Novel DNA virus isolated from samples showing endothelial cell necrosis in the Japanese eel, Anguilla japonica

Economic loss due to viral endothelial cell necrosis of eel (VECNE) of Anguilla japonica is a serious problem for the cultured Japanese eel market. However, the viral genome responsible for VECNE is unknown. We recently developed a rapid determination system for viral nucleic acid sequences (RDV) to determine viral genome sequences. In this study, viral DNA fragments were obtained using RDV, and approximately 15-kbp circular full genome sequences were determined using a next-generation sequencing system, overlapping PCR, and Southern blot analysis. One open reading frame (ORF) was homologous to the large T-antigen of polyomavirus; other ORFs have no homology with any nucleic or amino acid sequences of polyomavirus. Therefore, as this DNA virus might comprise a novel virus family, we provisionally named it Japanese eel endothelial cells-infecting virus (JEECV). JEECV was detected in both naturally and experimentally infected eels, suggesting that JEECV potentially causes VECNE.     

Torsion of the gallbladder in a 3-year-old infant

A 3-year-old girl was admitted to hospital with complaints of severe upper abdominal pain and vomiting. On admission, a board-like rigidity in the right hypochondrium was noted, along with a high white blood cell count and a high C-reactive protein value. Abdominal ultrasonography (US) revealed a left-sided enlarged gallbladder with a thickened wall, without gallstones. Contrast-enhanced computed tomography (CT) demonstrated an enlarged gallbladder without enhancement effects and a cystic duct located on the right side of the gallbladder. The patient underwent an emergency operation following a diagnosis of torsion of the gallbladder. The gallbladder appeared gangrenous, and 180° clockwise torsion was found at the neck of the gallbladder. The gallbladder was straightened and then removed without difficulty because there was only slight inflammation around Calot’s triangle and the gallbladder was not fixed to the liver. Histopathological examination revealed an acute bleeding infarction of the gallbladder. The patient was discharged on the ninth day after surgery, without any complications. The present case suggested that abdominal US and contrast-enhanced CT examinations are helpful in making a correct diagnosis of torsion of the gallbladder even in an infant; in the event of this diagnosis, prompt cholecystectomy is necessary.     

The development of a gene vector electrostatically assembled with a polysaccharide capsule

The purpose of this study was to develop a gene vector electrostatically assembled with a polysaccharide capsule. We used pDNA/polyethylenimine (PEI) complexes as efficient non-viral vectors. The pDNA/PEI complex was electrostatically encapsulated with various polysaccharides such as fucoidan, λ-carrageenan, xanthan gum, alginic acid, hyaluronic acid, and chondroitin sulfate (CS). The pDNA/PEI complex was shown as nanoparticles with positive ζ-potential, although the ternary complexes encapsulated with polysaccharides were shown as nanoparticles with negative ζ-potential. The pDNA/PEI complex showed high agglutination activity and cytotoxicity, although the ternary complexes encapsulated with polysaccharides had no agglutination activities and lower cytotoxicities. The pDNA/PEI complex showed high uptake and high transgene efficiency in B16-F10 cells. On the other hand, most of the ternary complexes show little uptake and gene expression. The ternary complex encapsulated by CS, however, showed comparable transgene efficiency to the pDNA/PEI complex. The uptake and gene expression of the ternary complex encapsulated by CS were significantly inhibited by hypothermia and the addition of CS, suggesting that the ternary complex was taken by CS-specific receptor-mediated energy-dependent process.     

Immortalized ovarian surface epithelial cells acquire tumorigenicity by Acrogranin gene overexpression

Malignant transformation is caused by multi-step genetic mutations, and growth factors are believed to play important roles in developing and maintaining malignant phenotype. However, there is no direct evidence that a specific growth factor contributes to malignant transformation of phenotypically normal cells. In order to assess the function of Acrogranin (also known as granulin epithelial precursor; GEP) in ovarian carcinogenesis, ovarian surface epithelial (OSE) cells, which are supposed to be the origin of primary ovarian epithelial cancer, were transfected with combined genes of hTERT, SV40 LT, and Acrogranin. Introduction of hTERT and SV40 LT was sufficient for immortalizing OSE cells but not enough for tumor formation in nude mice. In contrast, transfection and overexpression of Acrogranin in immortalized OSE cells showed augmented clonogenicity in soft agar and obvious tumorigenicity in nude mice. This is the first study showing evidence that a specific growth factor plays a direct role in malignant transformation in ovarian cancer development.     

So-called biological dressing effects of cultured epidermal sheets are mediated by the production of EGF family, TGF-beta and VEGF

BACKGROUND: Cultured epidermal sheet (CES) grafts accelerate wound healing as a result of so-called biological dressing effect, which is thought to be mediated by various growth factors. However, the profile of growth factor expression in CESs is unclear. OBJECTIVE: To investigate whether CESs produce growth factors along with stratification we investigated the production of growth factors and their regulation in CESs. METHODS: CESs conditioned medium was harvested and the concentration of TGF-alpha, TGF-beta1, TGF-beta2, and VEGF was measured using ELISA. The mRNA of EGF family, TGF-beta family and VEGF was detected by Northern blot or RNase protection assay. RESULTS: The concentration of TGF-alpha was 100 pg/ml in the monolayer culture, but dramatically increased to 600 pg/ml 2 days after stratification. It decreased to baseline, and then gradually increased to 300 pg/ml in the presence of EGF and remained at that level until day 20. TGF-beta1 increased from 50 to 400 pg/ml after stratification, and remained at that level day 20. TGF-beta2 was undetectable in the monolayer culture, but dramatically increased to 200 pg/ml 2 days after stratification. Unlike TGF-beta1, TGF-beta2 gradually increased over time after stratification. VEGF increased with stratification from 500 to 1500 pg/ml. The addition of EGF upregulated EGF family, TGF-beta, and VEGF production in CESs, as confirmed by ELISA, Northern blot, and RNase protection assay. CONCLUSION: These results indicate that so-called biological dressing effect of CESs is mediated by production of the EGF family, TGF-beta, and VEGF. Our results also demonstrate the ability of EGF to enhance growth factor production in CESs.     

