Prevention of aberrant protein aggregation by anchoring the molecular chaperone αB-crystallin to the endoplasmic reticulum.

The chaperone αB-crystallin (αBC) is a member of the small heat shock protein family and its point or truncated mutants cause the muscular disorder α-crystallinopathy. The illness is histologically characterized by accumulation of protein aggregates in muscle cells. Expression of the myopathy-causing R120G mutant of αBC, harboring an arginine-to-glycine mutation at position 120, results in aggregate formation. We demonstrated that tethering αBC to the endoplasmic reticulum (ER) membrane represses the protein aggregation mediated by the R120G mutant. ER-anchored αBC decreased the amount of the R120G mutant through autophagic proteolysis. In contrast, knockdown of ATG5, an E3 ligase essential for autophagy, in ER-anchored αBC-transfected cells restored the quantity of the R120G mutant. In this context, aggregate formation was still suppressed, indicating that ER-anchored αBC profoundly constrains aggregation competency of the R120G mutant separately from downregulating the abundance of the mutant. We have proposed that protein aggregation is prevented by manipulation of the ER microenvironment with αBC, and have shed light on a novel aspect of the ER as a therapeutic target.

Synthesis and evaluation of water-soluble resveratrol and piceatannol via BGLation.

Synthesis of four water-soluble resveratrol and piceatannol derivatives bearing symmetrically branched glyceryl trimer (BGL003) with a non-biocleavable linkage, and their biological evaluation as a mitochondrial fusion-inducing agent with cellular fat-reducing effect from cells, is described. The effect of Piceatannol-BGL003 conjugate was as high as that of original stilbenoids.

Triple combinations of lower and longer alkyl gallates and oxacillin improve antibiotic synergy against methicillin-resistant Staphylococcus aureus.

Using liposome systems, we found that gallates with short alkyl chains were located in the external medium and those with longer alkyl chains were located in the surface region of lipid bilayer. Combinations of these gallates remarkably reduced oxacillin MICs against methicillin-resistant Staphylococcus aureus to below the antibiotic breakpoint (< or = 2 microg/ml).

Regulation of mitochondrial morphology and cell survival by Mitogenin I and mitochondrial single-stranded DNA binding protein.

We found that a mouse homolog of human DNA polymerase delta interacting protein 38, referred to as Mitogenin I in this paper, and mitochondrial single-stranded DNA-binding protein (mtSSB), identified as upregulated genes in the heart of mice with juvenile visceral steatosis, play a role in the regulation of mitochondrial morphology. We demonstrated that overexpression of Mitogenin I or mtSSB increased elongated or fragmented mitochondria in mouse C2C12 myoblast cells, respectively. On the other hand, the silencing of Mitogenin I or mtSSB by RNA interference led to an increase in fragmented or elongated mitochondria in the cells, respectively, suggesting that Mitogenin I and mtSSB are involved in the processes of mitochondrial fusion and fission, respectively. In addition, we showed that the silencing of Mitogenin I resulted in an increase in the number of trypan blue-positive cells and the silencing of mtSSB resulted in an enhancement of the sensitivity of the cells to apoptotic stimulation by etoposide. The present results demonstrated that these proteins play a role in cell survival.

Cell-surface H+-ATP synthase as a potential molecular target for anti-obesity drugs.

Here we show that the cell-surface expression of the alpha subunit of H(+)-ATP synthase is markedly increased during adipocyte differentiation. Treatment of differentiated adipocytes with small molecule inhibitors of H(+)-ATP synthase or antibodies against alpha and beta subunits of H(+)-ATP synthase leads to a decrease in cytosolic lipid droplet accumulation. Apolipoprotein A-I, which has been shown to bind to the ectopic beta-chain of H(+)-ATP synthase and inhibit the activity of cell-surface H(+)-ATP synthase, also was found to inhibit cytosolic lipid accumulation. These results suggest that the cell-surface H(+)-ATP synthase has a previously unsuspected role in lipid metabolism in adipocytes.

Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells.

Extracellular ATP synthesis on human umbilical vein endothelial cells (HUVECs) was examined, and it was found that HUVECs possess high ATP synthesis activity on the cell surface. Extracellular ATP generation was detected within 5 s after addition of ADP and inorganic phosphate and reached a maximal level at 15 s. This type of ATP synthesis was almost completely inhibited by mitochondrial H(+)-ATP synthase inhibitors (e.g., efrapeptins, resveratrol, and piceatannol), which target the F(1) catalytic domain. Oligomycin and carbonyl cyanide m-chlorophenylhydrazone, but not potassium cyanide, also inhibited extracellular ATP synthesis on HUVECs, suggesting that cell surface ATP synthase employs the transmembrane electrochemical potential difference of protons to synthesize ATP as well as mitochondrial H(+)-ATP synthase. The F(1)-targeting H(+)-ATP synthase inhibitors markedly inhibited the proliferation of HUVECs, but intracellular ATP levels in HUVECs treated with these inhibitors were only slightly affected, as shown by comparison with the control cells. Interestingly, piceatannol inhibited only partially the activation of Syk (a nonreceptor tyrosine kinase), which has been shown to play a role in a number of endothelial cell functions, including cell growth and migration. These findings suggest that H(+)-ATP synthase-like molecules on the surface of HUVECs play an important role not only in extracellular ATP synthesis but also in the proliferation of HUVECs. The present results demonstrate that the use of small molecular H(+)-ATP synthase inhibitors targeting the F(1) catalytic domain may lead to significant advances in potential antiangiogenic cancer therapies.

Contribution of extracellular ATP on the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation.

Cell-surface F1F0-ATP synthase was involved in the cell signaling mediating various biological functions. Recently, we found that cell-surface F1F0-ATP synthase plays a role on intracellular triacylglycerol accumulation in adipocytes, and yet, the underlying mechanisms remained largely unknown. In this study, we investigated the role of extracellular ATP on the intracellular triacylglycerol accumulation. We demonstrated that significant amounts of ATP were produced extracellularly by cultured 3T3-L1 adipocytes and that the antibodies against α and ß subunits of F1F0-ATP synthase inhibited the extracellular ATP production. Piceatannol, a F1F0-ATP synthase inhibitor, and apyrase, an enzyme which degrades extracellular ATP, suppressed triacylglycerol accumulation. The selective P2Y1 receptor antagonist MRS2500 significantly inhibited triacylglycerol accumulation, whereas the selective P2X receptor antagonist NF279 has less effect. The present results indicate that cell-surface F1F0-ATP synthase on adipocytes is functional in extracellular ATP production and that the extracellular ATP produced contributes, at least in part, to the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation in adipocytes through P2Y1 receptor.

Variation in synergistic activity by flavone and its related compounds on the increased susceptibility of various strains of methicillin-resistant Staphylococcus aureus to beta-lactam antibiotics.

We found that some flavonoids had a weak antibacterial effect on methicillin-resistant Staphylococcus aureus (MRSA), but that at sub-MIC concentrations they greatly increased the susceptibility of these strains to beta-lactam antibiotics. Flavone showed diverse synergistic effects on the susceptibility of MRSA to beta-lactam antibiotics. The variation of the synergistic effects of the flavones to increase the susceptibility of strains of MRSA to beta-lactam antibiotics coincided with their varying effects on growth-inhibition of these strains. Based on these findings, we have proposed a model for the mechanisms of high resistance of MRSA to beta-lactams and the massive reduction in the beta-lactams MIC caused by flavones.

Involvement of reactive oxygen species in osteoblastic differentiation of MC3T3-E1 cells accompanied by mitochondrial morphological dynamics.

