Serum amyloid A (SAA)-induced remodeling of CSF-HDL

Inflammation is a risk factor for Alzheimer's disease. Serum amyloid A (SAA) is an acute phase protein that dissociates apolipoprotein AI (apoAI) from plasma HDL. In cerebrospinal fluid (CSF), the SAA concentration is much higher in subjects with Alzheimer's disease than in controls. CSF-HDL is rich in apoE, which plays an important role as a ligand for lipoprotein receptors in the central nervous system (CNS). To clarify whether SAA dissociates apoE from CSF-HDL, we added recombinant SAA to CSF and determined the apoE distribution in the CSF using native two-dimensional gel electrophoresis. We found that SAA dissociated apoE from CSF-HDL in a dose-dependent manner. This effect was more evident in apoE4 carriers than in apoE3 or apoE2 carriers. After a 24-h incubation at 37 degrees C, SAA continuously dissociated apoE from CSF-HDL. Amyloid beta (Abeta) fragments (1-42) were bound to large CSF-HDL but not to apoE dissociated by SAA. In conclusion, SAA dissociates apoE from CSF-HDL. We postulate that inflammation in the CNS may impair Abeta clearance due to the loss of apoE from CSF-HDL.     

Oxidative stress augments toll-like receptor 8 mediated neutrophilic responses in healthy subjects

Background

Excessive oxidative stress has been reported to be generated in inflamed tissues and contribute to the pathogenesis of inflammatory lung diseases, exacerbations of which induced by viral infections are associated with toll-like receptor (TLR) activation. Among these receptors, TLR8 has been reported as a key receptor that recognizes single-strand RNA virus. However, it remains unknown whether TLR8 signaling is potentiated by oxidative stress. The aim of this study is to examine whether oxidative stress modulates TLR8 signaling in vitro.

Methods

Human peripheral blood neutrophils were obtained from healthy non-smokers and stimulated with TLR 7/8 agonist imidazoquinoline resiquimod (R848) in the presence or absence of hydrogen peroxide (H₂O₂). Neutrophilic responses including cytokine release, superoxide production and chemotaxis were examined, and the signal transduction was also analyzed.

Results

Activation of TLR8, but not TLR7, augmented IL-8 release. The R848-augmented IL-8 release was significantly potentiated by pretreatment with H₂O₂ (p < 0.01), and N-acetyl-L-cysteine reversed this potentiation. The combination of H₂O₂ and R848 significantly potentiated NF-kB phosphorylation and IkBα degradation. The H₂O₂-potentiated IL-8 release was suppressed by MG-132, a proteosome inhibitor, and by dexamethasone. The expressions of TLR8, myeloid differentiation primary response gene 88 (MyD88), and tumor necrosis factor receptor-associated factor 6 (TRAF6) were not affected by H₂O₂.

Conclusion

TLR8-mediated neutrophilic responses were markedly potentiated by oxidative stress, and the potentiation was mediated by enhanced NF-kB activation. These results suggest that oxidative stress might potentiate the neutrophilic inflammation during viral infection.     

Nifedipine prevents vascular endothelial dysfunction in a mouse model of obesity and type 2 diabetes, by improving eNOS dysfunction and dephosphorylation

The effect of calcium channel blockers (CCBs) on type 2 diabetes is still unclear. The present study was undertaken to examine the efficacy of nifedipine, a dihydropyridine CCB, on obesity, glucose intolerance and vascular endothelial dysfunction in db/db mice (a mouse model of obesity and type 2 diabetes). db/db mice, fed high-fat diet (HFD) were treated with vehicle, nifedipine (10 mg kg(-1) day(-1)) or hydralazine (5 mg kg(-1) day(-1)) for 4 weeks, and the protective effects were compared. Although nifedipine and hydralazine exerted similar blood pressure lowering in db/db mice, neither affected body weight, fat weight, and glucose intolerance of db/db mice. However, nifedipine, but not hydralazine, significantly improved vascular endothelial function in db/db mice, being accompanied by more attenuation of vascular superoxide by nifedipine than hydralazine. These protective effects of nifedipine were attributed to the attenuation of eNOS uncoupling as shown by the prevention of vascular endothelial nitric oxide synthase (eNOS) dimer disruption, and the prevention of dihydrofolate reductase (DHFR) downregulation, the key enzyme responsible for eNOS uncoupling. Moreover, nifedipine, but not hydralazine, significantly prevented the decreases in phosphorylation of vascular akt and eNOS in db/db mice. Our work provided the first evidence that nifedipine prevents vascular endothelial dysfunction, through the inhibition of eNOS uncoupling and the enhancement of eNOS phosphorylation, independently of blood pressure-lowering effect. We propose that nifedipine may be a promising therapeutic agent for cardiovascular complications in type 2 diabetes.     

