Comparative Intracellular (THP-1 Macrophage) and Extracellular Activities of β-Lactams, Azithromycin, Gentamicin, and Fluoroquinolones against Listeria monocytogenes at Clinically Relevant Concentrations

ABSTRACT The activities of ampicillin, meropenem, azithromycin, gentamicin, ciprofloxacin, and moxifloxacin against intracellular hemolysin-positive Listeria monocytogenes were measured in human THP-1 macrophages and were compared with the extracellular activities observed in broth. All extracellular concentrations were adjusted to explore ranges that are clinically achievable in human serum upon conventional therapy. In broth, ampicillin, meropenem, and azithromycin were only bacteriostatic, whereas gentamicin, ciprofloxacin, and moxifloxacin were strongly bactericidal in a concentration-dependent manner. In cells, ampicillin, meropenem, azithromycin, and ciprofloxacin were slightly bactericidal (0.3- to 0.8-log CFU reductions), moxifloxacin was strongly bactericidal (2.1-log CFU reduction), and gentamicin was virtually inactive. The difference in the efficacies of moxifloxacin and ciprofloxacin in cells did not result from a difference in levels of accumulation in cells (6.96 ± 1.05 versus 7.75 ± 1.03) and was only partially explainable by the difference in the MICs (0.58 ± 0.04 versus 1.40 ± 0.17 mg/liter). Further analysis showed that intracellular moxifloxacin expressed only approximately 1/7 of the activity demonstrated against extracellular bacteria and ciprofloxacin expressed only 1/15 of the activity demonstrated against extracellular bacteria. Gentamicin did not increase the intracellular activities of the other antibiotics tested. The data suggest (i) that moxifloxacin could be of potential interest for eradication of the intracellular forms of L. monocytogenes, (ii) that the cellular accumulation of an antibiotic is not the only determinant of its intracellular activity (for fluoroquinolones, it is actually a self-defeating process as far as activity is concerned), and (iii) that pharmacodynamics (activity-to-concentration relationships) need to be considered for the establishment of efficacy against intracellular bacteria, just as they are for the establishment of efficacy against extracellular infections.

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Sphingosine Kinase as a Regulator of Calcium Entry through Autocrine Sphingosine 1-Phosphate Signaling in Thyroid FRTL-5 Cells

Endocrinology ◽

10.1210/en.2009-0288 ◽

2009 ◽

Vol 150 (11) ◽

pp. 5125-5134 ◽

Cited By ~ 11

Author(s):

Dan Gratschev ◽

Christoffer Löf ◽

Jari Heikkilä ◽

Anders Björkbom ◽

Pramod Sukumaran ◽

...

Keyword(s):

Intracellular Signaling ◽

Sphingosine Kinase ◽

Calcium Entry ◽

Dominant Negative ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Entry Pathway ◽

Sphingosine 1 Phosphate ◽

S1p Receptor ◽

In Cells

Calcium entry is one of the main regulators of intracellular signaling. Here, we have described the importance of sphingosine, sphingosine kinase 1 (SK1), and sphingosine 1-phosphate (S1P) in regulating calcium entry in thyroid FRTL-5 cells. In cells incubated with the phosphatase inhibitor calyculin A, which evokes calcium entry without mobilizing sequestered intracellular calcium, sphingosine inhibited calcium entry in a concentration-dependent manner. Furthermore, inhibiting SK1 or the ATP-binding cassette ABCC1 multidrug transporter attenuated calcium entry. The addition of exogenous S1P restored calcium entry. Neither sphingosine nor inhibition of SK1 attenuated thapsigargin-evoked calcium entry. Blocking S1P receptor 2 or phospholipase C attenuated calcium entry, whereas blocking S1P receptor 3 did not. Overexpression of wild-type SK1, but not SK2, enhanced calyculin-evoked calcium entry compared with mock-transfected cells, whereas calcium entry was decreased in cells transfected with the dominant-negative G82D SK1 mutant. Exogenous S1P restored calcium entry in G82D cells. Our results suggest that the calcium entry pathway is blocked by sphingosine and that activation of SK1 and the production of S1P, through an autocrine mechanism, facilitate calcium entry through activation of S1P receptor 2. This is a novel mechanism by which the sphingosine-S1P rheostat regulates cellular calcium homeostasis.

