site of its synthesis to the plasmodesmata of cellular walls. The MP conformation during its intracellular movement and fulfilling the transport function still remains unknown. In this study, we describe the preparation of murine monoclonal antibodies (MAs) to TMV MP and mapping of the MP epitopes. Stable hybridoma lines that produce MAs to the partially denatured recombinant MP (MPr) were obtained. MAs were tested by the immunoblotting and ELISA with the use of deletion variations of MPr. The epitopes of TMV MPr that recognize specific MAs were determined.

Role of C- and N-terminal mutations of the movement protein of tobacco mosaic virus in activation of complexes between the transport protein and viral RNA that are not translated in vitro.

Most plant viruses encode one or more proteins required for intercellular and systemic transport of virus. The movement protein (MP) 30K of tobacco mosaic virus (TMV) ensures translocation of viral genome from primary infected cells to neighboring healthy cells through plasmodesms. In infected cells, as well as in transgenic plants, TMV MP is accumulated in plasmodesms, increasing their carrying capacity. The movement protein of TMV nonspecifically binds to single-stranded RNA in vitro in a cooperative manner, forming linear ribonucleoproteins (RNPs) [1, 2]. These observations have given rise to the idea that viral infection spreads from one cell to another in the form of RNP containing viral RNA in the complex with MP (viral RNA temporarily may be not involved in translation and replication). Earlier, we showed that TMV TP is a specific repressor of RNA translation in vitro [3]. It is known that protein kinases associated with tobacco cell walls can phosphorylate the C-terminal region (Ser258, Thr261, and Ser265) of TMV TP [4]. The phosphorylation of TMV TP by protein kinases of cell walls or protein kinases of animal origin converts the MP–RNA complexes of TMV into in vitro translated complexes, which are infective for isolated protoplasts [5, 6]. To determine the functional domains of TMV TP, which are responsible for its activity as a translational repressor, in this work we obtained (1) C-terminal TMV TP mutants CT11, CT33, and CT84 (containing deletions of amino acids 258–268, 236–268, and 185– 268, respectively); (2) N-terminal deletion mutants NT30, NT96, and NT133 (lacking 30, 96, and 133 Nterminal amino acids, respectively); (3) C-terminal MP mutants, in which alanine substituted for the following amino acid residues: Ser267 (mutant Sb1A); Ser258, Thr261, and Ser265 (mutant Sb3A); and Ser258, Thr261, Ser265, and Ser267 (mutant Sb4A).