Note: This page contains sample records for the topic requires multiple pathways from Science.gov.
While these samples are representative of the content of Science.gov,
they are not comprehensive nor are they the most current set.
We encourage you to perform a real-time search of Science.gov
to obtain the most current and comprehensive results.
Last update: August 15, 2014.
1

Correct biological timing in Arabidopsis requires multiple light-signaling pathways  

PubMed Central

Circadian oscillators provide rhythmic temporal cues for a range of biological processes in plants and animals, enabling anticipation of the day/night cycle and enhancing fitness-associated traits. We have used engineering models to understand the control principles of a plant’s response to seasonal variation. We show that the seasonal changes in the timing of circadian outputs require light regulation via feed-forward loops, combining rapid light-signaling pathways with entrained circadian oscillators. Linear time-invariant models of circadian rhythms were computed for 3,503 circadian-regulated genes and for the concentration of cytosolic-free calcium to quantify the magnitude and timing of regulation by circadian oscillators and light-signaling pathways. Bioinformatic and experimental analysis show that rapid light-induced regulation of circadian outputs is associated with seasonal rephasing of the output rhythm. We identify that external coincidence is required for rephasing of multiple output rhythms, and is therefore important in general phase control in addition to specific photoperiod-dependent processes such as flowering and hypocotyl elongation. Our findings uncover a fundamental design principle of circadian regulation, and identify the importance of rapid light-signaling pathways in temporal control.

Dalchau, Neil; Hubbard, Katharine E.; Robertson, Fiona C.; Hotta, Carlos T.; Briggs, Helen M.; Stan, Guy-Bart; Goncalves, Jorge M.; Webb, Alex A. R.

2010-01-01

2

Requirement of multiple signaling pathways for the augmented production of hyaluronan by v-Src.  

PubMed

Malignant transformation of cells is frequently associated with an augmented production of hyaluronan and the subsequent formation of a hyaluronan-matrix. In v-Src-transformed cells, hyaluronan directly activate cell motility in a tumor-specific manner. Despite its importance, the mechanism by which v-Src activates hyaluronan production remains unclear. Here we report that multiple signaling pathways are required for the augmented production of hyaluronan. Either the expression of a dominant negative Ras or the treatment of cells with manumycin A, a Ras farnesyltransferase inhibitor, was able to suppress hyaluronan production. In contrast, expression of MEK1EE, a constitutive form of MEK1, activated both hyaluronan synthase expression and hyaluronan production. AG-490, a Jak-2 inhibitor, or LY294002, a PI3K inhibitor, similarly suppressed the augmented production of hyarulonan. Taken together, our results suggest the involvement of multiple signaling pathways, including Ras-dependent and independent ones, in augmented hyaluronan production by v-Src. PMID:17375476

Naito, Yuko; Suzuki, Noriko; Huang, Pengyu; Hasegawa, Hitoki; Sohara, Yasuyoshi; Iwamoto, Takashi; Hamaguchi, Michinari

2005-06-01

3

Atg6 is required for multiple vesicle trafficking pathways and hematopoiesis in Drosophila  

PubMed Central

Atg6 (beclin 1 in mammals) is a core component of the Vps34 complex that is required for autophagy. Beclin 1 (Becn1) functions as a tumor suppressor, and Becn1+/- tumors in mice possess elevated cell stress and p62 levels, altered NF-?B signaling and genome instability. The tumor suppressor function of Becn1 has been attributed to its role in autophagy, and the potential functions of Atg6/Becn1 in other vesicle trafficking pathways for tumor development have not been considered. Here, we generate Atg6 mutant Drosophila and demonstrate that Atg6 is essential for autophagy, endocytosis and protein secretion. By contrast, the core autophagy gene Atg1 is required for autophagy and protein secretion, but it is not required for endocytosis. Unlike null mutants of other core autophagy genes, all Atg6 mutant animals possess blood cell masses. Atg6 mutants have enlarged lymph glands (the hematopoietic organ in Drosophila), possess elevated blood cell numbers, and the formation of melanotic blood cell masses in these mutants is not suppressed by mutations in either p62 or NF?B genes. Thus, like mammals, altered Atg6 function in flies causes hematopoietic abnormalities and lethality, and our data indicate that this is due to defects in multiple membrane trafficking processes.

Shravage, Bhupendra V.; Hill, Jahda H.; Powers, Christine M.; Wu, Louisa; Baehrecke, Eric H.

2013-01-01

4

Multiple Pathways for All Students  

ERIC Educational Resources Information Center

Maine has been focusing on the importance of postsecondary training. Maine's Skowhegan Area High School (SAHS) and Somerset Career and Technical Center (SCTC) have partnered in a Multiple Pathways initiative (funded by the Nellie Mae Education Foundation) to increase students' high school completion rate and to increase enrollment in postsecondary…

Stirling, Lee Anna

2012-01-01

5

rugose (rg), a Drosophila A kinase anchor protein, is required for retinal pattern formation and interacts genetically with multiple signaling pathways.  

PubMed Central

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways.

Shamloula, Hoda K; Mbogho, Mkajuma P; Pimentel, Angel C; Chrzanowska-Lightowlers, Zosia M A; Hyatt, Vanneta; Okano, Hideyuki; Venkatesh, Tadmiri R

2002-01-01

6

On the Time Required to Perform Multiplication  

Microsoft Academic Search

The time required to perform multiplication is investigated. A lower bound on the time required to perform multiplication, as well as multiplication modulo N, is derived and it is shown that these lower bounds can be approached. Then a lower bound on the amount of time required to perform the most significant part of multiplication (⌞xy\\/N⌟) is derived.

Shmuel Winograd; Yorktown Heights

1967-01-01

7

Demonstration of differential quantitative requirements for NSF among multiple vesicle fusion pathways of GLUT4 using a dominant-negative ATPase-deficient NSF  

SciTech Connect

In this study, we investigated the relative participation of N-ethylmaleimide-sensitive factor (NSF) in vivo in a complex multistep vesicle trafficking system, the translocation response of GLUT4 to insulin in rat adipose cells. Transfections of rat adipose cells demonstrate that over-expression of wild-type NSF has no effect on total, or basal and insulin-stimulated cell-surface expression of HA-tagged GLUT4. In contrast, a dominant-negative NSF (NSF-D1EQ) can be expressed at a low enough level that it has little effect on total HA-GLUT4, but does reduce both basal and insulin-stimulated cell-surface HA-GLUT4 by {approx}50% without affecting the GLUT4 fold-translocation response to insulin. However, high expression levels of NSF-D1EQ decrease total HA-GLUT4. The inhibitory effect of NSF-D1EQ on cell-surface HA-GLUT4 is reversed when endocytosis is inhibited by co-expression of a dominant-negative dynamin (dynamin-K44A). Moreover, NSF-D1EQ does not affect cell-surface levels of constitutively recycling GLUT1 and TfR, suggesting a predominant effect of low-level NSF-D1EQ on the trafficking of GLUT4 from the endocytic recycling compared to the intracellular GLUT4-specific compartment. Thus, our data demonstrate that the multiple fusion steps in GLUT4 trafficking have differential quantitative requirements for NSF activity. This indicates that the rates of plasma and intracellular membrane fusion reactions vary, leading to differential needs for the turnover of the SNARE proteins.

Chen Xiaoli [Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, NIDDK, NIH, Bethesda, MD (United States); Matsumoto, Hideko [Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, NIDDK, NIH, Bethesda, MD (United States); Hinck, Cynthia S. [Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, NIDDK, NIH, Bethesda, MD (United States); Al-Hasani, Hadi [Institute of Biochemistry, University of Cologne, Cologne (Germany); St-Denis, Jean-Francois [Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, NIDDK, NIH, Bethesda, MD (United States); Whiteheart, Sidney W. [Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY (United States); Cushman, Samuel W. [Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, NIDDK, NIH, Bethesda, MD (United States)]. E-mail: sam_cushman@nih.gov

2005-07-22

8

Multiple signaling pathways induced by granulocyte colony-stimulating factor involving activation of JAKs, STAT5, and/or STAT3 are required for regulation of three distinct classes of immediate early genes.  

PubMed

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of proliferation and differentiation of neutrophilic granulocyte precursor cells. G-CSF activates multiple signaling molecules, including the JAK1 and JAK2 kinases and the STAT transcription factors. To investigate G-CSF signaling events regulated by the JAK-STAT pathway, we have generated UT7-epo cells stably expressing either wild-type (wt) G-CSF receptor or a series of C-terminal deletion mutants. Gel mobility shift and immunoprecipitation/Western analysis showed that STAT5 is rapidly activated by G-CSF in cells expressing the wt G-CSF receptor, in addition to the previously reported STAT3 and STAT1. Mutants lacking any tyrosine residues in the cytoplasmic domain maintain their ability to activate STAT5 and STAT1 but cannot activate STAT3, implying that STAT5 and STAT1 activation does not require receptor tyrosine phosphorylation. We also observed significant changes in the ratio of STAT1:STAT3:STAT5 activated by various G-CSF receptor C-terminal deletion mutants. These mutant receptors were further used to investigate the role of JAKs and STATs in G-CSF-mediated responses in these cells. We found that JAK activation correlates with G-CSF-induced cell proliferation, whereas STAT activation is not required. We have also identified three classes of G-CSF immediate early genes, whose activation correlates with the activation of distinct JAK-STAT pathways. Our data show that, whereas c-fos is regulated through a pathway independent of STAT activation, oncostatin M, IRF-1, and egr-1 are regulated by an STAT5-dependent pathway and fibrinogen is regulated by an STAT3-dependent pathway. In conclusion, our results suggest that G-CSF regulates its complex biologic activities by selectively activating distinct early response genes through different JAK-STAT signaling molecules. PMID:8977235

Tian, S S; Tapley, P; Sincich, C; Stein, R B; Rosen, J; Lamb, P

1996-12-15

9

Complementary identification of multiple fluxdistributions and multiple metabolic pathways  

Microsoft Academic Search

Cell robustness and complexity have been recognized as unique features of biological systems. Such robustness and complexity of metabolic-reaction systems can be explored by discovering, or identifying, the multiple flux distributions (MFD) and redundant pathways that lead to a given external state; however, this is exceedingly cumbersome to accomplish. It is, therefore, highly desirable to establish an effective computational method

Dong-Yup Lee; L. T. Fan; Sang Yup Lee; Shahram Shafie; Botond Bertok; Ferenc Friedler

10

Beyond Tracking: Multiple Pathways to College, Career, and Civic Participation  

ERIC Educational Resources Information Center

"Beyond Tracking" responds to the a sobering assessment of American high schools by delineating and promoting an innovative and well-defined notion of multiple pathways. The book's authors clearly distinguish their use of the term "multiple pathways" from any updated version of the tracking system that marked so many American high schools during…

Oakes, Jeannie, Ed.; Saunders, Marisa, Ed.

2008-01-01

11

Janus kinases: components of multiple signaling pathways  

Microsoft Academic Search

Cytoplasmic Janus protein tyrosine kinases (JAKs) are crucial components of diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. Evidence to date, indicates that JAK kinase function may integrate components of diverse signaling cascades. While it is likely that activation of STAT proteins may be an important function attributed to the JAK kinases, it is certainly not

Sushil G Rane; E Premkumar Reddy

2000-01-01

12

Multiple-camera tracking: UK government requirements  

NASA Astrophysics Data System (ADS)

The Imagery Library for Intelligent Detection Systems (i-LIDS) is the UK government's new standard for Video Based Detection Systems (VBDS). The standard was launched in November 2006 and evaluations against it began in July 2007. With the first four i-LIDS scenarios completed, the Home Office Scientific development Branch (HOSDB) are looking toward the future of intelligent vision in the security surveillance market by adding a fifth scenario to the standard. The fifth i-LIDS scenario will concentrate on the development, testing and evaluation of systems for the tracking of people across multiple cameras. HOSDB and the Centre for the Protection of National Infrastructure (CPNI) identified a requirement to track targets across a network of CCTV cameras using both live and post event imagery. The Detection and Vision Systems group at HOSDB were asked to determine the current state of the market and develop an in-depth Operational Requirement (OR) based on government end user requirements. Using this OR the i-LIDS team will develop a full i-LIDS scenario to aid the machine vision community in its development of multi-camera tracking systems. By defining a requirement for multi-camera tracking and building this into the i-LIDS standard the UK government will provide a widely available tool that developers can use to help them turn theory and conceptual demonstrators into front line application. This paper will briefly describe the i-LIDS project and then detail the work conducted in building the new tracking aspect of the standard.

Hosmer, Paul

2007-10-01

13

Endocytic pathway is required for Drosophila Toll innate immune signaling  

PubMed Central

The Toll signaling pathway is required for the innate immune response against fungi and Gram-positive bacteria in Drosophila. Here we show that the endosomal proteins Myopic (Mop) and Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) are required for the activation of the Toll signaling pathway. This requirement is observed in cultured cells and in flies, and epistasis experiments show that the Mop protein functions upstream of the MyD88 adaptor and the Pelle kinase. Mop and Hrs, which are critical components of the ESCRT-0 endocytosis complex, colocalize with the Toll receptor in endosomes. We conclude that endocytosis is required for the activation of the Toll signaling pathway.

Huang, Hon-Ren; Chen, Zhijian J.; Kunes, Sam; Chang, Geen-Dong; Maniatis, Tom

2010-01-01

14

Simultaneous Identification of Multiple Driver Pathways in Cancer  

PubMed Central

Distinguishing the somatic mutations responsible for cancer (driver mutations) from random, passenger mutations is a key challenge in cancer genomics. Driver mutations generally target cellular signaling and regulatory pathways consisting of multiple genes. This heterogeneity complicates the identification of driver mutations by their recurrence across samples, as different combinations of mutations in driver pathways are observed in different samples. We introduce the Multi-Dendrix algorithm for the simultaneous identification of multiple driver pathways de novo in somatic mutation data from a cohort of cancer samples. The algorithm relies on two combinatorial properties of mutations in a driver pathway: high coverage and mutual exclusivity. We derive an integer linear program that finds set of mutations exhibiting these properties. We apply Multi-Dendrix to somatic mutations from glioblastoma, breast cancer, and lung cancer samples. Multi-Dendrix identifies sets of mutations in genes that overlap with known pathways – including Rb, p53, PI(3)K, and cell cycle pathways – and also novel sets of mutually exclusive mutations, including mutations in several transcription factors or other genes involved in transcriptional regulation. These sets are discovered directly from mutation data with no prior knowledge of pathways or gene interactions. We show that Multi-Dendrix outperforms other algorithms for identifying combinations of mutations and is also orders of magnitude faster on genome-scale data. Software available at: http://compbio.cs.brown.edu/software.

Leiserson, Mark D. M.; Blokh, Dima

2013-01-01

15

Multiple Cholinergic Signaling Pathways in Pituitary Gonadotrophs  

PubMed Central

Acetylcholine (ACh) has been established as a paracrine factor in the anterior pituitary gland, but the receptors mediating ACh action and the cell types bearing these receptors have not been identified. Our results showed that the expression of the nicotinic subunits mRNAs followed the order ?2 > ?1 = ?9 > ?4 in cultured rat pituitary cells. The expression of the subunits in immortalized L?T2 mouse gonadotrophs followed the order ?2 > ?4 = ?1. M4 > M3 muscarinic receptor mRNA were also identified in pituitary and L?T2 cells. The treatment of cultured pituitary cells with GnRH down-regulated the expression of ?9 and ?4 mRNAs, without affecting the expression of M3 and M4 receptor mRNAs, and ACh did not alter the expression of GnRH receptor mRNA. We also performed double immunostaining to show the expression of ?2-subunit and M4 receptor proteins in gonadotrophs. Functional nicotinic channels capable of generating an inward current, facilitation of electrical activity, and Ca2+ influx were identified in single gonadotrophs and L?T2 cells. In both cell types, the M3 receptor-mediated, phospholipase C-dependent Ca2+ mobilization activated an outward apamin-sensitive K+ current and caused hyperpolarization. The activation of M4 receptors by ACh inhibited cAMP production and GnRH-induced LH release in a pertussis toxin-sensitive manner. We concluded that multiple cholinergic receptors are expressed in gonadotrophs and that the main secretory action of ACh is inhibitory through M4 receptor-mediated down-regulation of cAMP production. The expression of nicotinic receptors in vitro compensates for the lack of regular GnRH stimulation of gonadotrophs.

Kucka, Marek; Bjelobaba, Ivana; Tomic, Melanija

2013-01-01

16

The Frizzled family: receptors for multiple signal transduction pathways  

Microsoft Academic Search

SUMMARY: Frizzled genes encode integral membrane proteins that function in multiple signal transduction pathways. They have been identified in diverse animals, from sponges to humans. The family is defined by conserved structural features, including seven hydrophobic domains and a cysteine-rich ligand-binding domain. Frizzled proteins are receptors for secreted Wnt proteins, as well as other ligands, and also play a critical

Hui-Chuan Huang; Peter S Klein

2004-01-01

17

Costs of California Multiple Pathway Programs. Policy Report  

ERIC Educational Resources Information Center

There is widespread agreement that many of California's high schools are doing a poor job of preparing their students for college and careers. The James Irvine Foundation is sponsoring a major initiative to develop "Multiple Pathways"--now called the Linked Learning approach--as a strategy for improving the performance of California high schools.…

Parsi, Ace; Plank, David; Stern, David

2010-01-01

18

Electrophysiology of motor pathways for sphincter control in multiple sclerosis.  

PubMed

The central and peripheral motor pathways serving striated sphincter muscle function were studied using cortical and lumbar transcutaneous electrical stimulation, pudendal nerve stimulation and sphincter electromyography in 23 patients with multiple sclerosis (MS), and sphincter disturbance, including incontinence of urine or faeces, urinary voiding dysfunction, or constipation. The central motor conduction time was significantly increased in the MS group compared to controls (p less than 0.05). Damage to both the upper and lower motor neuron pathways can contribute to sphincter disturbance in MS. The latter may be due to coexisting pathology or to involvement of the conus medullaris by MS. PMID:2178181

Mathers, S E; Ingram, D A; Swash, M

1990-11-01

19

Raf/MEK/ERK pathway activation is required for Junín virus replication.  

PubMed

In the present work we investigated the importance of the Raf/MEK/ERK signalling pathway in the multiplication of the arenavirus Junín (JUNV) in monkey and human cell cultures. We established that JUNV induces a biphasic activation of ERK and we proved that a specific inhibitor of the ERK pathway, U0126, impairs viral replication. Furthermore, U0126 exerted inhibitory action against the arenaviruses Tacaribe and Pichinde. Moreover, treatment with known ERK activators such as phorbol 12-myristate 13-acetate and serum increased viral yields whereas ERK silencing by small interfering RNAs caused the inhibition of viral multiplication. Therefore, activation of the Raf/MEK/ERK signalling pathway is required to ensure efficient JUNV replication and may constitute a host target for the development of novel effective therapeutic strategies to deal with arenavirus infections. PMID:24421112

Rodríguez, María Eugenia; Brunetti, Jesús Emanuel; Wachsman, Mónica Beatriz; Scolaro, Luis Alberto; Castilla, Viviana

2014-04-01

20

Tocotrienols fight cancer by targeting multiple cell signaling pathways  

Microsoft Academic Search

Cancer cells are distinguished by several distinct characteristics, such as self-sufficiency in growth signal, resistance\\u000a to growth inhibition, limitless replicative potential, evasion of apoptosis, sustained angiogenesis, and tissue invasion and\\u000a metastasis. Tumor cells acquire these properties due to the dysregulation of multiple genes and associated cell signaling\\u000a pathways, most of which are linked to inflammation. For that reason, rationally designed

Ramaswamy Kannappan; Subash C. Gupta; Ji Hye Kim; Bharat B. Aggarwal

21

?-Haloarylsulfonamides: Multiple Cyclization Pathways to Skeletally Diverse Benzofused Sultams  

PubMed Central

The development of new methods to skeletally diverse sultams based on a central ?-halo benzene sulfonamide building block is reported. Several salient features of this building block are utilized in multiple reaction pathways, including the Heck reaction, C- and O-arylation, Sonogashira-Pauson-Khand, Sonogashira-intramolecular hydroamination, lithiative cyclization and domino aza-Michael Heck for the generation of 5-, 6- and 7-membered benzofused bicyclic and tricyclic sultams.

Rayabarapu, Dinesh Kumar; Zhou, Aihua; Jeon, Kyu Ok; Samarakoon, Thiwanka; Rolfe, Alan; Siddiqui, Hina

2009-01-01

22

Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?  

NASA Technical Reports Server (NTRS)

PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

Lecker, Stewart H.; Goldberg, A. L. (Principal Investigator)

2003-01-01

23

Multiple parietal-frontal pathways mediate grasping in macaque monkeys  

PubMed Central

The nodes of a parietal-frontal pathway that mediates grasping in primates are in anterior intraparietal area (AIP) and ventral premotor cortex (PMv). Nevertheless, multiple somatosensory and motor representations of the hand, respectively in parietal and frontal cortex, suggest that additional pathways remain unrealized. We explored this possibility in macaque monkeys by injecting retrograde tracers into grasp zones identified in M1, PMv, and area 2 with long train electrical stimulation. The M1 grasp zone was densely connected with other frontal cortex motor regions. The remainder of the connections originated from somatosensory areas 3a and S2/PV, and from the medial bank and fundus of the intraparietal sulcus (IPS). The PMv grasp zone was also densely connected with frontal cortex motor regions, albeit to a lesser extent than the M1 grasp zone. The remainder of the connections originated from areas S2/PV and aspects of the inferior parietal lobe such as PF, PFG, AIP, and the tip of the IPS. The area 2 grasp zone was densely connected with the hand representations of somatosensory areas 3b, 1, and S2/PV. The remainder of the connections was with areas 3a and 5 and the medial bank and fundus of the IPS. Connections with frontal cortex were relatively weak and concentrated in caudal M1. Thus, the three grasp zones may be nodes of parallel parietal-frontal pathways. Differential points of origin and termination of each pathway suggest varying functional specializations. Direct and indirect connections between those parietal-frontal pathways likely coordinate their respective functions into an accurate grasp.

Gharbawie, Omar A.; Stepniewska, Iwona; Qi, Huixin; Kaas, Jon H.

2011-01-01

24

Mycobacterial Esx-3 requires multiple components for iron acquisition.  

PubMed

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ?mycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ?mycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems. IMPORTANCE Mycobacteria have several paralogous type VII secretion systems, Esx-1 through Esx-5. Whereas Esx-1 is required for pathogenic mycobacteria to grow within an infected host, Esx-3 is essential for growth in vitro. We and others have shown that Esx-3 is required for siderophore-mediated iron acquisition. In this work, we identify individual Esx-3 components that contribute to this process. As in the Esx-1 system, most mutations that abolish Esx-3 protein export also disrupt its function. Unexpectedly, however, ultrasensitive quantitation of Esx-3 secretion by multiple-reaction-monitoring mass spectrometry (MRM-MS) revealed that very low levels of export were sufficient for iron acquisition under similar conditions. Although protein export clearly contributes to type VII function, the relationship is not absolute. PMID:24803520

Siegrist, M Sloan; Steigedal, Magnus; Ahmad, Rushdy; Mehra, Alka; Dragset, Marte S; Schuster, Brian M; Philips, Jennifer A; Carr, Steven A; Rubin, Eric J

2014-01-01

25

Mask is required for the activity of the Hippo pathway effector Yki/YAP  

PubMed Central

Summary The Drosophila Yorkie (Yki) protein and its mammalian homolog Yes-associated protein (YAP) are potent growth promoters and YAP overexpression is associated with multiple types of cancer [1,2]. Yki and YAP are transcriptional co-activators and function as downstream effectors of the Hippo tumor suppressor pathway [1–4]. The regulation of Yki and YAP by the Hippo signaling pathway has been extensively investigated, however, how they regulate gene expression is poorly understood. To identify additional regulators of Yki activity we performed a genome-wide RNAi screen in Drosophila S2 cells. In this screen, we identified the conserved protein Mask (Multiple ankyrin repeats single KH domain) as a novel promoter of Yki activity in vitro and validated this function in vivo in Drosophila. We found that Mask is required downstream of the Hippo pathway for Yki to induce target gene expression and that Mask forms complexes with Yki. The human Mask homolog MASK1 complexes with YAP and it is required for full activity of YAP and elevated MASK1 expression is associated with worsened outcomes for breast cancer patients. We conclude that Mask is a novel cofactor for Yki/YAP required for optimal Yki/YAP activity during development and oncogenesis.

Sansores-Garcia, Leticia; Atkins, Mardelle; Moya, Ivan M.; Shahmoradgoli, Maria; Tao, Chunyao; Mills, Gordon B.; Halder, Georg

2014-01-01

26

Evidence for Multiple Pathways to Deuterium Enhancements in Protoplanetary Disks  

NASA Astrophysics Data System (ADS)

The distributions of deuterated molecules in protoplanetary disks are expected to depend on the molecular formation pathways. We use observations of spatially resolved DCN emission from the disk around TW Hya, acquired during ALMA science verification with a ~3'' synthesized beam, together with comparable DCO+ observations from the Submillimeter Array, to investigate differences in the radial distributions of these species and hence differences in their formation chemistry. In contrast to DCO+, which shows an increasing column density with radius, DCN is better fit by a model that is centrally peaked. We infer that DCN forms at a smaller radii and thus at higher temperatures than DCO+. This is consistent with chemical network model predictions of DCO+ formation from H2D+ at T < 30 K and DCN formation from additional pathways involving CH2D+ at higher temperatures. We estimate a DCN/HCN abundance ratio of ~0.017, similar to the DCO+/HCO+ abundance ratio. Deuterium fractionation appears to be efficient at a range of temperatures in this protoplanetary disk. These results suggest caution in interpreting the range of deuterium fractions observed in solar system bodies, as multiple formation pathways should be taken into account.

Öberg, Karin I.; Qi, Chunhua; Wilner, David J.; Hogerheijde, Michiel R.

2012-04-01

27

EVIDENCE FOR MULTIPLE PATHWAYS TO DEUTERIUM ENHANCEMENTS IN PROTOPLANETARY DISKS  

SciTech Connect

The distributions of deuterated molecules in protoplanetary disks are expected to depend on the molecular formation pathways. We use observations of spatially resolved DCN emission from the disk around TW Hya, acquired during ALMA science verification with a {approx}3'' synthesized beam, together with comparable DCO{sup +} observations from the Submillimeter Array, to investigate differences in the radial distributions of these species and hence differences in their formation chemistry. In contrast to DCO{sup +}, which shows an increasing column density with radius, DCN is better fit by a model that is centrally peaked. We infer that DCN forms at a smaller radii and thus at higher temperatures than DCO{sup +}. This is consistent with chemical network model predictions of DCO{sup +} formation from H{sub 2}D{sup +} at T < 30 K and DCN formation from additional pathways involving CH{sub 2}D{sup +} at higher temperatures. We estimate a DCN/HCN abundance ratio of {approx}0.017, similar to the DCO{sup +}/HCO{sup +} abundance ratio. Deuterium fractionation appears to be efficient at a range of temperatures in this protoplanetary disk. These results suggest caution in interpreting the range of deuterium fractions observed in solar system bodies, as multiple formation pathways should be taken into account.

Oeberg, Karin I.; Qi, Chunhua; Wilner, David J. [Harvard-Smithsonian Center for Astrophysics, 60 Garden Street, Cambridge, MA 02138 (United States); Hogerheijde, Michiel R., E-mail: koberg@cfa.harvard.edu [Leiden Observatory, Leiden University, P.O. Box 9513, 2300 RA Leiden (Netherlands)

2012-04-20

28

Reovirus Uses Multiple Endocytic Pathways for Cell Entry  

PubMed Central

Entry of reovirus virions has been well studied in several tissue culture systems. After attachment to junctional adhesion molecule A (JAM-A), virions undergo clathrin-mediated endocytosis followed by proteolytic disassembly of the capsid and penetration to the cytoplasm. However, during in vivo infection of the intestinal tract, and likely in the tumor microenvironment, capsid proteolysis (uncoating) is initiated extracellularly. We used multiple approaches to determine if uncoated reovirus particles, called intermediate subviral particles (ISVPs), enter cells by directly penetrating the limiting membrane or if they take advantage of endocytic pathways to establish productive infection. We found that entry and infection by reovirus ISVPs was inhibited by dynasore, an inhibitor of dynamin-dependent endocytosis, as well as by genistein and dominant-negative caveolin-1, which block caveolar endocytosis. Inhibition of caveolar endocytosis also reduced infection by reovirus virions. Extraction of membrane cholesterol with methyl-?-cyclodextrin inhibited infection by virions but had no effect when infection was initiated with ISVPs. We found this pathway to be independent of both clathrin and caveolin. Together, these data suggest that reovirus virions can use both dynamin-dependent and dynamin-independent endocytic pathways during cell entry, and they reveal that reovirus ISVPs can take advantage of caveolar endocytosis to establish productive infection.

Schulz, Wade L.; Haj, Amelia K.

2012-01-01

29

Targeting multiple key signaling pathways in melanoma using leelamine.  

PubMed

Melanoma is a highly drug-resistant cancer with resistance developing to agents targeting single proteins. To circumvent this problem, a new class of agent inhibiting multiple key pathways important in this disease is being developed to reduce the likelihood of developing resistant disease. The phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and STAT3 pathways are constitutively activated in 50% to 70% of melanomas, promoting disease development. To identify a drug simultaneously targeting the PI3K, MAPK, and STAT3 cascades, a natural product library was screened to identify leelamine as a potential inhibitor. Leelamine was 4.5-fold more effective at inhibiting cultured melanoma cell survival than normal cells, with average IC50 values of 2 and 9.3 ?mol/L, respectively. It inhibited cellular proliferation at a concentration of 2.5 ?mol/L by 40% to 80% and longer exposure increased apoptosis 600%. Leelamine inhibited the growth of preexisting xenografted melanoma tumors by an average of 60% by targeting the PI3K, MAPK, and STAT3 pathways without affecting animal body weight or blood markers of major organ function. The mechanism of action of leelamine is mediated by disruption of cholesterol transport, causing decreased cellular proliferation and consequently leading to increased tumor cell apoptosis as well as decreased tumor vascularization. Thus, a unique agent and novel mechanism of action has been identified for the treatment of melanoma that acts by inhibiting the activity of three major signaling pathways regulating the development of this disease. Mol Cancer Ther; 13(7); 1679-89. ©2014 AACR. PMID:24688050

Gowda, Raghavendra; Madhunapantula, SubbaRao V; Kuzu, Omer F; Sharma, Arati; Robertson, Gavin P

2014-07-01

30

Multiple sweet receptors and transduction pathways revealed in knockout mice by temperature dependence and gurmarin sensitivity.  

PubMed

Sweet taste transduction involves taste receptor type 1, member 2 (T1R2), taste receptor type 1, member 3 (T1R3), gustducin, and TRPM5. Because knockout (KO) mice lacking T1R3, gustducin's Galpha subunit (Galphagust), or TRPM5 exhibited greatly reduced, but not abolished responses of the chorda tympani (CT) nerve to sweet compounds, it is likely that multiple sweet transduction pathways exist. That gurmarin (Gur), a sweet taste inhibitor, inhibits some but not all mouse CT responses to sweet compounds supports the existence of multiple sweet pathways. Here, we investigated Gur inhibition of CT responses to sweet compounds as a function of temperature in KO mice lacking T1R3, Galphagust, or TRPM5. In T1R3-KO mice, responses to sucrose and glucose were Gur sensitive (GS) and displayed a temperature-dependent increase (TDI). In Galphagust-KO mice, responses to sucrose and glucose were Gur-insensitive (GI) and showed a TDI. In TRPM5-KO mice, responses to glucose were GS and showed a TDI. All three KO mice exhibited no detectable responses to SC45647, and their responses to saccharin displayed neither GS nor a TDI. For all three KO mice, the lingual application of pronase, another sweet response inhibitor, almost fully abolished responses to sucrose and glucose but did not affect responses to saccharin. These results provide evidence for 1) the existence of multiple transduction pathways underlying responses to sugars: a T1R3-independent GS pathway for sucrose and glucose, and a TRPM5-independent temperature sensitive GS pathway for glucose; 2) the requirement for Galphagust in GS sweet taste responses; and 3) the existence of a sweet independent pathway for saccharin, in mouse taste cells on the anterior tongue. PMID:19211717

Ohkuri, Tadahiro; Yasumatsu, Keiko; Horio, Nao; Jyotaki, Masafumi; Margolskee, Robert F; Ninomiya, Yuzo

2009-04-01

31

Multiple sweet receptors and transduction pathways revealed in knockout mice by temperature dependence and gurmarin sensitivity  

PubMed Central

Sweet taste transduction involves taste receptor type 1, member 2 (T1R2), taste receptor type 1, member 3 (T1R3), gustducin, and TRPM5. Because knockout (KO) mice lacking T1R3, gustducin's G? subunit (G?gust), or TRPM5 exhibited greatly reduced, but not abolished responses of the chorda tympani (CT) nerve to sweet compounds, it is likely that multiple sweet transduction pathways exist. That gurmarin (Gur), a sweet taste inhibitor, inhibits some but not all mouse CT responses to sweet compounds supports the existence of multiple sweet pathways. Here, we investigated Gur inhibition of CT responses to sweet compounds as a function of temperature in KO mice lacking T1R3, G?gust, or TRPM5. In T1R3-KO mice, responses to sucrose and glucose were Gur sensitive (GS) and displayed a temperature-dependent increase (TDI). In G?gust-KO mice, responses to sucrose and glucose were Gur-insensitive (GI) and showed a TDI. In TRPM5-KO mice, responses to glucose were GS and showed a TDI. All three KO mice exhibited no detectable responses to SC45647, and their responses to saccharin displayed neither GS nor a TDI. For all three KO mice, the lingual application of pronase, another sweet response inhibitor, almost fully abolished responses to sucrose and glucose but did not affect responses to saccharin. These results provide evidence for 1) the existence of multiple transduction pathways underlying responses to sugars: a T1R3-independent GS pathway for sucrose and glucose, and a TRPM5-independent temperature sensitive GS pathway for glucose; 2) the requirement for G?gust in GS sweet taste responses; and 3) the existence of a sweet independent pathway for saccharin, in mouse taste cells on the anterior tongue.

Ohkuri, Tadahiro; Yasumatsu, Keiko; Horio, Nao; Jyotaki, Masafumi; Margolskee, Robert F.; Ninomiya, Yuzo

2009-01-01

32

Assessing multiple pathway exposures: Variability, uncertainty, and ignorance  

SciTech Connect

Human populations contact environmental pollutants through food, water, and air in varying amounts each day throughout a lifetime. Thus, a realistic strategy for managing the potential health risks of municipal incinerator emissions requires a comprehensive approach with adequate attention to uncertainties. Using contaminant transfers from air to food as a case study, this paper considers two important issues in exposure assessment--completeness of the exposure model and the treatment of uncertainty in exposure estimates. This case study is used to distinguish between variability (inherent randomness in data), ignorance (incomplete data and/or lack of scientific understanding) and uncertainty (the variance in exposure estimates attributable to the combination of variability and ignorance). For the air/food pathways, I explore the use of pathway exposure factors (PEFs) that combine information on environmental partitioning ( fugacity,'' biotransfer factors, deposition, etc.) with data on human diet, behavior patterns, and physiology into a numerical expression that links ambient air concentrations in mg/m{sup 3} into daily exposure in mg/kg-d. Following EPA protocol, exposure expresses human contact with contaminants through the lungs, the gut wall, and skin surface. I describe and assess the uncertainty for exposure estimates of incinerator emissions through the air/milk and air/meat pathways. I consider the advantages and disadvantages of various methods for propagating and analyzing uncertainties. 23 refs., 4 figs., 2 tabs.

McKone, T.E.

1989-08-01

33

Multiple Pathways Promote Dynamical Coupling between Catalytic Domains in Escherichia coli Prolyl-tRNA Synthetase  

PubMed Central

Aminoacyl-tRNA synthetases are multi-domain enzymes that catalyze covalent attachment of amino acids to their cognate tRNA. Cross-talk between functional domains is a prerequisite for this process. In the present study, we investigate the molecular mechanism of site-to-site communication in Escherichia coli prolyl-tRNA synthetase (Ec ProRS). Earlier studies have demonstrated that evolutionarily conserved/co-evolved residues that are engaged in correlated motion are critical for the propagation of functional conformational changes from one site to another in modular proteins. Here, molecular simulation and bioinformatics-based analysis was performed to identify dynamically coupled and evolutionarily constrained residues that form contiguous pathways of residue-residue interactions between the aminoacylation and editing domains of Ec ProRS. The results of this study suggest that multiple pathways exist between these two domains to maintain the dynamic coupling essential for enzyme function. Moreover, residues in these interaction networks are generally highly conserved. Site-directed changes of on-pathway residues have a significant impact on enzyme function and dynamics suggesting that any perturbation along these pathways disrupts the native residue-residue interactions that are required for effective communication between the two functional domains. Free energy analysis revealed that communication between residues within a pathway, as well as cross-talk between pathways are important to coordinate functions of different domains of Ec ProRS for efficient catalysis.

Johnson, James M.; Sanford, Brianne L.; Strom, Alexander M.; Tadayon, Stephanie N.; Lehman, Brent P.; Zirbes, Arrianna M.; Bhattacharyya, Sudeep; Musier-Forsyth, Karin; Hati, Sanchita

2013-01-01

34

BH3 mimetics activate multiple pro-autophagic pathways.  

PubMed

The BH3 mimetic ABT737 induces autophagy by competitively disrupting the inhibitory interaction between the BH3 domain of Beclin 1 and the anti-apoptotic proteins Bcl-2 and Bcl-X(L), thereby stimulating the Beclin 1-dependent allosteric activation of the pro-autophagic lipid kinase VPS34. Here, we examined whether ABT737 stimulates other pro-autophagic signal-transduction pathways. ABT737 caused the activating phosphorylation of AMP-dependent kinase (AMPK) and of the AMPK substrate acetyl CoA carboxylase, the activating phosphorylation of several subunits of the inhibitor of NF-?B (I?B) kinase (IKK) and the hyperphosphorylation of the IKK substrate I?B, inhibition of the activity of mammalian target of rapamycin (mTOR) and consequent dephosphorylation of the mTOR substrate S6 kinase. In addition, ABT737 treatment dephosphorylates (and hence likewise inhibits) p53, glycogen synthase kinase-3 and Akt. All these effects were shared by ABT737 and another structurally unrelated BH3 mimetic, HA14-1. Functional experiments revealed that pharmacological or genetic inhibition of IKK, Sirtuin and the p53-depleting ubiquitin ligase MDM2 prevented ABT737-induced autophagy. These results point to unexpected and pleiotropic pro-autophagic effects of BH3 mimetics involving the modulation of multiple signalling pathways. PMID:21460857

Malik, S A; Orhon, I; Morselli, E; Criollo, A; Shen, S; Mariño, G; BenYounes, A; Bénit, P; Rustin, P; Maiuri, M C; Kroemer, G

2011-09-15

35

Rac3-Mediated Transformation Requires Multiple Effector Pathways  

Microsoft Academic Search

Ourinitial characterizationof Rac3, aclose relative ofthesmall GTPase Rac1, established its ability to promote membrane ruffling, transformation, and activation of c-jun transcription- al activity. The finding that Rac3 is transforming, and its similarity to Rac1, a protein that has a well-established connection to many processes important for cancer progres- sion, prompted further investigation into Rac3 transformation. We used effector domain mutants

Patricia J. Keller; Michele R. Wing

2005-01-01

36

Estrogen Signaling Multiple Pathways to Impact Gene Transcription  

PubMed Central

Steroid hormones exert profound effects on cell growth, development, differentiation, and homeostasis. Their effects are mediated through specific intracellular steroid receptors that act via multiple mechanisms. Among others, the action mechanism starting upon 17?-estradiol (E2) binds to its receptors (ER) is considered a paradigmatic example of how steroid hormones function. Ligand-activated ER dimerizes and translocates in the nucleus where it recognizes specific hormone response elements located in or near promoter DNA regions of target genes. Behind the classical genomic mechanism shared with other steroid hormones, E2 also modulates gene expression by a second indirect mechanism that involves the interaction of ER with other transcription factors which, in turn, bind their cognate DNA elements. In this case, ER modulates the activities of transcription factors such as the activator protein (AP)-1, nuclear factor-?B (NF-?B) and stimulating protein-1 (Sp-1), by stabilizing DNA-protein complexes and/or recruiting co-activators. In addition, E2 binding to ER may also exert rapid actions that start with the activation of a variety of signal transduction pathways (e.g. ERK/MAPK, p38/MAPK, PI3K/AKT, PLC/PKC). The debate about the contribution of different ER-mediated signaling pathways to coordinate the expression of specific sets of genes is still open. This review will focus on the recent knowledge about the mechanism by which ERs regulate the expression of target genes and the emerging field of integration of membrane and nuclear receptor signaling, giving examples of the ways by which the genomic and non-genomic actions of ERs on target genes converge.

Marino, Maria; Galluzzo, Paola; Ascenzi, Paolo

2006-01-01

37

Meta-analysis for pathway enrichment analysis when combining multiple genomic studies  

PubMed Central

Motivation: Many pathway analysis (or gene set enrichment analysis) methods have been developed to identify enriched pathways under different biological states within a genomic study. As more and more microarray datasets accumulate, meta-analysis methods have also been developed to integrate information among multiple studies. Currently, most meta-analysis methods for combining genomic studies focus on biomarker detection and meta-analysis for pathway analysis has not been systematically pursued. Results: We investigated two approaches of meta-analysis for pathway enrichment (MAPE) by combining statistical significance across studies at the gene level (MAPE_G) or at the pathway level (MAPE_P). Simulation results showed increased statistical power of meta-analysis approaches compared to a single study analysis and showed complementary advantages of MAPE_G and MAPE_P under different scenarios. We also developed an integrated method (MAPE_I) that incorporates advantages of both approaches. Comprehensive simulations and applications to real data on drug response of breast cancer cell lines and lung cancer tissues were evaluated to compare the performance of three MAPE variations. MAPE_P has the advantage of not requiring gene matching across studies. When MAPE_G and MAPE_P show complementary advantages, the hybrid version of MAPE_I is generally recommended. Availability: http://www.biostat.pitt.edu/bioinfo/ Contact: ctseng@pitt.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Shen, Kui; Tseng, George C.

2010-01-01

38

Multiple pathways regulate 3' overhang generation at S. cerevisiae telomeres.  

PubMed

Generation of 3' G strand overhangs at telomere ends may play a role in regulating telomerase action and occurs by still unclear mechanisms. We show by an inducible short telomere assay that Sae2 and the Sgs1 RecQ helicase control two distinct but partially complementary pathways for nucleolytic processing of S. cerevisiae telomeres, with Sae2 function requiring its serine 267 phosphorylation. No processing activity is detectable in sae2Delta sgs1Delta cells, while the Exo1 exonuclease contributes to telomere end processing and elongation in both sae2Delta and sgs1Delta cells, suggesting that Exo1 telomeric function requires either Sgs1 or Sae2 action. Moreover, Dna2 might also support Sgs1 activity, as it acts redundantly with Exo1, but not with Sgs1. Finally, both length maintenance and G strand overhang generation at native telomeres are affected in sae2Delta sgs1Delta cells, further supporting the notion that Sae2 and Sgs1 combined activities control telomere length by regulating telomere processing. PMID:19595717

Bonetti, Diego; Martina, Marina; Clerici, Michela; Lucchini, Giovanna; Longhese, Maria Pia

2009-07-10

39

ErbB2-Dependent Chemotaxis Requires Microtubule Capture and Stabilization Coordinated by Distinct Signaling Pathways  

PubMed Central

Activation of the ErbB2 receptor tyrosine kinase stimulates breast cancer cell migration. Cell migration is a complex process that requires the synchronized reorganization of numerous subcellular structures including cell-to-matrix adhesions, the actin cytoskeleton and microtubules. How the multiple signaling pathways triggered by ErbB2 coordinate, in time and space, the various processes involved in cell motility, is poorly defined. We investigated the mechanism whereby ErbB2 controls microtubules and chemotaxis. We report that activation of ErbB2 increased both cell velocity and directed migration. Impairment of the Cdc42 and RhoA GTPases, but not of Rac1, prevented the chemotactic response. RhoA is a key component of the Memo/ACF7 pathway whereby ErbB2 controls microtubule capture at the leading edge. Upon Memo or ACF7 depletion, microtubules failed to reach the leading edge and cells lost their ability to follow the chemotactic gradient. Constitutive ACF7 targeting to the membrane in Memo-depleted cells reestablished directed migration. ErbB2-mediated activation of phospholipase C gamma (PLC?) also contributed to cell guidance. We further showed that PLC? signaling, via classical protein kinases C, and Memo signaling converged towards a single pathway controlling the microtubule capture complex. Finally, inhibiting the PI3K/Akt pathway did not affect microtubule capture, but disturbed microtubule stability, which also resulted in defective chemotaxis. PI3K/Akt-dependent stabilization of microtubules involved repression of GSK3 activity on the one hand and inhibition of the microtubule destabilizing protein, Stathmin, on the other hand. Thus, ErbB2 triggers distinct and complementary pathways that tightly coordinate microtubule capture and microtubule stability to control chemotaxis.

Benseddik, Khedidja; Sen Nkwe, Nadine; Daou, Pascale; Verdier-Pinard, Pascal; Badache, Ali

2013-01-01

40

Multiple sensors ensure guide strand selection in human RNAi pathways  

PubMed Central

Small RNAs guide RNA-induced silencing complexes (RISCs) to bind to cognate mRNA transcripts and trigger silencing of protein expression during RNA interference (RNAi) in eukaryotes. A fundamental aspect of this process is the asymmetric loading of one strand of a short interfering RNA (siRNA) or microRNA (miRNA) duplex onto RISCs for correct target recognition. Here, we use a reconstituted system to determine the extent to which the core components of the human RNAi machinery contribute to RNA guide strand selection. We show that Argonaute2 (Ago2), the endonuclease that binds directly to siRNAs and miRNAs within RISC, has intrinsic but substrate-dependent RNA strand selection capability. This activity can be enhanced substantially when Ago2 is in complex with the endonuclease Dicer and the double-stranded RNA-binding proteins (dsRBPs)—trans-activation response (TAR) RNA-binding protein (TRBP) or protein activator of PKR (PACT). The extent to which human Dicer/dsRBP complexes contribute to strand selection is dictated by specific duplex parameters such as thermodynamics, 5? nucleotide identity, and structure. Surprisingly, our results also suggest that strand selection for some miRNAs is enhanced by PACT-containing complexes but not by those containing TRBP. Furthermore, overall mRNA targeting by miRNAs is disfavored for complexes containing TRBP but not PACT. These findings demonstrate that multiple proteins collaborate to ensure optimal strand selection in humans and reveal the possibility of delineating RNAi pathways based on the presence of TRBP or PACT.

Noland, Cameron L.; Doudna, Jennifer A.

2013-01-01

41

Multiple genetic pathways for restarting DNA replication forks in Escherichia coli K-12.  

PubMed Central

In Escherichia coli, the primosome assembly proteins, PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG, are thought to help to restart DNA replication forks at recombinational intermediates. Redundant functions between priB and priC and synthetic lethality between priA2::kan and rep3 mutations raise the possibility that there may be multiple pathways for restarting replication forks in vivo. Herein, it is shown that priA2::kan causes synthetic lethality when placed in combination with either Deltarep::kan or priC303:kan. These determinations were made using a nonselective P1 transduction-based viability assay. Two different priA2::kan suppressors (both dnaC alleles) were tested for their ability to rescue the priA-priC and priA-rep double mutant lethality. Only dnaC809,820 (and not dnaC809) could rescue the lethality in each case. Additionally, it was shown that the absence of the 3'-5' helicase activity of both PriA and Rep is not the critical missing function that causes the synthetic lethality in the rep-priA double mutant. One model proposes that replication restart at recombinational intermediates occurs by both PriA-dependent and PriA-independent pathways. The PriA-dependent pathways require at least priA and priB or priC, and the PriA-independent pathway requires at least priC and rep. It is further hypothesized that the dnaC809 suppression of priA2::kan requires priC and rep, whereas dnaC809,820 suppression of priA2::kan does not.

Sandler, S J

2000-01-01

42

Why do personality traits predict divorce? Multiple pathways through satisfaction.  

PubMed

While previous studies indicate that personality traits influence the likelihood of divorce, the processes that drive this relationship have yet to be examined. Accordingly, the current study utilized a nationally representative, longitudinal sample (N = 8,206) to test whether relationship satisfaction is a pathway by which personality traits influence relationship dissolution. Specifically, we examined 2 different pathways: the enduring dynamics and emergent distress pathways. The enduring dynamics pathway specifies that the association between personality and relationship satisfaction reflects ongoing relationship dynamics, which are presumed to be stable across a relationship. In contrast, the emergent distress pathway proposes that personality leads to worsening dynamics across the course of a relationship, which is indicated by changes in satisfaction. For each pathway, we assessed actor, partner, and combined effects for the Big Five. Results replicate previous research in that personality traits prospectively predict relationship dissolution. Both the enduring dynamics and emergent distress pathways served to explain this relationship, though the enduring dynamics model evidenced the largest effects. The emergent distress pathway was stronger for couples who experienced certain life events, suggesting that personality plays a role in adapting to changing life circumstances. Moreover, results suggest that the personality of the dyad is important in this process: Above and beyond actor effects, partner effects influenced relationship functioning (although the influence of combined effects was less clear). In sum, the current study demonstrates that personality traits shape the overall quality of one's relationship, which in turn influences the likelihood of relationship dissolution. PMID:24841100

Solomon, Brittany C; Jackson, Joshua J

2014-06-01

43

Cytolethal Distending Toxins Require Components of the ER-Associated Degradation Pathway for Host Cell Entry.  

PubMed

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins. PMID:25078082

Eshraghi, Aria; Dixon, Shandee D; Tamilselvam, Batcha; Kim, Emily Jin-Kyung; Gargi, Amandeep; Kulik, Julia C; Damoiseaux, Robert; Blanke, Steven R; Bradley, Kenneth A

2014-07-01

44

Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition  

PubMed Central

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ?mycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ?mycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems.

Siegrist, M. Sloan; Steigedal, Magnus; Ahmad, Rushdy; Mehra, Alka; Dragset, Marte S.; Schuster, Brian M.; Philips, Jennifer A.; Carr, Steven A.

2014-01-01

45

Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution.  

PubMed Central

Although specific proteinases play a critical role in the active phase of apoptosis, their substrates are largely unknown. We previously identified poly(ADP-ribose) polymerase (PARP) as an apoptosis-associated substrate for proteinase(s) related to interleukin 1 beta-converting enzyme (ICE). Now we have used a cell-free system to characterize proteinase(s) that cleave the nuclear lamins during apoptosis. Lamin cleavage during apoptosis requires the action of a second ICE-like enyzme, which exhibits kinetics of cleavage and a profile of sensitivity to specific inhibitors that is distinct from the PARP proteinase. Thus, multiple ICE-like enzymes are required for apoptotic events in these cell-free extracts. Inhibition of the lamin proteinase with tosyllysine "chloromethyl ketone" blocks nuclear apoptosis prior to the packaging of condensed chromatin into apoptotic bodies. Under these conditions, the nuclear DNA is fully cleaved to a nucleosomal ladder. Our studies reveal that the lamin proteinase and the fragmentation nuclease function in independent parallel pathways during the final stages of apoptotic execution. Neither pathway alone is sufficient for completion of nuclear apoptosis. Instead, the various activities cooperate to drive the disassembly of the nucleus. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

Lazebnik, Y A; Takahashi, A; Moir, R D; Goldman, R D; Poirier, G G; Kaufmann, S H; Earnshaw, W C

1995-01-01

46

RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways  

PubMed Central

Background & Aims Our previous results showed that the knockdown of woodchuck hepatitis virus (WHV) by RNA interference (RNAi) led to upregulation of interferon stimulated genes (ISGs) in primary hepatocytes. In the present study, we tested the hypothesis that the cellular signaling pathways recognizing RNA molecules may be involved the ISG stimulation by RNAi. Methods Primary murine hepatocytes (PMHs) from wild type mice and WHV transgenic (Tg) mice were prepared and treated with defined siRNAs. The mRNA levels of target genes and ISGs were detected by real-time RT-PCR. The involvement of the signaling pathways including RIG-I/MDA5, PKR, and TLR3/7/8/9 was examined by specific inhibition and the analysis of their activation by Western blotting. Results In PMHs from WHV Tg mice, specific siRNAs targeting WHV, mouse ?-actin, and GAPDH reduced the levels of targeted mRNAs and increased the mRNA expression of IFN-?, MxA, and IP-10. The enhanced ISG expression by siRNA transfection were abolished by siRNA-specific 2?-O-methyl antisense RNA and the inhibitors 2-AP and chloroquine blocking PKR and other TLR-mediated signaling pathways. Furthermore, Western blotting revealed that RNAi results in an increase in PKR phosphorylation and nuclear translocation of IRF3 and NF-êB, indicating the possible role of IRF3 in the RNAi-directed induction of ISGs. In contrast, silencing of RIG-I and MDA5 failed to block RNAi-mediated MxA induction. Conclusions RNAi is capable of enhancing innate immune responses through the PKR- and TLR-dependent signaling pathways in primary hepatocytes. The immune stimulation by RNAi may contribute to the antiviral activity of siRNAs in vivo.

Wu, Jun; Pei, Rongjuan; Xu, Yang; Yang, Dongliang; Roggendorf, Michael; Lu, Mengji

2013-01-01

47

Ras signals to the cell cycle machinery via multiple pathways to induce anchorage-independent growth.  

PubMed

Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergized with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth. PMID:9566878

Yang, J J; Kang, J S; Krauss, R S

1998-05-01

48

Multiple kinetic components and the Ca2+ requirements of exocytosis.  

PubMed Central

The use of caged-Ca2+ compounds to stimulate Ca(2+)-dependent exocytosis has substantially increased our understanding of this complex process. By this approach, the existence of multiple kinetic components of exocytosis has been established. These components may correspond to a series of sequential steps that lead to a single fusion-ready state (sequential mechanism) or, alternatively, to heterogeneity in secretory vesicles or in fusion-ready states (parallel mechanism). It is suggested that both of these mechanisms can underlie exocytosis of a single type of vesicle (mixed sequential-parallel mechanism). Studies with caged-Ca2+ compounds have also indicated that the Ca2+ requirement for exocytosis is substantially greater than that suggested by conventional methodologies. This discrepancy is mainly attributable to the underestimation, by imaging studies with high-affinity Ca2+ indicators (due to dye saturation), of the local increases in cytosolic Ca2+ concentration that trigger the exocytosis of individual vesicles. The effects of local saturation of such indicators are explored by means of a simple theory.

Kasai, H; Takahashi, N

1999-01-01

49

Multiple phosphorylation of Rad9 by CDK is required for DNA damage checkpoint activation  

PubMed Central

The DNA damage checkpoint controls cell cycle arrest in response to DNA damage, and activation of this checkpoint is in turn cell cycle-regulated. Rad9, the ortholog of mammalian 53BP1, is essential for this checkpoint response and is phosphorylated by the cyclin-dependent kinase (CDK) in the yeast Saccharomyces cerevisiae. Previous studies suggested that the CDK consensus sites of Rad9 are important for its checkpoint activity. However, the precise CDK sites of Rad9 involved have not been determined. Here we show that CDK consensus sites of Rad9 function in parallel to its BRCT domain toward checkpoint activation, analogous to its fission yeast ortholog Crb2. Unlike Crb2, however, mutation of multiple rather than any individual CDK site of Rad9 is required to completely eliminate its checkpoint activity in vivo. Although Dpb11 interacts with CDK-phosphorylated Rad9, we provide evidence showing that elimination of this interaction does not affect DNA damage checkpoint activation in vivo, suggesting that additional pathway(s) exist. Taken together, these findings suggest that the regulation of Rad9 by CDK and the role of Dpb11 in DNA damage checkpoint activation are more complex than previously suggested. We propose that multiple phosphorylation of Rad9 by CDK may provide a more robust system to allow Rad9 to control cell cycle-dependent DNA damage checkpoint activation.

Wang, Guoliang; Tong, Xiangyan; Weng, Stephanie; Zhou, Huilin

2012-01-01

50

Multiple degradation pathways of phenanthrene by Stenotrophomonas maltophilia C6.  

PubMed

Stenotrophomonas maltophilia strain C6, capable of utilizing phenanthrene as a sole source of carbon and energy, was isolated from creosote-contaminated sites at Hilo, Hawaii. Twenty-two metabolites of phenanthrene, covering from dihydrodiol to protocatechuic acid, were isolated and characterized. Phenanthrene was degraded via an initial dioxygenation on 1,2-, 3,4-, and 9,10-C, where the 3,4-dioxygenation and subsequent metabolisms were most dominant. The metabolic pathways were further branched by ortho- and meta-cleavage of phenanthrenediols to produce 1-hydroxy-2-naphthoic acid, 2-hydroxy-1-naphthoic acid, and naphthalene-1,2-dicarboxylic acid. These intermediates were then transformed to naphthalene-1,2-diol. 1-Hydroxy-2-naphthoic acid was also degraded via a direct ring cleavage. Naphthalene-1,2-diol underwent primarily ortho-cleavage to produce trans-2-carboxycinnamic acid and then to form phthalic acid, 4,5-dihydroxyphthalic acid and protocatechuic acid. Accumulation of salicylic acid in prolonged incubation indicated that a limited extent of meta-cleavage of naphthalene-1, 2-diol also occurred. This is the first study of detailed phenanthrene metabolic pathways by Stenotrophomonas maltophilia. PMID:23539472

Gao, Shumei; Seo, Jong-Su; Wang, Jun; Keum, Young-Soo; Li, Jianqiang; Li, Qing X

2013-04-01

51

Multiple degradation pathways of phenanthrene by Stenotrophomonas maltophilia C6  

PubMed Central

Stenotrophomonas maltophilia strain C6, capable of utilizing phenanthrene as a sole source of carbon and energy, was isolated from creosote-contaminated sites at Hilo, Hawaii. Twenty-two metabolites of phenanthrene, covering from dihydrodiol to protocatechuic acid, were isolated and characterized. Phenanthrene was degraded via an initial dioxygenation on 1,2-, 3,4-, and 9,10-C, where the 3,4-dioxygenation and subsequent metabolisms were most dominant. The metabolic pathways were further branched by ortho- and meta-cleavage of phenanthrenediols to produce 1-hydroxy-2-naphthoic acid, 2-hydroxy-1-naphthoic acid, and naphthalene-1,2-dicarboxylic acid. These intermediates were then transformed to naphthalene-1,2-diol. 1-Hydroxy-2-naphthoic acid was also degraded via a direct ring cleavage. Naphthalene-1,2-diol underwent primarily ortho-cleavage to produce trans-2-carboxycinnamic acid and then to form phthalic acid, 4,5-dihydroxyphthalic acid and protocatechuic acid. Accumulation of salicylic acid in prolonged incubation indicated that a limited extent of meta-cleavage of naphthalene-1, 2-diol also occurred. This is the first study of detailed phenanthrene metabolic pathways by Stenotrophomonas maltophilia.

Gao, Shumei; Seo, Jong-Su; Wang, Jun; Keum, Young-Soo; Li, Jianqiang; Li, Qing X.

