Tamiflu-resistant but HA-mediated cell-to-cell transmission through apical membranes of cell-associated influenza viruses

The infection of viruses to a neighboring cell is considered to be beneficial in terms of evasion from host anti-virus defense systems. There are two pathways for viral infection to "right next door": one is the virus transmission through cell-cell fusion by forming syncytium without production of progeny virions, and the other is mediated by virions without virus diffusion, generally designated cell-to-cell transmission. Influenza viruses are believed to be transmitted as cell-free virus from infected cells to uninfected cells. Here, we demonstrated that influenza virus can utilize cell-to-cell transmission pathway through apical membranes, by handover of virions on the surface of an infected cell to adjacent host cells. Live cell imaging techniques showed that a recombinant influenza virus, in which the neuraminidase gene was replaced with the green fluorescence protein gene, spreads from an infected cell to adjacent cells forming infected cell clusters. This type of virus spreading requires HA activation by protease treatment. The cell-to-cell transmission was also blocked by amantadine, which inhibits the acidification of endosomes required for uncoating of influenza virus particles in endosomes, indicating that functional hemagglutinin and endosome acidification by M2 ion channel were essential for the cell-to-cell influenza virus transmission. Furthermore, in the cell-to-cell transmission of influenza virus, progeny virions could remain associated with the surface of infected cell even after budding, for the progeny virions to be passed on to adjacent uninfected cells. The evidence that cell-to-cell transmission occurs in influenza virus lead to the caution that local infection proceeds even when treated with neuraminidase inhibitors.     

Novel cap-independent translation with the 5′-noncoding region of L-A virus mRNA

A novel cap-independent translation has been performed where the ribosome entry is regulated by the 5-noncoding region (NCR) of L-A virus mRNA. Despite L-A virus mRNA containing neither cap structure nor a poly(A) tail, the reconstructed mRNA encoding the 5 NCR of L-A virus mRNA and a reporter gene (luciferase) was translated, in yeast lysate, 60 times more efficiently than control mRNA. The 5 NCR from L-A virus was effective in regulating the recruitment of ribosome in vitro. A possible mechanism in Saccharomyces cerevisiae is also suggested, whereby the ribosome entry is regulated by the 5 NCR of L-A virus mRNA.     

A novel hairless mouse model on an atopic dermatitis-prone genetic background generated by receptor-mediated transgenesis

Current mouse models for atopic dermatitis (AD) have a serious drawback, being the existence of dense hair on the body. Thus, a hairless animal model on an AD-prone genetic background will be a powerful tool to investigate the basis of and therapy for this complex disease. We applied the Toxin Receptor-mediated Cell Knockout (TRECK) method to generate a hairless transgenic (Tg) mice on the NC/Nga background, an AD-prone inbred strain. A minigene with the mouse Keratin71 (Krt71) promoter and human diphtheria toxin receptor, which intrinsically functions as the heparin-binding EGF-like growth factor, was introduced into the pronucleus of NC/Nga oocytes. Unexpectedly NCN24, one NC/Nga Tg line, showed a dominant hairless phenotype without diphtheria toxin administration. Furthermore, the atopic dermatitis-like predisposition and IgE elevation was observed in both NCN24 and the NC/Nga wildtype strain. NCN24 mice, which we have newly developed, will be useful to assess drugs for AD therapy, being able to monitor skin inflammation without shaving. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Control of cell polarity and motility by the PtdIns(3,4,5)P3 phosphatase SHIP1

Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) regulate cell migration, but the role of PtdIns(3,4,5)P(3)-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a PtdIns(3,4,5)P(3) phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the PtdIns(3,4,5)P(3) phosphatase PTEN had no impact on neutrophil chemotaxis. To study PtdIns(3,4,5)P(3) metabolism in living primary cells, we generated a novel transgenic mouse (AktPH-GFP Tg) expressing a bioprobe for PtdIns(3,4,5)P(3.) Time-lapse footage showed rapid, localized binding of AktPH-GFP to the leading edge membrane of chemotaxing ship1(+/+)AktPH-GFP Tg neutrophils, but only diffuse localization in ship1(-/-)AktPH-GFP Tg neutrophils. By directing where PtdIns(3,4,5)P(3) accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.     

Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange

Genetically engineered pigs with cell markers such as fluorescent proteins are highly useful in lines of research that include the tracking of transplanted cells or tissues. In this study, we produced transgenic-cloned pigs carrying a gene for the newly developed red fluorescent protein, humanized Kusabira-Orange (huKO), which was cloned from the coral stone Fungia concinna. The nuclear transfer embryos, reconstructed with fetal fibroblast cells that had been transduced with huKO cDNA using retroviral vector DDeltaNsap, developed efficiently in vitro into blastocysts (28.0%, 37/132). Nearly all (94.6%, 35/37) of the cloned blastocysts derived from the transduced cells exhibited clear huKO gene expression. A total of 429 nuclear transfer embryos were transferred to four recipients, all of which became pregnant and gave birth to 18 transgenic-cloned offspring in total. All of the pigs highly expressed huKO fluorescence in all of the 23 organs and tissues analyzed, including the brain, eyes, intestinal and reproductive organs, skeletal muscle, bone, skin, and hoof. Furthermore, such expression was also confirmed by histological analyses of various tissues such as pancreatic islets, renal corpuscles, neuronal and glial cells, the retina, chondrocytes, and hematopoietic cells. These data demonstrate that transgenic-cloned pigs exhibiting systemic red fluorescence expression can be efficiently produced by nuclear transfer of somatic cells retrovirally transduced with huKO gene.     

Genetic evidence for key roles of decorin and biglycan in dentin mineralization

Targeted disruption of the dentin sialophosphoprotein (DSPP) gene in the mice (Dspp^?/?) results in dentin mineralization defects with enlarged predentin phenotype similar to human dentinogenesis imperfecta type III. Using DSPP/biglycan (Dspp^?/?Bgn^?/0) and DSPP/decorin (Dspp^?/?Dcn^?/?) double knockout mice, here we determined that the enlarged predentin layer in Dspp^?/? teeth is rescued in the absence of decorin, but not in the absence of biglycan. However, Fourier transform infrared (FTIR) spectroscopy analysis reveals similar hypomineralization of dentin in both Dspp^?/?Bgn^?/0 and Dspp^?/?Dcn^?/? teeth. Atomic force microscopy (AFM) analysis of collagen fibrils in dentin shows subtle differences in the collagen fibril morphology in these genotypes. The reduction of enlarged predentin in Dspp^?/?Dcn^?/? mice suggests that the elevated level of decorin in Dspp^?/? predentin interferes with the mineralization process at the dentin mineralization front. On the other hand, the lack of DSPP and biglycan leads to the increased number of calcospherites in Dspp^?/?Bgn^?/0 predentin, suggesting that a failure in coalescence of calcospherites was augmented in Dspp^?/?Bgn^?/0 teeth as compared to Dspp^?/? teeth. These findings indicate that normal expression of small leucine rich proteoglycans, such as biglycan and decorin, plays an important role in the highly orchestrated process of dentin mineralization.     

On Edge-Corrected Probability Density Function Estimators for Point Processes

FIKSEL (1988) provides an asymptotically unbiased kernel estimator for the density h_s (r) of the spherical contact distribution function of stationary and isotropic point processes. This paper proposes alternative estimators of h_s (r) for use with regular grid of locations. The existing estimator of h_s (r) and the alternatives proposed in this paper are then tested out in a simulation study.     

Sheep Red Blood Cell Instillation at Palatine Tonsil Effectively Induces Specific IgA Class Antibody in Saliva in Rabbits

We have shown that the palatine tonsil effectively incorporates exogenous foreign substances instilled at its surface. It is not clear whether antigen-specific IgA can be induced by the instillation. Sheep red blood cells (SRBC) were instilled at the palatine tonsil every three days as the antigen, and the agglutination titer of specific IgA in saliva was examined. Nasal or intragastric administration, which have been shown to induce specific antibody in saliva, were done as control experiments. Anti-SRBC antibody in saliva from the tonsillar instillation group was detected in the second week, and the agglutination titer reached a maximum in the 6th week after the instillation. The maximum titers in the tonsillar instillation group and nasal administration group were 16 (P<0.01, n=7) and 4 times (P<0.01, n = 7) higher, respectively, than that in the intragastric administration group. In the tonsillar instillation group, the number of specific antibody-producing cells per 10⁵ lymphocytes was the highest in the parotid glands compared with the lymphoid tissues such as the retropharyngeal lymph nodes, nasal mucosa, mesenteric lymph nodes, Peyer's patches, cervical lymph nodes, palatine tonsil and spleen. In the nasal administration group, the number of lymphocytes was the highest in the nasal mucosa. The results indicate that tonsillar instillation was more effective than nasal administration in inducing specific IgA in saliva.