Two isoforms of ferredoxin:NADP+ oxidoreductase from wheat leaves: purification and initial biochemical characterization

Ferredoxin:NADP⁺ oxidoreductase is an enzyme associated with the stromal side of the thylakoid membrane in the chloroplast. It is involved in photosynthetic linear electron transport to produce NADPH and is supposed to play a role in cyclic electron transfer, generating a transmembrane pH gradient allowing ATP production, if photosystem II is non-functional or no NADP⁺ is available for reduction. Different FNR isoforms have been described in non-photosynthetic tissues, where the enzyme catalyses the NADPH-dependent reduction of ferredoxin (Fd), necessary for some biosynthetic pathways. Here, we report the isolation and purification of two FNR isoproteins from wheat leaves, called FNR-A and FNR-B. These forms of the enzyme were identified as products of two different genes, as confirmed by mass spectrometry. The molecular masses of FNR-A and FNR-B were 34.3 kDa and 35.5 kDa, respectively. The isoelectric point of both FNR-A and FNR-B was about 5, but FNR-B appeared more acidic (of about 0.2 pH unit) than FNR-A. Both isoenzymes were able to catalyse a NADPH-dependent reduction of dibromothymoquinone and the mixture of isoforms catalysed reduction of cytochrome c in the presence of Fd. For the first time, the pH- and ionic strength dependent oligomerization of FNRs is observed. No other protein was necessary for complex formation. The putative role of the two FNR isoforms in photosynthesis is discussed based on current knowledge of electron transport in chloroplasts.     

Mitochondrial diseases in children including Leigh syndrome--biochemical and molecular background

Mitochondrial diseases in children are more frequently caused by mutations in nuclear DNA then in mtDNA. Special clinical phenotypes are associated with the mutations in SURF1 gene, in SCO2 gene and with mtDNA depletion syndromes. Leigh syndrome is the most common clinical presentation of various mitochondrial disorders during childhood. Elevation of lactate in blood, cerebrospinal fluid and urine is a simple biochemical marker of mitochondrial disorders but its specificity and sensitivity are low. Biochemical investigation of muscle biopsy and search for mitochondrial mutations remain a gold standard in the diagnosis. The standarized diagnostic criteria to establish level of diagnostic certainty (possible, probable, definite) are proposed to be used in practice; these include clinical features, neuroimaging and muscle biopsy investigations. Further research directions to improve our understanding of mitochondrial pathologies in children are suggested.     

Decreased expression of p16 in ovarian cancers represents an unfavourable prognostic factor

Decreased expression of p16 may result from hypermethylation of the promoter or from deletion of the gene. It can lead to intensified proliferation of neoplastic cells and to cytostatic drug resistance. The study was aimed at the examination of prognostic value of p16 expression in relation to Ki67 and caspase-3 in ovarian cancers using immunohistochemistry. The immunohistochemical studies were performed on 73 paraffin-embedded samples of ovarian cancers from 43 patients and samples from 6 healthy ovaries. We have used monoclonal antibodies against p16. ABC method and DAB were used for antigens visualisation. The intensity of the immunohistochemical reactions was appraised using the semi-quantitative IRS scale. In healthy ovaries we have shown strong reaction in the nuclei of surface epithelium. In the case of studied ovarian cancers, the reaction of a nuclear and cytoplasmic localization was obtained. The mean overall immunoreactivity score of nuclear p16 expression amounted to 5.30+/-3.44 SD in primary laparotomy material and 6.61+/-4.34 SD in secondary cytoreduction material. Statistical analysis demonstrated that lower p16 expression was typical of the younger patients and the patients who died. Kaplan-Meier's analysis proved that lower expression of p16 was characteristic of cases with shorter overall survival. In the present study we have demonstrated that lowered p16 expression represented an unfavourable prognostic index in ovarian cancer. Lowered p16 expression was also typical for chemotherapy-resistant ceases (cases of lower caspase-3 and higher Ki67 at secondary cytoreduction expression).     

Model of two noise source dependent Ornstein-Uhlenbeck processes applied to postural sway

In this study the postural control system is modeled in terms of two counteracting bio-subsystems. Their activities are described by two Ornstein-Uhlenbeck processes with different-in-magnitude noise sources. The model is constructed as a sum of these processes, where in each of them the same noise source with opposite sign of the noise coefficients was introduced. Since the friction coefficients are also different for these processes, the delay of a crossover from ballistic to diffusive motion for one of the subsystems is greater than for its counterpart. It turns out that for smaller time intervals a superdiffusive behavior is observed, whereas, counteraction of subsystems is called into play for a larger time intervals, what for investigated range of data, is exhibited as a slow, subdiffusive behavior.     

