Oxidized low-density lipoprotein (ox-LDL) is involved in the pathogenesis of atherosclerosis and atherosclerotic plaque rupture by promoting lipid accumulation, proinflammatory responses, and cell death. LDL is mainly oxidized in the subendothelial layer of the vascular wall and then can be taken up by vascular endothelial cells. However, little is known about the intracellular processing of the damaged LDL. Previous studies found that autophagy is involved in degrading oxidized proteins under oxidative stress conditions in Arabidopsis thaliana, while ox-LDL can activate autophagy in EA.hy926 endothelial cells, suggesting a possible role of autophagy in the degradation of ox-LDL by endothelial cells. The present study showed that ox-LDL aggregated in human umbilical vein endothelial cells (HUVECs) and brought about an increase in the formation of autophagosomes and autolysosomes. Ox-LDL-induced increase in the autophagic level was blocked by treatment with the autophagy inhibitor 3-methyladenine and increased by the autophagy inducer rapamycin, while the aggregation of Dil-labled ox-LDL was increased by 3-methyladenine and decreased by rapamycin. In addition, Dil-labeled ox-LDL colocalized with the autophagy marker MDC, microtubule-associated protein light chain 3 (MAP1-LC3), and lysosome-associated membrane protein 2a (lamp2a). HUVECs treated with Dil-labeled-ox-LDL showed a much greater degree of overlap of MAP1-LC3 and Lamp2a than control. The results suggest that ox-LDL activates the autophagic lysosome pathway in HUVECs through the LC3/beclin1 pathway, leading to the degradation of ox-LDL.

Several members of the transient receptor potential (TRP) channel superfamily have been shown to assemble as tetramers. Here we have determined the subunit stoichiometry of the transient receptor potential M8 (TRPM8) channel using atomic force microscopy (AFM). TRPM8 channels were isolated from transfected cells, and complexes were formed between the channels and antibodies against a V5 epitope tag present on each subunit. The complexes were then subjected to AFM imaging. A frequency distribution of the molecular volumes of antibody decorated channels had a peak at 1305 nm(3), close to the expected size of a TRPM8 tetramer. The frequency distribution of angles between pairs of bound antibodies had two peaks, at 93 degrees and 172 degrees , confirming that the channel assembles as a tetramer. We suggest that this assembly pattern is common to all members of the TRP channel superfamily.

The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.

Prothrombin is converted to thrombin by factor Xa in the cell-associated prothrombinase complex. Prothrombin is present in calcified bone matrix and thrombin exerts effects on osteoblasts as well as on bone resorption by osteoclasts. We investigated whether 1) osteoclasts display factor Xa- dependent prothrombinase activity 2) osteoclasts express critical regulatory components upstream of the prothrombinase complex. The osteoclast differentiation factor RANKL induced formation of multinucleated TRAP positive cells concomitant with induction of prothrombinase activity in cultures of RAW 264.7 cells and bone marrow osteoclast progenitors. Expression analysis of extrinsic coagulation factors revealed that RANKL enhanced protein levels of factor Xa as well as of coagulation factor III (tissue factor). Inhibition assays indicated that factor Xa and tissue factor were involved in the control of prothrombinase activity in RANKL-differentiated osteoclasts, presumably at two stages 1) conversion of prothrombin to thrombin and 2) conversion of factor X to factor Xa, respectively. Activation of the extrinsic coagulation pathway during osteoclast differentiation through induction of tissue factor and factor Xa by a RANKL-dependent pathway indicates a novel role for osteoclasts in converting prothrombin to thrombin.