2013-01-01

52

Catheter Ablation of Multiple Accessory Pathways in Duchenne Muscular Dystrophy  

PubMed Central

A 23-year-old male with Duchenne muscular dystrophy (DMD) experienced self-limiting palpitations at age 19 years for the first time. Palpitations recurred not earlier than at age 23 years, and were attributed to narrow complex tachycardia, which could be terminated with adenosine. Since electrocardiography showed a delta-wave, Wolff-Parkinson-White (WPW) syndrome was diagnosed, ajmaline prescribed and radio-frequency catheter ablation of three accessory pathways carried out one week later. One day after ablation, however, a relapse of the supraventricular tachycardia occurred and was terminated with ajmaline. Re-entry tachycardia occurred a second time six days after ablation, and as before, it was stopped only with ajmaline. Despite administration of verapamil to prevent tachycardia, it occurred a third time four months after ablation. This case shows that cardiac involvement in DMD may manifest also as WPW-syndrome. In these patients, repeated radio-frequency catheter ablation of accessory pathways may be necessary to completely block the re-entry mechanism.

Stollberger, Claudia; Steger, Christine; Gatterer, Edmund

2013-01-01

53

Targeting Cell-Death Pathways in Multiple Myeloma: Therapeutic Implications  

Microsoft Academic Search

Multiple myeloma (MM) remains fatal despite available therapies. The median survival of MM patients is 3–4 years with standard therapy and 4–5 years with high dose and transplantation therapy. Drug resistance occurs despite the use of novel anti-MM therapies. Mechanisms conferring chemoresistance include chromosomal abnormalities, the interaction of MM cells with their bone marrow (BM) microenvironment, and defects in apoptotic

Dharminder Chauhan; Kenneth Anderson

54

The multiple causal pathways between performance measures' use and effects.  

PubMed

In recent decades, there has been a growing interest in the design and implementation of systems using public reporting of performance measures to improve performance. In their simplest form, such interventions rest on the market-based logic of consumers using publicly released information to modify their behavior, thereby penalizing poor performers. However, evidence from large-scale efforts to use public reporting of performance measures as an instrumental performance improvement tool suggests that the causal mechanisms involved are much more complex. This article offers a typology of four different plausible causal pathways linking public reporting of performance measures and performance improvement. This typology rests on a variety of conceptual models and a review of available empirical evidence. We then use this typology to discuss the core elements that need to be taken into account in efforts to use public reporting of performance measures as a performance improvement tool. PMID:23877955

Contandriopoulos, Damien; Champagne, François; Denis, Jean-Louis

2014-02-01

55

Screening Reactive Metabolites Bioactivated by Multiple Enzyme Pathways Using a Multiplexed Microfluidic System  

PubMed Central

A multiplexed, microfluidic platform to detect reactive metabolites is described, and its performance is illustrated for compounds metabolized by oxidative and bioconjugation enzymes in multi-enzyme pathways to mimic natural human drug metabolism. The device features four 8-electrode screen printed carbon arrays coated with thin films of DNA, a ruthenium-polyvinylpyridine (RuPVP) catalyst, and multiple enzyme sources including human liver microsomes (HLM), cytochrome P450 (cyt P450) 1B1 supersomes, microsomal epoxide hydrolase (EH), human S9 liver fractions (Hs9) and N-acetyltransferase (NAT). Arrays are arranged in parallel to facilitate multiple compound screening, enabling up to 32 enzyme reactions and measurements in 20–30 min. In the first step of the assay, metabolic reactions are achieved under constant flow of oxygenated reactant solutions by electrode driven natural catalytic cycles of cyt P450s and cofactor-supported bioconjugation enzymes. Reactive metabolites formed in the enzyme reactions can react with DNA. Relative DNA damage is measuring in the second assay step using square wave voltammetry (SWV) with RuPVP as catalyst. Studies were done on chemicals known to require metabolic activation to induce genotoxicity, and results reproduced known features of metabolite DNA-reactivity for the test compounds. Metabolism of benzo[a]pyrene (B[a]P) by cyt P450s and epoxide hydrolase showed an enhanced relative DNA damage rate for DNA damage compared to cyt P450s alone. DNA damage rates for arylamines by pathways featuring both oxidative and conjugative enzymes at pH 7.4 gave better correlation with rodent genotoxicity metric TD50. Results illustrate the broad utility of the reactive metabolite screening device.

Wasalathanthri, Dhanuka P.; Faria, Ronaldo C.; Malla, Spundana; Joshi, Amit A.; Schenkman, John B.; Rusling, James F.

2012-01-01

56

Overlapping roles and collective requirement for the co-receptors Gas1, Cdo and Boc in Shh pathway function  

PubMed Central

Summary Secreted Hedgehog (Hh) ligands signal through the canonical receptor Patched (Ptch1). However, recent studies implicate three additional Hh-binding, cell surface proteins, Gas1, Cdo and Boc, as putative co-receptors for Hh ligands. A central question is to what degree these co-receptors function similarly and their collective requirement in Hh signal transduction. Here we provide evidence that Gas1, Cdo, and Boc, play overlapping and essential roles during Hh-mediated ventral neural patterning of the mammalian neural tube. Specifically, we demonstrate two important roles for these molecules: an early role in cell fate specification of multiple neural progenitors, and a later role in motor neuron progenitor maintenance. Most strikingly, genetic loss-of-function experiments indicate an obligatory requirement for Gas1, Cdo and Boc in Hh pathway activity in multiple tissues.

Allen, Benjamin L.; Song, Jane Y.; Izzi, Luisa; Althaus, Irene W.; Kang, Jong-Sun; Charron, Frederic; Krauss, Robert S.; McMahon, Andrew P.

2011-01-01

57

PPARdelta regulates multiple proinflammatory pathways to suppress atherosclerosis  

Microsoft Academic Search

Lipid homeostasis and inflammation are key determinants in atherogenesis, exemplified by the requirement of lipid-laden, foam cell macrophages for atherosclerotic lesion formation. Although the nuclear receptor PPARdelta has been implicated in both systemic lipid metabolism and macrophage inflammation, its role as a therapeutic target in vascular disease is unclear. We show here that orally active PPARdelta agonists significantly reduce atherosclerosis

Grant D. Barish; Annette R. Atkins; Michael Downes; Peter Olson; Ling-Wa Chong; Mike Nelson; Yuhua Zou; Hoosang Hwang; Heonjoong Kang; Linda Curtiss; Ronald M. Evans; Chih-Hao Lee

2008-01-01

58

Multiple modes of proepicardial cell migration require heartbeat  

PubMed Central

Background The outermost layer of the vertebrate heart, the epicardium, forms from a cluster of progenitor cells termed the proepicardium (PE). PE cells migrate onto the myocardium to give rise to the epicardium. Impaired epicardial development has been associated with defects in valve development, cardiomyocyte proliferation and alignment, cardiac conduction system maturation and adult heart regeneration. Zebrafish are an excellent model for studying cardiac development and regeneration; however, little is known about how the zebrafish epicardium forms. Results We report that PE migration occurs through multiple mechanisms and that the zebrafish epicardium is composed of a heterogeneous population of cells. Heterogeneity is first observed within the PE and persists through epicardium formation. Using in vivo imaging, histology and confocal microscopy, we show that PE cells migrate through a cellular bridge that forms between the pericardial mesothelium and the heart. We also observed the formation of PE aggregates on the pericardial surface, which were released into the pericardial cavity. It was previously reported that heartbeat-induced pericardiac fluid advections are necessary for PE cluster formation and subsequent epicardium development. We manipulated heartbeat genetically and pharmacologically and found that PE clusters clearly form in the absence of heartbeat. However, when heartbeat was inhibited the PE failed to migrate to the myocardium and the epicardium did not form. We isolated and cultured hearts with only a few epicardial progenitor cells and found a complete epicardial layer formed. However, pharmacologically inhibiting contraction in culture prevented epicardium formation. Furthermore, we isolated control and silent heart (sih) morpholino (MO) injected hearts prior to epicardium formation (60 hpf) and co-cultured these hearts with “donor” hearts that had an epicardium forming (108 hpf). Epicardial cells from donor hearts migrated on to control but not sih MO injected hearts. Conclusions Epicardial cells stem from a heterogeneous population of progenitors, suggesting that the progenitors in the PE have distinct identities. PE cells attach to the heart via a cellular bridge and free-floating cell clusters. Pericardiac fluid advections are not necessary for the development of the PE cluster, however heartbeat is required for epicardium formation. Epicardium formation can occur in culture without normal hydrodynamic and hemodynamic forces, but not without contraction.

2014-01-01

59

The Toll and Imd Pathways Are Not Required for Wolbachia-Mediated Dengue Virus Interference  

PubMed Central

Wolbachia blocks dengue virus replication in Drosophila melanogaster as well as in Aedes aegypti. Using the Drosophila model and mutations in the Toll and Imd pathways, we showed that neither pathway is required for expression of the dengue virus-blocking phenotype in the Drosophila host. This provides additional evidence that the mechanistic basis of Wolbachia-mediated dengue virus blocking in insects is more complex than simple priming of the host insect innate immune system.

Rances, Edwige; Johnson, Travis K.; Popovici, Jean; Iturbe-Ormaetxe, Inaki; Zakir, Tasnim; Warr, Coral G.

2013-01-01

60

Multiple Use of Magma Pathways: Mechanism for Hybridization  

NASA Astrophysics Data System (ADS)

In the Karakoram Shear Zone, Ladakh, NW India, Miocene leucogranitic dykes form an extensive, varied and complex network, linking the Pangong Range anatectic terrane with leucogranites of the Karakoram Batholith. Water-fluxed Miocene anatexis occurs at upper amphibolite conditions, and was contemporaneous with shearing. The network is characterized by continuous and interconnected leucosomes and dykes, with only rare cross-cutting relationships, forming dyke swarms and more chaotic injection complexes where magmatic rocks cover up to 50% of the outcrop area. Despite this volume of magma, the system was always controlled by solid framework suggesting that it did not flow en masse and that the magma network was not all liquid simultaneously. Leucogranites in this network, carry an isotopic signature intermediate between the two main anatectic rocks in the source, suggesting efficient homogenization of the magmatic products. This meso- to macroscale complex network is also reflected at microscale. Microstructural observations indicate that these magmatic rocks consist dominantly of Qtz, Plg and Kfs in two very distinct appearances, as large irregularly-shaped grains with cuspate boundaries, or/and as fine-grained minerals with lobate boundaries. These two show intimate spatial relationship with fine-grained material forming semi- to continuous corridors to wide channels that links together and form an extensive network branching around large grains. We suggest, that the large minerals represent early formed solid granitic framework that was later invaded by a new melt batch that exploits microfractures in between and through the framework forming crystals giving rise to this interconnected network. The presence of later crystallized melt and its interaction with the solid rock was inferred from the following microstructures: (i) narrow, tortuous corridors of fine-grained minerals cutting across or lining the boundaries of larger grains, interpreted to be remnants of magma-filled cracks cutting across a pre-existing magmatic rock; (ii) compositional zoning of early-crystallized plagioclase and K-feldspar; (iii) quartz overgrows documented by CL imaging; (iv) corrosion of early-formed grains; and (v) different CPO of early-formed quartz and its overgrowths. In summary, the early formed dykes provided a pathway exploited by new magma batches. Once formed, the magma channels remained open either intermittently or continuously and the new melt batches migrated through following predominantly grain boundaries along an S-C fabric related to syn-magmatic shearing. Accordingly, hybrid signature results from the microscopic interaction between previously crystallized magmatic rock and new magma batch, through local equilibration, not from magma mixing. We conclude that leucosomes and magmatic bodies formed by the magma that flushed through them have a complex origin and composition that is reflected in the geochemistry and isotope chemistry. Final composition is a result of the accumulation of magma residue. This in turn depends on compositional changes of magma influx, P-T conditions, and the interaction of new magma with early crystallized magmatic products.

Hasalova, P.; Weinberg, R. F.; Reichardt, H.

2010-12-01

61

Humidity sensation requires both mechanosensory and thermosensory pathways in Caenorhabditis elegans  

PubMed Central

All terrestrial animals must find a proper level of moisture to ensure their health and survival. The cellular-molecular basis for sensing humidity is unknown in most animals, however. We used the model nematode Caenorhabditis elegans to uncover a mechanism for sensing humidity. We found that whereas C. elegans showed no obvious preference for humidity levels under standard culture conditions, worms displayed a strong preference after pairing starvation with different humidity levels, orienting to gradients as shallow as 0.03% relative humidity per millimeter. Cell-specific ablation and rescue experiments demonstrate that orientation to humidity in C. elegans requires the obligatory combination of distinct mechanosensitive and thermosensitive pathways. The mechanosensitive pathway requires a conserved DEG/ENaC/ASIC mechanoreceptor complex in the FLP neuron pair. Because humidity levels influence the hydration of the worm’s cuticle, our results suggest that FLP may convey humidity information by reporting the degree that subcuticular dendritic sensory branches of FLP neurons are stretched by hydration. The thermosensitive pathway requires cGMP-gated channels in the AFD neuron pair. Because humidity levels affect evaporative cooling, AFD may convey humidity information by reporting thermal flux. Thus, humidity sensation arises as a metamodality in C. elegans that requires the integration of parallel mechanosensory and thermosensory pathways. This hygrosensation strategy, first proposed by Thunberg more than 100 y ago, may be conserved because the underlying pathways have cellular and molecular equivalents across a wide range of species, including insects and humans.

Russell, Joshua; Vidal-Gadea, Andres G.; Makay, Alex; Lanam, Carolyn; Pierce-Shimomura, Jonathan T.

2014-01-01

62

Humidity sensation requires both mechanosensory and thermosensory pathways in Caenorhabditis elegans.  

PubMed

All terrestrial animals must find a proper level of moisture to ensure their health and survival. The cellular-molecular basis for sensing humidity is unknown in most animals, however. We used the model nematode Caenorhabditis elegans to uncover a mechanism for sensing humidity. We found that whereas C. elegans showed no obvious preference for humidity levels under standard culture conditions, worms displayed a strong preference after pairing starvation with different humidity levels, orienting to gradients as shallow as 0.03% relative humidity per millimeter. Cell-specific ablation and rescue experiments demonstrate that orientation to humidity in C. elegans requires the obligatory combination of distinct mechanosensitive and thermosensitive pathways. The mechanosensitive pathway requires a conserved DEG/ENaC/ASIC mechanoreceptor complex in the FLP neuron pair. Because humidity levels influence the hydration of the worm's cuticle, our results suggest that FLP may convey humidity information by reporting the degree that subcuticular dendritic sensory branches of FLP neurons are stretched by hydration. The thermosensitive pathway requires cGMP-gated channels in the AFD neuron pair. Because humidity levels affect evaporative cooling, AFD may convey humidity information by reporting thermal flux. Thus, humidity sensation arises as a metamodality in C. elegans that requires the integration of parallel mechanosensory and thermosensory pathways. This hygrosensation strategy, first proposed by Thunberg more than 100 y ago, may be conserved because the underlying pathways have cellular and molecular equivalents across a wide range of species, including insects and humans. PMID:24843133

Russell, Joshua; Vidal-Gadea, Andrés G; Makay, Alex; Lanam, Carolyn; Pierce-Shimomura, Jonathan T

2014-06-01

63

Multiple variants aggregate in the neuregulin signaling pathway in a subset of schizophrenia patients  

PubMed Central

Despite the strongly held view that schizophrenia (SZ) shows substantial genetic heterogeneity, pathway heterogeneity, as seen in cancer where different pathways are affected in similar tumors, has not been explored. We explore this possibility in a case-only study of the neuregulin signaling pathway (NSP), which has been prominently implicated in SZ and for which there is detailed knowledge on the ligand- and receptor-processing steps through ?- and ?-secretase cleavage. We hypothesize that more than one damaging variants in the NSP genes might be necessary to cause disease, leading to an apparent clustering of such variants in only the few patients with affected NSP. We analyze linkage and next-generation sequencing results for the genes encoding components of the pathway, including NRG1, NRG3, ERBB4, ?-secretase and the ?-secretase complex. We find multiple independent examples of supporting evidence for this hypothesis: (i) increased linkage scores over NSP genes, (ii) multiple positive interlocus correlations of linkage scores across families suggesting each family is linked to either many or none of the genes, (iii) aggregation of predicted damaging variants in a subset of individuals and (iv) significant phenotypic differences of the subset of patients carrying such variants. Collectively, our data strongly support the hypothesis that the NSP is affected by multiple damaging variants in a subset of phenotypically distinct patients. On the basis of this, we propose a general model of pathway heterogeneity in SZ, which, in part, may explain its phenotypic variability and genetic complexity.

Hatzimanolis, A; McGrath, J A; Wang, R; Li, T; Wong, P C; Nestadt, G; Wolyniec, P S; Valle, D; Pulver, A E; Avramopoulos, D

2013-01-01

64

Language Learning in Children Who Are Deaf and Hard of Hearing: Multiple Pathways.  

ERIC Educational Resources Information Center

This text on teaching language to students with hearing impairments stresses the use of multiple language learning pathways to meet the individual needs of students. The introductory chapter looks at language issues in the context of history, instruction, technology, culture, and the law. Chapter 2, on language acquisition, discusses the nature of…

Easterbrooks, Susan R.; Baker, Sharon

65

SUMOylation of ATRIP potentiates DNA damage signaling by boosting multiple protein interactions in the ATR pathway.  

PubMed

The ATR (ATM [ataxia telangiectasia-mutated]- and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR-Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11-RAD50-NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway. PMID:24990965

Wu, Ching-Shyi; Ouyang, Jian; Mori, Eiichiro; Nguyen, Hai Dang; Maréchal, Alexandre; Hallet, Alexander; Chen, David J; Zou, Lee

2014-07-01

66

Smoldering multiple myeloma requiring treatment: time for a new definition?  

PubMed

Smoldering multiple myeloma (SMM) bridges the gap between monoclonal gammopathy of undetermined significance (a mostly premalignant disorder) and active multiple myeloma (MM). Until recently, no interventional study in patients with SMM showed improved overall survival (OS) with therapy as compared with observation. A report from the PETHEMA-GEM (Programa Español de Tratamientos en Hematologica) group described both fewer myeloma-related events and better OS among patients with high-risk SMM who were treated with lenalidomide and dexamethasone. This unique study prompted us to review current knowledge about SMM and address the following questions: (1) Are there patients currently defined as SMM who should be treated routinely? (2) Should the definitions of SMM and MM be reconsidered? (3) Has the time come when not treating is more dangerous than treating? (4) Could unintended medical harm result from overzealous intervention? Our conclusion is that those patients with the highest-risk SMM (extreme bone marrow plasmacytosis, extremely abnormal serum immunoglobulin free light chain ratio, and multiple bone lesions detected only by modern imaging) should be reclassified as active MM so that they can receive MM-appropriate therapy and the paradigm of careful observation for patients with SMM can be preserved. PMID:24144641

Dispenzieri, Angela; Stewart, A Keith; Chanan-Khan, Asher; Rajkumar, S Vincent; Kyle, Robert A; Fonseca, Rafael; Kapoor, Prashant; Bergsagel, P Leif; McCurdy, Arleigh; Gertz, Morie A; Lacy, Martha Q; Lust, John A; Russell, Stephen J; Zeldenrust, Steven R; Reeder, Craig; Roy, Vivek; Buadi, Francis; Dingli, David; Hayman, Suzanne R; Leung, Nelson; Lin, Yi; Mikhael, Joseph; Kumar, Shaji K

2013-12-19

67

Regulation of multiple DNA repair pathways by the Fanconi anemia protein SLX4.  

PubMed

SLX4, the newly identified Fanconi anemia protein, FANCP, is implicated in repairing DNA damage induced by DNA interstrand cross-linking (ICL) agents, topoisomerase I (TOP1) inhibitors, and in Holliday junction resolution. It interacts with and enhances the activity of XPF-ERCC1, MUS81-EME1, and SLX1 nucleases, but the requirement for the specific nucleases in SLX4 function is unclear. Here, by complementing a null FA-P Fanconi anemia cell line with SLX4 mutants that specifically lack the interaction with each of the nucleases, we show that the SLX4-dependent XPF-ERCC1 activity is essential for ICL repair but is dispensable for repairing TOP1 inhibitor-induced DNA lesions. Conversely, MUS81-SLX4 interaction is critical for resistance to TOP1 inhibitors but is less important for ICL repair. Mutation of SLX4 that abrogates interaction with SLX1 results in partial resistance to both cross-linking agents and TOP1 inhibitors. These results demonstrate that SLX4 modulates multiple DNA repair pathways by regulating appropriate nucleases. PMID:23093618

Kim, Yonghwan; Spitz, Gabriella S; Veturi, Uma; Lach, Francis P; Auerbach, Arleen D; Smogorzewska, Agata

2013-01-01

68

A critical role for the NFkB pathway in multiple myeloma.  

PubMed

NFkB transcription factors play a key role in the survival and proliferation of many kinds of B-cell tumors, including multiple myeloma (MM). It was shown that NFkB activation in MM tumors results mainly from extrinsic signaling by APRIL and BAFF ligands that stimulate receptors on normal plasma cells as well as on pre-malignant monoclonal gammopathy of undetermined significance (MGUS) and MM tumors. However, the mutations that occur during MM progression and that constitutively activate NFkB would be expected to decrease dependence of tumor cells on the bone marrow microenvironment. These mutations can activate the classical or alternative NFkB pathways selectively, but usually both pathways are activated in MM. Significantly, activation of either NFkB pathway leads to a similar response of MM cell lines. This frequent activation of the alternative pathway distinguishes MM from other B-cell tumors, which more frequently have mutations that are predicted to activate only the classical NFkB pathway. Given the strong dependence of MGUS and MM tumors on NFkB pathway activation, inhibition by a combination of targeting extrinsic signaling plus both NFkB pathways appears to be an attractive therapeutic approach in MM tumors. PMID:20890394

Demchenko, Yulia N; Kuehl, W Michael

2010-05-01

69

Genetic and Epigenetic Events Generate Multiple Pathways in Colorectal Cancer Progression  

PubMed Central

Colorectal cancer (CRC) is one of the most common causes of death, despite decades of research. Initially considered as a disease due to genetic mutations, it is now viewed as a complex malignancy because of the involvement of epigenetic abnormalities. A functional equivalence between genetic and epigenetic mechanisms has been suggested in CRC initiation and progression. A hallmark of CRC is its pathogenetic heterogeneity attained through at least three distinct pathways: a traditional (adenoma-carcinoma sequence), an alternative, and more recently the so-called serrated pathway. While the alternative pathway is more heterogeneous and less characterized, the traditional and serrated pathways appear to be more homogeneous and clearly distinct. One unsolved question in colon cancer biology concerns the cells of origin and from which crypt compartment the different pathways originate. Based on molecular and pathological evidences, we propose that the traditional and serrated pathways originate from different crypt compartments explaining their genetic/epigenetic and clinicopathological differences. In this paper, we will discuss the current knowledge of CRC pathogenesis and, specifically, summarize the role of genetic/epigenetic changes in the origin and progression of the multiple CRC pathways. Elucidation of the link between the molecular and clinico-pathological aspects of CRC would improve our understanding of its etiology and impact both prevention and treatment.

Pancione, Massimo; Remo, Andrea; Colantuoni, Vittorio

2012-01-01

70

Hedgehog signaling is required at multiple stages of zebrafish tooth development  

PubMed Central

Background The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood. Results We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region. Conclusion We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution.

2010-01-01

71

Initiation of Rhombomeric Hoxb4 Expression Requires Induction by Somites and a Retinoid Pathway  

Microsoft Academic Search

Anteroposterior (AP) patterning in the vertebrate hindbrain is dependent upon the establishment of segmental domains of Hox expression. We investigated the mechanism that governs the early expression of Hoxb4 and found that transient signaling from the paraxial mesoderm induces expression in the hindbrain. Induction involves a retinoid pathway requiring retinoic acid receptor (RAR) function within the neural plate. Characterization of

Alex Gould; Nobue Itasaki; Robb Krumlauf

1998-01-01

72

The Multiple Interacting Pathways model - A scale-independent approach to modelling transport and flow in real soils and catchments  

NASA Astrophysics Data System (ADS)

Whilst classic continuum-based models of hydrological processes are in common usage, their applicability to structured soils at practical scales is questionable. Continuum equations require local equilibration of potentials and fluxes. If complex heterogeneities and preferential pathways that are present in most real soils are to be considered, then this requirement leads to impractically fine grid-scales. The Multiple Interacting Pathways (MIPs) modelling concept circumvents these issues through use of a discrete methodology. Water in the catchment is represented as a large set of discrete particles, each representing a volume of water that enters the catchment at the same time and spatial locality, and subsequently travels through the same pathways to the outlet. Random particle tracking is used to simulate the particles movement according to mechanistically-based equations. Localised densities of particles, combined with porosity characteristics, determine levels of saturation and soil moisture across the catchment, which in turn dynamically determine whether particles move as saturated or unsaturated flow. However, the particles within these flows do not all move with the same velocity. Instead, a distribution of velocities is applied to the particles, which attempts to represent the range of flow pathways available. Movement between the pathways can also be achieved using pathway exchange probabilities. In this way, the model is able to directly acknowledge the presence of heterogeneities in the soil, in a scale-independent manner. The exchange probabilities can also be used to simulate plant root uptake, evaporation and bedrock losses. An important feature of this methodology is that there is integrated simulation of flow and transport. Information such as age, origin, and chemistry can be associated with each particle, leading to the ability to analyse input/output/storage residence times and source contributions. This formulation provides a platform for testing our understanding of catchment processes, however, to identify models of temporally and spatially variable processes, then high frequency data over long periods of time, with possibly multiple observation points is ultimately needed. In the poster, the capabilities and potential of the MIPs methodology for characterisation of complex, non-stationary, spatially variable run-off generation processes are illustrated and discussed, and data requirements of such a model are also highlighted.

Davies, J.; Beven, K.

2012-04-01

73

The Toll pathway is required in the epidermis for muscle development in the Drosophila embryo  

NASA Technical Reports Server (NTRS)

The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. Here, we demonstrate that this pathway is required in the epidermis for proper muscle development. Previously, we showed that the zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, M.S., Hashimoto, C., and Keshishian, H., Dev. Biol. 169, 151-167, 1995). We have now also analyzed the requirements for easter, spatzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway. We find that spatzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, we have examined the spatial requirements for Toll expression during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. Our results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process.

Halfon, M. S.; Keshishian, H.

1998-01-01

74

Genome-Wide Pathway Association Studies of Multiple Correlated Quantitative Phenotypes Using Principle Component Analyses  

PubMed Central

Genome-wide pathway association studies provide novel insight into the biological mechanism underlying complex diseases. Current pathway association studies primarily focus on single important disease phenotype, which is sometimes insufficient to characterize the clinical manifestations of complex diseases. We present a multi-phenotypes pathway association study(MPPAS) approach using principle component analysis(PCA). In our approach, PCA is first applied to multiple correlated quantitative phenotypes for extracting a set of orthogonal phenotypic components. The extracted phenotypic components are then used for pathway association analysis instead of original quantitative phenotypes. Four statistics were proposed for PCA-based MPPAS in this study. Simulations using the real data from the HapMap project were conducted to evaluate the power and type I error rates of PCA-based MPPAS under various scenarios considering sample sizes, additive and interactive genetic effects. A real genome-wide association study data set of bone mineral density (BMD) at hip and spine were also analyzed by PCA-based MPPAS. Simulation studies illustrated the performance of PCA-based MPPAS for identifying the causal pathways underlying complex diseases. Genome-wide MPPAS of BMD detected associations between BMD and KENNY_CTNNB1_TARGETS_UP as well as LONGEVITYPATHWAY pathways in this study. We aim to provide a applicable MPPAS approach, which may help to gain deep understanding the potential biological mechanism of association results for complex diseases.

Zhang, Feng; Guo, Xiong; Wu, Shixun; Han, Jing; Liu, Yongjun; Shen, Hui; Deng, Hong-Wen

2012-01-01

75

Modeling of nitrous oxide production by autotrophic ammonia-oxidizing bacteria with multiple production pathways.  

PubMed

Autotrophic ammonia oxidizing bacteria (AOB) have been recognized as a major contributor to N2O production in wastewater treatment systems. However, so far N2O models have been proposed based on a single N2O production pathway by AOB, and there is still a lack of effective approach for the integration of these models. In this work, an integrated mathematical model that considers multiple production pathways is developed to describe N2O production by AOB. The pathways considered include the nitrifier denitrification pathway (N2O as the final product of AOB denitrification with NO2(-) as the terminal electron acceptor) and the hydroxylamine (NH2OH) pathway (N2O as a byproduct of incomplete oxidation of NH2OH to NO2(-)). In this model, the oxidation and reduction processes are modeled separately, with intracellular electron carriers introduced to link the two types of processes. The model is calibrated and validated using experimental data obtained with two independent nitrifying cultures. The model satisfactorily describes the N2O data from both systems. The model also predicts shifts of the dominating pathway at various dissolved oxygen (DO) and nitrite levels, consistent with previous hypotheses. This unified model is expected to enhance our ability to predict N2O production by AOB in wastewater treatment systems under varying operational conditions. PMID:24571180

Ni, Bing-Jie; Peng, Lai; Law, Yingyu; Guo, Jianhua; Yuan, Zhiguo

2014-04-01

76

Classical and/or alternative NF-?B pathway activation in multiple myeloma  

PubMed Central

Mutations involving the nuclear factor-?B (NF-?B) pathway are present in at least 17% of multiple myeloma (MM) tumors and 40% of MM cell lines (MMCLs). These mutations, which are apparent progression events, enable MM tumors to become less dependent on bone marrow signals that activate NF-?B. Studies on a panel of 51 MMCLs provide some clarification of the mechanisms through which these mutations act and the significance of classical versus alternative activation of NF-?B. First, only one mutation (NFKB2) selectively activates the alternative pathway, whereas several mutations (CYLD, NFKB1, and TACI) selectively activate the classical pathway. However, most mutations affecting NF-?B–inducing kinase (NIK) levels (NIK, TRAF2, TRAF3, cIAP1&2, and CD40) activate the alternative but often both pathways. Second, we confirm the critical role of TRAF2 in regulating NIK degradation, whereas TRAF3 enhances but is not essential for cIAP1/2-mediated proteasomal degradation of NIK in MM. Third, using transfection to selectively activate the classical or alternative NF-?B pathways, we show virtually identical changes in gene expression in one MMCL, whereas the changes are similar albeit nonidentical in a second MMCL. Our results suggest that MM tumors can achieve increased autonomy from the bone marrow microenvironment by mutations that activate either NF-?B pathway.

Demchenko, Yulia N.; Glebov, Oleg K.; Zingone, Adriana; Keats, Jonathan J.; Bergsagel, P. Leif

2010-01-01

77

Orexins stimulate steroidogenic acute regulatory protein expression through multiple signaling pathways in human adrenal H295R cells.  

PubMed

Orexins mediate a variety of physiological processes, including feeding behavior, the circadian pathway, and cortisol secretion. Steroidogenesis is regulated by a variety of neuropeptides, and one of the key rate-limiting steps is cholesterol transport across the mitochondrial membrane by the steroidogenic acute regulatory protein (StAR). StAR expression can be regulated through several different signaling pathways. Despite the clear link between orexins and steroid production, the actions of the orexin family of hormones on steroid biosynthesis are not fully understood. We present data showing that 100 nm of both orexins A and B for 4 or 24 h significantly up-regulates StAR, in H295R pluripotent adrenocortical cells. We present the dose-dependent and time-dependent characteristics of StAR up-regulation at the protein level, showing significant increases after 4 h at a relatively low agonist concentration (1 nm). We have provided a key analysis of the precise G protein-coupled signaling pathways required for the up-regulation of StAR in response to orexins A and B. This has involved dominant-negative G protein analysis, and the direct inhibition of the protein kinase A, protein kinase C, ERK1/2, and p38 pathways. This shows a fundamental role for multiple G protein-coupled and MAPK-mediated signaling pathways leading to StAR expression. Antagonist analysis also showed that orexin effects on StAR were primarily, but not exclusively, acting through the orexin receptor type 1. This is the first study linking orexin action on StAR expression and comprehensively describes the signaling pathways involved in regulating the complexity of hormone biosynthesis. PMID:18450961

Ramanjaneya, Manjunath; Conner, Alex C; Chen, Jing; Stanfield, Peter R; Randeva, Harpal S

2008-08-01

78

Promiscuous Mutations Activate the Non-Canonical NF-kB Pathway in Multiple Myeloma  

PubMed Central

Summary Activation of NF-kB has been noted in many tumor types, however only rarely has this been linked to an underlying genetic mutation. An integrated analysis of high-density oligonucleotide array CGH and gene expression profiling data from 155 multiple myeloma samples identified a promiscuous array of abnormalities contributing to the dysregulation of NF-kB in approximately 20% of patients. We report mutations in ten genes causing the inactivation of TRAF2, TRAF3, CYLD, cIAP1/cIAP2, and activation of NFKB1, NFKB2, CD40, LTBR, TACI, and NIK that result primarily in constitutive activation of the non-canonical NF-kB pathway, with the single most common abnormality being inactivation of TRAF3. These results highlight the critical importance of the NF-kB pathway in the pathogenesis of multiple myeloma.

Keats, Jonathan J.; Fonseca, Rafael; Chesi, Marta; Schop, Roelandt; Baker, Angela; Chng, Wee-Joo; Van Wier, Scott; Tiedemann, Rodger; Shi, Chang-Xin; Sebag, Michael; Braggio, Esteban; Henry, Travis; Zhu, Yuan-Xiao; Fogle, Homer; Price-Troska, Tammy; Ahmann, Gregory; Mancini, Catherine; Brents, Leslie A.; Kumar, Shaji; Greipp, Philip; Dispenzieri, Angela; Bryant, Barb; Mulligan, George; Bruhn, Laurakay; Barrett, Michael; Valdez, Riccardo; Trent, Jeff; Stewart, A. Keith; Carpten, John; Bergsagel, P. Leif

2007-01-01

79

Negative Energy: Why Interdisciplinary Physics Requires Multiple Ontologies  

NSDL National Science Digital Library

Much recent work in physics education research has focused on ontological metaphors for energy, particularly the substance ontology and its pedagogical affordances. The concept of negative energy problematizes the substance ontology for energy, but in many instructional settings, the specific difficulties around negative energy are outweighed by the general advantages of the substance ontology. However, we claim that our interdisciplinary setting (a physics class that builds deep connections to biology and chemistry) leads to a different set of considerations and conclusions. In a course designed to draw interdisciplinary connections, the centrality of chemical bond energy in biology necessitates foregrounding negative energy from the beginning. We argue that the emphasis on negative energy requires a combination of substance and location ontologies. The location ontology enables energies both "above" and "below" zero. We present preliminary student data that illustrate difficulties in reasoning about negative energy, and the affordances of the location metaphor.

Dreyfus, Benjamin W.; Geller, Benjamin D.; Gouvea, Julia; Sawtelle, Vashti; Turpen, Chandra; Redish, Edward F.

2014-01-31

80

Multiple gustatory receptors required for the caffeine response in Drosophila.  

PubMed

The ability of insects to detect and avoid ingesting naturally occurring repellents and insecticides is essential for their survival. Nevertheless, the gustatory receptors enabling them to sense toxic botanical compounds are largely unknown. The only insect gustatory receptor shown to be required for avoiding noxious compounds is the Drosophila caffeine receptor, Gr66a. However, this receptor is not sufficient for the caffeine response, suggesting that Gr66a may be a subunit of a larger receptor. Here, we report that mutations in the gene encoding the gustatory receptor, Gr93a, result in a phenotype identical to that caused by mutations in Gr66a. This includes an inability to avoid caffeine or the related methylxanthine present in tea, theophylline. Caffeine-induced action potentials were also eliminated in Gr93a-mutant animals, while the flies displayed normal responses to other aversive compounds or to sugars. The Gr93a protein was coexpressed with Gr66a in avoidance-gustatory receptor neurons (GRNs), and functioned in the same GRNs as Gr66a. However, misexpression of both receptors in GRNs that normally do not express either Gr93a or Gr66a does not confer caffeine sensitivity to these GRNs. Because Gr93a- and Gr66a-mutant animals exhibit the identical phenotypes and function in the same cells, we propose that they may be caffeine coreceptors. In contrast to mammalian and Drosophila olfactory receptors and mammalian taste receptors, which are monomeric or dimeric receptors, we propose that Drosophila taste receptors that function in avoidance of bitter compounds are more complex and require additional subunits that remain to be identified. PMID:19246397

Lee, Youngseok; Moon, Seok Jun; Montell, Craig

2009-03-17

81

Iron and the folate-vitamin B12-methylation pathway in multiple sclerosis  

Microsoft Academic Search

Some subjects with multiple sclerosis (MS) present with low blood iron parameters. Anecdotal reports and a single patient\\u000a study suggest that iron supplementation may be beneficial in these subjects. Myelin is regenerated continually, but prerequisites\\u000a for this process are iron and a functional folate-vitamin B12-methylation pathway. The aim of this study was to determine\\u000a iron status, folate and homocysteine in

S. J. van Rensburg; M. J. Kotze; D. Hon; P. Haug; J. Kuyler; M. Hendricks; J. Botha; F. C. V. Potocnik; T. Matsha; R. T. Erasmus

2006-01-01

82

Induction of Cancer Cell Death by Isoflavone: The Role of Multiple Signaling Pathways  

PubMed Central

Soy isoflavones have been documented as dietary nutrients broadly classified as “natural agents” which plays important roles in reducing the incidence of hormone-related cancers in Asian countries, and have shown inhibitory effects on cancer development and progression in vitro and in vivo, suggesting the cancer preventive or therapeutic activity of soy isoflavones against cancers. Emerging experimental evidence shows that isoflavones could induce cancer cell death by regulating multiple cellular signaling pathways including Akt, NF-?B, MAPK, Wnt, androgen receptor (AR), p53 and Notch signaling, all of which have been found to be deregulated in cancer cells. Therefore, homeostatic regulation of these important cellular signaling pathways by isoflavones could be useful for the activation of cell death signaling, which could result in the induction of apoptosis of both pre-cancerous and/or cancerous cells without affecting normal cells. In this article, we have attempted to summarize the current state-of-our-knowledge regarding the induction of cancer cell death pathways by isoflavones, which is believed to be mediated through the regulation of multiple cellular signaling pathways. The knowledge gained from this article will provide a comprehensive view on the molecular mechanism(s) by which soy isoflavones may exert their effects on the prevention of tumor progression and/or treatment of human malignancies, which would also aid in stimulating further in-depth mechanistic research and foster the initiation of novel clinical trials.

Li, Yiwei; Kong, Dejuan; Bao, Bin; Ahmad, Aamir; Sarkar, Fazlul H.

2011-01-01

83

Multiple functions of MRN in end-joining pathways during isotype class switching.  

PubMed

The Mre11-Rad50-NBS1 (MRN) complex has many roles in response to DNA double-strand breaks, but its functions in repair by nonhomologous end joining (NHEJ) pathways are poorly understood. We have investigated requirements for MRN in class switch recombination (CSR), a programmed DNA rearrangement in B lymphocytes that requires NHEJ. To this end, we have engineered mice that lack the entire MRN complex in B lymphocytes or that possess an intact complex that harbors mutant Mre11 lacking DNA nuclease activities. MRN deficiency confers a strong defect in CSR, affecting both the classic and the alternative NHEJ pathways. In contrast, absence of Mre11 nuclease activities causes a milder phenotype, revealing a separation of function within the complex. We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the classical and alternative NHEJ pathways. PMID:19633670

Dinkelmann, Maria; Spehalski, Elizabeth; Stoneham, Trina; Buis, Jeffrey; Wu, Yipin; Sekiguchi, JoAnn M; Ferguson, David O

2009-08-01

84

Requirements of the cytosolic iron-sulfur cluster assembly pathway in Arabidopsis  

PubMed Central

The assembly of iron–sulfur (Fe–S) clusters requires dedicated protein factors inside the living cell. Striking similarities between prokaryotic and eukaryotic assembly proteins suggest that plant cells inherited two different pathways through endosymbiosis: the ISC pathway in mitochondria and the SUF pathway in plastids. Fe–S proteins are also found in the cytosol and nucleus, but little is known about how they are assembled in plant cells. Here, we show that neither plastid assembly proteins nor the cytosolic cysteine desulfurase ABA3 are required for the activity of cytosolic aconitase, which depends on a [4Fe–4S] cluster. In contrast, cytosolic aconitase activity depended on the mitochondrial cysteine desulfurase NFS1 and the mitochondrial transporter ATM3. In addition, we were able to complement a yeast mutant in the cytosolic Fe–S cluster assembly pathway, dre2, with the Arabidopsis homologue AtDRE2, but only when expressed together with the diflavin reductase AtTAH18. Spectroscopic characterization showed that purified AtDRE2 could bind up to two Fe–S clusters. Purified AtTAH18 bound one flavin per molecule and was able to accept electrons from NAD(P)H. These results suggest that the proteins involved in cytosolic Fe–S cluster assembly are highly conserved, and that dependence on the mitochondria arose before the second endosymbiosis event leading to plastids.

Bernard, Delphine G.; Netz, Daili J. A.; Lagny, Thibaut J.; Pierik, Antonio J.; Balk, Janneke

2013-01-01

85

Multiple functions of MRN in end-joining pathways during isotype class switching  

Microsoft Academic Search

The Mre11–Rad50–NBS1 (MRN) complex has many roles in response to DNA double-strand breaks, but its functions in repair by nonhomologous end joining (NHEJ) pathways are poorly understood. We have investigated requirements for MRN in class switch recombination (CSR), a programmed DNA rearrangement in B lymphocytes that requires NHEJ. To this end, we have engineered mice that lack the entire MRN

Maria Dinkelmann; Elizabeth Spehalski; Trina Stoneham; Jeffrey Buis; Yipin Wu; JoAnn M Sekiguchi; David O Ferguson

2009-01-01

86

Multiple signaling pathways leading to the activation of interferon regulatory factor 3.  

PubMed

Virus infection of susceptible cells activates multiple signaling pathways that orchestrate the activation of genes, such as cytokines, involved in the antiviral and innate immune response. Among the kinases induced are the mitogen-activated protein (MAP) kinases, Jun-amino terminal kinases (JNK) and p38, the IkappaB kinase (IKK) and DNA-PK. In addition, virus infection also activates an uncharacterized VAK responsible for the C-terminal phosphorylation and subsequent activation of interferon regulatory factor 3 (IRF-3). Virus-mediated activation of IRF-3 through VAK is dependent on viral entry and transcription, since replication deficient virus failed to induce IRF-3 activity. The pathways leading to VAK activation are not well characterized, but IRF-3 appears to represent a novel cellular detection pathway that recognizes viral nucleocapsid (N) structure. Recently, the range of inducers responsible for IRF-3 activation has increased. In addition to virus infection, recognition of bacterial infection mediated through lipopolysaccharide by Toll-like receptor 4 has also been reported. Furthermore, MAP kinase kinase kinase (MAP KKK)-related pathways and DNA-PK induce N-terminal phosphorylation of IRF-3. This review summarizes recent observations in the identification of novel signaling pathways leading to IRF-3 activation. PMID:12213596

Servant, Marc J; Grandvaux, Nathalie; Hiscott, John

2002-09-01

87

A DNA repair pathway score predicts survival in human multiple myeloma: the potential for therapeutic strategy.  

PubMed

DNA repair is critical to resolve extrinsic or intrinsic DNA damage to ensure regulated gene transcription and DNA replication. These pathways control repair of double strand breaks, interstrand crosslinks, and nucleotide lesions occurring on single strands. Distinct DNA repair pathways are highly inter-linked for the fast and optimal DNA repair. A deregulation of DNA repair pathways may maintain and promote genetic instability and drug resistance to genotoxic agents in tumor cells by specific mechanisms that tolerate or rapidly bypass lesions to drive proliferation and abrogate cell death. Multiple Myeloma (MM) is a plasma cell disorder characterized by genetic instability and poor outcome for some patients, in which the compendium of DNA repair pathways has as yet not been assessed for a disease-specific prognostic relevance. We design a DNA repair risk score based on the expression of genes coding for proteins involved in DNA repair in MM cells. From a consensus list of 84 DNA repair genes, 17 had a bad prognostic value and 5 a good prognostic value for both event-free and overall survival of previously-untreated MM patients. The prognostic information provided by these 22 prognostic genes was summed within a global DNA repair score (DRScore) to take into account the tight linkage of repair pathways. DRscore was strongly predictive for both patients' event free and overall survivals. Also, DRscore has the potential to identify MM patients whose tumor cells are dependent on specific DNA repair pathways to design treatments that induce synthetic lethality by exploiting addiction to deregulated DNA repair pathways. PMID:24809299

Kassambara, Alboukadel; Gourzones-Dmitriev, Claire; Sahota, Surinder; Rème, Thierry; Moreaux, Jérôme; Goldschmidt, Hartmut; Constantinou, Angelos; Pasero, Philippe; Hose, Dirk; Klein, Bernard

2014-05-15

88

A DNA repair pathway score predicts survival in human multiple myeloma: the potential for therapeutic strategy  

PubMed Central

DNA repair is critical to resolve extrinsic or intrinsic DNA damage to ensure regulated gene transcription and DNA replication. These pathways control repair of double strand breaks, interstrand crosslinks, and nucleotide lesions occurring on single strands. Distinct DNA repair pathways are highly inter-linked for the fast and optimal DNA repair. A deregulation of DNA repair pathways may maintain and promote genetic instability and drug resistance to genotoxic agents in tumor cells by specific mechanisms that tolerate or rapidly bypass lesions to drive proliferation and abrogate cell death. Multiple Myeloma (MM) is a plasma cell disorder characterized by genetic instability and poor outcome for some patients, in which the compendium of DNA repair pathways has as yet not been assessed for a disease-specific prognostic relevance. We design a DNA repair risk score based on the expression of genes coding for proteins involved in DNA repair in MM cells. From a consensus list of 84 DNA repair genes, 17 had a bad prognostic value and 5 a good prognostic value for both event-free and overall survival of previously-untreated MM patients. The prognostic information provided by these 22 prognostic genes was summed within a global DNA repair score (DRScore) to take into account the tight linkage of repair pathways. DRscore was strongly predictive for both patients' event free and overall survivals. Also, DRscore has the potential to identify MM patients whose tumor cells are dependent on specific DNA repair pathways to design treatments that induce synthetic lethality by exploiting addiction to deregulated DNA repair pathways.

Kassambara, Alboukadel; Gourzones-Dmitriev, Claire; Sahota, Surinder; Reme, Thierry; Moreaux, Jerome; Goldschmidt, Hartmut; Constantinou, Angelos; Pasero, Philippe; Hose, Dirk; Klein, Bernard

2014-01-01

89

ADI1, a methionine salvage pathway enzyme, is required for Drosophila fecundity  

PubMed Central

Background Methionine, an essential amino acid, is required for protein synthesis and normal cell metabolism. The transmethylation pathway and methionine salvage pathway (MTA cycle) are two major pathways regulating methionine metabolism. Recently, methionine has been reported to play a key role in Drosophila fecundity. Results Here, we revealed that the MTA cycle plays a crucial role in Drosophila fecundity using the mutant of aci-reductone dioxygenase 1 (DADI1), an enzyme in the MTA cycle. In dietary restriction condition, the egg production of adi1 mutant flies was reduced compared to that of control flies. This fecundity defect in mutant flies was rescued by reintroduction of Dadi1 gene. Moreover, a functional homolog of human ADI1 also recovered the reproduction defect, in which the enzymatic activity of human ADI1 is required for normal fecundity. Importantly, methionine supply rescued the fecundity defect in Dadi1 mutant flies. The detailed analysis of Dadi1 mutant ovaries revealed a dramatic change in the levels of methionine metabolism. In addition, we found that three compounds namely, methionine, SAM and Methionine sulfoxide, respectively, may be required for normal fecundity. Conclusions In summary, these results suggest that ADI1, an MTA cycle enzyme, affects fly fecundity through the regulation of methionine metabolism.

2014-01-01

90

THE 5-LIPOXYGENASE PATHWAY IS REQUIRED FOR ACUTE LUNG INJURY FOLLOWING HEMORRHAGIC SHOCK  

PubMed Central

The cellular and biochemical mechanisms leading to acute lung injury and subsequent multiple organ failure are only partially understood. In order to study the potential role of eicosanoids, particularly leukotrienes, as possible mediators of acute lung injury, we used a murine experimental model of acute lung injury induced by hemorrhagic shock after blood removal via cardiac puncture. Neutrophil sequestration as shown by immunofluorescence, and protein leakage into the alveolar space, were measured as markers of injury. We used liquid chromatography coupled to tandem mass spectrometry to unequivocally identify several eicosanoids in the bronchoalveolar lavage fluid of experimental animals. MK886, a specific inhibitor of the 5-lipoxygenase pathway, as well as transgenic mice deficient in 5-lipoxygenase, were used to determine the role of this enzymatic pathway in this model. Leukotriene B4 and leukotriene C4 were consistently elevated in shock-treated mice compared to sham-treated mice. MK886 attenuated neutrophil infiltration and protein extravasation induced by hemorrhagic shock. 5-lipoxygenase-deficient mice showed reduced neutrophil infiltration and protein extravasation after shock treatment, indicating greatly reduced lung injury. These results support the hypothesis that 5-lipoxygenase, most likely through the generation of leukotrienes, plays an important role in the pathogenesis of acute lung injury induced by hemorrhagic shock in mice. This pathway could represent a new target for pharmacological intervention to reduce lung damage following severe primary injury.

Eun, John C.; Moore, Ernest E.; Mauchley, David C.; Johnson, Chris A.; Meng, Xianzhong; Banerjee, Anirban; Wohlauer, Max V.; Zarini, Simona; Gijon, Miguel A.; Murphy, Robert C.

2012-01-01

91

The 5-lipoxygenase pathway is required for acute lung injury following hemorrhagic shock.  

PubMed

The cellular and biochemical mechanisms leading to acute lung injury (ALI) and subsequent multiple organ failure are only partially understood. To study the potential role of eicosanoids, particularly leukotrienes, as possible mediators of ALI, we used a murine experimental model of ALI induced by hemorrhagic shock after blood removal via cardiac puncture. Neutrophil sequestration, as shown by immunofluorescence and protein leakage into the alveolar space were measured as markers of injury. We used liquid chromatography coupled to tandem mass spectrometry to unequivocally identify several eicosanoids in the bronchoalveolar lavage fluid of experimental animals. MK886, a specific inhibitor of the 5-lipoxygenase (5-LO) pathway, and transgenic mice deficient in 5-LO were used to determine the role of this enzymatic pathway in this model. Leukotriene B4 and leukotriene C4 were consistently elevated in shock-treated mice compared with sham-treated mice. MK886 attenuated neutrophil infiltration and protein extravasation induced by hemorrhagic shock. 5-Lipoxygenase-deficient mice showed reduced neutrophil infiltration and protein extravasation after shock treatment, indicating greatly reduced lung injury. These results support the hypothesis that 5-LO, most likely through the generation of leukotrienes, plays an important role in the pathogenesis of ALI induced by hemorrhagic shock in mice. This pathway could represent a new target for pharmacological intervention to reduce lung damage following severe primary injury. PMID:22392149

Eun, John C; Moore, Ernest E; Mauchley, David C; Johnson, Chris A; Meng, Xianzhong; Banerjee, Anirban; Wohlauer, Max V; Zarini, Simona; Gijón, Miguel A; Murphy, Robert C

2012-06-01

92

Durable adoptive immunotherapy for leukemia produced by manipulation of multiple regulatory pathways of CD8+ T cell tolerance  

PubMed Central

Tolerizing mechanisms within the host and tumor microenvironment inhibit T cell effector functions that can control cancer. These mechanisms blunt adoptive immunotherapy with infused T cells due to a complex array of signals that determine T cell tolerance, survival, or deletion. Ligation of the negative regulatory receptors CTLA4, PD-1(PDCD1) or LAG3 on T cells normally hinders their response to antigen through non-redundant biochemical processes that interfere with stimulatory pathways. In this study, we used an established mouse model of T cell tolerance to define the roles of these inhibitory receptors in regulating CD8+ T cell tolerance during adoptive immunotherapy to treat leukemia. Blocking CTLA4 and PD-1 in vivo combined to promote survival of transferred T cells despite powerful deletional signals that mediate Bim (BCL2L11)-dependent apoptosis. However, this dual blockade was not optimal for stimulating effector function by responding T cells, which required the additional blockade of LAG3 to induce full expansion and allow the acquisition of robust cytolytic activity. Thus, the cooperation of multiple distinct regulatory pathways was needed for the survival and effector differentiation of adoptively transferred tumor-reactive CD8+ T cells. Our work defines the immune escape pathways where simultaneous blockade could yield durable immunotherapeutic responses that can eradicate disseminated leukemia.

Berrien-Elliott, Melissa M.; Jackson, Stephanie R.; Meyer, Jennifer M.; Rouskey, Craig J.; Nguyen, Thanh-Long M.; Yagita, Hideo; Greenberg, Philip D.; DiPaolo, Richard J.; Teague, Ryan M.

2012-01-01

93

Evolutionary study of the isoflavonoid pathway based on multiple copies analysis in soybean  

PubMed Central

Background Previous studies suggest that the metabolic pathway structure influences the selection and evolution rates of involved genes. However, most of these studies have exclusively considered a single gene copy encoding each enzyme in the metabolic pathway. Considering multiple-copy encoding enzymes could provide direct evidence of gene evolution and duplication patterns in metabolic pathways. We conducted a detailed analysis of the phylogeny, synteny, evolutionary rate and selection pressure of the genes in the isoflavonoid metabolic pathway of soybeans. Results The results revealed that 1) only the phenylalanine ammonia-lyase (PAL) gene family most upstream from the pathway preserved all of the ancient and recent segmental duplications and maintained a strongly conserved synteny among these duplicated segments; gene families encoding branch-point enzymes with higher pleiotropy tended to retain more types of duplication; and genes encoding chalcone reductase (CHR) and isoflavone synthase (IFS) specific for legumes retained only recent segmental duplications; 2) downstream genes evolved faster than upstream genes and were subject to positive selection or relaxed selection constraints; 3) gene members encoding enzymes with high pleiotropy at the branching points were more likely to have undergone evolutionary differentiation, which may correspond to their functional divergences. Conclusions We reconciled our results with existing controversies and proposed that gene copies at branch points with higher connectivity might be under stronger selective constraints and that the gene copies controlling metabolic flux allocation underwent positive selection. Our analyses demonstrated that the structure and function of a metabolic pathway shapes gene duplication and the evolutionary constraints of constituent enzymes.

2014-01-01

94

Multiple W\\/O\\/W emulsions—Using the required HLB for emulsifier evaluation  

Microsoft Academic Search

Stable emulsions are best formulated with emulsifiers or combinations of emulsifiers, which possess HLB values close to the required HLB of the oil phase. In this work, we have investigated the application of this established method to the development of multiple emulsions. This is of particular interest, since multiple emulsions are highly sensitive in terms of variations of the individual

T. Schmidts; D. Dobler; A.-C. Guldan; N. Paulus; F. Runkel

2010-01-01

95

Endoplasmic reticulum stress pathway required for immune homeostasis is neurally controlled by arrestin-1.  

PubMed

In response to pathogen infection, the host innate immune system activates microbial killing pathways and cellular stress pathways that need to be balanced because insufficient or excessive immune responses have deleterious consequences. Recent studies demonstrate that two G protein-coupled receptors (GPCRs) in the nervous system of Caenorhabditis elegans control immune homeostasis. To investigate further how GPCR signaling controls immune homeostasis at the organismal level, we studied arrestin-1 (ARR-1), which is the only GPCR adaptor protein in C. elegans. The results indicate that ARR-1 is required for GPCR signaling in ASH, ASI, AQR, PQR, and URX neurons, which control the unfolded protein response and a p38 mitogen-activated protein kinase signaling pathway required for innate immunity. ARR-1 activity also controlled immunity through ADF chemosensory and AFD thermosensory neurons that regulate longevity. Furthermore, we found that although ARR-1 played a key role in the control of immunity by AFD thermosensory neurons, it did not control longevity through these cells. However, ARR-1 partially controlled longevity through ADF neurons. PMID:22875856

Singh, Varsha; Aballay, Alejandro

2012-09-28

96

Targeting of NF-kappaB signaling pathway, other signaling pathways and epigenetics in therapy of multiple myeloma.  

PubMed

Multiple myeloma (MM) remains an incurable disease, at least for the big majority of patients, in spite of the great progress with new drugs in the last years. New treatment strategies are needed to improve the outcome of patients. NF-?B activation in MM is caused by mutations in the factors involved in the NF-?B pathways contributing to their dysregulation and by signals from the bone marrow microenvironment. Agents with NF-?B inhibitory activity enhance the anti-MM effects of conventional chemotherapeutic agents. Bortezomib was the first approved member of a new class of anti-MM agents, the proteasome inhibitors. At least, five proteasome inhibitors of the next generation with greater efficacy (carfilzomib, marizomib (salinosporamide A, NPI-0052), threonine boronic acid-derived proteasome inhibitor CEP-18770, the peptide-semicarbazone S-2209, the tripeptide mimetic BSc2118, and MLN9708/2238) have been recently tested in preclinical models of MM. Carfilzomib has been recently approved for the treatment of patients with MM who have received at least two prior therapies, including bortezomib and immunomodulatory derivatives (IMiDs, thalidomide, lenalidomide or pomalidomide). More specific I?B kinase inhibitors were also used in preclinical studies. The analysis of MM genomes revealed also mutations in genes for histone methyltransferases (HMTases), histone demethylase (UTX) and serine/threonine protein kinase BRAF. Aberrant histone 3 lysine 27 trimethylation (H3K27me3) by mutant HMTases or UTX induces overexpression of the homeobox A9 (HOXA9) gene. HOXA9 is normally expressed in primitive bone marrow cells and is silenced when cells differentiate. HOXA9 is a MM oncogene and targeting of its expression by histone deacetylases inhibitors or by a phosphoinositide 3-kinase (PI3K) inhibitors through an epigenetic mechanism involving H3K27me3. Mutant BRAF kinase small-molecule, ATP-competitive, a highly selective, potent and orally bioavailable inhibitors (GDC-0879, PLX 4032 and PLX 4720) are already under investigation and PLX 4032 is in phase II and phase III clinical trials. Two key signaling pathways involved in the regulation of MM cell growth are the Ras/Raf/MEK/ERK and PI3K/Akt/mTOR pathways and their inhibition are anti-proliferative and pro-apoptotic and can overcome the development of resistance to common drugs. PMID:23534949

Fuchs, Ota

2013-03-01

97

Integrated modeling of flow and residence times at the catchment scale with multiple interacting pathways  

NASA Astrophysics Data System (ADS)

There is still a need for catchment hydrological and transport models that properly integrate the effects of preferential flows while accounting for differences in velocities and celerities. A modeling methodology is presented here which uses particle tracking methods to simulate both flow and transport in multiple pathways in a single consistent solution. Water fluxes and storages are determined by the volume and density of particles and transport is attained by labeling the particles with information that may be tracked throughout the lifetime of that particle in the catchment. The methodology allows representation of preferential flows through the use of particle velocity distributions, and mixing between pathways can be achieved with pathway transition probabilities. A transferable 3-D modeling methodology is presented for the first time and applied to a unique step-shift isotope experiment that was carried out at the 0.63 ha G1 catchment in Gårdsjön, Sweden. This application highlights the importance of combining flow and transport in hydrological representations, and the importance of pathway velocity distributions and interactions in obtaining a satisfactory representation of the observations.

Davies, J.; Beven, K.; Rodhe, A.; Nyberg, L.; Bishop, K.

