Influence of the postantibiotic effect and postantibiotic Sub-MICs effect of netilmicin,tobramycin, ciprofloxacin and pefloxacin on alginate production byPseudomonas aeruginosa

The postantibiotic effect (PAE) (postantibiotic phase induced by 2× or 4×MIC) as well as the postantibiotic effect of subinhibitory concentrations (0.1×, 0.2× and 0.3× MIC) (PA SME) of netilmicin, tobramycin, ciprofloxacin and pefloxacin affected the production of the virulence factor alginate by aP. aeruginosa strain. Aminoglycosides and ciprofloxacin at a concentration of 4× MIC inhibited the alginate production more significantly than 2× MIC. Suprainhibitory concentrations of aminoglycosides were more effective than pefloxacin (2× or 4× MIC) and ciprofloxacin (2× MIC). PA SME demonstrated by the above antibiotics (with the exception of ciprofloxacin 2× MIC +00.1× MIC) suppressed alginate production more efficiently.     

Distinct properties of CRAC and MIC channels in RBL cells

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca(2+) release-activated Ca(2+) (CRAC) channels open in response to passive Ca(2+) store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg(2+) exposes an outwardly rectifying current (Mg(2+)-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg(2+), spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg(2+). Removal of internal Mg(2+) induced MIC current despite widely varying Ca(2+) and EGTA levels, suggesting that Ca(2+)-store depletion is not involved in activation of MIC channels. Increasing internal Mg(2+) from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg(2+) and Cs(+) carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg(2+) blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels.     

Toxoplasma gondii-Induced Activation of EGFR Prevents Autophagy Protein-Mediated Killing of the Parasite

Toxoplasma gondii resides in an intracellular compartment (parasitophorous vacuole) that excludes transmembrane molecules required for endosome - lysosome recruitment. Thus, the parasite survives by avoiding lysosomal degradation. However, autophagy can re-route the parasitophorous vacuole to the lysosomes and cause parasite killing. This raises the possibility that T. gondii may deploy a strategy to prevent autophagic targeting to maintain the non-fusogenic nature of the vacuole. We report that T. gondii activated EGFR in endothelial cells, retinal pigment epithelial cells and microglia. Blockade of EGFR or its downstream molecule, Akt, caused targeting of the parasite by LC3⁺ structures, vacuole-lysosomal fusion, lysosomal degradation and killing of the parasite that were dependent on the autophagy proteins Atg7 and Beclin 1. Disassembly of GPCR or inhibition of metalloproteinases did not prevent EGFR-Akt activation. T. gondii micronemal proteins (MICs) containing EGF domains (EGF-MICs; MIC3 and MIC6) appeared to promote EGFR activation. Parasites defective in EGF-MICs (MIC1 ko, deficient in MIC1 and secretion of MIC6; MIC3 ko, deficient in MIC3; and MIC1-3 ko, deficient in MIC1, MIC3 and secretion of MIC6) caused impaired EGFR-Akt activation and recombinant EGF-MICs (MIC3 and MIC6) caused EGFR-Akt activation. In cells treated with autophagy stimulators (CD154, rapamycin) EGFR signaling inhibited LC3 accumulation around the parasite. Moreover, increased LC3 accumulation and parasite killing were noted in CD154-activated cells infected with MIC1-3 ko parasites. Finally, recombinant MIC3 and MIC6 inhibited parasite killing triggered by CD154 particularly against MIC1-3 ko parasites. Thus, our findings identified EGFR activation as a strategy used by T. gondii to maintain the non-fusogenic nature of the parasitophorous vacuole and suggest that EGF-MICs have a novel role in affecting signaling in host cells to promote parasite survival.     

