Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain

Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using 1?N-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme-product relationships and analysis of the P450 measurements based on different signature peptides revealed a possibility of retina-specific post-translational modification of CYP27A1. The data obtained provide important insights into the mechanisms of cholesterol elimination from different neural tissues.     

Mechanism of free radical nitric oxide-mediated Ras guanine nucleotide dissociation

Ras proteins cycle between GDP-bound and GTP-bound states to modulate a diverse array of cellular growth processes. In this study, we have elucidated a mechanism by which nitric oxide, in the presence of oxygen (NO/O2), regulates Ras activity. We show that treatment of Ras with NO/O2 causes conversion of Ras-bound GDP into a free 463.3 Da nucleotide-nitration product. Mass and UV/visible spectroscopic analyses suggest that this nitration product is 5-guanidino-4-nitroimidazole diphosphate (NIm-DP), a degradation product of 5-nitro-GDP. These results indicate that NO/O2 mediates Ras guanine nucleotide exchange (GNE) by conversion of Ras-bound GDP into an unstable 5-nitro-GDP. 5-Nitro-GDP can be produced by radical-based reaction of the GDP guanine base with nitrogen dioxide (*NO2). We also provide evidence that the Ras Phe28 side-chain plays a key role in the formation of a NO/O2-induced Ras 5-nitro-GDP product. We previously proposed a mechanism of NO/O2-mediated Ras GNE, in which *NO2, formed by the reaction of NO with O2, generates a Ras Cys118 thiyl radical (Ras-S118) intermediate. In the present study, we provide evidence for a radical-based mechanism of NO/O2-mediated Ras GNE. According to this mechanism, reaction of NO with O2 produces *NO2. *NO2 then reacts with Ras to produce Ras-S118, which withdraws an electron from the Ras-bound guanine nucleotide base to produce a guanine nucleotide diphosphate cation radical (G(+)-DP) via the Phe28 side-chain. G(+)-DP is subsequently converted to a neutral radical, and can react with another *NO2 to produce 5-nitro-GDP. This radical-based reaction process disrupts key binding interactions between Ras and the guanine base, resulting in release of GDP from Ras and its conversion to free 5-nitro-GDP. This mechanism is likely to be common to other NKCD motif-containing Ras superfamily GTPases, as NO/O2 also facilitates GNE on the redox-active Rap1A and Rab3A GTPases.     

Antimicrobial Air Filters Using Natural Euscaphis japonica Nanoparticles

Controlling bioaerosols has become more important with increasing participation in indoor activities. Treatments using natural-product nanomaterials are a promising technique because of their relatively low toxicity compared to inorganic nanomaterials such as silver nanoparticles or carbon nanotubes. In this study, antimicrobial filters were fabricated from natural Euscaphis japonica nanoparticles, which were produced by nebulizing E. japonica extract. The coated filters were assessed in terms of pressure drop, antimicrobial activity, filtration efficiency, major chemical components, and cytotoxicity. Pressure drop and antimicrobial activity increased as a function of nanoparticle deposition time (590, 855, and 1150 µg/cm2_filter at 3-, 6-, and 9-min depositions, respectively). In filter tests, the antimicrobial efficacy was greater against Staphylococcus epidermidis than Micrococcus luteus; ~61, ~73, and ~82% of M. luteus cells were inactivated on filters that had been coated for 3, 6, and 9 min, respectively, while the corresponding values were ~78, ~88, and ~94% with S. epidermidis. Although statistically significant differences in filtration performance were not observed between samples as a function of deposition time, the average filtration efficacy was slightly higher for S. epidermidis aerosols (~97%) than for M. luteus aerosols (~95%). High-performance liquid chromatography (HPLC) and electrospray ionization-tandem mass spectrometry (ESI/MS) analyses confirmed that the major chemical compounds in the E. japonica extract were 1(ß)-O-galloyl pedunculagin, quercetin-3-O-glucuronide, and kaempferol-3-O-glucoside. In vitro cytotoxicity and disk diffusion tests showed that E. japonica nanoparticles were less toxic and exhibited stronger antimicrobial activity toward some bacterial strains than a reference soluble nickel compound, which is classified as a human carcinogen. This study provides valuable information for the development of a bioaerosol control system that is environmental friendly and suitable for use in indoor environments.     

