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Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K(d)=3.62+/-2.1 x 10(-8)M) or the RNA corresponding sequence (K(d)=2.7+/-0.82 x 10(-8) M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.

The severe acute respiratory syndrome virus (SARS) is a coronavirus that instigated regional epidemics in Canada and several Asian countries in 2003. The newly identified SARS coronavirus (SARS-CoV) can be transmitted among humans and cause severe or even fatal illnesses. As preventive vaccine development takes years to complete and adverse reactions have been reported to some veterinary coronaviral vaccines, anti-viral compounds must be relentlessly pursued. In this study, we analyzed the effect of aurintricarboxylic acid (ATA) on SARS-CoV replication in cell culture, and found that ATA could drastically inhibit SARS-CoV replication, with viral production being 1000-fold less than that in the untreated control. Importantly, when compared with IFNs alpha and beta, viral production was inhibited by more than 1000-fold as compared with the untreated control. In addition, when compared with IFNs alpha and beta, ATA was approximately 10 times more potent than IFN alpha and 100 times more than interferon beta at their highest concentrations reported in the literature previously. Our data indicated that ATA should be considered as a candidate anti-SARS compound for future clinical evaluation.

Gene transfer into muscle upon injection of plasmid DNA is feasible but occurs with low frequency. However, by using electroporation after injection of plasmid DNA into mouse muscle it has been demonstrated that gene expression can be increased more than 150-fold. In this communication, we have used this technique in combination with plasmids containing a tandem repeat of three 72-bp DNA elements from the SV40 enhancer to study gene expression. Our results show that the combination of electroporation and a plasmid vector carrying these DNA elements results in increased and more persistent gene expression of the luciferase reporter gene in BALB/c mouse muscle. At 14 days after gene delivery, the gene expression was 16-fold higher in muscles injected and electroporated with the plasmid carrying the SV40 enhancers than with control plasmid. We have also studied the effects of the vehicle in which the plasmid was delivered, and the DNase inhibitor aurintricarboxylic acid (ATA), on gene expression. By combining ATA with 150 mM sodium phosphate buffer we were able to obtain a 2-fold increase in gene expression compared to delivery of the plasmid in physiological saline. These results are of importance for the development of efficient delivery techniques for naked DNA.

Laminin stimulates neurite outgrowth in rat pheochromocytoma cells (PC12 cells). Here, we investigated laminin signal transduction mechanisms by adding the tyrosine kinase/phosphatase modulators, genistein, quercetin, aurin tricarboxylic acid (ATA), and vanadate to PC12 cells. At 10 microM both genistein and quercetin enhanced laminin-mediated neurite outgrowth by 1.7- and 2.3-fold, respectively, while at 10 microM, ATA inhibited laminin-mediated neurite outgrowth by 92%. Vanadate inhibited neurite outgrowth by 63% at 10 microM. Immunoblot analysis revealed four proteins of approximately 240, 22, 110, and 35 kDa, which were dephosphorylated on tyrosine residues in laminin-treated PC12 cells, but not in NIH 3T3 cells. These results demonstrate that laminin-mediated neurite outgrowth involves protein tyrosine dephosphorylation and suggests that this mechanism may have specificity to neuronal cells., (Copyright 1999 Academic Press.)

Aurintricarboxylic acid (ATA) has been reported to protect PC12 cells and cultured neuronal cells from serum starvation-induced cell death, and hippocampal neurons from N-methyl D-aspartate- or ischemia-induced cell death in vivo. We have found that ATA activated tyrosine phosphorylation cascade in PC12 cells as growth factors. Here, we report that ATA prevents cell death under serum starvation and induces tyrosine phosphorylation also in human neuroblastoma SH-SY5Y cells. Furthermore, it was found that erbB4, a member of epidermal growth factor receptor family, is tyrosine-phosphorylated in response to ATA. Both, erbB4 and its ligand, neu differentiation factor (NDF)/ heregulin family, have been reported to be expressed abundantly in nervous system. Thus, tyrosine phosphorylation of erbB4 might explain the neuro-protective activity of ATA.

Cytosine arabinoside, a potent antimitotic agent, is used clinically as an anticancer drug with the side effect of severe neurotoxicity. Earlier reports suggested that this agent blocks the signaling pathway of nerve growth factor in sympathetic neurons, which results in neuronal death. The present study demonstrated that neuronal PC12 cells deprived of nerve growth factor showed a decrease of neurite outgrowth, which was not seen in cytosine arabinoside-treated neuronal PC12 cells, suggesting that this agent may not interfere with the part of the NGF signaling pathway that leads to neurite outgrowth. On the other hand, treatment of neuronal PC12 cells with cytosine arabinoside led to a dose- and time-dependent decrease of viability. These results suggest that there is a distinction between the neurite outgrowth and the survival promoting activities of nerve growth factor.

