scholarly journals Cellular Responses to Postsegregational Killing by Restriction-Modification Genes

2000 ◽  
Vol 182 (8) ◽  
pp. 2218-2229 ◽  
Author(s):  
Naofumi Handa ◽  
Asao Ichige ◽  
Kohji Kusano ◽  
Ichizo Kobayashi
Keyword(s):  
Viable Cell ◽  
Underlying Mechanism ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Gene Complex ◽  
Cellular Processes ◽  
Cell Counts ◽  
Gene Complexes ◽  
Restriction Modification

ABSTRACT Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in anEscherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC,recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted “nonself” DNA and repair restricted “self” DNA.

scholarly journals Use of a Rapid Throughput In Vivo Screen To Investigate Inhibitors of Eukaryotic Topoisomerase II Enzymes

1998 ◽  
Vol 42 (4) ◽  
pp. 889-894 ◽  
Author(s):  
Timothy R. Hammonds ◽  
Anthony Maxwell ◽  
John R. Jenkins
Keyword(s):  
Topoisomerase Ii ◽  
Antitumor Agents ◽  
Viable Cell ◽  
Cytotoxic Agents ◽  
New Method ◽  
Temperature Sensitive ◽  
Cell Counts ◽  
Similar Drug ◽  
In Vivo Screen

ABSTRACT Topoisomerase II catalyzes the passage of one DNA helix through another via a transient double-stranded break. The essential nature of this enzyme in cell proliferation and its mechanism of action make it an ideal target for cytotoxic agents. Saccharomyces cerevisiae topoisomerase II has been frequently used as a model for testing potential inhibitors of eukaryotic topoisomerase II as antitumor agents. The standard in vivo method of estimating the sensitivity of S. cerevisiae to the antitopoisomerase drugs is via inhibition or kill curves which rely on viable-cell counts and is labor intensive. We present an alternative to this, a high-throughput in vivo screen. This method makes use of a drug-permeable S. cerevisiae strain lacking endogenous topoisomerase II, which is modified to express either human topoisomerase IIα or IIβ or S. cerevisiae topoisomerase II carried on plasmids. Each modified strain expresses a full-length topoisomerase II enzyme, as opposed to the more commonly used temperature-sensitive S. cerevisiae mutant expressing yeast or yeast/human hybrid enzymes. A comparison of this new method with a plating-and-counting method gave similar drug sensitivity results, with increased accuracy and reduced manual input for the new method. The information generated has highlighted the sensitivities of different topoisomerase II enzymes and isoenzymes to several different classes of topoisomerase II inhibitor.

scholarly journals Evaluation of T-3811ME (BMS-284756), a New Des-F(6)-Quinolone, for Treatment of Meningitis Caused by Penicillin-Resistant Streptococcus pneumoniae in Rabbits

2002 ◽  
Vol 46 (6) ◽  
pp. 1760-1765 ◽  
Author(s):  
Masahiro Takahata ◽  
Hiroshi Yamada ◽  
Teiichi Morita ◽  
Shinichi Furubou ◽  
Shinzaburo Minami ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Viable Cell ◽  
Treated Group ◽  
Dependent Manner ◽  
Free Base ◽  
Time Curve ◽  
Gram Positive Bacteria ◽  
Cell Counts ◽  
Concentration Time ◽  
Dose Dependent

ABSTRACT T-3811ME (BMS-284756) is a new des-F(6)-quinolone with high levels of activity against gram-positive bacteria, including penicillin-resistant Streptococcus pneumoniae (PRSP) strains. T-3811, the free base of T-3811ME, exhibited potent activity against 28 clinical strains of PRSP isolated clinically (MIC at which 90% of the isolates tested are inhibited, 0.0625 μg/ml). After the intravenous dosing of T-3811ME (20 mg/kg of body weight as T-3811) in rabbits with meningitis caused by PRSP, the area under the concentration-time curve (AUC) of T-3811 in cerebrospinal fluid (CSF) was 5.79 μg · h/ml and was 4.5-fold higher than that of T-3811in the CSF of rabbits without meningitis. In addition, the AUC/MIC for T-3811ME (20 mg/kg as T-3811) in CSF was 185, which was 4.3-fold higher than that for ceftriaxone (administered intravenously at 100 mg/kg). After the administration of any dose of T-3811ME (5, 10, and 20 mg/kg as T-3811), the viable cell counts in CSF decreased in a dose-dependent manner. In particular, after dosing of 20 mg/kg (as T-3811), the viable cell counts in CSF were significantly less than those in the nontreated group (P < 0.01). By histopathological evaluation, 6 h after the administration of T-3811ME (20 mg/kg as T-3811), the thickening of the cerebral meninx and the infiltration of neutrophils into the cerebral meninx were less severe in the treated group than in the nontreated group. T-3811ME (BMS-284756) may be expected to be evaluated for the management of meningitis caused by highly penicillin-resistant pneumococci.

