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Antimicrobial Agents and Chemotherapy May 2011The prevalence and impact of the overexpression of AmpC and efflux pumps were evaluated with a collection of 190 Pseudomonas aeruginosa isolates recovered from...
Overexpression of AmpC and efflux pumps in Pseudomonas aeruginosa isolates from bloodstream infections: prevalence and impact on resistance in a Spanish multicenter study.
The prevalence and impact of the overexpression of AmpC and efflux pumps were evaluated with a collection of 190 Pseudomonas aeruginosa isolates recovered from bloodstream infections in a 2008 multicenter study (10 hospitals) in Spain. The MICs of a panel of 13 antipseudomonal agents were determined by microdilution, and the expressions of ampC, mexB, mexY, mexD, and mexF were determined by real-time reverse transcription (RT)-PCR. Up to 39% of the isolates overexpressed at least one of the mechanisms. ampC overexpression (24.2%) was the most prevalent mechanism, followed by mexY (13.2%), mexB (12.6%), mexF (4.2%), and mexD (2.2%). The overexpression of mexB plus mexY, documented for 5.3% of the isolates, was the only combination showing a significantly (P=0.02) higher prevalence than expected from the frequencies of the individual mechanisms (1.6%). Additionally, all imipenem-resistant isolates studied (25 representative isolates) showed inactivating mutations in oprD. Most of the isolates nonsusceptible to piperacillin-tazobactam (96%) and ceftazidime (84%) overexpressed ampC, while mexB (25%) and mexY (29%) overexpressions gained relevance among cefepime-nonsusceptible isolates. Nevertheless, the prevalence of mexY overexpression was highest among tobramycin-nonsusceptible isolates (37%), and that of mexB was highest among meropenem-nonsusceptible isolates (33%). Regarding ciprofloxacin-resistant isolates, besides the expected increased prevalence of efflux pump overexpression, a highly significant link to ampC overexpression was documented for the first time: up to 52% of ciprofloxacin-nonsusceptible isolates overexpressed ampC, sharply contrasting with the 24% documented for the complete collection (P<0.001). In summary, mutation-driven resistance was frequent in P. aeruginosa isolates from bloodstream infections, whereas metallo-β-lactamases, detected in 2 isolates (1%) producing VIM-2, although with increasing prevalences, were still uncommon.
Topics: Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Bacterial Proteins; Carbapenems; Cefepime; Ceftazidime; Cephalosporins; Imipenem; Membrane Transport Proteins; Meropenem; Microbial Sensitivity Tests; Porins; Pseudomonas aeruginosa; Reverse Transcriptase Polymerase Chain Reaction; Thienamycins; beta-Lactamases
PubMed: 21357294
DOI: 10.1128/AAC.01645-10 -
The Journal of Biological Chemistry Mar 1993We have determined the patterns of mRNA and protein expression of 7 protein kinase C (PKC) isozymes in NIH 3T3 cells. Only PKC-alpha is expressed abundantly in NIH 3T3...
We have determined the patterns of mRNA and protein expression of 7 protein kinase C (PKC) isozymes in NIH 3T3 cells. Only PKC-alpha is expressed abundantly in NIH 3T3 cells; endogenous levels of the other 6 PKC isozymes are low or undetectable. We have overexpressed PKC-delta and -epsilon in these cells to observe activation/translocation of these two isozymes and the biological consequences of overexpression. Both PKC-delta and -epsilon, but not PKC-alpha, are partially associated with the insoluble fraction even in the absence of phorbol 12-myristate 13-acetate (PMA). Upon PMA stimulation, both PKC-delta and -epsilon translocate to the insoluble fraction of cell homogenates, as can be observed with the endogenous PKC-alpha. Overexpression of PKC-delta induces significant changes in morphology and causes the cells to grow more slowly and to a decreased cell density in confluent cultures. These changes are accentuated by treatment with PMA. Overexpression of PKC-epsilon does not lead to morphological changes, but causes increased growth rates and higher cell densities in monolayers. None of the PKC-delta overexpressers grow in soft agar with or without PMA, but all the cell lines that overexpress PKC-epsilon grow in soft agar in the absence of PMA, but not in its presence. NIH 3T3 cells that overexpress PKC-epsilon also form tumors in nude mice with 100% incidence. This indicates that high expression of PKC-epsilon contributes to neoplastic transformation.
