Publications by authors named "Ryosuke Nakashima"

19 Publications

  • Page 1 of 1

Efficacy of thromboelastography in the management of anticoagulation for veno-venous extracorporeal membrane oxygenation in a coronavirus disease 2019 patient: A case report.

Medicine (Baltimore) 2021 Jun;100(23):e26313

Department of Cardiovascular Medicine, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan.

Rationale: In coronavirus disease 2019 (COVID-19) patients with acute respiratory distress syndrome refractory to optimal conventional management, we should consider the indication for veno-venous extracorporeal membrane oxygenation (V-V ECMO). Growing evidence indicates that COVID-19 frequently causes coagulopathy, presenting as hypercoagulation and incidental thrombosis. For these reasons, a multifactorial approach with several anticoagulant markers should be considered in the management of anticoagulation using heparin in COVID-19 patients on V-V ECMO.

Patient Concerns: A 48-year-old man was infected with COVID-19 with a worsening condition manifesting as acute respiratory distress syndrome.

Diagnoses: He was refractory to conventional therapy, thus we decided to introduce V-V ECMO. We used heparin as an anticoagulant therapy for V-V ECMO and adjusted the doses of heparin by careful monitoring of the activated clotting time (ACT) and activated partial thromboplastin time (APTT) to avoid both hemorrhagic and thrombotic complications. We controlled the doses of heparin in the therapeutic ranges of ACT and APTT, but clinical hemorrhaging and profound elevation of coagulant marker became apparent.

Interventions: Using thromboelastography (TEG; Haemonetics) in addition to ACT and APTT, we were able to clearly detect not only sufficient coagulability of COVID19 on V-V ECMO (citrated rapid thromboelastography-R 0.5 min, angle 75.5°, MA 64.0 mm, citrated functional fibrinogen-MA 20.7 mm) but also an excessive effect of heparin (citrated kaolin -R 42.7 min, citrated kaolin with heparinase 11.7 min).

Outcomes: Given the TEG findings indicating an excessive heparin effect, the early withdrawal of ECMO was considered. After an evaluation of the patient's respiratory capacity, withdrawal from V-V ECMO was achieved and then anticoagulation was stopped. The hemorrhagic complications and elevated thrombotic marker levels dramatically decreased.

Lessons: TEG monitoring might be a useful option for managing anticoagulation in COVID-19 patients on V-V ECMO frequently showing a hypercoagulative state and requiring massive doses of heparin, to reduce both hemorrhagic and thrombotic complications.
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http://dx.doi.org/10.1097/MD.0000000000026313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8202565PMC
June 2021

Undiagnosed Cardiac Sarcoidosis Causing Refractory Heart Failure After Acute Myocardial Infarction due to Thromboembolism.

Int Heart J 2021 Mar 17;62(2):437-440. Epub 2021 Mar 17.

Department of Cardiovascular Medicine, Faculty of Medical Sciences, Kyushu University.

A 61-year-old woman suffered chest pain and was admitted to a nearby hospital emergency department. She was diagnosed with acute myocardial infarction probably due to thromboembolism in the left anterior descending coronary artery and aspiration thrombectomy was performed. Afterwards, she developed refractory heart failure with severe global left ventricular dysfunction and was transferred to our hospital. An F-FDG-PET/CT scan revealed abnormal F-FDG uptake in non-infarcted regions of the left ventricle. Non-caseating granulomas were detected by biopsy from a skin eruption. She was diagnosed with cardiac sarcoidosis. In cases of refractory heart failure which cannot be explained only by myocardial infarction, evaluation of other undiagnosed cardiomyopathies is important for optimal management.
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http://dx.doi.org/10.1536/ihj.20-586DOI Listing
March 2021

Development of a structure determination method using a multidrug-resistance regulator protein as a framework.

Biochem Biophys Res Commun 2019 10 17;518(2):402-408. Epub 2019 Aug 17.

Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan.