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L-Lysine: A Modulator for the Relative Activity of High Molecular Weight (HMW) and Low Molecular Weight(LHW) Urokinase(UK)

10.1055/s-0039-1687222 ◽

1979 ◽

Author(s):

L.L. Shen ◽

W.H. Holleman

Keyword(s):

Molecular Weight ◽

Caproic Acid ◽

Fibrin Clot ◽

Relative Activity ◽

Test Tube ◽

Clot Lysis ◽

Dependent Manner ◽

Plasma Clot ◽

Concentration Dependent Manner ◽

The Difference

L-Lysine(Lys), in a concentration dependent manner, progressively inhibited UK-activated lysis of human plasma clots as demonstrated by Ploug test-tube method and elastometric measurements. Lys was more effective with HMW UK than LMW UK, and the effect of Lys with LMW UK from tissue culture and urine sources was the same. Epsilon amino caproic acid(EACA) and tranexamic acid(TXA) were stronger inhibitors but inhibited HMW and LMW UK-induced lysis to the same degree. Elastometric measurements showed that Lys inhibition was not due to its interference with the initial clotting process nor to the reduction of clot rigidity. Amidolytic assays using chromogenic substrates showed that Lys had no direct effect, on UK, and that Lys enhanced the activation of the native Glu-plasminogen(Pg) by LMW UK, but not the activation by HMW UK. When the substrate was human fibrin clots, Lys enhanced the lysis induced by LMW UK while the lysis induced by HMW UK was inhibited; however, the extent of enhancement and inhibition was limited. We concluded that the mode of Lys action is not identical to that of EACA or TXA, and that the stronger Lys inhibition of plasma clot lysis as compared to fibrin clot lysis is due to the potentiation of plasma fibrinolytic inhibitors by Lys. The difference In effect of Lys on HMW and LMW UK-induced lyels is likely due to a partial conformation change of Glu-Pg molecule upon Lys binding. The relatively moderate interaction of Lys with Glu-Fg results In a mildly modified UK substrate which reacts preferentially with the enzyme smaller in size.

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Metal Ions in Activated Carbon Improve the Detection Efficiency of Aflatoxin-Producing Fungi

Toxins ◽

10.3390/toxins11030140 ◽

2019 ◽

Vol 11 (3) ◽

pp. 140 ◽

Cited By ~ 1

Author(s):

Tadahiro Suzuki ◽

Masatoshi Toyoda

Keyword(s):

Activated Carbon ◽

Metal Ions ◽

Detection Efficiency ◽

Trace Element Analysis ◽

The Other ◽

Dependent Manner ◽

Element Analysis ◽

Concentration Dependent Manner ◽

Key Factor ◽

Co Addition

Aflatoxins (AF), produced by several Aspergillus species, are visible under ultraviolet light if present in high amounts. AF detection can be improved by adding activated carbon, which enhances the observation efficiency of weakly AF-producing fungi. However, commercial activated carbon products differ in their characteristics, making it necessary to investigate which characteristics affect method reproducibility. Herein, the addition of 10 activated carbon products resulted in different AF production rates in each case. The differences in the production of aflatoxin G1 (AFG1) were roughly correlated to the observation efficiency in the plate culture. Trace element analysis showed that the concentrations of several metal ions differed by factors of >100, and the carbons that most effectively increased AFG1 production contained higher amounts of metal ions. Adding 5 mg L−1 Fe or Mg ions increased AFG1 production even without activated carbon. Furthermore, co-addition of both ions increased AFG1 production stably with the addition of carbon. When varying the concentration of additives, only AFG1 production increased in a concentration-dependent manner, while the production of all the other AFs decreased or remained unchanged. These findings suggest that a key factor influencing AF production is the concentration of several metal ions in activated carbon and that increasing AFG1 production improves AF detectability.

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The effect of endotoxin on functional parameters of mammary CID-9 cells

Reproduction ◽

10.1530/rep.1.00135 ◽

2004 ◽

Vol 127 (3) ◽

pp. 397-406 ◽

Cited By ~ 13

Author(s):

B Safieh-Garabedian ◽

G M Mouneimne ◽

W El-Jouni ◽

M Khattar ◽

R Talhouk

Keyword(s):

Active Form ◽

Nuclear Factor Κb ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Gelatinase Activity ◽