Internucleosomal DNA fragmentation (DNA laddering) and formation of apoptotic bodies have long been considered characteristic features of apoptosis. However, recent work has shown that formation of high molecular weight DNA fragments precedes internucleosomal cleavage and may involve mechanisms that differ from those responsible for DNA laddering. Here, we show that glucocorticoid treatment of human thymocytes stimulated the formation of high molecular weight DNA fragments by Ca(2+)- and endonuclease-mediated mechanisms. Either the removal of Ca2+ from the medium or pretreatment of the cells with the intracellular Ca2+ chelator, BAPTA-AM, prevented the formation of large DNA fragments. Further, treatment of the thymocytes with the microsomal Ca(2+)-ATPase inhibitor, thapsigargin, which caused a sustained increase in intracellular Ca2+ concentration, was in itself sufficient to activate high molecular weight DNA fragmentation. Our results show that Ca(2+)-dependent mechanisms promote the multistep chromatin cleavage in human thymocyte apoptosis.

The protooncogene bcl-2 was studied as a candidate gene for N-methyl-D-aspartate-(NMDA)-induced excitoprotection of cerebellar granule neurons. Exposure of these neurons to excitoprotective concentrations of NMDA and to excitotoxic concentrations of glutamate decreased bcl-2 mRNA levels. Preexposure to NMDA failed to modify the glutamate-evoked decrease in bcl-2 mRNA but increased neuronal survival from 20% to 100%. Neither the intracellular mechanisms underlying the cascade of excitoprotection induced by NMDA nor those underlying the cascade of glutamate toxicity of cerebellar granule neurons seem to specifically involve changes in bcl-2 mRNA levels. Glutamate did not induce nucleosomal DNA fragmentation but glutamate toxicity was inhibited by aurintricarboxylic acid. Glutamate toxicity of cerebellar granule neurons differs considerably from classical programmed cell death.

Several aurintricarboxylic acid (ATA) monomers, monomer analogs, and polymer fractions have been tested as inhibitors of HIV-1 integration protein (IN). Both of the ATA monomers and all of the ATA polymer fractions inhibited a selective DNA cleavage reaction catalyzed by IN. The ATA monomer analogs were inactive or had low activity. The activities of the substances as inhibitors of HIV IN correlated in a positive way with their activities as inhibitors of the cytopathic effect of HIV-1 in CEM and HIV-2 in MT4 cells. These results suggest that inhibition of HIV IN may contribute to the antiviral activity of the ATA monomers and monomer analogs in cell culture.

Glutamate neurotoxicity is responsible for neuronal loss associated with numerous obstinate disorders. In this report, the mechanism of glutamate neurotoxicity was investigated on a viewpoint of DNA degradation. We found that chromosomal DNA of cultured neurons was degraded into nucleosomal-sized DNA fragments by the addition of glutamate, prior to the glutamate-induced neuronal death. Both the neuronal death and DNA fragmentation were prevented by the inhibitors of endonucleases and mRNA synthesis. Furthermore, an injection of glutamate into the rat hippocampi resulted in DNA fragmentation with the similar time course observed in neuronal death in vitro. These results suggest that the glutamate neurotoxicity involves an active suicide process which leads to neuronal death through internucleosomal DNA cleavage.

Binding of HIV to its receptor, the CD4 molecule of lymphocytes, can be prevented by chemical agents. These agents could be considered as potential anti-AIDS drugs. We have shown that aurin tricarboxylic acid (ATA, 3 microM) specifically blocks the binding of gp120, the HIV coat protein, to the CD4 molecule. We have also found that ATA prevents the binding of interferon-alpha to its receptor in a dose-dependent manner (12-50 microM range). Membrane potential shift, associated with binding of interferon-alpha to its receptor, was also blocked by ATA in a dose-dependent fashion. Our results indicate that potential anti-AIDS drugs should be screened for such undesired side effects.

Aurintricarboxylic acid, an anionic triphenylmethane dye, and Evans Blue, an anionic compound structurally related to suramin, are, like suramin itself, inhibitors of human T-cell lymphotropic virus type III (HTLV-III)/-lymphadenopathy-associated virus (LAV) in vitro. These compounds may be targeted, at least in part, at the HTLV-III/LAV reverse transcriptase. The lack of any appreciable cytostatic action of aurintricarboxylic acid, Evans Blue and suramin against several murine and human cell lines, their inability to inhibit cellular DNA, RNA and protein synthesis, and their high lethal dose-50 (greater than or equal to 0.340 g/kg) for NMRI mice point to the selectivity of the compounds as inhibitors of HTLV-III/LAV.

We have prepared a submitochondrial fraction from Ehrlich ascites tumor cell mitochondria which shows transcription and translation activities. The antibiotic aurin tricarboxylic acid (ATA) at low concentrations induces both RNA and protein synthetic activities of the mitochondrial lysate by several fold. At high concentrations, however, ATA inhibits the translation activity but continues to stimulate the transcription activity in a dose dependent manner up to 0.5 mM concentration tested. The lysate system transcribes endogenous DNA yielding RNA species resembling control mitochondrial RNA and synthesizes authentic cytochrome oxydase I and cytochrome oxidase II subunits.