The structure determination of organic compounds is desirable for the development of medicines, aroma chemicals, and agricultural chemicals. However, the crystallization of organic compounds is often troublesome, because crystallization requires a relatively large quantity of high purity compounds and crystallization trials often need to be performed repetitively using different conditions. Some proteins are known to be able to bind to various organic compounds. The multidrug-resistance regulator protein RamR is one such protein. We have developed a structure determination method for organic compounds using RamR. RamR bound to organic compounds, including one compound that was not a known ligand for RamR, and the structures of the complexes were successfully determined. Because the RamR crystal is hydrophilic, this method may be useful for compounds that cannot be handled by the crystalline sponge method.
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http://dx.doi.org/10.1016/j.bbrc.2019.08.070DOI Listing
October 2019

Acid-Tolerant Reversibly Switchable Green Fluorescent Protein for Super-resolution Imaging under Acidic Conditions.

Cell Chem Biol 2019 10 15;26(10):1469-1479.e6. Epub 2019 Aug 15.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan; The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan. Electronic address:

Reversibly switchable fluorescent proteins (RSFPs) are crucial tags for super-resolution observation of protein localization and dynamics inside living cells. However, due to the high fluorescence pK (∼5-6) of most RSFPs, their usage in acidic conditions (pH 4.5-6.0) has been limited. Here, we investigated a new photochromic mechanism in Gamillus, a recently developed green fluorescent protein with acid tolerance. Gamillus exhibits negative switching with especially high contrast in acidic conditions, and its off switching is caused by trans-to-cis isomerization of the chromophore hydroxyphenyl ring that accompanies protonation. Through a combination of rational design and saturation mutagenesis, we developed two variants with enhanced switching contrasts and off-switching speeds, designated rsGamillus-S and rsGamillus-F, respectively. The fluorescence intensity, off-switching speed, and switching contrast of the rsGamillus variants are only slightly affected by changes in pH between 4.5 and 7.5. Exploiting these properties, we succeeded in high-contrast super-resolution imaging of cellular architectures in acidic conditions.
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http://dx.doi.org/10.1016/j.chembiol.2019.07.012DOI Listing
October 2019

Crystal structures of multidrug efflux pump MexB bound with high-molecular-mass compounds.

Sci Rep 2019 03 13;9(1):4359. Epub 2019 Mar 13.

Laboratory of Cell Membrane Structural Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, 567-0047, Japan.

RND-type multidrug efflux pumps have two voluminous multisite drug-binding pockets named the proximal and distal binding pocket. High- and low-molecular-mass drugs bind to these proximal and distal pocket, respectively. Here, we report the crystal structures of MexB of Pseudomonas aeruginosa bound with high-molecular-mass compounds. Contrary to the expectations, lauryl maltose neopentyl glycol (LMNG, MW 1,005), which is a surfactant larger than the proximal pocket-binding drugs, was found to bind to the distal pocket: one of the two hydrophobic alkyl chains was inserted into the hydrophobic pit, which is the binding site of the efflux pump inhibitor ABI-PP. LMNG is a substrate of the MexAB-OprM system and competitively inhibits the export of other substrates by this system. However, LMNG does not inhibit the export of other substrates by the inhibitor-binding-pit mutant F178W, which retains the export activity of LMNG. The crystal structure of this mutant suggested that the alkyl chain of LMNG could no longer be inserted into the pit because of steric hindrance. We also determined the crystal structure of MexB containing the high-molecular-mass compound neopentyl glycol derivative C7NG (MW 1,028), the binding site of which overlapped with LMNG in the distal pocket, indicating that whether a substrate binds to the distal or proximal pockets is controlled not only by its molecular weight but also by its individual molecular characteristic.
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http://dx.doi.org/10.1038/s41598-019-40232-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416280PMC
March 2019

Crystal structure of the multidrug resistance regulator RamR complexed with bile acids.

Sci Rep 2019 01 17;9(1):177. Epub 2019 Jan 17.

Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, 565-0871, Osaka, Japan.

