Homogeneous tetracycline-regulatable gene expression in mammalian fibroblasts

The expression of transfected genes in mammalian cells is rapidly repressed by epigenetic mechanisms such that, within a matter of weeks, only a fraction of the cells in most clonal populations still exhibit detectable expression. This problem can become prohibitive when one wants to express two ectopically introduced genes, as is necessary to establish cell lines that harbor genes regulated by the tetracycline-controlled transactivators. We describe an approach to establish Chinese hamster ovary (CHO) cell lines that stably induce a tet-responsive reporter gene in all cells of a transfected clonal population. Screening of more than 100 colonies resulting from a standard co-transfection of the tetracycline transactivator (tTA) with a green fluorescent protein (GFP) reporter plasmid failed to identify a single colony that could induce GFP in more than 20% of cells. The presence of chromatin insulator sequences, previously shown to protect some transfected genes from epigenetic silencing, moderately improved stability but was not sufficient to produce homogeneous transformants. However, when cell lines were first established in which selection could be maintained either for the expression of tTA activity (co-transfection with a tTA-responsive selectable marker) or the presence of tTA mRNA (bicistronic message encoding a selectable marker), these cell lines could be subsequently transfected with the GFP reporter construct, and nearly 10% of the resulting colonies exhibited stable homogeneous tet-responsive GFP expression in 100% of the expanded clonal cell population.     

A 20-kDa protein with the GTP-binding and trypsin inhibitory activities from Glycine max

A 20-kDa protein (p20) with a GTP binding activity was purified from the cultured cells of Glycine max (soybean). The amino acid sequence of p20 showed 65% identity in a 23 amino acid overlap against the Kunitz-type trypsin inhibitor of soybean reported. Furthermore, it was found that a Kunitz-type soybean trypsin inhibitor of commercial origin also binds GTP.     

Asymmetric reduction of ethyl 2-methyl 3-oxobutanoate by Chlorella

Chlorella pyrenoidosa Chick reduced ethyl 2-methyl 3-oxobutanoate to the corresponding alcohols with the diastereomer (anti/syn) ratio of 53/47. The enantiomer excesses of anti-(2S, 3S)- and syn-(2S, 3R)-hydroxy esters were 89 and > 99ee% respectively. C. vulgaris and C. regularis afforded predominantly the syn-isomer, contrary to C. pyrenoidosa. The differences in the activity of reducing ethyl 2-methyl 3-oxobutanoate were observed among three strains of Chlorella. Addition of 2% metal salts slightly increased the chemical yield of the hydroxy ester.     

The residues between the two transformation effector sites of Epstein-Barr virus latent membrane protein 1 are not critical for B-lymphocyte growth transformation

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation of primary B lymphocytes. LMP1 spontaneously aggregates in the plasma membrane and enables two transformation effector sites (TES1 and TES2) within the 200-amino-acid cytoplasmic carboxyl terminus to constitutively engage the tumor necrosis factor receptor (TNFR)-associated factors TRAF1, TRAF2, TRAF3, and TRAF5 and the TNFR-associated death domain proteins TRADD and RIP, thereby activating NF-kappaB and c-Jun N-terminal kinase (JNK). To investigate the importance of the 60% of the LMP1 carboxyl terminus that lies between the TES1-TRAF and TES2-TRADD and -RIP binding sites, an EBV recombinant was made that contains a specific deletion of LMP1 codons 232 to 351. Surprisingly, the deletion mutant was similar to wild-type (wt) LMP1 EBV recombinants in its efficiency in transforming primary B lymphocytes into lymphoblastoid cell lines (LCLs). Mutant and wt EBV-transformed LCLs were similarly efficient in long-term outgrowth and in regrowth after endpoint dilution. Mutant and wt LMP1 proteins were also similar in their constitutive association with TRAF1, TRAF2, TRAF3, TRADD, and RIP. Mutant and wt EBV-transformed LCLs were similar in steady-state levels of Bcl2, JNK, and activated JNK proteins. The wt phenotype of recombinants with LMP1 codons 232 to 351 deleted further demarcates TES1 and TES2, underscores their central importance in B-lymphocyte growth transformation, and provides a new perspective on LMP1 sequence variation between TES1 and TES2.     

