Viral susceptibility, transfection and growth of SPB--a fish neural progenitor cell line from the brain of snubnose pompano, Trachinotus blochii (Lacépède).

This study investigates the susceptibilities of the SPB cell line to fish viruses including giant seaperch iridovirus (GSIV-K1), red sea bream iridovirus (RSIV-Ku), grouper nervous necrosis virus (GNNV-K1), chum salmon reovirus (CSV) and eel herpesvirus (HVA). GSIV-K1, RSIV-Ku and CSV replicated well in SPB cells, with a significant cytopathic effect and virus production. However, the cells were HVA and GNNV refractory. To examine the ability of SPB cells to stably express foreign protein, expression vectors encoding GNNV B1 and B2 fused to enhanced green fluorescent protein (EGFP) and GSIV ORF35L fused to DsRed were constructed and introduced by transfection into SPB cells. Stable transfectants displayed different morphologies compared with SPB and with each other. EGFP-B1 was predominantly localized in the nuclei, EFPF-B2 was distributed throughout the cytoplasm and nucleus, and granular 35L-DsRed was localized with secreted vesicles. The expression of EFPF-B2 in SPB cells produced blebs on the surface, but the cells showing stable expression of EGFP, EGFP-B1 or 35L-DsRed showed normal morphologies. Results show the SPB cells and the transfected cells grow well at temperatures between 20 and 35 °C and with serum-dependent growth. SPB cells are suitable for studies on foreign protein expression and virology.

Establishment and characterization of a fibroblast cell line derived from the dorsal fin of red sea bream, Pagrus major (Temminck & Schlegel).

The establishment and partial characterization of a continuous cell line from the dorsal fin of red sea bream, Pagrus major, are described. The cell line, designated RSBF-2, has been subcultured for more than 100 passages since its initiation in November 2000. It was optimally maintained at 28 degrees C in Leibovitz L-15 medium with 10% foetal bovine serum. Propagation of RSBF-2 cells was serum dependent and exhibited low plating efficiency (<1.7%). Aside from long-term cryopreservation, the cells could also be kept at 4 degrees C for 72 days. The distribution of the chromosome number was 38-98 with a mode of 48. The RSBF-2 cell line was susceptible to red sea bream iridovirus but only produced a few rounded and refractory cells. Virus-inoculated RSBF-2 cells were then subcultured to generate a persistently infected cell line. RSBF-2 was also very sensitive to the extracellular products of Photobacterium damselae ssp. piscicida and produced significant fluorescent signals after transfection with pEGFP-C3. Analysis of mitochondrial cytochrome b gene sequences revealed 99% identity between the cell line and Pagrus major.

Control of homeostasis of CD8+ memory T cells by opposing cytokines.

Memory T cells maintain their numbers for long periods after antigen exposure. Here we show that CD8+ T cells of memory phenotype divide slowly in animals. This division requires interleukin-15 and is markedly increased by inhibition of interleukin-2 (IL-2). Therefore, the numbers of CD8+ memory T cells in animals are controlled by a balance between IL-15 and IL-2.

PCR amplification and sequence analysis of the major capsid protein gene of megalocytiviruses isolated in Taiwan.

Viruses belonging to the genus Megalocytivirus in the family Iridoviridae are one of the major agents causing mass mortalities in marine and freshwater fish in Asian countries. Outbreaks of iridovirus disease have been reported among various fish species in Taiwan. However, the genotypes of these iridoviruses have not yet been determined. In this study, seven megalocytivirus isolates from four fish species: king grouper, Epinephelus lanceolatus (Bloch), barramundi perch, Lates calcarifer (Bloch), silver sea bream, Rhabdosargus sarba (Forsskal), and common ponyfish, Leiognathus equulus (Forsskal), cultured in three different regions of Taiwan were collected. The full open reading frame encoding the viral major capsid protein gene was amplified using PCR. The PCR products of approximately 1581 bp were cloned and the nucleotide sequences were phylogenetically analysed. Results showed that all seven PCR products contained a unique open reading frame with 1362 nucleotides and encoded a structural protein with 453 amino acids. Even though the nucleotide sequences were not identical, these seven megalocytiviruses were classified into one cluster and showed very high homology with red sea bream iridovirus (RSIV) with more than 97% identity. Thus, the seven iridovirus strains isolated from cultured marine fish in Taiwan were closer to the RSIV genotype than the infectious spleen and kidney necrosis virus genotype.

Characteristics and maintenance of CD8+ T-cell clones found in old mice.

Old mice, like old human beings, contain large clones of CD8+ T-cells. These cells grow poorly in tissue culture, therefore it is difficult to maintain the cells in vitro. The cells can be grown after transfer to sublethally irradiated mice. This technique will be useful in further studies on the properties of the cells. Based on observations from such transfer experiments, we conclude that: (1) expansion of the T-cell clones in recipients is dramatic but slow; (2) chance events caused by endogenous antigens or gene mutations rather than exogenous antigens may account for the expansion of these clones; and (3) the expanded T-cell clones are benign and do not cause malignancies.

