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Chemical modifications were used to identify some of the functionally important amino acid residues of the potato plant uncoupling protein (StUCP). The proton-dependent swelling of potato mitochondria in K(+)-acetate in the presence of linoleic acid and valinomycin was inhibited by mersalyl (K(i) = 5 microM) and other hydrophilic SH reagents such as Thiolyte MB, iodoacetate and 5, 5'-dithio-bis-(2-nitrobenzoate), but not by hydrophobic N-ethylmaleimide. This pattern of inhibition by SH reagents was similar to that of brown adipose tissue uncoupling protein (UCP1). As with UCP1, the arginine reagent 2,3-butadione, but not N-ethylmaleimide or other hydrophobic SH reagents, prevented the inhibition of StUCP-mediated transport by ATP in isolated potato mitochondria or with reconstituted StUCP. The results indicate that the most reactive amino acid residues in UCP1 and StUCP are similar, with the exception of N-ethylmaleimide-reactive cysteines in the purine nucleotide-binding site.

The suitability of a liposomal form of hydrophobic nonsulfonated meso-tetraphenyl porphyrin (TPP) for the photodynamic therapy of tumors was investigated. TPP was solubilized in small unilamellar lipid vesicles prepared by extrusion on a LIPOSOFAST apparatus. These samples were studied by laser-excited time resolved luminescence and triplet-triplet absorption spectroscopy. In this lipid environment TPP was still an efficient singlet oxygen producer, as indicated by the characteristic singlet oxygen phosphorescence at 1270 nm in D2O, when excited with a 28 ns laser pulse at 412 nm. Moreover, unlike with sulfonated TPP (TPPS4), liposomal TPP showed the reduced decay rates of TPP triplet-states with the increasing time of pre-illumination by a Xenon lamp. This was shown in an indirect way, based upon the appearance of a second component of the luminescence decay at 1270 nm in D2O; and by direct TPP triplet state monitoring, detecting triplet-triplet absorption at 440 nm in H2O. The deactivation of higher triplet states was delayed upon pre-illumination. This reflects an irreversible interaction of singlet oxygen with membrane lipids, thus demonstrating the potential of the liposomal form of TPP to efficiently disintegrate tumor cell membranes and to be a suitable preparation for the photodynamic therapy.

The mammalian uncoupling protein (UCP-1) from the gene family of mitochondrial carriers is a dimer of identical 33 kDa subunits, each containing six membrane-spanning alpha-helices. Its expression, restricted to brown fat, occurs upon birth, cold acclimation and overfeeding. UCP-1 dissipates redox energy and thereby provides heat to the animal. Two additional isoforms have recently been discovered, 59% homologous UCP-2, widely expressed (heart, kidney, lung, placenta, lymphocytes, white fat); and UCP-3 (57% homologous), found in brown fat and skeletal muscle. Their physiological roles are unknown, but may include the regulation of body weight and energy balance, muscle nonshivering thermogenesis, fever, and defense against generation of reactive oxygen species. Consequently, great pharmacological potential is expected in revealing their biochemical and hormonal regulators. UCP-1 mediates a purine-nucleotide-sensitive uniport of monovalent unipolar anions, including fatty acids, that lead to fatty acid cycling and uncoupling. UCP-2 and UCP-3 are expected to share a similar mechanism.

Potato and tomato plant uncoupling mitochondrial protein (PUMP) was reconstituted into liposomes, and K+ or H+ fluxes associated with fatty acid (FA)-induced ion movement were measured using fluorescent ion indicators potassium binding benzofuraneisophthalate and 6-methoxy-N-(3-sulfopropyl)-quinolinium. We suggest that PUMP, like its mammalian counterpart, the uncoupling protein of brown adipose tissue mitochondria (Garlid, K. D., Orosz, D. E., Modrianský, M., Vassanelli, S., and Jeek, P. (1996), J. Biol. Chem. 271, 2615-2702), allows for H+ translocation via a FA cycling mechanism. Reconstituted PUMP translocated anionic linoleic and heptylbenzoic acids, undecanesulfonate, and hexanesulfonate, but not phenylvaleric and abscisic acids or Cl-. Transport was inhibited by ATP and GDP. Internal acidification of protein-free liposomes by linoleic or heptylbenzoic acid indicated that H+ translocation occurs by FA flip-flopping across the lipid bilayer. However, addition of valinomycin after FA-initiated GDP-sensitive H+ efflux solely in proteoliposomes, indicating that influx of anionic FA via PUMP precedes a return of protonated FA carrying H+. Phenylvaleric acid, unable to flip-flop, was without effect. Kinetics of FA and undecanesulfonate uniport suggested the existence of an internal anion binding site. Exponential flux-voltage characteristics were also studied. We suggest that regulated uncoupling in plant mitochondria may be important during fruit ripening, senescence, and seed dormancy.

