Immunohistochemical Artifact for Nitrotyrosine in Eosinophils or Eosinophil Containing Tissue

Immunohistochemical artifacts for nitrotyrosine were investigated in eosinophils with regard to fixatives. Immunoreactivity for nitrotyrosine was revealed in separated eosinophils and in gastric mucosa fixed with periodate, lysine-paraformaldehyde (PLP). The increase in immunoreactivity by PLP was due to periodate itself, a component of PLP. Nitrotyrosine formed by peroxidase using NO 2 &#109 and H 2 O 2 or by peroxynitrite was not completely inhibited by 100 mM dithionite but the immunoreactivity for nitrotyrosine antibodies by PLP was completely inhibited by 5.7 mM dithionite. Although untreated eosinophils or ovalbumin (OVA) did not show protein tyrosine nitration in a standard Western blot, the treatment of the blotted membrane with PLP increased the reactivities of proteins from eosinophils with anti-nitrotyrosine antibodies. The increase in immunoreactivity of OVA with anti-nitrotyrosine antibodies by PLP did not change with pre-treatment with dithionite but was abolished by treatment with dithionite after PLP fixation. In HPLC assays, periodate did not generate nitrotyrosine from l -tyrosine and aminotyrosine. These results suggest that the treatment of eosinophils or eosinophil-containing tissues with PLP fixative augments the immunoreactivity of nitrotyrosine antibodies with eosinophils due to the formation of epitopes similar to nitrotyrosine by an oxidation reaction of periodate, which evokes an artifact in nitrotyrosine immunohistochemistry.     

The Effects of Electronic Cigarette Emissions on Systemic Cotinine Levels,Weight and Postnatal Lung Growth in Neonatal Mice

Background/ObjectiveElectronic cigarette (E-cigarettes) emissions present a potentially new hazard to neonates through inhalation, dermal and oral contact. Exposure to nicotine containing E-cigarettes may cause significant systemic absorption in neonates due to the potential for multi-route exposure. Systemic absorption of nicotine and constituents of E-cigarette emissions may adversely impact weight and lung development in the neonate. To address these questions we exposed neonatal mice to E-cigarette emissions and measured systemic cotinine levels and alveolar lung growth.

Methods/Main Results

Neonatal mice were exposed to E-cigarettes for the first 10 days of life. E-cigarette cartridges contained either 1.8% nicotine in propylene glycol (PG) or PG vehicle alone. Daily weights, plasma and urine cotinine levels and lung growth using the alveolar mean linear intercept (MLI) method were measured at 10 days of life and compared to room air controls. Mice exposed to 1.8% nicotine/PG had a 13.3% decrease in total body weight compared to room air controls. Plasma cotinine levels were found to be elevated in neonatal mice exposed to 1.8% nicotine/PG E-cigarettes (mean 62.34± 3.3 ng/ml). After adjusting for sex and weight, the nicotine exposed mice were found to have modestly impaired lung growth by MLI compared to room air control mice (p<.054 trial 1; p<.006 trial 2). These studies indicate that exposure to E-cigarette emissions during the neonatal period can adversely impact weight gain. In addition exposure to nicotine containing E-cigarettes can cause detectable levels of systemic cotinine, diminished alveolar cell proliferation and a modest impairment in postnatal lung growth.     

Development and evaluation of human T‐cell leukemia virus‐1 and ‐2 multiplex quantitative PCR

The diagnosis of human T ‐cell leukemia virus type 1 (HTLV‐1) infection in Japan is usually performed by serological testing, but the high rate of indeterminate results from western blotting makes it difficult to assess the infection accurately. Nucleic acid tests for HTLV‐1 and/or HTLV‐2 are used to confirm infection with HTLV‐1 and/or HTLV‐2 and are also used for the follow‐up of HTLV‐1 related diseases. To prepare a highly sensitive method that can discern infection with HTLV‐1 and HTLV‐2, a multiplex quantitative polymerase chain reaction (qPCR) by large‐scale primer screening was developed. Sensitivity and specificity were evaluated by serial dilution of cell lines and by testing with known clinical samples. The resulting multiplex qPCR can detect about four copies of HTLV‐1 provirus per 10⁵ cells. Moreover, HTLV‐1 provirus could be detected in 97.2% (205 of 211) of HTLV‐1 seropositive clinical samples. These sensitivities were sufficiently high compared with the methods reported previously. Also, all the HTLV‐2 seropositive clinical samples tested were found to be positive by this method (three of three). In conclusion, this method can successfully and simultaneously detect both types of HTLV‐1 and HTLV‐2 provirus with extremely high sensitivity.     