scholarly journals Hyperoside Alleviates High Glucose-Induced Proliferation of Mesangial Cells through the Inhibition of the ERK/CREB/miRNA-34a Signaling Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Le Zhang ◽  
Qian Dai ◽  
Lanlan Hu ◽  
Hua Yu ◽  
Jing Qiu ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Signaling Pathway ◽  
High Glucose ◽  
Mesangial Cells ◽  
Viable Cell ◽  
Underlying Mechanism ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Glomerular Mesangial Cells

Purpose. Hyperoside, a flavonoid isolated from conventional medicinal herbs, has been demonstrated to exert a significant protective effect in diabetic nephropathy. This study aimed to determine the underlying mechanisms, by which hyperoside inhibits high glucose-(HG-) induced proliferation in mouse renal mesangial cells. Methods. Mouse glomerular mesangial cells line (SV40-MES13) was used to study the inhibitory effect of hyperoside on cell proliferation induced by 30 mM glucose, which was used to simulate a diabetic condition. Viable cell count was assessed using the Cell Counting Kit-8 and by the 5-ethynyl-20-deoxyuridine incorporation assay. The underlying mechanism involving miRNA-34a was further investigated by quantitative RT-PCR and transfection with miRNA-34a agomir. The phosphorylation levels of extracellular signal-regulated kinases (ERKs) and cAMP-response element-binding protein (CREB) were measured by Western blotting. The binding region and the critical binding sites of CREB in the miRNA-34a promoter were investigated by the chromatin immunoprecipitation assay and luciferase reporter assay, respectively. Results. We found that hyperoside could significantly decrease HG-induced proliferation of SV40-MES13 cells in a dose-dependent manner, without causing obvious cell death. In addition, hyperoside inhibited the activation of ERK pathway and phosphorylation of its downstream transcriptional factor CREB, as well as the miRNA-34a expression. We further confirmed that CREB-mediated regulation of miRNA-34a is dependent on the direct binding to specific sites in the promoter region of miRNA-34a. Conclusion. Our cumulative results suggested that hyperoside inhibits the proliferation of SV40-MES13 cells through the suppression of the ERK/CREB/miRNA-34a signaling pathway, which provides new insight to the current investigation on therapeutic strategies for diabetic nephropathy.

scholarly journals Bactericidal Activity of ACH-702 against Nondividing and Biofilm Staphylococci

2012 ◽  
Vol 56 (7) ◽  
pp. 3812-3818 ◽  
Author(s):  
Steven D. Podos ◽  
Jane A. Thanassi ◽  
Melissa Leggio ◽  
Michael J. Pucci
Keyword(s):  
Protein Synthesis ◽  
Stationary Phase ◽  
Bactericidal Activity ◽  
Bacterial Infections ◽  
De Novo ◽  
Viable Cell ◽  
Bacterial Cells ◽  
Protein Synthesis Inhibitors ◽  
Content Type ◽  
Cell Counts