Genes encoding staphylococcal enterotoxins ( sea to see , seg , and seh ), toxic shock syndrome toxin 1 ( tst ), and exfoliative toxins ( eta and etb ) were not detected in a large panel of 48 Staphylococcus sciuri group isolates tested. This strongly suggests that production of the staphylococcal exotoxins by these bacteria is highly unlikely.

The protonophoretic function of uncoupling protein (UCP) is activated by fatty acids. According to the “docking site” hypothesis (Jezek, P., and Garlid, K. D., J. Biol. Chem. 265, 19303-19311, 1990), the fatty acid binding site is identical with the anion channel of UCP. Skulachev (Skulachev, V. P. (1991) FEBS Lett. 294, 158-162) extended this hypothesis by suggesting that fatty acid anions are transported by UCP and that H are delivered by back-diffusion of the protonated fatty acid through the lipid bilayer. In this model, UCP does not transport H at all but rather enables fatty acids to act as cycling protonophores. New evidence supports this mechanism (Garlid, K. D., Orosz, D. E., Modrianský, M., Vassanelli, S., and Jezek, P. (1996) J. Biol. Chem. 271, 2615-2620). To help elucidate these hypotheses, we synthesized a photoreactive analog of dodecanoic acid, 12-(4-azido-2-nitrophenylamino)dodecanoic acid (AzDA), and studied its effect on transport in mitochondria and proteoliposomes. AzDA behaved in every respect like a typical fatty acid. In micromolar doses, AzDA activated H translocation and inhibited Cl and hexanesulfonate uniport through UCP. After UV light exposure, however, activation of H transport was inhibited, whereas inhibition of anion transport was preserved. These effects were irreversible. Photolabeling of mitochondria with [3H]AzDA resulted in a prominent 32 kDa band of UCP, and few other proteins were labeled. The results indicate that AzDA can be ligated to the protein at or near the docking site, causing irreversible inhibition of both H and anion transport. The finding that fatty acid-induced H transport disappears along with anion transport supports the fatty acid-protonophore mechanism of H transport by UCP.

We hypothesize that fatty acid-induced uncoupling serves in bioenergetic systems to set the optimum efficiency and tune the degree of coupling of oxidative phosphorylation. Uncoupling results from fatty acid cycling, enabled by several phylogenetically specialized proteins and, to a lesser extent, by other mitochondrial carriers. It is suggested that the regulated uncoupling in mammalian mitochondria is provided by uncoupling proteins UCP-1, UCP-2 and UCP-3, whereas in plant mitochondria by PUMP and StUCP, all belonging to the gene family of mitochondrial carriers. UCP-1, and hypotheticaly UCP-3, serve mostly to provide nonshivering thermogenesis in brown adipose tissue and skeletal muscle, respectively. Fatty acid cycling was documented for UCP-1, PUMP and ADP/ATP carrier, and is predicted also for UCP-2 and UCP-3. UCP-1 mediates a purine nucleotide-sensitive uniport of monovalent unipolar anions, including anionic fatty acids. The return of protonated fatty acid leads to H+ uniport and uncoupling. UCP-2 is probably involved in the regulation of body weight and energy balance, in fever, and defense against generation of reactive oxygen species. PUMP has been discovered in potato tubers and immunologically detected in fruits and corn, whereas StUCP has been cloned and sequenced from a potato gene library. PUMP is supposed to act in the termination of synthetic processes in mature fruits and during the climacteric respiratory rise.

Thermogenic uncoupling has been proven only for UCP1 in brown adipose tissue. All other isoforms of UCPs are potentially acting in suppression of mitochondrial reactive oxygen species (ROS) production. In this contribution we show that BAT mitochondria can be uncoupled by lauric acid in the range of ∼100 nM when endogenous fatty acids are combusted by carnitine cycle and β-oxidation is properly separated from the uncoupling effect. Respiration increased up to 3 times when related to the lowest fatty acid content (BSA present plus carnitine cycle). We also illustrated that any effect leading to more coupled states leads to enhanced H 2 O 2 generation and any effect resulting in uncoupling gives reduced H 2 O 2 generation in BAT mitochondria. Finally, we report doubling of plant UCP transcript in cells as well as amount of protein detected by 3 H-GTP-binding sites in mitochondria of shoots and roots of maize seedlings subjected to the salt stress.

