Pitavastatin prevents NMDA-induced retinal ganglion cell death by suppressing leukocyte recruitment

Excitotoxicity is a major cause of retinal ganglion cell (RGC) death during ischemic diseases such as vessel occlusion and diabetic retinopathy. However, the underlying mechanisms are not well understood. Statins, inhibitors of the HMG-CoA reductase, have neuroprotective effects in addition to their original role in lowering cholesterol. We hypothesize that pitavastatin, a recently introduced potent statin, is protective against N-methyl-d-aspartic acid (NMDA)-induced RGC death. Pitavastatin, administered by gavage, abolished NMDA-induced loss of RGCs. To elucidate the mechanisms underlying the neuroprotective effect of pitavastatin, we investigated its impact on inflammation. NMDA increased the expression of interleukin-1beta and TNF-alpha, and endothelial adhesion molecules, including ICAM-1, and induced leukocyte accumulation in the retinal vessels. Pitavastatin significantly reduced NMDA-induced leukocyte accumulation and up-regulation of endothelial adhesion molecules, whereas cytokine expression was unaffected. Systemic blockade of ICAM-1 in wild-type mice or absence of CD18 in gene-deficient (CD18(-/-)) mice significantly suppressed NMDA-induced leukocyte accumulation and RGC death. These findings suggest a novel and causative role for inflammatory leukocyte recruitment in NMDA-induced excitotoxicity. Furthermore, we show the novel neuroprotective effect of statins against excitotoxicity-induced RGC death. Statins or other anti-inflammatory agents may thus have therapeutic benefits in excitotoxicity-associated neuronal diseases through blockade of leukocyte recruitment.     

La autoantigen translocates to cytoplasm after cleavage during granzyme B-mediated cytotoxicity

Our recent report demonstrated that apoptosis-specific autoantibodies against granzyme B-induced cleavage fragments of SS-B (La) were found in the sera from patients with primary Sj?gren's syndrome. The objective of this study was identified by the intracellular redistribution of La autoantigen during granzyme B-induced apoptosis. We developed green fluorescence protein (GFP)-La and GFP-LaDelta220 (generation of granzyme B-specific cleavage of La protein) fusion proteins. GFP-La protein was localized in the nucleus, whereas the GFP-LaDelta220 protein predominantly existed in the cytoplasm in transformed A293T cells. Nuclear GFP-La protein was translocated to cytoplasm after granzyme B enriched YT cells incubation. La protein in human salivary grand HSG cells is cleaved and translocated from the nucleus to the cytoplasm after YT cell co-cultivation. These results suggest that La protein is cleaved by granzyme B and N-terminal La fragment (27 kD) translocated to the cytoplasm, thus leading to a novel autoantibody production during granzyme B-mediated cytotoxicity.     

Association of Dishevelled with the clathrin AP-2 adaptor is required for Frizzled endocytosis and planar cell polarity signaling

Upon activation by Wnt, the Frizzled receptor is internalized in a process that requires the recruitment of Dishevelled. We describe a novel interaction between Dishevelled2 (Dvl2) and micro2-adaptin, a subunit of the clathrin adaptor AP-2; this interaction is required to engage activated Frizzled4 with the endocytic machinery and for its internalization. The interaction of Dvl2 with AP-2 requires simultaneous association of the DEP domain and a peptide YHEL motif within Dvl2 with the C terminus of micro2. Dvl2 mutants in the YHEL motif fail to associate with micro2 and AP-2, and prevent Frizzled4 internalization. Corresponding Xenopus Dishevelled mutants show compromised ability to interfere with gastrulation mediated by the planar cell polarity (PCP) pathway. Conversely, a Dvl2 mutant in its DEP domain impaired in PCP signaling exhibits defective AP-2 interaction and prevents the internalization of Frizzled4. We suggest that the direct interaction of Dvl2 with AP-2 is important for Frizzled internalization and Frizzled/PCP signaling.     

On-chip identification and interaction analysis of gel-resolved proteins using a diamond-like carbon-coated plate

We developed a novel protein chip made of a diamond-like, carbon-coated stainless steel plate (DLC plate), the surface of which is chemically modified with N-hydroxysuccinimide ester. To produce a high-density protein chip using the DLC plate, proteins separated by SDS gel electrophoresis or two-dimensional electrophoresis were electroblotted onto the DLC plate and immobilized covalently. A high blotting efficiency (25-70%) for transferring proteins from the gels onto the DLC plates was achieved by improvement of the electrophoresis device and electroblotting techniques. With the use of the DLC plate, we developed novel techniques to identify proteins immobilized on the chip and to detect protein-protein interactions on the chip by mass spectrometric analysis. We also developed a technique to identify post-translationally modified proteins, such as glycoproteins, on the protein chip.     

Effects of different nitrogen rich substrates and their combination to the yield performance of oyster mushroom (<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>)

This study was conducted to assess the effect of nitrogen (N) variations from lignocellulosic substrates of empty fruit bunch (EFB), palm oil frond (POF), palm oil trunks (POT), A.mangium bark (AB) and Cogongrass (IC), and combinations among them using biological supplements (POF and POT) for suitability as a substrate of P. ostreatus. Three different sets of substrates were evaluated in response to different parameters of P. ostreatus including spawn running, primordial initiation, fruit body yield and biological efficiency (BE). The supplementation of POT and POF generally revealed significant differences on the crop performance of P. ostreatus compared to the un-supplemented substrate. POT demonstrated reduced production time, while POF was the best supplement to promote fresh weight and BE. The most rapid spawn running took place within 18.6 ± 0.9 days, while the primordial initiation was observed within 23.2 ± 0.5 days in the combination of IC + POT. The most suitable substrate for higher mushroom weight and BE was IC + POF (19.6 ± 1.3 g; 70.5 ± 5.5%). In addition, the interval between primordial formations was found to be fastest in the un-supplemented EFB and the combination of EFB + POF, which supported a shortened production time. Therefore, IC and EFB appeared to be the better alternative materials for growing P. ostreatus, either with or without any supplementation.     

