Design, synthesis and structure-activity relationship studies of novel indazole analogues as DNA gyrase inhibitors with Gram-positive antibacterial activity

In this study, we report the design, synthesis and structure-activity relationships of novel indazole derivatives as DNA gyrase inhibitors with Gram-positive antibacterial activity. Our results show that selected compounds from this series exhibit potent antibacterial activity against Gram-positive bacteria including multi-drug resistant strains that is methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE).     

Kinetic characteristics and toxic effects of benzalkonium chloride following intravascular and oral administration in rats

Kinetic characteristics and toxic effects of benzalkonium chloride (BZK) following injection via jugular vein (JV), femoral artery (FA) and oral administration (PO) were experimentally investigated using rats. The BZK concentrations in blood and tissues (lung, liver and kidney) were determined by high-performance liquid chromatography with solid phase extraction. Toxic doses of 15 and 250 mg/kg of BZK were used for intravascular (JV and FA) and PO administration, respectively. The fatal effects appeared soon after the dose in JV-rats, while delayed in FA- or PO-rats. The blood BZK concentrations and the elimination half-lives were similar between JV- and FA-rats, while the distribution of BZK in tissues was slightly different. In PO administration, the rats that aspirated BZK into their lungs had some symptoms, while the rats that did not aspirate BZK appeared to be normal. The BZK concentrations in blood and tissues were significantly higher in the aspirated PO-rats. The toxic degree of BZK was correlated with the BZK concentration in orally dosed rats. Lung and kidney had higher BZK concentrations compared to blood or liver, and they could be the target organs of BZK.Keyword: Benzalkonium chloride     

The pheromone production of female Plodia interpunctella is inhibited by tyraminergic antagonists

Several compounds were found to suppress the calling behavior and in vitro pheromone biosynthesis of the Indian meal moth, Plodia interpunctella. The compounds were screened by means of a calling-behavior bioassay with female P. interpunctella. Five derivatives with activities in the nanomolar range were identified, in order of decreasing pheromonostatic activity: 4-hydroxybenzaldehyde semicarbazone (42) > 5-(4-methoxyphenyl)-1,3-oxazole (38) > 5-[4-(tert-butyl)phenyl]-1,3-oxazole (40) > 5-(3-methoxyphenyl)-1,3-oxazole (35) > 5-(4-cyanophenyl)-1,3-oxazole (36). These compounds also showed in vitro inhibitory activity in intracellular de novo pheromone biosynthesis, as determined with isolated pheromone-gland preparations that incorporated [1-(14)C]sodium acetate in the presence of the so-called pheromone-biosynthesis-activating neuropeptide (PBAN). The non-additive effect of the inhibitor with antagonist (yohimbine) for the tyramine (TA) receptor suggests that it could be a tyraminergic antagonist. Three-dimensional (3D) computer models were built from a set of compounds. Among the common-featured models generated by the program Catalyst/HipHop, aromatic-ring (AR) and H-bond-acceptor-lipophilic (HBAl) features were considered to be essential for inhibitory activity in the calling behavior and in vitro pheromone biosynthesis. Active compounds, including yohimbine, mapped well onto all the AR and HBAl features of the hypothesis. Less-active compounds were shown to be unable to achieve an energetically favorable conformation, consistent with our 3D common-feature pharmacophore models. The present hypothesis demonstrates that calling behavior and PBAN-stimulated incorporation of radioactivity are inhibited by tyraminergic antagonists.     

