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Sample records for gene homologue treated

  1. Detection of a Yersinia pestis gene homologue in rodent samples

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    Timothy A. Giles

    2016-08-01

    Full Text Available A homologue to a widely used genetic marker, pla, for Yersinia pestis has been identified in tissue samples of two species of rat (Rattus rattus and Rattus norvegicus and of mice (Mus musculus and Apodemus sylvaticus using a microarray based platform to screen for zoonotic pathogens of interest. Samples were from urban locations in the UK (Liverpool and Canada (Vancouver. The results indicate the presence of an unknown bacterium that shares a homologue for the pla gene of Yersinia pestis, so caution should be taken when using this gene as a diagnostic marker.

  2. Homologue Pairing in Flies and Mammals: Gene Regulation When Two Are Involved

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    Manasi S. Apte

    2012-01-01

    Full Text Available Chromosome pairing is usually discussed in the context of meiosis. Association of homologues in germ cells enables chromosome segregation and is necessary for fertility. A few organisms, such as flies, also pair their entire genomes in somatic cells. Most others, including mammals, display little homologue pairing outside of the germline. Experimental evidence from both flies and mammals suggests that communication between homologues contributes to normal genome regulation. This paper will contrast the role of pairing in transmitting information between homologues in flies and mammals. In mammals, somatic homologue pairing is tightly regulated, occurring at specific loci and in a developmentally regulated fashion. Inappropriate pairing, or loss of normal pairing, is associated with gene misregulation in some disease states. While homologue pairing in flies is capable of influencing gene expression, the significance of this for normal expression remains unknown. The sex chromosomes pose a particularly interesting situation, as females are able to pair X chromosomes, but males cannot. The contribution of homologue pairing to the biology of the X chromosome will also be discussed.

  3. Identification and Characterisation of the Murine Homologue of the Gene Responsible for Cystinosis, Ctns

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    Poras Isabelle

    2000-12-01

    Full Text Available Abstract Background Cystinosis is an autosomal recessive disorder characterised by an intralysosomal accumulation of cystine, and affected individuals progress to end-stage renal failure before the age of ten. The causative gene, CTNS, was cloned in 1998 and the encoded protein, cystinosin, was predicted to be a lysosomal membrane protein. Results We have cloned the murine homologue of CTNS, Ctns, and the encoded amino acid sequence is 92.6% similar to cystinosin. We localised Ctns to mouse chromosome 11 in a region syntenic to human chromosome 17 containing CTNS. Ctns is widely expressed in all tissues tested with the exception of skeletal muscle, in contrast to CTNS. Conclusions We have isolated, characterised and localised Ctns, the murine homologue of CTNS underlying cystinosis. Furthermore, our work has brought to light the existence of a differential pattern of expression between the human and murine homologues, providing critical information for the generation of a mouse model for cystinosis.

  4. NBS-LRR resistance gene homologues in rice

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Twenty three DNA fragments with a size of about 520 bp have been cloned from rice genome by PCR amplification using primers designed according to the conserved region of most plant resistance (R) genes which have Nucleotide Binding Site (NBS) and Leucine-Rich Repeat (LRR) domains. Homologous comparison showed that these fragments contained typical motifs of the NBS-LRR resistance gene class, kinase 1a, kinase 2a, kinase 3a and domain 2. Thus they were named R gene homologous sequences (RS). These RS were divided into 4 groups by clustering analysis and mapped onto chromosomes 1, 3, 4, 7, 8, 9, 10 and 11, respectively, by genetic mapping. Ten RS were located in the chromosomal intervals where known R genes had been mapped. Further RFLP analysis of an RS, RS13, near the bacterial blight resistance gene Xa4 locus on chromosome 11 among near isogenic lines and pyramiding lines of Xa4 showed that RS13 was possibly amplified from the gene family of Xa4.

  5. Natural replacement of vertically inherited lux-rib genes of Photobacterium aquimaris by horizontally acquired homologues.

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    Urbanczyk, Henryk; Furukawa, Takashi; Yamamoto, Yuki; Dunlap, Paul V

    2012-08-01

    We report here the first instance of a complete replacement of vertically inherited luminescence genes by horizontally acquired homologues. Different strains of Photobacterium aquimaris contain homologues of the lux-rib genes that have a different evolutionary history. Strain BS1 from the Black Sea contains a vertically inherited lux-rib operon, which presumably arose in the ancestor of this species, whereas the type strain NBRC 104633(T) , from Sagami Bay, lacks the vertically inherited lux-rib operon and instead carries a complete and functional lux-rib operon acquired horizontally from a bacterium related to Photobacterium mandapamensis. The results indicate that the horizontal acquisition of the lux genes expanded the pan-genome of P. aquimaris, but it did not influence the phylogenetic divergence of this species.

  6. The opiorphin gene (ProL1) and its homologues function in erectile physiology.

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    Tong, Yuehong; Tar, Moses; Melman, Arnold; Davies, Kelvin

    2008-09-01

    To determine if ProL1, a member of the opiorphin family of genes, can modulate erectile physiology, as it encodes a peptide which acts as a neutral endopeptidase inhibitor, other examples of which (Vcsa1, hSMR3A) modulate erectile physiology. We cloned members of the opiorphin family of genes into the same mammalian expression backbone (pVAX); 100 microg of these plasmids (pVAX-Vcsa1, -hSMR3A, -hSMR3B and -ProL1) were injected intracorporally into retired breeder rats and the affect on erectile physiology assessed visually, by histology and by measuring the intracavernous pressure (ICP) and blood pressure (BP). As a positive control, rats were treated with pVAX-hSlo (expressing the MaxiK potassium channel) and as a negative control the empty backbone plasmid was injected (pVAX). We also compared the level of expression of ProL1 in corporal tissue of patients not reporting erectile dysfunction (ED), ED associated with diabetes and ED not caused by diabetes. Gene transfer of plasmids expressing all members of the opiorphin family had a similar and significant effect on erectile physiology. At the concentration used in these experiments (100 microg) they resulted in higher resting ICP, and histological and visual analysis showed evidence of a priapic-like condition. After electrostimulation of the cavernous nerve, rats had significantly better ICP/BP than the negative control (pVAX). Gene transfer of pVAX-hSlo increased the ICP/BP ratio to a similar extent to the opiorphin homologues, but with no evidence for a priapic-like condition. Corpora cavernosa tissue samples obtained from men with ED, regardless of underlying causes, had significant down-regulation of both hSMR3A and ProL1. All members of the human opiorphin family of genes can potentially modulate erectile physiology. Both hSMR3 and ProL1 are down-regulated in the corpora of men with ED, and therefore both genes can potentially act as markers of ED.

  7. IDENTIFICATION OF HUMAN MURR1, THE HOMOLOGUE OF MOUSE IMPRINTED Murr1 GENE

    Institute of Scientific and Technical Information of China (English)

    Zhang Zhongming; Wang Youdong; Hitomi Yatsuki; Keiichiro Joh; Tsuyoshi Iwasaka; Tsunehiro Mukai

    2006-01-01

    Objective To identify the mRNA sequence, genetic construction, imprinting status, and expression profile of human MURR1 gene, the homologue of mouse imprinted Murr1 gene. Methods The MURR1 mRNA sequence was identified by colony hybridization screening of human cDNA library and the 5'-RACE analyses; Absence of U2AF1-RS1 gene within MURR1 was confirmed by Southern Blotting; Expression profile of MURR1 was examined by Northern Blotting; The imprinting status of MURR1 were revealed by SNP investigation and RT-PCR followed by sequencings and RFLP analyses. Results The full-length mRNA sequence of MURR1 spans 711 bp, transcribed from 3 exons, encodes predicted MURR1 protein of 190 amino acids. The gene was expressed in all the 12 kinds of human adult tissues and 6 kinds of fetal tissues. It showed biallelic expression in all 32 investigated samples including 6 kinds of human fetal tissues and 8 adult brains. Unlike mouse imprinted U2af1-rs1 gene existing in the intron of Murr1, the human U2AF1-RS1 gene was not located in the MURR1 locus. Conclusion Human MURR1 gene is not imprinted and the non-imprinting is possible due to the absence of human homologue of mouse U2af1-rs1 within MURR1 locus.

  8. Molecular Cloning of a Novel Bovine Homologue of the Drosophila Tumor Suppressor Gene, Lats

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Pervious studies demonstrate that lats, also known as warts, is a tumor suppressor gene in Drosophila[1,2]. Mutations of lats lead to an increase in cell number and organ size in Drosophila, indicating lats may be involved in organ size control. Furthermore, the high conservation of sequence and tumor suppression function of lats between Drosophila and human suggests that it may be also involved in organ size control of higher animals[3]. So here we isolated the bovine homologue of Drosophila lats. Sequence analysis indicates the bovine LATS1 to be very similar to other lats proteins.

  9. TRAUCO, a Trithorax-group gene homologue, is required for early embryogenesis in Arabidopsis thaliana.

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    Aquea, Felipe; Johnston, Amal J; Cañon, Paola; Grossniklaus, Ueli; Arce-Johnson, Patricio

    2010-02-01

    Embryogenesis is a critical stage during the plant life cycle in which a unicellular zygote develops into a multicellular organism. Co-ordinated gene expression is thus necessary for proper embryo development. Polycomb and Trithorax group genes are members of evolutionarily conserved machinery that maintains the correct expression patterns of key developmental regulators by repressing and activating gene transcription. TRAUCO (TRO), a gene homologous to the Trithorax group of genes that can functionally complement a BRE2P yeast mutant, has been identified in Arabidopsis thaliana. It is demonstrated that TRO is a nuclear gene product expressed during embryogenesis, and loss of TRO function leads to impaired early embryo development. Embryos that arrested at the globular stage in the tro-1 mutant allele were fully rescued by a TRO expression clone, a demonstration that the tro-1 mutation is a true loss-of-function in TRO. Our data have established that TRO is the first trithorax-group gene homologue in plants that is required for early embryogenesis.

  10. Positive selection in AvrP4 avirulence gene homologues across the genus Melampsora.

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    Van der Merwe, Marlien M; Kinnear, Mark W; Barrett, Luke G; Dodds, Peter N; Ericson, Lars; Thrall, Peter H; Burdon, Jeremy J

    2009-08-22

    Pathogen genes involved in interactions with their plant hosts are expected to evolve under positive Darwinian selection or balancing selection. In this study a single copy avirulence gene, AvrP4, in the plant pathogen Melampsora lini, was used to investigate the evolution of such a gene across species. Partial translation elongation factor 1-alpha sequences were obtained to establish phylogenetic relationships among the Melampsora species. We amplified AvrP4 homologues from species pathogenic on hosts from different plant families and orders, across the inferred phylogeny. Translations of the AvrP4 sequences revealed a predicted signal peptide and towards the C-terminus of the protein, six identically spaced cysteines were identified in all sequences. Maximum likelihood analysis of synonymous versus non-synonymous substitution rates indicated that positive selection played a role in the evolution of the gene during the diversification of the genus. Fourteen codons under significant positive selection reside in the C-terminal 28 amino acid region, suggesting that this region interacts with host molecules in most sequenced accessions. Selection pressures on the gene may be either due to the pathogenicity or avirulence function of the gene or both.

  11. The oil palm SHELL gene controls oil yield and encodes a homologue of SEEDSTICK.

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    Singh, Rajinder; Low, Eng-Ti Leslie; Ooi, Leslie Cheng-Li; Ong-Abdullah, Meilina; Ting, Ngoot-Chin; Nagappan, Jayanthi; Nookiah, Rajanaidu; Amiruddin, Mohd Din; Rosli, Rozana; Manaf, Mohamad Arif Abdul; Chan, Kuang-Lim; Halim, Mohd Amin; Azizi, Norazah; Lakey, Nathan; Smith, Steven W; Budiman, Muhammad A; Hogan, Michael; Bacher, Blaire; Van Brunt, Andrew; Wang, Chunyan; Ordway, Jared M; Sambanthamurthi, Ravigadevi; Martienssen, Robert A

    2013-08-15

    A key event in the domestication and breeding of the oil palm Elaeis guineensis was loss of the thick coconut-like shell surrounding the kernel. Modern E. guineensis has three fruit forms, dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), a hybrid between dura and pisifera. The pisifera palm is usually female-sterile. The tenera palm yields far more oil than dura, and is the basis for commercial palm oil production in all of southeast Asia. Here we describe the mapping and identification of the SHELL gene responsible for the different fruit forms. Using homozygosity mapping by sequencing, we found two independent mutations in the DNA-binding domain of a homologue of the MADS-box gene SEEDSTICK (STK, also known as AGAMOUS-LIKE 11), which controls ovule identity and seed development in Arabidopsis. The SHELL gene is responsible for the tenera phenotype in both cultivated and wild palms from sub-Saharan Africa, and our findings provide a genetic explanation for the single gene hybrid vigour (or heterosis) attributed to SHELL, via heterodimerization. This gene mutation explains the single most important economic trait in oil palm, and has implications for the competing interests of global edible oil production, biofuels and rainforest conservation.

  12. Cloning the mouse homologue of the human lysosomal acid {alpha}-glucosidase gene

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    Ding, J.H.; Yang, B.Z.; Liu, H.M. [Duke Univ. Medical Center, Durham, NC (United States)] [and others

    1994-09-01

    Pompe disease (GSD II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid {alpha}-glucosidase (GAA). In an attempt to create a mouse model for Pompe disease, we isolated and characterized the gene encoding the mouse homologue of the human GAA. Twenty clones that extend from exon 2 to the poly(A) tail were isolated from a mouse liver cDNA library, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-2 were cloned by RACE from mouse liver RNA. The full-length liver GAA cDNA contains 3365 nucleotides with a coding region of 2859 nucleotides and a 394 base pair 3{prime}-nontranslated region. The deduced amino acid sequence of the mouse GAA shows 84% identity to the human GAA. Southern blot analysis demonstrated that the mouse GAA was encoded by a single copy gene. Then six bacteriophages containing DNA from the GAA gene were isolated by screening 10{sup 6} phage plaques of a mouse 129 genomic library using a mouse GAA cDNA as a probe. From one of these bacteriophages, an 11-kilobase EcoRI fragment containing exons 3 to 15 was subcloned and sequenced. Work is in progress using this genomic clone to disrupt the GAA gene in murine embryonic stem cells in order to create GSD II mice.

  13. Development and mapping of SSR markers linked to resistance-gene homologue clusters in common bean

    Institute of Scientific and Technical Information of China (English)

    Luz; Nayibe; Garzon; Matthew; Wohlgemuth; Blair

    2014-01-01

    Common bean is an important but often a disease-susceptible legume crop of temperate,subtropical and tropical regions worldwide. The crop is affected by bacterial, fungal and viral pathogens. The strategy of resistance-gene homologue(RGH) cloning has proven to be an efficient tool for identifying markers and R(resistance) genes associated with resistances to diseases. Microsatellite or SSR markers can be identified by physical association with RGH clones on large-insert DNA clones such as bacterial artificial chromosomes(BACs). Our objectives in this work were to identify RGH-SSR in a BAC library from the Andean genotype G19833 and to test and map any polymorphic markers to identify associations with known positions of disease resistance genes. We developed a set of specific probes designed for clades of common bean RGH genes and then identified positive BAC clones and developed microsatellites from BACs having SSR loci in their end sequences. A total of 629 new RGH-SSRs were identified and named BMr(bean microsatellite RGH-associated markers). A subset of these markers was screened for detecting polymorphism in the genetic mapping population DOR364 × G19833. A genetic map was constructed with a total of 264 markers,among which were 80 RGH loci anchored to single-copy RFLP and SSR markers. Clusters of RGH-SSRs were observed on most of the linkage groups of common bean and in positions associated with R-genes and QTL. The use of these new markers to select for disease resistance is discussed.

  14. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli

    Science.gov (United States)

    Paproski, Robert J.; Li, Yan; Barber, Quinn; Lewis, John D.; Campbell, Robert E.; Zemp, Roger

    2015-10-01

    To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9× dilution sample was 55, suggesting that ˜20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and chorioallantoic membrane. Our results suggest that melA is a useful photoacoustic reporter gene for visualizing bacteria, and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted.

  15. Novel Type Ⅱ Peroxiredoxin Gene Homologue from Chinese Wildrye Enhancing Salt Stress Tolerance of Transgenic Yeast

    Institute of Scientific and Technical Information of China (English)

    YU Ying; LIU Xiang-guo; LU Yang; DOU Yao; WANG Hu-yi; HAN Si-ping; FENG Shu-dan; HAO Dong-yun

    2011-01-01

    Peroxiredoxins(Prxs)are a large family of antioxidant enzymes of various types that take part in signal transduction via decomposing reactive oxygen species(ROS).Although extensive efforts have been made over the last decades in understanding the structures and functions of Prxs,type Ⅱ Prxs in monocots are hardly studied.In this work,a monocot type Ⅱ Prx gene homologue from Chinese wildrye(Leymus Chinensis),designated as LcTpxⅡ,was isolated and characterized.LcTpxⅡ encoding a 162-amino acid protein contains a thioredoxin domain and a cysteine residue at position 51,suggesting that it is a member of the Prxs family.The LcTpxⅡ is capable of decomposing H2O2 and protecting plasmid DNA from damage caused by ROS.The expression of LcTpxⅡ in Chinese wildrye was induced by 400 mmol/L NaCl and 100 mmol/L Na2CO3 in the experiment.The overexpression of LcTpxⅡ enhances the tolerance of transgenic yeast to 1.6 mol/L NaCl and 10 mmol/L Na2CO3.

  16. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli.

    Science.gov (United States)

    Paproski, Robert J; Li, Yan; Barber, Quinn; Lewis, John D; Campbell, Robert E; Zemp, Roger

    2015-10-01

    To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9×dilution sample was 55, suggesting that ∼20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between Photoacoustic imaging could resolve the two tubes of melA-expressing bacteria even when the tubes were less than 1 mm from each other, while bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and chorioallantoic membrane. Our results suggest that melA is a useful photoacoustic reporter gene

  17. Allele mining in the gene pool of wild Solanum species for homologues of late blight resistance gene RB/Rpi-blb1

    Science.gov (United States)

    Solanum bulbocastanum comprising a CC-NBS-LRR gene RB/Rpi-blb1 confers broad-spectrum resistance to Phytophthora infestans and is currently employed in potato breeding for durable late blight (LB) resistance. Genomes of several Solanum species were reported to contain RB homologues with confirmed b...

  18. Pepsin homologues in bacteria

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    Bateman Alex

    2009-09-01

    Full Text Available Abstract Background Peptidase family A1, to which pepsin belongs, had been assumed to be restricted to eukaryotes. The tertiary structure of pepsin shows two lobes with similar folds and it has been suggested that the gene has arisen from an ancient duplication and fusion event. The only sequence similarity between the lobes is restricted to the motif around the active site aspartate and a hydrophobic-hydrophobic-Gly motif. Together, these contribute to an essential structural feature known as a psi-loop. There is one such psi-loop in each lobe, and so each lobe presents an active Asp. The human immunodeficiency virus peptidase, retropepsin, from peptidase family A2 also has a similar fold but consists of one lobe only and has to dimerize to be active. All known members of family A1 show the bilobed structure, but it is unclear if the ancestor of family A1 was similar to an A2 peptidase, or if the ancestral retropepsin was derived from a half-pepsin gene. The presence of a pepsin homologue in a prokaryote might give insights into the evolution of the pepsin family. Results Homologues of the aspartic peptidase pepsin have been found in the completed genomic sequences from seven species of bacteria. The bacterial homologues, unlike those from eukaryotes, do not possess signal peptides, and would therefore be intracellular acting at neutral pH. The bacterial homologues have Thr218 replaced by Asp, a change which in renin has been shown to confer activity at neutral pH. No pepsin homologues could be detected in any archaean genome. Conclusion The peptidase family A1 is found in some species of bacteria as well as eukaryotes. The bacterial homologues fall into two groups, one from oceanic bacteria and one from plant symbionts. The bacterial homologues are all predicted to be intracellular proteins, unlike the eukaryotic enzymes. The bacterial homologues are bilobed like pepsin, implying that if no horizontal gene transfer has occurred the duplication

  19. Gene targeting by RNAi-mediated knockdown of potent DNA ligase IV homologue in the cellulase-producing fungus Talaromyces cellulolyticus.

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    Hayata, Koutarou; Asada, Seiya; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Sawayama, Shigeki

    2014-11-01

    The genome of the cellulase-producing fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) was screened for a potent DNA ligase IV gene (ligD homologue). Homologous recombination efficiency in T. cellulolyticus is very low. Therefore, suppression of a non-homologous end-joining system was attempted to enable specific gene knockouts for molecular breeding. The transcript levels of ligD homologue were 0.037 of those of the parental YP-4 strain in the Li20 transformant carrying the RNAi construct targeting the ligD homologue. Transformation of the hairpin-type RNAi vector into T. cellulolyticus could be useful in fungal gene knockdown experiments. Cellulase production and protein secretion were similar in the parental YP-4 strain and the Li20 transformant. Knockout transformation of ligD homologue using the Li20 transformant led to 23.1 % double crossover gene targeting. Our results suggest that the potent DNA ligase IV gene of T. cellulolyticus is related to non-homologous end joining and that the knockdown of the ligD homologue is useful in gene targeting.

  20. Identification and characterisation of the angiotensin converting enzyme-3 (ACE3 gene: a novel mammalian homologue of ACE

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    Phelan Anne

    2007-06-01

    Full Text Available Abstract Background Mammalian angiotensin converting enzyme (ACE plays a key role in blood pressure regulation. Although multiple ACE-like proteins exist in non-mammalian organisms, to date only one other ACE homologue, ACE2, has been identified in mammals. Results Here we report the identification and characterisation of the gene encoding a third homologue of ACE, termed ACE3, in several mammalian genomes. The ACE3 gene is located on the same chromosome downstream of the ACE gene. Multiple sequence alignment and molecular modelling have been employed to characterise the predicted ACE3 protein. In mouse, rat, cow and dog, the predicted protein has mutations in some of the critical residues involved in catalysis, including the catalytic Glu in the HEXXH zinc binding motif which is Gln, and ESTs or reverse-transcription PCR indicate that the gene is expressed. In humans, the predicted ACE3 protein has an intact HEXXH motif, but there are other deletions and insertions in the gene and no ESTs have been identified. Conclusion In the genomes of several mammalian species there is a gene that encodes a novel, single domain ACE-like protein, ACE3. In mouse, rat, cow and dog ACE3, the catalytic Glu is replaced by Gln in the putative zinc binding motif, indicating that in these species ACE3 would lack catalytic activity as a zinc metalloprotease. In humans, no evidence was found that the ACE3 gene is expressed and the presence of deletions and insertions in the sequence indicate that ACE3 is a pseudogene.

  1. A WD40-repeat gene from Malus x domestica is a functional homologue of Arabidopsis thaliana TRANSPARENT TESTA GLABRA1.

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    Brueggemann, Julian; Weisshaar, Bernd; Sagasser, Martin

    2010-03-01

    The WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) is involved in a multitude of developmental and biochemical reactions in Arabidopsis thaliana such as the production of seed coat colour and mucilage, pigmentation by anthocyanins as well as the formation of trichomes and root hairs. In this study, a putative TTG1 homologue was isolated from apple (Malus x domestica Borkh.) showing 80.2% identity to A. thaliana TTG1 on nucleotide and 90.7% similarity on amino acid level. The MdTTG1 candidate was able to activate the AtBAN promoter in cooperation with the A. thaliana transcription factors TT2 and TT8 in A. thaliana protoplasts. This indicates that the encoded protein can be integrated into the complex that activates BAN in A. thaliana, and that a similar complex might also be present in apple. When transformed into ttg1 mutants of A. thaliana, the apple sequence was able to restore trichome growth, anthocyanin production in young seedlings as well as proanthocyanidin production in seeds. Additionally, roots of complemented mutant plants showed root hair formation resembling wild type. These results show that the studied apple WD40 gene is a functional homologue of AtTTG1 and we refer to this gene as MdTTG1.

  2. Equine herpesvirus 1 gene 12, the functional homologue of herpes simplex virus VP16, transactivates via octamer sequences in the equine herpesvirus IE gene promoter.

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    Elliott, G; O'Hare, P

    1995-10-20

    The HSV-1 transactivator of immediate-early (IE) gene expression, VP16, has several functional homologues among the alphaherpesviruses which have not yet been extensively studied in relation to their modes of action. To date, nothing is known of the exact sites or mechanism of interaction of the equine herpesvirus type 1 (EHV-1) homologue, the gene 12 protein, with the EHV-1 IE promoter. We show that the gene 12 protein utilises the promoter proximal region of the IE gene to induce activation and identify four potential octamer DNA binding sites within that region. Although there was divergence from its consensus, Oct-1 bound to each of these sites in an in vitro complex formation assay, and in the presence of the gene 12 product a second complex of slower migration, which was also dependent on Oct-1, was detected. When each site was inserted into a basal promoter, two conferred activation by gene 12 with a resulting increase in expression of up to 50-fold compared to basal levels. These results show that, despite the differences between the two proteins, the mechanism of interaction of the gene 12 protein with its target is analogous to that of VP16.

  3. The leukemia associated nuclear corepressor ETO homologue genes MTG16 and MTGR1 are regulated differently in hematopoietic cells

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    Ajore Ram

    2012-03-01

    Full Text Available Abstract Background MTG16, MTGR1 and ETO are nuclear transcriptional corepressors of the human ETO protein family. MTG16 is implicated in hematopoietic development and in controlling erythropoiesis/megakaryopoiesis. Furthermore, ETO homologue genes are 3'participants in leukemia fusions generated by chromosomal translocations responsible of hematopoietic dysregulation. We tried to identify structural and functional promoter elements of MTG16 and MTGR1 genes in order to find associations between their regulation and hematopoiesis. Results 5' deletion examinations and luciferase reporter gene studies indicated that a 492 bp sequence upstream of the transcription start site is essential for transcriptional activity by the MTG16 promoter. The TATA- and CCAAT-less promoter with a GC box close to the start site showed strong reporter activity when examined in erythroid/megakaryocytic cells. Mutation of an evolutionary conserved GATA -301 consensus binding site repressed promoter function. Furthermore, results from in vitro antibody-enhanced electrophoretic mobility shift assay and in vivo chromatin immunoprecipitation indicated binding of GATA-1 to the GATA -301 site. A role of GATA-1 was also supported by transfection of small interfering RNA, which diminished MTG16 expression. Furthermore, expression of the transcription factor HERP2, which represses GATA-1, produced strong inhibition of the MTG16 promoter reporter consistent with a role of GATA-1 in transcriptional activation. The TATA-less and CCAAT-less MTGR1 promoter retained most of the transcriptional activity within a -308 to -207 bp region with a GC-box-rich sequence containing multiple SP1 binding sites reminiscent of a housekeeping gene with constitutive expression. However, mutations of individual SP1 binding sites did not repress promoter function; multiple active SP1 binding sites may be required to safeguard constitutive MTGR1 transcriptional activity. The observed repression of MTG16

  4. A plasmid-encoded UmuD homologue regulates expression of Pseudomonas aeruginosa SOS genes.

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    Díaz-Magaña, Amada; Alva-Murillo, Nayeli; Chávez-Moctezuma, Martha P; López-Meza, Joel E; Ramírez-Díaz, Martha I; Cervantes, Carlos

    2015-07-01

    The Pseudomonas aeruginosa plasmid pUM505 contains the umuDC operon that encodes proteins similar to error-prone repair DNA polymerase V. The umuC gene appears to be truncated and its product is probably not functional. The umuD gene, renamed umuDpR, possesses an SOS box overlapped with a Sigma factor 70 type promoter; accordingly, transcriptional fusions revealed that the umuDpR gene promoter is activated by mitomycin C. The predicted sequence of the UmuDpR protein displays 23 % identity with the Ps. aeruginosa SOS-response LexA repressor. The umuDpR gene caused increased MMC sensitivity when transferred to the Ps. aeruginosa PAO1 strain. As expected, PAO1-derived knockout lexA-  mutant PW6037 showed resistance to MMC; however, when the umuDpR gene was transferred to PW6037, MMC resistance level was reduced. These data suggested that UmuDpR represses the expression of SOS genes, as LexA does. To test whether UmuDpR exerts regulatory functions, expression of PAO1 SOS genes was evaluated by reverse transcription quantitative PCR assays in the lexA-  mutant with or without the pUC_umuD recombinant plasmid. Expression of lexA, imuA and recA genes increased 3.4-5.3 times in the lexA-  mutant, relative to transcription of the corresponding genes in the lexA+ strain, but decreased significantly in the lexA- /umuDpR transformant. These results confirmed that the UmuDpR protein is a repressor of Ps. aeruginosa SOS genes controlled by LexA. Electrophoretic mobility shift assays, however, did not show binding of UmuDpR to 5' regions of SOS genes, suggesting an indirect mechanism of regulation.

  5. Human Jk recombination signal binding protein gene (IGKJRB): Comparison with its mouse homologue

    Energy Technology Data Exchange (ETDEWEB)

    Amakawa, Ryuichi; Jing, Wu; Matsunami, Norisada; Hamaguchi, Yasushi; Matsuda, Fumihiko; Kawaichi, Masashi; Honjo, Tasuku (Kyoto Univ., Sakyo-ku, Kyoto (Japan)); Ozawa, Kazuo (Tsukuba Life Science Center, Tsukuba, Ibraraki (Japan))

    1993-08-01

    The mouse Igkjrb protein specifically binds to the immunoglobulin Jk recombination signal sequence. The IGKJRB gene is highly conserved among many species such as human, Xenopus, and Drosophila. Using cDNA fragments of the mouse Igkjrb gene, the authors isolated its human counterpart, IGKJRB. The human genome contains one functional IGKJRB gene and two types of processed pseudogenes. In situ chromosome hybridization analysis demonstrated that the functional gene is localized at chromosome 3q25, and the pseudogenes (IGKJRBP1 and IGKJRBP2, respectively) are located at chromosomes 9p13 and 9q13. The functional gene is composed of 13 exons spanning at least 67 kb. Three types of cDNA with different 5[prime] sequences were isolated by rapid amplification of cDNA ends, suggesting the presence of three proteins. The aPCR-1 protein, which possessed the exon 1 sequence, was the counterpart of the mouse RBP-2 type protein. The aPCR-2 and 3 proteins may be specific to human cells because the mouse counterparts were not detected. The amino acid sequences of the human and mouse IGKJRB genes were 98% homologous in exons 2-11, whereas the homology of the human and mouse exon 1 sequences was 75%. 40 refs., 7 figs.

  6. Cloning and expression of the rat homologue of the Huntington disease gene

    Energy Technology Data Exchange (ETDEWEB)

    Schmitt, I.; Epplen, J.T.; Riess, O. [Ruhr-Univ. Bochum (Germany)] [and others

    1994-09-01

    Huntington`s disease (HD) is an autosomal dominant neurodegenerative disorder which is manifested usually in adult life. The age of onset is variable and leads to progressive symptoms including involuntary choreatic movements and various cognitive and psychiatric disturbances. Recently, a gene (IT15) was cloned containing a (CAG){sub n} repeat which is elongated and unstable in HD patients. IT15 is widely expressed in human tissues but unrelated to any known deduced protein sequence. To further investigate the HD gene, 15 rat cDNA libraries were screened. 24 clones have been identified covering the Huntingtin gene. Comparison of the Huntingtin gene between human and rat revealed homologies between 80% and 87% at the DNA level and about 90% at the protein level. These analyses will help to define biologically important sequence regions, e.g., via evolutionary conservation. One clone contains the (CAG){sub n} repeat which consists of eight triplets compared to seven triplets in the mouse and a median of 17 in human. As in humans there are two transcripts arising from differential 3{prime}-polyadenylation. In the 3{prime}UTR a stretch of about 280 bp is exchanged for a 250 bp fragment with no homology in rodents and man. The cDNA clones are currently used to study Huntingtin gene expression during development in rodent tissues. RNA in situ hybridization of embryonic sections shows predominant signals in all neuronal tissues. In contrast to previously published data Huntingtin mRNA expression in testis is increased in spermatocytes vs. spermatogonia.

  7. Activation of pur Gene Expression by a Homologue of the Bacillus subtilis PurR repressor:

    DEFF Research Database (Denmark)

    Kilstrup, Mogens; Martinussen, Jan

    1998-01-01

    . We have identified a PurBox sequence overlapping the -35 region of the L. lactis purR promoter and found, by studies of a purR-lacLM fusion plasmid, that purR is autoregulated. Because of the high similarity of the PurR proteins from B. subtilis and L. lactis, we looked for PurBox sequences...... in the promoter regions of the PurR regulated genes in B. subtilis, and identified a perfectly matching PurBox in the purA promoter region, and slightly degenerate PurBox like sequences in the promoter regions for the pur operon and the purR gene....

  8. An X-linked homologue of the autosomal inprinted gene ZNF127 escapes X inactivation

    Energy Technology Data Exchange (ETDEWEB)

    Longstreet, M.; Nicholls, R.D.; Willard, H.F. [Case Western Reserve Univ., Cleveland, OH (United States)] [and others

    1994-09-01

    The ZNF127 gene has been shown to be subject to parental imprinting in both humans and the mouse and maps to within the Prader-Willi/Angelman Syndrome critical region on chromosome 15. We have cloned two X-linked related loci, one of which, ZNFXp is a transcribed gene while the other, ZNFXq, is an untranscribed pseudogene. ZNFXp is 83.6% identical to ZNFXq and 65.4% identical to ZNF127 over 1.4 kb of open reading frame they share in common, Like ZNF127, the predicted protein sequence of ZNFXp contains a C{sub 3}HC{sub 4} zinc finger domain and C{sub 3}H zinc finger-like motifs. Whereas ZNF127 has three C{sub 3}H motifs, ZNFXp has four. A strong CpG island is located within 1 kb 5{prime} of the predicted amino terminus of ZNFXp. Expression of ZNFXp has been detected from mouse/human somatic cell hybrids containing either an active (n=2) or an inactive (n=4) chromosome, and thus escapes X inactivation. Probes made from the 3{prime} UTR of ZNFXp detect a number of related loci in both human and murine DNA, none of which is the ZNF127 locus on chromosome 15. None of the detectable murine bands shows dosage differences between males and females as would be expected for X-linked loci. This raises the possibility that ZNFXp inserted into the human X chromosome after its divergence from a common ancestor with the murine X. We have mapped ZNFXp to Xp11.4 by Southern blotting and PCR of hybrid DNAs and by fluorescence in situ hybridization (FISH). ZNFXq maps within the X Inactivation Center (XIC) region on Xq13.2, approximately 300 kb distal to the XIST gene. We find it intriguing, and perhaps significant, that two members of this gene family are subject to epigenetic regulation -- one autosomal imprinting, and the other escape from X inactivation. These results could imply an evolutionary and mechanistic relationship between these two processes.

  9. Mouse Homologue of the Schizophrenia Susceptibility Gene ZNF804A as a Target of Hoxc8

    Directory of Open Access Journals (Sweden)

    Hyun Joo Chung

    2010-01-01

    Full Text Available Using a ChIP-cloning technique, we identified a Zinc finger protein 804a (Zfp804a as one of the putative Hoxc8 downstream target genes. We confirmed binding of Hoxc8 to an intronic region of Zfp804a by ChIP-PCR in F9 cells as well as in mouse embryos. Hoxc8 upregulated Zfp804a mRNA levels and augmented minimal promoter activity in vitro. In E11.5 mouse embryos, Zfp804a and Hoxc8 were coexpressed. Recent genome-wide studies identified Zfp804a (or ZNF804A in humans as a plausible marker for schizophrenia, leading us to hypothesize that this embryogenic regulatory control might also exert influence in development of complex traits such as psychosis.

  10. IL-2R{gamma} gene microdeletion demonstrates that canine X-linked severe combined immunodeficiency is a homologue of the human disease

    Energy Technology Data Exchange (ETDEWEB)

    Henthorn, P.S.; Fimiani, V.M.; Patterson, D.F. [Univ. of Pennsylvania School of Veterinary Medicine, Philadelphia, PA (United States)] [and others

    1994-09-01

    X-linked severe combined immunodeficiency (SCID) is characterized by profound defects in cellular and humoral immunity and, in humans, is associated with mutations in the gene for the {gamma} chain of the IL-2 receptor (IL-2R{gamma}). We have examined this gene in a colony of dogs established from a single X-linked SCID carrier female. Affected dogs have a 4-bp deletion in the first exon of the IL-2R{gamma} gene, which precludes the production of a functional protein, demonstrating that the canine disease is a true homologue of human X-linked SCID. 37 refs., 3 figs.

  11. Sugar beet contains a large CONSTANS-LIKE gene family including a CO homologue that is independent of the early-bolting (B) gene locus.

    Science.gov (United States)

    Chia, T Y P; Müller, A; Jung, C; Mutasa-Göttgens, E S

    2008-01-01

    Floral transition in the obligate long-day (LD) plant sugar beet (Beta vulgaris ssp. vulgaris) is tightly linked to the B gene, a dominant early-bolting quantitative trait locus, the expression of which is positively regulated by LD photoperiod. Thus, photoperiod regulators like CONSTANS (CO) and CONSTANS-LIKE (COL) genes identified in many LD and short-day (SD)-responsive plants have long been considered constituents and/or candidates for the B gene. Until now, the photoperiod response pathway of sugar beet (a Caryophyllid), diverged from the Rosids and Asterids has not been identified. Here, evidence supporting the existence of a COL gene family is provided and the presence of Group I, II, and III COL genes in sugar beet, as characterized by different zinc-finger (B-box) and CCT (CO, CO-like, TOC) domains is demonstrated. BvCOL1 is identified as a close-homologue of Group 1a (AtCO, AtCOL1, AtCOL2) COL genes, hence a good candidate for flowering time control and it is shown that it maps to chromosome II but distant from the B gene locus. The late-flowering phenotype of A. thaliana co-2 mutants was rescued by over-expression of BvCOL1 thereby suggesting functional equivalence with AtCO, and it is shown that BvCOL1 interacts appropriately with the endogenous downstream genes, AtFT and AtSOC1 in the transgenic plants. Curiously, BvCOL1 has a dawn-phased diurnal pattern of transcription, mimicking that of AtCOL1 and AtCOL2 while contrasting with AtCO. Taken together, these data suggest that BvCOL1 plays an important role in the photoperiod response of sugar beet.

  12. A gonococcal homologue of meningococcal γ-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent

    Directory of Open Access Journals (Sweden)

    Watanabe Haruo

    2005-10-01

    Full Text Available Abstract Background It has been speculated that the γ-glutamyl transpeptidase (ggt gene is present only in Neisseria meningitidis and not among related species such as Neisseria gonorrhoeae and Neisseria lactamica, because N. meningitidis is the only bacterium with GGT activity. However, nucleotide sequences highly homologous to the meningococcal ggt gene were found in the genomes of N. gonorrhoeae isolates. Results The gonococcal homologue (ggt gonococcal homologue; ggh was analyzed. The nucleotide sequence of the ggh gene was approximately 95 % identical to that of the meningococcal ggt gene. An open reading frame in the ggh gene was disrupted by an ochre mutation and frameshift mutations induced by a 7-base deletion, but the amino acid sequences deduced from the artificially corrected ggh nucleotide sequences were approximately 97 % identical to that of the meningococcal ggt gene. The analyses of the sequences flanking the ggt and ggh genes revealed that both genes were localized in a common DNA region containing the fbp-ggt (or ggh-glyA-opcA-dedA-abcZ gene cluster. The expression of the ggh RNA could be detected by dot blot, RT-PCR and primer extension analyses. Moreover, the truncated form of ggh-translational product was also found in some of the gonococcal isolates. Conclusion This study has shown that the gonococcal ggh gene is a pseudogene of the meningococcal ggt gene, which can also be designated as Ψggt. The gonococcal ggh (Ψggt gene is the first identified bacterial pseudogene that is transcriptionally active but phenotypically silent.

  13. Genetic analysis of the spindle checkpoint genes san-1, mdf-2, bub-3 and the CENP-F homologues hcp-1 and hcp-2 in Caenorhabditis elegans.

    Science.gov (United States)

    Hajeri, Vinita A; Stewart, Anil M; Moore, Landon L; Padilla, Pamela A

    2008-02-04

    The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role san-1, the MAD3 homologue, has in Caenorhabditis elegans embryos we used RNA interference (RNAi) to identify genes synthetic lethal with the viable san-1(ok1580) deletion mutant. The san-1(ok1580) animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of san-1(ok1580) embryos is significantly reduced when HCP-1 (CENP-F homologue), MDF-1 (MAD-1 homologue), MDF-2 (MAD-2 homologue) or BUB-3 (predicted BUB-3 homologue) are reduced by RNAi. Interestingly, the viability of san-1(ok1580) embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of san-1(ok1580);hcp-1(RNAi) embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging). Several of the san-1(ok1580);hcp-1(RNAi) animals displayed abnormal kinetochore (detected by MPM-2) and microtubule structure. The survival of mdf-2(RNAi);hcp-1(RNAi) embryos but not bub-3(RNAi);hcp-1(RNAi) embryos was also compromised. Finally, we found that san-1(ok1580) and bub-3(RNAi), but not hcp-1(RNAi) embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein. Together, these data suggest that in the C. elegans embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that san-1(ok1580);hcp-1(RNAi) animals had a severe viability defect whereas in the san-1(ok1580);hcp-2(RNAi) and san-1(ok1580);hcp-2(ok1757) animals the viability defect was not as severe suggesting that hcp-1 and hcp-2 are not completely redundant.

  14. Genetic analysis of the spindle checkpoint genes san-1, mdf-2, bub-3 and the CENP-F homologues hcp-1 and hcp-2 in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Moore Landon L

    2008-02-01

    Full Text Available Abstract Background The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role san-1, the MAD3 homologue, has in Caenorhabditis elegans embryos we used RNA interference (RNAi to identify genes synthetic lethal with the viable san-1(ok1580 deletion mutant. Results The san-1(ok1580 animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of san-1(ok1580 embryos is significantly reduced when HCP-1 (CENP-F homologue, MDF-1 (MAD-1 homologue, MDF-2 (MAD-2 homologue or BUB-3 (predicted BUB-3 homologue are reduced by RNAi. Interestingly, the viability of san-1(ok1580 embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of san-1(ok1580;hcp-1(RNAi embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging. Several of the san-1(ok1580;hcp-1(RNAi animals displayed abnormal kinetochore (detected by MPM-2 and microtubule structure. The survival of mdf-2(RNAi;hcp-1(RNAi embryos but not bub-3(RNAi;hcp-1(RNAi embryos was also compromised. Finally, we found that san-1(ok1580 and bub-3(RNAi, but not hcp-1(RNAi embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein. Conclusion Together, these data suggest that in the C. elegans embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that san-1(ok1580;hcp-1(RNAi animals had a severe viability defect whereas in the san-1(ok1580;hcp-2(RNAi and san-1(ok1580;hcp-2(ok1757 animals the viability defect was not as severe suggesting that hcp-1 and hcp-2 are not completely redundant.

  15. Six3, a medaka homologue of the Drosophila homeobox gene sine oculis is expressed in the anterior embryonic shield and the developing eye.

    Science.gov (United States)

    Loosli, F; Köster, R W; Carl, M; Krone, A; Wittbrodt, J

    1998-06-01

    homologue Six3 (Oliver, G., Mailhos, A., Wehr, R., Copeland, N.G., Jenkins, N.A., Gruss, P., 1995. Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121, 4045-4055). sine oculis (so) is essential for the development of the larval and adult visual system (Cheyette, B.N.R., Green, P.J., Martin, K., Garren, H., Hartenstein, V., Zipursky, S.L., 1994. The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron l2, 977-996). Six3 is expressed in the anterior neural plate and optic vesicles, lens, olfactory placodes and ventral forebrain (Oliver, G., Mailhos, A., Wehr, R., Copeland, N.G., Jenkins, N.A., Gruss, P., 1995. Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121, 4045-4055). Overexpression of mouse Six3 gene in medaka fish embryos (Orvzias latipes) results in the formation of an ectopic lens, indicating that Six3 activity can trigger the genetic pathway leading to lens formation (Oliver, G., Loosli, F., Koster, R., Wittbrodt, J., Gruss, P., 1996. Ectopic lens induction in fish in response to the murine homeobox gene Six3. Mech. Dev. 60, 233-239). We isolated the medaka Six3 homologue and analyzed its expression pattern in the medaka embryo. It is expressed initially in the anterior embryonic shield and later in the developing eye and prosencephalon. The early localized expression of Six3 suggests a role in the regionalization of the rostral head.

  16. Analysis of a homologue of the adducin head gene which is a potential target for the Dictyostelium STAT protein Dd-STATa.

    Science.gov (United States)

    Aoshima, Ryota; Hiraoka, Rieko; Shimada, Nao; Kawata, Takefumi

    2006-01-01

    A Dd-STATa-null mutant, which is defective in expression of a Dictyostelium homologue of the metazoan STAT (signal transducers and activators of transcription) proteins, fails to culminate and this phenotype correlates with the loss of expression of various prestalk (pst) genes. An EST clone, SSK395, encodes a close homologue of the adducin amino-terminal head domain and harbors a putative actin-binding domain. We fused promoter fragments of the cognate gene, ahhA (adducin head homologue A), to a lacZ reporter and determined their expression pattern. The proximal promoter region is necessary for the expression of ahhA at an early (pre-aggregative) stage of development and this expression is Dd-STATa independent. The distal promoter region is necessary for expression at later stages of development in pstA cells, of the slug and in upper cup and pstAB cells during culmination. The distal region is partly Dd-STATa-dependent. The ahhA-null mutant develops almost normally until culmination, but it forms slanting culminants that tend to collapse on to the substratum. The mutant also occasionally forms fruiting bodies with swollen papillae and with constrictions in the prestalk region. The AhhA protein localizes to the stalk tube entrance and also to the upper cup cells and in cells at or near to the constricted region where an F-actin ring is localized. These findings suggest that Dd-STATa regulates culmination and may be necessary for straight downward elongation of the stalk, via the putative actin-binding protein AhhA.

  17. Cloning of the cDNA for a human homologue of the Drosophila white gene and mapping to chromosome 21q22.3

    Energy Technology Data Exchange (ETDEWEB)

    Haiming Chen; Lalioti, M.D.; Perrin, G.; Antonarakis, S.E. [Univ. of Geneva Medical School (Switzerland)] [and others

    1996-07-01

    In an effort to contribute to the transcript map of human chromosome 21 and the understanding of the pathophysiology of trisomy 21, we have used exon trapping to identify fragments of chromosome 21 genes. Two trapped exons, from pools of chromosome 21-specific cosmids, showed homology to the Drosophila white (w) gene. We subsequently cloned the corresponding cDNA for a human homologue of the Drosophila w gene (hW) from human retina and fetal brain cDNA libraries. The gene belongs to the ATP-binding cassette transporter gene family and is homologous to Drosophila w (and to 2 genes from other species) and to a lesser extent to Drosophila brown (bw) and scarlet (st) genes that are all involved in the transport of eye pigment precursor molecules. A DNA polymorphism with 62% heterozygosity due to variation of a poly (T) region in the 3{prime} UTR of the hW has been identified and used for the incorporation of this gene to the genetic map of chromosome 21. The hW is located at 21q22.3 between DNA markers D21S212 and D21S49 in a P1 clone that also contains marker BCEI. The gene is expressed at various levels in many human tissues. The contributions of this gene to the Down syndrome phenotypes, to human eye color, and to the resulting phenotypes of null or missense mutations are presently unknown. 56 refs., 8 figs., 1 tab.

  18. Isolation of new genes in distal Xq28: transcriptional map and identification of a human homologue of the ARD1 N-acetyl transferase of Saccharomyces cerevisiae.

    Science.gov (United States)

    Tribioli, C; Mancini, M; Plassart, E; Bione, S; Rivella, S; Sala, C; Torri, G; Toniolo, D

    1994-07-01

    In this paper, we describe the physical and transcriptional organization of a region of 140 kb in Xq28, 5' to the L1CAM gene. By isolation and mapping of CpG islands to the physical map of the region, isolation of cDNAs, determination of partial nucleotide sequences and study of the pattern of expression and of the orientation of the transcripts identified we have established a transcriptional map of this region. In this map, previously identified genes (L1CAM, V2R, HCF1 and RnBP) have been positioned as well as 3 new genes. All genes in the region are rather small, ranging in size from 2 to 30 kb, and very close to one another. With the exception of the V2R gene, they are housekeeping, have a CpG island at their 5' end and the same orientation of transcription. This kind of organization is consistent with the one previously described for the more distal portion of Xq28, between the Color Vision (CV) and the G6PD genes and indicates that genes with housekeeping and tissue specific pattern of expression are interspersed in the genome but they are probably found in different 'transcriptional domains'. Among the new genes, TE2 demonstrated 40% identity with the protein N-acetyl transferase ARD1 of S. cerevisiae: TE2 may be the human homologue of the S. cerevisiae gene.

  19. Histopathological variables and biomarkers enhancer of zeste homologue 2, Ki-67 and minichromosome maintenance protein 7 as prognosticators in primarily endocrine-treated prostate cancer.

    Science.gov (United States)

    Tolonen, Teemu T; Tammela, Teuvo L J; Kujala, Paula M; Tuominen, Vilppu J; Isola, Jorma J; Visakorpi, Tapio

    2011-11-01

    • To evaluate the prognostic value of histopathological variables and immunostainings of biomarkers enhancer of zeste homologue 2 (EZH2), Ki-67 and minichromosome maintenance protein 7 (MCM7) from core biopsies of hormonally treated patients with prostate cancer. • Biopsies of 247 primarily endocrine-treated patients were analysed for histopathological characteristics and Gleason scores (GS) according to the revised guidelines of International Society of Urologic Pathology (ISUP) consensus conference 2005. • Immunohistochemical stainings were analysed with the aid of digital image analysis. • The prognostic value of the histopathological variables and the biomarkers was analysed with univariate and multivariate Cox regression analysis, with biochemical recurrence as an endpoint. • Biomarkers EZH2 (relative risk [RR] 2.0, 95% confidence interval 1.2-3.3), Ki-67 (3.4, 2.1-5.5) and MCM7 (2.4, 1.5-3.9) were significantly associated with progression-free survival in a univariate analysis. • Ki-67 immunostaining index detected high-risk patients with GS of 7 (9.1, 8.0-10.3). • In a multivariate analysis with non-conventional GS groups 5-7 (3 + 4), 7(4 + 3)-8, and 9-10, the independent prognostic markers were pretreatment GS (2.2, 1.5-3.2), prostate-specific antigen (PSA) level (2.1, 1.1-4.2), perineural invasion (PNI) (1.6, 1.2-2.2), and clinical T-stage (cT) (1.9, 1.0-3.7). • Combination of the independent markers (PSA level > 20 ng/mL or GS >3 + 4 or PNI >3 or cT >2) yielded best risk stratification (RR 11.6, 10.4-12.7). • GS remains one of the most important prognostic factors in prostate cancer. However, the refined guidelines by ISUP 2005 might have shifted the threshold between low-grade and high-grade cancers from GS 6 vs 7 to GS 3 + 4 vs 4 + 3. • PNI is an independent prognostic marker superior to cT. • Ki-67 is the most useful biomarker in detecting patients with GS = 7 at high risk for progression. © 2011 THE AUTHORS. BJU INTERNATIONAL

  20. A homologue of the defender against the apoptotic death gene (dad1) in UV-exposed Chlamydomonas cells is downregulated with the onset of programmed cell death

    Indian Academy of Sciences (India)

    Swati Moharikar; Jacinta S D’souza; Basuthkar J Rao

    2007-03-01

    We report here the isolation of a homologue of the potential anti-apoptotic gene, defender against apoptotic death (dad1) from Chlamydomonas reinhardtii cells. Using polymerase chain reaction (PCR), we investigated its expression in the execution process of programmed cell death (PCD) in UV-C exposed dying C. reinhardtii cells. Reverse-transcriptase (RT)-PCR showed that C. reinhardtii dad1 amplification was drastically reduced in UV-C exposed dying C. reinhardtii cells. We connect the downregulation of dad1 with the upregulation of apoptosis protease activating factor-1 (APAF-1) and the physiological changes that occur in C. reinhardtii cells upon exposure to 12 J/m2 UV-C in order to show a reciprocal relationship between proapoptotic and inhibitor of apoptosis factors. The temporal changes indicate a correlation between the onset of cell death and dad1 downregulation. The sequence of the PCR product of the cDNA encoding the dad1 homologue was aligned with the annotated dad1 (C_20215) from the Chlamydomonas database (http://genome.jgi-psf.org:8080/annotator/servlet/jgi.annotation.Annotation?pDb=chlre2); Annotation?pDb=chlre2); this sequence was found to show 100% identity, both at the nucleotide and amino acid level. The 327 bp transcript showed an open reading frame of 87 amino acid residues. The deduced amino acid sequence of the putative C. reinhardtii DAD1 homologue showed 54% identity with Oryza sativa, 56% identity with Drosophila melanogaster, 66% identity with Xenopus laevis, and 64% identity with Homo sapiens, Sus scrofa, Gallus gallus, Rattus norvegicus and Mus musculus.