ABSTRACTMany bacterial infections involve slow or nondividing bacterial growth states and localized high cell densities. Antibiotics with demonstrated bactericidal activity rarely remain bactericidal at therapeutic concentrations under these conditions. The isothiazoloquinolone (ITQ) ACH-702 is a potent, bactericidal compound with activity against many antibiotic-resistant pathogens, including methicillin-resistantStaphylococcus aureus(MRSA). We evaluated its bactericidal activity under conditions where bacterial cells were not dividing and/or had slowed their growth. AgainstS. aureuscultures in stationary phase, ACH-702 showed concentration-dependent bactericidal activity and achieved a 3-log-unit reduction in viable cell counts within 6 h of treatment at ≥16× MIC values; in comparison, the bactericidal quinolone moxifloxacin and the additional comparator compounds vancomycin, linezolid, and rifampin at 16× to 32× MICs showed little or no bactericidal activity against stationary-phase cells. ACH-702 at 32× MIC retained bactericidal activity against stationary-phaseS. aureusacross a range of inoculum densities. ACH-702 did not kill cold-arrested cells yet remained bactericidal against cells arrested by protein synthesis inhibitors, suggesting that its bactericidal activity against nondividing cells requires active metabolism but notde novoprotein synthesis. ACH-702 also showed a degree of bactericidal activity at 16× MIC againstS. epidermidisbiofilm cells that was superior to that of moxifloxacin, rifampin, and vancomycin. The bactericidal activity of ACH-702 against stationary-phase staphylococci and biofilms suggests potential clinical utility in infections containing cells in these physiological states.

scholarly journals Vancomycin Acts Synergistically with Gentamicin against Penicillin-Resistant Pneumococci by Increasing the Intracellular Penetration of Gentamicin

2003 ◽  
Vol 47 (1) ◽  
pp. 144-147 ◽  
Author(s):  
P. Cottagnoud ◽  
M. Cottagnoud ◽  
M. G. Täuber
Keyword(s):  
Viable Cell ◽  
Underlying Mechanism ◽  
Intracellular Concentration ◽  
Combination Regimen ◽  
Cell Counts ◽  
Short Period ◽  
Before And After ◽  
Gentamicin Concentration

ABSTRACT Vancomycin and gentamicin act synergistically against penicillin-resistant pneumococci in vitro and in experimental rabbit meningitis. The aim of the present study was to investigate the underlying mechanism of this synergism. The intracellular concentration of gentamicin was measured by using the following experimental setting. Bacterial cultures were incubated with either gentamicin alone or gentamicin plus vancomycin for a short period (15 min). The gentamicin concentration was determined before and after grinding of the cultures by using the COBAS INTEGRA fluorescence polarization system (Roche). The grinding efficacies ranged between 44 and 54%, as determined by viable cell counts. In the combination regimen the intracellular concentration of gentamicin increased to 186% compared to that achieved with gentamicin monotherapy. These data suggest that the synergy observed in vivo and in vitro is based on an increased intracellular penetration of the aminoglycoside, probably due to the effect of vancomycin on the permeability of the cell wall.

Antibacterial and antibiofilm effects of iron chelators against Prevotella intermedia

2013 ◽  
Vol 62 (9) ◽  
pp. 1307-1316 ◽  
Author(s):  
Ji-Hoi Moon ◽  
Cheul Kim ◽  
Hee-Su Lee ◽  
Sung-Woon Kim ◽  
Jin-Yong Lee
Keyword(s):  
Bacterial Growth ◽  
Biofilm Formation ◽  
Therapeutic Agent ◽  
Viable Cell ◽  
Iron Chelators ◽  
Prevotella Intermedia ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cell Counts ◽  
Strong Antimicrobial Activity

Prevotella intermedia, a major periodontopathogen, has been shown to be resistant to many antibiotics. In the present study, we examined the effect of the FDA-approved iron chelators deferoxamine (DFO) and deferasirox (DFRA) against planktonic and biofilm cells of P. intermedia in order to evaluate the possibility of using these iron chelators as alternative control agents against P. intermedia. DFRA showed strong antimicrobial activity (MIC and MBC values of 0.16 mg ml−1) against planktonic P. intermedia. At subMICs, DFRA partially inhibited the bacterial growth and considerably prolonged the bacterial doubling time. DFO was unable to completely inhibit the bacterial growth in the concentration range tested and was not bactericidal. Crystal violet binding assay for the assessment of biofilm formation by P. intermedia showed that DFRA significantly decreased the biofilm-forming activity as well as the biofilm formation, while DFO was less effective. DFRA was chosen for further study. In the ATP-bioluminescent assay, which reflects viable cell counts, subMICs of DFRA significantly decreased the bioactivity of biofilms in a concentration-dependent manner. Under the scanning electron microscope, P. intermedia cells in DFRA-treated biofilm were significantly elongated compared to those in untreated biofilm. Further experiments are necessary to show that iron chelators may be used as a therapeutic agent for periodontal disease.

