Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells

Nitric oxide (NO) inhibits mitochondrial respiration by decreasing the apparent affinity of cytochrome c oxidase (CcO) for oxygen. Using iNOS-transfected HEK 293 cells to achieve regulated intracellular NO production, we determined NO and O₂ concentrations and mitochondrial O₂ consumption by high-resolution respirometry over a range of O₂ concentrations down to nanomolar. Inhibition of respiration by NO was reversible, and complete NO removal recovered cell respiration above its routine reference values. Respiration was observed even at high NO concentrations, and the dependence of IC₅₀ on [O₂] exhibits a characteristic but puzzling parabolic shape; both these features imply that CcO is protected from complete inactivation by NO and are likely to be physiologically relevant. We present a kinetic model of CcO inhibition by NO that efficiently predicts experimentally determined respiration at physiological O₂ and NO concentrations and under hypoxia, and accurately predicts the respiratory responses under hyperoxia. The model invokes competitive and uncompetitive inhibition by binding of NO to the reduced and oxidized forms of CcO, respectively, and suggests that dissociation of NO from reduced CcO may involve its O₂-dependent oxidation. It also explains the non-linear dependence of IC₅₀ on O₂ concentration, and the hyperbolic increase of c₅₀ as a function of NO concentration.     

Retinoic acid effects on thyroid function of female rats

AimsRetinoic acid is widely used in dermatological treatment and thyroid cancer management; however its possible side-effects on normal thyroid function remains unknown. We aimed to determine the effects of retinoic acid on thyroid function of adult female rats.Main methodsFemale Wistar rats were treated with all-trans-retinoic acid and 13-cis retinoic acid for 14 and 28 days. Then, rats were killed and thyroid function was evaluated.Key findingsSerum T4 and thyrotropin levels remained unchanged, while serum T3 increased in animals treated with all-trans-retinoic acid for 14 days. No changes were observed in hepatic or renal type 1 iodothyronine deiodinase (D1) activities, while thyroid D1 was higher in animals treated for 14 days with all-trans-retinoic acid, which could be related to the increased serum T3 levels. 13-cis retinoic acid increased thyroid iodide uptake after 28 days. These results show effects of retinoic acid treatment on these thyroid proteins: sodium/iodide symporter and deiodinase.SignificanceRetinoic acid is able to interfere with normal thyroid function, increasing thyroid type 1 deiodinase activity, serum T3 levels and sodium/iodide symporter function. However, the effects are time- and retinoic acid isomer-dependent. Since serum thyrotropin levels did not change in any group, the effects observed are probably mediated by a direct retinoic acid effect on the normal thyroid.     

Protecting capacity against malaria of chemically defined tetramer forms based on the Plasmodium falciparum apical sushi protein as potential vaccine components

Developing novel generations of subunit-based antimalarial vaccines in the form of chemically-defined macromolecule systems for multiple antigen presentation represents a classical problem in the field of vaccine development. Many efforts involving synthesis strategies leading to macromolecule constructs have been based on dendrimer-like systems, the condensation of large building blocks and conventional asymmetric double dimer constructs, all based on lysine cores.     

Measurement of selective effect of insulin on glucose disposal from labeled glucose oral test minimal model

The oral glucose minimal model (OMM) measures insulin sensitivity (S(I)) and the glucose rate of appearance (R(a)) of ingested glucose in the presence of physiological changes of insulin and glucose concentrations. However, S(I) of OMM measures the overall effect of insulin on glucose utilization and glucose production. In this study we show that, by adding a tracer to the oral dose, e.g., of a meal, and by using the labeled version of OMM, OMM* to interpret the data, one can measure the selective effect of insulin on glucose disposal, S(I)*. Eighty-eight individuals underwent both a triple-tracer meal with the tracer-to-tracee clamp technique, providing a model-independent reference of the R(a) of ingested glucose (R(a meal)(ref)) and an insulin-modified labeled intravenous glucose tolerance test (IVGTT*). We show that OMM* provides not only a reliable means of tracing the R(a) of ingested glucose (R(a meal)) but also accurately measures S(I)*. We do so by comparing OMM* R(a meal) with the model-independent R(a meal)(ref) provided by the tracer-to-tracee clamp technique, while OMM* S(I)* is compared with both S(I)(* ref), obtained by using as known input R(a meal)(ref), and with S(I)* measured during IVGTT*.     

Cell proliferation in the Rana catesbeiana auditory medulla over metamorphic development

During metamorphic development, bullfrogs (Rana catesbeiana) undergo substantial morphological, anatomical, and physiological changes as the animals prepare for the transition from a fully-aquatic to a semi-terrestrial existence. Using BrdU incorporation and immunohistochemistry, we quantify changes in cell proliferation in two key auditory brainstem nuclei, the dorsolateral nucleus and the superior olivary nucleus, over the course of larval and early postmetamorphic development. From hatchling through early larval stages, numbers of proliferating cells increase in both nuclei, paralleling the overall increase in total numbers of cells available for labeling. Numbers of proliferating cells in the superior olivary nucleus decrease during the late larval and deaf periods, and significantly increase during metamorphic climax. Proliferating cells in the dorsolateral nucleus increase in number from hatchling to late larval stages, decrease during the deaf period, and increase during climax. In both nuclei, numbers of proliferating cells decrease during the postmetamorphic froglet stage, despite increases in the number of cells available for label. Newly generated cells express either glial- or neural-specific phenotypes beginning between 1 week and 1 month post-BrdU injection, respectively, while some new cells express gamma-aminobutyric acid within 2 days of mitosis. Our data show that these auditory nuclei dramatically up-regulate mitosis immediately prior to establishment of a transduction system based on atmospheric hearing.     