Bone remodeling is regulated by local factors that regulate bone-forming osteoblasts and bone-resorbing osteoclasts, in addition to hormonal activity. Recent studies have shown that reactive oxygen species (ROS) act as an intracellular signal mediator for osteoclast differentiation. However the role of ROS on osteoblast differentiation is poorly understood. Here, we investigated the impact of ROS on osteoblastic differentiation of MC3T3-E1 cells. Osteogenic induction resulted in notable enhancement of mineralization and expression of osteogenic marker gene alkaline phosphatase, which were accompanied by an increase in ROS production. Additionally, we found that mitochondrial morphology dynamically changed from tubular reticulum to fragmented structures during the differentiation, suggesting that mitochondrial morphological transition is a novel osteoblast differentiation index. The antioxidant N-acetyl cysteine prevented not only ROS production but also mineralization and mitochondrial fragmentation. It is therefore suggested that the ROS-dependent signaling pathways play a role in osteoblast differentiation accompanied by mitochondrial morphological transition.

Simvastatin represses translocation of Pseudomonas aeruginosa across Madin-Darby canine kidney cell monolayers.

Pseudomonas aeruginosa causes both invasive (bacteremic) and chronic noninvasive infections. An increase in intestinal epithelial permeability is a characteristic of severe sepsis. Alterations in the normal barrier function of the gut mucosa may result in the translocation of microbial cells and products. On the otherhand, it has been demonstrated that statin use is associated with a lower risk of mortality from bloodstream infections. Therefore, we investigated the ability of P. aeruginosa PAO1 to translocate across the Madin-Darby canine kidney (MDCK) cell monolayers in the presence and absence of simvastatin. The bacteria readily translocated across MDCK cell monolayers after 3 h of infection irrespective of the presence or absence of the drug in the medium. However, the bacteria were less able to penetrate the MDCK monolayers in the presence of simvastatin than in its absence. A gentamicin survival assay demonstrated that simvastatin did not affect the bacteria's invasive behavior in the MDCK cells.

Pigment epithelium-derived factor binds to cell-surface F(1)-ATP synthase.

Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a approximately 60 kDa PEDF-binding protein in bovine retina with Bos taurus F(1)-ATP synthase beta-subunit, and that F(1)F(o)-ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding of PEDF to F(1). Size-exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F(1). Real-time binding as determined by surface plasmon resonance demonstrated that yeast F(1) interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F(1) and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF-binding and angiostatin-binding sites on F(1). Surfaces of endothelial cells exhibited affinity for PEDF-binding proteins of approximately 60 kDa. Antibodies to F(1)beta-subunit specifically captured PEDF-binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell-surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell-surface F(1), these results show that PEDF is a ligand for endothelial cell-surface F(1)F(o)-ATP synthase. They suggest that PEDF-mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity.

Possible role of mitochondrial remodelling on cellular triacylglycerol accumulation.

Mitochondrial fusion and fission processes play a role in a variety of cell functions, including energy metabolism, cell differentiation and programmed cell death. Still, it is not clear how these processes contribute to the cell functions. Here, we investigated the role of mitochondrial remodelling on lipid metabolism in adipocytes. In 3T3-L1 pre-adipocytes, the morphology of mitochondria is organized as a continuous reticulum. Upon differentiation of adipocytes manifested by cellular triacylglycerol (TG) accumulation, mitochondrial morphology altered from filamentous to fragmented and/or punctate structures. When the mitochondrial fusion was induced in adipocytes by silencing of mitochondrial fission proteins including Fis1 and Drp1, the cellular TG content was decreased. In contrast, the silencing of mitochondrial fusion proteins including mitofusin 2 and Opa1 increased the cellular TG content followed by fragmentation of mitochondria. It also appears that polyphenolic phytochemicals, negative regulators of lipid accumulation, have mitochondrial fusion activity and that there is a good correlation between mitochondrial fusion activity and the cellular TG accumulation-reducing activity of the phytochemicals. These results suggest that cellular TG accumulation is regulated, at least in part, via mitochondrial fusion and fission processes.

No relationship exists between PBP 2a amounts expressed in different MRSA strains obtained clinically and their beta-lactam MIC values.

