Replication stress by Py-Im polyamides induces a non-canonical ATR-dependent checkpoint response.

Pyrrole-imidazole polyamides targeted to the androgen response element were cytotoxic in multiple cell lines, independent of intact androgen receptor signaling. Polyamide treatment induced accumulation of S-phase cells and of PCNA replication/repair foci. Activation of a cell cycle checkpoint response was evidenced by autophosphorylation of ATR, the S-phase checkpoint kinase, and by recruitment of ATR and the ATR activators RPA, 9-1-1, and Rad17 to chromatin. Surprisingly, ATR activation was accompanied by only a slight increase in single-stranded DNA, and the ATR targets RPA2 and Chk1, a cell cycle checkpoint kinase, were not phosphorylated. However, ATR activation resulted in phosphorylation of the replicative helicase subunit MCM2, an ATR effector. Polyamide treatment also induced accumulation of monoubiquitinated FANCD2, which is recruited to stalled replication forks and interacts transiently with phospho-MCM2. This suggests that polyamides induce replication stress that ATR can counteract independently of Chk1 and that the FA/BRCA pathway may also be involved in the response to polyamides. In biochemical assays, polyamides inhibit DNA helicases, providing a plausible mechanism for S-phase inhibition.

Biosynthesis of the sesquiterpene botrydial in Botrytis cinerea. Mechanism and stereochemistry of the enzymatic formation of presilphiperfolan-8beta-ol.

Presilphiperfolan-8beta-ol synthase, encoded by the BcBOT2 gene from the necrotrophic plant pathogen Botrytis cinerea, catalyzes the multistep cyclization of farnesyl diphosphate (2) to the tricyclic sesquiterpene alcohol presilphiperfolan-8beta-ol (3), the preursor of the phytotoxin botrydial, a strain-dependent fungal virulence factor. Incubation of (1R)-[1-(2)H]farnesyl diphosphate (2b) with recombinant presilphiperfolan-8beta-ol synthase gave exclusively (5R)-[5alpha-(2)H]-3b, while complementary incubation of (1S)-[1-(2)H]FPP (2c) gave (5S)-[5beta-(2)H]-3c. These results established that cyclization of farnesyl diphosphate involves displacement of the diphosphate group from C-1 with net inversion of configuration and ruled out the proposed intermediacy of the cisoid conformer of nerolidyl diphosphate (9) in the cyclization. While not a mandatory intermediate, (3R)-nerolidyl diphosphate was shown to act as a substrate surrogate. Cyclization of [13,13,13-(2)H(3)] farnesyl diphosphate (2d) gave [14,14,14-(2)H(3)]-3d, thereby establishing that electrophilic attack takes place exclusively on the si face of the 12,13-double bond of 2. The combined results provide a detailed picture of the conformation of enzyme-bound farnesyl diphosphate at the active site of presilphiperfolan-8beta-ol synthase.

Biochemistry and molecular genetics of the biosynthesis of the earthy odorant methylisoborneol in Streptomyces coelicolor.

Methylisoborneol (2) is a volatile organic compound produced by a wide variety of Actinomycete soil organisms, myxobacteria, and cyanobacteria. It has an unusually low odor threshold and, together with geosmin, is responsible for the characteristic smell of moist soil as well as unpleasant taste and odor episodes associated with public water supplies and contamination of various foodstuffs, including fish, wine, and beer. Despite considerable interest in detection and remediation of methylisoborneol, the biosynthesis of this methylated monoterpene has been obscure. In Streptomyces coelicolor, the sco7700 and sco7701 genes are shown to correspond to a two-gene operon responsible for methylisoborneol biosynthesis. Both genes have been amplified by PCR and the resulting DNA has been cloned and expressed in Escherichia coli. Incubation of recombinant SCO7701 protein, annotated as a possible C-methyltransferase, with geranyl diphosphate (1) and S-adenosylmethionine gave the previously unknown compound, (E)-2-methylgeranyl diphosphate (3). Incubation of 3 in the presence of Mg2+ with recombinant SCO7700, previously annotated only as a possible metal-binding protein or terpenoid synthase, resulted in the formation of 2-methylisoborneol (2). The steady-state kinetic parameters for both biochemical reactions have been determined. Incubation of geranyl diphosphate and S-adenosylmethionine with a mixture of both SCO7700 and SCO7701 resulted in formation of methylisoborneol (2). Cyclization of 2-methylgeranyl diphosphate (3) to methylisoborneol (2) likely involves the intermediacy of 2-methyllinalyl diphosphate.

Expression of programmed death 1 ligand 1 on periodontal tissue cells as a possible protective feedback mechanism against periodontal tissue destruction.