Lack of evidence for TARC/CCL17 production by normal human keratinocytes in vitro

BACKGROUND: thymus and activation-regulated chemokine (TARC)/CCL17 is a CC chemokine that selectively attracts Th2-type lymphocytes. Immunohistochemical analyses have revealed that TARC is expressed in the epidermal keratinocytes of atopic dermatitis (AD), suggesting TARC involvement in the pathogenesis of the disease. However, keratinocyte TARC production has been described only in the transformed keratinocyte cell line HaCaT. OBJECTIVE: to examine TARC production in normal human epidermal keratinocytes (NHEK) in vitro. METHODS: the expression of TARC mRNA and protein were examined in NHEK and HaCaT cells stimulated with various cytokines. RESULTS: stimulation with inflammatory cytokines, including interleukin (IL)-1, IL-4, IL-6, IL-10, interferon (IFN)-alpha, IFN-beta, IFN-gamma, and tumor necrosis factor (TNF)-alpha failed to induce TARC mRNA expression in NHEK. However, stimulation with IFN-gamma and TNF-alpha together enhanced expression slightly. ELISA analysis failed to detect TARC protein in NHEK culture supernatant, even following stimulation with IFN-gamma and TNF-alpha. In contrast, HaCaT cells produced TARC protein even without stimulation of cytokines. CONCLUSION: these results indicate that production of TARC by HaCaT cells is a phenomenon specific to the cell line and the observation on TARC in HaCaT cells can not be generalized. NHEK do not produce TARC protein in vitro.     

Muscle relaxants in ambulatory anesthesia

Neuromuscular blocking agents are used to facilitate tracheal intubation and surgical procedure in ambulatory anesthesia. However, the ideal neuromuscular blocking agents for ambulatory anesthesia are not yet available. The only depolarizing neuromuscular blocking agent, suxamethonium, is still widely used by its rapid onset and short duration of action producing excellent intubating conditions, in spite of its numerous adverse effects. Nondepolarizing neuromuscular agents have proved to be associated with postoperative residual block more frequently than it was thought before. The use of neostigmine for reversal and the measurement of the TOF ratio during recovery are recommended after intermediate-acting neuromuscular blocking agents. Some studies have shown that tracheal intubation without neuromuscular agents may be associated with postoperative hoarseness and vocal cord injuries. Sugammadex will resolve many issues in using nondepolarizing neuromuscular agents in ambulatory anesthesia.     

Involvement of dopamine D1 receptors and alpha1-adrenoceptors in the antidepressant-like effect of chlorpheniramine in the mouse tail suspension test

It has been reported that chlorpheniramine, a classical antihistamine, has antidepressant-like effects in animal models of depression. In this study, we examined the involvement of dopaminergic (dopamine D(1) and dopamine D(2) receptors), noradrenergic (alpha(1)- and beta-adrenoceptors) and serotonergic (5-HT(1A) and 5-HT(2) receptors) receptors in the antidepressant-like effect of chlorpheniramine in the mouse tail suspension test. We also investigated the involvement of these monoamine receptors in the antidepressant-like effect of imipramine for comparison with the mechanisms of the effect of chlorpheniramine. Both imipramine and chlorpheniramine significantly reduced the duration of immobility in the tail suspension test without affecting spontaneous locomotor activity in mice. The anti-immobility effect of imipramine (30 mg/kg, i.p.) was significantly antagonized by the selective dopamine D(1) receptor antagonist SCH23390 but not by the other receptor antagonists. In contrast, the anti-immobility effect of chlorpheniramine was significantly inhibited by SCH23390 and the selective alpha(1)-adrenoceptor antagonist prazosin, but not by the other receptor antagonists. In conclusion, these results suggest that chlorpheniramine exerts an antidepressant-like effect in the mouse tail suspension test that is mediated by at least the activation of dopamine D(1) receptors and alpha(1)-adrenoceptors. In addition, the antidepressant-like effect of chlorpheniramine may be induced by several mechanisms that are different from those involved in the antidepressant-like effect of imipramine.     

Role of cyclin-dependent kinase 5 in capsaicin-induced cough

The role of cyclin-dependent kinase 5 (Cdk5) in the capsaicin-induced cough reflex was examined in mice. Pretreatment with inhaled roscovitine, a selective Cdk5 inhibitor, at concentrations of 0.3 to 3 mM inhibited the number of capsaicin-induced coughs in a concentration-dependent manner. Pretreatment with inhaled roscovitine, at a concentration of 3 mM also slightly but significantly inhibited the number of citric acid-induced coughs. The number of capsaicin-induced coughs was significantly reduced when C-fibers were desensitized by the pretreatment with capsaicin. The number of citric acid-induced coughs was slightly but significantly reduced in capsaicin-pretreated mice as compared with that in naive mice. Although the inhalation of roscovitine at a concentration of 3 mM significantly reduced the number of citric acid-induced coughs in naive mice to the level observed in capsaicin-pretreated mice, roscovitine had no effect on the number of citric acid-induced coughs in capsaicin-pretreated mice. These results suggest that Cdk5-dependent factors are involved in C-fiber-mediated cough signaling.