2013-08-01

98

Investigating sources and pathways of perfluoroalkyl acids (PFAAs) in aquifers in Tokyo using multiple tracers.  

PubMed

We employed a multi-tracer approach to investigate sources and pathways of perfluoroalkyl acids (PFAAs) in urban groundwater, based on 53 groundwater samples taken from confined aquifers and unconfined aquifers in Tokyo. While the median concentrations of groundwater PFAAs were several ng/L, the maximum concentrations of perfluorooctane sulfonate (PFOS, 990 ng/L), perfluorooctanoate (PFOA, 1800 ng/L) and perfluorononanoate (PFNA, 620 ng/L) in groundwater were several times higher than those of wastewater and street runoff reported in the literature. PFAAs were more frequently detected than sewage tracers (carbamazepine and crotamiton), presumably owing to the higher persistence of PFAAs, the multiple sources of PFAAs beyond sewage (e.g., surface runoff, point sources) and the formation of PFAAs from their precursors. Use of multiple methods of source apportionment including principal component analysis-multiple linear regression (PCA-MLR) and perfluoroalkyl carboxylic acid ratio analysis highlighted sewage and point sources as the primary sources of PFAAs in the most severely polluted groundwater samples, with street runoff being a minor source (44.6% sewage, 45.7% point sources and 9.7% street runoff, by PCA-MLR). Tritium analysis indicated that, while young groundwater (recharged during or after the 1970s, when PFAAs were already in commercial use) in shallow aquifers (<50 m depth) was naturally highly vulnerable to PFAA pollution, PFAAs were also found in old groundwater (recharged before the 1950s, when PFAAs were not in use) in deep aquifers (50-500 m depth). This study demonstrated the utility of multiple uses of tracers (pharmaceuticals and personal care products; PPCPs, tritium) and source apportionment methods in investigating sources and pathways of PFAAs in multiple aquifer systems. PMID:24814036

Kuroda, Keisuke; Murakami, Michio; Oguma, Kumiko; Takada, Hideshige; Takizawa, Satoshi

2014-08-01

99

Use of multiple dispersal pathways facilitates amphibian persistence in stream networks  

PubMed Central

Although populations of amphibians are declining worldwide, there is no evidence that salamanders occupying small streams are experiencing enigmatic declines, and populations of these species seem stable. Theory predicts that dispersal through multiple pathways can stabilize populations, preventing extinction in habitat networks. However, empirical data to support this prediction are absent for most species, especially those at risk of decline. Our mark-recapture study of stream salamanders reveals both a strong upstream bias in dispersal and a surprisingly high rate of overland dispersal to adjacent headwater streams. This evidence of route-dependent variation in dispersal rates suggests a spatial mechanism for population stability in headwater-stream salamanders. Our results link the movement behavior of stream salamanders to network topology, and they underscore the importance of identifying and protecting critical dispersal pathways when addressing region-wide population declines.

Campbell Grant, Evan H.; Nichols, James D.; Lowe, Winsor H.; Fagan, William F.

2010-01-01

100

Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae  

PubMed Central

The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination.

Paques, Frederic; Haber, James E.

1999-01-01

101

Yessotoxin as a tool to study induction of multiple cell death pathways.  

PubMed

This work proposes to use the marine algal toxin yessotoxin (YTX) to establish reference model experiments to explore medically valuable effects from induction of multiple cell death pathways. YTX is one of few toxins reported to make such induction. It is a small molecule compound which at low concentrations can induce apoptosis in primary cultures, many types of cells and cell lines. It can also induce a non-apoptotic form of programmed cell death in BC3H1 myoblast cell lines. The present contribution reviews arguments that this type of induction may have principal interest outside this particular example. One principal effect of medical interest may be that cancer cells will not so easily adapt to the synergistic effects from induction of more than one death pathway as compared to induction of only apoptosis. PMID:22852069

Korsnes, Mónica Suárez

2012-07-01

102

At the crossroads of differentiation and proliferation: Precise control of cell-cycle changes by multiple signaling pathways in Drosophila follicle cells  

PubMed Central

Here, we discuss the findings to date about genes and pathways required for regulation of somatic follicle-cell proliferation and differentiation during Drosophila oogenesis and demonstrate how loss of these genes contributes to the tumorigenic potential of mutant cells. Follicle cells undergo cell-fate determination through stepwise activation of multiple signaling pathways, including the Notch, Hedgehog, Wingless, janus kinase/STAT, and JNK pathways. In addition, changes in DNA replication and cellular growth depend on the spatial and temporal activation of the mitotic cycle-endocycle and endocycle-gene amplification cell-cycle switches and insulin-dependent monitoring of cellular health; systemic loss of these pathways contributes to loss of controlled cellular proliferation, loss of differentiation/growth, and aberrant cell polarity in follicle cells. We also highlight the effects of the neoplastic and Hippo pathways on the cell cycle and cellular proliferation in promoting normal development and conclude that lack of coordination of multiple signaling pathways promotes conditions favorable for tumorigenesis.

Klusza, Stephen; Deng, Wu-Min

2013-01-01

103

Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways  

PubMed Central

Cholangiocarcinoma (CCA) is a tumor with poor prognosis that is resistant to all currently available treatments. Whether curcumin, a nutraceutical derived from turmeric (Curcuma longa), has potential therapeutic activity against human CCA was investigated using three CCA cell lines (KKU100, KKU-M156 and KKU-M213). Examination of mitochondrial dehydrogenase activity, phosphatidylserine externalization, esterase staining, caspase activation and poly-adenosine diphosphate ribose polymerase cleavage demonstrated that curcumin inhibited proliferation of and induced apoptosis in these biliary cancer cells. Colony-formation assay confirmed the growth-inhibitory effect of curcumin on CCA cells. When examined for the mechanism, curcumin was found to activate multiple cell signaling pathways in these cells. First, all CCA cells exhibited constitutively active nuclear factor (NF)-?B, and treatment with curcumin abolished this activation as indicated by DNA binding, nuclear translocation and p65 phosphorylation. Second, curcumin suppressed activation of signal transducer and activator of transcription-3 as indicated by decreased phosphorylation at both tyrosine705 and serine727 and inhibition of janus kinase-1 phosphorylation. Third, curcumin induced expression of peroxisome proliferator-activated receptor gamma. Fourth, curcumin upregulated death receptors, DR4 and DR5. Fifth, curcumin suppressed the Akt activation pathway. Sixth, curcumin inhibited expression of cell survival proteins such as B-cell lymphoma-2, B-cell leukemia protein xL, X-linked inhibitor of apoptosis protein, c-FLIP, cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2 and survivin and proteins linked to cell proliferation, such as cyclin D1 and c-Myc. Seventh, the growth inhibitory effect of curcumin was enhanced in the I?B kinase-deficient cells, the enzyme required for nuclear factor-kappaB activation. Overall, our results indicate that curcumin mediates its antiproliferative and apoptotic effects through activation of multiple cell signaling pathways, and thus, its activity against CCA should be further investigated.

Prakobwong, Suksanti; Gupta, Subash C.; Kim, Ji Hye; Sung, Bokyung; Pinlaor, Porntip; Hiraku, Yusuke; Wongkham, Sopit; Sripa, Banchob; Pinlaor, Somchai

2011-01-01

104

Secretion is required for late events in the cell-fusion pathway of mating yeast.  

PubMed

Secretory vesicles accumulate adjacent to the contact site between the two cells of a yeast mating pair before they fuse, but there is no direct evidence that secretion is required to complete fusion. In this study, temperature-sensitive secretion (sec(ts)) mutants were used to investigate the role of secretion in yeast cell fusion. Cell fusion arrested less than 5 minutes after inhibiting secretion. This rapid fusion arrest was not an indirect consequence of reduced mating pheromone signaling, mating-pair assembly or actin polarity. Furthermore, secretion was required to complete cell fusion when it was transiently inhibited by addition and removal of the lipophilic styryl dye, FM4-64. These results indicate that ongoing secretion is required for late events in the cell-fusion pathway, which include plasma-membrane fusion and the completion of cell-wall remodeling, and they demonstrate a just-in-time delivery mechanism for the cell-fusion machinery. PMID:20460435

Grote, Eric

2010-06-01

105

Sigma-2 ligands induce tumour cell death by multiple signalling pathways  

PubMed Central

Background: The sigma-2 receptor has been identified as a biomarker of proliferating cells in solid tumours. In the present study, we studied the mechanisms of sigma-2 ligand-induced cell death in the mouse breast cancer cell line EMT-6 and the human melanoma cell line MDA-MB-435. Methods: EMT-6 and MDA-MB-435 cells were treated with sigma-2 ligands. The modulation of multiple signaling pathways of cell death was evaluated. Results: Three sigma-2 ligands (WC-26, SV119 and RHM-138) induced DNA fragmentation, caspase-3 activation and PARP-1 cleavage. The caspase inhibitor Z-VAD-FMK partially blocked DNA fragmentation and cytotoxicity caused by these compounds. These data suggest that sigma-2 ligand-induced apoptosis and caspase activation are partially responsible for the cell death. WC-26 and siramesine induced formation of vacuoles in the cells. WC-26, SV119, RHM-138 and siramesine increased the synthesis and processing of microtubule-associated protein light chain 3, an autophagosome marker, and decreased the expression levels of the downstream effectors of mammalian target of rapamycin (mTOR), p70S6K and 4EBP1, suggesting that sigma-2 ligands induce autophagy, probably by inhibition of the mTOR pathway. All four sigma-2 ligands decreased the expression of cyclin D1 in a time-dependent manner. In addition, WC-26 and SV119 mainly decreased cyclin B1, E2 and phosphorylation of retinoblastoma protein (pRb); RHM-138 mainly decreased cyclin E2; and 10?? siramesine mainly decreased cyclin B1 and pRb. These data suggest that sigma-2 ligands also impair cell-cycle progression in multiple phases of the cell cycle. Conclusion: Sigma-2 ligands induce cell death by multiple signalling pathways.

Zeng, C; Rothfuss, J; Zhang, J; Chu, W; Vangveravong, S; Tu, Z; Pan, F; Chang, K C; Hotchkiss, R; Mach, R H

2012-01-01

106

TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and -independent pathways  

PubMed Central

TonEBP (tonicity-responsive enhancer binding protein) is a transcription factor that promotes cellular accumulation of organic osmolytes in the hypertonic renal medulla by stimulating expression of its target genes. Genetically modified animals with deficient TonEBP activity in the kidney suffer from severe medullary atrophy in association with cell death, demonstrating that TonEBP is essential for the survival of the renal medullary cells. Using both TonEBP knockout cells and RNA interference of TonEBP, we found that TonEBP promoted cellular adaptation to hypertonic stress. Microarray analyses revealed that the genetic response to hypertonicity was dominated by TonEBP in that expression of totally different sets of genes was increased by hypertonicity in those cells with TonEBP vs. those without TonEBP activity. Of over 100 potentially new TonEBP-regulated genes, we selected seven for further analyses and found that their expressions were all dependent on TonEBP. RNA interference experiments showed that some of these genes, asporin, insulin-like growth factor-binding protein-5 and -7, and an extracellular lysophospholipase D, plus heat shock protein 70, a known TonEBP target gene, contributed to the adaptation to hypertonicity without promoting organic osmolyte accumulation. We conclude that TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress in addition to organic osmolyte accumulation.

Lee, Sang Do; Choi, Soo Youn; Lim, Sun Woo; Lamitina, S. Todd; Ho, Steffan N.; Go, William Y.

2011-01-01

107

Multiple pathways in nuclear transport: the import of U2 snRNP occurs by a novel kinetic pathway  

PubMed Central

Protein import to the nucleus is a signal-mediated process that exhibits saturation kinetics. We investigated whether signal bearing proteins compete with U2 and U6 snRNPs during import. When injected into Xenopus oocytes, saturating concentrations of P(Lys)-BSA, a protein bearing multiple nuclear localization signals from SV40 large T- antigen, reduce the rate of [125I]P(Lys)-BSA and of [125I]nucleoplasmin import, consistent with their competing for and sharing the same limiting component of the import apparatus. In contrast, saturating concentrations of P(Lys)-BSA do not reduce the rate of HeLa [32P]U2 snRNP assembly or import. The import of U6 snRNP is also competed by P(Lys)-BSA. We conclude that U2 snRNP is imported into oocyte nuclei by a kinetic pathway that is distinct from the one followed by P(Lys)-BSA, nucleoplasmin, and U6 snRNP.

1991-01-01

108

Suppressed RNA-polymerase 1 pathway is associated with benign multiple sclerosis.  

PubMed

Benign multiple sclerosis (BMS) occurs in about 15% of patients with relapsing-remitting multiple sclerosis (RRMS) that over time do not develop significant neurological disability. The molecular events associated with BMS are not clearly understood. This study sought to underlie the biological mechanisms associated with BMS. Blood samples obtained from a cohort of 31 patients with BMS and 36 patients with RRMS were applied for gene expression microarray analysis using HG-U133A-2 array (Affymetrix). Data were analyzed by Partek and pathway reconstruction was performed by Ingenuity for the most informative genes (MIGs). We identified a differing gene expression signature of 406 MIGs between BMS patients, mean±SE age 44.5±1.5 years, 24 females, 7 males, EDSS 1.9±0.2, disease duration 17.0±1.3 years, and RRMS patients, age 40.3±1.8 years, 24 females, 12 males, EDSS 3.5±0.2, disease duration 10.9±1.4 years. The signature was enriched by genes related RNA polymerase I (POL-1) transcription, general inflammatory response and activation of cell death. The most significant under-expressed pathway operating in BMS was the POL-1 pathway (p = 4.0*10(-5)) known while suppressed to activate P53 dependent apoptosis and to suppress NF?B induced inflammation. In accordance, of the 30 P53 target genes presented within the BMS signature, 19 had expression direction consistent with P53 activation. The transcripts within the pathway include POL-1 transcription factor 3 (RRN3, p = 4.8*10(-5)), POL-1 polypeptide D (POLR1D, p = 2.2*10(-4)), leucine-rich PPR-motif containing protein (LRPPRC p = 2.3*10(-5)), followed by suppression of the downstream family of ribosomal genes like RPL3, 6,13,22 and RPS6. In accordance POL-1 transcript and release factor PTRF that terminates POL-1 transcription, was over-expressed (p = 4.4*10(-3)). Verification of POL-1 pathway key genes was confirmed by qRT-PCR, and RRN3 silencing resulted in significant increase in the apoptosis level of PBMC sub-populations in RRMS patients. Our findings demonstrate that suppression of POL-1 pathway induce the low disease activity of BMS. PMID:23077530

Achiron, Anat; Feldman, Anna; Magalashvili, David; Dolev, Mark; Gurevich, Michael

2012-01-01

109

Role of the tissue factor pathway in the pathogenesis and management of multiple organ failure.  

PubMed

Sepsis is caused by a dysregulated immune response to infection and, without intervention, can lead to septic shock and multiple organ failure. A leading cause of morbidity and mortality in intensive care units worldwide, severe sepsis is also associated with a considerable cost burden that places significant strain on global healthcare budgets. The development of an efficacious and cost-effective treatment strategy is therefore of vital importance to today's intensive care physicians. This paper will examine the pathophysiology of sepsis and multiple organ dysfunction before reviewing trials recently undertaken to investigate three potential anticoagulant therapies: antithrombin III, activated protein C, and tissue factor pathway inhibitor. Finally, other recent developments in the care of sepsis patients will be briefly examined. PMID:15166928

Smithies, Mark N; Weaver, Christine B

2004-05-01

110

Induction of Multiple Immune Regulatory Pathways with Differential Impact in HCV/HIV Coinfection  

PubMed Central

Persistent viral infections including HCV, HBV, and HIV are associated with increased immune regulatory pathways including the extrinsic FoxP3+CD4+ regulatory T cells (Tregs) and intrinsic inhibitory pathways such as programed death-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) with potentially reversible suppression of antiviral effector T cells (1–12). Immunological consequences of viral coinfections relative to these immune regulatory pathways and their interplay are not well-defined. In this study, we examined the frequency, phenotype, and effector function of circulating T cell subsets in patients with chronic HCV and/or HIV infection, hypothesizing that HCV/HIV coinfection will result in greater immune dysregulation with pathogenetic consequences (13, 14). We show that multiple T cell inhibitory pathways are induced in HCV/HIV coinfection including FoxP3+ Tregs, PD-1, and CTLA-4 in inverse association with overall CD4 T cell frequency but not with liver function or HCV RNA titers. The inverse association between CD4 T cell frequency and their FoxP3, PD-1, or CTLA-4 expression remained significant in all subjects combined regardless of HCV and/or HIV infection, suggesting a global homeostatic mechanism to maintain immune regulation relative to CD4 T cell frequency. PD-1 blockade rescued T cell responses to HIV but not HCV without significant impact by CTLA-4 blockade in vitro. Collectively, these findings highlight complex immune interactions in viral coinfections and differential regulatory pathways influencing virus-specific T cells that are relevant in immunotherapeutic development.

Cho, Hyosun; Kikuchi, Masahiro; Li, Yun; Nakamoto, Nobuhiro; Amorosa, Valerianna K.; Valiga, Mary E.; Chang, Kyong-Mi

2014-01-01

111

Lipopolysaccharide activates innate immune responses in murine intestinal myofibroblasts through multiple signaling pathways  

PubMed Central

Myofibroblasts (MF) play an important role in intestinal wound healing. A compromised epithelial barrier exposes intestinal subepithelial MF to luminal bacterial products. However, responses of murine intestinal MF to bacterial adjuvants and potential roles of intestinal MF in innate immune responses are not well defined. Our aims in this study were to determine innate immune responses and intracellular signaling pathways of intestinal MF exposed to LPS, a prototypic Toll-like receptor (TLR) ligand. Expression of TLR4 in primary murine intestinal MF cultures was confirmed by RT-PCR and Western blotting. LPS-induced secretion of prostaglandin E2 (PGE2), interleukin (IL)-6, and keratinocyte-derived chemokines (KC) was measured by ELISA. Intracellular responses to LPS were assessed by Western blotting for NF-?B p65, I?-B?, Akt, p38 MAP kinase, and cyclooxygenase-2 (COX-2). LPS induced rapid phosphorylation of NF-?B p65, Akt, and p38 MAPK and degradation of I?-B?. LPS induced expression of COX-2 and secretion of PGE2 (2.0 ± 0.8-fold induction vs. unstimulated cells), IL-6 (6.6 ± 0.4-fold induction), and KC (12.5 ± 0.4-fold induction). Inhibition of phosphoinositide-3 (PI3)-kinase, p38 MAPK, or NF-?B pathways reduced LPS-induced PGE2, IL-6, and KC secretion. These studies show that primary murine intestinal MF respond to LPS, evidenced by activation of NF-?B, PI3-kinase, and MAPK signaling pathways and secretion of proinflammatory molecules. Inhibition of these pathways attenuated LPS-dependent PGE2, IL-6, and KC production, indicating that LPS activates MF by multiple signaling pathways. These data support the hypothesis that MF are a component of the innate immune system and may exert paracrine effects on adjacent epithelial and immune cells by responding to luminal bacterial adjuvants.

Walton, Kristen L. W.; Holt, Lisa; Sartor, R. Balfour

2009-01-01

112

Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways  

PubMed Central

Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live HeLa cells. Using real time confocal fluorescence microscopy imaging, we show that MCoTI-I is readily internalized in live HeLa cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I in HeLa cells is achieved primarily through fluid-phase endocytosis, as evidenced by its significant colocalization with 10K-dextran, but also through other pathways as well, as evidenced by its colocalization with markers for cholesterol-dependent and clathrin-mediated endocytosis, cholera toxin B and EGF respectively. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway by Latrunculin B-induced disassembly of actin filaments did not affect MCoTI-I uptake and treatment with EIPA which also seemed to inhibit other pathways collectively inhibited approximately 80% of cellular uptake. As well, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments and MCoTI-I-containing vesicles continue to exhibit directed movements. These findings demonstrate internalization of MCoTI-I through multiple endocytic pathways that are dominant in the cell type investigated, suggesting that this cyclotide has ready access to general endosomal/lysosomal pathways but could readily be re-targeted to specific receptors through addition of targeting ligands.

Contreras, Janette; Elnagar, Ahmed Y. O.; Hamm-Alvarez, Sarah F.; Camarero, Julio A.

2011-01-01

113

Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways.  

PubMed

Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live HeLa cells. Using real time confocal fluorescence microscopy imaging, we show that MCoTI-I is readily internalized in live HeLa cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I in HeLa cells is achieved primarily through fluid-phase endocytosis, as evidenced by its significant colocalization with 10K-dextran, but also through other pathways as well, as evidenced by its colocalization with markers for cholesterol-dependent and clathrin-mediated endocytosis, cholera toxin B and EGF respectively. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway by Latrunculin B-induced disassembly of actin filaments did not affect MCoTI-I uptake and treatment with EIPA which also seemed to inhibit other pathways collectively inhibited approximately 80% of cellular uptake. As well, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments and MCoTI-I-containing vesicles continue to exhibit directed movements. These findings demonstrate internalization of MCoTI-I through multiple endocytic pathways that are dominant in the cell type investigated, suggesting that this cyclotide has ready access to general endosomal/lysosomal pathways but could readily be re-targeted to specific receptors through addition of targeting ligands. PMID:21906641

Contreras, Janette; Elnagar, Ahmed Y O; Hamm-Alvarez, Sarah F; Camarero, Julio A

2011-10-30

114

Activation of NF-kB Pathway by Virus Infection Requires Rb Expression  

PubMed Central

The retinoblastoma protein Rb is a tumor suppressor involved in cell cycle control, differentiation, and inhibition of oncogenic transformation. Besides these roles, additional functions in the control of immune response have been suggested. In the present study we investigated the consequences of loss of Rb in viral infection. Here we show that virus replication is increased by the absence of Rb, and that Rb is required for the activation of the NF-kB pathway in response to virus infection. These results reveal a novel role for tumor suppressor Rb in viral infection surveillance and further extend the concept of a link between tumor suppressors and antiviral activity.

Garcia, Maria A.; Gallego, Pedro; Campagna, Michela; Gonzalez-Santamaria, Jose; Martinez, Gloria; Marcos-Villar, Laura; Vidal, Anxo; Esteban, Mariano; Rivas, Carmen

2009-01-01

115

Bacterial decolorization of textile dyes is an extracellular process requiring a multicomponent electron transfer pathway  

PubMed Central

Summary Many studies have reported microorganisms as efficient biocatalysts for colour removal of dye?containing industrial wastewaters. We present the first comprehensive study to identify all molecular components involved in decolorization by bacterial cells. Mutants from the model organism Shewanella oneidensis MR?1, generated by random transposon and targeted insertional mutagenesis, were screened for defects in decolorization of an oxazine and diazo dye. We demonstrate that decolorization is an extracellular reduction process requiring a multicomponent electron transfer pathway that consists of cytoplasmic membrane, periplasmic and outer membrane components. The presence of melanin, a redox?active molecule excreted by S. oneidensis, was shown to enhance the dye reduction rates. Menaquinones and the cytochrome CymA are the crucial cytoplasmic membrane components of the pathway, which then branches off via a network of periplasmic cytochromes to three outer membrane cytochromes. The key proteins of this network are MtrA and OmcB in the periplasm and outer membrane respectively. A model of the complete dye reduction pathway is proposed in which the dye molecules are reduced by the outer membrane cytochromes either directly or indirectly via melanin.

Brige, Ann; Motte, Bart; Borloo, Jimmy; Buysschaert, Geraldine; Devreese, Bart; Van Beeumen, Jozef J.

2008-01-01

116

Efficient Herpes Simplex Virus 1 Replication Requires Cellular ATR Pathway Proteins  

PubMed Central

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of the host cell and is known to interact with several components of the cellular DNA-damage-signaling machinery. We have previously reported that the DNA damage response kinase, ATR, is specifically inactivated in HSV-1-infected cells. On the other hand, we have also shown that ATR and its scaffolding protein, ATRIP, are recruited to viral replication compartments, where they play beneficial roles during HSV-1 replication. In order to better understand this apparent discrepancy, we tested the hypothesis that some of the components of the ATR pathway may exert an antiviral effect on infection. In fact, we learned that all 10 of the canonical ATR pathway proteins are stable in HSV-infected cells and are recruited to viral replication compartments; furthermore, short hairpin RNA (shRNA) knockdown shows that several, including ATRIP, RPA70, TopBP1, Claspin, and CINP, are required for efficient HSV-1 replication. We also determined that activation of the ATR kinase prior to infection did not affect virus yield but did result in reduced levels of recombination between coinfecting viruses. Together, these data suggest that ATR pathway proteins are not antiviral per se but that activation of ATR signaling may have negative consequences during viral replication, such as inhibiting recombination.

Mohni, Kareem N.; Dee, Alexander R.; Smith, Samantha; Schumacher, April J.

2013-01-01

117

The intrinsic apoptotic pathway is required for lipopolysaccharide-induced lung endothelial cell death.  

PubMed

LPS has been implicated in the pathogenesis of endothelial cell death associated with Gram-negative bacterial sepsis. The binding of LPS to the TLR-4 on the surface of endothelial cells initiates the formation of a death-inducing signaling complex at the cell surface. The subsequent signaling pathways that result in apoptotic cell death remain unclear and may differ among endothelial cells in different organs. We sought to determine whether LPS and cycloheximide-induced cell death in human lung microvascular endothelial cells (HmVECs) was dependent upon activation of the intrinsic apoptotic pathway and the generation of reactive oxygen species. We found that cells overexpressing the anti-apoptotic protein Bcl-X(L) were resistant to LPS and cycloheximide-induced death and that the proapoptotic Bcl-2 protein Bid was cleaved following treatment with LPS. The importance of Bid was confirmed by protection of Bid-deficient (bid(-/-)) mice from LPS-induced lung injury. Neither HmVECs treated with the combined superoxide dismutase/catalase mimetic EUK-134 nor HmVECs depleted of mitochondrial DNA (rho(0) cells) were protected against LPS and cycloheximide-induced death. We conclude that LPS and cycloheximide-induced death in HmVECs requires the intrinsic cell death pathway, but not the generation of reactive oxygen species. PMID:17641050

Wang, Helena L; Akinci, I Ozkan; Baker, Christina M; Urich, Daniela; Bellmeyer, Amy; Jain, Manu; Chandel, Navdeep S; Mutlu, Gökhan M; Budinger, G R Scott

2007-08-01

118

Multiple motor pathways to single smooth muscle cells in the ferret trachea.  

PubMed Central

1. We investigated the distribution and characteristics of motor pathways to individual smooth muscle cells activated by electrical stimulation of either, single nerves which enter the tracheal plexus (inlet nerves), or a longitudinal nerve trunk (LNT) located near the entrance of an inlet nerve into the plexus. Excitatory junction potentials (EJPs) were recorded using intracellular microelectrodes as an index of smooth muscle cell activation. In all experiments EJPs were completely blocked by tetrodotoxin and by atropine. 2. In smooth muscle fields located in the caudal direction from the point of inlet or LNT nerve stimulation, neural input decreased as a function of distance. There was evidence of a demarcated area innervated by neurons entering the plexus in one inlet nerve. In smooth muscle fields located in the rostral or transverse direction from the site of nerve stimulation, no such demarcated area could be identified. 3. Of the smooth muscle cells located within the innervated fields studied, 83-95% were activated following stimulation of a single inlet nerve or LNT. Evoked EJPs were similar in different innervated cells or units of electrically coupled cells located within the same 1 mm2 'field'. 4. There was overlapping cholinergic motor input to single smooth muscle cells originating from neurons present in different inlet nerves or different neurons present in the same inlet nerve or region of the LNT. Multiple small step increases in the voltage used to stimulate a LNT resulted in three or four step increases in EJP amplitudes. This gives a minimal value for the number of motor pathways that can be activated by neurons in a region of LNT leading to a single smooth muscle cell. 5. Motor pathways to smooth muscle cells located in caudal and rostral fields ran initially in the LNT and exited in proximity to the smooth muscle cell studied. 6. Motor pathways used in transmitting signals to smooth muscle cells to different areas of trachealis muscle varied in their sensitivity to hexamethonium or curare. EJPs evoked in fields located in the caudal direction from the stimulating electrode were abolished by these drugs. Muscle cells located in different rostral fields showed EJPs that were either sensitive or resistant to these drugs. 7. The rostral hexamethonium-resistant pathway ran initially in the LNT but it exited from the LNT several millimetres before reaching the level of the smooth muscle field innervated. This pathway followed stimulation frequencies up to 25 Hz. The final neuron in this pathway released acetylcholine and evoked EJPs were entirely inhibited by atropine.(ABSTRACT TRUNCATED AT 400 WORDS)

Mitchell, H W; Coburn, R F

1992-01-01

119

Gene profiling of keloid fibroblasts shows altered expression in multiple fibrosis-associated pathways  

PubMed Central

Keloids are benign tumors of the dermis that form during a protracted wound healing process. Susceptibility to keloid formation occurs predominantly in people of African and Asian descent. The key alteration(s) responsible for keloid formation has not been identified and there is no satisfactory treatment for this disorder. The altered regulatory mechanism is limited to dermal wound healing, although several diseases characterized by an exaggerated response to injury are prevalent in individuals of African ancestry. We have observed a complex pattern of phenotypic differences in keloid fibroblasts grown in standard culture medium or induced by hydrocortisone. In this study Affymetrix-based microarray was performed on RNA obtained from fibroblasts cultured from normal scars and keloids grown in the absence and presence of hydrocortisone. We observed differential regulation of approximately 500 genes of the 38,000 represented on the Affymetrix chip. Of particular interest was increased expression of several IGF-binding and IGF-binding related proteins and decreased expression of a subset of Wnt pathway inhibitors and multiple IL-1-inducible genes. Increased expression of CTGF and IGFBP-3 was observed in keloid fibroblasts only in the presence of hydrocortisone. These findings support a role for multiple fibrosis-related pathways in the pathogenesis of keloids.

Smith, Joan C.; Boone, Braden E.; Opalenik, Susan R.; Williams, Scott M.; Russell, Shirley B.

2010-01-01

120

Erect wing regulates synaptic growth in Drosophila by integration of multiple signaling pathways  

PubMed Central

Background Formation of synaptic connections is a dynamic and highly regulated process. Little is known about the gene networks that regulate synaptic growth and how they balance stimulatory and restrictive signals. Results Here we show that the neuronally expressed transcription factor gene erect wing (ewg) is a major target of the RNA binding protein ELAV and that EWG restricts synaptic growth at neuromuscular junctions. Using a functional genomics approach we demonstrate that EWG acts primarily through increasing mRNA levels of genes involved in transcriptional and post-transcriptional regulation of gene expression, while genes at the end of the regulatory expression hierarchy (effector genes) represent only a minor portion, indicating an extensive regulatory network. Among EWG-regulated genes are components of Wingless and Notch signaling pathways. In a clonal analysis we demonstrate that EWG genetically interacts with Wingless and Notch, and also with TGF-? and AP-1 pathways in the regulation of synaptic growth. Conclusion Our results show that EWG restricts synaptic growth by integrating multiple cellular signaling pathways into an extensive regulatory gene expression network.

Haussmann, Irmgard U; White, Kalpana; Soller, Matthias

2008-01-01

121

TFAP2C controls hormone response in breast cancer cells through multiple pathways of estrogen signaling.  

PubMed

Breast cancers expressing estrogen receptor-alpha (ERalpha) are associated with a favorable biology and are more likely to respond to hormonal therapy. In addition to ERalpha, other pathways of estrogen response have been identified including ERbeta and GPR30, a membrane receptor for estrogen, and the key mechanisms regulating expression of ERs and hormone response remain controversial. Herein, we show that TFAP2C is the key regulator of hormone responsiveness in breast carcinoma cells through the control of multiple pathways of estrogen signaling. TFAP2C regulates the expression of ERalpha directly by binding to the ERalpha promoter and indirectly via regulation of FoxM1. In so doing, TFAP2C controls the expression of ERalpha target genes, including pS2, MYB, and RERG. Furthermore, TFAP2C controlled the expression of GPR30. In distinct contrast, TFAP2A, a related factor expressed in breast cancer, was not involved in estrogen-mediated pathways but regulated expression of genes controlling cell cycle arrest and apoptosis including p21(CIP1) and IGFBP-3. Knockdown of TFAP2C abrogated the mitogenic response to estrogen exposure and decreased hormone-responsive tumor growth of breast cancer xenografts. We conclude that TFAP2C is a central control gene of hormone response and is a novel therapeutic target in the design of new drug treatments for breast cancer. PMID:17875680

Woodfield, George W; Horan, Annamarie D; Chen, Yizhen; Weigel, Ronald J

2007-09-15

122

A Phosphotyrosine Proteomic Screen Identifies Multiple Tyrosine Kinase Signaling Pathways Aberrantly Activated in Malignant Mesothelioma  

PubMed Central

Malignant mesothelioma (MM) is a highly aggressive cancer that is refractory to all current chemotherapeutic regimens. Therefore, uncovering new rational therapeutic targets is imperative in the field. Tyrosine kinase signaling pathways are aberrantly activated in many human cancers and are currently being targeted for chemotherapeutic intervention. Thus, we sought to identify tyrosine kinases hyperactivated in MM. An unbiased phosphotyrosine proteomic screen was employed to identify tyrosine kinases activated in human MM cell lines. From this screen, we have identified novel signaling molecules, such as JAK1, STAT1, cortactin (CTTN), FER, p130Cas (BCAR1), SRC, and FYN as tyrosine phosphorylated in human MM cell lines. Additionally, STAT1 and SRC family kinases (SFK) were confirmed to be active in primary MM specimens. We also confirmed that known signal transduction pathways previously implicated in MM, such as EGFR and MET signaling axes, are coactivated in the majority of human MM specimens and cell lines tested. EGFR, MET, and SFK appear to be coactivated in a significant proportion of MM cell lines, and dual inhibition of these kinases was demonstrated to be more efficacious for inhibiting MM cell viability and downstream effector signaling than inhibition of a single tyrosine kinase. Consequently, these data suggest that tyrosine kinase inhibitor monotherapy may not represent an efficacious strategy for the treatment of MM due to multiple tyrosine kinases potentially signaling redundantly to cellular pathways involved in tumor cell survival and proliferation.

Menges, Craig W.; Chen, Yibai; Mossman, Brooke T.; Chernoff, Jonathan; Yeung, Anthony T.; Testa, Joseph R.

2010-01-01

123

Simultaneous Reconstruction of Multiple Signaling Pathways via the Prize-Collecting Steiner Forest Problem  

PubMed Central

Abstract Signaling and regulatory networks are essential for cells to control processes such as growth, differentiation, and response to stimuli. Although many “omic” data sources are available to probe signaling pathways, these data are typically sparse and noisy. Thus, it has been difficult to use these data to discover the cause of the diseases and to propose new therapeutic strategies. We overcome these problems and use “omic” data to reconstruct simultaneously multiple pathways that are altered in a particular condition by solving the prize-collecting Steiner forest problem. To evaluate this approach, we use the well-characterized yeast pheromone response. We then apply the method to human glioblastoma data, searching for a forest of trees, each of which is rooted in a different cell-surface receptor. This approach discovers both overlapping and independent signaling pathways that are enriched in functionally and clinically relevant proteins, which could provide the basis for new therapeutic strategies. Although the algorithm was not provided with any information about the phosphorylation status of receptors, it identifies a small set of clinically relevant receptors among hundreds present in the interactome.

Tuncbag, Nurcan; Braunstein, Alfredo; Pagnani, Andrea; Huang, Shao-Shan Carol; Chayes, Jennifer; Borgs, Christian; Zecchina, Riccardo

2013-01-01

124

Genistein inhibits tumor invasion by suppressing multiple signal transduction pathways in human hepatocellular carcinoma cells  

PubMed Central

Background Genistein (Gen) exhibits anti-mutagenic and anti-metastatic activities in hepatoma cell lines. Gen has suppressive effects on tumor growth and angiogenesis in nude mice. Gen suppresses the enzymatic activity of matrix metalloproteinase (MMP)-9; however, the mechanism underlying its anti-invasive activity on hepatocellular carcinoma (HCC) cells is unclear. Methods In this study, the possible mechanisms underlying Gen-mediated reduction of 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced cell invasion and inhibition of secreted and cytosolic MMP-9 production in human hepatoma cells (HepG2, Huh-7, and HA22T) and murine embryonic liver cells (BNL CL2) were investigated. Results Gen suppressed MMP-9 transcription by inhibiting activator protein (AP)-1 and nuclear factor-? B (NF-?B) activity. Gen suppressed TPA-induced AP-1 activity through inhibitory phosphorylation of extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways, and TPA-stimulated inhibition of NF-?B nuclear translocation through I?B inhibitory signaling pathways. Moreover, Gen suppressed TPA-induced activation of ERK/phosphatidylinositol 3-kinase/Akt upstream of NF-?B and AP-1. Conclusions Gen and its inhibition of multiple signal transduction pathways can control the invasiveness and metastatic potential of HCC.

2014-01-01

125

Xenoestrogen-Induced ERK-1 and ERK-2 Activation via Multiple Membrane-Initiated Signaling Pathways  

PubMed Central

Xenoestrogens can mimic or antagonize the activity of physiological estrogens, and the suggested mechanism of xenoestrogen action involves binding to estrogen receptors (ERs). However, the failure of various in vitro or in vivo assays to show strong genomic activity of xenoestrogens compared with estradiol (E2) makes it difficult to explain their ability to cause abnormalities in animal (and perhaps human) reproductive functions via this pathway of steroid action. E2 has also been shown to initiate rapid intracellular signaling, such as changes in levels of intracellular calcium, cAMP, and nitric oxide, and activations of a variety of kinases, via action at the membrane. In this study, we demonstrate that several xenoestrogens can rapidly activate extracellular-regulated kinases (ERKs) in the pituitary tumor cell line GH3/B6/F10, which expresses high levels of the membrane receptor for ER-?(mER). We tested a phytoestrogen (coumestrol), organochlorine pesticides or their metabolites (endosulfan, dieldrin, and DDE), and detergent by-products of plastics manufacturing (p-nonylphenol and bisphenol A). These xenoestrogens (except bisphenol A) produced rapid (3–30 min after application), concentration (10?14–10?8 M)-dependent ERK-1/2 phosphorylation but with distinctly different activation patterns. To identify signaling pathways involved in ERK activation, we used specific inhibitors of ERs, epidermal growth factor receptors, Ca2+ signaling, Src and phosphoinositide-3 kinases, and a membrane structure disruption agent. Multiple inhibitors blocked ERK activation, suggesting simultaneous use of multiple pathways and complex signaling web interactions. However, inhibitors differentially affected each xenoestrogen response examined. These actions may help to explain the distinct abilities of xenoestrogens to disrupt reproductive functions at low concentrations.

Bulayeva, Nataliya N.; Watson, Cheryl S.

2004-01-01

126

Genetic Evidence Implicating Multiple Genes in the MET Receptor Tyrosine Kinase Pathway in Autism Spectrum Disorder  

PubMed Central

A functional promoter variant of the gene encoding the MET receptor tyrosine kinase alters SP1 and SUB1 transcription factor binding, and is associated with autism spectrum disorder (ASD). Recent analyses of postmortem cerebral cortex from ASD patients revealed altered expression of MET protein and three transcripts encoding proteins that regulate MET signaling, hepatocyte growth factor (HGF), urokinase plasminogen activator receptor (PLAUR) and plasminogen activator inhibitor-1 (SERPINE1). To address potential risk conferred by multiple genes in the MET signaling pathway, we screened all exons and 5? promoter regions for variants in the five genes encoding proteins that regulate MET expression and activity. Identified variants were genotyped in 664 families (2,712 individuals including 1,228 with ASD) and 312 unrelated controls. Replicating our initial findings, family-based association test (FBAT) analyses demonstrated that the MET promoter variant rs1858830 C allele was associated with ASD in 101 new families (P=0.033). Two other genes in the MET signaling pathway also may confer risk. A haplotype of the SERPINE1 gene exhibited significant association. In addition, the PLAUR promoter variant rs344781 T allele was associated with ASD by both FBAT (P=0.006) and case-control analyses (P=0.007). The PLAUR promoter rs344781 relative risk was 1.93 (95% Confidence Interval [CI]: 1.12?3.31) for genotype TT and 2.42 (95% CI: 1.38?4.25) for genotype CT compared to genotype CC. Gene-gene interaction analyses suggested a significant interaction between MET and PLAUR. These data further support our hypothesis that genetic susceptibility impacting multiple components of the MET signaling pathway contributes to ASD risk.

Campbell, Daniel B.; Li, Chun; Sutcliffe, James S.; Persico, Antonio M.; Levitt, Pat

2008-01-01

127

Aspergillus sporulation and mycotoxin production both require inactivation of the FadA G? protein-dependent signaling pathway  

Microsoft Academic Search

The filamentous fungus Aspergillus nidulans contains a cluster of 25 genes that encode enzymes required to synthesize a toxic and carcinogenic secondary metabolite called sterigmatocystin (ST), a precursor of the better known fungal toxin aflatoxin (AF). One ST Cluster (stc) gene, aflR, functions as a pathway-specific transcriptional regulator for activation of other genes in the ST pathway. However, the mechanisms

Julie K. Hicks; Jae-Hyuk Yu; Nancy P. Keller; Thomas H. Adams

1997-01-01

128

Multiple Accessory Pathways in the Young: The Impact of Structural Heart Disease  

PubMed Central

Background The presence of multiple accessory pathways (multAP) is described in structural heart disease (SHD) such as Ebstein’s anomaly and cardiomyopathies. Structural defects can impact the tolerability of tachyarrhythmia and can complicate both medical management and ablation. In a large cohort of pediatric patients with and without SHD undergoing invasive electrophysiology study, we examined the prevalence of multAP and the effect of both multAP and SHD on ablation outcomes. Methods Accessory pathway number and location, presence of SHD, ablation success, and recurrence were analyzed in consecutive patients from our center over a 16 year period. Results In 1088 patients, 1228 pathways (36% retrograde only) were mapped to the right side (TV) in 18%, septum(S) in 39%, and left side (MV) in 43%. MultAP were present in 111 pts (10%), involving 250 distinct pathways. SHD tripled the risk of multAP (26% SHD vs 8% no SHD, p<0.001). Multivariable adjusted risk factors for multAP included Ebstein’s(OR 8.7[4.4–17.5],p<0.001) and cardiomyopathy (OR13.3[5.1–34.5], p<0.001). Of 1306 ablation attempts, 94% were acutely successful with an 8% recurrence rate. Ablation success was affected by SHD (85% vs 95% for no SHD, p<0.01) but not by multAP (91% vs. 94% for single, p= 0.24). Recurrence rate was higher for SHD (17% SHD vs 8% no SHD, p<0.05) and multAP (19% multAP vs. 8% single, p<0.001). Conclusions MultAP are found in 10% of pediatric patients, and are more common in SHD compared to those with normal hearts. Both the presence of multAP and SHD negatively influence ablation outcomes.

Zachariah, Justin P; Walsh, Edward P; Triedman, John K; Berul, Charles I; Cecchin, Frank; Alexander, Mark E; Bevilacqua, Laura M

2012-01-01

129

Airborne fine particulate matter induces multiple cell death pathways in human lung epithelial cells.  

PubMed

Our group was the first one reporting that autophagy could be triggered by airborne fine particulate matter (PM) with a mean diameter of less than 2.5 ?m (PM2.5) in human lung epithelial A549 cells, which could potentially lead to cell death. In the present study, we further explored the potential interactions between autophagy and apoptosis because it was well documented that PM2.5 could induce apoptosis in A549 cells. Much to our surprise, we found that PM2.5-exposure caused oxidative stress, resulting in activation of multiple cell death pathways in A549 cells, that is, the tumor necrosis factor-alpha (TNF-?)-induced pathway as evidenced by TNF-? secretion and activation of caspase-8 and -3, the intrinsic apoptosis pathway as evidenced by increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic protein Bcl-2, disruption of mitochondrial membrane potential, and activation of caspase-9 and -3, and autophagy as evidenced by an increased number of double-membrane vesicles, accompanied by increases of conversion and punctuation of microtubule-associated proteins light chain 3 (LC3) and expression of Beclin 1. It appears that reactive oxygen species (ROS) function as signaling molecules for all the three pathways because pretreatment with N-acetylcysteine, a scavenger of ROS, almost completely abolished TNF-? secretion and significantly reduced the number of apoptotic and autophagic cells. In another aspect, inhibiting autophagy with 3-methyladenine, a specific autophagy inhibitor, enhanced PM2.5-induced apoptosis and cytotoxicity. Intriguingly, neutralization of TNF-? with an anti-TNF-? special antibody not only abolished activation of caspase-8, but also drastically reduced LC3-II conversion. Thus, the present study has provided novel insights into the mechanism of cytotoxicity and even pathogenesis of diseases associated with PM2.5 exposure. PMID:24722831

Deng, Xiaobei; Zhang, Fang; Wang, Lijuan; Rui, Wei; Long, Fang; Zhao, Yong; Chen, Deliang; Ding, Wenjun

2014-07-01

130

The nucleolar protein Viriato/Nol12 is required for the growth and differentiation progression activities of the Dpp pathway during Drosophila eye development.  

PubMed

Drosophila Decapentaplegic (Dpp), a member of the BMP2/4 class of the TGF-?s, is required for organ growth, patterning and differentiation. However, much remains to be understood about the mechanisms acting downstream of these multiple roles. Here we investigate this issue during the development of the Drosophila eye. We have previously identified viriato (vito) as a dMyc-target gene encoding a nucleolar protein that is required for proper tissue growth in the developing eye. By carrying out a targeted in vivo double-RNAi screen to identify genes and pathways functioning with Vito during eye development, we found a strong genetic interaction between vito and members of the Dpp signaling pathway including the TGF-? receptors tkv (type I), put (type II), and the co-Smad medea (med). Analyzing the expression of the Dpp receptor Tkv and the activation pattern of the pathway's transducer, p-Mad, we found that vito is required for a correct signal transduction in Dpp-receiving cells. Overall, we validate the use of double RNAi to find specific genetic interactions and, in particular, we uncover a link between the Dpp pathway and Vito, a nucleolar component. vito would act genetically downstream of Dpp, playing an important role in maintaining a sufficient level of Dpp activity for the promotion of eye disc growth and regulation of photoreceptor differentiation in eye development. PMID:23416177

Marinho, Joana; Martins, Torcato; Neto, Marta; Casares, Fernando; Pereira, Paulo S

2013-05-01

131

Multiple small RNA pathways regulate the silencing of repeated and foreign genes in C. elegans  

PubMed Central

Gene segments from other organisms, such as viruses, are detected as foreign and targeted for silencing by RNAi pathways. A deep-sequencing map of the small RNA response to repeated transgenes introduced to Caenorhabditis elegans revealed that specific segments are targeted by siRNAs. Silencing of the foreign gene segments depends on an antiviral response that involves changes in active and silent chromatin modifications and altered levels of antisense siRNAs. Distinct Argonaute proteins target foreign genes for silencing or protection against silencing. We used a repeated transgene in a genome-wide screen to identify gene disruptions that enhance silencing of foreign genetic elements and identified 69 genes. These genes cluster in four groups based on overlapping sets of coexpressed genes, including a group of germline-expressed genes that are likely coregulated by the E2F transcription factor. Many of the gene inactivations enhance exogenous RNAi. About half of the 69 genes have roles in endogenous RNAi pathways that regulate diverse processes, including silencing of duplicated genes and transposons and chromosome segregation. Of these newly identified genes, several are required for siRNA biogenesis or stability in the oocyte-specific ERGO-1 pathway, including eri-12, encoding an interactor of the RNAi-defective protein RDE-10, and ntl-9/CNOT9, one of several CCR4/NOT complex genes that we identified. The conserved ARF-like small GTPase ARL-8 is required specifically for primary siRNA biogenesis or stability in the sperm-specific ALG-3/4 endogenous RNAi pathway.

Fischer, Sylvia E.J.; Pan, Qi; Breen, Peter C.; Qi, Yan; Shi, Zhen; Zhang, Chi; Ruvkun, Gary

2013-01-01

132

p120 serine and threonine phosphorylation is controlled by multiple ligand-receptor pathways but not cadherin ligation.  

PubMed

p120-catenin (p120) regulates cadherin turnover and is required for cadherin stability. Extensive and dynamic phosphorylation on tyrosine, serine and threonine residues in the N-terminal regulatory domain has been postulated to regulate p120 function, possibly through modulation of the efficiency of p120/cadherin interaction. Here we have utilized novel phospho-specific monoclonal antibodies to four major p120 serine and threonine phosphorylation sites to monitor individual phosphorylation events and their consequences. Surprisingly, membrane-localization and not cadherin interaction is the main determinant in p120 serine and threonine phosphorylation and dephosphorylation. Furthermore, the phospho-status of these four residues had no obvious effect on p120's role in cadherin complex stabilization or cell-cell adhesion. Interestingly, dephosphorylation was dramatically induced by PKC activation, but PKC-independent pathways were also evident. The data suggest that p120 dephosphorylation at these sites is modulated by multiple cell surface receptors primarily through PKC-dependent pathways, but these changes do not seem to reduce p120/cadherin affinity. PMID:16935280

Xia, Xiaobo; Carnahan, Robert H; Vaughan, Meredith H; Wildenberg, Gregg A; Reynolds, Albert B

2006-10-15

133

Ursolic acid inhibits colorectal cancer angiogenesis through suppression of multiple signaling pathways.  

PubMed

Angiogenesis plays a critical role in the development of solid tumors by supplying nutrients and oxygen to support continuous growth of tumor as well as providing an avenue for hematogenous metastasis. Tumor angiogenesis is highly regulated by multiple intracellular signaling transduction cascades such as Hedgehog, STAT3, Akt and p70S6K pathways that are known to malfunction in many types of cancer including colorectal cancer (CRC). Therefore, suppression of tumor angiogenesis through targeting these signaling pathways has become a promising strategy for cancer chemotherapy. Ursolic acid (UA) is a major active compound present in many medicinal herbs that have long been used in China for the clinical treatment of various types of cancer. Although previous studies have demonstrated an antitumor effect for UA, the precise mechanisms of its anti-angiogenic activity are not well understood. To further elucidate the mechanism(s) of the tumorcidal activity of UA, using a CRC mouse xenograft model, chick embryo chorioallantoic membrane (CAM) model, the human colon carcinoma cell line HT-29 and human umbilical vein endothelial cells (HUVECs), in the present study we evaluated the efficacy of UA against tumor growth and angiogenesis in vivo and in vitro and investigated the underlying molecular mechanisms. We found that administration of UA significantly inhibited tumor volume but had no effect on body weight changes in CRC mice, suggesting that UA can suppress colon cancer growth in vivo without noticeable signs of toxicity. In addition, UA treatment reduced intratumoral microvessel density (MVD) in CRC mice, decreased the total number of blood vessels in the CAM model, and dose and time-dependently inhibited the proliferation, migration and tube formation of HUVECs, demonstrating UA's antitumor angiogenesis in vivo and in vitro. Moreover, UA treatment inhibited the expression of critical angiogenic factors, such as VEGF-A and bFGF. Furthermore, UA suppressed the activation of sonic hedgehog (SHH), STAT3, Akt and p70S6K pathways. Collectively, our findings suggest that inhibition of tumor angiogenesis via suppression of multiple signaling pathways might be one of the mechanisms whereby UA can be effective in cancer treatment. PMID:24042330

Lin, Jiumao; Chen, Youqin; Wei, Lihui; Hong, Zhenfeng; Sferra, Thomas J; Peng, Jun

2013-11-01

134

Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary  

PubMed Central

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary.

Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

2014-01-01

135

Role of costimulatory pathways in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis  

Microsoft Academic Search

Multiple sclerosis is an immune-mediated disorder of the central nervous system. T lymphocytes are thought to play a central role in the initiation and potentially in the propagation of this disease. Two signals are required for T-cell activation. The first signal consists of the interaction of the T-cell receptor with antigen presented by the MHC molecule on antigen-presenting cells. The

Tanuja Chitnis; Samia J Khoury

2003-01-01

136

The WWOX gene inhibits the growth of U266 multiple myeloma cells by triggering the intrinsic apoptotic pathway.  

PubMed

The role of the WW domain-containing oxidoreductase (WWOX) gene in multiple types of solid human cancers has been documented extensively. However, the functional role of WWOX in human multiple myeloma has not yet been fully elucidated. The present study aimed to investigate the effects of exogenous WWOX expression on the biological properties of U266 multiple myeloma cells, as well as the possible molecular mechanisms involved. In vitro experiments revealed that exogenous WWOX cDNA transfection resulted in marked growth arrest and the induction of apoptosis in the U266 multiple myeloma cells, accompanied by the activation of the intrinsic apoptotic pathway. Our data provide evidence that WWOX also plays a role as a tumor suppressor gene in multiple myeloma, possibly by suppressing cell proliferation and promoting apoptosis by triggering the intrinsic apoptotic pathway. PMID:24968878

Zhang, Hongsheng; Kong, Lingying; Cui, Zhaolei; Du, Wei; He, Yihui; Yang, Zhi; Wang, Li; Chen, Xiaoyan

2014-09-01

137

A novel pathway of HMGB1-mediated inflammatory cell recruitment that requires Mac-1-integrin  

PubMed Central

High-mobility group box 1 (HMGB1) is released extracellularly upon cell necrosis acting as a mediator in tissue injury and inflammation. However, the molecular mechanisms for the proinflammatory effect of HMGB1 are poorly understood. Here, we define a novel function of HMGB1 in promoting Mac-1-dependent neutrophil recruitment. HMGB1 administration induced rapid neutrophil recruitment in vivo. HMGB1-mediated recruitment was prevented in mice deficient in the ?2-integrin Mac-1 but not in those deficient in LFA-1. As observed by bone marrow chimera experiments, Mac-1-dependent neutrophil recruitment induced by HMGB1 required the presence of receptor for advanced glycation end products (RAGE) on neutrophils but not on endothelial cells. In vitro, HMGB1 enhanced the interaction between Mac-1 and RAGE. Consistently, HMGB1 activated Mac-1 as well as Mac-1-mediated adhesive and migratory functions of neutrophils in a RAGE-dependent manner. Moreover, HMGB1-induced activation of nuclear factor-?B in neutrophils required both Mac-1 and RAGE. Together, a novel HMGB1-dependent pathway for inflammatory cell recruitment and activation that requires the functional interplay between Mac-1 and RAGE is described here.

Orlova, Valeria V; Choi, Eun Young; Xie, Changping; Chavakis, Emmanouil; Bierhaus, Angelika; Ihanus, Eveliina; Ballantyne, Christie M; Gahmberg, Carl G; Bianchi, Marco E; Nawroth, Peter P; Chavakis, Triantafyllos

2007-01-01

138

Multiple pathways to identification: Exploring the multidimensionality of academic identity formation in ethnic minority males.  

PubMed

Empirical trends denote the academic underachievement of ethnic minority males across various academic domains. Identity-based explanations for this persistent phenomenon describe ethnic minority males as disidentified with academics, alienated, and oppositional. The present work interrogates these theoretical explanations and empirically substantiates a multidimensional lens for discussing academic identity formation within 330 African American and Latino early-adolescent males. Both hierarchical and iterative person-centered methods were utilized and reveal 5 distinct profiles derived from 6 dimensions of academic identity. These profiles predict self-reported classroom grades, mastery orientation, and self-handicapping in meaningful and varied ways. The results demonstrate multiple pathways to motivation and achievement, challenging previous oversimplified stereotypes of marginalized males. This exploratory study triangulates unique interpersonal and intrapersonal attributes for promoting healthy identity development and academic achievement among ethnic minority adolescent males. (PsycINFO Database Record (c) 2014 APA, all rights reserved). PMID:24447039

Matthews, Jamaal S

2014-04-01

139

Carbon emission limits required to satisfy future representative concentration pathways of greenhouse gases  

NASA Astrophysics Data System (ADS)

The response of the second-generation Canadian earth system model (CanESM2) to historical (1850-2005) and future (2006-2100) natural and anthropogenic forcing is assessed using the newly-developed representative concentration pathways (RCPs) of greenhouse gases (GHGs) and aerosols. Allowable emissions required to achieve the future atmospheric CO2 concentration pathways, are reported for the RCP 2.6, 4.5 and 8.5 scenarios. For the historical 1850-2005 period, cumulative land plus ocean carbon uptake and, consequently, cumulative diagnosed emissions compare well with observation-based estimates. The simulated historical carbon uptake is somewhat weaker for the ocean and stronger for the land relative to their observation-based estimates. The simulated historical warming of 0.9°C compares well with the observation-based estimate of 0.76 ± 0.19°C. The RCP 2.6, 4.5 and 8.5 scenarios respectively yield warmings of 1.4, 2.3, and 4.9°C and cumulative diagnosed fossil fuel emissions of 182, 643 and 1617 Pg C over the 2006-2100 period. The simulated warming of 2.3°C over the 1850-2100 period in the RCP 2.6 scenario, with the lowest concentration of GHGs, is slightly larger than the 2°C warming target set to avoid dangerous climate change by the 2009 UN Copenhagen Accord. The results of this study suggest that limiting warming to roughly 2°C by the end of this century is unlikely since it requires an immediate ramp down of emissions followed by ongoing carbon sequestration in the second half of this century.

Arora, V. K.; Scinocca, J. F.; Boer, G. J.; Christian, J. R.; Denman, K. L.; Flato, G. M.; Kharin, V. V.; Lee, W. G.; Merryfield, W. J.

2011-03-01

140

Resistance to TRAIL in non-transformed cells is due to multiple redundant pathways  

PubMed Central

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine and a selective inducer of apoptosis in a range of tumour cells, but not in normal, untransformed cells. A large number of chemotherapeutics as well as biological agents are being tested for their potential to sensitise resistant tumour cells to TRAIL as a means to broaden the range of tumours treatable with TRAIL. However, because of the incomplete understanding of the mechanism(s) underlying TRAIL resistance in non-malignant cells, it is unpredictable whether the effect of these sensitisers will be restricted to tumour cells or they would also sensitise non-transformed cells causing unwanted toxicity. In this study, we carried out a systematic analysis of the mechanisms driving TRAIL resistance in non-transformed cells. We found that cellular FLICE-like inhibitory protein, anti-apoptotic B-cell lymphoma 2 proteins, and X-linked inhibitor of apoptosis protein were independently able to provide resistance to TRAIL. Deficiency of only one of these proteins was not sufficient to elicit TRAIL sensitivity, demonstrating that in non-transformed cells multiple pathways control TRAIL resistance and they act in a redundant manner. This is contrary to the resistance mechanisms found in tumour cell types, many of them tend to rely on a single mechanism of resistance. Supporting this notion we found that 76% of TRAIL-resistant cell lines (13 out of 17) expressed only one of the above-identified anti-apoptotic proteins at a high level (?1.2-fold higher than the mean expression across all cell lines). Furthermore, inhibition or knockdown of the single overexpressed protein in these tumour cells was sufficient to trigger TRAIL sensitivity. Therefore, the redundancy in resistance pathways in non-transformed cells may offer a safe therapeutic window for TRAIL-based combination therapies where selective sensitisation of the tumour to TRAIL can be achieved by targeting the single non-redundant resistance pathway.

van Dijk, M; Halpin-McCormick, A; Sessler, T; Samali, A; Szegezdi, E

2013-01-01

141

Identifiability and estimation of multiple transmission pathways in cholera and waterborne disease.  