Phenology of annual kelp Eckloniopsis (Phaeophyceae,Laminariales) forest on a Diadema barren in Uchiura Bay,Central Pacific Coast of Honshu,Japan

On the southern coast of Uchiura Bay, central Pacific, Japan, Diadema barrens have expanded since the 1980s but Eckloniopsis radicosa (annual kelp endemic to Japan) has remained in deeper waters (>10 m in depth). Phenology of the kelp was studied on isolated boulders from December 2011 for a year. Young sporophytes appeared in December and rapid growth from April brought the maximum blade length (83.3?±?13.9 cm) and width (56.8?±?12.7 cm) and standing crop (7.4 kg m^?2) in May and June, respectively. Sorus formation began in June and spore release occurred from July to September. Blade length decreased from August and disappeared in November though holdfast remained. The unique holdfast-like spiny ball was found to provide habitats for mobile animals; its forests have an important role to maintain the biodiversity on barrens. During the period, water temperature was between 14.6 and 27.8 °C, salinity was stable around 34–35?‰, and nutrients were never depleted. Tolerance to large and frequent fluctuation of water temperature (7 °C in a day), rapid growth in winter to spring, and occurrence on limited boulders in soft substrata may be the reasons for the success in the maintenance of its forest in Diadema barrens.     

Crystal Structure of Methanocaldococcus jannaschii Trm4 Complexed with Sinefungin

tRNA:m⁵C methyltransferase Trm4 generates the modified nucleotide 5-methylcytidine in archaeal and eukaryotic tRNA molecules, using S-adenosyl-l-methionine (AdoMet) as methyl donor. Most archaea and eukaryotes possess several Trm4 homologs, including those related to diseases, while the archaeon Methanocaldococcus jannaschii has only one gene encoding a Trm4 homolog, MJ0026. The recombinant MJ0026 protein catalyzed AdoMet-dependent methyltransferase activity on tRNA in vitro and was shown to be the M. jannaschii Trm4. We determined the crystal structures of the substrate-free M. jannaschii Trm4 and its complex with sinefungin at 1.27 Å and 2.3 Å resolutions, respectively. This AdoMet analog is bound in a negatively charged pocket near helix α8. This helix can adopt two different conformations, thereby controlling the entry of AdoMet into the active site. Adjacent to the sinefungin-bound pocket, highly conserved residues form a large, positively charged surface, which seems to be suitable for tRNA binding. The structure explains the roles of several conserved residues that were reportedly involved in the enzymatic activity or stability of Trm4p from the yeast Saccharomyces cerevisiae. We also discuss previous genetic and biochemical data on human NSUN2/hTrm4/Misu and archaeal PAB1947 methyltransferase, based on the structure of M. jannaschii Trm4.     

Loss of ovule identity induced by overexpression of the fertilization-related kinase 2 (ScFRK2), a MAPKKK from Solanum chacoense

In order to gain information about protein kinases acting during plant fertilization and embryogenesis, a reverse genetic approach was used to determine the role of protein kinases expressed in reproductive tissues. Two cDNA clones named ScFRK1 and ScFRK2 (Solanum chacoense fertilization-related kinase 1 and 2) were isolated from an expressed sequence tag (EST) library normalized for weakly expressed genes in fertilized ovaries. These showed significant sequence similarities to members of the mitogen-activated protein kinase kinase kinase (MAPKKK) family. RNA gel blot and RNA in situ hybridization analyses confirmed the strong up-regulation of ScFRK2 in ovules after fertilization. In addition, ScFRK2 mRNAs accumulate during early ovule development in the megasporocyte and in the integument of developing ovules. Overexpression of ScFRK2 led to the production of fruits with a severely reduced number of seeds. The seeds that were produced also exhibited developmental retardation. Analysis of ovaries prior to fertilization showed that the seedless phenotype was caused by a homeotic conversion of ovules into carpel-like structures. The present observations are consistent with the role of ScFRK2 in pre- and post-fertilization events. Furthermore, overexpression of ScFRK2 led to changes in the expression of the class D floral homeotic gene ScFBP11, suggesting that the ScFRK2 kinase may interact, directly or indirectly, with the FBP7/11 pathway that directs establishment of ovule identity.     

The significance of multiple mating and male substance transferred to females at mating in the white grub beetle, <Emphasis Type="Italic">Dasylepida ishigakiensis</Emphasis> (Coleoptera: Scarabaeidae)

Males of the white grub beetle, Dasylepida ishigakiensis Niijima et Kinoshita, transfer a large amount of a colloidal substance to females during mating. In this study we investigated the effect of the male substance on the reproductive performance of mated females and the significance of multiple mating of this beetle. Females artificially separated from the males 5 min after the start of mating produced fewer eggs than those separated after 30 min or those that were undisturbed and separated spontaneously, suggesting that the male substance is used as a nutrient for egg production by the females. When females were allowed to mate with 1–4 males, multiple mating had no clear effect on reproductive performance. The amount of male substance stored in the bursa copulatrix (BC) was not significantly increased by a second mating. The functions of multiple mating of this species may be to provide a chance for females to obtain sufficient amounts of male substance when the first male to mate has only small amounts of this substance, and to increase the genetic heterogeneity of the progeny. The presence of a serine proteinase and its possible involvement in the dynamics of the BC contents are reported.