Double-stranded DNA-templated cleavage of oligonucleotides containing a P3'->N5' linkage triggered by triplex formation: the effects of chemical modifications and remarkable enhancement in reactivity.

We recently reported double-stranded DNA-templated cleavage of oligonucleotides as a sequence-specific DNA-detecting method. In this method, triplex-forming oligonucleotides (TFOs) modified with 5'-amino-2',4'-BNA were used as a DNA-detecting probe. This modification introduced a P3'→N5' linkage (P-N linkage) in the backbone of the TFO, which was quickly cleaved under acidic conditions when it formed a triplex. The prompt fission of the P-N linkage was assumed to be driven by a conformational strain placed on the linkage upon triplex formation. Therefore, chemical modifications around the P-N linkage should change the reactivity by altering the microenvironment. We synthesized 5'-aminomethyl type nucleic acids, and incorporated them into TFOs instead of 5'-amino-2',4'-BNA to investigate the effect of 5'-elongation. In addition, 2',4'-BNA/LNA or 2',5'-linked DNA were introduced at the 3'- and/or 5'-neighboring residues of 5'-amino-2',4'-BNA to reveal neighboring residual effects. We evaluated the triplex stability and reaction properties of these TFOs, and found out that chemical modifications around the P-N linkage greatly affected their reaction properties. Notably, 2',5'-linked DNA at the 3' position flanking 5'-amino-2',4'-BNA brought significantly higher reactivity, and we succeeded in indicating that a TFO with this modification is promising as a DNA analysis tool.

CpG oligonucleotides activate the immune response in burned mice.

BACKGROUND: Immunosuppression after burn injury increases the risk of sepsis and multiple organ failure. We examined changes of immune function in mice after burn injury and investigated the immunostimulatory effect of oligodeoxynucleotides containing CpG motifs. MATERIALS AND METHODS: Male BALB/c mice (8-10 wk old) received a full-thickness burn to 20% of their body surface area, after which the immunological parameters of splenic macrophages were evaluated. To assess the immunostimulatory effect of oligodeoxynucleotide treatment, splenic macrophages harvested from burned mice were incubated with oligodeoxynucleotides. Then cytokine production and major histocompatibility complex class II antigen expression were measured. To assess the in vivo effect of oligodeoxynucleotides, intraperitoneal administration was done on day 4 after burn injury, and class II antigen expression by splenic macrophages was measured 10 d later. RESULTS: Class II antigen expression and the synthesis of cytokines (interleukin-12, tumor necrosis factor-alpha, interleukin-6, and interleukin-1) by splenic macrophages were significantly reduced after burn injury, while incubation of splenic macrophages from burned mice with oligodeoxynucleotides partially enhanced the production of interleukin-12, tumor necrosis factor-alpha, interleukin-6, and interleukin-1. In addition, intraperitoneal administration of oligodeoxynucleotides enhanced class II antigen expression by splenic macrophages. CONCLUSIONS: The reduction of class II antigen expression and synthesis of cytokines (interleukin-12, tumor necrosis factor-alpha, interleukin-6, and interleukin-1) by splenic macrophages after burn injury was partially reversed by oligodeoxynucleotide treatment. Therefore, immunostimulatory oligodeoxynucleotides may be a potential treatment for post-burn immunosuppression.

Immune dysfunction in endotoxicosis: role of nitric oxide produced by inducible nitric oxide synthase.

