Progesterone modulates extracellular heat-shock proteins and interlukin-1ß in human choriodecidual after Escherichia coli infection.

INTRODUCTION: Chorioamnionitis is an adverse condition in human pregnancy caused by many bacterial pathogens including Escherichia coli (E. coli); which has been associated with higher risk of preterm birth. We recently reported that human maternal decidua (MDec) tissue responds to E. coli infection by secreting extracellular heat-shock proteins (eHsp)-60, -70 and interlukin-1ß (IL-1ß). Previous studies have shown that progesterone (P4) regulates the immune response, but it is unknown whether P4 inhibits the secretion of eHsp. The aim of this investigation was to determine the role of P4 on the secretion of eHsp-27, -60, -70 and IL-1ß in MDec after 3, 6, and 24 h of E. coli infection. METHODS: Nine human feto-maternal interface (HFMi) tissues were included and mounted in the Transwell culture system. Only the maternal decidua (MDec) was stimulated for 3, 6 and 24 h with E. coli alone or in combination with progesterone and RU486. After each treatment, the HFMi tissue was recovered to determine histological changes and the culture medium recovered to evaluate the levels of eHsp-27, -60, -70 and IL-1ß by ELISA and mRNA expression by RT-PCR. RESULTS: No structural changes were observed in the HFMi tissue treated with P4 and RU486. However, stimulation with E. coli produces diffuse inflammation and ischemic necrosis. E. coli induced infection decreases, in time- and dose-dependent manner, eHsp-27 and increases eHsp-60, eHsp-70 and IL-1ß levels. In contrast, incubation of HFMi tissue with E. coli + P4 reversed eHsp and IL-1ß secretion levels relative to E. coli stimulation group but not relative to the control group. The same profile was observed on the expression of eHsp-27 and eHsp-60. DISCUSSION: we found that progesterone modulates the anti-inflammatory (eHsp-27) and pro-inflammatory (eHsp-60 and eHsp-70) levels of eHsp induced by E. coli infection in human choriodecidual tissue. eHsp-60 and eHsp-70 levels were not completely reversed; maintaining the secretion of IL-1ß, which has been associated with adverse events during pregnancy.

The mRNA and protein levels of the glycolytic enzymes lactate dehydrogenase A (LDHA) and phosphofructokinase platelet (PFKP) are good predictors of survival time, recurrence, and risk of death in cervical cancer patients.

INTRODUCTION: Patients with cervical cancer (CC) may experience local recurrence very often after treatment; when only clinical parameters are used, most cases are diagnosed in late stages, which decreases the chance of recovery. Molecular markers can improve the prediction of clinical outcome. Glycolysis is altered in 70% of CCs, so molecular markers of this pathway associated with the aggressiveness of CC can be identified. METHODS: The expression of 14 glycolytic genes was analyzed in 97 CC and 29 healthy cervical tissue (HCT) with microarray; only LDHA and PFKP were validated at the mRNA and protein levels in 36 of those CC samples and in 109 new CC samples, and 31 HCT samples by qRT-PCR, Western blotting, or immunohistochemistry. A replica analysis was performed on 295 CC from The Cancer Genome Atlas (TCGA) database. RESULTS: The protein expression of LDHA and PFKP was associated with poor overall survival [OS: LDHA HR = 4.0 (95% CI = 1.4-11.1); p = 8.0 × 10-3 ; PFKP HR = 3.3 (95% CI = 1.1-10.5); p = 4.0 × 10-2 ] and disease-free survival [DFS: LDHA HR = 4.5 (95% CI = 1.9-10.8); p = 1.0 × 10-3 ; PFKP HR = 3.2 (95% CI = 1.2-8.2); p = 1.8 × 10-2 ] independent of FIGO clinical stage, and the results for mRNA expression were similar. The risk of death was greater in patients with overexpression of both biomarkers than in patients with advanced FIGO stage [HR = 8.1 (95% CI = 2.6-26.1; p = 4.3 × 10-4 ) versus HR = 7 (95% CI 1.6-31.1, p = 1.0 × 10-2 )] and increased exponentially as the expression of LDHA and PFKP increased. CONCLUSIONS: LDHA and PFKP overexpression at the mRNA and protein levels was associated with poor OS and DFS and increased risk of death in CC patients regardless of FIGO stage. The measurement of these two markers could be very useful for evaluating clinical evolution and the risk of death from CC and could facilitate better treatment decision making.