The mechanochemical forces that move and position intracellular organelles and their intermediates in eukaryotic cells are provided by molecular motor proteins which include the cytoplasmic dynein-1 motor complex. Recently, we identified the Rab11 GTPase effector protein Rab11-FIP3 (henceforth, FIP3) as a novel binding partner for dynein light intermediate chain 1 (DLIC-1, gene symbol DYNC1LI1), a subunit of cytoplasmic dynein-1. Here, we show that FIP3 also binds the dynein light intermediate chain 2 subunit (DLIC-2, gene symbol DYNC1LI2). We show that like DLIC-1, DLIC-2 binds the amino-terminal 435 amino acids of FIP3 and that FIP3 links Rab11a to DLIC-2. We also show that FIP3 recruits DLIC-2 onto membranes and that DLIC-2 is necessary for the accumulation of endocytosed-transferrin (Tfn) at the pericentrosomal endosomal-recycling compartment (ERC). Finally, we demonstrate that overexpression of FIP3 fragments the Golgi complex by sequestering cytoplasmic dynein-1. In conclusion, we have identified FIP3 as the first membrane-associated interacting-partner for DLIC-2 and propose that this interaction serves to control endosomal trafficking from sorting endosomes to the ERC.

Mesenchymal stem cells (MSCs) have been induced to differentiate successfully from human embryonic stem cells (hES-MSCs), which could serve as an in vitro source of MSCs. However, the homing behaviors of such cells and their potential utility for liver regeneration in vivo have not been reported. We investigated factors that influenced early homing and the hepatic-directed differentiation potency of hES-MSCs in a mouse model of acute liver injury. The hES-MSCs could be detected 36 h after cell infusion and this was unaffected by the number of cell passages in culture. Pretreatment of hES-MSCs with TNF-alpha resulted in higher rates of homing of these cells to the injured liver. Interestingly most of the cells homing at an early stage expressed alpha-fetoprotein (AFP), indicating hepatic differentiation. Thus, hES-MSCs can home to the acutely injured liver at high efficiency and undergo hepatic differentiation, suggesting that these cells could be useful for treating acute human liver injury.

Previous studies have assumed that amelogenin is responsible for the therapeutic effect of the enamel matrix derivative (EMD) in periodontal tissue healing and regeneration. However, it is difficult to confirm this hypothesis because both the EMD and the amelogenins are complex mixtures of multiple proteins. Further adding to the difficulties is the fact that periodontal tissue regeneration involves various types of cells and a sequence of associated cellular events including the attachment, migration and proliferation of various cells. In this study, we investigated the potential effect of a 25 kDa recombinant porcine amelogenin on primarily cultured periodontal ligament fibroblasts (PDLF), gingival fibroblasts (GF) and gingival epithelial cells (GEC). The cells were treated with 25 kDa recombinant porcine amelogenin (rPAm) at a concentration of 10 mug/mL. We found that rPAm significantly promoted the proliferation and migration of PDLF, but not their adhesion. Similarly, the proliferation and adhesion of GF were significantly enhanced by treatment with rPAm, while migration was greatly inhibited. Interestingly, this recombinant protein inhibited the growth rate, cell adhesion and migration of GEC. These data suggest that rPAm may play an essential role in periodontal regeneration through the activation of periodontal fibroblasts and inhibition of the cellular behaviors of gingival epithelial cells.

Factors involved in transition from the immunotolerant to immunoactive phase in chronic hepatitis B virus (HBV) infection remain unclear. We investigated viral mutations occurring during transition and elucidated their virological and immunological significance. Full-length HBV DNA sequences were serially determined in a chronic HBV carrier from the immunotolerant to immunoactive phase. Viral replicative competence was examined by transfection analysis. HBV-specific CD8(+) T cell response was evaluated by coculture of CD8(+) T cells with autologous dendritic cells followed by interferon-gamma Elispot assay. Eleven point mutations and two deletions appeared around the onset of the immunoactive phase. Viral replicative competence declined significantly after the onset of active hepatitis. Examination of the CD8(+) T cell response against two putative T cell epitopes, which contained substituted amino acids from the immunotolerant to immunoactive phase, showed that mutant HBV epitopes gave a lesser T cell response than wild-type HBV ones. In summary, point mutations and deletions may occur prior to or concurrent with the onset of the immunoactive phase during chronic HBV infection. These mutations may result in a significant decrease in both viral replicative competence and HBV-specific CD8(+) T cell response, suggesting a possible adaptation for the maintenance of viral persistence.

