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Journal of Cell Science Sep 2017This study was designed to identify bioactive compounds that alter the cellular shape of the fission yeast by affecting functions involved in the cell cycle or cell...
This study was designed to identify bioactive compounds that alter the cellular shape of the fission yeast by affecting functions involved in the cell cycle or cell morphogenesis. We used a multidrug-sensitive fission yeast strain, SAK950 to screen a library of 657 actinomycete bacteria and identified 242 strains that induced eight different major shape phenotypes in These include the typical cell cycle-related phenotype of elongated cells, and the cell morphology-related phenotype of rounded cells. As a proof of principle, we purified four of these activities, one of which is a novel compound and three that are previously known compounds, leptomycin B, streptonigrin and cycloheximide. In this study, we have also shown novel effects for two of these compounds, leptomycin B and cycloheximide. The identification of these four compounds and the explanation of the phenotypes in terms of their known, or predicted bioactivities, confirm the effectiveness of this approach.
Topics: Actinomyces; Biological Products; Cell Shape; Checkpoint Kinase 1; Cycloheximide; DNA Damage; Drug Evaluation, Preclinical; Fatty Acids, Unsaturated; Phenotype; Schizosaccharomyces; Spectrometry, Mass, Electrospray Ionization
PubMed: 28775153
DOI: 10.1242/jcs.194571 -
Molecular Carcinogenesis Jul 2017Quinone oxidoreductase (NQO1) functions as an important part of cellular antioxidant defense by detoxifying quinones, thus preventing the formation of reactive oxygen...
Quinone oxidoreductase (NQO1) functions as an important part of cellular antioxidant defense by detoxifying quinones, thus preventing the formation of reactive oxygen species. The aims of our study were to determine if NQO1 is elevated in pancreatic cancer specimens and pancreatic cancer cell lines and if so, would compounds previously demonstrated to redox cycle with NQO1 be effective in killing pancreatic cancer cells. Immunohistochemistry of resected pancreatic specimens demonstrated an increased immunoreactivity for NQO1 in pancreatic cancer and pancreatic intraepithelial neoplasia (PanIN) specimens versus normal human pancreas. Immunocytochemistry and Western immunoblots demonstrated inceased immunoreactivity in pancreatic cancer cells when compared to a near normal immortalized human pancreatic ductal epithelial cell line and a colonic epithelial cell line. Streptonigrin, a compound known to cause redox cycling in the presence of NQO1, decreased clonogenic survival and decreased anchorage-independent growth in soft agar. Streptonigrin had little effect on cell lines with absent or reduced levels of NQO1. The effects of streptonigrin were reversed in pancreatic cancer cells pretreated with dicumarol, a known inhibitor of NQO1. NQO1 may be a therapeutic target in pancreatic cancer where survival is measured in months. © 2006 Wiley-Liss, Inc.
PubMed: 28639725
DOI: 10.1002/mc.20199 -
Cell Death & Disease May 2017The role of connexin proteins (Cx), which form gap junctions (GJ), in progression and chemotherapeutic sensitivity of cervical cancer (CaCx), is unclear. Using cervix...
The role of connexin proteins (Cx), which form gap junctions (GJ), in progression and chemotherapeutic sensitivity of cervical cancer (CaCx), is unclear. Using cervix specimens (313 CaCx, 78 controls) and CaCx cell lines, we explored relationships among Cx expression, prognostic variables and mechanisms that may link them. In CaCx specimens, Cx32 was upregulated and cytoplasmically localized, and three other Cx downregulated, relative to controls. Cx32 expression correlated with advanced FIGO staging, differentiation and increased tumor size. In CaCx cell lines, Cx32 expression suppressed streptonigrin/cisplatin-induced apoptosis in the absence of functional GJ. In CaCx specimens and cell lines, expression of Cx32 upregulated epidermal growth factor receptor (EGFR) expression. Inhibition of EGFR signaling abrogated the anti-apoptotic effect of Cx32 expression. In conclusion, upregulated Cx32 in CaCx cells produces anti-apoptotic, pro-tumorigenic effects in vivo and vitro. Abnormal Cx32 expression/localization in CaCx appears to be both a mechanism and biomarker of chemotherapeutic resistance.