Functional Parameters ◽

Nuclear Extracts ◽

Cell Growth And Viability ◽

Factor Α ◽

In Cells

The effect of endotoxin on mammary CID-9 cells, which differentiate in culture and express β-casein, was investigated. Cells in culture supplemented with lactogenic hormones and dripped with EMS-Matrix (EMS-drip), were treated daily with endotoxin (0.5–500 μg/ml). Endotoxin at concentrations of less or equal to 10 μg/ml did not affect cell growth and viability up to 5 days post endotoxin treatment. Endotoxin (0.01–10 μg/ml) was added to the culture medium, upon confluence, and functional parameters were examined within 48 h post endotoxin treatment. Nuclear factor-κB (NF-κB) (p52) increased in nuclear extracts from endotoxin-stimulated cells within 1 h of treatment, while β-casein mRNA and protein expression decreased in a concentration-dependent manner at 24 and 48 h post treatment. Zymography showed that the 72 and 92 kDa gelatinase activity increased in cells at 24 and 48 h post endotoxin treatment at 10 and 50 μg/ml. At the latter concentration, the active form of 72 kDa gelatinase was induced at 48 h. Interleukin-6 and tumor necrosis factor-α levels increased at 1–3 h post endotoxin treatment and peaked at 6 h in cells on plastic and EHS-drip. Nerve growth factor (NGF) levels increased in control and endotoxin-treated cells in a time-dependent manner, and endotoxin increased NGF levels in culture at 6 and 9 h post endotoxin treatment. This study shows that endotoxin activated NF-κB, suppressed β-casein expression and upregulated gelatinases, cytokines and NGF. This model could be used to investigate the role of mammary cells in initiating and propagating inflammation and to test candidate molecules for potential anti-inflammatory properties.

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Dual-specific Src and Abl kinase inhibitors, PP1 and CGP76030, inhibit growth and survival of cells expressing imatinib mesylate–resistant Bcr-Abl kinases

Blood ◽

10.1182/blood-2002-01-0288 ◽

2003 ◽

Vol 101 (2) ◽

pp. 664-672 ◽

Cited By ~ 100

Author(s):

Markus Warmuth ◽

Nicola Simon ◽

Olga Mitina ◽

Ruth Mathes ◽

Doriano Fabbro ◽

...

Keyword(s):

Imatinib Mesylate ◽

Kinase Inhibitors ◽

Clonal Evolution ◽

Src Kinase ◽

Dependent Manner ◽

Binding Modes ◽

Concentration Dependent Manner ◽

Growth And Survival ◽

Abl Kinase ◽

In Cells

The leukemogenic tyrosine kinase Bcr-Abl contains a highly conserved inhibitor-binding pocket (IBP), which serves as a binding site for imatinib mesylate. Mutations at the IBP may lead to resistance of the Abl kinase against imatinib mesylate. To examine the mechanisms of imatinib mesylate binding and resistance in more detail, we created several point mutations at amino acid positions 315 and 380 of Abl, blocking the access to the IBP and rendering Bcr-Abl imatinib mesylate–resistant. Moreover, introduction of a mutation destabilizing the inactive conformation of Abl (Asp276Ser/Glu279Ser) also led to imatinib mesylate resistance, suggesting that the inhibitor required inactivation of the kinase prior to binding. These Bcr-Abl mutants were then used to evaluate the binding mode and specificity of 2 compounds, PP1 and CGP76030, originally characterized as Src kinase inhibitors. Both compounds inhibited Bcr-Abl in a concentration-dependent manner by overlapping binding modes. However, in contrast to imatinib mesylate, PP1 and CGP76030 blocked cell growth and survival in cells expressing various inhibitor-resistant Abl mutants. Studies on the potential signaling mechanisms demonstrated that in cells expressing inhibitor-resistant Bcr-Abl mutants, PP1 and CGP76030 inhibited the activity of Src family tyrosine kinases and Akt but not signal transducer and activator of transcription–5 (STAT5) and JUN kinase (Jnk). The results suggest that the use of Src kinase inhibitors is a potential strategy to prevent or overcome clonal evolution of imatinib mesylate resistance in Bcr-Abl+ leukemia.

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Inhibitory effect of Sphagnum palustre extract and its bioactive compounds on aromatase activity

Bangladesh Journal of Pharmacology ◽

10.3329/bjp.v11i3.26776 ◽

2016 ◽

Vol 11 (3) ◽

pp. 661

Author(s):

Hee Jeong Eom ◽

Yong Joo Park ◽

Hee Rae Kang ◽

Ha Ryong Kim ◽

In Jae Bang ◽

...

