Mitochondrial Physiology of Cellular Redox Regulations.

Mitochondria (mt) represent the vital hub of the molecular physiology of the cell, being decision-makers in cell life/death and information signaling, including major redox regulations and redox signaling. Now we review recent advances in understanding mitochondrial redox homeostasis, including superoxide sources and H2O2 consumers, i.e., antioxidant mechanisms, as well as exemplar situations of physiological redox signaling, including the intramitochondrial one and mt-to-cytosol redox signals, which may be classified as acute and long-term signals. This review exemplifies the acute redox signals in hypoxic cell adaptation and upon insulin secretion in pancreatic beta-cells. We also show how metabolic changes under these circumstances are linked to mitochondrial cristae narrowing at higher intensity of ATP synthesis. Also, we will discuss major redox buffers, namely the peroxiredoxin system, which may also promote redox signaling. We will point out that pathological thresholds exist, specific for each cell type, above which the superoxide sources exceed regular antioxidant capacity and the concomitant harmful processes of oxidative stress subsequently initiate etiology of numerous diseases. The redox signaling may be impaired when sunk in such excessive pro-oxidative state.

Corynebacterium sp. isolated from blood culture of a bacteremic patient. Will the assumptions about a new corynebacterium be confirmed?

A case report is presented of a patient with suspected septicaemia from whose blood culture a new strain of Corynebacterium sp. was isolated. Until now, no report of this strain isolated from human clinical materials has been available in the literature. In addition to a brief clinical description of the case, the article also features morphological, biochemical properties as well as antibiogram of the bacterium. It describes also methods used for the identification of this isolate. The aim of the work was to highlight a novel and rare coryneform strain.

Background levels of neomorphic 2-hydroxyglutarate facilitate proliferation of primary fibroblasts.

Each cell types or tissues contain certain "physiological" levels of R-2-hydroxyglutarate (2HG), as well as enzymes for its synthesis and degradation. 2HG accumulates in certain tumors, possessing heterozygous point mutations of isocitrate dehydrogenases IDH1 (cytosolic) or IDH2 (mitochondrial) and contributes to strengthening their malignancy by inhibiting 2-oxoglutarate-dependent dioxygenases. By blocking histone de-methylation and 5-methyl-cytosine hydroxylation, 2HG maintains cancer cells de-differentiated and promotes their proliferation. However, physiological 2HG formation and formation by non-mutant IDH1/2 in cancer cells were neglected. Consequently, low levels of 2HG might play certain physiological roles. We aimed to elucidate this issue and found that compared to highest 2HG levels in hepatocellular carcinoma HepG2 cells and moderate levels in neuroblastoma SH-SY5Y cells, rat primary fibroblast contained low basal 2HG levels at early passages. These levels increased at late passage and likewise 2HG/2OG ratios dropped without growth factors and enormously increased at hypoxia, reaching levels compared to cancer HepG2 cells. Responses in SH-SY5Y cells were opposite. Moreover, external 2HG supplementation enhanced fibroblast growth. Hence, we conclude that low 2HG levels facilitate cell proliferation in primary fibroblasts, acting via hypoxia-induced factor regulations and epigenetic changes.

Silica-modified monodisperse hexagonal lanthanide nanocrystals: synthesis and biological properties.

Oleic acid-stabilized hexagonal NaYF4:Yb(3+)/Er(3+) nanocrystals, emitting green and red luminescence, were prepared by the high-temperature co-precipitation of lanthanide chlorides. By varying the reaction time and the Ln(3+)/Na(+) ratio, the nanocrystal size can be controlled within the range 16-270 nm. The maximum upconversion quantum yield is achieved under 970 nm excitation. The reverse microemulsion technique using hydrolysis and condensation of tetraethoxysilane is a suitable method to coat the nanocrystal surface with a silica shell to make the particles dispersible and colloidally stable in aqueous media. During the subsequent functionalization, (3-aminopropyl)trimethoxysilane introduced amino groups onto the silica to enable future bioconjugation with the target molecules. All specimens were characterized by TEM microscopy, electron and X-ray diffraction, ATR FT-IR spectroscopy, and upconversion luminescence. Finally, in vitro cytotoxicity and intracellular nanoparticle uptake (using confocal microscopy) were determined with human cervical carcinoma HeLa and mRoGFP HeLa cells, respectively. From the investigated particles, amino-functionalized NaYF4:Yb(3+)/Er(3+) nanocrystals internalized into the cells most efficiently. The nanoparticles proved to be nontoxic at moderate concentrations, which is important when considering their prospective application in biolabeling and luminescence imaging of various cell types.