Mersalyl, 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) and fluorescent Thiolyte DB react with SH groups in the H+ channel (SHc) of the uncoupling protein of brown adipose tissue mitochondria, as inferred from their inhibition of H+ transport. Cl- transport by the uncoupling protein was unaffected. Using these modifiers and N-ethylmaleimide (MalNEt), distinct SH groups (SHB) in the purine nucleotide binding site were identified. Nbs2 reacts more readily with the SHB than with the SHc groups, but mersalyl and Thiolyte DB are more reactive with the SHc groups. MalNEt reacts exclusively with the SHB. GDP inhibition is fully prevented after sufficient modification of the SHB. Pretreatment with p-diazobenzenesulfonate (N2PhSO2) suppresses only 20-25% of fluorescence of Thiolyte-DB-labeled uncoupling protein on SDS/PAGE gels, while MalNEt suppresses 66% and Nbs2 80-90%. Since N2PhSO2 also affects the GDP binding site, these results demonstrate that the N2PhSO2-reactive residue is not identical with the SHB.

The physiological role of monocarboxylate transport in brown adipose tissue mitochondria has been reevaluated. We studied pyruvate, α-ketoisovalerate, α-ketoisocaproate, and phenylpyruvate uniport via the uncoupling protein (UCP1) as a GDP-sensitive swelling in K+salts induced by valinomycin or by monensin and carbonyl cyanide- p-(trifluoromethoxy)phenylhydrazone in Na+salts. We have demonstrated that this uniport is inhibited by fatty acids. GDP inhibition in K+salts was not abolished by an uncoupler, indicating a negligible monocarboxylic acid penetration via the lipid bilayer. In contrast, the electroneutral pyruvate uptake (swelling in ammonium pyruvate or potassium pyruvate induced by change in pH) mediated by the pyruvate carrier was inhibited by its specific inhibitor α-cyano-4-hydroxycinnamate but not by fatty acids. Moreover, α-cyano-4-hydroxycinnamate enhanced the energization of brown adipose tissue mitochondria, which was monitored fluorometrically by 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide and safranin O. Consequently, we suggest that UCP1 might participate in futile cycling of unipolar ketocarboxylates under certain physiological conditions while expelling these anions from the matrix. The cycle is completed on their return via the pyruvate carrier in an H+symport mode.

Upon certain insults and/or pathological states fragmented mitochondrial (mt) network persists in cells, with spheroid mt remnants containing clustered nucleoids of mtDNA (1).To image nucleoid distribution within mt network, we employed 3D super-resolution fluorescent photoactivable localization microscopy (FPALM)(2), in conjunction with photoconvertible fluorescent protein conjugates of marker proteins such as mitochondrial (mt) transcription factor A (TFAM) or mt single-stranded-DNA-binding protein (mtSSB). We have shown that nucleoids in hepatocellular HepG2 cells exhibit a rather wide size distribution ranging between 50 to 300 nm. This was confirmed using 3D TFAM/mtSSB immunocytochemistry in conjunction with direct stochastic optical reconstruction microscopy (dSTORM). Positioning in TIM23 dSTORM visualized membranes have confirmed our previous model for nucleoid clustering in mt network fragments (1), which were induced by mtDNA replication/transcription inhibition or other pathogenic simulations.Supported by grants grant 13-02033S, P305/12/P388 and P305/12/1247 of GACR.(1) Tauber J, Dlaskova A, Santorova J, Smolkova K, Alan L, Spacek T, Plecita-Hlavata L, Jabůrek M, Ježek, P Distribution of mitochondrial nucleoids upon mitochondrial network fragmentation and network reintegration in HEPG2 cells. Int J Biochem Cell Biol 45:593-603, 2013.(2) Mlodzianoski MJ, Schreiner JM, Callahan SP, Smolkova K, Dlaskova A, Santorova J, Ježek P, Bewersdorf J. Opt Express 2011; 19:15009-19.

Mitochondrial (mt) network undergoes locally frequent fragmentation and fusion events. When integrated over time, its basic morphology encompasses highly interconnected mt reticulum, a single mitochondrion within the cell (1). Upon certain insults and/or pathological states fragmented network persists. MtDNA is organized in nucleoids containing assessor proteins and recruited proteins of mt replication/ transcription machinery. It is debated on their uniform size and whether a single nucleoid contains a single mtDNA molecule or up to average 6 mtDNA molecules. To image nucleoid distribution within mt network, we employed 3D super-resolution fluorescent photactivable localization microscopy of Biplane schema (1). Mt network of hepatocellular carcinoma HepG2 cells was imaged first by its matrix space using mtEos2 or as outer mitochondrial membrane contour using Eos2-conjugates of truncated FIS1 protein (not inducing massive fission, Eos2-FIS1tr). Resulting 3D images confirm the existence of highly-connected mt network and unlike conventional confocal microscopy, 3D BiplaneFPALM distinguished a hollow character of mt reticulum tubules when visualized by Eos2-FIS1tr. Upon network fragmentation, hollow max ∼2 micrometer spheres occurred. Imaging of mt nucleoids confirmed the existence of ∼1000 nucleoids per cell with size distribution from 50 nm to 300 nm. Optimized dual transfection strategy had to be employed for simultaneous imaging of network (mtEos or Eos2-FIS1tr) and nucleoids (mtSSB-PSCFP2). Images revealed an equidistant nucleoid distribution of an average distance of ∼1 micrometer between nucleoids. Fragmentation by different agents led to observations of clusters of mt nucleoids within the spherical fragmented objects thus formed.Supported by grants P302/10/0346, P305/12/P388 and P305/12/1247 of GACR and ME09029 (Czech Ministry of Education); and 1R01GM091791-02 (NIH).(1) Mlodzianoski MJ, Schreiner JM, Callahan SP, Smolkova K, Dlaskova A, Santorova J, Ježek P, Bewersdorf J. Opt Express 2011;19:15009–19.