During infection, Salmonella senses and responds to harsh environments within the host. Persistence in a bile-rich environment is important for Salmonella to infect the small intestine or gallbladder and the multidrug efflux system AcrAB-TolC is required for bile resistance. The genes encoding this system are mainly regulated by the ramRA locus, which is composed of the divergently transcribed ramA and ramR genes. The acrAB and tolC genes are transcriptionally activated by RamA, whose encoding gene is itself transcriptionally repressed by RamR. RamR recognizes multiple drugs; however, the identity of the environmental signals to which it responds is unclear. Here, we describe the crystal structures of RamR in complexes with bile components, including cholic acid and chenodeoxycholic acid, determined at resolutions of 2.0 and 1.8 Å, respectively. Both cholic and chenodeoxycholic acids form four hydrogen bonds with Tyr59, Thr85, Ser137 and Asp152 of RamR, instead of π-π interactions with Phe155, a residue that is important for the recognition of multiple compounds including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6 G. Binding of these compounds to RamR reduces its DNA-binding affinity, resulting in the increased transcription of ramA and acrAB-tolC. Our results reveal that Salmonella senses bile acid components through RamR and then upregulates the expression of RamA, which can lead to induction of acrAB-tolC expression with resulting tolerance to bile-rich environments.
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http://dx.doi.org/10.1038/s41598-018-36025-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336783PMC
January 2019

Multiple entry pathways within the efflux transporter AcrB contribute to multidrug recognition.

Nat Commun 2018 01 9;9(1):124. Epub 2018 Jan 9.

Laboratory of Cell Membrane Structural Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, 567-0047, Japan.

AcrB is the major multidrug exporter in Escherichia coli. Although several substrate-entrances have been identified, the specificity of these various transport paths remains unclear. Here we present evidence for a substrate channel (channel 3)  from the central cavity of the AcrB trimer, which is connected directly to the deep pocket without first passing the switch-loop and the proximal pocket . Planar aromatic cations, such as ethidium, prefer channel 3 to channels 1 and 2. The efflux through channel 3 increases by targeted mutations and is not in competition with the export of drugs such as minocycline and erythromycin through channels 1 and 2. A switch-loop mutant, in which the pathway from the proximal to the deep pocket is hindered, can export only channel 3-utilizing drugs. The usage of multiple entrances thus contributes to the recognition and transport of a wide range of drugs with different physicochemical properties.
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http://dx.doi.org/10.1038/s41467-017-02493-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760665PMC
January 2018

Acid-Tolerant Monomeric GFP from Olindias formosa.

Cell Chem Biol 2018 03 28;25(3):330-338.e7. Epub 2017 Dec 28.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan; The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan. Electronic address:

The fluorescent protein (FP) color palette has greatly contributed to the visualization of molecular and cellular processes. However, most FPs lose fluorescence at a pH lower than their neutral pK (∼6), and this has hampered their application in acidic organelles (pH ∼4.5-6.0). Currently, several cyan- and red-colored acid-tolerant FPs are available; however, there are few reports of acid-tolerant green FPs (GFPs) that are practically applicable to bioimaging. Here, we developed the acid-tolerant monomeric GFP "Gamillus" from the jellyfish Olindias formosa, with excellent brightness, maturation speed, and photostability. Results from X-ray crystallography and point mutagenesis suggest that across a broad pH range the acid tolerance is attributed to stabilization of deprotonation in the chromophore phenyl ring by forming a unique trans configuration. We demonstrate that Gamillus can serve as a molecular tag suitable for imaging in acidic organelles through autophagy-mediated molecular tracking to lysosomes.
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http://dx.doi.org/10.1016/j.chembiol.2017.12.005DOI Listing
March 2018

Crystallographic Analysis of Drug and Inhibitor-Binding Structure of RND-Type Multidrug Exporter AcrB in Physiologically Relevant Asymmetric Crystals.

Methods Mol Biol 2018 ;1700:25-36

Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan.