Deletion of the ATP20 gene in Ustilago maydis produces an unstable dimer of F1FO-ATP synthase associated with a decrease in mitochondrial ATP synthesis and a high H2O2 production.

The F1FO-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a monomer and dimer. The dimer shows higher ATPase activity than the monomer and is essential for cristae folding. The monomer-monomer interface is constituted by subunits a, i/j, e, g, and k. The role of the subunit g in a strict respiratory organism is unknown. A gene knockout was generated in Ustilago maydis to study the role of subunit g on mitochondrial metabolism and cristae architecture. Deletion of the ATP20 gene, encoding the g subunit, did not affect cell growth or glucose consumption, but biomass production was lower in the mutant strain (gΔ strain). Ultrastructure observations showed that mitochondrial size and cristae shape were similar in wild-type and gΔ strains. The mitochondrial membrane potential in both strains had a similar magnitude, but oxygen consumption was higher in the WT strain. ATP synthesis was 20 % lower in the gΔ strain. Additionally, the mutant strain expressed the alternative oxidase in the early stages of growth (exponential phase), probably as a response to ROS stress. Dimer from mutant strain was unstable to digitonin solubilization, avoiding its isolation and kinetic characterization. The isolated monomeric state activated by n-dodecyl-ß-D-maltopyranoside showed similar kinetic constants to the monomer from the WT strain. A decrease in mitochondrial ATP synthesis and the presence of the AOX during the exponential growth phase suggests that deletion of the g gene induces ROS stress.

Moderate exercise combined with metformin-treatment improves mitochondrial bioenergetics of the quadriceps muscle of old female Wistar rats.

Sarcopenia is a syndrome that leads to physical disability and that deteriorates elderly people´s life quality. The etiology of sarcopenia is multifactorial, but mitochondrial dysfunction plays a paramount role in this pathology. Our research group has shown that the combined treatment of metformin (MTF) and exercise has beneficial effects for preventing muscle loss and fat accumulation, by modulating the redox state. To get an insight into the mechanism of the combined treatment, the mitochondrial bioenergetics was studied in the mitochondria isolated from old female Wistar rats quadriceps muscles. The animals were divided into six groups; three performed exercise on a treadmill for 5 days/week for 20 months, and the other three were sedentary. Also, two groups of each were treated with MTF for 6 or 12 months. The rats were euthanized at 24 months. The mitochondria were isolated and supercomplexes formation along with oxygen consumption, ATP synthesis, and ROS generation were evaluated. Our results showed that the combined treatment for 12 months increased the complex I and IV activities associated with the supercomplexes, simultaneously, ATP synthesis increased while ROS production decreased, indicating a tightly coupled mitochondria. The role of exercise plus the MTF treatment against sarcopenia in old muscles is discussed.

Isolation of Mitochondria from Ustilago maydis Protoplasts.

Ustilago maydis, a basidiomycete that infects Zea mays, is one of the top ten fungal models for studying DNA repair, signal transduction pathways, and dimorphic transitions, among other processes. From a metabolic point of view, U. maydis lacks fermentative capacity, pointing to mitochondria as a key player in central metabolism. Oxidative phosphorylation, synthesis of heme groups, Krebs cycle, ß-oxidation of fatty acids, and synthesis of amino acids are some of the processes that take place in mitochondria. Given the importance of this organelle in eukaryotic cells in general, and in fungal cells in particular, we present a protocol for the isolation of U. maydis mitochondria based on the enzymatic disruption of U. maydis cell wall and differential centrifugation. The method can easily be extrapolated to other fungal species, by using appropriate lytic enzymes.

Pseudomonas aeruginosa induces spatio-temporal secretion of IL-1ß, TNFα, proMMP-9, and reduction of epithelial E-cadherin in human alveolar epithelial type II (A549) cells.

