Potassium Ions Decrease Mitochondrial Matrix pH: Implications for ATP Production and Reactive Oxygen Species Generation.

Potassium (K+) is the most abundant cation in the cytosol and is maintained at high concentrations within the mitochondrial matrix through potassium channels. However, many effects of K+ at such high concentrations on mitochondria and the underlying mechanisms remain unclear. This study aims to elucidate these effects and mechanisms by employing fluorescence imaging techniques to distinguish and precisely measure signals inside and outside the mitochondria. We stained the mitochondrial matrix with fluorescent dyes sensitive to K+, pH, reactive oxygen species (ROS), and membrane potential in plasma membrane-permeabilized C6 cells and isolated mitochondria from C6 cells. Fluorescence microscopy facilitated the accurate measurement of fluorescence intensity inside and outside the matrix. Increasing extramitochondrial K+ concentration from 2 mM to 127 mM led to a reduction in matrix pH and a decrease in the generation of highly reactive ROS. In addition, elevated K+ levels electrically polarized the inner membrane of the mitochondria and promoted efficient ATP synthesis via FoF1-ATPase. Introducing protons (H+) into the matrix through phosphate addition led to further mitochondrial polarization, and this effect was more pronounced in the presence of K+. K+ at high concentrations, reaching sub-hundred millimolar levels, increased H+ concentration within the matrix, suppressing ROS generation and boosting ATP synthesis. Although this study does not elucidate the role of specific types of potassium channels in mitochondria, it does suggest that mitochondrial K+ plays a beneficial role in maintaining cellular health.

Intramolecular Palladium Catalyst Transfer on Benzoheterodiazoles as Acceptor Monomers and Discovery of Catalyst Transfer Inhibitors.

Intramolecular catalyst transfer on benzoheterodiazoles was investigated in Suzuki-Miyaura coupling reactions and polymerization reactions with t Bu3 PPd precatalyst. In the coupling reactions of dibromobenzotriazole, dibromobenzoxazole, and dibromobenzothiadiazole with pinacol phenylboronate, the product ratios of monosubstituted product to disubstituted product were 0/100, 27/73, and 89/11, respectively, indicating that the Pd catalyst undergoes intramolecular catalyst transfer on dibromobenzotriazole, whereas intermolecular transfer occurs in part in the case of dibromobenzoxazole and is predominant for dibromobenzothiadiazole. The polycondensation of 1.3 equivalents of dibromobenzotriazole with 1.0 equivalent of para- and meta-phenylenediboronates afforded high-molecular-weight polymer and cyclic polymer, respectively. In the case of dibromobenzoxazole, however, para- and meta-phenylenediboronates afforded moderate-molecular-weight polymer with bromine at both ends and cyclic polymer, respectively. In the case of dibromobenzothiadiazole, they afforded low-molecular-weight polymers with bromine at both ends. Addition of benzothiadiazole derivatives interfered with catalyst transfer in the coupling reactions.

Effects of proton pumping on the structural rigidity of cristae in mitochondria.

Mitochondria change their morphology and inner membrane structure depending on their activity. Since mitochondrial activity also depends on their structure, it is important to elucidate the interrelationship between the activity and structure of mitochondria. However, the mechanism by which mitochondrial activity affects the structure of cristae, the folded structure of the inner membrane, is not well understood. In this study, the effect of the mitochondrial activity on the cristae structure was investigated by examining the structural rigidity of cristae. Taking advantage of the fact that unfolding of cristae induces mitochondrial swelling, we investigated the relationship between mitochondrial activity and the susceptibility to swelling. The swelling of individual isolated mitochondria exposed to a hypotonic solution was observed with an optical microscope. The presence of respiratory substrates (malate and glutamate) increased the percentage of mitochondria that underwent swelling, and the further addition of rotenone or KCN (inhibitors of proton pumps) reversed the increase. In the absence of respiratory substrates, acidification of the buffer surrounding the mitochondria also increased the percentage of swollen mitochondria. These observations suggest that acidification of the outer surface of inner membranes, especially intracristal space, by proton translocation from the matrix to the intracristal space, decreases the structural rigidity of the cristae. This interpretation was verified by the observation that ADP or CCCP, which induces proton re-entry to the matrix, suppressed the mitochondrial swelling in the presence of respiratory substrates. The addition of CCCP to the cells induced a morphological change in mitochondria from an initial elongated structure to a largely curved structure at pH 7.4, but there were no morphological changes when the pH of the cytosol dropped to 6.2. These results suggest that a low pH in the intracristal space may be helpful in maintaining the elongated structure of mitochondria. The present study shows that proton pumping by the electron transfer chain is the mechanism underlying mitochondrial morphology and the flexibility of cristae structure.

