Simian virus 40 (SV40) T antigen binds specifically to double-stranded DNA but not to single-stranded DNA or DNA/RNA hybrids containing the SV40 regulatory sequences.

Simian virus 40 T antigen has been shown previously to bind specifically with high affinity to sites within the regulatory region of double-stranded simian virus 40 DNA. Using competition filter binding and the DNA-binding immunoassay, we show that T antigen did not bind specifically to either early or late single-stranded DNA containing these binding sites. Moreover, T antigen did not bind these sequences present in single-stranded RNA, RNA/RNA duplexes, or RNA/DNA hybrids. T antigen did, however, bind as efficiently to single-stranded DNA-cellulose as to double-stranded DNA-cellulose. This binding was nonspecific because it was independent of the presence of T-antigen-binding sites. The implications of these observations are discussed.     

Heavy metal intracellular balance and relationship with metallothionein induction in the liver of carp after contamination by silver,cadmium and mercury following or not pretreatment by zinc

Determination of metal levels (copper, zinc, cadmium, silver and mercury) in soluble and insoluble fractions of liver homogenates has been performed after 7 days exposure of carps (Cyprinus carpio) to moderate concentrations of cadmium, silver and mercury in water. Metallothionein (MT) levels have been quantified by a polarographic method before and after the contamination and a subsequent decontamination phase (7 days). The influence of pretreatment by zinc (7 days) has also been evaluated. MT level variations have been interpreted as having regard to inter-related flows of metal between subcellular fractions. Special interest has been focused on heat-stable compound (HSC)-bound heavy metal flows within the cytosol, taking in account that MT is the major component of these ligands. Our data showed differences between the ability of metals to bind cytosolic ligands and HSCs, and their respective potency for MT induction in liver. Regardless of pretreatment, mercury gave the highest increase of liver MT, but the MT level decreased during the decontamination step, especially after pretreatment by zinc. Cadmium and silver gave similar increases, but a significant difference with the control appeared only after the decontamination step with cadmium, while 1 week of contamintion was enough for silver. However, silver binding with MT was achieved only by the end of the decontamination step, while cadmium depicted the highest ratio for HSC-bound toxic metals after the contamination. Our experimental conditions gave the following order of potency for MT induction in liver: mercury silver > cadmium > zinc. Results are discussed comparatively with data obtained with carp gills.     

Discriminative uptake of metals by the liver and its relation to induction of metallothionein by cadmium, copper and zinc

1. Disappearance from plasma and uptake by the liver of cadmium (Cd), copper (Cu) and zinc (Zn) were examined with a view to studying the biological discrimination between essential and non-essential heavy metals. 2. Cd injected intravenously at a single dose of 0.8 mg/kg body wt disappeared from rat plasma rapidly within about 10 min, while Cu and Zn injected at the same dose disappeared slowly in plasma and decreased to the control level after about 3 hr. 3. Uptake of Cd by the liver corresponded well with the rapid disappearance from plasma, while Cu and Zn accumulated slowly in the liver and their concentrations started to increase after their plasma concentrations had decreased. 4. Metallothionein was induced in the liver at a similar time course for the three metals, suggesting the presence of discriminative uptake processes by the liver with similar or the same detoxification mechanisms through induction of metallothionein.     

Selective induction of c-jun and jun-B but not c-fos or c-myc during mitogenesis in SV40-transformed cells at the predifferentiation growth arrest state.

Insulin has recently been reported to function as a complete mitogen for SV40 large T antigen-transformed 3T3 T-cells, designated CSV3-1, but not for nontransformed 3T3 T-cells (H. Wang and R. E. Scott, J. Cell. Physiol., 147: 102-110, 1991). It is now reported that sodium orthovanadate mimics this effect of insulin. For example, when exposed to 1-5 microM vanadate, most predifferentiation growth-arrested CSV3-1 cells undergo DNA synthesis within 24 h, but neither vanadate nor insulin induces mitogenesis in nontransformed 3T3 T-cells. To investigate the possible mechanisms by which mitogenesis is induced in CSV3-1 cells, the effects of insulin and vanadate on the expression of growth-related genes were examined. Whereas insulin and vanadate had no effect on the expression of c-fos, c-myc, c-jun, jun-B, or ornithine decarboxylase activity in nontransformed 3T3 T-cells, insulin and vanadate showed different effects on the expression of these genes in CSV3-1 cells. Insulin induced a rapid and transient accumulation of c-fos mRNA followed by induction of c-myc, c-jun, jun-B, and ornithine decarboxylase. In contrast, vanadate induced the expression of c-jun, jun-B, and ornithine decarboxylase without inducing c-fos and c-myc. These observations suggest that SV40 large T antigen may play an important role in insulin- and vanadate-induced mitogenesis and that insulin and vanadate may mediate their mitogenic effects by different signal transduction pathways.     