Pseudomonas aeruginosa, is an opportunistic bacterium with a high prevalence in diverse pulmonary infections. Although several genes are involved in the system of resistance and evasion of the immunological response of the host, little is known about the inflammatory, degradative, and cell-binding response induced by P. aeruginosa in human lung alveolar epithelial cells. The purpose of this study was to determine the cytokine expression (IL-1ß and TNFα), pro matrix metalloproteinases activation (proMMP-2 and proMMP-9), and the effects on the cell-binding adhesion protein (E-cadherin) in an in vitro model of human lung alveolar epithelial cells. A549 cells were stimulated with a different number of colony-forming units of P. aeruginosa for 3, 6, and 24 hours. Subsequently, the culture medium was collected, IL-1ß and TNFα levels were evaluated by ELISA; proMMP-2 and -9 levels were determined by substrate gel zymography, and the MMP-9 and E-cadherin assessed by immunostaining of A549 cells. Our results demonstrated that P. aeruginosa induces mainly the secretion of TNFα, increases actMMP-9 level, and significantly reduces the level of E-cadherin in the A549 cells. In summary, the inflammatory/degradative process induced by P. aeruginosa modulates the expression of the E-cadherin protein. The probable clinical implications of this study suggest the use of inhibitors that reduce the degradative activity of proMMP-9 which will be further explored in the next phase of this study.

Steady-state persistence of respiratory syncytial virus in a macrophage-like cell line and sequence analysis of the persistent viral genome.

Long-term infection by human respiratory syncytial virus (hRSV) has been reported in immunocompromised patients. Cell lines are valuable in vitro model systems to study mechanisms associated with viral persistence. Persistent infections in cell cultures have been categorized at least as in "carrier-state", where there exist a low proportion of cells infected by a lytic virus, and as in "steady-state", where most of cells are infected, but in absence of cytophatic effect. Here, we showed that hRSV maintained a steady-state persistence in a macrophage-like cell line after 120 passages, since the viral genome was detected in all of the cells analyzed by fluorescence in situ hybridization, whereas only defective viruses were identified by sucrose gradients and titration assay. Interestingly, eight percent of cells harboring the hRSV genome revealed undetectable expression of the viral nucleoprotein N; however, when this cell population was sorted by flow cytometry and independently cultured, viral protein expression was induced at detectable levels since the first post-sorting passage, supporting that sorted cells harbored the viral genome. Sequencing of the persistent hRSV genome obtained from virus collected from cell-culture supernatants, allowed assembling of a complete genome that displayed 24 synonymous and 38 nonsynonymous substitutions in coding regions, whereas extragenic and intergenic regions displayed 12 substitutions, two insertions and one deletion. Previous reports characterizing mutations in extragenic regulatory sequences of hRSV, suggested that some mutations localized at the 3' leader region of our persistent virus might alter viral transcription and replication, as well as assembly of viral nucleocapsids. Besides, substitutions in P, F and G proteins might contribute to altered viral assembly, budding and membrane fusion, reducing the cytopathic effect and in consequence, contributing to host-cell survival. Full-length mutant genomes might be part of the repertoire of defective viral genomes formed during hRSV infections, contributing to the establishment and maintenance of virus persistence.

Carbon and Nitrogen Sources Have No Impact on the Organization and Composition of Ustilago maydis Respiratory Supercomplexes.

Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in principle should increase the respiratory chain efficiency and ATP synthesis. In addition to the four classic respiratory complexes and the ATP synthase, U. maydis mitochondria contain three type II NADH dehydrogenases (NADH for reduced nicotinamide adenine dinucleotide) and the alternative oxidase. Changes in the composition of the respiratory supercomplexes due to energy requirements have been reported in certain organisms. In this study, we addressed the organization of the mitochondrial respiratory complexes in U. maydis under diverse energy conditions. Supercomplexes were obtained by solubilization of U. maydis mitochondria with digitonin and separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). The molecular mass of supercomplexes and their probable stoichiometries were 1200 kDa (I1:IV1), 1400 kDa (I1:III2), 1600 kDa (I1:III2:IV1), and 1800 kDa (I1:III2:IV2). Concerning the ATP synthase, approximately half of the protein is present as a dimer and half as a monomer. The distribution of respiratory supercomplexes was the same in all growth conditions. We did not find evidence for the association of complex II and the alternative NADH dehydrogenases with other respiratory complexes.

Rapamycin induces morphological and physiological changes without increase in lipid content in Ustilago maydis.

The evolutionarily conserved serine/threonine kinase TOR recruits different subunits to assemble the Target of Rapamycin Complex 1 (TORC1), which is inhibited by rapamycin and regulates ribosome biogenesis, autophagy, and lipid metabolism by regulating the expression of lipogenic genes. In addition, TORC1 participates in the cell cycle, increasing the length of the G2 phase. In the present work, we investigated the effect of rapamycin on cell growth, cell morphology and neutral lipid metabolism in the phytopathogenic fungus Ustilago maydis. Inhibition of TORC1 by rapamycin induced the formation of septa that separate the nuclei that were formed after mitosis. Regarding neutral lipid metabolism, a higher accumulation of triacylglycerols was not detected, but the cells did contain large lipid bodies, which suggests that small lipid bodies became fused into big lipid droplets. Vacuoles showed a similar behavior as the lipid bodies, and double labeling with Blue-CMAC and BODIPY indicates that vacuoles and lipid bodies were independent organelles. The results suggest that TORC1 has a role in cell morphology, lipid metabolism, and vacuolar physiology in U. maydis.