Xenobiotic extruding pumps have recently been known to be widely distributed in living organisms from mammalian to bacteria as a host-defense mechanism in cellular level. These pumps not only confer multidrug resistance of cancer cells and pathogenic bacteria but also cause hereditary diseases through the mutation. Our purposes are to elucidate the molecular structures and mechanisms of these xenobiotic exporters.We had succeeded to determine the crystal structure of bacterial major multidrug exporter AcrB at 3.5 Å resolution (Murakami et al., Nature 419:587-593, 2002) and elucidated the structural bases of substrate recognition that the pump recognize the places and thus act as a "membrane vacuum cleaner." After that we also determined the crystal structure of the drug-binding form of AcrB in space group C2 in which asymmetric unit contains structurally asymmetric homo-trimer of AcrB (Murakami et al., Nature 443:173-179, 2006; Nakashima et al., Nature 480:565-569, 2011; Nakashima et al., Nature 500:120-126, 2013). Analyses revealed the existence of a specific mechanism to recognize numerous substrates that the multisite binding is the base of multidrug recognition rather than induced-fit, and functional-rotation mechanism in which three monomers undergo a strictly coordinated sequential conformational change cycle of access, binding, and extrusion. Determination of physiological asymmetric AcrB structure was crucially important to understand these transport mechanisms.
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http://dx.doi.org/10.1007/978-1-4939-7454-2_2DOI Listing
July 2018

Hoisting-Loop in Bacterial Multidrug Exporter AcrB Is a Highly Flexible Hinge That Enables the Large Motion of the Subdomains.

Front Microbiol 2017 25;8:2095. Epub 2017 Oct 25.

Department of Cell Membrane Structural Biology, Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan.

The overexpression of RND-type exporters is one of the main causes of multidrug resistance (MDR) in Gram-negative pathogens. In RND transporters, such as 's main efflux pump AcrB, drug efflux occurs in the porter domain, while protons flow through the transmembrane domain: remote conformational coupling. At the border of a transmembrane helix (TM8) and subdomain PC2, there is a loop which makes a hoisting movement by a random-coil-to-α-helix change, and opens and closes a drug channel entrance. This loop is supposed to play a key role in the allosteric conformational coupling between the transmembrane and porter domain. Here we show the results of a series of flexibility loop-mutants of AcrB. We determined the crystal structure of a three amino acid truncated loop mutant, which is still a functional transporter, and show that the short α-helix between Cβ15 and the loop unwinds to a random coil in the access and binding monomers and in the extrusion monomer it makes a partially stretched coil-to-helix change. The loop has undergone compensatory conformational changes and still facilitates the opening and closing of the channel. In addition, more flexible mutated loops (proline mutated and significantly elongated) can still function during export. The flexibility in this region is however limited, as an even more truncated mutant (six amino acid deletion) becomes mostly inactive. We found that the hoisting-loop is a highly flexible hinge that enables the conformational energy transmission passively.
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http://dx.doi.org/10.3389/fmicb.2017.02095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661021PMC
October 2017

Structural Analysis and New Drug Development against Multidrug Efflux Pumps.

Yakugaku Zasshi 2017 ;137(4):377-382

Institute of Scientific and Industrial Research, Osaka University.

Multidrug efflux pumps are important in the multidrug resistance of Gram-negative pathogens. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. ABI-PP (a pryridopyrimidine derivative) is a MexB-specific inhibitor that does not inhibit MexY; MexB and MexY are principal pumps in Pseudomonas aeruginosa. We previously found that drugs were exported through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of pumps. ABI-PP binds tightly to a narrow pit located in the distal pocket and sterically hinders the functional rotation. Phenylalanine is located at the edge of this pit in MexB and contributes to the tight binding of the inhibitor molecule. On the other hand, the voluminous side chain of tryptophan located at the corresponding position in MexY prevents inhibitor binding. For the development of universal inhibitors of MexB and MexY, it is important to avoid the steric hindrance of tryptophan in MexY. Now we are developing clinically useful inhibitors on the basis of the structural information obtained. Started from the ABI-PP structure, we designed many compounds that can bind to the inhibitor-binding pits of MexB and MexY. Some of designed compounds were actually synthesized and their inhibitory activity determined. Finally, we obtained some lead compounds that showed complete prevention of the growth of strains expressing MexB and MexY with low concentrations of antibiotics.
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http://dx.doi.org/10.1248/yakushi.16-00235-2DOI Listing
May 2017

AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters.

