Formation of cholesterol ozonolysis products in vitro and in vivo through a myeloperoxidase-dependent pathway

3β-Hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (secosterol-B) were recently detected in human atherosclerotic tissues and brain specimens, and they may play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. However, as their origin remains unidentified, we examined the formation mechanism, the stability, and the fate of secosterols in vitro and in vivo. About 40% of secosterol-A remained unchanged after 3 h incubation in the FBS-free medium, whereas 20% and 40% were converted to its aldehyde-oxidation product, 3β-hydroxy-5-oxo-secocholestan-6-oic acid, and secosterol-B, respectively. In the presence of FBS, almost all secosterol-A was converted immediately to these compounds. Secosterol-B in the medium, with and without FBS, was relatively stable, but ～30% was converted to its aldehyde-oxidation product, 3β-hydroxy-5β-hydroxy-B-norcholestane-6-oic acid (secoB-COOH). When neutrophil-like differentiated human leukemia HL-60 (nHL-60) cells activated with PMA were cultured in the FBS-free medium containing cholesterol, significantly increased levels of secosterol-A and its aldehyde-oxidation product, but not secosterol-B, were formed. This secosterol-A formation was decreased in the culture of PMA-activated nHL-60 cells containing several reactive oxygen species (ROS) inhibitors and scavengers or in the culture of PMA-activated neutrophils isolated from myeloperoxidase (MPO)-deficient mice. Our results demonstrate that secoterol-A is formed by an ozone-like oxidant generated with PMA-activated neutrophils through the MPO-dependent mechanism.     

Anti-hyperlipidemic sesquiterpenes and new sesquiterpene glycosides from the leaves of artichoke (Cynara scolymus L.): structure requirement and mode of action

The methanolic extract from the leaves of artichoke (Cynara scolymus L.) was found to suppress serum triglyceride elevation in olive oil-loaded mice. Through bioassay-guided separation, sesquiterpenes (cynaropicrin, aguerin B, and grosheimin) were isolated as the active components together with new sesquiterpene glycosides (cynarascolosides A, B, and C). The oxygen functional groups at the 3- and 8-positions and exo-methylene moiety in alpha-methylene-gamma-butyrolactone ring were found to be essential for the anti-hyperlipidemic activity of guaiane-type sesquiterpene. In addition, inhibition of gastric emptying was shown to be partly involved in anti-hyperlipidemic activity.     

Biological and serological comparisons of eel herpesvirus in Formosa (EHVF) and Herpesvirus anguillae (HVA)

A comparative study between EHVF (eel herpesvirus in Formosa) and HVA ( Herpesvirus anguillae ) was performed. Similar syncytia were produced by each virus in cell lines infected with the viruses and similar viral yields were obtained. Significant cross-reactivity was observed in neutralization tests between HVA and EHVF but HVA and EFVF were distinctly different from channel catfish virus (CCV). An electrophoretic analysis of the structural proteins of EHVF and HVA produced similar electrophoretic patterns. Western blotting analysis of the viral proteins, using antiserum against EHVF, supported the results obtained in the cross-neutralization test. In conclusion, the isolates of HVA and EHVF are tightly clustered.     

Mechanism of Deep-Sea Fish α-Actin Pressure Tolerance Investigated by Molecular Dynamics Simulations

The pressure tolerance of monomeric α-actin proteins from the deep-sea fish Coryphaenoides armatus and C. yaquinae was compared to that of non-deep-sea fish C. acrolepis, carp, and rabbit/human/chicken actins using molecular dynamics simulations at 0.1 and 60 MPa. The amino acid sequences of actins are highly conserved across a variety of species. The actins from C. armatus and C. yaquinae have the specific substitutions Q137K/V54A and Q137K/L67P, respectively, relative to C. acrolepis, and are pressure tolerant to depths of at least 6000 m. At high pressure, we observed significant changes in the salt bridge patterns in deep-sea fish actins, and these changes are expected to stabilize ATP binding and subdomain arrangement. Salt bridges between ATP and K137, formed in deep-sea fish actins, are expected to stabilize ATP binding even at high pressure. At high pressure, deep-sea fish actins also formed a greater total number of salt bridges than non-deep-sea fish actins owing to the formation of inter-helix/strand and inter-subdomain salt bridges. Free energy analysis suggests that deep-sea fish actins are stabilized to a greater degree by the conformational energy decrease associated with pressure effect.     