After establishing a linear relationship between the amount of penicillin-binding protein (PBP) 2a and membrane proteins of methicillin-resistant Staphylococcus aureus (MRSA) COL by dot-blot analysis using an antibody against PBP 2a, we determined the PBP 2a quantities in membrane fractions prepared from 14 different MRSA cells. Methicillin-sensitive S. aureus ATCC 6538P was used as a quality control strain. The amounts of PBP 2a diverged among the strains, and no relationship to beta-lactam MIC values were observed in the corresponding strains.

Dynamics of mitochondria during the cell cycle.

Mitochondria are highly dynamic organelles in eukaryotic cells. Although the role of mitochondria in metabolism, ATP production and apoptosis is more widely recognized, alterations in mitochondrial morphology and abundance are also important for cellular functions. Here we investigated mitochondrial dynamics in synchronized HeLa cells in which the major stages of the cell cycle of the observed cells were resolved by staining phosphorylate histones H1 and H3, and showed that mitochondria exist as filamentous network structures throughout the cell cycle progression, changing their morphology, distribution, and abundance. The current results suggest that mitochondrial condensation occurred at prophase is required for the proper progression of mitochondrial division.

Alkyl gallates, intensifiers of beta-lactam susceptibility in methicillin-resistant Staphylococcus aureus.

We found that ethyl gallate purified from a dried pod of tara (Caesalpinia spinosa) intensified beta-lactam susceptibility in methicillin-resistant and methicillin-sensitive strains of Staphylococcus aureus (MRSA and MSSA strains, respectively). This compound and several known alkyl gallates were tested with MRSA and MSSA strains to gain new insights into their structural functions in relation to antimicrobial and beta-lactam susceptibility-intensifying activities. The maximum activity of alkyl gallates against MRSA and MSSA strains occurred at 1-nonyl and 1-decyl gallate, with an MIC at which 90% of the isolates tested were inhibited of 15.6 microg/ml. At concentrations lower than the MIC, alkyl gallates synergistically elevated the susceptibility of MRSA and MSSA strains to beta-lactam antibiotics. Such a synergistic activity of the alkyl gallates appears to be specific for beta-lactam antibiotics, because no significant changes were observed in the MICs of other classes of antibiotics examined in this study. The length of the alkyl chain was also associated with the modifying activity of the alkyl gallates, and the optimum length was C5 to C6. The present work clearly demonstrates that the length of the alkyl chain has a key role in the elevation of susceptibility to beta-lactam antibiotics.

6,7-dihydroxyflavone dramatically intensifies the susceptibility of methicillin-resistant or -sensitive Staphylococcus aureus to beta-lactams.

We have demonstrated that 6,7-dihydroxyflavone by itself has only a weak antibacterial effect on methicillin-resistant Staphylococcus aureus (MRSA) but that at concentrations less than MIC it synergistically elevates the susceptibility of clinically isolated MRSA and methicillin-sensitive S. aureus strains to beta-lactam antibiotics from 8- to 32,800-fold.

Induction of G1 cell cycle arrest in human umbilical vein endothelial cells by flavone's inhibition of the extracellular signal regulated kinase cascade.

Dietary flavonoids have demonstrated anti-carcinogenic activity in several animal models, but their mechanisms of action have not yet been clearly established. Here, we show that flavone, a parent compound of flavonoids, inhibits the proliferation, migration, and capillary tube formation of human umbilical vein endothelial cells (HUVECs). Flow cytometric analysis showed that flavone arrests the cell cycle progression at G(1) phase in HUVECs. We observed the down-regulation of the hyperphosphorylated form of retinoblastoma gene product and cyclin-dependent kinases 2 and 4 in flavone-treated cells, but it had no affect on the expression of p53 and cyclin-dependent kinase inhibitors p21(CIP/Waf1) and p27(Kip). Flavone almost completely inhibited the activation of extracellular signal regulated kinase 1. The present results suggest that the flavone moiety of flavonoids is required for anti-proliferative activity of flavonoids and that anti-carcinogenic action of flavonoids in vivo was mediated, at least in part, by inhibiting angiogenesis.