Effect of an Artificial Caudal Fin on the Performance of a Biomimetic Fish Robot Propelled by Piezoelectric Actuators

This paper addresses the design of a biomimetic fish robot actuated by piezoeeramic actuators and the effect of artificial caudal fins on the fish robot＇s performance. The limited bending displacement produced by a lightweight piezocomposite actuator was amplified and transformed into a large tail beat motion by means of a linkage system. Caudal fins that mimic the shape of a mackerel fin were fabricated for the purpose of examining the effect of caudal fm characteristics on thrust production at an operating frequency range. The thickness distribution of a real mackerel＇s fin was measured and used to design artificial caudal fins. The thrust performance of the biomimetic fish robot propelled by fins of various thicknesses was examined in terms of the Strouhal number, the Froude number, the Reynolds number, and the power consumption. For the same fm area and aspect ratio, an artificial caudal fin with a distributed thickness shows the best forward speed and the least power consumption.     

Aberrant immune responses in a mouse with behavioral disorders

BTBR T+tf/J (BTBR) mice have recently been reported to have behaviors that resemble those of autistic individuals, in that this strain has impairments in social interactions and a restricted repetitive and stereotyped pattern of behaviors. Since immune responses, including autoimmune responses, are known to affect behavior, and individuals with autism have aberrant immune activities, we evaluated the immune system of BTBR mice, and compared their immunity and degree of neuroinflammation with that of C57BL/6 (B6) mice, a highly social control strain, and with F1 offspring. Mice were assessed at postnatal day (pnd) 21 and after behavioral analysis at pnd70. BTBR mice had significantly higher amounts of serum IgG and IgE, of IgG anti-brain antibodies (Abs), and of IgG and IgE deposited in the brain, elevated expression of cytokines, especially IL-33 IL-18, and IL-1β in the brain, and an increased proportion of MHC class II-expressing microglia compared to B6 mice. The F1 mice had intermediate levels of Abs and cytokines as well as social activity. The high Ab levels of BTBR mice are in agreement with their increased numbers of CD40(hi)/I-A(hi) B cells and IgG-secreting B cells. Upon immunization with KLH, the BTBR mice produced 2-3 times more anti-KLH Abs than B6 mice. In contrast to humoral immunity, BTBR mice are significantly more susceptible to listeriosis than B6 or BALB/c mice. The Th2-like immune profile of the BTBR mice and their constitutive neuroinflammation suggests that an autoimmune profile is implicated in their aberrant behaviors, as has been suggested for some humans with autism.     

Redox regulation of RhoA

We have previously shown that redox agents including superoxide anion radical and nitrogen dioxide can react with GXXXXGK(S/T)C motif-containing GTPases (i.e., Rac1, Cdc42, and RhoA) to stimulate guanine nucleotide release. We now show that the reaction of RhoA with redox agents leads to different functional consequences from that of Rac1 and Cdc42 due to the presence of an additional cysteine (GXXXCGK(S/T)C) in the RhoA redox-active motif. While reaction of redox agents with RhoA stimulates guanine nucleotide dissociation, RhoA is subsequently inactivated through formation of an intramolecular disulfide that prevents guanine nucleotide binding thereby causing RhoA inactivation. Thus, redox agents may function to downregulate RhoA activity under conditions that stimulate Rac1 and Cdc42 activity. The opposing functions of these GTPases may be due in part to their differential redox regulation. In addition, the results presented herein suggest that the platinated-chemotherapeutic agent, cisplatin, which is known for targeting nucleic acids, reacts with RhoA to produce a RhoA thiol-cisplatin-thiol adduct, leading to inactivation of RhoA. Similarly, certain arsenic complexes (i.e., arsenate and arsenic trioxide) may inactivate RhoA by bridging the cysteine residues in the GXXXCGK(S/T)C motif. Thus, in addition to redox agents, platinated-chemotherapeutic agents and arsenic complexes may modulate the activity of GTPases containing the GXXXCGK(S/T)C motif (i.e., RhoA and RhoB).     

Immunization with major outer membrane protein of Vibrio vulnificus elicits protective antibodies in a murine model

Sera from rabbits were infected with Vibrio vulnificus containing an antibody against major outer membrane protein (MOMP). MOMP of V. vulnificus ATCC 27562 were isolated and purified by Sarkosyl and TritonX-100 dual treatment. Molecular size of MOMP was identified as 36-kDa on 13% SDS-PAGE. The sequence of the first 26 amino acid residues from the N-terminal end of the protein is AELYNQDGTSLDMGGRAEARLSMKDG , which is a perfect match with OmpU of V. vulnificus CMCP6 and YJ016. MOMP specific IgM and IgG were investigated in groups of mice. The group of mice immunized with MOMP and Alum showed higher levels of IgG2b than the group immunized with only MOMP. Vaccination with MOMP resulted in protective antibodies in the mouse infection experiment.     

Recording of elapsed time and temporal information about biological events using Cas9

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