PubMed

Cholera and many waterborne diseases exhibit multiple characteristic timescales or pathways of infection, which can be modeled as direct and indirect transmission. A major public health issue for waterborne diseases involves understanding the modes of transmission in order to improve control and prevention strategies. An important epidemiological question is: given data for an outbreak, can we determine the role and relative importance of direct vs. environmental/waterborne routes of transmission? We examine whether parameters for a differential equation model of waterborne disease transmission dynamics can be identified, both in the ideal setting of noise-free data (structural identifiability) and in the more realistic setting in the presence of noise (practical identifiability). We used a differential algebra approach together with several numerical approaches, with a particular emphasis on identifiability of the transmission rates. To examine these issues in a practical public health context, we apply the model to a recent cholera outbreak in Angola (2006). Our results show that the model parameters-including both water and person-to-person transmission routes-are globally structurally identifiable, although they become unidentifiable when the environmental transmission timescale is fast. Even for water dynamics within the identifiable range, when noisy data are considered, only a combination of the water transmission parameters can practically be estimated. This makes the waterborne transmission parameters difficult to estimate, leading to inaccurate estimates of important epidemiological parameters such as the basic reproduction number (R0). However, measurements of pathogen persistence time in environmental water sources or measurements of pathogen concentration in the water can improve model identifiability and allow for more accurate estimation of waterborne transmission pathway parameters as well as R0. Parameter estimates for the Angola outbreak suggest that both transmission pathways are needed to explain the observed cholera dynamics. These results highlight the importance of incorporating environmental data when examining waterborne disease. PMID:23333764

Eisenberg, Marisa C; Robertson, Suzanne L; Tien, Joseph H

2013-05-01

142

Multiple Pudendal Sensory Pathways Reflexly Modulate Bladder and Urethral Activity in Persons with Spinal Cord Injury  

PubMed Central

Purpose Electrical stimulation of pudendal afferents can evoke reflex bladder contractions with relaxation of the external urethral sphincter in cats. This voiding reflex is mediated by pudendal sensory fibers innervating the penile and prostatic urethra that engage either a spinal or spinobulbospinal micturition pathway, respectively. However, the clinical translation of this potential therapy in persons with spinal cord injury (SCI) is limited by the lack of evidence demonstrating analogous reflex mechanisms in humans. Materials and Methods We investigated the presence of excitatory pudendal-to-bladder reflexes in seven individuals with chronic SCI. The isovolumetric bladder pressure and perineal electromyogram (EMG) were recorded in response to intraurethral (IU) electrical stimulation at varying amplitudes and frequencies. Results Selective electrical stimulation of the proximal and distal segments of the urethra evoked sustained reflex bladder contractions in different subsets of participants: 24.9 ± 13.9 cmH2O (n = 3) and 23.3 ± 16.1 cmH2O (n = 3), respectively. In contrast, the corresponding reflex perineal EMG exhibited a differential activation pattern between proximal and distal IU stimulation (normalized EMG, p < 0.05): 1.3 ± 0.2 and 0.3 ± 0.1, respectively. Conclusions This study presents the first clinical evidence of two independent excitatory pudendal-to-bladder reflex pathways that can, in turn, differentially modulate efferent pudendal output. Both reflex mechanisms involve a complex interaction of multiple sensory inputs and may provide a neural substrate for restoring micturition following SCI.

Yoo, Paul B.; Horvath, Eric E.; Amundsen, Cindy L.; Webster, George D.; Grill, Warren M.

2011-01-01

143

Select spinal lesions reveal multiple ascending pathways in the rat conveying input from the male genitalia  

PubMed Central

The specific white matter location of all the spinal pathways conveying penile input to the rostral medulla is not known. Our previous studies using rats demonstrated the loss of low but not high threshold penile inputs to medullary reticular formation (MRF) neurons after acute and chronic dorsal column (DC) lesions of the T8 spinal cord and loss of all penile inputs after lesioning the dorsal three-fifths of the cord. In the present study, select T8 lesions were made and terminal electrophysiological recordings were performed 45–60 days later in a limited portion of the nucleus reticularis gigantocellularis (Gi) and Gi pars alpha. Lesions included subtotal dorsal hemisections that spared only the lateral half of the dorsal portion of the lateral funiculus on one side, dorsal and over-dorsal hemisections, and subtotal transections that spared predominantly just the ventromedial white matter. Electrophysiological data for 448 single unit recordings obtained from 32 urethane-anaesthetized rats, when analysed in groups based upon histological lesion reconstructions, revealed (1) ascending bilateral projections in the dorsal, dorsolateral and ventrolateral white matter of the spinal cord conveying information from the male external genitalia to MRF, and (2) ascending bilateral projections in the ventrolateral white matter conveying information from the pelvic visceral organs (bladder, descending colon, urethra) to MRF. Multiple spinal pathways from the penis to the MRF may correspond to different functions, including those processing affective/pleasure/motivational, nociception, and mating-specific (such as for erection and ejaculation) inputs.

Hubscher, C H; Reed, W R; Kaddumi, E G; Armstrong, J E; Johnson, R D

2010-01-01

144

Multiple Regression Analysis of mRNA-miRNA Associations in Colorectal Cancer Pathway  

PubMed Central

Background. MicroRNA (miRNA) is a short and endogenous RNA molecule that regulates posttranscriptional gene expression. It is an important factor for tumorigenesis of colorectal cancer (CRC), and a potential biomarker for diagnosis, prognosis, and therapy of CRC. Our objective is to identify the related miRNAs and their associations with genes frequently involved in CRC microsatellite instability (MSI) and chromosomal instability (CIN) signaling pathways. Results. A regression model was adopted to identify the significantly associated miRNAs targeting a set of candidate genes frequently involved in colorectal cancer MSI and CIN pathways. Multiple linear regression analysis was used to construct the model and find the significant mRNA-miRNA associations. We identified three significantly associated mRNA-miRNA pairs: BCL2 was positively associated with miR-16 and SMAD4 was positively associated with miR-567 in the CRC tissue, while MSH6 was positively associated with miR-142-5p in the normal tissue. As for the whole model, BCL2 and SMAD4 models were not significant, and MSH6 model was significant. The significant associations were different in the normal and the CRC tissues. Conclusion. Our results have laid down a solid foundation in exploration of novel CRC mechanisms, and identification of miRNA roles as oncomirs or tumor suppressor mirs in CRC.

Wang, Fengfeng; Wong, S. C. Cesar; Chan, Lawrence W. C.; Cho, William C. S.; Yip, S. P.; Yung, Benjamin Y. M.

2014-01-01

145

The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy.  

PubMed

AMP-activated protein kinase (AMPK) is a crucial energy sensor and plays a key role in integration of cellular functions to maintain homeostasis. Despite this, it is largely unknown whether targeting the AMPK pathway can be used as a therapeutic strategy for infectious diseases. Herein, we show that AMPK activation robustly induces antibacterial autophagy, which contributes to antimicrobial defense against Mycobacterium tuberculosis (Mtb). AMPK activation led to inhibition of Mtb-induced phosphorylation of the mechanistic target of rapamycin (MTOR) in macrophages. In addition, AMPK activation increased the genes involved in oxidative phosphorylation, mitochondrial ATP production, and biogenesis in Mtb-infected macrophages. Notably, peroxisome proliferator-activated receptor-gamma, coactivator 1? (PPARGC1A) was required for AMPK-mediated antimicrobial activity, as well as enhancement of mitochondrial function and biogenesis, in macrophages. Further, the AMPK-PPARGC1A pathway was involved in the upregulation of multiple autophagy-related genes via CCAAT/enhancer binding protein (C/EBP), ? (CEBPB). PPARGC1A knockdown inhibited the AMPK-mediated induction of autophagy and impaired the fusion of phagosomes with MAP1LC3B (LC3B) autophagosomes in Mtb-infected macrophages. The link between autophagy, mitochondrial function, and antimicrobial activity was further demonstrated by studying LysMCre-mediated knockout of atg7, demonstrating mitochondrial ultrastructural defects and dysfunction, as well as blockade of antimicrobial activity against mycobacteria. Collectively, our results identify the AMPK-PPARGC1A axis as contributing to autophagy activation leading to an antimicrobial response, as a novel host defense mechanism. PMID:24598403

Yang, Chul-Su; Kim, Jwa-Jin; Lee, Hye-Mi; Jin, Hyo Sun; Lee, Sang-Hee; Park, Ji-Hoon; Kim, Soung Jung; Kim, Jin-Man; Han, Yong-Mahn; Lee, Myung-Shik; Kweon, Gi Ryang; Shong, Minho; Jo, Eun-Kyeong

2014-05-01

146

A Targeted Spatial-Temporal Proteomics Approach Implicates Multiple Cellular Trafficking Pathways in Human Cytomegalovirus Virion Maturation*  

PubMed Central

The assembly of infectious virus particles is a complex event. For human cytomegalovirus (HCMV) this process requires the coordinated expression and localization of at least 60 viral proteins that comprise the infectious virion. To gain insight into the mechanisms controlling this process, we identified protein binding partners for two viral proteins, pUL99 (also termed pp28) and pUL32 (pp150), which are essential for HCMV virion assembly. We utilized HCMV strains expressing pUL99 or pUL32 carboxyl-terminal green fluorescent protein fusion proteins from their native location in the HCMV genome. Based on the presence of ubiquitin in the pUL99 immunoisolation, we discovered that this viral protein colocalizes with components of the cellular endosomal sorting complex required for transport (ESCRT) pathway during the initial stages of virion assembly. We identified the nucleocapsid and a large number of tegument proteins as pUL32 binding partners, suggesting that events controlling trafficking of this viral protein in the cytoplasm regulate nucleocapsid/tegument maturation. The finding that pUL32, but not pUL99, associates with clathrin led to the discovery that the two viral proteins traffic via distinct pathways during the early stages of virion assembly. Additional investigation revealed that the majority of the major viral glycoprotein gB initially resides in a third compartment. Analysis of the trafficking of these three viral proteins throughout a time course of virion assembly allowed us to visualize their merger into a single large cytoplasmic structure during the late stages of viral assembly. We propose a model of HCMV virion maturation in which multiple components of the virion traffic independently of one another before merging.

Moorman, Nathaniel J.; Sharon-Friling, Ronit; Shenk, Thomas; Cristea, Ileana M.

2010-01-01

147

Yap1, transcription regulator in the Hippo signaling pathway, is required for Xenopus limb bud regeneration.  

PubMed

The Hippo signaling pathway is conserved from insects to mammals and is important for multiple processes, including cell proliferation, apoptosis and tissue homeostasis. Hippo signaling is also crucial for regeneration, including intercalary regeneration, of the whole body in the flatworm and of the leg in the cricket. However, its role in vertebrate epimorphic regeneration is unknown. Therefore, to identify principles of regeneration that are conserved among bilaterians, we investigated the role of Hippo signaling in the limb bud regeneration of an anuran amphibian, Xenopus laevis. We found that a transcription factor, Yap1, an important downstream effector of Hippo signaling, is upregulated in the regenerating limb bud. To evaluate Yap1?s function in limb bud regeneration, we made transgenic animals that expressed a dominant-negative form of Yap under a heat-shock promoter. Overexpression of a dominant-negative form of Yap in tadpoles reduced cell proliferation, induced ectopic apoptosis, perturbed the expression domains of limb-patterning genes including hoxa13, hoxa11, and shh in the regenerating limb bud. Transient expression of a dominant-negative Yap in transgenic tadpoles also caused limb bud regeneration defects, and reduced intercalary regeneration. These results indicate that Yap1 has a crucial role in controlling the limb regenerative capacity in Xenopus, and suggest that the involvement of Hippo signaling in regeneration is conserved between vertebrates and invertebrates. This finding provides molecular evidence that common principles underlie regeneration across phyla, and may contribute to the development of new therapies in regenerative medicine. PMID:24491818

Hayashi, Shinichi; Tamura, Koji; Yokoyama, Hitoshi

2014-04-01

148

Preclinical renal cancer chemopreventive efficacy of geraniol by modulation of multiple molecular pathways.  

PubMed

In the present study, we have evaluated the chemopreventive potential of geraniol (GOH), an acyclic monoterpene alcohol against ferric nitrilotriacetate (Fe-NTA) induced renal oxidative stress and carcinogenesis in Wistar rats. Chronic treatment of Fe-NTA induced oxidative stress, inflammation and cellular proliferation in Wistar rats. The chemopreventive efficacy of GOH was studied in terms of xenobiotic metabolizing enzyme activities, LPO, redox status, serum toxicity markers and the expression of putative nephrotoxicity biomarker Kim-1, tumor suppressor gene P53, inflammation, cell proliferation and apoptosis related genes in the kidney tissue. Oral administration of GOH at doses of 100 and 200mg/kg b wt effectively suppressed renal oxidative stress and tumor incidence. Chemopreventive effects of GOH were associated with upregulation of xenobiotic metabolizing enzyme activities and down regulation of serum toxicity markers. GOH was able to down regulate expression of Kim-1, NF?B, PCNA, P53 along with induction of apoptosis. However, higher dose of GOH was more effective in modulating these multiple molecular targets both at transcriptional and protein level. These results provide a powerful evidence for the chemopreventive efficacy of GOH against renal carcinogenesis possibly by modulation of multiple molecular pathways. PMID:21907755

Ahmad, Shiekh Tanveer; Arjumand, Wani; Seth, Amlesh; Nafees, Sana; Rashid, Summya; Ali, Nemat; Sultana, Sarwat

2011-11-28

149

The nitric oxide-cyclic GMP pathway is required for nociceptive signalling at specific loci within the somatosensory pathway  

Microsoft Academic Search

The involvement of nitric oxide in nociceptive processing was examined at the main loci of synaptic transmission within the rat somatosensory pathway from the caudal sural cutaneous nerve. Intrathecal (lumbar 1–3) administration of the nitric oxide synthase inhibitor,N?-nitro-l-arginine methyl ester (30 ?g), inhibited nitric oxide synthase in this region of the spinal cord by greater than 80% but had no

M. Salter; P. J. L. M. Strijbos; S. Neale; C. Duffy; R. L. Follenfant; J. Garthwaite

1996-01-01

150

The MRN-CtIP pathway is required for metaphase chromosome alignment.  

PubMed

Faithful duplication of the genome in S phase followed by its accurate segregation in mitosis is essential to maintain genomic integrity. Recent studies have suggested that proteins involved in DNA transactions are also required for whole-chromosome stability. Here we demonstrate that the MRN (Mre11, Rad50, and Nbs1) complex and CtIP are required for accurate chromosome segregation. Depletion of Mre11 or CtIP, antibody-mediated inhibition of Mre11, or small-molecule inhibition of MRN using mirin results in metaphase chromosome alignment defects in Xenopus egg extracts. Similarly, loss of MRN function adversely affects spindle assembly around DNA-coated beads in egg extracts. Inhibition of MRN function in mammalian cells triggers a metaphase delay and disrupts the RCC1-dependent RanGTP gradient. Addition of the Mre11 inhibitor mirin to egg extracts and mammalian cells reduces RCC1 association with mitotic chromosomes. Thus, the MRN-CtIP pathway contributes to Ran-dependent mitotic spindle assembly by modulating RCC1 chromosome association. PMID:23434370

Rozier, Lorene; Guo, Yige; Peterson, Shaun; Sato, Mai; Baer, Richard; Gautier, Jean; Mao, Yinghui

2013-03-28

151

Replication-independent activation of human plasmacytoid dendritic cells by the paramyxovirus SV5 Requires TLR7 and autophagy pathways.  

PubMed

The paramyxovirus Simian Virus 5 (SV5) is a poor inducer of interferon (IFN) secretion in all cell types tested so far, including primary epithelial cells and primary human myeloid dendritic cells. SV5 is hypothesized to limit induction of antiviral responses through control of viral gene expression and production of the V protein antagonist. Plasmacytoid dendritic cells (pDCs) are known to uniquely express toll-like receptor (TLR)-7 and are a main producer of IFN-alpha among peripheral blood mononuclear cells in response to many viruses. Here, we tested whether SV5 would remain a poor inducer of IFN in primary human pDCs. The efficiency of SV5 infection of pDCs could be increased by an increasing multiplicity of infection. pDCs infected by both live and UV-inactivated SV5 induced large amounts of IFN-alpha secretion and resulted in upregulation of maturation markers CD80 and CD86. However, IL-6 secretion was not induced by SV5 infection. When TLR7 signaling was inhibited, SV5 induced less IFN secretion and CD80 expression, and there was a corresponding increase in number of infected cells. Similar effects were seen with inhibitors of cellular autophagy pathways, suggesting that the SV5 activation of pDC requires access to the cytoplasm and autophagic sampling of cytoplasmic contents. These results have implications for control of SV5 infections in vivo and for development of SV5 as a vaccine vector. PMID:20605567

Manuse, Mary J; Briggs, Caitlin M; Parks, Griffith D

2010-09-30

152

Replication-Independent Activation of Human Plasmacytoid Dendritic Cells by the Paramyxovirus SV5 Requires TLR7 and Autophagy Pathways  

PubMed Central

The paramyxovirus Simian Virus 5 (SV5) is a poor inducer of interferon (IFN) secretion in all cell types tested so far, including primary epithelial cells and primary human myeloid dendritic cells. SV5 is hypothesized to limit induction of antiviral responses through control of viral gene expression and production of the V protein antagonist. Plasmacytoid dendritic cells (pDCs) are known to uniquely express toll-like receptor (TLR)-7 and are a main producer of IFN-alpha among peripheral blood mononuclear cells in response to many viruses. Here, we tested whether SV5 would remain a poor inducer of IFN in primary human pDCs. The efficiency of SV5 infection of pDCs could be increased by an increasing multiplicity of infection. pDCs infected by both live and UV-inactivated SV5 induced large amounts of IFN-alpha secretion and resulted in upregulation of maturation markers CD80 and CD86. However, IL-6 secretion was not induced by SV5 infection. When TLR7 signaling was inhibited, SV5 induced less IFN secretion and CD80 expression, and there was a corresponding increase in number of infected cells. Similar effects were seen with inhibitors of cellular autophagy pathways, suggesting that the SV5 activation of pDC requires access to the cytoplasm and autophagic sampling of cytoplasmic contents. These results have implications for control of SV5 infections in vivo and for development of SV5 as a vaccine vector.

Manuse, Mary J.; Briggs, Caitlin M.; Parks, Griffith D.

2010-01-01

153

Ten-m3 Is Required for the Development of Topography in the Ipsilateral Retinocollicular Pathway  

PubMed Central

Background The alignment of ipsilaterally and contralaterally projecting retinal axons that view the same part of visual space is fundamental to binocular vision. While much progress has been made regarding the mechanisms which regulate contralateral topography, very little is known of the mechanisms which regulate the mapping of ipsilateral axons such that they align with their contralateral counterparts. Results Using the advantageous model provided by the mouse retinocollicular pathway, we have performed anterograde tracing experiments which demonstrate that ipsilateral retinal axons begin to form terminal zones (TZs) in the superior colliculus (SC), within the first few postnatal days. These appear mature by postnatal day 11. Importantly, TZs formed by ipsilaterally-projecting retinal axons are spatially offset from those of contralaterally-projecting axons arising from the same retinotopic location from the outset. This pattern is consistent with that required for adult visuotopy. We further demonstrate that a member of the Ten-m/Odz/Teneurin family of homophilic transmembrane glycoproteins, Ten-m3, is an essential regulator of ipsilateral retinocollicular topography. Ten-m3 mRNA is expressed in a high-medial to low-lateral gradient in the developing SC. This corresponds topographically with its high-ventral to low-dorsal retinal gradient. In Ten-m3 knockout mice, contralateral ventrotemporal axons appropriately target rostromedial SC, whereas ipsilateral axons exhibit dramatic targeting errors along both the mediolateral and rostrocaudal axes of the SC, with a caudal shift of the primary TZ, as well as the formation of secondary, caudolaterally displaced TZs. In addition to these dramatic ipsilateral-specific mapping errors, both contralateral and ipsilateral retinocollicular TZs exhibit more subtle changes in morphology. Conclusions We conclude that important aspects of adult visuotopy are established via the differential sensitivity of ipsilateral and contralateral axons to intrinsic guidance cues. Further, we show that Ten-m3 plays a critical role in this process and is particularly important for the mapping of the ipsilateral retinocollicular pathway.

Dharmaratne, Nuwan; Glendining, Kelly A.; Young, Timothy R.; Tran, Heidi; Sawatari, Atomu; Leamey, Catherine A.

2012-01-01

154

Multiple Signaling Pathways Regulate Yeast Cell Death during the Response to Mating Pheromones  

PubMed Central

Mating pheromones promote cellular differentiation and fusion of yeast cells with those of the opposite mating type. In the absence of a suitable partner, high concentrations of mating pheromones induced rapid cell death in ?25% of the population of clonal cultures independent of cell age. Rapid cell death required Fig1, a transmembrane protein homologous to PMP-22/EMP/MP20/Claudin proteins, but did not require its Ca2+ influx activity. Rapid cell death also required cell wall degradation, which was inhibited in some surviving cells by the activation of a negative feedback loop involving the MAP kinase Slt2/Mpk1. Mutants lacking Slt2/Mpk1 or its upstream regulators also underwent a second slower wave of cell death that was independent of Fig1 and dependent on much lower concentrations of pheromones. A third wave of cell death that was independent of Fig1 and Slt2/Mpk1 was observed in mutants and conditions that eliminate calcineurin signaling. All three waves of cell death appeared independent of the caspase-like protein Mca1 and lacked certain “hallmarks” of apoptosis. Though all three waves of cell death were preceded by accumulation of reactive oxygen species, mitochondrial respiration was only required for the slowest wave in calcineurin-deficient cells. These findings suggest that yeast cells can die by necrosis-like mechanisms during the response to mating pheromones if essential response pathways are lacking or if mating is attempted in the absence of a partner.

Zhang, Nan-Nan; Dudgeon, Drew D.; Paliwal, Saurabh; Levchenko, Andre; Grote, Eric

2006-01-01

155

The nitric oxide-cyclic GMP pathway is required for nociceptive signalling at specific loci within the somatosensory pathway.  

PubMed

The involvement of nitric oxide in nociceptive processing was examined at the main loci of synaptic transmission within the rat somatosensory pathway from the caudal sural cutaneous nerve. Intrathecal (lumbar 1-3) administration of the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (30 micrograms), inhibited nitric oxide synthase in this region of the spinal cord by greater than 80% but had no significant effect on nitric oxide synthase in parietal cerebral cortex, thalamus or medulla/pons. In a rat model of peripheral neuropathy (one to two week ligation of the caudal sural cutaneous nerve), intrathecal administration of the same dose of N omega-nitro-L-arginine methyl ester prevented the hyperalgesic response to thermal stimuli. Administration of 30 micrograms N omega-nitro-L-arginine methyl ester into the lateral ventricle had no effect on nitric oxide synthase in the lumbar 1-3 region of the spinal cord but gave substantial inhibition in higher areas of the somatosensory pathway (parietal cerebral cortex, thalamus and medulla/pons). Nitric oxide synthase in the parietal cerebral cortex (but not thalamus) was inhibited to a greater extent in the hemisphere ipsilateral to the site of administration. Administration of 30 micrograms N omega-nitro-L-arginine methyl ester into the lateral ventricle decreased thermal hyperalgesia, but only when N omega-nitro-L-arginine methyl ester was administered contralateral to the ligated caudal sural cutaneous nerve and therefore ipsilateral to the cortical nociceptive processing from this nerve. Intrathecal and intracerebroventricular administration of the selective inhibitor of nitric oxide-sensitive guanylyl cyclase, 1-H-[1,2,4]oxadiazalo[4,3-a]quinoxalin-1-one, also decreased the hyperalgesic response to thermal stimuli. These data demonstrate that, in a model of neuropathic pain, nitric oxide is involved in nociceptive processing at spinal and cerebrocortical synaptic loci of the somatosensory pathway and that its actions appear to be mediated through guanylyl cyclase. PMID:8809786

Salter, M; Strijbos, P J; Neale, S; Duffy, C; Follenfant, R L; Garthwaite, J

1996-08-01

156

Isoprenoid biosynthetic pathway inhibition disrupts monoclonal protein secretion and induces the unfolded protein response pathway in multiple myeloma cells  

PubMed Central

Myeloma is characterized by the overproduction and secretion of monoclonal protein. Inhibitors of the isoprenoid biosynthetic pathway (IBP) have pleiotropic effects in myeloma cells. To investigate whether IBP inhibition interferes with monoclonal protein secretion, human myeloma cells were treated with specific inhibitors of the IBP or prenyltransferases. These studies demonstrate that agents that inhibit Rab geranylgeranylation disrupt light chain trafficking, lead to accumulation of light chain in the endoplasmic reticulum, activate the unfolded protein response pathway and induce apoptosis. These studies provide a novel mechanism of action for IBP inhibitors and suggest that further exploration of Rab-targeted agents in myeloma is warranted.

Holstein, Sarah A.; Hohl, Raymond J.

2010-01-01

157

Insulin Stimulates the Expression of the SHARP-1 Gene via Multiple Signaling Pathways.  

PubMed

The rat enhancer of split- and hairy-related protein-1 (SHARP-1) is a basic helix-loop-helix transcription factor. An issue of whether SHARP-1 is an insulin-inducible transcription factor was examined. Insulin rapidly increased the level of SHARP-1 mRNA both in vivo and in vitro. Then, signaling pathways involved with the increase of SHARP-1 mRNA by insulin were determined in H4IIE rat hepatoma cells. Pretreatments with LY294002, wortmannin, and staurosporine completely blocked the induction effect, suggesting the involvement of both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) pathways. In fact, overexpression of a dominant negative form of atypical protein kinase C lambda (aPKC?) significantly decreased the induction of the SHARP-1 mRNA. In addition, inhibitors for the small GTPase Rac or Jun N-terminal kinase (JNK) also blocked the induction of SHARP-1 mRNA by insulin. Overexpression of a dominant negative form of Rac1 prevented the activation by insulin. Furthermore, actinomycin D and cycloheximide completely blocked the induction of SHARP-1 mRNA by insulin. Finally, when a SHARP-1 expression plasmid was transiently transfected with various reporter plasmids into H4IIE cells, the promoter activity of PEPCK reporter plasmid was specifically decreased. Thus, we conclude that insulin induces the SHARP-1 gene expression at the transcription level via a both PI 3-K/aPKC?/JNK- and a PI 3-K/Rac/JNK-signaling pathway; protein synthesis is required for this induction; and that SHARP-1 is a potential repressor of the PEPCK gene expression. PMID:24446161

Takagi, K; Asano, K; Haneishi, A; Ono, M; Komatsu, Y; Yamamoto, T; Tanaka, T; Ueno, H; Ogawa, W; Tomita, K; Noguchi, T; Yamada, K

2014-06-01

158

Multiple signal transduction pathways are involved in G2/M growth arrest and apoptosis induced by the immunomodulator AS101 in multiple myeloma.  

PubMed

The organotellurium compound, AS101, induces G(2)/M growth arrest and apoptosis in multiple myeloma (MM) cell lines. To characterize the mechanism by which AS101 promotes these effects, an antibody microarray analysis was performed, comparing levels of proteins and phosphoproteins in untreated versus AS101-treated mouse 5T33 MM cells. We found that AS101 down-regulated Ilk-1, Cdc25C and phosphorylation of Plk-1 on Thr210, all of which can affect the onset of mitosis or cell survival. In addition, AS101 inhibited the activity of a high molecular weight matrix metalloproteinase complex corresponding to the MMP-9/NGAL complex. Another signaling pathway that was affected by AS101 involves p53 and p65/RelA. Levels of both proteins were elevated upon treatment with AS101. Thus, multiple signaling pathways are involved in the G(2)/M growth arrest and apoptosis induced by AS101 in multiple myeloma, suggesting that if one pathway becomes unresponsive, the therapeutic effect of AS101 might persist through alternative pathways. PMID:22712839

Naor, Yaniv; Hayun, Michal; Sredni, Benjamin; Don, Jeremy

2013-01-01

159

Retinoid signaling is required to complete the vertebrate cardiac left/right asymmetry pathway.  

PubMed

Vitamin A-deficient (VAD) quail embryos have severe abnormalities, including a high incidence of reversed cardiac situs. Using this model we examined in vivo the physiological function of vitamin A in the left/right (L/R) cardiac asymmetry pathway. Molecular analysis reveals the expression of early asymmetry genes activin receptor IIa, sonic hedgehog, Caronte, Lefty-1, and Fgf8 to be unaffected by the lack of retinoids, while expression of the downstream genes nodal-related, snail-related (cSnR), and Pitx2 is altered. In VAD embryos nodal expression in left lateral plate mesoderm (LPM) is severely downregulated and the expression domain altered during neurulation. Similarly, the expression of cSnR in the right LPM and of Pitx2 in the left side posterior heart-forming region (HFR) is downregulated in the VAD embryos. The lack of retinoids does not cause randomization or ectopic expression of nodal, cSnR, or Pitx2. At the six- to eight-somite stage nodal is expressed transiently in the left posterior HFR of normal quail embryos; this expression is missing in VAD embryos and may be linked to the loss of Pitx2 expression in this region of VAD quail embryos. Administration of retinoids to VAD embryos prior to the six-somite stage rescues the expression of nodal, cSnR, and Pitx2 as well as the randomized VAD cardiac phenotype. There is an absolute requirement for retinoids at the four- to five-somite developmental window for cardiogenesis and cardiac L/R specification to proceed normally. We conclude that retinoids do not regulate the left/right-specific sidedness assignments for expression of genes on the vertebrate cardiac asymmetry pathway, but are required during neurulation for the maintenance of adequate levels of their expression and for the development of the posterior heart tube and a loopable heart. Cardiac asymmetry may be but one of several critical events regulated by retinoid signaling in the retinoid-sensitive developmental window. PMID:10882519

Zile, M H; Kostetskii, I; Yuan, S; Kostetskaia, E; St Amand, T R; Chen, Y; Jiang, W

2000-07-15

160

Rapamycin inhibits hepatic fibrosis in rats by attenuating multiple profibrogenic pathways.  

PubMed

Hepatic stellate cell transdifferentiation, epithelial-mesenchymal cell transition, and the ductular reaction each contribute to the development of hepatic fibrosis in cholestatic liver diseases. Inhibitors of mammalian target of rapamycin have antifibrotic properties. We evaluated the hypothesis that the antifibrotic action of rapamycin is due to attenuated myofibroblast proliferation in addition to an inhibitory effect on epithelial-mesenchymal transition and the ductular reaction. Hepatic fibrosis was induced by bile duct ligation, and rodents received 1.5 mg/kg/day rapamycin by subcutaneous infusion for 21 days. The expression of various markers of hepatic fibrosis, stellate cell transactivation, epithelial-mesenchymal transition, and the ductular reaction was compared between treated and untreated animals. Hepatic fibrosis, hepatic procollagen type 1 messenger RNA, and alpha-smooth muscle actin expression were significantly reduced in treated animals. Hepatic stellate cell procollagen expression and proliferation were also reduced by rapamycin. The following markers of epithelial-mesenchymal transition--vimentin protein expression, S100 calcium binding protein A4 and transforming growth factor beta 1 messenger RNA, and the mothers against decapentaplegic homolog signaling pathway--were all reduced after rapamycin treatment. The intensity of the ductular reaction was reduced by rapamycin as assessed by histopathological scoring and by reduced cytokeratin 19 expression. Rapamycin caused a reduction in hepatic progenitor cell proliferation. Together, these data show that multiple profibrogenic pathways are activated in an animal model of cholestasis and that rapamycin attenuates epithelial-mesenchymal transition and the ductular reaction as well as hepatic stellate cell activation. PMID:19790156

Bridle, Kim R; Popa, Claudia; Morgan, Maelle L; Sobbe, Amy L; Clouston, Andrew D; Fletcher, Linda M; Crawford, Darrell H G

2009-10-01

161

Multiple Forms of Plant Phosphoenolpyruvate Carboxylase Associated with Different Metabolic Pathways 1  

PubMed Central

The physical and kinetic properties of multiple forms of phosphoenolpyruvate carboxylase were studied in leaves of C4 and C3 species, their F1 and F3 hybrids, in greening maize leaves, in Crassulacean acid metabolism plants, and in nongreen root tissues. Four different forms are suggested: a C4 photosynthetic phosphoenolpyruvate carboxylase with high Km for phosphoenolpyruvate (?0.59 mm), Km Mg (?0.5 mm), and Vmax (?29 micromoles per minute per milligram of chlorophyll); a C3 photosynthetic phosphoenolpyruvate carboxylase with low Km for phosphoenolpyruvate (?0.14 mm), Km for Mg (?0.097 mm), and Vmax (1.5); a Crassulacean acid metabolism type with low Km for phosphoenolpyruvate (0.14 mm), and high Vmax (14 micromoles per minute per milligram of chlorophyll); and a nongreen or nonautotrophic type with low Km for phosphoenolpyruvate, Km for Mg, and low Vmax. In closely related species or within species, the types can be differentiated by anion exchange column chromatography. Each of the four forms is associated with a different metabolic pathway: the phosphoenolpyruvate carboxylase of C4 species for malate generation as a photosynthetic intermediate, the phosphoenolpyruvate carboxylase of C3 species in malate generation as a photosynthetic product, the phosphoenolpyruvate carboxylase of Crassulacean acid metabolism species in malate generation as a CO2 donor for photosynthesis during the subsequent light period, and a nongreen or root type producing malate for ionic balance and reduced nicotinamide adenine dinucleotide phosphate generation. The data in this paper in conjunction with published information support the notion of different molecular forms of a protein functioning in different metabolic pathways which have common enzymic steps.

Ting, Irwin P.; Osmond, C. B.

1973-01-01

162

Antenatal inflammation reduces expression of caveolin-1 and influences multiple signaling pathways in preterm fetal lungs.  

PubMed

Bronchopulmonary dysplasia (BPD), associated with chorioamnionitis, results from the simultaneous effects of disrupted lung development, lung injury, and repair superimposed on the developing lung. Caveolins (Cavs) are implicated as major modulators of lung injury and remodeling by multiple signaling pathways, although Cavs have been minimally studied in the injured developing lung. We hypothesized that chorioamnionitis-associated antenatal lung inflammation would decrease the expression of Cav-1 in preterm fetal lungs. We tested whether changes occurred in the transcription factors Smad2/3, Smad1/5, Stat3, and Stat1, and we also studied the activation of acid-sphingomyelinase (a-SMase) with the generation of ceramide, along with changes in the expression of heme oxygenase-1 (HO-1) as indicators of possible Cav-1-mediated effects. Fetal sheep were exposed to 10 mg of intra-amniotic endotoxin or saline for 2, 7, or 2 + 7 days before preterm delivery at 124 days of gestation. The expression of Cav-1 and HO-1 and the phosphorylation of Smad and Stat were evaluated by real-time PCR, Western blotting, and/or immunohistochemistry. The activity of a-SMase and the concentrations of ceramide were measured. Intra-amniotic endotoxin decreased Cav-1 mRNA and protein expression in the lungs, with a maximum reduction of Cav-1 mRNA to 50% ± 7% of the control value (P < 0.05), and of Cav-1 protein expression to 20% ± 5% of the control value (P < 0.05). Decreased concentrations of Cav-1 were associated with the elevated phosphorylation of Smad2/3, Stat3, and Stat1, but not of Smad1/5. The expression of HO-1, a-SMase activity, and ceramide increased. Antenatal inflammation decreased the expression of Cav-1 in the preterm fetal lung. The decreased expression of Cav-1 was associated with the activation of the Smad2/3, Stat, and a-SMase/ceramide pathways, and with the increased expression of HO-1. The decreased concentrations of Cav-1 and changes in other signaling pathways may contribute to BPD. PMID:21562314

Kunzmann, Steffen; Collins, Jennifer J P; Yang, Yang; Uhlig, Stefan; Kallapur, Suhar G; Speer, Christian P; Jobe, Alan H; Kramer, Boris W

2011-11-01

163

Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum  

PubMed Central

Background Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (<30?Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis has been reported under different growth conditions. To elucidate P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P) and whole-genome transcripts were monitored over time via RNA-sequence determination. Results The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases). Conclusions The results indicate that P. tricornutum continued carbon dioxide reduction when population growth was arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels. Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up of precursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellular responses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.

2012-01-01

164

Antenatal Inflammation Reduces Expression of Caveolin-1 and Influences Multiple Signaling Pathways in Preterm Fetal Lungs  

PubMed Central

Bronchopulmonary dysplasia (BPD), associated with chorioamnionitis, results from the simultaneous effects of disrupted lung development, lung injury, and repair superimposed on the developing lung. Caveolins (Cavs) are implicated as major modulators of lung injury and remodeling by multiple signaling pathways, although Cavs have been minimally studied in the injured developing lung. We hypothesized that chorioamnionitis-associated antenatal lung inflammation would decrease the expression of Cav-1 in preterm fetal lungs. We tested whether changes occurred in the transcription factors Smad2/3, Smad1/5, Stat3, and Stat1, and we also studied the activation of acid-sphingomyelinase (a-SMase) with the generation of ceramide, along with changes in the expression of heme oxygenase–1 (HO-1) as indicators of possible Cav-1–mediated effects. Fetal sheep were exposed to 10 mg of intra-amniotic endotoxin or saline for 2, 7, or 2 + 7 days before preterm delivery at 124 days of gestation. The expression of Cav-1 and HO-1 and the phosphorylation of Smad and Stat were evaluated by real-time PCR, Western blotting, and/or immunohistochemistry. The activity of a-SMase and the concentrations of ceramide were measured. Intra-amniotic endotoxin decreased Cav-1 mRNA and protein expression in the lungs, with a maximum reduction of Cav-1 mRNA to 50% ± 7% of the control value (P < 0.05), and of Cav-1 protein expression to 20% ± 5% of the control value (P < 0.05). Decreased concentrations of Cav-1 were associated with the elevated phosphorylation of Smad2/3, Stat3, and Stat1, but not of Smad1/5. The expression of HO-1, a-SMase activity, and ceramide increased. Antenatal inflammation decreased the expression of Cav-1 in the preterm fetal lung. The decreased expression of Cav-1 was associated with the activation of the Smad2/3, Stat, and a-SMase/ceramide pathways, and with the increased expression of HO-1. The decreased concentrations of Cav-1 and changes in other signaling pathways may contribute to BPD.

Collins, Jennifer J. P.; Yang, Yang; Uhlig, Stefan; Kallapur, Suhar G.; Speer, Christian P.; Jobe, Alan H.; Kramer, Boris W.

2011-01-01

165

TNIK is required for postsynaptic and nuclear signalling pathways and cognitive function  

PubMed Central

Traf2 and NcK interacting Kinase (TNiK) contains serine-threonine kinase and scaffold domains and has been implicated in cell proliferation and glutamate receptor regulation in vitro. Here we report its role in vivo using mice carrying a knockout mutation. TNiK binds protein complexes in the synapse linking it to the NMDA receptor (NMDAR) via AKAP9. NMDAR and metabotropic receptors bidirectionally regulate TNiK phosphorylation and TNiK was required for AMPA expression and synaptic function. TNiK also organises nuclear complexes and in the absence of TNiK, there was a marked elevation in GSK3? and phosphorylation levels of its cognate phosphorylation sites on NeuroD1 with alterations in Wnt pathway signalling. We observed impairments in dentate gyrus neurogenesis in TNiK knockout mice and cognitive testing using the touchscreen apparatus revealed impairments in pattern separation on a test of spatial discrimination. Object-location paired associates learning, which is dependent on glutamatergic signalling was also impaired. Additionally, TNiK knockout mice displayed hyperlocomotor behavior that could be rapidly reversed by GSK3? inhibitors, indicating the potential for pharmacological rescue of a behavioral phenotype. These data establish TNiK as a critical regulator of cognitive functions and suggest it may play a regulatory role in diseases impacting on its interacting proteins and complexes.

Coba, M.P.; Komiyama, N.H.; Nithianantharajah, J.; Kopanitsa, M.V.; Indersmitten, T.; Skene, N.G.; Tuck, E.J.; Fricker, D.G.; Elsegood, K.A.; Stanford, L.E.; Afinowi, N.; Saksida, L.M.; Bussey, T.J.; O'Dell, T.J.; Grant, S.G.N.

2014-01-01

166

Densovirus Infectious Pathway Requires Clathrin-Mediated Endocytosis Followed by Trafficking to the Nucleus ?  

PubMed Central

Junonia coenia densovirus (JcDNV) is an ambisense insect parvovirus highly pathogenic for lepidopteran pests at larval stages. The potential use of DNVs as biological control agents prompted us to reinvestigate the host range and cellular mechanisms of infection. In order to understand the early events of infection, we set up a functional infection assay in a cell line of the pest Lymantria dispar to determine the intracellular pathway undertaken by JcDNV to infect a permissive lepidopteran cell line. Our results show that JcDNV particles are rapidly internalized into clathrin-coated vesicles and slowly traffic within early and late endocytic compartments. Blocking late-endocytic trafficking or neutralizing the pH with drugs inhibited infection. During internalization, disruption of the cytoskeleton, and inhibition of phosphatidylinositol 3-kinase blocked the movement of vesicles containing the virus to the nucleus and impaired infection. In summary, our results define for the first time the early endocytic steps required for a productive DNV infection.

Vendeville, Agnes; Ravallec, Marc; Jousset, Francoise-Xaviere; Devise, Micheline; Mutuel, Doriane; Lopez-Ferber, Miguel; Fournier, Philippe; Dupressoir, Thierry; Ogliastro, Mylene

2009-01-01

167

Densovirus infectious pathway requires clathrin-mediated endocytosis followed by trafficking to the nucleus.  

PubMed

Junonia coenia densovirus (JcDNV) is an ambisense insect parvovirus highly pathogenic for lepidopteran pests at larval stages. The potential use of DNVs as biological control agents prompted us to reinvestigate the host range and cellular mechanisms of infection. In order to understand the early events of infection, we set up a functional infection assay in a cell line of the pest Lymantria dispar to determine the intracellular pathway undertaken by JcDNV to infect a permissive lepidopteran cell line. Our results show that JcDNV particles are rapidly internalized into clathrin-coated vesicles and slowly traffic within early and late endocytic compartments. Blocking late-endocytic trafficking or neutralizing the pH with drugs inhibited infection. During internalization, disruption of the cytoskeleton, and inhibition of phosphatidylinositol 3-kinase blocked the movement of vesicles containing the virus to the nucleus and impaired infection. In summary, our results define for the first time the early endocytic steps required for a productive DNV infection. PMID:19225003

Vendeville, Agnès; Ravallec, Marc; Jousset, Françoise-Xavière; Devise, Micheline; Mutuel, Doriane; López-Ferber, Miguel; Fournier, Philippe; Dupressoir, Thierry; Ogliastro, Mylène

2009-05-01

168

Proteasome Function Is Required for DNA Damage Response and Fanconi Anemia Pathway Activation  

Microsoft Academic Search

Proteasome inhibitors sensitize tumor cells to DNA-damaging agents, including ionizing radiation (IR), and DNA cross- linking agents (melphalan and cisplatin) through unknown mechanisms. The Fanconi anemia pathway is a DNA damage- activated signaling pathway, which regulates cellular resis- tance to DNA cross-linking agents. Monoubiquitination and nuclear foci formation of FANCD2 are critical steps of the Fanconi anemia pathway. Here, we

Celine Jacquemont; Toshiyasu Taniguchi

2007-01-01

169

Application of Multidimensional Selective Item Response Regression Model for Studying Multiple Gene Methylation in SV40 Oncogenic Pathways  

PubMed Central

Alteration of gene methylation patterns has been reported to be involved in the early onsets of many human malignancies. Many exogenous risk factors, such as cigarette smoke, dietary additives, chemical exposures, radiation, and biologic agents including viral infection, are involved in the methylation pathways of cancers. We propose a multidimensional selective item response regression model to describe and test how a risk factor may alter molecular pathways involving aberrant methylation of multiple genes in oncogenesis. Our modeling framework is built on an item response model for multivariate dichotomous responses of high dimension, such as aberrant methylation of multiple tumor-suppressor genes, but we allow risk factors such as SV40 viral infection to alter the distribution of the latent factors that subsequently affect the outcome of cancer. We postulate empirical identification conditions under our model formulation. Moreover, we do not prespecify the links between the multiple dichotomous methylation responses and the latent factors, but rather conduct specification searches with a genetic algorithm to discover the links. Parameter estimation through maximum likelihood and specification searches in models with multidimensional latent factors for multivariate binary responses have become practical only recently, due to modern statistical computing development. We illustrate our proposal with the biological finding that simultaneous methylation of multiple tumor-suppressor genes is associated with the presence of SV40 viral sequences and with the cancer status of lymphoma/leukemia.We are able to test whether the data are consistent with the causal hypothesis that SV40 induces aberrant methylation of multiple genes in its oncogenic pathways. At the same time, we are able to evaluate the role of SV40 in the methylation pathway and to determine whether the methylation pathway is responsible for the development of leukemia/lymphoma.

Lin, Haiqun; Feng, Ziding; Yu, Yan; Zheng, Yingye; Shivapurkar, Narayan; Gazdar, Adi F.

2009-01-01

170

A type II protein secretory pathway required for levansucrase secretion by Gluconacetobacter diazotrophicus.  

PubMed

The endophytic diazotroph Gluconacetobacter diazotrophicus secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose. LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway. We identified an unusually organized gene cluster encoding at least the components LsdG, -O, -E, -F, -H, -I, -J, -L, -M, -N, and -D of a type II secretory system required for LsdA translocation across the outer membrane. Another open reading frame, designated lsdX, is located between the operon promoter and lsdG, but it was not identified in BLASTX searches of the DDBJ/EMBL/GenBank databases. The lsdX, -G, and -O genes were isolated from a cosmid library of strain SRT4 by complementation of an ethyl methanesulfonate mutant unable to transport LsdA across the outer membrane. The downstream genes lsdE, -F, -H, -I, -J, -L, -M, -N, and -D were isolated through chromosomal walking. The high G+C content (64 to 74%) and the codon usage of the genes identified are consistent with the G+C content and codon usage of the standard G. diazotrophicus structural gene. Sequence analysis of the gene cluster indicated that a polycistronic transcript is synthesized. Targeted disruption of lsdG, lsdO, or lsdF blocked LsdA secretion, and the bacterium failed to grow on sucrose. Replacement of Cys(162) by Gly at the C terminus of the pseudopilin LsdG abolished the protein functionality, suggesting that there is a relationship with type IV pilins. Restriction fragment length polymorphism analysis revealed conservation of the type II secretion operon downstream of the levansucrase-levanase (lsdA-lsdB) locus in 14 G. diazotrophicus strains representing 11 genotypes recovered from four different host plants in diverse geographical regions. To our knowledge, this is the first report of a type II pathway for protein secretion in the Acetobacteraceae. PMID:15262940

Arrieta, Juan G; Sotolongo, Mailin; Menéndez, Carmen; Alfonso, Dubiel; Trujillo, Luis E; Soto, Melvis; Ramírez, Ricardo; Hernández, Lázaro

2004-08-01

171

TNFR1 and TNFR2 regulate the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms  

PubMed Central

The huge majority of myeloma cell lines express TNFR2 while a substantial subset of them failed to show TNFR1 expression. Stimulation of TNFR1 in the TNFR1-expressing subset of MM cell lines had no or only a very mild effect on cellular viability. Surprisingly, however, TNF stimulation enhanced cell death induction by CD95L and attenuated the apoptotic effect of TRAIL. The contrasting regulation of TRAIL- and CD95L-induced cell death by TNF could be traced back to the concomitant NF?B-mediated upregulation of CD95 and the antiapoptotic FLIP protein. It appeared that CD95 induction, due to its strength, overcompensated a rather moderate upregulation of FLIP so that the net effect of TNF-induced NF?B activation in the context of CD95 signaling is pro-apoptotic. TRAIL-induced cell death, however, was antagonized in response to TNF because in this context only the induction of FLIP is relevant. Stimulation of TNFR2 in myeloma cells leads to TRAF2 depletion. In line with this, we observed cell death induction in TNFR1-TNFR2-costimulated JJN3 cells. Our studies revealed that the TNF-TNF receptor system adjusts the responsiveness of the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms that generate a highly context-dependent net effect on myeloma cell survival.

Rauert, H; Stuhmer, T; Bargou, R; Wajant, H; Siegmund, D

2011-01-01

172

Small molecules affect human dental pulp stem cell properties via multiple signaling pathways.  

PubMed

One fundamental issue regarding stem cells for regenerative medicine is the maintenance of stem cell stemness. The purpose of the study was to test whether small molecules can enhance stem cell properties of mesenchymal stem cells (MSCs) derived from human dental pulp (hDPSCs), which have potential for multiple clinical applications. We identified the effects of small molecules (Pluripotin (SC1), 6-bromoindirubin-3-oxime and rapamycin) on the maintenance of hDPSC properties in vitro and the mechanisms involved in exerting the effects. Primary cultures of hDPSCs were exposed to optimal concentrations of these small molecules. Treated hDPSCs were analyzed for their proliferation, the expression levels of pluripotent and MSC markers, differentiation capacities, and intracellular signaling activations. We found that small molecule treatments decreased cell proliferation and increased the expression of STRO-1, NANOG, OCT4, and SOX2, while diminishing cell differentiation into odonto/osteogenic, adipogenic, and neurogenic lineages in vitro. These effects involved Ras-GAP-, ERK1/2-, and mTOR-signaling pathways, which may preserve the cell self-renewal capacity, while suppressing differentiation. We conclude that small molecules appear to enhance the immature state of hDPSCs in culture, which may be used as a strategy for adult stem cell maintenance and extend their capacity for regenerative applications. PMID:23573877

Al-Habib, Mey; Yu, Zongdong; Huang, George T-J

2013-09-01

173

Multiple signaling pathways regulate the transcriptional activity of the orphan nuclear receptor NURR1  

PubMed Central

The orphan nuclear receptor nurr1 (NR4A2) is an essential transcription factor for the acquisition and maintenance of the phenotype of dopamine (DA)-synthesizing neurons in the mesencephalon. Although structurally related to ligand-regulated nuclear receptors, nurr1 is functionally atypical due to its inability to bind a cognate ligand and to activate transcription following canonical nuclear receptor (NR) rules. Importantly, the physiological stimuli that activate this NR and the signaling proteins that regulate its transcriptional activity in mesencephalic neurons are unknown. We used an affinity chromatography approach and CSM14.1 cells of mesencephalic origin to isolate and identify several proteins that interact directly with nurr1 and regulate its transcriptional activity. Notably, we demonstrate that the mitogen-activated protein kinases, ERK2 and ERK5, elevate, whereas LIM Kinase 1 inhibits nurr1 transcriptional activity. Furthermore, nurr1 recruits ERK5 to a NBRE-containing promoter and is a potential substrate for this kinase. We have identified amino acids in the A/B domain of nurr1 important for mediating the ERK5 activating effects on nurr1 transcriptional activity. Our results suggest that nurr1 acts as a point of convergence for multiple signaling pathways that likely play a critical role in differentiation and phenotypic expression of dopaminergic (DAergic) neurons.

Sacchetti, Paola; Carpentier, Rodolphe; Segard, Pascaline; Olive-Cren, Cecile; Lefebvre, Philippe

2006-01-01

174

Identification of multiple pathways involved in the malignant transformation of endometriosis (Review)  

PubMed Central

The association between endometriosis and malignant transformation has often been described in the medical literature. A search was conducted between 1966 and 2010 through the English language literature (online Medline PubMed database) using the keywords endometriosis combined with malignant transformation. The search revealed an increase in reports describing endometriosis and malignancy. Approximately 1.0% of women with endometriosis have lesions that undergo malignant transformation. The malignant processes that are associated with endometriosis may be classified into three groups: i) epithelial ovarian cancers (endometrioid adenocarcinoma and clear cell carcinoma), ii) other Müllerian-type tumors, including Müllerian-type mucinous borderline tumor and serous borderline tumor and iii) sarcomas such as adenosarcoma and endometrial stromal sarcoma in the female pelvic cavity. Persistent oxidative stress induced by endometriosis-dependent hemorrhage may be associated with carcinogenesis. In conclusion, the malignant transformation of endometriosis has multiple pathways of development and may share a common pathogenic mechanism; iron-induced oxidative stress derived from repeated hemorrhage.

HIGASHIURA, YUMI; KAJIHARA, HIROTAKA; SHIGETOMI, HIROSHI; KOBAYASHI, HIROSHI

2012-01-01

175

Mycophenolic Acid Inhibits Migration and Invasion of Gastric Cancer Cells via Multiple Molecular Pathways  

PubMed Central

Mycophenolic acid (MPA) is the metabolized product and active element of mycophenolate mofetil (MMF) that has been widely used for the prevention of acute graft rejection. MPA potently inhibits inosine monophosphate dehydrogenase (IMPDH) that is up-regulated in many tumors and MPA is known to inhibit cancer cell proliferation as well as fibroblast and endothelial cell migration. In this study, we demonstrated for the first time MPA’s antimigratory and anti-invasion abilities of MPA-sensitive AGS (gastric cancer) cells. Genome-wide expression analyses using Illumina whole genome microarrays identified 50 genes with ?2 fold changes and 15 genes with > 4 fold alterations and multiple molecular pathways implicated in cell migration. Real-time RT-PCR analyses of selected genes also confirmed the expression differences. Furthermore, targeted proteomic analyses identified several proteins altered by MPA treatment. Our results indicate that MPA modulates gastric cancer cell migration through down-regulation of a large number of genes (PRKCA, DOCK1, INF2, HSPA5, LRP8 and PDGFRA) and proteins (PRKCA, AKT, SRC, CD147 and MMP1) with promigratory functions as well as up-regulation of a number of genes with antimigratory functions (ATF3, SMAD3, CITED2 and CEAMCAM1). However, a few genes that may promote migration (CYR61 and NOS3) were up-regulated. Therefore, MPA’s overall antimigratory role on cancer cells reflects a balance between promigratory and antimigratory signals influenced by MPA treatment.

Dun, Boying; Sharma, Ashok; Teng, Yong; Liu, Haitao; Purohit, Sharad; Xu, Heng; Zeng, Lingwen; She, Jin-Xiong

2013-01-01

176

The history and visions of African American psychology: multiple pathways to place, space, and authority.  

PubMed

The author describes the multiple pathways of events and strategies that served to nurture African American psychology in the United States. Special attention is given to strategies for inclusion and empowerment used in 4 psychological professional and scholarly associations: the American Counseling Association, the American Psychological Association, the Association of Black Psychologists, and the Society for Research in Child Development. In addition, the author describes 4 major intellectual traditions that informed not only the strategies of inclusion but also the theoretical, research, and intervention perspectives and other professional and academic efforts of African American psychologists. Those perspectives are the Afrocentric/African-centered tradition derived from longstanding nationalist/Pan-African and culturally centered traditions within African American communities; the social contextual/multidisciplinary research tradition of the University of Chicago School of Social Science; the empirical social science research tradition of the University of Michigan; and the Black scholar/activist tradition of Howard University. This article also presents a chronological timeline of major events in the history of African American psychology. PMID:19916668

Holliday, Bertha Garrett

2009-10-01

177

In Silico Reconstruction of the Metabolic Pathways of Lactobacillus plantarum: Comparing Predictions of Nutrient Requirements with Those from Growth Experiments  

PubMed Central

On the basis of the annotated genome we reconstructed the metabolic pathways of the lactic acid bacterium Lactobacillus plantarum WCFS1. After automatic reconstruction by the Pathologic tool of Pathway Tools (http://bioinformatics.ai.sri.com/ptools/), the resulting pathway-genome database, LacplantCyc, was manually curated extensively. The current database contains refinements to existing routes and new gram-positive bacterium-specific reactions that were not present in the MetaCyc database. These reactions include, for example, reactions related to cell wall biosynthesis, molybdopterin biosynthesis, and transport. At present, LacplantCyc includes 129 pathways and 704 predicted reactions involving some 670 chemical species and 710 enzymes. We tested vitamin and amino acid requirements of L. plantarum experimentally and compared the results with the pathways present in LacplantCyc. In the majority of cases (32 of 37 cases) the experimental results agreed with the final reconstruction. LacplantCyc is the most extensively curated pathway-genome database for gram-positive bacteria and is open to the microbiology community via the World Wide Web (www.lacplantcyc.nl). It can be used as a reference pathway-genome database for gram-positive microbes in general and lactic acid bacteria in particular.

Teusink, Bas; van Enckevort, Frank H. J.; Francke, Christof; Wiersma, Anne; Wegkamp, Arno; Smid, Eddy J.; Siezen, Roland J.

2005-01-01

178

Presynaptic UNC-31 (CAPS) Is Required to Activate the G?s Pathway of the Caenorhabditis elegans Synaptic Signaling Network  

PubMed Central

C. elegans mutants lacking the dense-core vesicle priming protein UNC-31 (CAPS) share highly similar phenotypes with mutants lacking a neuronal G?s pathway, including strong paralysis despite exhibiting near normal levels of steady-state acetylcholine release as indicated by drug sensitivity assays. Our genetic analysis shows that UNC-31 and neuronal G?s are different parts of the same pathway and that the UNC-31/G?s pathway is functionally distinct from the presynaptic G?q pathway with which it interacts. UNC-31 acts upstream of G?s because mutations that activate the G?s pathway confer similar levels of strongly hyperactive, coordinated locomotion in both unc-31 null and (+) backgrounds. Using cell-specific promoters, we show that both UNC-31 and the G?s pathway function in cholinergic motor neurons to regulate locomotion rate. Using immunostaining we show that UNC-31 is often concentrated at or near active zones of cholinergic motor neuron synapses. Our data suggest that presynaptic UNC-31 activity, likely acting via dense-core vesicle exocytosis, is required to locally activate the neuronal G?s pathway near synaptic active zones.

Charlie, Nicole K.; Schade, Michael A.; Thomure, Angela M.; Miller, Kenneth G.

2006-01-01

179

The degradation of apolipoprotein B100: multiple opportunities to regulate VLDL triglyceride production by different proteolytic pathways  

PubMed Central

Very low density lipoproteins (VLDL) are a major secretory product of the liver. They serve to transport endogenously synthesized lipids, mainly triglycerides (but also some cholesterol and cholesteryl esters) to peripheral tissues. VLDL is also the precursor of LDL. ApoB100 is absolutely required for VLDL assembly and secretion. The amount of VLDL triglycerides secreted by the liver depends on the amount loaded onto each lipoprotein particle, as well as the number of particles. Each VLDL has one apoB100 molecule, making apoB100 availability a key determinant of the number of VLDL particles, and hence, triglycerides, that can be secreted by hepatic cells. Surprisingly, the pool of apoB100 in the liver is typically regulated not by its level of synthesis, which is relatively constant, but by its level of degradation. It is now recognized that there are multiple opportunities for the hepatic cell to intercept apoB100 molecules and to direct them to distinct degradative processes. This mini-review will summarize progress in understanding these processes, with an emphasis on autophagy, the most recently described pathway of apoB100 degradation, and the one with possibly the most physiologic relevance to common metabolic perturbations affecting VLDL production.

Fisher, Edward A.

2013-01-01

180

Self-assembly of VPS41 promotes sorting required for biogenesis of the regulated secretory pathway.  