Topics: Apoptosis; Biomarkers, Tumor; Connexins; Drug Resistance, Neoplasm; ErbB Receptors; Female; HeLa Cells; Humans; Neoplasm Proteins; Uterine Cervical Neoplasms; Gap Junction beta-1 Protein
PubMed: 28492539
DOI: 10.1038/cddis.2017.183 -
Biochemical and Biophysical Research... May 2017Citrullination is an important posttranslational modification that occurs during retinal gliosis. We examined the expression of peptidyl arginine deiminases (PADs) to...
Citrullination is an important posttranslational modification that occurs during retinal gliosis. We examined the expression of peptidyl arginine deiminases (PADs) to identify the PADs that mediate citrullination in a model of alkali-induced retinal gliosis. Mouse corneas were exposed to 1.0 N NaOH and posterior eye tissue from injured and control uninjured eyes was evaluated for transcript levels of various PADs by reverse-transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR (qPCR). Retinas were also subjected to immunohistochemistry (IHC) for glial fibrillary acidic protein (GFAP), citrullinated species, PAD2, and PAD4 and tissue levels of GFAP, citrullinated species, and PAD4 were measured by western blots. In other experiments, the PAD4 inhibitor streptonigrin was injected intravitreally into injured eyes ex vivo to test inhibitory activity in an organ culture system. We found that uninjured retina and choroid expressed Pad2 and Pad4 transcripts. Pad4 transcript levels increased by day 7 post-injury (p < 0.05), whereas Pad2 levels did not change significantly (p > 0.05) by qPCR. By IHC, PAD2 was expressed in uninjured eyes along ganglion cell astrocytes, but in injured retina PAD2 was downregulated at 7 days. On the other hand, PAD4 showed increased staining in the retina upon injury revealing a pattern that overlapped with filamentous GFAP staining in Müller glial processes by 7 days. Injury-induced citrullination and soluble GFAP protein levels were reduced by PAD4 inhibition in western blot experiments of organ cultures. Together, our findings for the first time identify PAD4 as a novel injury-inducible druggable target for retinal gliosis.
Topics: Animals; Arginine; Burns, Chemical; Citrulline; Eye Burns; Female; Gliosis; Hydrolases; Male; Mice; Protein-Arginine Deiminase Type 4; Retina; Retinal Diseases; Sodium Hydroxide
PubMed: 28400047
DOI: 10.1016/j.bbrc.2017.04.031 -
Infection and Immunity Jun 2017(group A [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and,...
(group A [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA (erR-regulated etal ransporter ), a P-type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448Δ deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448Δ deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen.
Topics: Adenosine Triphosphatases; Animals; Bacterial Proteins; Gene Expression Regulation, Bacterial; Homeostasis; Humans; Hydrogen Peroxide; Iron; Membrane Transport Proteins; Mice; Mice, Transgenic; Mutation; Oxidative Stress; Streptococcal Infections; Streptococcus pyogenes; Streptonigrin
PubMed: 28373352
DOI: 10.1128/IAI.00140-17 -
Scientific Reports Jan 2017Streptonigrin methylesterase A (StnA) is one of the tailoring enzymes that modify the aminoquinone skeleton in the biosynthesis pathway of Streptomyces species. Although...
Streptonigrin methylesterase A (StnA) is one of the tailoring enzymes that modify the aminoquinone skeleton in the biosynthesis pathway of Streptomyces species. Although StnA has no significant sequence homology with the reported α/β-fold hydrolases, it shows typical hydrolytic activity in vivo and in vitro. In order to reveal its functional characteristics, the crystal structures of the selenomethionine substituted StnA (SeMet-StnA) and the complex (S185A mutant) with its substrate were resolved to the resolution of 2.71 Å and 2.90 Å, respectively. The overall structure of StnA can be described as an α-helix cap domain on top of a common α/β hydrolase domain. The substrate methyl ester of 10'-demethoxystreptonigrin binds in a hydrophobic pocket that mainly consists of cap domain residues and is close to the catalytic triad Ser185-His349-Asp308. The transition state is stabilized by an oxyanion hole formed by the backbone amides of Ala102 and Leu186. The substrate binding appears to be dominated by interactions with several specific hydrophobic contacts and hydrogen bonds in the cap domain. The molecular dynamics simulation and site-directed mutagenesis confirmed the important roles of the key interacting residues in the cap domain. Structural alignment and phylogenetic tree analysis indicate that StnA represents a new subfamily of lipolytic enzymes with the specific binding pocket located at the cap domain instead of the interface between the two domains.