RANKL-induced TRPV2 expression regulates osteoclastogenesis via calcium oscillations

The receptor activator of NFκB ligand (RANKL) induces Ca(2+) oscillations and activates the Nuclear Factor of Activated T cells 1 (NFATc1) during osteoclast differentiation (osteoclastogenesis). Ca(2+) oscillations are an important trigger signal for osteoclastogenesis, however the molecular basis of Ca(2+) permeable influx pathways serving Ca(2+) oscillations has not yet been identified. Using a DNA microarray, we found that Transient Receptor Potential Vanilloid channels 2 (TRPV2) are expressed significantly in RANKL-treated RAW264.7 cells (preosteoclasts) compared to untreated cells. Therefore, we further investigated the expression and functional role of TRPV2 on Ca(2+) oscillations and osteoclastogenesis. We found that RANKL dominantly up-regulates TRPV2 expression in preosteoclasts, and evokes spontaneous Ca(2+) oscillations and a transient inward cation current in a time-dependent manner. TRPV inhibitor ruthenium red and tetracycline-induced TRPV2 silencing significantly decreased both the frequency of Ca(2+) oscillations and the transient inward currents in RANKL-treated preosteoclasts. Silencing of store-operated Ca(2+) entry (SOCE) proteins similarly suppressed both RANKL-induced oscillations and currents in preosteoclasts. Furthermore, suppression of TRPV2 also reduced RANKL-induced NAFTc1 expression, its nuclear translocation, and osteoclastogenesis. In summary, Ca(2+) oscillations in preosteoclasts are triggered by RANKL-dependent TRPV2 and SOCE activation and intracellular Ca(2+) release. Subsequent activation of NFATc1 promotes osteoclastogenesis.     

Circularized chromosome with a large palindromic structure in Streptomyces griseus mutants

Streptomyces linear chromosomes display various types of rearrangements after telomere deletion, including circularization, arm replacement, and amplification. We analyzed the new chromosomal deletion mutants Streptomyces griseus 301-22-L and 301-22-M. In these mutants, chromosomal arm replacement resulted in long terminal inverted repeats (TIRs) at both ends; different sizes were deleted again and recombined inside the TIRs, resulting in a circular chromosome with an extremely large palindrome. Short palindromic sequences were found in parent strain 2247, and these sequences might have played a role in the formation of this unique structure. Dynamic structural changes of Streptomyces linear chromosomes shown by this and previous studies revealed extraordinary strategies of members of this genus to keep a functional chromosome, even if it is linear or circular.     

Down-regulation of beta2-adrenergic receptor expression by exercise training increases IL-12 production by macrophages following LPS stimulation

Three-week exercise training decreased the steady state level of beta(2)-adrenergic receptor (beta(2)AR) mRNA in peritoneal macrophages from BALB/c mice. When peritoneal macrophages from both exercise-trained and sedentary control mice were stimulated with lipopolysaccharide (LPS), interleukin (IL)-12 mRNA and protein expression was markedly higher in trained mice than in control mice. To determine whether enhanced production of IL-12 was associated with decreased expression of beta(2)AR, we transfected the macrophage cell line, RAW264, with a eukaryotic expression vector containing beta(2)ar cDNA, establishing a cell line overexpressing beta(2)AR (RAWar). Following LPS stimulation, IL-12 mRNA and protein expression was significantly lower in RAWar cells than in RAW264 cells transfected with vector alone (RAWvec). Furthermore, when the expression of transfected beta(2)AR in RAWar cells was down-regulated by a tetracycline repressor-regulated mammalian expression system, expression of IL-12 mRNA and protein following LPS stimulation tended to return to the levels in RAWvec cells. These findings indicate that macrophage production of IL-12 following LPS stimulation is regulated by the expression level of beta(2)AR, suggesting that the down-regulation of beta(2)AR expression associated with exercise training improves IL-12-induced type 1 helper T cell-mediated immune responses.     

Asymmetric chloronicotinyl insecticide, 1-[1-(6-chloro-3-pyridyl)ethyl]-2-nitroiminoimidazolidine: preparation,resolution and biological activities toward insects and their nerve preparations

The asymmetric chloronicotinyl insecticide, 1-[1-(6-chloro-3-pyridyl)ethyl]-2-nitroiminoimidazolidine, was prepared, and the absolute configurations of the enantiomers were determined by an X-ray analysis. The insecticidal activity against the housefly measured with metabolic inhibitors showed the (S) enantiomer to be slightly more active than the (R) isomer. Electrophysiological measurements on the American cockroach central nerve cord showed the compounds to elicite the impulses and subsequently blocked them. The neuroblocking potency of the (S) isomer was 5.9 microM, while that of the (R) isomer was as high as 73 microM. The molar concentrations required for 50% inhibition of the specific binding of [3H]imidacloprid to the housefly head membrane preparation were respectively 0.19 microM and 0.95 microM for the (S) and (R) isomers. This enatioselectivity ratio was smaller than 35 for nicotine isomers but greater than 2 for epibatidine isomers.