PubMed

The regulated release of polypeptides has a central role in physiology, behavior, and development, but the mechanisms responsible for production of the large dense core vesicles (LDCVs) capable of regulated release have remained poorly understood. Recent work has implicated cytosolic adaptor protein AP-3 in the recruitment of LDCV membrane proteins that confer regulated release. However, AP-3 in mammals has been considered to function in the endolysosomal pathway and in the biosynthetic pathway only in yeast. We now find that the mammalian homolog of yeast VPS41, a member of the homotypic fusion and vacuole protein sorting (HOPS) complex that delivers biosynthetic cargo to the endocytic pathway in yeast, promotes LDCV formation through a common mechanism with AP-3, indicating a conserved role for these proteins in the biosynthetic pathway. VPS41 also self-assembles into a lattice, suggesting that it acts as a coat protein for AP-3 in formation of the regulated secretory pathway. PMID:24210660

Asensio, Cédric S; Sirkis, Daniel W; Maas, James W; Egami, Kiyoshi; To, Tsz-Leung; Brodsky, Frances M; Shu, Xiaokun; Cheng, Yifan; Edwards, Robert H

2013-11-25

181

The 3-hydroxy-2-butanone pathway is required for Pectobacterium carotovorum pathogenesis.  

PubMed

Pectobacterium species are necrotrophic bacterial pathogens that cause soft rot diseases in potatoes and several other crops worldwide. Gene expression data identified Pectobacterium carotovorum subsp. carotovorum budB, which encodes the ?-acetolactate synthase enzyme in the 2,3-butanediol pathway, as more highly expressed in potato tubers than potato stems. This pathway is of interest because volatiles produced by the 2,3-butanediol pathway have been shown to act as plant growth promoting molecules, insect attractants, and, in other bacterial species, affect virulence and fitness. Disruption of the 2,3-butanediol pathway reduced virulence of P. c. subsp. carotovorum WPP14 on potato tubers and impaired alkalinization of growth medium and potato tubers under anaerobic conditions. Alkalinization of the milieu via this pathway may aid in plant cell maceration since Pectobacterium pectate lyases are most active at alkaline pH. PMID:21876734

Marquez-Villavicencio, Maria del Pilar; Weber, Brooke; Witherell, R Andrews; Willis, David K; Charkowski, Amy O

2011-01-01

182

Epithelial barrier assembly requires coordinated activity of multiple domains of the tight junction protein ZO-1  

PubMed Central

Summary Tight junctions (TJs) regulate the paracellular movement of ions, macromolecules and immune cells across epithelia. Zonula occludens (ZO)-1 is a multi-domain polypeptide required for the assembly of TJs. MDCK II cells lacking ZO-1, and its homolog ZO-2, have three distinct phenotypes: reduced localization of occludin and some claudins to the TJs, increased epithelial permeability, and expansion of the apical actomyosin contractile array found at the apical junction complex (AJC). However, it is unclear exactly which ZO-1 binding domains are required to coordinate these activities. We addressed this question by examining the ability of ZO-1 domain-deletion transgenes to reverse the effects of ZO depletion. We found that the SH3 domain and the U5 motif are required to recruit ZO-1 to the AJC and that localization is a prerequisite for normal TJ and cytoskeletal organization. The PDZ2 domain is not required for localization of ZO-1 to the AJC, but is necessary to establish the characteristic continuous circumferential band of ZO-1, occludin and claudin-2. PDZ2 is also required to establish normal permeability, but is not required for normal cytoskeletal organization. Finally, our results demonstrate that PDZ1 is crucial for the normal organization of both the TJ and the AJC cytoskeleton. Our results establish that ZO-1 acts as a true scaffolding protein and that the coordinated activity of multiple domains is required for normal TJ structure and function.

Rodgers, Laurel S.; Beam, M. Tanner; Anderson, James M.; Fanning, Alan S.

2013-01-01

183

Autocrine prolactin induced by the Pten-Akt pathway is required for lactation initiation and provides a direct link between the Akt and Stat5 pathways  

PubMed Central

Extrapituitary prolactin (Prl) is produced in humans and rodents; however, little is known about its in vivo regulation or physiological function. We now report that autocrine prolactin is required for terminal mammary epithelial differentiation during pregnancy and that its production is regulated by the Pten–PI3K–Akt pathway. Conditional activation of the PI3K–Akt pathway in the mammary glands of virgin mice by either Akt1 expression or Pten deletion rapidly induced terminal mammary epithelial differentiation accompanied by the synthesis of milk despite the absence of lobuloalveolar development. Surprisingly, we found that mammary differentiation was due to the PI3K–Akt-dependent synthesis and secretion of autocrine prolactin and downstream activation of the prolactin receptor (Prlr)–Jak–Stat5 pathway. Consistent with this, Akt-induced mammary differentiation was abrogated in Prl?/?, Prlr?/?, and Stat5?/? mice. Furthermore, cells treated with conditioned medium from mammary glands in which Akt had been activated underwent rapid Stat5 phosphorylation in a manner that was blocked by inhibition of Jak2, treatment with an anti-Prl antibody, or deletion of the prolactin gene. Demonstrating a physiological requirement for autocrine prolactin, mammary glands from lactation-defective Akt1?/?;Akt2+/? mice failed to express autocrine prolactin or activate Stat5 during late pregnancy despite normal levels of circulating serum prolactin and pituitary prolactin production. Our findings reveal that PI3K–Akt pathway activation is necessary and sufficient to induce autocrine prolactin production in the mammary gland, Stat5 activation, and terminal mammary epithelial differentiation, even in the absence of the normal developmental program that prepares the mammary gland for lactation. Together, these findings identify a function for autocrine prolactin during normal development and demonstrate its endogenous regulation by the PI3K–Akt pathway.

Chen, Chien-Chung; Stairs, Douglas B.; Boxer, Robert B.; Belka, George K.; Horseman, Nelson D.; Alvarez, James V.; Chodosh, Lewis A.

2012-01-01

184

NFAT5 regulates the canonical Wnt pathway and is required for cardiomyogenic differentiation  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer NFAT5 protein expression is downregulated during cardiomyogenesis. Black-Right-Pointing-Pointer Inhibition of NFAT5 function suppresses canonical Wnt signaling. Black-Right-Pointing-Pointer Inhibition of NFAT5 function attenuates mesodermal induction. Black-Right-Pointing-Pointer NFAT5 function is required for cardiomyogenesis. -- Abstract: While nuclear factor of activated T cells 5 (NFAT5), a transcription factor implicated in osmotic stress response, is suggested to be involved in other processes such as migration and proliferation, its role in cardiomyogenesis is largely unknown. Here, we examined the role of NFAT5 in cardiac differentiation of P19CL6 cells, and observed that it was abundantly expressed in undifferentiated P19CL6 cells, and its protein expression was significantly downregulated by enhanced proteasomal degradation during DMSO-induced cardiomyogenesis. Expression of a dominant negative mutant of NFAT5 markedly attenuated cardiomyogenesis, which was associated with the inhibition of mesodermal differentiation. TOPflash reporter assay revealed that the transcriptional activity of canonical Wnt signaling was activated prior to mesodermal differentiation, and this activation was markedly attenuated by NFAT5 inhibition. Pharmacological activation of canonical Wnt signaling by [2 Prime Z, 3 Prime E]-6-bromoindirubin-3 Prime -oxime (BIO) restored Brachyury expression in NFAT5DN-expressing cells. Inhibition of NFAT5 markedly attenuated Wnt3 and Wnt3a induction. Expression of Dkk1 and Cerberus1, which are secreted Wnt antagonists, was also inhibited by NFAT5 inhibition. Thus, endogenous NFAT5 regulates the coordinated expression of Wnt ligands and antagonists, which are essential for cardiomyogenesis through the canonical Wnt pathway. These results demonstrated a novel role of NFAT5 in cardiac differentiation of stem cells.

Adachi, Atsuo [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan)] [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan); Takahashi, Tomosaburo, E-mail: ttaka@koto.kpu-m.ac.jp [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan)] [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan); Ogata, Takehiro; Imoto-Tsubakimoto, Hiroko; Nakanishi, Naohiko [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan)] [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan); Ueyama, Tomomi, E-mail: toueyama-circ@umin.ac.jp [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan)] [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan); Matsubara, Hiroaki [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan)] [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan)

2012-09-28

185

Fungal morphogenetic pathways are required for the hallmark inflammatory response during Candida albicans vaginitis.  

PubMed

Vulvovaginal candidiasis, caused primarily by Candida albicans, presents significant health issues for women of childbearing age. As a polymorphic fungus, the ability of C. albicans to switch between yeast and hyphal morphologies is considered its central virulence attribute. Armed with new criteria for defining vaginitis immunopathology, the purpose of this study was to determine whether the yeast-to-hypha transition is required for the hallmark inflammatory responses previously characterized during murine vaginitis. Kinetic analyses of vaginal infection with C. albicans in C57BL/6 mice demonstrated that fungal burdens remained constant throughout the observation period, while polymorphonuclear leukocyte (PMN), S100A8, and interleukin-1? levels obtained from vaginal lavage fluid increased by day 3 onward. Lactate dehydrogenase activity was also positively correlated with increased effectors of innate immunity. Additionally, immunodepletion of neutrophils in infected mice confirmed a nonprotective role for PMNs during vaginitis. Determination of the importance of fungal morphogenesis during vaginitis was addressed with a two-pronged approach. Intravaginal inoculation of mice with C. albicans strains deleted for key transcriptional regulators (bcr1?/?, efg1?/?, cph1?/?, and efg1?/? cph1?/?) controlling the yeast-to-hypha switch revealed a crucial role for morphogenetic signaling through the Efg1 and, to a lesser extent, the Bcr1 pathways in contributing to vaginitis immunopathology. Furthermore, overexpression of transcription factors NRG1 and UME6, to maintain yeast and hyphal morphologies, respectively, confirmed the importance of morphogenesis in generating innate immune responses in vivo. These results highlight the yeast-to-hypha switch and the associated morphogenetic response as important virulence components for the immunopathogenesis of Candida vaginitis, with implications for transition from benign colonization to symptomatic infection. PMID:24478069

Peters, Brian M; Palmer, Glen E; Nash, Andrea K; Lilly, Elizabeth A; Fidel, Paul L; Noverr, Mairi C

2014-02-01

186

Maximal adamantyl-substituted retinoid-related molecule-induced apoptosis requires NF-?B noncanonical and canonical pathway activation  

PubMed Central

NF-?B transcription factors have a critical role in regulating cell survival and apoptosis. We have previously shown that 4-(3-Cl-(1-adamantyl)-4-hydroxyphenyl)-3-chlorocinnamic acid (3-Cl-AHPC), an adamantyl-substituted retinoid molecule, induced apoptosis and required NF-?B activation in prostate and breast carcinoma cells. Here, we show that 3-Cl-AHPC activated both I?B kinase (IKK)? and IKK? with subsequent activation of the canonical and noncanonical NF-?B pathways in the human breast carcinoma and leukemia cell lines. 3-Cl-AHPC-mediated activation of the NF-?B canonical pathway occurred within 6?h, whereas maximal activation of the NF-?B noncanonical pathway required 48?h. Knockout of IKK? or IKK? expression in mouse embryonic fibroblast cells and knockdown of IKK? or IKK? in MDA-MB-468 cells resulted in the inhibition of 3-Cl-AHPC-mediated apoptosis, indicating that activation of canonical and noncanonical pathways are required for maximal 3-Cl-AHPC-mediated apoptosis. 3-Cl-AHPC activation of the noncanonical pathway was preceded by caspase-mediated decrease in the E3-ligase c-IAP1 with subsequent stabilization of NF-?B-inducing kinase (NIK) expression, increased binding of NIK by TRAF3, activation of IKK?, and the resultant increased levels of RelB and p52. Increased expression of c-IAP1 blocked 3-Cl-AHPC-mediated stabilization of NIK levels and 3-Cl-AHPC-mediated apoptosis. Cdc37 expression was required for activation of IKK? and IKK? by 3-Cl-AHPC. These findings suggest that NF-?B pathways have an important role in 3-Cl-AHPC-mediated apoptosis.

Farhana, L; Dawson, M I; Murshed, F; Fontana, J A

2011-01-01

187

Axon regeneration requires coordinate activation of p38 and JNK MAPK pathways  

PubMed Central

Signaling pathways essential for axon regeneration, but not for neuron development or function, are particularly well suited targets for therapeutic intervention. We find that the parallel PMK-3(p38) and KGB-1(JNK) MAPK pathways must be coordinately activated to promote axon regeneration. Axon regeneration fails if the activity of either pathway is absent. These two MAPKs are coregulated by the E3 ubiquitin ligase RPM-1(Phr1) via targeted degradation of the MAPKKKs DLK-1 and MLK-1 and by the MAPK phosphatase VHP-1(MKP7), which negatively regulates both PMK-3(p38) and KGB-1(JNK).

Nix, Paola; Hisamoto, Naoki; Matsumoto, Kunihiro; Bastiani, Michael

2011-01-01

188

Axon regeneration requires coordinate activation of p38 and JNK MAPK pathways.  

PubMed

Signaling pathways essential for axon regeneration, but not for neuron development or function, are particularly well suited targets for therapeutic intervention. We find that the parallel PMK-3(p38) and KGB-1(JNK) MAPK pathways must be coordinately activated to promote axon regeneration. Axon regeneration fails if the activity of either pathway is absent. These two MAPKs are coregulated by the E3 ubiquitin ligase RPM-1(Phr1) via targeted degradation of the MAPKKKs DLK-1 and MLK-1 and by the MAPK phosphatase VHP-1(MKP7), which negatively regulates both PMK-3(p38) and KGB-1(JNK). PMID:21670305

Nix, Paola; Hisamoto, Naoki; Matsumoto, Kunihiro; Bastiani, Michael

2011-06-28

189

Precise lamination of retinal axons generates multiple parallel input pathways in the tectum  

PubMed Central

The axons of retinal ganglion cells (RGCs) form topographic connections in the optic tectum, recreating a two-dimensional map of the visual field in the midbrain. RGC axons are also targeted to specific positions along the laminar axis of the tectum. Understanding the sensory transformations performed by the tectum requires identification of the rules that control the formation of synaptic laminae by RGC axons. However, there is little information regarding the spatial relationships between multiple axons as they establish laminar and retinotopic arborization fields within the same region of neuropil. Moreover, the contribution of RGC axon lamination to the processing of visual information is unknown. We have utilized Brainbow genetic labeling to visualize groups of individually identifiable axons during the assembly of a precise laminar map in the tectum. Live imaging of multiple RGCs revealed that axons target specific sublaminar positions during initial innervation and maintain their relative laminar positions throughout early larval development, ruling out a model for lamina selection based on iterative refinements. During this period of laminar stability, RGC arbors undergo structural rearrangements that shift their relative retinotopic positions. Analysis of cell type-specific lamination patterns revealed that distinct combinations of RGCs converge to form each sublamina, and this input heterogeneity correlates with different functional responses to visual stimuli. These findings suggest that lamina-specific sorting of retinal inputs provides an anatomical blueprint for the integration of visual features in the tectum.

Robles, Estuardo; Filosa, Alessandro; Baier, Herwig

2013-01-01

190

Identification of Common Biological Pathways and Drug Targets Across Multiple Respiratory Viruses Based on Human Host Gene Expression Analysis  

PubMed Central

Background Pandemic and seasonal respiratory viruses are a major global health concern. Given the genetic diversity of respiratory viruses and the emergence of drug resistant strains, the targeted disruption of human host-virus interactions is a potential therapeutic strategy for treating multi-viral infections. The availability of large-scale genomic datasets focused on host-pathogen interactions can be used to discover novel drug targets as well as potential opportunities for drug repositioning. Methods/Results In this study, we performed a large-scale analysis of microarray datasets involving host response to infections by influenza A virus, respiratory syncytial virus, rhinovirus, SARS-coronavirus, metapneumonia virus, coxsackievirus and cytomegalovirus. Common genes and pathways were found through a rigorous, iterative analysis pipeline where relevant host mRNA expression datasets were identified, analyzed for quality and gene differential expression, then mapped to pathways for enrichment analysis. Possible repurposed drugs targets were found through database and literature searches. A total of 67 common biological pathways were identified among the seven different respiratory viruses analyzed, representing fifteen laboratories, nine different cell types, and seven different array platforms. A large overlap in the general immune response was observed among the top twenty of these 67 pathways, adding validation to our analysis strategy. Of the top five pathways, we found 53 differentially expressed genes affected by at least five of the seven viruses. We suggest five new therapeutic indications for existing small molecules or biological agents targeting proteins encoded by the genes F3, IL1B, TNF, CASP1 and MMP9. Pathway enrichment analysis also identified a potential novel host response, the Parkin-Ubiquitin Proteasomal System (Parkin-UPS) pathway, which is known to be involved in the progression of neurodegenerative Parkinson's disease. Conclusions Our study suggests that multiple and diverse respiratory viruses invoke several common host response pathways. Further analysis of these pathways suggests potential opportunities for therapeutic intervention.

Smith, Steven B.; Dampier, William; Tozeren, Aydin; Brown, James R.; Magid-Slav, Michal

2012-01-01

191

Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development  

PubMed Central

Background The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC). Genetic studies in zebrafish and mice have established that the Hedgehog (Hh)-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE), which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia [1,2]. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA) skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1) for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study chameleon mutants, lacking a functional disp1(con/disp1). Results con/disp1 mutants display reduced and dysmorphic mandibular and hyoid arch cartilages and lack all ceratobranchial cartilage elements. CNCC specification and migration into the PA primorida occurs normally in con/disp1 mutants, however disp1 is necessary for post-migratory CNCC patterning and differentiation. We show that disp1 is required for post-migratory CNCC to become properly patterned within the first arch, while the gene is dispensable for CNCC condensation and patterning in more posterior arches. Upon residing in well-formed pharyngeal epithelium, neural crest condensations in the posterior PA fail to maintain expression of two transcription factors essential for chondrogenesis, sox9a and dlx2a, yet continue to robustly express other neural crest markers. Histology reveals that posterior arch residing-CNCC differentiate into fibrous-connective tissue, rather than becoming chondrocytes. Treatments with Cyclopamine, to inhibit Hh-signaling at different developmental stages, show that Hh-signaling is required during gastrulation for normal patterning of CNCC in the first PA, and then during the late pharyngula stage, to promote CNCC chondrogenesis within the posterior arches. Further, loss of disp1 disrupted normal expression of bapx1 and gdf5, markers of jaw joint patterning, thus resulting in jaw joint defects in con/disp1 mutant animals. Conclusion This study reveals novel requirements for Hh-signaling in the zebrafish PA skeleton and highlights the functional diversity and differential sensitivity of craniofacial tissues to Hh-signaling throughout the face, a finding that may help to explain the spectrum of human facial phenotypes characteristic of HPE.

2009-01-01

192

Integrated QSAR study for inhibitors of hedgehog signal pathway against multiple cell lines:a collaborative filtering method  

PubMed Central

Background The Hedgehog Signaling Pathway is one of signaling pathways that are very important to embryonic development. The participation of inhibitors in the Hedgehog Signal Pathway can control cell growth and death, and searching novel inhibitors to the functioning of the pathway are in a great demand. As the matter of fact, effective inhibitors could provide efficient therapies for a wide range of malignancies, and targeting such pathway in cells represents a promising new paradigm for cell growth and death control. Current research mainly focuses on the syntheses of the inhibitors of cyclopamine derivatives, which bind specifically to the Smo protein, and can be used for cancer therapy. While quantitatively structure-activity relationship (QSAR) studies have been performed for these compounds among different cell lines, none of them have achieved acceptable results in the prediction of activity values of new compounds. In this study, we proposed a novel collaborative QSAR model for inhibitors of the Hedgehog Signaling Pathway by integration the information from multiple cell lines. Such a model is expected to substantially improve the QSAR ability from single cell lines, and provide useful clues in developing clinically effective inhibitors and modifications of parent lead compounds for target on the Hedgehog Signaling Pathway. Results In this study, we have presented: (1) a collaborative QSAR model, which is used to integrate information among multiple cell lines to boost the QSAR results, rather than only a single cell line QSAR modeling. Our experiments have shown that the performance of our model is significantly better than single cell line QSAR methods; and (2) an efficient feature selection strategy under such collaborative environment, which can derive the commonly important features related to the entire given cell lines, while simultaneously showing their specific contributions to a specific cell-line. Based on feature selection results, we have proposed several possible chemical modifications to improve the inhibitor affinity towards multiple targets in the Hedgehog Signaling Pathway. Conclusions Our model with the feature selection strategy presented here is efficient, robust, and flexible, and can be easily extended to model large-scale multiple cell line/QSAR data. The data and scripts for collaborative QSAR modeling are available in the Additional file 1.

2012-01-01

193

Identification of Pathways Required for the Coordination of Late Mitotic Events in Animal Cells.  

National Technical Information Service (NTIS)

Telomeres are specialized chromatin structures that protect chromosomes ends from the DNA repair pathways. Telomeres are re-formed after each round of DNA replication. The molecular details of telomere maturation have been well characterized. Many of the ...

B. Baumgartner

2006-01-01

194

The Requirement for Sodium as a Micronutrient by Species Having the C4 Dicarboxylic Photosynthetic Pathway  

PubMed Central

Six species having characteristics of plants with the C4 dicarboxylic photosynthetic pathway, Echinochloa utilis L. Ohwi et Yabuno (Japanese millet), Cynodon dactylon L. (Bermuda grass), Kyllinga brevifolia Rottb., Amaranthus tricolor L. cv. Early splendour, Kochia childsii Hort., and Portulaca grandiflora Hook (rose moss), responded decisively to 0.1 milliequivalent per liter NaCl supplied to their culture solutions initially containing less than 0.08 microequivalent per liter Na. Chlorosis and necrosis occurred in leaves of plants not receiving sodium. Portulaca failed to set flower in the sodium-deficient cultures. Under similar conditions Poa pratensis L. (Kentucky blue grass) having characteristics of the C3 photosynthetic pathway made normal growth and did not respond to the addition of sodium. It is concluded from these results and previously reported work that sodium is generally essential for species having the C4 pathway but not for species with the C3 pathway. Images

Brownell, P. F.; Crossland, C. J.

1972-01-01

195

The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells  

SciTech Connect

Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-specific inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.

Xiao Wei; Yang Yi; Weng Qingbei; Lin Tiehao; Yuan Meijin [State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275 (China); Yang Kai, E-mail: yangkai@mail.sysu.edu.c [State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275 (China); Pang Yi [State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275 (China)

2009-08-15

196

Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis  

Microsoft Academic Search

The tumour-suppressor phosphatase with tensin homology (PTEN) is the most important negative regulator of the cell-survival signalling pathway initiated by phosphatidylinositol 3-kinase (PI3K). Although PTEN is mutated or deleted in many tumours, deregulation of the PI3K–PTEN network also occurs through other mechanisms. Crosstalk between the PI3K pathways and other tumorigenic signalling pathways, such as those that involve Ras, p53, TOR

Megan Cully; Han You; Arnold J. Levine; Tak W. Mak

2006-01-01

197

Assessing structure and function of the afferent visual pathway in multiple sclerosis and associated optic neuritis  

Microsoft Academic Search

The afferent visual pathway is commonly affected in MS. Assessment of the afferent visual pathway using clinical, imaging\\u000a and electrophysiological methods not only provides insights into the pathophysiology of MS, but also provides a method of\\u000a investigating potential therapeutic measures in MS. This review summarises the various assessment methods, in particular imaging\\u000a techniques of the visual pathway. Retinal nerve fibre

Madhan Kolappan; Andrew P. D. Henderson; Thomas M. Jenkins; Claudia A. M. Wheeler-Kingshott; Gordon T. Plant; Alan J. Thompson; David H. Miller

2009-01-01

198

Multiple and Interconnected Pathways for L-Lysine Catabolism in Pseudomonas putida KT2440  

Microsoft Academic Search

L-Lysine catabolism in Pseudomonas putida KT2440 was generally thought to occur via the aminovalerate pathway. In this study we demonstrate the operation of the alternative aminoadipate pathway with the intermediates D-lysine, L-pipecolate, and aminoadipate. The simultaneous operation of both pathways for the use of L-lysine as the sole carbon and nitrogen source was confirmed genetically. Mutants with mutations in either

Olga Revelles; Manuel Espinosa-Urgel; Tobias Fuhrer; Uwe Sauer; Juan L. Ramos

2005-01-01

199

Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways  

PubMed Central

Estradiol (E2) and other steroids have recently been shown to initiate various intracellular signaling cascades from the plasma membrane, including those stimulating mitogen-activated protein kinases (MAPKs), and particularly extracellular-regulated kinases (ERKs). In this study we demonstrated the ability of E2 to activate ERKs in the GH3/B6/F10 pituitary tumor cell line, originally selected for its enhanced expression of membrane estrogen receptor-? (mER?). We compared E2 to its cell-impermeable analog (E2 conjugated to peroxidase, E2–P), and to the synthetic estrogen diethylstilbestrol (DES). Time-dependent ERK activation was quantified with a novel fixed cell-based immunoassay developed to efficiently determine activation by multiple compounds over multiple parameters. Both E2 and DES produced bimodal responses, but with distinctly different time courses of enzyme phosphorylation (activation) and inactivation; E2–P induced a monophasic ERK activation. E2 also phosphorylated ERKs in concentration-dependent manner with two concentration optima (10?14 and 10?8 M). Inhibitors were employed to determine pathway (ER, EGFR, membrane organization, PI3 kinase, Src kinase, Ca2+) involvement and timing of pathway activations; all affected ERK activation as early as 3–6 min, suggesting simultaneous, not sequential, activation. Therefore, E2 and other estrogenic compounds can produce rapid ERK phosphorylations via nongenomic pathways, using more than one pathway for signal generation.

Bulayeva, Nataliya N.; Gametchu, Bahiru; Watson, Cheryl S.

2005-01-01

200

mir-30d regulates multiple genes in the autophagy pathway and impairs autophagy process in human cancer cells  

PubMed Central

In human epithelial cancers, the microRNA (miRNA) mir-30d is amplified with high frequency and serves as a critical oncomir by regulating metastasis, apoptosis, proliferation, and differentiation. Autophagy, a degradation pathway for long-lived protein and organelles, regulates the survival and death of many cell types. Increasing evidence suggests that autophagy plays an important function in epithelial tumor initiation and progression. Using a combined bioinformatics approach, gene set enrichment analysis and miRNA target prediction, we found that mir-30d might regulate multiple genes in the autophagy pathway including BECN1, BNIP3L, ATG12, ATG5, ATG2. Our further functional experiments demonstrated that the expression of these core proteins in the autophagy pathway was directly suppressed by mir-30d in cancer cells. Finally, we showed that mir-30d regulated the autophagy process by inhibiting autophagosome formation and LC3B-I conversion to LC3B-II. Taken together, our results provide evidence that the oncomir mir-30d impairs the autophagy process by targeting multiple genes in the autophagy pathway. This result will contribute to understanding the molecular mechanism of mir-30d in tumorigenesis and developing novel cancer therapy strategy.

Yang, Xiaojun; Zhong, Xiaomin; Tanyi, Janos L.; Shen, Jianfeng; Xu, Congjian; Gao, Peng; Zheng, Tim M.; DeMichele, Angela M.; Zhang, Lin

2013-01-01

201

Incidence and distribution of extravascular compression of extracranial venous pathway in patients with chronic cerebrospinal venous insufficiency and multiple sclerosis.  

PubMed

OBJECTIVE: To examine the incidence and distribution of extravascular compression of the extracranial venous pathway (the jugular and/or azygous veins) in multiple sclerosis patients with chronic cerebrospinal venous insufficiency evaluated by mulitislice computer tomographic angiography. METHODS AND RESULTS: Study group consisted of 51 consecutive patients with multiple sclerosis in whom chronic cerebrospinal venous insufficiency was diagnosed by color Doppler sonography (CDS). Mulitislice computer tomographic angiography was performed in all patients, and it revealed significant extravascular compression (>70%) of extracranial venous pathway in 26 patients (51%), while in 25 patients (49%) no significant extravascular compression was seen. Extracranial compression due to transverse processus of cervical vertebrae was seen in 23 patients, carotid bulb compression was seen in two patients, and in one case, compression presented as a thoracic outlet syndrome. CONCLUSION: Our data indicate that extravascular compression of the extracranial venous pathway is frequent in multiple sclerosis patients with chronic cerebrospinal venous insufficiency, and that it is mainly due to compression caused by transverse processus of cervical vertebrae. Further studies are needed to evaluate potential clinical implications of this phenomenon. PMID:23761866

Djordje, Radak; Nenad, Ilijevski; Jovo, Kolar; Dragan, Sagic; Zelimir, Antonic; Slobodan, Tanaskovic; Nikola, Aleksic; Srdjan, Babic; Petar, Otasevic

2013-05-01

202

Multiple components of eIF4F are required for protein synthesis-dependent hippocampal long-term potentiation  

PubMed Central

Persistent forms of synaptic plasticity are widely thought to require the synthesis of new proteins. This feature of long-lasting forms of plasticity largely has been demonstrated using inhibitors of general protein synthesis, such as either anisomycin or emetine. However, these drugs, which inhibit elongation, cannot address detailed questions about the regulation of translation initiation, where the majority of translational control occurs. Moreover, general protein synthesis inhibitors cannot distinguish between cap-dependent and cap-independent modes of translation initiation. In the present study, we took advantage of two novel compounds, 4EGI-1 and hippuristanol, each of which targets a different component of the eukaryotic initiation factor (eIF)4F initiation complex, and investigated their effects on long-term potentiation (LTP) at CA3-CA1 synapses in the hippocampus. We found that 4EGI-1 and hippuristanol both attenuated long-lasting late-phase LTP induced by two different stimulation paradigms. We also found that 4EGI-1 and hippuristanol each were capable of blocking the expression of newly synthesized proteins immediately after the induction of late-phase LTP. These new pharmacological tools allow for a more precise dissection of the role played by translational control pathways in synaptic plasticity and demonstrate the importance of multiple aspects of eIF4F in processes underlying hippocampal LTP, laying the foundation for future studies investigating the role of eIF4F in hippocampus-dependent memory processes.

Hoeffer, Charles A.; Santini, Emanuela; Ma, Tao; Arnold, Elizabeth C.; Whelan, Ashley M.; Wong, Helen; Pierre, Philippe; Pelletier, Jerry

2013-01-01

203

RAB5 activation is required for multiple steps in Arabidopsis thaliana root development.  

PubMed

Rab GTPases regulate the tethering and fusion of transport vesicles to target membranes in membrane trafficking by acting as a molecular switch, cycling between GDP- and GTP-bound states. RAB5 is a member of the Rab GTPase family, the members of which have been shown to perform various functions in the endocytic pathway, including the regulation of endosomal fusion and motility in animal cells. RAB5-mediated endosomal trafficking has also been found to play important roles in various higher order plant functions, which include the regulation of the polar transport of auxin and responses to environmental conditions. The regulatory mechanisms and functions of plant RAB5 have also been investigated at the molecular and cellular levels. However, the significance of RAB5 activity at the tissue and organ levels has hardly been investigated thus far. In the present study, we examined the effect of a mutation in VPS9a, which encodes the sole guanine nucleotide exchange factor for all RAB5s in the vegetative stages of Arabidopsis thaliana. We found that multiple developmental processes were impaired in the mutant plants, including the growth and pattern formation of the roots and establishment of auxin maxima. Our results indicate that RAB5 plays distinctive pivotal roles in the development of plants. PMID:23893024

Inoue, Takeshi; Kondo, Yuki; Naramoto, Satoshi; Nakano, Akihiko; Ueda, Takashi

2013-10-01

204

Multiple pathways for steel regulation suggested by genomic and sequence analysis of the murine Steel gene  

SciTech Connect

The Steel (Sl) locus encodes mast cell growth factor (Mgf) that is required for the development of germ cells, hematopoietic cells and melanocytes. Although the expression patterns of the Mgf gene are well characterized, little is known of the factors which regulate its expression. Here, we describe the cloning and sequence of the full-length transcription unit and the 5{prime} flanking region of the murine Mgf gene. The full-length Mgf mRNA consists of a short 5{prime} untranslated region (UTR), a 0.8-kb ORF and a long 3{prime} UTR. A single transcription initiation site is used in a number of mouse tissues and is located just downstream of binding sites for several known transcription factors. In the 5{prime} UTR, two ATGs were found upstream of the initiator methionine and are conserved among different species, suggesting that Mgf may be translationally regulated. At least two Mgf mRNAs are produced by alternative use of polyadenylation sites, but numerous other potential polyadenylation sites were found in the 3{prime} UTR. In addition, the 3{prime} UTR contains numerous sequence motifs that may regulate Mgf mRNA stability. These studies suggest multiple ways in which expression of Mgf may be regulated. 39 refs., 4 figs.

Bedell, M.A.; Copeland, N.G.; Jenkins, N.A. [NCI-Frederick Cancer Research and Development Center, Frederick, MD (United States)

1996-03-01

205

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses  

PubMed Central

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.

Gillespie, Eugene J.; Ho, Chi-Lee C.; Balaji, Kavitha; Clemens, Daniel L.; Deng, Gang; Wang, Yao E.; Elsaesser, Heidi J.; Tamilselvam, Batcha; Gargi, Amandeep; Dixon, Shandee D.; France, Bryan; Chamberlain, Brian T.; Blanke, Steven R.; Cheng, Genhong; de la Torre, Juan Carlos; Brooks, David G.; Jung, Michael E.; Colicelli, John; Damoiseaux, Robert; Bradley, Kenneth A.

2013-01-01

206

Assessing data quality for a federal environmental restoration project: Rationalizing the requirements of multiple clients  

SciTech Connect

Most environmental restoration projects at federal facilities face the difficult task of melding the quality assurance (QA) requirements of multiple clients, as well as dealing with historical data that are often of unknown quality. At Lawrence Livermore National Laboratory (LLNL), we have successfully integrated the requirements of our multiple clients by carefully developing a QA program that efficiently meets our clients` needs. The Site 300 Experimental Test Site is operated by LLNL in support of its national defense program. The responsibility for conducting environmental contaminant investigations and restoration at Site 300 is vested in the Site 300 Environmental Restoration Project (Site 300 ERP) of LLNL`s Environmental Restoration Division. LLNL Site 300 ERP must comply with the QA requirements of several clients, which include: the LLNL Environmental Protection Department, the DOE, the US Environmental Protection Agency-Region IX (EPA), the California Regional Water Quality Control Board -- Central Valley Region, and the California Department of Toxic Substances Control. This comprehensive QA program was used to determine the acceptability of historical data. The Site 300 ERP began soil and ground water investigations in 1982. However, we did not begin receiving analytical quality assurance/quality control (QA/QC) data until 1989; therefore, the pre-1989 data that were collected are of unknown quality. The US EPA QAMS-005/80 defines data quality as the totality of features and characteristics of data that bears on its ability to satisfy a given purpose. In the current context, the characteristics of major importance are accuracy, precision, completeness, representativeness, and comparability. Using our established QA program, we determined the quality of this historical data based on its comparability to the post-1989 data. By accepting this historical data, we were able to save a considerable amount of money in recharacterization costs.

Kiszka, V.R.; Carlsen, T.M.

1994-07-01

207

The root hair assay facilitates the use of genetic and pharmacological tools in order to dissect multiple signalling pathways that lead to programmed cell death.  

PubMed

The activation of programmed cell death (PCD) is often a result of complex signalling pathways whose relationship and intersection are not well understood. We recently described a PCD root hair assay and proposed that it could be used to rapidly screen genetic or pharmacological modulators of PCD. To further assess the applicability of the root hair assay for studying multiple signalling pathways leading to PCD activation we have investigated the crosstalk between salicylic acid, autophagy and apoptosis-like PCD (AL-PCD) in Arabidopsis thaliana. The root hair assay was used to determine rates of AL-PCD induced by a panel of cell death inducing treatments in wild type plants treated with chemical modulators of salicylic acid synthesis or autophagy, and in genetic lines defective in autophagy or salicylic acid signalling. The assay demonstrated that PCD induced by exogenous salicylic acid or fumonisin B1 displayed a requirement for salicylic acid signalling and was partially dependent on the salicylic acid signal transducer NPR1. Autophagy deficiency resulted in an increase in the rates of AL-PCD induced by salicylic acid and fumonisin B1, but not by gibberellic acid or abiotic stress. The phenylalanine ammonia lyase-dependent salicylic acid synthesis pathway contributed only to death induced by salicylic acid and fumonisin B1. 3-Methyladenine, which is commonly used as an inhibitor of autophagy, appeared to influence PCD induction in all treatments suggesting a possible secondary, non-autophagic, effect on a core component of the plant PCD pathway. The results suggest that salicylic acid signalling is negatively regulated by autophagy during salicylic acid and mycotoxin-induced AL-PCD. However, this crosstalk does not appear to be directly involved in PCD induced by gibberellic acid or abiotic stress. This study demonstrates that the root hair assay is an effective tool for relatively rapid investigation of complex signalling pathways leading to the activation of PCD. PMID:24755572

Kacprzyk, Joanna; Devine, Aoife; McCabe, Paul F

2014-01-01

208

The Root Hair Assay Facilitates the Use of Genetic and Pharmacological Tools in Order to Dissect Multiple Signalling Pathways That Lead to Programmed Cell Death  

PubMed Central

The activation of programmed cell death (PCD) is often a result of complex signalling pathways whose relationship and intersection are not well understood. We recently described a PCD root hair assay and proposed that it could be used to rapidly screen genetic or pharmacological modulators of PCD. To further assess the applicability of the root hair assay for studying multiple signalling pathways leading to PCD activation we have investigated the crosstalk between salicylic acid, autophagy and apoptosis-like PCD (AL-PCD) in Arabidopsis thaliana. The root hair assay was used to determine rates of AL-PCD induced by a panel of cell death inducing treatments in wild type plants treated with chemical modulators of salicylic acid synthesis or autophagy, and in genetic lines defective in autophagy or salicylic acid signalling. The assay demonstrated that PCD induced by exogenous salicylic acid or fumonisin B1 displayed a requirement for salicylic acid signalling and was partially dependent on the salicylic acid signal transducer NPR1. Autophagy deficiency resulted in an increase in the rates of AL-PCD induced by salicylic acid and fumonisin B1, but not by gibberellic acid or abiotic stress. The phenylalanine ammonia lyase-dependent salicylic acid synthesis pathway contributed only to death induced by salicylic acid and fumonisin B1. 3-Methyladenine, which is commonly used as an inhibitor of autophagy, appeared to influence PCD induction in all treatments suggesting a possible secondary, non-autophagic, effect on a core component of the plant PCD pathway. The results suggest that salicylic acid signalling is negatively regulated by autophagy during salicylic acid and mycotoxin-induced AL-PCD. However, this crosstalk does not appear to be directly involved in PCD induced by gibberellic acid or abiotic stress. This study demonstrates that the root hair assay is an effective tool for relatively rapid investigation of complex signalling pathways leading to the activation of PCD.

Kacprzyk, Joanna; Devine, Aoife; McCabe, Paul F.

2014-01-01

209

Determining the elastic properties of aptamer-ricin single molecule multiple pathway interactions  

NASA Astrophysics Data System (ADS)

We report on the elastic properties of ricin and anti-ricin aptamer interactions, which showed three stable binding conformations, each of which has its special elastic properties. These different unbinding pathways were investigated by the dynamic force spectroscopy. A series-spring model combining the worm-like-chain model and Hook's law was used to estimate the apparent spring constants of the aptamer and linker molecule polyethylene glycol. The aptamer in its three different unbinding pathways showed different apparent spring constants. The two reaction barriers in the unbinding pathways also influence the apparent spring constant of the aptamer. This special elastic behavior of aptamer was used to distinguish its three unbinding pathways under different loading rates. This method also offered a way to distinguish and discard the non-specific interactions in single molecule experiments.

Wang, Bin; Park, Bosoon; Kwon, Yongkuk; Xu, Bingqian

2014-05-01

210

mir-30d Regulates multiple genes in the autophagy pathway and impairs autophagy process in human cancer cells  

SciTech Connect

Highlights: ? Gene set enrichment analysis indicated mir-30d might regulate the autophagy pathway. ? mir-30d represses the expression of BECN1, BNIP3L, ATG12, ATG5 and ATG2. ? BECN1, BNIP3L, ATG12, ATG5 and ATG2 are direct targets of mir-30d. ? mir-30d inhibits autophagosome formation and LC3B-I conversion to LC3B-II. ? mir-30d regulates the autophagy process. -- Abstract: In human epithelial cancers, the microRNA (miRNA) mir-30d is amplified with high frequency and serves as a critical oncomir by regulating metastasis, apoptosis, proliferation, and differentiation. Autophagy, a degradation pathway for long-lived protein and organelles, regulates the survival and death of many cell types. Increasing evidence suggests that autophagy plays an important function in epithelial tumor initiation and progression. Using a combined bioinformatics approach, gene set enrichment analysis, and miRNA target prediction, we found that mir-30d might regulate multiple genes in the autophagy pathway including BECN1, BNIP3L, ATG12, ATG5, and ATG2. Our further functional experiments demonstrated that the expression of these core proteins in the autophagy pathway was directly suppressed by mir-30d in cancer cells. Finally, we showed that mir-30d regulated the autophagy process by inhibiting autophagosome formation and LC3B-I conversion to LC3B-II. Taken together, our results provide evidence that the oncomir mir-30d impairs the autophagy process by targeting multiple genes in the autophagy pathway. This result will contribute to understanding the molecular mechanism of mir-30d in tumorigenesis and developing novel cancer therapy strategy.

Yang, Xiaojun [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States) [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Department of General Surgery, Gansu Provincial Hospital, Lanzhou, Gansu 710000 (China); Zhong, Xiaomin [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States) [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011 (China); Tanyi, Janos L.; Shen, Jianfeng [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States)] [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Xu, Congjian [Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011 (China)] [Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011 (China); Gao, Peng [Department of General Surgery, Gansu Provincial Hospital, Lanzhou, Gansu 710000 (China)] [Department of General Surgery, Gansu Provincial Hospital, Lanzhou, Gansu 710000 (China); Zheng, Tim M. [Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104 (United States)] [Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104 (United States); DeMichele, Angela [Division of Hematology and Oncology, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104 (United States)] [Division of Hematology and Oncology, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104 (United States); Zhang, Lin, E-mail: linzhang@mail.med.upenn.edu [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States)] [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States)

2013-02-15

211

Cross-Sectional Relations of Multiple Biomarkers From Distinct Biological Pathways to Brachial Artery Endothelial Function  

Microsoft Academic Search

Background—Endothelial dysfunction is a critical intermediate phenotype in the pathogenesis of cardiovascular disease. We evaluated the relative contributions of distinct biological pathways to interindividual variation in endothelial function by relating prototype biomarkers (representing these pathways) to brachial artery vasodilator function. Methods and Results—We investigated the cross-sectional relations of a panel of 7 biomarkers measured at a routine examination to brachial

Sekar Kathiresan; Philimon Gona; Martin G. Larson; Joseph A. Vita; Gary F. Mitchell; Geoffrey H. Tofler; Daniel Levy; Christopher Newton-Cheh; Thomas J. Wang; Emelia J. Benjamin; Ramachandran S. Vasan

2010-01-01

212

The RdDM pathway is required for basal heat tolerance in Arabidopsis.  

PubMed

Heat stress affects epigenetic gene silencing in Arabidopsis. To test for a mechanistic involvement of epigenetic regulation in heat-stress responses, we analyzed the heat tolerance of mutants defective in DNA methylation, histone modifications, chromatin-remodeling, or siRNA-based silencing pathways. Plants deficient in NRPD2, the common second-largest subunit of RNA polymerases IV and V, and in the Rpd3-type histone deacetylase HDA6 were hypersensitive to heat exposure. Microarray analysis demonstrated that NRPD2 and HDA6 have independent roles in transcriptional reprogramming in response to temperature stress. The misexpression of protein-coding genes in nrpd2 mutants recovering from heat correlated with defective epigenetic regulation of adjacent transposon remnants which involved the loss of control of heat-stress-induced read-through transcription. We provide evidence that the transcriptional response to temperature stress, at least partially, relies on the integrity of the RNA-dependent DNA methylation pathway. PMID:23376771

Popova, Olga V; Dinh, Huy Q; Aufsatz, Werner; Jonak, Claudia

2013-03-01

213

The RdDM Pathway Is Required for Basal Heat Tolerance in Arabidopsis  

PubMed Central

Heat stress affects epigenetic gene silencing in Arabidopsis. To test for a mechanistic involvement of epigenetic regulation in heat-stress responses, we analyzed the heat tolerance of mutants defective in DNA methylation, histone modifications, chromatin-remodeling, or siRNA-based silencing pathways. Plants deficient in NRPD2, the common second-largest subunit of RNA polymerases IV and V, and in the Rpd3-type histone deacetylase HDA6 were hypersensitive to heat exposure. Microarray analysis demonstrated that NRPD2 and HDA6 have independent roles in transcriptional reprogramming in response to temperature stress. The misexpression of protein-coding genes in nrpd2 mutants recovering from heat correlated with defective epigenetic regulation of adjacent transposon remnants which involved the loss of control of heat-stress-induced read-through transcription. We provide evidence that the transcriptional response to temperature stress, at least partially, relies on the integrity of the RNA-dependent DNA methylation pathway.

Jonak, Claudia

2013-01-01

214

Modular control of multiple pathways using engineered orthogonal T7 polymerases.  

PubMed

Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a 'controller' plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure. PMID:22743271

Temme, Karsten; Hill, Rena; Segall-Shapiro, Thomas H; Moser, Felix; Voigt, Christopher A

2012-09-01

215

Modular control of multiple pathways using engineered orthogonal T7 polymerases  

PubMed Central

Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a ‘controller’ plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure.

Temme, Karsten; Hill, Rena; Segall-Shapiro, Thomas H.; Moser, Felix; Voigt, Christopher A.

2012-01-01

216

ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway  

Microsoft Academic Search

We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive ?360 kDa protein\\/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active

Antti Nykänen; Benjamin Haley; Phillip D. Zamore

2001-01-01

217

Autographa californica multiple nucleopolyhedrovirus EXON0 (ORF141) is required for efficient egress of nucleocapsids from the nucleus.  

PubMed

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) exon0 (orf141) has been shown to be required for the efficient production of budded virus (BV). The deletion of exon0 reduces the level of BV production by up to 99% (X. Dai, T. M. Stewart, J. A. Pathakamuri, Q. Li, and D. A. Theilmann, J. Virol. 78:9633-9644, 2004); however, the function or mechanism by which EXON0 affects BV production is unknown. In this study, we further elucidated the function of EXON0 by investigating the localization of EXON0 in infected Sf9 cells and in virions and by identifying interactions between EXON0 and other viral proteins. In addition, electron microscopy was used to study the cellular localization of nucleocapsids in cells transfected with an exon0 knockout (KO) virus. The results showed that EXON0 was localized to both the cytoplasm and the nuclei of infected Sf9 cells throughout the infection. Western blotting results also showed that EXON0 was purified along with BV and occlusion-derived virus (ODV). The fractionation of BV into the nucleocapsid and envelope components showed that EXON0 localized to the BV nucleocapsid. Yeast two-hybrid screening, coimmunoprecipitation, and confocal microscopy revealed that it interacted with nucleocapsid proteins FP25 and BV/ODV-C42. Cells transfected with the exon0 KO virus exhibited normally appearing nucleocapsids in the nuclei in numbers equal to those in the nuclei of cells transfected with the EXON0 repaired virus. In contrast, the numbers of nucleocapsids in the cytoplasm of cells transfected with the exon0 KO virus were significantly lower than those in the cytoplasm of cells transfected with the repaired virus. These results support the conclusion that EXON0 is required in the BV pathway for the efficient egress of nucleocapsids from the nucleus to the cytoplasm. PMID:17626083

Fang, Minggang; Dai, Xiaojiang; Theilmann, David A

2007-09-01

218

The SUMO Pathway Is Developmentally Regulated and Required for Programmed DNA Elimination in Paramecium tetraurelia† ‡  

PubMed Central

Extensive genome-wide remodeling occurs during the formation of the somatic macronuclei from the germ line micronuclei in ciliated protozoa. This process is limited to sexual reproduction and includes DNA amplification, chromosome fragmentation, and the elimination of internal segments of DNA. Our efforts to define the pathways regulating these events revealed a gene encoding a homologue of ubiquitin activating enzyme 2 (UBA2) that is upregulated at the onset of macronuclear development in Paramecium tetraurelia. Uba2 enzymes are known to activate the protein called small ubiquitin-related modifier (SUMO) that is covalently attached to target proteins. Consistent with this relationship, Northern analysis showed increased abundance of SUMO transcripts during sexual reproduction in Paramecium. RNA interference (RNAi) against UBA2 or SUMO during vegetative growth had little effect on cell survival or fission rates. In contrast, RNAi of mating cells resulted in failure to form a functional macronucleus. Despite normal amplification of the genome, excision of internal eliminated sequences was completely blocked. Additional experiments showed that the homologous UBA2 and SUMO genes in Tetrahymena thermophila are also upregulated during conjugation. These results provide evidence for the developmental regulation of the SUMO pathway in ciliates and suggest a key role for the pathway in controlling genome remodeling.

Matsuda, Atsushi; Forney, James D.

2006-01-01

219

Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression.  

PubMed Central

Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen-activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti-SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable.

Bennett, A M; Hausdorff, S F; O'Reilly, A M; Freeman, R M; Neel, B G

1996-01-01

220

Vid30 is required for the association of Vid vesicles and actin patches in the vacuole import and degradation pathway  

PubMed Central

When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are induced. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. Recent evidence suggests that the Vid pathway merges with the endocytic pathway at actin patches where endocytic vesicles are formed. The convergence of the Vid pathway with the endocytic pathway allows cells to remove intracellular and extracellular proteins simultaneously. However, the genes that regulate this step of the convergence have not been identified previously. Here we show that VID30 plays a critical role for the association of Vid vesicles and actin patches. Vid30 is constitutively expressed and interacts with Vid vesicle proteins Vid24 and Sec28 but not with the cargo protein FBPase. In the absence of SEC28 or VID24, Vid30 association with actin patches was prolonged. In cells lacking the VID30 gene, FBPase and Vid24 were not localized to actin patches, suggesting that Vid30 has a role in the association of Vid vesicles and actin patches. Vid30 contains a LisH and a CTLH domain, both of which are required for FBPase degradation. When these domains were deleted, FBPase trafficking to the vacuole was impaired. We suggest that Vid30 also has a role in the Vid pathway at a later step in a process that is mediated by the LisH and CTLH domains.

Alibhoy, Abbas A; Giardina, Bennett J; Dunton, Danielle D

2012-01-01

221

mediator of paramutation1 Is Required for Establishment and Maintenance of Paramutation at Multiple Maize Loci  

PubMed Central

Paramutation is the directed, heritable alteration of the expression of one allele when heterozygous with another allele. Here, the isolation and characterization of a mutation affecting paramutation, mediator of paramutation1-1 (mop1-1), are described. Experiments demonstrate that the wild-type gene Mop1 is required for establishment and maintenance of the paramutant state. The mop1-1 mutation affects paramutation at the multiple loci tested but has no effect on alleles that do not participate in paramutation. The mutation does not alter the amounts of actin and ubiquitin transcripts, which suggests that the mop1 gene does not encode a global repressor. Maize plants homozygous for mop1-1 can have pleiotropic developmental defects, suggesting that mop1-1 may affect more genes than just the known paramutant ones. The mop1-1 mutation does not alter the extent of DNA methylation in rDNA and centromeric repeats. The observation that mop1 affects paramutation at multiple loci, despite major differences between these loci in their gene structure, correlations with DNA methylation, and stability of the paramutant state, suggests that a common mechanism underlies paramutation. A protein-based epigenetic model for paramutation is discussed.

Dorweiler, Jane E.; Carey, Charles C.; Kubo, Kenneth M.; Hollick, Jay B.; Kermicle, Jerry L.; Chandler, Vicki L.

2000-01-01

222

Wolfberry Water Soluble Phytochemicals Down-Regulate ER Stress Biomarkers and Modulate Multiple Signaling Pathways Leading To Inhibition of Proliferation and Induction of Apoptosis in Jurkat Cells  

PubMed Central

Phytochemicals have received much recent attention in cancer prevention through simultaneous targeting multiple pathways in the disease progression. Here we determined that wolfberry phytochemicals was chemopreventive on the leukemic Jurkat cell. The water soluble wolfberry fractions (i.e., wolfberry phytochemicals) were enriched in carbohydrates (73.4 ± 4.5 % (w/w)), polyphenolics (1555 ± 112 mg quercetin equivalent/100 g freeze dry powder, including 213 mg rutin/100 g freeze dry powder), and had enhanced antioxidant activity (7771 ± 207 ?M Trolox equivalent/100 g freeze dry powder). Wolfberry phytochemicals, but not purified wolfberry polysaccharide fractions, inhibited Jurkat cell proliferation, induced cycle arrest at the G2/M phase in a dose dependent manner starting at 1 mg/ml for 48 h. Wolfberry phytochemicals eliminated cellular reactive oxygen species, declined expression of endoplasmic reticulum (ER) stress biomarkers, including glucose regulated protein 78, inositol-requiring protein 1(IRE1), activating transcription factor 6 (ATF6), protein kinase RNA-like ER kinase (PERK), and c/EBP-homologous protein, and induced activation of AMP activated protein kinase, stabilization of ?-catenin, and inhibition of NF?B, and AKT activity. Simultaneous siRNA knockdown of ATF6, IRE1 and PERK caused inhibition of cell proliferation and induction of apoptosis. Data suggested that ER stress and multiple survival/apoptosis signaling pathways were modulated by wolfberry phytochemicals during the apoptotic progression. Consumption of wolfberry could be an efficacious dietary strategy for preventing leukemia.

Jiang, Yu; Zhang, Yunong; Wark, Logan; Ortiz, Edlin; Lim, Soyoung; He, Hui; Wang, Weiqun; Medeiros, Denis; Lin, Dingbo

2012-01-01

223

Identification of Shared Genes and Pathways: A Comparative Study of Multiple Sclerosis Susceptibility, Severity and Response to Interferon Beta Treatment  

PubMed Central

Recent genome-wide association studies (GWAS) have successfully identified several gene loci associated with multiple sclerosis (MS) susceptibility, severity or interferon-beta (IFN-ß) response. However, due to the nature of these studies, the functional relevance of these loci is not yet fully understood. We have utilized a systems biology based approach to explore the genetic interactomes of these MS related traits. We hypothesised that genes and pathways associated with the 3 MS related phenotypes might interact collectively to influence the heterogeneity and unpredictable clinical outcomes observed. Individual genetic interactomes for each trait were constructed and compared, followed by prioritization of common interactors based on their frequencies. Pathway enrichment analyses were performed to highlight shared functional pathways. Biologically relevant genes ABL1, GRB2, INPP5D, KIF1B, PIK3R1, PLCG1, PRKCD, SRC, TUBA1A and TUBA4A were identified as common to all 3 MS phenotypes. We observed that the highest number of first degree interactors were shared between MS susceptibility and MS severity (p?=?1.34×10?79) with UBC as the most prominent first degree interactor for this phenotype pair from the prioritisation analysis. As expected, pairwise comparisons showed that MS susceptibility and severity interactomes shared the highest number of pathways. Pathways from signalling molecules and interaction, and signal transduction categories were found to be highest shared pathways between 3 phenotypes. Finally, FYN was the most common first degree interactor in the MS drugs-gene network. By applying the systems biology based approach, additional significant information can be extracted from GWAS. Results of our interactome analyses are complementary to what is already known in the literature and also highlight some novel interactions which await further experimental validation. Overall, this study illustrates the potential of using a systems biology based approach in an attempt to unravel the biological significance of gene loci identified in large GWAS.

Mahurkar, Sunil; Moldovan, Max; Suppiah, Vijayaprakash; O'Doherty, Catherine

2013-01-01

224

Multiple Sequence-Specific DNA-Binding Proteins Mediate Estrogen Receptor Signaling through a Tethering Pathway  

PubMed Central

The indirect recruitment (tethering) of estrogen receptors (ERs) to DNA through other DNA-bound transcription factors (e.g. activator protein 1) is an important component of estrogen-signaling pathways, but our understanding of the mechanisms of ligand-dependent activation in this pathway is limited. Using proteomic, genomic, and gene-specific analyses, we demonstrate that a large repertoire of DNA-binding transcription factors contribute to estrogen signaling through the tethering pathway. In addition, we define a set of endogenous genes for which ER? tethering through activator protein 1 (e.g. c-Fos) and cAMP response element-binding protein family members mediates estrogen responsiveness. Finally, we show that functional interplay between c-Fos and cAMP response element-binding protein 1 contributes to estrogen-dependent regulation through the tethering pathway. Based on our results, we conclude that ER? recruitment in the tethering pathway is dependent on the ligand-induced formation of transcription factor complexes that involves interplay between the transcription factors from different protein families.

Heldring, Nina; Isaacs, Gary D.; Diehl, Adam G.; Sun, Miao; Cheung, Edwin; Ranish, Jeffrey A.

2011-01-01

225

AtGSNOR1 function is required for multiple developmental programs in Arabidopsis.  

PubMed

Nitric oxide (NO) has been proposed to regulate a diverse array of activities during plant growth, development and immune function. S-nitrosylation, the addition of an NO moiety to a reactive cysteine thiol, to form an S-nitrosothiol (SNO), is emerging as a prototypic redox-based post-translational modification. An ARABIDOPSIS THALIANA S-NITROSOGLUTATHIONE (GSNO) REDUCTASE (AtGSNOR1) is thought to be the major regulator of total cellular SNO levels in this plant species. Here, we report on the impact of loss- and gain-of-function mutations in AtGSNOR1 upon plant growth and development. Loss of AtGSNOR1 function in atgsnor1-3 plants increased the number of initiated higher order axillary shoots that remain active, resulting in a loss of apical dominance relative to wild type. In addition atgsnor1-3 affected leaf shape, germination, 2,4-D sensitivity and reduced hypocotyl elongation in both light and dark grown seedlings. Silique size and seed production were also decreased in atgsnor1-3 plants and the latter was reduced in atgsnor1-1 plants, which overexpress AtGSNOR1. Overexpression of AtGSNOR1 slightly delayed flowering time in both long and short days, whereas atgsnor1-3 showed early flowering compared to wild type. In the atgsnor1-3 line, FLOWERING LOCUS C (FLC) expression was reduced, whereas transcription of CONSTANS (CO) was enhanced. Therefore, AtGSNOR1 may negatively regulate the autonomous and photoperiod flowering time pathways. Both overexpression and loss of AtGSNOR1 function also reduced primary root growth, while root hair development was increased in atgsnor1-1 and reduced in atgsnor1-3 plants. Collectively, our findings imply that AtGSNOR1 controls multiple genetic networks integral to plant growth and development. PMID:22767201

Kwon, Eunjung; Feechan, Angela; Yun, Byung-Wook; Hwang, Byung-Ho; Pallas, Jacqueline A; Kang, Jeong-Gu; Loake, Gary J

2012-09-01

226

Dissection of Biological Property of Chinese Acupuncture Point Zusanli Based on Long-Term Treatment via Modulating Multiple Metabolic Pathways  

PubMed Central

Acupuncture has a history of over 3000 years and is a traditional Chinese medical therapy that uses hair-thin metal needles to puncture the skin at specific points on the body to promote wellbeing, while its molecular mechanism and ideal biological pathways are still not clear. High-throughput metabolomics is the global assessment of endogenous metabolites within a biologic system and can potentially provide a more accurate snap shot of the actual physiological state. We hypothesize that acupuncture-treated human would produce unique characterization of metabolic phenotypes. In this study, UPLC/ESI-HDMS coupled with pattern recognition methods and system analysis were carried out to investigate the mechanism and metabolite biomarkers for acupuncture treatment at “Zusanli” acupoint (ST-36) as a case study. The top 5 canonical pathways including alpha-linolenic acid metabolism, d-glutamine and d-glutamate metabolism, citrate cycle, alanine, aspartate, and glutamate metabolism, and vitamin B6 metabolism pathways were acutely perturbed, and 53 differential metabolites were identified by chemical profiling and may be useful to clarify the physiological basis and mechanism of ST-36. More importantly, network construction has led to the integration of metabolites associated with the multiple perturbation pathways. Urine metabolic profiling might be a promising method to investigate the molecular mechanism of acupuncture.