OBJECTIVE: To investigate whether stimulation with lipopolysaccharide (LPS) alters cytokine production by splenocytes in mice and whether it changes the T-helper 1 (Th1)/Th2 balance. The role of nitric oxide in such immunologic changes was also explored using mice with genetic lack of inducible nitric oxide synthase (iNOS). DESIGN: : Prospective animal study with concurrent controls. SETTING: University research laboratory. SUBJECTS: iNOS knockout mice and wild-type littermates. INTERVENTIONS: iNOS knockout mice or wild-type mice were injected with LPS or saline with or without anti-interleukin (IL)-6 antibody, and survival was monitored for 7 days. MEASUREMENTS AND MAIN RESULTS: At 24 and 48 hrs after administration, blood samples and splenocytes were obtained to examine immunologic variables. Cell surface markers and cytokine expression of splenocytes were used to characterize the Th1/Th2 balance and were measured by flow cytometry. At 48 hrs after LPS administration, the Th1/Th2 balance shifted toward Th2 predominance in wild-type mice, irrespective of the IL-6 level, whereas it showed Th1 predominance in iNOS knockout mice, and the increase of IL-6 and IL-10 in response to LPS persisted in these animals. After LPS administration, the mortality rate was significantly higher in iNOS knockout mice than in wild-type mice, irrespective of the IL-6 level. CONCLUSIONS: These findings suggest that nitric oxide produced by iNOS during endotoxemia may be involved in down-regulation of Th1 cytokines and up-regulation of Th2 cytokines, whereas IL-6 has no such role. The increased lethality of LPS in iNOS knockout mice suggests that nitric oxide may be protective against proinflammatory cytokine-induced damage. Nitric oxide excess may increase susceptibility to nosocomial infections, so-called immunoparalysis.

The involvement of p53 in paraquat-induced apoptosis in human lung epithelial-like cells.

To investigate the possible role of p53 in the progression of paraquat-induced apoptosis, the authors used two cell lines that were wild-type p53-expressing human lung epithelial-like cell line (L132) and a p53-deficient human promyelocytic leukemia cell line (U937) and explored the linkage between p53, DNA damage, and apoptosis. Following paraquat exposure to L132 cells, the percentage of S-phase cells decreased significantly and the expression of p53 protein increased, suggesting that entry into S phase from G1 phase was blocked. U937 cells showed complete resistance to paraquat, although paraquat-evoked initial single-stranded DNA breaks was shown equally in either L132 or U937 cells, as assessed by single-cell gel electrophoresis. U937 and L132 cells die normally with similar kinetics when exposed to tumor necrosis factor in the presence of cycloheximide, indicating that their capacity to undergo p53-independent mechanisms of inducing apoptosis has an equal rate. These results suggest that paraquat-induced DNA damage caused G1 arrest and apoptosis only in L132 cells, and that p53 protein accumulation is required for the induction of apoptosis by paraquat.

Mitochondrial permeability transition and cytochrome c release in ischemia-reperfusion injury of the rat liver.

BACKGROUND: We investigated whether ischemia-reperfusion causes activation of caspases and whether this activation is related to cytochrome c release from the mitochondria into the cytosol as a result of the mitochondrial inner membrane permeability transition. MATERIALS AND METHODS: Rats were subjected to 30 min to 120 min of hepatic ischemia followed by 6 h of reperfusion. Cyclosporin A or ruthenium red (inhibitors of the mitochondrial inner membrane permeability transition) was given intravenously at 60 and 30 min before ischemia, respectively. RESULTS: Reperfusion after ischemia caused the release of liver enzymes accompanied by mitochondrial membrane depolarization, DNA fragmentation, and translocation of cytochrome c from the mitochondria into the cytosol. Accumulation of cytochrome c in the cytosol and activation of caspase-3-like protease was already detected during ischemia and before reperfusion. Pretreatment with cyclosporin A or ruthenium red significantly ameliorated the loss of the mitochondrial membrane potential, the increase of plasma membrane permeability, the cytosolic accumulation of cytochrome c, DNA fragmentation, and caspase-3-like protease activation. CONCLUSIONS: The mitochondrial inner membrane permeability transition occurs during ischemia and/or after reperfusion, resulting in translocation of cytochrome c and activation of caspases.

[Relapse of Creosote poisoning: report of a case taking Seirogan tablets].

A thirty-eight year old man took about 180 tablets of Seirogan. He was unconscious and had dyspnea with dark brown urine on admission. He recovered gradually after initial treatment. Seirogan contains a phenolic component. Symptoms and signs of poisoning are unconsciousness, convulsion, digestive tract disorder, pulmonary edema, hepatic failure, renal failure, and miosis. Clinical features include dark brown urine. On day 7, he again showed signs of creosote poisoning: relapse of unconsciousness and dark colored urine. Plasma concentration of phenol determined on the day before the relapse was much higher than that expected from the half-life of blood phenol. It is reported that Creosote poisoning results in a decrease in the intestinal peristalsis, or paralytic ileus. We would like to emphasize that a relapse of Creosote poisoning may occur due to possible delayed absorption of the Seirogan tablets.