Effects of cadmium, copper and metallothionein synthesis inhibiting and stimulating compounds on zinc uptake and accumulation in rat hepatoma HTC cells.

Experiments were carried out to investigate the uptake and accumulation of Zn in rat hepatoma HTC cells, as affected by interfering metals (Cd, Cu), metallothionein synthesis inhibiting compounds (Actinomycin D, cycloheximide) and metallothionein synthesis stimulating compounds (dexamethasone, dibu-cAMP). Cell viability was tested under all experimental conditions by the measurement of LDH leakage, K+ uptake and total cell protein. Determinations of Zn were performed by AAS (total Zn) or by gamma-ray spectrometry (65Zn). Metallothionein analysis was carried out by Cd-saturation tests. The results indicate that cellular responses in rat hepatoma HTC cells with respect to the uptake and accumulation of 65Zn are fully comparable with literature data existing for 65Zn accumulation in rat hepatocytes, under all experimental conditions applied. Cu2+ and dibutyryl-cAMP did not significantly affect rates of 65Zn accumulation. Cd2+, Actinomycin D and cycloheximide reduced 65Zn uptake, but dexamethasone additions resulted in increased 65Zn accumulation in the cells. Effects on 65Zn were shown both in cytosolic and in the membranes/organelles cell fractions. HPLC chromatography in control cells suggested that newly accumulated cytosolic 65Zn was predominantly MT-associated. Dexamethasone-induced 65Zn accumulation could not be related to elevated cellular MT levels, nor were the total cytosolic Zn levels significantly affected. Non-specific attenuations in MT levels (Actinomycin D, cycloheximide and dibu-cAMP) yielded linear relations between cytosolic 65Zn and MT levels, without any change in cytosolic Zn (AAS). Combined addition of Cd and dexamethasone yielded elevated MT levels, but severely reduced total cytosolic Zn and 65Zn concentrations. The results further indicate the non-Zn-specific nature of dexamethasone-action and suggest the relatively easy Zn-complexing and Zn-release of MT. The simultaneous determinations of total cytosolic zinc and cytosolic 65Zn levels showed that the application and sole measurement of radiotracers may yield only one-sided views of what is actually present or occurring in the cells.     

Ultraviolet-A irradiation but not ultraviolet-B or infrared-A irradiation leads to a disturbed zinc homeostasis in cells

Changes of the redox balance in cells alter the availability of intracellular free Zn(2+). Here, cells were exposed to ultraviolet (UV)-A, UV-B, or infrared (IR)-A light irradiation, and the intracellular free zinc pool was monitored. Under sublethal conditions only UV-A irradiation resulted in a transient cytoplasmic and nuclear increase of intracellular free Zn(2+). Likewise, tert-butyl hydroperoxide and singlet oxygen, but not H(2)O(2) or intracellular generation of O(2)(*-) by redox cyclers, mimicked the effects of UV-A irradiation, while disulfide stress by diamide only led to a transient cytoplasmic zinc release. These results show that only certain types of subtoxic cellular stress massively disturb the zinc homeostasis in cells.     

Overexpression of poplar GSTU51 confers selective tolerance to both mercury and methyl viologen but not to CDNB or cadmium in transgenic poplars

Glutathione S-transferases belong to a large ancient gene family and are thought to be one of the effective detoxification systems. To elucidate the function of the gene in poplar, a tau class gst gene (PatgGSTU51) was cloned from poplar cell suspension cDNA library and its expression was examined. The gene was not expressed in normal conditions, but significantly induced by toxic heavy metals like cadmium and inorganic mercury. However, the highest expression was observed when treated with its synthetic substrate, 1-chloro-2,4-dinitrobenzene (CDNB). Several transgenic poplar lines harboring a chimeric p35S-PatgGSTU51 were developed to understand its function in plant defense. Real-time quantitative PCR, western blot and cellular GST activities consistently showed the transgene was highly expressed in the transgenic lines. The transgenic lines showed increased tolerance to methyl viologen as well as to inorganic mercury but not to cadmium. Furthermore, they did not show any significant tolerance against CDNB, the electrophilic substrate of GST isozymes. Thus, the results suggest that, although PatgGSTU51 is induced by a number of stress agents, it confers a selective tolerance to the toxicity of heavy metal mercury over those of other stress agents and also to oxidation stress caused by MV. Tolerance to CDNB that induces the gene to high level may require such an extra supplemental gene action as vacuolar sequestration.     