Topics: 3T3 Cells; Animals; Biological Transport; Blotting, Northern; Blotting, Western; Brain; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cloning, Molecular; Enzyme Activation; Enzyme Stability; Indoles; Isoenzymes; Kinetics; Maleimides; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasms, Experimental; Protein Kinase C; Tetradecanoylphorbol Acetate
PubMed: 8454583
DOI: No ID Found -
Oncotarget Jul 2017Chromosomal instability (CIN) is a hallmark of many cancers and a major contributor to tumorigenesis. Centromere and kinetochore associated proteins such as the...
Chromosomal instability (CIN) is a hallmark of many cancers and a major contributor to tumorigenesis. Centromere and kinetochore associated proteins such as the evolutionarily conserved centromeric histone H3 variant CENP-A, associate with centromeric DNA for centromere function and chromosomal stability. Stringent regulation of cellular CENP-A levels prevents its mislocalization in yeast and flies to maintain genome stability. CENP-A overexpression and mislocalization are observed in several cancers and reported to be associated with increased invasiveness and poor prognosis. We examined whether there is a direct relationship between mislocalization of overexpressed CENP-A and CIN using HeLa and chromosomally stable diploid RPE1 cell lines as model systems. Our results show that mislocalization of overexpressed CENP-A to chromosome arms leads to chromosome congression defects, lagging chromosomes, micronuclei formation and a delay in mitotic exit. CENP-A overexpressing cells showed altered localization of centromere and kinetochore associated proteins such as CENP-C, CENP-T and Nuf2 leading to weakened native kinetochores as shown by reduced interkinetochore distance and CIN. Importantly, our results show that mislocalization of CENP-A to chromosome arms is one of the major contributors for CIN as depletion of histone chaperone DAXX prevents CENP-A mislocalization and rescues the reduced interkinetochore distance and CIN phenotype in CENP-A overexpressing cells. In summary, our results establish that CENP-A overexpression and mislocalization result in a CIN phenotype in human cells. This study provides insights into how overexpression of CENP-A may contribute to CIN in cancers and underscore the importance of understanding the pathways that prevent CENP-A mislocalization for genome stability.
Topics: Cell Line; Centromere; Centromere Protein A; Chromosomal Instability; Chromosome Segregation; Diploidy; Gene Expression; HeLa Cells; Histones; Humans; Kinetochores; Micronuclei, Chromosome-Defective; Models, Biological; Neoplasms; Phenotype; Protein Binding
PubMed: 28596481
DOI: 10.18632/oncotarget.18108 -
Scientific Reports Sep 2020Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a...
Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.
Topics: Cell Cycle; DNA Damage; Gene Expression Regulation, Fungal; Metabolome; Phosphoprotein Phosphatases; Phosphorylation; Reactive Oxygen Species; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcriptome
PubMed: 32973189
DOI: 10.1038/s41598-020-72391-y -
PLoS Genetics Aug 2023Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for efficient mRNA m6A methylation, which regulates meiotic entry. Kar4p is also...
Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for efficient mRNA m6A methylation, which regulates meiotic entry. Kar4p is also required for a second seemingly non-catalytic function during meiosis. Overexpression of the early meiotic transcription factor, IME1, can bypass the requirement for Kar4p in meiotic entry but the additional overexpression of the translational regulator, RIM4, is required to permit sporulation in kar4Δ/Δ. Using microarray analysis and RNA sequencing, we sought to determine the impact of removing Kar4p and consequently mRNA methylation on the early meiotic transcriptome in a strain background (S288c) that is sensitive to the loss of early meiotic regulators. We found that kar4Δ/Δ mutants have a largely wild type transcriptional profile with the exception of two groups of genes that show delayed and reduced expression: (1) a set of Ime1p-dependent early genes as well as IME1, and (2) a set of late genes dependent on the mid-meiotic transcription factor, Ndt80p. The early gene expression defect is likely the result of the loss of mRNA methylation and is rescued by overexpressing IME1, but the late defect is only suppressed by overexpression of both IME1 and RIM4. The requirement for RIM4 led us to predict that the non-catalytic function of Kar4p, like methyltransferase complex orthologs in other systems, may function at the level of translation. Mass spectrometry analysis identified several genes involved in meiotic recombination with strongly reduced protein levels, but with little to no reduction in transcript levels in kar4Δ/Δ after IME1 overexpression. The low levels of these proteins were rescued by overexpression of RIM4 and IME1, but not by the overexpression of IME1 alone. These data expand our understanding of the role of Kar4p in regulating meiosis and provide key insights into a potential mechanism of Kar4p's later meiotic function that is independent of mRNA methylation.
Topics: Animals; Cytoplasm; DNA-Binding Proteins; Gene Expression; Meiosis; Methyltransferases; RNA, Messenger; Saccharomyces cerevisiae Proteins; Transcription Factors; Gene Expression Regulation, Fungal
PubMed: 37639444
DOI: 10.1371/journal.pgen.1010898 -
Genetics Jun 2018Mismatch repair (MMR) proteins act in spellchecker roles to excise misincorporation errors that occur during DNA replication. Curiously, large-scale analyses of a...
Mismatch repair (MMR) proteins act in spellchecker roles to excise misincorporation errors that occur during DNA replication. Curiously, large-scale analyses of a variety of cancers showed that increased expression of MMR proteins often correlated with tumor aggressiveness, metastasis, and early recurrence. To better understand these observations, we used The Cancer Genome Atlas and Gene Expression across Normal and Tumor tissue databases to analyze MMR protein expression in cancers. We found that the MMR genes and are overexpressed more frequently than , and that and are often cooverexpressed as a result of copy number amplifications of these genes. These observations encouraged us to test the effects of upregulating MMR protein levels in baker's yeast, where we can sensitively monitor genome instability phenotypes associated with cancer initiation and progression. Msh6 overexpression (two- to fourfold) almost completely disrupted mechanisms that prevent recombination between divergent DNA sequences by interacting with the DNA polymerase processivity clamp PCNA and by sequestering the Sgs1 helicase. Importantly, cooverexpression of Msh2 and Msh6 (∼eightfold) conferred, in a PCNA interaction-dependent manner, several genome instability phenotypes including increased mutation rate, increased sensitivity to the DNA replication inhibitor HU and the DNA-damaging agents MMS and 4-nitroquinoline N-oxide, and elevated loss-of-heterozygosity. Msh2 and Msh6 cooverexpression also altered the cell cycle distribution of exponentially growing cells, resulting in an increased fraction of unbudded cells, consistent with a larger percentage of cells in G1. These novel observations suggested that overexpression of MSH factors affected the integrity of the DNA replication fork, causing genome instability phenotypes that could be important for promoting cancer progression.
Topics: Cell Cycle; DNA Mismatch Repair; DNA Replication; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genomic Instability; Humans; MutS Homolog 2 Protein; MutS Homolog 3 Protein; Proliferating Cell Nuclear Antigen; Protein Binding; RecQ Helicases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Up-Regulation
PubMed: 29654124
DOI: 10.1534/genetics.118.300923 -
Molecular Medicine Reports May 2017Distal-less homeobox 2 (Dlx2) is a member of the homeodomain family of transcription factors and is important for the development of cranial neural crest cells...
