Penetrating Cations Enhance Uncoupling Activity of Anionic Protonophores in Mitochondria

Protonophorous uncouplers causing a partial decrease in mitochondrial membrane potential are promising candidates for therapeutic applications. Here we showed that hydrophobic penetrating cations specifically targeted to mitochondria in a membrane potential-driven fashion increased proton-translocating activity of the anionic uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide-p-trifluorophenylhydrazone (FCCP). In planar bilayer lipid membranes (BLM) separating two compartments with different pH values, DNP-mediated diffusion potential of H⁺ ions was enhanced in the presence of dodecyltriphenylphosphonium cation (C₁₂TPP). The mitochondria-targeted penetrating cations strongly increased DNP- and carbonylcyanide m-chlorophenylhydrazone (CCCP)-mediated steady-state current through BLM when a transmembrane electrical potential difference was applied. Carboxyfluorescein efflux from liposomes initiated by the plastoquinone-containing penetrating cation SkQ1 was inhibited by both DNP and FCCP. Formation of complexes between the cation and CCCP was observed spectophotometrically. In contrast to the less hydrophobic tetraphenylphosphonium cation (TPP), SkQ1 and C₁₂TPP promoted the uncoupling action of DNP and FCCP on isolated mitochondria. C₁₂TPP and FCCP exhibited a synergistic effect decreasing the membrane potential of mitochondria in yeast cells. The stimulating action of penetrating cations on the protonophore-mediated uncoupling is assumed to be useful for medical applications of low (non-toxic) concentrations of protonophores.     

Antiviral Activity of Antisense Oligonucleotides Against Various Targets of Herpes Simplex Virus 1 (Hsv1) and Coxsackievirus B3 (Cvb3) Genome

Abstract

The influence of chemical modification on the antiviral activity of oligonucleotides was studied on Green monkey kidney cells (GMK) using a known antisense oligodeoxynucleotide (AS-ODN) directed against the IE-110 gene of HSVl. The highest antiviral activity was observed with ODNs carrying exclusively phosphothioate internucleotide linkages. CVB3-specific ODNs of this type were synthesized and successfully tested for antiviral activity on HeLa cells.     

Intraduplex Photo-cross-linking of p-Azidoaniline Residue and Amino Acid Side Chains Linked to the Complementary Oligonucleotides via a New Phosphorylating Intermediate Formed in the Mukaiyama System

Abstract

Oligonucleotide derivatives carrying a side chain of either lysine or histidine at the 3′-end and their complementary oligonucleotides having photoreactive groups a p-azidophenyl-NH(CH2)_nNH- (n = 4, 6) residue at the 5′-end were prepared by using new phosphorylating species formed by treatment of oligonucleotides with Ph3P and (PyS)₂ or (PyrS)₂. in DMF, DMSO or their mixture. Efficient cross-linking of duplexes occurred under UV-irradiation (λ > 300 nm).     

Synthesis and characterization of fluorinated homocysteine derivatives as potential molecular probes for ¹⁹F magnetic resonance spectroscopy and imaging

A N-trifluoroacetyl-protected amino acid containing a thioester function, 2,2,2-trifluoro-N-(2-oxo-tetrahydrothiophen-3-yl)acetamide (TFA-tHcy), has been synthesized and characterized. It was then used to prepare a fluorine-labeled N-homocysteinylated protein, ¹⁹F-Hcy-εN-Lys-albumin, that was characterized by SDS-PAGE, MALDI-TOF-MS, UV-vis and ¹⁹F NMR spectroscopy. On average, four N-trifluoroacetylhomocysteine residues were covalently conjugated to human serum albumin through the N-substituted homocysteine thiolactone. The in situ homocysteinylation of human plasma proteins with TFA-tHcy has also been performed and has led to the formation of N-homocysteinylated proteins, with albumin modification accounting for ca. 75% of all fluorine-labeled human plasma proteins. The synthesized fluorinated molecular probes can be potentially used as informative molecular probes for in vivo ¹⁹F magnetic resonance spectroscopy and imaging.     

In utero protein restriction causes growth delay and alters sperm parameters in adult male rats

Background

Hyperglycemia can impair the male reproductive system in experimental animals and in men during reproductive age. Studies have shown that vitamin C has some good effects on male reproductive system, and therefore vitamin C treatment could attenuate the dysfunctions in this system caused by hyperglycemia. Thus, the objective of this work was to evaluate whether vitamin C treatment could attenuate reproductive dysfunctions in hyperglycemic male rats.