  1. Gene therapy to treat cardiac arrhythmias.

    Science.gov (United States)

    Bongianino, Rossana; Priori, Silvia G

    2015-09-01

    Gene therapy to treat electrical dysfunction of the heart is an appealing strategy because of the limited therapeutic options available to manage the most-severe cardiac arrhythmias, such as ventricular tachycardia, ventricular fibrillation, and asystole. However, cardiac genetic manipulation is challenging, given the complex mechanisms underlying arrhythmias. Nevertheless, the growing understanding of the molecular basis of these diseases, and the development of sophisticated vectors and delivery strategies, are providing researchers with adequate means to target specific genes and pathways involved in disorders of heart rhythm. Data from preclinical studies have demonstrated that gene therapy can be successfully used to modify the arrhythmogenic substrate and prevent life-threatening arrhythmias. Therefore, gene therapy might plausibly become a treatment option for patients with difficult-to-manage acquired arrhythmias and for those with inherited arrhythmias. In this Review, we summarize the preclinical studies into gene therapy for acquired and inherited arrhythmias of the atria or ventricles. We also provide an overview of the technical advances in the design of constructs and viral vectors to increase the efficiency and safety of gene therapy and to improve selective delivery to target organs.

  2. Treating Immunodeficiency through HSC Gene Therapy.

    Science.gov (United States)

    Booth, Claire; Gaspar, H Bobby; Thrasher, Adrian J

    2016-04-01

    Haematopoietic stem cell (HSC) gene therapy has been successfully employed as a therapeutic option to treat specific inherited immune deficiencies, including severe combined immune deficiencies (SCID) over the past two decades. Initial clinical trials using first-generation gamma-retroviral vectors to transfer corrective DNA demonstrated clinical benefit for patients, but were associated with leukemogenesis in a number of cases. Safer vectors have since been developed, affording comparable efficacy with an improved biosafety profile. These vectors are now in Phase I/II clinical trials for a number of immune disorders with more preclinical studies underway. Targeted gene editing allowing precise DNA correction via platforms such as ZFNs, TALENs and CRISPR/Cas9 may now offer promising strategies to improve the safety and efficacy of gene therapy in the future.

  3. Increased frequency of DNA deletions in pink-eyed unstable mice carrying a mutation in the Werner syndrome gene homologue.

    Science.gov (United States)

    Lebel, Michel

    2002-01-01

    Werner syndrome (WS) is a rare autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases, including cancers. Accumulating evidence indicates that the WS gene product is involved in resolving aberrant DNA structures that may arise during the process of DNA replication and/or transcription. To estimate the frequency of DNA deletions directly in the skin of mouse embryos, mice with a deletion of part of the murine WRN helicase domain were created. These mutant mice were then crossed to the pink-eyed unstable animals, which have a 70 kb internal duplication at the pink-eyed dilution (p) gene. This report indicates that the frequency of deletion of the duplicated sequence at the p locus is elevated in mice with a mutation in the WRN allele when compared with wild-type mice. In addition, the inhibitor of topoisomerase I camptothecin also increases the frequency of deletion at the p locus. This frequency is even more elevated in WRN mutant mice treated with camptothecin. In contrast, while the inhibition of poly(ADP-ribose) polymerase (PARP) activity by 3-aminobenzamide increases the frequency of DNA deletion, mutant WRN mice are not significantly more sensitive to the inhibition of PARP activity than wild-type animals.

  4. Molecular cloning and identification of mouse epididymis-specific gene mHong1, the homologue of rat HongrES1

    Institute of Scientific and Technical Information of China (English)

    Shuang-Gang Hu; Han Du; Guang-Xin Yao; Yong-Lian Zhang

    2012-01-01

    Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility.In this study,we cloned the mouse homologue gene by sequence alignment and RT-PCR methods and designated it as mHong1.The mHong1 gene is located on chromosome 12p14,spanning five exons.The cDNA sequence consists of 1257 nucleotides and encodes a 419 amino-acid protein with a predicted N-terminal signal peptide of 20 amino acids.The mHong1 mRNA shows similarity with HongrES1 in the expression patterns:(i) specific expression in epididymal tissue,especially in the cauda region; and (ii) androgen-dependence but testicular fluid factor independence.Its protein product shows 71% similarity with HongrES 1 and contains a classical serpin domain as does HongrES1.A polyclonal antibody against mHong1 with high specificity and sensitivity was raised.Like HongrES1,the mHong1 protein shows a checker-board expression pattern in the epididymal epithelium and is secreted into the epididymal lumen.The mHong1 protein shows higher glycosylation than HongrES1.Although both of them are deposited onto the sperm head surface,mHong 1 is localized to the equatorial segment,which is different from that of HongrES 1.The mHong1 protein can be removed from the sperm membrane by high ionic strength and therefore can be classed as an extrinsic membrane protein.Collectively,we conclude that mHong1 is the homologue of HongrES1 and the present work paves the way for establishing animal models to elucidate the precise functions of HongrES1 and mHong1.

  5. Characterization and mapping to human chromosome 8q24.3 of Ly-6-related gene 9804 encoding an apparent homologue of mouse TSA-1.

    Science.gov (United States)

    Shan, X; Bourdeau, A; Rhoton, A; Wells, D E; Cohen, E H; Landgraf, B E; Palfree, R G

    1998-01-01

    The 9804 gene, which encodes a human Ly-6 protein most similar to mouse differentiation Ag TSA-1/Sca-2, has also been called RIG-E. Like mouse TSA-1, it has a broad tissue distribution with varied expression levels in normal human tissues and tumor cell lines. Like some members of the murine Ly-6 family, the 9804 gene is responsive to IFNs, particularly IFN-alpha. Overlapping genomic fragments spanning the 9804 gene (5543 bp) have been isolated and characterized. The gene organization is analogous to that of known mouse Ly-6 genes. The first exon, 2296 bp upstream from exon II, is entirely untranslated. The three coding exons (II, III, and IV) are separated by short introns of 321 and 131 bp, respectively. Primers were developed for specific amplification of 9804 gene fragments. Screening of human-hamster somatic cell hybrids and yeast artificial chromosomes (YACs) indicated that the gene is distal to c-Myc, located in the q arm of human chromosome 8. No positives were detected from the Centre d'Etude du Polymorphisme Humain mega-YAC A or B panels, nor from bacterial artificial chromosome libraries; two positive cosmids (c101F1 and c157F6) were isolated from a human chromosome 8 cosmid library (LA08NC01). Fluorescence in situ hybridization of metaphase spreads of chromosome 8, containing hybrid cell line 706-B6 clone 17 (CL-17) with cosmid c101F1, placed the 9804 gene close to the telomere at 8q24.3. This mapping is significant, since the region shares a homology with a portion of mouse chromosome 15, which extends into band E where Ly-6 genes reside. Moreover, the gene encoding E48, the homologue of mouse Ly-6 molecule ThB, has also been mapped to 8q24.

  6. Constitutive Expression of Sense & Antisense PtAP3, an AP3 Homologue Gene of Populus tomentosa, Affects Growth and Flowering Time in Transgenic Tobacco

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To analyze the function of PtAP3, an APETALA3 (AP3) homologue gene isolated from Populus tomentosa Carr., the full length sequence (1 797 bp) and a fragment (870 bp) of PtAP3 were fused to a CaMV 35S promoter of pBI121 to generate the sense and antisense constructs of PtAP3. These constructs were transformed into tobacco by Agrobacterium infection of leaf disks and selection on kanamycin medium. Some sense and antisense transgenic tobacco plants were obtained by PCR and Southern blot analysis. Great phenotypic differences in transgenic tobacco plants were observed. Almost all of sense PtAP3 to transgenic tobaccos showed a higher growth rate than those of antisense transformants and a few developed pregnancy earlier than wild type seedlings and antisense transformants under the same conditions.

  7. Characterization of the Distal-less gene homologue, NlDll, in the brown planthopper, Nilaparvata lugens (Stål).

    Science.gov (United States)

    Lin, Xinda; Yao, Yun; Jin, Minna; Li, Qilin

    2014-02-10

    The brown planthopper, Nilaparvata lugens (Stål), is a globally devastating insect pest of rice, particularly in eastern Asia. Distal-less or Dll is a highly conserved and well studied transcription factor required for limb formation in invertebrates and vertebrates. We have identified a homologue of this gene, NlDll, and demonstrated that it is expressed in all life stages of N. lugens, particularly in adult brachypterous females. When we compared between specific adult tissues it was expressed most strongly in wings. Using RNAi techniques we demonstrated that downregulation of NlDll in the 3rd instar larvae led to the disrupted development of the leg, while downregulation of NlDll in the 5th instar larvae led to abnormal wing formation. Ectopic over-expression of NlDll in Drosophila melanogaster using the GAL4-UAS system led to fatal or visible phenotypic changes such as the loss of normal wing structure and disrupted haltere structure. Our work suggests that NlDll is a conserved homologue of Distal-less and is required for both leg development and wing structure. Since researches have shown that Dll is required for wing morphogenesis, understanding the role of NlDll during the wing development will further provide a basis for revealing the molecular mechanism of the wing dimorphism in brown planthopper. In the future, NlDll could be used as a target gene for brown planthopper pest management in the field. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  8. A wheat homologue of PHYTOCLOCK 1 is a candidate gene conferring the early heading phenotype to einkorn wheat.

    Science.gov (United States)

    Mizuno, Nobuyuki; Nitta, Miyuki; Sato, Kazuhiro; Nasuda, Shuhei

    2012-01-01

    An X-ray mutant showing an early flowering phenotype has been identified in einkorn wheat (Triticum monococcum L.), for which a major QTL for heading time was previously mapped in the telomeric region on the long arm of chromosome 3A. Recent advances in Triticeae genomics revealed that the gene order in this region is highly conserved between wheat and barley. Thus, we adopted a hypothetical gene order in barley, the so-called GenomeZipper, to develop DNA markers for fine mapping the target gene in wheat. We identified three genes tightly linked to the early heading phenotype. PCR analysis revealed that early-flowering is associated with the deletion of two genes in the mutant. Of the two deleted genes, one is an ortholog of the LUX ARRHYTHMO (LUX)/PHYTOCLOCK 1 (PCL1) gene found in Arabidopsis, which regulates the circadian clock and flowering time. We found distorted expression patterns of two clock genes (TOC1 and LHY) in the einkorn pcl1 deletion mutant as was reported for the Arabidopsis lux mutant. Transcript accumulation levels of photoperiod-response related genes, a photoperiod sensitivity gene (Ppd-1) and two wheat CONSTANS-like genes (WCO1 and TaHd1), were significantly higher in the einkorn wheat mutant. In addition, transcripts of the wheat florigen gene (WFT) accumulated temporally under short-day conditions in the einkorn wheat mutant. These results suggest that deletion of WPCL1 leads to abnormally higher expression of Ppd-1, resulting in the accumulation of WFT transcripts that triggers flowering even under short-day conditions. Our observations from gene mapping, gene deletions, and expression levels of flowering related genes strongly suggest that WPCL1 is the most likely candidate gene for controlling the early flowering phenotype in the einkorn wheat mutant.

  9. Light and auxin responsive cytochrome P450s from Withania somnifera Dunal: cloning, expression and molecular modelling of two pairs of homologue genes with differential regulation.

    Science.gov (United States)

    Srivastava, Sudhakar; Sangwan, Rajender Singh; Tripathi, Sandhya; Mishra, Bhawana; Narnoliya, L K; Misra, L N; Sangwan, Neelam S

    2015-11-01

    Cytochrome P450s (CYPs) catalyse a wide variety of oxygenation/hydroxylation reactions that facilitate diverse metabolic functions in plants. Specific CYP families are essential for the biosynthesis of species-specialized metabolites. Therefore, we investigated the role of different CYPs related to secondary metabolism in Withania somnifera, a medicinally important plant of the Indian subcontinent. In this study, complete complementary DNAs (cDNAs) of four different CYP genes were isolated and christened as WSCYP93Id, WSCYP93Sm, WSCYP734B and WSCYP734R. These cDNAs encoded polypeptides comprising of 498, 496, 522 and 550 amino acid residues with their deduced molecular mass of 56.7, 56.9, 59.4 and 62.2 kDa, respectively. Phylogenetic study and molecular modelling analysis of the four cloned WSCYPs revealed their categorization into two CYP families (CYP83B1 and CYP734A1) belonging to CYP71 and CYP72 clans, respectively. BLASTp searches showed similarity of 75 and 56 %, respectively, between the two CYP members of CYP83B1 and CYP734A1 with major variances exhibited in their N-terminal regions. The two pairs of homologues exhibited differential expression profiles in the leaf tissues of selected chemotypes of W. somnifera as well as in response to treatments such as methyl jasmonate, wounding, light and auxin. Light and auxin regulated two pairs of WSCYP homologues in a developing seedling in an interesting differential manner. Their lesser resemblance and homology with other CYP sequences suggested these genes to be more specialized and distinct ones. The results on chemotype-specific expression patterns of the four genes strongly suggested their key/specialized involvement of the CYPs in the biosynthesis of chemotype-specific metabolites, though their further biochemical characterization would reveal the specificity in more detail. It is revealed that WSCYP93Id and WSCYP93Sm may be broadly involved in the oxygenation reactions in the plant and, thereby, control

  10. Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens.

    Science.gov (United States)

    Eklund, D Magnus; Thelander, Mattias; Landberg, Katarina; Ståldal, Veronika; Nilsson, Anders; Johansson, Monika; Valsecchi, Isabel; Pederson, Eric R A; Kowalczyk, Mariusz; Ljung, Karin; Ronne, Hans; Sundberg, Eva

    2010-04-01

    The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation, delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants. Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.

  11. The shf gene of a Shigella flexneri homologue on the virulent plasmid pAA2 of enteroaggregative Escherichia coli 042 is required for firm biofilm formation.

    Science.gov (United States)

    Fujiyama, Rika; Nishi, Junichiro; Imuta, Naoko; Tokuda, Koichi; Manago, Kunihiro; Kawano, Yoshifumi

    2008-05-01

    Enteroaggregative Escherichia coli (EAEC) is an increasingly important cause of diarrhea in both developing and industrialized countries, and is characterized by strong biofilm formation on the intestinal mucosa. Sequencing of the virulent plasmid pAA2 of the prototype EAEC 042 revealed a cluster of three open reading frames (ORFs; shf, capU, and virK) ca. 93% identical to a similar cluster located in Shigella flexneri. The function of the first ORF Shf protein is not known, but the closest well-characterized homologue is the IcaB protein of Staphylococcus epidermidis, which plays a crucial role in exopolysaccharide modification in bacterial biofilm formation. To investigate the role of this cluster in the virulence of EAEC, we mutated three genes at this locus. All the mutants maintained the aggregative phenotype in the liquid phase. However, the insertional mutant of shf formed a less abundant biofilm in a microtiter plate assay than did the wild type, while the capU mutant and the virK mutant did not. The complementation of the shf mutant with this cluster restored the thick biofilm similar to that of the wild type. The shf transcriptional level decreased in the transcriptional regulator aggR mutant and was restored when the mutant was complemented with aggR. These results suggest that the shf gene is required for the firm biofilm formation of EAEC 042, and transcription of the shf gene is dependent on AggR.

  12. DFL1, an auxin-responsive GH3 gene homologue, negatively regulates shoot cell elongation and lateral root formation, and positively regulates the light response of hypocotyl length.

    Science.gov (United States)

    Nakazawa, M; Yabe, N; Ichikawa, T; Yamamoto, Y Y; Yoshizumi, T; Hasunuma, K; Matsui, M

    2001-01-01

    A novel dominant mutant designated 'dwarf in light 1' (dfl1-D) was isolated from screening around 1200 Arabidopsis activation-tagged lines. dfl1-D has a shorter hypocotyl under blue, red and far-red light, but not in darkness. Inhibition of cell elongation in shoots caused an exaggerated dwarf phenotype in the adult plant. The lateral root growth of dfl1-D was inhibited without any reduction of primary root length. The genomic DNA adjacent to the right border of T-DNA was cloned by plasmid rescue. The rescued genomic DNA contained a gene encoding a GH3 homologue. The transcript was highly accumulated in the dfl1-D. The dfl1-D phenotype was confirmed by over-expression of the gene in the wild-type plant. The dfl1-D showed resistance to exogenous auxin treatment. Moreover, over-expression of antisense DFL1 resulted in larger shoots and an increase in the number of lateral roots. These results indicate that the gene product of DFL1 is involved in auxin signal transduction, and inhibits shoot and hypocotyl cell elongation and lateral root cell differentiation in light.

  13. Multiple splice variants within the bovine silver homologue (SILV gene affecting coat color in cattle indicate a function additional to fibril formation in melanophores

    Directory of Open Access Journals (Sweden)

    Weikard Rosemarie

    2007-09-01

    Full Text Available Abstract Background The silver homologue(SILV gene plays a major role in melanosome development. SILV is a target for studies concerning melanoma diagnostics and therapy in humans as well as on skin and coat color pigmentation in many species ranging from zebra fish to mammals. However, the precise functional cellular mechanisms, in which SILV is involved, are still not completely understood. While there are many studies addressing SILV function upon a eumelaneic pigment background, there is a substantial lack of information regarding the further relevance of SILV, e.g. for phaeomelanosome development. Results In contrast to previous results in other species reporting SILV expression exclusively in pigmented tissues, our experiments provide evidence that the bovine SILV gene is expressed in a variety of tissues independent of pigmentation. Our data show that the bovine SILV gene generates an unexpectedly large number of different transcripts occurring in skin as well as in non-pigmented tissues, e.g. liver or mammary gland. The alternative splice sites are generated by internal splicing and primarily remove complete exons. Alternative splicing predominantly affects the repeat domain of the protein, which has a functional key role in fibril formation during eumelanosome development. Conclusion The expression of the bovine SILV gene independent of pigmentation suggests SILV functions exceeding melanosome development in cattle. This hypothesis is further supported by transcript variants lacking functional key elements of the SILV protein relevant for eumelanosome development. Thus, the bovine SILV gene can serve as a model for the investigation of the putative additional functions of SILV. Furthermore, the splice variants of the bovine SILV gene represent a comprehensive natural model to refine the knowledge about functional domains in the SILV protein. Our study exemplifies that the extent of alternative splicing is presumably much higher than

  14. Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in Vibrio anguillarum strain 775.

    Science.gov (United States)

    Di Lorenzo, Manuela; Stork, Michiel; Crosa, Jorge H

    2011-08-01

    Anguibactin, the siderophore produced by Vibrio anguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. In this work we report the identification of a homologue of the plasmid-encoded angB on the chromosome of strain 775. The product of both genes harbor an isochorismate lyase (ICL) domain that converts isochorismic acid to 2,3-dihydro-2,3-dihydroxybenzoic acid, one of the steps of DHBA synthesis. We show in this work that both ICL domains are functional in the production of DHBA in V. anguillarum as well as in E. coli. Substitution by alanine of the aspartic acid residue in the active site of both ICL domains completely abolishes their isochorismate lyase activity in vivo. The two proteins also carry an aryl carrier protein (ArCP) domain. In contrast with the ICL domains only the plasmid encoded ArCP can participate in anguibactin production as determined by complementation analyses and site-directed mutagenesis in the active site of the plasmid encoded protein, S248A. The site-directed mutants, D37A in the ICL domain and S248A in the ArCP domain of the plasmid encoded AngB were also tested in vitro and clearly show the importance of each residue for the domain function and that each domain operates independently.

  15. Deep mRNA sequencing of the Tritonia diomedea brain transcriptome provides access to gene homologues for neuronal excitability, synaptic transmission and peptidergic signalling.

    Directory of Open Access Journals (Sweden)

    Adriano Senatore

    Full Text Available The sea slug Tritonia diomedea (Mollusca, Gastropoda, Nudibranchia, has a simple and highly accessible nervous system, making it useful for studying neuronal and synaptic mechanisms underlying behavior. Although many important contributions have been made using Tritonia, until now, a lack of genetic information has impeded exploration at the molecular level.We performed Illumina sequencing of central nervous system mRNAs from Tritonia, generating 133.1 million 100 base pair, paired-end reads. De novo reconstruction of the RNA-Seq data yielded a total of 185,546 contigs, which partitioned into 123,154 non-redundant gene clusters (unigenes. BLAST comparison with RefSeq and Swiss-Prot protein databases, as well as mRNA data from other invertebrates (gastropod molluscs: Aplysia californica, Lymnaea stagnalis and Biomphalaria glabrata; cnidarian: Nematostella vectensis revealed that up to 76,292 unigenes in the Tritonia transcriptome have putative homologues in other databases, 18,246 of which are below a more stringent E-value cut-off of 1x10-6. In silico prediction of secreted proteins from the Tritonia transcriptome shotgun assembly (TSA produced a database of 579 unique sequences of secreted proteins, which also exhibited markedly higher expression levels compared to other genes in the TSA.Our efforts greatly expand the availability of gene sequences available for Tritonia diomedea. We were able to extract full length protein sequences for most queried genes, including those involved in electrical excitability, synaptic vesicle release and neurotransmission, thus confirming that the transcriptome will serve as a useful tool for probing the molecular correlates of behavior in this species. We also generated a neurosecretome database that will serve as a useful tool for probing peptidergic signalling systems in the Tritonia brain.

  16. Cloning, comparative mapping, and RNA expression of the mouse homologues of the Saccharomyces cerevisiae nucleotide excision repair gene RAD23.

    NARCIS (Netherlands)

    P.J. van der Spek (Peter); C.E. Visser (Cécile); F. Hanaoka (Fumio); B. Smit (Bep); A. Hagemeijer (Anne); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1996-01-01

    textabstractThe Saccharomyces cerevisiae RAD23 gene is involved in nucleotide excision repair (NER). Two human homologs of RAD23, HHR23A and HHR23B (HGMW-approved symbols RAD23A and RAD23B), were previously isolated. The HHR23B protein is complexed with the protein defective in the cancer-prone

  17. Isolation of a cotton CAP gene: a homologue of adenylyl cyclase-associated protein highly expressed during fiber elongation.

    Science.gov (United States)

    Kawai, M; Aotsuka, S; Uchimiya, H

    1998-12-01

    The cDNA encoding CAP (adenylyl cyclase-associated protein) was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. The cDNA (GhCAP) contained an open reading frame that encoded 471 amino acid residues. RNA blot analysis showed that the cotton CAP gene was expressed mainly in young fibers.

  18. Over-expression of HOX-8, the human homologue of the mouse Hox-8 homeobox gene, in human tumors.

    Science.gov (United States)

    Suzuki, M; Tanaka, M; Iwase, T; Naito, Y; Sugimura, H; Kino, I

    1993-07-15

    A human ovarian yolk sac tumor cDNA library was screened for homeobox genes with an oligonucleotide probe under low stringent condition. Three homeobox genes were isolated, two of which were identified as HHO.c1 and HB24. The third was highly homologous with the mouse Hox-8 gene and was designated as HOX-8. Studies on RNAs from 25 human tumor tissues and cell lines showed that the profile of HOX-8 expression was different from those of HHO.c1 and HB24. The expression of HOX-8 was not detected in hematopoietic tumor cells, in which HHO.c1 and HB24 were highly expressed. HOX-8 was expressed at higher levels in a variety of tumors of epithelial origin than in their corresponding normal tissues more frequently than HHO.c1 and HB24. All three homeobox genes were highly expressed in a yolk sac tumor, an immature tumor of gonadal origin. These results suggest that HOX-8 plays a more important role in human tumors of epithelial origin than those of hematopoietic origin.

  19. Capturing sequence variation among flowering-time regulatory gene homologues in the allopolyploid crop species Brassica napus

    Directory of Open Access Journals (Sweden)

    Sarah eSchiessl

    2014-08-01

    Full Text Available Flowering, the transition from the vegetative to the generative phase, is a decisive time point in the lifecycle of a plant. Flowering is controlled by a complex network of transcription factors, photoreceptors, enzymes and miRNAs. In recent years, several studies gave rise to the hypothesis that this network is also strongly involved in the regulation of other important lifecycle processes ranging from germination and seed development through to fundamental developmental and yield-related traits. In the allopolyploid crop species Brassica napus, (genome AACC, homoeologous copies of flowering time regulatory genes are implicated in major phenological variation within the species, however the extent and control of intraspecific and intergenomic variation among flowering-time regulators is still unclear. To investigate differences among B. napus morphotypes in relation to flowering-time gene variation, we performed targeted deep sequencing of 29 regulatory flowering-time genes in four genetically and phenologically diverse B. napus accessions. The genotype panel included a winter-type oilseed rape, a winter fodder rape, a spring-type oilseed rape (all B. napus ssp. napus and a swede (B. napus ssp. napobrassica, which show extreme differences in winter-hardiness, vernalization requirement and flowering behaviour. A broad range of genetic variation was detected in the targeted genes for the different morphotypes, including non-synonymous SNPs, copy number variation and presence-absence variation. The results suggest that this broad variation in vernalisation, clock and signaling genes could be a key driver of morphological differentiation for flowering-related traits in this recent allopolyploid crop species.

  20. The PAM1 gene of petunia, required for intracellular accommodation and morphogenesis of arbuscular mycorrhizal fungi, encodes a homologue of VAPYRIN.

    Science.gov (United States)

    Feddermann, Nadja; Muni, Rajasekhara Reddy Duvvuru; Zeier, Tatyana; Stuurman, Jeroen; Ercolin, Flavia; Schorderet, Martine; Reinhardt, Didier

    2010-11-01

    Most terrestrial plants engage into arbuscular mycorrhizal (AM) symbiosis with fungi of the phylum Glomeromycota. The initial recognition of the fungal symbiont results in the activation of a symbiosis signalling pathway that is shared with the root nodule symbiosis (common SYM pathway). The subsequent intracellular accommodation of the fungus, and the elaboration of its characteristic feeding structures, the arbuscules, depends on a genetic programme in the plant that has recently been shown to involve the VAPYRIN gene in Medicaco truncatula. We have previously identified a mutant in Petunia hybrida, penetration and arbuscule morphogenesis 1 (pam1), that is defective in the intracellular stages of AM development. Here, we report on the cloning of PAM1, which encodes a VAPYRIN homologue. PAM1 protein localizes to the cytosol and the nucleus, with a prominent affinity to mobile spherical structures that are associated with the tonoplast, and are therefore referred to as tonospheres. In mycorrhizal roots, tonospheres were observed in the vicinity of intracellular hyphae, where they may play an essential role in the accommodation and morphogenesis of the fungal endosymbiont.

  1. Nutritional and hormonal factors control the gene expression of FoxOs, the mammalian homologues of DAF-16.

    Science.gov (United States)

    Imae, M; Fu, Z; Yoshida, A; Noguchi, T; Kato, H

    2003-04-01

    Transcription factors of the FoxO family in mammals are orthologues of the Caenorhabditis elegans forkhead factor DAF-16, which has been characterized as a target of insulin-like signalling. Three members of this family have been identified in rodents: FoxO1, FoxO3 and FoxO4, originally termed FKHR, FKHRL1 and AFX respectively. A number of in vitro studies have revealed that FoxOs are regulated through phosphorylation in response to insulin and related growth factors, resulting in their nuclear exclusion and inactivation. To clarify the mechanisms involved in the regulation of these factors in vivo, we investigated in the present study whether or not, and if so how, their mRNA levels in rat liver respond to the stimuli of several nutritional and hormonal factors. Imposed fasting for 48 h significantly elevated mRNA levels of FoxO1 (1.5-fold), FoxO3 (1.4-fold), and FoxO4 (1.6-fold). Refeeding for 3 h recovered the induced mRNA levels of FoxO1 and FoxO3 to the control levels, but did not affect that of FoxO4. FoxO1 and FoxO4 mRNA levels were proved to be highly reflective of their protein levels measured by Western immunoblotting. Of the three FoxO genes, FoxO4 only showed altered levels of mRNA (a 1.5-fold increase) in response to a protein-free diet. Streptozotocin-induced diabetes for 28 days decreased hepatic mRNA levels of FoxO1 and FoxO3 and increased the level of FoxO4 mRNA, but short-term (7 days) diabetes had fewer effects on the expression of these genes. Insulin replacement partially restored the FoxO1 and FoxO4 mRNA levels, but had no effect on the FoxO3 mRNA level. Daily administration for 1 week of dexamethasone, a synthetic glucocorticoid, increased the mRNA levels of FoxO1 (1.8-fold) and FoxO3 (2.4-fold). These results show that the FoxO genes respond differently to nutritional and hormonal factors, suggesting a new mechanism for the regulation of FoxO-dependent gene expression by these factors. Moreover, changes of FoxO1 and FoxO4 in the nucleus in

  2. The TIR Homologue Lies near Resistance Genes in Staphylococcus aureus, Coupling Modulation of Virulence and Antimicrobial Susceptibility.

    Directory of Open Access Journals (Sweden)

    Sabine Patot

    2017-01-01

    Full Text Available Toll/interleukin-1 receptor (TIR domains in Toll-like receptors are essential for initiating and propagating the eukaryotic innate immune signaling cascade. Here, we investigate TirS, a Staphylococcus aureus TIR mimic that is part of a novel bacterial invasion mechanism. Its ectopic expression in eukaryotic cells inhibited TLR signaling, downregulating the NF-kB pathway through inhibition of TLR2, TLR4, TLR5, and TLR9. Skin lesions induced by the S. aureus knockout tirS mutant increased in a mouse model compared with wild-type and restored strains even though the tirS-mutant and wild-type strains did not differ in bacterial load. TirS also was associated with lower neutrophil and macrophage activity, confirming a central role in virulence attenuation through local inflammatory responses. TirS invariably localizes within the staphylococcal chromosomal cassettes (SCC containing the fusC gene for fusidic acid resistance but not always carrying the mecA gene. Of note, sub-inhibitory concentration of fusidic acid increased tirS expression. Epidemiological studies identified no link between this effector and clinical presentation but showed a selective advantage with a SCCmec element with SCC fusC/tirS. Thus, two key traits determining the success and spread of bacterial infections are linked.

  3. The TIR Homologue Lies near Resistance Genes in Staphylococcus aureus, Coupling Modulation of Virulence and Antimicrobial Susceptibility

    Science.gov (United States)

    Patot, Sabine; RC Imbert, Paul; Baude, Jessica; Martins Simões, Patricia; Campergue, Jean-Baptiste; Louche, Arthur; Bès, Michèle; Tristan, Anne; Laurent, Frédéric; Fischer, Adrien; Schrenzel, Jacques; François, Patrice; Lina, Gérard

    2017-01-01

    Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors are essential for initiating and propagating the eukaryotic innate immune signaling cascade. Here, we investigate TirS, a Staphylococcus aureus TIR mimic that is part of a novel bacterial invasion mechanism. Its ectopic expression in eukaryotic cells inhibited TLR signaling, downregulating the NF-kB pathway through inhibition of TLR2, TLR4, TLR5, and TLR9. Skin lesions induced by the S. aureus knockout tirS mutant increased in a mouse model compared with wild-type and restored strains even though the tirS-mutant and wild-type strains did not differ in bacterial load. TirS also was associated with lower neutrophil and macrophage activity, confirming a central role in virulence attenuation through local inflammatory responses. TirS invariably localizes within the staphylococcal chromosomal cassettes (SCC) containing the fusC gene for fusidic acid resistance but not always carrying the mecA gene. Of note, sub-inhibitory concentration of fusidic acid increased tirS expression. Epidemiological studies identified no link between this effector and clinical presentation but showed a selective advantage with a SCCmec element with SCC fusC/tirS. Thus, two key traits determining the success and spread of bacterial infections are linked. PMID:28060920

  4. A pomegranate (Punica granatum L.) WD40-repeat gene is a functional homologue of Arabidopsis TTG1 and is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit development.

    Science.gov (United States)

    Ben-Simhon, Zohar; Judeinstein, Sylvie; Nadler-Hassar, Talia; Trainin, Taly; Bar-Ya'akov, Irit; Borochov-Neori, Hamutal; Holland, Doron

    2011-11-01

    Anthocyanins are the major pigments responsible for the pomegranate (Punica granatum L.) fruit skin color. The high variability in fruit external color in pomegranate cultivars reflects variations in anthocyanin composition. To identify genes involved in the regulation of anthocyanin biosynthesis pathway in the pomegranate fruit skin we have isolated, expressed and characterized the pomegranate homologue of the Arabidopsis thaliana TRANSPARENT TESTA GLABRA1 (TTG1), encoding a WD40-repeat protein. The TTG1 protein is a regulator of anthocyanins and proanthocyanidins (PAs) biosynthesis in Arabidopsis, and acts by the formation of a transcriptional regulatory complex with two other regulatory proteins: bHLH and MYB. Our results reveal that the pomegranate gene, designated PgWD40, recovered the anthocyanin, PAs, trichome and seed coat mucilage phenotype in Arabidopsis ttg1 mutant. PgWD40 expression and anthocyanin composition in the skin were analyzed during pomegranate fruit development, in two accessions that differ in skin color intensity and timing of appearance. The results indicate high positive correlation between the total cyanidin derivatives quantity (red pigments) and the expression level of PgWD40. Furthermore, strong correlation was found between the steady state levels of PgWD40 transcripts and the transcripts of pomegranate homologues of the structural genes PgDFR and PgLDOX. PgWD40, PgDFR and PgLDOX expression also correlated with the expression of pomegranate homologues of the regulatory genes PgAn1 (bHLH) and PgAn2 (MYB). On the basis of our results we propose that PgWD40 is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit development and that expression of PgWD40, PgAn1 and PgAn2 in the pomegranate fruit skin is required to regulate the expression of downstream structural genes involved in the anthocyanin biosynthesis.

  5. The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking.

    Science.gov (United States)

    Warner, T S; Sinclair, D A; Fitzpatrick, K A; Singh, M; Devlin, R H; Honda, B M

    1998-04-01

    Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.

  6. GENE TECHNOLOGY: A NEW WAY TO TREAT CANCER

    Directory of Open Access Journals (Sweden)

    Prajapati P M.

    2012-06-01

    Full Text Available Gene therapy is the process of introducing genetic material RNA or DNA into a person's cells to fight disease. Gene therapy treats disease by either replacing damaged or missing genes with normal ones, or by providing new genes. The concept of gene therapy was born more than thirty years ago; however, new technology is opening the door to dramatically new possibilities in the treatment of cancers of all kinds. The long-term goal of cancer gene therapy is to develop treatments that attack only cancer cells, thereby eliminating adverse effects on the body and improving the possibility to cure disease. Gene therapy may someday soon make cancer a manageable disease with nominal side effects to the patients. Furthermore, since gene therapy has potential for other diseases such as cystic fibrosis, hemophilia, sickle-cell anemia, muscular dystrophy and Parkinson's, the value of research and discovery has broad applications.

  7. Molecular and functional characterization of a human ATM gene analogue at Arabidopsis thaliana; Caracterisation moleculaire et Fonctionnelle d'un Homologue du gene humain ATM chez Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, V.

    2001-12-15

    The human ATM gene, whose inactivation is responsible for the human disease ataxia telangiectasia is conserved throughout the Eukaryotes and plays an important role in the cellular responses to DNA damage, in particular to DNA double-strand breaks (DSBs). Here we describe the identification of an Arabidopsis thaliana homologue of ATM (AtATM), and the molecular and cytological characterization of plants, hereafter called atm, carrying a disrupting T-DNA insertion in this gene. AtATM covers a 32 kb region on chromosome 3. The AtATM transcript has a complex structure, is 12 kb long and formed by 79 exons. The transcriptional level of AtATM is very low in all the tissues tested, and does not vary after exposure to ionizing radiations (IR). In atm plants, the protein is not detected suggesting the mutants are null. The atm mutants are partially sterile. Aberrant segregation of chromosomes during meiosis I on both male and female sides account for this sterility. However, meiotic recombination frequency is normal. Mutant plants are also hypersensitive to gamma rays and methyl methane sulfonate, but not to UV-B, pointing to a specific defect of atm mutants in the response to DNA DSBs. In plants, ionizing radiations induce a strong, rapid and transient transcriptional activation of genes involved in the cellular response to or the repair of DSBs. This transcriptional regulation of AtRAD51, AtPARP1, atGR1 and AtL1G4 is lost in the atm mutants . The absence of AtRAD51 induction associated with ionizing radiation sensitivity suggest that AtAtm play an important function in DSB repair by homologous recombination. In addition we show that homologous intra-chromosomal recombination frequency is elevated in the mutant comparing to wild-type, with or without gamma irradiation. These results show the implication of AtAtm in the genomic stability. (author)

  8. The human homologue of the Drosophila melanogaster flightless-I gene (fliI) maps within the Smith-Magenis microdeletion critical region in 17p11.2

    Energy Technology Data Exchange (ETDEWEB)

    Chen, K.S.; Gunaratne, P.H.; Greenberg, F.; Shaffer, L.G.; Lupski, J.R. [Baylor College of Medicine, Houston, TX (United States); Hoheisel, J.D. [German Cancer Research Center, Heidelberg (Germany); Young, I.G.; Miklos, G.L.G.; Campbell, H.D. [Australian National Univ., Canberra (Australia)

    1995-01-01

    The Smith-Magenis syndrome (SMS) appears to be a contiguous-gene-deletion syndrome associated with a proximal deletion of the short arm of chromosome 17 in band p11.2. The spectrum of clinical findings includes short stature, brachydactyly, developmental delay, dysmorphic features, sleep disturbances, and behavioral problems. The complex phenotypic features suggest deletion of several contiguous genes. However, to date, no protein-encoding gene has been mapped to the SMS critical region. Recently, the Drosophila melanogaster flightless-I gene, fliI, and the homologous human cDNA have been isolated. Mutations in fliI result in loss of flight ability and, when severe, cause lethality due to incomplete cellularization with subsequent abnormal gastrulation. Here, we demonstrate that the human homologue (FLI) maps within the SMS critical region. Genomic cosmids were used as probes for FISH, which localized this gene to the 17p11.2 region. Somatic-cell hybrid-panel mapping further localized this gene to the SMS critical region. Southern blot analysis of somatic-cell hybrids and/or FISH analysis of lymphoblastoid cell lines from 12 SMS patients demonstrates the deletion of one copy of FLI in all SMS patients analyzed. 47 refs., 4 figs., 1 tab.

  9. The SPR3 gene encodes a sporulation-specific homologue of the yeast CDC3/10/11/12 family of bud neck microfilaments and is regulated by ABFI.

    Science.gov (United States)

    Ozsarac, N; Bhattacharyya, M; Dawes, I W; Clancy, M J

    1995-10-16

    The SPR3 gene is selectively activated only during the sporulation phase of the Saccharomyces cerevisiae (Sc) life cycle. The predicted amino acid (aa) sequence has homology to microfilament proteins that are involved in cytokinesis and other proteins of unknown function. These include the products of Sc cell division cycle (CDC) genes involved in bud formation (Cdc3p, Cdc10p, Cdc11p and Cdc12p), Candida albicans proteins that accumulate in the hyphal phase (CaCdc3p and CaCdc10p), mouse brain-specific (H5p) and lymphocyte (Diff6p) proteins, Drosophila melanogaster (Dm) protein Pnutp (which is localized to the cleavage furrow of dividing cells), a Diff6p homologue (DmDiff6p), and the Sc septin protein (Sep1hp), a homologue of the 10-nm filament proteins of Sc. One strongly conserved region contains a potential ATP-GTP-binding domain. Primer extension analysis revealed six major transcription start points (tsp) beginning at -142 relative to the ATG start codon. The sequence immediately upstream from the tsp contains consensus binding sites for the HAP2/3/4 and ABFI transcription factors, a T-rich sequence and two putative novel elements for mid to late sporulation, termed SPR3 and PAL. Electrophoretic mobility shift assay (EMSA) and footprint analyses demonstrated that the ABFI protein binds to a region containing the putative ABFI site in vitro, and site-directed mutagenesis showed that the ABFI motif is essential for expression of SPR3 at the appropriate stage in sporulating cells.

  10. 人类p53和c-myc同源基因在玉米颖果发育过程中的表达%Expressions of Human p53 and c-myc Gene Homologues During Caryopsis Development in Maize

    Institute of Scientific and Technical Information of China (English)

    亓翠英; 宁顺斌; 王宁; 李立家; 宋运淳

    2003-01-01

    肿瘤抑制基因p53和原癌基因c-myc已被证明在动物中高度保守并参与许多PCD过程.这两个基因编码的同源蛋白及其RNA在玉米中的存在已有报道,并且其DNA同源序列已利用荧光原位杂交定位在玉米相应的染色体上.利用免疫组织化学方法探测了与人类p53和c-myc基因同源的玉米基因在玉米颖果发育过程中的时空表达模式.结果发现,在授粉后的一定阶段,在反足细胞、珠被、未成熟的胚乳、子房壁、导管组织和糊粉层中,p53同源基因表达强烈,c-myc同源基因的表达相反,在授粉后的这些组织中基本不表达,而在授粉前的中央细胞的极核中表达水平较高.TUNEL检测显示,在p53同源基因呈现高水平表达的地方,DNA断裂信号强烈.在动物细胞中,p53和c-myc起相反的调节作用,这与其同源基因在玉米中的作用模式相似.由此说明p53和c-myc同源基因可能在玉米颖果发育PCD过程中起重要作用,并进一步推论高等植物PCD和动物细胞凋亡存在一定的保守性机制.%Tumor suppressor gene p53 and proto-oncogene c-myc have been proved to be highly conserved and participate in many PCD processes in animals.In maize,proteins and RNAs related to p53 and c-myc have already been reported and the sequences homologous to these two genes have also been localized onto maize chromosomes by FISH.In this study,using immunohistochemistry we investigated the expression patterns of maize genes homologous to human p53 and c-myc during caryopsis development stages in maize.In a giving stage after pollination,p53 homologue showed high levels in the antipodal cells,integument,immature endosperm,ovary wall,tracheary elements,and aleurone layer,while c-myc homologue showed low levels in these tissues,only before pollination showed high expression in polar nucleus.The results of TUNEL assay demonstrated that TUNEL positive signals were detected where p53 homologue showed high expression

  11. Treating psoriasis by targeting its susceptibility gene Rel.

    Science.gov (United States)

    Fan, Tingting; Wang, Shaowen; Yu, Linjiang; Yi, Huqiang; Liu, Ruiling; Geng, Wenwen; Wan, Xiaochun; Ma, Yifan; Cai, Lintao; Chen, Youhai H; Ruan, Qingguo

    2016-04-01

    Psoriasis is a chronic inflammatory disorder of the skin. Accumulating evidence indicates that the Rel gene, a member of the NF-κB family, is a risk factor for the disease. We sought to investigate whether psoriasis can be prevented by directly targeting the Rel gene transcript, i.e., the c-Rel mRNA. Using chemically-modified c-Rel specific siRNA (siRel) and poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-leucine) (PEG-PLL-PLLeu) micelles, we successfully knocked down the expression of c-Rel, and showed that the expression of cytokine IL-23, a direct target of c-Rel that can drive the development of IL-17-producing T cells, was markedly inhibited. More importantly, treating mice with siRel not only prevented but also ameliorated imiquimod (IMQ)-induced psoriasis. Mechanistic studies showed that siRel treatment down-regulated the expression of multiple inflammatory cytokines. Taken together, these results indicate that the susceptibility gene Rel can be targeted to treat and prevent psoriasis.

  12. The human homologue of the Drosophila melanogaster flightless-I gene (fliI) maps within the Smith-Magenis microdeletion critical region in 17p11.2

    Energy Technology Data Exchange (ETDEWEB)

    Chen, K.S.; Nguyen, D.; Greenberg, F. [Baylor College of Medicing, Houston, TX (United States)] [and others

    1994-09-01

    The Smith-Magenis syndrome (SMS) appears to be a contiguous gene deletion syndrome associated with a proximal deletion of the short arm of chromosome 17 in band p11.2. The spectrum of clinical findings includes short stature, brachydactyly, developmental delay, dysmorphic features, sleep disturbances and behavioral problems. The complex phenotypic features suggest deletion of several contiguous genes. However, to date no protein encoding gene has been mapped to the SMS critical region. Recently, Campbell described the cloning and characterization of D. melanogaster fli cDNAs and of homologous cDNAs from caenorhabditis elegans and from humans. Mutations in fliI result in loss of flight ability and, when severe, cause lethality due to incomplete cellularization with subsequent abnormal gastrulation. The amino acid sequence deduced from the FLI cDNA has 52% similarity to the human gelsolin protein and also has a N-terminal leucine-rich domain with 16 consecutive leucine-rich repeats (LRR). Here, we demonstrate that the human homologue (FLI) maps within the SMS critical region. Genomic cosmids were used as probes for fluorescence in situ hybridization (FISH) and localized this gene to the 17p11.2 region. Somatic cell hybrids and/or FISH analysis of lymphoblastoid cell lines form 12 SMS patients demonstrate that one copy of the FLI gene is deleted in all SMS patients analyzed with the common deletion. Further studies are required to determine if haploinsufficiency of FLI or other as yet unidentified genes is important for the expression of the SMS phenotype.

  13. Isolation and characterization of an AGAMOUS homologue from cocoa

    NARCIS (Netherlands)

    Chaidamsari, T.; Sugiarit, H.; Santoso, D.; Angenent, G.C.; Maagd, de R.A.

    2006-01-01

    We report the cloning of a cDNA from TcAG, an AG (Arabidopsis thaliana MADS-box C-type transcription factor gene AGAMOUS) homologue from cocoa (Theobroma cacao L.). TcAG was in the cocoa flower expressed primarily in stamens and ovaries, comparable to AG in Arabidopsis. Additionally, we found that T

  14. The Solanum demissumR8 late blight resistance gene is an Sw-5 homologue that has been deployed worldwide in late blight resistant varieties

    NARCIS (Netherlands)

    Vossen, Jack H.; Arkel, van Gert; Bergervoet-van Deelen, Marjan; Jo, Kwang Ryong; Jacobsen, Evert; Visser, Richard G.F.

    2016-01-01

    The potato late blight resistance geneR8has been cloned.R8is found in five late blight resistant varieties deployed in three different continents. R8 recognises Avr8 and is homologous to the NB-LRR protein Sw-5 from tomato.Abstract: The broad spectrum late blight resistance gene R8 from Solanum

  15. Dataset of the human homologues and orthologues of lipid-metabolic genes identified as DAF-16 targets their roles in lipid and energy metabolism

    Directory of Open Access Journals (Sweden)

    Lavender Yuen-Nam Fan

    2017-04-01

    Full Text Available The data presented in this article are related to the review article entitled ‘Unravelling the role of fatty acid metabolism in cancer through the FOXO3-FOXM1 axis’ (Saavedra-Garcia et al., 2017 [24]. Here, we have matched the DAF-16/FOXO3 downstream genes with their respective human orthologues and reviewed the roles of these targeted genes in FA metabolism. The list of genes listed in this article are precisely selected from literature reviews based on their functions in mammalian FA metabolism. The nematode Caenorhabditis elegans gene orthologues of the genes are obtained from WormBase, the online biological database of C. elegans. This dataset has not been uploaded to a public repository yet.

  16. The DNA sequence of the equine herpesvirus 4 gene encoding glycoprotein gp17/18, the homologue of herpes simplex virus glycoprotein gD.

    Science.gov (United States)

    Cullinane, A A; Neilan, J; Wilson, L; Davison, A J; Allen, G

    1993-09-01

    The nucleotide sequence of the gene to the left of the gI gene of equine herpesvirus 4 (EHV-4) was determined. The gene encodes a peptide of 402 amino acids with an unprocessed M(r) of 45,323. The predicted polypeptide has several features of a glycoprotein including a hydrophobic signal sequence, a membrane spanning domain and four potential N-linked glycosylation sites within the proposed external domain. The predicted amino acid sequence of EHV-4 gD shows 83% identity with that of equine herpesvirus 1 gD. Conservation of the tertiary structure is suggested by the alignment of six cysteine residues with those of the gD of six other alphaherpesviruses. Screening a lambda gt11/EHV-4 expression library with monoclonal antibodies against several of the most abundant EHV-4 glycoproteins unequivocally identified the protein encoded by the EHV-4 gD gene as gp17/18.

  17. Mating type gene homologues and putative sex pheromone-sensing pathway in arbuscular mycorrhizal fungi, a presumably asexual plant root symbiont.

    Science.gov (United States)

    Halary, Sébastien; Daubois, Laurence; Terrat, Yves; Ellenberger, Sabrina; Wöstemeyer, Johannes; Hijri, Mohamed

    2013-01-01

    The fungal kingdom displays a fascinating diversity of sex-determination systems. Recent advances in genomics provide insights into the molecular mechanisms of sex, mating type determination, and evolution of sexual reproduction in many fungal species in both ancient and modern phylogenetic lineages. All major fungal groups have evolved sexual differentiation and recombination pathways. However, sexuality is unknown in arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota, an ecologically vital group of obligate plant root symbionts. AMF are commonly considered an ancient asexual lineage dating back to the Ordovician, approximately 460 M years ago. In this study, we used genomic and transcriptomic surveys of several AMF species to demonstrate the presence of conserved putative sex pheromone-sensing mitogen-activated protein (MAP) kinases, comparable to those described in Ascomycota and Basidiomycota. We also find genes for high mobility group (HMG) transcription factors, homologous to SexM and SexP genes in the Mucorales. The SexM genes show a remarkable sequence diversity among multiple copies in the genome, while only a single SexP sequence was detected in some isolates of Rhizophagus irregularis. In the Mucorales and Microsporidia, the sexM gene is flanked by genes for a triosephosphate transporter (TPT) and a RNA helicase, but we find no evidence for synteny in the vicinity of the Sex locus in AMF. Nonetheless, our results, together with previous observations on meiotic machinery, suggest that AMF could undergo a complete sexual reproduction cycle.

  18. Mating type gene homologues and putative sex pheromone-sensing pathway in arbuscular mycorrhizal fungi, a presumably asexual plant root symbiont.

    Directory of Open Access Journals (Sweden)

    Sébastien Halary

    Full Text Available The fungal kingdom displays a fascinating diversity of sex-determination systems. Recent advances in genomics provide insights into the molecular mechanisms of sex, mating type determination, and evolution of sexual reproduction in many fungal species in both ancient and modern phylogenetic lineages. All major fungal groups have evolved sexual differentiation and recombination pathways. However, sexuality is unknown in arbuscular mycorrhizal fungi (AMF of the phylum Glomeromycota, an ecologically vital group of obligate plant root symbionts. AMF are commonly considered an ancient asexual lineage dating back to the Ordovician, approximately 460 M years ago. In this study, we used genomic and transcriptomic surveys of several AMF species to demonstrate the presence of conserved putative sex pheromone-sensing mitogen-activated protein (MAP kinases, comparable to those described in Ascomycota and Basidiomycota. We also find genes for high mobility group (HMG transcription factors, homologous to SexM and SexP genes in the Mucorales. The SexM genes show a remarkable sequence diversity among multiple copies in the genome, while only a single SexP sequence was detected in some isolates of Rhizophagus irregularis. In the Mucorales and Microsporidia, the sexM gene is flanked by genes for a triosephosphate transporter (TPT and a RNA helicase, but we find no evidence for synteny in the vicinity of the Sex locus in AMF. Nonetheless, our results, together with previous observations on meiotic machinery, suggest that AMF could undergo a complete sexual reproduction cycle.

  19. Homologues of Neisserial Heme Oxygenase in Gram-Negative Bacteria: Degradation of Heme by the Product of the pigA Gene of Pseudomonas aeruginosa

    OpenAIRE

    2001-01-01

    The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The pigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of Neisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced with pigA, we demonstrated that pigA could functionally replace hemO and allow for heme util...

  20. Characterization of mouse Dach2, a homologue of Drosophila dachshund.

    Science.gov (United States)

    Davis, R J; Shen, W; Sandler, Y I; Heanue, T A; Mardon, G

    2001-04-01

    The Drosophila genes eyeless, eyes absent, sine oculis and dachshund cooperate as components of a network to control retinal determination. Vertebrate homologues of these genes have been identified and implicated in the control of cell fate. We present the cloning and characterization of mouse Dach2, a homologue of dachshund. In situ hybridization studies demonstrate Dach2 expression in embryonic nervous tissues, sensory organs and limbs. This pattern is similar to mouse Dach1, suggesting a partially redundant role for these genes during development. In addition, we determine that Dach2 expression in the forebrain of Pax6 mutants and dermamyotome of Pax3 mutants is not detectably altered. Finally, genetic mapping experiments place mouse Dach2 on the X chromosome between Xist and Esx1. The identification of human DACH2 sequences at Xq21 suggests a possible role for this gene in Allan-Herndon syndrome, Miles-Carpenter syndrome, X-linked cleft palate and/or Megalocornea.

  1. Treating hearing disorders with cell and gene therapy

    Science.gov (United States)

    Gillespie, Lisa N.; Richardson, Rachael T.; Nayagam, Bryony A.; Wise, Andrew K.

