The Beclin 1 gene is a haplo-insufficient tumor suppressor and plays an essential role in autophagy. However, the molecular mechanism by which Beclin 1 functions remains largely unknown. Here we report the crystal structure of the evolutionarily conserved domain (ECD) of Beclin 1 at 1.6 Å resolution. Beclin 1 ECD exhibits a previously unreported fold, with three structural repeats arranged symmetrically around a central axis. Beclin 1 ECD defines a novel class of membrane-binding domain, with a strong preference for lipid membrane enriched with cardiolipin. The tip of a surface loop in Beclin 1 ECD, comprising three aromatic amino acids, acts as a hydrophobic finger to associate with lipid membrane, consequently resulting in the deformation of membrane and liposomes. Mutation of these aromatic residues rendered Beclin 1 unable to stably associate with lipid membrane in vitro and unable to fully rescue autophagy in Beclin 1-knockdown cells in vivo. These observations form an important framework for deciphering the biological functions of Beclin 1.

The immortalization process is a fundamental step in the development of most (if not all) human cancers, including the aggressive endothelial cell (EC)-derived malignancy angiosarcoma. Inactivation of the tumor suppressor p16(INK4a) and the development of multiple chromosomal abnormalities are features of angiosarcoma that are recapitulated during telomerase-mediated immortalization of human ECs in vitro. The present study used a panel of telomerase-immortalized bone marrow EC (BMEC) lines to define the consequences of inactivation of p16(INK4a) on EC function and to identify molecular changes associated with repression of p16(INK4a). In a comparison of two immortalized BMEC mass cultures and six clones, the cell lines that repressed p16(INK4a) showed a higher rate of proliferation and an impaired ability to undergo morphogenic differentiation and form vessel-like structures in vitro. Proteomic comparison of a p16(INK4a)-negative and a p16(INK4a)-positive BMEC mass culture at early- and late-passage time points following transduction with telomerase reverse transcriptase (hTERT) revealed altered expression of cytoskeletal proteins, including vimentin and α-tropomyosin (αTm), in the immortal cells. Immunoblot analyses of a panel of 11 immortal clones showed that cells that lacked p16(INK4a) expression tended to accumulate more dramatic changes in these cytoskeletal proteins than cells that retained p16(INK4a) expression. This corresponded with aberrant cytoskeletal architectures among p16(INK4a)-negative clones, which featured thicker actin stress fibers and less fluid membrane ruffles than p16(INK4a)-positive cells. A direct link between p16(INK4a) repression and defective EC function was confirmed by analysis of normal cells transfected with small interfering RNA (siRNA) targeting p16(INK4a). siRNA-mediated repression of p16(INK4a) significantly impaired random motility and vessel formation in vitro. This report is the first to demonstrate that ECs that repress the expression of p16(INK4a) are prone to defects in motility, morphogenesis and cytoskeletal organization. These defects are likely to reflect alterations that occur during the development of EC-derived malignancies.

Hypoxic and oxidant stresses can coexist in biological systems, and oxidant stress has been proposed to activate hypoxia pathways through the inactivation of the 'oxygen-sensing' hypoxia-inducible factor (HIF) prolyl and asparaginyl hydroxylases. Here, we show that despite reduced sensitivity to cellular hypoxia, the HIF asparaginyl hydroxylase--known as FIH, factor inhibiting HIF--is strikingly more sensitive to peroxide than the HIF prolyl hydroxylases. These contrasting sensitivities indicate that oxidant stress is unlikely to signal hypoxia directly to the HIF system, but that hypoxia and oxidant stress can interact functionally as distinct regulators of HIF transcriptional output.