Alzheimer's disease (AD) is the most common cause of dementia affecting the elderly. Treatment for effective cure of this complex neurodegenerative disease does not yet exist. In AD, otherwise soluble, monomeric form of amyloid ss (Ass) peptide converts into toxic, fibrillar form rich in ss-sheet content. Several immunological approaches that prevent this conversion of Ass into pathological form or that accelerate its clearance are being actively pursued worldwide. As part of these attempts, we report here, the design and characterization of a non-amyloidogenic homologue of Ass (Ass-KEK). We demonstrate that this peptide is helical in nature and retains the immunoneutralizing epitopes of native Ass. More importantly, Fab fragments of the polyclonal anti-Ass-KEK antibodies interfere with formation of Ass fibrils as well as dissociate the preformed Ass aggregates in vitro. These results suggest that non-amyloidogenic Ass-KEK may serve as a safer alternative vaccine for Alzheimer's disease.

The mechanism of cisplatin resistance in cancer cells is not fully understood. Here, we show that the Akt/mTOR survival pathway plays an important role in cisplatin resistance in human ovarian cancer cells. Specifically, we found that cisplatin treatment activates the Akt/mTOR survival pathway and that inhibition of this pathway by the PI3K inhibitor LY294002 or knockdown of Akt sensitizes ovarian cancer cells to cisplatin. Furthermore, we generated cisplatin resistant cells and found that resistant cells express a higher level of activated Akt as compared to their cisplatin sensitive counterparts. Importantly, inhibition of Akt or mTOR sensitized resistant cells to cisplatin-induced apoptosis. Taken together, our data indicate that activation of the Akt/mTOR pathway prevents cisplatin-induced apoptosis, leading to cisplatin resistance. Therefore, our study suggests that cisplatin resistance can be overcome by targeting the Akt/mTOR survival pathway in human ovarian cancer cells.

Leptin is an appetite regulatory hormone that is secreted into the blood circulation by adipose tissue, and functions in the central nerve system (i.e. hypothalamus) by crossing the blood brain barrier (BBB). In the present study, we investigated the function of a leptin-derived peptide (Lep(70-89)) as a ligand for mouse brain-derived endothelial cells (MBEC4). Lep(70-89)-modified liposomes, prepared with a polyethyleneglycol (PEG) spacer (Lep(70-89)-PEG-LPs) exhibited a significantly higher cellular uptake than peptide-unmodified liposomes (PEG-LPs). Furthermore, cellular uptake was inhibited by amiloride, while no significant inhibitory effect was observed by the presence of chlorpromazine and filipin III, suggesting that macropinocytosis largely contributed to the cellular uptake of Lep(70-89)-PEG-LPs. Imaging studies revealed that Lep(70-89)-PEG-LPs were not colocalized with endosome/lysosomes, whereas neutral dextran (70kDa) was predominantly co-localized with these compartments. This indicates that Lep(70-89)-PEG-LPs are taken up via macropinocytosis and are subject to non-classical intracellular trafficking, resulting in the circumvention of lysosomal degradation in endothelial cells.

Quantum dots (QDs) are rapidly emerging as an important class of nanoparticles (NPs) with potential applications in medicine. However, little is known about penetration of QDs through human skin. This study investigated skin penetration of QDs in both in vivo and in vitro human skin. Using the tape stripping method, this study demonstrates for the first time that QDs can actually penetrate through the stratum corneum (SC) of human skin. Transmission electron microscope (TEM) and energy diverse X-ray (EDX) analysis showed accumulation of QDs in the SC of a human skin equivalent model (HSEM) after dermal exposure to QDs. These findings suggest possible transdermal absorption of QDs after dermal exposure over a relatively long period of time.

