Lysophospholipids and ATP mutually suppress maturation and release of IL-1 beta in mouse microglial cells using a Rho-dependent pathway

The P2X7 receptor (P2X7R), an ATP-gated ion channel, plays essential roles in the release and maturation of IL-1beta in microglial cells in the brain. Previously, we found that lysophosphatidylcholine (LPC) potentiated P2X7R-mediated intracellular signals in microglial cells. In this study, we determined whether the lysophospholipids, i.e., LPC and sphingosylphosphorylcholine (SPC), modulate the ATP-induced release and processing of IL-1beta mediated by P2X7R in mouse MG6 microglial cells. LPC or SPC alone induced the release of precursor (pro-IL-1beta) and mature IL-1beta (mIL-1beta) from LPS-primed MG6 cells, possibly due to lytic functions. However, these lysophospholipids inhibited ATP-induced caspase-1 activation that is usually followed by the release of mIL-1beta. Conversely, ATP inhibited the release of pro-IL-1beta and mIL-1beta induced by LPC/SPC. This suggests that lysophospholipids and ATP mutually suppressed each function to release IL-1beta. P2X7R activation resulted in microtubule reorganization in the MG6 cells that was blocked in the presence of LPC and SPC. LPC/SPC reduced the amount of activated RhoA after stimulation with ATP, implying that these lysophospholipids block ATP-induced microtubule reorganization by interfering with RhoA activation. In addition, the microtubule inhibitor colchicine inhibited ATP-induced release of mIL-1beta similar to that of LPC and SPC. This suggests that the impairment of the microtubule reassembly may be associated with the inhibitory effects of LPC/SPC on ATP-induced mIL-1beta release. Mutual suppression by ATP and LPC/SPC on the maturation of IL-1beta was observed in LPS-primed primary microglia. Collectively, these data suggest opposing functions by lysophospholipids, either proinflammatory or anti-inflammatory, in regard to the maturation and release of IL-1beta from microglial cells.     

Restriction on conjugational transfer of pLS20 in Bacillus subtilis 168

Conjugational transfer of pLS20 in Bacillus subtilis Marburg 168 is restricted by the BsuM restriction-modification system. Restriction efficiency was measured using pLS20 derivatives possessing various numbers of XhoI sites, which are known to be recognized by BsuM. An increase in XhoI sites clearly reduced the conjugational efficiency of pLS20 as compared with that of pUB110 plasmid lacking XhoI.     

Characterization of fibrillation process of alpha-synuclein at the initial stage

α-Synuclein is the major component of the filamentous Lewy bodies and Lewy-related neurites, neuropathological hallmarks of Parkinson’s disease. Although numerous studies on α-synuclein fibrillation have been reported, the molecular mechanisms of aggregation and fibrillation at the initial stage are still unclear. In the present study, structural properties and propensities to form fibrils of α-synuclein at the initial stage were investigated using 2D ¹H-¹⁵N NMR spectroscopy, electron microscope, and small angle X-ray scattering (SAXS). Observation of the 2D ¹H-¹⁵N HSQC spectra indicated significant attenuation of many cross peak intensities in the regions of KTKEGV-type repeats and the non-Aβ component of Alzheimer’s disease amyloid (NAC), suggesting that these regions contributed fibril formation. Oligomerization comprising heptamer was successfully monitored at the initial stage using the time-dependent SAXS measurements.     

Development of amphiphilic gamma-PGA-nanoparticle based tumor vaccine: potential of the nanoparticulate cytosolic protein delivery carrier

Nanoscopic therapeutic systems that incorporate biomacromolecules, such as protein and peptides, are emerging as the next generation of nanomedicine aimed at improving the therapeutic efficacy of biomacromolecular drugs. In this study, we report that poly(γ-glutamic acid)-based nanoparticles (γ-PGA NPs) are excellent protein delivery carriers for tumor vaccines that delivered antigenic proteins to antigen-presenting cells and elicited potent immune responses. Importantly, γ-PGA NPs efficiently delivered entrapped antigenic proteins through cytosolic translocation from the endosomes, which is a key process of γ-PGA NP-mediated anti-tumor immune responses. Our findings suggest that the γ-PGA NP system is suitable for the intracellular delivery of protein-based drugs as well as tumor vaccines.     

NFBD1/MDC1 stabilizes oncogenic MDM2 to contribute to cell fate determination in response to DNA damage

In response to DNA damage, NFBD1/MDC1 induces the accumulation of DNA repair machinery such as MRN complex at the sites of damaged DNA to form nuclear foci. In this study, we found that NFBD1 directly interacts with MDM2 and increases its stability. During adriamycin (ADR)-mediated apoptosis, expression levels of NFBD1 reduced in association with the down-regulation of MDM2. Enforced expression of NFBD1 resulted in a significant stabilization of MDM2. Consistent with these observations, siRNA-mediated knockdown of the endogenous NFBD1 decreased the amounts of the endogenous MDM2. Immunoprecipitation and in vitro pull-down assays demonstrated that NFBD1 interacts with MDM2 through its COOH-terminal BRCT domains. In accordance with our recent results, enforced expression of NFBD1 rendered cells resistant to DNA damage. Similar results were also obtained in cells expressing exogenous MDM2. Taken together, our present findings suggest that NFBD1-mediated stabilization contributes to cell survival in response to DNA damage.     