Direct Analysis of Mitochondrial Damage Caused by Misfolded/Destabilized Proteins.

Protein quality control is essential for cellular homeostasis. In this study, we examined the effect of improperly folded proteins that do not form amyloid fibrils on mitochondria, which play important roles in ATP production and cell death. First, we prepared domain 3 of the dengue envelope protein in wild type and four mutants with widely different biophysical properties in misfolded/aggregated or destabilized states. The effects of the different proteins were detected using fluorescence microscopy and Western blotting, which revealed that three of the five proteins disrupted both inner and outer membrane integrity, while the other two proteins, including the wild type, did not. Next, we examined the common characteristics of the proteins that displayed toxicity against mitochondria by measuring oligomer size, molten globule-like properties, and thermal stability. The common feature of all three toxic proteins was thermal instability. Therefore, our data strongly suggest that thermally unstable proteins generated in the cytosol can cause cellular damage by coming into direct contact with mitochondria. More importantly, we revealed that this damage is not amyloid-specific.

Intramolecular Catalyst Transfer on a Variety of Functional Groups between Benzene Rings in a Suzuki-Miyaura Coupling Reaction.

Suzuki-Miyaura coupling reaction of BrC6 H4 -X-C6 H4 Br 1 (X=CH2 , CO, N-Bu, O, S, SO, and SO2 ) with arylboronic acid 2 was investigated in the presence of tBu3 PPd precatalyst and CsF/[18]crown-6 as a base to establish whether or not the Pd catalyst can undergo catalyst transfer on these functional groups. In the reaction of 1 (X=CH2 , CO, N-Bu, O, and SO2 ) with 2, aryl-disubstituted product 3 (Ar-C6 H4 -X-C6 H4 -Ar) was exclusively obtained, indicating that the Pd catalyst undergoes catalyst transfer on these functional groups. On the other hand, the reaction of 1 e (X=S) and 1 f (X=SO) with 2 afforded only aryl-monosubstituted product 4 (Ar-C6 H4 -X-C6 H4 -Br) and a mixture of 3 and 4, respectively, indicating that S and SO interfere with intramolecular catalyst transfer. Furthermore, we found that Suzuki-Miyaura polycondensation of 1 (X=CH2 , CO, N-Bu, O, and SO2 ) and phenylenediboronic acid 5 in the presence of tBu3 PPd precatalyst afforded high-molecular-weight polymer even when excess 1 was used. The polymers obtained from 1 (X=CH2 , N-Bu, and O) and 5 turned out to be cyclic.

Time-lapse imaging of Ca2+-induced swelling and permeability transition: Single mitochondrion study.

Mitochondrial functions are closely related to the membrane structure. Mitochondrial swelling, which is accompanied with dissipation of the crista structure and rupture of the outer membrane, have been observed as mitochondrial damage when mitochondria are under Ca2+-overload or oxidative stress. Although these phenomena have been well studied, the detailed behaviors of individual mitochondria upon swelling remain unknown. The aim of this study was to investigate the detailed behavior of mitochondrial volume upon addition of Ca2+. Here, we report for the first time, time-lapse measurements of single mitochondrion swelling and permeability transition induced by Ca2+ by optical microscopy. We added 220 µM Ca2+ to mitochondria, and found that 1) the swelling rate depended on the mitochondrion, 2) a small number of mitochondria showed step-like swelling, 3) cyclosporin A decreased the percentage of mitochondria that underwent swelling induced by Ca2+, but did not affect the amplitude of swelling, 4) permeability transition is necessary but not sufficient for Ca2+-induced swelling, 5) permeability transition is more sensitive to Ca2+ than swelling, 6) Ca2+ stimulated mitochondrial swelling after permeability transition. These results suggest that single mitochondrion measurement of swelling is a powerful tool for examining the regulation of mitochondrial structure.

Exclusive Synthesis of Poly(3-hexylthiophene) with an Ethynyl Group at Only One End for Effective Block Copolymerization.

Well-controlled synthesis of ethynyl-functionalized poly(3-hexylthiophene) (P3HT) is crucial for preparation of block copolymers containing the P3HT segment by means of click coupling reaction. A well-known chain end modification method, in which Kumada-Tamao catalyst-transfer polymerization is quenched with ethynylmagnesium chloride, under various conditions is re-examined, but in all cases not only P3HT with an ethynyl group at one end but also P3HT di-ethynylated at both ends is obtained. Accordingly, Sonogashira coupling reaction of P3HT having H/Br ends with trimethylsilylacetylene is tried, followed by removal of the trimethylsilyl group, and it is found that this protocol affords exclusively P3HT with an ethynyl group at one end. This post end-modification method is applied to the synthesis of an amphiphilic diblock copolymer of P3HT and poly(2-ethyl-2-oxazoline) (PEtOx) by means of click reaction between ethynylated P3HT and PEtOx with an azide group at one end, and the product is confirmed to be free from contamination with triblock copolymer. Micellization of this block copolymer is confirmed in tetrahydrofuran (THF)/water and THF/methanol mixtures.