J Bacteriol 2016 01 2;198(2):332-42. Epub 2015 Nov 2.

The Institute of Scientific and Industrial Research (ISIR), Osaka University, Ibaraki, Osaka, Japan CREST, Japan Science and Technology Agency, Saitama, Japan

Unlabelled: The AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB-deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function.

Importance: The structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB-deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for considering the structure and mechanism of AcrAB-TolC-mediated multidrug export.
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http://dx.doi.org/10.1128/JB.00587-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751787PMC
January 2016

Structural basis of RND-type multidrug exporters.

Front Microbiol 2015 20;6:327. Epub 2015 Apr 20.

Laboratory of Cell Membrane Structural Biology, Institute of Scientific and Industrial Research, Osaka University Ibaraki, Japan.

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.
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http://dx.doi.org/10.3389/fmicb.2015.00327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403515PMC
May 2015

Structural basis for the inhibition of bacterial multidrug exporters.

Nature 2013 Aug 30;500(7460):102-6. Epub 2013 Jun 30.

Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.

The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa. We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics. Drugs were shown to be exported by a functionally rotating mechanism through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π-π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa.
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http://dx.doi.org/10.1038/nature12300DOI Listing
August 2013

The crystal structure of multidrug-resistance regulator RamR with multiple drugs.

Nat Commun 2013 ;4:2078

Laboratory of Microbiology and Infectious Diseases, Division of Special Projects, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.

RamR is a transcriptional repressor of the gene-encoding RamA protein, which controls the expression of the multidrug efflux system genes acrAB-tolC. RamR is an important multidrug-resistance factor, however, its structure and the identity of the molecules to which it responds have been unknown. Here, we report the crystal structure of RamR in complex with multiple drugs, including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6G. All compounds are found to interact with Phe155 of RamR, and each compound is surrounded by different amino acid residues. Binding of these compounds to RamR reduces its DNA-binding affinity, which results in the increased expression of ramA. Our results reveal significant flexibility in the substrate-recognition region of RamR, which regulates the bacterial efflux participating in multidrug resistance.
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http://dx.doi.org/10.1038/ncomms3078DOI Listing
December 2013

Exposure to DEHP decreased four fatty acid levels in plasma of prepartum mice.

Toxicology 2013 Jul 22;309:52-60. Epub 2013 Apr 22.

Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

Maternal exposure to di(2-ethylhexyl) phthalate (DEHP) decreased the plasma triglyceride in prepartum mice. To identify the fatty acid (FA) species involved and to understand the underlying mechanisms, pregnant Sv/129 wild-type (mPPARα), peroxisome proliferator-activated receptor α-null (Pparα-null) and humanized PPARα (hPPARα) mice were treated with diets containing 0%, 0.01%, 0.05% or 0.1% DEHP. Dams were dissected on gestational day 18 together with fetuses, and on postnatal day 2 together with newborns. n-3/n-6 polyunsaturated, saturated, and monounsaturated FAs in maternal plasma and in liver of wild-type offspring, and representative enzymes for FA desaturation and elongation in maternal liver, were measured. The plasma levels of linoleic acid, α-linolenic acid, palmitic acid and oleic acid were higher in the pregnant control mPPARa mice than in Ppara-null and hPPARa mice. DEHP exposure significantly decreased the levels of these four FAs only in pregnant mPPARα mice. Plasma levels of many FAs were higher in pregnant mice than in postpartum ones in a genotype-independent manner, while it was lower in the livers of fetuses than pups. DEHP exposure slightly increased hepatic arachidonic acid, α-linolenic acid, palmitoleic acid and oleic acid in fetuses, but not in pups. However, DEHP exposure did not clearly influence FA desaturase 1 and 2 nor elongase 2 and 5 expressions in the liver of all maternal mice. Taken together, the levels of plasma four FAs with shorter carbon chains were higher in pregnant mPPARα mice than in other genotypes, and DEHP exposure decreased these specific FA concentrations only in mPPARα mice, similarly to triglyceride levels.
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http://dx.doi.org/10.1016/j.tox.2013.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413867PMC
July 2013