Isocitrate dehydrogenase type 2 (IDH2) is part of a multiprotein complex for placental steroidogenesis.

BACKGROUND: Human syncytiotrophoblast mitochondria require the activity of the isocitrate dehydrogenase type 2 (IDH2) to obtain reduced coenzymes for progesterone (P4) synthesis. Data from the literature indicate that mitochondrial steroidogenic contact sites transform efficiently cholesterol into P4. In this research, we identified the IDH2 as a member of the steroidogenic contact site and analyzed the steroidogenic role of its activity. METHOD: Human syncytiotrophoblast mitochondria were isolated by differential centrifugation, and steroidogenic contact sites were obtained by osmotic shock and sucrose gradient ultracentrifugation. In-gel native activity assay, mass spectroscopy, and western blot were used to identify the association of proteins and their activities. P4 was determined by immunofluorescence. RESULTS: The IDH2 was mainly identified in steroidogenic contact sites, and its activity was associated with a complex of proteins with an apparent molecular mass of ~590 kDa. Mass spectroscopy showed many groups of proteins with several metabolic functions, including steroidogenesis and ATP synthesis. The IDH2 activity was coupled to P4 synthesis since in the presence of Ca2+ or Na2SeO3, inhibitors of the IDH2, the P4 production decreased. CONCLUSIONS: The human syncytiotrophoblast mitochondria build contact sites for steroidogenesis. The IDH2, a non-membrane protein, supplies the NADPH required for the synthesis of P4 in a complex (steroidosome) that associate the proteins required to transform efficiently cholesterol into P4, which is necessary in pregnancy to maintain the relationship between mother and fetus. GENERAL SIGNIFICANCE: The IDH2 is proposed as a check point in the regulation of placental steroidogenesis.

Mitochondrial respirasome works as a single unit and the cross-talk between complexes I, III2 and IV stimulates NADH dehydrogenase activity.

Ustilago maydis is an aerobic basidiomycete that depends on oxidative phosphorylation for its ATP supply, pointing to the mitochondrion as a key player in its energy metabolism. Mitochondrial respiratory complexes I, III2, and IV occur in supramolecular structures named respirasome. In this work, we characterized the subunit composition and the kinetics of NADH:Q oxidoreductase activity of the digitonine-solubilized respirasome (1600 kDa) and the free-complex I (990 kDa). In the presence of 2,6-dimethoxy-1,4-benzoquinone (DBQ) and cytochrome c, both the respirasome NADH:O2 and the NADH:DBQ oxidoreductase activities were inhibited by rotenone, antimycin A or cyanide. A value of 2.4 for the NADH oxidized/oxygen reduced ratio was determined for the respirasome activity, while ROS production was less than 0.001% of the oxygen consumption rate. Analysis of the NADH:DBQ oxidoreductase activity showed that respirasome was 3-times more active and showed higher affinity than free-complex I. The results suggest that the contacts between complexes I, III2 and IV in the respirasome increase the catalytic efficiency of complex I and regulate its activity to prevent ROS production.

The relevance of the supramolecular arrangements of the respiratory chain complexes in human diseases and aging.

Mitochondrial dysfunction, a common factor in several diseases is accompanied with reactive oxygen species (ROS) production. These molecules react with proteins and lipids at their site of generation, establishing a vicious cycle which might result in further mitochondrial injury. It is well established that mitochondrial respiratory complexes can be organized into supramolecular structures called supercomplexes (SCs) or respirasomes; yet, the physiological/pathological relevance of these structures remains unresolved. Changes in their stabilization and content have been documented in Barth's syndrome, degenerative diseases such as Parkinson's and Alzheimer, cardiovascular diseases including heart failure and ischemia-reperfusion damage, as well as in aging. Under pathological conditions, SCs stability could have relevant biomedical implications or might be used as a reliable marker of mitochondrial damage. The purpose of this review is to recapitulate the current state of the significance on mitochondrial bioenergetics of these structures and their possible role in pathophysiologies related with ROS increase.