Long-term inhibition of cyclophilin D results in intracellular translocation of calcein AM from mitochondria to lysosomes.

Cyclophilin D is a peptidyl-prolyl cis-trans isomerase localized in the mitochondrial matrix. Although its effects on mitochondrial characteristics have been well studied, its relation to the uptake of molecules by mitochondria remains unknown. Here, we demonstrated the effects of cyclophilin D on the intracellular translocation of calcein AM. Following addition of calcein AM to control cells or cells overexpressing wild-type cyclophilin D, calcein fluorescence was observed in mitochondria. However, long-term inhibition of cyclophilin D in these cells altered the localization of calcein fluorescence from mitochondria to lysosomes without changing mitochondrial esterase activity. In addition, depletion of glucose from the medium recovered calcein localization from lysosomes to mitochondria. This is the first demonstration of the effects of cyclophilin D on the intracellular translocation of molecules other than proteins and suggests that cyclophilin D may modify mitochondrial features by inducing the translocation of molecules to the mitochondria through the mechanism associated with cellular energy metabolism.

Mechanistic Investigation of Catalyst-Transfer Suzuki-Miyaura Condensation Polymerization of Thiophene-Pyridine Biaryl Monomers with the Aid of Model Reactions.

We have investigated the requirements for efficient Pd-catalyzed Suzuki-Miyaura catalyst-transfer condensation polymerization (Pd-CTCP) reactions of 2-alkoxypropyl-6-(5-bromothiophen-2-yl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (12) as a donor-acceptor (D-A) biaryl monomer. As model reactions, we first carried out the Suzuki-Miyaura coupling reaction of X-Py-Th-X' (Th=thiophene, Py=pyridine, X, X'=Br or I) 1 with phenylboronic acid ester 2 by using tBu3 PPd0 as the catalyst. Monosubstitution with a phenyl group at Th-I mainly took place in the reaction of Br-Py-Th-I (1 b) with 2, whereas disubstitution selectively occurred in the reaction of I-Py-Th-Br (1 c) with 2, indicating that the Pd catalyst is intramolecularly transferred from acceptor Py to donor Th. Therefore, we synthesized monomer 12 by introduction of a boronate moiety and bromine into Py and Th, respectively. However, examination of the relationship between monomer conversion and the Mn of the obtained polymer, as well as the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra, indicated that Suzuki-Miyaura coupling polymerization of 12 with (o-tolyl)tBu3 PPdBr initiator 13 proceeded in a step-growth polymerization manner through intermolecular transfer of the Pd catalyst. To understand the discrepancy between the model reactions and polymerization reaction, Suzuki-Miyaura coupling reactions of 1 c with thiopheneboronic acid ester instead of 2 were carried out. This resulted in a decrease of the disubstitution product. Therefore, step-growth polymerization appears to be due to intermolecular transfer of the Pd catalyst from Th after reductive elimination of the Th-Pd-Py complex formed by transmetalation of polymer Th-Br with (Pin)B-Py-Th-Br monomer 12 (Pin=pinacol). Catalysts with similar stabilization energies of metal-arene η2 -coordination for D and A monomers may be needed for CTCP reactions of biaryl D-A monomers.

Alkannin inhibits CCL3 and CCL5 production in human periodontal ligament cells.

Alkannin, which is found in Alkanna tinctoria, a member of the borage family, is used as a food coloring. Alkannin has recently been reported to have certain biological functions, such as anti-microbial and anti-oxidant effects. It is known that CC chemokine receptor (CCR) 5-positive leukocytes contribute to alveolar bone resorption in periodontal lesions. The aim of this study was to examine whether alkannin inhibits the production of CC chemokine ligand (CCL) 3 and CCL5, which are CCR5 ligands, in human periodontal ligament cells (HPDLC). Interleukin (IL)-1ß induced CCL3 and CCL5 production in HPDLC. Alkannin inhibited IL-1ß-mediated CCL3 and CCL5 production in HPDLC in a dose-dependent manner. Moreover, we revealed that alkannin suppressed inhibitor of kappa B-α degradation in IL-1ß-stimulated HPDLC. In addition, a nuclear factor (NF)-κB inhibitor significantly inhibited CCL3 and CCL5 production in IL-1ß-stimulated HPDLC. These results demonstrate that alkannin inhibits CCR5 ligand production in IL-1ß-stimulated HPDLC by attenuating the NF-κB signaling pathway.

