Myogenic differentiation of p53- and Rb-deficient immortalized and transformed bovine fibroblasts in response to MyoD

We have established in culture a spontaneously immortalized bovine embryonic fibroblast (BEF) cell line that has lost p53 and p16(INK4a) functions. MyoD is a muscle-specific regulator capable of inducing myogenesis in a number of cell types. When the BEF cells were transduced with MyoD they differentiated efficiently to desmin-positive myofibers in the presence of 2% horse serum and 1.7 nM insulin. The myogenic differentiation of this cell line was more rapid and obvious than that of C2C12 cells, as judged by morphological changes and expression of various muscle regulatory factors. To confirm that lack of the p53 and p16(INK4a) pathway does not prevent MyoD-mediated myogenesis, we established a cell line transformed with SV40LT (BEFV) and introduced MyoD into it. In the presence of 2% horse serum and 1.7 nM insulin, the MyoD-transduced BEFV cells differentiated like the MyoD-transduced BEFS cells, and displayed a similar pattern of expression of muscle regulatory proteins. Taken together, our results indicate that MyoD overexpression overcomes the defect in muscle differentiation associated with immortalization and cell transformation caused by the loss of p53 and Rb functions.     

Establishment of immortal swine kidney epithelial cells

Using normal swine kidney epithelial (SKE) cells that were shown to be senescent at passages 12 to 14, we have established one lifespan-extended cell line and two lifespan-extended cell lines by exogenous introduction of the human catalytic subunit of telomerase (hTERT) and simian virus 40 large T-antigen (SV40LT), all of which maintain epithelial morphology and express cytokeratin, a marker of epithelial cells. SV40LT- and hTERT-transduced immortal cell lines appeared to be smaller and exhibited more uniform morphology relative to primary and spontaneously immortalized SKE cells. We determined the in vitro lifespan of primary SKE cells using a standard 3T6 protocol. There were two steps of the proliferation barrier at 12 and 20, in which a majority of primary SKE cells appeared enlarged, flattened, vacuolated, and ss-galactosidase-positive, all phenotypical characteristics of senescent cells. Lifespan-extended SKE cells were eventually established from most of the cellular foci, which is indicative of spontaneous cellular conversion at passage 23. Beyond passage 25, the rate of population doubling of the established cells gradually increased. At passage 30, immortal cell lines grew faster than primary counterpart cells in 10% FBS-DMEM culture conditions, and only SV40LT-transduced immortal cells grew faster than primary and other SKE immortal cells in 0.5% FBS-DMEM. These lifespan-extended SKE cell lines failed to grow in an anchorage-independent manner in soft-agar dishes. Hence, three immortalized swine kidney epithelial cells that are not transformed would be valuable biological tools for virus propagation and basic kidney epithelial cell research.     

Membrane-delimited regulation of novel background K+ channels by MgATP in murine immature B cells

In WEHI-231, a representative immature B cell line, Ca(2+) entry is paradoxically augmented by treatment with 2-aminoethoxydiphenyl borate (2-APB), a blocker of inositol 1,4,5-trisphosphate receptor and of nonselective cation channels (Nam, J. H., Yun, S. S., Kim, T. J., Uhm, D.-Y., and Kim, S. J. (2003) FEBS Lett. 535, 113-118). The initial goal of the present study was to elucidate the effects of 2-APB on membrane currents, which revealed the presence of novel K(+) channels in WEHI-231 cells. Under whole-cell patch clamp conditions, 2-APB induced background K(+) current (I(K,bg)) and hyperpolarization in WEHI-231 cells. Lowering of intracellular MgATP also induced the I(K,bg). The I(K,bg) was blocked by micromolar concentrations of quinidine but not by tetraethylammonium. In a single channel study, two types of voltage-independent K(+) channels were found with large (346 picosiemens) and medium conductance (112 picosiemens), named BK(bg) and MK(bg), respectively. The excision of membrane patches (inside-out (i-o) patches) greatly increased the P(o) of BK(bg). In i-o patches, cytoplasmic MgATP (IC(50) = 0.18 mm) decreased the BK(bg) activity, although non-hydrolyzable adenosine 5'-(beta,gamma-imino)triphosphate had no effect. A pretreatment with Al(3+) or wortmannin (50 microm) blocked the inhibitory effects of MgATP. A direct application of phosphoinositide 4,5-bisphosphate (10 microm) inhibited the BK(bg) activity. Meanwhile, the activity of MK(bg) was unaffected by MgATP. In cell-attached conditions, the BK(bg) activity was largely increased by 2-APB. In i-o patches, however, the MgATP-induced inhibition of BK(bg) was weakly reversed by the addition of 2-APB. In summary, WEHI-231 cells express the unique background K(+) channels. The BK(bg)s are inhibited by membrane-delimited elevation of phosphoinositide 4,5-bisphosphate. The activation of BK(bg) would hyperpolarize the membrane, which augments the calcium influx in WEHI-231 cells.     