Distal-less homeobox 2 (Dlx2) is a member of the homeodomain family of transcription factors and is important for the development of cranial neural crest cells (CNCCs)‑derived craniofacial tissues. Previous studies revealed that Dlx2 was expressed in the cementum and a targeted null mutation disrupted tooth development in mice. However, whether Dlx2 overexpression may impair in vivo tooth morphogenesis remains to be elucidated. The present study used a transgenic mouse model to specifically overexpress Dlx2 in neural crest cells in order to identify the dental phenotypes in mice by observation, micro‑computed tomography and histological examination. The Dlx2‑overexpressed mice exhibited tooth abnormalities including incisor cross‑bite, shortened tooth roots, increased cementum deposition, periodontal ligament disorganization and osteoporotic alveolar bone. Therefore, Dlx2 overexpression may alter the alveolar bone, cementum and periodontal ligament (PDL) phenotypes in mice.
Topics: Animals; Gene Expression Regulation, Developmental; Homeodomain Proteins; Mice; Mice, Transgenic; Organogenesis; Periodontium; Tooth; Transcription Factors
PubMed: 28447749
DOI: 10.3892/mmr.2017.6315 -
Cancer Science Mar 2013Overexpression of the ErbB2/HER2 receptor tyrosine kinase contributes to tumorigenesis. However, mechanisms regulating ErbB2 protein levels remain largely unclear. Here,...
Overexpression of the ErbB2/HER2 receptor tyrosine kinase contributes to tumorigenesis. However, mechanisms regulating ErbB2 protein levels remain largely unclear. Here, we identified novel mechanisms of ErbB2 downregulation. ErbB2 constitutively binds to an adaptor protein FRS2β. We found that FRS2β bound to CD2AP and CIN85, which induces endosomal trafficking that targets lysosomes. FRS2β colocalized with CIN85 in the cytoplasm. Expression of wild type FRS2β but not its CIN85 non-binding mutant, downregulated the ErbB2 protein and inhibited anchorage-independent cell growth. Moreover, the E3 ubiquitin-protein ligase Cbl was contained within a complex of FRS2β and CIN85. Knockdown of both CIN85 and CD2AP or of Cbl, or treatment with lysosomal degradation inhibitors diminished FRS2β downregulation of ErbB2. In addition, knockdown of endogenous FRS2β caused upregulation of ErbB2 in primary neural cells. Finally, immunohistochemical analysis showed that human breast cancer tissues that overexpress ErbB2 expressed low levels of FRS2β. Thus, an FRS2β-CIN85/CD2AP-Cbl axis for downregulation of ErbB2 may regulate ErbB2 protein levels in physiological and pathological settings. Molecular targeting drugs that can increase or stabilize the ErbB2-FRS2β-CIN85/CD2AP-Cbl axis may have promise for the control of ErbB2-overexpressing tumors.
Topics: Adaptor Proteins, Signal Transducing; Amino Acid Motifs; Breast Neoplasms; Cells, Cultured; Down-Regulation; Female; Gene Knockdown Techniques; Humans; Neurons; Phosphoproteins; Proto-Oncogene Proteins c-cbl; Receptor, ErbB-2; Signal Transduction
PubMed: 23279575
DOI: 10.1111/cas.12086 -
Arthritis and Rheumatism Mar 2013Hyperactivation of innate immunity by Toll-like receptors (TLRs) can contribute to the development of autoinflammatory or autoimmune diseases. This study evaluated the...
OBJECTIVE
Hyperactivation of innate immunity by Toll-like receptors (TLRs) can contribute to the development of autoinflammatory or autoimmune diseases. This study evaluated the activation of Tyro3, Axl, Mer (TAM) receptors, physiologic negative regulators of TLRs, by their agonists, growth arrest-specific protein 6 (GAS-6) and protein S, in the prevention of collagen-induced arthritis (CIA).
