Aldehyde-Based Inhibitors of the Peptidoglycan O-Acetylesterase Ape.

The O-acetylation of the muramic acid residues in peptidoglycan (PG) is a modification that protects the bacteria from lysis due to the action of lysozyme. In Gram-negative bacteria, deacetylation is required to allow lytic transglycosylases to promote PG cleavage during cell growth and division. This deacetylation is catalyzed by O-acetylpeptidoglycan esterase (Ape) which is a serine esterase and employs covalent catalysis via a serine-linked acyl enzyme intermediate. Loss of Ape activity affects the size and shape of bacteria and dramatically reduces virulence. In this work, we report the first rationally designed aldehyde-based inhibitors of Ape from Campylobacter jejuni. The most potent of these acts as a competitive inhibitor with a Ki value of 13 µM. We suspect that the inhibitors are forming adducts with the active site serine that closely mimic the tetrahedral intermediate of the normal catalytic cycle. Support for this notion is found in the observation that reduction of the aldehyde to an alcohol effectively abolishes the inhibition.

Singular Spectrum Analysis for Modal Estimation from Stationary Response Only.

Conventional experimental modal analysis uses excitation and response information to estimate the frequency response function. However, many engineering structures face excitation signals that are difficult to measure, so output-only modal estimation is an important issue. In this paper, singular spectrum analysis is employed to construct a Hankel matrix of appropriate dimensions based on the measured response data, and the observability of the system state space model is used to treat the Hankel matrix as three components containing system characteristics, excitation and noise. Singular value decomposition is used to factorize the data matrix and use the characteristics of the left and right singular matrices to reduce the dimension of the data matrix to improve calculation efficiency. Furthermore, the singular spectrum is employed to estimate the minimum order to reconstruct the Hankel matrix; then, the excitation and noise components can be removed, and the system observability matrix can be obtained. By appropriately a factorizing system observability matrix, we obtain the system matrix to estimate the modal parameters. In addition, the fictitious modes produced by increasing the order of the matrix can be eliminated through the stabilization diagram.

Peptidoglycan binding by a pocket on the accessory NTF2-domain of Pgp2 directs helical cell shape of Campylobacter jejuni.

The helical morphology of Campylobacter jejuni, a bacterium involved in host gut colonization and pathogenesis in humans, is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by trimming of peptide stems by the LD-carboxypeptidase Pgp2 within the periplasm. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. We determined a 1.6 Å resolution crystal structure of Pgp2, which contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues and located ∼40 Å from the LD-carboxypeptidase active site. Expression of pgp2 in trans with substitutions of charged (Lys257, Lys307, Glu324) and hydrophobic residues (Phe242 and Tyr233) within the pocket did not restore helical morphology to a pgp2 deletion strain. Muropeptide analysis indicated a decrease of murotripeptides in the deletion strain expressing these mutants, suggesting reduced Pgp2 catalytic activity. Pgp2 but not the K307A mutant was pulled down by C. jejuni Δpgp2 PG sacculi, supporting a role for the pocket in PG binding. NMR spectroscopy was used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase domain and the accessory NTF2 domain to induce a helical cell shape.

Peptides Containing meso-Oxa-Diaminopimelic Acid as Substrates for the Cell-Shape-Determining Proteases Csd6 and Pgp2.

The enzymes Csd6 and Pgp2 are peptidoglycan (PG) proteases found in the pathogenic bacteria Helicobacter pylori and Campylobacter jejuni, respectively. These enzymes are involved in the trimming of non-crosslinked PG sidechains and catalyze the cleavage of the bond between meso-diaminopimelic acid (meso-Dap) and d-alanine, thus converting a PG tetrapeptide into a PG tripeptide. They are known to be cell-shape-determining enzymes, because deletion of the corresponding genes results in mutant strains that have lost the normal helical phenotype and instead possess a straight-rod morphology. In this work, we report two approaches directed towards the synthesis of the tripeptide substrate Ac-iso-d-Glu-meso-oxa-Dap-d-Ala, which serves as a mimic of the terminus of an non-crosslinked PG tetrapeptide substrate. The isosteric analogue meso-oxa-Dap was utilized in place of meso-Dap to simplify the synthetic procedure. The more efficient synthesis involved ring opening of a peptide-embedded aziridine by a serine-based nucleophile. A branched tetrapeptide was also prepared as a mimic of the terminus of a crosslinked PG tetrapeptide. We used MS analysis to demonstrate that the tripeptide serves as a substrate for both Csd6 and Pgp2 and that the branched tetrapeptide serves as a substrate for Pgp2, albeit at a significantly slower rate.