Identification of proteins that bind to the neuroprotective agent neoechinulin A

Neoechinulin A is an indole alkaloid with several biological activities. We previously reported that this compound protects neuronal PC12 cells from cytotoxicity induced by the peroxynitrite generator 3-morpholinosydnonimine (SIN-1), but the target proteins and precise mechanism of action of neoechinulin A were unclear. Here, we employed a phage display screen to identify proteins that bind directly with neoechinulin A. Our findings identified two proteins, chromogranin B and glutaredoxin 3, as candidate target binding partners for the alkaloid. QCM analyses revealed that neoechinulin A displays high affinity for both chromogranin B and glutaredoxin 3. RNA interference-mediated depletion of chromogranin B decreased the sensitivity of PC12 cells against SIN-1. Our results suggested chromogranin B is a plausible target of neoechinulin A.     

LotA,a Legionella deubiquitinase,has dual catalytic activity and contributes to intracellular growth

The intracellular bacterial pathogen, Legionella pneumophila, establishes the replicative niche as a result of the actions of a large array of effector proteins delivered via the Legionella Type 4 secretion system. Many effector proteins are expected to be involved in biogenesis and regulation of the Legionella‐containing vacuole (LCV) that is highly decorated with ubiquitin. Here, we identified a Legionella deubiquitinase, designated LotA, by carrying out a genome analysis to find proteins resembling the eukaryotic ovarian tumour superfamily of cysteine proteases. LotA exhibits a dual ability to cleave ubiquitin chains that is dependent on 2 distinctive catalytic cysteine residues in the eukaryotic ovarian tumour domains. One cysteine dominantly contributes to the removal of ubiquitin from the LCVs by its polyubiquitin cleavage activity. The other specifically cleaves conjugated Lys6‐linked ubiquitin. After delivered by the Type 4 secretion system, LotA localises on the LCVs via its PI(3)P‐binding domain. The lipid‐binding ability of LotA is crucial for ubiquitin removal from the vacuoles. We further analysed the functional interaction of the protein with the recently reported noncanonical ubiquitin ligases of L. pneumophila, revealing that the effector proteins are involved in coordinated regulation that contributes to bacterial growth in the host cells.     

DNA helicase activity in Werner's syndrome gene product synthesized in a baculovirus system.

The gene responsible for Werner's syndrome (WRN) contains a region homologous to the Escherichia coli RecQ type DNA helicase and was thought to code for a DNA helicase belonging to this helicase family. However, no evidence has been shown before to substantiate this prediction. Here, we show data that the product of the WRN gene is indeed a DNA helicase. The gene product, a polypeptide with a relative molecular mass of 170 kDa, expressed in the insect Spodoptera frugiperda (Sf21) cell and purified by affinity column chromatography contained both the ATPase and DNA unwinding activities characteristic of DNA helicase. Expressions in Sf21, as well as in HeLa cells, showed that the WRN DNA helicase is exclusively transported to the nucleoplasm, which is consistent with its function in DNA metabolism. Our studies on strand displacement suggest that WRN helicase can unwind not only a duplex DNA, but also an RNA-DNA heteroduplex, while the latter reaction seems less efficient. Enzymological features learned from the purified WRN helicase are discussed with respect to the biological function, which remains to be clarified.     

Essential Contribution of CD4+ T Cells to Antigen-Induced Nasal Hyperresponsiveness in Experimental Allergic Rhinitis

Nasal hyperresponsiveness (NHR) is a characteristic feature of allergic rhinitis (AR); however, the pathogenesis of NHR is not fully understood. In this study, during the establishment of an experimental AR model using ovalbumin-immunized and -challenged mice, augmentation of the sneezing reaction in response to nonspecific proteins as well as a chemical stimulant was detected. Whether NHR is independent of mast cells and eosinophils was determined by using mast cell- and eosinophil-deficient mice. NHR was suppressed by treatment with anti-CD4 antibody, suggesting the pivotal contribution of CD4⁺ T cells. Furthermore, antigen challenge to mice to which in vitro-differentiated Th1, Th2, and Th17 cells but not naïve CD4⁺ T cells had been adoptively transferred led to the development of equivalent NHR. Since antigen-specific IgE and IgG were not produced in these mice and since antigen-specific IgE-transgenic mice did not develop NHR even upon antigen challenge, humoral immunity would be dispensable for NHR. CD4⁺ T cells play a crucial role in the pathogenesis of AR via induction of NHR, independent of IgE-, mast cell-, and eosinophil-mediated responses.     

Inhibition of the mutagenicity of amino acid pyrolysis products by hemin and other biological pyrrole pigments

Inhibition of the mutagenic activities of the amino acid pyrolysis products by hemin and other biological pyrrole pigments was investigated using the Ames' $\text{Salmonella}$/microsome system. Hemin, biliverdin and chlorophyllin showed inhibition to all the six mutagens tested, and protoporphyrin to three of them. Hemin was the most effective among these pigments; e.g., the mutageniciti of 1.8 nmole Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole) was suppressed to 50 % by 1–3 nmole of hemin. Hemin appears to interact with the metabolically activated form of Trp-P-1 and as a result to inhibit the mutagenicity.