    2014-12-01

    Hearing loss is an increasing problem for a substantial number of people and, with an aging population, the incidence and severity of hearing loss will become more significant over time. There are very few therapies currently available to treat hearing loss, and so the development of new therapeutic strategies for hearing impaired individuals is of paramount importance to address this unmet clinical need. Most forms of hearing loss are progressive in nature and therefore an opportunity exists to develop novel therapeutic approaches to slow or halt hearing loss progression, or even repair or replace lost hearing function. Numerous emerging technologies have potential as therapeutic options. This paper details the potential of cell- and gene-based therapies to provide therapeutic agents to protect sensory and neural cells from various insults known to cause hearing loss; explores the potential of replacing lost sensory and nerve cells using gene and stem cell therapy; and describes the considerations for clinical translation and the challenges that need to be overcome.

  2. The hexA gene of Erwinia carotovora encodes a LysR homologue and regulates motility and the expression of multiple virulence determinants.

    Science.gov (United States)

    Harris, S J; Shih, Y L; Bentley, S D; Salmond, G P

    1998-05-01

    We have identified a gene important for the regulation of exoenzyme virulence factor synthesis in the plant pathogen Erwinia carotovora ssp. carotovora (Ecc) and virulence and motility in Erwinia carotovora ssp. atroseptica (Eca). This gene, hexA (hyperproduction of exoenzymes), is a close relative of the Erwinia chrysanthemi (Echr) gene pecT and encodes a member of the LysR family of transcriptional regulators. hexA mutants in both Ecc and Eca produce abnormally high levels of the exoenzyme virulence factors pectate lyase, cellulase and protease. In addition, Eca hexA mutants show increased expression of the fliA and fliC genes and hypermotility. Consistent with a role as a global regulator, expression of hexA from even a low-copy plasmid can suppress exoenzyme production in Ecc and Eca and motility in Eca. Production of the quorum-sensing pheromone OHHL in Ecc hexA is higher throughout the growth curve compared with the wild-type strain. Overexpression of Ecc hexA also caused widespread effects in several strains of the opportunistic human pathogen, Serratia. Low-copy hexA expression resulted in repression of exoenzyme, pigment and antibiotic production and repression of the spreading phenotype. Finally, mutations in hexA were shown to increase Ecc or Eca virulence in planta.

  3. Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in Vibrioanguillarum strain 775

    NARCIS (Netherlands)

    Di Lorenzo, M.; Stork, M.; Crosa, J.H.

    2011-01-01

    Anguibactin, the siderophore produced by Vibrio anguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. I

  4. USE OF GENE EXPRESSION ANALYSIS INCORPORATING OPERON-TRANSCRIPTIONAL COUPLING AND TOXICANT DOSE RESPONSE TO DISTINGUISH AMONG STRUCTURAL HOMOLOGUES OF MX

    Science.gov (United States)

    We recently described a general method that can improve microarray analysis of toxicant-exposed cells that uses the intrinsic power of transcriptional coupling and toxicant concentration-expression response data. In this analysis, we characterized changes in global gene expressio...

  5. Characterization of a pollen-preferential gene OSIAGP from rice (Oryza sativa L. subspecies indica) coding for an arabinogalactan protein homologue, and analysis of its promoter activity during pollen development and pollen tube growth.

    Science.gov (United States)

    Anand, Saurabh; Tyagi, Akhilesh K

    2010-06-01

    During differential screening of inflorescence-specific cDNA libraries from Oryza sativa indica, an arabinogalactan protein (OSIAGP) cDNA (586 bp) expressing preferentially in the inflorescence has been isolated. It encodes an arabinogalactan protein of 59 amino acids (6.4 kDa) with a transmembrane domain and a secretory domain at the N terminus. The protein shows homology with AGP23 from Arabidopsis, and its homologue in japonica rice is located on chromosome 6. OSIAGP transcripts also accumulate in shoots and roots of rice seedling grown in the dark, but light represses expression of the gene. Analysis of a genomic clone of OSIAGP revealed that its promoter contains several pollen-specificity and light-regulatory elements. The promoter confers pollen-preferential activity on gus, starting from the release of microspores to anther dehiscence in transgenic tobacco, and is also active during pollen tube growth. Analysis of pollen preferential activity of the promoter in the transgenic rice system revealed that even the approximately 300 bp fragment has activity in pollen and the anther wall and further deletion down to approximately 100 bp completely abolishes this activity, which is consistent with in-silico analysis of the promoter. Arabinogalactan proteins have been shown to be involved in the cell elongation process. The homology of OSIAGP with AGP23 and the fact that seedling growth in the dark and pollen tube growth are events based on cell elongation strengthen the possibility of OSIAGP performing a similar function.

  6. Gene dosage effects of the imprinted delta-like homologue 1 (dlk1/pref1 in development: implications for the evolution of imprinting.

    Directory of Open Access Journals (Sweden)

    Simao Teixeira da Rocha

    2009-02-01

    Full Text Available Genomic imprinting is a normal process that causes genes to be expressed according to parental origin. The selective advantage conferred by imprinting is not understood but is hypothesised to act on dosage-critical genes. Here, we report a unique model in which the consequences of a single, double, and triple dosage of the imprinted Dlk1/Pref1, normally repressed on the maternally inherited chromosome, can be assessed in the growing embryo. BAC-transgenic mice were generated that over-express Dlk1 from endogenous regulators at all sites of embryonic activity. Triple dosage causes lethality associated with major organ abnormalities. Embryos expressing a double dose of Dlk1, recapitulating loss of imprinting, are growth enhanced but fail to thrive in early life, despite the early growth advantage. Thus, any benefit conferred by increased embryonic size is offset by postnatal lethality. We propose a negative correlation between gene dosage and survival that fixes an upper limit on growth promotion by Dlk1, and we hypothesize that trade-off between growth and lethality might have driven imprinting at this locus.

  7. A homologue of the yeast SHE4 gene is essential for the transition between the syncytial and cellular stages during sexual reproduction of the fungus Podospora anserina.

    Science.gov (United States)

    Berteaux-Lecellier, V; Zickler, D; Debuchy, R; Panvier-Adoutte, A; Thompson-Coffe, C; Picard, M

    1998-01-01

    The Podospora anserina cro1 gene was identified as a gene required for sexual sporulation. Crosses homozygous for the cro1-1 mutation yield fruiting bodies which produce few asci due to the formation of giant plurinucleate cells instead of dikaryotic cells after fertilization. This defect does not impair karyogamy, but meioses of the resultant polyploid nuclei are most often abortive. Cytological studies suggest that the primary defect of the mutant is its inability to form septa between the daughter nuclei after each mitosis, a step specific for normal dikaryotic cell divisions. The cro1-1 mutant would thus be unable to leave the syncytial vegetative state while abiding by the meiotic programme. cro1-1 also shows defects in ascospore germination and growth rate. GFP-tagging of the CRO1 protein reveals that it is a cytosolic protein mainly expressed at the beginning of the dikaryotic stage and at the time of ascospore maturation. The CRO1 protein exhibits significant similarity to the SHE4 protein, which is required for asymmetric mating-type switching in budding yeast cells. Thus, a gene involved in asymmetric cell divisions in a unicellular organism plays a key role at the transition between the syncytial (vegetative) state and the cellular (sexual) state in a filamentous fungus. PMID:9482722

  8. Structure-based computational study of two disease resistance gene homologues (Hm1 and Hm2) in maize (Zea mays L.) with implications in plant-pathogen interactions.

    Science.gov (United States)

    Dehury, Budheswar; Patra, Mahesh Chandra; Maharana, Jitendra; Sahu, Jagajjit; Sen, Priyabrata; Modi, Mahendra Kumar; Choudhury, Manabendra Dutta; Barooah, Madhumita

    2014-01-01

    The NADPH-dependent HC-toxin reductases (HCTR1 and 2) encoded by enzymatic class of disease resistance homologous genes (Hm1 and Hm2) protect maize by detoxifying a cyclic tetrapeptide, HC-toxin, secreted by the fungus Cochliobolus carbonum race 1(CCR1). Unlike the other classes' resistance (R) genes, HCTR-mediated disease resistance is an inimitable mechanism where the avirulence (Avr) component from CCR1 is not involved in toxin degradation. In this study, we attempted to decipher cofactor (NADPH) recognition and mode of HC-toxin binding to HCTRs through molecular docking, molecular dynamics (MD) simulations and binding free energy calculation methods. The rationality and the stability of docked complexes were validated by 30-ns MD simulation. The binding free energy decomposition of enzyme-cofactor complex was calculated to find the driving force behind cofactor recognition. The overall binding free energies of HCTR1-NADPH and HCTR2-NADPH were found to be -616.989 and -16.9749 kJ mol-1 respectively. The binding free energy decomposition revealed that the binding of NADPH to the HCTR1 is mainly governed by van der Waals and nonpolar interactions, whereas electrostatic terms play dominant role in stabilizing the binding mode between HCTR2 and NADPH. Further, docking analysis of HC-toxin with HCTR-NADPH complexes showed a distinct mode of binding and the complexes were stabilized by a strong network of hydrogen bond and hydrophobic interactions. This study is the first in silico attempt to unravel the biophysical and biochemical basis of cofactor recognition in enzymatic class of R genes in cereal crop maize.

  9. TTYH2, a human homologue of the Drosophila melanogaster gene tweety, is up-regulated in colon carcinoma and involved in cell proliferation and cell aggregation

    Institute of Scientific and Technical Information of China (English)

    Yuji Toiyama; Akira Mizoguchi; Kazushi Kimura; Junichirou Hiro; Yasuhiro Inoue; Tomonari Tutumi; Chikao Miki; Masato Kusunoki

    2007-01-01

    AIM: To investigate the expression patterns of TTYH2 in the human colon cancer and colon cancer cell lines and to evaluate the inhibitory effect of small interfering RNA (siRIMA) on the expression of TTYH2 in colon cancer cell lines.METHODS: We investigated the expression patterns of TTYH2 in colon cancer, adjacent non-tumorous colon mucosa, and cancer cell lines (DLD-1, caco-2, and Lovo) by RT-PCR. Furthermore, a siRNA plasmid expression vector against TTYH2 was constructed and transfected into DLD-1 and Caco-2 with LipofectamineTM 2000. The down regulation of TTYH2 expression was detected by RT-PCR and the role of siRNA in inducing cell proliferation and cell aggregation was evaluated by MTT and aggregation assay.RESULTS: TTYH2 gene expression in colon cancer tissue was significantly up-regulated compared with normal colonic mucosa (1.23 ± 0.404 vs 0.655 ± 0.373, P=0.0103). Colon cancer derived cell lines including DLD-1, Caco-2, and Lovo also expressed high levels of TTYH2. In contrast, transfection with siRNA-TTYH2 significantly inhibited both proliferation and scattering of these cancer cell lines.CONCLUSION: The present work demonstrates, for the first time, that the TTYH2 gene expression is significantly up-regulated in colon cancer. The TTYH2 gene may play an important role in regulating both proliferating and metastatic potentials of colorectal cancer.

  10. Combination of hypomorphic mutations of the Drosophila homologues of aryl hydrocarbon receptor and nucleosome assembly protein family genes disrupts morphogenesis, memory and detoxification.

    Directory of Open Access Journals (Sweden)

    Boris A Kuzin

    Full Text Available Aryl hydrocarbon receptor is essential for biological responses to endogenous and exogenous toxins in mammals. Its Drosophila homolog spineless plays an important role in fly morphogenesis. We have previously shown that during morphogenesis spineless genetically interacts with CG5017 gene, which encodes a nucleosome assembly factor and may affect cognitive function of the fly. We now demonstrate synergistic interactions of spineless and CG5017 in pathways controlling oxidative stress response and long-term memory formation in Drosophila melanogaster. Oxidative stress was induced by low doses of X-ray irradiation of flies carrying hypomorphic mutation of spineless, mutation of CG5017, and their combination. To determine the sensitivity of these mutants to pharmacological modifiers of the irradiation effect, we irradiated flies growing on standard medium supplemented by radiosensitizer furazidin and radioprotector serotonin. The effects of irradiation were investigated by analyzing leg and antenna morphological structures and by using real-time PCR to measure mRNA expression levels for spineless, Cyp6g1 and Gst-theta genes. We also examined long-term memory in these mutants using conditioned courtship suppression paradigm. Our results show that the interaction of spineless and CG5017 is important for regulation of morphogenesis, long-term memory formation, and detoxification during oxidative stress. Since spineless and CG5017 are evolutionary conserved, these results must be considered when evaluating the risk of combining similar mutations in other organisms, including humans.

  11. Liver steatosis study_PFAA treated mouse gene array data

    Data.gov (United States)

    U.S. Environmental Protection Agency — This file contains a link for Gene Expression Omnibus and the GSE designations for the publicly available gene expression data used in the study and reflected in...

  12. Identification and analysis of cation channel homologues in human pathogenic fungi.

    Directory of Open Access Journals (Sweden)

    David L Prole

    Full Text Available Fungi are major causes of human, animal and plant disease. Human fungal infections can be fatal, but there are limited options for therapy, and resistance to commonly used anti-fungal drugs is widespread. The genomes of many fungi have recently been sequenced, allowing identification of proteins that may become targets for novel therapies. We examined the genomes of human fungal pathogens for genes encoding homologues of cation channels, which are prominent drug targets. Many of the fungal genomes examined contain genes encoding homologues of potassium (K(+, calcium (Ca(2+ and transient receptor potential (Trp channels, but not sodium (Na(+ channels or ligand-gated channels. Some fungal genomes contain multiple genes encoding homologues of K(+ and Trp channel subunits, and genes encoding novel homologues of voltage-gated K(v channel subunits are found in Cryptococcus spp. Only a single gene encoding a homologue of a plasma membrane Ca(2+ channel was identified in the genome of each pathogenic fungus examined. These homologues are similar to the Cch1 Ca(2+ channel of Saccharomyces cerevisiae. The genomes of Aspergillus spp. and Cryptococcus spp., but not those of S. cerevisiae or the other pathogenic fungi examined, also encode homologues of the mitochondrial Ca(2+ uniporter (MCU. In contrast to humans, which express many K(+, Ca(2+ and Trp channels, the genomes of pathogenic fungi encode only very small numbers of K(+, Ca(2+ and Trp channel homologues. Furthermore, the sequences of fungal K(+, Ca(2+, Trp and MCU channels differ from those of human channels in regions that suggest differences in regulation and susceptibility to drugs.

  13. Gene expression profile analysis of genes in rat hippocampus from antidepressant treated rats using DNA microarray

    Directory of Open Access Journals (Sweden)

    Shin Minkyu

    2010-11-01

    Full Text Available Abstract Background The molecular and biological mechanisms by which many antidepressants function are based on the monoamine depletion hypothesis. However, the entire cascade of mechanisms responsible for the therapeutic effect of antidepressants has not yet been elucidated. Results We used a genome-wide microarray system containing 30,000 clones to evaluate total RNA that had been isolated from the brains of treated rats to identify the genes involved in the therapeutic mechanisms of various antidepressants, a tricyclic antidepressant (imipramine. a selective serotonin reuptake inhibitor (fluoxetine, a monoamine oxidase inhibitor (phenelzine and psychoactive herbal extracts of Nelumbinis Semen (NS. To confirm the differential expression of the identified genes, we analyzed the amount of mRNA that was isolated from the hippocampus of rats that had been treated with antidepressants by real-time RT-PCR using primers specific for selected genes of interest. These data demonstrate that antidepressants interfere with the expression of a large array of genes involved in signaling, survival and protein metabolism, suggesting that the therapeutic effect of these antidepressants is very complex. Surprisingly, unlike other antidepressants, we found that the standardized herbal medicine, Nelumbinis Semen, is free of factors that can induce neurodegenerative diseases such as caspase 8, α-synuclein, and amyloid precursor protein. In addition, the production of the inflammatory cytokine, IFNγ, was significantly decreased in rat hippocampus in response to treatment with antidepressants, while the inhibitory cytokine, TGFβ, was significantly enhanced. Conclusions These results suggest that antidepressants function by regulating neurotransmission as well as suppressing immunoreactivity in the central nervous system.

  14. Advances in gene therapy technologies to treat retinitis pigmentosa

    OpenAIRE

    2013-01-01

    Hilda Petrs-Silva, Rafael LindenInstitute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, BrazilAbstract: Retinitis pigmentosa (RP) is a class of diseases that leads to progressive degeneration of the retina. Experimental approaches to gene therapy for the treatment of inherited retinal dystrophies have advanced in recent years, inclusive of the safe delivery of genes to the human retina. This review is focused on the development of gene therapy for RP using recombinant a...

  15. Crystal chemistry of sartorite homologues and related sulfosalts

    DEFF Research Database (Denmark)

    Berlepsch, Peter; Makovicky, Emil; Balic-Zunic, Tonci

    2001-01-01

    sartorite homologues, sulfosalt, crystal chemistry, coordination polyhedra, bond-pairs, crankshaft chains......sartorite homologues, sulfosalt, crystal chemistry, coordination polyhedra, bond-pairs, crankshaft chains...

  16. Advances in gene therapy technologies to treat retinitis pigmentosa.

    Science.gov (United States)

    Petrs-Silva, Hilda; Linden, Rafael

    2014-01-01

    Retinitis pigmentosa (RP) is a class of diseases that leads to progressive degeneration of the retina. Experimental approaches to gene therapy for the treatment of inherited retinal dystrophies have advanced in recent years, inclusive of the safe delivery of genes to the human retina. This review is focused on the development of gene therapy for RP using recombinant adenoassociated viral vectors, which show a positive safety record and have so far been successful in several clinical trials for congenital retinal disease. Gene therapy for RP is under development in a variety of animal models, and the results raise expectations of future clinical application. Nonetheless, the translation of such strategies to the bedside requires further understanding of the mutations and mechanisms that cause visual defects, as well as thorough examination of potential adverse effects.

  17. ALTERATION OF GENE EXPRESSION IN LEUKOCYTES FROM RECOMBINANT SOMATOTROPIN TREATED ANIMALS: SEARCHING FOR INSPECTION INDICATORS

    Directory of Open Access Journals (Sweden)

    NR Brizioli

    2008-12-01

    Full Text Available Besides immunochemical approaches, biomolecular studies can be carried out in order to discover a greater number of biological indicators to be exploited for the identification of bovines treated with recombinant somatotropin (rbST. With this aim, we analysed the expression of a number of genes related to the somatotropic axis in leucocytes from rbST treated cows and non-treated animals. Significant differences were observed in the genes IGF-1,IGFBP-1, IGFBP-4 and the I- 5’UTR variant of the GHR gene.

  18. Gene ontology study of methyl jasmonate-treated and non-treated hairy roots of Panax ginseng to identify genes involved in secondary metabolic pathway.

    Science.gov (United States)

    Sathiyamoorthy, S; In, J G; Gayathri, S; Kim, Y Ju; Yang, D Ch

    2010-07-01

    The roots of Panax ginseng C.A. Meyer, known as Korean ginseng have been a valuable and important folk medicine in East Asian countries. It mainly used to maintain the homeostasis of the human body, with the presence ofginsenosides and non-saponin compounds like phenol compounds, acidic polysaccharides and polyethylene compounds. Functional genomics aid to annotate based on gene ontology. In this study, we focused on the genes involving in secondary metabolic pathways and to visualize temporal changes of gene expression in ginseng hairy roots with methyl ester methyl jasmonate (MeJA) along with non-treated hairy roots. A 5.774 EST clones were clustered and assembled as 501 contigs and 2.955 singletons. Annotations categorized with molecular functions, biological processes, cellular compounds of gene ontological terms and biochemical functions, enzyme commission to sequences were assigned to metabolic pathways of Kyoto Encyclopedia of Genes and Genomes database. Comparatively, EST sequences are assigned to cellular process, metabolic process, biotic and abiotic stress stimuli, developmental and biological regulations and transports are up-regulated 2-3 fold in MeJA treated hairy roots. 46 different sub groups of enzymes found in the MeJA treated plants. These annotated ESTs represents a significant proportion of the P. ginseng and provides molecular resource for developmental of microarrays for gene expression studies concerning development, metabolism and reproduction.

  19. PROMISES FOR TREATING COLON CANCER PATIENTS WITH BRAF GENE MUTATION

    Directory of Open Access Journals (Sweden)

    M. Yu. Fedyanin

    2014-01-01

    Full Text Available Colon cancer represents a heterogenous disease group, which differ by cancerogenesis mechanisms, molecular alterations, prognosis and treatment possibilities. In modern clinical practice assessment of KRAS and NRAS genes status is already necessary in order to prescribe anti-EGFR treatment for metastatic disease. A separate poor prognosis group are patients with BRAF mutation. In this review we will focus on biological features of BRAF-mutant colorectal cancer, its epidemiology, clinical features on different stages, treatment choice and promising new treatment possibilities.

  20. Long-term follow-up of cancer patients treated with gene therapy medicinal products.

    Science.gov (United States)

    Galli, Maria Cristina

    2012-06-01

    European Union requirements are discussed for the long-term follow-up of advanced therapy medicinal products, as well as how they can be applied to cancer patients treated with gene therapy medicinal products in the context of clinical trials, as described in a specific guideline issued by Gene Therapy Working Party at the European Medicine Agency.

  1. Transcriptional modulation of squalene synthase genes in barley treated with PGPR

    OpenAIRE

    Yousaf, Anam; Qadir, Abdul; Anjum, Tehmina; Ahmad, Aqeel

    2015-01-01

    Phytosterol contents and food quality of plant produce is directly associated with transcription of gene squalene synthase (SS). In current study, barley plants were treated with different rhizobacterial strains under semi controlled (27 ± 3°C) greenhouse conditions in order to modulate expression of SS gene. Plant samples were analyzed through semi-quantitative PCR to evaluate effect of rhizobacterial application on transcriptional status of SS. Results revealed that among four SS genes (i.e...

  2. Combining Cytotoxic and Immune-Mediated Gene Therapy to Treat Brain Tumors

    OpenAIRE

    Curtin, James; King, Gwendalyn; Candolfi, Marianela; Greeno, Remy; Kroeger, Kurt; Lowenstein, Pedro; Castro,Maria

    2005-01-01

    Glioblastoma (GBM) is a type of intracranial brain tumor, for which there is no cure. In spite of advances in surgery, chemotherapy and radiotherapy, patients die within a year of diagnosis. Therefore, there is a critical need to develop novel therapeutic approaches for this disease. Gene therapy, which is the use of genes or other nucleic acids as drugs, is a powerful new treatment strategy which can be developed to treat GBM. Several treatment modalities are amenable for gene therapy implem...

  3. Cloning of an agr homologue of Staphylococcus saprophyticus.

    Science.gov (United States)

    Sakinc, Türkan; Kulczak, Pawel; Henne, Karsten; Gatermann, Sören G

    2004-08-01

    An agr homologue of Staphylococcus saprophyticus was identified, cloned and sequenced. The gene locus shows homologies to other staphylococcal agr systems, especially to those of S. epidermidis and S. lugdunensis. A putative RNAIII was identified and found to be differentially expressed during the growth phases. In contrast to the RNAIII molecules of S. epidermidis and S. aureus it does not contain an open reading frame that codes for a protein with homologies to the delta-toxin. Using PCR, the agr was found to be present in clinical isolates of S. saprophyticus.

  4. GENE EXPRESSION PROFILING OF HUMAN PROMYELOCYTIC LEUKEMIA HL-60 CELL TREATED BY AJOENE

    Institute of Scientific and Technical Information of China (English)

    方志俊; 黄文秀; 黄明辉; 梁润松; 崔景荣; 王夔; 杨梦苏

    2002-01-01

    Objective: Ajoene, a major compound extracted from crashed garlic, has been shown to have antitumor, antimycotic, antimicrobial, antimutagenic functions in vivo or in vitro and treated as a potential antitumor drug. However, the molecular mechanisms underlying the tumor cytotoxicity of ajoene and even garlic substances are poorly defined. In the present study, we aimed to generate gene expression profiles of HL-60 cell treated by ajoene. Methods: A cDNA microarray presenting 2400 of genes amplified from human leukocyte cDNA library was constructed and the gene expression profiles of HL-60 cell induced by ajoene were generated. Results: After data analysis, 28 differentially expressed genes were identified and sequenced. These genes include 21 known genes and 7 ESTs. Most of the known genes are related to cell apoptosis, such as secretory granule (PRG1), beta-2 microglobulin (B2M), 16S ribosomal RNA gene and ribosomal protein S12. Several genes are related to cell differentiation, including the genes similar to H3 histone and ribosomal protein L31. Northern blot analysis was used to verify and quantify the expression of selected genes. Conclusion: Ajoene can induce HL-60 cell apoptosis significantly and may play a role in differentiation. cDNA microarray technology can be a valuable tool to gain insight into molecular events of pharmacological mechanism of herbal medicine.

  5. Gene therapy as a potential tool for treating neuroblastoma-a focused review.

    Science.gov (United States)

    Kumar, M D; Dravid, A; Kumar, A; Sen, D

    2016-05-01

    Neuroblastoma, a solid tumor caused by rapid division of undifferentiated neuroblasts, is the most common childhood malignancy affecting children aged genes is restored to normalcy. Gene therapy is a powerful tool with the potential to inhibit the deleterious effects of oncogenes by inserting corrected/normal genes into the genome. Both viral and non-viral vector-based gene therapies have been developed and adopted to deliver the target genes into neuroblastoma cells. These attempts have given hope to bringing in a new regime of treatment against neuroblastoma. A few gene-therapy-based treatment strategies have been tested in limited clinical trials yielding some positive results. This mini review is an attempt to provide an overview of the available options of gene therapy to treat neuroblastoma.

  6. Comparison of gene expression profiles predicting progression in breast cancer patients treated with tamoxifen.

    Science.gov (United States)

    Kok, Marleen; Linn, Sabine C; Van Laar, Ryan K; Jansen, Maurice P H M; van den Berg, Teun M; Delahaye, Leonie J M J; Glas, Annuska M; Peterse, Johannes L; Hauptmann, Michael; Foekens, John A; Klijn, Jan G M; Wessels, Lodewyk F A; Van't Veer, Laura J; Berns, Els M J J

    2009-01-01

    Molecular signatures that predict outcome in tamoxifen treated breast cancer patients have been identified. For the first time, we compared these response profiles in an independent cohort of (neo)adjuvant systemic treatment naïve breast cancer patients treated with first-line tamoxifen for metastatic disease. From a consecutive series of 246 estrogen receptor (ER) positive primary tumors, gene expression profiling was performed on available frozen tumors using 44K oligoarrays (n = 69). A 78-gene tamoxifen response profile (formerly consisting of 81 cDNA-clones), a 21-gene set (microarray-based Recurrence Score), as well as the HOXB13-IL17BR ratio (Two-Gene-Index, RT-PCR) were analyzed. Performance of signatures in relation to time to progression (TTP) was compared with standard immunohistochemical (IHC) markers: ER, progesterone receptor (PgR) and HER2. In univariate analyses, the 78-gene tamoxifen response profile, 21-gene set and HOXB13-IL17BR ratio were all significantly associated with TTP with hazard ratios of 2.2 (95% CI 1.3-3.7, P = 0.005), 2.3 (95% CI 1.3-4.0, P = 0.003) and 4.2 (95% CI 1.4-12.3, P = 0.009), respectively. The concordance among the three classifiers was relatively low, they classified only 45-61% of patients in the same category. In multivariate analyses, the association remained significant for the 78-gene profile and the 21-gene set after adjusting for ER and PgR. The 78-gene tamoxifen response profile, the 21-gene set and the HOXB13-IL17BR ratio were all significantly associated with TTP in an independent patient series treated with tamoxifen. The addition of multigene assays to ER (IHC) improves the prediction of outcome in tamoxifen treated patients and deserves incorporation in future clinical studies.

  7. Gene Expression Profile of Multiple Myeloma Cell Line Treated by Arsenic Trioxide

    Institute of Scientific and Technical Information of China (English)

    WANG Mengchang; LIU Shaanxi; LIU Pengbo

    2007-01-01

    cDNA microarray was used to compare the gone expression profiles of multiple myeloma cell line RPMI8226 24 h before and after treatment with arsenic trioxide. Two eDNA probes were prepared by mRNA reverse transcription of both arsenic trioxide-treated and untreated RPMI8226 cells. The probes were labeled with Cy3 and Cy5 fluorescence dyes separately, hybridized with cDNA microarray representing 4096 different human genes, and scanned for fluorescence intensity. The differences in gene expression were calculated on the basis of the ratios of signal intensity of treated and untreated samples. The up- and down-regulated genes were screened through the analysis of gene expression ratios. The results showed that 273 genes were differentially altered at mRNA level, 121 genes were up-regulated and 152 were down-regulated. It is concluded that the treatment with arsenic trioxide can induce a variety of gene changes in RPMI8226 cell line. Many genes may be involved in the pathogenesis of multiple myeloma. ALK-1 and TXNIP genes may play an impor- tant role in the apoptosis and partial differentiation of RPMI8226 cells.

  8. Gene expression profiles in BCL11B-siRNA treated malignant T cells

    Directory of Open Access Journals (Sweden)

    Grabarczyk Piotr

    2011-05-01

    Full Text Available Abstract Background Downregulation of the B-cell chronic lymphocytic leukemia (CLL/lymphoma11B (BCL11B gene by small interfering RNA (siRNA leads to growth inhibition and apoptosis of the human T-cell acute lymphoblastic leukemia (T-ALL cell line Molt-4. To further characterize the molecular mechanism, a global gene expression profile of BCL11B-siRNA -treated Molt-4 cells was established. The expression profiles of several genes were further validated in the BCL11B-siRNA -treated Molt-4 cells and primary T-ALL cells. Results 142 genes were found to be upregulated and 109 genes downregulated in the BCL11B-siRNA -treated Molt-4 cells by microarray analysis. Among apoptosis-related genes, three pro-apoptotic genes, TNFSF10, BIK, BNIP3, were upregulated and one anti-apoptotic gene, BCL2L1 was downregulated. Moreover, the expression of SPP1 and CREBBP genes involved in the transforming growth factor (TGF-β pathway was down 16-fold. Expression levels of TNFSF10, BCL2L1, SPP1, and CREBBP were also examined by real-time PCR. A similar expression pattern of TNFSF10, BCL2L1, and SPP1 was identified. However, CREBBP was not downregulated in the BLC11B-siRNA -treated Molt-4 cells. Conclusion BCL11B-siRNA treatment altered expression profiles of TNFSF10, BCL2L1, and SPP1 in both Molt-4 T cell line and primary T-ALL cells.

  9. Gene expression and pathway analysis of human hepatocellular carcinoma cells treated with cadmium

    Science.gov (United States)

    Cartularo, Laura; Laulicht, Freda; Sun, Hong; Kluz, Thomas; Freedman, Jonathan H.; Costa, Max

    2015-01-01

    Cadmium (Cd) is a toxic and carcinogenic metal naturally occurring in the earth’s crust. A common route of human exposure is via diet and cadmium accumulates in the liver. The effects of Cd exposure on gene expression in human hepatocellular carcinoma (HepG2) cells were examined in this study. HepG2 cells were acutely-treated with 0.1, 0.5, or 1.0 μM Cd for 24 hours; or chronically-treated with 0.01, 0.05, or 0.1 μM Cd for three weeks and gene expression analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST Arrays. Acute and chronic exposures significantly altered the expression of 333 and 181 genes, respectively. The genes most upregulated by acute exposure included several metallothioneins. Downregulated genes included the monooxygenase CYP3A7, involved in drug and lipid metabolism. In contrast, CYP3A7 was upregulated by chronic Cd exposure, as was DNAJB9, an anti-apoptotic J protein. Genes downregulated following chronic exposure included the transcriptional regulator early growth response protein 1. Ingenuity Pathway Analysis revealed that the top networks altered by acute exposure were lipid metabolism, small molecule biosynthesis, and cell morphology, organization, and development; while top networks altered by chronic exposure were organ morphology, cell cycle, cell signaling, and renal and urological diseases/cancer. Many of the dysregulated genes play important roles in cellular growth, proliferation, and apoptosis, and may be involved in carcinogenesis. In addition to gene expression changes, HepG2 cells treated with cadmium for 24 hours indicated a reduction in global levels of histone methylation and acetylation that persisted 72 hours post-treatment. PMID:26314618

  10. Transcriptional Modulation of Squalene Synthase Genes in Barley Treated with PGPR

    OpenAIRE

    Anam eYousaf; Abdul eQadir; Tehmina eAnjum; Aqeel eAhmad

    2015-01-01

    Phytosterol contents and food quality of plant produce is directly associated with transcription of gene Squalene Synthase (SS). In current study, barley plants were treated with different rhizobacterial strains under semi controlled (27±3°C) greenhouse conditions in order to modulate expression of SS gene. Plant samples were analysed through semi-quantitative PCR to evaluate effect of rhizobacterial application on transcriptional status of squalene synthase. Results revealed that among four ...

  11. Arabidopsis homologues of the autophagy protein Atg8 are a novel family of microtubule binding proteins

    NARCIS (Netherlands)

    Ketelaar, T.; Voss, C.; Dimmock, S.A.; Thumm, M.; Hussey, P.J.

    2004-01-01

    Autophagy is the non-selective transport of proteins and superfluous organelles destined for degradation to the vacuole in fungae, or the lysosome in animal cells. Some of the genes encoding components of the autophagy pathway are conserved in plants, and here we show that Arabidopsis homologues of

  12. CRIP Homologues Maintain Apical Cytoskeleton to Regulate Tubule Size in C. elegans

    OpenAIRE

    Tong, Xiangyan; Buechner, Matthew

    2008-01-01

    Maintenance of the shape and diameter of biological tubules is a critical task in the development and physiology of all metazoan organisms. We have cloned the exc-9 gene of C. elegans, which regulates the diameter of the single-cell excretory canal tubules. exc-9 encodes a homologue of the highly expressed mammalian intestinal LIM-domain protein CRIP, whose function has not previously been determined. A second well-conserved CRIP homologue functions in multiple valves of C. elegans. EXC-9 sho...

  13. Gene Expression Analysis of Human Vascular Endothelial Cells Treated by Ouabain in Pathological Concentration

    Institute of Scientific and Technical Information of China (English)

    任延平; 吕卓人

    2004-01-01

    Objectives To study the gene expression of human vascular endothelial cells (HUVEC) treated by ouabain in pathological concentration. Methods The response of endothelial cells to ouabain of 1.8 nmol/L was explored with a complementary DNA microarray representing 8 464 different human genes. Results The results of mRNA profiles analysis indicated that 129 of the genes were differently expressed, 26 were upregulated. Conclusions The pathological role of ouabain on HUVEC may be involved in the controlling of DNA transcription、protein translation、 metabolism and signal transduction.

  14. Predictive value of MSH2 gene expression in colorectal cancer treated with capecitabine

    DEFF Research Database (Denmark)

    Jensen, Lars H; Danenberg, Kathleen D; Danenberg, Peter V;

    2007-01-01

    was associated with a hazard ratio of 0.5 (95% confidence interval, 0.23-1.11; P = 0.083) in survival analysis. CONCLUSION: The higher gene expression of MSH2 in responders and the trend for predicting overall survival indicates a predictive value of this marker in the treatment of advanced CRC with capecitabine.......PURPOSE: The objective of the present study was to evaluate the gene expression of the DNA mismatch repair gene MSH2 as a predictive marker in advanced colorectal cancer (CRC) treated with first-line capecitabine. PATIENTS AND METHODS: Microdissection of paraffin-embedded tumor tissue, RNA...

  15. Gene Polymorphisms Affect the Effectiveness of Atorvastatin in Treating Ischemic Stroke Patients

    Directory of Open Access Journals (Sweden)

    Yun-Hua Yue

    2016-07-01

    Full Text Available Background/Aims: The aim of the present study is to investigate whether the single nucleotide polymorphism (SNP in lipid metabolism related genes would affect the effectiveness of atorvastatin in both Han and Uighur populations. Methods: 200 ischemic stroke patients were treated with atorvastatin. The differences of blood lipid level and their ratios were measured. Six lipid related genes, HMGCR, APOA5, LPL, CETP, LDLR and PCSK9 were selected as candidate genes. And nine SNP loci in these six genes were genotyped by SNaPshot technique. Results: In all patients treated with atorvastatin, the SNP rs662799 significantly affected the ratio of ΔLDL and ΔLDL/LDL (p < 0.05; the SNP rs320 significantly affected the ratio of ΔLDL/LDL and Δ(LDL/HDL/(LDL/HDL (p < 0.01 and the SNP rs708272 significantly affected the ratio of ΔLDL (p < 0.05. In Han population treated with atorvastatin, the SNP rs662799 significantly affected the ratio of ΔTG (p < 0.05; the SNP rs320 significantly affected the ratio of ΔLDL/LDL and Δ(LDL/HDL/(LDL/HDL (p < 0.01. In Uighur population treated with atorvastatin, the SNP rs2266788 significantly affected the ratio of ΔHDL (p < 0.05; the SNP rs662799 significantly affected the ratio of ΔLDL/LDL (p < 0.05 and the SNP rs708272 significantly affected the ratio of ΔLDL (p < 0.05. Conclusion: Polymorphisms of rs662799 and rs2266788 in APOA5 gene, rs320 in LPL gene and rs708272 in CETP gene had significant association with the effect of the lipid-lowering therapy via atorvastatin calcium on ischemic stroke patients.

  16. Gene delivery in conjunction with gold nanoparticle and tumor treating electric field

    Science.gov (United States)

    Tiwari, Pawan K.; Soo Lee, Yeon

    2013-08-01

    The advances in electrotherapy to treat the diseased biological cell instigate its extension in gene therapy through the delivery of gene into the nucleus. The objective of this study is to investigate the application of moderate intensity alternating electric field, also known as tumor treating electric field on a carrier system consisting of a charged gene complex conjugated to the surface of a gold nanoparticle. The gene delivery mechanism relies on the magnitude and direction of the induced electric field inside the cytoplasm in presence of carrier system. The induced electric field strength is significant in breaking the gene complex-gold nanoparticle bonding, and exerting an electric force pushing the charged gene into the nucleus. The electric force orientation is dependent on the aspect ratio (AR) of the gold nanoparticle and a relationship between them is studied via Maxwell two-dimensional (2D) finite element simulation analyzer. The development of charge density on the surface of carrier system and the required electric field strength to break the bonding are investigated utilizing the Gouy-Chapman-Grahame-Stern (GCGS) theoretical model. A carrier system having the aspect ratio of the gold nanoparticle in the range 1 < AR ≤ 5 and AR = 1 are substantial delivering cationic and anionic genes into the nucleus, respectively.

  17. CRIP homologues maintain apical cytoskeleton to regulate tubule size in C. elegans.

    Science.gov (United States)

    Tong, Xiangyan; Buechner, Matthew

    2008-05-01

    Maintenance of the shape and diameter of biological tubules is a critical task in the development and physiology of all metazoan organisms. We have cloned the exc-9 gene of Caenorhabditis elegans, which regulates the diameter of the single-cell excretory canal tubules. exc-9 encodes a homologue of the highly expressed mammalian intestinal LIM-domain protein CRIP, whose function has not previously been determined. A second well-conserved CRIP homologue functions in multiple valves of C. elegans. EXC-9 shows genetic interactions with other EXC proteins, including the EXC-5 guanine exchange factor that regulates CDC-42 activity. EXC-9 and its nematode homologue act in polarized epithelial cells that must maintain great flexibility at their apical surface; our results suggest that CRIPs function to maintain cytoskeletal flexibility at the apical surface.

  18. Evaluation of neural gene expression in serum treated embryonic stem cells in Alzheimer′s patients

    Directory of Open Access Journals (Sweden)

    Leila Dehghani

    2013-01-01

    Full Text Available Background: Previous studies confirmed that neural gene expression in embryonic stem cells (ESC could influence by chemical compounds through stimulating apoptotic pathway. We aimed to use ESCs-derived neural cells by embryoid body formation as an in vitro model for determination of neural gene expression changes in groups that treated by sera from Alzheimer′s patients and compare with healthy individuals. Materials and Methods: ESC line which was derived from the C57BL/6 mouse strain was used throughout this study. ESC-derived neural cells were treated with serum from Alzheimer′s patient and healthy individual. Neural gene expression was assessed in both groups by quantitative real-time polymerase chain reaction analysis. The data was analyzed by SPSS Software (version 18. Results: Morphologically, the reducing in neurite out-growth was observed in neural cells in group, which treated by serum from Alzheimer′s patient, while neurite growth was natural in appearance in control group. Microtubule-associated protein 2 and glial fibrillary acidic protein expression significantly reduced in the Alzheimer′s patient group compared with the control group. Nestin expression did not significantly differ among the groups. Conclusion: Neural gene expression could be reduced in serum treated ESC in Alzheimer′s patients.

  19. Crystal chemical formula for sartorite homologues

    DEFF Research Database (Denmark)

    Makovicky, Emil; Topa, Dan

    2015-01-01

    The members of the sartorite homologous series are complex sulfides Me 2+ 8N–16–2X Me 3+ 16+X Me + XS8N+8where Me 2+ is Pb and Me 3+ is As and Sb, whereas Me + is Ag and/or Tl. This paper presents calculation formulae for the homologue order N and for the separate substitution percentages for Tl ...... + (As,Sb) ↔ 2Pb and Ag + (As,Sb) ↔ 2Pb substitutions. This enables one to evaluate the crystal chemistry and build a systematic classification of the sartorite homologues...

  20. HerDing: herb recommendation system to treat diseases using genes and chemicals.

    Science.gov (United States)

    Choi, Wonjun; Choi, Chan-Hun; Kim, Young Ran; Kim, Seon-Jong; Na, Chang-Su; Lee, Hyunju

    2016-01-01

    In recent years, herbs have been researched for new drug candidates because they have a long empirical history of treating diseases and are relatively free from side effects. Studies to scientifically prove the medical efficacy of herbs for target diseases often spend a considerable amount of time and effort in choosing candidate herbs and in performing experiments to measure changes of marker genes when treating herbs. A computational approach to recommend herbs for treating diseases might be helpful to promote efficiency in the early stage of such studies. Although several databases related to traditional Chinese medicine have been already developed, there is no specialized Web tool yet recommending herbs to treat diseases based on disease-related genes. Therefore, we developed a novel search engine, HerDing, focused on retrieving candidate herb-related information with user search terms (a list of genes, a disease name, a chemical name or an herb name). HerDing was built by integrating public databases and by applying a text-mining method. The HerDing website is free and open to all users, and there is no login requirement. Database URL: http://combio.gist.ac.kr/herding.

  1. Profiling gene expression in citrus fruit calyx abscission zone (AZ-C) treated with ethylene.

    Science.gov (United States)

    Cheng, Chunzhen; Zhang, Lingyun; Yang, Xuelian; Zhong, Guangyan

    2015-10-01

    On-tree storage and harvesting of mature fruit account for a large proportion of cost in the production of citrus, and a reduction of the cost would not be achieved without a thorough understanding of the mechani sm of the mature fruit abscission. Genome-wide gene expression changes in ethylene-treated fruit calyx abscission zone (AZ-C) of Citrus sinensis cv. Olinda were therefore investigated using a citrus genome array representing up to 33,879 citrus transcripts. In total, 1313 and 1044 differentially regulated genes were identified in AZ-C treated with ethylene for 4 and 24 h, respectively. The results showed that mature citrus fruit abscission commenced with the activation of ethylene signal transduction pathway that led to the activation of ethylene responsive transcription factors and the subsequent transcriptional regulation of a large set of ethylene responsive genes. Significantly down-regulated genes included those of starch/sugar biosynthesis, transportation of water and growth promoting hormone synthesis and signaling, whereas significantly up-regulated genes were those involved in defense, cell wall degradation, and secondary metabolism. Our data unraveled the underlying mechanisms of some known important biochemical events occurring at AZ-C and should provide informative suggestions for future manipulation of the events to achieve a controllable abscission for mature citrus fruit.

  2. Gene expression profiling of liver from dairy cows treated intra-mammary with lipopolysaccharide

    DEFF Research Database (Denmark)

    Jiang, Li; Sørensen, Peter; Røntved, Christine;

    2008-01-01

    also seemed to participate in APR. CONCLUSIONS: Performing global gene expression analysis on liver tissue from IM LPS treated cows verified that the liver plays a major role in the APR of E. coli mastitis, and that the bovine hepatic APR follows the same pattern as other mammals when......-dependent expression profile and consisted of genes involved in different biological processes. Our findings suggest that APR in the liver is triggered by the activation of signaling pathways that are involved with common and hepatic-specific transcription factors and pro-inflammatory cytokines. These mediators...

  3. Gene expression of panaxydol-treated human melanoma cells using radioactive cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Joong Youn; Yu, Su Jin; Soh, Jeong Won; Kim, Meyoung Kon [College of Medicine, Korea Univ., Seoul (Korea, Republic of)

    2001-07-01

    Polyacetylenic alcohols derived from Panax ginseng have been studied to be an anticancer reagent previously. One of the Panax ginseng polyacetylenic alcohols, i.e., panaxydol, has been studied to possess an antiproliferative effect on human melanoma cell line (SK-MEL-1). In ths study, radioactive cDNA microarrays enabled an efficient approach to analyze the pattern of gene expression (3.194 genes in a total) simultaneously. The bioinformatics selection of human cDNAs, which is specifically designed for immunology, apoptosis and signal transduction, were arrayed on nylon membranes. Using with {sup 33}P labeled probes, this method provided highly sensitive gene expression profiles of our interest including apoptosis, cell proliferation, cell cycle, and signal transduction. Gene expression profiles were also classified into several categories in accordance with the duration of panaxydol treatment. Consequently, the gene profiles of our interest were significantly up (199 genes, > 2.0 of Z-ratio) or down-(196 genes, < 2.0 of Z-ratio) regulated in panaxydol-treated human melanoma cells.

  4. Isolation and characterization of the human MRE11 homologue

    Energy Technology Data Exchange (ETDEWEB)

    Petrini, J.H.J.; Walsh, M.E.; DiMare, C. [Harvard Medical School, Boston, MA (United States)] [and others

    1995-09-01

    Mutation of the Saccharomyces cerevisiae RAD52 epistasis group gene, MRE11, blocks meiotic recombination, confers profound sensitivity to double-strand break damage, and has a hyperrecombinational phenotype in mitotic cells. We isolated a highly conserved human MRE11 homologue using a two-hybrid screen for DNA ligase I-interacting proteins. Human MRE11 shares approximately 50% identity with its yeast counterpart over the N-terminal half of the protein. MRE11 is expressed at the highest levels in proliferating tissues, but is also observed in other tissues. The MRE11 locus maps to human chromosome 11q21 in a region frequently associated with cancer-related chromosomal abnormalities. A MRE11-related locus was found on chromosome 7q11.2-q11.3. 60 refs., 4 figs.

  5. Differential expression and alternative splicing of cell cycle genes in imatinib-treated K562 cells.

    Science.gov (United States)

    Liu, Jing; Lin, Jin; Huang, Lin-Feng; Huang, Bo; Xu, Yan-Mei; Li, Jing; Wang, Yan; Zhang, Jing; Yang, Wei-Ming; Min, Qing-Hua; Wang, Xiao-Zhong

    2015-09-01

    Cancer progression often involves the disorder of the cell cycle, and a number of effective chemotherapeutic drugs have been shown to induce cell cycle arrest. The purpose of this study was to comprehensively investigate the effects of imatinib on the expression profile of cell cycle genes in the chronic myeloid leukemia (CML) K562 cell line. In addition, we also investigated alternative splicing of the cell cycle genes affected by imatinib, since an important relationship has been shown to exist between RNA splicing and cell cycle progression. Exon array analysis was performed using total RNA purified from normal and imatinib-treated K562 cells. We identified 185 differentially expressed genes and 277 alternative splicing events between the two cell groups. A detailed analysis by reverse transcription-PCR (RT-PCR) of key genes confirmed the experimental results of the exon array. These results suggested that treatment of K562 cells with imatinib shifts the expression and alternative splicing profiles of several cell cycle-related genes. Importantly, these findings may help improve imatinib treatment strategies in patients with CML and may be useful for imatinib resistance research and CML drug development.

  6. Profiling of hepatic gene expression in rats treated with fibric acid analogs

    Energy Technology Data Exchange (ETDEWEB)

    Cornwell, Paul D.; Souza, Angus T. de; Ulrich, Roger G

    2004-05-18

    Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptors whose ligands include fatty acids, eicosanoids and the fibrate class of drugs. In humans, fibrates are used to treat dyslipidemias. In rodents, fibrates cause peroxisome proliferation, a change that might explain the observed hepatomegaly. In this study, rats were treated with multiple dose levels of six fibric acid analogs (including fenofibrate) for up to two weeks. Pathological analysis identified hepatocellular hypertrophy as the only sign of hepatotoxicity, and only one compound at the highest dose caused any significant increase in serum ALT or AST activity. RNA profiling revealed that the expression of 1288 genes was related to dose or length of treatment and correlated with hepatocellular hypertrophy. This gene list included expression changes that were consistent with increased mitochondrial and peroxisomal {beta}-oxidation, increased fatty acid transport, increased hepatic uptake of LDL-cholesterol, decreased hepatic uptake of glucose, decreased gluconeogenesis and decreased glycolysis. These changes are likely linked to many of the clinical benefits of fibrate drugs, including decreased serum triglycerides, decreased serum LDL-cholesterol and increased serum HDL-cholesterol. In light of the fact that all six compounds stimulated similar or identical changes in the expression of this set of 1288 genes, these results indicate that hepatomegaly is due to PPAR{alpha} activation, although signaling through other receptors (e.g. PPAR{gamma}, RXR) or through non-receptor pathways cannot be excluded.

  7. Gene expression profile of colon cancer cell lines treated with SN-38

    DEFF Research Database (Denmark)

    Wallin, A; Francis, P; Nilbert, M

    2010-01-01

    Colorectal cancer is the third most common form of cancer in the industrial countries. Due to advances regarding the treatments, primarily development of improved surgical methods and the ability to make the earlier diagnosis, the mortality has remained constant during the past decades even though...... the incidence in fact has increased. To improve chemotherapy and enable personalised treatment, the need of biomarkers is of great significance. In this study, we evaluated the gene expression profiles of the colon cancer cell lines treated with SN-38, the active metabolite of topoisomerase-1 inhibitor...

  8. Gene expression profile of colon cancer cell lines treated with SN-38

    DEFF Research Database (Denmark)

    Wallin, A; Francis, P; Nilbert, M;

    2010-01-01

    the incidence in fact has increased. To improve chemotherapy and enable personalised treatment, the need of biomarkers is of great significance. In this study, we evaluated the gene expression profiles of the colon cancer cell lines treated with SN-38, the active metabolite of topoisomerase-1 inhibitor......Colorectal cancer is the third most common form of cancer in the industrial countries. Due to advances regarding the treatments, primarily development of improved surgical methods and the ability to make the earlier diagnosis, the mortality has remained constant during the past decades even though...

  9. A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen.

    Science.gov (United States)

    Ma, Xiao-Jun; Wang, Zuncai; Ryan, Paula D; Isakoff, Steven J; Barmettler, Anne; Fuller, Andrew; Muir, Beth; Mohapatra, Gayatry; Salunga, Ranelle; Tuggle, J Todd; Tran, Yen; Tran, Diem; Tassin, Ana; Amon, Paul; Wang, Wilson; Wang, Wei; Enright, Edward; Stecker, Kimberly; Estepa-Sabal, Eden; Smith, Barbara; Younger, Jerry; Balis, Ulysses; Michaelson, James; Bhan, Atul; Habin, Karleen; Baer, Thomas M; Brugge, Joan; Haber, Daniel A; Erlander, Mark G; Sgroi, Dennis C

    2004-06-01

    Tamoxifen significantly reduces tumor recurrence in certain patients with early-stage estrogen receptor-positive breast cancer, but markers predictive of treatment failure have not been identified. Here, we generated gene expression profiles of hormone receptor-positive primary breast cancers in a set of 60 patients treated with adjuvant tamoxifen monotherapy. An expression signature predictive of disease-free survival was reduced to a two-gene ratio, HOXB13 versus IL17BR, which outperformed existing biomarkers. Ectopic expression of HOXB13 in MCF10A breast epithelial cells enhances motility and invasion in vitro, and its expression is increased in both preinvasive and invasive primary breast cancer. The HOXB13:IL17BR expression ratio may be useful for identifying patients appropriate for alternative therapeutic regimens in early-stage breast cancer.

  10. Defense gene expression in elicitor-treated cell suspension cultures of french bean cv. Imuna.

    Science.gov (United States)

    Ellis, J S; Jennings, A C; Edwards, L A; Mavandad, M; Lamb, C J; Dixon, R A

    1989-12-01

    Cell suspension cultures of bean (Phaseolus vulgaris) cv. Imuna accumulated isoflavonoid phytoalexins on exposure to elicitor from the phytopathogenic fungus Colletotrichum lindemuthianum (CL). This was preceeded by rapid increases in the activities of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS). However, the patterns of expression of PAL and CHS genes differed from those observed in cultures of a previously studied bean cultivar. The relative levels of transcripts from individual members of the CHS multigene family differed significantly at 1.5 h compared to 22.5 h after elicitation. More strikingly, three PAL genes were expressed in cultivar Imuna in response to fungal elicitor, whereas two are expressed in elicitor-treated cell cultures of cultivar Canadian Wonder.