Prevalence study on carbapenemase-producing Escherichia coli and Klebsiella pneumoniae isolates in Czech hospitals--results from Czech Part of European Survey on Carbapenemase--Producing Enterobacteriaceae (EuSCAPE).

OBJECTIVE: One of the most important threats of current medicine is the spread of multiresistant Gram-negative bacteria. We report here data from a six-month prevalence study on carbapenemase-producing K. pneumoniae and E. coli performed in Czech hospitals participating on European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE). METHODS: Ten hospitals covering all regions of the Czech Republic were selected. During the study period (1st November 2013 to 30th April 2014), first ten carbapenem non-susceptible isolates of K. pneumoniae or E. coli isolated from non-surveillance specimens (i.e., blood, lower respiratory tract secretions, urine, puncture fluids, and wound secretions) of single successive patients were collected. Successive carbapenem-susceptible isolates of the same species were also preserved as controls. Susceptibility to 15 antibiotics was determined using EUCAST recommendations. Carbapenemase activity was detected by MALDI-TOF MS meropenem hydrolysis assay. Positive isolates were subjected for molecular typing (multi-locus sequence typing, identification of carbapenemase gene). RESULTS: During the study period, thirty non-susceptible isolates (K. pneumoniae n=28, E. coli n=2) were identified in 5 hospitals. Only two of them were confirmed to be carbapenemase producers. A NDM-1-producing K. pneumoniae ST11 was recovered from a patient, transferred from Ukraine, being injured during a Maidan revolution. The second isolate, an OXA-48-producing K. pneumoniae, belonging to ST101, was recovered from a patient admitted to a hospital for an ischemic stroke. CONCLUSIONS: This study again confirmed that the Czech Republic still belongs to the countries with low prevalence of carbapenemase-producing Enterobacteriaceae (CPE). Cases of CPE are usually restricted to an import from high-prevalence countries or countries with unknown epidemiological situation.

Antioxidant and regulatory role of mitochondrial uncoupling protein UCP2 in pancreatic beta-cells.

Research on brown adipose tissue and its hallmark protein, mitochondrial uncoupling protein UCP1, has been conducted for half a century and has been traditionally studied in the Institute of Physiology (AS CR, Prague), likewise UCP2 residing in multiple tissues for the last two decades. Our group has significantly contributed to the elucidation of UCP uncoupling mechanism, fully dependent on free fatty acids (FFAs) within the inner mitochondrial membrane. Now we review UCP2 physiological roles emphasizing its roles in pancreatic beta-cells, such as antioxidant role, possible tuning of redox homeostasis (consequently UCP2 participation in redox regulations), and fine regulation of glucose-stimulated insulin secretion (GSIS). For example, NADPH has been firmly established as being a modulator of GSIS and since UCP2 may influence redox homeostasis, it likely affects NADPH levels. We also point out the role of phospholipase iPLA2 isoform gamma in providing FFAs for the UCP2 antioxidant function. Such initiation of mild uncoupling hypothetically precedes lipotoxicity in pancreatic beta-cells until it reaches the pathological threshold, after which the antioxidant role of UCP2 can be no more cell-protective, for example due to oxidative stress-accumulated mutations in mtDNA. These mechanisms, together with impaired autocrine insulin function belong to important causes of Type 2 diabetes etiology.

Relative prevalence of Mycobacterium marinum in fish collected from aquaria and natural freshwaters in central Europe.

