Identification of a chemical that inhibits the mycobacterial UvrABC complex in nucleotide excision repair.

Bacterial DNA can be damaged by reactive nitrogen and oxygen intermediates (RNI and ROI) generated by host immunity, as well as by antibiotics that trigger bacterial production of ROI. Thus a pathogen's ability to repair its DNA may be important for persistent infection. A prominent role for nucleotide excision repair (NER) in disease caused by Mycobacterium tuberculosis (Mtb) was suggested by attenuation of uvrB-deficient Mtb in mice. However, it was unknown if Mtb's Uvr proteins could execute NER. Here we report that recombinant UvrA, UvrB, and UvrC from Mtb collectively bound and cleaved plasmid DNA exposed to ultraviolet (UV) irradiation or peroxynitrite. We used the DNA incision assay to test the mechanism of action of compounds identified in a high-throughput screen for their ability to delay recovery of M. smegmatis from UV irradiation. 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo[f]chromen-3-one) (ATBC) but not several closely related compounds inhibited cleavage of damaged DNA by UvrA, UvrB, and UvrC without intercalating in DNA and impaired recovery of M. smegmatis from UV irradiation. ATBC did not affect bacterial growth in the absence of UV exposure, nor did it exacerbate the growth defect of UV-irradiated mycobacteria that lacked uvrB. Thus, ATBC appears to be a cell-penetrant, selective inhibitor of mycobacterial NER. Chemical inhibitors of NER may facilitate studies of the role of NER in prokaryotic pathobiology.

HSP70 and EndoG modulate cell death by heat in human skin keratinocytes in vitro.

We examined how young and old keratinocytes died from heat stress in vitro. We found that keratinocyte cell death was not due to oxidative stress as neither Mn-SOD nor Cu-Zn-SOD was produced in either young or old heated keratinocytes. Instead, analysis of the anti-apoptotic factors, Bcl2 and HSP70, and the pro-apoptotic factors, caspase 3, caspase 8, Apaf-1, cytochrome c, AIF, and EndoG, indicated that keratinocyte cell death occurred via the caspase-independent EndoG apoptotic pathway. We found that both young and old keratinocytes died via the same pathway, and that we could specifically reduce both young and old keratinocyte death by addition of the EndoG inhibitor NEM. Further analysis suggested that the difference between young and old keratinocyte death was due to the synthesis of HSP70 protein, with the increase in response to heat more pronounced in young keratinocytes than in old keratinocytes. When we inhibited HSP70 by adding quercetin, death was increased in both young and old keratinocytes, but more so in old keratinocytes. These data suggest that old keratinocytes may die more readily than young keratinocytes when heated because they synthesize HSP70 at a lower efficiency. Such findings suggest that HSP70 production may be age-dependent.

Tetrandrine-induced apoptosis in rat primary hepatocytes is initiated from mitochondria: caspases and endonuclease G (Endo G) pathway.

Tetrandrine, a bisbenylisoquinoline alkaloid isolated from the dried root of Stephenia tetrandra (S Moore), possesses a remarkable pharmacological profile. However, the mechanisms of tetrandrine hepatotoxicity remain to be elucidated. In this study, we first proved apoptosis and mitochondrial dysfunction induced by tetrandrine in Sprague-Dawley rat liver in vivo. By further assuming apoptosis as an important mechanism in tetrandrine-induced hepatotoxicity, we focused on mitochondria-initiated apoptosis in primary hepatocytes isolated from Sprague-Dawley male rats. Tetrandrine treatment led to significant release of cytochrome c and downregulation of Bcl-X(L) accompanied by caspase 3 activation, and ultimately, DNA fragmentation. Caspase 3 activation was markedly inhibited by cyclosporin A (CsA) and Ac-DEVD-CHO. Furthermore, Endo G, a caspase-independent apoptotic protein, was detected for its expression and DNase activity. CsA blocked the release both of Endo G and cytochrome c significantly. Additionally, the generation of reactive oxygen species (ROS) increased in a time-dependent manner corresponding with a fall in intracellular GSH content after 10 microM tetrandrine treatment in 4h. Tetrandrine also induced mitochondrial dysfunction indicated by transition of mitochondrial transmembrane potential and decrease of intracellular ATP level. The findings indicated that the caspase-dependent mitochondrial apoptosis pathway was primarily involved in tetrandrine-induced apoptosis in rat primary hepatocytes. In addition, a caspase-independent pathway indicated by Endo G also contributed to apoptosis caused by tetrandrine. Meanwhile, ROS was proved an important inducer in this apoptosis process.

[New antivirals for herpesviruses].

The long-term treatment of herpesvirus infections with current antivirals leads to the development of drug-resistant viruses. Because currently available antivirals finally target the viral DNA polymerase, mutant resistant to one drug often shows cross-resistance to other drugs. This evidence highlights the need for the development of new antivirals that have the different viral targets. Recently, high-through-put screening of large compound collections for inhibiting specific viral enzymes, or in vitro cell culture assay, has identified several new antivirals. These include the inhibitors of helicase/primase complex, terminase complex, portal protein and UL97 protein kinase. This review will focus on these new compounds that directly inhibit viral replication.

Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA.

Cationic polymers have the potential for DNA complexation and it is recognised that they may be useful as non-viral vectors for gene delivery. Highly purified chitosan fractions of < 5000 Da (N1), 5000-10,000 Da (N2) and > 10,000 Daltons (N3) were prepared and characterised in respect of their cytotoxicity, ability to cause haemolysis, ability to complex DNA as well as to protect DNA from nuclease degradation. Also the biodistribution of 125I-labelled chitosans was followed at 5 and 60 min after intravenous injection into male Wistar rats. All chitosan fractions displayed little cytotoxicity against CCRF-CEM and L132 cells (IC50 > 1 mg/ml), and they were not haemolytic (< 15% lysis after 1 and 5 h). Chitosan-DNA interaction at a charge ration of 1:1 was much greater than seen for poly(L-lysine) and complexation resulted in inhibition of DNA degradation by DNase II: 99.9 +/- 0.1, 99.1 +/- 1.5 and 98.5 +/- 2.0% for N1, N2 and N3, respectively. After intravenous injection, all the chitosans showed rapid blood clearance, the plasma levels at 1 h being 32.2 +/- 10.5% of recovered dose for N1 and 2.6 +/- 0.5% of recovered dose for N3. Liver accumulation was molecular mass dependent, being 26.5 +/- 4.9% of the recovered dose for N1 and 82.7 +/- 1.9% of the recovered dose for N3. The observations that the highly purified chitosan fractions used were neither toxic nor haemolytic, that they have the ability to complex DNA and protect against nuclease degradation and that low molecular weight chitosan can be administered intravenously without liver accumulation suggest there is potential to investigate further low molecular weight chitosans as components of a synthetic gene delivery system.

Effect of systemic zinc administration on delayed neuronal death in the gerbil hippocampus.

The divalent cation zinc has been reported to possess several physiological properties such as blocking apoptotic cell death through an inhibitory effect on Ca(2+)-Mg2+ endonuclease activity, or modulating the neurotoxicity via glutamate receptor subtypes. In the present study, we investigated the effect of peripherally injected zinc on delayed neuronal death seen in the hippocampus after transient global ischemia, in order to elucidate a possible beneficial role on zinc in ischemic neuronal cell death. Forty-five adult Mongolian gerbils of both sexes underwent transient bilateral clipping of the common carotid arteries for 3 min. In the pretreated animals, ZnCl2 (20 mg/kg) was injected subcutaneously once, 1 h before ischemia (superacute group; n = 6) or twice at 24 and 48 h before ischemia (subacute group; n = 14). Histological survey was carried out 3 days later by in situ DNA fragmentation method and 4 days later by hematoxylin-eosin staining by semiquantatively counting dead neurons in the CA1 sector. Subacute zinc pre-administration significantly reduced the nuclear damage and subsequent neuronal death; however, superacutely pre-administered zinc did not protect hippocampal neurons against ischemia but it did not aggravate the effect of ischemia, either. The present study suggested that transfer of exogenous zinc into the intracellular space is required for neuroprotection, presumably via the anti-endonuclease activity.

Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cells through the inactivation of a pH-dependent endonuclease.

Protein isoprenylation is a post-translational modification essential for the biological activity of G-proteins. Inhibition of protein isoprenylation by lovastatin (LOV) induces apoptosis in HL-60 cells, a process of active cell death characterized by the internucleosomal degradation of genomic DNA. In this article we show that LOV-induced apoptosis is associated with intracellular acidification and that activation of the Na+/H+ antiporter induces a raise in pHi which is sufficient to prevent or arrest DNA digestion. First, LOV induced a decrease in pHi which was dose-dependent and correlated with the extent of DNA degradation. Flow cytometry analysis revealed that this acidification was due to the appearance of a subpopulation of cells whose pHi was 0.9 pH units below control values. Cell sorting experiments demonstrated that DNA degradation had occurred only in those cells which had suffered intracellular acidification. LOV-induced apoptosis could be suppressed by mevalonate supplementation, inhibition of protein synthesis, and protein kinase C activation by phorbol myristate acetate. In all three cases, intracellular acidification was abolished. Inhibition of the Na+/H+ antiporter by 5-N-ethyl-N-isopropyl amiloride induced DNA degradation in HL-60 cells per se and suppressed the protective effect of phorbol myristate acetate. LOV-induced intracellular acidification was not due to a complete inhibition of the Na+/H+ antiporter. In fact, LOV-treated cells were able to respond to phorbol myristate acetate stimulation of the Na+/H+ antiporter with a marked increase in pHi. This effect was accompanied by a rapid arrest of DNA digestion. These observations illustrate the strong pH dependence of LOV-induced DNA degradation, thus providing a connection between the activation of the Na+/H+ antiporter and the suppression of apoptosis.