  11. Alteration of gene expression in human cells treated with the agricultural chemical diazinon: possible interaction in fetal development.

    Science.gov (United States)

    Mankame, T; Hokanson, R; Fudge, R; Chowdhary, R; Busbee, D

    2006-05-01

    Agricultural chemicals frequently alter human health or development, typically because they have endocrine agonist or antagonist activities and alter hormone-regulation of gene expression. The insecticide, diazinon, was evaluated for gene expression disrupting activity using MCF-7 cells, an estrogen-dependent human cell line, to examine the capacity of the insecticide to disrupt gene expression essential for morphological development, immune system development or function, and/or central nervous system development and function. MCF-7 cells were treated with 30, 50 or 67 ppm diazinon, and gene expression was measured in treated cells compared to expression in untreated or estrogen-treated cells. DNA microarray analysis of diazinon-treated cells showed significant up- or down-regulation of a large number of genes compared to untreated cells. Of the 600 human genes on the Phase 1 chip utilized for these studies, two specific genes--calreticulin and TGF-beta3--were selected for corroboration using quantitative real time PCR (qrtPCR). qrtPCR, completed to assess gene expression levels for calreticulin and TGFbeta3, confirmed results showing significant up-regulation of these two genes obtained from the microarray data. These studies were designed to provide baseline data on the gene expression-altering capacity of a specific chemical, diazinon, and allow a partial assessment of the potentially deleterious effects associated with exposure of human cells to this chemical. Currently, it is not known whether results from cells in vitro can be extrapolated to human health consequences of chemical exposure.

  12. Gene expression profiling of liver from dairy cows treated intra-mammary with lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Vels Lotte

    2008-09-01

    Full Text Available Abstract Background Liver plays a profound role in the acute phase response (APR observed in the early phase of acute bovine mastitis caused by Escherichia coli (E. coli. To gain an insight into the genes and pathways involved in hepatic APR of dairy cows we performed a global gene expression analysis of liver tissue sampled at different time points before and after intra-mammary (IM exposure to E. coli lipopolysaccharide (LPS treatment. Results Approximately 20% target transcripts were differentially expressed and eight co-expression clusters were identified. Each cluster had a unique time-dependent expression profile and consisted of genes involved in different biological processes. Our findings suggest that APR in the liver is triggered by the activation of signaling pathways that are involved with common and hepatic-specific transcription factors and pro-inflammatory cytokines. These mediators in turn stimulated or repressed the expression of genes encoding acute phase proteins (APP, collectins, complement components, chemokines, cell adhesion molecules and key metabolic enzymes during the APR. Hormones, anti-inflammatory and other hypothalamus-pituitary-adrenal axis (HPAA linked mediators also seemed to participate in APR. Conclusion Performing global gene expression analysis on liver tissue from IM LPS treated cows verified that the liver plays a major role in the APR of E. coli mastitis, and that the bovine hepatic APR follows the same pattern as other mammals when they are challenged with LPS. Our work presents the first insight into the dynamic changes in gene expression in the liver that influences the induction, kinetics and clinical outcome of the APR in dairy cows.

  13. Gene expression profiling of MPP+-treated MN9D cells: a mechanism of toxicity study.

    Science.gov (United States)

    Wang, Jianyong; Xu, Zengjun; Fang, Hong; Duhart, Helen M; Patterson, Tucker A; Ali, Syed F

    2007-09-01

    Parkinson's disease (PD) is a common neurodegenerative disease characterized by progressive loss of midbrain dopaminergic neurons with unknown etiology. MPP+ (1-methyl-4-phenylpyridinium) is the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which induces Parkinson's-like syndromes in humans and animals. MPTP/MPP+ treatment produces selective dopaminergic neuronal degeneration, therefore, these agents are commonly used to study the pathogenesis of PD. However, the mechanisms of their toxicity have not been elucidated. In order to gain insights into MPP+-induced neurotoxicity, a gene expression microarray study was performed using a midbrain-derived dopaminergic neuronal cell line, MN9D. Utilizing a two-color reference design, Agilent mouse oligonucleotide microarrays were used to examine relative gene expression changes in MN9D cells treated with 40microM MPP+ compared with controls. Bioinformatics tools were used for data evaluation. Briefly, raw data were imported into the NCTR ArrayTrack database, normalized using a Lowess method and data quality was assessed. The Student's t-test was used to determine significant changes in gene expression (set as p1.5). Gene Ontology for Function Analysis (GOFFA) and Ingenuity Pathway Analysis were employed to analyze the functions and roles of significant genes in biological processes. Of the 51 significant genes identified, 44 were present in the GOFFA or Ingenuity database. These data indicate that multiple pathways are involved in the underlying mechanisms of MPP+-induced neurotoxicity, including apoptosis, oxidative stress, iron binding, cellular metabolism, and signal transduction. These data also indicate that MPP+-induced toxicity shares common molecular mechanisms with the pathogenesis of PD and further pathway analyses will be conducted to explore these mechanisms.

  14. Profile of stress and toxicity gene expression in human hepatic cells treated with Efavirenz.

    Science.gov (United States)

    Gomez-Sucerquia, Leysa J; Blas-Garcia, Ana; Marti-Cabrera, Miguel; Esplugues, Juan V; Apostolova, Nadezda

    2012-06-01

    Hepatic toxicity and metabolic disorders are major adverse effects elicited during the pharmacological treatment of the human immunodeficiency virus (HIV) infection. Efavirenz (EFV), the most widely used non-nucleoside reverse transcriptase inhibitor (NNRTI), has been associated with these events, with recent studies implicating it in stress responses involving mitochondrial dysfunction and oxidative stress in human hepatic cells. To expand these findings, we analyzed the influence of EFV on the expression profile of selected stress and toxicity genes in these cells. Significant up-regulation was observed with Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), which indicated metabolic stress. Several genes directly related to oxidative stress and damage exhibited increased expression, including Methalothionein 2A (MT2A), Heat shock 70kDa protein 6 (HSPA6), Growth differentiation factor 15 (GDF15) and DNA-damage-inducible transcript 3 (DDIT3). In addition, Early growth response protein 1 (EGR1) was enhanced, whereas mRNA levels of the inflammatory genes Chemokine (C-X-C motif) ligand 10 (CXCL10) and Serpin peptidase inhibitor (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1) decreased and increased, respectively. This profile of gene expression supports previous data demonstrating altered mitochondrial function and presence of oxidative stress/damage in EFV-treated hepatic cells, and may be of relevance in the search for molecular targets with therapeutic potential to be employed in the prevention, diagnosis and treatment of the hepatic toxicity associated with HIV therapy.

  15. An Approach for Treating the Hepatobiliary Disease of Cystic Fibrosis by Somatic Gene Transfer

    Science.gov (United States)

    Yang, Yiping; Raper, Steven E.; Cohn, Jonathan A.; Engelhardt, John F.; Wilson, James M.

    1993-05-01

    Cystic fibrosis (CF) is an inherited disease of epithelial cell ion transport that is associated with pathology in multiple organ systems, including lung, pancreas, and liver. As treatment of the pulmonary manifestations of CF has improved, management of CF liver disease has become increasingly important in adult patients. This report describes an approach for treating CF liver disease by somatic gene transfer. In situ hybridization and immunocytochemistry analysis of rat liver sections indicated that the endogenous CFTR (cystic fibrosis transmembrane conductance regulator) gene is primarily expressed in the intrahepatic biliary epithelial cells. To specifically target recombinant genes to the biliary epithelium in vivo, recombinant adenoviruses expressing lacZ or human CFTR were infused retrograde into the biliary tract through the common bile duct. Conditions were established for achieving recombinant gene expression in virtually all cells of the intrahepatic bile ducts in vivo. Expression persisted in the smaller bile ducts for the duration of the experiment, which was 21 days. These studies suggest that it may be feasible to prevent CF liver disease by genetically reconstituting CFTR expression in the biliary tract, using an approach that is clinically feasible.

  16. Adipose tissue gene expression profiles in ob/ob mice treated with leptin.

    Science.gov (United States)

    Zhang, Wei; Della-Fera, Mary Anne; Hartzell, Diane L; Hausman, Dorothy; Baile, Clifton A

    2008-07-04

    Leptin plays a critical role in regulating body weight, lipid metabolism, apoptosis and microvasculature of adipose tissue. To explore multiple signaling pathways of leptin action on adipose tissue, real-time PCR utilizing TaqMan low-density arrays was performed to compare mRNA expression in adipose tissue of ob/ob mice treated with vehicle or leptin (2.5 microg/d or 10 microg/d) for 14 days via subcutaneous osmotic minipumps. Of the 24 target genes selected for characterization, many were differentially expressed between control ob/ob mice and leptin-treated ob/ob mice. Increases in mRNA expression were found for hormone sensitive lipase (HSL), uncoupling protein 2 (UCP2), adrenergic receptor 3 (ADR3), mitofusin 2 (Mfn2), sirtuin 3 (Sirt3), transcription factor sterol regulatory element binding factor 1 (SREBF1), Bcl-2, Bax, Caspase 3, tumor necrosis factor alpha (TNFalpha), adiponectin and angiopoietin 2 (Ang-2). Decreases in expression were found for stearoyl-coenzyme A desaturase 1 (SCD1), fatty acid synthase (FAS), and retinol binding protein 4 (RBP4). There were no changes in expression of transcription factors involved in adipocyte differentiation (C/EBPalpha, PPARalpha, and PPARgamma). These results confirm that alterations in the expression of specific adipose tissue genes including those associated with the promotion of lipid mobilization, energy dissipation, and apoptosis may mediate leptin-induced fat loss in ob/ob mice.

  17. Functional Characterization of Aspergillus nidulans ypkA, a Homologue of the Mammalian Kinase SGK

    Science.gov (United States)

    Colabardini, Ana Cristina; Brown, Neil Andrew; Savoldi, Marcela; Goldman, Maria Helena S.; Goldman, Gustavo Henrique

    2013-01-01

    The serum- and glucocorticoid-regulated protein kinase (SGK) is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The Saccharomyces cerevisiae ypk1 and ypk2 genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the Aspergillus nidulans YPK1 homologue, YpkA, representing the first filamentous fungal YPK1 homologue. Two conditional mutant strains were constructed by replacing the endogenous ypk1 promoter with two different regulatable promoters, alcA (from the alcohol dehydrogenase gene) and niiA (from the nitrate reductase gene). Both constructs confirmed that ypkA was an essential gene in A. nidulans. Repression of ypkA caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by ypkA repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The A. nidulans Pkh1 homologue pkhA was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of pkhA or epistatic to pkhA, rather, ypkA and pkhA are genetically independent or in parallel. BarA is a homologue of the yeast Lag1 acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When barA was absent, ypkA repression was lethal to the cell. Therefore, there appears to be a genetic interaction between ypkA, barA, and the sphingolipid synthesis. Transcriptional profiling of ypkA overexpression and down-regulation revealed several putative YpkA targets associated with the

  18. Functional characterization of Aspergillus nidulans ypkA, a homologue of the mammalian kinase SGK.

    Science.gov (United States)

    Colabardini, Ana Cristina; Brown, Neil Andrew; Savoldi, Marcela; Goldman, Maria Helena S; Goldman, Gustavo Henrique

    2013-01-01

    The serum- and glucocorticoid-regulated protein kinase (SGK) is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The Saccharomyces cerevisiae ypk1 and ypk2 genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the Aspergillus nidulans YPK1 homologue, YpkA, representing the first filamentous fungal YPK1 homologue. Two conditional mutant strains were constructed by replacing the endogenous ypk1 promoter with two different regulatable promoters, alcA (from the alcohol dehydrogenase gene) and niiA (from the nitrate reductase gene). Both constructs confirmed that ypkA was an essential gene in A. nidulans. Repression of ypkA caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by ypkA repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The A. nidulans Pkh1 homologue pkhA was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of pkhA or epistatic to pkhA, rather, ypkA and pkhA are genetically independent or in parallel. BarA is a homologue of the yeast Lag1 acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When barA was absent, ypkA repression was lethal to the cell. Therefore, there appears to be a genetic interaction between ypkA, barA, and the sphingolipid synthesis. Transcriptional profiling of ypkA overexpression and down-regulation revealed several putative YpkA targets associated with the

  19. Functional characterization of Aspergillus nidulans ypkA, a homologue of the mammalian kinase SGK.

    Directory of Open Access Journals (Sweden)

    Ana Cristina Colabardini

    Full Text Available The serum- and glucocorticoid-regulated protein kinase (SGK is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The Saccharomyces cerevisiae ypk1 and ypk2 genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the Aspergillus nidulans YPK1 homologue, YpkA, representing the first filamentous fungal YPK1 homologue. Two conditional mutant strains were constructed by replacing the endogenous ypk1 promoter with two different regulatable promoters, alcA (from the alcohol dehydrogenase gene and niiA (from the nitrate reductase gene. Both constructs confirmed that ypkA was an essential gene in A. nidulans. Repression of ypkA caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by ypkA repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The A. nidulans Pkh1 homologue pkhA was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of pkhA or epistatic to pkhA, rather, ypkA and pkhA are genetically independent or in parallel. BarA is a homologue of the yeast Lag1 acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When barA was absent, ypkA repression was lethal to the cell. Therefore, there appears to be a genetic interaction between ypkA, barA, and the sphingolipid synthesis. Transcriptional profiling of ypkA overexpression and down-regulation revealed several putative YpkA targets

  20. A gene expression signature that can predict the recurrence of tamoxifen-treated primary breast cancer.

    Science.gov (United States)

    Chanrion, Maïa; Negre, Vincent; Fontaine, Hélène; Salvetat, Nicolas; Bibeau, Frédéric; Mac Grogan, Gaëtan; Mauriac, Louis; Katsaros, Dionyssios; Molina, Franck; Theillet, Charles; Darbon, Jean-Marie

    2008-03-15

    The identification of a molecular signature predicting the relapse of tamoxifen-treated primary breast cancers should help the therapeutic management of estrogen receptor-positive cancers. A series of 132 primary tumors from patients who received adjuvant tamoxifen were analyzed for expression profiles at the whole-genome level by 70-mer oligonucleotide microarrays. A supervised analysis was done to identify an expression signature. We defined a 36-gene signature that correctly classified 78% of patients with relapse and 80% of relapse-free patients (79% accuracy). Using 23 independent tumors, we confirmed the accuracy of the signature (78%) whose relevance was further shown by using published microarray data from 60 tamoxifen-treated patients (63% accuracy). Univariate analysis using the validation set of 83 tumors showed that the 36-gene classifier is more efficient in predicting disease-free survival than the traditional histopathologic prognostic factors and is as effective as the Nottingham Prognostic Index or the "Adjuvant!" software. Multivariate analysis showed that the molecular signature is the only independent prognostic factor. A comparison with several already published signatures demonstrated that the 36-gene signature is among the best to classify tumors from both training and validation sets. Kaplan-Meier analyses emphasized its prognostic power both on the whole cohort of patients and on a subgroup with an intermediate risk of recurrence as defined by the St. Gallen criteria. This study identifies a molecular signature specifying a subgroup of patients who do not gain benefits from tamoxifen treatment. These patients may therefore be eligible for alternative endocrine therapies and/or chemotherapy.

  1. Gene expression analysis in the hippocampal formation of tree shrews chronically treated with cortisol.

    Science.gov (United States)

    Alfonso, Julieta; Agüero, Fernán; Sanchez, Daniel O; Flugge, Gabriele; Fuchs, Eberhard; Frasch, Alberto C C; Pollevick, Guido D

    2004-12-01

    Adrenal corticosteroids influence the function of the hippocampus, the brain structure in which the highest expression of glucocorticoid receptors is found. Chronic high levels of cortisol elicited by stress or through exogenous administration can cause irreversible damage and cognitive deficits. In this study, we searched for genes expressed in the hippocampal formation after chronic cortisol treatment in male tree shrews. Animals were treated orally with cortisol for 28 days. At the end of the experiments, we generated two subtractive hippocampal hybridization libraries from which we sequenced 2,246 expressed sequenced tags (ESTs) potentially regulated by cortisol. To validate this approach further, we selected some of the candidate clones to measure mRNA expression levels in hippocampus using real-time PCR. We found that 66% of the sequences tested (10 of 15) were differentially represented between cortisol-treated and control animals. The complete set of clones was subjected to a bioinformatic analysis, which allowed classification of the ESTs into four different main categories: 1) known proteins or genes (approximately 28%), 2) ESTs previously published in the database (approximately 16%), 3) novel ESTs matching only the reference human or mouse genome (approximately 5%), and 4) sequences that do not match any public database (50%). Interestingly, the last category was the most abundant. Hybridization assays revealed that several of these clones are indeed expressed in hippocampal tissue from tree shrew, human, and/or rat. Therefore, we discovered an extensive inventory of new molecular targets in the hippocampus that serves as a reference for hippocampal transcriptional responses under various conditions. Finally, a detailed analysis of the genomic localization in human and mouse genomes revealed a survey of putative novel splicing variants for several genes of the nervous system.

  2. Evaluation of Bcl-2 Family Gene Expression in Hippocampus of 3, 4-methylenedioxymethamphetamine Treated Rats

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    Hamed Hashemi-Nasl

    2012-01-01

    Full Text Available Objective: 3,4-methylenedioxymethamphetamine (MDMA is an illicit, recreational drugthat causes cellular death and neurotoxicity. This study evaluates the effects of differentdoses of MDMA on the expression of apoptosis–related proteins and genes in the hippocampusof adult rats.Materials and Methods: In this expremental study,a total of 20 male Sprague Dawley rats(200-250 g were treated with MDMA (0, 5, 10, 20 mg/kg i.p. twice daily for 7 days. Sevendays after the last administration of MDMA, the rats were killed. Bax and Bcl-2 genesin addition to protein expressions were detected by western blot and reverse transcriptionpolymerasechain reaction (RT-PCR.Results were analyzed using one-way ANOVA andp≤0.05 was considered statistically significant.Results: Our results showed that MDMA caused dose dependent up-regulation of Baxand down-regulation of Bcl-2 in the hippocampus. There was a significant alteration inbcl-2 and bax genes density.Conclusion: Changes in apoptosis-related proteins and respective genes relating to Baxand Bcl-2 might be involved in the molecular mechanism of MDMA-induced apoptosis.

  3. Isolation of Crb1, a mouse homologue of Drosophila crumbs, and analysis of its expression pattern in eye and brain.

    NARCIS (Netherlands)

    Hollander, A.I. den; Ghiani, M.; Kok, Y.J.M. de; Wijnholds, J.; Ballabio, A.; Cremers, F.P.M.; Broccoli, V.

    2002-01-01

    Mutations in the human Crumbs homologue 1 (CRB1) gene cause severe retinal dystrophies. CRB1 is homologous to Drosophila Crumbs, a protein essential for establishing and maintaining epithelial polarity. We have isolated the mouse orthologue, Crb1, and analyzed its expression pattern in embryonic and

  4. Characterization of the promoter and extended C-terminal domain of Arabidopsis WRKY33 and functional analysis of tomato WRKY33 homologues in plant stress responses.

    Science.gov (United States)

    Zhou, Jie; Wang, Jian; Zheng, Zuyu; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2015-08-01

    Arabidopsis AtWRKY33 plays a critical role in broad plant stress responses. Whether there are evolutionarily conserved homologues of AtWRKY33 in other plants and what make AtWRKY33 such an important protein in plant stress responses are largely unknown. We compared AtWRKY33 with its close homologues to identify AtWRKY33-specific regulatory and structural elements, which were then functionally analysed through complementation. We also performed phylogenetic analysis to identify structural AtWRKY33 homologues in other plants and functionally analysed two tomato homologues through complementation and gene silencing. AtWRKY33 has an extended C-terminal domain (CTD) absent in its close homologue AtWRKY25. Both its CTD and the strong pathogen/stress-responsive expression of AtWRKY33 are necessary to complement the critical phenotypes of atwrky33. Structural AtWRKY33 homologues were identified in both dicot and monocot plants including two (SlWRKY33A and SlWRKY33B) in tomato. Molecular complementation and gene silencing confirmed that the two tomato WRKY genes play a critical role similar to that of AtWRKY33 in plant stress responses. Thus, WRKY33 proteins are evolutionarily conserved with a critical role in broad plant stress responses. Both its CTD and promoter are critical for the uniquely important roles of WRKY33 in plant stress responses.

  5. Chemoprevention gene therapy (CGT): novel combinatorial approach for preventing and treating pancreatic cancer.

    Science.gov (United States)

    Sarkar, S; Azab, B M; Das, S K; Quinn, B A; Shen, X; Dash, R; Emdad, L; Thomas, S; Dasgupta, S; Su, Z-Z; Wang, X-Y; Sarkar, D; Fisher, P B

    2013-08-01

    Pancreatic cancer remains one of the deadliest of all cancers despite aggressive surgical treatment combined with adjuvant radiotherapy and chemotherapy. Chemoresistance and radioresistance are the principal causes of failure of pancreatic cancer patients to respond to therapy. Conditionally replication competent adenovirus (CRCA)-based cancer gene therapy is an innovative strategy for treating cancers displaying inherent resistance to treatment. Limitations of current adenovirus (Ad)-based gene therapies for malignant tumors include lack of cancer-specificity, and effective and targeted delivery. To remedy this situation, CRCAs have been designed that express E1A, necessary for Ad replication, under the control of a cancer-specific progression elevated gene-3 promoter (PEG-Prom) with concomitant expression of an immunomodulatory cytokine, such as mda-7/IL-24 or interferon-γ (IFN-γ), under the control of a ubiquitous and strong cytomegalovirus promoter (CMV-Prom) from the E3 region. These bipartite CRCAs, when armed with a transgene, are called cancer terminator viruses (CTVs), i.e., Ad.PEG-E1A-CMV-mda-7 (CTV-M7) and Ad.PEG-E1A-CMV-IFN-γ (CTV-γ), because of their universal effectiveness in cancer treatment irrespective of p53/pRb/p16 or other genetic alterations in tumor cells. In addition to their selective oncolytic effects in tumor cells, the potent 'bystander antitumor' properties of MDA-7/IL-24 and IFN-γ embody the CTVs with expanded treatment properties for both primary and distant cancers. Pancreatic cancer cells display a "translational block" of mda-7/IL-24 mRNA, limiting production of MDA-7/IL-24 protein and cancer-specific apoptosis. Specific chemopreventive agents abrogate this "translational block" resulting in pancreatic cancer-specific killing. This novel chemoprevention gene therapy (CGT) strategy holds promise for both prevention and treatment of pancreatic cancers where all other strategies have proven ineffective.

  6. Characterization of the C. elegans erlin homologue

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    Hoegg Maja B

    2012-01-01

    Full Text Available Abstract Background Erlins are highly conserved proteins associated with lipid rafts within the endoplasmic reticulum (ER. Biochemical studies in mammalian cell lines have shown that erlins are required for ER associated protein degradation (ERAD of activated inositol-1,4,5-trisphosphate receptors (IP3Rs, implying that erlin proteins might negatively regulate IP3R signalling. In humans, loss of erlin function appears to cause progressive intellectual disability, motor dysfunction and joint contractures. However, it is unknown if defects in IP3R ERAD are the underlying cause of this disease phenotype, whether ERAD of activated IP3Rs is the only function of erlin proteins, and what role ERAD plays in regulating IP3R-dependent processes in the context of an intact animal or embryo. In this study, we characterize the erlin homologue of the nematode Caenorhabditis elegans and examine erlin function in vivo. We specifically set out to test whether C. elegans erlin modulates IP3R-dependent processes, such as egg laying, embryonic development and defecation rates. We also explore the possibility that erlin might play a more general role in the ERAD pathway of C. elegans. Results We first show that the C. elegans erlin homologue, ERL-1, is highly similar to mammalian erlins with respect to amino acid sequence, domain structure, biochemical properties and subcellular location. ERL-1 is present throughout the C. elegans embryo; in adult worms, ERL-1 appears restricted to the germline. The expression pattern of ERL-1 thus only partially overlaps with that of ITR-1, eliminating the possibility of ERL-1 being a ubiquitous and necessary regulator of ITR-1. We show that loss of ERL-1 does not affect overall phenotype, or alter brood size, embryonic development or defecation cycle length in either wild type or sensitized itr-1 mutant animals. Moreover we show that ERL-1 deficient worms respond normally to ER stress conditions, suggesting that ERL-1 is not an

  7. Combining cytotoxic and immune-mediated gene therapy to treat brain tumors.

    Science.gov (United States)

    Curtin, James F; King, Gwendalyn D; Candolfi, Marianela; Greeno, Remy B; Kroeger, Kurt M; Lowenstein, Pedro R; Castro, Maria G

    2005-01-01

    Glioblastoma (GBM) is a type of intracranial brain tumor, for which there is no cure. In spite of advances in surgery, chemotherapy and radiotherapy, patients die within a year of diagnosis. Therefore, there is a critical need to develop novel therapeutic approaches for this disease. Gene therapy, which is the use of genes or other nucleic acids as drugs, is a powerful new treatment strategy which can be developed to treat GBM. Several treatment modalities are amenable for gene therapy implementation, e.g. conditional cytotoxic approaches, targeted delivery of toxins into the tumor mass, immune stimulatory strategies, and these will all be the focus of this review. Both conditional cytotoxicity and targeted toxin mediated tumor death, are aimed at eliminating an established tumor mass and preventing further growth. Tumors employ several defensive strategies that suppress and inhibit anti-tumor immune responses. A better understanding of the mechanisms involved in eliciting anti-tumor immune responses has identified promising targets for immunotherapy. Immunotherapy is designed to aid the immune system to recognize and destroy tumor cells in order to eliminate the tumor burden. Also, immune-therapeutic strategies have the added advantage that an activated immune system has the capability of recognizing tumor cells at distant sites from the primary tumor, therefore targeting metastasis distant from the primary tumor locale. Pre-clinical models and clinical trials have demonstrated that in spite of their location within the central nervous system (CNS), a tissue described as 'immune privileged', brain tumors can be effectively targeted by the activated immune system following various immunotherapeutic strategies. This review will highlight recent advances in brain tumor immunotherapy, with particular emphasis on advances made using gene therapy strategies, as well as reviewing other novel therapies that can be used in combination with immunotherapy. Another important

  8. C21orf57 is a human homologue of bacterial YbeY proteins.

    Science.gov (United States)

    Ghosal, Anubrata; Köhrer, Caroline; Babu, Vignesh M P; Yamanaka, Kinrin; Davies, Bryan W; Jacob, Asha I; Ferullo, Daniel J; Gruber, Charley C; Vercruysse, Maarten; Walker, Graham C

    2017-03-11

    The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria.

  9. Is the Prosthetic Homologue Necessary for Embodiment?

    Science.gov (United States)

    Dornfeld, Chelsea; Swanston, Michelle; Cassella, Joseph; Beasley, Casey; Green, Jacob; Moshayev, Yonatan; Wininger, Michael

    2016-01-01

    Embodiment is the process by which patients with limb loss come to accept their peripheral device as a natural extension of self. However, there is little guidance as to how exacting the prosthesis must be in order for embodiment to take place: is it necessary for the prosthetic hand to look just like the absent hand? Here, we describe a protocol for testing whether an individual would select a hand that looks like their own from among a selection of five hands, and whether the hand selection (regardless of homology) is consistent across multiple exposures to the same (but reordered) set of candidate hands. Pilot results using healthy volunteers reveals that hand selection is only modestly consistent, and that selection of the prosthetic homologue is atypical (61 of 192 total exposures). Our protocol can be executed in minutes, and makes use of readily available equipment and softwares. We present both a face-to-face and a virtual protocol, for maximum flexibility of implementation. PMID:28066228

  10. Is the prosthetic homologue necessary for embodiment?

    Directory of Open Access Journals (Sweden)

    Chelsea Dornfeld

    2016-12-01

    Full Text Available Embodiment is the process by which patients with limb loss come to accept their peripheral device as a natural extension of self. However, there is little guidance as to how exacting the prosthesis must be in order for embodiment to take place: is it necessary for the prosthetic hand to look just like the absent hand? Here, we describe a protocol for testing whether an individual would select a hand that looks like their own from among a selection of 5 hands, and whether the hand selection (regardless of homology is consistent across multiple exposures to the same (but reordered set of candidate hands. Pilot results using healthy volunteers reveals that hand selection is only modestly consistent, and that selection of the prosthetic homologue is atypical (61 of 192 total exposures. Our protocol can be executed in minutes, and makes use of readily available equipment and softwares. We present both a face-to-face and a virtual protocol, for maximum flexibility of implementation.

  11. Gene expression profiles of hepatocytes treated with La(NO3)3 of rare earth in rats

    Institute of Scientific and Technical Information of China (English)

    Hui Zhao; Wei-Dong Hao; Hou-En Xu; Lan-Qin Shang; You-Yong Lu

    2004-01-01

    AIM: To compare the gene expression between La(NO3)3-exposed and control rats in vivo.METHODS: Rats were fed La(NO3)3 once daily at a dose of 20 mg/kg for one month by gavage. Gene expression of hepatocytes was detected using mRNA differential display (DD) technique and cDNA microarray and compared between treated and control groups.RESULTS: Six differentially expressed sequence tags were cloned by DD, of which five were up regulated and one was down regulated in treated rats. Two sequences were determined. One band was novel. The other shared 100%sequence homology with AU080263 Sugano mouse brain mncb Mus musculus cDNA clone MNCb-5435 5′. With DNA microarray, 136 differentially expressed genes were identified including 131 over-expressed genes and 5 under-expressed genes. Most of these differentially expressed genes were cell signal and transmission genes, genes associated with metabolism, protein translation and synthesis.CONCLUSION: La(NO3)3 could change the expression levels of some kinds of genes. Further analysis of the differentially expressed genes would be helpful for understanding the wide biological effect spectrum of rare earth elements.

  12. Phosphatase and tensin homologue deleted on chromosome 10

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    Imran Haruna Abdulkareem

    2013-01-01

    Full Text Available Phosphatase and tensin homologue deleted on chromosome 10 (PTEN is a tumor suppressor gene deleted or mutated in many human cancers such as glioblastoma, spinal tumors, prostate, bladder, adrenals, thyroid, breast, endometrium, and colon cancers. They result from loss of heterozygosity (LOH for the PTEN gene on chromosome 10q23. Previous studies reported that various drugs, chemicals, and foods can up-regulate PTEN mRNA and protein expression in different cell lines, and they may be useful in the future prevention and/or treatment of these cancers. PTEN has also been observed to have prognostic significance and is gradually being accepted as an independent prognostic factor. This will help in monitoring disease progression and/or recurrence, with a view to improving treatment outcomes and reducing the associated morbidity and mortality from these cancers. Neprilysin (NEP is a zinc-dependent metallopeptidase that cleaves and inactivates some biologically active peptides thus switching off signal transduction at the cell surface. Decreased NEP expression in many cancers has been reported. NEP can form a complex with PTEN and enhance PTEN recruitment to the plasma membrane as well as stabilize its phosphatase activity. MicroRNA-21 (miR-21 post-transcriptionally down-regulates the expression of PTEN and stimulates growth and invasion in non-small cell lung cancer (NSCLC (lung Ca, suggesting that this may be a potential therapeutic target in the future treatment of NSCLC. PTEN is a tumor suppressor gene associated with many human cancers. This has diagnostic, therapeutic, and prognostic significance in the management of many human cancers, and may be a target for new drug development in the future.

  13. Use of serial analysis of gene expression to reveal the specific regulation of gene expression profile in asthmatic rats treated by acupuncture

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    Xu Yu-Dong

    2009-05-01

    Full Text Available Abstract Background Asthma has become an important public health issue and approximately 300 million people have suffered from the disease worldwide. Nowadays, the use of acupuncture in asthma is increasing. This study intended to systematically analyze and compare the gene expression profiles between the asthmatic and acupuncture-treated asthmatic rat lung, and tried to gain insight into the molecular mechanism underlying the early airway response (EAR phase of asthma treated by acupuncture. Methods Four tag libraries of serial analysis of gene expression (SAGE were established from lung tissues of control rats (CK, asthmatic rats (AS, asthmatic rats treated by acupuncture (ASAC, and control rats treated by acupuncture (CKAC. Bioinformatic analyses were carried out by using the methods including unsupervised hierarchical clustering, functional annotation tool of the database for annotation, visualization, and integrated discovery (DAVID, gene ontology (GO tree machine, and Kyoto encyclopedia of genes and genomes (KEGG pathway analysis. Results There were totally 186 differentially expressed tags (P CK/AS between the libraries of CK and AS, 130 differentially expressed tags between libraries of AS/ASAC (P AS/ASAC, and 144 differentially expressed tags between libraries of CK/CKAC (P CK/CKAC. The gene expression profiles of AS and ASAC were more similar than other libraries via unsupervised SAGE clustering. By comparison of PCK/AS and PAS/ASAC, the DAVID genes functional classification was found to be changed from "immune response" to "response to steroid hormone stimulus", and the GO term "antigen processing and presentation of peptide antigen" disappeared in PAS/ASAC. Totally 3 same KEGG pathways were found among the three groups. Moreover, 21 specific tags of the acupuncture in treating asthma were detected using Venn diagrams. Conclusion Our SAGE research indicates that the gene expression profile of the EAR phase of asthma could be

  14. Osmotic stress-dependent serine phosphorylation of the histidine kinase homologue DokA

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    Oehme Felix

    2001-03-01

    Full Text Available Abstract Background Two-component systems consisting of histidine kinases and their corresponding receivers are widespread in bacterial signal transduction. In the past few years, genes coding for homologues of two-component systems were also discovered in eukaryotic organisms. DokA, a homologue of bacterial histidine kinases, is an element of the osmoregulatory pathway in the amoeba Dictyostelium. The work described here addresses the question whether DokA is phosphorylated in vivo in response to osmotic stress. Results We have endogenously overexpressed individual domains of DokA to investigate post-translational modification of the protein in response to osmotic shock in vivo. Dictyostelium cells were labeled with [32P]-orthophosphate, exposed to osmotic stress and DokA fragments were subsequently isolated by immunoprecipitation. Thus, a stress-dependent phosphorylation could be demonstrated, with the site of phosphorylation being located in the kinase domain. We demonstrate biochemically that the phosphorylated amino acid is serine, and by mutational analysis that the phosphorylation reaction is not due to an autophosphorylation of DokA. Furthermore, mutation of the conserved histidine did not affect the osmostress-dependent phosphorylation reaction. Conclusions A stimulus-dependent serine phosphorylation of a eukaryotic histidine kinase homologue was demonstrated for the first time in vivo. That implies that DokA, although showing typical structural features of a bacterial two-component system, might be part of a eukaryotic signal transduction pathway that involves serine/threonine kinases.

  15. Arabidopsis thaliana CENTRORADIALIS homologue (ATC) acts systemically to inhibit floral initiation in Arabidopsis.

    Science.gov (United States)

    Huang, Nien-Chen; Jane, Wann-Neng; Chen, Jychian; Yu, Tien-Shin

    2012-10-01

    Floral initiation is orchestrated by systemic floral activators and inhibitors. This remote-control system may integrate environmental cues to modulate floral initiation. Recently, FLOWERING LOCUS T (FT) was found to be a florigen. However, the identity of systemic floral inhibitor or anti-florigen remains to be elucidated. Here we show that Arabidopsis thaliana CENTRORADIALIS homologue (ATC), an Arabidopsis FT homologue, may act in a non-cell autonomous manner to inhibit floral initiation. Analysis of the ATC null mutant revealed that ATC is a short-day-induced floral inhibitor. Cell type-specific expression showed that companion cells and apex that express ATC are sufficient to inhibit floral initiation. Histochemical analysis showed that the promoter activity of ATC was mainly found in vasculature but under the detection limit in apex, a finding that suggests that ATC may move from the vasculature to the apex to influence flowering. Consistent with this notion, Arabidopsis seedling grafting experiments demonstrated that ATC moved over a long distance and that floral inhibition by ATC is graft transmissible. ATC probably antagonizes FT activity, because both ATC and FT interact with FD and affect the same downstream meristem identity genes APETALA1, in an opposite manner. Thus, photoperiodic variations may trigger functionally opposite FT homologues to systemically influence floral initiation.

  16. Elements of lentiviral vector design toward gene therapy for treating mucopolysaccharidosis I

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    Li Ou

    2016-09-01

    Full Text Available Mucopolysaccharidosis type I (MPS I is a lysosomal disease caused by α-l-iduronidase (IDUA deficiency and accumulation of glycosaminoglycans (GAG. Lentiviral vector encoding correct IDUA cDNA could be used for treating MPS I. To optimize the lentiviral vector design, 9 constructs were designed by combinations of various promoters, enhancers, and codon optimization. After in vitro transfection into 293FT cells, 5 constructs achieved the highest IDUA activities (5613 to 7358 nmol/h/mg protein. These 5 candidate vectors were then tested by injection (1 × 107 TU/g into neonatal MPS I mice. After 30 days, one vector, CCEoIDW, achieved the highest IDUA levels: 2.6% of wildtype levels in the brain, 9.9% in the heart, 200% in the liver and 257% in the spleen. CCEoIDW achieved the most significant GAG reduction: down 49% in the brain, 98% in the heart, 100% in the liver and 95% in the spleen. Further, CCEoIDW had the lowest transgene frequency, especially in the gonads (0.03 ± 0.01 copies/100 cells, reducing the risk of insertional mutagenesis and germ-line transmission. Therefore, CCEoIDW is selected as the optimal lentiviral vector for treating MPS I disease and will be applied in large animal preclinical studies. Further, taken both in vitro and in vivo comparisons together, codon optimization, use of EF-1α promoter and woodchuck hepatitis virus posttranscriptional response element (WPRE could enhance transgene expression. These results provided a better understanding of factors contributing efficient transgene expression in lentiviral gene therapies.

  17. Bypassing hazard of housekeeping genes: Their evaluation in rat granule neurons treated with cerebrospinal fluid of Multiple Sclerosis subjects

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    Deepali eMathur

    2015-09-01

    Full Text Available Gene expression studies employing real-time PCR has become an intrinsic part of biomedical research. Appropriate normalization of target gene transcript(s based on stably expressed housekeeping genes is crucial in individual experimental conditions to obtain accurate results. In multiple sclerosis (MS, several gene expression studies have been undertaken, however, the suitability of housekeeping genes to express stably in this disease is not yet explored. Recent research suggests that their expression level may vary under different experimental conditions. Hence it is indispensible to evaluate their expression stability to accurately normalize target gene transcripts. The present study aims to evaluate the expression stability of seven housekeeping genes in rat granule neurons treated with cerebrospinal fluid of MS patients. The selected reference genes were quantified by real time PCR and their expression stability was assessed using GeNorm and NormFinder algorithms. Both methods reported transferrin receptor (Tfrc and microglobulin beta-2 (B2m the most stable genes whereas beta-actin (ActB and glyceraldehyde-3-phosphate-dehydrogenase (Gapdh the most fluctuated ones. Altogether our data demonstrate the significance of pre-validation of housekeeping genes for accurate normalization and indicates Tfrc and B2m as best endogenous controls in MS. ActB and Gapdh are not recommended in gene expression studies related to the current one.

  18. 含有额外拷贝黄曲霉cyp51同源基因的烟曲霉对抗真菌药物的敏感性测定%Antifungal susceptibility of the A.fumigatus transformants containing extra copies of A.flavus cyp51 gene homologues to the common antifungal drugs

    Institute of Scientific and Technical Information of China (English)

    刘伟霞; 孙毅; 万喆; 李若瑜; 刘伟

    2011-01-01

    Objective To investigate the effect of Aspergillus flavus cyp51 genes on antifungal susceptibility by cloning and constucting the extra copies of Aspergillus flavus cyp51 genes. Methods A.flavus cyp5l gene homologues were identified by tblastn searches inA. flavus genome database. PCR fragments composed of the 5'flanking sequence (approximately 1 000 bp) ofcyp51 ,cyp51 ORF, and its 3'flanking sequence (approximately 1 000 bp), were subcloned into shuttle plasmid pRG3-AMAl-NotI to produce recombinant plasmids. These plasmids and empty plasmid pRG3-AMA1-Notl were transformed into A.fumigatus strain AF293.1 (pyrG-) respectively to produce transformants. The Clinical Laboratory Standard Institute broth microdilution method M38-A2 and E-test method were used to assay the minimal inhibitory concentrations (MICs) of itraconazole ( ITC), voriconazole ( VRC), amphotericin B (AMB), and posaconazole (POS), or minimal effect concentration (MEC) of caspofungin (CAS), against these transformants. Results A. flavus genome contains three cyp51 gene homologues, cyp51A ,cyp51B and cyp51 C, of which the ORF size are 1 400-2 000 bp. When these genes were subcloned into shuttle plasmid pRG3-AMA1-NotI, we get plasmids pRG3-AMA1-CYP51 A, pRG3-AMA1-CYP51B and pRG3-AMA1-CYP51C. These plasmids and empty plasmid were transformed into A.fumigatus strain AF293.1 (pyrG-) to produce transformants rCYP51A, rCYP51B, rCYP51C and rpRG. The antifungal susceptibility of these A.fumigatus transformants to the antifungal drugs by broth microdilution assaying and E-test method showed that, rCYP51A and rCYP51B were cross-resistant to VRC and ITC, susceptible to both AM B and CAS; rCYP51C and rpRG were intermediate to ITC and VRC, susceptible to both A MB and CAS. Conclusion In A. fumigatus , extra copies of A.flavus ' cyp51A gene or cyp51B gene have effect on antifungal susceptibility to azoles, have no effect on AMB and CAS. Extra copy ofcyp51C has no obvious effect on all the tested drugs.%目的

  19. Hepatic insulin gene therapy prevents diabetic enteropathy in STZ-treated CD-1 mice

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    Shuo You

    2015-01-01

    Full Text Available Depending on the population examined, from 6 to 83% of people with diabetes mellitus exhibit symptoms of altered gut motility, manifesting as dysphagia, reflux, early satiety, nausea, abdominal pain, diarrhea, or constipation. Hyperglycemia-induced cell loss within the enteric nervous system has been demonstrated in both diabetic rodents and patients with diabetes. Glycemic control is recommended to prevent diabetic gastroenteropathy but is often difficult to achieve with current treatment modalities. We asked if hepatic insulin gene therapy (HIGT could inhibit the development of diabetic gastroenteropathy in mice. Bowel length, bowel transit, colonic muscle relaxation, and the numbers of both stimulatory and inhibitory neurons in the colonic myenteric plexus were compared in groups of diabetic mice (DM, control nondiabetic mice (Con, and diabetic mice treated with HIGT (HIGT. Delivery of a metabolically responsive insulin transgene to the liver of STZ-diabetic mice with an adeno-associated virus, sero-type 8 (AAV8 produced near-normal blood sugars for over 1 month and prevented anatomic, functional, and neurohistologic changes observed in diabetic mice. We conclude that in addition to normalizing oxidative metabolism in diabetic rodents, HIGT is sufficient to prevent the development of diabetic gastroenteropathy.

  20. Gene Expression Profiling of IL-17A-Treated Synovial Fibroblasts from the Human Temporomandibular Joint

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    Toshio Hattori

    2015-01-01

    Full Text Available Synovial fibroblasts contribute to the inflammatory temporomandibular joint under pathogenic stimuli. Synovial fibroblasts and T cells participate in the perpetuation of joint inflammation in a mutual activation feedback, via secretion of cytokines and chemokines that stimulate each other. IL-17 is an inflammatory cytokine produced primarily by Th17 cells which plays critical role in the pathogenesis of numerous autoimmune and inflammatory diseases. Here, we investigated the roles of IL-17A in temporomandibular joint disorders (TMD using genome-wide analysis of synovial fibroblasts isolated from patients with TMD. IL-17 receptors were expressed in synovial fibroblasts as assessed using real-time PCR. Microarray analysis indicated that IL-17A treatment of synovial fibroblasts upregulated the expression of IL-6 and chemokines. Real-time PCR analysis showed that the gene expression of IL-6, CXCL1, IL-8, and CCL20 was significantly higher in IL-17A-treated synovial fibroblasts compared to nontreated controls. IL-6 protein production was increased by IL-17A in a time- and a dose-dependent manner. Additionally, IL-17A simulated IL-6 protein production in synovial fibroblasts samples isolated from three patients. Furthermore, signal inhibitor experiments indicated that IL-17-mediated induction of IL-6 was transduced via activation of NFκB and phosphatidylinositol 3-kinase/Akt. These results suggest that IL-17A is associated with the inflammatory progression of TMD.

  1. Gene-gene interactions of the INSIG1 and INSIG2 in metabolic syndrome in schizophrenic patients treated with atypical antipsychotics.

    Science.gov (United States)

    Liou, Y-J; Bai, Y M; Lin, E; Chen, J-Y; Chen, T-T; Hong, C-J; Tsai, S-J

    2012-02-01

    The use of atypical antipsychotics (AAPs) is associated with increasing the risk of the metabolic syndrome (MetS), which is an important risk factor for cardiovascular disease and diabetes. Two insulin-induced gene (INSIG) isoforms, designated INSIG-1 and INSIG-2 encode two proteins that mediate feedback control of lipid metabolism. In this genetic case-control study, we investigated whether the common variants in INSIG1 and INSIG2 genes were associated with MetS in schizophrenic patients treated with atypical antipsychctics. The study included 456 schizophrenia patients treated with clozapine (n=171), olanzapine (n=91) and risperidone (n=194), for an average of 45.5±27.6 months. The prevalence of MetS among all subjects was 22.8% (104/456). Two single-nucleotide polymorphisms (SNPs) of the INSIG1 gene and seven SNPs of the INSIG2 gene were chosen as haplotype-tagging SNPs. In single-marker-based analysis, the INSIG2 rs11123469-C homozygous genotype was found to be more frequent in the patients with MetS than those without MetS (P=0.001). In addition, haplotype analysis showed that the C-C-C haplotype of rs11123469-rs10185316- rs1559509 of the INSIG2 gene significantly increased the risk of MetS (P=0.0023). No significant associations were found between polymorphisms of INSIG1 gene and MetS, however, INSIG1 and INSIG2 interactions were found in the significant 3-locus and 4-locus gene-gene interaction models (P=0.003 and 0.012, respectively). The results suggest that the INSIG2 gene may be associated with MetS in patients treated with AAPs independently or in an interactive manner with INSIG1.

  2. NBS-LRR resistance gene homologues in rice

    Institute of Scientific and Technical Information of China (English)

    ZHENG; Xianwu

    2001-01-01

    [1]Henglein, A., Small-particle research: physical-chemical properties of extremely small colloidal metal and semiconductor particles, Chem. Rev., 1989, 89: 1861-1873.[2]Serpone, N., Pelizzetti, E., eds., Photocatalysis-fundamentals and applications, New York: Wiley, 1989.[3]Kamat, P. V., Photoelectrochemistry in particulate systems. 9. Photosensitized reduction in a colloidal TiO2 system using anthracene-9-carboxylic acid as the sensitizer, J. Phys. Chem., 1989, 93: 859-864.[4]Usami, A., Theoretical study of charge transportation in dye-sensitized nanocrystalline TiO2 electrodes, Chemical Physics Letters, 1998, 292: 223-228.[5]Jiang Lijin, Structures, properties, photochemistry reactions and its mechanisms (I)--The structures and properties of hypocrellins, Chinese Science Bulletin, 1990, 35(21): 1608-1616.[6]Pivera, J. L., Gracia-jimenez, J. M., Silva Gonzalez, R. et al., Characterization of thin CdS films grown by the gradient re-crystallization and growth technique, J. Appl. Phys., 1990, 68(3): 1375-1377.[7]Henglein, A., Mechanism of reactions on colloidal microelectrodes and size quantization effects, Topics in Current Chemistry, 1988, 143: 113-180.[8]Kamat, P. V., Chanvet, J. P., Fessenden, R. W., Photoelectrochemistry in particulate systems. 4. Photosensitization of a TiO2 semiconductor with a chlorophyll analogue, J. Phys. Chem., 1986, 90: 1389-1394.[9]Rossetti, R., Brus, L. E., Time-resolved Raman scattering study of adsorbed, semiosidized Eosin Y formed by excited-stated electron transfer into colloidal TiO2 particles, J. Am. Chem. Soc., 1984, 106: 4336-4340.[10]Yang, J. J., Feng, L. B., Zhang, Z. J. et al., Study of photoreduction of MV2+ taking place on the surface of nanosized CdS particle, Photographic Science and Photochemistry (in Chinese), 1995, 13(3): 227-233.[11]He, H. Z., Jiang, X. F., Wang, D. Y., The photophysical characters of hypocrellin A in the capsule solution, Photographic Science and Photochemistry (in Chinese), 1992, 10(3): 193-199.[12]Diwu Zhenjun, William, L. J., Photosensitization by anticancer agents. 12. Perylene quinonoid pigments, a novel type of singlet oxygen sensitizer, J. Photochem Photobiol A: Chem., 1992, 64: 273-287.[13]Ira N Leveine, Physics Chemistry, 2nd ed., New York: McGraw-Hill, 1983.[14]Wang Nenghui, Zhang, Z. Y., Relationship between photosensitizing activities and chemical structure of Hypocrellin A and B, J. Photochem. Photobiol. B: Biol., 1992, 14: 207-217.[15]Zhang, M. H., Weng, M., Chen, S. et al., Study of electron transfer interaction between hypocrellin and N,N-diethylaniline by UV-visible, fluorescence, electron spin resonance spectra and time-resolved transient absorption spectra, J. Photochem. Photobiol. A: Chem., 1996, 96: 57-63.[16]Xia, W. L., Zhang, M. H., Jiang, L. J. et al., Study of hypocrellin A by nanosecond transient absorption spectra, Science in China (in Chinese), Ser. B, 1992, (3): 230-235.

  3. The OxyR homologue in Tannerella forsythia regulates expression of oxidative stress responses and biofilm formation.

    Science.gov (United States)

    Honma, Kiyonobu; Mishima, Elina; Inagaki, Satoru; Sharma, Ashu

    2009-06-01

    Tannerella forsythia is an anaerobic periodontal pathogen that encounters constant oxidative stress in the human oral cavity due to exposure to air and reactive oxidative species from coexisting dental plaque bacteria as well as leukocytes. In this study, we sought to characterize a T. forsythia ORF with close similarity to bacterial oxidative stress response sensor protein OxyR. To analyse the role of this OxyR homologue, a gene deletion mutant was constructed and characterized. Aerotolerance, survival after hydrogen peroxide challenge and transcription levels of known bacterial antioxidant genes were then determined. Since an association between oxidative stress and biofilm formation has been observed in bacterial systems, we also investigated the role of the OxyR protein in biofilm development by T. forsythia. Our results showed that aerotolerance, sensitivity to peroxide challenge and the expression of oxidative stress response genes were significantly reduced in the mutant as compared with the wild-type strain. Moreover, the results of biofilm analyses showed that, as compared with the wild-type strain, the oxyR mutant showed significantly less autoaggregation and a reduced ability to form mixed biofilms with Fusobacterium nucleatum. In conclusion, a gene annotated in the T. forsythia genome as an oxyR homologue was characterized. Our studies showed that the oxyR homologue in T. forsythia constitutively activates antioxidant genes involved in resistance to peroxides as well as oxygen stress (aerotolerance). In addition, the oxyR deletion attenuates biofilm formation in T. forsythia.

  4. Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa.

    Science.gov (United States)

    Beltran, William A; Cideciyan, Artur V; Lewin, Alfred S; Iwabe, Simone; Khanna, Hemant; Sumaroka, Alexander; Chiodo, Vince A; Fajardo, Diego S; Román, Alejandro J; Deng, Wen-Tao; Swider, Malgorzata; Alemán, Tomas S; Boye, Sanford L; Genini, Sem; Swaroop, Anand; Hauswirth, William W; Jacobson, Samuel G; Aguirre, Gustavo D

    2012-02-07

    Hereditary retinal blindness is caused by mutations in genes expressed in photoreceptors or retinal pigment epithelium. Gene therapy in mouse and dog models of a primary retinal pigment epithelium disease has already been translated to human clinical trials with encouraging results. Treatment for common primary photoreceptor blindness, however, has not yet moved from proof of concept to the clinic. We evaluated gene augmentation therapy in two blinding canine photoreceptor diseases that model the common X-linked form of retinitis pigmentosa caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, which encodes a photoreceptor ciliary protein, and provide evidence that the therapy is effective. After subretinal injections of adeno-associated virus-2/5-vectored human RPGR with human IRBP or GRK1 promoters, in vivo imaging showed preserved photoreceptor nuclei and inner/outer segments that were limited to treated areas. Both rod and cone photoreceptor function were greater in treated (three of four) than in control eyes. Histopathology indicated normal photoreceptor structure and reversal of opsin mislocalization in treated areas expressing human RPGR protein in rods and cones. Postreceptoral remodeling was also corrected: there was reversal of bipolar cell dendrite retraction evident with bipolar cell markers and preservation of outer plexiform layer thickness. Efficacy of gene therapy in these large animal models of X-linked retinitis pigmentosa provides a path for translation to human treatment.