Interaction of model IgG complexes with lectin from Ricinus communis in solutions studied by light scattering method]

In order to demonstrate the participation of galactose-containing carbohydrate epitopes on the surface of model IgG complexes (MIC) during their interaction with high molecular weight ligands, MIC were obtained. The interaction of MIC with Ricinus communis agglutinin (RCA) was studied. The time-dependent changes in the intensity of light scattering in solutions containing MIC of different molecular masses were measured after addition of RCA. It was shown that the efficiency of MIC interaction with RCA depends on the molecular mass of the former. The binding of RCA to MIC is highly specific, it being completely abolished after addition of lactose (1-15 mM). It was found that the final lactose concentration necessary for the complete inhibition of MIC interaction with RCA to take place, depends on the molecular mass of MIC. The data obtained point to the accessibility of IgG oligosaccharide antennae within the composition of MIC for the binding to high molecular weight ligands as well as to the increased density of galactose-containing epitopes on the surface of MIC resulting from the increase in their molecular mass.     

Toxoplasma gondii micronemal protein MIC1 is a lactose-binding lectin.

Host cell invasion by Toxoplasma gondii is a multistep process with one of the first steps being the apical release of micronemal proteins that interact with host receptors. We demonstrate here that micronemal protein 1 (MIC1) is a lactose-binding lectin. MIC1 and MIC4 were recovered in the lactose-eluted (Lac(+)) fraction on affinity chromatography on immobilized lactose of the soluble antigen fraction from tachyzoites of the virulent RH strain. MIC1 and MIC4 were both identified by N-terminal microsequencing. MIC4 was also identified by sequencing cDNA clones isolated from an expression library following screening with mouse polyclonal anti-60/70 kDa (Lac(+) proteins) serum. This antiserum localized the Lac(+) proteins on the apical region of T. gondii tachyzoites by confocal microscopy. The Lac(+) fraction induced hemagglutination (mainly type A human erythrocytes), which was inhibited by beta-galactosides (3 mM lactose and 12 mM galactose) but not by up to 100 mM melibiose (alpha-galactoside), fucose, mannose, or glucose or 0.2 mg/ml heparin. The lectin activity of the Lac(+) preparation was attributed to MIC1, because blotted MIC1, but not native MIC4, bound human erythrocyte type A and fetuin. The copurification of MIC1 and MIC4 may have been due to their association, as reported by others. These data suggest that MIC1 may act through its lectin activity during T. gondii infection.     

Conserved GxxxG and WN motifs of MIC13 are essential for bridging two MICOS subcomplexes

Mitochondrial ultrastructure is highly adaptable and undergoes dynamic changes upon physiological and energetic cues. MICOS (mitochondrial contact site and cristae organizing system), a large oligomeric protein complex, maintains mitochondrial ultrastructure as it is required for formation of crista junctions (CJs) and contact sites. MIC13 acts as a critical bridge between two MICOS subcomplexes. Deletion of MIC13 causes loss of CJs resulting in cristae accumulating as concentric rings and specific destabilization of the MIC10-subcomplex. Mutations in MIC13 are associated with infantile lethal mitochondrial hepato-encephalopathy, yet functional regions within MIC13 were not known. To identify and characterize such regions, we systemically generated 20 amino-acids deletion variants across the length of MIC13. While deletion of many of these regions of MIC13 is dispensable for its stability, the N-terminal region and a stretch between amino acid residues 84 and 103 are necessary for the stability and functionality of MIC13. We could further locate conserved motifs within these regions and found that a GxxxG motif in the N-terminal transmembrane segment and an internal WN motif are essential for stability of MIC13, formation of the MIC10-subcomplex, interaction with MIC10- and MIC60-subcomplexes and maintenance of cristae morphology. The GxxxG motif is required for membrane insertion of MIC13. Overall, we systematically found important conserved residues of MIC13 that are required to perform the bridging between the two MICOS subcomplexes. The study improves our understanding of the basic molecular function of MIC13 and has implications for its role in the pathogenesis of a severe mitochondrial disease.     

Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii

The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.     