The Biophysical Basis for Phosphorylation-Enhanced DNA-Binding Autoinhibition of the ETS1 Transcription Factor.

The eukaryotic transcription factor ETS1 is regulated by an intrinsically disordered serine-rich region (SRR) that transiently associates with the adjacent ETS domain to inhibit DNA binding. In this study, we further elucidated the physicochemical basis for ETS1 autoinhibition by characterizing the interaction of its ETS domain with a series of synthetic peptides corresponding to the SRR. Binding is driven by the hydrophobic effect and enhanced electrostatically by phosphorylation of serines adjacent to aromatic residues in the amphipathic SRR. Structural characterization of the dynamic peptide/protein complex by NMR spectroscopy and X-ray crystallography revealed multiple modes of binding that lead to autoinhibition by synergistically blocking the DNA-binding interface of the ETS domain and stabilizing an appended helical inhibitory module against allosterically induced unfolding. Consistent with these conclusions, the SRR peptide does not interact with DNA-bound ETS1. In addition, we found that the ETS1 SRR phosphopeptide binds to distantly related PU.1 in vitro, indicating that autoinhibition exploits features of the ETS domain that are conserved across this family of transcription factors.

Evaluating the Prognostic Value of ERCC1 and Thymidylate Synthase Expression and the Epidermal Growth Factor Receptor Mutation Status in Adenocarcinoma Non-Small-Cell Lung Cancer.

The present study evaluated the prognostic value of the epidermal growth factor receptor (EGFR) mutation status, and excision repair cross-complementation group 1 (ERCC1) and thymidylate synthase (TS) expression following intercalated tyrosine kinase inhibitor (TKI) therapy and platinum- and pemetrexed-based chemotherapies (subsequent second-line treatment) for patients with adenocarcinoma non-small-cell lung cancer (AC-NSCLC). In total, 131 patients with AC-NSCLC were enrolled. The EGFR mutation status and ERCC1 and TS expression were evaluated through direct DNA sequencing and immunohistochemical analyses, respectively. The EGFR mutation status and ERCC1 and TS expression were the significant predictors of clinical outcomes. The EGFR mutation status was the main outcome predictor for overall survival (OS) benefits in the overall population. Further exploratory ERCC1 and TS expression analyses were conducted to provide additional insights. Low TS expression was predictive of improved OS of patients with negative EGFR-mutated advanced AC-NSCLC, whereas high ERCC1 expression resulted in poor OS in patients with positive EGFR-mutated advanced AC-NSCLC. TS and ERCC1 expression levels were effective prognostic factors for negative and positive EGFR-mutated AC-NSCLC, respectively. In conclusion, the present results indicate that the EGFR mutation status and TS and ERCC1 expression can be used as the predictors of OS after subsequent second-line treatments for AC-NSCLC.

Independent Prognostic Value of Hypoxia-inducible Factor 1-alpha Expression in Small Cell Lung Cancer.

Hypoxia is an important factor in tumor angiogenesis, metastasis, and resistance to chemotherapy or radiotherapy, and may be an indicator of poor prognosis. The transcription factor hypoxia-inducible factor 1 (HIF-1) is the key regulator of the hypoxic state. This study was designed to evaluate the prognostic value of HIF-1α expression in small cell lung cancer (SCLC). Forty-three paraffin-embedded biopsy materials were examined using immunohistochemistry. Our results indicated that the expression of HIF-1α was high in males, and patients with poor Eastern Cooperative Oncology Group (ECOG) performance status and metastases. To elucidate the prognostic value of HIF-1α expression, Kaplan-Meier analysis was carried out and the results showed that patients with high HIF-1α expression had a poorer prognosis than patients with low expression of HIF-1α. After adjusting clinical parameters by the Cox proportional hazards model, our results demonstrated that high HIF-1α expression is an independent prognostic factor for SCLC with a 39.2-fold risk of death (p<0.003). In conclusion, we have provided evidence that HIF-1α expression has significant value in predicting survival of patients with SCLC and is an independent prognostic factor beyond ECOG performance and metastasis status.