An Epstein-Barr virus that expresses only the first 231 LMP1 amino acids efficiently initiates primary B-lymphocyte growth transformation

An Epstein-Barr virus (EBV) recombinant (MS231) that expresses the first 231 amino acids (aa) of LMP1 and is truncated 155 aa before the carboxyl terminus transformed resting B lymphocytes into lymphoblastoid cell lines (LCLs) only when the infected cells were grown on fibroblast feeder cells (K. M. Kaye et al., J. Virol. 69:675-683, 1995). Higher-titer MS231 virus has now been compared to wild-type (WT) EBV recombinants for the ability to cause resting primary B-lymphocyte transformation. Unexpectedly, MS231 is as potent as WT EBV recombinants in causing infected B lymphocytes to proliferate in culture for up to 5 weeks. When more than one transforming event is initiated in a microwell, the MS231 recombinant supports efficient long-term LCL outgrowth and fibroblast feeder cells are not required. However, with limited virus input, MS231-infected cells differed in their growth from WT virus-infected cells as early as 6 weeks after infection. In contrast to WT virus-infected cells, most MS231-infected cells could not be grown into long-term LCLs. Thus, the LMP1 amino-terminal 231 aa are sufficient for initial growth transformation but the carboxyl-terminal 155 aa are necessary for efficient long-term outgrowth. Despite the absence of the carboxyl-terminal 155 aa, MS231- and WT-transformed LCLs are similar in latent EBV gene expression, in ICAM-1 and CD23 expression, and in NF-kappaB and c-jun N-terminal kinase activation. MS231 recombinant-infected LCLs, however, require 16- to 64-fold higher cell density than WT-infected LCLs for regrowth after limiting dilution. These data indicate that the LMP1 carboxyl-terminal 155 aa are important for growth at lower cell density and appear to reduce dependence on paracrine growth factors.     

Bleomycin stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activity

We determined whether human lung fibroblasts might release chemotactic activity for neutrophils (NCA) and monocytes (MCA) in response to bleomycin. The human lung fibroblasts supernatant fluids were evaluated for chemotactic activity by a blind well chamber technique. Human lung fibroblasts released NCA and MCA in a dose- and time-dependent manner in response to bleomycin. Checkerboard analysis of supernatant fluids revealed that both NCA and MCA were chemotactic. Partial characterization revealed that NCA was partly heat labile, trypsin sensitive, and predominantly ethyl acetate extractable. In contrast, MCA was partly trypsin sensitive and ethyl acetate extractable. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular sieve column chromatography revealed that both NCA and MCA had multiple chemotactic peaks. NCA was inhibited by leukotriene B4 receptor antagonist and anti-IL-8 and G-CSF Abs. MCA was attenuated by leukotriene B4 receptor antagonist, and monocyte chemoattractant protein-1, GM-CSF, and TGF-beta Abs. Leukotriene B4 receptor antagonist and these Abs inhibited the corresponding m.w. chemotactic activity separated by column chromatography. The concentrations of IL-8, G-CSF, monocyte chemoattractant protein-1, GM-CSF, and TGF-beta in the supernatant fluids significantly increased in response to bleomycin. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to bleomycin.     

Overexpression of regucalcin suppresses cell death and apoptosis in cloned rat hepatoma H4-II-E cells induced by lipopolysaccharide, PD 98059, dibucaine, or Bay K 8644