  5. Citrus nobiletin suppresses inducible nitric oxide synthase gene expression in interleukin-1β-treated hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshigai, Emi [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Machida, Toru [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Okuyama, Tetsuya [Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Mori, Masatoshi; Murase, Hiromitsu; Yamanishi, Ryota [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Okumura, Tadayoshi [Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Shiga (Japan); Department of Surgery, Kansai Medical University, Hirakata, Osaka (Japan); Ikeya, Yukinobu [Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga (Japan); Nishino, Hoyoku [Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto (Japan); Nishizawa, Mikio, E-mail: nishizaw@sk.ritsumei.ac.jp [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan)

    2013-09-13

    Highlights: •Nobiletin is a polymethoxylated flavone that is abundant in citrus peels. •Nobiletin is a major constituent of the Citrus unshiu peel extract. •Nobiletin suppresses induction of NO and reduces iNOS expression in hepatocytes. •Nobiletin reduces the iNOS promoter activity and the DNA-binding activity of NF-κB. -- Abstract: Background: Nobiletin is a polymethoxylated flavone that is abundant in the peels of citrus fruits, such as Citrus unshiu (Satsuma mandarin) and Citrus sinensis. The dried peels of C. unshiu (chinpi) have been included in several formulae of Japanese Kampo medicines. Nobiletin may suppress the induction of inducible nitric oxide synthase (iNOS), which synthesizes the inflammatory mediator nitric oxide (NO) in hepatocytes. Methods: A C. unshiu peel (CUP) extract was prepared. Primary cultured rat hepatocytes were treated with the CUP extract or nobiletin in the presence of interleukin 1β (IL-1β), which induces iNOS expression. NO production and iNOS gene expression were analyzed. Results: High-performance liquid chromatography analyses revealed that the nobiletin content in the CUP extract was 0.14%. Nobiletin dose-dependently reduced the NO levels and decreased iNOS expression at the protein, mRNA and antisense transcript levels. Flavone, which does not contain any methoxy groups, also suppressed iNOS induction. Nobiletin reduced the transcriptional activity of iNOS promoter-luciferase constructs and the DNA-binding activity of nuclear factor κB (NF-κB) in the nuclei. Conclusions: The suppression of iNOS induction by nobiletin suggests that nobiletin may be responsible for the anti-inflammatory effects of citrus peels and have a therapeutic potential for liver diseases.

  6. Enhanced food intake by progesterone-treated female rats is related to changes in neuropeptide genes expression in hypothalamus.

    Science.gov (United States)

    Stelmańska, Ewa; Sucajtys-Szulc, Elżbieta

    2014-01-01

    Progesterone-treated females eat more food, but the mechanism underlying this effect is not well understood. The aim of the study was to analyse the effect of progesterone on neuropeptide genes expression in rat hypothalamus. Experiments were carried out on female and male Wistar rats. Animals were treated with progesterone (100 mg per rat) for 28 days. NPY and CART mRNA levels in hypothalamus were quantified by real-time PCR. The serum progesterone concentration was determined by radioimmunoassay. Progesterone administration to females caused an increase in food intake, body mass, and white adipose tissue mass. Elevated circulating progesterone concentration up-regulated NPY and down-regulated CART genes expression in hypothalamus of females. In males, elevated blood progesterone concentration had no effect on food intake, body and fat mass and on the neuropeptide genes expression in hypothalamus. Moreover, administration of progesterone in females resulted in decrease of PR mRNA level in hypothalamus. No effect of progesterone administration on PR mRNA level in hypothalamus of males was found. The changes in neuropeptide genes expression in hypothalamus may lead to stimulation of appetite and might explain the observed increase in food intake, body and adipose tissue mass in progesterone-treated females.

  7. Prediction of key genes in ovarian cancer treated with decitabine based on network strategy.

    Science.gov (United States)

    Wang, Yu-Zhen; Qiu, Sheng-Chun

    2016-06-01

    The objective of the present study was to predict key genes in ovarian cancer before and after treatment with decitabine utilizing a network approach and to reveal the molecular mechanism. Pathogenic networks of ovarian cancer before and after treatment were identified based on known pathogenic genes (seed genes) and differentially expressed genes (DEGs) detected by Significance Analysis of Microarrays (SAM) method. A weight was assigned to each gene in the pathogenic network and then candidate genes were evaluated. Topological properties (degree, betweenness, closeness and stress) of candidate genes were analyzed to investigate more confident pathogenic genes. Pathway enrichment analysis for candidate and seed genes were conducted. Validation of candidate gene expression in ovarian cancer was performed by reverse transcriptase-polymerase chain reaction (RT-PCR) assays. There were 73 nodes and 147 interactions in the pathogenic network before treatment, while 47 nodes and 66 interactions after treatment. A total of 32 candidate genes were identified in the before treatment group of ovarian cancer, of which 16 were rightly candidate genes after treatment and the others were silenced. We obtained 5 key genes (PIK3R2, CCNB1, IL2, IL1B and CDC6) for decitabine treatment that were validated by RT-PCR. In conclusion, we successfully identified 5 key genes (PIK3R2, CCNB1, IL2, IL1B and CDC6) and validated them, which provides insight into the molecular mechanisms of decitabine treatment and may be potential pathogenic biomarkers for the therapy of ovarian cancer.

  8. The TFL1 homologue KSN is a regulator of continuous flowering in rose and strawberry.

    Science.gov (United States)

    Iwata, Hikaru; Gaston, Amèlia; Remay, Arnaud; Thouroude, Tatiana; Jeauffre, Julien; Kawamura, Koji; Oyant, Laurence Hibrand-Saint; Araki, Takashi; Denoyes, Béatrice; Foucher, Fabrice

    2012-01-01

    Flowering is a key event in plant life, and is finely tuned by environmental and endogenous signals to adapt to different environments. In horticulture, continuous flowering (CF) is a popular trait introduced in a wide range of cultivated varieties. It played an essential role in the tremendous success of modern roses and woodland strawberries in gardens. CF genotypes flower during all favourable seasons, whereas once-flowering (OF) genotypes only flower in spring. Here we show that in rose and strawberry continuous flowering is controlled by orthologous genes of the TERMINAL FLOWER 1 (TFL1) family. In rose, six independent pairs of CF/OF mutants differ in the presence of a retrotransposon in the second intron of the TFL1 homologue. Because of an insertion of the retrotransposon, transcription of the gene is blocked in CF roses and the absence of the floral repressor provokes continuous blooming. In OF-climbing mutants, the retrotransposon has recombined to give an allele bearing only the long terminal repeat element, thus restoring a functional allele. In OF roses, seasonal regulation of the TFL1 homologue may explain the seasonal flowering, with low expression in spring to allow the first bloom. In woodland strawberry, Fragaria vesca, a 2-bp deletion in the coding region of the TFL1 homologue introduces a frame shift and is responsible for CF behaviour. A diversity analysis has revealed that this deletion is always associated with the CF phenotype. Our results demonstrate a new role of TFL1 in perennial plants in maintaining vegetative growth and modifying flowering seasonality.

  9. Time Dependent Gene Expression Changes in the Liver of Mice Treated with Benzene

    Directory of Open Access Journals (Sweden)

    S.V.S. Rana

    2008-01-01

    Full Text Available Benzene is used as a general purpose solvent. Benzene metabolism starts from phenol and ends with p-benzoquinone and o-benzoquinone. Liver injury inducted by benzene still remains a toxicologic problem. Tumor related genes and immune responsive genes have been studied in patients suffering from benzene exposure. However, gene expression profiles and pathways related to its hepatotoxicity are not known. This study reports the results obtained in the liver of BALB/C mice (SLC, Inc., Japan administered 0.05 ml/100 g body weight of 2% benzene for six days. Serum, ALT, AST and ALP were determined using automated analyzer (Fuji., Japan. Histopathological observations were made to support gene expression data. c-DNA microarray analyses were performed using Affymetrix Gene-chip system. After six days of benzene exposure, twenty five genes were down regulated whereas nineteen genes were up-regulated. These gene expression changes were found to be related to pathways of biotransformation, detoxification, apoptosis, oxidative stress and cell cycle. It has been shown for the first time that genes corresponding to circadian rhythms are affected by benzene. Results suggest that gene expression profile might serve as potential biomarkers of hepatotoxicity during benzene exposure.

  10. Digital gene expression profiling (DGE) of cadmium-treated Drosophila melanogaster.

    Science.gov (United States)

    Guan, Delong; Mo, Fei; Han, Yan; Gu, Wei; Zhang, Min

    2015-01-01

    Cadmium is highly toxic and can cause oxidative damage, metabolic disorders, and reduced lifespan and fertility in animals. In this study, we investigated the effects of cadmium in Drosophila melanogaster, performing transcriptome analysis by using tag-based digital gene expression (DGE) profiling. Among 1970 candidate genes, 1443 were up-regulated and 527 were down-regulated following cadmium exposure. Using Gene Ontology analysis, we found that cadmium stress affects three processes: transferase activity, stress response, and the cell cycle. Furthermore, we identified five differentially expressed genes (confirmed by real-time PCR) involved in all three processes: Ald, Cdc2, skpA, tefu, and Pvr. Pathway analysis revealed that these genes were involved in the cell cycle pathway and fat digestion and absorption pathway. This study reveals the gene expression response to cadmium stress in Drosophila, it provides insights into the mechanisms of this response, and it could contribute to our understanding of cadmium toxicity in humans.

  11. Modulation of DNA methylation and gene expression in cultured sycamore cells treated by hypomethylating base analog.

    Science.gov (United States)

    Ngernprasirtsiri, J; Akazawa, T

    1990-12-12

    The selective suppression of photosynthetic genes in both the nuclear and plastid genomes of the nonphotosynthetic white wild-type cell line of sycamore (Acer pseudoplatanus) has been found to be inversely related to the presence of a variety of methylated bases, especially 5-methylcytosine (5-MeCyt) and N6-methyladenine (N6-MeAde), localized in regions of the plastid genome containing silent genes. We used hypomethylating base analogs to manipulate the level of cytosine and adenine methylation in the white cells of sycamore, and examined the effects of changes in methylation on gene expression. Treatment with 5-azacytidine (5-AzaCyd) and N6-benzyladenine (N6-BzlAde) decreased cytosine and adenine methylation. This was accompanied by restoration of transcriptional activity in photosynthetic genes which are usually suppressed. Both 5-MeCyt and N6-MeAde suppressed nuclear gene expression, but only 5-MeCyt suppressed plastid gene expression.

  12. Xenopus BTBD6 and its Drosophila homologue lute are required for neuronal development.

    Science.gov (United States)

    Bury, Frédéric J; Moers, Virginie; Yan, Jiekun; Souopgui, Jacob; Quan, Xiao-Jiang; De Geest, Natalie; Kricha, Sadia; Hassan, Bassem A; Bellefroid, Eric J

    2008-11-01

    BBP proteins constitute a subclass of CUL3 interacting BTB proteins whose in vivo function remains unknown. Here, we show that the Xenopus BBP gene BTBD6 and the single Drosophila homologue of mammalian BBP genes lute are strongly expressed in the developing nervous system. In Xenopus, BTBD6 expression responds positively to proneural and negatively to neurogenic gene overexpression. Knockdown of BTBD6 in Xenopus or loss of Drosophila lute result in embryos with strong defects in late neuronal markers and strongly reduced and disorganized axons while early neural development is unaffected. XBTBD6 knockdown in Xenopus also affects muscle development. Together, these data indicate that BTBD6/lute is required for proper embryogenesis and plays an essential evolutionary conserved role during neuronal development.

  13. Altered Gene Transcription in Human Cells Treated with Ludox® Silica Nanoparticles

    Directory of Open Access Journals (Sweden)

    Caterina Fede

    2014-08-01

    Full Text Available Silica (SiO2 nanoparticles (NPs have found extensive applications in industrial manufacturing, biomedical and biotechnological fields. Therefore, the increasing exposure to such ultrafine particles requires studies to characterize their potential cytotoxic effects in order to provide exhaustive information to assess the impact of nanomaterials on human health. The understanding of the biological processes involved in the development and maintenance of a variety of pathologies is improved by genome-wide approaches, and in this context, gene set analysis has emerged as a fundamental tool for the interpretation of the results. In this work we show how the use of a combination of gene-by-gene and gene set analyses can enhance the interpretation of results of in vitro treatment of A549 cells with Ludox® colloidal amorphous silica nanoparticles. By gene-by-gene and gene set analyses, we evidenced a specific cell response in relation to NPs size and elapsed time after treatment, with the smaller NPs (SM30 having higher impact on inflammatory and apoptosis processes than the bigger ones. Apoptotic process appeared to be activated by the up-regulation of the initiator genes TNFa and IL1b and by ATM. Moreover, our analyses evidenced that cell treatment with LudoxÒ silica nanoparticles activated the matrix metalloproteinase genes MMP1, MMP10 and MMP9. The information derived from this study can be informative about the cytotoxicity of Ludox® and other similar colloidal amorphous silica NPs prepared by solution processes.

  14. Altered gene transcription in human cells treated with Ludox® silica nanoparticles.

    Science.gov (United States)

    Fede, Caterina; Millino, Caterina; Pacchioni, Beniamina; Celegato, Barbara; Compagnin, Chiara; Martini, Paolo; Selvestrel, Francesco; Mancin, Fabrizio; Celotti, Lucia; Lanfranchi, Gerolamo; Mognato, Maddalena; Cagnin, Stefano

    2014-08-28

    Silica (SiO2) nanoparticles (NPs) have found extensive applications in industrial manufacturing, biomedical and biotechnological fields. Therefore, the increasing exposure to such ultrafine particles requires studies to characterize their potential cytotoxic effects in order to provide exhaustive information to assess the impact of nanomaterials on human health. The understanding of the biological processes involved in the development and maintenance of a variety of pathologies is improved by genome-wide approaches, and in this context, gene set analysis has emerged as a fundamental tool for the interpretation of the results. In this work we show how the use of a combination of gene-by-gene and gene set analyses can enhance the interpretation of results of in vitro treatment of A549 cells with Ludox® colloidal amorphous silica nanoparticles. By gene-by-gene and gene set analyses, we evidenced a specific cell response in relation to NPs size and elapsed time after treatment, with the smaller NPs (SM30) having higher impact on inflammatory and apoptosis processes than the bigger ones. Apoptotic process appeared to be activated by the up-regulation of the initiator genes TNFa and IL1b and by ATM. Moreover, our analyses evidenced that cell treatment with LudoxÒ silica nanoparticles activated the matrix metalloproteinase genes MMP1, MMP10 and MMP9. The information derived from this study can be informative about the cytotoxicity of Ludox® and other similar colloidal amorphous silica NPs prepared by solution processes.

  15. Isolation and characterization of AtMLH1, a MutL homologue from Arabidopsis thaliana.

    Science.gov (United States)

    Jean, M; Pelletier, J; Hilpert, M; Belzile, F; Kunze, R

    1999-12-01

    DNA mismatch repair systems play an essential role in the maintenance of genetic information in living organisms and are also implicated in genetic recombination and genome stability. Using degenerate primers, we have cloned the first plant homologue of the E. coli MutL gene, which we have called AtMLH1 for Arabidopsis thaliana MutL-homologue 1. AtMLH1 is present as a single-copy gene in the Arabidopsis genome and is located on the top arm of chromosome 4. Sequence analysis revealed that the product of this gene shows extensive sequence homology with other eukaryotic MLH1 proteins. As mlh1-deficient lines would be useful for studying the biological function of this gene, several populations that had been mutagenized using T-DNA and transposon insertions were screened to identify such mutants. One line that carries a T-DNA insertion in the promoter region of the AtMLH1 gene was isolated. Surprisingly, although the insertion occurred only approximately 80 bp upstream of the putative transcription start site, Northern analyses revealed very low but similar amounts of AtMLH1 transcript in both the wild type and the T-DNA insertion lines. RT-PCR analyses suggest, however, that transcription is initiated further upstream in the insertion line and that the T-DNA may supply this novel initiation site. Finally, no increase in microsatellite instability - a phenotype often associated with mutations in mismatch repair genes - was observed in plants homozygous for this insertion.

  16. Cloning and characterization of Arabidopsis thaliana AtNAP57--a homologue of yeast pseudouridine synthase Cbf5p.

    Science.gov (United States)

    Maceluch, J; Kmieciak, M; Szweykowska-Kulińska, Z; Jarmołowski, A

    2001-01-01

    Rat Nap57 and its yeast homologue Cbf5p are pseudouridine synthases involved in rRNA biogenesis, localized in the nucleolus. These proteins, together with H/ACA class of snoRNAs compose snoRNP particles, in which snoRNA guides the synthase to direct site-specific pseudouridylation of rRNA. In this paper we present an Arabidopsis thaliana protein that is highly homologous to Cbf5p (72% identity and 85% homology) and NAP57 (67% identity and 81% homology). Moreover, the plant protein has conserved structural motifs that are characteristic features of pseudouridine synthases of the TruB class. We have named the cloned and characterized protein AtNAP57 (Arabidopsis thaliana homologue of NAP57). AtNAP57 is a 565 amino-acid protein and its calculated molecular mass is 63 kDa. The protein is encoded by a single copy gene located on chromosome 3 of the A. thaliana genome. Interestingly, the AtNAP57 gene does not contain any introns. Mutations in the human DKC1 gene encoding dyskerin (human homologue of yeast Cbf5p and rat NAP57) cause dyskeratosis congenita a rare inherited bone marrow failure syndrome characterized by abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia.

  17. Pescadillo homologue 1 and Peter Pan function during Xenopus laevis pronephros development.

    Science.gov (United States)

    Tecza, Aleksandra; Bugner, Verena; Kühl, Michael; Kühl, Susanne J

    2011-10-01

    pes1 (pescadillo homologue 1) and ppan (Peter Pan) are multifunctional proteins involved in ribosome biogenesis, cell proliferation, apoptosis, cell migration and regulation of gene expression. Both proteins are required for early neural development in Xenopus laevis, as previously demonstrated. We show that the expression of both genes in the developing pronephros depends on wnt4 and fzd3 (frizzled homologue 3) function. Loss of pes1 or ppan by MO (morpholino oligonucleotide)-based knockdown approaches resulted in strong malformations during pronephric tubule formation. Defects were already notable during specification of pronephric progenitor cells, as shown by lhx1 expression. Moreover, we demonstrated that Xenopus pes1 and ppan interact physically and functionally and that pes1 and ppan can cross-rescue the loss of function phenotype of one another. Interference with rRNA synthesis, however, did not result in a similar early pronephros phenotype. These results demonstrate that pes1 and ppan are required for Xenopus pronephros development and indicate that their function in the pronephros is independent of their role in ribosome biosynthesis.

  18. Removal of bacterial cells, antibiotic resistance genes and integrase genes by on-site hospital wastewater treatment plants: surveillance of treated hospital effluent quality

    KAUST Repository

    Timraz, Kenda

    2016-12-15

    This study aims to evaluate the removal efficiency of microbial contaminants, including total cell counts, antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs, e.g. tetO, tetZ, sul1 and sul2) and integrase genes (e.g. intl1 and intl2), by wastewater treatment plants (WWTPs) operated on-site of two hospitals (i.e., SH WWTP and IH WWTP). Both SH and IH WWTPs utilize the conventional activated sludge process but differences in the removal efficiencies were observed. Over the 2 week sampling period, IH WWTP outperformed SH WWTP, and achieved an approximate 0.388 to 2.49-log log removal values (LRVs) for total cell counts compared to the 0.010 to 0.162-log removal in SH WWTP. Although ARB were present in the hospital influent, the treatment process of both hospitals effectively removed ARB from most of the effluent samples. In instances where ARB were recovered in the effluent, none of the viable isolates were identified to be opportunistic pathogenic species based on 16S rRNA gene sequencing. However, sul1 and intl1 genes remained detectable at up to 105 copies per mL or 8 x 10(-1) copies per 16S rRNA gene in the treated effluent, with an LRV of less than 1.2. When the treated effluent is discharged from hospital WWTPs into the public sewer for further treatment as per requirement in many countries, the detected amount of ARGs and integrase genes in the hospital effluent can become a potential source of horizontal gene dissemination in the municipal WWTP. Proper on-site wastewater treatment and surveillance of the effluent quality for emerging contaminants are therefore highly recommended.

  19. Estrogen receptor alpha augments changes in hemostatic gene expression in HepG2 cells treated with estradiol and phytoestrogens.

    Science.gov (United States)

    Kelly, Lynne A; Seidlova-Wuttke, Dana; Wuttke, Wolfgang; O'Leary, John J; Norris, Lucy A

    2014-01-15

    Phytoestrogens are popular alternatives to estrogen therapy however their effects on hemostasis in post-menopausal women are unknown. The aim of this study was to determine the effect of the phytoestrogens, genistein, daidzein and equol on the expression of key genes from the hemostatic system in human hepatocyte cell models and to determine the role of estrogen receptors in mediating any response seen. HepG2 cells and Hep89 cells (expressing estrogen receptor alpha (ERα)) were incubated for 24 h with 50 nM 17β-estradiol, genistein, daidzein or equol. Tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), Factor VII, fibrinogen γ, protein C and protein S mRNA expression were determined using TaqMan PCR. Genistein and equol increased tPA and PAI-1 expression in Hep89 cells with fold changes greater than those observed for estradiol. In HepG2 cells (which do not express ERα), PAI-1 and tPA expression were unchanged. Increased expression of Factor VII was observed in phytoestrogen treated Hep89 cells but not in similarly treated HepG2s. Prothrombin gene expression was increased in equol and daidzein treated HepG2 cells in the absence of the classical estrogen receptors. These data suggest that phytoestrogens can regulate the expression of coagulation and fibrinolytic genes in a human hepatocyte cell line; an effect which is augmented by ERα.

  20. Gene expression alterations associated with outcome in aromatase inhibitor-treated ER+ early-stage breast cancer patients.

    Science.gov (United States)

    Thomsen, Karina G; Lyng, Maria B; Elias, Daniel; Vever, Henriette; Knoop, Ann S; Lykkesfeldt, Anne E; Lænkholm, Anne-Vibeke; Ditzel, Henrik J

    2015-12-01

    Aromatase inhibitors (AI), either alone or together with chemotherapy, have become the standard adjuvant treatment for postmenopausal, estrogen receptor-positive (ER+) breast cancer. Although AIs improve overall survival, resistance is still a major clinical problem, thus additional biomarkers predictive of outcome of ER+ breast cancer patients treated with AIs are needed. Global gene expression analysis was performed on ER+ primary breast cancers from patients treated with adjuvant AI monotherapy; half experienced recurrence (median follow-up 6.7 years). Gene expression alterations were validated by qRT-PCR, and functional studies evaluating the effect of siRNA-mediated gene knockdown on cell growth were performed. Twenty-six genes, including TFF3, DACH1, RGS5, and GHR, were shown to exhibit altered expression in tumors from patients with recurrence versus non-recurrent (fold change ≥1.5, p proliferation, growth, and development. TFF3, which encodes for trefoil factor 3 and is an estrogen-responsive oncogene shown to play a functional role in tamoxifen resistance and metastasis of ER+ breast cancer, was also shown to be upregulated in an AI-resistant cell line model, and reduction of TFF3 levels using TFF3-specific siRNAs decreased the growth of both the AI-resistant and -sensitive parental cell lines. Moreover, overexpression of TFF3 in parental AI-sensitive MCF-7/S0.5 cells resulted in reduced sensitivity to the AI exemestane, whereas TFF3 overexpression had no effect on growth in the absence of exemestane, indicating that TFF3 mediates growth and survival signals that abrogate the growth inhibitory effect of exemestane. We identified a panel of 26 genes exhibiting altered expression associated with disease recurrence in patients treated with adjuvant AI monotherapy, including TFF3, which was shown to exhibit a growth- and survival-promoting effect in the context of AI treatment.

  1. Gene expression changes in peripheral mononuclear cells from schizophrenic patients treated with a combination of antipsychotic with fluvoxamine.

    Science.gov (United States)

    Chertkow, Yael; Weinreb, Orly; Youdim, Moussa B H; Silver, Henry

    2007-10-01

    Antipsychotic treatment combined with Selective Serotonin Reuptake Inhibitor (SSRI) antidepressant can improve negative symptoms in schizophrenic patients that are unresponsive to antipsychotic drugs alone. The mechanism of this therapeutic effect is not clear. The current study examined molecular changes induced by the combined treatment in human peripheral mononuclear cells (PMC) in order to get insight into its mechanism of action. Gene expression profile of PMC from antipsychotic-treated patients was examined before addition of the SSRI fluvoxamine, and 3 and 6 weeks after. Gene expression patterns screened with a cDNA array, comprising 1176 genes, revealed homologous changes in a range of transcripts related to G-protein coupled receptors (GPCR). Genes related to GPCR-family were assayed using customized cDNA array and the results verified by real-time RT-PCR. The mRNA expression of chemokine receptors, IL8RA and CCR1, and of RGS7 was significantly down-regulated following fluvoxamine augmentation. The clinical assessments showed improvement in negative symptoms following the combined treatment. The transcriptional analysis suggests that the therapeutic mechanism of the combined antipsychotic-fluvoxamine treatment may involve genes associated with G-protein coupled receptors (GPCR). Our findings suggest that gene expression changes in PMC may be useful in investigating the mechanism of drug action in schizophrenia.

  2. Association of polymorphisms in oxidative stress genes with clinical outcomes for bladder cancer treated with Bacillus Calmette-Guerin.

    Directory of Open Access Journals (Sweden)

    Hua Wei

    Full Text Available Genetic polymorphisms in oxidative stress pathway genes may contribute to carcinogenesis, disease recurrence, treatment response, and clinical outcomes. We applied a pathway-based approach to determine the effects of multiple single nucleotide polymorphisms (SNPs within this pathway on clinical outcomes in non-muscle-invasive bladder cancer (NMIBC patients treated with Bacillus Calmette-Guérin (BCG. We genotyped 276 SNPs in 38 genes and evaluated their associations with clinical outcomes in 421 NMIBC patients. Twenty-eight SNPs were associated with recurrence in the BCG-treated group (P<0.05. Six SNPs, including five in NEIL2 gene from the overall and BCG group remained significantly associated with recurrence after multiple comparison adjustments (q<0.1. Cumulative unfavorable genotype analysis showed that the risk of recurrence increased with increasing number of unfavorable genotypes. In the analysis of risk factors associated with progression to disease, rs3890995 in UNG, remained significant after adjustment for multiple comparison (q<0.1. These results support the hypothesis that genetic variations in host oxidative stress genes in NMIBC patients may affect response to therapy with BCG.

  3. Knockdown of the putative Lifeguard homologue CG3814 in neurons of Drosophila melanogaster.

    Science.gov (United States)

    M'Angale, P G; Staveley, B E

    2016-12-19

    Lifeguard is an integral transmembrane protein that modulates FasL-mediated apoptosis by interfering with the activation of caspase 8. It is evolutionarily conserved, with homologues present in plants, nematodes, zebra fish, frog, chicken, mouse, monkey, and human. The Lifeguard homologue in Drosophila, CG3814, contains the Bax inhibitor-1 family motif of unknown function. Downregulation of Lifeguard disrupts cellular homeostasis and disease by sensitizing neurons to FasL-mediated apoptosis. We used bioinformatic analyses to identify CG3814, a putative homologue of Lifeguard, and knocked down CG3814/LFG expression under the control of the Dopa decarboxylase (Ddc-Gal4) transgene in Drosophila melanogaster neurons to investigate whether it possesses neuroprotective activity. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons resulted in a shortened lifespan and impaired locomotor ability, phenotypes that are strongly associated with the degeneration and loss of dopaminergic neurons. Lifeguard interacts with anti-apoptotic Bcl-2 proteins and possibly pro-apoptotic proteins to exert its neuroprotective function. The co-expression of Buffy, the sole anti-apoptotic Bcl-2 gene family member in Drosophila, and CG3814/LFG by stable inducible RNA interference, suppresses the shortened lifespan and the premature age-dependent loss in climbing ability. Suppression of CG3814/LFG in the Drosophila eye reduces the number of ommatidia and increases disruption of the ommatidial array. Overexpression of Buffy, along with the knockdown of CG3814/LFG, counteracts the eye phenotypes. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons in Drosophila diminishes its neuroprotective ability and results in a shortened lifespan and loss of climbing ability, phenotypes that are improved upon overexpression of the pro-survival Buffy.

  4. Dynamics of antibiotic resistance genes in microbial fuel cell-coupled constructed wetlands treating antibiotic-polluted water.

    Science.gov (United States)

    Zhang, Shuai; Song, Hai-Liang; Yang, Xiao-Li; Huang, Shan; Dai, Zhe-Qin; Li, Hua; Zhang, Yu-Yue

    2017-07-01

    Microbial fuel cell-coupled constructed wetlands (CW-MFCs) use electrochemical, biological, and ecological functions to treat wastewater. However, few studies have investigated the risks of antibiotic resistance genes (ARGs) when using such systems to remove antibiotics. Therefore, three CW-MFCs were designed to assess the dynamics of ARGs in filler biofilm and effluent over 5000 h of operation. The experimental results indicated that relatively high steady voltages of 605.8 mV, 613.7 mV, and 541.4 mV were obtained at total influent antibiotic concentrations of 400, 1,000, and 1600 μg L(-1), respectively. The 16S rRNA gene level in the cathode layer was higher than those in the anode and two middle layers, but the opposite trend was observed for the sul and tet genes. The relative abundance of the three tested sul genes were in the order sulI > sulII > sulIII, and those of the five tet genes were in the order tetA > tetC > tetW > tetO > tetQ. The levels of sul and tet genes in the media biofilm showed an increase over the treatment period. The effluent water had relatively low abundances of sul and tet genes compared with the filler biofilm. No increases were observed for most ARGs over the treatment period, and no significant correlations were observed between the ARGs and 16S rRNA gene copy numbers, except for sulI and tetW in the effluent. However, significant correlations were observed among most of the ARG copy numbers. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Temporal changes of antibiotic-resistance genes and bacterial communities in two contrasting soils treated with cattle manure.

    Science.gov (United States)

    Hu, Hang-Wei; Han, Xue-Mei; Shi, Xiu-Zhen; Wang, Jun-Tao; Han, Li-Li; Chen, Deli; He, Ji-Zheng

    2016-02-01

    The emerging environmental spread of antibiotic-resistance genes (ARGs) and their subsequent acquisition by clinically relevant microorganisms is a major threat to public health. Animal manure has been recognized as an important reservoir of ARGs; however, the dissemination of manure-derived ARGs and the impacts of manure application on the soil resistome remain obscure. Here, we conducted a microcosm study to assess the temporal succession of total bacteria and a broad spectrum of ARGs in two contrasting soils following manure application from cattle that had not been treated with antibiotics. High-capacity quantitative PCR detected 52 unique ARGs across all the samples, with β-lactamase as the most dominant ARG type. Several genes of soil indigenous bacteria conferring resistance to β-lactam, which could not be detected in manure, were found to be highly enriched in manure-treated soils, and the level of enrichment was maintained over the entire course of 140 days. The enriched β-lactam resistance genes had significantly positive relationships with the relative abundance of the integrase intI1 gene, suggesting an increasing mobility potential in manure-treated soils. The changes in ARG patterns were accompanied by a significant effect of cattle manure on the total bacterial community compositions. Our study indicates that even in the absence of selective pressure imposed by agricultural use of antibiotics, manure application could still strongly impact the abundance, diversity and mobility potential of a broad spectrum of soil ARGs. Our findings are important for reliable prediction of ARG behaviors in soil environment and development of appropriate strategies to minimize their dissemination.

  6. Isolation of a cotton NADP(H oxidase homologue induced by drought stress

    Directory of Open Access Journals (Sweden)

    NEPOMUCENO ALEXANDRE LIMA

    2000-01-01

    Full Text Available The aim of this study was to identify and isolate genes that are differentially expressed in four selected cotton (Gossypium hirsutum L. genotypes contrasting according to their tolerance to water deficit. The genotypes studied were Siokra L-23, Stoneville 506, CS 50 and T-1521. Physiological, morphological and developmental changes that confer drought tolerance in plants must have a molecular genetic basis. To identify and isolate the genes, the mRNA Differential Display (DD technique was used. Messenger RNAs differentially expressed during water deficit were identified, isolated, cloned and sequenced. The cloned transcript A12B15-5, a NADP(H oxidase homologue, was up regulated only during the water deficit stress and only in Siokra L-23, a drought tolerant genotype. Ribonuclease protection assay confirmed that transcription.

  7. Indoleamine 2,3-dioxygenase attenuates inhibitor development in gene-therapy-treated hemophilia A mice.

    Science.gov (United States)

    Liu, L; Liu, H; Mah, C; Fletcher, B S

    2009-06-01

    A serious impediment to gene and protein replacement therapy in hemophilia A is the development of inhibitors. Mechanisms responsible for inhibitor development include T-cell-dependent adaptive immune responses and the CD28-B7 signaling pathway that eventually leads to the formation of antibodies directed against factor VIII (FVIII). Indoleamine 2,3-dioxygenase (IDO) is a potent immunosuppressive enzyme that can inhibit T-cell responses and induce T-cell apoptosis by regulation of tryptophan metabolism. Kynurenine, one of the metabolites of tryptophan, has been implicated as an immune modulator. Here we hypothesize that co-delivery of the genes for FVIII and IDO can attenuate inhibitor formation. Using transposon-based gene delivery, we observed long-term therapeutic FVIII expression and significantly reduced inhibitor titers when the genes were co-delivered. Co-expression of FVIII and IDO in the liver was associated with increased plasma kynurenine levels, an inhibition of T-cell infiltration and increased apoptosis of T cells within the liver. These experiments suggest that modulation of tryptophan catabolism through IDO expression provides a novel strategy to reduce inhibitor development in hemophilia gene/protein therapy.

  8. Plasmodium falciparum Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling

    Institute of Scientific and Technical Information of China (English)

    QiFan; LijiaAn; LiwangCui

    2005-01-01

    The yeast transcriptional coactivator GCN5 (yGCN5), a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcriptional activation. Like other eukaryotes, the malaria parasite DNA is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Here we show that GCN5 is conserved in Plasmodium species and that the most homologous regions are within the HAT domain and the bromodomain. The Plasmodiumfalclparum GCN5 homologue (PfGCN5) is spliced with three introns, encoding a protein of 1,464 residues. Mapping of the ends of the PfGCN5 transcript suggests that the mRNA is 5.2 to 5.4 kb, consistent with the result from Northern analysis. Using free core histones, we determined that recombinant PfGCN5 proteins have conserved HAT activity with a substrate preference for histone H3. Using substrate-specific antibodies, we determined that both Lys-8 and -14 of H3 were acetylated by the recombinant PfGCN5. In eukaryotes, GCN5 homologues interact with yeast ADA2 homologues and form large multiprotein HAT complexes. We have identified an ADA2 homologue in P. falciparum, PfADA2. Yeast two-hybrid and in vitro binding assays verified the interactions between PfGCN5 and PfADA2, suggesting that they may be associated with each other in vivo. The conserved function of the HAT domain in PfGCN5 was further illustrated with yeast complementation experiments, which showed that the PfGCN5 region corresponding to the full-length yGCN5 could partially complement the yGCN5 deletion mutation. Furthermore, a chimera comprising the PfGCN5 HAT domain fused to the remainder of yeast GCN5 (yGCN5) fully rescued the yGCN5 deletion mutant. These data demonstrate that PfGCN5 is an authentic GCN5 family member and may exist in chromatin-remodeling complexes to regulate gene expression in P. falciparum.

  9. NIH researchers use gene therapy to treat a soft tissue tumor

    Science.gov (United States)

    Results of an intermediate stage clinical trial of several dozen people provides evidence that a method that has worked for treating patients with metastatic melanoma can also work for patients with metastatic synovial cell sarcoma, one of the most common soft tissue tumors in adolescents and young adults.

  10. Identification of differentially expressed genes in aflatoxin B1-treated cultured primary rat hepatocytes and Fischer 344 rats.

    Science.gov (United States)

    Harris, A J; Shaddock, J G; Manjanatha, M G; Lisenbey, J A; Casciano, D A

    1998-08-01

    Aflatoxin B1 (AFB1), a mutagen and hepatocarcinogen in rats and humans, is a contaminant of the human food supply, particularly in parts of Africa and Asia. AFB1-induced changes in gene expression may play a part in the development of the toxic, immunosuppressive and carcinogenic properties of this fungal metabolite. An understanding of the-role of AFB1 in modulating gene regulation should provide insight regarding mechanisms of AFB1-induced carcinogenesis. We used three PCR-based subtractive techniques to identify AFB1-responsive genes in cultured primary rat hepatocyte RNA: differential display PCR (DD-PCR), representational difference analysis (RDA) and suppression subtractive hybridization (SSH). Each of the three techniques identified AFB1-responsive genes, although no individual cDNA was isolated by more than one technique. Nine cDNAs isolated using DD-PCR, RDA or SSH were found to represent eight genes that are differentially expressed as a result of AFB1 exposure. Genes whose mRNA levels were increased in cultured primary rat hepatocytes after AFB1 treatment were corticosteroid binding globulin (CBG), cytochrome P450 4F1 (CYP4F1), alpha-2 microglobulin, C4b-binding protein (C4BP), serum amyloid A-2 and glutathione S-transferase Yb2 (GST). Transferrin and a small CYP3A-like cDNA had reduced mRNA levels after AFB1 exposure. Full-length CYP3A mRNA levels were increased. When liver RNA from AFB1-treated male F344 rats was evaluated for transferrin, CBG, GST, CYP3A and CYP4F1 expression, a decrease in transferrin mRNA and an increase in CBG, GST, CYP3A and CYP4F1 mRNA levels was also seen. Analysis of the potential function of these genes in maintaining cellular homeostasis suggests that their differential expression could contribute to the toxicity associated with AFB1 exposure.

  11. Characterization of Fabry mice treated with recombinant adeno-associated virus 2/8-mediated gene transfer

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    Choi Jin-Ok

    2010-04-01

    Full Text Available Abstract Background Enzyme replacement therapy (ERT with α-galactosidase A (α-Gal A is currently the most effective therapeutic strategy for patients with Fabry disease, a lysosomal storage disease. However, ERT has limitations of a short half-life, requirement for frequent administration, and limited efficacy for patients with renal failure. Therefore, we investigated the efficacy of recombinant adeno-associated virus (rAAV vector-mediated gene therapy for a Fabry disease mouse model and compared it with that of ERT. Methods A pseudotyped rAAV2/8 vector encoding α-Gal A cDNA (rAAV2/8-hAGA was prepared and injected into 18-week-old male Fabry mice through the tail vein. The α-Gal A expression level and globotriaosylceramide (Gb3 levels in the Fabry mice were examined and compared with Fabry mice with ERT. Immunohistochemical and ultrastructural studies were conducted. Results Treatment of Fabry mice with rAAV2/8-hAGA resulted in the clearance of accumulated Gb3 in tissues such as liver, spleen, kidney, heart, and brain with concomitant elevation of α-Gal A enzyme activity. Enzyme activity was elevated for up to 60 weeks. In addition, expression of the α-Gal A protein was identified in the presence of rAAV2/8-hAGA at 6, 12, and 24 weeks after treatment. α-Gal A activity was significantly higher in the mice treated with rAAV2/8-hAGA than in Fabry mice that received ERT. Along with higher α-Gal A activity in the kidney of the Fabry mice treated with gene therapy, immunohistochemical studies showed more α-Gal A expression in the proximal tubules and glomerulus, and less Gb3 deposition in Fabry mice treated with this gene therapy than in mice given ERT. The α-gal A gene transfer significantly reduced the accumulation of Gb3 in the tubules and podocytes of the kidney. Electron microscopic analysis of the kidneys of Fabry mice also showed that gene therapy was more effective than ERT. Conclusions The rAAV2/8-hAGA mediated α-Gal A gene

  12. Recent advances in use of gene therapy to treat hepatitis B virus infection.

    Science.gov (United States)

    Bloom, Kristie; Ely, Abdullah; Arbuthnot, Patrick

    2015-01-01

    Chronic infection with hepatitis B virus (HBV) occurs in approximately 5 % of the world's human population and persistence of the virus is associated with serious complications of cirrhosis and liver cancer. Currently available treatments are modestly effective and advancing novel therapeutic strategies is a medical priority. Stability of the viral cccDNA replication intermediate is a major factor that has impeded the development of therapies that are capable of eliminating chronic infection. Recent advances that employ gene therapy strategies offer useful advantages over current therapeutics. Silencing of HBV gene expression by harnessing the RNA interference pathway has been shown to be highly effective in cell culture and in vivo. However, a potential limitation of this approach is that the post-transcriptional mechanism of gene silencing does not disable cccDNA. Early results using designer transcription activator-like effector nucleases (TALENs) and repressor TALEs (rTALEs) have shown potential as a mode of inactivating cccDNA. In this article, we review the recent advances that have been made in HBV gene therapy, with a particular emphasis on the potential anti-HBV therapeutic utility of designed sequence-specific DNA binding proteins and their derivatives.

  13. Treating TRAPS Syndrome with a Previously Undescribed TNF α Gene Receptor Mutation Successfully with Canakinumab

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    T. V. Sleptsova

    2016-01-01

    Full Text Available The article presents an observation of one of the most common autoinflammatory syndromes — TRAPS (periodic syndrome associated with a mutation in the TNF α receptor gene. During a molecular-genetic examination of a 9-year-old child, a c.337_339del deletion in the heterozygous state of the TNFRSF1A gene exon 04, leading to a p.Glu113del amino acid deletion, was found. This mutation has not been described previously in TRAPS patients, and according to computer analysis (Alamut Visual the issue is pathogenic. This observation indicates the presence of families with TRAPS in the Russian population, who can have «atypical» TNFRSF1A gene mutations. A successful use of monoclonal antibodies to interleukin 1 — canakinumab — in the patient is described. As a result, fever and abdominal syndromes have completely stopped, while knee joints pain decreased a day later. After a week of treatment, the child’s disease activity laboratory indices returned to normal (ESR, C-reactive protein. No exacerbations were fixed over the next 32 weeks. No adverse effects were registered during canakinumab therapy. Thus, canakinumab has demonstrated a high level of effectiveness and safety for the patient suffering from a periodic syndrome associated with a mutation in the TNF α  gene receptor. This indicates therapeutic use prospects for the interleukin 1 β blocker in TRAPS syndrome patients.

  14. Monolayer structures of alkyl aldehydes: Odd-membered homologues

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, T.K. [BP Institute, Department of Chemistry, University of Cambridge, Cambridge (United Kingdom); Clarke, S.M., E-mail: stuart@bpi.cam.ac.u [BP Institute, Department of Chemistry, University of Cambridge, Cambridge (United Kingdom); Bhinde, T. [BP Institute, Department of Chemistry, University of Cambridge, Cambridge (United Kingdom); Castro, M.A.; Millan, C. [Instituto Ciencia de los Materiales de Sevilla, Departamento de Quimica Inorganica (CSIC-Universidad de Sevilla) (Spain); Medina, S. [Centro de Investigacion, Tecnologia e Innovacion de la Universidad de Sevilla (CITIUS), Sevilla (Spain)

    2011-03-01

    Crystalline monolayers of three aldehydes with an odd number of carbon atoms in the alkyl chain (C{sub 7}, C{sub 9} and C{sub 11}) at low coverages are observed by a combination of X-ray and neutron diffraction. Analysis of the diffraction data is discussed and possible monolayer crystal structures are proposed; although unique structures could not be ascertained for all molecules. We conclude that the structures are flat on the surface, with the molecules lying in the plane of the layer. The C{sub 11} homologue is determined to have a plane group of either p2, pgb or pgg, and for the C{sub 7} homologue the p2 plane group is preferred.

  15. Mammalian homologue of the calcium-sensitive phosphoglycoprotein, parafusin.

    Science.gov (United States)

    Wyroba, E; Widding Høyer, A; Storgaard, P; Satir, B H

    1995-12-01

    Three specific antipeptide antibodies and oligonucleotide probes synthesized to internal sequences of parafusin have been used to search for mammalian counterpart(s) of this protein. Parafusin is an exocytic-sensitive phosphoglycoprotein from a unicellular eukaryote Paramecium that was recently cloned and sequenced (Subramanian et al., Proc. Natl. Acad. Sci. USA 91, 9832-9836 (1994)). Western and Southern blot analyses, polymerase chain reaction (PCR) and reverse transcriptase coupled PCR (RT-PCR) techniques have been used to examine rat liver and pancreas, human pancreas and a murine pancreatic beta-cell line (beta TC3) arising in transgenic mice. The parafusin-specific antibodies showed cross-reaction with a protein at approximately 63 kDa in 4 tissues, whereas a phosphoglucomutase-specific antibody also detected a second band of similar molecular weight in the beta TC3 cells. The presence of two bands shows that parafusin homologue(s) and phosphoglucomutase are separate entities. beta TC3 cells were shown to incorporate [beta 35]UDPGlc into the parafusin homologue in a Ca(++)-sensitive manner characteristic of parafusin. Southern blot analysis revealed that the parafusin-specific probe hybridized with restriction enzyme digests of rat DNA in distinct patterns different from those observed with a phosphoglucomutase-specific probe. Rat genomic DNA and mRNA from the beta TC3 cells were used as the templates for PCR and RT-PCR using internal parafusin primers. In both cases similarly sized products were obtained which hybridized in Southern analysis with a specific parafusion probe located within the amplified region. These results indicate that a parafusin homologue exists in mammalian cells.

  16. Importance of MutL homologue MLH1 and MutS homologue MSH2 expression in Turkish patients with sporadic colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Sibel Erdamar; Esra Ucaryilmaz; Gokhan Demir; Tayfun Karahasanoglu; Gulen Dogusoy; Ahmet Dirican; Suha Goksel

    2007-01-01

    AIM: To assess the incidence of MLH1 (the human MutL homologue) and MSH2 (the human MutS homologue)protein expression in Turkish patients with sporadic colorectal cancers and to compare their survival and clinicopathological features.METHODS: We validated the tissue microarray technology in 77 colorectal carcinomas by analyzing the immunohistochemical expression of proteins involved in two main pathways of colorectal carcinogenesis: p53 protein for loss of heterozygosity tumors; MLH1 and MSH2 proteins for microsatellite instability (MSI).RESULTS: Our analysis showed that 29 (39.2%) had loss of MLH1 expression, 5 (6.8%) had loss of MSH2 expression and 2 cases had loss of expression of both proteins. We found that 60% of MSH2-negative tumors were located in the right side of the colon; all MSH2-negative cases were women. In addition, the loss of MSH2 expression was correlated with low p53 expression. Neither MLH1 nor MSH2 expressions were associated with prognosis, although there seemed a tendency of longer survival (71.7 ± 8.65 mo vs 47.08 ± 5.26 mo) for the patients with MLH1-negative versus MLH1-positive carcinomas. There were not significant differences in overall and recurrence-free survival among MLH1/MSH2-positive and -negative cases.CONCLUSION: Our data supports that Turkish patients with MLH1- and MSH2-defective tumors have some distinct features from each other. Although prognostic importance remains controversial, immunohistochemical analysis of mismatch repair genes may be used as a routine histopathological examination of sporadic colorectal carcinomas.

  17. Neural stem cell transplantation with Nogo-66 receptor gene silencing to treat severe traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    Dong Wang; Jianjun Zhang; Jingjian Ma; Yuan Mu; Yinghui Zhuang

    2011-01-01

    Inhibition of neurite growth, which is mediated by the Nogo-66 receptor (NgR), affects nerve regeneration following neural stem cell (NSC) transplantation. The present study utilized RNA interference to silence NgR gene expression in NSCs, which were subsequently transplanted into rats with traumatic brain injury. Following transplantation of NSCs transfected with small interfering RNA,typical neural cell-like morphology was detected in injured brain tissues, and was accompanied by absence of brain tissue cavity, increased growth-associated protein 43 mRNA and protein expression,and improved neurological function compared with NSC transplantation alone. Results demonstrated that NSC transplantation with silenced NgR gene promoted functional recovery following brain injury.

  18. Identification of possible targets of the Aspergillus fumigatus CRZ1 homologue, CrzA

    Directory of Open Access Journals (Sweden)

    Goldman Gustavo H

    2010-01-01

    Full Text Available Abstract Background Calcineurin, a serine/threonine-specific protein phosphatase, plays an important role in the control of cell morphology and virulence in fungi. Calcineurin regulates localization and activity of a transcription factor called CRZ1. Recently, we characterize Aspergillus fumigatus CRZ1 homologue, AfCrzA. Here, we investigate which pathways are influenced by A. fumigatus AfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile of A. fumigatus wild type and ΔAfcrzA mutant strains. Results We were able to observe 3,622 genes modulated in at least one timepoint in the mutant when compared to the wild type strain (3,211 and 411 at 10 and 30 minutes, respectively. Decreased mRNA abundance in the ΔcrzA was seen for genes encoding calcium transporters, transcription factors and genes that could be directly or indirectly involved in calcium metabolism. Increased mRNA accumulation was observed for some genes encoding proteins involved in stress response. AfCrzA overexpression in A. fumigatus increases the expression of several of these genes. The deleted strain of one of these genes, AfRcnA, belonging to a class of endogenous calcineurin regulators, calcipressins, had more calcineurin activity after exposure to calcium and was less sensitive to menadione 30 μM, hydrogen peroxide 2.5 mM, EGTA 25 mM, and MnCl2 25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely related A. nidulans AnRcnA. GFP::RcnA was mostly detected along the germling, did not accumulate in the nuclei and its location is not affected by the cellular response to calcium chloride. Conclusion We have performed a transcriptional profiling analysis of the A. fumigatus ΔAfcrzA mutant strain exposed to calcium stress. This provided an excellent opportunity to identify genes and pathways that are under the influence of AfCrzA. AfRcnA, one of these selected genes, encodes a modulator of calcineurin

  19. Clinical translation of TALENS: Treating SCID-X1 by gene editing in iPSCs.

    Science.gov (United States)

    Biffi, Alessandra

    2015-04-02

    Mutations causing X-linked severe combined immunodeficiency (SCID-X1) reduce immune cell populations and function and may be amenable to targeted gene correction strategies. Now in Cell Stem Cell, Menon et al. (2015) correct SCID-X1-related blood differentiation defects by TALEN-mediated genome editing in patient-derived iPSCs, suggesting a possible strategy for autologous cell therapy of SCID-X1.

  20. Molecular anthropology meets genetic medicine to treat blindness in the North African Jewish population: human gene therapy initiated in Israel.

    Science.gov (United States)

    Banin, Eyal; Bandah-Rozenfeld, Dikla; Obolensky, Alexey; Cideciyan, Artur V; Aleman, Tomas S; Marks-Ohana, Devora; Sela, Malka; Boye, Sanford; Sumaroka, Alexander; Roman, Alejandro J; Schwartz, Sharon B; Hauswirth, William W; Jacobson, Samuel G; Hemo, Itzhak; Sharon, Dror

    2010-12-01

    The history of the North African Jewish community is ancient and complicated with a number of immigration waves and persecutions dramatically affecting its population size. A decade-long process in Israel of clinical-molecular screening of North African Jews with incurable autosomal recessive blindness led to the identification of a homozygous splicing mutation (c.95-2A > T; IVS2-2A > T) in RPE65, the gene encoding the isomerase that catalyzes a key step in the retinoid-visual cycle, in patients from 10 unrelated families. A total of 33 patients (four now deceased) had the severe childhood blindness known as Leber congenital amaurosis (LCA), making it the most common cause of retinal degeneration in this population. Haplotype analysis in seven of the patients revealed a shared homozygous region, indicating a population-specific founder mutation. The age of the RPE65 founder mutation was estimated to have emerged 100-230 (mean, 153) generations ago, suggesting it originated before the establishment of the Jewish community in North Africa. Individuals with this RPE65 mutation were characterized with retinal studies to determine if they were candidates for gene replacement, the recent and only therapy to date for this otherwise incurable blindness. The step from molecular anthropological studies to application of genetic medicine was then taken, and a representative of this patient subgroup was treated with subretinal rAAV2-RPE65 gene therapy. An increase in vision was present in the treated area as early as 15 days after the intervention. This process of genetically analyzing affected isolated populations as a screen for gene-based therapy suggests a new paradigm for disease diagnosis and treatment.

  1. Enhanced thermogenic program by non-viral delivery of combinatory browning genes to treat diet-induced obesity in mice.