The production rate of dengue viruses (DENVs), especially low-passage virus isolates, is low, and, therefore, the isolates are generally used only after several passages. However, in vitro passages could induce mutation(s). In this study, we established a system for the characterization of low-passage viral isolates using an infectious cDNA clone. We used R05-624, a plaque derived from type 2 (DENV-2) Thai strain, for the construction of the cDNA clone, named pmMW/R05-624. We found that transfection of both of mammalian Vero cells and mosquito C6/36 cells with viral RNA derived from the cDNA clone produced a significant amount of progeny virus: 3.2 x 10(6) focus-forming units (FFU) production per ml of cultured fluid only 3 days after transfection with 2 mug RNA. Conversely, no detectable level of viruses was produced by conventional methods using a single cell line, Vero or C6/36. When this system was applied for the characterization of 8 low-passage clinical viral isolates by placing their 5'-half or 3'-half in the above cDNA clone, we found that all the isolates, except for L04-225, produced similar levels of progeny virus. Among a total of 8 cDNA clones reconstructed with the NS4A-3'NCR region derived from L04-225, one clone carried an insertion and produced a low level of progeny virus. Thus, our system to efficiently rescue clinical samples or low-passage viral isolates could be useful for assessing the virological and molecular characteristics of DENV that could be related to disease pathogenesis.

Microphthalmia-associated transcription factor, Mitf, has been shown to be necessary for regulating genes involved in osteoclast differentiation. Previously it was shown by others that Mitf translocates from the cytoplasm to the nucleus upon M-CSF/RANKL signaling in osteoclasts. Mitf's movement is regulated by its interaction with 14-3-3 and the kinase C-TAK1. Here we demonstrate that the related family member, Tfe3, does not shuttle from the cytoplasm to the nucleus and does not interact with C-TAK1. We also demonstrate that overexpression of C-TAK1 inhibits the expression of Acp5 while a kinase dead C-TAK1 or a Mitf mutant that cannot interact with C-TAK1 increased expression of Acp5. Finally, we show that the catalytic subunit of protein phosphatase 2A is upregulated in osteoclasts with M-CSF/RANKL signaling indicating a possible mechanism for dephosphorylating Mitf on its 14-3-3 binding site and allowing Mitf to be translocated to the nucleus of osteoclasts.

Differentiation agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA) engage cell signaling pathways that activate downstream transcriptional programs necessary for cell differentiation. Recent evidence has indicated microRNAs (miRNAs) are an integral part of these transcriptional programs, which target key proteins and impact cell growth thereby facilitating changes required for differentiation. To further investigate the role of miRNAs in cell growth and differentiation, we focused on miR-22, a miRNA induced by TPA in the HL60 leukemia cell line model of monocytic differentiation. TPA-induced miR-22 transcription was found to be downstream of the protein kinase c (PKC)-extracellular regulated kinase (ERK) signaling module, a pathway central to the growth and differentiation of many different cell types. Enforced miR-22 expression inhibited the growth of several different cancer cell lines, causing an accumulation of cells in the G1 phase of the cell cycle. The mechanism of miR-22's inhibitory effects involves targeting of the obligate c-Myc binding partner Max. Enforced miR-22 expression presumably lowers Max levels available for Myc binding, which differentially influenced the transcription of downstream targets of the Myc-Max complex. Our study provides additional support for miRNAs targeting key cellular regulatory microcircuits such as those governed by the Myc-Max transcriptional complex as well as their being active participants in cell growth and differentiation.

Cannabinoid CB(1) receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB(1) receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB(1) receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB(1) receptors in both binding (K(i)=0.7 nM) and functional assays (K(i)=0.2 nM). The compound has low affinity (K(i)=7600 nM) for human CB(2) receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10 mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB(1) receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB(1) receptor competitive antagonist that may further our understanding of the endocannabinoid system.