Chronic Influences of Obstructive Sleep Apnea on Cerebral Venous Flow.

OBJECTIVES: Evidence reveals a pathophysiologic link between sleep apnea syndrome and cerebrovascular diseases. It is known that obstructive sleep apnea (OSA) may cause serial hemodynamic changes and structural abnormalities in the cerebral and cardiac arterial systems, but its effect on the cerebral venous system has remained unclear. The purpose of this study was to compare internal jugular vein hemodynamics between patients with OSA and healthy individuals. METHODS: Patients with OSA and age-, body mass index-, and sex-matched healthy control participants were recruited for a jugular venous duplex study and neurologic examination. The luminal area of the internal jugular vein, jugular venous flow volume, time-averaged mean velocity, and presence of jugular venous reflux were recorded. These flow characteristics were obtained at different respiratory statuses, and we analyzed the differences between patients and controls. RESULTS: In the OSA group, there was an increasing flow volume in total internal jugular veins at rest. The frequency of venous reflux in patients compared with controls was significantly decreased (26.7% versus 53.3%, respectively; P < .05). The internal jugular vein drainage dominance was greater on the left side in the OSA group (right versus left: 48.8% versus 51.2%), whereas it was greater on the right side in the control group (right versus left: 61.7% versus 38.3%). CONCLUSIONS: Our data showed peculiar internal jugular vein hemodynamics at baseline and different respiratory statuses in patients with OSA. These characteristics imply that cerebral venous drainage conditions might be involved in the pathophysiologic mechanisms of OSA syndrome.

Distinct structural features of the peroxide response regulator from group A Streptococcus drive DNA binding.

Group A streptococcus (GAS, Streptococcus pyogenes) is a strict human pathogen that causes severe, invasive diseases. GAS does not produce catalase, but has an ability to resist killing by reactive oxygen species (ROS) through novel mechanisms. The peroxide response regulator (PerR), a member of ferric uptake regulator (Fur) family, plays a key role for GAS to cope with oxidative stress by regulating the expression of multiple genes. Our previous studies have found that expression of an iron-binding protein, Dpr, is under the direct control of PerR. To elucidate the molecular interactions of PerR with its cognate promoter, we have carried out structural studies on PerR and PerR-DNA complex. By combining crystallography and small-angle X-ray scattering (SAXS), we confirmed that the determined PerR crystal structure reflects its conformation in solution. Through mutagenesis and biochemical analysis, we have identified DNA-binding residues suggesting that PerR binds to the dpr promoter at the per box through a winged-helix motif. Furthermore, we have performed SAXS analysis and resolved the molecular architecture of PerR-DNA complex, in which two 30 bp DNA fragments wrap around two PerR homodimers by interacting with the adjacent positively-charged winged-helix motifs. Overall, we provide structural insights into molecular recognition of DNA by PerR and define the hollow structural arrangement of PerR-30bpDNA complex, which displays a unique topology distinct from currently proposed DNA-binding models for Fur family regulators.

The CYP1B1 Leu432Val polymorphism and risk of urinary system cancers.