Insight into the Mechanistic Basis of the Hysteretic-Like Kinetic Behavior of Thioredoxin-Glutathione Reductase (TGR).

A kinetic study of thioredoxin-glutathione reductase (TGR) from Taenia crassiceps metacestode (cysticerci) was carried out. The results obtained from both initial velocity and product inhibition experiments suggest the enzyme follows a two-site ping-pong bi bi kinetic mechanism, in which both substrates and products are bound in rapid equilibrium fashion. The substrate GSSG exerts inhibition at moderate or high concentrations, which is concomitant with the observation of hysteretic-like progress curves. The effect of NADPH on the apparent hysteretic behavior of TGR was also studied. At low concentrations of NADPH in the presence of moderate concentrations of GSSG, atypical time progress curves were observed, consisting of an initial burst-like stage, followed by a lag whose amplitude and duration depended on the concentration of both NADPH and GSSG. Based on all the kinetic and structural evidence available on TGR, a mechanism-based model was developed. The model assumes a noncompetitive mode of inhibition by GSSG in which the disulfide behaves as an affinity label-like reagent through its binding and reduction at an alternative site, leading the enzyme into an inactive state. The critical points of the model are the persistence of residual GSSG reductase activity in the inhibited GSSG-enzyme complexes and the regeneration of the active form of the enzyme by GSH. Hence, the hysteretic-like progress curves of GSSG reduction by TGR are the result of a continuous competition between GSH and GSSG for driving the enzyme into active or inactive states, respectively. By using an arbitrary but consistent set of rate constants, the experimental full progress curves were successfully reproduced in silico.

Secretion of heat shock -60, -70 kD protein, IL-1ß and TNFα levels in serum of a term normal pregnancy and patients with pre-eclampsia development.

The extracellular heat shock proteins (eHsp) family act as molecular chaperones regulating folding, transporting protein and are associated with immune modulation in different physiological and pathological processes. They have been localized in different gestational tissues and their concentration in amniotic fluid and serum has been determined. In the present study, we proposed to determine the concentration of eHsp-60, -70, IL-1ß and TNFα in the serum of pregnant patients with 34 weeks of gestation with and without clinical evidences of preeclampsia (PE). Our results indicate significant increase of these markers in patients with PE with respect to healthy pregnant patients without active labor. Finally, the concentration of eHsp-60 and -70 correlated positively with the hepatic dysfunction markers uric acid, lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT) glutamic pyruvic transaminase (GPT), and inflammatory IL-1ß and TNFα response. In conclusion, our results demonstrate a strong associated between Hsp and marker of hepatic dysfunction.

Effects of Moringa oleifera Leaves Extract on High Glucose-Induced Metabolic Changes in HepG2 Cells.

Mitochondrial dysfunction is a hallmark of diabetes, but the metabolic alterations during early stages of the disease remain unknown. The ability of liver cells to rearrange their metabolism plays an important role in compensating the energy shortage and may provide cell survival. Moringa oleifera leaves have been studied for its health properties against diabetes, insulin resistance, and non-alcoholic liver disease. We postulated that M. oleifera executes a protective function on mitochondrial functionality in HepG2 treated with high glucose. We evaluated the effect of high glucose treatment on the mitochondrial function of HepG2 cells using a Seahorse extracellular flux analyzer (Agilent, Santa Clara, CA, USA), blue native polyacrylamide gel electrophoresis (BN-PAGE), and western blot analysis. For assessment of mitochondrial abnormalities, we measured the activity of mitochondrial Complex I and IV as well as uncoupling protein 2, and sirtuin 3 protein contents. Our results demonstrate that, under conditions mimicking the hyperglycemia, Complex I activity, UCP2, Complex III and IV subunits content, supercomplex formation, and acetylation levels are modified with respect to the control condition. However, basal oxygen consumption rate was not affected and mitochondrial reactive oxygen species production remained unchanged in all groups. Treatment of HepG2 cells with M. oleifera extract significantly increased both protein content and mitochondrial complexes activities. Nonetheless, control cells’ respiratory control ratio (RCR) was 4.37 compared to high glucose treated cells’ RCR of 15.3, and glucose plus M. oleifera treated cells’ RCR of 5.2, this indicates high-quality mitochondria and efficient oxidative phosphorylation coupling. Additionally, the state app was not altered between different treatments, suggesting no alteration in respiratory fluxes. These findings enhance understanding of the actions of M. oleifera and suggest that the known antidiabetic property of this plant, at least in part, is mediated through modulating the mitochondrial respiratory chain.