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 12-72 h in medium without FBS containing either vehicle or lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml). The number of wild-type cells was significantly decreased by culture for 24 or 48 h in the presence of LPS (0.1 or 1.0 microg/ml). The effect of LPS (0.1 or 1.0 microg/ml) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. However, the culture with LPS (0.1 or 1.0 microg/ml) for 72 h caused a significant decrease in cell number of transfectants. Ca(2+)/calmodulin-dependent nitric oxide (NO) synthase activity was significantly decreased by culture with LPS (1.0 microg/ml) for 24-72 h of wild-type cells. This decrease was significantly prevented in transfectants. LPS (0.1 or 1.0 microg/ml)-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M). Moreover, the number of wild-type cells was significantly decreased by culture with PD 98059 (10(-6) M), dibucaine (10(-6) M), or staurosporine (10(-6) M), which is an inhibitor of various protein kinases. The effect of PD 98059 or dibucaine on the number of wild-type cells was not observed in transfectants, although the effect of staurosporine was seen in transfectants. Culture with Bay K 8644 (2.5 x 10(-6) M), an agonist of Ca(2+) entry in cells, caused a significant decrease in the number of wild-type cells. Such an effect was not seen in transfectants. The presence of LPS did not significantly decrease the number of wild-type cells in the presence of Bay K 8644. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with Bay K 8644, and this DNA fragmentation was significantly prevented in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by LPS or various intracellular signaling-related factors.     

Regeneration failure of lakeshore plants under an artificially altered water regime

To reveal the effects of artificial alteration of water level regime on the regeneration of lakeshore plants from seeds, we examined the factors causing regeneration failure in Lake Kasumigaura, Japan. A survey of microtopography within and around a remnant fragment of lakeshore vegetation revealed that, over a large range, the habitat is frequently inundated in spring under the current water regime, although it was rarely inundated under past water regimes. Analysis of the patterns of seedling emergence and establishment at microsites at various elevations revealed a significant negative correlation between number of inundation days and abundance or species-richness of seedlings that emerged in the spring. Most seedling deaths occurred when the study site was inundated. We suggest that regeneration failure caused by the artificial raising of the lakes water level is one of the principal mechanisms of the recent vegetational decline in the lake.     

Role of taurine supplementation to prevent exercise-induced oxidative stress in healthy young men

Summary. To evaluate the protective effects of taurine supplementation on exercise-induced oxidative stress and exercise performance, eleven men aged 18–20 years were selected to participate in two identical bicycle ergometer exercises until exhaustion. Single cell gel assay (SCG assay) was used to study DNA damage in white blood cells (WBC). Pre-supplementation of taurine, a significant negative correlation was found between plasma taurine concentration before exercise and plasma thiobaribituric-acid reactive substance (TBARS) 6hr after exercise (r=–0.642, p<0.05). WBC showed a significant increase in DNA strand breakage 6hr and 24hr after exercise. Seven-day taurine supplementation reduced serum TBARS before exercise (p<0.05) and resulted in a significantly reduced DNA migration 24hr after exercise (p<0.01). Significant increases were also found in VO₂max, exercise time to exhaustion and maximal workload in test with taurine supplementation (p<0.05). After supplementation, the change in taurine concentration showed positive correlations with the changes in exercise time to exhaustion and maximal workload. The results suggest that taurine may attenuate exercise-induced DNA damage and enhance the capacity of exercise due to its cellular protective properties.     

Identification of beta-tubulin isoform V as an autoantigen in allergic rhinitis by a proteomic approach

Autoantibodies to IgE and beta2-adrenergic receptor have been reported in patients with allergic rhinitis. To investigate whether autoimmunity in allergic rhinitis is directed to such limited molecules or directed to a wide range of self proteins, we here attempted to survey autoantigens/autoantibodies comprehensively, using proteomics. Specifically, we separated proteins extracted from peripheral blood mononuclear cells by 2-dimensional electrophoresis and then detected autoantigens by subsequent western blotting with sera from patients with allergic rhinitis. As a result, we detected multiple autoantigens, some of which were further identified by mass fingerprinting. Next, we confirmed antigenicity of one of the identified autoantigens, beta-tubulin isoform V (beta-tubV), using a recombinant protein and then measured prevalence of the anti-beta-tubV autoantibodies. As a result, 52% of the tested patients with allergic rhinitis were found to possess anti-beta-tubV autoantibodies. Our study indicates that autoimmunity is a common phenomena and beta-tubV is one of the major autoantigens in allergic rhinitis.