C5a and TNF-alpha up-regulate the expression of tissue factor in intra-alveolar neutrophils of patients with the acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is characterized by the presence of fibrin-rich inflammatory exudates in the intra-alveolar spaces and the extensive migration of neutrophils into alveoli of the lungs. Tissue factor (TF)-dependent procoagulant properties of bronchoalveaolar lavage fluid (BALF) obtained from ARDS patients favor fibrin deposition, and are likely the result of cross-talk between inflammatory mediators and hemostatic mechanisms. However, the regulation of these interactions remains elusive. Prompted by previous findings suggesting that neutrophils, under certain inflammatory conditions, can express functional TF, we investigated the contribution of intra-alveolar neutrophils to the procoagulant properties of BALF from patients with ARDS. Our results confirm that the procoagulant properties of BALF from ARDS patients are the result of TF induction, and further indicate that BALF neutrophils are a main source of TF in intra-alveolar fluid. We also found that BALF neutrophils in these patients express significantly higher levels of TF than peripheral blood neutrophils. These results suggest that the alveolar microenvironment contributes to TF induction in ARDS. Additional experiments indicated that the ability of BALF to induce TF expression in neutrophils from healthy donors can be abolished by inhibiting C5a or TNF-alpha signaling, suggesting a primary role for these inflammatory mediators in the up-regulation of TF in alveolar neutrophils in ARDS. This cross-talk between inflammatory mediators and the induction of TF expression in intra-alveolar neutrophils may be a potential target for novel therapeutic strategies to limit ARDS-associated disturbances of coagulation.     

Role for calcium in the development of ovarial patency in Heliothis virescens

Insect oocytes sequester nutritive proteins from the hemolymph under the regulation by juvenile hormone (JH), in a process called patency. Here, a pharmacological approach was used to decipher the role for calcium in ovarial patency in the moth, Heliothis virescens. Follicular epithelial cells were exposed in calcium-free or calcium-containing media to JH I, JH II or JH III alone, or in combination with various inhibitors of signal transduction. Protein kinase inhibitors, Na(+)/K(+) -ATPase inhibitor, ouabain, an inhibitor of voltage-dependent calcium channels in plasma membrane, omega-Conotoxin MVII, endoplasmic reticulum (ER) Ca(2+) -ATPase inhibitor, thapsigargin, ER inositol 1,4,5-triphosphate receptor (IP(3)R) inhibitor, 2-ABP and ER ryanodine receptor (RyR) inhibitor, ryanodine, were used. The results of our study suggest that JH II evokes patency via protein kinase C-dependent signaling pathway, and activation of Na(+)/K(+) -ATPase, similar to JH III. Response to JH II and JH III predominantly relies upon external and internal calcium stores, using voltage-dependent calcium channels, IP(3)Rs and RyRs. In contrast, regulation of patency by JH I appears to be largely calcium independent, and the calcium-dependent component of the signaling pathway likely does not use IP(3)Rs, but RyRs only. The JH II, JH III and calcium-dependent component of JH I signaling pathway probably utilize calcium/calmodulin-dependent kinase II for activation of Na(+)/K(+) -ATPase.     

Homologous recombination is very rare or absent in human influenza A virus

To determine the extent of homologous recombination in human influenza A virus, we assembled a data set of 13,852 sequences representing all eight segments and both major circulating subtypes, H3N2 and H1N1. Using an exhaustive search and a nonparametric test for mosaic structure, we identified 315 sequences (approximately 2%) in five different RNA segments that, after a multiple-comparison correction, had statistically significant mosaic signals compatible with homologous recombination. Of these, only two contained recombinant regions of sufficient length (>100 nucleotides [nt]) that the occurrence of homologous recombination could be verified using phylogenetic methods, with the rest involving very short sequence regions (15 to 30 nt). Although this secondary analysis revealed patterns of phylogenetic incongruence compatible with the action of recombination, neither candidate recombinant was strongly supported. Given our inability to exclude the occurrence of mixed infection and template switching during amplification, laboratory artifacts provide an alternative and likely explanation for the occurrence of phylogenetic incongruence in these two cases. We therefore conclude that, if it occurs at all, homologous recombination plays only a very minor role in the evolution of human influenza A virus.     

Towards malaria elimination in Savannakhet,Lao PDR: mathematical modelling driven strategy design

Background

The number of Plasmodium falciparum malaria cases around the world has decreased substantially over the last 15 years, but with the spread of resistance against anti-malarial drugs and insecticides, this decline may not continue. There is an urgent need to consider alternative, accelerated strategies to eliminate malaria in countries like Lao PDR, where there are a few remaining endemic areas. A deterministic compartmental modelling tool was used to develop an integrated strategy for P. falciparum elimination in the Savannakhet province of Lao PDR. The model was designed to include key aspects of malaria transmission and integrated control measures, along with a user-friendly interface.

Results

Universal coverage was the foundation of the integrated strategy, which took the form of the deployment of community health workers who provided universal access to early diagnosis, treatment and long-lasting insecticidal nets. Acceleration was included as the deployment of three monthly rounds of mass drug administration targeted towards high prevalence villages, with the addition of three monthly doses of the RTS,S vaccine delivered en masse to the same high prevalence sub-population. A booster dose of vaccine was added 1 year later. The surveillance-as-intervention component of the package involved the screening and treatment of individuals entering the simulated population.

Conclusions

In this modelling approach, the sequential introduction of a series of five available interventions in an integrated strategy was predicted to be sufficient to stop malaria transmission within a 3-year period. These interventions comprised universal access to early diagnosis and adequate treatment, improved access to long-lasting insecticidal nets, three monthly rounds of mass drug administration together with RTS,S vaccination followed by a booster dose of vaccine, and screening and treatment of imported cases.