    Science.gov (United States)

    Park, Hongsuk; Cho, Sungpil; Janat-Amsbury, Margit M; Bae, You Han

    2015-12-01

    Thermogenic program (also known as browning) is a promising and attractive anti-obesity approach. Islet amyloid polypeptide (IAPP) and irisin have emerged as potential browning hormones that hold high potential to treat obesity. Here, we have constructed a dual browning gene system containing both IAPP and irisin (derived from fibronectin type III domain containing 5; FNDC5) combined with 2A and furin self-cleavage sites. Intraperitoneal administration of the construct complexed with a linear polyethylenimine into diet-induced obese mice demonstrated the elevation of anti-obesogenic effects characterized as the decreased body weight, adiposity, and levels of glucose and insulin. In addition, the construct delivery increased energy expenditure and the expression of core molecular determinants associated with browning. The additional advantages of the dual browning gene construct delivery compared to both single gene construct delivery and dual peptide delivery can be emphasized on efficacy and practicability. Hence, we have concluded that dual browning gene delivery makes it therapeutically attractive for diet-induced obesity treatment.

  2. Occurrence of virulence genes among Vibrio cholerae and Vibrio parahaemolyticus strains from treated wastewaters.

    Science.gov (United States)

    Khouadja, Sadok; Suffredini, Elisabetta; Baccouche, Besma; Croci, Luciana; Bakhrouf, Amina

    2014-10-01

    Pathogenic Vibrio species are an important cause of foodborne illnesses. The aim of this study was to describe the occurrence of potentially pathogenic Vibrio species in the final effluents of a wastewater treatment plant and the risk that they may pose to public health. During the 1-year monitoring, a total of 43 Vibrio strains were isolated: 23 Vibrio alginolyticus, 1 Vibrio cholerae, 4 Vibrio vulnificus, and 15 Vibrio parahaemolyticus. The PCR investigation of V. parahaemolyticus and V. cholerae virulence genes (tlh, trh, tdh, toxR, toxS, toxRS, toxT, zot, ctxAB, tcp, ace, vpi, nanH) revealed the presence of some of these genes in a significant number of strains. Intraspecies variability and genetic relationships among the environmental isolates were analyzed by random amplified polymorphic DNA-PCR (RAPD-PCR). We report the results of the first isolation and characterization of an environmental V. cholerae non-O1 non-O139 and of a toxigenic V. parahaemolyticus strain in Tunisia. We suggest that non-pathogenic Vibrio might represent a marine reservoir of virulence genes that can be transmitted between strains by horizontal transfer.

  3. Baseline Gene Expression Signatures in Monocytes from Multiple Sclerosis Patients Treated with Interferon-beta

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    Bustamante, Marta F.; Nurtdinov, Ramil N.; Río, Jordi; Montalban, Xavier; Comabella, Manuel

    2013-01-01

    Background A relatively large proportion of relapsing-remitting multiple sclerosis (RRMS) patients do not respond to interferon-beta (IFNb) treatment. In previous studies with peripheral blood mononuclear cells (PBMC), we identified a subgroup of IFNb non-responders that was characterized by a baseline over-expression of type I IFN inducible genes. Additional mechanistic experiments carried out in IFNb non-responders suggested a selective alteration of the type I IFN signaling pathway in the population of blood monocytes. Here, we aimed (i) to investigate whether the type I IFN signaling pathway is up-regulated in isolated monocytes from IFNb non-responders at baseline; and (ii) to search for additional biological pathways in this cell population that may be implicated in the response to IFNb treatment. Methods Twenty RRMS patients classified according to their clinical response to IFNb treatment and 10 healthy controls were included in the study. Monocytes were purified from PBMC obtained before treatment by cell sorting and the gene expression profiling was determined with oligonucleotide microarrays. Results and discussion Purified monocytes from IFNb non-responders were characterized by an over-expression of type I IFN responsive genes, which confirms the type I IFN signature in monocytes suggested from previous studies. Other relevant signaling pathways that were up-regulated in IFNb non-responders were related with the mitochondrial function and processes such as protein synthesis and antigen presentation, and together with the type I IFN signaling pathway, may also be playing roles in the response to IFNb. PMID:23637780

  4. Calcitonin gene-related peptide targeted immunotherapy for migraine: progress and challenges in treating headache.

    Science.gov (United States)

    Peroutka, Stephen J

    2014-06-01

    A role for calcitonin gene-related peptide (CGRP) in the pathophysiology of migraine has been established over the past 25 years. There have now been at least five different small-molecule CGRP antagonists that have demonstrated statistical proof of efficacy in the acute treatment of migraine. At present, multiple clinical trials are underway that are assessing the ability of long-acting antibodies against CGRP to prevent frequent migraine attacks. This review summarizes the existing data concerning the role of CGRP in migraine and attempts to highlight some possible outcomes from the ongoing anti-CGRP antibody trials.

  5. Identification of NoxD/Pro41 as the homologue of the p22phox NADPH oxidase subunit in fungi.

    Science.gov (United States)

    Lacaze, Isabelle; Lalucque, Hervé; Siegmund, Ulrike; Silar, Philippe; Brun, Sylvain

    2015-03-01

    NADPH oxidases (Nox) are membrane complexes that produce O2(-). Researches in mammals, plants and fungi highlight the involvement of Nox-generated ROS in cell proliferation, differentiation and defense. In mammals, the core enzyme gp91(phox)/Nox2 is associated with p22(phox) forming the flavocytochrome b558 ready for activation by a cytosolic complex. Intriguingly, no homologue of the p22(phox) gene has been found in fungal genomes, questioning how the flavoenzyme forms. Using whole genome sequencing combined with phylogenetic analysis and structural studies, we identify the fungal p22(phox) homologue as being mutated in the Podospora anserina mutant IDC(509). Functional studies show that the fungal p22(phox), PaNoxD, acts along PaNox1, but not PaNox2, a second fungal gp91(phox) homologue. Finally, cytological analysis of functional tagged versions of PaNox1, PaNoxD and PaNoxR shows clear co-localization of PaNoxD and PaNox1 and unravel a dynamic assembly of the complex in the endoplasmic reticulum and in the vacuolar system.

  6. Docosahexaenoic acid regulates gene expression in HUVEC cells treated with polycyclic aromatic hydrocarbons.

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    Gdula-Argasińska, Joanna; Czepiel, Jacek; Totoń-Żurańska, Justyna; Jurczyszyn, Artur; Perucki, William; Wołkow, Paweł

    2015-07-16

    The molecular mechanism of inflammation and carcinogenesis induced by exposure of polycyclic aromatic hydrocarbons (PAHs) is not clearly understood. Our study was undertaken due to the strong pro-carcinogenic potential and reactivity of PAH-metabolites, as well as the susceptibility of polyunsaturated fatty acids to oxidation. The aim of this study was to evaluate the pro- or anti-inflammatory impact of n-3 docosahexaenoic acid on human primary umbilical vein endothelial cells (HUVEC) exposed to polycyclic aromatic hydrocarbons. We analysed the influence of docosahexaenoic acid (DHA) and/or PAHs supplementation on the fatty acid profile of cell membranes, on cyclooxygenase-2 (COX-2), aryl hydrocarbon receptor (AHR), and glutathione S transferase Mu1 (GSTM1) protein expression as well as on the prostaglandin synthase 2 (PTGS2), AHR, GSTM1, PLA2G4A, and cytochrome P450 CYP1A1 gene expression. We observed that COX-2 and AHR protein expression was increased while GSTM1 expression was decreased in cells exposed to DHA and PAHs. Docosahexaenoic acid down-regulated CYP1A1 and up-regulated the AHR and PTGS2 genes. Our findings suggested that DHA contributes significantly to alleviate the harmful effects caused by PAHs in endothelial cells. Moreover, these results suggest that a diet rich in n-3 fatty acids is helpful to reduce the harmful effects of PAHs exposure on human living in heavily polluted areas.

  7. AtNFXL1, an Arabidopsis homologue of the human transcription factor NF-X1, functions as a negative regulator of the trichothecene phytotoxin-induced defense response.

    Science.gov (United States)

    Asano, Tomoya; Masuda, Daisuke; Yasuda, Michiko; Nakashita, Hideo; Kudo, Toshiaki; Kimura, Makoto; Yamaguchi, Kazuo; Nishiuchi, Takumi

    2008-02-01

    Trichothecenes are a closely related family of phytotoxins that are produced by phytopathogenic fungi. In Arabidopsis, expression of AtNFXL1, a homologue of the putative human transcription repressor NF-X1, was significantly induced by application of type A trichothecenes, such as T-2 toxin. An atnfxl1 mutant growing on medium lacking trichothecenes showed no phenotype, whereas a hypersensitivity phenotype was observed in T-2 toxin-treated atnfxl1 mutant plants. Microarray analysis indicated that several defense-related genes (i.e. WRKYs, NBS-LRRs, EDS5, ICS1, etc.) were upregulated in T-2 toxin-treated atnfxl1 mutants compared with wild-type plants. In addition, enhanced salicylic acid (SA) accumulation was observed in T-2 toxin-treated atnfxl1 mutants, which suggests that AtNFXL1 functions as a negative regulator of these defense-related genes via an SA-dependent signaling pathway. We also found that expression of AtNFXL1 was induced by SA and flg22 treatment. Moreover, the atnfxl1 mutant was less susceptible to a compatible phytopathogen, Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000). Taken together, these results indicate that AtNFXL1 plays an important role in the trichothecene response, as well as the general defense response in Arabidopsis.

  8. Gene expression analysis of forskolin treated basilar papillae identifies microRNA181a as a mediator of proliferation.

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    Corey S Frucht

    Full Text Available BACKGROUND: Auditory hair cells spontaneously regenerate following injury in birds but not mammals. A better understanding of the molecular events underlying hair cell regeneration in birds may allow for identification and eventually manipulation of relevant pathways in mammals to stimulate regeneration and restore hearing in deaf patients. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression was profiled in forskolin treated (i.e., proliferating and quiescent control auditory epithelia of post-hatch chicks using an Affymetrix whole-genome chicken array after 24 (n = 6, 48 (n = 6, and 72 (n = 12 hours in culture. In the forskolin-treated epithelia there was significant (ptwo-fold change upregulation of many genes thought to be relevant to cell cycle control and inner ear development. Gene set enrichment analysis was performed on the data and identified myriad microRNAs that are likely to be upregulated in the regenerating tissue, including microRNA181a (miR181a, which is known to mediate proliferation in other systems. Functional experiments showed that miR181a overexpression is sufficient to stimulate proliferation within the basilar papilla, as assayed by BrdU incorporation. Further, some of the newly produced cells express the early hair cell marker myosin VI, suggesting that miR181a transfection can result in the production of new hair cells. CONCLUSIONS/SIGNIFICANCE: These studies have identified a single microRNA, miR181a, that can cause proliferation in the chicken auditory epithelium with production of new hair cells.

  9. Multi-parametric MRI at 14T for muscular dystrophy mice treated with AAV vector-mediated gene therapy.

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    Joshua Park

    Full Text Available The objective of this study was to investigate the efficacy of using quantitative magnetic resonance imaging (MRI as a non-invasive tool for the monitoring of gene therapy for muscular dystrophy. The clinical investigations for this family of diseases often involve surgical biopsy which limits the amount of information that can be obtained due to the invasive nature of the procedure. Thus, other non-invasive tools may provide more opportunities for disease assessment and treatment responses. In order to explore this, dystrophic mdx4cv mice were systemically treated with a recombinant adeno-associated viral (AAV vector containing a codon-optimized micro-dystrophin gene. Multi-parametric MRI of T2, magnetization transfer, and diffusion effects alongside 3-D volume measurements were then utilized to monitor disease/treatment progression. Mice were imaged at 10 weeks of age for pre-treatment, then again post-treatment at 8, 16, and 24 week time points. The efficacy of treatment was assessed by physiological assays for improvements in function and quantification of expression. Tissues from the hindlimbs were collected for histological analysis after the final time point for comparison with MRI results. We found that introduction of the micro-dystrophin gene restored some aspects of normal muscle histology and pathology such as decreased necrosis and resistance to contraction-induced injury. T2 relaxation values showed percentage decreases across all muscle types measured (tibialis anterior, gastrocnemius, and soleus when treated groups were compared to untreated groups. Additionally, the differences between groups were statistically significant for the tibialis anterior as well. The diffusion measurements showed a wider range of percentage changes and less statistical significance while the magnetization transfer effect measurements showed minimal change. MR images displayed hyper-intense regions of muscle that correlated with muscle pathology in

  10. Mutations in the Reverse Transcriptase and Protease Genes of Human Immunodeficiency Virus-1 from Antiretroviral Naïve and Treated Pediatric Patients

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    Dinesh Bure

    2015-02-01

    Full Text Available The success of highly active antiretroviral therapy (HAART is challenged by the emergence of resistance-associated mutations in human immunodeficiency virus-1 (HIV-1. In this study, resistance associated mutations in the reverse transcriptase (RT and protease (PR genes in antiretroviral therapy (ART  naïve and treated HIV-1 infected pediatric patients from North India were evaluated. Genotyping was successfully performed in 46 patients (30 ART naive and 16 treated for the RT gene and in 53 patients (27 ART naive and 26 treated for PR gene and mutations were identified using Stanford HIV Drug Resistance Database. A major drug resistant mutation in RT gene, L74I (NRTI, and two such mutations, K101E and G190A (NNRTI, were observed in two ART naïve patients, while M184V was detected in two ART treated patients. Overall, major resistance associated mutations in RT gene were observed in nine (30% and seven (36% of ART naïve and treated children respectively. Minor mutations were identified in PR gene in five children. Few non-clade C viral strains (≈30% were detected, although subtype C was most predominant. The screening of ART naïve children for mutations in HIV-1 RT and protease genes, before and after initiation of ART is desirable for drug efficacy and good prognosis.

  11. GET_HOMOLOGUES, a versatile software package for scalable and robust microbial pangenome analysis.

    Science.gov (United States)

    Contreras-Moreira, Bruno; Vinuesa, Pablo

    2013-12-01

    GET_HOMOLOGUES is an open-source software package that builds on popular orthology-calling approaches making highly customizable and detailed pangenome analyses of microorganisms accessible to nonbioinformaticians. It can cluster homologous gene families using the bidirectional best-hit, COGtriangles, or OrthoMCL clustering algorithms. Clustering stringency can be adjusted by scanning the domain composition of proteins using the HMMER3 package, by imposing desired pairwise alignment coverage cutoffs, or by selecting only syntenic genes. The resulting homologous gene families can be made even more robust by computing consensus clusters from those generated by any combination of the clustering algorithms and filtering criteria. Auxiliary scripts make the construction, interrogation, and graphical display of core genome and pangenome sets easy to perform. Exponential and binomial mixture models can be fitted to the data to estimate theoretical core genome and pangenome sizes, and high-quality graphics can be generated. Furthermore, pangenome trees can be easily computed and basic comparative genomics performed to identify lineage-specific genes or gene family expansions. The software is designed to take advantage of modern multiprocessor personal computers as well as computer clusters to parallelize time-consuming tasks. To demonstrate some of these capabilities, we survey a set of 50 Streptococcus genomes annotated in the Orthologous Matrix (OMA) browser as a benchmark case. The package can be downloaded at http://www.eead.csic.es/compbio/soft/gethoms.php and http://maya.ccg.unam.mx/soft/gethoms.php.

  12. Biofilm processes in treating mariculture wastewater may be a reservoir of antibiotic resistance genes.

    Science.gov (United States)

    Li, Shuai; Zhang, Shenghua; Ye, Chengsong; Lin, Wenfang; Zhang, Menglu; Chen, Lihua; Li, Jinmei; Yu, Xin

    2017-05-15

    Antibiotics are heavily used in Chinese mariculture, but only a small portion of the added antibiotics are absorbed by living creatures. Biofilm processes are universally used in mariculture wastewater treatment. In this study, removal of antibiotics (norfloxacin, rifampicin, and oxytetracycline) from wastewater by moving bed biofilm reactors (MBBRs) and the influence of antibiotics on reactor biofilm were investigated. The results demonstrated that there was no significant effect of sub-μg/L-sub-mg/L concentrations of antibiotics on TOC removal. Moreover, the relative abundance of antibiotic resistance genes (ARGs) and antibiotic resistance bacteria (ARB) in MBBR biofilm increased because of selective pressure of antibiotics. In addition, antibiotics decreased the diversity of the biofilm bacterial community and altered bacterial community structure. These findings provide an empirical basis for the development of appropriate practices for mariculture, and suggest that disinfection and advanced oxidation should be applied to eliminate antibiotics, ARGs, and ARB from mariculture wastewater. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Endogenous type C viral gene expression in cultures of fetal diploid baboon cells treated with 5'-bromodeoxyuridine

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    Lavelle, G.; Kennel, S.J.; Foote, L.J.

    1981-04-30

    Cultures of fetal diploid baboon fibroblasts treated with 5-bromodeoxyuridine synthesized protein antigenically related to baboon endogenous type C viral gag gene product, p28. Radioimmunoassays detected p28 antigenic specificities indistinguishable from those of purified virus. However, viral RNA-dependent DNA polymerase was not detected in culture fluids, and infectious virus was rarely recovered by cocultivation with susceptible heterologous cells. Extracellular particles containing p28 were not readily detected, further indicating that viral gag gene-coded proteins were synthesized independently of whole virus. Normal cultures of the same baboon cells exhibited endogenous expression of a glycoprotein antigenically related to BEV gp70, suggesting differential regulation of the endogenous gag and env gene-coded products. Baboon cell cultures exogenously infected with BEV produced extracellular particles having viral p28 and gp70 as measured by radioimmunoassays of culture fluids. Since induced cultures have about 10% positive cells versus close to 100% for infected culture, the amount of p28 per producing cell was about the same in both cell populations.

  14. Astragaloside IV Inhibits NF-κB Activation and Inflammatory Gene Expression in LPS-Treated Mice

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    Wei-Jian Zhang

    2015-01-01

    Full Text Available In this study we investigated the role of astragaloside IV (AS-IV, one of the major active constituents purified from the Chinese medicinal herb Astragalus membranaceus, in LPS-induced acute inflammatory responses in mice in vivo and examined possible underlying mechanisms. Mice were assigned to four groups: vehicle-treated control animals; AS-IV-treated animals (10 mg/kg b.w. AS-IV daily i.p. injection for 6 days; LPS-treated animals; and AS-IV plus LPS-treated animals. We found that AS-IV treatment significantly inhibited LPS-induced increases in serum levels of MCP-1 and TNF by 82% and 49%, respectively. AS-IV also inhibited LPS-induced upregulation of inflammatory gene expression in different organs. Lung mRNA levels of cellular adhesion molecules, MCP-1, TNFα, IL-6, and TLR4 were significantly attenuated, and lung neutrophil infiltration and activation were strongly inhibited, as reflected by decreased myeloperoxidase content, when the mice were pretreated with AS-IV. Similar results were observed in heart, aorta, kidney, and liver. Furthermore, AS-IV significantly suppressed LPS-induced NF-κB and AP-1 DNA-binding activities in lung and heart. In conclusion, our data provide new in vivo evidence that AS-IV effectively inhibits LPS-induced acute inflammatory responses by modulating NF-κB and AP-1 signaling pathways. Our results suggest that AS-IV may be useful for the prevention or treatment of inflammatory diseases.

  15. RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.

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    B Alex Merrick

    Full Text Available Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1, a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL. Paired-end reads were mapped to the rat genome (Rn4 with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005 compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c

  16. In vitro dentine remineralization with a potential salivary phosphoprotein homologue.

    Science.gov (United States)

    Romero, Maria Jacinta Rosario H; Nakashima, Syozi; Nikaido, Toru; Sadr, Alireza; Tagami, Junji

    2016-08-01

    Advantages of introducing a salivary phosphoprotein homologue under standardized in vitro conditions to simulate the mineral-stabilizing properties of saliva have been proposed. This study longitudinally investigates the effects of casein, incorporated as a potential salivary phosphoprotein homologue in artificial saliva (AS) solutions with/without fluoride (F) on in vitro dentine lesion remineralization. Thin sections of bovine root dentine were demineralized and allocated randomly into 6 groups (n=18) having equivalent mineral loss (ΔZ) after transverse microradiography (TMR). The specimens were remineralized using AS solutions containing casein 0μg/ml, F 0ppm (C0-F0); casein 0μg/ml, F 1ppm (C0-F1); casein 10μg/ml, F 0ppm (C10-F0); casein 10μg/ml, F 1ppm (C10-F1); casein 100μg/ml, F 0ppm (C100-F0) or casein 100μg/ml, F 1ppm (C100-F1) for 28days with TMR taken every 7 days. Surface mineral precipitation, evident in group C0-F1, was apparently inhibited in groups with casein incorporation. Repeated measures ANOVA with Bonferroni correction revealed higher ΔZ for non-F and non-casein groups than for their counterparts (p<0.001). Subsequent multiple comparisons showed that mineral gain was higher (p<0.001) with 10μg/ml casein than with 100μg/ml when F was present in the earlier stages of remineralization, with both groups achieving almost complete remineralization after 28 days. Casein is a potential salivary phosphoprotein homologue that could be employed for in vitro dentine remineralization studies. Concentration related effects may be clinically significant and thus must be further examined. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. A 1.4-Mb interval RH map of horse chromosome 17 provides detailed comparison with human and mouse homologues.

    Science.gov (United States)

    Lee, Eun-Joon; Raudsepp, Terje; Kata, Srinivas R; Adelson, David; Womack, James E; Skow, Loren C; Chowdhary, Bhanu P

    2004-02-01

    Comparative genomics has served as a backbone for the rapid development of gene maps in domesticated animals. The integration of this approach with radiation hybrid (RH) analysis provides one of the most direct ways to obtain physically ordered comparative maps across evolutionarily diverged species. We herein report the development of a detailed RH and comparative map for horse chromosome 17 (ECA17). With markers distributed at an average interval of every 1.4 Mb, the map is currently the most informative among the equine chromosomes. It comprises 75 markers (56 genes and 19 microsatellites), of which 50 gene specific and 5 microsatellite markers were generated in this study and typed to our 5000-rad horse x hamster whole genome RH panel. The markers are dispersed over six RH linkage groups and span 825 cR(5000). The map is among the most comprehensive whole chromosome comparative maps currently available for domesticated animals. It finely aligns ECA17 to human and mouse homologues (HSA13 and MMU1, 3, 5, 8, and 14, respectively) and homologues in other domesticated animals. Comparisons provide insight into their relative organization and help to identify evolutionarily conserved segments. The new ECA17 map will serve as a template for the development of clusters of BAC contigs in regions containing genes of interest. Sequencing of these regions will help to initiate studies aimed at understanding the molecular mechanisms for various diseases and inherited disorders in horse as well as human.

  18. Application of a cDNA microarray for profiling the gene expression of Echinococcus granulosus protoscoleces treated with albendazole and artemisinin.

    Science.gov (United States)

    Lü, Guodong; Zhang, Wenbao; Wang, Jianhua; Xiao, Yunfeng; Zhao, Jun; Zhao, Jianqin; Sun, Yimin; Zhang, Chuanshan; Wang, Junhua; Lin, Renyong; Liu, Hui; Zhang, Fuchun; Wen, Hao

    2014-12-01

    Cystic echinoccocosis (CE) is a neglected zoonosis that is caused by the dog-tapeworm Echinococcus granulosus. The disease is endemic worldwide. There is an urgent need for searching effective drug for the treatment of the disease. In this study, we sequenced a cDNA library constructed using RNA isolated from oncospheres, protoscoleces, cyst membrane and adult worms of E. granulosus. A total of 9065 non-redundant or unique sequences were obtained and spotted on chips as uniEST probes to profile the gene expression in protoscoleces of E. granulosus treated with the anthelmintic drugs albendazole and artemisinin, respectively. The results showed that 7 genes were up-regulated and 38 genes were down-regulated in the protoscoleces treated with albendazole. Gene analysis showed that these genes are responsible for energy metabolism, cell cycle and assembly of cell structure. We also identified 100 genes up-regulated and 6 genes down-regulated in the protoscoleces treated with artemisinin. These genes play roles in the transduction of environmental signals, and metabolism. Albendazole appeared its drug efficacy in damaging cell structure, while artemisinin was observed to increase the formation of the heterochromatin in protoscolex cells. Our results highlight the utility of using cDNA microarray methods to detect gene expression profiles of E. granulosus and, in particular, to understand the pharmacologic mechanism of anti-echinococcosis drugs.

  19. Alkylresorcinol content and homologue composition in durum wheat (Triticum durum) kernels and pasta products.

    Science.gov (United States)

    Landberg, Rikard; Kamal-Eldin, Afaf; Andersson, Roger; Aman, Per

    2006-04-19

    The total alkylresorcinol (AR) content and relative homologue composition of 21 durum wheat (Triticum durum) kernel samples, as well as 5 pasta products and the corresponding flour mixtures, were determined. Durum wheat contained on average 455 microg/g ARs, and the average relative homologue composition was C17:0 (0.4%), C19:0 (14%), C21:0 (58%), C23:0 (21%), and C25:0 (6.5%). The homologue composition was found to be relatively consistent among samples, with durum wheat being different from common wheat by having a higher proportion of the longer homologues. No differences in content or homologue composition were observed in pasta products compared to flour ingredients, showing that alkylresorcinols are stable during pasta processing. The ratio of the homologues C17:0 to C21:0 was wheat products, which is different from those of common wheat (0.1) and rye (0.9).

  20. Epidermal growth factor receptor gene copy number in 101 advanced colorectal cancer patients treated with chemotherapy plus cetuximab

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    Zeuli Massimo

    2010-04-01

    Full Text Available Abstract Background Responsiveness to Cetuximab alone can be mediated by an increase of Epidermal Growth factor Receptor (EGFR Gene Copy Number (GCN. Aim of this study was to assess the role of EGFR-GCN in advanced colorectal cancer (CRC patients receiving chemotherapy plus Cetuximab. Methods One hundred and one advanced CRC patients (43 untreated- and 58 pre-treated were retrospectively studied by fluorescence in situ hybridization (FISH to assess EGFR-GCN and by immunohistochemistry (IHC to determine EGFR expression. Sixty-one out of 101 patients were evaluated also for k-ras status by direct sequencing. Clinical end-points were response rate (RR, progression-free survival (PFS and overall survival (OS. Results Increased EGFR-GCN was found in 60/101 (59% tumor samples. There was no correlation between intensity of EGFR-IHC and EGFR-GCN (p = 0.43. Patients receiving chemotherapy plus Cetuximab as first line treatment had a RR of 70% (30/43 while it was 18% (10/56 in the group with previous lines of therapy (p Conclusion In metastatic CRC patients treated with chemotherapy plus Cetuximab number of chemotherapy lines and increased EGFR-GCN were significantly associated with a better clinical outcome, independent of k-ras status.

  1. Structure of a DsbF homologue from Corynebacterium diphtheriae.

    Science.gov (United States)

    Um, Si-Hyeon; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2014-09-01

    Disulfide-bond formation, mediated by the Dsb family of proteins, is important in the correct folding of secreted or extracellular proteins in bacteria. In Gram-negative bacteria, disulfide bonds are introduced into the folding proteins in the periplasm by DsbA. DsbE from Escherichia coli has been implicated in the reduction of disulfide bonds in the maturation of cytochrome c. The Gram-positive bacterium Mycobacterium tuberculosis encodes DsbE and its homologue DsbF, the structures of which have been determined. However, the two mycobacterial proteins are able to oxidatively fold a protein in vitro, unlike DsbE from E. coli. In this study, the crystal structure of a DsbE or DsbF homologue protein from Corynebacterium diphtheriae has been determined, which revealed a thioredoxin-like domain with a typical CXXC active site. Structural comparison with M. tuberculosis DsbF would help in understanding the function of the C. diphtheriae protein.

  2. Mitochondrial dysfunction and transactivation of p53-dependent apoptotic genes in BaP-treated human fetal lung fibroblasts.

    Science.gov (United States)

    Yang, Guangtao; Jiang, Ying; Rao, Kaimin; Chen, Xi; Wang, Qian; Liu, Ailin; Xiong, Wei; Yuan, Jing

    2011-12-01

    Benzo(a)pyrene (BaP) has been shown to be an inducer of apoptosis. However, mechanisms involved in BaP-induced mitochondrial dysfunction are not well-known. In this study, human fetal lung fibroblasts cells were treated with BaP (8, 16, 32, 64 and 128 μM) for 4 and 12 h. Cell viability, intracellular level of reactive oxygen species (ROS), total antioxidant capacity (T-AOC), mitochondrial membrane potential (ΔΨ(m)) and cytochrome c release were determined. Changes in transcriptional levels of p53-dependent apoptotic genes (p53, APAF1, CASPASE3, CASPASE9, NOXA and PUMA) were measured. At time point of 4 h, BaP induced the intracellular ROS generation in 64 (p BaP groups (p BaP groups (p BaP groups (p BaP group (p BaP groups (p BaP group a relatively little expression of p53 mRNA was observed (p BaP promoted the generation of excessive ROS and subsequently the mitochondrial depolarization, whereas transactivations of the p53-dependent apoptotic genes were significantly induced at the later period.

  3. Hepatic gene expression in multiparous Holstein cows treated with bovine somatotropin and fed n-3 fatty acids in early lactation.

    Science.gov (United States)

    Carriquiry, M; Weber, W J; Fahrenkrug, S C; Crooker, B A

    2009-10-01

    Multiparous cows were fed supplemental dietary fat and treated with bST to assess effects of n-3 fatty acid supply, bovine somatotropin (bST), and stage of lactation on hepatic gene expression. Cows were blocked by expected calving date and previous milk yield and assigned randomly to treatment. Supplemental dietary fat was provided from calving as either whole high-oil sunflower seeds (SS; 10% of dietary dry matter; n-6/n-3 ratio of 4.6) as a source of linoleic acid or a mixture of Alifet-High Energy and Alifet-Repro (AF; 3.5 and 1.5% of dietary dry matter, respectively; n-6/n-3 ratio of 2.6) as a source of protected n-3 fatty acids. Cows were treated with 0 (SSN, AFN) or 500 (SSY, AFY) mg of bST every 10 d from 12 to 70 d in milk (DIM) and at 14-d intervals thereafter. Liver biopsies were collected on -12, 10, 24, and 136 DIM for gene expression analysis. Growth hormone receptor (GHR), insulin-like growth factor-I (IGF-I), IGF-binding protein-3 (IGFBP3), hepatic nuclear factor 4alpha (HNF4alpha), fibroblast growth factor-21 (FGF-21), and peroxisome proliferator-activated receptor alpha (PPARalpha) were the target genes and hypoxanthine phosphoribosyltransferase (HPRT) was used as an endogenous control gene. Expression was measured by quantitative real-time reverse transcription-PCR analyses of 4 samples from each of 32 cows (8 complete blocks). Amounts of hepatic HPRT mRNA were not affected by bST or diet but were increased by approximately 3.8% in early lactation (3.42, 3.52, 3.54, and 3.41 x 10(4) message copies for -12, 10, 24, and 136 DIM, respectively). This small change had little detectable impact on the ability of HPRT to serve as an internal control gene. Amounts of hepatic GHR, IGF-I, and IGFBP3 mRNA were reduced by 1.5 to 2-fold after calving. Expression of GHR and IGF-I increased and IGFBP3 tended to increase within 12 d (by 24 DIM) of bST administration. These effects of bST persisted through 136 DIM. Hepatic HNF4alpha mRNA was not altered by DIM or

  4. Concordance among gene expression-based predictors for ER-positive breast cancer treated with adjuvant tamoxifen.

    Science.gov (United States)

    Prat, A; Parker, J S; Fan, C; Cheang, M C U; Miller, L D; Bergh, J; Chia, S K L; Bernard, P S; Nielsen, T O; Ellis, M J; Carey, L A; Perou, C M

    2012-11-01

    ER-positive (ER+) breast cancer includes all of the intrinsic molecular subtypes, although the luminal A and B subtypes predominate. In this study, we evaluated the ability of six clinically relevant genomic signatures to predict relapse in patients with ER+ tumors treated with adjuvant tamoxifen only. Four microarray datasets were combined and research-based versions of PAM50 intrinsic subtyping and risk of relapse (PAM50-ROR) score, 21-gene recurrence score (OncotypeDX), Mammaprint, Rotterdam 76 gene, index of sensitivity to endocrine therapy (SET) and an estrogen-induced gene set were evaluated. Distant relapse-free survival (DRFS) was estimated by Kaplan-Meier and log-rank tests, and multivariable analyses were done using Cox regression analysis. Harrell's C-index was also used to estimate performance. All signatures were prognostic in patients with ER+ node-negative tumors, whereas most were prognostic in ER+ node-positive disease. Among the signatures evaluated, PAM50-ROR, OncotypeDX, Mammaprint and SET were consistently found to be independent predictors of relapse. A combination of all signatures significantly increased the performance prediction. Importantly, low-risk tumors (>90% DRFS at 8.5 years) were identified by the majority of signatures only within node-negative disease, and these tumors were mostly luminal A (78%-100%). Most established genomic signatures were successful in outcome predictions in ER+ breast cancer and provided statistically independent information. From a clinical perspective, multiple signatures combined together most accurately predicted outcome, but a common finding was that each signature identified a subset of luminal A patients with node-negative disease who might be considered suitable candidates for adjuvant endocrine therapy alone.

  5. Histamine homologues discriminating between two functional H3 receptor assays. Evidence for H3 receptor heterogeneity?.

    Science.gov (United States)

    Leurs, R; Kathmann, M; Vollinga, R C; Menge, W M; Schlicker, E; Timmerman, H

    1996-03-01

    We studied several histamine homologues as potential ligands for the histamine H3 receptor in two binding assays ([125l]iodophenpropit and N alpha-[3H]methylhistamine binding to rat brain cortex membranes) and two functional H3 receptor models (inhibition of the neurogenic contraction in the guinea pig jejunum and of [3H]noradrenaline release in mouse brain cortex slices). The histamine homologues acted all as competitive H3 antagonists at the guinea pig jejunum. The potency in this model and/or the affinity for N alpha-[3H]methylhistamine binding was higher for the butylene (pA2 = 7.7; pKi = 9.4) and pentylene homologue (impentamine, pA2 = 8.4; pKi = 9.1) than for the propylene, hexylene and octylene homologues (pA2 = 5.9-7.8; pKi = 6.1-7.6). In the mouse brain cortex the propylene, butylene and pentylene homologues acted as partial agonists (alpha = 0.3-0.6) and the hexylene and octylene homologues acted as antagonists. [125I]Iodophenpropit binding was displaced monophasically by the propylene, hexylene and octylene homologues and biphasically by the butylene and pentylene homologues. Biphasic displacement curves were converted to monophasic ones by 10 microM guanosine-5'-O-(3-thiotriphosphate. In conclusion, the homologue of histamine with five methylene groups is a more potent H3 receptor antagonist in the guinea pig jejunum than the other homologues tested. Furthermore, the propylene, butylene and pentylene homologues can discriminate between the two functional H3 receptor models in the guinea pig jejunum and mouse brain. These data are discussed in relation to the efficiency of receptor coupling and receptor heterogeneity.

  6. Characterization and expression analysis of an allograft inflammatory factor-1 homologue in yellow grouper (Epinephelus awoara)

    Institute of Scientific and Technical Information of China (English)

    WANG Li; SHI Dawei; WU Xinzhong

    2008-01-01

    Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic calcium-binding protein involved in iullammatory response-related dis-eases in mammals.Previously an identified AIF-1 gene was simply reported in yellow grouper.The characterization of AIF-1 gene and its expression at the gene and protein level are further described.Yellow grouper AIF-1 is composed of 147 amino acids,and 64% ~ 84% identical to other homologues.Basal level AIF-I mRNA expression was noted in spleen,anterior kid-ney and kidney,using reverse-transcription polymerase chain reaction (RT-PCR).After stimulation of LPS,the AIF-1 mRNA expression was up-regulated in tissues examined:spleen,anterior kidney,kidney,heart and liver,but not in muscle.The re-combinant AIF-1 protein was expressed in Escherichia coli,and then purified for the development of antiserum.Western blotting analysis revealed a band with a molecular mass of about 17 ku.

  7. A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

    Science.gov (United States)

    Wang, W.; Takezawa, D.; Poovaiah, B. W.

    1996-01-01

    A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

  8. Comparative analysis of global gene expression profiles between diabetic rat wounds treated with vacuum-assisted closure therapy, moist wound healing or gauze under suction.

    Science.gov (United States)

    Derrick, Kathleen L; Norbury, Kenneth; Kieswetter, Kris; Skaf, Jihad; McNulty, Amy K

    2008-12-01

    How differential gene expression affects wound healing is not well understood. In this study, Zucker diabetic fatty (fa/fa) male inbred rats were used to investigate gene expression during wound healing in an impaired wound-healing model. Whole genome microarray surveys were used to gain insight into the biological pathways and healing processes in acute excisional wounds treated with vacuum-assisted closure (V.A.C.). Therapy, moist wound healing (MWH) or gauze under suction (GUS). Global gene expression analyses after 2 days of healing indicated major differences with respect to both number of genes showing fold changes and pathway regulation between the three different wound treatments. Statistical analysis of expression profiles indicated that 5072 genes showed a >1.6-fold change with V.A.C. Therapy compared with 3601 genes with MWH and 3952 genes with GUS. Pathways and related genes associated with the early phases of wound healing diverged between treatment groups. For example, pathways involving angiogenesis, cytoskeletal regulation and inflammation were associated with elevated gene expression following V.A.C. Therapy. This study is the first to assess wound healing by whole genome interrogation in a diabetic rat model treated with different healing modalities.

  9. A Biotin Biosynthesis Gene Restricted to Helicobacter.

    Science.gov (United States)

    Bi, Hongkai; Zhu, Lei; Jia, Jia; Cronan, John E

    2016-02-12

    In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the BioC methyltransferase allows synthesis of the pimelate moiety by a modified fatty acid biosynthetic pathway. Analyses of the extant bacterial genomes showed that bioH is absent from many bioC-containing bacteria and is replaced by other genes. Helicobacter pylori lacks a gene encoding a homologue of the known pimeloyl-ACP methyl ester cleavage enzymes suggesting that it encodes a novel enzyme that cleaves this intermediate. We isolated the H. pylori gene encoding this enzyme, bioV, by complementation of an E. coli bioH deletion strain. Purified BioV cleaved the physiological substrate, pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a catalytic triad, each member of which was essential for activity. The role of BioV in biotin biosynthesis was demonstrated using a reconstituted in vitro desthiobiotin synthesis system. BioV homologues seem the sole pimeloyl-ACP methyl ester esterase present in the Helicobacter species and their occurrence only in H. pylori and close relatives provide a target for development of drugs to specifically treat Helicobacter infections.

  10. Gene expression signatures that predict outcome of tamoxifen-treated estrogen receptor-positive, high-risk, primary breast cancer patients: a DBCG study.

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    Maria B Lyng

    Full Text Available BACKGROUND: Tamoxifen significantly improves outcome for estrogen receptor-positive (ER+ breast cancer, but the 15-year recurrence rate remains 30%. The aim of this study was to identify gene profiles that accurately predicted the outcome of ER+ breast cancer patients who received adjuvant Tamoxifen mono-therapy. METHODOLOGY/PRINCIPAL FINDINGS: Post-menopausal breast cancer patients diagnosed no later than 2002, being ER+ as defined by >1% IHC staining and having a frozen tumor sample with >50% tumor content were included. Tumor samples from 108 patients treated with adjuvant Tamoxifen were analyzed for the expression of 59 genes using quantitative-PCR. End-point was clinically verified recurrence to distant organs or ipsilateral breast. Gene profiles were identified using a model building procedure based on conditional logistic regression and leave-one-out cross-validation, followed by a non-parametric bootstrap (1000x re-sampling. The optimal profiles were further examined in 5 previously-reported datasets containing similar patient populations that were either treated with Tamoxifen or left untreated (n = 623. Three gene signatures were identified, the strongest being a 2-gene combination of BCL2-CDKN1A, exhibiting an accuracy of 75% for prediction of outcome. Independent examination using 4 previously-reported microarray datasets of Tamoxifen-treated patient samples (n = 503 confirmed the potential of BCL2-CDKN1A. The predictive value was further determined by comparing the ability of the genes to predict recurrence in an additional, previously-published, cohort consisting of Tamoxifen-treated (n = 58, p = 0.015 and untreated patients (n = 62, p = 0.25. CONCLUSIONS/SIGNIFICANCE: A novel gene expression signature predictive of outcome of Tamoxifen-treated patients was identified. The validation suggests that BCL2-CDKN1A exhibit promising predictive potential.

  11. Polymorphisms of homologous recombination genes and clinical outcomes of non-small cell lung cancer patients treated with definitive radiotherapy.

    Directory of Open Access Journals (Sweden)

    Ming Yin

    Full Text Available The repair of DNA double-strand breaks (DSBs is the major mechanism to maintain genomic stability in response to irradiation. We hypothesized that genetic polymorphisms in DSB repair genes may affect clinical outcomes among non-small cell lung cancer (NSCLC patients treated with definitive radio(chemotherapy. We genotyped six potentially functional single nucleotide polymorphisms (SNPs (i.e., RAD51 -135G>C/rs1801320 and -172G>T/rs1801321, XRCC2 4234G>C/rs3218384 and R188H/rs3218536 G>A, XRCC3 T241M/rs861539 and NBN E185Q/rs1805794 and estimated their associations with overall survival (OS and radiation pneumonitis (RP in 228 NSCLC patients. We found a predictive role of RAD51 -135G>C SNP in RP development (adjusted hazard ratio [HR] = 0.52, 95% confidence interval [CI], 0.31-0.86, P = 0.010 for CG/CC vs. GG. We also found that RAD51 -135G>C and XRCC2 R188H SNPs were independent prognostic factors for overall survival (adjusted HR = 1.70, 95% CI, 1.14-2.62, P = 0.009 for CG/CC vs. GG; and adjusted HR = 1.70; 95% CI, 1.02-2.85, P = 0.043 for AG vs. GG, respectively and that the SNP-survival association was most pronounced in the presence of RP. Our study suggests that HR genetic polymorphisms, particularly RAD51 -135G>C, may influence overall survival and radiation pneumonitis in NSCLC patients treated with definitive radio(chemotherapy. Large studies are needed to confirm our findings.

  12. Structure of a bacterial homologue of vitamin K epoxide reductase

    Energy Technology Data Exchange (ETDEWEB)

    Li, Weikai; Schulman, Sol; Dutton, Rachel J.; Boyd, Dana; Beckwith, Jon; Rapoport, Tom A. (Harvard-Med); (HHMI)

    2010-03-19

    Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain {gamma}-carboxylation of many blood coagulation factors. Here, we report the 3.6 {angstrom} crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.

  13. Validation of housekeeping genes for the normalization of RT-qPCR expression studies in oral squamous cell carcinoma cell line treated by 5 kinds of chemotherapy drugs.

    Science.gov (United States)

    Song, W; Zhang, W H; Zhang, H; Li, Y; Zhang, Y; Yin, W; Yang, Q

    2016-11-30

    Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become a frequently used strategy in gene expression studies. The relative quantification method is an important and commonly used method for the evaluation of RT-qPCR data. The key of this method is to identify an applicable internal control gene because the usage of different internal control genes may lead to distinct conclusions. Herein, we report the validation of 12 common housekeeping genes for RT-qPCR for gene expression analysis in the Oral squamous cell carcinoma (OSCC) cell line (KB and Tca-8113) treated by 5 kinds of Chemotherapy Drugs. The gene expression stability and applicability of the 12 housekeeping gene candidates were determined using the geNorm, NormFinder, and BestKeeper software programs. Comprehensive analyzing the results of the three software, ALAS1/GAPDH, ALAS1 and GUSB were suggested to be the most stable candidate genes for the study of both KB and Tca-8113 cell line together, KB cell line, and Tca-8113 cell line, respectively. This study provides useful information to normalize gene expression accurately for the investigation of target gene profiling in cell lines of OSCC. Further clarification of tumor molecular expression markers with our recommended housekeeping genes may improve the accuracy of diagnosis and estimation of prognostic factors as well as provide novel personalized treatments for OSCC patients.

  14. Mitochondrial and oxidative stress genes are differentially expressed in neutrophils of sJIA patients treated with tocilizumab: a pilot microarray study

    OpenAIRE

    Omoyinmi, E; Hamaoui, R.; Bryant, A.; Jiang, M. C.; Athigapanich, T.; Eleftheriou, D; Hubank, M; Brogan, P.; Woo, P.

    2016-01-01

    Background Various pathways involved in the pathogenesis of sJIA have been identified through gene expression profiling in peripheral blood mononuclear cells (PBMC), but not in neutrophils. Since neutrophils are important in tissue damage during inflammation, and are elevated as part of the acute phase response, we hypothesised that neutrophil pathways could also be important in the pathogenesis of sJIA. We therefore studied the gene profile in both PBMC and neutrophils of sJIA patients treat...

  15. Effects of AGTR1 A1166C gene polymorphism in patients with heart failure treated with candesartan.

    Science.gov (United States)

    de Denus, Simon; Zakrzewski-Jakubiak, Marcin; Dubé, Marie-Pierre; Bélanger, François; Lepage, Serge; Leblanc, Marie-Hélène; Gossard, Denis; Ducharme, Anique; Racine, Normand; Whittom, Lucette; Lavoie, Joel; Touyz, Rhian M; Turgeon, Jacques; White, Michel

    2008-07-01

    The benefits of angiotensin II receptor blockers (ARBs) in patients with heart failure who are treated with standard pharmacotherapy, including an angiotensin-converting enzyme (ACE) inhibitor, were demonstrated in 2 large randomized trials. It is currently impossible to determine which patient will benefit from the addition of an ARB. To explore the impact of selected candidate genes on the hemodynamic, neurohormonal, and antiinflammatory effects of candesartan in patients with heart failure who are already being treated with an ACE inhibitor. We investigated the impact of 10 candidate genetic polymorphisms on the effects of candesartan in patients with heart failure who are treated with an ACE inhibitor. We evaluated their impact on acute (2 wk) and long-term (24 wk) changes in blood pressure and N-terminal proB-type natriuretic peptide (NT-proBNP) and high sensitivity C-reactive protein (hsCRP) during treatment with candesartan. Thirty-one patients were included. Homozygotes of the AGTR1 A1166 allele (n = 13) had a greater decrease in systolic (-9.1 +/- 4.7 vs 1.1 +/- 3.3 mm Hg; p = 0.04 by analysis of variance [ANOVA], adjusting for dose) and diastolic blood pressure (-5.1 +/- 1.5 vs 1.9 +/- 1.9 mm Hg; p = 0.005 by ANOVA, adjusting for dose) compared with C1166 allele carriers (n = 18) following 2 weeks of treatment. After 6 months of treatment, C1166 carriers experienced a greater decrease in NT-proBNP (-151.4 [-207; -19.8] ng/L vs 147.3 [-61.3; 882.9] ng/L; p = 0.03) and hsCRP (-0.8 [-2.2; -0.03] mg/L) vs 0.2 [-1.8; 5.3] mg/L; p = 0.09) compared with patients carrying the AA1166 genotype. No other significant association was found. The results of this proof-of concept study provide the first evidence that the AGTR1 A1166C polymorphism could influence the response to candesartan in patients with heart failure who are receiving ACE inhibitors. Validation of these exploratory findings in larger populations is required before use of the AGTR1 A1166C genotype can

  16. Profiling of differentially expressed chemotactic-related genes in MCP-1 treated macrophage cell line using human cDNA arrays

    Institute of Scientific and Technical Information of China (English)

    Guang-Xing Bian; Hong Miao; Lei Qiu; Dong-Mei Cao; Bao-Yu Guo

    2005-01-01

    AIM: To study the global gene expression of chemotactic genes in macrophage line U937 treated with human monocyte chemoattractant protein-1 (MCP-1) through the use of ExpreeChipTMHO2 cDNA array.METHODS: Total RNA was extracted from MCP-1 treated macrophage line U937 and normal U937 cells, reversely transcribed to cDNA, and then screened in parallel with HO2 human cDNA array chip. The scanned result was additionally validated using RT-PCR.RESULTS: The result of cDNA array showed that one chemotactic-related gene was up-regulated more than two-fold (RANTES) and seven chemotactic-related genes were down-regulated more than two-fold (CCR1, CCR5,ccl16, GROβ, GROγ, IL-8 and granulocyte chemotactic protein 2) in MCP-1 treated U937 cells at mRNA level.RT-PCR analysis of four of these differentially expressed genes gave results consistent with cDNA array findings.CONCLUSION: MCP-1 could influence some chemokine and receptor expressions in macrophages in vitro. MCP-1mainly down-regulates the expression of chemotactic genes influencing neutrophilic granulocyte expression (GROβ, GROγ, IL-8 and granulocyte chemotactic protein 2), and the mRNA level of CCR5, which plays a critical role in many disorders and illnesses.

  17. Regulatory elements and transcriptional control of chicken vasa homologue (CVH) promoter in chicken primordial germ cells.

    Science.gov (United States)

    Jin, So Dam; Lee, Bo Ram; Hwang, Young Sun; Lee, Hong Jo; Rim, Jong Seop; Han, Jae Yong

    2017-01-01

    Primordial germ cells (PGCs), the precursors of functional gametes, have distinct characteristics and exhibit several unique molecular mechanisms to maintain pluripotency and germness in comparison to somatic cells. They express germ cell-specific RNA binding proteins (RBPs) by modulating tissue-specific cis- and trans-regulatory elements. Studies on gene structures of chicken vasa homologue (CVH), a chicken RNA binding protein, involved in temporal and spatial regulation are thus important not only for understanding the molecular mechanisms that regulate germ cell fate, but also for practical applications of primordial germ cells. However, very limited studies are available on regulatory elements that control germ cell-specific expression in chicken. Therefore, we investigated the intricate regulatory mechanism(s) that governs transcriptional control of CVH. We constructed green fluorescence protein (GFP) or luciferase reporter vectors containing the various 5' flanking regions of CVH gene. From the 5' deletion and fragmented assays in chicken PGCs, we have identified a CVH promoter that locates at -316 to +275 base pair fragment with the highest luciferase activity. Additionally, we confirmed for the first time that the 5' untranslated region (UTR) containing intron 1 is required for promoter activity of the CVH gene in chicken PGCs. Furthermore, using a transcription factor binding prediction, transcriptome analysis and siRNA-mediated knockdown, we have identified that a set of transcription factors play a role in the PGC-specific CVH gene expression. These results demonstrate that cis-elements and transcription factors localizing in the 5' flanking region including the 5' UTR and an intron are important for transcriptional regulation of the CVH gene in chicken PGCs. Finally, this information will contribute to research studies in areas of reproductive biology, constructing of germ cell-specific synthetic promoter for tracing primordial germ cells as well as

  18. Transcriptome and proteome analysis of tyrosine kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes

    Directory of Open Access Journals (Sweden)

    Klopfleisch Robert

    2012-06-01

    Full Text Available Abstract Background Canine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Inhibitors of the KIT tyrosine kinase have recently been introduced and successfully applied as a therapeutic agent for this tumour type. However, little is known on the downstream target genes of this signaling pathway and molecular changes after inhibition. Results Transcriptome analysis of the canine mast cell tumour cell line C2 treated for up to 72 hours with the tyrosine kinase inhibitor masitinib identified significant changes in the expression levels of approximately 3500 genes or 16% of the canine genome. Approximately 40% of these genes had increased mRNA expression levels including genes associated with the pro-proliferative pathways of B- and T-cell receptors, chemokine receptors, steroid hormone receptors and EPO-, RAS and MAP kinase signaling. Proteome analysis of C2 cells treated for 72 hours identified 24 proteins with changed expression levels, most of which being involved in gene transcription, e.g. EIA3, EIA4, TARDBP, protein folding, e.g. HSP90, UCHL3, PDIA3 and protection from oxidative stress, GSTT3, SELENBP1. Conclusions Transcriptome and proteome analysis of neoplastic canine mast cells treated with masitinib confirmed the strong important and complex role of KIT in these cells. Approximately 16% of the total canine genome and thus the majority of the active genes were significantly transcriptionally regulated. Most of these changes were associated with reduced proliferation and metabolism of treated cells. Interestingly, several pro-proliferative pathways were up-regulated, which may represent attempts of masitinib treated cells to activate alternative pro-proliferative pathways. These pathways may contain hypothetical targets for a combination therapy with masitinib to further improve its therapeutic effect.