Regulation of magnesium-inhibited cation current by actin cytoskeleton rearrangement

Magnesium-inhibited, non-selective cation current (I(MIC)) is activated by depletion of intracellular Mg(2+) and ATP. I(MIC) transports various divalent cations including Mg(2+) and Ca(2+), and is involved in cell viability. We investigated the effect of actin dynamics on I(MIC). Formation of a stable cortical actin network by calyculin A inhibited the activation of I(MIC), while the actin depolymerizing reagent, cytochalasin D, reversed the inhibition. Induction of a dense cortical actin layer by transfecting the constitutively active form of RhoA also inhibited the activation of I(MIC). These results suggest that the activation of I(MIC) may be dynamically regulated by actin cytoskeleton rearrangement.     

Postantibiotic effects and postantibiotic sub-MIC effects of ciprofloxacin, pefloxacin and amikacin on the biological properties ofSalmonella strains

The postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PASME) of ciprofloxacin, pefloxacin and amikacin were studied forSalmonella typhimurium andS. enteritidis strains. PAE was induced by 2× and 4×MIC of antibiotics studied for 0.5 h. After PAE and PASME their effect on prophage induction of a lysogenicS. typhimurium strain and on Congo red binding for both strains as a marker of their surface hydrophobicity was examined. The longest PAE was found after treatment with ciprofloxacin, higher values being observed withS. typhimurium. PAEs of pefloxacin and amikacin were much lower, except for the suprainhibitory concentration 4×MIC of amikacin withS. enteritidis (6.9 h). PASMEs of ciprofloxacin did not allow any regrowth of either strain. For other antibiotics the PASME's were different while concentrations of 2×MIC+0.2×MIC and 0.3×MIC, and of 4×MIC+0.1×MIC, 0.2×MIC and 0.3×MIC of amikacin did not allow any regrowth ofS. enteritidis. PAEs of the antibiotics tested did not affect the Congo red binding by bothSalmonella strains, but the PAEs of ciprofloxacin and pefloxacin expressively induced a prophage of lysogenicS. typhimurium strain. We noted the influence of Congo red binding after applying 4×MIC+0.1×MIC, 0.2×MIC and 0.3×MIC of amikacin forS. typhinurium and 2×MIC+0.1×MIC forS. enteritidis.     

Effects of subinhibitory concentrations of antibiotics on biological properties ofSalmonella typhimurium

We followed the effects of subinhibitory concentrations (sub-MICs) of 7 antibiotics (ticarcilin, cefotaxim, streptomycin, gentamicin, ciprofloxacin, pefloxacin, mitomycin C) on the sensitivity of aSalmonella typhimurium strain to standard bacteriophages, on the phage DNA as well as on the factors of virulence (permeability and cytotoxic activity). The phage type was not changed by the sub-MICs of the tested antibiotics. However, differences were found in culture filtrates prepared from the bacterial suspensions of the strain cultivated with the sub-MICs. Marked inducing effects on phage DNA were exhibited by mitomycin C (1/2, 1/4, 1/8 of the MIC), pefloxacin (1/2, 1/4, 1/8 of the MIC) and ciprofloxacin (1/2, 1/4, weakly also 1/8 of the MIC). Ticarcilin (1/2 of the MIC), like the aminoglycosides streptomycin and gentamicin (1/2, 1/4, 1/8 of the MIC), had a weak effect. Sub-MICs of the studied antibiotics (with the exception of 1/8 of the MIC of ciprofloxacin and 1/4 of the MIC of ticarcilin) decreased the permeability reaction in rabbit skin. Most effective was streptomycin (1/2 of the MIC). Sub-MICs of the tested antibiotics (with the exception of 1/4 and 1/8 of the MIC of ciprofloxacin and 1/4 of the MIC of pefloxacin) caused also an inhibition of the factor responsible for morphological changes on Vero cells. Gentamicin and streptomycin were effective at all the sub-MICs tested.     

