Neonatal administration of synthetic estrogen, diethylstilbestrol to mice up-regulates inflammatory Cxclchemokines located in the 5qE1 region in the vaginal epithelium.

A synthetic estrogen, diethylstilbestrol (DES), is known to cause adult vaginal carcinoma by neonatal administration of DES to mice. However, the carcinogenic process remains unclear. By Cap Analysis of Gene Expression method, we found that neonatal DES exposure up-regulated inflammatory Cxcl chemokines 2, 3, 5, and 7 located in the 5qE1 region in the vaginal epithelium of mice 70 days after birth. When we examined the gene expressions of these genes much earlier stages, we found that neonatal DES exposure increased these Cxcl chemokine genes expression even after 17 days after birth. It implies the DES-mediated persistent activation of inflammatory genes. Intriguingly, we also detected DES-induced non-coding RNAs from a region approximately 100 kb far from the Cxcl5 gene. The non-coding RNA up-regulation by DES exposure was confirmed on the 17-day vagina and continued throughout life, which may responsible for the activation of Cxcl chemokines located in the same region, 5qE1. This study shows that neonatal administration of DES to mice causes long-lasting up-regulation of inflammatory Cxcl chemokines in the vaginal epithelium. DES-mediated inflammation may be associated with the carcinogenic process.

Class†III Polyphosphate Kinase†2 Enzymes Catalyze the Pyrophosphorylation of Adenosine-5'-Monophosphate.

Polyphosphate kinase 2 (PPK2) transfer phosphate from inorganic polyphosphate to nucleotides. According to their activity, PPK2 enzymes are classified into three groups. Among them, class III enzymes catalyze both the phosphorylation of nucleotide mono- to diphosphates and di- to triphosphates by using polyphosphate, which is a very inexpensive substrate. Therefore, class III enzymes are very attractive for use in biotechnological applications. Despite several studies on class III enzymes, a detailed mechanism of how phosphate is transferred from the polyphosphate to the nucleotide remains to be elucidated. Herein, it is reported that PPK2 class III enzymes from two different bacterial species catalyze the phosphorylation of adenosine mono- (AMP) into triphosphate (ATP) not only through step-by-step phosphorylation, but also by pyrophosphorylation. These are the first PPK2 enzymes that have been shown to possess polyphosphate-dependent pyrophosphorylation activity.

A Case of Cardiac Arrest for 31 Seconds During Recovery After Intravenous Sedation.

A 26-year-old woman with a history of feeling nauseated during dental local anesthesia presented to our clinic for tooth extraction under intravenous sedation. Although she had experienced episodes of neurally-mediated syncope, her symptoms were controlled well with drug therapy, stopped 3 years earlier. No syncope episodes developed over the previous 2 years. Tooth extraction was performed under intravenous sedation without incident. When she was returned to a sitting position after being roused, convulsion, loss of consciousness, and cardiac arrest developed. One week later, similar symptoms occurred immediately after suture removal. We suspect that the change in body position triggered these episodes. It is important to avoid abrupt changes in body position and any other triggers and to administer preventive drugs in patients at high risk of syncope.

Comparison of the Contact Force Exerted on Teeth by Conventional Macintosh Laryngoscope Versus Video Laryngoscopes.

During laryngoscopy, the laryngoscope blade sometimes comes in contact with the teeth, fracturing or dislocating them. However, no studies have compared the effects of newly marketed video laryngoscopes and the Macintosh laryngoscope (Mac) on teeth. In this study, we measured and compared the force exerted on the teeth of an intubating manikin by the Mac, the Airway Scope (Pentax), and the McGrath MAC (Covidien). The mean force exerted was 141.1 ± 15.7 kg by the Mac, 39.2 ± 10.3 kg by the Airway Scope, and 48.7 ± 6.7 kg by the McGrath MAC. No significant difference was observed between the Airway Scope and the McGrath MAC. When the Mac is inserted, the glottis has to be visually located from outside the oral cavity. However, a significant force is not necessary when inserting video laryngoscopes because a camera is mounted on the blade tip. In this laboratory model, the lower force exerted by the video laryngoscopes should contribute to a reduction in their impact on fracture or dislocation of teeth.

Genomic integration and ligand-dependent activation of the human estrogen receptor α in the crustacean Daphnia magna.