Keyword(s):

Video Clip ◽

Inhibitory Effect ◽

The Other ◽

Aromatase Activity ◽

Dependent Manner ◽

Inhibitory Effects ◽

Aromatase Inhibition ◽

Concentration Dependent Manner ◽

Cardiac Pain ◽

Sphagnum Palustre

Sphagnum palustre (a moss) has been traditionally used in Korea for the cure of several diseases such as cardiac pain and stroke. In this research, the inhibitory effect of S. palustre on aromatase (cytochrome P450 19, CYP19) activity was studied. [1β-3H] androstenedione was used as a substrate and incubated with S. palustre extract and recombinant human CYP19 in the presence of NADPH. S. palustre extract inhibited aromatase in a concentration-dependent manner (IC50 value: 36.4 ± 8.1 µg/mL). To elucidate the major compounds responsible for the aromatase inhibitory effects of S. palustre extract, nine compounds were isolated from the extract and tested for their inhibition of aromatase activity. Compounds 1, 6, and 7 displayed aromatase inhibition, while the inhibition by the other compounds was negligible.Video Clip<a href="https://youtube.com/v/n6xeo3RXJVY">Aromatase enzyme activity:</a> 4 min 16 sec

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Antiausterity Activity of Arctigenin Enantiomers: Importance of (2R,3R)-Absolute Configuration

Natural Product Communications ◽

10.1177/1934578x1400900123 ◽

2014 ◽

Vol 9 (1) ◽

pp. 1934578X1400900 ◽

Cited By ~ 1

Author(s):

Suresh Awale ◽

Mamoru Kato ◽

Dya Fita Dibwe ◽

Feng Li ◽

Chika Miyoshi ◽

...

Keyword(s):

Cytotoxic Activity ◽

Cancer Cells ◽

Absolute Configuration ◽

The Other ◽

Dependent Manner ◽

Meoh Extract ◽

Concentration Dependent Manner ◽

Akt Activation ◽

Other Hand

From a MeOH extract of powdered roots of Wikstroema indica, six dibenzyl-γ-butyrolactone-type lignans with (2 S,3 S)-absolute configuration [(+)-arctigenin (1), (+)-matairesinol (2), (+)-trachelogenin (3), (+)-nortrachelogenin (4), (+)-hinokinin (5), and (+)-kusunokinin (6)] were isolated, whereas three dibenzyl-γ-butyrolactone-type lignans with (2 R,3 R)-absolute configuration [(-)-arctigenin (1), (-)-matairesinol (2), (-)-trachelogenin (3)] were isolated from Trachelospermum asiaticum. The in vitro preferential cytotoxic activity of the nine compounds was evaluated against human pancreatic PANC-1 cancer cells in nutrient-deprived medium (NDM), but none of the six lignans (1–6) with (2 S,3 S)-absolute configuration showed preferential cytotoxicity. On the other hand, three lignans (1*–3*) with (2 R,3 R)-absolute configuration exhibited preferential cytotoxicity in a concentration-dependent manner with PC50 values of 0.54, 6.82, and 5.85 μM, respectively. Furthermore, the effect of (-)- and (+)-arctigenin was evaluated against the activation of Akt, which is a key process in the tolerance to nutrition starvation. Interestingly, only (-)-arctigenin (1*) strongly suppressed the activation of Akt. These results indicate that the (2 R,3 R)-absolute configuration of (-)-enantiomers should be required for the preferential cytotoxicity through the inhibition of Akt activation.

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Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00535.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. H2790-H2800 ◽

Cited By ~ 14

Author(s):

Harjot K. Saini ◽

Naranjan S. Dhalla

Keyword(s):

Sarcoplasmic Reticulum ◽

Intracellular Calcium ◽

Calcium Concentration ◽

Extracellular Ph ◽

The Other ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Rat Cardiomyocytes ◽

Intracellular Ca ◽

Isolated Rat

Although the Na+/H+ exchanger (NHE) is considered to be involved in regulation of intracellular Ca2+ concentration ([Ca2+]i) through the Na+/Ca2+ exchanger, the exact mechanisms of its participation in Ca2+ handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca2+ movements in cardiomyocytes and exposed to an NHE inhibitor, 5-( N-methyl- N-isobutyl)amiloride (MIA). [Ca2+]i in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca2+]i and augmented the KCl-induced increase in [Ca2+]i in a concentration-dependent manner. The MIA-induced increase in basal [Ca2+]i was unaffected by extracellular Ca2+, antagonists of the sarcolemmal (SL) L-type Ca2+ channel, and inhibitors of the SL Na+/Ca2+ exchanger, SL Ca2+ pump ATPase and mitochondrial Ca2+ uptake. However, the MIA-induced increase in basal [Ca2+]i was attenuated by inhibitors of SL Na+-K+-ATPase and sarcoplasmic reticulum (SR) Ca2+ transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca2+ concentration and attenuated by agents that inhibit SL L-type Ca2+ channels, the SL Na+/Ca2+ exchanger, SL Na+-K+-ATPase, and SR Ca2+ release channels and the SR Ca2+ pump. However, the effect of MIA on the KCl-induced increase in [Ca2+]i remained unaffected by treatment with inhibitors of SL Ca2+ pump ATPase and mitochondrial Ca2+ uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca2+]i, whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca2+]i in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca2+]i and that MIA-induced increases in basal [Ca2+]i, as well as augmentation of the KCl-induced increase in [Ca2+]i, in cardiomyocytes are regulated differentially.