Programmed death 1 ligand 1 (PD­L1) is a negative co­stimulatory molecule in immune responses. Previous reports have indicated that inflammatory cytokines can upregulate the expression of PD­L1 in tumor cells, which in turn suppresses host immune responses. Periodontitis is characterized by persistent inflammation of the periodontium, which is initiated by infection with oral bacteria and results in damage to cells and the matrices of the periodontal connective tissues. In the present study, the expression and function of PD­L1 in periodontal tissue destruction were examined. Periodontal ligament cells (PDLCs) were stimulated by inflammatory cytokines and periodontal pathogens. The expression and function of PD­L1 on the surface of PDLCs was investigated using flow cytometry in vitro. Periodontal disease was induced by the injection of Porphyromonas gingivalis in mouse models. The expression levels of PD­L1 in the periodontal tissues of the mice were analyzed using flow cytometry and immunohistochemistry. PD­L1 was inducibly expressed on the PDLCs by the inflammatory cytokines and periodontal pathogens. The inflammation­induced expression of PD­L1 was shown to cause the apoptosis of activated T lymphocytes and improve the survival of PDLCs. Furthermore, in the mouse model of experimental periodontitis, the expression of PD­L1 in severe cases of periodontitis was significantly lower, compared with that in mild cases. By contrast, no significant differences were observed between the healthy control and severe periodontitis groups. The results of the present study showed that the expression of PD­L1 may inhibit the destruction of periodontal tissues, indicating the involvement of a possible protective feedback mechanism against periodontal infection.

Which subtype of functional dyspepsia patients responses better to acupuncture? A retrospective analysis of a randomized controlled trial.

BACKGROUND: Whether subgroups of functional dyspepsia (FD) should be treated with different approaches is controversially discussed in research. As our previous study has demonstrated the effect of acupuncture in FD treatment, we now further analyze the therapeutic effect of acupuncture in the treatment of postprandial distress syndrome (PDS) and epigastric pain syndrome (EPS). METHODS: A retrospective analysis was conducted in 465 eligible PDS patients and 241 EPS patients. 4 acupuncture groups (group A: specific acupoints along the stomach meridian; group B: non-specific acupoints along the stomach meridian; group C: alarm and transport acupoints; group D: specific acupoints along the gallbladder meridian) were compared with a non-acupoint sham acupuncture group and an itopride group. The patients were treated in 5 consecutive sessions per week for 4 weeks and were followed-up for 12 weeks afterwards. Primary outcome of the study was defined as response rate and symptom improvement as measured by the Symptom Index of Dyspepsia, while secondary outcome was designated as improvement in quality of life (QoL) as determined by the Nepean Dyspepsia Index. RESULTS: Symptoms of dyspepsia and QoL were improved from baseline in all groups. In EPS patients, no statistically significant differences could be observed in response rate (p = 0.239) and symptoms improvement (p = 0.344 for epigastric pain; p = 0.465 for epigastric burning). In contrast, PDS patients of the acupuncture group A showed higher response rate (53.2% vs. 19.7%, p<0.001; 53.2% vs. 35.1%, p = 0.025) and score change in postprandial fullness (1.01 vs. 0.27, p<0.001; 1.01 vs. 0.57, p<0.001), early satiation (0.81 vs. 0.21, p<0.001; 0.81 vs. 0.39, p=0.001), and QoL (14.5 vs. 4.33, p<0.001; 14.5 vs. 8.5, p<0.001) compared to the sham acupuncture and itopride group. CONCLUSIONS: FD patients with PDS responded better to the acupuncture therapies, especially at the specific acupoints along the stomach meridian. The positive therapeutic effect of acupuncture on PDS was correlated with the improvement in postprandial fullness. TRIAL REGISTRATION: ClinicalTrial.gov NCT00599677.

Animal toxicity of hairpin pyrrole-imidazole polyamides varies with the turn unit.

A hairpin pyrrole-imidazole polyamide (1) targeted to the androgen receptor consensus half-site was found to exert antitumor effects against prostate cancer xenografts. A previous animal study showed that 1, which has a chiral amine at the α-position of the γ-aminobutyric acid turn (γ-turn), did not exhibit toxicity at doses less than 10 mg/kg. In the same study, a polyamide with an acetamide at the ß-position of the γ-turn resulted in animal morbidity at 2.3 mg/kg. To identify structural motifs that cause animal toxicity, we synthesized polyamides 1-4 with variations at the α- and ß-positions in the γ-turn. Weight loss, histopathology, and serum chemistry were analyzed in mice post-treatment. While serum concentration was similar for all four polyamides after injection, dose-limiting liver toxicity was only observed for three polyamides. Polyamide 3, with an α-acetamide, caused no significant evidence of rodent toxicity and retains activity against LNCaP xenografts.

Sesquiterpene synthase from the botrydial biosynthetic gene cluster of the phytopathogen Botrytis cinerea.

The fungus Botrytis cinerea is the causal agent of the economically important gray mold disease that affects more than 200 ornamental and agriculturally important plant species. B. cinerea is a necrotrophic plant pathogen that secretes nonspecific phytotoxins, including the sesquiterpene botrydial and the polyketide botcinic acid. The region surrounding the previously characterized BcBOT1 gene has now been identified as the botrydial biosynthetic gene cluster.Five genes including BcBOT1 and BcBOT2 were shown by quantitative reverse transcription-PCR to be co-regulated through the calcineurin signaling pathway. Inactivation of the BcBOT2 gene, encoding a putative sesquiterpene cyclase, abolished botrydial biosynthesis, which could be restored by in trans complementation.Inactivation of BcBOT2 also resulted in overproduction of botcinic acid that was observed to be strain-dependent. Recombinant BcBOT2 protein converted farnesyl diphosphate to the parent sesquiterpene of the botrydial biosynthetic pathway, the tricyclic alcohol presilphiperfolan-8beta-ol.