Diagonal recurrent neural networks for dynamic systems control.

A new neural paradigm called diagonal recurrent neural network (DRNN) is presented. The architecture of DRNN is a modified model of the fully connected recurrent neural network with one hidden layer, and the hidden layer comprises self-recurrent neurons. Two DRNN's are utilized in a control system, one as an identifier called diagonal recurrent neuroidentifier (DRNI) and the other as a controller called diagonal recurrent neurocontroller (DRNC). A controlled plant is identified by the DRNI, which then provides the sensitivity information of the plant to the DRNC. A generalized dynamic backpropagation algorithm (DBP) is developed and used to train both DRNC and DRNI. Due to the recurrence, the DRNN can capture the dynamic behavior of a system. To guarantee convergence and for faster learning, an approach that uses adaptive learning rates is developed by introducing a Lyapunov function. Convergence theorems for the adaptive backpropagation algorithms are developed for both DRNI and DRNC. The proposed DRNN paradigm is applied to numerical problems and the simulation results are included.

Jun dimerization protein 2 is a critical component of the Nrf2/MafK complex regulating the response to ROS homeostasis.

Oxidative stress and reactive oxygen species (ROS) are associated with diseases such as cancer, cardiovascular complications, inflammation and neurodegeneration. Cellular defense systems must work constantly to control ROS levels and to prevent their accumulation. We report here that the Jun dimerization protein 2 (JDP2) has a critical role as a cofactor for transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) and small Maf protein family K (MafK) in the regulation of the antioxidant-responsive element (ARE) and production of ROS. Chromatin immunoprecipitation-quantitative PCR (qPCR), electrophoresis mobility shift and ARE-driven reporter assays were carried out to examine the role of JDP2 in ROS production. JDP2 bound directly to the ARE core sequence, associated with Nrf2 and MafK (Nrf2-MafK) via basic leucine zipper domains, and increased DNA-binding activity of the Nrf2-MafK complex to the ARE and the transcription of ARE-dependent genes. In mouse embryonic fibroblasts from Jdp2-knockout (Jdp2 KO) mice, the coordinate transcriptional activation of several ARE-containing genes and the ability of Nrf2 to activate expression of target genes were impaired. Moreover, intracellular accumulation of ROS and increased thickness of the epidermis were detected in Jdp2 KO mice in response to oxidative stress-inducing reagents. These data suggest that JDP2 is required to protect against intracellular oxidation, ROS activation and DNA oxidation. qPCR demonstrated that several Nrf2 target genes such as heme oxygenase-1, glutamate-cysteine ligase catalytic and modifier subunits, the notch receptor ligand jagged 1 and NAD(P)H dehydrogenase quinone 1 are also dependent on JDP2 for full expression. Taken together, these results suggest that JDP2 is an integral component of the Nrf2-MafK complex and that it modulates antioxidant and detoxification programs by acting via the ARE.

Androgen receptor-mediated apoptosis in bovine testicular induced pluripotent stem cells in response to phthalate esters.

The androgen receptor (AR) has a critical role in promoting androgen-dependent and -independent apoptosis in testicular cells. However, the molecular mechanisms that underlie the ligand-independent apoptosis, including the activity of AR in testicular stem cells, are not completely understood. In the present study, we generated induced pluripotent stem cells (iPSCs) from bovine testicular cells by electroporation of octamer-binding transcription factor 4 (OCT4). The cells were supplemented with leukemia inhibitory factor and bone morphogenetic protein 4, which maintained and stabilized the expression of stemness genes and pluripotency. The iPSCs were used to assess the apoptosis activity following exposure to phthalate esters, including di (2-ethyhexyl) phthalates, di (n-butyl) phthalate, and butyl benzyl phthalate. Phthalate esters significantly reduced the expression of AR in iPSCs and induced a higher ratio of BAX/BCL-2, thereby favoring apoptosis. Phthalate esters also increased the expression of cyclin-dependent kinase inhibitor 1 (p21(Cip1)) in a p53-dependent manner and enhanced the transcriptional activity of p53. The forced expression of AR and knockdown of p21(Cip1) led to the rescue of the phthalate-mediated apoptosis. Overall, this study suggests that testicular iPSCs are a useful system for screening the toxicity of environmental disruptors and examining their effect on the maintenance of stemness and pluripotency, as well as for identifying the iPSC signaling pathway(s) that are deregulated by these chemicals.

CD8+ T-cell clones in old mice.

Most old mice and human beings contain large clones of CD8+ alpha beta TCR+ T cells. In mice, clones bearing V beta 7 appear more frequently in animals infected with mouse hepatitis virus than in uninfected animals. This property is controlled by some non-MHC gene in the animals. The frequency of old mice containing such clones is affected by the origin of the animals. Although the clones are relatively anergic to acute stimuli in vitro, they can divide in vivo since in old animals they divide and turnover with about the same kinetics as other, non-clonally expanded CD8+ T cells. Moreover the clones expand slowly but inexorably after transfer into recipient animals. These data suggest that the CD8+ alpha beta TCR clones arise because they are specific for some exogenous or auto antigen to which the cells are continuously exposed in vivo.

