Dual Role of Plasma Membrane Electron Transport Systems in Defense

Bcause oxidative stress is one of the main sources of severe cellular damage, cells have different defense weapons against reactive oxygen species. Ubiquitous plasma membrane redox systems play a role in defense against oxidative stress damage. On the other hand, a tightly controlled and localized production of reactive oxygen species by a plasma membrane NADPH oxidase can be used as a potent microbicidal weapon. This dual, prooxidant and antioxidant role of plasma membrane electron transport systems in defense is studied and discussed.     

Solution structures of Mycobacterium tuberculosis thioredoxin C and models of intact thioredoxin system suggest new approaches to inhibitor and drug design

Here, we report the NMR solution structures of Mycobacterium tuberculosis (M. tuberculosis) thioredoxin C in both oxidized and reduced states, with discussion of structural changes that occur in going between redox states. The NMR solution structure of the oxidized TrxC corresponds closely to that of the crystal structure, except in the C‐terminal region. It appears that crystal packing effects have caused an artifactual shift in the α4 helix in the previously reported crystal structure, compared with the solution structure. On the basis of these TrxC structures, chemical shift mapping, a previously reported crystal structure of the M. tuberculosis thioredoxin reductase (not bound to a Trx) and structures for intermediates in the E. coli thioredoxin catalytic cycle, we have modeled the complete M. tuberculosis thioredoxin system for the various steps in the catalytic cycle. These structures and models reveal pockets at the TrxR/TrxC interface in various steps in the catalytic cycle, which can be targeted in the design of uncompetitive inhibitors as potential anti‐mycobacterial agents, or as chemical genetic probes of function. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1^−/−) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca²⁺ cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1^−/− compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca²⁺ transient (Δ[Ca²⁺]_i) responses in NOS1^−/− cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca²⁺]_i across the range of pacing frequencies and increased myofilament Ca²⁺ sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca²⁺ reuptake, and restored SR Ca²⁺ content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca²⁺ leak, HYD improves Ca²⁺ cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.     

Virological Efficacy in Cerebrospinal Fluid and Neurocognitive Status in Patients with Long-Term Monotherapy Based on Lopinavir/Ritonavir: An Exploratory Study

Background

Data on suppression of HIV replication in the CNS and on the subsequent risk of neurocognitive impairment using monotherapy with boosted protease inhibitors are limited.

Methods

Ours was an exploratory cross-sectional study in patients on lopinavir/ritonavir-based monotherapy (LPV/r-MT) or standard triple therapy (LPV/r-ART) for at least 96 weeks who maintained a plasma viral load <50 copies/mL. HIV-1 RNA in CSF was determined by HIV-1 SuperLow assay (lower limit of detection, 1 copy/mL). Neurocognitive functioning was assessed using a recommended battery of neuropsychological tests covering 7 areas. Neurocognitive impairment (NCI) was determined and also a global deficit score (GDS) for study comparisons.

Results

Seventeen patients on LPV/r-MT and 17 on LPV/r-ART were included. Fourteen (82.4%) patients on LPV/r-MT and 16 (94.1%) on LPV/r-ART had HIV-1 RNA <1 copy/mL in CSF (p = 0.601). NCI was observed in 7 patients on LPV/r-MT and in 10 on LPV/r-ART (41% vs 59%; p = 0.494). Mean (SD) GDS was 0.22 (0.20) in patients on LPV/r-MT and 0.47 (0.34) in those on LPV/r-ART (p = 0.012).

Conclusions

Suppression of HIV in CSF is similar in individuals with durable plasma HIV-1 RNA suppression who are receiving LPV/r-MT or LPV/r-ART for at least 96 weeks. Findings for HIV-1 replication in CSF and neurocognitive status indicate that this strategy seems to be safe for CNS functioning.     

Adrenaline stimulates H2O2 generation in liver via NADPH oxidase

It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O₂ into superoxide radical, later transformed into H₂O₂. This enzymatic activity requires previous activation with either 3 mM Mn²⁺ or guanosine 5′-0-(3-thiotriphosphate) (GTPγS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S_0.5 for NADPH was 44 μM; S_0.5 for FAD was 8 μM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPγS plus adrenaline was dose- and Ca²⁺-dependent and proceeded through α₁-adrenergic receptors (AR), whereas β-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H₂O₂ as additional transduction signal for adrenaline response in hepatic cells.     

Novel Series of [ddN]-[TSAO-T] Heterodimers as Potential Bi-Functional Inhibitors of HIV-1 RT. Studies in the Linker and ddN Region

Abstract

Novel series of [ddN]-(CH₂)_n-[TSAO-T] heterodimers have been prepared and tested for their anti-HIV-1 and HIV-2 activity. The most active compound of this series was the [d4T]-(CH₂)₃-[TSAO-T] heterodimer (EC₅₀ = 0.018 ± 0.03 μM).     

The highly thermostable arginine repressor of Bacillus stearothermophilus: gene cloning and repressor–operator interactions

We report here the cloning of the arginine repressor gene argR of Bacillus stearothermophilus and the characterization and purification to homogeneity of its product. The deduced amino acid sequence of the 16.8-kDa ArgR subunit shares 72% identity with its mesophilic homologue AhrC of Bacilus subtilis . Sequence analysis of B. stearothermophilus ArgR and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N-terminal DNA-binding and a C-terminal oligomerization and arginine-binding region. B. stearothermophilus ArgR has been overexpressed in E. coli and purified as a 48.0-kDa trimeric protein. The repressor inhibits the expression of a B. stearothermophilus argC–lacZ fusion in E. coli cells. In the presence of arginine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter. The purified B. stearothermophilus repressor proved to be very thermostable with a half-life of approximately 30 min at 90°C, whereas B. subtilis AhrC was largely inactivated at 65°C. Moreover, ArgR operator complexes were found to be remarkably thermostable and could be formed efficiently at up to 85°C, well above the optimal growth temperature of the moderate thermophile B. stearothermophilus . This pronounced resistance of the repressor–operator complexes to heat treatment suggests that the same type of regulatory mechanism could operate in extreme thermophiles.     

Topological Progression in Proliferating Epithelia Is Driven by a Unique Variation in Polygon Distribution

Morphogenesis is consequence of lots of small coordinated variations that occur during development. In proliferating stages, tissue growth is coupled to changes in shape and organization. A number of studies have analyzed the topological properties of proliferating epithelia using the Drosophila wing disc as a model. These works are based in the existence of a fixed distribution of these epithelial cells according to their number of sides. Cell division, cell rearrangements or a combination of both mechanisms have been proposed to be responsible for this polygonal assembling. Here, we have used different system biology methods to compare images from two close proliferative stages that present high morphological similarity. This approach enables us to search for traces of epithelial organization. First, we show that geometrical and network characteristics of individual cells are mainly dependent on their number of sides. Second, we find a significant divergence between the distribution of polygons in epithelia from mid-third instar larva versus early prepupa. We show that this alteration propagates into changes in epithelial organization. Remarkably, only the variation in polygon distribution driven by morphogenesis leads to progression in epithelial organization. In addition, we identify the relevant features that characterize these rearrangements. Our results reveal signs of epithelial homogenization during the growing phase, before the planar cell polarity pathway leads to the hexagonal packing of the epithelium during pupal stages.