Streptozotocin-Induced Adaptive Modification of Mitochondrial Supercomplexes in Liver of Wistar Rats and the Protective Effect of Moringa oleifera Lam.

The increasing prevalence of diabetes continues to be a major health issue worldwide. Alteration of mitochondrial electron transport chain is a recognized hallmark of the diabetic-associated decline in liver bioenergetics; however, the molecular events involved are only poorly understood. Moringa oleifera is used for the treatment of diabetes. However, its role on mitochondrial functionality is not yet established. This study was aimed to evaluate the effect of M. oleifera extract on supercomplex formation, ATPase activity, ROS production, GSH levels, lipid peroxidation, and protein carbonylation. The levels of lipid peroxidation and protein carbonylation were increased in diabetic group. However, the levels were decreased in Moringa-treated diabetic rats. Analysis of in-gel activity showed an increase in all complex activities in the diabetic group, but spectrophotometric determinations of complex II and IV activities were unaffected in this treatment. However, we found an oxygen consumption abolition through complex I-III-IV pathway in the diabetic group treated with Moringa. While respiration with succinate feeding into complex II-III-IV was increased in the diabetic group. These findings suggest that hyperglycemia modifies oxygen consumption, supercomplexes formation, and increases ROS levels in mitochondria from the liver of STZ-diabetic rats, whereas M. oleifera may have a protective role against some alterations.

Microbiological comparison of blood culture and amplification of 16S rDNA methods in combination with DGGE for detection of neonatal sepsis in blood samples.

It is estimated that 15% of all newborns admitted to the neonatal intensive care unit (NICU) for suspected sepsis receive multiple broad-spectrum antibiotics without pathogen identification. The gold standard for bacterial sepsis detection is blood culture, but the sensitivity of this method is very low. Recently, amplification and analysis of the 16S ribosomal DNA (rDNA) bacterial gene in combination with denaturing gradient gel electrophoresis (DGGE) has proven to be a useful approach for identifying bacteria that are difficult to isolate by standard culture methods. The main goal of this study was to compare two methods used to identify bacteria associated with neonatal sepsis: blood culture and broad range 16S rDNA-DGGE. Twenty-two blood samples were obtained from newborns with (n = 15) or without (n = 7) signs and symptoms of sepsis. Blood samples were screened to identify pathogenic bacteria with two different methods: (1) bacteriological culture and (2) amplification of the variable V3 region of 16S rDNA-DGGE. Blood culture analysis was positive in 40%, whereas 16S rDNA-DGGE was positive in 87% of neonatal sepsis cases. All 16S rDNA-DGGE positive samples were associated with some other signs of neonatal sepsis. CONCLUSION: Our study shows that the molecular approach with 16S rDNA-DGGE identifies twofold more pathogenic bacteria than bacteriological culture, including complex bacterial communities associated with the development of bacterial sepsis in neonates. What is Known: • Neonatal sepsis affects 2.3% of birth in the NICU with a high mortality risk. • Evidence supports the use of molecular methods as an alternative to blood culture for identification of bacterial associated neonatal sepsis. What is New: • The DGGE gel is a good methodological approach for the identification of bacterial in neonatal blood samples. • This study describes the pattern of electrophoretic mobility obtained by DGGE gels and allows to determine the type of bacteria associated in the development of neonatal sepsis.

Structural and kinetics characterization of the F1F0-ATP synthase dimer. New repercussion of monomer-monomer contact.

Ustilago maydis is an aerobic basidiomycete that fully depends on oxidative phosphorylation for its supply of ATP, pointing to mitochondria as a key player in the energy metabolism of this organism. Mitochondrial F1F0-ATP synthase occurs in supramolecular structures. In this work, we isolated the monomer (640kDa) and the dimer (1280kDa) and characterized their subunit composition and kinetics of ATP hydrolysis. Mass spectrometry revealed that dimerizing subunits e and g were present in the dimer but not in the monomer. Analysis of the ATPase activity showed that both oligomers had Michaelis-Menten kinetics, but the dimer was 7 times more active than the monomer, while affinities were similar. The dimer was more sensitive to oligomycin inhibition, with a Ki of 24nM, while the monomer had a Ki of 169nM. The results suggest that the interphase between the monomers in the dimer state affects the catalytic efficiency of the enzyme and its sensitivity to inhibitors.

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala).

A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.