Irreversibly glycated LDL induce oxidative and inflammatory state in human endothelial cells; added effect of high glucose

In diabetes, hyperglycemia and the associated formation of advanced glycation end-products (AGE) and AGE-modified low density lipoproteins (AGE-LDL) can directly affect the cells of the vascular wall. We hypothesize that AGE-LDL may act directly and induce oxidant and inflammatory alterations in human endothelial cells (HEC), this effect being amplified by high glucose. To test this assumption, the activity of NADPH oxidase (NADPHox) was evaluated and the expression of its subunits (p22^phox, NOX4, and p67^phox), of the AGE receptor (RAGE), and of the monocyte chemoattractant protein-1 (MCP-1) were assessed by real-time PCR and Western blot in confluent EA.hy926 cells incubated with AGE-LDL for 24 and 48 h, in normal and high glucose conditions. Exposure of HEC for 48 h to AGE-LDL in 5 mM glucose induced an increase of RAGE expression (50%), NADPHox activity (107%), p22^phox and NOX4 mRNA (50% and 188%, respectively) and MCP-1 expression (80%). AGE-LDL-stimulated p22^phox expression by activating p38 MAP kinase and NF-kB, and MCP-1 expression by activating NF-kB, as demonstrated by the use of specific inhibitors (SB203580 and Bay11-7085). The addition of 25 mM glucose in the culture medium enhanced the effect of AGE-LDL, but also of nLDL, on RAGE, p22^phox, NOX4, p67^phox, and MCP-1 gene expression. In conclusion, AGE-LDL induce an oxidative stress and a pro-inflammatory state in human endothelial cells. Both AGE-LDL and nLDL in the presence of high glucose amplify their effect, revealing a link between hyperlipidemia, diabetes, and endothelial cell dysfunction.     

Antibiotics LL-Z1272 identified as novel inhibitors discriminating bacterial and mitochondrial quinol oxidases

To counter antibiotic-resistant bacteria, we screened the Kitasato Institute for Life Sciences Chemical Library with bacterial quinol oxidase, which does not exist in the mitochondrial respiratory chain. We identified five prenylphenols, LL-Z1272β, γ, δ, ? and ζ, as new inhibitors for the Escherichia coli cytochrome bd. We found that these compounds also inhibited the E. coli bo-type ubiquinol oxidase and trypanosome alternative oxidase, although these three oxidases are structurally unrelated. LL-Z1272β and ? (dechlorinated derivatives) were more active against cytochrome bd while LL-Z1272γ, δ, and ζ (chlorinated derivatives) were potent inhibitors of cytochrome bo and trypanosome alternative oxidase. Thus prenylphenols are useful for the selective inhibition of quinol oxidases and for understanding the molecular mechanisms of respiratory quinol oxidases as a probe for the quinol oxidation site. Since quinol oxidases are absent from mammalian mitochondria, LL-Z1272β and δ, which are less toxic to human cells, could be used as lead compounds for development of novel chemotherapeutic agents against pathogenic bacteria and African trypanosomiasis.     

Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast

The Sonic Hedgehog (Shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. Inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors Patched (Ptc) and Smoothened (Smo), which lead to birth defects, cancer or neurodegenerative diseases. However, little is known about Ptc, the receptor of the Shh protein, and the way Ptc regulates Smo, the receptor responsible for the transduction of the signal. To develop structure-function studies of these receptors, we expressed human Ptc (hPtc) in the yeast Saccharomyces cerevisiae. We demonstrated that hPtc expressed in a yeast membrane fraction is able to interact with its purified ligand Shh, indicating that hPtc is produced in yeast in its native conformational state. Using Surface Plasmon Resonance technology, we showed that fluorinated surfactants preserve the ability of hPtc to interact with its ligand after purification. This is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor Ptc. This work will allow the scale-up of hPtc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by Shh signalling dysfunction.     

Relationship between calcium release and NADPH oxidase inhibition in human neutrophils

The aim of this study was to investigate the possible relationship between NADPH oxidase activity and changes in cytosolic Ca²⁺ in response to different agonists. Treatment of neutrophils with leukotriene B4 (LTB₄) demonstrated characteristic changes to cytoslic Ca²⁺ yielding an EC₅₀ of 4 nM. The pA₂ values for the specific LTB₄ receptor (BLT) antagonists, U-75302 and LY-255283 were 6.32 and 6.38, respectively. Similarly, neutrophils treated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and platelet activating factor (PAF) exhibited changes in cytoslic Ca²⁺ in a dose dependant manner with pD₂ values of 9.0 and 9.9, respectively. The phorbol ester PMA prevented elevations in cytosolic Ca²⁺ in response to LTB₄, FMLP and PAF with IC₅₀ values of 5.88, 1.44 and 5.71 nM, respectively. In addition, potent NADPH oxidase inhibitors apocynin and diphenyleneiodonium (DPI) inhibited FMLP mediated cytosolic Ca²⁺ release. These results demonstrate that inhibition of the NADPH oxidase suppresses cytosolic Ca²⁺ release in FMLP activated human neutrophils.     