Methods

Adult male rats were divided into 3 groups: a normoglycemic (n = 10) and two hyperglycemic (that received a single dose of streptozotocin - 40 mg/kg BW). The two last groups (n = 10 per group) were divided into: hyperglycemic control (Hy) and hyperglycemic + 150 mg of vitamin C (HyC), by gavage during 30 consecutive days. The normoglycemic and hyperglycemic control groups received the vehicle (water). The first day after the treatment, the rats were anesthetized and killed to evaluate oxidative stress biomarkers (TBARS, SOD, GSHt and GSH-Px) in the erythrocytes, body and reproductive organ weights, sperm parameters, plasma hormone levels (FSH, LH and testosterone), testicular and epididymal histo-morphometry and histopathology.

Results

Compared with the normoglycemic animals, hyperglycemic control rats showed reduced weight of the body and reproductive organ but testis weight was maintained. It was also observed reduction of testosterone and LH levels, seminiferous tubular diameter, sperm motility and sperm counts in the epididymis. In addition, there was an increase in morphological abnormalities on spermatozoa as well as in oxidative stress level. Vitamin C reduced the oxidative stress level, diminished the number of abnormal sperm, and increased testosterone and LH levels and seminiferous tubular diameter but did not show improvement of sperm motility in relation to the hyperglycemic control group. Hyperglycemia caused a rearrangement in the epididymal tissue components (stroma, ephitelium and lumen) as demonstrated by the stereological analysis results. However, this alteration was partially prevented by vitamin C treatment.

Conclusions

We conclude that vitamin C partially attenuated some male reproductive system dysfunctions in hyperglycemic rats.     

Functional degradation of myelin basic protein. The proteomic approach

Proteolytic degradation of autoantigens is of prime importance in current biochemistry and immunology. The fundamental issue is the functional role of peptides produced in the process of change of the hydrolysis specificity during the transition from the normal to a pathologic state. In some cases identification of specific peptide fragments can be a diagnostic and prognostic criterion of the pathology progress. The subject of this work is the comparative study of degradation peculiarities of one of the major neuroantigens, myelin basic protein, by proteases activated upon the development of a pathological demyelinating process, and by proteasomes of different origin. Comparison of the specificity of the tested biocatalysts in some cases demonstrated critical changes in the set of myelin basic protein fragments capable of being presented on the major histocompatibility complex class I upon neurodegeneration, which may promote the development of autoimmune pathological processes.     

Functional analysis of MFS protein CefT involved in the transport of beta-lactam antibiotics in Acremonium chrysogenum and Saccharomyces cerevisiae

Vectors for the expression of MFS transporter CefT of Acremonium chrysogenum—a producer of beta-lactam antibiotic cephalosporin C—and in Saccharomyces cerevisiae as a fusion with the cyan fluorescent protein (CFP) have been generated. The subcellular localization of the CefT-CFP hybrid protein in yeast cells has been investigated. It was shown that the CefT-CFP hybrid protein is capable of complementation of the qdr3, tpo1, and tpo3 genes encoding for orthologous MFS transporters of Saccharomycetes, making the corresponding strains resistant to spermidine, ethidium bromide, and hygromycin B. High-producing strain A. chrysogenum VKM F 4081D, expressing the cefT-cfp fusion construct, was obtained by an agrobacteria mediated transformation. It was also shown that the constitutive expression of cefT in A. chrysogenum VKM F-4081D led to a change in the biosynthetic profiles of cephalosporin C and its precursors. This resulted in a 25–35% decrease in the amount of the final product accumulated in the cultural liquid with a simultaneous increase in the concentration of its intermediates.     

Molecular divergence and phylogeny: rates and patterns of cytochrome b evolution in cranes

Analyses of complete cytochrome b sequences from all species of cranes (Aves: Gruidae) reveal aspects of sequence evolution in the early stages of divergence. These DNA sequences are > or = 89% identical, but expected departures from random substitution are evident. Silent, third- position pyrimidine transitions are the dominant substitution type, with transversion comprising only a small fraction of sequence differences. Substitution patterns are not clearly manifested until divergence has reached a moderate level (> 3%), as expected for a stochastic process. Variation in the frequency of mismatch types among lineages decreases at larger divergences, but the level of bias does not decay. Divergence varies up to fivefold among gene regions but is not correlated with structural domain. All protein structural domains except extramembrane 4 display < 20% variable residues. Regions corresponding to putative functional domains show the excepted conservation of amino acids, although the C-terminal portion of the Q0 reaction center displays several nonconservative replacements. Phylogenetic analyses incorporating substitution asymmetries produced mixed results. Distances estimated with multiple parameters (transition, codon-position, composition, and pyrimidine-transition biases) yielded identical additive tree topologies with comparable bootstrap values, all consistent with uncontroversial species relationships. Maximum likelihood analysis incorporating these biases, as well as equally weighted parsimony analysis, produced similar results. Static, differential weighting for parsimony did not improve the phylogenetic signal but produced unusual trees with low bootstraps. The overall rate of nucleotide substitution varies slightly but significantly among cranes, and calibration of distances against fossil dates suggests divergence rates of 0.7%-1.7% per million years.