Yan, Guangli; Zhang, Aihua; Sun, Hui; Cheng, Weiping; Meng, Xiangcai; Liu, Li; Zhang, Yingzhi; Xie, Ning; Wang, Xijun

2013-01-01

227

Combined biosynthetic pathway for de novo production of UDP-galactose: catalysis with multiple enzymes immobilized on agarose beads.  

PubMed

Regeneration of sugar nucleotides is a critical step in the biosynthetic pathway for the formation of oligosaccharides. To alleviate the difficulties in the production of sugar nucleotides, we have developed a method to produce uridine diphosphate galactose (UDP-galactose). The combined biosynthetic pathway, which involves seven enzymes, is composed of three parts: i) the main pathway to form UDP-galactose from galactose, with the enzymes galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophosphorylase, and inorganic pyrophosphatase, ii) the uridine triphosphate supply pathway catalyzed by uridine monophosphate (UMP) kinase and nucleotide diphosphate kinase, and iii) the adenosine triphosphate (ATP) regeneration pathway catalyzed by polyphosphate kinase with polyphosphate added as an energy resource. All of the enzymes were expressed individually and immobilized through their hexahistidine tags onto nickel agarose beads ("super beads"). The reaction requires a stoichiometric amount of UMP and galactose, and catalytic amounts of ATP and glucose 1-phosphate, all inexpensive starting materials. After continuous circulation of the reaction mixture through the super-bead column for 48 h, 50 % of the UMP was converted into UDP-galactose. The results show that de novo production of UDP-galactose on the super-bead column is more efficient than in solution because of the stability of the immobilized enzymes. PMID:11933236

Liu, Ziye; Zhang, Jianbo; Chen, Xi; Wang, Peng G

2002-04-01

228

Requirement of IS911 replication before integration defines a new bacterial transposition pathway  

PubMed Central

Movement of transposable elements is often accompanied by replication to ensure their proliferation. Replication is associated with both major classes of transposition mechanisms: cut-and-paste and cointegrate formation (paste-and-copy). Cut-and-paste transposition is often activated by replication of the transposon, while in cointegrate formation replication completes integration. We describe a novel transposition mechanism used by insertion sequence IS911, which we call copy-and-paste. IS911 transposes using a circular intermediate (circle), which then integrates into a target. We demonstrate that this is derived from a branched intermediate (figure-eight) in which both ends are joined by a single-strand bridge after a first-strand transfer. In vivo labelling experiments show that the process of circle formation is replicative. The results indicate that the replication pathway not only produces circles from figure-eight but also regenerates the transposon donor plasmid. To confirm the replicative mechanism, we have also used the Escherichia coli terminators (terC) which, when bound by the Tus protein, inhibit replication forks in a polarised manner. Finally, we demonstrate that the primase DnaG is essential, implicating a host-specific replication pathway.

Duval-Valentin, G; Marty-Cointin, B; Chandler, M

2004-01-01

229

Requirement of IS911 replication before integration defines a new bacterial transposition pathway.  

PubMed

Movement of transposable elements is often accompanied by replication to ensure their proliferation. Replication is associated with both major classes of transposition mechanisms: cut-and-paste and cointegrate formation (paste-and-copy). Cut-and-paste transposition is often activated by replication of the transposon, while in cointegrate formation replication completes integration. We describe a novel transposition mechanism used by insertion sequence IS911, which we call copy-and-paste. IS911 transposes using a circular intermediate (circle), which then integrates into a target. We demonstrate that this is derived from a branched intermediate (figure-eight) in which both ends are joined by a single-strand bridge after a first-strand transfer. In vivo labelling experiments show that the process of circle formation is replicative. The results indicate that the replication pathway not only produces circles from figure-eight but also regenerates the transposon donor plasmid. To confirm the replicative mechanism, we have also used the Escherichia coli terminators (terC) which, when bound by the Tus protein, inhibit replication forks in a polarised manner. Finally, we demonstrate that the primase DnaG is essential, implicating a host-specific replication pathway. PMID:15359283

Duval-Valentin, G; Marty-Cointin, B; Chandler, M

2004-10-01

230

Single-target RNA interference for the blockade of multiple interacting proinflammatory and profibrotic pathways in cardiac fibroblasts.  

PubMed

Therapeutic targets of broad relevance are likely located in pathogenic pathways common to disorders of various etiologies. Screening for targets of this type revealed CCN genes to be consistently upregulated in multiple cardiomyopathies. We developed RNA interference (RNAi) to silence CCN2 and found this single-target approach to block multiple proinflammatory and profibrotic pathways in activated primary cardiac fibroblasts (PCFBs). The RNAi-strategy was developed in murine PCFBs and then investigated in "individual" human PCFBs grown from human endomyocardial biopsies (EMBs). Screening of short hairpin RNA (shRNA) sequences for high silencing efficacy and specificity yielded RNAi adenovectors silencing CCN2 in murine or human PCFBs, respectively. Comparison of RNAi with CCN2-modulating microRNA (miR) vectors expressing miR-30c or miR-133b showed higher efficacy of RNAi. In murine PCFBs, CCN2 silencing resulted in strongly reduced expression of stretch-induced chemokines (Ccl2, Ccl7, Ccl8), matrix metalloproteinases (MMP2, MMP9), extracellular matrix (Col3a1), and a cell-to-cell contact protein (Cx43), suggesting multiple signal pathways to be linked to CCN2. Immune cell chemotaxis towards CCN2-depleted PCFBs was significantly reduced. We demonstrate here that this RNAi strategy is technically applicable to "individual" human PCFBs, too, but that these display individually strikingly different responses to CCN2 depletion. Either genomically encoded factors or stable epigenetic modification may explain different responses between individual PCFBs. The new RNAi approach addresses a key regulator protein induced in cardiomyopathies. Investigation of this and other molecular therapies in individual human PCBFs may help to dissect differential pathogenic processes between otherwise similar disease entities and individuals. PMID:24239602

Tank, Juliane; Lindner, Diana; Wang, Xiaomin; Stroux, Andrea; Gilke, Leona; Gast, Martina; Zietsch, Christin; Skurk, Carsten; Scheibenbogen, Carmen; Klingel, Karin; Lassner, Dirk; Kühl, Uwe; Schultheiss, Heinz-Peter; Westermann, Dirk; Poller, Wolfgang

2014-01-01

231

Synergistic Heterozygosity: Disease Resulting from Multiple Partial Defects in One or More Metabolic Pathways  

Microsoft Academic Search

Inborn errors of metabolism show considerable variation in the severity of symptoms. This is often ascribed to the differential effects of specific mutations on gene\\/enzyme function; however, such genotype\\/phenotype correlations are usually imprecise. In addition, in some patients with clinical and biochemical findings consistent with a defect in a particular metabolic pathway, it is ultimately impossible to arrive at a

Jerry Vockley; Piero Rinaldo; Michael J. Bennett; Dietrich Matern; Georgirene D. Vladutiu

2000-01-01

232

Multiple phytohormone signalling pathways modulate susceptibility of tomato plants to Alternaria alternata f. sp. lycopersici  

PubMed Central

Three phytohormone molecules – ethylene (ET), jasmonic acid (JA) and salicylic acid (SA) – play key roles in mediating disease response to necrotrophic fungal pathogens. This study investigated the roles of the ET, JA, and SA pathways as well as their crosstalk during the interaction between tomato (Solanum lycopersicum) plants and a necrotrophic fungal pathogen Alternaria alternata f. sp. lycopersici (AAL). Both the ET and JASMONIC ACID INSENSITIVE1 (JAI1) receptor-dependent JA signalling pathways are necessary for susceptibility, while SA response promotes resistance to AAL infection. In addition, the role of JA in susceptibility to AAL is partly dependent on ET biosynthesis and perception, while the SA pathway enhances resistance to AAL and antagonizes the ET response. Based on these results, it is proposed that ET, JA, and SA each on their own can influence the susceptibility of tomato to AAL. Furthermore, the functions of JA and SA in susceptibility to the pathogen are correlated with the enhanced or decreased action of ET, respectively. This study has revealed the functional relationship among the three key hormone pathways in tomato defence against AAL.

Jia, Chengguo; Zhang, Liping; Wang, Qiaomei

2013-01-01

233

Multiple Signaling Pathways Converge to Regulate Bone-Morphogenetic-Protein-Dependent Glial Gene Expression  

Microsoft Academic Search

A fundamental problem in developmental neuroscience is understanding how extracellular cues link to complex intracellular signaling pathways to drive stage-specific developmental decisions. During the formation of the mammalian peripheral nervous system, bone morphogenetic proteins (BMPs) promote neuronal differentiation. BMPs also maintain the expression of early glial genes such as GFAP, while blocking the acquisition of a mature, myelinating Schwann cell

Justin J. Dore; John C. DeWitt; Nithya Setty; Mareshia D. Donald; Esther Joo; Melissa A. Chesarone; Susan J. Birren

2009-01-01

234

ups1 ,a nArabidopsis thaliana camalexin accumulation mutant defective in multiple defence signalling pathways  

Microsoft Academic Search

Summary We report the characterization of an Arabidopsis thaliana mutant, ups1, isolated on the basis of reduced expression of phosphoribosylanthranilate transferase, a tryptophan biosynthetic enzyme. ups1 also exhibits defects in a wide range of defence responses. After infection with Pseudomonas syringae or Botrytis cinerea, the expression of genes regulated by both the salicylic acid and jasmonic acid\\/ethylene pathways is reduced

Katherine J. Denby; Laure J. M. Jason; Shane L. Murray; Robert L. Last

235

Targeting Multiple Signaling Pathways by Green Tea Polyphenol ()-Epigallocatechin-3Gallate  

Microsoft Academic Search

Cell signaling pathways, responsible for maintaining a balance between cell proliferation and death, have emerged as rational targets for the management of cancer. Emerging data amassed from various laboratories around the world suggests that green tea, particularly its major polyphenolic constituent ()-epigallocatechin-3-gallate (EGCG), possesses remarkable cancer chemopreventive and therapeutic poten- tial against various cancer sites in animal tumor bioassay systems

Naghma Khan; Farrukh Afaq; Mohammad Saleem; Nihal Ahmad; Hasan Mukhtar

236

Multiple Smaller Missions as a Direct Pathway to Mars Sample Return.  

National Technical Information Service (NTIS)

Recent discoveries by the Mars Exploration Rovers, Mars Express, Mars Odyssey, and Mars Reconnaissance Orbiter spacecraft include multiple, tantalizing astrobiological targets representing both past and present environments on Mars. The most desirable pat...

A. M. Baldridge A. T. Knudson C. A. Evans C. H. Seaman C. M. Weitz D. Ming D. Vaniman D. A. Crown D. S. Draper E. K. Gibson E. N. Dobrea J. Andrews-Hanna J. Michalski J. F. Bell J. H. Jones L. D. Graham L. P. Knauth M. Fries M. C. Bourke P. B. Niles P. D. Archer R. M. E. Williams S. M. Lederer

2012-01-01

237

Gene expression profiles from discordant monozygotic twins suggest that molecular pathways are shared among multiple systemic autoimmune diseases  

Microsoft Academic Search

Introduction  The objective of this study is to determine if multiple systemic autoimmune diseases (SAID) share gene expression pathways\\u000a that could provide insights into pathogenic mechanisms common to these disorders.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  RNA microarray analyses (Agilent Human 1A(V2) 20K oligo arrays) were used to quantify gene expression in peripheral blood\\u000a cells from 20 monozygotic (MZ) twin pairs discordant for SAID. Six affected probands

Terrance P O’Hanlon; Lisa G Rider; Lu Gan; Rick Fannin; Richard S Paules; David M Umbach; Clarice R Weinberg; Ruchir R Shah; Deepak Mav; Mark F Gourley; Frederick W Miller

2011-01-01

238

Elevating calcium in Th2 cells activates multiple pathways to induce IL-4 transcription and mRNA stabilization  

PubMed Central

PMA and ionomycin cause T cell cytokine production. We report that ionomycin alone induces IL-4 and IFN?, but not IL-2, from in vivo and in vitro generated murine Th2 and Th1 cells. Ionomycin-induced cytokine production requires nuclear factor of activated T cells (NFAT), p38; and calmodulin-dependent kinase IV (CaMKIV). Ionomycin induces p38 phosphorylation through a calcium-dependent, cyclosporine A-inhibitable pathway. “Knocking-down” apoptosis signal-regulating kinase 1 (ASK1) inhibits ionomycin-induced p38 phosphorylation and IL-4 production. Ionomycin also activates CaMKIV, which, together with p38, induces AP-1. Cooperation between AP-1 and NFAT leads to Il4 gene transcription. p38 also regulates IL-4 production by mRNA stabilization. T cell receptor stimulation also phosphorylates p38, partially through the calcium-dependent pathway; activated p38 is required for optimal IL-4 and IFN?.

Guo, Liying; Urban, Joseph F.; Zhu, Jinfang; Paul, William E.

2008-01-01

239

Multiplication  

NSDL National Science Digital Library

How sharp are your multiplication skills? Give these great math games a try ! Play Asteroids blaster and test your multiplication skills. How fast can you solve the problem... play a round of Baseball multiplication and see! Multiplication is fun and delicious with Crazy Cones. Help Lemonade Larry determine the correct amount! Test your multiplication skills with Tic Tac Toe! ...

Ms.roberts

2009-02-24

240

Secretion of the chlamydial virulence factor CPAF requires the Sec-dependent pathway  

PubMed Central

The chlamydial protease/proteasome-like activity factor (CPAF) is secreted into the host cytosol to degrade various host factors that benefit chlamydial intracellular survival. Although the full-length CPAF is predicted to contain a putative signal peptide at its N terminus, the secretion pathway of CPAF is still unknown. Here, we have provided experimental evidence that the N-terminal sequence covering the M1–G31 region was cleaved from CPAF during chlamydial infection. The CPAF N-terminal sequence, when expressed in a phoA gene fusion construct, was able to direct the export of the mature PhoA protein across the inner membrane of wild-type Escherichia coli. However, E. coli mutants deficient in SecB failed to support the CPAF signal-peptide-directed secretion of PhoA. Since native PhoA secretion was known to be independent of SecB, this SecB dependence must be rendered by the CPAF leader peptide. Furthermore, lack of SecY function also blocked the CPAF signal-peptide-directed secretion of PhoA. Most importantly, CPAF secretion into the host cell cytosol during chlamydial infection was selectively inhibited by an inhibitor specifically targeting type I signal peptidase but not by a type III secretion-system-specific inhibitor. Together, these observations have demonstrated that the chlamydial virulence factor CPAF relies on Sec-dependent transport for crossing the chlamydial inner membrane, which has provided essential information for further delineating the pathways of CPAF action and understanding chlamydial pathogenic mechanisms.

Chen, Ding; Lei, Lei; Lu, Chunxue; Flores, Rhonda; DeLisa, Matthew P.; Roberts, Tucker C.; Romesberg, Floyd E.; Zhong, Guangming

2010-01-01

241

Exome Capture Sequencing of Adenoma Reveals Genetic Alterations in Multiple Cellular Pathways at the Early Stage of Colorectal Tumorigenesis  

PubMed Central

Most of colorectal adenocarcinomas are believed to arise from adenomas, which are premalignant lesions. Sequencing the whole exome of the adenoma will help identifying molecular biomarkers that can predict the occurrence of adenocarcinoma more precisely and help understanding the molecular pathways underlying the initial stage of colorectal tumorigenesis. We performed the exome capture sequencing of the normal mucosa, adenoma and adenocarcinoma tissues from the same patient and sequenced the identified mutations in additional 73 adenomas and 288 adenocarcinomas. Somatic single nucleotide variations (SNVs) were identified in both the adenoma and adenocarcinoma by comparing with the normal control from the same patient. We identified 12 nonsynonymous somatic SNVs in the adenoma and 42 nonsynonymous somatic SNVs in the adenocarcinoma. Most of these mutations including OR6X1, SLC15A3, KRTHB4, RBFOX1, LAMA3, CDH20, BIRC6, NMBR, GLCCI1, EFR3A, and FTHL17 were newly reported in colorectal adenomas. Functional annotation of these mutated genes showed that multiple cellular pathways including Wnt, cell adhesion and ubiquitin mediated proteolysis pathways were altered genetically in the adenoma and that the genetic alterations in the same pathways persist in the adenocarcinoma. CDH20 and LAMA3 were mutated in the adenoma while NRXN3 and COL4A6 were mutated in the adenocarcinoma from the same patient, suggesting for the first time that genetic alterations in the cell adhesion pathway occur as early as in the adenoma. Thus, the comparison of genomic mutations between adenoma and adenocarcinoma provides us a new insight into the molecular events governing the early step of colorectal tumorigenesis.

Zheng, Liangtao; Ge, Weiting; Li, Dan; Zhang, Yong; Hu, Xueda; Gao, Zhibo; Xu, Jinghong; Huang, Yanqin; Hu, Hanguang; Zhang, Hang; Zhang, Hao; Liu, Mingming; Yang, Huanming; Zheng, Lei; Zheng, Shu

2013-01-01

242

Kinetochore genes are required to fully activate secretory pathway expansion in S. cerevisiae under induced ER stress.  

PubMed

Basal ER stress occurs when proteins misfold in normal physiological conditions and are corrected by the unfolded protein response (UPR). Elevated ER stress occurs when misfolding is refractory as found in numerous diseases such as atherosclerosis, Type II diabetes and some cancers. In elevated ER stress it is unclear whether cells utilise the same or different networks of genes as in basal levels of ER stress. To probe this question, we used secretory pathway reporters Yip3p-GFP, Erv29p-GFP, Orm2p-GFP and UPREpr-GFP placed on the yeast deletion mutant array (DMA) genetic background. The reporter's expression levels, measured by automated microscopy, at basal versus elevated ER stress induced by the over-expression of CPY* were compared. A novel group of kinetochore genes (CTF19 complex) were found to be uniquely required for full induction of all four ER stress reporters in elevated stress. A follow-up reporter screen was developed by mating the ctf19? kinetochore gene deletion strain into the genome-wide XXXp-GFP tagged library then testing with over-expressed CPY*. This screen identified Bcy1p and Bfr1p as possible signalling points that down-regulate the UPR and secretory pathway when kinetochore proteins are absent under elevated stress conditions. Bfr1p appears to be a checkpoint that monitors the integrity of kinetochores at increased levels of ER stress. This study concludes that functional kinetochores are required for full activation of the secretory pathway in elevated ER stress and that the responses to basal and elevated levels of ER stress require different networks of genes. PMID:24722431

Low, Yee S; Bircham, Peter W; Maass, David R; Atkinson, Paul H

2014-07-01

243

Activation of the MEK pathway is required for complete scattering of MCF7 cells stimulated with heregulin-?1.  

PubMed

Rac1 is important for dissociation of cells during scattering, but whether its activation alone is sufficient to induce complete scattering is not known. To test this, we created an inducible MCF7 cell line that expresses dominant active Rac1. Although induction of dominant active Rac1 resulted in dissociation of cells, their scattering was incomplete. We co-expressed dominant active MKK1a, an activator of ERK, and dominant active Rac1. In this case, cells completely scattered. These results suggest that not only Rac1 but also the MEK1 pathway is required for dissociation and complete scattering of MCF7 cells treated with HRG-?1. PMID:23510995

Chang, Yu-Tsu; Shu, Chung-Li; Fukui, Yasuhisa

2013-04-12

244

Fas Activation of the p38 Mitogen-Activated Protein Kinase Signalling Pathway Requires ICE\\/CED3 Family Proteases  

Microsoft Academic Search

p38andJNK,within2hinJurkatTlymphocytesbutnotthemitogen-responsivekinaseERK1orpp70 S6k .Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3(glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involve- ment of the MKK3\\/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1b-convertingenzyme)familyproteasestoinduceapoptosisfromstudiesutilizingthecowpoxICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the

PETER JUO; CALVIN J. KUO; SUSAN E. REYNOLDS; RICHARD F. KONZ; JOEL RAINGEAUD; ROGER J. DAVIS; HANS-PETER BIEMANN; ANDJOHN BLENIS

1997-01-01

245

Molecular Pathways Controlling Heart Development  

Microsoft Academic Search

Heart formation requires complex interactions among cells from multiple embryonic origins. Recent studies have begun to reveal the genetic pathways that control cardiac morphogenesis. Many of the genes within these pathways are conserved across vast phylogenetic distances, which has allowed cardiac development to be dissected in organisms ranging from flies to mammals. Studies of cardiac development have also revealed the

Eric N. Olson; Deepak Srivastava

1996-01-01

246

Bidirectional signaling between calcium channels of skeletal muscle requires multiple direct and indirect interactions  

PubMed Central

We have defined regions of the skeletal muscle ryanodine receptor (RyR1) essential for bidirectional signaling with dihydropyridine receptors (DHPRs) and for the organization of DHPR into tetrad arrays by expressing RyR1–RyR3 chimerae in dyspedic myotubes. RyR1–RyR3 constructs bearing RyR1 residues 1–1681 restored wild-type DHPR tetrad arrays and, in part, skeletal-type excitation–contraction (EC) coupling (orthograde signaling) but failed to enhance DHPR Ca2+ currents (retrograde signaling) to WT RyR1 levels. Within this region, the D2 domain (amino acids 1272–1455), although ineffective on its own, dramatically enhanced the formation of tetrads and EC coupling rescue by constructs that otherwise are only partially effective. These findings suggest that the orthograde signal and DHPR tetrad formation require the contributions of numerous RyR regions. Surprisingly, we found that RyR3, although incapable of supporting EC coupling or tetrad formation, restored a significant level of Ca2+ current, revealing a functional interaction with the skeletal muscle DHPR. Thus, our data support the hypotheses that (i) the structural/functional link between RyR1 and the skeletal muscle DHPR requires multiple interacting regions, (ii) the D2 domain of RyR1 plays a key role in stabilizing this interaction, and (iii) a form of retrograde signaling from RyR3 to the DHPR occurs in the absence of direct protein–protein interactions.

Sheridan, David C.; Takekura, Hiroaki; Franzini-Armstrong, Clara; Beam, Kurt G.; Allen, Paul D.; Perez, Claudio F.

2006-01-01

247

Multiple signaling pathways control nitrogen-mediated root elongation in maize.  

PubMed

Response of root system architecture to nutrient availability is an essential way for plants to adapt to soil environments. Nitrogen can affect root development either as a result of changes in the external concentration, or through changes in the internal nutrient status of the plant. Low soil N stimulates root elongation in maize. Recent evidence suggests that plant hormones auxin and cytokinin, as well as NO signaling pathway, are involved in the regulation of root elongation by low nitrogen nutrition. PMID:19704443

Mi, Guohua; Chen, Fanjun; Zhang, Fusuo

2008-11-01

248

PEP-1-CAT protects hypoxia/reoxygenation-induced cardiomyocyte apoptosis through multiple sigaling pathways  

PubMed Central

Background Catalase (CAT) breaks down H2O2 into H2O and O2 to protects cells from oxidative damage. However, its translational potential is limited because exogenous CAT cannot enter living cells automatically. This study is aimed to investigate if PEP-1-CAT fusion protein can effectively protect cardiomyocytes from oxidative stress due to hypoxia/reoxygenation (H/R)-induced injury. Methods H9c2 cardomyocytes were pretreated with catalase (CAT) or PEP-1-CAT fusion protein followed by culturing in a hypoxia and re-oxygenation condition. Cell apoptosis were measured by Annexin V and PI double staining and Flow cytometry. Intracellular superoxide anion level was determined, and mitochondrial membrane potential was measured. Expression of apoptosis-related proteins including Bcl-2, Bax, Caspase-3, PARP, p38 and phospho-p38 was analyzed by western blotting. Results PEP-1-CAT protected H9c2 from H/R-induced morphological alteration and reduced the release of lactate dehydrogenase (LDH) and malondialdehyde content. Superoxide anion production was also decreased. In addition, PEP-1-CAT inhibited H9c2 apoptosis and blocked the expression of apoptosis stimulator Bax while increased the expression of Bcl-2, leading to an increased mitochondrial membrane potential. Mechanistically, PEP-1-CAT inhibited p38 MAPK while activating PI3K/Akt and Erk1/2 signaling pathways, resulting in blockade of Bcl2/Bax/mitochondrial apoptotic pathway. Conclusion Our study has revealed a novel mechanism by which PEP-1-CAT protects cardiomyocyte from H/R-induced injury. PEP-1-CAT blocks Bcl2/Bax/mitochondrial apoptotic pathway by inhibiting p38 MAPK while activating PI3K/Akt and Erk1/2 signaling pathways.

2013-01-01

249

Multispecific drug transporter Slc22a8 (Oat3) regulates multiple metabolic and signaling pathways.  

PubMed

Multispecific drug transporters of the solute carrier and ATP-binding cassette families are highly conserved through evolution, but their true physiologic role remains unclear. Analyses of the organic anion transporter 3 (OAT3; encoded by Slc22a8/Oat3, originally Roct) knockout mouse have confirmed its critical role in the renal handling of common drugs (e.g., antibiotics, antivirals, diuretics) and toxins. Previous targeted metabolomics of the knockout of the closely related Oat1 have demonstrated a central metabolic role, but the same approach with Oat3 failed to reveal a similar set of endogenous substrates. Nevertheless, the Oat3 knockout is the only Oat described so far with a physiologically significant phenotype, suggesting the disturbance of metabolic or signaling pathways. Here we analyzed global gene expression in Oat3 knockout tissue, which implicated OAT3 in phase I and phase II metabolism (drug metabolizing enzymes or DMEs), as well as signaling pathways. Metabolic reconstruction with the recently developed "mouse Recon1" supported the involvement of Oat3 in the aforementioned pathways. Untargeted metabolomics were used to determine whether the predicted metabolic alterations could be confirmed. Many significant changes were observed; several metabolites were tested for direct interaction with mOAT3, whereas others were supported by published data. Oat3 thus appears critical for the handling of phase I (hydroxylation) and phase II (glucuronidation) metabolites. Oat3 also plays a role in bioenergetic pathways (e.g., the tricarboxylic acid cycle), as well as those involving vitamins (e.g., folate), steroids, prostaglandins, gut microbiome products, uremic toxins, cyclic nucleotides, amino acids, glycans, and possibly hyaluronic acid. The data seemingly consistent with the Remote Sensing and Signaling Hypothesis (Ahn and Nigam, 2009; Wu et al., 2011), also suggests that Oat3 is essential for the handling of dietary flavonoids and antioxidants. PMID:23920220

Wu, Wei; Jamshidi, Neema; Eraly, Satish A; Liu, Henry C; Bush, Kevin T; Palsson, Bernhard O; Nigam, Sanjay K

2013-10-01

250

Human melanocytes expressing MC1R variant alleles show impaired activation of multiple signaling pathways  

Microsoft Academic Search

Variant alleles of the human MC1R gene are strongly associated with red hair color, fair skin and poor tanning ability (RHC-trait). Recently, we demonstrated that melanocytes harboring RHC-associated alleles have markedly reduced surface expression and\\/or impaired G-protein coupling of the corresponding receptor protein. The consequences of such a deficit on MC1R-mediated signaling pathways have now been quantitatively evaluated utilizing strains

Richard A. Newton; Donald W. Roberts; J. Helen Leonard; Richard A. Sturm

2007-01-01

251

The Glycolytic Inhibitor 2-Deoxyglucose Activates Multiple Prosurvival Pathways through IGF1R*  

PubMed Central

Recent molecular studies indicate that aerobic glycolysis plays an important role in tumorigenesis and is a valid target for cancer therapy. Although 2-deoxyglucose (2-DG) is well characterized as a glycolytic inhibitor, we recently discovered that it activates a prosurvival oncoprotein, AKT, through PI3K. In this study, we discovered that 2-DG treatments disrupted the binding between insulin-like growth factor 1 (IGF-1) and IGF-binding protein 3 (IGFBP3) so that the free form of IGF-1 could be released from the IGF-1·IGFBP3 complex to activate IGF-1 receptor (IGF1R) signaling. Because IGF1R signaling is involved, PI3K/AKT constitutes only one of the prosurvival pathways that are activated by 2-DG treatment; we validated that MEK-ERK signaling was also induced in an IGF1R-dependent manner in some cancer cell lines. Furthermore, our phospho-specific antibody microarray analysis indicated that 2-DG up-regulated the phosphorylation of 64 sites within various signaling pathways in H460 cells. Chemical inhibition of IGF1R reduced 57 of these up-regulations. These data suggest that 2-DG-induced activation of many survival pathways can be jointly attenuated through IGF1R inhibition. Our in vitro analysis demonstrated that treatment with a combination of subtoxic doses of 2-DG and the IGF1R inhibitor II reduced cancer cell proliferation 90% and promoted significant apoptosis.

Zhong, Diansheng; Xiong, Li; Liu, Tongrui; Liu, Xiuju; Liu, Xiangguo; Chen, Jing; Sun, Shi-Yong; Khuri, Fadlo R.; Zong, Yaping; Zhou, Qinghua; Zhou, Wei

2009-01-01

252

Multiple GAL pathway gene clusters evolved independently and by different mechanisms in fungi  

PubMed Central

A notable characteristic of fungal genomes is that genes involved in successive steps of a metabolic pathway are often physically linked or clustered. To investigate how such clusters of functionally related genes are assembled and maintained, we examined the evolution of gene sequences and order in the galactose utilization (GAL) pathway in whole-genome data from 80 diverse fungi. We found that GAL gene clusters originated independently and by different mechanisms in three unrelated yeast lineages. Specifically, the GAL cluster found in Saccharomyces and Candida yeasts originated through the relocation of native unclustered genes, whereas the GAL cluster of Schizosaccharomyces yeasts was acquired through horizontal gene transfer from a Candida yeast. In contrast, the GAL cluster of Cryptococcus yeasts was assembled independently from the Saccharomyces/Candida and Schizosaccharomyces GAL clusters and coexists in the Cryptococcus genome with unclustered GAL paralogs. These independently evolved GAL clusters represent a striking example of analogy at the genomic level. We also found that species with GAL clusters exhibited significantly higher rates of GAL pathway loss than species with unclustered GAL genes. These results suggest that clustering of metabolic genes might facilitate fungal adaptation to changing environments both through the acquisition and loss of metabolic capacities.

Slot, Jason C.; Rokas, Antonis

2010-01-01

253

Evaluations of the trans-sulfuration pathway in multiple liver toxicity studies  

SciTech Connect

Drug-induced liver injury has been associated with the generation of reactive metabolites, which are primarily detoxified via glutathione conjugation. In this study, it was hypothesized that molecules involved in the synthesis of glutathione would be diminished to replenish the glutathione depleted through conjugation reactions. Since S-adenosylmethionine (SAMe) is the primary source of the sulfur atom in glutathione, UPLC/MS and NMR were used to evaluate metabolites involved with the transulfuration pathway in urine samples collected during studies of eight liver toxic compounds in Sprague-Dawley rats. Urinary levels of creatine were increased on day 1 or day 2 in 8 high dose liver toxicity studies. Taurine concentration in urine was increased in only 3 of 8 liver toxicity studies while SAMe was found to be reduced in 4 of 5 liver toxicity studies. To further validate the results from the metabonomic studies, microarray data from rat liver samples following treatment with acetaminophen was obtained from the Gene Expression Omnibus (GEO) database. Some genes involved in the trans-sulfuration pathway, including guanidinoacetate N-methyltransferase, glycine N-methyltransferase, betaine-homocysteine methyltransferase and cysteine dioxygenase were found to be significantly decreased while methionine adenosyl transferase II, alpha increased at 24 h post-dosing, which is consistent with the SAMe and creatine findings. The metabolic and transcriptomic results show that the trans-sulfuration pathway from SAMe to glutathione was disturbed due to the administration of heptatotoxicants.

Schnackenberg, Laura K. [Division of Systems Toxicology, National Center for Toxicological Research, Food and Drug Administration, FDA, Jefferson, AR 72079 (United States)], E-mail: richard.beger@fda.hhs.gov; Chen Minjun [Environmental Bioinformatics Computational Toxicology Center, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854 (United States); Sun, Jinchun; Holland, Ricky D.; Dragan, Yvonne; Tong Weida [Division of Systems Toxicology, National Center for Toxicological Research, Food and Drug Administration, FDA, Jefferson, AR 72079 (United States); Welsh, William [Environmental Bioinformatics Computational Toxicology Center, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854 (United States); Beger, Richard D. [Division of Systems Toxicology, National Center for Toxicological Research, Food and Drug Administration, FDA, Jefferson, AR 72079 (United States)

2009-02-15

254

Proper Actin Ring Formation and Septum Constriction Requires Coordinated Regulation of SIN and MOR Pathways through the Germinal Centre Kinase MST-1  

PubMed Central

Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in ?mst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1-dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes.

Heilig, Yvonne; Dettmann, Anne; Mourino-Perez, Rosa R.; Schmitt, Kerstin; Valerius, Oliver; Seiler, Stephan

2014-01-01

255

Proper actin ring formation and septum constriction requires coordinated regulation of SIN and MOR pathways through the germinal centre kinase MST-1.  

PubMed

Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in ?mst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1-dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes. PMID:24762679

Heilig, Yvonne; Dettmann, Anne; Mouriño-Pérez, Rosa R; Schmitt, Kerstin; Valerius, Oliver; Seiler, Stephan

2014-04-01

256

Adenovirus RID? uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1.  

PubMed

Niemann-Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RID? rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RID? reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RID? pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RID?/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RID? as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation. PMID:24025716

Cianciola, Nicholas L; Greene, Diane J; Morton, Richard E; Carlin, Cathleen R

2013-11-01

257

Adenovirus RID? uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1  

PubMed Central

Niemann–Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RID? rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RID? reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RID? pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RID?/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RID? as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

Cianciola, Nicholas L.; Greene, Diane J.; Morton, Richard E.; Carlin, Cathleen R.

2013-01-01

258

Nuclear localization of actin requires AC102 in Autographa californica multiple nucleopolyhedrovirus-infected cells  

PubMed Central

Autographa californica multiple nucleopolyhedrovirus requires nuclear actin for progeny virus production and thereby encodes viral products that ensure actin’s translocation to and retention within the nucleus. Current evidence suggests that the ie0–ie1 gene complex along with five nuclear localization of actin (NLA) genes are sufficient for NLA in transient transfection experiments. Here we report that, during infection, only one of the five NLA genes, Ac102, was essential for NLA, and that AC102 had at least one other activity critical for budded virus (BV) production. Viral deletion mutants in the other four NLA genes were viable, with only two having replication phenotypes different from that of the wild type. Infection with Ac?pe38 revealed a delay in both BV production and NLA. Infection with Ac?152 revealed a delay in BV production, but no corresponding delay in NLA. Infection with either Ac?pe38 or Ac?152 resulted in slightly reduced BV titres. Deletion of Ac004 or he65 had no impact on actin translocation kinetics, timing of BV production or BV titres. These results implicate AC102 as a key player in baculovirus manipulation of actin.

Ohkawa, Taro; Volkman, Loy E.

2012-01-01

259

CAP defines a second signalling pathway required for insulin-stimulated glucose transport  

NASA Astrophysics Data System (ADS)

Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl protooncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.

Baumann, Christian A.; Ribon, Vered; Kanzaki, Makoto; Thurmond, Debbie C.; Mora, Silvia; Shigematsu, Satoshi; Bickel, Perry E.; Pessin, Jeffrey E.; Saltiel, Alan R.

2000-09-01

260

Cryptococcus neoformans Requires a Functional Glycolytic Pathway for Disease but Not Persistence in the Host  

PubMed Central

ABSTRACT Cryptococcus neoformans is an important fungal pathogen of immunocompromised individuals, with a close relative, Cryptococcus gattii, emerging as a serious threat for the immunocompetent. During initial infection, C. neoformans colonizes the airspaces of the lungs, resulting in pneumonia, and subsequently migrates to the central nervous system (CNS). We sought to understand fungal carbon utilization during colonization of these fundamentally different niches within the host, in particular the roles of gluconeogenesis and glycolysis. We created mutants at key points in the gluconeogenesis/glycolysis metabolic pathways that are restricted for growth on lactate and glucose, respectively. A phosphoenolpyruvate carboxykinase mutant (the pck1? mutant), blocked for entry of 2- and 3-carbon substrates into gluconeogenesis and attenuated for virulence in a murine inhalation model, showed wild-type (WT) persistence in a rabbit cerebrospinal fluid (CSF) model of cryptococcosis. Conversely, both the pyruvate kinase (pyk1?) and the hexose kinase I and II (hxk1?/hxk2?) mutants, which show impaired glucose utilization, exhibited severely attenuated virulence in the murine inhalation model of cryptococcosis and decreased persistence in the CNS in both the rabbit CSF and the murine inhalation models while displaying adequate persistence in the lungs of mice. These data suggest that glucose utilization is critical for virulence of C. neoformans and persistence of the yeast in the CNS.

Price, Michael S.; Betancourt-Quiroz, Marisol; Price, Jennifer L.; Toffaletti, Dena L.; Vora, Haily; Hu, Guanggan; Kronstad, James W.; Perfect, John R.

2011-01-01

261

CAP defines a second signalling pathway required for insulin-stimulated glucose transport.  

PubMed

Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl proto-oncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake. PMID:11001060

Baumann, C A; Ribon, V; Kanzaki, M; Thurmond, D C; Mora, S; Shigematsu, S; Bickel, P E; Pessin, J E; Saltiel, A R

2000-09-14

262

Antigen Processing and Remodeling of the Endosomal Pathway: Requirements for Antigen Cross-Presentation  

PubMed Central

Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8+ T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

Compeer, Ewoud Bernardus; Flinsenberg, Thijs Willem Hendrik; van der Grein, Susanna Geertje; Boes, Marianne

2012-01-01

263

JNK pathway is required for TNCB-induced IL-18 expression in murine keratinocytes.  

PubMed

The epidermal tissue of the skin is the first line of defense against exposure to microbial, chemical, and physical agents that cause cutaneous immune responses. Epidermal epithelial cells (keratinocytes) produce pro-inflammatory cytokine interleukin-18 (IL-18) potentially relevant for the skin immune responses. Expression of IL-18 was investigated after exposure of murine keratinocytes PAM212 to pro-inflammatory stimuli, allergen TNCB (Trinitrichlorobenzen), LPS (lipopolysaccharide) or PMA (phorbol myristate acetate). IL-18 mRNA transcription and IL-18 secretion were detected by real time-PCR and ELISA, respectively. The results showed that TNCB-induced IL-18 expression in cultured murine keratinocytes in a dose-dependent manner, and it significantly stimulated IL-18 mRNA and protein expression at 10 microM. In addition, both LPS and PMA could increase the expression of IL-18 in murine keratinocytes. To determine the molecular mechanism involved, keratinocytes were pretreated with JNK, p38 or ERK MAPK inhibitors SP600125, SB203580, PD98059. We found that JNK inhibitors could significantly suppress IL-18 expression enhanced by TNCB. Western blot results showed that TNCB could induce the phosphorylation of JNK, but not p38 or ERK1/2. These results suggest that TNCB has an up-regulation effect on IL-18 production in murine keratinocyte cell line PAM212, and that the activation of the JNK signal pathway is the mechanism responsible for TNCB-induced IL-18 gene expression in murine keratinocytes. PMID:20381601

Yun, Wang; Li, Chunfeng

2010-06-01

264

A novel piperazine derivative potently induces caspase-dependent apoptosis of cancer cells via inhibition of multiple cancer signaling pathways  

PubMed Central

Despite rapid progress in anticancer drug development and improvement in clinical outcomes, the survival rate for many types of cancer is still unacceptably low. Therefore, it is crucial to discover novel anticancer drugs to both prevent and treat the disease. In recent years, the advent of combinatorial chemistry allows the design and parallel synthesis of millions of small compounds that have drug-like properties. In vitro high throughput screening of such compound libraries has allowed the identification of many new drug candidates that may be further evaluated for their efficacy and mechanism of action. The overall objective of this study was to identify small molecule compounds as candidates for anti-cancer drug development. We first used cell proliferation and cytotoxicity assays to identify compounds exhibiting anti-cancer activity in vitro in a leukemia cell line (K562). Six top compounds selected from the initial screening of a library of 2,560 compounds were further evaluated in multiple cancer cell lines to rank the drug candidates. The top candidate was further investigated to elucidate the molecular mechanism underlying its anticancer activity. Our studies suggest that this piperazine derivative effectively (GI50 = 0.06-0.16 ?M) inhibits cancer cell proliferation and induces caspase-dependent apoptosis via inhibiting multiple cancer signaling pathways including the PI3K/AKT, the Src family kinases and the BCR-ABL pathways.

She, Edward X; Hao, Zhonglin

2013-01-01

265

Sorting of carboxypeptidase E to the regulated secretory pathway requires interaction of its transmembrane domain with lipid rafts.  

PubMed Central

Carboxypeptidase E (CPE) functions as a regulated secretory pathway sorting receptor for several prohormones, including pro-opiomelanocortin (POMC), proenkephalin and proinsulin. The association of CPE with lipid rafts in the trans -Golgi network and secretory granule membranes is necessary for its sorting receptor function. We now provide evidence that a domain within the C-terminal 25 residues of CPE functions as a signal for both raft association and the sorting of CPE to the regulated secretory pathway. A fusion protein containing the extracellular domain of the human interleukin-2 receptor Tac (N-Tac) and the C-terminal 25 amino acids of CPE was transfected into Neuro2A cells. This fusion protein floated in sucrose density gradients, indicating raft association, and co-localized with chromogranin A (CGA), a secretory granule marker. To define further a minimum sequence required for raft association and sorting, deletion mutants of CPE that lacked the C-terminal four or 15 residues (CPE-Delta4 and CPE-Delta15 respectively) were transfected into a clone of CPE-deficient Neuro2A cells. In contrast with full-length CPE, neither CPE-Delta4 nor CPE-Delta15 floated in sucrose density gradients. The sorting of both CPE-Delta4 and CPE-Delta15 to the regulated secretory pathway was impaired, as indicated by significantly increased basal secretion and a lack of response to stimulation. Additionally, there was a significant decrease in the co-localization of mutant CPE immunofluorescence with CGA when compared with full-length CPE. Finally, the sorting of the prohormone POMC to the regulated pathway was impaired in cells transfected with either CPE-Delta4 or CPE-Delta15. We conclude that the sorting of CPE to the regulated secretory pathway in endocrine cells is mediated by lipid rafts, and that the C-terminal four residues of CPE, i.e. Thr(431)-Leu-Asn-Phe(434), are required for raft association and sorting.

Zhang, Chun-Fa; Dhanvantari, Savita; Lou, Hong; Loh, Y Peng

2003-01-01

266

Hepatocyte growth factor pathway upregulation in the bone marrow microenvironment in multiple myeloma is associated with lytic bone disease.  

PubMed

Lytic bone disease (LBD) in multiple myeloma (MM) is caused by osteoclast hyperactivation and osteoblast inhibition. Based on in vitro studies, the hepatocyte growth factor (HGF) pathway is thought to be central in osteoblast inhibition. We evaluated the gene expression of the HGF pathway in vivo using bone marrow biopsies (BMBs) of patients with MM and monoclonal gammopathy of undetermined significance (MGUS), and healthy volunteers (HV). BMBs (N = 110) obtained at diagnosis were snap-frozen and used to evaluate gene expression by quantitative reverse transcription polymerase chain reaction. LBD was evaluated using standard radiographs. Enzyme-linked immunosorbent assay (ELISA) was performed on matched bone marrow plasma and immunohistochemistry on matched formalin-fixed paraffin-embedded biopsies. Gene expression of HGF, SDC1, and MET in BMBs were significantly altered in MM versus HV and MGUS, and HGF and MET correlated with the extent of LBD. A significant correlation between gene and protein expression levels was observed for SDC1 (Syndecan-1) and HGF. The HGF bone marrow plasma level was significantly lower in MM patients with no/limited versus advanced LBD. Our novel approach using snap-frozen BMBs seems generally applicable because it allows evaluation of gene expression independent of the extent of MM plasma-cell infiltration. Our study highlights the importance of the HGF pathway in MM LBD. PMID:23431957

Kristensen, Ida B; Christensen, Jacob H; Lyng, Maria B; Møller, Michael B; Pedersen, Lise; Rasmussen, Lars M; Ditzel, Henrik J; Abildgaard, Niels

2013-05-01

267

Small molecule inhibitors of the Candida albicans budded-to-hyphal transition act through multiple signaling pathways.  

PubMed

The ability of the pathogenic yeast Candida albicans to interconvert between budded and hyphal growth states, herein termed the budded-to-hyphal transition (BHT), is important for C. albicans development and virulence. The BHT is under the control of multiple cell signaling pathways that respond to external stimuli, including nutrient availability, high temperature, and pH. Previous studies identified 21 small molecules that could inhibit the C. albicans BHT in response to carbon limitation in Spider media. However, the studies herein show that the BHT inhibitors had varying efficacies in other hyphal-inducing media, reflecting their varying abilities to block signaling pathways associated with the different media. Chemical epistasis analyses suggest that most, but not all, of the BHT inhibitors were acting through either the Efg1 or Cph1 signaling pathways. Notably, the BHT inhibitor clozapine, a FDA-approved drug used to treat atypical schizophrenia by inhibiting G-protein-coupled dopamine receptors in the brain, and several of its functional analogs were shown to act at the level of the Gpr1 G-protein-coupled receptor. These studies are the first step in determining the target and mechanism of action of these BHT inhibitors, which may have therapeutic anti-fungal utility in the future. PMID:21966518

Midkiff, John; Borochoff-Porte, Nathan; White, Dylan; Johnson, Douglas I

2011-01-01

268

Small Molecule Inhibitors of the Candida albicans Budded-to-Hyphal Transition Act through Multiple Signaling Pathways  

PubMed Central

The ability of the pathogenic yeast Candida albicans to interconvert between budded and hyphal growth states, herein termed the budded-to-hyphal transition (BHT), is important for C. albicans development and virulence. The BHT is under the control of multiple cell signaling pathways that respond to external stimuli, including nutrient availability, high temperature, and pH. Previous studies identified 21 small molecules that could inhibit the C. albicans BHT in response to carbon limitation in Spider media. However, the studies herein show that the BHT inhibitors had varying efficacies in other hyphal-inducing media, reflecting their varying abilities to block signaling pathways associated with the different media. Chemical epistasis analyses suggest that most, but not all, of the BHT inhibitors were acting through either the Efg1 or Cph1 signaling pathways. Notably, the BHT inhibitor clozapine, a FDA-approved drug used to treat atypical schizophrenia by inhibiting G-protein-coupled dopamine receptors in the brain, and several of its functional analogs were shown to act at the level of the Gpr1 G-protein-coupled receptor. These studies are the first step in determining the target and mechanism of action of these BHT inhibitors, which may have therapeutic anti-fungal utility in the future.

Midkiff, John; Borochoff-Porte, Nathan; White, Dylan; Johnson, Douglas I.

2011-01-01

269

Multiple signaling pathways regulate contractile activity-mediated PGC-1? gene expression and activity in skeletal muscle cells.  

PubMed

PGC-1? is an important transcriptional coactivator that plays a key role in mediating mitochondrial biogenesis. Within seconds of the onset of contractile activity, a number of rapid cellular events occur that form part of the initial signaling processes involved in PGC-1? gene regulation, such as elevations in cytoplasmic calcium, AMPK and p38 activation, and elevated ROS production. We observed that basal levels of PGC-1? promoter activity were more sensitive to resting Ca(2+) levels, compared to ROS, p38 or, AMPK signaling. Moreover, enhanced PGC-1? transcription and post-translational activity on DNA were a result of the activation of multiple signal transduction pathways during contractile activity of myotubes. AMPK, ROS, and Ca(2+) appear to be necessary for the regulation of contractile activity-induced PGC-1? gene expression, governed partly through p38 MAPK and CaMKII activity. Whether these signaling pathways are arranged as a linear sequence of events, or as largely independent pathways during contractile activity, remains to be determined. PMID:24843073

Zhang, Yuan; Uguccioni, Giulia; Ljubicic, Vladimir; Irrcher, Isabella; Iqbal, Sobia; Singh, Kaustabh; Ding, Shuzhe; Hood, David A

2014-05-01

270

Plasma proteomic profiles from disease-discordant monozygotic twins suggest that molecular pathways are shared in multiple systemic autoimmune diseases*  

PubMed Central

Introduction Although systemic autoimmune diseases (SAID) share many clinical and laboratory features, whether they also share some common features of pathogenesis remains unclear. We assessed plasma proteomic profiles among different SAID for evidence of common molecular pathways that could provide insights into pathogenic mechanisms shared by these diseases. Methods Differential quantitative proteomic analyses (one-dimensional reverse-phase liquid chromatography-mass spectrometry) were performed to assess patterns of plasma protein expression. Monozygotic twins (four pairs discordant for systemic lupus erythematosus, four pairs discordant for juvenile idiopathic arthritis and two pairs discordant for juvenile dermatomyositis) were studied to minimize polymorphic gene effects. Comparisons were also made to 10 unrelated, matched controls. Results Multiple plasma proteins, including acute phase reactants, structural proteins, immune response proteins, coagulation and transcriptional factors, were differentially expressed similarly among the different SAID studied. Multivariate Random Forest modeling identified seven proteins whose combined altered expression levels effectively segregated affected vs. unaffected twins. Among these seven proteins, four were also identified in univariate analyses of proteomic data (syntaxin 17, ?-glucosidase, paraoxonase 1, and the sixth component of complement). Molecular pathway modeling indicated that these factors may be integrated through interactions with a candidate plasma biomarker, PON1 and the pro-inflammatory cytokine IL-6. Conclusions Together, these data suggest that different SAID may share common alterations of plasma protein expression and molecular pathways. An understanding of the mechanisms leading to the altered plasma proteomes common among these SAID may provide useful insights into their pathogeneses.

2011-01-01

271

Oxygen and hydroxyl species induce multiple reaction pathways for the partial oxidation of allyl alcohol on gold.  

PubMed

Partial oxidation of alcohols is a topic of great interest in the field of gold catalysis. In this work, we provide evidence that the partial oxidation of allyl alcohol to its corresponding aldehyde, acrolein, over oxygen-precovered gold surfaces occurs via multiple reaction pathways. Utilizing temperature-programmed desorption (TPD) with isotopically labeled water and oxygen species, reactive molecular beam scattering, and density functional theory (DFT) calculations, we demonstrate that the reaction mechanism for allyl alcohol oxidation is influenced by the relative proportions of atomic oxygen and hydroxyl species on the gold surface. Both atomic oxygen and hydroxyl species are shown to be active for allyl alcohol oxidation, but each displays a different pathway of oxidation, as indicated by TPD measurements and DFT calculations. The hydroxyl hydrogen of allyl alcohol is readily abstracted by either oxygen adatoms or adsorbed hydroxyl species on the gold surface to generate a surface-bound allyloxide intermediate, which then undergoes ?-dehydrogenation via interaction with an oxygen adatom or surface hydroxyl species to generate acrolein. Mediation of a second allyloxide with the hydroxyl species lowers the activation barrier for the ?-dehydrogenation process. A third pathway exists in which two hydroxyl species recombine to generate water and an oxygen adatom, which subsequently dehydrogenates allyloxide. This work may aid in the understanding of oxidative catalysis over gold and the effect of water therein. PMID:24702503

Mullen, Gregory M; Zhang, Liang; Evans, Edward J; Yan, Ting; Henkelman, Graeme; Mullins, C Buddie

2014-04-30

272

The Hippo signaling pathway is required for salivary gland development and its dysregulation is associated with Sjogren's syndrome.  

PubMed

Sjogren's syndrome (SS) is a complex autoimmune disease that primarily affects salivary and lacrimal glands and is associated with high morbidity. Although the prevailing dogma is that immune system pathology drives SS, increasing evidence points to structural defects, including defective E-cadherin adhesion, to be involved in its etiology. We have shown that E-cadherin has pivotal roles in the development of the mouse salivary submandibular gland (SMG) by organizing apical-basal polarity in acinar and ductal progenitors and by signaling survival for differentiating duct cells. Recently, E-cadherin junctions have been shown to interact with effectors of the Hippo signaling pathway, a core pathway regulating the organ size, cell proliferation, and differentiation. We now show that Hippo signaling is required for SMG-branching morphogenesis and is involved in the pathophysiology of SS. During SMG development, a Hippo pathway effector, TAZ, becomes increasingly phosphorylated and associated with E-cadherin and ?-catenin, consistent with the activation of Hippo signaling. Inhibition of Lats2, an upstream kinase that promotes TAZ phosphorylation, results in dysmorphogenesis of the SMG and impaired duct formation. SMGs from non-obese diabetic mice, a mouse model for SS, phenocopy the Lats2-inhibited SMGs and exhibit a reduction in E-cadherin junctional components, including TAZ. Importantly, labial specimens from human SS patients display mislocalization of TAZ from junctional regions to the nucleus, coincident with accumulation of extracellular matrix components, fibronectin and connective tissue growth factor, known downstream targets of TAZ. Our studies show that Hippo signaling has a crucial role in SMG-branching morphogenesis and provide evidence that defects in this pathway are associated with SS in humans. PMID:24080911

Enger, Tone B; Samad-Zadeh, Arman; Bouchie, Meghan P; Skarstein, Kathrine; Galtung, Hilde K; Mera, Toshiyuki; Walker, Janice; Menko, A Sue; Varelas, Xaralabos; Faustman, Denise L; Jensen, Janicke L; Kukuruzinska, Maria A

2013-11-01

273

The Hippo signaling pathway is required for salivary gland development and its dysregulation is associated with Sjogren's-like disease  

PubMed Central

Sjogren's syndrome (SS) is a complex autoimmune disease that primarily affects salivary and lacrimal glands and is associated with high morbidity. Although the prevailing dogma is that immune system pathology drives SS, increasing evidence points to structural defects, including defective E-cadherin adhesion, to be involved in its etiology. We have shown that E-cadherin plays pivotal roles in the development of the mouse salivary submandibular gland (SMG) by organizing apical-basal polarity in acinar and ductal progenitors and by signaling survival for differentiating duct cells. Recently, E-cadherin junctions have been shown to interact with effectors of the Hippo signaling pathway, a core pathway regulating organ size, cell proliferation and differentiation. We now show that Hippo signaling is required for SMG branching morphogenesis and is involved in the pathophysiology of SS. During SMG development, a Hippo pathway effector, TAZ, becomes increasingly phosphorylated and associated with E-cadherin and ?-catenin, consistent with the activation of Hippo signaling. Inhibition of Lats2, an upstream kinase that promotes TAZ phosphorylation, results in dysmorphogenesis of the SMG and impaired duct formation. SMGs from NOD mice, a mouse model for SS, phenocopy the Lats2-inhibited SMGs and exhibit a reduction in E-cadherin junctional components, including TAZ. Importantly, labial specimens from human SS patients display mislocalization of TAZ from junctional regions to the nucleus, coincident with accumulation of extracellular matrix components, fibronectin and CTGF, known downstream targets of TAZ. Our studies show that Hippo signaling plays a crucial role in SMG branching morphogenesis and provide evidence that defects in this pathway are associated with SS in humans.

Enger, Tone Berge; Samad-Zadeh, Arman; Bouchie, Meghan; Skarstein, Kathrine; Galtung, Hilde Kanli; Mera, Toshiyuki; Walker, Janice; Menko, A. Sue; Varelas, Xaralabos; Faustman, Denise L.; Jensen, Janicke Liaaen; Kukuruzinska, Maria

2013-01-01

274

Inducible pathway is required for mutagenesis in Salmonella typhimurium LT2  

SciTech Connect

UV mutability of Salmonella typhimurium LT2 was eliminated in the presence of a multicopy plasmid carrying the Escherichia coli lexA/sup +/ gene. This result suggests that inducible, SOS-like functions are required for UV mutagenesis in S. typhimurium. S. typhimurium strains carrying either point or deletion mutations in topA had previously been shown to lose their mutability by UV or methyl methanesulfonate. Mitomycin C induction of the Phi(mucB'-lacZ') fusion (a DNA damage-inducible locus carried on plasmid pSE205) in S. typhimurium topA was normal, suggesting that RecA is activated in topA mutants. These observations lead the authors deduce that S. typhimurium has at least one DNA damage-inducible locus in addition to recA that is required for UV mutability.

Orrego, C.; Eisenstadt, E.

1987-06-01

275

The Multiple Intelligences Pathways to Literacy: Making SMILIES. Pre-K-3.  

ERIC Educational Resources Information Center

This handbook draws from Howard Gardner's multiple intelligences theory and explains it through a teacher- and student-friendly acronym: SMILIES (Strategy, Musical, Intrapersonal, Linguistic, Interpersonal, Exercise and Environmental, and Spatial). The handbook helps teachers explore innovative techniques to teaching reading and writing. According…

Freed, Shirley A.; Moon, Louise

276

Fumonisin B1-Induced Cell Death in Arabidopsis Protoplasts Requires Jasmonate-, Ethylene-, and Salicylate-Dependent Signaling Pathways  

PubMed Central

We have established an Arabidopsis protoplast model system to study plant cell death signaling. The fungal toxin fumonisin B1 (FB1) induces apoptosis-like programmed cell death (PCD) in wild-type protoplasts. FB1, however, only marginally affects the viability of protoplasts isolated from transgenic NahG plants, in which salicylic acid (SA) is metabolically degraded; from pad4-1 mutant plants, in which an SA amplification mechanism is thought to be impaired; or from jar1-1 or etr1-1 mutant plants, which are insensitive to jasmonate (JA) or ethylene (ET), respectively. FB1 susceptibility of wild-type protoplasts decreases in the dark, as does the cellular content of phenylalanine ammonia-lyase, a light-inducible enzyme involved in SA biosynthesis. Interestingly, however, FB1-induced PCD does not require the SA signal transmitter NPR1, given that npr1-1 protoplasts display wild-type FB1 susceptibility. Arabidopsis cpr1-1, cpr6-1, and acd2-2 protoplasts, in which the SA signaling pathway is constitutively activated, exhibit increased susceptibility to FB1. The cpr6-1 and acd2-2 mutants also constitutively express the JA and ET signaling pathways, but only the acd2-2 protoplasts undergo PCD in the absence of FB1. These results demonstrate that FB1 killing of Arabidopsis is light dependent and requires SA-, JA-, and ET-mediated signaling pathways as well as one or more unidentified factors activated by FB1 and the acd2-2 mutation.

Asai, Tsuneaki; Stone, Julie M.; Heard, Jacqueline E.; Kovtun, Yelena; Yorgey, Peter; Sheen, Jen; Ausubel, Frederick M.

2000-01-01

277

Curcumin suppresses multiple DNA damage response pathways and has potency as a sensitizer to PARP inhibitor.  