Isolation and propagation of a wild type phage, isolate AP-423, from an apparently lysogenic strain of Pseudomonas aeruginosa, resistant to a series of anti-pseudomonadal antibiotics, and its use for transduction of resistance determinants is described. The phage isolate AP-423 showed a phenomenon of host restriction, i.e. it was lysogenic only for some of the recipient strains tested. Its transduction capacity, both in sets of genes transduced and frequency of transduction, was different in two recipient strains of P. aeruginosa. This phage showed also some restriction in titers, to which it could be propagated, only in certain recipient strains.

Cell death-inducing DFF[DNA fragmentation factor]-like effector-a (CIDEa), may initiate apoptosis by disrupting a complex consisting of 40-kDa caspase-3-activated nuclease (DFF40/CAD) and its 45-kDa inhibitor (DFF45/ICAD). CIDEa, however, was found to be localized in mitochondria. We have performed immunodetection of CIDEa in whole cells and subcellular fractions of HeLa cells adapted for a tetracycline-inducible CIDEa expression. Using immunocytochemistry we observed redistribution, enhanced upon treatment with camptothecin or valinomycin, of CIDEa to nucleus. Similarly, CIDEa content increased in the nuclear fraction but decreased in cytosolic fraction in cells treated to initiate apoptosis. We hypothesize that CIDEa is sequestered in mitochondria while transfer of this potentially dangerous protein from mitochondria into nucleus intensifies or even initiates apoptosis.

To determine antibiotic resistance and incidence of multidrug resistance among Nontyphoidal salmonellae serovars isolated from humans.Consecutive Salmonella isolates from patients, recovered in 48 microbiology laboratories in May 2012, were analyzed in the respective reference laboratories at the National Institute of Public Health. Strains were re-identified and differentiated into serovars. Their minimum inhibitory concentrations (MICs) to 11 antibiotics were determined by the microdilution method.Of 25 serovars identified among 637 strains of Salmonella enterica, the most frequent were Enteritidis (87.0 %), Typhimurium (4.9 %), and monophasic Typhimurium 4,[5],12:i:- (2.0 %) and Mbandaka (0.6 %); other serovars were rare. Altogether 558 strains (87.6 %) were susceptible to all antibiotics tested and the remaining 79 strains were resistant to one or more antibiotics. The prevalence rates of resistance to individual antibiotics among 637 study strains were as follows: ampicillin 8.5%, tetracycline 5.7%, sulfamethoxazole 5.2%, cipro-floxacin 3.8%, and chloramphenicol 2.5%. Resistance to gentamicin, trimethoprim, and third and fourth generation cephalosporins was rare ( 0.5%) and none of the study strains showed resistance to meropenem. Three producers of extended spectrum beta-lactamase were multidrug resistant and two of them recovered from twins exhibited a different pattern of resistance. Resistant strains were most often assigned to the following serovars: Enteritidis (49.4%), Typhimurium (26.6%), and monophasic Typhimurium (15.2%). While only 7% (39 of 554 strains) of Enteritidis strains were resistant, the serovars Typhimurium and its monophasic variant 4,[5],12:i:- showed high rates of resistance, i.e. 66.7 and 92.3%, respectively. Furthermore, resistance was revealed in all strains of the serovars Virchow (n = 3), Kentucky (n = 1), and Newport (n = 1), in two of three strains of the serovar Infantis, and in one of two strains of the serovar Stanley. All five blood isolates were assigned to the serovar Enteritidis and one of them showed resistance to ciprofloxacin. Of 79 resistant strains, 26.6% showed resistance to ampicillin only and 24.1% to ciprofloxacin only, with multidrug resistance, i.e. resistance to three or more antibiotics, confirmed in 43.0% of strains.Despite a relatively low prevalence of resistance to the antibiotics tested among 637 study strains, the following alarming findings were made: Detection of Salmonella enterica strains resistant to ciprofloxacin as the drug of choice or to higher generation cephalosporins and multidrug resistance revealed in two thirds of the strains of the serovar Typhimurium and in all but one strains of its monophasic variant 4,[5],12:i:-.