Thymidine kinase deficient human cells have increased UV sensitivity in their capacity to support herpes simplex virus but normal UV sensitivity for colony formation

A thymidine kinase deficient (tk-) and two thymidine kinase proficient (tk+) human cell lines were compared for UV sensitivity using colony-forming ability as well as their capacity to support the plaque formation of herpes simplex type 1 (HSV-1). The tk- line (143 cells) was a derivative of one of the tk+ lines (R970-5), whereas the other tk+ line (AC4 cells) was a derivative of the 143 cells obtained by transfection with purified sheared HSV-2 DNA encoding the viral tk gene. 143, R970-5 and AC4 cells showed a similar UV sensitivity for colony-forming ability. In contrast, the capacity to support HSV-1 plaque formation immediately (within 1 h) after UV-irradiation was reduced to a greater extent in the 143 cells compared to the R970-5 and AC4 cells. Capacity curves for plaque formation of the HSV-1: KOS wild-type (tk+) strain were similar to those for the HSV-1: PTK3B mutant (tk-) strain in the 3 cell strains, indicating that the viral tk gene does not influence the ability of HSV-1 to form plaques in UV-irradiated compared to unirradiated human cells. Cellular capacity for HSV-1 plaque formation was found to recover in both tk- and tk+ cells for cultures infected 24 h after UV-irradiation. These results suggest that repair of UV-damaged DNA takes place to a similar extent in both tk- and tk+ human cells, but the kinetics of repair are initially slower in tk- compared to tk+ human cells.     

Short-term and long-term effects of cadmium,chromium, copper,nickel, lead and zinc on soil microbial respiration in relation to abiotic soil factors

Summary The inhibition of the respiration rate by the heavy metals, Cd, Cr, Cu, Pb, Ni and Zn was investigated in five Dutch soil types in relation to the length of time these heavy metals were present in the soil. The amounts of heavy metal added as chloride salts to the soils were 0, 55, 150, 400, 1000, 3000 and 8000 g·g^–1, respectively. The measurements were carried out both immediately after the addition of the heavy metals and approximately 18 months later. The inhibition during the first two to eight weeks was not obscured by an extra nutrient flush to drying. During the 18 months, the toxicity decreased but was still significant. Inhibition was greatest in the sandy soil and least in the clay soil. In a loam soil and in a sandy peat soil, the inhibiting effects were intermediate, but distinct. The main abiotic factors responsible for these different degrees of inhibition were the clay fraction for Cd, the Fe content for Cu, Pb and Zn and the pH for Ni. Although clay, Fe, and Mn together with the organic matter fraction, determine the total cation exchange capacity of soil, their contribution to the toxicity of heavy metals may be antagonistic. The latter may increase the mobility due to chelation and therefore possibly increase the toxicity, while the other factors may bind the heavy metals and therefore decrease the toxicity.     

CD40 engagement on dendritic cells, but not on B or T cells, is required for long-term control of murine gammaherpesvirus 68

CD4 T cells are not essential for primary clearance of replicating murine gammaherpesvirus 68 (MHV-68) but are required for effective long-term control. The virus reactivates in the lungs of major histocompatibility complex class II-deficient (CII-/-) mice that lack functional CD4 T cells. CD40 ligand (CD40L) is upregulated on activated CD4 T cells, and it is thought that CD40-CD40L interactions are an important component of CD4 T-cell help. Our previous studies have shown that agonistic antibodies to CD40 can substitute for CD4 T-cell function in the long-term control of MHV-68. In the present study, we sought to identify the CD40-positive cell type mediating this effect. To address this question, we adoptively transferred MHV-68 peptide-pulsed CII(-/-) dendritic cells (DC) that had been treated with an agonistic antibody to CD40 into MHV-68-infected CII(-/-) recipients. Viral reactivation was significantly lower in mice injected with anti-CD40-treated DC than in those injected with control DC or in mice that did not receive any DC. However, in similar experiments with B cells, anti-CD40 treatment had no effect. We also investigated the requirement for CD40 expression on T cells by adoptive transfer of T cells from CD40(+/+) or CD40(-/-) mice into T-cell-deficient recipients that were subsequently infected with MHV-68. The results showed that CD40 expression on T cells is not necessary for preventing viral reactivation. Taken together, our data suggest that CD40 engagement on DC, but not on T or B cells, is essential for effective long-term control of MHV-68.     