Topics: Antibiotics, Antineoplastic; Bacterial Proteins; Carboxylic Ester Hydrolases; Catalytic Domain; Escherichia coli; Molecular Conformation; Molecular Dynamics Simulation; Sequence Analysis, Protein; Streptonigrin; Substrate Specificity
PubMed: 28074848
DOI: 10.1038/srep40254 -
Molecular Oral Microbiology Dec 2016SloR, a 25-kDa metalloregulatory protein in Streptococcus mutans modulates the expression of multiple genes, including the sloABC operon that encodes essential Mn...
SloR, a 25-kDa metalloregulatory protein in Streptococcus mutans modulates the expression of multiple genes, including the sloABC operon that encodes essential Mn transport and genes that promote cariogenesis. In this study, we report on SloC- and SloR-deficient strains of S. mutans (GMS284 and GMS584, respectively) that demonstrate compromised survivorship compared with their UA159 wild-type progenitor and their complemented strains (GMS285 and GMS585, respectively), when challenged with streptonigrin and/or in growth competition experiments. The results of streptonigrin assays revealed significantly larger zones of inhibition for GMS584 than for either UA159 or GMS585, indicating weakened S. mutans survivorship in the absence of SloR. Competition assays revealed a compromised ability for GMS284 and GMS584 to survive peroxide challenge compared with their SloC- and SloR-proficient counterparts. These findings are consistent with a role for SloC and SloR in S. mutans aerotolerance. We also predicted differential expression of oxidative stress tolerance genes in GMS584 versus UA159 and GMS585 when grown aerobically. The results of quantitative RT-PCR experiments revealed S. mutans sod, tpx, and sloC expression that was upregulated in GMS584 compared with UA159 and GMS585, indicating that the impact of oxidative stress on S. mutans is more severe in the absence of SloR than in its presence. The results of electrophoretic mobility shift assays indicate that SloR does not bind to the sod or tpx promoter regions directly, implicating intermediaries that may arbitrate the SloR response to oxidative stress.
Topics: Bacterial Proteins; DNA, Bacterial; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Hydrogen Peroxide; Metals; Mutation; Oxidative Stress; Streptococcus mutans; Streptonigrin; Superoxide Dismutase-1; Virulence
PubMed: 26577188
DOI: 10.1111/omi.12147 -
Infection Control and Hospital... Oct 2016OBJECTIVE To evaluate the new adult Centers for Disease Control and Prevention (CDC) ventilator-associated event (VAE) module in critically ill children and compare with...
OBJECTIVE To evaluate the new adult Centers for Disease Control and Prevention (CDC) ventilator-associated event (VAE) module in critically ill children and compare with the traditionally used CDC definition for ventilator-associated pneumonia (VAP). DESIGN Retrospective observational study of mechanically ventilated children in a pediatric intensive care unit in Greece January 1-December 31, 2011. METHODS Assessment of new adult CDC VAE module including 3 definition tiers: ventilator-associated condition (VAC), infection-related VAC, and possible/probable ventilator-associated pneumonia (VAE-VAP); comparison with traditional CDC criteria for clinically defined pneumonia in mechanically ventilated children (PNEU-VAP). We recorded Pediatric Risk of Mortality score at admission (PRISM III), number of ventilator-days, and outcome. RESULTS Among 119 patients with mechanical ventilation (median [range] number of ventilator-days, 7 [1-183]), 19 patients experienced VAC. Criteria for VAE-VAP were fulfilled in 12 of 19 patients with VAC (63%). Children with either VAC or VAE-VAP were on ventilation more days than patients without these conditions (16.5 vs 5 d, P=.0006 and 18 vs 5 d, P<.001, respectively), whereas PRISM-III score was similar between them. Mortality was significant higher in patients with new VAE-VAP definition (50%), but not in patients with VAC (31.6%), than the patients without new VAE-VAP (14%, P=.007) or VAC (15%, P=.1), respectively. No significant association was found between PNEU-VAP and death. Incidences of PNEU-VAP and VAE-VAP were similar, but the agreement was poor. CONCLUSIONS VAE-VAP and PNEU-VAP found similar prevalence in critically ill children but with poor agreement. However, excess of death was significantly associated only with VAE-VAP. Infect Control Hosp Epidemiol 2016:1-5.