scholarly journals Microencapsulation of Lactococcus lactis Gh1 with Gum Arabic and Synsepalum dulcificum via Spray Drying for Potential Inclusion in Functional Yogurt

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1422 ◽  
Author(s):  
Nurul Farhana Fazilah ◽  
Nurmelissa Hanani Hamidon ◽  
Arbakariya B. Ariff ◽  
Mohd Ezuan Khayat ◽  
Helmi Wasoh ◽  
...  
Keyword(s):  
Spray Drying ◽  
Lactococcus Lactis ◽  
Gum Arabic ◽  
Streptococcus Thermophilus ◽  
Viable Cell ◽  
Starter Cultures ◽  
Lactobacillus Delbrueckii ◽  
Bacterial Cells ◽  
Cell Counts

There has been an explosion of probiotic incorporated based product. However, many reports indicated that most of the probiotics have failed to survive in high quantity, which has limited their effectiveness in most functional foods. Thus, to overcome this problem, microencapsulation is considered to be a promising process. In this study, Lactococcus lactis Gh1 was encapsulated via spray-drying with gum Arabic together with Synsepalum dulcificum or commonly known as miracle fruit. It was observed that after spray-drying, high viability (~109 CFU/mL) powders containing L. lactis in combination with S. dulcificum were developed, which was then formulated into yogurt. The tolerance of encapsulated bacterial cells in simulated gastric juice at pH 1.5 was tested in an in-vitro model and the result showed that after 2 h, cell viability remained high at 1.11 × 106 CFU/mL. Incubation of encapsulated cells in the presence of 0.6% (w/v) bile salts showed it was able to survive (~104 CFU/mL) after 2 h. Microencapsulated L. lactis retained a higher viability, at ~107 CFU/mL, when incorporated into yogurt compared to non-microencapsulated cells ~105 CFU/mL. The fortification of microencapsulated and non-microencapsulated L. lactis in yogurts influenced the viable cell counts of yogurt starter cultures, Lactobacillus delbrueckii subs. bulgaricus and Streptococcus thermophilus.

scholarly journals Persister cells mediate tolerance to metal oxyanions in Escherichia coli

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3181-3195 ◽  
Author(s):  
Joe J. Harrison ◽  
Howard Ceri ◽  
Nicole J. Roper ◽  
Erin A. Badry ◽  
Kimberley M. Sproule ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Viable Cell ◽  
Small Population ◽  
Water Soluble ◽  
Bacterial Cells ◽  
Persister Cells ◽  
Term Survival ◽  
E Coli ◽  
Cell Counts ◽  
High Concentrations

Bacterial cultures produce subpopulations of cells termed ‘persisters’, reputedly known for high tolerance to killing by antibiotics. Ecologically, antibiotics produced by competing microflora are only one potential stress encountered by bacteria. Another pressure in the environment is toxic metals that are distributed ubiquitously by human pollution, volcanic activity and the weathering of minerals. This study evaluated the time- and concentration-dependent killing of Escherichia coli planktonic and biofilm cultures by the water-soluble metal(loid) oxyanions chromate (), arsenate (), arsenite (), selenite (), tellurate () and tellurite (). Correlative to previous reports in the literature, control antibiotic assays indicated that a small proportion of E. coli biofilm populations remained recalcitrant to killing by antibiotics (even with 24 h exposure). In contrast, metal oxyanions presented a slow, bactericidal action that eradicated biofilms. When exposed for 2 h, biofilms were up to 310 times more tolerant to killing by metal oxyanions than corresponding planktonic cultures. However, by 24 h, planktonic cells and biofilms were eradicated at approximately the same concentration in all instances. Coloured complexes of metals and chelators could not be generated in biofilms exposed to or , suggesting that the extracellular polymeric matrix of E. coli may have a low binding affinity for metal oxyanions. Viable cell counts at 2 and 24 h exposure revealed that, at high concentrations, all of the metal oxyanions had killed 99 % (or a greater proportion) of the bacterial cells in biofilm populations. It is suggested here that the short-term survival of <1 % of the bacterial population corresponds well with the hypothesis that a small population of persister cells may be responsible for the time-dependent tolerance of E. coli biofilms to high concentrations of metal oxyanions.