A survey was carried out on occurrence of Mycobacterium marinum in fish kept in aquaria and those living in their natural environment. Species-specific qPCR targeting the erp and IS2404 genes together with the conventional culture method were used. The analysis of 72 ornamental fish (n = 216 samples: gills, muscle and intestine) collected from aquaria revealed the presence of M. marinum in 30 individuals (41.7%) of whom 17 (23.6%) were later culture positive. Culture-independent detection revealed the presence of M. marinum in 16 of 83 environmental samples (19.3%) collected in aquaria. The presence of viable M. marinum cells was later confirmed in 5 samples (6.0%). No qPCR or culture positivity was observed when 123 groundwater fish and their corresponding environmental samples (n = 142) were analysed.

[Antibiotic resistance in nontyphoidal salmonellae serovars in the Czech Republic]. / Antibiotická rezistence u netyfových sérovaru Salmonella spp. v Ceské republice.

STUDY AIM: To determine antibiotic resistance and incidence of multidrug resistance among Nontyphoidal salmonellae serovars isolated from humans. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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:-.

Assessment of mitochondrial DNA as an indicator of islet quality: an example in Goto Kakizaki rats.

BACKGROUND: Diabetic Goto Kakizaki (GK) rats represent an established model of type 2 diabetes that exhibit an onset of pancreatic islet (PI) pathology characterized by islet hypertrophy with a decreased number of insulin-secreting ß-cells. Among the remaining ß-cells, oxidative phosphorylation (OXPHOS) and consequently glucose-stimulated insulin secretion (GSIS) are impaired, perhaps owing to a deficit in mitochondrial DNA (mtDNA). We sought to identify this abnormality. METHODS: ß-Cells were obtained from Accutase-dissolved PI isolated from GK or Wistar rats and sorted based on the positive Zn(2+) signal of Newport Green. The mtDNA copy number per cell was quantified as the amplicon ratio by polymerase chain reaction using specific primers against the rat ND5 mt gene and UCP2 nuclear gene. RESULTS: The 12-month-old GK rats exhibited drastically reduced copy numbers per remaining ß-cell, from 7,400 ± 600 in 12-month old Wistar rats (100%) to 24 ± 4%; mtDNA content in heart and liver was 70 ± 25% and 60 ± 20%, respectively. Versus age-paired Wistar rats, 6- and 4-month-old GK rats showed reductions to 60 ± 15% and 50 ± 20%, respectively. CONCLUSIONS: OXPHOS of remnant ß-cells in diabetic GK was drastically impaired due to the lack of sufficient mtDNA levels. We suggest the use of mtDNA quantification to quickly assess PI quality before transplantation.

Mycobacterium marinum epididymoorchitis: case report and literature review.

Mycobacterium marinum is the most frequent non-tuberculous Mycobacterium in humans. We report the first ever described case of epididymoorchitis resulting from hematogenous spread of M. marinum from hand oligoarthritis. This was initially mistaken for rheumatoid disease and methylprednisolone-induced immunosuppression led to hematogenous spread of infection to the testis and epididymis.

[Filamentous "contaminants" in the mycobacteriology laboratory; their culture, identification and clinical significance]. / Vlálmoté "kontaminanty" v mykobakteriologické laboratori, jejich kultivace, identifikace a klinický význam.

Frequent "contaminants" detected during mycobacterial culture of decontaminated samples are bacteria of the order Actinomycetales. These are usually bacteria classified as the family Corynebacterineae, genera Corynebacterium, Dietzia, Gordonia, Nocardia, Rhodococcus and Tsukamurella. These bacteria frequently colonize the airways and, under certain circumstances, they may cause life-threatening diseases. In severely immunocompromised patients, they regularly cause life-threatening infections with bacteria of the genus Nocardia. These filamentous bacteria, developing aerial mycelium in the culture, are partly acid-resistant and resistant to lysozyme. They cause nocardiosis, a rare but serious disease in patients with various types of immune deficiency. Differential diagnosis must distinguish between the genera Streptomyces, Actinomadura and Nocardiopsis and other soil saprophytes that are not acid-resistant, sensitive to lysozyme and faster growing. They frequently colonize the airways of patients with lung disease but very rarely cause diseases. The diagnosis of aerobic actinomycetes and determination of their sensitivity to antibiotics are problematic since they grow longer, are difficult to stain and are involved in atypical biochemical reactions. Precise identification of the genera and species requires polyphasic identification of isolates using molecular microbiology methods. If diagnosed early, infections caused by aerobic actinomycetes are easy to treat with targeted antibiotic therapy.