Lymphatic vessels (LVs) are important structures for antigen presentation, for lipid metabolism, and as conduits for tumor metastases, but they have been difficult to visualize in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in ontogeny and the maintenance of LVs. To visualize LVs in the lymph node of a living mouse in real time, we made the ProxTom transgenic mouse in a C57BL/6 background using red fluorescent LVs that are suitable for in vivo imaging. The ProxTom transgene contained all Prox1 regulatory sequences and was faithfully expressed in LVs coincident with endogenous Prox1 expression. The progenies of a ProxTom × Hec6stGFP cross were imaged using two-photon laser scanning microscopy, allowing the simultaneous visualization of LVs and high endothelial venules in a lymph node of a living mouse for the first time. We confirmed the expression of Prox1 in the adult liver, lens, and dentate gyrus. These intensely fluorescent mice revealed the expression of Prox1 in three novel sites: the neuroendocrine cells of the adrenal medulla, megakaryocytes, and platelets. The novel sites identified herein suggest previously unknown roles for Prox1. The faithful expression of the fluorescent reporter in ProxTom LVs indicates that these mice have potential utility in the study of diseases as diverse as lymphedema, filariasis, transplant rejection, obesity, and tumor metastasis.

Adenosine deaminases that act on RNA bind double-stranded and structured RNAs and convert adenosines to inosines by hydrolytic deamination. Inosines are recognized as guanosines, and, hence, RNA editing alters the sequence information but also structure of RNAs. Editing by ADARs is widespread and essential for normal life and development. Precursors of miRNAs are abundantly edited by ADARs, but neither the abundance nor the consequences of miRNA editing has been firmly established. Using transgenic mouse embryos that are deficient in the two enzymatically active editing enzymes ADAR and ADARB1, we compare relative frequencies but also sequence composition of miRNAs in these genetically modified backgrounds to wild-type mice by "next-generation sequencing." Deficiency of ADARB1 leads to a reproducible change in abundance of specific miRNAs and their predicted targets. Changes in miRNA abundance seem unrelated to editing events. Additional deletion of ADAR has surprisingly little impact on the mature miRNA repertoire, indicating that miRNA expression is primarily dependent on ADARB1. A-to-G transitions reflecting A-to-I editing events can be detected at few sites and at low frequency during the early embryonic stage investigated. Again, most editing events are ADARB1-dependent with only few editing sites being specifically edited by ADAR. Besides known editing events in miRNAs, a few novel, previously unknown editing events were identified. Some editing events are located to the seed region of miRNAs, opening the possibility that editing leads to their retargeting.

Interest in binuclear ruthenium(II) polypyridyl complexes as luminescent cellular imaging agents and for biomedical applications is increasing rapidly. We have investigated the cellular localization, uptake, and biomolecular interactions of the pure enantiomers of two structural isomers of [μ-bipb(phen)(4)Ru(2)](4+) (bipb is bis(imidazo[4,5-f]-1,10-phenanthrolin-2-yl)benzene and phen is 1,10-phenanthroline) using confocal laser scanning microscopy, emission spectroscopy, and linear dichroism. Both complexes display distinct enantiomeric differences in the staining pattern of fixed cells, which are concluded to arise from chiral discrimination in the binding to intracellular components. Uptake of complexes in live cells is efficient and nontoxic at 5 μM, and occurs through an energy-dependent mechanism. No differences in uptake are observed between the structural isomers or the enantiomers, suggesting that the interactions triggering uptake are rather insensitive to structural variations. Altogether, these findings show that the complexes investigated are promising for future applications as cellular imaging probes. In addition, linear dichroism shows that the complexes exhibit DNA-condensing properties, making them interesting as potential gene delivery vectors.

We quantified absolute myocardial blood flow (MBF) using a spin-labeling MRI (SL-MRI) method after transplantation of endothelial cells (ECs) into the infarcted heart. Our aims were to study the temporal changes in MBF in response to EC transplantation and to compare regional MBF with contractile function (wall motion) and microvascular density.

