Random Mutational Analysis Targeting Residue K155 within the Transmembrane β-Hairpin of the Mosquitocidal Mpp46Ab Toxin

Mpp46Ab is a mosquito-larvicidal pore-forming toxin derived from Bacillus thuringiensis TK-E6. Pore formation is believed to be a central mode of Mpp46Ab action, and the cation selectivity of the channel pores, in particular, is closely related to its mosquito-larvicidal activity. In the present study, we constructed a mutant library in which residue K155 within the transmembrane β-hairpin was randomly replaced with other amino acid residues. Upon mutagenesis and following primary screening using Culex pipiens mosquito larvae, we obtained 15 mutants in addition to the wild-type toxin. Bioassays using purified proteins revealed that two mutants, K155E and K155I, exhibited toxicity significantly higher than that of the wild-type toxin. Although increased cation selectivity was previously reported for K155E channel pores, we demonstrated in the present study that the cation selectivity of K155I channel pores was also significantly increased. Considering the characteristics of the amino acids, the charge of residue 155 may not directly affect the cation selectivity of Mpp46Ab channel pores. Replacement of K155 with glutamic acid or isoleucine may induce a similar conformational change in the region associated with the ion selectivity of the Mpp46Ab channel pores. Mutagenesis targeting the transmembrane β-hairpin may be an effective strategy for enhancing the ion permeability of the channel pores and the resulting mosquito-larvicidal activity of Mpp46Ab

A Case of Death After Glue Sniffing

A 25-year-old male, a thinner and/or glue sniffer, was found dead in the bathtub of his house approximately 24 hr after abusing a glue. As the cause of death was unknown, an autopsy of the deceased was performed. The ingredients of the glue in the tissue and organs were analyzed by gas chromatography and gas chromatographymass spectrometry. Toluene, n-hexane and methylcyclopentane in the mesenterial fat were confirmed by gas chromatography-mass spectrometry, and the toluene concentration analyzed by gas chromatography was 10.9 μ,gig in the blood and 50.0 μ,gig in the brain. The cause of death was considered to be suffocation or acute cardiac failure under the effect of toluene

Evaluation of Latex Agglutination Inhibition Reaction Test for Urinary Methamphetamine

A latex agglutination inhibition reaction test for a screening method of methamphetamine in urine has been reported by Aoki et al. An evaluation of this method was attempted by using gas chromatography and chemical ionization mass fragmentography. Some false negative and false positive cases were found. It seems that this method needs some modification for false negative cases and also it needs a confirmation test

Channel-pore cation selectivity is a major determinant of Bacillus thuringiensis Cry46Ab mosquitocidal activity

Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type architecture, and it is expected to be used as a novel bioinsecticide. Cry46Ab acts as a functional pore-forming toxin, and characteristics of the resulting channel pores, including ion selectivity, have been analyzed. However, the relationship between channel-pore ion selectivity and insecticidal activity remains to be elucidated. To clarify the effects of charged amino acid residues on the ion permeability of channel-pores and the resulting insecticidal activity, in the present study, we constructed Cry46Ab mutants in which a charged amino acid residue within a putative transmembrane β-hairpin region was replaced with an oppositely charged residue. Bioassays using Culex pipiens mosquito larvae revealed that the mosquitocidal activity was altered by the mutation. A K155E Cry46Ab mutant exhibited toxicity apparently higher than that of wild-type Cry46Ab, but the E159K and E163K mutants exhibited decreased toxicity. Ions selectivity measurements demonstrated that the channel pores formed by both wild-type and mutant Cry46Abs were cation selective, and their cation preference was also similar. However, the degree of cation selectivity was apparently higher in channel pores formed by the K155E mutant, and reduced selectivity was observed with the E159K and E163K mutants. Our data suggest that channel-pore cation selectivity is a major determinant of Cry46Ab mosquitocidal activity and that cation selectivity can be controlled via mutagenesis targeting the transmembrane β-hairpin region

Genetic diversity of group A rotaviruses associated with repeated outbreaks of diarrhea in a farrow-to-finish farm: identification of a porcine rotavirus strain bearing a novel VP7 genotype, G26

Group A rotaviruses (GARs) are one of the most common causes of diarrhea in suckling pigs. Although a number of G and P genotypes have been identified in porcine GARs, few attempts have been made to study the molecular epidemiology of these viruses associated with diarrhea outbreaks within a farm over an extended period of time. Here, we investigated the molecular characteristics of GARs that caused four outbreaks of diarrhea among suckling pigs in a farrow-to-finish farm over the course of a year. G and P genotyping of GARs detected at each outbreak demonstrated genetic diversity in this farm as follows: G9P[23] was detected at the first outbreak, G9P[13]/[22] and G9P[23] at the second, G3P[7] at the third, and G9P[23], G5P[13]/[22], and P[7] combined with an untypeable G genotype at the fourth. Sequence analysis of the detected GARs revealed that such genetic diversity could have resulted not only from the introduction of new GAR strains, but also from gene reassortment between GAR strains within the farm. Further, the GAR strain carrying the untypeable G genotype was shown to be a novel porcine GAR bearing a new G26 genotype, as confirmed by the Rotavirus Classification Working Group

A Rapid and Sensitive Method for Detecting Fenitrothion in Biological Fluids Using the Phosphorus-Sulfur Selective Detector: a fenitrothion intoxication case