Alternating Intramolecular and Intermolecular Catalyst-Transfer Suzuki-Miyaura Condensation Polymerization: Synthesis of Boronate-Terminated π-Conjugated Polymers Using Excess Dibromo Monomers.

The Suzuki-Miyaura coupling polymerization of dibromoarene 1 and arylenediboronic acid (ester) 2 with a Pd catalyst having a high propensity for intramolecular catalyst transfer is reported. The polymerization of excess 1 with 2 affords high-molecular-weight π-conjugated polymer having boronic acid (ester) moieties at both ends, contrary to Flory's principle. This unstoichiometric polycondensation behavior is accounted for by intramolecular transfer of the Pd catalyst on 1. In the polymerization of 1 and 2 having different aryl residues, high-molecular-weight polymer is obtained when the stronger donor aromatic is used as the dibromo monomer and the weaker donor or acceptor aromatic is used as diboronic acid (ester) monomer. The pinacol boronate moieties at both ends of the obtained poly(p-phenylene) (PPP) can be converted to benzoic acid ester, hydroxyl group, and bromine. Furthermore, the reaction of the pinacol boronate-terminated PPP with poly(3-hexylthiophene) (P3HT) having bromine at one end yields a triblock copolymer of P3HT-b-PPP-b-P3HT.

Biochemical and biophysical characterization of an unexpected bacteriolytic activity of VanX, a member of the vancomycin-resistance vanA gene cluster.

VanX is a d-alanyl-d-alanine (d-Ala-d-Ala) dipeptidase encoded in the vancomycin-resistance vanA gene cluster. Here we report that strong bacteriolysis occurred when isolated VanX was expressed in Escherichia coli at temperatures lower than 30 °C, which was unexpected because the vanA operon confers vancomycin resistance by protecting the cell wall. Therefore, we monitored cell lysis by measuring sample turbidity with absorbance at 590 nm and VanX expression using SDS-PAGE. No cell lysis was observed when VanX was expressed, even in large quantities, in the cell inclusion bodies at 37 °C, suggesting that a natively folded VanX is required for lysis. In addition, VanX mutants with suppressed dipeptidase activity did not lyse E. coli cells, confirming that bacteriolysis originated from the dipeptidase activity of VanX. We also observed shape changes in E. coli cells undergoing VanX-mediated lysis with optical microscopy and classified these changes into three classes: bursting, deformation, and leaking fluid. Optical microscopic image analysis fully corroborated our interpretation of the turbidity changes in the samples. From a practical perspective, the finding that VanX expressed in isolation induces cell lysis suggests that inhibitors of VanA and VanH that act downstream from VanX could provide a new class of therapeutic chemicals against bacteria expressing the vancomycin-resistance gene cluster.

Structural requirements for palladium catalyst transfer on a carbon-carbon double bond.

Intramolecular transfer of (t)Bu3PPd(0) on a carbon-carbon double bond (C═C) was investigated by using Suzuki-Miyaura coupling reaction of dibromostilbenes with aryl boronic acid or boronic acid esters in the presence of various additives containing C═C as a model. Substituent groups at the ortho position of C═C of stilbenes are critical for selective intramolecular catalyst transfer and may serve to suppress formation of the bimolecular C═C-Pd-C═C complex that leads to intermolecular transfer of (t)Bu3PPd(0).

Epigenetically coordinated GATA2 binding is necessary for endothelium-specific endomucin expression.

GATA2 is well recognized as a key transcription factor and regulator of cell-type specificity and differentiation. Here, we carried out comparative chromatin immunoprecipitation with comprehensive sequencing (ChIP-seq) to determine genome-wide occupancy of GATA2 in endothelial cells and erythroids, and compared the occupancy to the respective gene expression profile in each cell type. Although GATA2 was commonly expressed in both cell types, different GATA2 bindings and distinct cell-specific gene expressions were observed. By using the ChIP-seq with epigenetic histone modifications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial-specific GATA2-mediated endomucin gene expression, that was regulated by the endothelial-specific chromatin loop with a GATA2-associated distal enhancer and core promoter. Knockdown of endomucin markedly attenuated endothelial cell growth, migration and tube formation. Moreover, abrogation of GATA2 in endothelium demonstrated not only a reduction of endothelial-specific markers, but also induction of mesenchymal transition promoting gene expression. Our findings provide new insights into the correlation of endothelial-expressed GATA2 binding, epigenetic modification, and the determination of endothelial cell specificity.