Rapidly activating and inactivating somatodendritic voltage-gated K(+) (Kv) currents, I(A), play critical roles in the regulation of neuronal excitability. Considerable evidence suggests that native neuronal I(A) channels function in macromolecular protein complexes comprising pore-forming (α) subunits of the Kv4 subfamily together with cytosolic, K(+) channel interacting proteins (KChIPs) and transmembrane, dipeptidyl peptidase 6 and 10 (DPP6/10) accessory subunits, as well as other accessory and regulatory proteins. Several recent studies have demonstrated a critical role for the KChIP subunits in the generation of native Kv4.2-encoded channels and that Kv4.2-KChIP complex formation results in mutual (Kv4.2-KChIP) protein stabilization. The results of the experiments here, however, demonstrate that expression of DPP6 in the mouse cortex is unaffected by the targeted deletion of Kv4.2 and/or Kv4.3. Further experiments revealed that heterologously expressed DPP6 and DPP10 localize to the cell surface in the absence of Kv4.2, and that co-expression with Kv4.2 does not affect total or cell surface DPP6 or DPP10 protein levels. In the presence of DPP6 or DPP10, however, cell surface Kv4.2 protein expression is selectively increased. Further addition of KChIP3 in the presence of DPP10 markedly increases total and cell surface Kv4.2 protein levels, compared with cells expressing only Kv4.2 and DPP10. Taken together, the results presented here demonstrate that the expression and localization of the DPP accessory subunits are independent of Kv4 α subunits and further that the DPP6/10 and KChIP accessory subunits independently stabilize the surface expression of Kv4.2.

The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors.

Identification of mitochondrial DNA (mtDNA) variations in the inherited cataract patients from south India.

Anti-oxidation and exocytosis are important for maintaining exocrine tissue homeostasis. During aging, functional and structural alterations occur in the lacrimal gland (LG), including oxidative damage to proteins, lipids, and DNA. The aims of the present study were to determine in the aging LG: a) the effects of aging on LG structure and secretory activity and b) changes in the expression of oxidative stress markers.

Obesity, a chronic metabolic disorder, is characterized by enlarged fat mass and dysregulation of lipid metabolism. The medicinal plant, Boesenbergia pandurata (Roxb.) Schltr., has been reported to possess anti-oxidative and anti-inflammatory properties; however, its anti-obesity activity is unexplored. The present study was conducted to determine whether B. pandurata extract (BPE), prepared from its rhizome parts, attenuated high-fat diet (HFD)-induced obesity in C57BL/6J mice. The molecular mechanism was investigated in 3T3-L1 adipocytes and HepG2 human hepatoma cells. BPE treatment decreased triglyceride accumulation in both 3T3-L1 adipocytes and HepG2 hepatocytes by activating AMP-activated protein kinase (AMPK) signaling and regulating the expression of lipid metabolism-related proteins. In the animal model, oral administration of BPE (200 mg/kg/day for 8 weeks) significantly reduced HFD-induced body weight gain without altering the amount of food intake. In addition, elevated serum levels of total cholesterol, low-density lipoprotein cholesterol, and triglycerides were suppressed by BPE administration. Fat pad masses were reduced in BPE-treated mice, as evidenced by reduced adipocyte size. Furthermore, BPE protected against the development of nonalcoholic fatty liver by decreasing hepatic triglyceride accumulation. BPE also activated AMPK signaling and altered the expression of lipid metabolism-related proteins in white adipose tissue and liver. Taken together, these findings indicate that BPE attenuates HFD-induced obesity by activating AMPK and regulating lipid metabolism, suggesting a potent anti-obesity agent.

PISTILLATA (PI)-like genes are crucial regulators of flowering in angiosperms. A homologue of PI, designated as AcPI (Genbank accession number HQ717796), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcPI is 907 bp in length and contains an open reading frame of 594 bp, which encodes a protein of 197 amino acids. The molecular weight was 2.29 kDa and the isoelectric point was 9.28. The alignment showed that AcPI had a high identity with CsPIC2 (78.6%), AoPI (77.4%), OrcPI (75.7%) and HPI2 (72.4%). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses in different tissues showed that the expression pattern of AcPI was different from the B-class genes in eudicots. AcPI was expressed in all the tissues investigated. The expression level was very low in fruit stems, bracts, leaves and sepals, high in petals and carpels, and moderate in apical meristems, flesh and stamens. The qRT-PCR analyses in different stages indicated that the expression of AcPI reached the highest level at 40 days after flower inducement, when the multiple fruit and floral organs were forming. It proved the important role of AcPI in floral organs and fruit development. The 35S::AcPI transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants.