Plasma L-cystine/L-glutamate imbalance increases tumor necrosis factor-alpha from CD14+ circulating monocytes in patients with advanced cirrhosis

Background and Aims

The innate immune cells can not normally respond to the pathogen in patients with decompensated cirrhosis. Previous studies reported that antigen-presenting cells take up L-Cystine (L-Cys) and secrete substantial amounts of L-Glutamate (L-Glu) via the transport system Xc- (4F2hc+xCT), and that this exchange influences the immune responses. The aim of this study is to investigate the influence of the plasma L-Cys/L-Glu imbalance observed in patients with advanced cirrhosis on the function of circulating monocytes.

Methods

We used a serum-free culture medium consistent with the average concentrations of plasma amino acids from patients with advanced cirrhosis (ACM), and examined the function of CD14+ monocytes or THP-1 under ACM that contained 0–300 nmol/mL L-Cys with LPS. In patients with advanced cirrhosis, we actually determined the TNF-alpha and xCT mRNA of monocytes, and evaluated the correlation between the plasma L-Cys/L-Glu ratio and TNF-alpha.

Results

The addition of L-Cys significantly increased the production of TNF alpha from monocytes under ACM. Monocytes with LPS and THP-1 expressed xCT and a high level of extracellular L-Cys enhanced L-Cys/L-Glu antiport, and the intracellular GSH/GSSG ratio was decreased. The L-Cys transport was inhibited by excess L-Glu. In patients with advanced cirrhosis (n = 19), the TNF-alpha and xCT mRNA of monocytes were increased according to the Child-Pugh grade. The TNF-alpha mRNA of monocytes was significantly higher in the high L-Cys/L-Glu ratio group than in the low ratio group, and the plasma TNF-alpha was significantly correlated with the L-Cys/L-Glu ratio.

Conclusions

A plasma L-Cys/L-Glu imbalance, which appears in patients with advanced cirrhosis, increased the TNF-alpha from circulating monocytes via increasing the intracellular oxidative stress. These results may reflect the immune abnormality that appears in patients with decompensated cirrhosis.     

Identification of putative metabolites of docosahexaenoic acid as potent PPARgamma agonists and antidiabetic agents

We found that putative metabolites of docosahexaenoic acid (DHA) are strong PPARgamma activators and potential antidiabetic agents. We designed DHA derivatives based on the crystal structure of PPARgamma, synthesized them and evaluated their activities in vitro and in vivo. The efficacy of 5E-4-hydroxy-DHA 2a as a PPARgamma activator was about fourfold stronger than that of pioglitazone. Furthermore, the 4-keto derivative (10b) showed antidiabetic activity in animal models without producing undesirable effects such as obesity and hepatotoxicity.     

Regulation of apoptosis by glutathione redox state in PC12 cells exposed simultaneously to iron and ascorbic acid

We previously reported that the levels of non-protein-bound iron (NPBI) and ascorbic acid (AA) are markedly increased in the cerebrospinal fluid of infants with perinatal asphyxia. The present study showed that FeSO4 and AA synergistically induced apoptosis of PC12 cells, which was prevented by alpha-tocopherol and glutathione (GSH) ethyl ester. Markers of free radical damage, such as ortho-tyrosine, meta-tyrosine, and F(2alpha)-isoprostane, showed a gradual increase. AA and ferrous NPBI disappeared rapidly from the culture medium, but exposure for only a few hours was sufficient to trigger apoptosis. Intracellular GSH decreased progressively along with a concomitant increase of glutathione disulfide (GSSG). The baseline half-cell reduction potential (Ehc) for GSSG, 2H+/2GSH couple was -246 mV and an Ehc of -200 mV was the critical level to switch on apoptosis, although some cells escaped this fate by transient increase of intracellular GSH. Once Ehc reached around -165 mV (81 mV oxidation from the baseline), all cells lost the ability to maintain an adequate intracellular GSH level and subsequently underwent apoptosis. These findings at least partly explain the mechanism of Fe-AA cytotoxicity, in that ferrous iron catalyzes hydroxyl radical generation and induces lipid peroxidation, after which subsequent depletion of GSH raises Ehc to the critical level for triggering or potentiating the apoptotic cascade.     

SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor

Sclerosteosis is an autosomal recessive disease that is characterized by overgrowth of bone tissue and is linked to mutations in the gene encoding the secreted protein SOST. Sclerosteosis shares remarkable similarities with "high bone mass" diseases caused by "gain-of-function" mutations in the LRP5 gene, which encodes a coreceptor for Wnt signaling proteins. We show here that SOST antagonizes Wnt signaling in Xenopus embryos and mammalian cells by binding to the extracellular domain of the Wnt coreceptors LRP5 and LRP6 and disrupting Wnt-induced Frizzled-LRP complex formation. Our findings suggest that SOST is an antagonist for Wnt signaling and that the loss of SOST function likely leads to the hyperactivation of Wnt signaling that underlies bone overgrowth seen in sclerosteosis patients.