The freshwater crustacean Daphnia have a long history in water quality assessments and now lend themselves to detection of targeted chemicals using genetically encoded reporter gene due to recent progress in the development of genome editing tools. By introducing human genes into Daphnia, we may be able to detect chemicals that affect the human system, or even apply it to screening potentially useful chemicals. Here, we aimed to develop a transgenic line of Daphnia magna that contains the human estrogen receptor alpha (hERα) and shows a fluorescence response to exposure of estrogens. We designed plasmids to express hERα in Daphnia (EF1α1:esr1) and to report estrogenic activity via red fluorescence (ERE:mcherry) under the control of estrogen response element (ERE). After confirmation of functionality of the plasmids by microinjection into embryos, the two plasmids were joined, a TALE site was added and integrated into the D. magna genome using TALEN. When the resulting transgenic Daphnia named the ES line was exposed to Diethylstilbestrol (DES) or 17ß-Estradiol (E2), the ES line could reliably expressed red fluorescence derived from mCherry in a ligand-dependent manner, indicating that an estrogen-responsive line of D. magna was established. This is the first time a human gene was expressed in Daphnia, showcasing potential for further research.

Development of a bicistronic expression system in the branchiopod crustacean Daphnia magna.

The viral 2A peptides have recently been used for bicistronic expression in various organisms. In this system, a single mRNA that codes for two proteins flanking the 2A peptide can be translated simultaneously into each protein by ribosomal skipping at this peptide sequence. Here, we tested the function of the Thosea asigna insect virus 2A (T2A) peptide in the branchiopod crustacean Daphnia magna-an emerging model of evolutionary developmental biology. First, we used transgenic Daphnia that expresses a potential bicistronic RNA containing mCherry and histone H2B- green fluorescent protein (GFP) open reading frames upstream and downstream of the T2A sequence, respectively. Microscopic observation revealed difference of localization of the two proteins in the cell, homogenous distribution of mCherry and nuclear localization of H2B-GFP. Second, we changed localization of mCherry from cytoplasm to plasma membrane by attachment of a consensus myristoylation motif in the bicistronic reporter. RNA that codes for this new bicistronic reporter was injected into eggs. At gastrulation stage, we found spectrally distinct fluorescence with enough intensity and resolution to detect membrane localized mCherry and nuclear GFP. These results indicate that the T2A peptide functions in D. magna and T2A-mediated bicistronic expression would be a promising tool for evo-devo studies of this species.

Organization and repression by juvenile hormone of a vitellogenin gene cluster in the crustacean, Daphnia magna.

Two Daphnia magna vitellogenin (VTG) genes in neighboring but opposite orientations were identified. One was the gene for DmagVTG1, a previously characterized VTG polypeptide with a superoxide dismutase (SOD)-like domain at its NH(2)-terminus [Kato et al., Gene 334 (2004) 157-165]. Both genes had a 17-exon and 16-intron structure in the same configuration. DmagVTG2, a polypeptide encoded by the other gene, also had a SOD-like domain at its NH(2)-terminus. The amino acid sequences of the two VTG domains were highly homologous (95.5% identity), while those of the SOD-like domains were less homologous (62.4% identity). The VTG domains are phylogenetically related to insect VTGs while the SOD-like domains are related to viral and bacterial SODs. The intergenic region of 2.6kb between the two genes contains sequences resembling known juvenile hormone (JH)-responsive and ecdysone-responsive elements. JH agonists, pyriproxyfen and fenoxycarb, strongly repressed the expression of VTG genes in neonate daphnids.

A vitellogenin chain containing a superoxide dismutase-like domain is the major component of yolk proteins in cladoceran crustacean Daphnia magna.

A cDNA encoding vitellogenin (VTG), a precursor of a major yolk protein, vitellin (VTN), was isolated from cladoceran crustacean Daphnia magna. The deduced amino acid sequence of DmagVTG1, the polypeptide encoded by the cDNA, contained a possible signal peptide sequence of 16 amino acid (aa) residues. The possible mature form of DmagVTG1 consists of 1985 aa residues with a calculated molecular mass of 223,070 Da. The large lipid transfer (LLT) module and a part of the von Willebrand factor D (VWD) module found in the aa sequences of VTGs of many other organisms are well conserved in DmagVTG1. Phylogenetic analysis suggested that the LLT module of DmagVTG1 is more closely related to those of insect VTGs than those of decapodan crustaceans. A unique feature of DmagVTG1 is that it has a superoxide dismutase (SOD)-like domain at its NH(2)-terminus. Antisera against the SOD-like domain, the NH(2)-terminal part of the VTG domain and the COOH-terminal part of the VTG domain, respectively, were prepared and used for analysis of D. magna yolk proteins. Six species (I to VI) of major protein complexes were found in D. magna parthenogenetic eggs isolated immediately after ovulation. Complexes IV and V were the most abundant. DmagVTG1 was a component of Complexes III, IV and V, and the most abundant polypeptide in D. magna eggs. The protein complexes underwent gradual proteolysis during development. One of the primary sites of cleavage was between the two successive Arg residues located at the 1454th and 1455th positions of DmagVTG1.