Collective motion of cell sheets plays a role not only in development and repair, but also in devastating diseases such as cancer. However, unlike single-cell motility, collective motion of cell sheets involves complex cell-cell communication during migration; therefore, its mechanism is largely unknown. To elucidate propagation of signaling transduced by cell-cell interaction, we designed a hydrogel substrate that can cause local mechanical stretching of cell sheets. Poly (N-isopropyl acrylamide) (PNIPAAm) hydrogel is a temperature-responsive polymer gel whose volume changes isotropically in response to temperature changes below 37 °C. We designed a combined hydrogel substrate consisting of collagen-immobilized PNIPAAm as the local stimulation side and polyacrylamide (PAAm) as the non-stimulation side to assess propagation of mechanical transduction. Mardin-Darby canine kidney (MDCK) cells adhered to the collagen-immobilized PNIPAAm gel increased it area and were flattened as the gel swelled with temperature decrease. E-cadherin in these cells became undetectable in some domains, and actin stress fibers were more clearly observed at the cell base. In contrast, E-cadherin in cells adhered to the collagen-immobilized PAAm side was equally stained as that in cells adhered to the collagen-immobilized PAAm side even after temperature decrease. ERK1/2 MAPK activation of cells on the non-stimulated substrate occurred after partial stretching of the cell sheet suggesting the propagation of signaling. These results indicate that a change in the balance of mechanical tension induced by partial stretching of cell sheets leads to activation and propagation of the cell signaling.

Invasion, metastasis and resistance to conventional chemotherapeutic agents are obstacles to successful treatment of pancreatic cancer, and a better understanding of the molecular basis of this malignancy may lead to improved therapeutics. In the present study, we investigated whether AKT2 silencing sensitized pancreatic cancer L3.6pl, BxPC-3, PANC-1 and MIAPaCa-2 cells to gemcitabine via regulating PUMA (p53-upregulated modulator of apoptosis) and nuclear factor (NF)-κB signaling pathway. MTT, TUNEL, EMSA and NF-κB reporter assays were used to detect tumor cell proliferation, apoptosis and NF-κB activity. Western blotting was used to detect different protein levels. Xenograft of established tumors was used to evaluate primary tumor growth and apoptosis after treatment with gemcitabine alone or in combination with AKT2 siRNA. Gemcitabine activated AKT2 and NF-κB in MIAPaCa-2 and L3.6pl cells in vitro or in vivo, and in PANC-1 cells only in vivo. Gemcitabine only activated NF-κB in BxPC-3 cells in vitro. The presence of PUMA was necessary for gemcitabine-induced apoptosis only in BxPC-3 cells in vitro. AKT2 inhibition sensitized gemcitabine-induced apoptosis via PUMA upregulation in MIAPaCa-2 cells in vitro, and via NF-κB activity inhibition in L3.6pl cells in vitro. In PANC-1 and MIAPaCa-2 cells in vivo, AKT2 inhibition sensitized gemcitabine-induced apoptosis and growth inhibition via both PUMA upregulation and NF-κB inhibition. We suggest that AKT2 inhibition abrogates gemcitabine-induced activation of AKT2 and NF-κB, and promotes gemcitabine-induced PUMA upregulation, resulting in chemosensitization of pancreatic tumors to gemcitabine, which is probably an important strategy for the treatment of pancreatic cancer.

Most of what is known about the Helicobacter pylori (H. pylori) cytotoxin, CagA, pertains to a much-vaunted role as a determinant of gastric inflammation and cancer. Little attention has been devoted to potential roles of CagA in the majority of H. pylori infected individuals not showing oncogenic progression, particularly in relation to host tolerance. Regenerating islet-derived (REG)3γ encodes a secreted C-type lectin that exerts direct bactericidal activity against Gram-positive bacteria in the intestine. Here, we extend this paradigm of lectin-mediated innate immunity, showing that REG3γ expression is triggered by CagA in the H. pylori-infected stomach.

