Interspecies transmission of the canine influenza H3N2 virus to domestic cats in South Korea, 2010.

In the past 4 years, incidences of endemic or epidemic respiratory diseases associated with canine influenza H3N2 virus in Asian dogs have been reported in countries such as South Korea and China. Canine species were considered to be the new natural hosts for this virus. However, at the beginning of 2010, influenza-like respiratory signs, such as dyspnoea, were also observed among cats as well as in dogs in an animal shelter located in Seoul, South Korea. The affected cats showed 100 % morbidity and 40 % mortality. We were able to isolate a virus from a lung specimen of a dead cat, which had suffered from the respiratory disease, in embryonated-chicken eggs. The eight viral genes isolated were almost identical to those of the canine influenza H3N2 virus, suggesting interspecies transmission of canine influenza H3N2 virus to the cat. Moreover, three domestic cats infected with intranasal canine/Korea/GCVP01/07 (H3N2) all showed elevated rectal temperatures, nasal virus shedding and severe pulmonary lesions, such as suppurative bronchopneumonia. Our study shows, for the first time, that cats are susceptible to canine influenza H3N2 infection, suggesting that cats may play an intermediate host role in transmitting the H3N2 virus among feline and canine species, which could lead to the endemic establishment of the virus in companion animals. Such a scenario raises a public health concern, as the possibility of the emergence of new recombinant feline or canine influenza viruses in companion animals with the potential to act as a zoonotic infection cannot be excluded.

Identification of tyrosine-nitrated proteins in HT22 hippocampal cells during glutamate-induced oxidative stress.

OBJECTIVES: Nitration of tyrosine residues in protein is a post-translational modification, which occurs under oxidative stress, and is associated with several neurodegenerative diseases. To understand the role of nitrated proteins in oxidative stress-induced cell death, we identified nitrated proteins and checked correlation of their nitration in glutamate-induced HT22 cell death. MATERIALS AND METHODS: Nitrated proteins were detected by western blotting using an anti-nitrotyrosine antibody, extracted from matching reference 2-dimensional electrophoresis gels, and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: Glutamate treatment induced apoptosis in HT22 cells, while reactive oxygen species (ROS) inhibitor or neuronal nitric oxide synthase (nNOS) inhibitor blocked glutamate-induced HT22 cell death. Nitration levels of 13 proteins were increased after glutamate stimulation; six of them were involved in regulation of energy production and two were related to apoptosis. The other nitrated proteins were associated with calcium signal modulation, ER dysfunction, or were of unknown function. CONCLUSIONS: The 13 tyrosine-nitrated proteins were detected in these glutamate-treated HT22 cells. Results demonstrated that cell death, ROS accumulation and nNOS expression were related to nitration of protein tyrosine in the glutamate-stimulated cells.

alpha-MSH inhibits TNF-alpha-induced matrix metalloproteinase-13 expression by modulating p38 kinase and nuclear factor kappaB signaling in human chondrosarcoma HTB-94 cells.

OBJECTIVE: Proinflammatory cytokine-induced expression of matrix metalloproteinases (MMPs) is a major cause of arthritic cartilage destruction. The neuropeptide, alpha-melanocyte-stimulating hormone (alpha-MSH), has been detected in the synovial fluid of arthritis patients, where it is thought to play an anti-inflammatory role. Here, we examined whether alpha-MSH acts via downregulation of MMP expression, and sought to elucidate the intracellular signal pathways underlying this effect. DESIGN: Human chondrosarcoma cell line, HTB-94 (SW1353) was pretreated with or without alpha-MSH and then treated with tumor necrosis factor-alpha (TNF-alpha). The effect of alpha-MSH on TNF-alpha-induced MMP-13 expression and mitogen-activated protein kinases' (MAPKs) activation were determined by reverse transcriptase-polymerase chain reaction and Western blot analysis. Additionally, the intracellular signaling of alpha-MSH was investigated using the inhibitors of MAPK and nuclear factor kappaB (NF-kappaB) and plasmids encoding dominant negative (dn) forms of inhibitor kappaB kinase-alpha (IKKalpha) and inhibitor kappaB kinase-beta (IKKbeta). RESULTS: We found that alpha-MSH pretreatment inhibited TNF-alpha-induced MMP-13 expression and p38 kinase phosphorylation in HTB-94 human chondrosarcoma cells. TNF-alpha-induced MMP-13 expression was not suppressed by extracellular signal-regulated kinase (ERK) inhibitors (PD98059 and U0126) or a c-jun terminal kinase (JNK) inhibitor (SP600125), but was inhibited by inhibitors of p38 kinase (SB203580) and NF-kappaB (SN-50 peptide) and dnIKKalpha and dnIKKbeta. CONCLUSIONS: Our results suggest that alpha-MSH regulates TNF-alpha-induced MMP-13 expression by decreasing p38 kinase phosphorylation and subsequent NF-kappaB activation in human chondrocytes and may be an effective inhibitor of MMP-13-mediated collagen degradation, providing new potential opportunities for the development of anti-arthritis therapeutics.