Topics: Adolescent; Algorithms; Centers for Disease Control and Prevention, U.S.; Child; Child, Preschool; Critical Illness; Female; Greece; Humans; Infant; Intensive Care Units, Pediatric; Male; Pneumonia, Ventilator-Associated; Respiration, Artificial; Severity of Illness Index; Streptonigrin; United States; Ventilator-Induced Lung Injury; Ventilators, Mechanical
PubMed: 27396590
DOI: 10.1017/ice.2016.135 -
The Journal of Antibiotics Jan 2017Xantholipin is a polycyclic xanthone antibiotic that exhibits potent cytotoxic and antibacterial activity. In this study, a new xanthone-type antibiotic, xantholipin B... (Comparative Study)
Comparative Study
Xantholipin is a polycyclic xanthone antibiotic that exhibits potent cytotoxic and antibacterial activity. In this study, a new xanthone-type antibiotic, xantholipin B (1), was isolated for the first time along with its known derivative, xantholipin (2), from strain WJN-1, an aminotransferase inactivation mutant of the streptonigrin-producer Streptomyces flocculus CGMCC 4.1223. The structure of 1 was established based on spectroscopic analysis and supports the previously proposed biosynthetic pathway as a key intermediate of 2. Moreover, 1 showed 3- to 10-fold greater cytotoxicity than 2 against a select panel of human cancer cell lines. In addition, 1 demonstrated powerful antimicrobial activity against both Gram-positive bacteria and fungi. Importantly, both 1 and 2 inhibited the methicillin-resistant strain Staphylococcus aureus Mu50, with the MIC value of 0.025 μg ml. The new structural features of 1 enrich the structural diversity of xantholipin family compounds and shed new light on the structure-activity relationship of 1 as a promising antitumor drug candidate.
Topics: Anti-Bacterial Agents; Antifungal Agents; Antineoplastic Agents; Cell Line, Tumor; Fungi; Gram-Positive Bacteria; Humans; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Neoplasms; Polyketides; Spectrum Analysis; Streptomyces; Structure-Activity Relationship
PubMed: 27328868
DOI: 10.1038/ja.2016.60 -
PloS One 2016An mbfA mutant of Bradyrhizobium japonicum defective in iron export is sensitive to short term exposure to high levels iron or H2O2. Here, we found that the mbfA strain...
Synthetic Lethality of the bfr and mbfA Genes Reveals a Functional Relationship between Iron Storage and Iron Export in Managing Stress Responses in Bradyrhizobium japonicum.
An mbfA mutant of Bradyrhizobium japonicum defective in iron export is sensitive to short term exposure to high levels iron or H2O2. Here, we found that the mbfA strain grown in elevated iron media (100 μM) became resistant to those treatments, suggesting a stress response adaptation. The bfr gene encodes the iron storage protein bacterioferritin, and its expression is derepressed by iron. An mbfA bfr double mutant showed a loss of stress adaptation, and had a severe growth phenotype in high iron media. Moreover, a bfrup allele in which bfr is constitutively derepressed conferred stress tolerance on an mbfA mutant without elevating the iron content in the growth media. The intracellular iron content of the mbfA bfr double mutant was substantially higher than that found in the wild type, even when grown in relatively low iron media (5 μM). Under that condition, iron-responsive gene expression was aberrant in the mbfA bfr strain. Moreover, the double mutant was sensitive to the iron-activated antibiotic streptonigrin. We conclude that MbfA and Bfr work in concert to manage iron and oxidative stresses. In addition, the need for iron detoxification is not limited to extreme environments, but is also required for normal cellular function.
Topics: Bacterial Proteins; Bradyrhizobium; Cytochrome b Group; Ferritins; Gene Expression Regulation, Bacterial; Hydrogen Peroxide; Iron; Membrane Transport Proteins; Oxidative Stress; Synthetic Lethal Mutations
PubMed: 27285822
DOI: 10.1371/journal.pone.0157250