Isolation of mitochondria by gentle cell membrane disruption, and their subsequent characterization.

Mitochondria play a key role in several physiological processes as in integrating signals in the cell. However, understanding of the mechanism by which mitochondria sense and respond to signals has been limited due to the lack of an appropriate model system. In this study, we developed a method to isolate and characterize mitochondria without cell homogenization. By gently pipetting cells treated with streptolysin-O, a pore-forming membrane protein, we disrupted the cell membrane and were able to isolate both elongated and spherical mitochondria. Fluorescence imaging combined with super resolution microscopy showed that both the outer and inner membranes of the elongated mitochondria isolated using the newly developed method were intact. In addition, a FRET-based ATP sensor expressed in the mitochondrial matrix demonstrated that ATP generation by FoF1-ATPase in the isolated elongated mitochondria was as high as that in intracellular mitochondria. On the other hand, some of the spherical mitochondria isolated with this method had the outer membrane that no longer encapsulated the inner membrane. In addition, all mitochondria isolated using conventional procedures involving homogenization were spherical, many of them had damaged membranes, and low levels of ATP generation. Our results suggest that elongated mitochondria isolated from cells through gentle cell membrane disruption using a pore-forming protein tend to be more similar to intracellular mitochondria, having an intact membrane system and higher activity than spherical mitochondria.

Analysis of mitochondrial mechanical dynamics using a confocal fluorescence microscope with a bent optical fibre.

The cells in the cardiovascular system are constantly subjected to mechanical forces created by blood flow and the beating heart. The effect of forces on cells has been extensively investigated, but their effect on cellular organelles such as mitochondria remains unclear. We examined the impact of nano-Newton forces on mitochondria using a bent optical fibre (BOF) with a flat-ended tip (diameter exceeding 2 µm) and a confocal fluorescence microscope. By indenting a single mitochondrion with the BOF tip, we found that the mitochondrial elastic modulus was proportional to the (-1/2) power of the mitochondrial radius in the 9.6-115 kPa range. We stained the mitochondria with a potential-metric dye (TMRE) and measured the changes in TMRE fluorescence intensity. We confirmed that more active mitochondria exhibit a higher frequency of repetitive transient depolarization. The same trend was observed at forces lower than 50 nN. We further showed that the depolarization frequency of mitochondria decreases under an extremely large force (nearly 100 nN). We conclude that mitochondrial function is affected by physical environmental factors, such as external forces at the nano-Newton level.

Influence of the boron moiety and water on suzuki-miyaura catalyst-transfer condensation polymerization.

Although water promotes Suzuki-Miyaura coupling reaction, it also induces side reactions such as deboronation and dehalogenation. Therefore, Suzuki-Miyaura polymerization of triolborate halothiophene monomer 1 with (t) Bu3 PPd(o-tolyl)Br (2) in dry tetrahydrofuran (THF) is investigated. However, the resultant poly(3-hexylthiophene) (P3HT) shows a broad molecular weight distribution and uncontrolled polymer ends. Model reactions of a number of boron reagents 3 with 2,5-dibromothiophene (4) in the presence or absence of water indicate that intramolecular transfer of the catalyst is hardly affected by the boron moiety of 3, whereas it is hindered in the absence of water. Indeed, polymerization of 1 with 2 in H2 O/THF affords P3HT with a narrower molecular weight distribution and controlled tolyl/H ends, as compared to the reaction in dry THF.

Fluorescent analog of OSW-1 and its cellular localization.

OSW-1 is a steroidal saponin, which has emerged as an attractive anticancer agent with highly cancer cell selective activity. A fluorescent analog was prepared from the natural product to analyze its cellular uptake and localization. We found that the fluorescent analog is rapidly internalized into cells and is primarily distributed in endoplasmic reticulum and Golgi apparatus.

Active RNA polymerases: mobile or immobile molecular machines?

It is widely assumed that active RNA polymerases track along their templates to produce a transcript. We test this using chromosome conformation capture and human genes switched on rapidly and synchronously by tumour necrosis factor alpha (TNFalpha); one is 221 kbp SAMD4A, which a polymerase takes more than 1 h to transcribe. Ten minutes after stimulation, the SAMD4A promoter comes together with other TNFalpha-responsive promoters. Subsequently, these contacts are lost as new downstream ones appear; contacts are invariably between sequences being transcribed. Super-resolution microscopy confirms that nascent transcripts (detected by RNA fluorescence in situ hybridization) co-localize at relevant times. Results are consistent with an alternative view of transcription: polymerases fixed in factories reel in their respective templates, so different parts of the templates transiently lie together.