METHODS
Adenoviruses overexpressing GAS-6 and protein S were injected intravenously or intraarticularly into mice during CIA. Splenic T helper cell subsets from intravenously injected mice were studied by flow cytometry, and the knee joints of mice injected intravenously and intraarticularly were assessed histologically. Synovium from mice injected intraarticularly was evaluated for cytokine and suppressor of cytokine signaling (SOCS) expression.
RESULTS
Protein S significantly reduced ankle joint swelling when overexpressed systemically. Further analysis of knee joints revealed a moderate reduction in pathologic changes in the joint and a significant reduction in the number of splenic Th1 cells when protein S was overexpressed systemically. Local overexpression of GAS-6 decreased joint inflammation and joint pathology. Protein S treatment showed a similar trend of protection. Consistently, GAS-6 and protein S reduced cytokine production in the synovium. Moreover, levels of messenger RNA for interleukin-12 (IL-12) and IL-23 were reduced by GAS-6 and protein S treatment, with a corresponding decrease in the production of interferon-γ and IL-17. TAM ligand overexpression was associated with an increase in SOCS-3 levels, which likely contributed to the amelioration of arthritis.
CONCLUSION
This study provides the first evidence that TAM receptor stimulation by GAS-6 and protein S can be used to ameliorate arthritis when applied systemically or locally. TAM receptor stimulation limits proinflammatory signaling and adaptive immunity. This pathway provides a novel strategy by which to combat rheumatoid arthritis.
Topics: Adenoviridae; Animals; Arthritis, Experimental; Cytokines; Genetic Therapy; Injections, Intra-Articular; Intercellular Signaling Peptides and Proteins; Knee Joint; Male; Mice; Mice, Inbred DBA; Protein S; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Severity of Illness Index; Signal Transduction; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Synovial Membrane; Th1 Cells; c-Mer Tyrosine Kinase; Axl Receptor Tyrosine Kinase
PubMed: 23203851
DOI: 10.1002/art.37786 -
Biomedical Research (Tokyo, Japan) 2021Overexpressed TBC1D8B, a GTPase-activating protein, significantly reduced cultured HCT116 human colon cancer cell number. We tested N-terminal TBC1D8B, which is...
Overexpressed TBC1D8B, a GTPase-activating protein, significantly reduced cultured HCT116 human colon cancer cell number. We tested N-terminal TBC1D8B, which is identical to wild type TBC1D8B from amino acid positions 1 to 427 and possesses a modified sequence from position 428 to 435 (ECGGLFLL) because of the introduction of a premature stop codon at position 436 to narrow down the minimum requirement element. The N-terminal TBC1D8B contains two GRAM domains but not the TBC domain essential for Rab-GTPase activity. The N-terminal TBC1D8B overexpression significantly reduced the cultured HCT116 cell number. When we tested C-terminal TBC1D8B, containing the portion of TBC1D8B absent in the N-terminal TBC1D8B, the cell number reduction was not observed. The N-terminal TBC1D8B overexpression significantly increased the coronin 1B expression and reduced the phosphorylation of serine 51 in eIF2α, respective markers of apoptosis and cell death/survival. Also, caspase 3 and poly ADP-ribose polymerase increased cleavage in suspended cells overexpressing the N-terminal TBC1D8B. Taken together, it is not the TBC domain for Rab-GTPase activity, but amino acids 1 to 435, including the two GRAM domains, that is enough for TBC1D8B to cause spontaneous apoptosis. TBC1D8B could be a potential anticancer therapeutic molecule.
Topics: Antineoplastic Agents; Apoptosis; Calcium-Binding Proteins; Cell Death; Cloning, Molecular; Codon, Terminator; Eukaryotic Initiation Factor-2; GTPase-Activating Proteins; HCT116 Cells; Humans; Phosphorylation; Protein Domains; Transfection; Vesicular Transport Proteins
PubMed: 34092754
DOI: 10.2220/biomedres.42.95