Transcriptome analysis and physiology of Bifidobacterium longum NCC2705 cells under continuous culture conditions

2012 ◽  
Vol 3 (4) ◽  
pp. 261-272 ◽  
Author(s):  
V. Mozzetti ◽  
F. Grattepanche ◽  
D. Moine ◽  
B. Berger ◽  
E. Rezzonico ◽  
...  
Keyword(s):  
Continuous Culture ◽  
Bifidobacterium Longum ◽  
Viable Cell ◽  
Culture Conditions ◽  
Stress Conditions ◽  
Stress Factors ◽  
Bacterial Cells ◽  
Continuous Cultures ◽  
Batch Cultures ◽  
Cell Counts

A central issue in the use of probiotics in food and food supplements is their sensitivity to many environmental stress factors. The resistance of probiotic cells to lethal stress can be improved by application of homologous or heterologous sub-lethal stress during culture. This screening procedure is generally performed using batch cultures. Continuous cultures could be a suitable and more efficient method to test different stress factors on one culture instead of repeating several batch cultures. However, before testing stresses using continuous cultures, the physiological stability of continuously produced cells over a considered time period must be first evaluated. A continuous culture of Bifidobacterium longum NCC2705 was maintained for 211 h at a dilution rate of 0.1 per h, mimicking a deceleration growth phase culture. Stable viable cell counts were measured over the culture period, decreasing only moderately from 8.8 to 8.6 log10 cfu/ml. A slight shift in metabolite production, characterized by increased lactate and decreased acetate, formate and ethanol concentrations was observed. Susceptibilities to antibiotics and stress conditions were stable (cefotaxim, ampicillin, ceftazidime) or moderately affected (simulated gastric juices, heat, bile salts, tetracycline, chloramphenicol, penicillin, vancomycin and neomycin) over culturing time. Comparison of gene transcription profiles between samples collected after 31 h of continuous culture and samples collected after 134 and 211 h revealed only limited changes in expression of 1.0 and 3.8% of total genes, respectively. Based on these results, we propose that continuous culture can be used to produce bacterial cells with stable physiological properties suitable for fast and efficient screening of sub-lethal stress conditions.

scholarly journals A DNA Methyltransferase Can Protect the Genome from Postdisturbance Attack by a Restriction-Modification Gene Complex

2002 ◽  
Vol 184 (22) ◽  
pp. 6100-6108 ◽  
Author(s):  
Noriko Takahashi ◽  
Yasuhiro Naito ◽  
Naofumi Handa ◽  
Ichizo Kobayashi
Keyword(s):  
Dna Methyltransferase ◽  
Dna Methyltransferases ◽  
Cell Killing ◽  
Gene Complex ◽  
Prokaryotic Genomes ◽  
A Cell ◽  
Gene Complexes ◽  
Potential Mobility ◽  
Restriction Modification

ABSTRACT In prokaryotic genomes, some DNA methyltransferases form a restriction-modification gene complex, but some others are present by themselves. Dcm gene product, one of these orphan methyltransferases found in Escherichia coli and related bacteria, methylates DNA to generate 5′-CmCWGG just as some of its eukaryotic homologues do. Vsr mismatch repair function of an adjacent gene prevents C-to-T mutagenesis enhanced by this methylation but promotes other types of mutation and likely has affected genome evolution. The reason for the existence of the dcm-vsr gene pair has been unclear. Earlier we found that several restriction-modification gene complexes behave selfishly in that their loss from a cell leads to cell killing through restriction attack on the genome. There is also increasing evidence for their potential mobility. EcoRII restriction-modification gene complex recognizes the same sequence as Dcm, and its methyltransferase is phylogenetically related to Dcm. In the present work, we found that stabilization of maintenance of a plasmid by linkage of EcoRII gene complex, likely through postsegregational cell killing, is diminished by dcm function. Disturbance of EcoRII restriction-modification gene complex led to extensive chromosome degradation and severe loss of cell viability. This cell killing was partially suppressed by chromosomal dcm and completely abolished by dcm expressed from a plasmid. Dcm, therefore, can play the role of a “molecular vaccine” by defending the genome against parasitism by a restriction-modification gene complex.

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