Use of the addressing sequence of yeast D-lactate dehydrogenase for insertion of CYP11A1p into the inner membrane of yeast mitochondria

Mammalian cytochrome P450scc (CYP11A1p) is a pseudointegral protein of the inner membrane of mitochondria with the active center exposed in the matrix. Upon import of the CYP11A1p precursor into yeast mitochondria, only a minor part was incorporated into the inner mitochondrial membrane and acquired catalytic activity (Kovaleva, I. E., Novikova, L. A., Nazarov, P. A., Grivennikov, S. I., and Luzikov, V. N. (2003) Eur. J. Biochem., 270, 222-229). The present work is an attempt to increase the efficiency of this process by substitution of the inherent N-terminal presequence of CYP11A1p by the addressing signal of D-lactate dehydrogenase (D-LD) of the yeast Saccharomyces cerevisiae. D-LD is known to be inserted into the inner membrane of mitochondria through its transmembrane domain located close to the N-terminus of the polypeptide chain in such a way that the protein globule is exposed in the intermembrane space. The hybrid protein D-LD(1-72)-mCYP11A1p synthesized in yeast cells was imported into yeast mitochondria, underwent processing, and was inserted into the inner membrane on the side of the intermembrane space. In the presence of adrenodoxin and adrenodoxin reductase, the hybrid protein exhibited cholesterol side-chain cleavage activity. Thus, CYP11A1p insertion into the inner membrane of mitochondria mediated by the D-LD topogenic signal resulted in the catalytically active mCYP11A1p domain in the hybrid protein.     

Ethylene synthesis in petunia stigma tissues governs the growth of pollen tubes in progamic phase of fertilization

In the pollen-pistil system of petunia (Petunia hybrida L.) self-compatible and self-incompatible clones within 7 h after self-pollination, we determined the content of ACC (1-aminocyclopropane-1-carboxylic acid), the activity of two enzymes (ACC synthase and ACC oxidase), and the rate of ethylene production. Depending on the type of pollination, germination of pollen on the stigma surface and the pollen tube growth in the tissues of style were accompanied by different levels of ACC and ethylene release. The pollen-pistil system of the self-compatible clone contained twice more ACC than in the self-incompatible clone, whereas the pollen-pistil system in the self-incompatible clone produced 4–5 times more ethylene than in the self-compatible clone. For both types of pollination, ACC and ethylene were predominantly produced in the stigma tissues. The rate of ethylene production therein was 50 times greater than in the styles and ovaries, and the content of ACC was 100 times higher than in the styles and ovaries. Germination of male gametophyte after both types of pollination was accompanied by elevated ACC synthase activity (especially in the case of compatible pollination), whereas notable increase in ACC oxidase activity was manifested in growing pollen tubes after self-incompatible pollination     

Dynamics of microtubular cytoskeleton in higher plant meiosis. V. Late prometaphase. The general scheme of anastral spindle formation

By means of morphological analyses of meiotic abnormalities in pollen mother cells (PMCs) of cereal wide hybrids, haploids and meiotic mutants, the processes involved in cytoskeleton cycle at late prometaphase (a sub-stage of transition from chaotic figure to bipolar spindle) were studied. A significance of the four processes of late prometaphase--axial orientation, lateral association, consolidation and convergance of spindle fibers--is discussed.     

Sporophyte-gametophyte interactions between anther and male gametophyte in petunia

Sporophyte-gametophyte interactions between anther and male gametophyte were investigated in two (fertile and sterile) clones of petunia (Petunia hybrida L.) with different reproductive strategies. Structural and functional reorganization of sporophyte tissues in the developing anther of fertile clone is closely coordinated with each of the successive stages of male gametophyte development (from meiosis to the formation of binuclear pollen) and comprises not only destruction of tapetum and three middle layers of the wall but also an activation of gas exchange and a rise in the content of sugars (sucrose, fructose, and glucose). In sterile clone, degradation of tapetum and anomalies in the development of sporogenic tissue were simultaneously observed in the prophase of meiosis. The death of microsporocytes and degeneration of tapetum were accompanied by a decrease in the level of sucrose delivered to the anther tissues and changes in the ratio between sucrose and hexoses in favor of glucose.     

Induction of permeability of the inner membrane of yeast mitochondria

The current view on apoptosis is given, with a special emphasis placed on apoptosis in yeasts. Induction of a non-specific permeability transition pore (mPTP) in mammalian and yeast mitochondria is described, particularly in mitochon-dria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, which are aerobes possessing the fully competent respiratory chain with all three points of energy conservation and well-structured mitochondria. They were examined for their ability to induce an elevated permeability transition of the inner mitochondrial membrane, being subjected to virtually all conditions known to induce the mPTP in animal mitochondria. Yeast mitochondria do not form Ca²⁺-dependent pores, neither the classical Ca²⁺/P_i-dependent, cyclosporin A-sensitive pore even under deenergization of mitochondria or depletion of the intramitochondrial nucleotide pools, nor a pore induced in mammalian mitochondria upon concerted action of moderate Ca²⁺ concentrations (in the presence of the Ca²⁺ ionophore ETH129) and saturated fatty acids. No pore formation was found in yeast mitochondria in the presence of elevated phosphate concentrations at acidic pH values. It is concluded that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

Novel conjugates of tacrine with 1,2,4,-thiadiazole as highly effective cholinesterase inhibitors,blockers of NMDA receptors,and antioxidants

Conjugates of tacrine with 1,2,4-thiadiazole derivatives were synthesized for the first time. Their esterase profile and effects on the key NMDA receptor-binding sites as well as antioxidant activity were investigated. The obtained compounds effectively inhibited cholinesterases (with a predominant effect on butyrylcholinesterase), simultaneously blocked two NMDA receptor-binding sites (allosteric and intrachannel sites, and exhibited a high radical-scavenging activity. Our study shows that the obtained compounds are promising to design drugs for the treatment of Alzheimer’s disease and other multifactorial neurodegenerative diseases.     