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Block of current through single calcium channels by Fe, Co, and Ni. Location of the transition metal binding site in the pore.

The Journal of General Physiology ◽

10.1085/jgp.97.2.351 ◽

1991 ◽

Vol 97 (2) ◽

pp. 351-367 ◽

Cited By ~ 54

Author(s):

B D Winegar ◽

R Kelly ◽

J B Lansman

Keyword(s):

Metal Binding ◽

The Other ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Coulombic Interactions ◽

Rate Limiting Step ◽

Ion Size ◽

Rate Limiting ◽

Kinetics Of ◽

Ca2 Channels

The blocking actions of Fe2+, Co2+, and Ni2+ on unitary currents carried by Ba2+ through single dihydropyridine-sensitive Ca2+ channels were recorded from cell-attached patches on myotubes from the mouse C2 cell line. Adding millimolar concentrations of blocker to patch electrodes containing 110 mM BaCl2 produced discrete excursions to the closed channel level. The kinetics of blocking and unblocking were well described with a simple model of open channel block. Hyperpolarization speeded the exit of all of the blockers from the channel, as expected if the blocking site resides within the pore. The block by Ni2+ differs from that produced by Fe2+ and Co2+ because Ni2+ enters the channel approximately 20 times more slowly and exits approximately 50 times more slowly. Ni2+ also differs from the other transition metals because at millimolar concentrations it reduces the amplitude of the unitary current in a concentration-dependent manner. The results are consistent with the idea that the rate-limiting step for ion entry into the channel is water loss at its inner coordination sphere; unblocking, on the other hand, cannot be explained in terms of simple coulombic interactions arising from differences in ion size.

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In vitroandin vivoactivities of a bi-aryl oxazolidinone RBx 11760 against Gram positive bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00453-16 ◽

2016 ◽

pp. AAC.00453-16 ◽

Cited By ~ 4

Author(s):

Tarani Kanta Barman ◽

Manoj Kumar ◽

Tarun Mathur ◽

Tridib Chaira ◽

G. Ramkumar ◽

...

Keyword(s):

Staphylococcus Epidermidis ◽

High Dose ◽

Dependent Manner ◽

Gram Positive ◽

Concentration Dependent Manner ◽

Gram Positive Bacteria ◽

Initial Control ◽

The Difference ◽

Time Kill ◽

Groin Abscess

RBx 11760, a bi-aryl oxazolidinone was investigated for antibacterial activity against Gram positive bacteria. The MIC90(mg/L) of RBx 11760 and linezolid againstStaphylococcus aureuswere: 2 and 4,Staphylococcus epidermidis: 0.5 and 2,Enterococcus: 1 and 4, respectively. Similarly againstStreptococcus pneumoniaeMIC90was: 0.5 and 2, respectively. In time-kill studies, RBx 11760, tedizolid and linezolid exhibited bacteriostatic effect exceptS. pneumoniae. RBx 11760 showed 2-log10kill at 4 X MIC while tedizolid and linezolid showed 2 log10and 1.4-log10kill at 16 X MIC, respectively against MRSA H-29. AgainstS. pneumoniae5051, RBx 11760 showed bactericidal activity with 4.6 log10kill at 4 X MIC compared to 2.42 log10and 1.95 log10kill of tedizolid and linezolid at 16 X MIC. RBx 11760 showed 3 h post antibiotic effects (PAE) at 4 mg/L against MRSA H-29 and linezolid showed same effect at 16mg/L. RBx 11760 inhibited the biofilm production against MRSE ATCC 35984 in concentration dependent manner. In foreign body model, linezolid and rifampicin resulted in no advantage over stasis, while same dose of RBx 11760 demonstrated a significant killing from initial control againstS. aureus(*p<0.05) and MRSE (**p<0.01). The difference in killing was statistically significant for the lower dose of RBx 11760 (*p<0.05) versus high dose of linezolid (nsp>0.05) in groin abscess model. In neutropenic mouse thigh infection, RBx 11760 showed stasis at 20 mg/kg whereas tedizolid showed same effect at 40 mg/kg. These data support the RBx 11760 as a promising investigational candidate.

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