An abnormal pK(a) value of internal histidine of the insulin molecule revealed by neutron crystallographic analysis

Insulin is stored in pancreatic β-cell as hexameric form with Zn²⁺ ions, while the hormonally active form is monomer. The hexamer requires the coordination of Zn²⁺ ions to the HisB10. In order to reveal the mechanism of the hexamerization of insulin, we investigated the Zn²⁺ free insulin at pD6.6 and pD9 by neutron crystallographic analyses. HisB10 is doubly protonated not only at pD6.6 but also at pD9, indicating an abnormal pK_a of this histidine. It is suggested that HisB10 acts on a strong cation capture and contributes to the high stability of the hexameric form in pancreas.     

Functional analysis of phenolsulfonphthalein transport system in Long-Evans Cinnamon rats

It has been reported that the transport function for organic anions on the kidney is maintained in multidrug resistance-associated protein 2 (Mrp2)-deficient rats. Different from Mrp2-deficient rats, Long-Evans Cinnamon (LEC) rats have impaired urinary excretion of Mrp2-substrate, phenolsulfonphthalein (PSP). PSP is transported by the potential-sensitive urate transport system in rat brush-border membranes. We analyzed the function of PSP transport system in LEC rats. Unlike Long-Evans Agouti (LEA) rats, the initial uptake of PSP and urate into the renal brush-border membrane vesicles of LEC rats were not significantly enhanced in the presence of positive intravesicular potential, suggesting that the potential-sensitive urate transport system is impaired in LEC rats. LEC rats should be useful for elucidating the potential-sensitive urate transport system in rats at the molecular level.     

PACAP provides colonic protection against dextran sodium sulfate induced colitis

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a crucial role in immunity and inflammation. Our aim was to obtain insight in the role of PACAP in experimental colitis in mice and thus its possible role in inflammatory bowel disease. PACAP-deficient (PACAP-/-) mice and wild-type control mice were challenged by colitis-inducing agent, dextran sulfate sodium (DSS). We monitored clinical symptoms, intestinal morphology, and difference of cytokine production in the proximal and distal colon. After DSS administration, mortality was more severe in PACAP-/- mice versus wild-type control mice. The histological score and the disease activity index of PACAP-/- mice were significantly higher than those of wild-type control mice. In proximal colon, production of IL-1beta and IL-6 in PACAP-/- mice were significantly upregulated on day 8 after DSS administration, compared to wild-type control mice. In distal colon, furthermore, production of IFNgamma, IL-1beta, IL-6, IL-12, and KC were significantly higher in PACAP-/- mice than in wild-type control mice on day 4. Our findings indicate that PACAP regulates the production of pro-inflammatory cytokine in the experimental colitis.     

Secreted acid phosphatase is expressed in cluster roots of lupin in response to phosphorus deficiency

The roots of white lupin (Lupinus albus L. cv. Kievskij mutant) secrete acid phosphatase, S-APase, when they grow under conditions of low available phosphorus (P). S-APases hydrolyze organic phosphate compounds in the rhizosphere and supply inorganic phosphate to the plants. Low phosphorus availability also induces vigorous growth of cluster roots. In this study, the function of cluster roots was investigated with reference to S-APase secretion. White lupins were grown in hydroponic culture in a greenhouse under P-deficient and P-sufficient conditions. S-APase in the excised roots after treatment was detected by staining with 4-methylumbelliferone phosphate (MUP). Gene expression of S-APase in cluster and normal roots was also investigated. Activity was greatest in the roots of plants grown under conditions of P -deficiency, particularly in cluster roots. S-APase gene expression was induced by a decrease in internal P concentrations, and was especially high in cluster roots formed under conditions of P -deficiency. It was suggested that decrease of internal P concentration stimulated both of the S-APase expression and cluster root formation.     

Accuracy of genotyping for single nucleotide polymorphisms by a microarray-based single nucleotide polymorphism typing method involving hybridization of short allele-specific oligonucleotides.

Advances in technologies for identifying genetic polymorphisms rapidly and accurately will dramatically accelerate the discovery of disease-related genes. Among a variety of newly described methods for rapid typing of single-nucleotide polymorphisms (SNPs), gene detection using DNA microarrays is gradually achieving widespread use. This method involves the use of short (11- to 13-mer) allele-specific oligonucleotides. This method allows simultaneous analysis of many SNPs in DNAs from a large number of individuals, in a single experiment. In this work, we evaluated the accuracy of a new microarray-based short allele-specific oligonucleotide (ASO) hybridization method. There is a 96-well formatted array on a single plate, in which up to 256 spots are included in each well. Fluorescent probes for our experiments were produced by multiplex PCR amplification often target SNP-containing regions. We genotyped 192 individuals across a panel of ten single base variations, which included an insertion/deletion polymorphism. For comparison, we genotyped the same individuals for the same SNPs by the method of single-base extension with fluorescence detection. The typing accuracies of the microarray-based PCR-ASO and single-base extension methods were calculated as 99.9% and 99.1%, respectively, on the basis of genotyping results determined by direct sequencing. We conclude that the microarray-based hybridization method using short ASO probes represents a potential breakthrough technology for typing large numbers of SNPs rapidly and efficiently.