The AtC-VPS protein complex is localized to the tonoplast and the prevacuolar compartment in arabidopsis

Plant cells contain several types of vacuoles with specialized functions. Although the biogenesis of these organelles is well understood at the morphological level, the machinery involved in plant vacuole formation is largely unknown. We have recently identified an Arabidopsis mutant, vcl1, that is deficient in vacuolar formation. VCL1 is homologous to a protein that regulates membrane fusion at the tonoplast in yeast. On the basis of these observations, VCL1 is predicted to play a direct role in vacuolar biogenesis and vesicular trafficking to the vacuole in plants. In this work, we show that VCL1 forms a complex with AtVPS11 and AtVPS33 in vivo. These two proteins are homologues of proteins that have a well-characterized role in membrane fusion at the tonoplast in yeast. VCL1, AtVPS11, and AtVPS33 are membrane-associated and cofractionate with tonoplast and denser endomembrane markers in subcellular fractionation experiments. Consistent with this, VCL1, AtVPS11, and AtVPS33 are found on the tonoplast and the prevacuolar compartment (PVC) by immunoelectron microscopy. We also show that a VCL1-containing complex includes SYP2-type syntaxins and is most likely involved in membrane fusion on both the PVC and tonoplast in vivo. VCL1, AtVPS11, and AtVPS33 are the first components of the vacuolar biogenesis machinery to be identified in plants.     

Arf6, RalA and BIRC5 protein expression in non small cell lung cancer

Evaluation of tumor markers expression pattern which determines individual progression parameters is one of the major topics in molecular oncopathology research. This work presents research on expression analysis of several Ras-Ral associated signal transduction pathway proteins (Arf6, RalA and BIRC5) in accordance with clinical criteria in non small cell lung cancer patients. Using Western-blot analysis and RT-PCR Arf6, RalA and BIRC5 expression has been analyzed in parallel in 53 non small cell lung cancer samples of different origin. Arf6 protein expression was elevated in 55% non small cell lung cancer tumor samples in comparison with normal tissue. In the group of squamous cell lung cancer Arf6 expression elevation was observed more often. RalA protein expression was decreased in comparison to normal tissue samples in 64% of non small cell lung cancer regardless to morphological structure. Correlation between RalA protein expression decrease and absence of regional metastases was revealed for squamous cell lung cancer. BIRC5 protein expression in tumor samples versus corresponding normal tissue was 1.3 times more often elevated in the squamous cell lung cancer group (in 76% tumor samples). At the same time elevation of BIRC5 expression was fixed only in 63% of adenocarcinoma tumor samples. A statistically significant decrease (p = 0.0158) of RalA protein expression and increase (p = 0.0498) of Arf6 protein expression in comparison with normal tissue was found for T1-2N0M0 and T1-2N1-2M0 groups of squamous cell lung cancer correspondingly.     

Complexes of antiparallel telomeric G-quadruplex d(TTAGGG)4 with carboxymethyl tetracationic porphyrins

Porphyrins are a chemical class that is widely used in drug design. Cationic porphyrins may bind to DNA guanine quadruplexes. We report the parameters of the binding of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium) porphyrin (P1) and 5,10,15,20-tetrakis(N-etoxycarbonylmethyl-4-pyridinium) porphyrin (P2) to antiparallel telomeric G-quadruplex formed by d(TTAGGG)₄ sequence (TelQ). The binding constants (K _i ) and the number of binding sites (N _j ) were determined from absorption isotherms generated from the absorption spectra of complexes of P1 and P2 with TelQ. Compound P1 demonstrated a high affinity to TelQ (K _i = (40 ± 6) × 10⁶ M^?1, N ₁ = 1; K ₂ = (5.4 ± 0.4) × 10⁶ M^?1, N ₂ = 2). In contrast, the binding constants of P2-TelQ complexes (K ₁ = (3.1 ± 0.2) × 10⁶ M^?1, N ₁ = 1; K ₂ = (1.2 ± 0.2) × 10⁶ M^?1, N ₂ = 2) were one order of magnitude smaller than the corresponding values for P2-TelQ complexes. Measurements of the quantum yield and fluorescence lifetime of the drug’s TelQ complexes revealed two types of binding sites for P1 and P2 on the quadruplex oligonucleotide. We concluded that strong complexes can result from the interaction of the porphyrins with TTA loops whereas the weaker complexes are formed with G-quartets. The altered TelQ conformation detected by the circular dichroism spectra of P1-TelQ complexes can be explained by the disruption of the G-quartet. We conclude that peripheral carboxy groups contribute to the high affinity of P1 for the antiparallel telomeric G-quadruplex.