Fenitrothion (sumithion) in biological fluids of a patient, who attempted suicide by ingesting of fenitrothion, was separated and purified by Extrelut® column extraction. A gas chromatograph equipped with a flame photometric detec1 or and a gas chromatograph-mass spectrometer were used for a detection of fenitrothion. A 41-year-old female, who attempted suicide by ingesting about 30 ml of Sumithion® (40% fenitrothion), started to vomit spontaneously and recurringly, and was transported to a hospital 3 hr after the ingestion. The patient was almost fully conscious and the diameter of her pupils was 3 mm on both sides. The fenitrothion concentration in the blood sample was 260 ng/g and was less than 6 ng/g in the urine sample both of which were collected 4 hr after ingestion. Aminofenitrothion, 4-nitro-3-methyl phenol, S-methylfenitrothion, phenobarbital and lidocaine were identified in the ethyl ether extract of the urine sample. After ingestion, the serum cholinesterase activity (normal range: 175-440 IU) was 104 at hr, 38 at 1 day, 85 at 2 days, 102 at 3 days and 137 at 4 days

Large-scale Filamentary Structure around the Protocluster at Redshift z=3.1

We report the discovery of a large-scale coherent filamentary structure of Lyman alpha emitters in a redshift space at z=3.1. We carried out spectroscopic observations to map the three dimensional structure of the belt-like feature of the Lyman alpha emitters discovered by our previous narrow-band imaging observations centered on the protocluster at z=3.1. The feature was found to consist of at least three physical filaments connecting with each other. The result is in qualitative agreement with the prediction of the 'biased' galaxy-formation theories that galaxies preferentially formed in large-scale filamentary or sheet-like mass overdensities in the early Universe. We also found that the two known giant Lyman alpha emission-line nebulae showing high star-formation activities are located near the intersection of these filaments, which presumably evolves into a massive cluster of galaxies in the local Universe. This may suggest that massive galaxy formation occurs at the characteristic place in the surrounding large-scale structure at high redshift.Comment: 11 pages, 3 figures, accepted for publication in ApJ Letter

Distinction of synthetic dl-α-tocopherol from natural vitamin E (d-α-tocopherol) by reversed-phase liquid chromatography. Enhanced selectivity of a polymeric C18 stationary phase at low temperature and/or at high pressure

© 2016 Elsevier B.V. Separation of diastereomers of DL-α-tocopherol was studied by reversed-phase liquid chromatography using three types of stationary phases, polymeric ODS, polymeric C30, and monomeric ODS. Polymeric ODS stationary phase (Inertsil ODS-P, 3 mmID, 20 cm) was effective for the separation of the isomers created by the presence of three chiral centers on the alkyl chain of synthetic DL-α-tocopherol. Considerable improvement of the separation of isomers was observed on ODS-P phase at high pressure and at low temperature. Complete separation of four pairs of diastereomers was achieved at 12.0 °C, 536 bar, while three peaks were observed when the separation was carried out either at 12.0 °C at low pressure or at 20 °C at 488 bar. Higher temperature (30.0 °C) with the ODS-P phase resulted in only partial separation of the diastereomers even at high pressure. Only slight resolution was observed for the mixture of diastereomers with the C30 stationary phase (Inertsil C30) at 12.0 °C and 441 bar, although the stationary phase afforded greater resolution for β- and γ-tocopherol than ODS-P. A monomeric C18 stationary phase did not show any separation at 12.0 °C and 463 bar. The results suggest that the binding site of the polymeric ODS-P phase is selective for flexible alkyl chains that provided the longest retention for the natural form, (R,R,R) form, and the enantiomer, (S,S,S) form, of DL-α-tocopherol

The Halpha Luminosity Function of the Galaxy Cluster Abell 521 at z = 0.25

We present an optical multicolor-imaging study of the galaxy cluster Abell 521 at $z = 0.25$, using Suprime-Cam on the Subaru Telescope, covering an area of $32 \times 20$ arcmin$^2$ ($9.4 \times 5.8 h_{50}^{-2}$ Mpc$^2$ at $z = 0.25$). Our imaging data taken with both a narrow-band filter, $NB816$ ($\lambda_0 = 8150$\AA and $\Delta \lambda = 120$\AA), and broad-band filters, $B,V,R_{\rm C}, i^\prime$, and $z^\prime$ allow us to find 165 H$\alpha$ emitters. We obtain the H$\alpha$ luminosity function (LF) for the cluster galaxies within 2 Mpc; the Schechter parameters are $\alpha = -0.75 \pm 0.23$, $\phi^\star = 10^{-0.25 \pm 0.20}$ Mpc$^{-3}$, and $L^\star = 10^{42.03 \pm 0.17}$ erg s$^{-1}$. Although the faint end slope, $\alpha$, is consistent with that of the local cluster H$\alpha$ LFs, the characteristic luminosity, $L^\star$, is about 6 times (or $\approx 2$ mag) brighter. This strong evolution implies that Abell 521 contains more active star-forming galaxies than the local clusters, being consistent with the observed Butcher-Oemler effect. However, the bright $L^\star$ of Abell 521 may be, at least in part, due to the dynamical condition of this cluster.Comment: 21 pages, 7 figures, ApJ, Part 1, in pres