  19. The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells.

    Science.gov (United States)

    Lavial, Fabrice; Acloque, Hervé; Bertocchini, Federica; Macleod, David J; Boast, Sharon; Bachelard, Elodie; Montillet, Guillaume; Thenot, Sandrine; Sang, Helen M; Stern, Claudio D; Samarut, Jacques; Pain, Bertrand

    2007-10-01

    Embryonic stem cells (ESC) have been isolated from pregastrulation mammalian embryos. The maintenance of their pluripotency and ability to self-renew has been shown to be governed by the transcription factors Oct4 (Pou5f1) and Nanog. Oct4 appears to control cell-fate decisions of ESC in vitro and the choice between embryonic and trophectoderm cell fates in vivo. In non-mammalian vertebrates, the existence and functions of these factors are still under debate, although the identification of the zebrafish pou2 (spg; pou5f1) and Xenopus Pou91 (XlPou91) genes, which have important roles in maintaining uncommitted putative stem cell populations during early development, has suggested that these factors have common functions in all vertebrates. Using chicken ESC (cESC), which display similar properties of pluripotency and long-term self-renewal to mammalian ESC, we demonstrated the existence of an avian homologue of Oct4 that we call chicken PouV (cPouV). We established that cPouV and the chicken Nanog gene are required for the maintenance of pluripotency and self-renewal of cESC. These findings show that the mechanisms by which Oct4 and Nanog regulate pluripotency and self-renewal are not exclusive to mammals.

  20. A novel lipocalin homologue from the venom gland of Deinagkistrodon acutus similar to mammalian lipocalins

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    CB Wei

    2012-01-01

    Full Text Available Lipocalins are involved in a variety of functions including retinol transport, cryptic coloration, olfaction, pheromone transport, prostaglandin synthesis, regulation of the immune response and cell homeostatic mediation. A full-length cDNA clone (named d-lipo, isolated from the venom gland cDNA library of Deinagkistrodon acutus, contained an insert of 664 bp including an open reading frame that encodes a lipocalin homologue of 177 amino acids. Comparison of d-lipo and other related proteins revealed an overall amino acid identity of less than 21.5%. Primary structures of d-lipo carried three structurally conserved regions (SCR showing homologies to those of lipocalins. The first conserved Cys residue - the essential amino acid residue for the catalytic activity and unique to lipocalin-type prostaglandin D synthase (L-PGDS in the lipocalin protein family - was identified in d-lipo at amino acid position 58. Phylogenetic tree analysis showed that d-lipo was in-between the large L-PGDS cluster and the small von Ebner's-gland proteins (VEGP cluster. Moreover, d-lipo gene presented a high-level expression in the venom gland and a low-level expression in the brain and its expression was significantly increased under pathological conditions, suggesting a possible relationship between d-lipo mRNA expression and the venom gland inflammatory disease. This is also the first report of a lipocalin homologous gene identified in the venom gland of a snake.

  1. Changes in diapause related gene expression pattern during early embryonic development in HCl-treated eggs of bivoltine silkworm Bombyx mori (Lepidoptera: Bombycidae

    Directory of Open Access Journals (Sweden)

    Sirigineedi Sasibhushan

    2013-02-01

    Full Text Available Investigation of differential expression of diapause related genes (five metabolic, five heat shock protein and one translational regulatory in HCl-treated (non-diapause and untreated (diapause eggs of B. mori during early embryogenesis (up to 48h following oviposition revealed the up-regulation of sorbitol dehydrogenase upon HCl treatment, indicating increased glycogen synthesis for further embryonic development but, down-regulation of phosphofructo kinase gene expression after 18h of oviposition indicating an arrest of glycerol and sorbitol conversion. The expression of poly A binding protein gene expression was higher upon HCl treatment, revealing the initiation of translation. The expression levels of other genes analyzed did not vary significantly, except for Hsp90 and Hsp40, which were up-regulated on acid treatment until 18h. Thus, Sorbitoldehydrogenase and phosphofructo kinasegenes have a crucial role in diapause termination as evidenced by HCl treatment, while the other genes did not have major roles.

  2. Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.

    Directory of Open Access Journals (Sweden)

    Lin-Lin Liu

    Full Text Available Reverse transcription-quantitative polymerase chain reaction (RT-qPCR is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct, and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2 expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.

  3. Expression of metallothionein gene at different time in testicular interstitial cells and liver of rats treated with cadmium

    Institute of Scientific and Technical Information of China (English)

    Xu-Yi Ren; Yong Zhou; Jian-Peng Zhang; Wei-Hua Feng; Bing-Hua Jiao

    2003-01-01

    AIM: Rodent testes are generally more susceptible to cadmium (Cd)-induced toxicity than liver. To clarify the molecular mechanism of Cd-induced toxicity in testes, we compared metallothionein (MT) gene expression, MT protein accumulation, and Cd retention at different time in freshly isolated testicular interstitial cells and liver of rats treated with Cd.METHODS: Adult male Sprague-Dawley rats weighing 250-280 g received a s.c injection of 4.0 μmol Cd/kg and were euthanized by CO2 asphyxiation 1h, 3 h, 6 h, or 24 h later.Tissue was sampled and testicular interstitial cells were isolated. There were three replicates per treatment and 3animals per replicate for RNA analyses, others, three replicates per treatment and one animal per replicate. MT1 and MT2 mRNA levels were determined by semi-quantitative RT-PCR analysis followed by densitometry scanning, and MT was estimated by the enzyme-linked immunosorbent assay (ELISA) method. Cadmium content was determined by atomic absorption spectrophotometry. The same parametersd were also analyzed in the liver, since this tissue unquestionably accumulate MT.RESULTS: The rat testis expressed MT1 and MT2, the major isoforms. We also found that untreated animals contained relatively high basal levels of both isoform mRNA, which were increased after Cd treatment in liver and peaked at 3 h, followed by a decline. In contrast, the mRNA levels in interstitial cells peaked at 6 h. Interestingly, the induction of MT1 mRNA was lower than MT2 mRNA in liver of rat treated with Cd, but it was opposite to interstitial cells. Cd exposure substantially increased hepatic MT (3.9-fold increase), but did not increase MT translation in interstitial cells. CONCLUSION: Cd-induced expression of MT isoforms is not only tissue dependent but also time-dependent. The inability to induce the metal-detoxicating MT-protein in response to Cd, may account for a higher susceptibility of testes to Cd toxicity and carcinogenesis compared to liver.

  4. Ionization potentials and electron affinities of the superheavy elements 115-117 and their sixth-row homologues Bi, Po, and At

    Science.gov (United States)

    Borschevsky, A.; Pašteka, L. F.; Pershina, V.; Eliav, E.; Kaldor, U.

    2015-02-01

    Calculations of the first and second ionization potentials and electron affinities of superheavy elements 115-117 are presented. The calculations are performed in the framework of the Dirac-Coulomb Hamiltonian, and the results are corrected for the Breit and QED contributions. Correlation is treated by the relativistic coupled cluster approach with single, double, and perturbative triple excitations [CCSD(T)]. The same approach is used to calculate the ionization potentials and electron affinities of the lighter homologues, Bi, Po, and At. Comparison of the available experimental values for these atoms, namely, the first ionization potentials (IPs) of Bi, Po, and At and the second IP and EA of Bi, with our results shows excellent agreement, within a few hundredths of an eV, lending credence to our predictions for their superheavy homologues. High-accuracy predictions are also made for the second ionization potentials and electron affinities of Po and At, where no experiment is available.

  5. Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides.

    Science.gov (United States)

    Thynne, Elisha; Saur, Isabel M L; Simbaqueba, Jaime; Ogilvie, Huw A; Gonzalez-Cendales, Yvonne; Mead, Oliver; Taranto, Adam; Catanzariti, Ann-Maree; McDonald, Megan C; Schwessinger, Benjamin; Jones, David A; Rathjen, John P; Solomon, Peter S

    2016-06-13

    In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1-like sequences were observed in most cases; however, RALF27-like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27-like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen-activated protein kinase activation). Gene expression analysis confirmed that a RALF-encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant-pathogen interactions.

  6. Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4

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    Kah Yan How

    2015-08-01

    Full Text Available Quorum sensing is a mechanism for regulating proteobacterial gene expression in response to changes in cell population. In proteobacteria, N-acyl homoserine lactone (AHL appears to be the most widely used signalling molecules in mediating, among others, the production of extracellular virulence factors for survival. In this work, the genome of B. cepacia strain GG4, a plasmid-free strain capable of AHL synthesis was explored. In silico analysis of the 6.6 Mb complete genome revealed the presence of a LuxI homologue which correspond to Type I quorum sensing. Here, we report the molecular cloning and characterization of this LuxI homologue, designated as BurI. This 609 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3. The purified protein was approximately 25 kDa and is highly similar to several autoinducer proteins of the LuxI family among Burkholderia species. To verify the AHL synthesis activity of this protein, high resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-hexanoylhomoserine lactone, N-octanoylhomoserine lactone and 3-hydroxy-octanoylhomoserine lactone from induced E. coli BL21 harboring the recombinant BurI. Our data show, for the first time, the cloning and characterization of the LuxI homologue from B. cepacia strain GG4 and confirmation of its AHL synthesis activity.

  7. Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4.

    Science.gov (United States)

    How, Kah Yan; Hong, Kar Wai; Chan, Kok-Gan

    2015-01-01

    Quorum sensing is a mechanism for regulating proteobacterial gene expression in response to changes in cell population. In proteobacteria, N-acyl homoserine lactone (AHL) appears to be the most widely used signalling molecules in mediating, among others, the production of extracellular virulence factors for survival. In this work, the genome of B. cepacia strain GG4, a plasmid-free strain capable of AHL synthesis was explored. In silico analysis of the 6.6 Mb complete genome revealed the presence of a LuxI homologue which correspond to Type I quorum sensing. Here, we report the molecular cloning and characterization of this LuxI homologue, designated as BurI. This 609 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was approximately 25 kDa and is highly similar to several autoinducer proteins of the LuxI family among Burkholderia species. To verify the AHL synthesis activity of this protein, high resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-hexanoylhomoserine lactone, N-octanoylhomoserine lactone and 3-hydroxy-octanoylhomoserine lactone from induced E. coli BL21 harboring the recombinant BurI. Our data show, for the first time, the cloning and characterization of the LuxI homologue from B. cepacia strain GG4 and confirmation of its AHL synthesis activity.

  8. A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae.

    Science.gov (United States)

    Kwon, T H; Kim, M S; Choi, H W; Joo, C H; Cho, M Y; Lee, B L

    2000-10-01

    Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor-I (PPAF-I) that encoded a member of the serine proteinase group with a disulfide-knotted motif at the N-terminus and a trypsin-like catalytic domain at the C-terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S. , Kawabata, S.I., Iwanaga, S. & Lee, B.L. (1998) Eur. J. Biochem. 257, 615-621]. PPAF-I is directly involved in the activation of pro-phenoloxidase (pro-PO) by limited proteolysis and the overall structure is highly similar to that of Drosophila easter serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development. Here, we report purification and molecular cloning of cDNA for another 45-kDa novel PPAF from the hemocyte lysate of Holotrichia diomphalia larvae. The gene encodes a serine proteinase homologue consisting of 415 amino-acid residues with a molecular mass of 45 256 Da. The overall structure of the 45-kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster. The 45-kDa protein contained a trypsin-like serine proteinase domain at the C-terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide-knotted domain at the N-terminus. A highly similar 45-kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran, Tenebrio molitor. By in vitro reconstitution experiments, we found that the purified 45-kDa serine proteinase homologue, the purified active PPAF-I and the purified pro-PO were necessary for expressing phenoloxidase activity in the Holotrichia pro-PO system. However, incubation of pro-PO with either PPAF-I or 45-kDa protein, no phenoloxidase activity was observed. Interestingly, when the 45-kDa protein was incubated with PPAF-I and pro-PO in the absence, but not in the presence of Ca2+, the 45-k

  9. Phosphatase and Tensin Homologue: Novel Regulation by Developmental Signaling

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    Travis J. Jerde

    2015-01-01

    Full Text Available Phosphatase and tensin homologue (PTEN is a critical cell endogenous inhibitor of phosphoinositide signaling in mammalian cells. PTEN dephosphorylates phosphoinositide trisphosphate (PIP3, and by so doing PTEN has the function of negative regulation of Akt, thereby inhibiting this key intracellular signal transduction pathway. In numerous cell types, PTEN loss-of-function mutations result in unopposed Akt signaling, producing numerous effects on cells. Numerous reports exist regarding mutations in PTEN leading to unregulated Akt and human disease, most notably cancer. However, less is commonly known about nonmutational regulation of PTEN. This review focuses on an emerging literature on the regulation of PTEN at the transcriptional, posttranscriptional, translational, and posttranslational levels. Specifically, a focus is placed on the role developmental signaling pathways play in PTEN regulation; this includes insulin-like growth factor, NOTCH, transforming growth factor, bone morphogenetic protein, wnt, and hedgehog signaling. The regulation of PTEN by developmental mediators affects critical biological processes including neuronal and organ development, stem cell maintenance, cell cycle regulation, inflammation, response to hypoxia, repair and recovery, and cell death and survival. Perturbations of PTEN regulation consequently lead to human diseases such as cancer, chronic inflammatory syndromes, developmental abnormalities, diabetes, and neurodegeneration.

  10. Characterization of Major Surface Protease Homologues of Trypanosoma congolense

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    Veronica Marcoux

    2010-01-01

    Full Text Available Trypanosomes encode a family of proteins known as Major Surface Metalloproteases (MSPs. We have identified six putative MSPs encoded within the partially sequenced T. congolense genome. Phylogenic analysis indicates that T. congolense MSPs belong to five subfamilies that are conserved among African trypanosome species. Molecular modeling, based on the known structure of Leishmania Major GP63, reveals subfamily-specific structural variations around the putative active site despite conservation of overall structure, suggesting that each MSP subfamily has evolved to recognize distinct substrates. We have cloned and purified a protein encoding the amino-terminal domain of the T. congolense homologue TcoMSP-D (most closely related to Leishmania GP63. We detect TcoMSP-D in the serum of T. congolense-infected mice. Mice immunized with the amino-terminal domain of TcoMSP-D generate a persisting IgG1 antibody response. Surprisingly, a low-dose challenge of immunized mice with T. congolense significantly increases susceptibility to infection, indicating that immunity to TcoMSP-D is a factor affecting virulence.

  11. A C. elegans stretch receptor neuron revealed by a mechanosensitive TRP channel homologue.

    Science.gov (United States)

    Li, Wei; Feng, Zhaoyang; Sternberg, Paul W; Xu, X Z Shawn

    2006-03-30

    The nematode Caenorhabditis elegans is commonly used as a genetic model organism for dissecting integration of the sensory and motor systems. Despite extensive genetic and behavioural analyses that have led to the identification of many genes and neural circuits involved in regulating C. elegans locomotion behaviour, it remains unclear whether and how somatosensory feedback modulates motor output during locomotion. In particular, no stretch receptors have been identified in C. elegans, raising the issue of whether stretch-receptor-mediated proprioception is used by C. elegans to regulate its locomotion behaviour. Here we have characterized TRP-4, the C. elegans homologue of the mechanosensitive TRPN channel. We show that trp-4 mutant worms bend their body abnormally, exhibiting a body posture distinct from that of wild-type worms during locomotion, suggesting that TRP-4 is involved in stretch-receptor-mediated proprioception. We show that TRP-4 acts in a single neuron, DVA, to mediate its function in proprioception, and that the activity of DVA can be stimulated by body stretch. DVA both positively and negatively modulates locomotion, providing a unique mechanism whereby a single neuron can fine-tune motor activity. Thus, DVA represents a stretch receptor neuron that regulates sensory-motor integration during C. elegans locomotion.

  12. Characterization of a chaperone ClpB homologue of Paracoccidioides brasiliensis.

    Science.gov (United States)

    Jesuino, Rosália S A; Azevedo, Maristela O; Felipe, M Sueli S; Pereira, Maristela; De Almeida Soares, Célia M

    2002-08-01

    We report the cloning and sequence analysis of a genomic clone encoding a Paracoccidioides brasiliensis ClpB chaperone homologue (PbClpB). The clpb gene was identified in a lambda Dash II library. Sequencing of Pbclpb revealed a long open reading frame capable of encoding a 792 amino acid, 87.9 kDa protein, pI of 5.34. The predicted polypeptide contains several consensus motifs of the ClpB proteins. Canonical sequences such as two putative nucleotide-binding sites, chaperonins ClpA/B signatures and highly conserved casein kinase phosphorylation domains are present. ClpB is 69% to 49% identical to members of the ClpB family from several organisms from prokaryotes to eukaryotes. The transcript of PbclpB was detected as a mRNA species of 3.0 kb, preferentially expressed in the yeast parasitic phase of the fungus. A 89 kDa protein was also detected in yeast cells of P. brasiliensis.

  13. Cyclophosphamide alters the gene expression profile in patients treated with high doses prior to stem cell transplantation.

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    Ibrahim El-Serafi

    Full Text Available BACKGROUND: Hematopoietic stem cell transplantation is a curative treatment for several haematological malignancies. However, treatment related morbidity and mortality still is a limiting factor. Cyclophosphamide is widely used in condition regimens either in combination with other chemotherapy or with total body irradiation. METHODS: We present the gene expression profile during cyclophosphamide treatment in 11 patients conditioned with cyclophosphamide for 2 days followed by total body irradiation prior to hematopoietic stem cell transplantation. 299 genes were identified as specific for cyclophosphamide treatment and were arranged into 4 clusters highly down-regulated genes, highly up-regulated genes, early up-regulated but later normalized genes and moderately up-regulated genes. RESULTS: Cyclophosphamide treatment down-regulated expression of several genes mapped to immune/autoimmune activation and graft rejection including CD3, CD28, CTLA4, MHC II, PRF1, GZMB and IL-2R, and up-regulated immune-related receptor genes, e.g. IL1R2, IL18R1, and FLT3. Moreover, a high and significant expression of ANGPTL1 and c-JUN genes was observed independent of cyclophosphamide treatment. CONCLUSION: This is the first investigation to provide significant information about alterations in gene expression following cyclophosphamide treatment that may increase our understanding of the cyclophosphamide mechanism of action and hence, in part, avoid its toxicity. Furthermore, ANGPTL1 remained highly expressed throughout the treatment and, in contrast to several other alkylating agents, cyclophosphamide did not influence c-JUN expression.

  14. Microarray and synchronization of neuronal differentiation with pathway changes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) databank in nerve growth factor-treated PC12 cells.

    Science.gov (United States)

    Lin, Chih-Ming; Feng, Wayne

    2012-08-01

    The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database creates networks from interrelations between molecular biology and underlying chemical elements. This allows for analysis of biologic networks, genomic information, and higher-order functional information at a systems level. We performed microarray experiments and used the KEGG database, systems biology analysis, and annotation of pathway function to study nerve growth factor (NGF)-induced differentiation of PC12 cells. Cells were cultured to 70%-80% confluence, treated with NGF for 1 or 3 hours (h), and RNA was extracted. Stage 1 data analysis involved analysis of variance (ANOVA), and stage 2 involved cluster analysis and heat map generation. We identified 2020 NGF-induced PC12 genes (1038 at 1 h and 1554 at 3 h). Results showed changes in gene expression over time. We compared these genes with 6035 genes from the KEGG database. Cross-matching resulted in 830 genes. Among these, we identified 395 altered genes (155 at 1 h and 301 at 3 h; 2-fold increase from 1 h to 3 h). We identified 191 biologic pathways in the KEGG database; the top 15 showed correlations with neuronal differentiation (mitogen-activated protein kinase [MAPK] pathway: 35 genes at 1 h, 54 genes at 3 h; genes associated with axonal guidance: 12 at 1 h, 26 at 3 h; Wnt pathway: 16 at 1 h, 25 at 3 h; neurotrophin pathway: 4 at 1 h, 14 at 3 h). Thus, we identified changes in neuronal differentiation pathways with the KEGG database, which were synchronized with NGF-induced differentiation.

  15. Functional Cross-Talking between Differentially Expressed and Alternatively Spliced Genes in Human Liver Cancer Cells Treated with Berberine.

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    Zhen Sheng

    Full Text Available Berberine has been identified with anti-proliferative effects on various cancer cells. Many researchers have been trying to elucidate the anti-cancer mechanisms of berberine based on differentially expressed genes. However, differentially alternative splicing genes induced by berberine might also contribute to its pharmacological actions and have not been reported yet. Moreover, the potential functional cross-talking between the two sets of genes deserves further exploration. In this study, RNA-seq technology was used to detect the differentially expressed genes and differentially alternative spliced genes in BEL-7402 cancer cells induced by berberine. Functional enrichment analysis indicated that these genes were mainly enriched in the p53 and cell cycle signalling pathway. In addition, it was statistically proven that the two sets of genes were locally co-enriched along chromosomes, closely connected to each other based on protein-protein interaction and functionally similar on Gene Ontology tree. These results suggested that the two sets of genes regulated by berberine might be functionally cross-talked and jointly contribute to its cell cycle arresting effect. It has provided new clues for further researches on the pharmacological mechanisms of berberine as well as the other botanical drugs.

  16. Functional Cross-Talking between Differentially Expressed and Alternatively Spliced Genes in Human Liver Cancer Cells Treated with Berberine.

    Science.gov (United States)

    Sheng, Zhen; Sun, Yi; Zhu, Ruixin; Jiao, Na; Tang, Kailin; Cao, Zhiwei; Ma, Chao

    2015-01-01

    Berberine has been identified with anti-proliferative effects on various cancer cells. Many researchers have been trying to elucidate the anti-cancer mechanisms of berberine based on differentially expressed genes. However, differentially alternative splicing genes induced by berberine might also contribute to its pharmacological actions and have not been reported yet. Moreover, the potential functional cross-talking between the two sets of genes deserves further exploration. In this study, RNA-seq technology was used to detect the differentially expressed genes and differentially alternative spliced genes in BEL-7402 cancer cells induced by berberine. Functional enrichment analysis indicated that these genes were mainly enriched in the p53 and cell cycle signalling pathway. In addition, it was statistically proven that the two sets of genes were locally co-enriched along chromosomes, closely connected to each other based on protein-protein interaction and functionally similar on Gene Ontology tree. These results suggested that the two sets of genes regulated by berberine might be functionally cross-talked and jointly contribute to its cell cycle arresting effect. It has provided new clues for further researches on the pharmacological mechanisms of berberine as well as the other botanical drugs.

  17. [Patterns of PCDD/Fs, PCBs and PCNs homologues in fly ash from cement kilns].

    Science.gov (United States)

    Zhang, Jing; Ni, Yu-Wen; Zhang, Hai-Jun; Zhang, Xue-Ping; Zhang, Qing; Chen, Ji-Ping

    2009-02-15

    The concentrations and toxic equivalent (TEQ) values of PCDD/Fs, PCBs and PCNs in fly ash collected from three types of cement kilns (vertical shaft kiln, wet-process rotary kiln and dry-process rotary kiln) and two types of waste incinerators were determined, and the patterns of homologues and congeners were compared. The results showed that the total TEQ of PCDD/Fs, PCBs and PCNs in cement kiln fly ash, which were in the range of 4.0-62, 0.069-3.9 and 0.47-2.8 ng x kg(-1) respectively, were much lower than that of fly ash from waste incinerators. In cement kiln fly ash, the predominating PCDD/Fs homologues were TCDFs, and the chief 2, 3, 7, 8-PCDD/Fs congeners were OCDD, 2, 3, 7, 8-TCDF and 1, 2, 3, 4, 6, 7, 8-HpCDF. The patterns of PCBs homologues in cement kiln fly ash were similar to those of waste incinerators in which TeCB were predominating homologues. PCB77, PCB105, PCB118 were at higher concentrations than other co-polar PCBs. Different types of cement kiln fly ash presented similar PCNs homologue patterns. The predominant homologues were TeCN, whereas OcCN were not detected. PCN 66/67 which has dioxin like toxity was the most abundant congener in all fly ash.

  18. The human homologue of macaque area V6A.

    Science.gov (United States)

    Pitzalis, S; Sereno, M I; Committeri, G; Fattori, P; Galati, G; Tosoni, A; Galletti, C

    2013-11-15

    In macaque monkeys, V6A is a visuomotor area located in the anterior bank of the POs, dorsal and anterior to retinotopically-organized extrastriate area V6 (Galletti et al., 1996). Unlike V6, V6A represents both contra- and ipsilateral visual fields and is broadly retinotopically organized (Galletti et al., 1999b). The contralateral lower visual field is over-represented in V6A. The central 20°-30° of the visual field is mainly represented dorsally (V6Ad) and the periphery ventrally (V6Av), at the border with V6. Both sectors of area V6A contain arm movement-related cells, active during spatially-directed reaching movements (Gamberini et al., 2011). In humans, we previously mapped the retinotopic organization of area V6 (Pitzalis et al., 2006). Here, using phase-encoded fMRI, cortical surface-based analysis and wide-field retinotopic mapping, we define a new cortical region that borders V6 anteriorly and shows a clear over-representation of the contralateral lower visual field and the periphery. As with macaque V6A, the eccentricity increases moving ventrally within the area. The new region contains a non-mirror-image representation of the visual field. Functional mapping reveals that, as in macaque V6A, the new region, but not the nearby area V6, responds during finger pointing and reaching movements. Based on similarity in position, retinotopic properties, functional organization and relationship with the neighboring extrastriate visual areas, we propose that the new cortical region is the human homologue of macaque area V6A.

  19. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

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    Singh,S.; Yamashita, A.; Gouaux, E.

    2007-01-01

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 {angstrom} above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the

  20. Two plant bacteria, S. meliloti and Ca. Liberibacter asiaticus, share functional znuABC homologues that encode for a high affinity zinc uptake system.

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    Cheryl M Vahling-Armstrong

    Full Text Available The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not been functionally characterized in a plant-associated bacterium. A single homologue of this system has been identified in the plant endosymbiont, Sinorhizobium meliloti, while two homologous systems were found in the destructive citrus pathogen, Candidatus Liberibacter asiaticus. To understand the role of these protein homologues, a complementation assay was devised allowing the individual genes that comprise the system to be assayed independently for their ability to reinstate a partially-inactivated Znu system. Results from the assays have demonstrated that although all of the genes from S. meliloti were able to restore activity, only one of the two Ca. Liberibacter asiaticus encoded gene clusters contained genes that were able to functionally complement the system. Additional analysis of the gene clusters reveals that distinct modes of regulation may also exist between the Ca. Liberibacter asiaticus and S. meliloti import systems despite the intracellular-plant niche common to both of these bacteria.

  1. Transcription analysis of stx1, marA, and eaeA genes in Escherichia coli O157:H7 treated with sodium benzoate.

    Science.gov (United States)

    Critzer, Faith J; Dsouza, Doris H; Golden, David A

    2008-07-01

    Expression of the multiple antibiotic resistance (mar) operon causes increased antimicrobial resistance in bacterial pathogens. The activator of this operon, MarA, can alter expression of >60 genes in Escherichia coli K-12. However, data on the expression of virulence and resistance genes when foodborne pathogens are exposed to antimicrobial agents are lacking. This study was conducted to determine transcription of marA (mar activator), stx1 (Shiga toxin 1), and eaeA (intimin) genes of E. coli O157:H7 EDL933 as affected by sodium benzoate. E. coli O157:H7 was grown in Luria-Bertani broth containing 0 (control) and 1% sodium benzoate at 37 degrees C for 24 h, and total RNA was extracted. Primers were designed for hemX (209 bp; housekeeping gene), marA (261 bp), and eaeA (223 bp) genes; previously reported primers were used for stx1. Tenfold dilutions of RNA were used in a real-time one-step reverse transcriptase PCR to determine transcription levels. All experiments were conducted in triplicate, and product detection was validated by gel electrophoresis. For marA and stx1, real-time one-step reverse transcriptase PCR products were detected at a 1-log-greater dilution in sodium benzoate-treated cells than in control cells, although cell numbers for each were similar (7.28 and 7.57 log CFU/ml, respectively). This indicates a greater (albeit slight) level of their transcription in treated cells than in control cells. No difference in expression of eaeA was observed. HemX is a putative uroporphyrinogen III methylase. The hemX gene was expressed at the same level in control and treated cells, validating hemX as an appropriate housekeeping marker. These data indicate that stx1 and marA genes could play a role in pathogen virulence and survival when treated with sodium benzoate, whereas eaeA expression is not altered. Understanding adaptations of E. coli O157:H7 during antimicrobial exposure is essential to better understand and implement methods to inhibit or control

  2. Impaired gene expression of beta 1-adrenergic receptor, but not stimulatory G-protein Gs alpha, in rat ventricular myocardium treated with isoproterenol.

    Science.gov (United States)

    Kizaki, Keiichiro; Momozaki, Masami; Akatsuka, Keiko; Fujimori, Yuuki; Uchide, Tsuyoshi; Temma, Kyosuke; Hara, Yukio

    2004-07-01

    We investigated the gene expression of beta(1)-adrenergic receptor (beta(1)AR) and stimulatory G-protein Gsalpha, important signal transduction elements for regulating heart rate and contractility, in ventricle after chronic treatment with isoproterenol (ISO) in rat. Rats were treated with ISO (4 mg/kg, intraperitoneal) twice a day for 4 d. Ventricle weight of the heart and ventricle weight/body weight ratio were increased by 23% and 25% compared with control, respectively. Positive inotropic responses to ISO in left atrial muscle preparations isolated from ISO-treated rats were markedly decreased. Northern blot hybridization showed that the mRNA transcript of beta(1)AR was significantly decreased in ventricle of ISO-treated rats, whereas Gsalpha mRNA level was unchanged. Present results demonstrate that the gene expression of myocardial beta(1)AR, but not Gsalpha, was decreased in rat myocardium of ISO-induced cardiac hypertrophy, and suggesting that decrease in the gene expression of beta(1)AR may be one of the mechanisms responsible for the diminished cardiac function.

  3. Analysis of the gene expression profile of curcumin-treated kidney on endotoxin-induced renal inflammation.

    Science.gov (United States)

    Zhong, Fang; Chen, Hui; Jin, Yuanmeng; Guo, Shanmai; Wang, Weiming; Chen, Nan

    2013-02-01

    Acute or chronic kidney inflammation is closely related to the progress of kidney diseases. Curcumin, a yellow pigment present in the rhizome of turmeric (Curcuma longa L. Zingiberaceae), was found to be a potential anti-inflammatory agent. The present study aimed to investigate the effects and explore the protective mechanism of curcumin on lipopolysaccharide (LPS)-induced kidney inflammation in mice using gene chip and pathological technology. Nine SPF Kunming mice (aged 6-8 weeks, weighing 20-25 g) were divided into three groups. Saline and LPS were injected intraperitoneally in a normal control group and a model group, respectively. Mice in the treatment group were first injected with curcumin (5 mg/kg) for 3 days before being injected with LPS (5 mg/kg). Kidney tissues were harvested at 6 h after treatment. Parts of kidney were fixed with 10 % formaldehyde for HE, Periodic acid-Schiff staining, and immunohistochemistry. Affymetrix gene chips (mouse 430 chip) were used to detect the renal gene expression profile, and the results were analyzed using bioinformatics methods. The renal gene expression profile showed that there are 148 Affy IDs (up-down group) whose levels of gene expression were increased after LPS stimulation and decreased by curcumin treatment and that there are 133 Affy IDs (down-up group) exhibiting the opposite trend. In the differentially expressed genes of the up-down group, 21 Gene Ontology (GO) genes were selected by screening function (P ≤ 0.01). In the biological processes, most of the genes were found to be related to the genes of regulation of macrophage activation and macrophage activation-associated genes. In the cellular localization, there were four functional GO genes (P ≤ 0.01); in the molecular structure, there were seven functional GO genes (P ≤ 0.01). In the down-up group, there were functional GO genes (P ≤ 0.01) and one functional GO gene (P ≤ 0.01) in the biological process and the cellular

  4. Mitochondrial and oxidative stress genes are differentially expressed in neutrophils of sJIA patients treated with tocilizumab: a pilot microarray study.

    Science.gov (United States)

    Omoyinmi, Ebun; Hamaoui, Raja; Bryant, Annette; Jiang, Mike Chao; Athigapanich, Trin; Eleftheriou, Despina; Hubank, Mike; Brogan, Paul; Woo, Patricia

    2016-02-09

    Various pathways involved in the pathogenesis of sJIA have been identified through gene expression profiling in peripheral blood mononuclear cells (PBMC), but not in neutrophils. Since neutrophils are important in tissue damage during inflammation, and are elevated as part of the acute phase response, we hypothesised that neutrophil pathways could also be important in the pathogenesis of sJIA. We therefore studied the gene profile in both PBMC and neutrophils of sJIA patients treated with tocilizumab. We studied the transcriptomes of peripheral blood mononuclear cells (PBMC) and neutrophils from eight paired samples obtained from 4 sJIA patients taken before and after treatment, selected on the basis that they achieved ACR90 responses within 12 weeks of therapy initiation with tocilizumab. RNA was extracted and gene expression profiling was performed using Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray platform. A longitudinal analysis using paired t-test (p ontology analysis in neutrophils revealed that response to tocilizumab significantly altered genes regulating mitochondrial dysfunction and oxidative stress (p = 4.6E-05). This was independently verified with GSEA, by identifying a set of oxidative genes whose expression correlated with response to tocilizumab. In PBMC, treatment of sJIA with tocilizumab appeared to affect genes in Oncostatin M signalling and B cell pathways. For the first time we demonstrate that neutrophils from sJIA patients responding to tocilizumab showed significantly different changes in gene expression. These data could highlight the importance of mitochondrial genes that modulate oxidative stress in the pathogenesis of sJIA.

  5. Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues.

    Science.gov (United States)

    Wyroba, E; Surmacz, L; Osinska, M; Wiejak, J

    2007-01-01

    Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein) and LAMP-2 (lysosomal membrane protein 2) as well as alpha7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2) in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100,000 x g) of equal load were quantified by immunoblotting. LAMP-2 cross-reacting polypeptide of approximately106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The alpha7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILP-related polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

  6. Phenotypic characterization of OmpX, an Ail homologue of Yersinia pestis KIM.

    Science.gov (United States)

    Kolodziejek, Anna M; Sinclair, Dylan J; Seo, Keun S; Schnider, Darren R; Deobald, Claudia F; Rohde, Harold N; Viall, Austin K; Minnich, Scott S; Hovde, Carolyn J; Minnich, Scott A; Bohach, Gregory A

    2007-09-01

    The goal of this study was to characterize the Yersinia pestis KIM OmpX protein. Yersinia spp. provide a model for studying several virulence processes including attachment to, and internalization by, host cells. For Yersinia enterocolitica and Yersinia pseudotuberculosis, Ail, YadA and Inv, have been implicated in these processes. In Y. pestis, YadA and Inv are inactivated. Genomic analysis of two Y. pestis strains revealed four loci with sequence homology to Ail. One of these genes, designated y1324 in the Y. pestis KIM database, encodes a protein designated OmpX. The mature protein has a predicted molecular mass of 17.47 kDa, shares approximately 70 % sequence identity with Y. enterocolitica Ail, and has an identical homologue, designated Ail, in the Y. pestis CO92 database. The present study compared the Y. pestis KIM6(+) parental strain with a mutant derivative having an engineered disruption of the OmpX structural gene. The parental strain (and a merodiploid control strain) expressed OmpX at 28 and 37 degrees C, and the protein was detectable throughout all phases of growth. OmpX was required for efficient adherence to, and internalization by, cultured HEp-2 cell monolayers and conferred resistance to the bactericidal effect of human serum. Deletion of ompX resulted in a significantly reduced autoaggregation phenotype and loss of pellicle formation in vitro. These results suggest that Y. pestis OmpX shares functional homology with Y. enterocolitica Ail in adherence, internalization into epithelial cells and serum resistance.

  7. Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues

    Directory of Open Access Journals (Sweden)

    E Wyroba

    2009-08-01

    Full Text Available Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein and LAMP-2 (lysosomal membrane protein 2 as well as a7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2 in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100 000 x g of equal load were quantified by immunoblotting. LAMP-2 crossreacting polypeptide of ~106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The a7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILPrelated polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

  8. Stress associated gene expression in blood cells is related to outcome in radiotherapy treated head and neck cancer patients

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    Bøhn Siv K

    2012-09-01

    Full Text Available Abstract Background We previously observed that a radiotherapy-induced biochemical response in plasma was associated with favourable outcome in head and neck squamous carcinoma cancer (HNSCC patients. The aim of the present study was to compare stress associated blood cell gene expression between two sub-groups of HNSCC patients with different biochemical responses to radiotherapy. Methods Out of 87 patients (histologically verified, 10 biochemical ‘responders’ having a high relative increase in plasma oxidative damage and a concomitant decrease in plasma antioxidants during radiotherapy and 10 ‘poor-responders’ were selected for gene-expression analysis and compared using gene set enrichment analysis. Results There was a significant induction of stress-relevant gene-sets in the responders following radiotherapy compared to the poor-responders. The relevance of the involvement of similar stress associated gene expression for HNSCC cancer and radioresistance was verified using two publicly available data sets of 42 HNSCC cases and 14 controls (GEO GSE6791, and radiation resistant and radiation sensitive HNSCC xenografts (E-GEOD-9716. Conclusions Radiotherapy induces a systemic stress response, as revealed by induction of stress relevant gene expression in blood cells, which is associated to favourable outcome in a cohort of 87 HNSCC patients. Whether these changes in gene expression reflects a systemic effect or are biomarkers of the tumour micro-environmental status needs further study. Trial registration Raw data are available at ArrayExpress under accession number E-MEXP-2460.

  9. Expression of pro-inflammatory genes in lesions, spleens and blood neutrophils after burn injuries in mice treated with silver sulfodiazine.

    Science.gov (United States)

    Akhzari, Soheyla; Rezvan, Hossein; Zolhavarieh, Seyed Masoud

    2017-07-01

    It is now supposed that cytokines released during the burn injuries have a great impact on the immunological and pathological responses after the burn. The main objective of this study was to investigate the expression of some pro-inflammatory genes in the wound, spleen and blood neutrophils during the healing process of burn wounds in a murine model. The expression of ten pro-inflammatory genes were examined in wounds, spleens and blood neutrophils of mice with burn injuries treated with either silver sulfodiazine or phosphate-buffered saline (PBS) using RT-PCR at the end of the first and second weeks after injuries. None of the pro-inflammatory genes were expressed in the skin, spleen and blood neutrophils of healthy mice. In the group control, IL-12P35, IL-12P40, CCR5, IL-1β and IFN-γ were expressed in the spleen and blood neutrophils in the first week. Instead, CCL5, CCR5, IL-1β and IFN-γ were expressed in the wound, but in the second week, the expression of the genes became similar. In the test group, in the first week, TNF-α, IL-12P35, IL-12P40 and IL-1β were expressed in the lesions, CCL4, IL-1α, IL-12P35, IL-12P40, CCR5 and IFN-γ were expressed in the spleen and no pro-inflammatory gene expression was detected in blood neutrophils. IL-1β and IFN-γ are expressed in wound, spleen and neutrophils of untreated mice, but not in silver sulfodiazine treated mice. Hence, treatment with silver sulfodiazine suppressed the expression of pro-inflammatory genes in some stages of healing.

  10. Gene expression profiling and pathway analysis data in MCF-7 and MDA-MB-231 human breast cancer cell lines treated with dioscin.

    Science.gov (United States)

    Aumsuwan, Pranapda; Khan, Shabana I; Khan, Ikhlas A; Walker, Larry A; Dasmahapatra, Asok K

    2016-09-01

    Microarray technology (Human OneArray microarray, phylanxbiotech.com) was used to compare gene expression profiles of non-invasive MCF-7 and invasive MDA-MB-231 breast cancer cells exposed to dioscin (DS), a steroidal saponin isolated from the roots of wild yam, (Dioscorea villosa). Initially the differential expression of genes (DEG) was identified which was followed by pathway enrichment analysis (PEA). Of the genes queried on OneArray, we identified 4641 DEG changed between MCF-7 and MDA-MB-231 cells (vehicle-treated) with cut-off log2 |fold change|≧1. Among these genes, 2439 genes were upregulated and 2002 were downregulated. DS exposure (2.30 μM, 72 h) to these cells identified 801 (MCF-7) and 96 (MDA-MB-231) DEG that showed significant difference when compared with the untreated cells (pMB-231 cells. Further comparison of DEG between MCF-7 and MDA-MB-231 cells exposed to DS identified 3626 DEG of which 1700 were upregulated and 1926 were down-regulated. Regarding to PEA, 12 canonical pathways were significantly altered between these two cell lines. However, there was no alteration in any of these pathways in MCF-7 cells, while in MDA-MB-231 cells only MAPK pathway showed significant alteration. When PEA comparison was made on DS exposed cells, it was observed that only 2 pathways were significantly affected. Further, we identified the shared DEG, which were targeted by DS and overlapped in both MCF-7 and MDA-MB-231 cells, by intersection analysis (Venn diagram). We found that 7 DEG were overlapped of which six are reported in the database. This data highlight the diverse gene networks and pathways in MCF-7 and MDA-MB-231 human breast cancer cell lines treated with dioscin.

  11. Expression of pro-inflammatory genes in lesions, spleens and blood neutrophils after burn injuries in mice treated with silver sulfodiazine

    Directory of Open Access Journals (Sweden)

    Soheyla Akhzari

    2017-07-01

    Full Text Available Objective(s: It is now supposed that cytokines released during the burn injuries have a great impact on the immunological and pathological responses after the burn. The main objective of this study was to investigate the expression of some pro-inflammatory genes in the wound, spleen and blood neutrophils during the healing process of burn wounds in a murine model. Materials and Methods: The expression of ten pro-inflammatory genes were examined in wounds, spleens and blood neutrophils of mice with burn injuries treated with either silver sulfodiazine or phosphate-buffered saline (PBS using RT-PCR at the end of the first and second weeks after injuries. Results: None of the pro-inflammatory genes were expressed in the skin, spleen and blood neutrophils of healthy mice. In the group control, IL-12P35, IL-12P40, CCR5, IL-1β and IFN- γ were expressed in the spleen and blood neutrophils in the first week. Instead, CCL5, CCR5, IL-1β and IFN- γ were expressed in the wound, but in the second week, the expression of the genes became similar. In the test group, in the first week, TNF-α, IL-12P35, IL-12P40 and IL-1β were expressed in the lesions, CCL4, IL-1α, IL-12P35, IL-12P40, CCR5 and IFN- γ were expressed in the spleen and no pro-inflammatory gene expression was detected in blood neutrophils.  Conclusion: IL-1β and IFN- γ are expressed in wound, spleen and neutrophils of untreated mice, but not in silver sulfodiazine treated mice. Hence, treatment with silver sulfodiazine suppressed the expression of pro-inflammatory genes in some stages of healing.

  12. Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy.

    Science.gov (United States)

    Aiuti, Alessandro; Cassani, Barbara; Andolfi, Grazia; Mirolo, Massimiliano; Biasco, Luca; Recchia, Alessandra; Urbinati, Fabrizia; Valacca, Cristina; Scaramuzza, Samantha; Aker, Memet; Slavin, Shimon; Cazzola, Matteo; Sartori, Daniela; Ambrosi, Alessandro; Di Serio, Clelia; Roncarolo, Maria Grazia; Mavilio, Fulvio; Bordignon, Claudio

    2007-08-01

    Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase-deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34(+) cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.

  13. Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy

    Science.gov (United States)

    Aiuti, Alessandro; Cassani, Barbara; Andolfi, Grazia; Mirolo, Massimiliano; Biasco, Luca; Recchia, Alessandra; Urbinati, Fabrizia; Valacca, Cristina; Scaramuzza, Samantha; Aker, Memet; Slavin, Shimon; Cazzola, Matteo; Sartori, Daniela; Ambrosi, Alessandro; Di Serio, Clelia; Roncarolo, Maria Grazia; Mavilio, Fulvio; Bordignon, Claudio

    2007-01-01

    Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase–deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34+ cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy. PMID:17671653

  14. Differential adipose tissue gene expression profiles in abacavir treated patients that may contribute to the understanding of cardiovascular risk: a microarray study.

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    Mohsen Shahmanesh

    Full Text Available To compare changes in gene expression by microarray from subcutaneous adipose tissue from HIV treatment naïve patients treated with efavirenz based regimens containing abacavir (ABC, tenofovir (TDF or zidovidine (AZT.Subcutaneous fat biopsies were obtained before, at 6- and 18-24-months after treatment, and from HIV negative controls. Groups were age, ethnicity, weight, biochemical profile, and pre-treatment CD4 count matched. Microarray data was generated using the Agilent Whole Human Genome Microarray. Identification of differentially expressed genes and genomic response pathways was performed using limma and gene set enrichment analysis.There were significant divergences between ABC and the other two groups 6 months after treatment in genes controlling cell adhesion and environmental information processing, with some convergence at 18-24 months. Compared to controls the ABC group, but not AZT or TDF showed enrichment of genes controlling adherence junction, at 6 months and 18-24 months (adjusted p<0.05 and focal adhesions and tight junction at 6 months (p<0.5. Genes controlling leukocyte transendothelial migration (p<0.05 and ECM-receptor interactions (p = 0.04 were over-expressed in ABC compared to TDF and AZT at 6 months but not at 18-24 months. Enrichment of pathways and individual genes controlling cell adhesion and environmental information processing were specifically dysregulated in the ABC group in comparison with other treatments. There was little difference between AZT and TDF.After initiating treatment, there is divergence in the expression of genes controlling cell adhesion and environmental information processing between ABC and both TDF and AZT in subcutaneous adipose tissue. If similar changes are also taking place in other tissues including the coronary vasculature they may contribute to the increased risk of cardiovascular events reported in patients recently started on abacavir-containing regimens.

  15. Inflammatory gene expression in monocytes of patients with schizophrenia: overlap and difference with bipolar disorder. A study in naturalistically treated patients.

    Science.gov (United States)

    Drexhage, Roosmarijn C; van der Heul-Nieuwenhuijsen, Leonie; Padmos, Roos C; van Beveren, Nico; Cohen, Dan; Versnel, Marjan A; Nolen, Willem A; Drexhage, Hemmo A

    2010-11-01

    Accumulating evidence indicates an activated inflammatory response system as a vulnerability factor for schizophrenia (SZ) and bipolar disorder (BD). We aimed to detect a specific inflammatory monocyte gene expression signature in SZ and compare such signature with our recently described inflammatory monocyte gene signature in BD. A quantitative-polymerase chain reaction (Q-PCR) case-control gene expression study was performed on monocytes of 27 SZ patients and compared to outcomes collected in 56 BD patients (all patients naturalistically treated). For Q-PCR we used nine 'SZ specific genes' (found in whole genome analysis), the 19 BD signature genes (previously found by us) and six recently described autoimmune diabetes inflammatory monocyte genes. Monocytes of SZ patients had (similar to those of BD patients) a high inflammatory set point composed of three subsets of strongly correlating genes characterized by different sets of transcription/MAPK regulating factors. Subset 1A, characterized by ATF3 and DUSP2, and subset 1B, characterized by EGR3 and MXD1, were shared between BD and SZ patients (up-regulated in 67% and 51%, and 34% and 41%, respectively). Subset 2, characterized by PTPN7 and NAB2 was up-regulated in the monocytes of 62% BD, but down-regulated in the monocytes of 48% of SZ patients. Our approach shows that monocytes of SZ and BD patients overlap, but also differ in inflammatory gene expression. Our approach opens new avenues for nosological classifications of psychoses based on the inflammatory state of patients, enabling selection of those patients who might benefit from an anti-inflammatory treatment.

  16. Quantitative analysis of protein and gene expression in salivary glands of Sjogren's-like disease NOD mice treated by bone marrow soup.

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    Kaori Misuno

    Full Text Available BACKGROUND: Bone marrow cell extract (termed as BM Soup has been demonstrated to repair irradiated salivary glands (SGs and restore saliva secretion in our previous study. In the present study, we aim to investigate if the function of damaged SGs in non-obese diabetic (NOD mice can be restored by BM Soup treatment and the molecular alterations associated with the treatment. METHODS: Whole BM cells were lysed and soluble intracellular contents ("BM Soup" were injected I.V. into NOD mice. Tandem mass tagging with 2-D liquid chromatography-mass spectrometry was used to quantify proteins in the submandibular glands (SMGs between untreated and BM Soup-treated mice. Quantitative PCR was used to identify genes with altered expression in the treated mice. RESULTS BM SOUP: restored salivary flow rates to normal levels and significantly reduced the focus scores of SMGs in NOD mice. More than 1800 proteins in SMG cells were quantified by the proteomic approach. Many SMG proteins involved in inflammation and apoptosis were found to be down-regulated whereas those involved in salivary gland biology and development/regeneration were up-regulated in the BM Soup-treated mice. qPCR analysis also revealed expression changes of growth factors and cytokines in the SMGs of the treated NOD mice. CONCLUSION: BM Soup treatment is effective to restore the function of damaged SGs in NOD mice. Through gene/protein expression analysis, we have found that BM Soup treatment might effectuate via inhibiting apoptosis, focal adhesion and inflammation whereas promoting development, regeneration and differentiation of the SG cells in NOD mice. These findings provide important insights on the potential mechanisms underlying the BM Soup treatment for functional restoration of damaged SGs in NOD mice. Additional studies are needed to further confirm the identified target genes and their related signaling pathways that are responsible for the BM Soup treatment.

  17. Interrelationship of Gene Expression, Polysome Prevalence, and Respiration during Ripening of Ethylene and/or Cyanide-Treated Avocado Fruit.

    Science.gov (United States)

    Tucker, M L; Laties, G G

    1984-02-01

    Upon initiation of ripening in avocado fruit (Persea americana Mill. cv Hass) with 10 microliters/liter ethylene, polysome prevalence and associated poly(A)(+) mRNA increase approximately 3-fold early in the respiratory climacteric and drop off to preclimacteric levels at the peak of the respiratory climacteric. The increase in poly(A)(+) mRNA on polysomes early in the respiratory climacteric constitutes a generic increase in constitutive mRNAs. New gene expression associated with ripening is minimal but evident after 10 hours of ethylene treatment and continues to increase relative to constitutive gene expression throughout the climacteric. The respiratory climacteric can be temporally separated into two phases. The first phase is associated with a general increase in protein synthesis, whereas the second phase reflects new gene expression and accumulation of corresponding proteins which may be responsible for softening and other ripening characteristics. A major new message on polysomes that arises concomitantly with the respiratory climacteric codes for an in vitro translation product of 53 kilodaltons which is immunoprecipitated by antiserum against avocado fruit cellulase.Cyanide at 500 microliters/liter fails to affect the change in polysome prevalance or new gene expression associated with the ethylene-evoked climacteric in avocado fruit. Treatment of fruit with 500 microliters/liter cyanide alone initiates a respiratory increase within 4 hours, ethylene biosynthesis within 18 hours, and new gene expression akin to that educed by ethylene within 20 hours of exposure to cyanide.

  18. Differential adipose tissue gene expression profiles in abacavir treated patients that may contribute to the understanding of cardiovascular risk: a microarray study.

    Science.gov (United States)

    Shahmanesh, Mohsen; Phillips, Kenneth; Boothby, Meg; Tomlinson, Jeremy W

    2015-01-01

    To compare changes in gene expression by microarray from subcutaneous adipose tissue from HIV treatment naïve patients treated with efavirenz based regimens containing abacavir (ABC), tenofovir (TDF) or zidovidine (AZT). Subcutaneous fat biopsies were obtained before, at 6- and 18-24-months after treatment, and from HIV negative controls. Groups were age, ethnicity, weight, biochemical profile, and pre-treatment CD4 count matched. Microarray data was generated using the Agilent Whole Human Genome Microarray. Identification of differentially expressed genes and genomic response pathways was performed using limma and gene set enrichment analysis. There were significant divergences between ABC and the other two groups 6 months after treatment in genes controlling cell adhesion and environmental information processing, with some convergence at 18-24 months. Compared to controls the ABC group, but not AZT or TDF showed enrichment of genes controlling adherence junction, at 6 months and 18-24 months (adjusted ptissue. If similar changes are also taking place in other tissues including the coronary vasculature they may contribute to the increased risk of cardiovascular events reported in patients recently started on abacavir-containing regimens.

  19. Effect of transfection with PLP2 antisense oligonucleotides on gene expression of cadmium-treated MDA-MB231 breast cancer cells.

    Science.gov (United States)

    Longo, Alessandra; Librizzi, Mariangela; Luparello, Claudio

    2013-02-01

    Emerging evidence indicates that cadmium (Cd) is able to regulate gene expression, drastically affecting the pattern of transcriptional activity in human normal and pathological cells. We have already shown that exposure of MDA-MB231 breast cancer cells to 5 μM CdCl(2) for 96 h, apart from significantly affecting mitochondrial metabolism, induces modifications of the expression level of genes coding for members of stress response-, mitochondrial respiration-, MAP kinase-, NF-κB-, and apoptosis-related pathways. In the present study, we have expanded the knowledge on the biological effects of Cd-breast cancer cell interactions, indicating PLP2 (proteolipid protein-2) as a novel member of the list of Cd-upregulated genes by MDA-MB231 cancer cells and, through the application of transfection techniques with specific antisense oligonucleotides, we have demonstrated that such over-expression may be an upstream event to some of the changes of gene expression levels already observed in Cd-treated cells, thus unveiling new possible molecular relationship between PLP2 and genes linked to the stress and apoptotic responses.

  20. In situ hybridization analysis of the expression of futsch, tau, and MESK2 homologues in the brain of the European honeybee (Apis mellifera L..