The growth of the very large CD8+ T cell clones in older mice is controlled by cytokines.

Older humans and mice frequently contain very large clones of CD8(+) T cells. In mice these cells are phenotypically very similar to memory CD8(+) T cells. Like memory CD8(+) T cells, most members of the clones are in continuous slow division, apparently independently of Ag stimulation. Proliferation of the CD8(+) clonal T cells is inhibited in mice treated with Ab to the IL-2R beta-chain that blocks signaling by either IL-2 or IL-15. However, inhibition of IL-2 increases the numbers of dividing clonal cells. Therefore, like normal memory CD8(+) T cells, expansion of the clones is driven by IL-15 and inhibited by IL-2 and is probably limited by the amounts of IL-15 and IL-2 present in the host. Control by these two cytokines may account for the fact that, although the clones can be very large, they do not overwhelm or kill their hosts. Nevertheless the clonal cells compete successfully with normal memory CD8(+) T cells for growth. Perhaps the clonal cells use IL-15 more effectively or are more resistant to the inhibitory effects of IL-2. Thus they might affect the immune response of their hosts by competing for factors that stimulate and inhibit normal CD8(+) memory T cells.

Studies on epizootic iridovirus infection among red sea bream, Pagrus major (Temminck & Schlegel), cultured in Taiwan.

Since 1993, an epizootic viral disease has occurred in net-cage cultured red sea bream, Pagrus major (Temminck & Schlegel), in Peng-hu Island located on the south-western coast of Taiwan. The diseased fish exhibited abnormal swimming and were lethargic, but few visible external signs were observed. The cumulative mortality because of the disease sometimes reached 50-90% over 2 months. Histopathogical studies of the affected fish showed enlarged basophilic cells in the gill, kidney, heart, liver and spleen. These necrotic cells were Feulgen-positive and stained blue using Giemsa. Transmission electron microscopy revealed icosahedral virions in the cytoplasm of the necrotic cells. The viral particles consisted of a central nucleocapsid (75-80 nm) and envelope, and were 120-150 nm in diameter. These results suggest that the virus belongs to the Iridoviridae. Using polymerase chain reaction (PCR), approximately 570 bp fragments were produced from the viral DNA using as a template 1-F and 1-R primers derived from red seabream iridovirus (RSIV) from red sea bream in Japan. Similar results were also obtained using nested-PCR with different primer sets (1-F, 2-R and 2-F, 1-R). Although the size and some features of epizootics of this virus differed from RSIV in Japan, it shows close genetic affinities with the latter and it is suggested that RSIV has been introduced to Taiwan.

Homologous and heterologous neutralization antibody responses after immunization with Japanese encephalitis vaccine among Taiwan children.

Because 21 immunized children (13%) among the 162 confirmed Japanese encephalitis (JE) cases during 1986-1991 occurred in Taiwan, we collected 320 serum samples from Taiwan children aged 15-31 and 27-44 months immediately before the 1st dose (n = 41) and 1-3 months after the 2nd dose (n = 78, 27 pairs), and immediately before (n = 58) and 1-3 months after the 3rd dose (n = 143, 44 pairs) to determine neutralization antibody (Nt Ab) against the Nakayama (N) and Beijing-1 (B) strains and two Taiwan wild type JE viruses (JEV): CC-27 and CH-1392. Our Nt results showed that (1) B vaccine stimulated a better homologous Ab response than N vaccine for Nt Ab seropositivity rate (NASR), produced a higher level of Nt titer after the primary immunization [2 doses = 100% vs. 91%, geometric mean titer (GMT) = 115 vs. 22], had a greater booster effect (3 doses: 100% vs. 95%; GMT = 320 vs 33), and showed a better capability to neutralize two local Taiwan JEV strains, particularly only after 3 doses (ave. NASR for B vs. N = 90% vs. 10%; and GMT for B vs. N = 154 vs. 1); (2) the two wild type JEV strains had different plaque morphology and antigenic variation and the CC-27 strain was not neutralized as well as the CH-1392 strain after 3 doses of vaccine (BBB or NNN or NNB); and (3) 30% of the children had lost JEV Nt Ab one year after the 2nd dose of N vaccine and natural infection with JE virus did occur among those children after immunization. In conclusion, (1) three doses of mouse-brain vaccine are the minimum requirement to protect children against the local Taiwan JEV-, (2) the best strain for a JE vaccine depends on level of Nt Ab it induced, the molecular epidemiology and antigenic variation of the JEV in each local area; and (3) future vaccine must produce better B- and T-cell memory.

Mutational analysis of the repeated open reading frames, ORFs 63 and 70 and ORFs 64 and 69, of varicella-zoster virus.

Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative AvrII site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.