Highly Purified Micro- and Macronuclei from Tetrahymena thermophila Isolated by Percoll Gradients1

ABSTRACT. A new procedure is described that utilizes Percoll gradients for purifying micronuclei (MIC) and macronuclei (MAC) from Tetrahymena thermophila. Separation of MIC from MAC during certrifugation in Percoll gradients occurs as a result of their difference in size rather than density. Three kinds of tests were used to evaluate the purity of the nuclei: visualization of the nuclei by light microscopy; examination of the nuclei by electron microscopy; and Southern blots of MIC and MAC DNA probed with the 5s rRNA genes or a fragment from the MAC extrachromosomal rDNA molecule. When examined under the light microscope, the isolated MIC and MAC have much lower nuclear cross contamination levels than previous methods have reported. MIC's contaminated with less than 1 MAC in 1000 MIC and MAC's contaminated with less than 1 MIC in 500 MAC can be routinely prepared. Quantitative analyses of electron micrographs gave higher estimates of cross contamination in our purified nuclei, which may, in part, be explained by the difficulty in identifying small MIC or MAC fragments. Southern blots of MIC and MAC DNA probed with 5s rDNA confirmed the level of MAC contamination in the MIC estimated by light microscopy during purification of the nuclei. The level of nucleolar contamination in the MIC was estimated at 10% by Southern blots of MIC and MAC DNA, derived from a heterokaryon with distinctive MIC and MAC Bam HI sites, using an rDNA probe.     

An six-amino acid motif in the α3 domain of MICA is the cancer therapeutic target to inhibit shedding

Expression of the MHC class I chain related molecules A and B (MICA/B) on tumor cell surface can signal the immune receptor NKG2D for tumor immune destruction. However, MIC was found to be shed by tumors in cancer patients, which negatively regulates host immunity and promotes tumor immune evasion and progression. The mechanisms by which tumors shed MIC are not well understood although diverse groups of enzymes are suggested to be involved. The functional complexity of these enzymes makes them unfeasible therapeutic targets for inhibiting MIC shedding. Here we identified an six-amino acid (6-aa) motif in the α3 domain of MIC that is critical for the interaction of MIC with ERp5 to enable shedding. Mutations in this motif prevented MIC shedding but did not interfere with NKG2D-mediated recognition of MIC. Our study suggests that the 6-aa motif is a feasible target to inhibit MIC shedding for cancer therapy.     

Isolation of methylcarbamoyl-adducts of adenine and cytosine following in vitro reaction of methyl isocyanate with calf thymus DNA

Methylisocyanate (MIC) is the direct-acting acylating compound involved in the Bhopal, India disaster which occurred on December 3rd, 1984. The accidental release of MIC resulted in at least 2000 deaths, thousands of injuries and exposure of at least 200,000 people to varying amounts of MIC. We have studied how MIC reacts with 2'-deoxyribonucleosides at pH 7.0 and 37 degrees C for 1 h. MIC acylates exocyclic amino groups resulting in the following methylcarbamoyl (MC) adducts: N6-MC-Ade (0.5% yield) and N4-MC-dCyd (6%). No adducts were detected with dThd and dGuo. UV, NMR and mass spectrometry were employed to spectroscopically characterize these adducts. MIC was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 1 h) and yielded N6-MC-Ade (0.3 nmol/mg DNA) and N4-MC-dCyd (2.0 nmol/mg DNA). The inability of others to observe genetic mutations by MIC in Salmonella and Drosophila is consistent with the exocyclic adducts at N4 of Cyt and N6 of Ade where normal hydrogen bonding can occur after rotation of the methylcarbamoyl group anti to the Watson-Crick side of the molecule assuming that MIC binds to DNA within the intact cell.     