Functional disorders of the oxidative phosphorylation system in the heart mitochondria of mice with juvenile visceral steatosis.

Mice with juvenile visceral steatosis (JVS) develop remarkable cardiac hypertrophy and exhibit an increased number of mitochondria in their heart. However, the biochemical characteristics and physiological functions of these mitochondria cardiac are little known. Here we show that the respiratory activities at state 3 with glutamate plus malate or succinate in the heart mitochondria of JVS mice were greatly decreased to 47% or 77%, respectively, compared with those of control mice. The contents of cytochromes a+a(3), b, and c+c(1) in the heart mitochondria of these mice were also decreased, to 51%, 45%, and 79%, respectively, of those of the control mice. Oligomycin-sensitive ATPase activitiy in these mitochondria, however, was increased to about 2 times over that of the control mice. Surprisingly, the ATP-Pi exchange activity of the heart mitochondria of JVS mice was greatly decreased, to 35% of that of control mice. On the other hand, the expression levels of 2 subunits of H(+)-ATP synthase, i.e., coupling factor 6 and alpha subunit, in heart mitochondria from control and JVS mice were almost the same. These results indicate that the coordinate regulation of mitochondrial proliferation and gene expression for components of the oxidative phosphorylation system was markedly defective in the heart of JVS mice. Our current results also suggest the presence of a novel regulatory mechanisms of ATP synthase activities in the heart.

Flavone markedly affects phenotypic expression of beta-lactam resistance in methicillin-resistant Staphylococcus aureus strains isolated clinically.

Flavone and its derivatives had very weak antibacterial effects on methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus, but dramatically intensified MRSA's susceptibility to beta-lactams. We named these compounds "ILSMR (intensifier of beta-lactam-susceptibility in MRSA)." We also found discrepancies among MRSA strains in their responses to flavone; some strains showed phenotypic susceptibility to methicillin while others showed phenotypic resistance to it. To understand the mechanism underlying this discrepancy, we characterized 20 MRSA strains in detail, analyzed their conventional and molecular typings, and examined each strain's resistance to beta-lactams, with COL serving as a reference. Neither SCCmec typing nor coagulase typing explained the diverse effects of flavone on the beta-lactam MICs of these strains. Likewise, changes in pulsed-field gel electrophoresis (PFGE) type were not associated with the profiles of ILSMR effects. However, the present observations suggest that the ILSMR effects on MRSA is strain-specific, and that this effect depends on an as-yet unknown mechanism that is essential for the expression of the phenotype conferring beta-lactam resistance to MRSA strains, independently of an interaction with the mecA-encoded penicillin-binding protein 2a or with the beta-lactamase.

Identification of the up- and down-regulated genes in the heart of juvenile visceral steatosis mice.

Juvenile visceral steatosis (JVS) mice, novel animal models of systemic carnitine deficiency, exhibit a remarkably increased number of mitochondria in their cardiac myocytes. To date, however, there has been no reported investigation of the molecular mechanism of this increased number of mitochondria. Here, we analyzed the gene expression profile from the hearts of JVS and control mice by Affymetrix GeneChip analysis representing 34323 genes. We found that 176 genes, containing 93 known genes and 83 novel genes, were up-regulated in JVS mice compared with control mice, and 167 genes, containing 67 known genes and 100 novel genes, were down-regulated in JVS mice compared with control mice. We found several interesting molecular aspects that have not yet been identified in the hearts of JVS mice, including down-regulation of a number of ion channels and up-regulation of regulators involved in cell cycle progression. This genome-wide analysis should contribute to a greater understanding of the molecular mechanism of mitochondrial biogenesis in the heart of JVS mouse and provide a strategy for identifying novel genes involved not only in mitochondrial biogenesis but also in cardiac hypertrophy.