The cytochrome P450 1B1 (CYP1B1) gene plays a key role in the metabolism of various carcinogens. The CYP1B1 Leu432Val polymorphism leads to leucine to valine substitution at codon 432. A lot of studies have shown that the CYP1B1 Leu432Val polymorphism was associated with urinary system cancers, especially prostate cancer. However, the results were still inconclusive. In this meta-analysis, by searching online databases and references of related reviews, we identified 17 eligible studies to assess the relationship between CYP1B1 Leu432Val polymorphism and urinary system cancers, including 7,783 cancer cases and 7,238 controls. By pooling all eligible studies, we found that the CYP1B1 Leu432Val polymorphism was not associated with overall urinary system cancers. However, in subgroup analyses, we found that the variant 432Val allele significantly increased the risk of prostate cancer (Val vs. Leu, odds ratio (OR) = 1.064, 95% confidence interval (CI) 0.981-1.154; Pheterogeneity = 0.002), while no association was found for bladder cancer (Val vs. Leu, OR = 0.942, 95% CI 0.853-1.041; Pheterogeneity = 0.504). No evidence of publication bias was found (Begg's test, P = 0.053; Egger's test, P = 0.073). In conclusion, based on 17 eligible studies, we found that the CYP1B1 Leu432Val polymorphism was associated with an increased risk of prostate cancer, while no association of bladder cancer was observed.

Control of Oxidative Stress and Generation of Induced Pluripotent Stem Cell-like Cells by Jun Dimerization Protein 2.

We report here that the Jun dimerization protein 2 (JDP2) plays a critical role as a cofactor for the transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) and MafK in the regulation of the antioxidants and production of reactive oxygen species (ROS). JDP2 associates with Nrf2 and MafK (Nrf2-MafK) to increase the transcription of antioxidant response element-dependent genes. Oxidative-stress-inducing reagent led to an increase in the intracellular accumulation of ROS and cell proliferation in Jdp2 knock-out mouse embryonic fibroblasts. In Jdp2-Cre mice mated with reporter mice, the expression of JDP2 was restricted to granule cells in the brain cerebellum. The induced pluripotent stem cells (iPSC)-like cells were generated from DAOY medulloblastoma cell by introduction of JDP2, and the defined factor OCT4. iPSC-like cells expressed stem cell-like characteristics including alkaline phosphatase activity and some stem cell markers. However, such iPSC-like cells also proliferated rapidly, became neoplastic, and potentiated cell malignancy at a later stage in SCID mice. This study suggests that medulloblastoma cells can be reprogrammed successfully by JDP2 and OCT4 to become iPSC-like cells. These cells will be helpful for studying the generation of cancer stem cells and ROS homeostasis.

Identification of furo[3', 2':3,4]naphtho[1,2-d]imidazole derivatives as orally active and selective inhibitors of microsomal prostaglandin E(2) synthase-1 (mPGES-1).

This study describes the synthesis and anti-inflammatory effects of furo[3', 2':3,4]naphtho[1,2-d] imidazole derivatives. Among these furo[3', 2':3,4]naphtho[1,2-d]imidazole derivatives, 2-(4-methoxyphenyl)furo [3', 2':3,4]naphtho[1,2-d]imidazole (12) exhibited a strong inhibitory activity against LPS-induced PGE(2) production, with an IC(50) value of 47 nM. Compound 12 is then further examined for its inhibitory effects in the protein expression of COX-2 and microsomal prostaglandin E(2) synthase-1 (mPGES-1) in Raw 264.7 cells. Our results indicate that compound 12 was capable against inhibiting LPS-induced mPGES-1 protein expression at a concentration of 1.0 µM and no inhibitory effect in COX-2 expression. The sepsis-induced PGE(2) production in rat serum decreased ~250% by the pretreatment of 12 at 10 mg/kg. These results are especially important since compound 12 exhibited good oral bioavailability (72%) and was not cytotoxic at a concentration of 10.0 µM. Therefore, compound 12 is a highly selective mPGES-1 inhibitor that can serve as a lead for the development of novel oral anti-inflammatory drug candidates.

A survey of allergic rhinitis in Taiwanese asthma patients.