Antimicrobial susceptibility patterns of unusual nonfermentative gram-negative bacilli isolated from Latin America: report from the SENTRY Antimicrobial Surveillance Program (1997-2002)

The antimicrobial susceptibility of 176 unusual non-fermentative gram-negative bacilli (NF-GNB) collected from Latin America region through the SENTRY Program between 1997 and 2002 was evaluated by broth microdilution according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. Nearly 74% of the NF-BGN belonged to the following genera/species: Burkholderia spp. (83), Achromobacter spp. (25), Ralstonia pickettii (16), Alcaligenes spp. (12), and Cryseobacterium spp. (12). Generally, trimethoprim/sulfamethoxazole (MIC50, < 0.5 microg/ml) was the most potent drug followed by levofloxacin (MIC50, 0.5 microg/ml), and gatifloxacin (MIC50, 1 microg/ml). The highest susceptibility rates were observed for levofloxacin (78.3%), gatifloxacin (75.6%), and meropenem (72.6%). Ceftazidime (MIC50, 4 microg/ml; 83.1% susceptible) was the most active beta-lactam against B. cepacia. Against Achromobacter spp. isolates, meropenem (MIC50, 0.25 microg/ml; 88% susceptible) was more active than imipenem (MIC50, 2 microg/ml). Cefepime (MIC50, 2 microg/ml; 81.3% susceptible), and imipenem (MIC50, 2 microg/ml; 81.3% susceptible) were more active than ceftazidime (MIC50, >16 microg/ml; 18.8% susceptible) and meropenem (MIC50, 8 microg/ml; 50% susceptible) against Ralstonia pickettii. Since selection of the most appropriate antimicrobial agents for testing and reporting has not been established by the NCCLS for many of NF-GNB species, results from large multicenter studies may help to guide the best empiric therapy.     

In-vitro activity of 5-fluorocytosine against 1,021 Spanish clinical isolates of Candida and other medically important yeasts

The aim of this study was to determine the prevalence of primary resistance to 5-fluorocytosine (5FC) among clinical isolates of yeasts in Spain where this drug is not currently available for therapy. We have tested the in vitro activity of 5FC against 1,021 recent yeast clinical isolates, including 522 Candida albicans, 140 Candida parapsilosis, 68 Candida glabrata, 41 Candida dubliniensis, 50 Candida guilliermondii, 34 Candida tropicalis, 28 Candida krusei, 20 Candida famata, 11 Cryptococcus neoformans, 5 Cryptococcus albidus, 43 Rhodotorula spp., 24 Trichosporon spp., 5 Saccharomyces cerevisiae, 9 Pichia spp., and 21 isolates from other 11 yeast species. The MICs were determined by the ATB Fungus agar microdilution test (bioMerieux, France) and the following interpretive breakpoints were used: susceptible, > 4 microg/ml; intermediate, 8 to 16 microg/ml; resistant, > 32 microg/ml. 5FC was very active against Candida spp. and other medically important yeasts as 852 (83.4%) of the studied isolates were susceptible (MIC < 4 microg/ml). The species most susceptible to 5FC were C. dubliniensis (100%of isolates; MIC90, 0.25 microg/ml), C. famata (100% of isolates; MIC90, 0.25 microg/ml), C. guilliermondii (98%of isolates; MIC90, 0.25 microg/ml), C. glabrata (95.5% of isolates; MIC90, 0.25 microg/ml), and C. neoformans (90.9% of isolates; MIC90, 2 microg/ml). Primary resistance to 5FC was very uncommon, and a MIC > 32 microg/ml, indicator of in vitro resistance, was observed in 106 isolates (10.4%): 77 C. albicans (16.5% of isolates; MIC90, > 128 microg/ml), 9 C. parapsilosis (6.4% of isolates; MIC90, 8 microg/ml), 4 C. albidus (80% of isolates, MIC50, > 128 microg/ml), 3 C. glabrata (4.4% of isolates; MIC90, 0.25 microg/ml), 3 C. tropicalis (8.8% of isolates; MIC90, 4 microg/ml), 2 C. krusei (7.1% of isolates; MIC90, 8 microg/ml), 2 Rhodotorula spp. (4.6% of isolates, MIC90, 1 microg/ml), 8 Trichosporon spp. (33.3% of isolates; MIC90, 64 microg/ml), and 1 C. lipolytica (50% of isolates). Interestingly, most C. albicans (67 out of 77 isolates) resistant to 5FC were serotype B isolates.     