Mitochondrial phospholipase A2 activated by reactive oxygen species in heart mitochondria induces mild uncoupling.

Homeostasis of reactive oxygen species (ROS) in cardiomyocytes is critical for elucidation of normal heart physiology and pathology. Mitochondrial phospholipases A2 (mt-PLA2) have been previously suggested to be activated by ROS. Therefore, we have attempted to elucidate physiological role of such activation. We have found that function of a specific i-isoform of mitochondrial phospholipase A2 (mt-iPLA2) is activated by tert-butylhydroperoxide in isolated rat heart mitochondria. Isoform specificity was judged from the inhibition by bromoenol lactone (BEL), a specific iPLA2 inhibitor. Concomitant uncoupling has been caused by free fatty acids, since it was inhibited by bovine serum albumin. The uncoupling was manifested as a respiration burst accompanied by a slight decrease in mitochondrial inner membrane potential. Since this uncoupling was sensitive to carboxyatractyloside and purine nucleotide di- and tri-phosphates, we conclude that it originated from the onset of fatty acid cycling mediated by the adenine nucleotide translocase (major contribution) and mitochondrial uncoupling protein(s) (minor contribution), respectively. Such a mild uncoupling may provide a feedback downregulation of oxidative stress, since it can further attenuate mitochondrial production of ROS. In conclusion, ROS-induced function of cardiac mt-iPLA2 may stand on a pro-survival side of ischemia-reperfusion injury.

Redistribution of cell death-inducing DNA fragmentation factor-like effector-a (CIDEa) from mitochondria to nucleus is associated with apoptosis in HeLa cells.

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.

In vitro assessment of pancreatic islet vitality by oxymetry.

In order to assess the quality of freshly isolated and cultivated pancreatic islets designed for experimental transplantation in rats we combined the vitality staining test, in vitro measurement of insulin secretion capacity, and assessment of islet respiration. Oxygen consumption was measured using the respirometer Oxygraph 2K equipped with polarographic oxygen sensors. The results of oxymetry demonstrated a linear correlation between islet number and oxygen consumption. Respiration per unit of viable islet tissue was constant. Oxygen consumption tests were in good correlation with the results of insulin release assays, with a correlation coefficient of 0.82. We found no significant differences in all three vitality-testing methods performed with fresh and 24-hour cultivated islets (P > .05). We conclude that polarographic oxymetry provides a fast and easy evaluation test of islet quality. After appropriate standardization, the oxymetric technique can be used for routine clinical pretransplant islet quality testing. In addition, cell membrane integrity and mitochondrial function could be assessed after addition of specific respiration inhibitors or stimulators.

Staphylococcus sciuri: an unusual cause of pelvic inflammatory disease.

We present the case of polymicrobial pelvic inflammatory disease (PID) that involved Staphylococcus sciuri, S. epidermidis, and Streptococcus agalactiae. In order to determine the frequency of S. sciuri isolation from the female lower genital tract, 3415 vaginal samples were analysed during the one-year study period. S. sciuri was isolated from three (0.09%) samples. In all the three cases, S. sciuri was obtained in mixed culture from outpatients without symptoms of infection. While the origin of S. sciuri in the female genital tract remains to be elucidated, the present study showed that this bacterium may colonize vagina and, moreover, may be involved in the pathogenesis of an infection as serious as PID. The low rate of isolation we established, however, indicates infrequent and, most probably, transient colonization of the female genital tract by S. sciuri.

Sequence similarities of protein kinase substrates and inhibitors with immunoglobulins and model immunoglobulin homologue: cell adhesion molecule from the living fossil sponge Geodia cydonium. Mapping of coherent database similarities and implications for evolution of CDR1 and hypermutation.