The human hepatitis B virus transactivator X gene product regulates Sp1 mediated transcription of an insulin-like growth factor II promoter 4.

Human hepatitis B virus (HBV) is one of the causative agents of hepatocellular carcinoma (HCC). The virus encodes a 17 kDa protein, X, which is known to be a causative agent in the formation of HCC. An insulin-like growth factor-II (IGF-II) is expressed during the formation of HCC. Among the four promoters of the IGF-II gene, promoters 2, 3 and 4 become activated during the formation of HCC. The high frequency of detection of hepatitis B virus X (HBV-X) antigen in liver cells from patients with chronic hepatitis, cirrhosis, and liver cancer suggested that the expressions of HBV-X and IGF-II are associated. Studies were carried out to test the relationship between the HBV-X gene product and the activation of IGF-II promoter 4. We demonstrated that the HBV-X protein increases the endogenous IGF-II expression from promoter 3 and 4 of IGF-II gene. Analysis of the fourth promoter of IGF-II gene showed that the HBV-X gene product positively regulates transcription. Two copies of a motif are responsible for conferring HBV-X regulation on the fourth promoter of IGF-II. These motifs have been identified as Sp1 binding sites. Sp1 binding to IGF-II P4 promoter was identified by gel mobility shift assay using purified Sp1. By using a GAL4-Sp1 fusion protein it was demonstrated that HBV-X positively regulates the Spl mediated transcriptional activity of IGF-II in vivo. A protein-affinity chromatography experiment showed that HBV-X protein does not bind directly to Sp1, but HBV-X does augment the DNA binding activity of the phosphorylated form of Sp1 in HepG2 cells. Sp1 was phosphorylated by HBV-X and its DNA-binding activity was up-regulated upon HBV-X transfections. Various HBV-X mutant expression vectors were used for the demonstration of specific interactions between Sp1 and HBV-X. These results indicate that HBV-X functions as a positive regulator of transcription, and that Sp1 is a direct target for the transcriptional regulation of IGF-II. Increasing the DNA binding ability of the phosphorylated form of Sp1 by HBV-X might be an important mechanism for regulating the IGF-II gene expression and possibly promoting cell division during hepatic carcinogenesis. Our experimental results suggest that expression of HBV-X might induce the expression of IGF-II and the IGF-II might play a role in hepatitis B virus pathogenesis during the formation of HCC.

Establishment and characterization of cell lines constitutively expressing hepatitis B virus X-protein.

We prepared human hepatoma cell lines, which expressed the human hepatitis B virus-X gene product. The plasmid pMAMneo-X, containing an HBV-X gene promoter, an enhancer and a structural gene was constructed. Transfected HBV-X gene integration and expression were detected by Southern and Northern blotting, as well as by chloramphenicol acetylase transferase (CAT) assay using various kinds of promoter-CAT reporter systems. HBV-X protein expression in stable transfectants was confirmed by immunofluorescence microscopy. Transfected cell lines showed permanent expression of HBV-X proteins. The HBV-X transfectant activated its target promoters in promoter-CAT constructs as reporters. The HBV-X transfectant enhanced AP-1 transcription factor binding to its target DNA. Therefore, X-transfectants are not only stable, but also have specific biological functions. Cell cycle analysis by flow cytometry showed that the majority of the transfectant cells are arrested in the G1 or G2 phase of the cell cycle. These cell lines may be useful in analyzing the biological functions of HBV-X and its functional role in the formation of hepatocellular carcinomas.

Interaction of Fc gamma receptor type IIIB with complement receptor type 3 in fibroblast transfectants: evidence from lateral diffusion and resonance energy transfer studies.