Nine human neurodegenerative diseases, including Huntington's disease and several spinocerebellar ataxia, are associated to the aggregation of proteins comprising an extended tract of consecutive glutamine residues (polyQs) once it exceeds a certain length threshold. This event is believed to be the consequence of the expansion of polyCAG codons during the replication process. This is in apparent contradiction with the fact that many polyQs-containing proteins remain soluble and are encoded by invariant genes in a number of eukaryotes. The latter suggests that polyQs expansion and/or aggregation might be counter-selected through a genetic and/or protein context. To identify this context, we designed a software that scrutinize entire proteomes in search for imperfect polyQs. The nature of residues flanking the polyQs and that of residues other than Gln within polyQs (insertions) were assessed. We discovered strong amino acid residue biases robustly associated to polyQs in the 15 eukaryotic proteomes we examined, with an over-representation of Pro, Leu and His and an under-representation of Asp, Cys and Gly amino acid residues. These biases are conserved amongst unrelated proteins and are independent of specific functional classes. Our findings suggest that specific residues have been co-selected with polyQs during evolution. We discuss the possible selective pressures responsible of the observed biases.

Extinction is an integral part of normal healthy fear responses, while it is compromised in several fear-related mental conditions in humans, such as post-traumatic stress disorder (PTSD). Although much research has recently been focused on fear extinction, its molecular and cellular underpinnings are still unclear. The development of animal models for extinction will greatly enhance our approaches to studying its neural circuits and the mechanisms involved. Here, we describe two gene-knockout mouse lines, one with impaired and another with enhanced extinction of learned fear. These mutant mice are based on fear memory-related genes, stathmin and gastrin-releasing peptide receptor (GRPR). Remarkably, both mutant lines showed changes in fear extinction to the cue but not to the context. We performed indirect imaging of neuronal activity on the second day of cued extinction, using immediate-early gene c-Fos. GRPR knockout mice extinguished slower (impaired extinction) than wildtype mice, which was accompanied by an increase in c-Fos activity in the basolateral amygdala and a decrease in the prefrontal cortex. By contrast, stathmin knockout mice extinguished faster (enhanced extinction) and showed a decrease in c-Fos activity in the basolateral amygdala and an increase in the prefrontal cortex. At the same time, c-Fos activity in the dentate gyrus was increased in both mutant lines. These experiments provide genetic evidence that the balance between neuronal activities of the amygdala and prefrontal cortex defines an impairment or facilitation of extinction to the cue while the hippocampus is involved in the context-specificity of extinction.

Cigarette smoke (CS) causes sustained lung inflammation, which is an important event in the pathogenesis of chronic obstructive pulmonary disease (COPD). We have previously reported that IKKα (I kappaB kinase alpha) plays a key role in CS-induced pro-inflammatory gene transcription by chromatin modifications; however, the underlying role of downstream signaling kinase is not known. Mitogen- and stress-activated kinase 1 (MSK1) serves as a specific downstream NF-κB RelA/p65 kinase, mediating transcriptional activation of NF-κB-dependent pro-inflammatory genes. The role of MSK1 in nuclear signaling and chromatin modifications is not known, particularly in response to environmental stimuli. We hypothesized that MSK1 regulates chromatin modifications of pro-inflammatory gene promoters in response to CS. Here, we report that CS extract activates MSK1 in human lung epithelial (H292 and BEAS-2B) cell lines, human primary small airway epithelial cells (SAEC), and in mouse lung, resulting in phosphorylation of nuclear MSK1 (Thr581), phospho-acetylation of RelA/p65 at Ser276 and Lys310 respectively. This event was associated with phospho-acetylation of histone H3 (Ser10/Lys9) and acetylation of histone H4 (Lys12). MSK1 N- and C-terminal kinase-dead mutants, MSK1 siRNA-mediated knock-down in transiently transfected H292 cells, and MSK1 stable knock-down mouse embryonic fibroblasts significantly reduced CS extract-induced MSK1, NF-κB RelA/p65 activation, and posttranslational modifications of histones. CS extract/CS promotes the direct interaction of MSK1 with RelA/p65 and p300 in epithelial cells and in mouse lung. Furthermore, CS-mediated recruitment of MSK1 and its substrates to the promoters of NF-κB-dependent pro-inflammatory genes leads to transcriptional activation, as determined by chromatin immunoprecipitation. Thus, MSK1 is an important downstream kinase involved in CS-induced NF-κB activation and chromatin modifications, which have implications in pathogenesis of COPD.

No abstract available