BACKGROUND: Allergic rhinitis (AR) often coexists with and can significantly worsen bronchial asthma (BA). We evaluated the proportion of patients with BA and concomitant AR (BA+AR) diagnosed and treated in an average clinical practice. METHODS: A cross-sectional study methodology was used to determine the prevalence of AR in patients more than 15 years of age with a documented history of asthma who experienced wheezing during the prior 12 months. The International Study of Asthma and Allergies in Childhood standardized written questionnaire was used and therapeutic class choices were recorded. RESULTS: Among 750 surveyed asthma patients, 524 patients (69.9%) experienced AR. Of those with BA and AR, there were 44 patients (8.4%) who were not diagnosed with AR, and the treatment rate for AR was 62.1%. The most frequent severity level of BA and AR were moderate persistent (38.9%) and intermittent mild (52.5%), respectively. There were no significant differences between patients with AR and without AR. CONCLUSIONS: The prevalence of AR in patients with asthma was 69.9% in this study. Despite Allergic Rhinitis and its Impact on Asthma guideline recommendations encouraging evaluation and treatment of AR among asthmatics, nearly 8.4% of asthmatics with AR were undiagnosed, and 37.9% of asthmatics with AR were untreated for AR.

Macrophage activation by gastric fluid suggests MMP involvement in aspiration-induced lung disease.

Asthma occurs in more than 5% of the population in industrialized countries and is now characterized as a chronic inflammatory disease. The chronic aspiration of gastric fluid is considered by many investigators to be a primary inflammatory factor exacerbating or predisposing patients to asthma, with more than 50 medical papers per year linking asthma with gastroesophageal reflux disease (GERD), which can lead to aspiration events. However, the mechanisms involved in the inflammatory effects caused by gastric-fluid aspiration are not clear at the present time. The role of macrophages in the pathogenesis of disease seems likely given the involvement of those cells in a variety of chronic inflammatory diseases. To investigate the potential role of gastric fluid and the mechanisms potentially underlying chronic aspiration-associated pathogenesis, we examined the activation of murine macrophages (Raw 264.7 cell line) with gastric fluid. Inflammatory cytokine production and activation of the NF-kappaB signaling pathway were observed. Toll-like receptor (TLR)-4-dependent activation was observed under some conditions, indicating that bacterial components within the gastric fluid are involved in macrophage activation. Matrix metalloproteinase-9 (MMP-9) expression by macrophages was enhanced by gastric fluid, suggesting a potential mechanism by which remodeling of airways might be induced by gastric-fluid aspiration.

Furo[3',2':3,4]naphtho[1,2-d]imidazole derivatives as potential inhibitors of inflammatory factors in sepsis.

Synthesis and anti-inflammatory effects of certain furo[3',2':3,4]naphtho[1,2-d]imidazole derivatives 12-18 were studied. These compounds were synthesized from naphtho[1,2-b]furan-4,5-dione (10) which in turn was prepared from the known 2-hydroxy-1,4-naphthoquinone (7) in a one pot reaction. Furo[3',2':3,4]naphtho[1,2-d]imidazole (12) was inactive (IC(50) value of >30 microM) while its 5-phenyl derivative 13, with an IC(50) value of 16.3 and 11.4 microM against lysozyme and beta-glucuronidase release, respectively, was comparable to the positive trifluoperazine. The same potency was observed for 5-furan derivative 16 with an IC(50) value of 19.5 and 11.3 microM against lysozyme and beta-glucuronidase release, respectively. An electron-withdrawing NO(2) substituted on 5-phenyl or 5-furanyl group led to the devoid of activity as in the cases of 14 and 17. Among them, compound 15 exhibited significant inhibitory effects, with an IC(50) value of 7.4 and 5.0 microM against lysozyme and beta-glucuronidase release, respectively. For the LPS-induced NO production, the phenyl derivatives 12-15 were inactive while the nitrofuran counterparts 17 and 18 suppress LPS-induced NO production significantly, with an IC(50) value of 1.5 and 1.3 microM, respectively, which are more active than that of the positive 1400 W. Compounds 16-18 were capable of inhibiting LPS-induced iNOS protein expression at a dose-dependent manner in which compound 18, with an IC(50) of 0.52 microM in the inhibition of iNOS expression, is approximately fivefold more potent than that of the positive 1400 W. In the CLP rat animal model, compound 18 was found to be more active than the positive hydrocortisone in the inhibition of the iNOS mRNA expression in rat lung tissue. The sepsis-induced PGE2 production in rat serum decreased 150% by the pretreatment of 18 in a dose of 10 mg/kg.