PubMed

Inhibitors of poly(ADP-ribose) polymerase (PARP) are promising anticancer drugs, particularly for the treatment of tumors deficient in the DNA damage response (DDR). However, it is challenging to design effective therapeutic strategies for use of these compounds against cancers without DDR deficiencies. In this context, combination therapies in which PARP inhibitors are used alongside DDR inhibitors have elicited a great deal of interest. Curcumin, a component of turmeric (Curcuma longa), has been tested in clinical studies for its chemosensitizing potential; however, the mechanisms of chemosensitization by curcumin have not been fully elucidated. This study demonstrates that curcumin suppresses three major DDR pathways: non-homologous end joining (NHEJ), homologous recombination (HR) and the DNA damage checkpoint. Curcumin suppresses the histone acetylation at DNA double-strand break (DSB) sites by inhibiting histone acetyltransferase activity, thereby reducing recruitment of the key NHEJ factor KU70/KU80 to DSB sites. Curcumin also suppresses HR by reducing expression of the BRCA1 gene, which regulates HR, by impairing histone acetylation at the BRCA1 promoter. Curcumin also inhibits ataxia telangiectasia and Rad3-related protein (ATR) kinase (IC50 in vitro = 493 nM), resulting in impaired activation of ATR-CHK1 signaling, which is necessary for HR and the DNA damage checkpoint pathway. Thus, curcumin suppresses three DDR pathways by inhibiting histone acetyltransferases and ATR. Concordantly, curcumin sensitizes cancer cells to PARP inhibitors by enhancing apoptosis and mitotic catastrophe via inhibition of both the DNA damage checkpoint and DSB repair. Our results indicate that curcumin is a promising sensitizer for PARP inhibitor-based therapy. PMID:23825154

Ogiwara, Hideaki; Ui, Ayako; Shiotani, Bunsyo; Zou, Lee; Yasui, Akira; Kohno, Takashi

2013-11-01

278

Ginseng saponin metabolite 20(S)-protopanaxadiol inhibits tumor growth by targeting multiple cancer signaling pathways  

PubMed Central

Plant-derived active constituents and their semi-synthetic or synthetic analogs have served as major sources of anticancer drugs. 20(S)-protopanaxadiol (PPD) is a metabolite of ginseng saponin of both American ginseng (Panax quinquefolius L.) and Asian ginseng (Panax ginseng C.A. Meyer). We previously demonstrated that ginsenoside Rg3, a glucoside precursor of PPD, exhibits anti-proliferative effects on HCT116 cells and reduces tumor size in a xenograft model. Our subsequent study indicated that PPD has more potent antitumor activity than that of Rg3 in vitro although the mechanism underlying the anticancer activity of PPD remains to be defined. Here, we investigated the mechanism underlying the anticancer activity of PPD in human cancer cells in vitro and in vivo. PPD was shown to inhibit growth and induce cell cycle arrest in HCT116 cells. The in vivo studies indicate that PPD inhibits xenograft tumor growth in athymic nude mice bearing HCT116 cells. The xenograft tumor size was significantly reduced when the animals were treated with PPD (30 mg/kg body weight) for 3 weeks. When the expression of previously identified Rg3 targets, A kinase (PRKA) anchor protein 8 (AKAP8L) and phosphatidylinositol transfer protein ? (PITPNA), was analyzed, PPD was shown to inhibit the expression of PITPNA while upregulating AKAP8L expression in HCT116 cells. Pathway-specific reporter assays indicated that PPD effectively suppressed the NF-?B, JNK and MAPK/ERK signaling pathways. Taken together, our results suggest that the anticancer activity of PPD in colon cancer cells may be mediated through targeting NF-?B, JNK and MAPK/ERK signaling pathways, although the detailed mechanisms underlying the anticancer mode of PPD action need to be fully elucidated.

GAO, JIAN-LI; LV, GUI-YUAN; HE, BAI-CHENG; ZHANG, BING-QIANG; ZHANG, HONGYU; WANG, NING; WANG, CHONG-ZHI; DU, WEI; YUAN, CHUN-SU; HE, TONG-CHUAN

2013-01-01

279

The requirement for recombination factors differs considerably between different pathways of homologous double-strand break repair in somatic plant cells.  

PubMed

In recent years, multiple factors involved in DNA double-strand break (DSB) repair have been characterised in Arabidopsis thaliana. Using homologous sequences in somatic cells, DSBs are mainly repaired by two different pathways: synthesis-dependent strand annealing (SDSA) and single-strand annealing (SSA). By applying recombination substrates in which recombination is initiated by the induction of a site-specific DSB by the homing endonuclease I-SceI, we were able to characterise the involvement of different factors in both pathways. The nucleases MRE11 and COM1, both involved in DSB end processing, were not required for either SDSA or SSA in our assay system. Both SDSA and SSA were even more efficient without MRE11, in accordance with the fact that a loss of MRE11 might negatively affect the efficiency of non-homologous end joining. Loss of the classical recombinase RAD51 or its two paralogues RAD51C and XRCC3, as well as the SWI2/SNF2 remodelling factor RAD54, resulted in a drastic deficiency in SDSA but had hardly any influence on SSA, confirming that a strand exchange reaction is only required for SDSA. The helicase FANCM, which is postulated to be involved in the stabilisation of recombination intermediates, is surprisingly not only needed for SDSA but to a lesser extent also for SSA. Both SSA and SDSA were affected only weakly when the SMC6B protein, implicated in sister chromatid recombination, was absent, indicating that SSA and SDSA are in most cases intrachromatid recombination reactions. PMID:22860689

Roth, Nadine; Klimesch, Jacqueline; Dukowic-Schulze, Stefanie; Pacher, Michael; Mannuss, Anja; Puchta, Holger

2012-12-01

280

A Dicer-independent miRNA biogenesis pathway that requires Ago catalysis  

PubMed Central

The nucleolytic activity of animal Argonaute proteins is deeply conserved, despite its having no obvious role in microRNA-directed gene regulation. In mice, Ago2 (also known as Eif2c2) is uniquely required for viability, and only this family member retains catalytic competence. To investigate the evolutionary pressure to conserve Argonaute enzymatic activity, we engineered a mouse with catalytically inactive Ago2 alleles. Homozygous mutants died shortly after birth with an obvious anaemia. Examination of microRNAs and their potential targets revealed a loss of miR-451, a small RNA important for erythropoiesis. Though this microRNA is processed by Drosha (also known as Rnasen), its maturation does not require Dicer. Instead, the pre-miRNA becomes loaded into Ago and is cleaved by the Ago catalytic centre to generate an intermediate 3? end, which is then further trimmed. Our findings link the conservation of Argonaute catalysis to a conserved mechanism of microRNA biogenesis that is important for vertebrate development.

Cheloufi, Sihem; Dos Santos, Camila O.; Chong, Mark M. W.; Hannon, Gregory J.

2010-01-01

281

Multiple Independent Fusions of Glucose-6-Phosphate Dehydrogenase with Enzymes in the Pentose Phosphate Pathway  

PubMed Central

Fusions of the first two enzymes in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconolactonase (6PGL), have been previously described in two distant clades, chordates and species of the malarial parasite Plasmodium. We have analyzed genome and expressed sequence data from a variety of organisms to identify the origins of these gene fusion events. Based on the orientation of the domains and range of species in which homologs can be found, the fusions appear to have occurred independently, near the base of the metazoan and apicomplexan lineages. Only one of the two metazoan paralogs of G6PD is fused, showing that the fusion occurred after a duplication event, which we have traced back to an ancestor of choanoflagellates and metazoans. The Plasmodium genes are known to contain a functionally important insertion that is not seen in the other apicomplexan fusions, highlighting this as a unique characteristic of this group. Surprisingly, our search revealed two additional fusion events, one that combined 6PGL and G6PD in an ancestor of the protozoan parasites Trichomonas and Giardia, and another fusing G6PD with phosphogluconate dehydrogenase (6PGD) in a species of diatoms. This study extends the range of species known to contain fusions in the pentose phosphate pathway to many new medically and economically important organisms.

Stover, Nicholas A.; Dixon, Thomas A.; Cavalcanti, Andre R. O.

2011-01-01

282

Thermal, Chemical and pH Induced Denaturation of a Multimeric ?-Galactosidase Reveals Multiple Unfolding Pathways  

PubMed Central

Background In this case study, we analysed the properties of unfolded states and pathways leading to complete denaturation of a multimeric chick pea ?-galactosidase (CpGAL), as obtained from treatment with guanidium hydrochloride, urea, elevated temperature and extreme pH. Methodology/Principal Findings CpGAL, a heterodimeric protein with native molecular mass of 85 kDa, belongs to ?+? class of protein. The conformational stability and thermodynamic parameters of CpGAL unfolding in different states were estimated and interpreted using circular dichroism and fluorescence spectroscopic measurements. The enzyme was found to be structurally and functionally stable in the entire pH range and upto 50°C temperature. Further increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were irreversible, non-coincidental and sigmoidal. Free energy of protein unfolding (?G0) and unfolding constant (Kobs) were also calculated for chemically denatured CpGAL. Significance The protein seems to use different pathways for unfolding in different environments and is a classical example of how the environment dictates the path a protein might take to fold while its amino acid sequence only defines its final three-dimensional conformation. The knowledge accumulated could be of immense biotechnological significance as well.

Kishore, Devesh; Kundu, Suman; Kayastha, Arvind M.

2012-01-01

283

The proinflammatory cytokine interleukin-18 alters multiple signaling pathways to inhibit natural killer cell death  

USGS Publications Warehouse

The proinflammatory cytokine, interleukin-18 (IL-18), is a natural killer (NK) cell activator that induces NK cell cytotoxicity and interferon-?? (IFN-??) expression. In this report, we define a novel role for IL-18 as an NK cell protective agent. Specifically, IL-18 prevents NK cell death initiated by different and distinct stress mechanisms. IL-18 reduces NK cell self-destruction during NK-targeted cell killing, and in the presence of staurosporin, a potent apoptotic inducer, IL-18 reduces caspase-3 activity. The critical regulatory step in this process is downstream of the mitochondrion and involves reduced cleavage and activation of caspase-9 and caspase-3. The ability of IL-18 to regulate cell survival is not limited to a caspase death pathway in that IL-18 augments tumor necrosis factor (TNF) signaling, resulting in increased and prolonged mRNA expression of c-apoptosis inhibitor 2 (cIAP2), a prosurvival factor and caspase-3 inhibitor, and TNF receptor-associated factor 1 (TRAF1), a prosurvival protein. The cumulative effects of IL-18 define a novel role for this cytokine as a molecular survival switch that functions to both decrease cell death through inhibition of the mitochondrial apoptotic pathway and enhance TNF induction of prosurvival factors. ?? Mary Ann Liebert, Inc.

Hodge, D. L.; Subleski, J. J.; Reynolds, D. A.; Buschman, M. D.; Schill, W. B.; Burkett, M. W.; Malyguine, A. M.; Young, H. A.

2006-01-01

284

Multiple degradation pathways for misfolded mutants of the yeast plasma membrane ATPase, Pma1.  

PubMed

To understand protein sorting and quality control in the secretory pathway, we have analyzed intracellular trafficking of the yeast plasma membrane ATPase, Pma1. Pma1 is ideal for such studies because it is a very abundant polytopic membrane protein, and its localization and activity at the plasma membrane are essential for cell viability and growth. We have tested whether the cytoplasmic amino- and carboxyl-terminal domains of Pma1 carry sorting information. As the sole copy of Pma1, mutants truncated at either NH2 or COOH termini are targeted at least partially to the plasma membrane and have catalytic activity to sustain cell viability. The mutants are also delivered to degradative pathways. Strikingly, NH2- and COOH-terminal Pma1 mutants are differentially recognized for degradation at distinct cellular locales. COOH-terminal mutants are recognized for destruction by endoplasmic reticulum-associated degradation. By contrast, NH2-terminal mutants escape detection by endoplasmic reticulum-associated degradation entirely, and undergo endocytosis for vacuolar degradation after apparently normal cell surface targeting. Both NH2- and COOH-terminal mutants are conformationally abnormal, as revealed by increased sensitivity to tryptic cleavage, but are able to assemble to form oligomers. We propose that different quality control mechanisms may assess discrete domains of Pma1 rather than a global conformational state. PMID:16928681

Liu, Yu; Sitaraman, Sujatha; Chang, Amy

2006-10-20

285

The Drosophila Nbs Protein Functions in Multiple Pathways for the Maintenance of Genome Stability  

PubMed Central

The Mre11/Rad50/Nbs (MRN) complex and the two protein kinases ATM and ATR play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. It has been previously shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. Here, we have analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations.

Ciapponi, Laura; Cenci, Giovanni; Gatti, Maurizio

2006-01-01

286

Estrogen receptors alpha and beta have similar activities in multiple endothelial cell pathways.  

PubMed

The presence of both estrogen receptor alpha (ERalpha) and ERbeta in vascular cells has greatly increased the complexity of potential estrogen regulatory pathways in the cardiovascular system. Here, human umbilical vein endothelial cells were engineered using adenovirus vectors to express either ERalpha or ERbeta. The activities of ERalpha and ERbeta were compared in three distinct gene regulatory pathways, including inhibition of IL-1beta induction of E-selectin expression, inhibition of basal endothelin-1 production, and the ability to induce two matrix-stabilizing enzymes: tissue transglutaminase and a novel member of the lysyl oxidase family. Both ERs were active on these end points, although ERbeta was typically less efficacious than ERalpha. As no class of gene regulation could differentiate ERalpha from ERbeta activity, we characterized a novel steroid (7alpha-thiophenyl-E2) that bound with similar affinities to ERalpha and ERbeta, but functioned as an ERalpha agonist and ERbeta antagonist for all of these endothelial responses. This pattern of receptor subtype-selective activity was not unique to endothelial cells, but was also seen in metabolically active HepG2 cells, suggesting potential in vivo utility. The panel of endothelial responses coupled with a selective modulator should provide a means to characterize the roles of ERalpha and ERbeta in endothelial cells in vivo. PMID:12239089

Evans, Mark J; Harris, Heather A; Miller, Chris P; Karathanasis, Sotirios K; Adelman, Steven J

2002-10-01

287

Multiple signalling pathways involved in ?2-adrenoceptor-mediated glucose uptake in rat skeletal muscle cells  

PubMed Central

?-adrenoceptor (AR) agonists increase 2-deoxy-[3H]-D-glucose uptake (GU) via ?2-AR in rat L6 cells. The ?-AR agonists, zinterol (?2-AR) and (?)-isoprenaline, increased cAMP accumulation in a concentration-dependent manner (pEC50=9.1±0.02 and 7.8±0.02). Cholera toxin (% max increase 141.8±2.5) and the cAMP analogues, 8-bromo-cAMP (8Br-cAMP) and dibutyryl cAMP (dbcAMP), also increased GU (196.8±13.5 and 196.4±17.3%). The adenylate cyclase inhibitor, 2?,5?-dideoxyadenosine (50??M), significantly reduced cAMP accumulation to zinterol (100?nM) (109.7+35.0 to 21.6+4.5?pmol?well?1), or forskolin (10??M) (230.1±58.0 to 107.2±26.3?pmol?well?1), and partially inhibited zinterol-stimulated GU (217±26.3 to 176.1±20.4%). The protein kinase A (PKA) inhibitor, 4-cyano-3-methylisoquinoline (100?nM), did not inhibit zinterol-stimulated GU. The PDE4 inhibitor, rolipram (10??M), increased cAMP accumulation to zinterol or forskolin, and sensitised the GU response to zinterol, indicating a stimulatory role of cAMP in GU. cAMP accumulation studies indicated that the ?2-AR was desensitised by prolonged stimulation with zinterol, but not forskolin, whereas GU responses to zinterol increased with time, suggesting that receptor desensitisation may be involved in GU. Receptor desensitisation was not reversed by inhibition of PKA or Gi. PTX pretreatment (100?ng?ml?1) inhibited insulin or zinterol-stimulated but not 8Br-cAMP or dbcAMP-stimulated GU. The PI3K inhibitor, LY294002 (1??M), inhibited insulin- (174.9±5.9 to 142.7±2.7%) and zinterol- (166.9±7.6 to 141.1±8.1%) but not 8 Br-cAMP-stimulated GU. In contrast to insulin, zinterol did not cause phosphorylation of Akt. The results suggest that GU in L6 cells involves three mechanisms: (1) an insulin-dependent pathway involving PI3K, (2) a ?2-AR-mediated pathway involving both cAMP and PI3K, and (3) a receptor-independent pathway suggested by cAMP analogues that increase GU independently of PI3K. PKA appears to negatively regulate ?2-AR-mediated GU.

Nevzorova, Julia; Evans, Bronwyn A; Bengtsson, Tore; Summers, Roger J

2006-01-01

288

The Drosophila gene fs(2)cup interacts with otu to define a cytoplasmic pathway required for the structure and function of germ-line chromosomes.  

PubMed

The Drosophila ovarian tumor gene (otu) encodes cytoplasmic proteins that are required in germ-line cells for cyst formation, nurse cell chromosome structure and egg maturation. We have analyzed a gene, fs(2)cup, that participates in many of the same processes and interacts with otu genetically. Both nurse cell and oocyte chromosomes require cup to attain a normal morphology. In addition, the gene is needed for the oocyte to grow normally by taking up materials transported from the nurse cells. The gene encodes a 1132-amino-acid protein containing a putative membrane-spanning domain. Cup protein (but not cup RNA) is transported selectively into the oocyte in germarial cysts, like the p104 Otu protein. It is strongly associated with large structures in the cytoplasm and perinuclear region of nurse cells and, like Otu, moves to the periphery of these cells in stages 9-10. Moreover, cup mutations dominantly disrupt meiotic chromosome segregation. We propose that cup, otu and another interacting gene, fs(2)B, take part in a common cytoplasmic pathway with multiple functions during oogenesis. PMID:9118812

Keyes, L N; Spradling, A C

1997-04-01

289

Multiple pathways for Epstein-Barr virus episome loss from nasopharyngeal carcinoma  

PubMed Central

Epstein-Barr virus (EBV) is the prototypical example for episomal persistence of genetic information. Yet, little is known about how this viral episome is lost. Episome loss occurs naturally in naso-pharyngeal carcinoma (NPC) upon explantation into culture. Using whole-genome profiling, we found evidence for 2 different pathways of episome loss: (i) rapid loss of the entire episome or (ii) successive mutation/deletion of the episome until at least 1 essential cis-element is destroyed. This second phenotype was seen in a clone of HONE-1 NPC cells that maintains the EBV episome for prolonged time in culture. The conceptual insights provided by our quantitative analysis should aid our understanding of mammalian episomes, as well as lead to designs to cure latent viral infection.

Dittmer, Dirk P.; Hilscher, Chelsey J.; Gulley, Margaret L.; Yang, Eric V.; Chen, Min; Glaser, Ronald

2010-01-01

290

Integrating Multiple Microarray Data for Cancer Pathway Analysis Using Bootstrapping K-S Test  

PubMed Central

Previous applications of microarray technology for cancer research have mostly focused on identifying genes that are differentially expressed between a particular cancer and normal cells. In a biological system, genes perform different molecular functions and regulate various biological processes via interactions with other genes thus forming a variety of complex networks. Therefore, it is critical to understand the relationship (e.g., interactions) between genes across different types of cancer in order to gain insights into the molecular mechanisms of cancer. Here we propose an integrative method based on the bootstrapping Kolmogorov-Smirnov test and a large set of microarray data produced with various types of cancer to discover common molecular changes in cells from normal state to cancerous state. We evaluate our method using three key pathways related to cancer and demonstrate that it is capable of finding meaningful alterations in gene relations.

Han, Bing; Chen, Xue-Wen; Wang, Xinkun; Michaelis, Elias K.

2009-01-01

291

Life Stress, Genes, and Depression: Multiple Pathways Lead to Increased Risk and New Opportunities for Intervention  

NSDL National Science Digital Library

This STKE Review with 2 figures and 122 references concerns the interaction between stress, genetic factors, and vulnerability to depression. Evidence suggests that the combination of genetics, early life stress, and ongoing stress determine how an individual responds to stress and his vulnerability to psychiatric disorders, such as depression. It is likely that genetic factors and life stress contribute not only to alterations in various neurotransmitter systems, but also to the impairments of cellular plasticity and resilience that are observed in depression. Increased understanding of the specific cellular and neurochemical alterations that contribute to depression, and of the intracellular signaling pathways that underlie cellular plasticity and resilience, may lead to the identification of novel therapeutic targets and, therefore, to the development of novel antidepressant therapies.

Dennis S. Charney (National Institute of Mental Health;Mood and Anxiety Disorders Research Program REV); Husseini K. Manji (National Institute of Mental Health;Laboratory of Molecular Pathophysiology REV)

2004-03-23

292

Epidermal growth factor induces phosphorylation of extracellular signal-regulated kinase 2 via multiple pathways.  

PubMed Central

Expression of p21rasAsn-17, a dominant negative mutant of p21ras that blocks p21ras activation by growth factors, inhibits activation of extracellular signal-regulated kinase 2 (ERK2) by insulin and platelet-derived growth factor in rat-1 cells [A. M. M. de Vries-Smits, B. M. T. Burgering, S. J. Leevers, C. J. Marshall, and J. L. Bos, Nature (London) 357:602-604, 1992]. Here we report that expression of p21rasAsn-17 does not abolish epidermal growth factor (EGF)-induced phosphorylation of ERK2 in fibroblasts. Since EGF activates p21ras in these cells, this indicates that EGF induces a p21ras-independent pathway for the phosphorylation of ERK2 as well. We investigated whether activation of protein kinase C (PKC) or increase in intracellular calcium could be involved in p21ras-independent signaling. In rat-1 cells, inhibition of either PKC, by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) pretreatment, or calcium influx, by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) pretreatment, did not abolish EGF-induced ERK2 phosphorylation. However, a combined inhibition of both p21ras and calcium influx, but not PKC, resulted in a complete inhibition of EGF-induced ERK2 phosphorylation. In contrast, in Swiss 3T3 cells, inhibition of both p21ras activation and TPA-sensitive PKC, but not calcium influx, inhibited EGF-induced ERK2 phosphorylation. These results demonstrate that in fibroblasts, EGF induces alternative pathways of ERK2 phosphorylation in a cell-type-specific manner. Images

Burgering, B M; de Vries-Smits, A M; Medema, R H; van Weeren, P C; Tertoolen, L G; Bos, J L

1993-01-01

293

Sealing of transected neurites of rat B104 cells requires a diacylglycerol PKC-dependent pathway and a PKA-dependent pathway.  

PubMed

To survive, neurons and other eukaryotic cells must rapidly repair (seal) plasmalemmal damage. Such repair occurs by an accumulation of intracellular vesicles at or near the plasmalemmal disruption. Diacylglycerol (DAG)-dependent and cAMP-dependent proteins are involved in many vesicle trafficking pathways. Although recent studies have implicated the signaling molecule cAMP in sealing, no study has investigated how DAG and DAG-dependent proteins affect sealing. By means of dye exclusion to assess Ca(2+)-dependent vesicle-mediated sealing of transected neurites of individually identifiable rat hippocampal B104 cells, we now report that, compared to non-treated controls, sealing probabilities and rates are increased by DAG and cAMP analogs that activate PKC and Munc13-1 and PKA. Sealing is decreased by inhibiting DAG-activated novel protein kinase C isozymes ? (nPKC?) and ? (nPKC?) and Munc13-1, the PKC effector myristoylated alanine rich PKC substrate (MARCKS) or phospholipase C (PLC). DAG-increased sealing is prevented by inhibiting MARCKS or protein kinase A (PKA). Sealing probability is further decreased by simultaneously inhibiting nPKC?, nPKC?, and PKA. Extracellular Ca(2+), DAG, or cAMP analogs do not affect this decrease in sealing. These and other data suggest that DAG increases sealing through MARCKS and that nPKC?, nPKC?, and PKA are all required to seal plasmalemmal damage in B104 and likely all eukaryotic cells. PMID:22865002

Zuzek, Aleksej; Fan, Jerry D; Spaeth, Christopher S; Bittner, George D

2013-01-01

294

An activated JAK/STAT3 pathway and CD45 expression are associated with sensitivity to Hsp90 inhibitors in multiple myeloma.  

PubMed

The molecular chaperone Hsp90 is required to maintain the activity of many signaling proteins, including members of the JAK/STAT and the PI3K pathways. Inhibitors of Hsp90 (Hsp90-Is) demonstrated varying activity against multiple myeloma (MM) in clinical trials. We aimed to determine which signaling pathways that account for the differential sensitivity to the Hsp90-I 17DMAG on a panel of MM cell lines and freshly obtained MM cells. Three CD45(+) cell lines with an activated JAK/STAT3 pathway were sensitive to 17DMAG and underwent prominent apoptosis upon treatment, while the majority of CD45(-) cell lines, that were dependent on the activated PI3K pathway, were more resistant to the drug. Culturing the most resistant cell line, LP1, in the presence of IL-6 resulted in up-regulation of CD45 and pSTAT3, and sensitized to 17DMAG-induced apoptosis, primarily in the induced CD45(+) sub-population of cells. The high CD45 expressers among primary myeloma cells also expressed significantly higher levels of pSTAT3, as compared to the low CD45 expressers. Ex vivo treatment of primary myeloma cells with 17DMAG resulted in a stronger caspase3 activation in tumor samples with the prevalence of high CD45 expressers. STAT3 activity was efficiently inhibited by Hsp90-Is in both cell lines and primary cells suggesting an importance of STAT3 inactivation for the pro-apoptotic effects of HSP90-Is. Indeed, over-expression of STAT3C, a variant with an increased DNA binding activity, in U266 cells protected them from 17DMAG-induced cell death. The down-regulation of the STAT3 target gene Mcl-1 at both the mRNA and protein levels following 17DMAG treatment was significantly attenuated in STAT3C-expressing cells, and transient over-expression of Mcl-1 protected U266 cells from 17DMAG-induced cell death. The finding that CD45(+) MM cells with an IL-6-activated JAK/STAT3 pathway are particularly sensitive to Hsp90-Is as compared to the low CD45 expressers may provide a rational basis for selection of MM patients amenable to Hsp90-I treatment. PMID:23246572

Lin, Huiqiong; Kolosenko, Iryna; Björklund, Ann-Charlotte; Protsyuk, Darya; Österborg, Anders; Grandér, Dan; Tamm, Katja Pokrovskaja

2013-03-10

295

Targeting angiogenesis via a c-Myc/hypoxia-inducible factor-1alpha-dependent pathway in multiple myeloma.  

PubMed

Bone marrow angiogenesis is associated with multiple myeloma (MM) progression. Here, we report high constitutive hypoxia-inducible factor-1alpha (Hif-1alpha) expression in MM cells, which is associated with oncogenic c-Myc. A drug screen for anti-MM agents that decrease Hif-1alpha and c-Myc levels identified a variety of compounds, including bortezomib, lenalidomide, enzastaurin, and adaphostin. Functionally, based on transient knockdowns and overexpression, our data delineate a c-Myc/Hif-1alpha-dependent pathway mediating vascular endothelial growth factor production and secretion. The antiangiogenic activity of our tool compound, adaphostin, was subsequently shown in a zebrafish model and translated into a preclinical in vitro and in vivo model of MM in the bone marrow milieu. Our data, therefore, identify Hif-1alpha as a novel molecular target in MM and add another facet to anti-MM drug activity. PMID:19509231

Zhang, Jing; Sattler, Martin; Tonon, Giovanni; Grabher, Clemens; Lababidi, Samir; Zimmerhackl, Alexander; Raab, Marc S; Vallet, Sonia; Zhou, Yiming; Cartron, Marie-Astrid; Hideshima, Teru; Tai, Yu-Tzu; Chauhan, Dharminder; Anderson, Kenneth C; Podar, Klaus

2009-06-15

296

Cell Entry of Borna Disease Virus Follows a Clathrin-Mediated Endocytosis Pathway That Requires Rab5 and Microtubules?  

PubMed Central

Borna disease virus (BDV), the prototypic member of the Bornaviridae family within the order Mononegavirales, exhibits high neurotropism and provides an important and unique experimental model system for studying virus-cell interactions within the central nervous system. BDV surface glycoprotein (G) plays a critical role in virus cell entry via receptor-mediated endocytosis, and therefore, G is a critical determinant of virus tissue and cell tropism. However, the specific cell pathways involved in BDV cell entry have not been determined. Here, we provide evidence that BDV uses a clathrin-mediated, caveola-independent cell entry pathway. We also show that BDV G-mediated fusion takes place at an optimal pH of 6.0 to 6.2, corresponding to an early-endosome compartment. Consistent with this finding, BDV cell entry was Rab5 dependent but Rab7 independent and exhibited rapid fusion kinetics. Our results also uncovered a key role for microtubules in BDV cell entry, whereas the integrity and dynamics of actin cytoskeleton were not required for efficient cell entry of BDV.

Clemente, Roberto; de la Torre, Juan C.

2009-01-01

297

Activation of multiple cancer pathways and tumor maintenance function of the 3q amplified oncogene FNDC3B.  

PubMed

FNDC3B was recently identified in an oncogenomic screen for amplified oncogenes in hepatocellular carcinoma. It is located at 3q26 and is amplified in over 20% of cancers, usually as part of a broad amplified region encompassing the entire 3q arm. Consistent with an oncogenic role in multiple cancer types, we show here that overexpression of FNDC3B is capable of malignantly transforming mammary and kidney epithelial cells in addition to hepatocytes. To explore how FNDC3B transforms cells, we determined the cellular localization of its gene product and the cancer pathways that it activates. We found that the FNDC3B oncoprotein localizes to the Golgi network, and that its correct localization is essential for its transforming function. We found that overexpression of FNDC3B induces the epithelial-to-mesenchymal transition (EMT) and activates several cancer pathways, including PI3-kinase/Akt, Rb1 and TGF? signaling. For TGF? signaling, we analyzed the point in the pathway at which FNDC3B operates and obtained evidence that it induces expression of all three TGF? ligands and also promotes TGFBR1 cell-surface localization. We found that RNAi-mediated knockdown of FNDC3B in cancer cells with 3q amplification suppressed their clonogenicity and tumorigenicity, but that the same RNAi knockdown had no effect on single-copy 3q cancer cells. These results indicate that FNDC3B is an important oncogenic driver gene of the 3q amplicon, adding to the growing list of oncogenic drivers within this commonly amplified region. PMID:22510613

Cai, Chunlin; Rajaram, Megha; Zhou, Xin; Liu, Qing; Marchica, John; Li, Jinyu; Powers, R Scott

2012-05-01

298

Paradoxical dysregulation of the neural stem cell pathway Sonic hedgehog-Gli1 in autoimmune encephalomyelitis and multiple sclerosis  

PubMed Central

Objective Neurovascular niches have been proposed as critical components of the neural stem cell (NSC) response to acute central nervous system (CNS) injury, however, it is unclear whether these potential reparative niches remain functional during chronic injury. Here we asked how CNS inflammatory injury regulates the intrinsic properties of NSCs and their niches. Methods We investigated the sonic hedgehog (Shh)-Gli1 pathway, an important signaling pathway for NSCs, in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) and its regulation of by inflammatory cytokines. Results We show that Shh is markedly up-regulated by reactive and perivascular astroglia in areas of injury in MS lesions and during EAE. Astroglia outside the subventricular zone (SVZ) niche can support NSC differentiation towards neurons and oligodendrocytes and Shh is a critical mediator of this effect. Shh induces differential upregulation of the transcription factor Gli1, which mediates Shh-induced NSC differentiation. However, despite the increase in Shh and the fact that Gli1 was initially increased during early inflammation of EAE and active lesions of MS, Gli1 was significantly decreased in spinal cord oligodendrocyte precursor cells (OPCs) after onset of EAE and in chronic active and inactive lesions from MS brain. The Th1 cytokine IFN-? was unique in inducing Shh expression in astroglia and NSCs, while paradoxically suppressing Gli1 expression in NSCs and inhibiting Shh-mediated NSC differentiation. Interpretation Our data suggest that endogenous repair potential during chronic injury appears to be limited by inflammation-induced alterations in intrinsic NSC molecular pathways such as Gli1.

Wang, Yue; Imitola, Jaime; Rasmussen, Stine; O'Connor, Kevin C.; Khoury, Samia J.

2009-01-01

299

Overcoming inherent resistance to histone deacetylase inhibitors in multiple myeloma cells by targeting pathways integral to the actin cytoskeleton  

PubMed Central

Histone deacetylase inhibitors (HDACi) are novel chemotherapeutics undergoing evaluation in clinical trials for the potential treatment of patients with multiple myeloma (MM). Although HDACi have demonstrable synergy when combined with proteasome inhibitors (PIs), recent evidence indicates that combination of HDACi and PI is beneficial only in a subset of patients with advanced MM, clearly indicating that other rational combinations should be explored. In this context we hypothesized that understanding the molecular signature associated with inherent resistance to HDACi would provide a basis for the identification of therapeutic combinations with improved clinical efficacy. Using human myeloma cell lines (HMCL) categorized as sensitive, intermediate or resistant to HDACi, gene expression profiling (GEP) and gene ontology enrichment analyses were performed to determine if a genetic signature associated with inherent resistance to HDACi-resistance could be identified. Correlation of GEP to increasing or decreasing sensitivity to HDACi indicated a unique 35-gene signature that was significantly enriched for two pathways – regulation of actin cytoskeleton and protein processing in endoplasmic reticulum. When HMCL and primary MM samples were treated with a combination of HDACi and agents targeting the signaling pathways integral to the actin cytoskeleton, synergistic cell death was observed in all instances, thus providing a rationale for combining these agents with HDACi for the treatment of MM to overcome resistance. This report validates a molecular approach for the identification of HDACi partner drugs and provides an experimental framework for the identification of novel therapeutic combinations for anti-MM treatment.

Mithraprabhu, S; Khong, T; Spencer, A

2014-01-01

300

Multiple protein kinase pathways are involved in gastrin-releasing peptide receptor-regulated secretion.  

PubMed

Gastrin-releasing peptide (GRP) and its amphibian homolog, bombesin, are potent secretogogues in mammals. We determined the roles of intracellular free Ca(2+) ([Ca(2+)](i)), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) in GRP receptor (GRP-R)-regulated secretion. Bombesin induced either [Ca(2+)](i) oscillations or a biphasic elevation in [Ca(2+)](i). The biphasic response was associated with peptide secretion. Receptor-activated secretion was blocked by removal of extracellular Ca(2+), by chelation of [Ca(2+)](i), and by treatment with inhibitors of phospholipase C, conventional PKC isozymes, and MAPK kinase (MEK). Agonist-induced increases in [Ca(2+)](i) were also inhibited by dominant negative MEK-1 and the MEK inhibitor, PD89059, but not by an inhibitor of PKC. Direct activation of PKC by a phorbol ester activated MAPK and stimulated peptide secretion without a concomitant increase in [Ca(2+)](i). Inhibition of MEK blocked both bombesin- and phorbol 12-myristate 13-acetate-induced secretion. GRP-R-regulated secretion is initiated by an increase in [Ca(2+)](i); however, elevated [Ca(2+)](i) is insufficient to stimulate secretion in the absence of activation of PKC and the downstream MEK/MAPK pathways. We demonstrated that the activity of MEK is important for maintaining elevated [Ca(2+)](i) levels induced by GRP-R activation, suggesting that MEK may affect receptor-regulated secretion by modulating the activity of Ca(2+)-sensitive PKC. PMID:10446156

Hellmich, M R; Ives, K L; Udupi, V; Soloff, M S; Greeley, G H; Christensen, B N; Townsend, C M

1999-08-20

301

Characterization of Mutants with Single and Multiple Defects in the Tryptophan Biosynthetic Pathway in Bacillus subtilis  

PubMed Central

Sixty-five tryptophan auxotrophs which map in a cluster on the genome of Bacillus subtilis were characterized on the basis of (i) growth response, (ii) accumulation of intermediate compounds, and (iii) determination of enzymatic defects. They could be placed into six phenotypic classes. Certain of the mutants exhibited pleiotropic effects on more than one enzymatic activity in a manner different from those effects reported for the tryptophan pathway in other organisms. Invariably, mutations in the second gene, that coding for phosphoribosyl transferase activity, were found to lack the indoleglycerol phosphate synthase activity specified by the third gene in the cluster; however, this polarity did not extend to genes more distal in the cluster. Furthermore, mutations in the gene which codes for phosphoribosyl-anthranilate isomerase not only led to a loss of this enzyme but also to a loss of phosphoribosyl transferase and indoleglycerol phosphate synthase. In contrast, mutations in either of the loci coding for these latter functions had no apparent effect on isomerase activity. No polarity of the conventional type was found, e.g., none of the mutations in any gene led to polarized effects on the levels of the enzymes specified by the other genes of the cluster. These observations indicated a possible in vivo aggregation involving the transferase, isomerase, and synthase enzymes, with the isomerase acting as the “key” enzyme in the aggregate.

Whitt, Dixie D.; Carlton, Bruce C.

1968-01-01

302

Overexpression of ubiquitin carboxyl terminal hydrolase impairs multiple pathways during eye development in Drosophila melanogaster.  

PubMed

UCH-L1 (ubiquitin carboxyl terminal hydrolase L1) is well known as an enzyme that hydrolyzes polyubiquitin at its C-terminal to release ubiquitin monomers. Although the overexpression of UCH-L1 inhibits proteasome activity in cultured cells, its biological significance in living organisms has not been clarified in detail. Here, we utilized Drosophila as a model system to examine the effects of the overexpression of dUCH, a Drosophila homologue of UCH-L1, on development. Overexpression in the eye imaginal discs induced a rough eye phenotype in the adult, at least partly resulting from the induction of caspase-dependent apoptosis followed by compensatory proliferation. Genetic crosses with enhancer trap lines marking the photoreceptor cells also revealed that the overexpression of dUCH specifically impaired R7 photoreceptor cell differentiation with a reduction in activated extracellular-signal-regulated kinase signals. Furthermore, the dUCH-induced rough eye phenotype was rescued by co-expression of the sevenless gene or the Draf gene, a downstream component of the mitogen-activated protein kinase (MAPK) cascade. These results indicate that the overexpression of dUCH impairs R7 photoreceptor cell differentiation by down-regulating the MAPK pathway. Interestingly, this process appears to be independent of its pro-apoptotic function. PMID:22526625

Thao, Dang Thi Phuong; An, Phan Nguyen Thuy; Yamaguchi, Masamitsu; LinhThuoc, Tran

2012-06-01

303

Multiple Signaling Pathways Coordinate to Induce a Threshold Response in a Chordate Embryo  

PubMed Central

In animal development, secreted signaling molecules evoke all-or-none threshold responses of target gene transcription to specify cell fates. In the chordate Ciona intestinalis, the neural markers Otx and Nodal are induced at early embryonic stages by Fgf9/16/20 signaling. Here we show that three additional signaling molecules act negatively to generate a sharp expression boundary for neural genes. EphrinA signaling antagonizes FGF signaling by inhibiting ERK phosphorylation more strongly in epidermal cells than in neural cells, which accentuates differences in the strength of ERK activation. However, even weakly activated ERK activates Otx and Nodal transcription occasionally, probably because of the inherently stochastic nature of signal transduction processes and binding of transcription factors to target sequences. This occasional and undesirable activation of neural genes by weak residual ERK activity is directly repressed by Smad transcription factors activated by Admp and Gdf1/3-like signaling, further sharpening the differential responses of cells to FGF signaling. Thus, these signaling pathways coordinate to evoke a threshold response that delineates a sharp expression boundary.

Ohta, Naoyuki; Satou, Yutaka

2013-01-01

304

Multiple pathways are involved in DNA degradation during keratinocyte terminal differentiation.  

PubMed

Loss of the nucleus is a critical step in keratinocyte terminal differentiation. To elucidate the mechanisms involved, we focused on two characteristic events: nuclear translocation of N-terminal fragment of profilaggrin and caspase-14-dependent degradation of the inhibitor of caspase-activated DNase (ICAD). First, we demonstrated that epidermal mesotrypsin liberated a 55-kDa N-terminal fragment of profilaggrin (FLG-N) and FLG-N was translocated into the nucleus. Interestingly, these cells became TUNEL positive. Mutation in the mesotrypsin-susceptible Arg-rich region between FLG-N and the first filaggrin domain abolished these changes. Furthermore, caspase-14 caused limited proteolysis of ICAD, followed by accumulation of caspase-activated DNase (CAD) in TUNEL-positive nuclei. Knockdown of both proteases resulted in a significant increase of remnant nuclei in a skin equivalent model. Immunohistochemical study revealed that both caspase-14 and mesotrypsin were markedly downregulated in parakeratotic areas of lesional skin from patients with atopic dermatitis and psoriasis. Collectively, our results indicate that at least two pathways are involved in the DNA degradation process during keratinocyte terminal differentiation. PMID:24743736

Yamamoto-Tanaka, M; Makino, T; Motoyama, A; Miyai, M; Tsuboi, R; Hibino, T

2014-01-01

305

Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions.  

PubMed

We have previously demonstrated that an intact PSAP motif in the ORF3 protein is required for the formation and release of membrane-associated hepatitis E virus (HEV) particles with ORF3 proteins on their surface. In this study, we investigated the direct interaction between the ORF3 protein and tumour susceptibility gene 101 (Tsg101), a cellular factor involved in the budding of viruses containing the P(T/S)AP late-domain, in PLC/PRF/5 cells expressing the wild-type or PSAP-mutated ORF3 protein and Tsg101 by co-immunoprecipitation. Tsg101 bound to wild-type ORF3 protein, but not to the PSAP-inactive ORF3 protein. To examine whether HEV utilizes the multivesicular body (MVB) pathway to release the virus particles, we analysed the efficiency of virion release from cells upon introduction of small interfering RNA (siRNA) against Tsg101 or dominant-negative (DN) mutants of Vps4 (Vps4A and Vps4B). The relative levels of virus particles released from cells depleted of Tsg101 decreased to 6.4?% of those transfected with negative control siRNA. Similarly, virion egress was significantly reduced by the overexpression of DN forms (Vps4AEQ or Vps4BEQ). The relative levels of virus particles released from cells expressing Vps4AEQ and Vps4BEQ were 19.2 and 15.6?%, respectively, while the overexpression of wild-type Vps4A and Vps4B did not alter the levels of virus release. These results indicate that the ORF3 protein interacts with Tsg101 through the PSAP motifs in infected cells, and that Tsg101 and the enzymic activities of Vps4A and Vps4B are involved in HEV release, thus suggesting that HEV requires the MVB pathway for egress of virus particles. PMID:21880841

Nagashima, Shigeo; Takahashi, Masaharu; Jirintai, Suljid; Tanaka, Toshinori; Nishizawa, Tsutomu; Yasuda, Jiro; Okamoto, Hiroaki

2011-12-01

306

P38 AND EGF RECEPTOR KINASE-MEDIATED ACTIVATION OF THE PHOSPHATIDYLINOSITOL 3-KINASE/AKT PATHWAY IS REQUIRED FOR ZN2+INDUCED CYCLOOXYGENASE-2 EXPRESSION  

EPA Science Inventory

Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus- and cell type-specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction...

307

Gray matter NG2 cells display multiple Ca2+-signaling pathways and highly motile processes.  

PubMed

NG2 cells, the fourth type of glia in the mammalian CNS, receive synaptic input from neurons. The function of this innervation is unknown yet. Postsynaptic changes in intracellular Ca(2+)-concentration ([Ca(2+)](i)) might be a possible consequence. We employed transgenic mice with fluorescently labeled NG2 cells to address this issue. To identify Ca(2+)-signaling pathways we combined patch-clamp recordings, Ca(2+)-imaging, mRNA-transcript analysis and focal pressure-application of various substances to identified NG2-cells in acute hippocampal slices. We show that activation of voltage-gated Ca(2+)-channels, Ca(2+)-permeable AMPA-receptors, and group I metabotropic glutamate-receptors provoke [Ca(2+)](i)-elevations in NG2 cells. The Ca(2+)-influx is amplified by Ca(2+)-induced Ca(2+)-release. Minimal electrical stimulation of presynaptic neurons caused postsynaptic currents but no somatic [Ca(2+)](i) elevations, suggesting that [Ca(2+)](i) elevations in NG2 cells might be restricted to their processes. Local Ca(2+)-signaling might provoke transmitter release or changes in cell motility. To identify structural prerequisites for such a scenario, we used electron microscopy, immunostaining, mRNA-transcript analysis, and time lapse imaging. We found that NG2 cells form symmetric and asymmetric synapses with presynaptic neurons and show immunoreactivity for vesicular glutamate transporter 1. The processes are actin-based, contain ezrin but not glial filaments, microtubules or endoplasmic reticulum. Furthermore, we demonstrate that NG2 cell processes in situ are highly motile. Our findings demonstrate that gray matter NG2 cells are endowed with the cellular machinery for two-way communication with neighboring cells. PMID:21455301

Haberlandt, Christian; Derouiche, Amin; Wyczynski, Alexandra; Haseleu, Julia; Pohle, Jörg; Karram, Khalad; Trotter, Jacqueline; Seifert, Gerald; Frotscher, Michael; Steinhäuser, Christian; Jabs, Ronald

2011-01-01

308

Gray Matter NG2 Cells Display Multiple Ca2+-Signaling Pathways and Highly Motile Processes  

PubMed Central

NG2 cells, the fourth type of glia in the mammalian CNS, receive synaptic input from neurons. The function of this innervation is unknown yet. Postsynaptic changes in intracellular Ca2+-concentration ([Ca2+]i) might be a possible consequence. We employed transgenic mice with fluorescently labeled NG2 cells to address this issue. To identify Ca2+-signaling pathways we combined patch-clamp recordings, Ca2+-imaging, mRNA-transcript analysis and focal pressure-application of various substances to identified NG2-cells in acute hippocampal slices. We show that activation of voltage-gated Ca2+-channels, Ca2+-permeable AMPA-receptors, and group I metabotropic glutamate-receptors provoke [Ca2+]i-elevations in NG2 cells. The Ca2+-influx is amplified by Ca2+-induced Ca2+-release. Minimal electrical stimulation of presynaptic neurons caused postsynaptic currents but no somatic [Ca2+]i elevations, suggesting that [Ca2+]i elevations in NG2 cells might be restricted to their processes. Local Ca2+-signaling might provoke transmitter release or changes in cell motility. To identify structural prerequisites for such a scenario, we used electron microscopy, immunostaining, mRNA-transcript analysis, and time lapse imaging. We found that NG2 cells form symmetric and asymmetric synapses with presynaptic neurons and show immunoreactivity for vesicular glutamate transporter 1. The processes are actin-based, contain ezrin but not glial filaments, microtubules or endoplasmic reticulum. Furthermore, we demonstrate that NG2 cell processes in situ are highly motile. Our findings demonstrate that gray matter NG2 cells are endowed with the cellular machinery for two-way communication with neighboring cells.

Haseleu, Julia; Pohle, Jorg; Karram, Khalad; Trotter, Jacqueline; Seifert, Gerald; Frotscher, Michael; Steinhauser, Christian; Jabs, Ronald

2011-01-01

309

Daphnetin attenuates microglial activation and proinflammatory factor production via multiple signaling pathways.  

PubMed

Daphnetin, a natural coumarin derivative, is known to display anti-inflammatory properties and has been used to treat inflammatory diseases. A novel finding suggested that daphnetin might have a neuroprotective effect in stressed mice, leading us to explore its role in the microglial inflammatory response, as well as its underlying mechanism of action. We found that the production of pro-inflammatory mediators, including interleukin-1? (IL-1?) and tumor necrosis factor-? (TNF-?), induced by lipopolysaccharide (LPS) or ?-amyloid (A?) was significantly suppressed by daphnetin in a dose-dependent manner in BV2 microglia. Also, daphnetin inhibited LPS-induced nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and NO formation by microglia. Mechanistically, daphnetin blunted the transcriptional activity of nuclear factor-kappa B (NF-?B), which was associated with the down-regulation of the phosphorylation and nuclear translocation of RelA/p65. Inhibitors of kappa B (I?B) phosphorylation and degradation were also affected by daphnetin, which was likely due to the reduced activation of I?B kinase (IKK). Additionally, LPS-induced activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK, were, to a varying extent, altered by daphnetin. Finally, daphnetin blocked phosphatidylinositol-3 kinase (PI-3K)/protein kinase B (Akt) signaling in LPS-activated microglia, which appeared to at least partially account for the reduction in NF-?B transcriptional activity. Thus, daphnetin inhibited microglial activation and proinflammatory responses by modulating a series of intracellular signaling pathways, including IKK/I?B, MAPKs and PI-3K/Akt. PMID:24747094

Yu, Wenwen; Wang, Huanhuan; Ying, Hangjie; Yu, Yingying; Chen, Dandan; Ge, Weihong; Shi, Liyun

2014-07-01

310

PPAR?2 NUCLEAR RECEPTOR CONTROLS MULTIPLE REGULATORY PATHWAYS OF OSTEOBLAST DIFFERENTIATION FROM MARROW MESENCHYMAL STEM CELLS  

PubMed Central

Rosiglitazone (Rosi), a member of the thiazolidinedione class of drugs used to treat type 2 diabetes, activates the adipocyte-specific transcription factor peroxisome proliferator-activated receptor gamma (PPAR?). This activation causes bone loss in animals and humans, at least in part due to suppression of osteoblast differentiation from marrow mesenchymal stem cells (MSC). In order to identify mechanisms by which PPAR?2 suppresses osteoblastogenesis and promotes adipogenesis in MSC, we have analyzed the PPAR?2 transcriptome in response to Rosi. A total of 4,252 transcriptional changes resulted when Rosi (1 ?M) was applied to the U-33 marrow stromal cell line stably transfected with PPAR?2 (U-33/?2) as compared to non-induced U-33/?2 cells. Differences between U-33/?2 and U-33 cells stably transfected with empty vector (U-33/c) comprised 7,928 transcriptional changes, independent of Rosi. Cell type-, time- and treatment-specific gene clustering uncovered distinct patterns of PPAR?2 transcriptional control of MSC lineage commitment. The earliest changes accompanying Rosi activation of PPAR?2 included effects on Wnt, TGF?/BMP and G-protein signaling activities, as well as sustained induction of adipocyte-specific gene expression and lipid metabolism. While suppression of osteoblast phenotype is initiated by a diminished expression of osteoblast-specific signaling pathways, induction of the adipocyte phenotype is initiated by adipocyte-specific transcriptional regulators. This indicates that distinct mechanisms govern the repression of osteogenesis and the stimulation of adipogenesis. The co-expression patterns found here indicate that PPAR?2 has a dominant role in controlling osteoblast differentiation and suggests numerous gene-gene interactions that could lead to the identification of a “master” regulatory scheme directing this process.

Shockley, Keith R.; Lazarenko, Oxana P.; Czernik, Piotr J.; Rosen, Clifford J.; Churchill, Gary A.; Lecka-Czernik, Beata

2009-01-01

311

Transgenic muscle-specific Nor-1 expression regulates multiple pathways that effect adiposity, metabolism, and endurance.  

PubMed

The mRNA encoding Nor-1/NR4A3 is rapidly and strikingly induced by ?2-adrenergic signaling in glycolytic and oxidative skeletal muscle. In skeletal muscle cells, Nor-1 expression is important for the regulation of oxidative metabolism. Transgenic skeletal muscle-specific expression of activated Nor-1 resulted in the acquisition of an endurance phenotype, an increase in type IIA/X oxidative muscle fibers, and increased numbers of mitochondria. In the current study, we used dual-energy x-ray absorptiometry and magnetic resonance imaging analysis to demonstrate decreased adiposity in transgenic (Tg) Nor-1 mice relative to that in wild-type littermates. Furthermore, the Tg-Nor-1 mice were resistant to diet-induced weight gain and maintained fasting glucose at normoglycemic levels. Expression profiling and RT-quantitative PCR analysis revealed significant increases in genes involved in glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid oxidation, and glycogen synthesis, in concordance with the lean phenotype. Moreover, expression profiling identified several Z-disc and sarcomeric binding proteins that modulate fiber type phenotype and endurance, eg, ?-actinin-3. In addition, we demonstrated that the Tg-Nor-1 mouse line has significantly higher glycogen content in skeletal muscle relative to that in wild-type littermates. Finally, we identified a decreased NAD(+)/NADH ratio with a concordant increase in peroxisome proliferator-activated receptor ? coactivator-1?1 protein/mRNA expression. Increased NADH was associated with an induction of the genes involved in the malate-aspartate shuttle and a decrease in the glycerol 3-phosphate shuttle, which maximizes aerobic ATP production. In conclusion, skeletal muscle-specific Nor-1 expression regulates genes and pathways that regulate adiposity, muscle fiber type metabolic capacity, and endurance. PMID:24065705

Pearen, Michael A; Goode, Joel M; Fitzsimmons, Rebecca L; Eriksson, Natalie A; Thomas, Gethin P; Cowin, Gary J; Wang, S-C Mary; Tuong, Zewen K; Muscat, George E O

2013-11-01

312

Visual Pathway Axonal Loss in Benign Multiple Sclerosis: A Longitudinal Study  

PubMed Central

Background Benign MS, traditionally defined as EDSS ?3 and ?15 years’ disease duration, is thought to follow a milder course. We determined the extent of visual pathway axonal loss by optical coherence tomography (OCT) retinal nerve fiber layer (RNFL) thickness in a benign MS cohort, and examined the relation to vision and quality of life (QOL). Methods In this longitudinal study of vision in MS at three academic centers, a subset of patients with EDSS, visual function, OCT, and QOL assessments was analyzed. Low- and high-contrast letter acuity were performed to assess visual function. RNFL thickness was determined using OCT-3. QOL scales included the 25-Item National Eye Institute Visual Functioning Questionnaire (NEI-VFQ-25) and SF-36. Results Among 68 patients (135 eyes) studied longitudinally, 13 (26 eyes) had benign MS using criteria of EDSS ?3 and ?15 years disease duration. Benign MS eyes had as much RNFL thinning (-3.6 ?m, P=0.0008 vs. baseline, paired t-test) as typical MS eyes (-3.3 ?m, P<0.0001). Both groups had significant low-contrast acuity loss. Prior history of optic neuritis (ON) was more frequent in benign MS (69% vs. 33% of eyes). History of ON distinguished benign vs. typical MS (P=0.002) and correlated with RNFL thickness at baseline (P=0.002) and disease duration (P=0.03), but not EDSS (P=0.32, logistic regression). NEI-VFQ-25 scores were also worse for benign MS, accounting for age (75±21 vs. 88±11, P=0.005). Conclusions Patients with benign MS have RNFL axonal loss that is as marked as that of typical MS, and have reduced vision and QOL. While overall neurologic impairment is mild, visual dysfunction, not well-captured by the EDSS, accounts for a substantial degree of disability in benign MS.

Galetta, Kristin M.; Graves, Jennifer; Talman, Lauren S.; Lile, Deacon J.; Frohman, Elliot M.; Calabresi, Peter A.; Galetta, Steven L.; Balcer, Laura J.

2012-01-01

313

AKT/mTOR and c-Jun N-terminal kinase signaling pathways are required for chrysotile asbestos-induced autophagy.  

PubMed

Chrysotile asbestos is closely associated with excess mortality from pulmonary diseases such as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms in which chrysotile asbestos fibers induce pulmonary disease have been identified, the role of autophagy in human lung epithelial cells has not been examined. In this study, we evaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelial cells and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos induced autophagy in A549 cells based on a series of biochemical and microscopic autophagy markers. We observed that asbestos increased expression of A549 cell microtubule-associated protein 1 light chain 3 (LC3-II), an autophagy marker, in conjunction with dephosphorylation of phospho-AKT, phospho-mTOR, and phospho-p70S6K. Notably, AKT1/AKT2 double-knockout murine embryonic fibroblasts (MEFs) had negligible asbestos-induced LC3-II expression, supporting a crucial role for AKT signaling. Chrysotile asbestos also led to the phosphorylation/activation of Jun N-terminal kinase (JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited the protein expression of LC3-II. Moreover, JNK2(-/-) MEFs but not JNK1(-/-) MEFs blocked LC3-II levels induced by chrysotile asbestos. In addition, N-acetylcysteine, an antioxidant, attenuated chrysotile asbestos-induced dephosphorylation of P-AKT and completely abolished phosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitor 3-methyladenine or autophagy-related gene 5 siRNA, indicating that the chrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findings demonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotile asbestos-induced autophagy. These data provide a mechanistic basis for possible future clinical applications targeting these signaling pathways in the management of asbestos-induced lung disease. PMID:24735948

Lin, Ziying; Liu, Tie; Kamp, David W; Wang, Yahong; He, Huijuan; Zhou, Xu; Li, Donghong; Yang, Lawei; Zhao, Bin; Liu, Gang

2014-07-01

314

MicroRNA regulate immune pathways in T-cells in multiple sclerosis (MS)  

PubMed Central

Background MicroRNA are small noncoding RNA molecules that are involved in the control of gene expression. To investigate the role of microRNA in multiple sclerosis (MS), we performed genome-wide expression analyses of mRNA and microRNA in T-cells from MS patients and controls. Methods Heparin-anticoagulated peripheral blood was collected from MS-patients and healthy controls followed by isolation of T-cells. MicroRNA and RNA from T-cells was prepared and hybridized to Affymetrix miR 2.0 array and Affymetrix U133Plus 2.0 Human Genome array (Santa Clara, CA), respectively. Verifications were performed with real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Results We identified 2,452 differentially expressed genes and 21 differentially expressed microRNA between MS patients and controls. By Kolmogorov-Smirnov test, 20 of 21 differentially expressed microRNA were shown to affect the expression of their target genes, many of which were involved in the immune system. Tumor necrosis factor ligand superfamily member 14 (TNFSF14) was a microRNA target gene significantly decreased in MS. The differential expression of mir-494, mir-197 and the predicted microRNA target gene TNFSF14 was verified by real-time PCR and ELISA. Conclusion These findings indicate that microRNA may be important regulatory molecules in T-cells in MS.

2013-01-01

315

Multiple pathways for uptake of paraquat, methylglyoxal bis(guanylhydrazone), and polyamines  

SciTech Connect

The uptake of polyamines, methylglyoxal bis(guanylhydrazone) (MGBG), and paraquat (N,N-dimethyl-4,4'-bipyridylium) into control Chinese hamster ovary (CHO) cells and a mutant CHO cell line selected for resistance to the toxicity of MGBG was examined. In contrast to control CHO cells, the mutant cells had no detectable uptake of (/sup 14/C)-MGBG or any of the polyamines. There was no difference between the two cell lines in the uptake of ..cap alpha..-aminoisobutyric (/sup 3/H-AIB), which indicates that there was no general change in membrane transport processes. The mutant cells were also found to be resistant to the toxicity of paraquat and to have a reduced capability to take up the herbicide. This finding confirms that the uptake of paraquat is necessary for the toxicity of this compound and that the paraquat is taken up by a transport system that also transports MGBG. Competition experiments showed that an excess of unlabeled paraquat inhibited uptake of MGBG and, to a lesser extent, uptake of putrescine and spermidine, but no inhibitory action on spermine uptake could be detected. Studies with type II cells isolated from rat lung also demonstrated uptake of paraquat and spermidine, but paraquat was only a weak inhibitor of spermidine uptake in this system. These results suggest that there may be multiple systems for the uptake of MGBG and polyamines and that paraquat is taken up by at least one but not by all of these systems.

Byers, T.L.; Kameji, R.; Rannels, D.E.; Pegg, A.E.