Hepatic metallothionein production and resistance to heavy metals by rainbow trout (Salmo gairdneri)--I. Exposed to an artificial mixture of zinc, copper and cadmium

Rainbow trout developed elevated hepatic metallothionein concentrations after 4 weeks in a solution containing zinc, copper and cadmium in a fixed ratio of 400:20:1. Resistance to a combination of these metals increased in proportion to the concentration to which they were exposed for 4 weeks. The concentration of copper but not zinc or cadmium in the low molecular weight proteins separated by gel filtration was related to the concentrations of metallothionein present. The combined toxicity of the metals in the mixture was additive.     

Dose-dependent changes in influenza virus-infected dendritic cells result in increased allogeneic T-cell proliferation at low, but not high, doses of virus

During the acute phase of infection with influenza A virus, the degree of lymphopenia correlates with severity of disease. Factors that contribute to T-cell activation during influenza virus infection may contribute to this observation. Since the immune response is initiated when dendritic cells (DC) interact with T cells, we have established an in vitro system to examine the effects of influenza virus infection on DC function. Our results show that allogeneic T-cell proliferation was dependent on the dose of A/PR/8/34 used to infect DC, with enhanced responses at low, but not high, multiplicities of infection. The lack of enhancement at high virus doses was not primarily due to the increased rate of DC apoptosis, but required viral replication and neuraminidase (NA) activity. Clusters that formed between DC or between DC and T cells were also dependent on the viral dose. This change in cellular interaction may oppose T-cell proliferation in response to DC infected with high doses of PR8, since the increased contact between DC resulted in the exclusion of T cells. The enhanced alloreactive T-cell response was restored by neutralization of transforming growth factor beta1 (TGF-beta1). It is likely that NA present on viral particles released from DC infected with high doses of PR8 activates TGF-beta1. Future studies will determine the mechanism by which TGF-beta1 modifies the in vitro T-cell response and address the contribution of this cytokine to the lymphopenia observed in severe disease.     

Cellular mutation mediates T-antigen-positive revertant cells resistant to simian virus 40 transformation but not to retransformation by polyomavirus and adenovirus type 2.

T-antigen-positive transformation revertant cell lines were isolated from fully simian virus 40 (SV40)-transformed Fisher rat embryo fibroblast cells (REF 52 cells) by methionine starvation. Reversion of the transformed cells (SV-52 cells) was caused by a mutation within the cellular genome. To demonstrate this, we isolated SV40 DNA from the host genome, inserted it into plasmid pSPT18 DNA, cloned it in Escherichia coli, and microinjected it into the nuclei of the REF 52 cells. Fully transformed cells were obtained with the same efficiency (20 to 25%) as after microinjection of wild-type SV40 DNA I. Furthermore, the revertant cells were resistant to retransformation by SV40. Following microinjection of wild-type SV40 DNA I, 42 independent cell lines were isolated. Cells of all analyzed lines acquired additional SV40 DNA copies, but changes in the cell morphology or growth characteristic were not demonstrable. However, the revertants were retransformable with a high efficiency after polyomavirus and adenovirus type 2 infections or microinjection. Also, fusion of the revertant cells with the grandparental REF 52 cells led to restoration of the transformed state.     

OX40-mediated differentiation to effector function requires IL-2 receptor signaling but not CD28, CD40, IL-12Rbeta2, or T-bet

Ag-specific CD4 T cells transferred into unirradiated Ag-bearing recipients proliferate, but survival and accumulation of proliferating cells is not extensive and the donor cells do not acquire effector functions. We previously showed that a single costimulatory signal delivered by an agonist Ab to OX40 (CD134) promotes accumulation of proliferating cells and promotes differentiation to effector CD4 T cells capable of secreting IFN-gamma. In this study, we determined whether OX40 costimulation requires supporting costimulatory or differentiation signals to drive acquisition of effector T cell function. We report that OX40 engagement drives effector T cell differentiation in the absence of CD28 and CD40 signals. Two important regulators of Th1 differentiation, IL-12R and T-bet, also are not required for acquisition of effector function in CD4 T cells responsive to OX40 stimulation. Finally, we show that CD25-deficient CD4 T cells produce little IFN-gamma in the presence of OX40 costimulation compared with wild type, suggesting that IL-2R signaling is required for efficient OX40-mediated differentiation to IFN-gamma secretion.     

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.