In vitro activities of antimicrobials against Brucella abortus isolates from cattle in Korea during 1998-2006

In vitro activities of 13 antibiotics were assessed against 85 Brucella abortus isolates from naturally infected cattle in the Republic of Korea during 1998-2006, using broth microdilution test. Tetracyclines showed the most excellent activity against B. abortus, displaying MIC values of 0.5 μg/ml or below. In particular, minocycline showed the lowest MIC??/?? values (0.125/0.125 μg/ml) in this study. Among four fluoroquinolones tested, ciprofloxacin (MIC??/??, 0.5/1 μg/ml) and norfloxacin (MIC??/??, 8/8 μg/ml) had the most and the least activities, respectively. Gentamicin (MIC??/??, 1/1 μg/ml) was more effective than streptomycin, erythromycin, rifampin, and chloramphenicol (MIC??/??, 2/2 μg/ml).     

Effect of bovine lactoferrin on the minimum inhibitory concentrations of ampicillin and trimethoprim-sulfamethoxazole for clinical Shigella spp. strains

Here, we determined the effect of bovine lactoferrin (bLF) on the minimum inhibitory concentration (MIC) of ampicillin and trimethoprim-sulfamethoxazole in Shigella . Using a microdilution method, the MIC was determined in the presence or absence of bovine lactoferrin (10 mg/mL) on 88 Shigella strains (56 Shigella flexneri , 15 Shigella boydii , 13 Shigella sonnei , and 4 Shigella dysenteriae ) previously isolated from peruvian children <2 years old. A fold change of 2 or more in MIC values was considered significant. For ampicillin, 67 (76%) strains were highly resistant; one-third of the strains (32%) showed a decrease in ampicillin MIC in the presence of LF. This was more typical of MIC values in less resistant strains. For 7 (8%) ampicillin-resistant strains, the decrease in the MIC resulted in the strains reaching the cutoff for susceptible in the presence of bLF. For trimethoprim-sulfamethoxazole, 93% of the isolates (n = 82) were highly resistant and only 4 isolates (5%) decreased their MIC in the presence of bLF. None of the trimethoprim-sulfamethoxazole resistant strains became susceptible in the presence of LF. The decrease in the MIC in the presence of bLF seems to depend on the mechanisms of action of each antibiotic. In vivo studies are needed to further evaluate bLF as a coadjuvant to antibiotic treatment of resistant Shigella.     

Reliability of the Etest in light of the correlation between an antibiotic's critical concentration (Cc) and MIC values.

The study relates to the theory of diffusion methods for antibiotic sensitivity testing. The aim of the study was to show the relationship between the antibiotic critical concentration (Cc) and its minimum inhibitory concentration (MIC). The results contribute to the explanation of the Etest's reliability and support the scientific basis for MIC determination using agar diffusion methods. Susceptibility among 90 clinical isolates of 12 common aerobic bacterial species to gentamicin, erythromycin, or oxacillin was assessed using the multidisc method (for Cc), by the agar dilution method (for MIC) and by the Etest. The results of all three methods were statistically compared and found to be closely related. The regression equation for Cc values and MIC was log2(MIC) = 0.99 x log2(Cc)-0.13; r = 0.99; p < 0.05; the regression equation for Cc values and Etest-MIC (Et) was log2(Et) = 0.86 x log2 (Cc)+0.34; r = 0.96; p < 0.05; the regression equation for Etest-MIC values and MIC was log2(MIC) = 1.12 x log2(Et)-0.50; r = 0.96; p < 0.05.