1987-06-01

316

Combination of multiple alignment analysis and surface mapping paves a way for a detailed pathway reconstruction--The case of VHL (von Hippel-Lindau) protein and angiogenesis regulatory pathway  

Microsoft Academic Search

Using the tumor suppressor VHL protein as an example, we show that detailed analysis of conservation versus variation pattern in the multiple alignment can be coupled with the genomic pathway\\/complex conservation analysis to provide a more complete picture of the entire interaction\\/regulatory network. Results from the present study have allowed us to hypothesize that two additional proteins are involved in

SERGEY SIKORA; ADAM GODZIK

2004-01-01

317

Assessing Visual Pathway Function in Multiple Sclerosis Patients with Multifocal Visual Evoked Potentials  

PubMed Central

Background Multifocal visual evoked potentials (mfVEP) provide topographic measure of visual response amplitude and latency. Objective To evaluate the sensitivity and specificity of mfVEP technique in detecting visual abnormalities in multiple sclerosis (MS) patients. Methods mfVEPs were recorded from 74 MS patients with history of optic neuritis (MS-ON, n=74 eyes) or without (MS-no-ON, n=71 eyes), and 50 normal subjects (controls, n=100 eyes) using a 60-sector pattern reversal dartboard stimuli (VERIS). Amplitude and latency for each sector were compared to normative data and assigned probabilities. Size and location of clusters of adjacent abnormal sectors (p < 5%) were examined. Results Mean response amplitudes were 0.39±0.02SE, 0.53±0.02, and 0.60±0.01 for MS-ON, MS-no-ON and control groups, respectively, with significant differences between all groups (p<0.0001). Mean latencies (ms; relative to normative data) were 12.7±1.3SE (MS-ON), 4.3±1.1 (MS-no-ON), and 0.3±0.4 (controls); group differences again significant (p<0.0001). Half the MS-ON eyes had clusters larger than 5 sectors compared to 13% in MS-no-ON and 2% in controls. Abnormal sectors were diffusely distributed, although the largest cluster was smaller than 15 sectors in two thirds of MS-ON eyes. Cluster criteria combining amplitude and latency showed an area of 0.96 under the receiver operating characteristic curve yielding a criterion with 91% sensitivity and 95% specificity. Conclusions The mfVEP provides high sensitivity and specificity in detecting abnormalities in visual function in MS patients.

Laron, Michal; Cheng, Han; Zhang, Bin; Schiffman, Jade S.; Tang, Rosa A.; Frishman, Laura J.

2010-01-01

318

Amyloid Precursor Protein Is Required for Normal Function of the Rod and Cone Pathways in the Mouse Retina  

PubMed Central

Amyloid precursor protein (APP) is a transmembrane glycoprotein frequently studied for its role in Alzheimer's disease. Our recent study in APP knockout (KO) mice identified an important role for APP in modulating normal neuronal development in the retina. However the role APP plays in the adult retina and whether it is required for vision is unknown. In this study we evaluated the role of APP in retinal function and morphology comparing adult wildtype (WT) and APP-KO mice. APP was expressed on neuronal cells of the inner retina, including horizontal, cone bipolar, amacrine and ganglion cells in WT mice. The function of the retina was assessed using the electroretinogram and although the rod photoreceptor responses were similar in APP-KO and WT mice, the post-photoreceptor, inner retinal responses of both the rod and cone pathways were reduced in APP-KO mice. These changes in inner retinal function did not translate to a substantial change in visual acuity as assessed using the optokinetic response or to changes in the gross cellular structure of the retina. These findings indicate that APP is not required for basic visual function, but that it is involved in modulating inner retinal circuitry.

Ho, Tracy; Vessey, Kirstan A.; Cappai, Roberto; Dinet, Virginie; Mascarelli, Frederic; Ciccotosto, Giuseppe D.; Fletcher, Erica L.

2012-01-01

319

Yeast dom34 mutants are defective in multiple developmental pathways and exhibit decreased levels of polyribosomes.  

PubMed Central

The DOM34 gene of Saccharomyces cerevisiae is similar to genes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminisehd. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34delta growth defect. dom34delta mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34delata mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

Davis, L; Engebrecht, J

1998-01-01

320

Evidence for Multiple Export Pathways of Mercury from the Inoperative New Idria Hg Mine, CA  

NASA Astrophysics Data System (ADS)

Understanding mercury transport from inoperative Hg mines is important for California because of the presence of nearly 2,000 abandoned Hg mines in the California Coast Range. Since its closure in 1972, the New Idria Hg mine has developed an extensive acid mine drainage (AMD) system (pH 3) that drains into the San Carlos Creek (pH 9) about 100m downstream of a mine tailings pile. Sediment samples along the AMD system were analyzed using synchrotron radiation-based X-ray fluorescence (XRF), ?-X-ray absorption near edge structure (?-XANES) spectroscopy, extended X-ray absorption fine structure (EXAFS) spectroscopy, and sequential chemical extractions (SCE). It was determined by XRF mapping that Hg within the AMD settling pond sediments occurs mostly as colloids ranging in size from 1-10 ?m. Hg speciation of the colloids, determined by ?-XANES and EXAFS, consisted of 80% ?-HgS and 20% ?-HgS. SCE analysis of sediments along the entire AMD system resulted in the HgS fraction comprising >95% of the total Hg, suggesting minor Hg adsorption. Even though liquid Hg(0) can be panned in the stream it was not detected by SCE, suggesting that liquid Hg(0) settles into deeper portions of the sediments than were sampled. Mercury volatilization to the atmosphere is the other main pathway for Hg release from the New Idria mine site. Analysis of three size fractions of calcine waste material exhibited an increase in Hg volatilization when exposed to light (>500 nm) over dark controls. Calcine size fractions of 500-2000 ?m, 75-125 ?m, and <45 ?m exhibited light:dark ratios of 1.7 ± 0.05, 3.7 ± 0.05, and 4.3 ± 0.1, respectively. A new low-temperature EXAFS technique to directly detect liquid Hg(0) within Hg-contaminated soils was used to determine that mercury speciation in the three size fractions consisted of ?-HgS, ?-HgS, Eglestonite, Montroydite, and liquid Hg(0). The samples with light:dark ratios of 3.7 and 4.3 contained 10% and 9% Hg(0), respectively, while the sample with the lowest ratio had no detectable liquid Hg(0). A plot of light:dark Hg flux ratios vs. % liquid Hg(0) of waste material from other Hg mine sites shows a linear relationship, suggesting that the light:dark ratio of gaseous Hg release from Hg mine sites is strongly influenced by the presence of elemental Hg in the sediments. Based on our results, Hg is being exported from the site as colloidal HgS in the AMD system and by volatilization of liquid Hg(0) present in waste material.

Jew, A. D.; Luong, P. N.; Kim, C. S.; Rytuba, J. J.; Gustin, M. S.; Brown, G. E.

2009-12-01

321

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice  

SciTech Connect

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-{kappa}B). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF's role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-{kappa}B and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-{kappa}B activity and phosphorylation of the inhibitor of kappa B (I{kappa}B{alpha}) increased in ischemic brains, but IRF3, inhibitor of {kappa}B kinase complex-{epsilon} (IKK{epsilon}), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-{kappa}B activity or p-I{kappa}B{alpha} induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-{kappa}B signaling and brain injury after acute cerebral I/R.

Hua, Fang, E-mail: fhua2@emory.edu [Department of Emergency Medicine, Brain Research Laboratory, Emory University School of Medicine, 1365B Clifton Road, Suite 5100, Atlanta, GA 30322 (United States)] [Department of Emergency Medicine, Brain Research Laboratory, Emory University School of Medicine, 1365B Clifton Road, Suite 5100, Atlanta, GA 30322 (United States); Wang, Jun; Sayeed, Iqbal; Ishrat, Tauheed; Atif, Fahim; Stein, Donald G. [Department of Emergency Medicine, Brain Research Laboratory, Emory University School of Medicine, 1365B Clifton Road, Suite 5100, Atlanta, GA 30322 (United States)] [Department of Emergency Medicine, Brain Research Laboratory, Emory University School of Medicine, 1365B Clifton Road, Suite 5100, Atlanta, GA 30322 (United States)

2009-12-18

322

Is multiple sclerosis a disease that requires frequent beta interferon dosing?  

Microsoft Academic Search

The three currently available beta interferon products for the treatment of patients with relapsing-remitting multiple sclerosis (RRMS) are administered according to different regimens. Placebo-controlled clinical trials support the efficacy of both alternate-day interferon beta-1b (Betaferon®) and once-a-week interferon beta-1a (Avonex™), but benefits to patients are probably dependent on the regimen used. Once-weekly administration, perceived to have fewer adverse events and

Luca Durelli

2004-01-01

323

LRP-6 is a coreceptor for multiple fibrogenic signaling pathways in pericytes and myofibroblasts that are inhibited by DKK-1.  

PubMed

Fibrosis of vital organs is a major public health problem with limited therapeutic options. Mesenchymal cells including microvascular mural cells (pericytes) are major progenitors of scar-forming myofibroblasts in kidney and other organs. Here we show pericytes in healthy kidneys have active WNT/?-catenin signaling responses that are markedly up-regulated following kidney injury. Dickkopf-related protein 1 (DKK-1), a ligand for the WNT coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP-5 and LRP-6) and an inhibitor of WNT/?-catenin signaling, effectively inhibits pericyte activation, detachment, and transition to myofibroblasts in vivo in response to kidney injury, resulting in attenuated fibrogenesis, capillary rarefaction, and inflammation. DKK-1 blocks activation and proliferation of established myofibroblasts in vitro and blocks pericyte proliferation to PDGF, pericyte migration, gene activation, and cytoskeletal reorganization to TGF-? or connective tissue growth factor. These effects are largely independent of inhibition of downstream ?-catenin signaling. DKK-1 acts predominantly by inhibiting PDGF-, TGF-?-, and connective tissue growth factor-activated MAPK and JNK signaling cascades, acting via LRP-6 with associated WNT ligand. Biochemically, LRP-6 interacts closely with PDGF receptor ? and TGF-? receptor 1 at the cell membrane, suggesting that it may have roles in pathways other than WNT/?-catenin. In summary, DKK-1 blocks many of the changes in pericytes required for myofibroblast transition and attenuates established myofibroblast proliferation/activation by mechanisms dependent on LRP-6 and WNT ligands but not the downstream ?-catenin pathway. PMID:23302695

Ren, Shuyu; Johnson, Bryce G; Kida, Yujiro; Ip, Colin; Davidson, Kathryn C; Lin, Shuei-Liong; Kobayashi, Akio; Lang, Richard A; Hadjantonakis, Anna-Katerina; Moon, Randall T; Duffield, Jeremy S

2013-01-22

324

Targeting the IL-6 pathway in multiple myeloma and its implications in cancer-associated gene hypermethylation.  

PubMed

Aberrant methylation of tumor suppressor genes (TSG) is an important epigenetic event in cancer, including multiple myeloma (MM). Interleukin-6 (IL-6), which plays a significant role in the pathogenesis of MM, also regulates DNA methylation. However, attempts to bring IL-6 blockade to the clinic have had limited success. We hypothesize that IL-6 regulation of hypermethylation may be an important pathway leading to rational chemotherapeutic/anti-IL-6 combinations. We first studied the correlation of IL-6 expression and dependence in MM cell lines: U266B1, RPMI8226, and KAS6/1. We confirmed that KAS6/1 is IL-6-dependent whereas U266B1 and RPMI8226 cells are IL-6-independent and that blocking IL-6 inhibited the growth of U266B1 (36% inhibition; p<0.05) and KAS6/1 (68% inhibition; p<0.01), but not the RPMI8226 cells. Using RT-PCR, we showed that U266B1 cells express IL-6, but RPMI8226 and KAS6/1 cells do not. This IL-6 expression pattern correlates with the anti-IL-6 inhibition findings. To correlate IL-6 sensitivity with hypermethylation of TSG, we investigated promoter methylation of CDH1 and DcR1. We found that the promoter of DcR1 and CDH1 is methylated in U266B1 cells and un-methylated in RPMI8226 cells. Furthermore, the DcR1 promoter was un-methylated in KAS6/1 cells. These data support our hypothesis that an IL-6-dependent pathway may regulate hypermethylation of TSG in MM. Newer chemotherapeutic agents that affect methylation are being studied in combination with IL-6 blockade. PMID:21801145

Ingersoll, Susan Blaydes; Ahmad, Sarfraz; Thoni, Natalie D; Ahmed, Farhana H; Monahan, Kimberly A; Edwards, John R

2011-09-01

325

Ursolic Acid Simultaneously Targets Multiple Signaling Pathways to Suppress Proliferation and Induce Apoptosis in Colon Cancer Cells  

PubMed Central

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid distributed in medical herbs, exerts antitumor effects and is emerging as a promising compound for cancer prevention and therapy, but its excise mechanisms of action in colon cancer cells remains largely unknown. Here, we identified the molecular mechanisms by which UA inhibited cell proliferation and induced apoptosis in human colon cancer SW480 and LoVo cells. Treatment with UA led to significant inhibitions in cell viability and clone formation and changes in cell morphology and spreading. UA also suppressed colon cancer cell migration by inhibiting MMP9 and upregulating CDH1 expression. Further studies showed that UA inhibited the phosphorylation of Akt and ERK proteins. Pretreatment with an Akt or ERK-specific inhibitor considerably abrogated the proliferation inhibition by UA. UA also significantly inhibited colon cancer cell COX-2 expression and PGE2 production. Pretreatment with a COX-2 inhibitor (celecoxib) abrogated the UA-induced cell proliferation. Moreover, we found that UA effectively promoted NF-?B and p300 translocation from cell nuclei to cytoplasm, and attenuated the p300-mediated acetylation of NF-?B and CREB2. Pretreatment with a p300 inhibitor (roscovitine) abrogated the UA-induced cell proliferation, which is reversed by p300 overexpression. Furthermore, UA treatment induced colon cancer cell apoptosis, increased the cleavage of PARP, caspase-3 and 9, and trigged the release of cytochrome c from mitochondrial inter-membrane space into cytosol. These results indicate that UA inhibits cell proliferation and induces apoptosis in colon cancer cells through simultaneous modulation of the multiple signaling pathways such as MMP9/CDH1, Akt/ERK, COX-2/PGE2, p300/NF-?B/CREB2, and cytochrome c/caspase pathways.

Zhang, Xiaohong; Guo, Wei; Chen, Wangbing; Tian, Yun; Fu, Lingyi; Shi, Dingbo; Cheng, Jianding; Huang, Wenlin; Deng, Wuguo

2013-01-01

326

Identification and Analyses of AUX-IAA target genes controlling multiple pathways in developing fiber cells of Gossypium hirsutum L  

PubMed Central

Technological development led to an increased interest in systems biological approaches in plants to characterize developmental mechanism and candidate genes relevant to specific tissue or cell morphology. AUX-IAA proteins are important plant-specific putative transcription factors. There are several reports on physiological response of this family in Arabidopsis but in cotton fiber the transcriptional network through which AUX-IAA regulated its target genes is still unknown. in-silico modelling of cotton fiber development specific gene expression data (108 microarrays and 22,737 genes) using Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) reveals 3690 putative AUX-IAA target genes of which 139 genes were known to be AUX-IAA co-regulated within Arabidopsis. Further AUX-IAA targeted gene regulatory network (GRN) had substantial impact on the transcriptional dynamics of cotton fiber, as showed by, altered TF networks, and Gene Ontology (GO) biological processes and metabolic pathway associated with its target genes. Analysis of the AUX-IAA-correlated gene network reveals multiple functions for AUX-IAA target genes such as unidimensional cell growth, cellular nitrogen compound metabolic process, nucleosome organization, DNA-protein complex and process related to cell wall. These candidate networks/pathways have a variety of profound impacts on such cellular functions as stress response, cell proliferation, and cell differentiation. While these functions are fairly broad, their underlying TF networks may provide a global view of AUX-IAA regulated gene expression and a GRN that guides future studies in understanding role of AUX-IAA box protein and its targets regulating fiber development.

Nigam, Deepti; Sawant, Samir V

2013-01-01

327

Model-Derived Dispersal Pathways from Multiple Source Populations Explain Variability of Invertebrate Larval Supply  

PubMed Central

Background Predicting the spatial and temporal patterns of marine larval dispersal and supply is a challenging task due to the small size of the larvae and the variability of oceanographic processes. Addressing this problem requires the use of novel approaches capable of capturing the inherent variability in the mechanisms involved. Methodology/Principal Findings In this study we test whether dispersal and connectivity patterns generated from a bio-physical model of larval dispersal of the crab Carcinus maenas, along the west coast of the Iberian Peninsula, can predict the highly variable daily pattern of wind-driven larval supply to an estuary observed during the peak reproductive season (March–June) in 2006 and 2007. Cross-correlations between observed and predicted supply were significant (p<0.05) and strong, ranging from 0.34 to 0.81 at time lags of ?6 to +5 d. Importantly, the model correctly predicted observed cross-shelf distributions (Pearson r?=?0.82, p<0.001, and r?=?0.79, p<0.01, in 2006 and 2007) and indicated that all supply events were comprised of larvae that had been retained within the inner shelf; larvae transported to the outer shelf and beyond never recruited. Estimated average dispersal distances ranged from 57 to 198 km and were only marginally affected by mortality. Conclusions/Significance The high degree of predicted demographic connectivity over relatively large geographic scales is consistent with the lack of genetic structuring in C. maenas along the Iberian Peninsula. These findings indicate that the dynamic nature of larval dispersal can be captured by mechanistic biophysical models, which can be used to provide meaningful predictions of the patterns and causes of fine-scale variability in larval supply to marine populations.

Domingues, Carla P.; Nolasco, Rita; Dubert, Jesus; Queiroga, Henrique

2012-01-01

328

Multiple organellar RNA editing factor (MORF) family proteins are required for RNA editing in mitochondria and plastids of plants  

PubMed Central

RNA editing in plastids and mitochondria of flowering plants changes hundreds of selected cytidines to uridines, mostly in coding regions of mRNAs. Specific sequences around the editing sites are presumably recognized by up to 200 pentatricopeptide repeat (PPR) proteins. The here identified family of multiple organellar RNA editing factor (MORF) proteins provides additional components of the RNA editing machinery in both plant organelles. Two MORF proteins are required for editing in plastids; at least two are essential for editing in mitochondria. The loss of a MORF protein abolishes or lowers editing at multiple sites, many of which are addressed individually by PPR proteins. In plastids, both MORF proteins are required for complete editing at almost all sites, suggesting a heterodimeric complex. In yeast two-hybrid and pull-down assays, MORF proteins can connect to form hetero- and homodimers. Furthermore, MORF proteins interact selectively with PPR proteins, establishing a more complex editosome in plant organelles than previously thought.

Takenaka, Mizuki; Zehrmann, Anja; Verbitskiy, Daniil; Kugelmann, Matthias; Hartel, Barbara; Brennicke, Axel

2012-01-01

329

Proteomic and functional analysis of NCS-1 binding proteins reveals novel signaling pathways required for inner ear development in zebrafish  

PubMed Central

Background The semicircular canals, a subdivision of the vestibular system of the vertebrate inner ear, function as sensors of angular acceleration. Little is currently known, however, regarding the underlying molecular mechanisms that govern the development of this intricate structure. Zebrafish represent a particularly tractable model system for the study of inner ear development. This is because the ear can be easily visualized during early embryogenesis, and both forward and reverse genetic techniques are available that can be applied to the discovery of novel genes that contribute to proper ear development. We have previously shown that in zebrafish, the calcium sensing molecule neuronal calcium sensor-1 (NCS-1) is required for semicircular canal formation. The function of NCS-1 in regulating semicircular canal formation has not yet been elucidated. Results We initiated a multistep functional proteomic strategy to identify neuronal calcium sensor-1 (NCS-1) binding partners (NBPs) that contribute to inner ear development in zebrafish. By performing a Y2H screen in combination with literature and database searches, we identified 10 human NBPs. BLAST searches of the zebrafish EST and genomic databases allowed us to clone zebrafish orthologs of each of the human NBPs. By investigating the expression profiles of zebrafish NBP mRNAs, we identified seven that were expressed in the developing inner ear and overlapped with the ncs-1a expression profile. GST pulldown experiments confirmed that selected NBPs interacted with NCS-1, while morpholino-mediated knockdown experiments demonstrated an essential role for arf1, pi4k?, dan, and pink1 in semicircular canal formation. Conclusion Based on their functional profiles, the hypothesis is presented that Ncs-1a/Pi4k?/Arf1 form a signaling pathway that regulates secretion of molecular components, including Dan and Bmp4, that are required for development of the vestibular apparatus. A second set of NBPs, consisting of Pink1, Hint2, and Slc25a25, are destined for localization in mitochondria. Our findings reveal a novel signalling pathway involved in development of the semicircular canal system, and suggest a previously unrecognized role for NCS-1 in mitochondrial function via its association with several mitochondrial proteins.

Petko, Jessica A; Kabbani, Nadine; Frey, Colleen; Woll, Matthew; Hickey, Katharine; Craig, Michael; Canfield, Victor A; Levenson, Robert

2009-01-01

330

Ablation of Vacuole Protein Sorting 18 (Vps18) Gene Leads to Neurodegeneration and Impaired Neuronal Migration by Disrupting Multiple Vesicle Transport Pathways to Lysosomes*?  

PubMed Central

Intracellular vesicle transport pathways are critical for neuronal survival and central nervous system development. The Vps-C complex regulates multiple vesicle transport pathways to the lysosome in lower organisms. However, little is known regarding its physiological function in mammals. We deleted Vps18, a central member of Vps-C core complex, in neural cells by generating Vps18F/F; Nestin-Cre mice (Vps18 conditional knock-out mice). These mice displayed severe neurodegeneration and neuronal migration defects. Mechanistic studies revealed that Vps18 deficiency caused neurodegeneration by blocking multiple vesicle transport pathways to the lysosome, including autophagy, endocytosis, and biosynthetic pathways. Our study also showed that ablation of Vps18 resulted in up-regulation of ?1 integrin in mouse brain probably due to lysosome dysfunction but had no effects on the reelin pathway, expression of N-cadherin, or activation of JNK, which are implicated in the regulation of neuronal migration. Finally, we demonstrated that knocking down ?1 integrin partially rescued the migration defects, suggesting that Vps18 deficiency-mediated up-regulation of ?1 integrin may contribute to the defect of neuronal migration in the Vps18-deficient brain. Our results demonstrate important roles of Vps18 in neuron survival and migration, which are disrupted in multiple neural disorders.

Peng, Chao; Ye, Jian; Yan, Shunfei; Kong, Shanshan; Shen, Ye; Li, Chenyu; Li, Qinyu; Zheng, Yufang; Deng, Kejing; Xu, Tian; Tao, Wufan

2012-01-01

331

Multiple Activities of LigB Potentiate Virulence of Leptospira interrogans: Inhibition of Alternative and Classical Pathways of Complement  

PubMed Central

Microbial pathogens acquire the immediate imperative to avoid or counteract the formidable defense of innate immunity as soon as they overcome the initial physical barriers of the host. Many have adopted the strategy of directly disrupting the complement system through the capture of its components, using proteins on the pathogen's surface. In leptospirosis, pathogenic Leptospira spp. are resistant to complement-mediated killing, in contrast to the highly vulnerable non-pathogenic strains. Pathogenic L. interrogans uses LenA/LfhA and LcpA to respectively sequester and commandeer the function of two regulators, factor H and C4BP, which in turn bind C3b or C4b to interrupt the alternative or classical pathways of complement activation. LigB, another surface-proximal protein originally characterized as an adhesin binding multiple host proteins, has other activities suggesting its importance early in infection, including binding extracellular matrix, plasma, and cutaneous repair proteins and inhibiting hemostasis. In this study, we used a recent model of ectopic expression of LigB in the saprophyte, L. biflexa, to test the hypothesis that LigB also interacts with complement proteins C3b and C4b to promote the virulence of L. interrogans. The surface expression of LigB partially rescued the non-pathogen from killing by 5% normal human serum, showing 1.3- to 48-fold greater survival 4 to 6 d following exposure to complement than cultures of the non-expressing parental strain. Recombinant LigB7?-12 comprising the LigB-specific immunoglobulin repeats binds directly to human complement proteins, C3b and C4b, with respective Kds of 43±26 nM and 69±18 nM. Repeats 9 to 11, previously shown to contain the binding domain for fibronectin and fibrinogen, are also important in LigB-complement interactions, which interfere with the alternative and classical pathways measured by complement-mediated hemolysis of erythrocytes. Thus, LigB is an adaptable interface for L. interrogans to efficiently counteract the multiple homeostatic processes of the host.

Choy, Henry A.

2012-01-01

332

Autographa californica Multiple Nucleopolyhedrovirus EXON0 (ORF141) Is Required for Efficient Egress of Nucleocapsids from the Nucleus  

Microsoft Academic Search

Received 20 March 2007\\/Accepted 2 July 2007 Autographa californica multiple nucleopolyhedrovirus (AcMNPV) exon0 (orf141) has been shown to be required for the efficient production of budded virus (BV). The deletion of exon0 reduces the level of BV production by up to 99% (X. Dai, T. M. Stewart, J. A. Pathakamuri, Q. Li, and D. A. Theilmann, J. Virol. 78:9633-9644, 2004);

Minggang Fang; Xiaojiang Dai; David A. Theilmann

2007-01-01

333

76 FR 14548 - Federal Acquisition Regulation; Requirements for Acquisitions Pursuant to Multiple-Award Contracts  

Federal Register 2010, 2011, 2012, 2013

...practicable, consistent with market research appropriate to the circumstances...Government's requirements and market research indicates other companies' similar...results from acceptance of an unsolicited research proposal that demonstrates a unique...

2011-03-16

334

Multiple Wnt genes are required for segmentation in the short-germ embryo of Tribolium castaneum.  

PubMed

wingless (wg)/Wnt family are essential to development in virtually all metazoans. In short-germ insects, including the red flour beetle (Tribolium castaneum), the segment-polarity function of wg is conserved [1]. Wnt signaling is also implicated in posterior patterning and germband elongation [2-4], but despite its expression in the posterior growth zone, Wnt1/wg alone is not responsible for these functions [1-3]. Tribolium contains additional Wnt family genes that are also expressed in the growth zone [5]. After depleting Tc-WntD/8 we found a small percentage of embryos lacking abdominal segments. Additional removal of Tc-Wnt1 significantly enhanced the penetrance of this phenotype. Seeking alternative methods to deplete Wnt signal, we performed RNAi with other components of the Wnt pathway including wntless (wls), porcupine (porc), and pangolin (pan). Tc-wls RNAi caused segmentation defects similar to Tc-Wnt1 RNAi, but not Tc-WntD/8 RNAi, indicating that Tc-WntD/8 function is Tc-wls independent. Depletion of Tc-porc and Tc-pan produced embryos resembling double Tc-Wnt1,Tc-WntD/8 RNAi embryos, suggesting that Tc-porc is essential for the function of both ligands, which signal through the canonical pathway. This is the first evidence of functional redundancy between Wnt ligands in posterior patterning in short-germ insects. This Wnt function appears to be conserved in other arthropods [6] and vertebrates [7-9]. PMID:18926702

Bolognesi, Renata; Farzana, Laila; Fischer, Tamara D; Brown, Susan J

2008-10-28

335

Multiple Wnt genes are required for posterior patterning in the short germ embryo of Tribolium castaneum  

PubMed Central

Summary wingless (wg)/Wnt family genes encode secreted glycoproteins essential for the development of virtually all metazoans. In short germ insects, including the red flour beetle, Tribolium castaneum, the segment-polarity function of wg is conserved [1]. Wnt signalling is also implicated in posterior patterning and germband elongation [2–4], but despite its expression in the posterior growth zone, Wnt1/wg alone is not responsible for these functions; [1–3]. Tribolium contains additional Wnt family genes of unknown function that are also expressed in the growth zone [5]. After depleting one of these, Tc-WntD/8, we found a small percentage of embryos lacking abdominal segments. Additional removal of Tc-Wnt1 significantly enhanced this phenotype, suggesting functional redundancy. Seeking alternative methods to deplete Wnt signal, we performed RNAi with other components of the Wnt pathway including wntless (wls) and porcupine (porc), which process Wnt ligands, and pangolin (pan), which transduces the signal to the nucleus. Tc-wls RNAi caused segmentation defects similar to Tc-Wnt1, but not Tc-WntD/8 RNAi, indicating that the effects of Tc-WntD/8 depletion are Tc-wls-independent. In contrast, depletion of Tc-porc and Tc-pan resulted in embryos resembling those of double Tc-Wnt1,Tc-WntD/8 RNAi, suggesting Tc-porc is essential for the function of both ligands and that they signal through the canonical pathway. Our results provide the first evidence of functional redundancy between Wnt ligands in posterior patterning in short germ insects. This Wnt function appears to be conserved in other arthropods [6] and vertebrates [7–9].

Bolognesi, Renata; Farzana, Laila; Fischer, Tamara D.; Brown, Susan J.

2008-01-01

336

A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants  

NASA Technical Reports Server (NTRS)

We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

Yang, Tianbao; Poovaiah, B. W.

2002-01-01

337

Delivery of endocytosed proteins to the cell-division plane requires change of pathway from recycling to secretion.  

PubMed

Membrane trafficking is essential to fundamental processes in eukaryotic life, including cell growth and division. In plant cytokinesis, post-Golgi trafficking mediates a massive flow of vesicles that form the partitioning membrane but its regulation remains poorly understood. Here, we identify functionally redundant Arabidopsis ARF guanine-nucleotide exchange factors (ARF-GEFs) BIG1-BIG4 as regulators of post-Golgi trafficking, mediating late secretion from the trans-Golgi network but not recycling of endocytosed proteins to the plasma membrane, although the TGN also functions as an early endosome in plants. In contrast, BIG1-4 are absolutely required for trafficking of both endocytosed and newly synthesized proteins to the cell-division plane during cytokinesis, counteracting recycling to the plasma membrane. This change from recycling to secretory trafficking pathway mediated by ARF-GEFs confers specificity of cargo delivery to the division plane and might thus ensure that the partitioning membrane is completed on time in the absence of a cytokinesis-interphase checkpoint. DOI: http://dx.doi.org/10.7554/eLife.02131.001. PMID:24714496

Richter, Sandra; Kientz, Marika; Brumm, Sabine; Nielsen, Mads Eggert; Park, Misoon; Gavidia, Richard; Krause, Cornelia; Voss, Ute; Beckmann, Hauke; Mayer, Ulrike; Stierhof, York-Dieter; Jürgens, Gerd

2014-01-01

338

Delivery of endocytosed proteins to the cell-division plane requires change of pathway from recycling to secretion  

PubMed Central

Membrane trafficking is essential to fundamental processes in eukaryotic life, including cell growth and division. In plant cytokinesis, post-Golgi trafficking mediates a massive flow of vesicles that form the partitioning membrane but its regulation remains poorly understood. Here, we identify functionally redundant Arabidopsis ARF guanine-nucleotide exchange factors (ARF-GEFs) BIG1–BIG4 as regulators of post-Golgi trafficking, mediating late secretion from the trans-Golgi network but not recycling of endocytosed proteins to the plasma membrane, although the TGN also functions as an early endosome in plants. In contrast, BIG1-4 are absolutely required for trafficking of both endocytosed and newly synthesized proteins to the cell–division plane during cytokinesis, counteracting recycling to the plasma membrane. This change from recycling to secretory trafficking pathway mediated by ARF-GEFs confers specificity of cargo delivery to the division plane and might thus ensure that the partitioning membrane is completed on time in the absence of a cytokinesis-interphase checkpoint. DOI: http://dx.doi.org/10.7554/eLife.02131.001

Richter, Sandra; Kientz, Marika; Brumm, Sabine; Nielsen, Mads Eggert; Park, Misoon; Gavidia, Richard; Krause, Cornelia; Voss, Ute; Beckmann, Hauke; Mayer, Ulrike; Stierhof, York-Dieter; Jurgens, Gerd

2014-01-01

339

Kindlin-3-mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption  

PubMed Central

The blood cell–specific kindlin-3 protein is required to activate leukocyte and platelet integrins. In line with this function, mutations in the KINDLIN-3 gene in man cause immunodeficiency and severe bleeding. Some patients also suffer from osteopetrosis, but the underlying mechanism leading to abnormal bone turnover is unknown. Here we show that kindlin-3–deficient mice develop severe osteopetrosis because of profound adhesion and spreading defects in bone-resorbing osteoclasts. Mechanistically, loss of kindlin-3 impairs the activation of ?1, ?2, and ?3 integrin classes expressed on osteoclasts, which in turn abrogates the formation of podosomes and sealing zones required for bone resorption. In agreement with these findings, genetic ablation of all integrin classes abolishes the development of podosomes, mimicking kindlin-3 deficiency. Although loss of single integrin classes gives rise to podosomes, their resorptive activity is impaired. These findings show that osteoclasts require their entire integrin repertoire to be regulated by kindlin-3 to orchestrate bone homeostasis.

Schmidt, Sarah; Nakchbandi, Inaam; Ruppert, Raphael; Kawelke, Nina; Hess, Michael W.; Pfaller, Kristian; Jurdic, Pierre; Fassler, Reinhard

2011-01-01

340

Processing of meiotic DNA double strand breaks requires cyclin-dependent kinase and multiple nucleases.  

PubMed

Meiotic recombination requires the formation of programmed Spo11-dependent DNA double strand breaks (DSBs). In Saccharomyces cerevisiae, the Sae2 protein and the Mre11-Rad50-Xrs2 complex are necessary to remove the covalently attached Spo11 protein from the DNA ends, which are then resected by so far unknown nucleases. Here, we demonstrate that phosphorylation of Sae2 Ser-267 by cyclin-dependent kinase 1 (Cdk1) is required to initiate meiotic DSB resection by allowing Spo11 removal from DSB ends. This finding suggests that Cdk1 activity is required for the processing of Spo11-induced DSBs, thus providing a mechanism for coordinating DSB resection with progression through meiotic prophase. Furthermore, the helicase Sgs1 and the nucleases Exo1 and Dna2 participate in lengthening the 5'-3' resection tracts during meiosis by controlling a step subsequent to Spo11 removal. PMID:20150422

Manfrini, Nicola; Guerini, Ilaria; Citterio, Andrea; Lucchini, Giovanna; Longhese, Maria Pia

2010-04-01

341

Multiple BiP genes of Arabidopsis thaliana are required for male gametogenesis and pollen competitiveness.  

PubMed

Immunoglobulin-binding protein (BiP) is a molecular chaperone of the heat shock protein 70 (Hsp70) family. BiP is localized in the endoplasmic reticulum (ER) and plays key roles in protein translocation, protein folding and quality control in the ER. The genomes of flowering plants contain multiple BiP genes. Arabidopsis thaliana has three BiP genes. BIP1 and BIP2 are ubiquitously expressed. BIP3 encodes a less well conserved BiP paralog, and it is expressed only under ER stress conditions in the majority of organs. Here, we report that all BiP genes are expressed and functional in pollen and pollen tubes. Although the bip1 bip2 double mutation does not affect pollen viability, the bip1 bip2 bip3 triple mutation is lethal in pollen. This result indicates that lethality of the bip1 bip2 double mutation is rescued by BiP3 expression. A decrease in the copy number of the ubiquitously expressed BiP genes correlates well with a decrease in pollen tube growth, which leads to reduced fitness of mutant pollen during fertilization. Because an increased protein secretion activity is expected to increase the protein folding demand in the ER, the multiple BiP genes probably cooperate with each other to ensure ER homeostasis in cells with active secretion such as rapidly growing pollen tubes. PMID:24486762

Maruyama, Daisuke; Sugiyama, Tomoyuki; Endo, Toshiya; Nishikawa, Shuh-Ichi

2014-04-01

342

Interpretation and applicability of empirical tissue enhancement metrics in dynamic contrast-enhanced MRI based on a multiple pathway model  

NASA Astrophysics Data System (ADS)

Computer simulations based on a physiologically realistic tracer kinetic model with multiple pathways was used to provide insights on the applicability and interpretation of tissue enhancement metrics such as the maximum slope, peak enhancement and area under curve, commonly used in dynamic contrast-enhanced (DCE) MRI. Results show that physiological conditions of the tissue that could affect the accuracy of the maximal slope method include a high blood flow, increased variability of flow within the vasculature or a low vascular volume. Interestingly, changes in permeability and interstitial volume might not affect the accuracy of the maximal slope method. Time-to-peak and peak value of the tissue enhancement curve are not strictly properties of the tissue alone, and they cannot be linearly related to intrinsic tissue parameters such as blood flow, blood volume, capillary permeability, interstitial volume and mean transit time. Similar to the normalized initial area under tissue concentration curve, an alternative estimate of the total tracer distribution volume can be simply given by the ratio of tracer concentration in the tissue and artery sampled at the final DCE scan.

Koh, T. S.; Shi, W.; Thng, C. H.; Kwek, J. W.; Bisdas, S.; Khoo, J. B. K.

2012-08-01

343

Streptococcus pneumoniae Invades Endothelial Host Cells via Multiple Pathways and Is Killed in a Lysosome Dependent Manner  

PubMed Central

Streptococcus pneumoniae is one of the major causative agents of pneumonia, sepsis, meningitis and other morbidities. In spite of its heavy disease burden, surprisingly little is known about the mechanisms involved in the switch of life style, from commensal colonizer of the nasopharynx to invasive pathogen. In vitro experiments, and mouse models have shown that S. pneumoniae can be internalized by host cells, which coupled with intracellular vesicle transport through the cells, i.e. transcytosis, is suggested to be the first step of invasive disease. To further dissect the process of S. pneumoniae internalization, we chemically inhibited discrete parts of the cellular uptake system. We show that this invasion of the host cells was facilitated via both clathrin- and caveolae-mediated endocytosis. After internalization we demonstrated that the bulk of the internalized S. pneumoniae was killed in the lysosome. Interestingly, inhibition of the lysosome altered transcytosis dynamics as it resulted in an increase in the transport of the internalized bacteria out of the cells via the basal side. These results show that uptake of S. pneumoniae into host cells occurs via multiple pathways, as opposed to the often proposed view of invasion being dependent on specific, and singular receptor-mediated endocytosis. This indicates that the endothelium not only has a critical role as a physical barrier against S. pneumoniae in the blood stream, but also in degrading S. pneumonia cells that have adhered to, and invaded the endothelial cells.

Gradstedt, Henrik; Iovino, Federico; Bijlsma, Jetta J. E.

2013-01-01

344

Betulinic acid suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase SHP-1 in human multiple myeloma cells.  

PubMed

STAT3 activation has been associated with survival, proliferation and invasion of various human cancers. Whether betulinic acid, a pentacyclic triterpene, can modulate the STAT3 pathway, was investigated in human multiple myeloma (MM) cells. We found that betulinic acid inhibited constitutive activation of STAT3, Src kinase, JAK1 and JAK2. Pervanadate reversed the betulinic acid-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, betulinic acid induced the expression of the PTP SHP-1 and silencing of the SHP-1 gene abolished the ability of betulinic acid to inhibit STAT3 activation and rescued betulinic acid-induced cell death. Betulinic acid also downregulated the expression of STAT3-regulated gene products such as bcl-xL, bcl-2, cyclin D1 and survivin. This correlated with an increase in apoptosis as indicated by an increase in the sub-G1 cell population and an increase in caspase-3-induced PARP cleavage. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the betulinic acid-induced apoptosis. Betulinic acid also enhanced the apoptosis induced by thalidomide (from 10 to 55%) and bortezomib (from 5 to 70%) in MM cells. Overall, our results suggest that betulinic acid downregulates STAT3 activation through upregulation of SHP-1, and this may have potential in sensitization of STAT3 overexpressing tumors to chemotherapeutic agents. PMID:19937797

Pandey, Manoj K; Sung, Bokyung; Aggarwal, Bharat B

2010-07-15

345

Betulinic Acid Suppresses STAT3 Activation Pathway Through Induction of Protein Tyrosine Phosphatase SHP-1 in Human Multiple Myeloma Cells  

PubMed Central

STAT3 activation has been associated with survival, proliferation and invasion of various human cancers. Whether betulinic acid, a pentacyclic triterpene, can modulates the STAT3 pathway, was investigated in human multiple myeloma (MM) cells. We found that betulinic acid inhibited constitutive activation of STAT3, Src kinase, JAK1 and JAK2. Pervanadate reversed the betulinic acid -induced down regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, betulinic acid induced the expression of the PTP SHP-1 and silencing of the SHP-1 gene abolished the ability of betulinic acid to inhibit STAT3 activation and rescues betulinic acid-induced cell death. Betulinic acid also downregulated the expression of STAT3-regulated gene products such as bcl-xL, bcl-2, cyclin D1, and survivin. This correlated with an increase in apoptosis as indicated by an increase in the sub-G1 cell population and an increase in caspase-3–induced PARP cleavage. Consistent with these results, over expression of constitutive active STAT3 significantly reduced the betulinic acid-induced apoptosis. Betulinic acid also enhanced the apoptosis induced by thalidomide (from 10% to 55%) and bortezomib (from 5% to 70%) in MM cells. Overall, our results suggest that betulinic acid down regulates STAT3 activation through upregulation of SHP-1 and this may have potential in sensitization of STAT3 over expressing tumors to chemotherapeutic agents.

Pandey, Manoj K.; Sung, Bokyung; Aggarwal, Bharat B.

2009-01-01

346

A genome-wide siRNA screen reveals multiple mTORC1 independent signaling pathways regulating autophagy under normal nutritional conditions  

PubMed Central

Summary Autophagy is a cellular catabolic mechanism that plays an essential function in protecting multicellular eukaryotes from neurodegeneration, cancer and other diseases. However, we still know very little about mechanisms regulating autophagy under normal homeostatic conditions when nutrients are not limiting. In a genome-wide human siRNA screen, we demonstrate that under normal nutrient conditions up regulation of autophagy requires the type III PI3 kinase, but not inhibition of mTORC1, the essential negative regulator of starvation-induced autophagy. We show that a group of growth factors and cytokines inhibit the type III PI3 kinase through multiple pathways, including the MAPK-ERK1/2, Stat3, Akt/Foxo3 and CXCR4/GPCR, which are all known to positively regulate cell growth and proliferation. Our study suggests that the type III PI3 kinase integrates diverse signals to regulate cellular levels of autophagy, and that autophagy and cell proliferation may represent two alternative cell fates that are regulated in a mutually exclusive manner.

Lipinski, Marta M.; Hoffman, Greg; Ng, Aylwin; Zhou, Wen; Py, Benedicte F.; Hsu, Emily; Liu, Xuxin; Eisenberg, Jason; Liu, Jun; Blenis, John; Xavier, Ramnik J.; Yuan, Junying

2010-01-01

347

49 CFR 173.423 - Requirements for multiple hazard limited quantity Class 7 (radioactive) materials.  

Code of Federal Regulations, 2012 CFR

(a) Except as provided in § 173.4, when a limited quantity radioactive material meets the definition of another hazard class or division, it must beâ (1) Classed for the additional hazard; (2) Packaged to conform with the requirements specified in §...

2013-10-01

348

Multiple Apoptotic Caspase Cascades Are Required in Nonapoptotic Roles for Drosophila Spermatid Individualization  

Microsoft Academic Search

Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER\\/HID\\/GRIM family proapoptotic proteins.

Jun R. Huh; Stephanie Y. Vernooy; Hong Yu; Nieng Yan; Yigong Shi; Ming Guo; Bruce A. Hay

2004-01-01

349

Sustained Post-Mating Response in Drosophila melanogaster Requires Multiple Seminal Fluid Proteins  

PubMed Central

Successful reproduction is critical to pass genes to the next generation. Seminal proteins contribute to important reproductive processes that lead to fertilization in species ranging from insects to mammals. In Drosophila, the male's accessory gland is a source of seminal fluid proteins that affect the reproductive output of males and females by altering female post-mating behavior and physiology. Protein classes found in the seminal fluid of Drosophila are similar to those of other organisms, including mammals. By using RNA interference (RNAi) to knock down levels of individual accessory gland proteins (Acps), we investigated the role of 25 Acps in mediating three post-mating female responses: egg production, receptivity to remating and storage of sperm. We detected roles for five Acps in these post-mating responses. CG33943 is required for full stimulation of egg production on the first day after mating. Four other Acps (CG1652, CG1656, CG17575, and CG9997) appear to modulate the long-term response, which is the maintenance of post-mating behavior and physiological changes. The long-term post-mating response requires presence of sperm in storage and, until now, had been known to require only a single Acp. Here, we discovered several novel Acps together are required which together are required for sustained egg production, reduction in receptivity to remating of the mated female and for promotion of stored sperm release from the seminal receptacle. Our results also show that members of conserved protein classes found in seminal plasma from insects to mammals are essential for important reproductive processes.

Ram, K. Ravi; Wolfner, Mariana F

2007-01-01

350

The Mechanism of Bacillus anthracis Intracellular Germination Requires Multiple and Highly Diverse Genetic Loci?  

PubMed Central

In an effort to better understand the mechanisms by which Bacillus anthracis establishes disease, experiments were undertaken to identify the genes essential for intracellular germination. Eighteen diverse genetic loci were identified via an enrichment protocol using a transposon-mutated library of B. anthracis spores, which was screened for mutants delayed in intracellular germination. Fourteen transposon mutants were identified in genes not previously associated with B. anthracis germination and included disruption of factors involved in membrane transport, transcriptional regulation, and intracellular signaling. Four mutants contained transposon insertions in gerHA, gerHB, gerHC, and pagA, respectively, each of which has been previously associated with germination or survival of B. anthracis within macrophages. Strain MIGD101 (named for macrophage intracellular germination defective 101) was of particular interest, since this mutant contained a transposon insertion in an intergenic region between BAs2807 and BAs2808, and was the most highly represented mutant in the enrichment. Analysis of B. anthracis MIGD101 by confocal microscopy and differential heat sensitivity following macrophage infection revealed ungerminated spores within the cell. Moreover, B. anthracis MIGD101 was attenuated in cell killing relative to the parent strain. Further experimental analysis found that B. anthracis MIGD101 was defective in five known B. anthracis germination pathways, supporting a mechanism wherein the intergenic region between BAs2807 and BAs2808 has a global affect on germination of this pathogen. Collectively, these findings provide insight into the mechanisms supporting B. anthracis germination within host cells.

Barua, Soumitra; McKevitt, Matthew; DeGiusti, Kevin; Hamm, Elaine E.; Larabee, Jason; Shakir, Salika; Bryant, Katie; Koehler, Theresa M.; Blanke, Steven R.; Dyer, David; Gillaspy, Allison; Ballard, Jimmy D.

2009-01-01

351

Inducible Expression of Human ?-Defensin 2 by Fusobacterium nucleatum in Oral Epithelial Cells: Multiple Signaling Pathways and Role of Commensal Bacteria in Innate Immunity and the Epithelial Barrier  

PubMed Central

Human gingival epithelial cells (HGE) express two antimicrobial peptides of the ?-defensin family, human ?-defensin 1 (hBD-1) and hBD-2, as well as cytokines and chemokines that contribute to innate immunity. In the present study, the expression and transcriptional regulation of hBD-2 was examined. HBD-2 mRNA was induced by cell wall extract of Fusobacterium nucleatum, an oral commensal microorganism, but not by that of Porphyromonas gingivalis, a periodontal pathogen. HBD-2 mRNA was also induced by the proinflammatory cytokine tumor necrosis factor alpha (TNF-?) and phorbol myristate acetate (PMA), an epithelial cell activator. HBD-2 mRNA was also expressed in 14 of 15 noninflamed gingival tissue samples. HBD-2 peptide was detected by immunofluorescence in HGE stimulated with F. nucleatum cell wall, consistent with induction of the mRNA by this stimulant. Kinetic analysis indicates involvement of multiple distinct signaling pathways in the regulation of hBD-2 mRNA; TNF-? and F. nucleatum cell wall induced hBD-2 mRNA rapidly (2 to 4 h), while PMA stimulation was slower (?10 h). In contrast, each stimulant induced interleukin 8 (IL-8) within 1 h. The role of TNF-? as an intermediary in F. nucleatum signaling was ruled out by addition of anti-TNF-? that did not inhibit hBD-2 induction. However, inhibitor studies show that F. nucleatum stimulation of hBD-2 mRNA requires both new gene transcription and new protein synthesis. Bacterial lipopolysaccharides isolated from Escherichia coli and F. nucleatum were poor stimulants of hBD-2, although they up-regulated IL-8 mRNA. Collectively, our findings show inducible expression of hBD-2 mRNA via multiple pathways in HGE in a pattern that is distinct from that of IL-8 expression. We suggest that different aspects of innate immune responses are differentially regulated and that commensal organisms have a role in stimulating mucosal epithelial cells in maintaining the barrier that contributes to homeostasis and host defense.

Krisanaprakornkit, Suttichai; Kimball, Janet R.; Weinberg, Aaron; Darveau, Richard P.; Bainbridge, Brian W.; Dale, Beverly A.

2000-01-01

352

49 CFR 234.306 - Multiple dispatching or maintaining railroads with respect to the same highway-rail or pathway...  

Code of Federal Regulations, 2013 CFR

...maintaining railroads with respect to the same highway-rail or pathway grade crossing; appointment...Telephonic Reporting of Unsafe Conditions at Highway-Rail and Pathway Grade Crossings ...maintaining railroads with respect to the same highway-rail or pathway grade crossing;...

2013-10-01

353

Lipopolysaccharide-induced activation of NF-{kappa}B non-canonical pathway requires BCL10 serine 138 and NIK phosphorylations  

SciTech Connect

Background and aims: B-cell lymphoma/leukemia (BCL)-10 and reactive oxygen species mediate two pathways of NF-{kappa}B (RelA) activation by lipopolysaccharide (LPS) in human colonic epithelial cells. The pathway for LPS activation of RelB by the non-canonical pathway (RelB) in non-myeloid cells was not yet reported, but important for understanding the range of potential microbial LPS-induced effects in inflammatory bowel disease. Methods: Experiments were performed in human colonic epithelial cells and in mouse embryonic fibroblasts deficient in components of the IkappaB kinase (IKK) signalosome, in order to detect mediators of the non-canonical pathway of NF-{kappa}B activation, including nuclear RelB and p52 and phospho- and total NF-{kappa}B inducing kinase (NIK). BCL10 was silenced by siRNA and effects of mutations of specific phosphorylation sites of BCL10 (Ser138Gly and Ser218Gly) were determined. Results: By the non-canonical pathway, LPS exposure increased nuclear RelB and p52, and phospho-NIK, with no change in total NIK. Phosphorylation of BCL10 serine 138 was required for NIK phosphorylation, since mutation of this residue eliminated the increases in phospho-NIK and nuclear RelB and p52. Mutations of either serine 138 or serine 218 reduced RelA, p50, and phospho-I{kappa}B{alpha} of the canonical pathway. Effects of LPS stimulation and BCL10 silencing on NIK phosphorylation were demonstrated in confocal images. Conclusions: LPS induces activation of both canonical and non-canonical pathways of NF-{kappa}B in human colonic epithelial cells, and the non-canonical pathway requires phosphorylations of BCL10 (serine 138) and NIK. These findings demonstrate the important role of BCL10 in mediating LPS-induced inflammation in human colonic epithelial cells and may open new avenues for therapeutic interventions.

Bhattacharyya, Sumit; Borthakur, Alip; Dudeja, Pradeep K. [Department of Medicine, University of Illinois at Chicago and Jesse Brown VA Medical Center, Chicago, IL 60612-7227 (United States)] [Department of Medicine, University of Illinois at Chicago and Jesse Brown VA Medical Center, Chicago, IL 60612-7227 (United States); Tobacman, Joanne K., E-mail: jkt@uic.edu [Department of Medicine, University of Illinois at Chicago and Jesse Brown VA Medical Center, Chicago, IL 60612-7227 (United States)

2010-11-15

354

Genetic polymorphisms of multiple DNA repair pathways impact age at diagnosis and TP53 mutations in breast cancer  

PubMed Central

Defective DNA repair may contribute to early age and late stage at time of diagnosis and mutations in critical tumor suppressor genes, such as TP53 in breast cancer. Using DNA samples from 436 breast cancer cases (374 Caucasians and 62 African-Americans), we tested these associations with 18 non-synonymous single-nucleotide polymorphisms (nsSNPs) in four DNA repair pathways: (i) base excision repair: ADPRT V762A, APE1 D148E, XRCC1 R194W/R280H/R399Q and POLD1 R119H; (ii) double-strand break repair: NBS1 E185Q and XRCC3 T241M; (iii) mismatch repair: MLH1 I219V, MSH3 R940Q/T1036A and MSH6 G39E and (iv) nucleotide excision repair: ERCC2 D312N/K751Q, ERCC4 R415Q, ERCC5 D1104H and XPC A499V/K939Q. Younger age at diagnosis (<50) was associated with ERCC2 312 DN/NN genotypes [odds ratio (OR) = 1.76; 95% confidence interval (CI) = 1.10, 2.81] and NBS1 185 QQ genotype (OR = 3.09; 95% CI = 1.47, 6.49). The XPC 939 QQ genotype was associated with TP53 mutations (OR = 5.80; 95% CI = 2.23, 15.09). There was a significant trend associating younger age at diagnosis (<50) with increasing numbers of risk genotypes for ERCC2 312 DN/NN, MSH6 39 EE and NBS1 185 QQ (Ptrend < 0.001). A similar significant trend was also observed associating TP53 mutations with increasing numbers of risk genotypes for XRCC1 399 QQ, XPC 939 QQ, ERCC4 415 QQ and XPC 499 AA (Ptrend < 0.001). Our pilot data suggest that nsSNPs of multiple DNA repair pathways are associated with younger age at diagnosis and TP53 mutations in breast cancer and larger studies are warranted to further evaluate these associations.

Smith, Tasha R.; Liu-Mares, Wen; Van Emburgh, Beth O.; Levine, Edward A.; Allen, Glenn O.; Hill, Jeff W.; Reis, Isildinha M.; Kresty, Laura A.; Pegram, Mark D.; Hu, Jennifer J.

2011-01-01

355

Glycosylphosphatidylinositol Anchor-Dependent Stimulation Pathway Required for Generation of Baculovirus-Derived Recombinant Scrapie Prion Protein? †  

PubMed Central

The pathogenic isoform (PrPSc) of the host-encoded cellular prion protein (PrPC) is considered to be an infectious agent of transmissible spongiform encephalopathy (TSE). The detailed mechanism by which the PrPSc seed catalyzes the structural conversion of endogenous PrPC into nascent PrPSc in vivo still remains unclear. Recent studies reveal that bacterially derived recombinant PrP (recPrP) can be used as a substrate for the in vitro generation of protease-resistant recPrP (recPrPres) by protein-misfolding cyclic amplification (PMCA). These findings imply that PrP modifications with a glycosylphosphatidylinositol (GPI) anchor and asparagine (N)-linked glycosylation are not necessary for the amplification and generation of recPrPSc by PMCA. However, the biological properties of PrPSc obtained by in vivo transmission of recPrPres are unique or different from those of PrPSc used as the seed, indicating that the mechanisms mediated by these posttranslational modifications possibly participate in reproductive propagation of PrPSc. In the present study, using baculovirus-derived recombinant PrP (Bac-PrP), we demonstrated that Bac-PrP is useful as a PrPC substrate for amplification of the mouse scrapie prion strain Chandler, and PrPSc that accumulated in mice inoculated with Bac-PrPres had biochemical and pathological properties very similar to those of the PrPSc seed. Since Bac-PrP modified with a GPI anchor and brain homogenate of Prnp knockout mice were both required to generate Bac-PrPres, the interaction of GPI-anchored PrP with factors in brain homogenates is essential for reproductive propagation of PrPSc. Therefore, the Bac-PMCA technique appears to be extremely beneficial for the comprehensive understanding of the GPI anchor-mediated stimulation pathway.

Imamura, Morikazu; Kato, Nobuko; Yoshioka, Miyako; Okada, Hiroyuki; Iwamaru, Yoshifumi; Shimizu, Yoshihisa; Mohri, Shirou; Yokoyama, Takashi; Murayama, Yuichi

2011-01-01

356

A New Pathway for Salvaging the Coenzyme B12 Precursor Cobinamide in Archaea Requires Cobinamide-Phosphate Synthase (CbiB) Enzyme Activity  

Microsoft Academic Search

The ability of archaea to salvage cobinamide has been under question because archaeal genomes lack orthologs to the bacterial nucleoside triphosphate:5-deoxycobinamide kinase enzyme (cobU in Salmonella enterica). The latter activity is required for cobinamide salvaging in bacteria. This paper reports evidence that archaea salvage cobinamide from the environment by using a pathway different from the one used by bacteria. These

Jesse D. Woodson; Carmen L. Zayas; Jorge C. Escalante-Semerena

2003-01-01

357

Evolution at increased error rate leads to the coexistence of multiple adaptive pathways in an RNA virus  

PubMed Central

Background When beneficial mutations present in different genomes spread simultaneously in an asexual population, their fixation can be delayed due to competition among them. This interference among mutations is mainly determined by the rate of beneficial mutations, which in turn depends on the population size, the total error rate, and the degree of adaptation of the population. RNA viruses, with their large population sizes and high error rates, are good candidates to present a great extent of interference. To test this hypothesis, in the current study we have investigated whether competition among beneficial mutations was responsible for the prolonged presence of polymorphisms in the mutant spectrum of an RNA virus, the bacteriophage Q?, evolved during a large number of generations in the presence of the mutagenic nucleoside analogue 5-azacytidine. Results The analysis of the mutant spectra of bacteriophage Q? populations evolved at artificially increased error rate shows a large number of polymorphic mutations, some of them with demonstrated selective value. Polymorphisms distributed into several evolutionary lines that can compete among them, making it difficult the emergence of a defined consensus sequence. The presence of accompanying deleterious mutations, the high degree of recurrence of the polymorphic mutations, and the occurrence of epistatic interactions generate a highly complex interference dynamics. Conclusions Interference among beneficial mutations in bacteriophage Q? evolved at increased error rate permits the coexistence of multiple adaptive pathways that can provide selective advantages by different molecular mechanisms. In this way, interference can be seen as a positive factor that allows the exploration of the different local maxima that exist in rugged fitness landscapes.

2013-01-01

358

Elevated hepatic fatty acid elongase-5 activity affects multiple pathways controlling hepatic lipid and carbohydrate composition*s?  

PubMed Central

Hepatic fatty acid elongase-5 (Elovl-5) plays an important role in long chain monounsaturated and polyunsaturated fatty acid synthesis. Elovl-5 activity is regulated during development, by diet, hormones, and drugs, and in chronic disease. This report examines the impact of elevated Elovl-5 activity on hepatic function. Adenovirus-mediated induction of Elovl5 activity in livers of C57BL/6 mice increased hepatic and plasma levels of dihomo-?-linolenic acid (20:3,n-6) while suppressing hepatic arachidonic acid (20:4,n-6) and docosahexaenoic acid (22:6,n-3) content. The fasting-refeeding response of peroxisome proliferator-activated receptor ?-regulated genes was attenuated in mice with elevated Elovl5 activity. In contrast, the fasting-refeeding response of hepatic sterol-regulatory element binding protein-1 (SREBP-1)-regulated and carbohydrate-regulatory element binding protein/Max-like factor X-regulated genes, Akt and glycogen synthase kinase (Gsk)-3? phosphorylation, and the accumulation of hepatic glycogen content and nuclear SREBP-1 were not impaired by elevated Elovl5 activity. Hepatic triglyceride content and the phosphorylation of AMP-activated kinase ? and Jun kinase 1/2 were reduced by elevated Elovl5 activity. Hepatic phosphoenolpyruvate carboxykinase expression was suppressed, while hepatic glycogen content and phosphorylated Gsk-3? were significantly increased, in livers of fasted mice with increased Elovl5 activity. As such, hepatic Elovl5 activity may affect hepatic glucose production during fasting. In summary, Elovl5-induced changes in hepatic fatty acid content affect multiple pathways regulating hepatic lipid and carbohydrate composition.

Wang, Yun; Torres-Gonzalez, Moises; Tripathy, Sasmita; Botolin, Daniela; Christian, Barbara; Jump, Donald B.

2008-01-01