Sequences of immunoglobulin (Ig) domains of adhesive molecule GSAMS from the living fossil sponge Geodia cydonium were compared with the important motif of peptide protein kinase substrates and inhibitors (PKSI), detail PKSI sequences, and a common template sequence, derived from structures determined previously. We found the site-restricted sequence similarities to these peptide sequences predominantly in the GSAM Ig1 domain of GSAMS in the domain region related to corresponding Ig similarities detected earlier. Additional sequence block-related analysis revealed the presence of CDR1-like segments within PKSI-related regions and resulted in the detection of increased numbers of hypermutation motifs just in the CDR1-like segment of GSAM Ig1 (GSAM(cdrl.1)). In the following database searches with PKSI-related regions and GSAM(cdr1.1) we looked for: (i) peptide similarities present in the context of Ig domains or related structures in a large range of species from Archaea to Vertebrata, and (ii) some special nucleotide similarities.

Mitochondrial uncoupling proteins--facts and fantasies.

Instead of a comprehensive review, we describe the basic undisputed facts and a modest contribution of our group to the fascinating area of the research on mitochondrial uncoupling proteins. After defining the terms uncoupling, leak, protein-mediated uncoupling, we discuss the assumption that due to their low abundance the novel mitochondrial uncoupling proteins (UCP2 to UCP5) can provide only a mild uncoupling, i.e. can decrease the proton motive force by several mV only. Contrary to this, the highly thermogenic role of UCP1 in brown adipose tissue is not given only by its high content (approximately 5 % of mitochondrial proteins) but also by the low ATP synthase content and high capacity respiratory chain. Fatty acid cycling mechanism as a plausible explanation for the protonophoretic function of all UCPs and some other mitochondrial carriers is described together with the experiments supporting it. The phylogenesis of all UCPs, estimated UCP2 content in several tissues, and details of UCP2 activation are described on the basis of our experiments. Functional activation of UCP2 is proposed to decrease reactive oxygen species (ROS) production. Moreover, reaction products of lipoperoxidation such as cleaved hydroperoxy-fatty acids and hydroxy-fatty acid can activate UCP2 and promote feedback down-regulation of mitochondrial ROS production.

First occurrence of transferable extended-spectrum beta-lactamase hydrolyzing cefoperazone in multiresistant nosocomial strains of Klebsiella pneumoniae from two hospitals in Czech and Slovak Republics.

The occurrence of positive synergy between antibiotic discs of amoxicillin/clavulanate and cefoperazone was registered in two Klebsiella pneumoniae strains, isolated from hospitals in Czech and Slovak Republic, indicating the presence of genes coding for an extended-spectrum beta-lactamase active also against cefoperazone, a broad-spectrum cephalosporin. Sulbactam inhibited the hydrolysis of cefoperazone by cell-free lysates of these strains which substantiates its use in combination with cefoperazone. Resistance to cephalothin, cefotaxime, ceftazidime, cefoperazone, cefepime and aztreonam was transferred from K. pneumoniae isolates to Escherichia coli K-12 3110 and to Proteus mirabilis P-38 recipient strains.

Possible basic and specific functions of plant uncoupling proteins (pUCP).

Evidence has been provided that the plant uncoupling proteins (pUCP) play basic physiological roles similar to the other uncoupling protein subfamily members (mammalian UCP1,2,3,4 and BMCP) and are effective in the situations of slight uncoupling that leads to: (1) accelerated respiration and metabolic rates that are beneficial to plant growth and development; (2) decreased formation of reactive oxygen species in mitochondria; and, (3) mild thermogenesis, inevitably accompanying the previous two phenomena. Hypothetically, specific physiological roles of pUCP such as cut off of ATP synthesis could be manifested in connection with climacteric respiratory rise during fruit ripening, seed dormancy, and plant senescence. pUCP might also facilitate growth under low temperatures, e.g., during seed germination or in roots. The existence of these specific roles is suggested by the immunochemical and functional localization of pUCP in mitochondria of fruits, seeds and roots of various plant species.