Unwinding by Local Strand Separation Is Critical for the Function of DEAD-Box Proteins as RNA Chaperones

The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and to activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements mitochondrial translation and group I and group II intron splicing defects in mss116Δ strains, stimulates the in vitro splicing of group I and group II introns, and functions indistinguishably from CYT-19 to resolve different nonnative secondary and/or tertiary structures in the Tetrahymena thermophila large subunit rRNA-ΔP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and group II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and the vaccinia virus DExH-box protein NPH-II gave little, if any, group I or group II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and group II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. In contrast, DbpA binds group I and group II intron RNAs nonspecifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and group II intron splicing correlates primarily with their RNA-unwinding activity, which, for the protein preparations used here, was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Furthermore, this correlation holds for all group I and group II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their distinctive RNA-unwinding mechanism, which enables refolding of localized RNA regions or structures without globally disrupting RNA structure.     

Different phylogeographic patterns in two Japanese Silpha species (Coleoptera: Silphidae) affected by climatic gradients and topography

To reveal differences in phylogeographic patterns of flightless insect species occurring in different regions of Japan, we studied the phylogeography and demographic history of Silpha beetles occurring in cool-temperate habitats of two major islands, Honshu and Hokkaido, using sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. Honshu has a more mountainous topography, and cool-temperate habitats occur discontinuously, whereas Hokkaido, located to the north of Honshu, has more continuous cool-temperate habitats. A species endemic to Honshu, S. longicornis occurs on Honshu, whereas S. perforata occurs on Hokkaido and the East Asian continent. Our results indicate that the ancestors of S. longicornis colonized Honshu via a south-west route c . 0.7 Mya and the species has highly divergent populations in isolated mountainous areas of Honshu, whereas S. perforata colonized Hokkaido via a northern route less than 90 000 years ago and has less divergent geographic populations. During the last glacial period, S. perforata was probably restricted to refugia in southern Hokkaido and later expanded into northern Hokkaido, whereas S. longicornis populations existed in many isolated refugia, probably because of the complex topography of Honshu. Thus, our study demonstrates that, even between closely related species, interactions among biology, latitudinal climatic gradients and topography can produce different phylogeographic patterns.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 452–467.     

Absence of TGFβ signaling in embryonic vascular smooth muscle leads to reduced lysyl oxidase expression,impaired elastogenesis,and aneurysm

To address the requirement for TGFβ signaling in the formation and maintenance of the vascular matrix, we employed lineage‐specific mutation of the type II TGFβ receptor gene (Tgfbr2) in vascular smooth muscle precursors in mice. In both neural crest‐ and mesoderm‐derived smooth muscle, absence of TGFβ receptor function resulted in a poorly organized vascular elastic matrix in late‐stage embryos which was prone to dilation and aneurysm. This defect represents a failure to initiate formation of the elastic matrix, rather than a failure to maintain a preexisting matrix. In mutant tissue, lysyl oxidase expression was substantially reduced, which may contribute to the observed pathology. genesis 47:115–121, 2009. © 2009 Wiley‐Liss, Inc.     

Emergence and transmission of visual awareness through optical coding in the brain: A redox molecular hypothesis on visual mental imagery

Does the primary visual cortex mediate consciousness for higher-level stages of information processing by providing an outlet for mental imagery? Evidence based on neural electrical activity is inconclusive as reflected in the “imagery debate” in cognitive science. Neural information and activity, however, also depend on regulated biophoton (optical) signaling. During encoding and retrieval of visual information, regulated electrical (redox) signals of neurons are converted into synchronized biophoton signals by bioluminescent radical processes. That is, visual information may be represented by regulated biophotons of mitochondrial networks in retinotopically organized cytochrome oxidase-rich neural networks within early visual areas. Therefore, we hypothesize that regulated biophotons can generate intrinsic optical representations in the primary visual cortex and then propagate variably degraded versions along cytochrome oxidase pathways during both perception and imagery. Testing this hypothesis requires to establish a methodology for measurement of in vivo and/or in vitro increases of biophoton emission in humans' brain during phosphene inductions by transcranial magnetic stimulation and to compare the decrease in phosphene thresholds during transcranial magnetic stimulation and imagery. Our hypothesis provides a molecular mechanism for the visual buffer and for imagery as the prevalent communication mode (through optical signaling) within the brain. If confirmed empirically, this hypothesis could resolve the imagery debate and the underlying issue of continuity between perception and abstract thought.     

Brain monoamine oxidase A in hyperammonemia is regulated by NMDA receptors

Mitochondrial enzyme monoamine oxidase A (MAO-A) generates hydrogen peroxide (H₂O₂) and is up-regulated by Ca²⁺ and presumably by ammonia. We hypothesized that MAO-A may be under the control of NMDA receptors in hyperammonemia. In this work, the in vivo effects of single dosing with ammonia and NMDA receptor antagonist MK-801 and the in vitro effect of Ca²⁺ on MAO-A activity in isolated rat brain mitochondria were studied employing enzymatic procedure. Intraperitoneal injection of rats with ammonia led to an increase in MAO-A activity in mitochondria indicating excessive H₂O₂ generation. Calcium added to isolated mitochondria stimulated MAO-A activity by as much as 84%. MK-801 prevented the in vivo effect of ammonia, implying that MAO-A activation in hyperammonemia is mediated by NMDA receptors. These data support the conclusion that brain mitochondrial MAO-A is regulated by the function of NMDA receptors. The enzyme can contribute to the oxidative stress associated with hyperammonemic conditions such as encephalopathy and Alzheimer’s disease. The attenuation of the oxidative stress highlights MAO-A inactivation and NMDA receptor antagonists as sources of novel avenues in the treatment of mental disorders.