S-Nitrosation regulates the activation of endogenous procaspase-9 in HT-29 human colon carcinoma cells

Nitric oxide-mediated signals have been suggested to regulate the activity of caspases negatively, yet literature has provided little direct evidence. We show in this paper that cytokines and nitric-oxide synthase (NOS) inhibitors regulate S-nitrosation of an initiator caspase, procaspase-9, in a human colon adenocarcinoma cell line, HT-29. A NOS inhibitor, N(G)-methyl-l-arginine, enhanced the tumor necrosis factor-alpha (TNF-alpha)-induced cleavage of procaspase-9, procaspase-3, and poly-(ADP-ribose) polymerase, as well as the level of apoptosis. N(G)-Methyl-l-arginine, however, did not affect the cleavage of procaspase-8. These results suggest that nitric oxide regulates the cleavage of procaspase-9 and its downstream proteins and, subsequently, apoptosis in HT-29 cells. Labeling S-nitrosated cysteines with a biotin tag enabled us to reveal S-nitrosation of endogenous procaspase-9 that was immunoprecipitated from the HT-29 cell extracts. Furthermore, the treatment with TNF-alpha, as well as NOS inhibitors, decreased interferon-gamma-induced S-nitrosation in procaspase-9. Our results show that S-nitrosation of endogenous procaspase-9 occurs in the HT-29 cells under normal conditions and that denitrosation of procaspase-9 enhances its cleavage and consequent apoptosis. We, therefore, suggest that S-nitrosation regulates activation of endogenous procaspase-9 in HT-29 cells.     

Teratological studies of prenatal exposure of mice to a 20 kHz sawtooth magnetic field

In order to evaluate the importance of gestational age in possible effects due to exposure to a 20 kHz sawtooth magnetic field, pregnant ICR mice at gestational 2.5-15.5 days post-coitus, which is the most sensitive stage for the induction of major congenital malformations, were exposed in a carrousel irradiator. The mice were exposed to a 20 kHz intermediate frequency (IF) sawtooth magnetic field had a 6.5 microT peak intensity for 8 h/day. The animals were sacrificed on the 18th day of gestation; and the fetuses were examined for mortality, growth retardation, changes in head size, and other morphological abnormalities. From the above conditions, it is concluded that the exposure to a 20 kHz sawtooth magnetic field with 6.5 microT peak intensity does not inflict any adverse effect on fetuses of pregnant mice.     

Evaluation of protective efficacy of respiratory syncytial virus vaccine against A and B subgroup human isolates in Korea

Human respiratory syncytial virus (HRSV) is a significant cause of upper and lower respiratory tract illness mainly in infants and young children worldwide. HRSV is divided into two subgroups, HRSV-A and HRSV-B, based on sequence variation within the G gene. Despite its importance as a respiratory pathogen, there is currently no safe and effective vaccine for HRSV. In this study, we have detected and identified the HRSV by RT-PCR from nasopharyngeal aspirates of Korean pediatric patients. Interestingly, all HRSV-B isolates exhibited unique deletion of 6 nucleotides and duplication of 60 nucleotides in the G gene. We successfully amplified two isolates ('KR/A/09-8' belonging to HRSV-A and 'KR/B/10-12' to HRSV-B) on large-scale, and evaluated the cross-protective efficacy of our recombinant adenovirus-based HRSV vaccine candidate, rAd/3xG, by challenging the immunized mice with these isolates. The single intranasal immunization with rAd/3xG protected the mice completely from KR/A/09-8 infection and partially from KR/B/10-12 infection. Our study contributes to the understanding of the genetic characteristics and distribution of subgroups in the seasonal HRSV epidemics in Korea and, for the first time, to the evaluation of the cross-protective efficacy of RSV vaccine against HRSV-A and -B field-isolates.