The Arf1 GTPase-activating protein ArfGAP1 regulates vesicular traffic through the COPI system. This protein consists of N-terminal catalytic domain, lipid packing sensors (the ALPS motifs) in the central region, and a carboxy part of unknown function. The carboxy part contains several diaromatic sequences that are reminiscent of motifs known to interact with clathrin adaptors. In pulldown experiments using GST-fused peptides from rat ArfGAP1, a peptide containing a (329)WETF sequence interacted strongly with clathrin adaptors AP-1 and AP-2, whereas a major coatomer-binding determinant was identified within the extreme carboxy terminal peptide ((405)AADEGWDNQNW). Mutagenesis and peptide competition experiments revealed that this determinant is required for coatomer binding to full-length ArfGAP1, and that interaction is mediated through the delta-subunit of the coatomer adaptor-like subcomplex. Evidence for a role of the carboxy motif in ArfGAP1-coatomer interaction in vivo is provided by means of a reporter fusion assay. Our findings point to mechanistic differences between ArfGAP1 and the other ArfGAPs known to function in the COPI system.

Apart from their well-established role in nitric oxide detoxification, flavohemoglobins (FHbs) are also believed to be involved in protection against oxidative stress in some yeast and bacteria. However, different studies have reported contradictory results in this regard. Here, we investigate the relationship between two FHbs in Aspergillus oryzae (cytosolic FHb1 and mitochondrial FHb2) and oxidative stress. The strains deficient in the two FHbs exhibited higher resistance to hydrogen peroxide than the wild type. In addition, the FHb2 overexpression strain showed hypersensitivity to hydrogen peroxide. Flavin reductase accompanied by the ferric reductase activities of the two FHbs were observed in correspondence with this expression. The reductase activities of the FHbs were attributed to their C-terminal flavin reductase domains. The reduced intracellular free iron can subsequently promote oxidative damage by accelerating the Fenton reaction in the cytosol and mitochondria (corresponding to the subcellular localizations of the two FHbs). This study is the first to show that fungal FHbs have a deleterious effect on oxidative protection, and suggests that the accelerated Fenton reaction induced by FHbs might be responsible for this effect.

Desipramine is a tricyclic antidepressant for psychiatric disorders that can induce QT prolongation, which may lead to torsades de pointes. Since blockade of cardiac human ether-a-go-go-related gene (hERG) channels is an important cause of acquired long QT syndrome, we investigated the acute effects of desipramine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. We examined the effects of desipramine on the hERG channels expressed in Xenopus oocytes using two-microelectrode voltage-clamp techniques. Desipramine induced concentration-dependent decreases in the current amplitude at the end of the voltage steps and hERG tail currents. The IC(50) for desipramine needed to block the hERG current in Xenopus oocytes decreased progressively relative to the degree of depolarization. Desipramine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations, Tyr-652 located in the S6 domain of the hERG channel reduced the potency of the channel block by desipramine more than a mutation of Phe-656 in the same region. These results suggest that desipramine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of desipramine.

Resveratrol has shown array of biological actions, and is under clinical development for various disease conditions. The etiology of diabetic neuropathy revolves around oxidative stress, AGE formation, lipid peroxidation etc. All these stimulate inflammatory processes and NF-kB cascade is considered as one of the major players of inflammatory response. Activation of NF-kB results in elevated levels of inflammatory mediators. COX-2 and TNF-alpha activity have also been correlated with inflammatory damage in the pathophysiology of diabetic neuropathy (DN). Therefore we investigated the effect of Resveratrol on NF-kappaB inflammatory cascade, COX-2, TNF-alpha and IL-6 levels in experimental DN. We found that resveratrol protected against various functional and behavioral deficits in diabetic neuropathy in line with our earlier published reports. In this study we found that the resveratrol treatment decreased the expression of p65 and IkB-alpha in treated rats. Treatment also ameliorated the elevated levels of TNF- alpha, IL-6 and COX-2. Resveratrol treatment produced significant decrease in nerve MDA levels in treated animals which may also be contributing to reduction in neuro-inflammation. This study confirms the NF-kB inhibitory activity and anti inflammatory activity of resveratrol which may contribute to neuroprotection in diabetic neuropathy apart from its antioxidant effect.