To explore potential inter-receptor interactions between Fc gamma RIIIB, a GPI-linked protein, and the leukocyte integrin CR3, we have prepared transfected 3T3 fibroblast cell lines expressing Fc gamma RIIIB, CR3, or both Fc gamma RIIIB and CR3. We test the hypothesis that Fc gamma RIIIB and CR3 are physically associated in membranes using fluorescence recovery after photobleaching (FRAP) and resonance energy transfer (r.e.t.) microscopy. Cells expressing Fc gamma RIIIB alone displayed a diffusion coefficient (D) of 3.4 x 10(-9) (+/- 2.9 x 10(-9) cm2/second and a mobile fraction (m.f.) of 0.73 (+/- 0.10). In contrast, Fc gamma RIIIB exhibited D = 2.5 x 10(-9) (+/- 1.4 x 10(-9) cm2/second (n.s.) and a m.f. of 0.48 (+/- 0.08) (p < 0.01) on cells expressing both Fc gamma RIIB and CR3, thus indicating that co-expression of CR3 constrains the lateral diffusion of Fc gamma RIIIB. To further test for a direct physical interaction between these gene products, (r.e.t.) microscopy was performed. Donor-labeled anti-CR3 and acceptor-labeled anti-Fc gamma RIIIB on cells expressing both receptors yielded a r.e.t. photon count rate of 8.9(+/- 6.4) kilocounts/second (kC/s), whereas CR3-to-CR3 measurements gave 1.6(+/- 0.6) kC/s (p < 0.01). Moreover, the addition of exogenous agents such as N-acetyl-D-glucosamine, but not indomethacin, diminished the magnitude of these interactions in transfectant membranes. These data support the notion that a subpopulation of Fc gamma RIIIB is physically associated with CR3 and that this association can be affected by exogeneous compounds.

Ligation of CD3 triggers transmembrane proximity between LFA-1 and cortical microfilaments in a cytotoxic T cell clone derived from tumor infiltrating lymphocytes: a quantitative resonance energy transfer microscopy study.

We have explored the transmembrane associations of leukocyte function associated antigen-1 (LFA-1) in response to T cell receptor ligation using resonance energy transfer (r.e.t.) microscopy to detect receptor to microfilament proximity. R.e.t. was detected using both imaging and photon counting techniques. T cells were labeled with fluorescein-conjugated F(ab')2 fragments of an anti-LFA-1 monoclonal antibody. Cells were incubated at 37 degrees C on unmodified glass surfaces and surfaces coated with anti-CD3 or anti-H-9 antibodies. Microfilaments of fixed cells were labeled with rhodamine-phalloidin. R.e.t. was not affected on unmodified (blank) or irrelevant antibody-treated (H-9) surfaces. However, both fluorescence images and photon count rates were significantly enhanced when cells bound to anti-CD3-coated surfaces. This enhancement was not due to a general effect of T cell activation on transmembrane cytoskeletal proximity since CD45-phalloidin r.e.t. was not affected by CD3 ligation. These experiments provide direct physical evidence that ligation of the CD3 complex specifically increases the proximity of LFA-1 and microfilaments, which may be relevant to T cell mediated adherence reactions.

Surface-bound immune complexes trigger transmembrane proximity between complement receptor type 3 and the neutrophil's cortical microfilaments.

To characterize the transmembrane associations participating in antibody-dependent binding, we have used resonance energy transfer (r.e.t.) microscopy to assess the molecular proximity of complement receptor type 3 (CR3) and Fc gamma R type II (Fc gamma RII) and type III (Fc gamma RIII) with microfilaments during neutrophil adherence to untreated surfaces, surfaces coated with BSA, surfaces coated with BSA/anti-BSA F(ab')2 complexes, or surfaces coated with BSA/anti-BSA rabbit IgG immune complexes. Receptors were labeled with fluorescein-conjugated antireceptor Fab fragments, whereas microfilaments were labeled with rhodamine-phalloidin. CR3-to-microfilament r.e.t. was dramatically increased in neutrophils adherent to IgG immune complex-coated surfaces but not untreated or control surfaces. However, the low level of r.e.t. between donor-labeled anti-Fc gamma RII Fab fragments and rhodamine-phalloidin was not affected by any condition including surface-bound immune complexes. Significant r.e.t. levels between Fc gamma RIII and microfilaments were not found under any condition. We suggest that CR3 plays an important role in tethering surface-attached immune complexes to the neutrophil's cytoskeleton.

[Modification of Stamey's endoscopic cervicopexy for stress urinary incontinence]. / Modificaciones de la cervicopexia endoscópica de Stamey para la incontinencia urinaria de estrés.

The modifications made by the authors to Stamey's endoscopic suspension of the vesical neck are described. These are analyzed, explained, and their advantages are highlighted. The modified technique has been utilized in 51 female patients with stress urinary incontinence with a success rate of 92.1% and furthermore, without detriment to the benefits afforded by the Stamey procedure.

Transformation of a filamentous cyanobacterium by electroporation.

The filamentous cyanobacterium Anabaena sp. strain M131 was transformed with the shuttle vector pRL6 by electroporation. Optimum conditions for electroporation required relatively high field strengths with short time constants. Restriction significantly lowered the efficiency of transformation. A plasmid containing a single unmodified AvaII restriction site transformed cells with about 100-fold-lower efficiency than did the same plasmid with a modified restriction site.