Stereoselective inhibition of glutamate carboxypeptidase by organophosphorus derivatives of glutamic acid

A series of alkyl and aryl phosphonyl, thiophosphonyl, and dithiophosphonyl derivatives of (S)- and (R)-glutamic acid were prepared and examined for inhibitory potency against glutamate carboxypeptidase (carboxypeptidase G). The acquisition of the phosphonamidodithioic acids and the individual phosphonamidothioic acid diastereomers was achieved through a common phosphonamidothiolate precursor, which also allowed for the chromatographic resolution of the chiral phosphorus center of the phosphonamidothioic acids. The most potent inhibitor of the series was the n-butylphosphonamidate derivative of the natural isomer of glutamic acid. Although each diastereomeric pair of three phosphonamidothionates exhibited stereoselective inhibition consistent with the configuration of the chiral phosphorus center, this effect was generally not remarkable. More important, was the effect of carbon stereochemistry upon glutamate carboxypeptidase inhibition as exemplified by a limited series of enantiomeric pairs of phosphonamidate and phosphonamidodithionate derivatives of glutamic acid. The phosphonamidate analogs derived from the unnatural stereoisomer of glutamic acid were devoid of inhibitory potency in contrast to their enantiomers. Surprisingly, the phosphonamidodithionates derived from the unnatural stereoisomer of glutamic acid demonstrated greater inhibitory potency than their naturally-derived antipodes.     

Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Image shift due to beam-induced specimen charging has become the most severe problem in electron microscopy for imaging two-dimensional (2D) crystals of biological macromolecules, especially in the case of highly tilted specimens. Image shift causes diffraction spots perpendicular to the tilt axis to disappear even at medium or low resolution. The yield of good images from tilted specimens prepared on a single layer of continuous carbon support film is therefore very low. In this paper, we have used 2D crystals of aquaporin-4 to investigate the effect of a carbon sandwich preparation method on specimen charging. We find that a larger number of images show sharp diffraction spots perpendicular to the tilt axis if crystals are placed in between two sheets of carbon film as compared to images taken from specimens prepared by the conventional single carbon support film technique. Our results demonstrate that the reproducible carbon sandwich preparation technique overcomes the severe specimen charging problem and thus has the potential to significantly speed up structure analysis by electron crystallography.     

Teprenone, but not H2-receptor blocker or sucralfate, suppresses corpus Helicobacter pylori colonization and gastritis in humans: teprenone inhibition of H. pylori-induced interleukin-8 in MKN28 gastric epithelial cell lines

Background. The role of teprenone in Helicobacter pylori‐associated gastritis has yet to be determined. To investigate the effect of teprenone on inflammatory cell infiltration, and on H. pylori colonization of the gastric mucosa in H. pylori‐infected patients, we first compared the effect of teprenone with that of both histamine H2 receptor antagonists (H2‐RA) and sucralfate on the histological scores of H. pylori gastritis. We then examined its in vitro effect on H. pylori‐induced interleukin (IL)‐8 production in MKN28 gastric epithelial cells. Materials and Methods. A total of 68 patients were divided into three groups, each group undergoing a 3‐month treatment with either teprenone (150 mg/day), H2‐RA (nizatidine, 300 mg/day), or sucralfate (3 g/day). All subjects underwent endoscopic examination of the stomach before and after treatment. IL‐8 production in MKN28 gastric epithelial cells was measured by enzyme‐linked immunosorbent assay (ELISA). Results. Following treatment, the teprenone group showed a significant decrease in both neutrophil infiltration and H. pylori density of the corpus (before vs. after: 2.49 ± 0.22 vs. 2.15 ± 0.23, p = .009; 2.36 ± 0.25 vs. 2.00 ± 0.24, p = .035, respectively), with no significant differences seen in either the sucralfate or H2‐RA groups. Teprenone inhibited H. pylori‐enhanced IL‐8 production in MKN28 gastric epithelial cells in vitro, in a dose‐dependent manner. Conclusions. Teprenone may modify corpus H. pylori‐associated gastritis through its effect on neutrophil infiltration and H. pylori density, in part by its inhibition of IL‐8 production in the gastric mucosa.     