Extraction of features from ultrasound acoustic emissions: a tool to assess the hydraulic vulnerability of Norway spruce trunkwood?

The aim of this study was to assess the hydraulic vulnerability of Norway spruce (Picea abies) trunkwood by extraction of selected features of acoustic emissions (AEs) detected during dehydration of standard size samples. The hydraulic method was used as the reference method to assess the hydraulic vulnerability of trunkwood of different cambial ages. Vulnerability curves were constructed by plotting the percentage loss of conductivity vs an overpressure of compressed air. Differences in hydraulic vulnerability were very pronounced between juvenile and mature wood samples; therefore, useful AE features, such as peak amplitude, duration and relative energy, could be filtered out. The AE rates of signals clustered by amplitude and duration ranges and the AE energies differed greatly between juvenile and mature wood at identical relative water losses. Vulnerability curves could be constructed by relating the cumulated amount of relative AE energy to the relative loss of water and to xylem tension. AE testing in combination with feature extraction offers a readily automated and easy to use alternative to the hydraulic method.     

Peroxynitrite mobilizes calcium ions from ryanodine-sensitive stores, a process associated with the mitochondrial accumulation of the cation and the enforced formation of species mediating cleavage of genomic DNA

Peroxynitrite does not directly cause strand scission of genomic DNA. Rather, as we previously reported, the DNA cleavage is largely mediated by H(2)O(2) resulting from the dismutation of superoxide generated in the mitochondria upon peroxynitrite-dependent inhibition of complex III. The present study demonstrates that this process is strictly controlled by the availability of Ca(2+) in the mitochondrial compartment. Experiments using intact as well as permeabilized U937 cells showed that the DNA-damaging response evoked by peroxynitrite is enhanced by treatments causing an increase in mitochondrial Ca(2+) uptake and remarkably reduced under conditions leading to inhibition of mitochondrial Ca(2+) accumulation. An additional, important observation was that the source of the Ca(2+) mobilized by peroxynitrite is the ryanodine receptor; preventing the mobilization of Ca(2+) with ryanodine suppressed the mitochondrial formation of reactive oxygen species and the ensuing DNA strand scission. Identical results were obtained using PC12, C6, and THP-1 cells. These results, along with our previous findings indicating that the DNA damage induced by peroxynitrite is also suppressed by inhibition of the electron flow through complex I, e.g., by rotenone, or by the respiration-deficient phenotype, demonstrate that the mitochondrial formation of DNA-damaging species is critically regulated by the inhibition of complex III and by the availability of Ca(2+).     

Discovery of Candidate Disease Genes in ENU–Induced Mouse Mutants by Large-Scale Sequencing,Including a Splice-Site Mutation in Nucleoredoxin

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.     

SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium‐triggered fusion

Synaptic vesicles fuse with the plasma membrane in response to Ca²⁺ influx, thereby releasing neurotransmitters into the synaptic cleft. The protein machinery that mediates this process, consisting of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) and regulatory proteins, is well known, but the mechanisms by which these proteins prime synaptic membranes for fusion are debated. In this study, we applied large‐scale, automated cryo‐electron tomography to image an in vitro system that reconstitutes synaptic fusion. Our findings suggest that upon docking and priming of vesicles for fast Ca²⁺‐triggered fusion, SNARE proteins act in concert with regulatory proteins to induce a local protrusion in the plasma membrane, directed towards the primed vesicle. The SNAREs and regulatory proteins thereby stabilize the membrane in a high‐energy state from which the activation energy for fusion is profoundly reduced, allowing synchronous and instantaneous fusion upon release of the complexin clamp.     

TGF-β1 suppresses IL-6-induced STAT3 activation through regulation of Jak2 expression in prostate epithelial cells

Chronic inflammation plays an important role in the initiation and progression of various human diseases including benign prostatic hyperplasia or prostate cancer. Here we show that the proinflammatory cytokine interleukin-6 (IL-6) has prosurvival effects and chronically activates the Jak2/STAT3 signalling pathway in a model of benign prostatic hyperplasia (BPH-1). We demonstrate that the antiinflammatory cytokine transforming growth factor-β1 (TGF-β1), which also permanently activates its canonical signalling pathway through SMAD proteins in BPH-1 cells, modifies the effects of IL-6 on cell proliferation. Importantly, TGF-β1 inhibits IL-6 signal transduction by decreasing the phosphorylation levels of STAT3. This effect is associated with decreased expression of Jak2 at both mRNA and protein levels. Moreover, we showed that TGF-β1 inhibits IL-6-induced expression of the cancer-associated gene MUC1. These observations demonstrated a novel interaction between TGF-β1 and IL-6 signalling and suggested another mechanism of how defects in TGF-β signalling, frequently associated with prostate pathologies, can contribute to the disruption of tissue homeostasis.