Structures of the multidrug exporter AcrB reveal a proximal multisite drug-binding pocket.

Nature 2011 Nov 27;480(7378):565-9. Epub 2011 Nov 27.

Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.

AcrB and its homologues are the principal multidrug transporters in Gram-negative bacteria and are important in antibiotic drug tolerance. AcrB is a homotrimer that acts as a tripartite complex with the outer membrane channel TolC and the membrane fusion protein AcrA. Minocycline and doxorubicin have been shown to bind to the phenylalanine cluster region of the binding monomer. Here we report the crystal structures of AcrB bound to the high-molecular-mass drugs rifampicin and erythromycin. These drugs bind to the access monomer, and the binding sites are located in the proximal multisite binding pocket, which is separated from the phenylalanine cluster region (distal pocket) by the Phe-617 loop. Our structures indicate that there are two discrete multisite binding pockets along the intramolecular channel. High-molecular-mass drugs first bind to the proximal pocket in the access state and are then forced into the distal pocket in the binding state by a peristaltic mechanism involving subdomain movements that include a shift of the Phe-617 loop. By contrast, low-molecular-mass drugs, such as minocycline and doxorubicin, travel through the proximal pocket without specific binding and immediately bind to the distal pocket. The presence of two discrete, high-volume multisite binding pockets contributes to the remarkably broad substrate recognition of AcrB.
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http://dx.doi.org/10.1038/nature10641DOI Listing
November 2011

Crystal structures of a multidrug transporter reveal a functionally rotating mechanism.

Nature 2006 Sep 16;443(7108):173-9. Epub 2006 Aug 16.

Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.

AcrB is a principal multidrug efflux transporter in Escherichia coli that cooperates with an outer-membrane channel, TolC, and a membrane-fusion protein, AcrA. Here we describe crystal structures of AcrB with and without substrates. The AcrB-drug complex consists of three protomers, each of which has a different conformation corresponding to one of the three functional states of the transport cycle. Bound substrate was found in the periplasmic domain of one of the three protomers. The voluminous binding pocket is aromatic and allows multi-site binding. The structures indicate that drugs are exported by a three-step functionally rotating mechanism in which substrates undergo ordered binding change.
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http://dx.doi.org/10.1038/nature05076DOI Listing
September 2006

Crystal structure of bacterial multidrug efflux transporter AcrB.

Nature 2002 Oct;419(6907):587-93

Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.

AcrB is a major multidrug exporter in Escherichia coli. It cooperates with a membrane fusion protein, AcrA, and an outer membrane channel, TolC. We have determined the crystal structure of AcrB at 3.5 A resolution. Three AcrB protomers are organized as a homotrimer in the shape of a jellyfish. Each protomer is composed of a transmembrane region 50 A thick and a 70 A protruding headpiece. The top of the headpiece opens like a funnel, where TolC might directly dock into AcrB. A pore formed by three alpha-helices connects the funnel with a central cavity located at the bottom of the headpiece. The cavity has three vestibules at the side of the headpiece which lead into the periplasm. In the transmembrane region, each protomer has twelve transmembrane alpha-helices. The structure implies that substrates translocated from the cell interior through the transmembrane region and from the periplasm through the vestibules are collected in the central cavity and then actively transported through the pore into the TolC tunnel.
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http://dx.doi.org/10.1038/nature01050DOI Listing
October 2002
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