Exhaustive identification of interaction domains using a high-throughput method based on two-hybrid screening and PCR-convergence: molecular dissection of a kinetochore subunit Spc34p

The Dam1 complex, also known as DASH complex, is the outer kinetochore protein complex of yeast that plays a crucial role in attachment of kinetochore to microtubule. The Dam1 complex is formed by at least nine proteins including Dam1p, Duo1p, Dad1p, Spc19p and Spc34p. In this study, domains of Spc34p that physically interact with other subunits of the complex were mapped using a high-throughput methodology. The method is a combination of two-hybrid screening of a random truncation library of the Spc34 gene and a unique PCR-based amplification that converge the selected DNA fragments to a few short fragments. Duo1p, Dam1p, Dad1p and Spc19p binding domains of Spc34p were mapped on M1-E59, M1-D47, M1-D47 or T207-E295 and S154-Q294, respectively. Most of the boundaries were located at less conserved regions among fungal Spc34p homologs, which is consistent with the boundaries of the putative secondary structures. The accuracy of the mapped domain boundaries was verified using truncated Spc34p polypeptides. The results and methodology we demonstrated herein not only shed light on the molecular architecture of the protein complex but also pave the road to the high-throughput identification of specific interaction domains of proteins whose possible interaction partners have been identified in genome-scale analyses.     

A series of immune responses leading to the induction ofT cell IL-12/IL-18 responsiveness in patientswith relatively large tumor burdens

The induction of interleukin-12 (IL-12) responsiveness in T cells depends on T cell receptor (TCR) triggering, and is regarded as a parameter of recently TCR-sensitized T cells. Here, we investigated whether IL-12 responsiveness could be detected in freshly prepared T cells from tumor-bearing patients, and if so whether such patients exhibited additional immunological parameters related to IL-12 responsiveness. CD4(+) and CD8(+) T cell populations from an appreciable proportion of tumor-bearing patients exhibited high levels of IL-12 responsiveness as evaluated by IL-12-stimulated interferon-gamma (IFN-gamma) production. T cell populations with high IL-12 responsiveness were observed in the group of patients with moderate to large tumor mass or tumor metastases rather than in patients with small tumors. The frequency of such a T cell population was also lower in post-surgery tumor-free patients, showing the correlation between IL-12 responsiveness and the presence of a certain extent of tumor burden. More importantly, a higher incidence of IL-12 responsiveness was observed in tumor-bearing patients exhibiting detectable plasma IL-12 levels, and correlated with IL-18 responsiveness. T cell IL-12 and IL-18 responsiveness is induced by TCR triggering and subsequent IL-12 stimulation respectively. Furthermore, TCR-triggered T cells stimulate antigen-presenting cells (APC) to produce IL-12. Therefore, the present observations suggest that an immune response loop from TCR sensitization to the induction of IL-12/IL-18 responsiveness via IL-12 production operates in tumor-bearing patients, particularly in those with relatively large tumor burdens.     

A combination effect of epigallocatechin gallate, a major compound of green tea catechins, with antibiotics on Helicobacter pylori growth in vitro

Since green tea catechins are known to have antimicrobial activity against a variety of microorganisms, their possible effects on Helicobacter pylori in combination with antibiotics were examined. Fifty-six clinical isolates of H. pylori, including 19 isolates highly resistant to metronidazole (MTZ) and/or clarithromycin (CLR), were used to determine in vitro sensitivity to tea catechins. The MIC90 of both epigallocatechin gallate (EGCg) and epicatechin gallate (ECg) was 100 microg/ml. However, other tea catechins tested did not show any anti-H. pylori activity. Highly antibiotic-resistant clinical isolates showed a similar sensitivity to both EGCg and ECg. The kinetic study of antibacterial activity in liquid cultures revealed a relatively slow but strong activity on the growth of H. pylori. In combination with sub-MIC of amoxicillin (AMX), the antibacterial activity of AMX was significantly enhanced by the presence of EGCg. To estimate the general combination effect between EGCg and other antibiotics, such as MTZ and CLR, on the antibacterial activity against clinical isolates, the fraction inhibitory concentration (FIC) was determined by checkerboard study. The FIC indexes showed additive effects between EGCg and antibiotics tested. These results indicatethat EGCg may be a valuable therapeutic agent against H. pylori infection.