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    Kumi Kaneko

    Full Text Available BACKGROUND: The importance of visual sense in Hymenopteran social behavior is suggested by the existence of a Hymenopteran insect-specific neural circuit related to visual processing and the fact that worker honeybee brain changes morphologically according to its foraging experience. To analyze molecular and neural bases that underlie the visual abilities of the honeybees, we used a cDNA microarray to search for gene(s expressed in a neural cell-type preferential manner in a visual center of the honeybee brain, the optic lobes (OLs. METHODOLOGY/PRINCIPAL FINDINGS: Expression analysis of candidate genes using in situ hybridization revealed two genes expressed in a neural cell-type preferential manner in the OLs. One is a homologue of Drosophila futsch, which encodes a microtubule-associated protein and is preferentially expressed in the monopolar cells in the lamina of the OLs. The gene for another microtubule-associated protein, tau, which functionally overlaps with futsch, was also preferentially expressed in the monopolar cells, strongly suggesting the functional importance of these two microtubule-associated proteins in monopolar cells. The other gene encoded a homologue of Misexpression Suppressor of Dominant-negative Kinase Suppressor of Ras 2 (MESK2, which might activate Ras/MAPK-signaling in Drosophila. MESK2 was expressed preferentially in a subclass of neurons located in the ventral region between the lamina and medulla neuropil in the OLs, suggesting that this subclass is a novel OL neuron type characterized by MESK2-expression. These three genes exhibited similar expression patterns in the worker, drone, and queen brains, suggesting that they function similarly irrespective of the honeybee sex or caste. CONCLUSIONS: Here we identified genes that are expressed in a monopolar cell (Amfutsch and Amtau or ventral medulla-preferential manner (AmMESK2 in insect OLs. These genes may aid in visualizing neurites of monopolar cells and ventral

  1. Fisetin up-regulates the expression of adiponectin in 3T3-L1 adipocytes via the activation of silent mating type information regulation 2 homologue 1 (SIRT1)-deacetylase and peroxisome proliferator-activated receptors (PPARs).

    Science.gov (United States)

    Jin, Taewon; Kim, Oh Yoen; Shin, Min-Jeong; Choi, Eun Young; Lee, Sung Sook; Han, Ye Sun; Chung, Ji Hyung

    2014-10-29

    Adiponectin, an adipokine, has been described as showing physiological benefits against obesity-related malfunctions and vascular dysfunction. Several natural compounds that promote the expression and secretion of adipokines in adipocytes could be useful for treating metabolic disorders. This study investigated the effect of fisetin, a dietary flavonoid, on the regulation of adiponectin in adipocytes using 3T3-L1 preadipocytes. The expression and secretion of adiponectin increased in 3T3-L1 cells upon treatment with fisetin in a dose-dependent manner. Fisetin-induced adiponectin secretion was inhibited by peroxisome proliferator-activated receptor (PPAR) antagonists. It was also revealed that fisetin increased the activities of PPARs and silent mating type information regulation 2 homologue 1 (SIRT1) in a dose-dependent manner. Furthermore, the up-regulation of adiponectin and the activation of PPARs induced by fisetin were prevented by a SIRT1 inhibitor. Fisetin also promoted deacetylation of PPAR γ coactivator 1 (PGC-1) and its interaction with PPARs. SIRT knockdown by siRNA significantly decreased both adiponectin production and PPARs-PGC-1 interaction. These results provide evidence that fisetin promotes the gene expression of adiponectin through the activation of SIRT1 and PPARs in adipocytes.

  2. Differential expression of metallothionein type-2 homologues in leaves and roots of Black pepper (Piper nigrum L

    Directory of Open Access Journals (Sweden)

    Susan M. Alex

    2008-01-01

    Full Text Available Black pepper (Piper nigrum L., member of the family Piperaceae is indigenous to India and is one of the most widely used spices in the world. In this paper we report the results of our attempts to identify a set of genes differentially expressed in the leaves of Piper nigrum, which could facilitate targeted engineering of this valuable crop. A PCR-based Suppression Subtractive Hybridization (SSH technique was used to generate a leaf-specific subtracted cDNA library of Piper nigrum. A tester population of leaf cDNA was subtracted with a root derived driver cDNA. The efficiency of subtraction was confirmed by PCR analysis using the housekeeping gene actin. On sequence analysis, almost 30% of the clones showed homology to metallothionein type-2 gene. The predominance of metallothionein transcripts in the leaf was further confirmed using Real-Time PCR analyses and Northern blot. The possible role of metallothionein type-2 homologues in the leaf is discussed along with the feasibility of using SSH technique for identification of more number of tissue-specific genes from Piper nigrum.

  3. Rearrangements of MYC gene facilitate risk stratification in diffuse large B-cell lymphoma patients treated with rituximab-CHOP

    DEFF Research Database (Denmark)

    Tzankov, Alexandar; Xu-Monette, Zijun Y; Gerhard, Marc;

    2014-01-01

    In order to address the debatable prognostic role of MYC rearrangements in diffuse large B-cell lymphoma patients treated with rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, we evaluated MYC rearrangements by fluorescence in situ hybridization in 563 cases using br...

  4. Separation of bacteriochlorophyll homologues from green photosynthetic sulfur bacteria by reversed-phase HPLC.

    Science.gov (United States)

    Borrego, C M; Garcia-Gil, L J

    1994-07-01

    A reversed-phase High Performance Liquid Cromatography (HPLC) method has been developed to accurately separate bacteriochlorophyllsc, d ande homologues in a reasonably short run time of 60 minutes. By using this method, two well-defined groups of bacteriochlorophyll homologue peaks can be discriminated. The first one consists of 4 peaks (min 24 to 30), which corresponds to the four main farnesyl homologues. The second peak subset is formed by a cluster of up to 10 minor peaks (min 33 to 40). These peaks can be related with series of several alcohol esters of the different chlorosome chlorophylls. The number of homologues was, however, quite variable depending on both, the bacteriochlorophyll and the bacterial species. The method hereby described, also provides a good separation of other photosynthetic pigments, either bacterial (Bacteriochlorophylla, chlorobactene, isorenieratene and okenone) or algal ones (Chlorophylla, Pheophytina and β-carotene). A preliminary screening of the homologue composition of several green photosynthetic bacterial species and isolates, has revealed different relative quantitative patterns. These differences seem to be related to physiological aspects rather than to taxonomic ones. The application of the method to the study of natural populations avoids the typical drawbacks on the pigment identification of overlapping eukaryotic and prokaryotic phototrophic microorganisms, giving further information about their physiological status.

  5. Different gene expressions in human periodontal cells after being treated with Emdogain%Emdogain作用下人牙周膜细胞的差异表达基因

    Institute of Scientific and Technical Information of China (English)

    张雪洋; 赵华; 章锦才

    2009-01-01

    Objective:To study the different gene expressions in human periodontal cells after being treated with Emdogain(EMD). Methods: The fourth passage periodontal cells were cultured. Cells in experimental group were cultured with 100 μg/ml EMD in non-serum DMEM for 3 days. Cells in control group were cultured in non-serum DMEM for 3 days. The gene expressions in human periodontal cells were screened with gene chip technique. Results: There were 112 differently expressed genes, 11 down-regulated genes and 101 up-regulated genes. Conclusion: After treated with Emdogain, expression of genes related to cell growth, cell proliferation, biosynthesis of extracellular matrix, angiogenesis and osteogenesis is up-regulated dramatically. Genes related to lipid metabolization and keratinocyte growth is down-regulated in human periodontal cells.%目的:筛选猪釉基质蛋白(EMD,Emdogain)作用于人牙周膜细胞后的差异表达基因.方法:培养第4 代牙周膜细胞,实验组加入终浓度为100 μg/ml EMD的无血清DMEM培养液,对照组为不含EMD的无血清DMEM培养液,培养3 d,采用基因芯片技术检测相关的差异表达基因.结果:共得到112 条差异表达基因,上调基因101 条,下调基因11 条.结论:EMD作用于人牙周膜细胞后,与细胞的生长、增殖,细胞外基质的合成,新生血管的形成,骨形成等功能相关的基因表达都显著提高,而脂蛋白代谢相关基因和角化细胞生长相关基因的表达下调.

  6. Analysis of the human Atox 1 homologue in Wilson patients

    Institute of Scientific and Technical Information of China (English)

    Isabel Simon; Mark Schaefer; Jürgen Reichert; Wolfgang Stremmel

    2008-01-01

    AIM: To analyze the metallochaperone antioxidant-1 (Atoxl) gene sequence in Wilson disease patients.METHODS: Mutation analysis of the four exons of the Atoxl gene including the intron- exon boundaries was performed in 63 Wilson disease patients by direct sequencing.RESULTS: From 63 selected patients no mutations were identified after the entire coding region including the intron- exon boundaries of Atoxl were sequenced.One known polymorphism within the Atoxl gene (5'UTR-99 T>C) in 31 (49%) of the Wilson patients as well as one previously undescribed variation (5'UTR -68 C>T)in 2 of the Wilson patients could be detected. Statistical analyses revealed that the existence of a variation within the Atoxl- gene showed a tendency towards an earlier onset of the disease.CONCLUSION: Based on the data of this study, no major role can be attributed to Atoxl in the pathophysiology or clinical variation of Wilson disease.

  7. Gene expression of human osteoblasts cells on chemically treated surfaces of Ti-6Al-4V-ELI.

    Science.gov (United States)

    Oliveira, D P; Palmieri, A; Carinci, F; Bolfarini, C

    2015-06-01

    Surface modifications of titanium alloys are useful methods to enhance the biological stability of intraosseous implants and to promote a well succeeded osseointegration in the early stages of implantation. This work aims to investigate the influence of chemically modified surfaces of Ti-6Al-4V-ELI (extra-low interstitial) on the gene expression of human osteoblastic (HOb) cells. The surface treatments by acid etching or acid etching plus alkaline treatment were carried out to modify the topography, effective area, contact angle and chemical composition of the samples. The surface morphology was investigated using: scanning electron microscopy (SEM) and confocal laser-scanning microscope (CLSM). Roughness measurements and effective surface area were obtained using the CLSM. Surface composition was analysed by energy dispersive X-ray spectroscopy (EDX) and by X-Ray Diffraction (XRD). The expression levels of some bone related genes (ALPL, COL1A1, COL3A1, SPP1, RUNX2, and SPARC) were analysed using real-time Reverse Transcription Polymerase Chain Reaction (real-time RT-PCR). The results showed that all the chemical modifications studied in this work influenced the surface morphology, wettability, roughness, effective area and gene expression of human osteoblasts. Acid phosphoric combined to alkaline treatment presented a more accelerated gene expression after 7days while the only phosphoric etching or chloride etching combined to alkaline treatment presented more effective responses after 15days.

  8. Longitudinal Transcriptome Analysis Reveals a Sustained Differential Gene Expression Signature in Patients Treated for Acute Lyme Disease.

    Science.gov (United States)

    Bouquet, Jerome; Soloski, Mark J; Swei, Andrea; Cheadle, Chris; Federman, Scot; Billaud, Jean-Noel; Rebman, Alison W; Kabre, Beniwende; Halpert, Richard; Boorgula, Meher; Aucott, John N; Chiu, Charles Y

    2016-02-12

    Lyme disease is a tick-borne illness caused by the bacterium Borrelia burgdorferi, and approximately 10 to 20% of patients report persistent symptoms lasting months to years despite appropriate treatment with antibiotics. To gain insights into the molecular basis of acute Lyme disease and the ensuing development of post-treatment symptoms, we conducted a longitudinal transcriptome study of 29 Lyme disease patients (and 13 matched controls) enrolled at the time of diagnosis and followed for up to 6 months. The differential gene expression signature of Lyme disease following the acute phase of infection persisted for at least 3 weeks and had fewer than 44% differentially expressed genes (DEGs) in common with other infectious or noninfectious syndromes. Early Lyme disease prior to antibiotic therapy was characterized by marked upregulation of Toll-like receptor signaling but lack of activation of the inflammatory T-cell apoptotic and B-cell developmental pathways seen in other acute infectious syndromes. Six months after completion of therapy, Lyme disease patients were found to have 31 to 60% of their pathways in common with three different immune-mediated chronic diseases. No differential gene expression signature was observed between Lyme disease patients with resolved illness to those with persistent symptoms at 6 months post-treatment. The identification of a sustained differential gene expression signature in Lyme disease suggests that a panel of selected human host-based biomarkers may address the need for sensitive clinical diagnostics during the "window period" of infection prior to the appearance of a detectable antibody response and may also inform the development of new therapeutic targets. Lyme disease is the most common tick-borne infection in the United States, and some patients report lingering symptoms lasting months to years despite antibiotic treatment. To better understand the role of the human host response in acute Lyme disease and the

  9. A transglutaminase homologue as a condensation catalyst in antibiotic assembly lines.

    Science.gov (United States)

    Fortin, Pascal D; Walsh, Christopher T; Magarvey, Nathan A

    2007-08-16

    The unrelenting emergence of antibiotic-resistant bacterial pathogens demands the investigation of antibiotics with new modes of action. The pseudopeptide antibiotic andrimid is a nanomolar inhibitor of the bacterial acetyl-CoA carboxylase that catalyses the first committed step in prokaryotic fatty acid biosynthesis. Recently, the andrimid (adm) biosynthetic gene cluster was isolated and heterologously expressed in Escherichia coli. This establishes a heterologous biological host in which to rapidly probe features of andrimid formation and to use biosynthetic engineering to make unnatural variants of this important and promising new class of antibiotics. Bioinformatic analysis of the adm cluster revealed a dissociated biosynthetic assembly system lacking canonical amide synthases between the first three carrier protein domains. Here we report that AdmF, a transglutaminase (TGase) homologue, catalyses the formation of the first amide bond, an N-acyl-beta-peptide link, in andrimid biosynthesis. Hence, AdmF is a newly discovered biosynthetic enzyme that acts as a stand-alone amide synthase between protein-bound, thiotemplated substrates in an antibiotic enzymatic assembly line. TGases (enzyme class (EC) 2.3.2.13) normally catalyse the cross-linking of (poly)peptides by creating isopeptidic bonds between the gamma-carboxamide group of a glutamine side chain of one protein and various amine donors, including lysine side chains. To the best of our knowledge, the present study constitutes the first report of a TGase-like enzyme recruited for the assembly of an antibiotic. Moreover, genome mining using the AdmF sequence yielded additional TGases in unassigned natural product biosynthetic pathways. With many more microbial genomes being sequenced, such a strategy could potentially unearth biosynthetic pathways producing new classes of antibiotics.

  10. Gene expression of human osteoblasts cells on chemically treated surfaces of Ti–6Al–4V–ELI

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, D.P., E-mail: dpedreira@ufscar.br [Department of Materials Engineering, Federal University of São Carlos, São Carlos (Brazil); Palmieri, A.; Carinci, F. [Department of D.M.C.C.C., Section of Maxillofacial and Plastic Surgery, University of Ferrara, Ferrara (Italy); Bolfarini, C. [Department of Materials Engineering, Federal University of São Carlos, São Carlos (Brazil)

    2015-06-01

    Surface modifications of titanium alloys are useful methods to enhance the biological stability of intraosseous implants and to promote a well succeeded osseointegration in the early stages of implantation. This work aims to investigate the influence of chemically modified surfaces of Ti–6Al–4V–ELI (extra-low interstitial) on the gene expression of human osteoblastic (HOb) cells. The surface treatments by acid etching or acid etching plus alkaline treatment were carried out to modify the topography, effective area, contact angle and chemical composition of the samples. The surface morphology was investigated using: scanning electron microscopy (SEM) and confocal laser-scanning microscope (CLSM). Roughness measurements and effective surface area were obtained using the CLSM. Surface composition was analysed by energy dispersive X-ray spectroscopy (EDX) and by X-Ray Diffraction (XRD). The expression levels of some bone related genes (ALPL, COL1A1, COL3A1, SPP1, RUNX2, and SPARC) were analysed using real-time Reverse Transcription Polymerase Chain Reaction (real-time RT-PCR). The results showed that all the chemical modifications studied in this work influenced the surface morphology, wettability, roughness, effective area and gene expression of human osteoblasts. Acid phosphoric combined to alkaline treatment presented a more accelerated gene expression after 7 days while the only phosphoric etching or chloride etching combined to alkaline treatment presented more effective responses after 15 days. - Highlights: • Chemical treatments were effective for surface modification of Ti–6Al–4V. • Alkaline and phosphoric treatments induced osteopontin up-regulation. • Topographic formation on surface can induce RUNX2 up regulation. • Acid etch plus alkaline treatment accelerated the expression of some bone related genes.

  11. Prognostic value of TP53 gene mutations in myelodysplastic syndromes and acute myeloid leukemia treated with azacitidine.

    Science.gov (United States)

    Bally, Cecile; Adès, Lionel; Renneville, Aline; Sebert, Marie; Eclache, Virginie; Preudhomme, Claude; Mozziconacci, Marie-Joelle; de The, Hugues; Lehmann-Che, Jacqueline; Fenaux, Pierre

    2014-07-01

    TP53 mutations are found in 5-10% of MDS and AML, where they are generally associated with complex karyotype and an overall poor prognosis. However, the impact of TP53 mutations in MDS treated with azacitidine (AZA) remains unclear. We analyzed TP53 mutations in 62 patients with high risk MDS or AML treated with AZA. A TP53 mutation was found in 23 patients (37.1%), associated with complex karyotype in 18 (78.3%) of them. TP53 mutations had no significant impact on response or complete response to AZA (p=0.60 and p=0.26, respectively). By univariate analysis, OS was negatively influenced by the presence of TP53 mutation (median OS 12.4 months versus 23.7 months, pmutational status (HR 2.89 (95% confidence interval 1.38-6.04; p=0.005) retained statistical significance for OS. Results were similar when the analysis was restricted to MDS and CMML patients, excluding AML (HR=2.46 (95% confidence interval: 1.1-6.4); p=0.04)). Thus, TP53 mutations strongly correlated with poorer survival in higher risk MDS and AML treated with AZA.

  12. Solid-Phase Synthesis of Amine/Carboxyl Substituted Prolines and Proline Homologues: Scope and Limitations.

    Science.gov (United States)

    Zhou, Ziniu; Scott, William L; O'Donnell, Martin J

    2016-03-15

    A solid-phase procedure is used to synthesize racemic peptidomimetics based on the fundamental peptide unit. The peptidomimetics are constructed around proline or proline homologues variably substituted at the amine and carbonyl sites. The procedure expands the diversity of substituted peptidomimetic molecules available to the Distributed Drug Discovery (D3) project. Using a BAL-based solid-phase synthetic sequence the proline or proline homologue subunit is both constructed and incorporated into the peptidomimetic by an α-alkylation, hydrolysis and intramolecular cyclization sequence. Further transformations on solid-phase provide access to a variety of piperazine derivatives representing a class of molecules known to exhibit central nervous system activity. The procedure works well with proline cores, but with larger six- and seven-membered ring homologues the nature of the carboxylic acid acylating the cyclic amine can lead to side reactions and result in poor overall yields.

  13. Peptidoglycan inducible expression of a serine proteinase homologue from kuruma shrimp (Marsupenaeus japonicus).

    Science.gov (United States)

    Rattanachai, Achara; Hirono, Ikuo; Ohira, Tsuyoshi; Takahashi, Yukinori; Aoki, Takashi

    2005-01-01

    A cDNA encoding a serine proteinase homologue of kuruma shrimp (Marsupenaeus japonicus) was cloned. The 1257 bp cDNA encodes a 339 amino acid putative peptide, with a signal sequence of 16 amino acid residues. The deduced amino acid sequence is 42-67% similar to the immune-related serine proteinases and serine proteinase homologues of arthropods. It contains catalytic triad residues in the putative catalytic domain except for one substitution of Ser by a Gly residue. The six cysteine residues that form three disulphide bridges in most serine proteinases were conserved. The M. japonicus serine proteinase homologue was mainly expressed in haemocytes, in which expression dramatically increased after 3 days feeding with peptidoglycan at 0.2 mg kg(-1) shrimp body weight per day.

  14. Expression pattern of INNER NO OUTER homologue in Nymphaea (water lily family, Nymphaeaceae).

    Science.gov (United States)

    Yamada, Toshihiro; Ito, Motomi; Kato, Masahiro

    2003-10-01

    Two homologues of INNER NO OUTER ( INO) in Nymphaea alba and N. colorata (Nymphaeaceae) were isolated and the expression pattern of the N. alba INO homologue NaINO was examined by in situ hybridization. The INO homologues obtained have a portion similar to INO in the predicted amino acid sequences between the conserved zinc finger-like and YABBY domains. In an in situ hybridization analysis, NaINO is expressed in the outer epidermis of the outer integument, inner integument, and the tip of the nucellus. The pattern observed in the outer integument is very similar to that of Arabidopsis thaliana, while the expression in the inner integument and nucellus is not observed in A. thaliana.

  15. Development of an Autologous Macrophage-based Adoptive Gene Transfer Strategy to Treat Posttraumatic Osteoarthritis (PTOA) and Osteoarithritis (OA)

    Science.gov (United States)

    2015-02-01

    of the diseases. The proposed study will test two hypotheses: 1) the autologous macrophage-based adoptive gene transfer strategy can effectively...12% of all OA are resulted from an acute trauma to the joint and are referred to as PTOA [1]. It is estimated that the total cost of OA cases is...and medications. Joint replacement surgery may also be required in eroding forms of arthritis . Medications can help reduce inflammation in the joint

  16. The association study of polymorphisms in DAT, DRD2, and COMT genes and acute extrapyramidal adverse effects in male schizophrenic patients treated with haloperidol.

    Science.gov (United States)

    Zivković, Maja; Mihaljević-Peles, Alma; Bozina, Nada; Sagud, Marina; Nikolac-Perkovic, Matea; Vuksan-Cusa, Bjanka; Muck-Seler, Dorotea

    2013-10-01

    Extrapyramidal symptoms (EPSs) are common adverse effects of antipsychotics. The development of acute EPSs could depend on the activity of dopaminergic system and its gene variants. The aim of this study was to determine the association between dopaminergic type 2 receptor (DRD2) dopamine transporter (SLC6A3) and catechol-O-methyltransferase (COMT) gene polymorphisms and acute EPSs in 240 male schizophrenic patients treated with haloperidol (15-mg/d) over a period of 2 weeks. Acute EPSs were assessed with Simpson-Angus Scale. Three dopaminergic gene polymorphisms, the DRD2 Taq1A, the SLC6A3 VNTR, and the COMT Val158Met, were determined. Extrapyramidal symptoms occurred in 116 (48.3%) of patients. Statistically significant associations were found for SLC6A3 VNTR and COMT Val158Met polymorphisms and EPS susceptibility. Patients with SLC6A3 9/10 genotype had almost twice the odds to develop EPSs compared with those with all other SLC6A3 genotypes (odds ratio, 1.9; 95% confidence interval, 1.13-3.30), and patients with COMT Val/Met genotype had 1.7 times greater odds to develop EPSs than those with all other COMT genotypes (odds ratio, 1.7; 95% confidence interval, 1.01-2.88). There was no statistically significant association between genotype and allele frequencies of DRD2, SLC6A3, or COMT polymorphisms and the development of particular EPSs.In conclusion, the results of the present study showed for the first time the association between acute haloperidol-induced EPSs and SLC6A3 VNTR and COMT Val158Met polymorphisms. Although the precise biological mechanisms underlying these findings are not yet understood, the results suggest that the dopaminergic gene variations could predict the vulnerability to the development of the acute EPSs in haloperidol-treated schizophrenic patients.

  17. A five-gene model predicts clinical outcome in ER+/PR+, early-stage breast cancers treated with adjuvant tamoxifen.

    Science.gov (United States)

    Kerr, Daniel Alan; Wittliff, James L

    2011-10-01

    Primary breast carcinomas expressing both estrogen and progesterone receptors are most likely to respond to tamoxifen therapy, especially in patients with early-stage lesions. However, certain patients exhibit clinicopathologic features suggesting good prognosis relapse within 10 years, justifying a search for biomarkers identifying patients at risk for recurrence. Nine candidate genes associated with estrogen signaling were selected from microarray studies and combined with those for conventional biomarkers (ESR1, PGR, ERBB2). Expression of this 12-gene subset was analyzed by RT-qPCR in frozen tissue specimens from 60 early-stage, estrogen receptor (ER)+/progestin receptor (PR)+ breast cancers from patients treated with adjuvant tamoxifen. A multivariate model was created by Cox regression using a training data set and applied to an independent validation set. A five-gene model was developed from the training set (n = 36) that exhibited significant correlations with both relapse-free and overall survival. Applying this model to Kaplan-Meier regression, patients were separated into low-risk (100% relapse-free at 150 months) and high-risk (60% relapse-free at 150 months) groups (P = 0.03). When this model was applied to the validation set (n = 24), similar risk stratification was achieved for both relapse-free and overall survival (P = 0.01 and 0.04, respectively). We developed a five-gene model composed of PgR, BCL2, ERBB4 JM-a, RERG, and CD34 that identified early-stage, ER+/PR+ breast cancers in patients treated with tamoxifen that relapsed, although they exhibited clinicopathologic features suggesting good prognosis. Within this multivariate model, increased expression of PgR, ERBB4 JM-a, RERG, and CD34 was associated with increased survival, while increased expression of BCL2 was associated with decreased survival.

  18. Plasmodium falciparum Bloom homologue, a nucleocytoplasmic protein, translocates in 3' to 5' direction and is essential for parasite growth.

    Science.gov (United States)

    Rahman, Farhana; Tarique, Mohammed; Tuteja, Renu

    2016-05-01

    Malaria caused by Plasmodium, particularly Plasmodium falciparum, is the most serious and widespread parasitic disease of humans. RecQ helicase family members are essential in homologous recombination-based error-free DNA repair processes in all domains of life. RecQ helicases present in each organism differ and several homologues have been identified in various multicellular organisms. These proteins are involved in various pathways of DNA metabolism by providing duplex unwinding function. Five members of RecQ family are present in Homo sapiens but P. falciparum contains only two members of this family. Here we report the detailed biochemical and functional characterization of the Bloom (Blm) homologue (PfBlm) from P. falciparum 3D7 strain. Purified PfBlm exhibits ATPase and 3' to 5' direction specific DNA helicase activity. The calculated average reaction rate of ATPase was ~13 pmol of ATP hydrolyzed/min/pmol of enzyme. The immunofluorescence assay results show that PfBlm is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain. In some stages of development in addition to nucleus PfBlm also localizes in the cytoplasm. The gene disruption studies of PfBlm by dsRNA showed that it is required for the ex-vivo intraerythrocytic development of the parasite P. falciparum 3D7 strain. The dsRNA mediated inhibition of parasite growth suggests that a variety of pathways are affected resulting in curtailing of the parasite growth. This study will be helpful in unravelling the basic mechanism of DNA transaction in the malaria parasite and additionally it may provide leads to understand the parasite specific characteristics of this protein.

  19. Expression Profiles and DNA-Binding Affinity of Five ERF Genes in Bunches of Vitis vinifera cv. Cardinal Treated with High Levels of CO2 at Low Temperature

    Science.gov (United States)

    Romero, Irene; Vazquez-Hernandez, Maria; Escribano, M. I.; Merodio, Carmen; Sanchez-Ballesta, M. T.

    2016-01-01

    Ethylene response factors (ERFs) play an important role in plants by regulating defense response through interaction with various stress pathways. After harvest, table grapes (Vitis vinifera L.) are subject to a range of problems associated with postharvest storage at 0°C, such as fungal attack, water loss and rachis browning. The application of a 3-day high CO2 treatment maintained fruit quality and activated the induction of transcription factors belonging to different families such as ERF. In this paper, we have isolated five VviERFs from table grapes cv. Cardinal, whose deduced amino acid sequence contained the conserved apetalous (AP2)/ERF domain. The phylogeny and putative conserved motifs in VviERFs were analyzed and compared with those previously reported in Vitis. VviERFs-c gene expression was studied by quantitative real-time RT-PCR in the different tissues of bunches stored at low temperature and treated with high levels of CO2. The results showed that in most of the tissues analyzed, VviERFs-c gene expression was induced by the storage under normal atmosphere although the application of high levels of CO2 caused a greater increase in the VviERFs-c transcript accumulation. The promoter regions of two PRs (pathogenesis related proteins), Vcchit1b and Vcgns1, were obtained and the in silico analysis revealed the presence of a cis-acting ethylene response element (GCC box). In addition, expression of these two PR genes was analyzed in the pulp and rachis of CO2-treated and non-treated table grapes stored at 0°C and results showed significant correlations with VviERF2-c and VviERF6L7-c gene expression in rachis, and between VviERF11-c and Vcchit1b in pulp. Finally by using electro mobility shift assays, we denoted differences in binding of VviERFs to the GCC sequences present in the promoters of both PRs, with VviERF6L7-c being the only member which did not bind to any tested probe. Overall, our results suggest that the beneficial effect of high CO2

  20. Gene expression profiles in human HepG2 cells treated with extracts of the Tamarindus indica fruit pulp.

    Science.gov (United States)

    Razali, Nurhanani; Aziz, Azlina A; Junit, Sarni M

    2010-12-01

    Tamarindus indicaL. (T. indica) or locally known as asam jawa belongs to the family of Leguminosae. The fruit pulp had been reported to have antioxidant activities and possess hypolipidaemic effects. In this study, we attempted to investigate the gene expression patterns in human hepatoma HepG2 cell line in response to treatment with low concentration of the fruit pulp extracts. Microarray analysis using Affymetrix Human Genome 1.0 S.T arrays was used in the study. Microarray data were validated using semi-quantitative RT-PCR and real-time RT-PCR. Amongst the significantly up-regulated genes were those that code for the metallothioneins (MT1M, MT1F, MT1X) and glutathione S-transferases (GSTA1, GSTA2, GST02) that are involved in stress response. APOA4, APOA5, ABCG5 and MTTP genes were also significantly regulated that could be linked to hypolipidaemic activities of the T. indica fruit pulp.

  1. Transcriptomic analysis of common carp anterior kidney during Cyprinid herpesvirus 3 infection: Immunoglobulin repertoire and homologue functional divergence

    Science.gov (United States)

    Neave, Matthew J.; Sunarto, Agus; McColl, Kenneth A.

    2017-01-01

    Cyprinid herpesvirus 3 (CyHV-3) infects koi and common carp and causes widespread mortalities. While the virus is a significant concern for aquaculture operations in many countries, in Australia the virus may be a useful biocontrol agent for pest carp. However, carp immune responses to CyHV-3, and the molecular mechanisms underpinning resistance, are not well understood. Here we used RNA-Seq on carp during different phases of CyHV-3 infection to detect the gene expression dynamics of both host and virus simultaneously. During acute CyHV-3 infection, the carp host modified the expression of genes involved in various immune systems and detoxification pathways. Moreover, the activated pathways were skewed toward humoral immune responses, which may have been influenced by the virus itself. Many immune-related genes were duplicated in the carp genome, and often these were expressed differently across the infection phases. Of particular interest were two interleukin-10 homologues that were not expressed synchronously, suggesting neo- or sub-functionalization. The carp immunoglobulin repertoire significantly diversified during active CyHV-3 infection, which was followed by the selection of high-affinity B-cells. This is indicative of a developing adaptive immune response, and is the first attempt to use RNA-Seq to understand this process in fish during a viral infection. PMID:28148967

  2. Transcriptomic analysis of common carp anterior kidney during Cyprinid herpesvirus 3 infection: Immunoglobulin repertoire and homologue functional divergence.

    Science.gov (United States)

    Neave, Matthew J; Sunarto, Agus; McColl, Kenneth A

    2017-02-02

    Cyprinid herpesvirus 3 (CyHV-3) infects koi and common carp and causes widespread mortalities. While the virus is a significant concern for aquaculture operations in many countries, in Australia the virus may be a useful biocontrol agent for pest carp. However, carp immune responses to CyHV-3, and the molecular mechanisms underpinning resistance, are not well understood. Here we used RNA-Seq on carp during different phases of CyHV-3 infection to detect the gene expression dynamics of both host and virus simultaneously. During acute CyHV-3 infection, the carp host modified the expression of genes involved in various immune systems and detoxification pathways. Moreover, the activated pathways were skewed toward humoral immune responses, which may have been influenced by the virus itself. Many immune-related genes were duplicated in the carp genome, and often these were expressed differently across the infection phases. Of particular interest were two interleukin-10 homologues that were not expressed synchronously, suggesting neo- or sub-functionalization. The carp immunoglobulin repertoire significantly diversified during active CyHV-3 infection, which was followed by the selection of high-affinity B-cells. This is indicative of a developing adaptive immune response, and is the first attempt to use RNA-Seq to understand this process in fish during a viral infection.

  3. Serotonin- and Dopamine-Related Gene Expression in db/db Mice Islets and in MIN6 β-Cells Treated with Palmitate and Oleate

    Science.gov (United States)

    Cataldo, L. R.; Olmos, P.; Galgani, J. E.; Valenzuela, R.; Aranda, E.; Cortés, V. A.; Santos, J. L.

    2016-01-01

    High circulating nonesterified fatty acids (NEFAs) concentration, often reported in diabetes, leads to impaired glucose-stimulated insulin secretion (GSIS) through not yet well-defined mechanisms. Serotonin and dopamine might contribute to NEFA-dependent β-cell dysfunction, since extracellular signal of these monoamines decreases GSIS. Moreover, palmitate-treated β-cells may enhance the expression of the serotonin receptor Htr2c, affecting insulin secretion. Additionally, the expression of monoamine-oxidase type B (Maob) seems to be lower in islets from humans and mice with diabetes compared to nondiabetic islets, which may lead to increased monoamine concentrations. We assessed the expression of serotonin- and dopamine-related genes in islets from db/db and wild-type (WT) mice. In addition, the effect of palmitate and oleate on the expression of such genes, 5HT content, and GSIS in MIN6 β-cell was determined. Lower Maob expression was found in islets from db/db versus WT mice and in MIN6 β-cells in response to palmitate and oleate treatment compared to vehicle. Reduced 5HT content and impaired GSIS in response to palmitate (−25%; p < 0.0001) and oleate (−43%; p < 0.0001) were detected in MIN6 β-cells. In conclusion, known defects of GSIS in islets from db/db mice and MIN6 β-cells treated with NEFAs are accompanied by reduced Maob expression and reduced 5HT content. PMID:27366756

  4. Advances with using CRISPR/Cas-mediated gene editing to treat infections with hepatitis B virus and hepatitis C virus.

    Science.gov (United States)

    Moyo, Buhle; Bloom, Kristie; Scott, Tristan; Ely, Abdullah; Arbuthnot, Patrick

    2017-01-10

    Chronic infections with hepatitis B and hepatitis C viruses (HBV and HCV) account for the majority of cases of cirrhosis and hepatocellular carcinoma. Current therapies for the infections have limitations and improved efficacy is necessary to prevent complications in carriers of the viruses. In the case of HBV persistence, the replication intermediate comprising covalently closed circular DNA (cccDNA) is particularly problematic. Licensed therapies have little effect on cccDNA and HBV replication relapses following treatment withdrawal. Disabling cccDNA is thus key to curing HBV infections and application of gene editing technology, such as harnessing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, has curative potential. Several studies have reported good efficacy when employing CRISPR/Cas technologies to disable HBV replication in cultured cells and in hydrodynamically injected mice. Recent advances with HCV drug development have revolutionized treatment of the infection. Nevertheless, individuals may be refractory to treatment. Targeting RNA from HCV with CRISPR/Cas isolated from Francisella novicida may have therapeutic utility. Although preclinical work shows that CRISPR/Cas technology has potential to overcome infection with HBV and HCV, significant challenges need to be met. Ensuring specificity for viral targets and efficient delivery of the gene editing sequences to virus-infected cells are particularly important. The field is at an interesting stage and the future of curative antiviral drug regimens, particularly for treatment of chronic HBV infection, may well entail use of combinations that include derivatives of CRISPR/Cas.

  5. Use of archived biopsy specimens to study gene expression in oral mucosa from chemotherapy-treated cancer patients.

    Science.gov (United States)

    Mougeot, Jean-Luc C; Mougeot, Farah K B; Peterson, Douglas E; Padilla, Ricardo J; Brennan, Michael T; Lockhart, Peter B

    2013-05-01

    Oral mucositis caused by cancer chemotherapy can result in significant clinical complications. There is a strategic need to accelerate the delineation of the pathobiology. This proof-of-principle study was designed to demonstrate the feasibility of studying archived oral mucosal specimens to further delineate oral mucositis pathobiology. Twenty-nine formalin-fixed and paraffin-embedded tissue blocks of 25-year-old oral mucosa autopsy specimens from cancer chemotherapy patients were studied. Standardized technology was utilized, including RNA isolation and amplification, array hybridization, and gene expression analysis. A predominance of DNA damage in buccal mucosal basal keratinocytes was observed. Data comparing basal cells from buccal vs. gingival mucosa identified differential gene expression of host responses in relation to pathways relevant to oral mucositis pathogenesis, including responses to cancer-associated inflammation. This proof-of-principle study demonstrated that archived oral mucosal specimens may be a potentially valuable resource for the study of oral mucositis in cancer patients. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Mammary gland morphology and gene expression differ in female rats treated with 17β-estradiol or fed soy protein isolate.

    Science.gov (United States)

    Ronis, Martin J J; Shankar, Kartik; Gomez-Acevedo, Horacio; Hennings, Leah; Singhal, Rohit; Blackburn, Michael L; Badger, Thomas M

    2012-12-01

    Soy foods have been suggested to have both positive health benefits and potentially adverse effects as a result of their content of phytoestrogens. However, studies on the estrogenicity of soy foods are lacking. Here we directly compared the effects of soy protein isolate (SPI), the protein in soy infant formula, with those of 17β-estradiol (E2), on global gene expression profiles and morphology in the female rat mammary gland. Rats were fed AIN-93G diets containing casein or SPI beginning on postnatal d 30. Rats were ovariectomized on postnatal d 50 and treated with 5 μg/kg/d E2 or vehicle for 14 d. Microarray analysis revealed that E2 treatment altered expression of 780 genes more than or equal to 2-fold (P < 0.05), whereas SPI feeding altered expression of only 53 genes more than or equal to 2-fold. Moreover, the groups had only 10 genes in common to increase more than or equal to 2-fold. The combination of SPI feeding and E2 altered expression of 422 genes and reversed E2 effects on many mRNAs, including those involved in the c-myc signaling pathway, cyclin D1, and Ki67. ERα binding to its response element on the Tie-2/Tek and progesterone receptor promoters was increased by E2, but not SPI, and this promoter binding was suppressed by the combination of E2 + SPI for the Tie-2/Tek promoter but increased for the progesterone receptor promoter (P < 0.05). SPI reduced the ratio of epithelial to fat pad area and E2 + SPI reduced both epithelial and fat pad area (P < 0.05). These data suggest that SPI is only minimally estrogenic in the rat mammary gland even in the absence of endogenous estrogens.

  7. Pharmacodynamic impact of carboxylesterase 1 gene variants in patients with congestive heart failure treated with angiotensin-converting enzyme inhibitors

    DEFF Research Database (Denmark)

    Nelveg-Kristensen, Karl Emil; Bie, Peter; Ferrero, Laura

    2016-01-01

    BACKGROUND: Variation in the carboxylesterase 1 gene (CES1) may contribute to the efficacy of ACEIs. Accordingly, we examined the impact of CES1 variants on plasma angiotensin II (ATII)/angiotensin I (ATI) ratio in patients with congestive heart failure (CHF) that underwent ACEI dose titrations......1Var, and three other CES1 variants were examined. METHODS: Patients with CHF, and clinical indication for ACEIs were categorized according to their CES1 genotype. Differences in mean plasma ATII/ATI ratios between genotype groups after ACEI dose titration, expressed as the least square mean (LSM......) with 95% confidence intervals (CIs), were assessed by analysis of variance. RESULTS: A total of 200 patients were recruited and 127 patients (63.5%) completed the study. The mean duration of the CHF drug dose titration was 6.2 (SD 3.6) months. After ACEI dose titration, there was no difference in mean...

  8. Inhibiting matrix metalloproteinase by cell-based timp-3 gene transfer effectively treats acute and chronic ischemic cardiomyopathy.

    Science.gov (United States)

    Tian, Hai; Huang, Ming-Li; Liu, Kai-Yu; Jia, Zhi-Bo; Sun, Lu; Jiang, Shu-Lin; Liu, Wei; McDonald Kinkaid, Heather Y; Wu, Jun; Li, Ren-Ke

    2012-01-01

    After a myocardial infarction (MI), an increase in the cardiac ratio of matrix metalloproteinases (MMPs) relative to their inhibitors (TIMPs) causes extracellular matrix modulation that leads to ventricular dilatation and congestive heart failure. Cell therapy can mitigate these effects. In this study, we tested whether increasing MMP inhibition via cell-based gene transfer of Timp-3 further preserved ventricular morphometry and cardiac function in a rat model of MI. We also measured the effect of treatment timing. We generated MI (coronary artery ligation) in adult rats. Three or 14 days later, we implanted medium (control) or vascular smooth muscle cells transfected with empty vector (VSMCs) or Timp-3 (C-TIMP-3) into the peri-infarct region (n = 15-24/group). We assessed MMP-2 and -9 expression and activity, TIMP-3, and TNF-α expression, cell apoptosis, infarct size and thickness, ventricular morphometry, and cardiac function (by echocardiography). Relative to medium, VSMCs delivered at either time point significantly reduced cardiac expression and activity of MMP-2 and -9, reduced expression of TNF-α, and increased expression of TIMP-3. Cell therapy also reduced apoptosis and scar area, increased infarct thickness, preserved ventricular structure, and reduced functional loss. All these effects were augmented by C-TIMP-3 treatment. Survival and cardiac function were significantly greater when VSMCs or C-TIMP-3 were delivered at 3 (vs. 14) days after MI. Upregulating post-MI cardiac TIMP-3 expression via cell-based gene therapy contributed additional regulation of MMP, TIMP, and TNF-α levels, thereby boosting the structural and functional effects of VSMCs transplanted at 3 or 14 days after an MI in rats. Early treatment may be superior to late, though both are effective.

  9. ANTITUMOR EFFECT OF INTRATUMORAL INJECTION OF LIPOSOME-ENCAPSULATED G-CSF GENE AND IN SITU BIOLOGICAL CHARACTERISTICS OF THE TREATED TUMOR CELLS

    Institute of Scientific and Technical Information of China (English)

    Sun Yanping; Cao Xuetao; Wang Quanxing; Wang Yuanhe; Shi Jinghua

    1998-01-01

    In order to investigate the antitumor effects of the in vivo G-CSF gene therapy mediated by liposome and its mechanisms, human G-CSF gene was encapsulated into liposome and was directly injected into tumor mass of C26 colon adenocarcinoma-bearing mice. After direct intratumoral injection of liposome encapsulated G-CSF DNA, the subcutaneous tumor growth was dramatically inhibited and the survival time was prolonged significantly. Tumor regression could be observed in about 30%of C-26-bearing mice. By the analysis of the antitumor mechanisms, we found that anti-G41s (600ug/ml) clone could be selected from the tumor cells freshly separated from the treated C-26 tumor mass, and secretion of GCSF in the supernatant could be detected. Northern-blot also confirmed the expression of hG-CSF by the tumor cells. Higher expressions of MHC class I(H-2kd) molecule and ICAM-1 on the tumor cells could be observed. The results demonstrated that liposome can effectively transfect G-CSF gene into tumor cellsin situ, and then increase the immunogenicity of the tumor cells which may contribute to the activation of the local antitumor immune responses effectively.

  10. Identification and cloning of a sequence homologue of dopamine β-hydroxylase

    NARCIS (Netherlands)

    Chambers, Kaylene J.; Tonkin, Leath A.; Chang, Edwin; Shelton, Dawne N.; Linskens, Maarten H.; Funk, Walter D.

    1998-01-01

    We have identified and cloned a cDNA encoding a new member of the monooxygenase family of enzymes. This novel enzyme, which we call MOX (monooxygenase X; unknown substrate) is a clear sequence homologue of the enzyme dopamine β-hydroxylase (DBH). MOX maintains many of the structural features of DBH,

  11. Structure of HLA-A*1101 in complex with a hepatitis B peptide homologue

    DEFF Research Database (Denmark)

    Blicher, Thomas; Kastrup, Jette Sandholm; Pedersen, Lars Østergaard

    2006-01-01

    A high-resolution structure of the human MHC-I molecule HLA-A*1101 is presented in which it forms a complex with a sequence homologue of a peptide that occurs naturally in hepatitis B virus DNA polymerase. The sequence of the bound peptide is AIMPARFYPK, while that of the corresponding natural...

  12. The actin homologue MreB organizes the bacterial cell membrane

    NARCIS (Netherlands)

    Strahl, H.; Burmann, F.; Hamoen, L.W.

    2014-01-01

    The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate

  13. Identification of Plant Homologues of Dual Specificity Yak1-Related Kinases

    Directory of Open Access Journals (Sweden)

    Pavel Karpov

    2014-01-01

    Full Text Available Currently, Dual Specificity YAK1-Related Kinases (MNB/DYRK were found in slime molds, protista, fungi, and animals, but the existence of plant homologues is still unclear. In the present study, we have identified 14 potential plant homologues with the previously unknown functions, based on the strong sequence similarity. The results of bioinformatics analysis revealed their correspondence to DYRK1A, DYRK1B, DYRK3, and DYRK4. For two plant homologues of animal DYRK1A from Physcomitrella patens and Arabidopsis thaliana spatial structures of catalytic domains were predicted, as well as their complexes with ADP and selective inhibitor d15. Comparative analysis of 3D-structures of the human DYRK1A and plant homologues, their complexes with the specific inhibitors, and results of molecular dynamics confirm their structural and functional similarity with high probability. Preliminary data indicate the presence of potential MNB/DYRK specific phosphorylation sites in such proteins associated with plant cytoskeleton as plant microtubule-associated proteins WVD2 and WDL1, and FH5 and SCAR2 involved in the organization and polarity of the actin cytoskeleton and some kinesin-like microtubule motor proteins.

  14. Congenital sideroblastic anemia due to mutations in the mitochondrial HSP70 homologue HSPA9

    DEFF Research Database (Denmark)

    Schmitz-Abe, Klaus; Ciesielski, Szymon J; Schmidt, Paul J

    2015-01-01

    The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases, characterized by defects in mitochondrial heme synthesis, iron-sulfur cluster (Fe-S) biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homologue located in the 5q...

  15. The Retention Behaviors of Benzene and Its Alkyl Homologues in Microemulsion Electrokinetic Chromatography

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The retention behaviors of benzene and its alkyl homologues in microemulsion electrokinetic chromatography were investigated in both anionic and cationic surfactant MEEKC systems. The effects of the composition of microemulsion on retention time and selectivity were studied. A good linear relationship was obtained between log k' and the carbon number of alkyl chain.

  16. Muscle cell migrations of C. elegans are mediated by the alpha-integrin INA-1, Eph receptor VAB-1, and a novel peptidase homologue MNP-1.

    Science.gov (United States)

    Tucker, Morgan; Han, Min

    2008-06-15

    Cell migration is a fundamental process occurring during embryonic development and tissue morphogenesis. In the nematode Caenorhabditis elegans, morphogenesis of the body-wall musculature involves short-range migrations of 81 embryonic muscle cells from the lateral surface of the embryo towards the dorsal and ventral midlines. This study shows that mutations in ina-1 (alpha-integrin), as well as vab-1 (Eph receptor), and vab-2 (ephrin), display defects in embryonic muscle cell migration. Furthermore, an RNAi-based enhancer screen in an ina-1 weak loss-of-function background identified mnp-1 (matrix non-peptidase homologue-1) as a previously uncharacterized gene required for promoting proper migration of the embryonic muscle cells. mnp-1 encodes a membrane associated metalloproteinase homologue that is predicted to be catalytically inactive. Our data suggest that MNP-1 is expressed in migrating muscle cells and localizes to the plasma membrane with the non-peptidase domain exposed to the extra-cellular environment. Double-mutant analysis between mnp-1(RNAi), ina-1, and vab-1 mutations; as well as tissue specific rescue experiments; indicated that each of these gene products function predominantly independent of each other and from different cell types to affect muscle cell migration. Together these results suggest complex interactions between the adjacent epidermal, neuronal, and muscle cells are required to promote proper muscle cell migration during embryogenesis.

  17. The effect of the Taq1A variant in the dopamine D2 receptor gene and common CYP2D6 alleles on prolactin levels in risperidone-treated boys

    NARCIS (Netherlands)

    Roke, Y.; Harten, P.N. van; Franke, B.; Galesloot, T.E.; Boot, A.M.; Buitelaar, J.K.

    2013-01-01

    OBJECTIVE: To investigate the effect of the Taq1A variant in the Dopamine D2 receptor gene (DRD2) and common functional genetic variants in the cytochrome P450 2D6 gene (CYP2D6) on prolactin levels in risperidone-treated boys with autism spectrum disorders and disruptive behavior disorders. METHODS:

  18. The effect of the Taq1A variant in the dopamine D-2 receptor gene and common CYP2D6 alleles on prolactin levels in risperidone-treated boys

    NARCIS (Netherlands)

    Roke, Yvette; van Harten, Peter N.; Franke, Barbara; Galesloot, Tessel E.; Boot, Annemieke M.; Buitelaar, Jan K.

    2013-01-01

    Objective To investigate the effect of the Taq1A variant in the Dopamine D2 receptor gene (DRD2) and common functional genetic variants in the cytochrome P450 2D6 gene (CYP2D6) on prolactin levels in risperidone-treated boys with autism spectrum disorders and disruptive behavior disorders.Methods Fo

  19. The effect of the Taq1A variant in the dopamine D-2 receptor gene and common CYP2D6 alleles on prolactin levels in risperidone-treated boys

    NARCIS (Netherlands)

    Roke, Yvette; van Harten, Peter N.; Franke, Barbara; Galesloot, Tessel E.; Boot, Annemieke M.; Buitelaar, Jan K.

    2013-01-01

    Objective To investigate the effect of the Taq1A variant in the Dopamine D2 receptor gene (DRD2) and common functional genetic variants in the cytochrome P450 2D6 gene (CYP2D6) on prolactin levels in risperidone-treated boys with autism spectrum disorders and disruptive behavior disorders.Methods Fo

  20. The effect of the Taq1A variant in the dopamine D2 receptor gene and common CYP2D6 alleles on prolactin levels in risperidone-treated boys

    NARCIS (Netherlands)

    Roke, Y.; Harten, P.N. van; Franke, B.; Galesloot, T.E.; Boot, A.M.; Buitelaar, J.K.

    2013-01-01

    OBJECTIVE: To investigate the effect of the Taq1A variant in the Dopamine D2 receptor gene (DRD2) and common functional genetic variants in the cytochrome P450 2D6 gene (CYP2D6) on prolactin levels in risperidone-treated boys with autism spectrum disorders and disruptive behavior disorders. METHODS:

  1. Treating Infertility

    Science.gov (United States)

    ... Education & Events Advocacy For Patients About ACOG Treating Infertility Home For Patients Search FAQs Treating Infertility Page ... Treating Infertility FAQ137, March 2015 PDF Format Treating Infertility Gynecologic Problems What is infertility? What treatment options ...

  2. Human Tregs Made Antigen Specific by Gene Modification: The Power to Treat Autoimmunity and Antidrug Antibodies with Precision

    Directory of Open Access Journals (Sweden)

    Patrick R. Adair

    2017-09-01

    Full Text Available Human regulatory CD4+ T cells (Tregs are potent immunosuppressive lymphocytes responsible for immune tolerance and homeostasis. Since the seminal reports identifying Tregs, vast research has been channeled into understanding their genesis, signature molecular markers, mechanisms of suppression, and role in disease. This research has opened the doors for Tregs as a potential therapeutic for diseases and disorders such as multiple sclerosis, type I diabetes, transplantation, and immune responses to protein therapeutics, like factor VIII. Seminal clinical trials have used polyclonal Tregs, but the frequency of antigen-specific Tregs among polyclonal populations is low, and polyclonal Tregs may risk non-specific immunosuppression. Antigen-specific Treg therapy, which uses genetically modified Tregs expressing receptors specific for target antigens, greatly mitigates this risk. Building on the principles of T-cell receptor cloning, chimeric antigen receptors (CARs, and a novel CAR derivative, called B-cell antibody receptors, our lab has developed different types of antigen-specific Tregs. This review discusses the current research and optimization of gene-modified antigen-specific human Tregs in our lab in several disease models. The preparations and considerations for clinical use of such Tregs also are discussed.

  3. Constant expression of cyclooxygenase-2 gene in prostate and the lower urinary tract of estrogen-treated male rats.

    Science.gov (United States)

    Luo, C; Strauss, L; Ristimäki, A; Streng, T; Santti, R

    2001-01-01

    Expression of cyclooxygenase-2 (E. C. 1.14.99.1) in prostate and the lower urinary tract (LUT) of the neonatally estrogenized male rat has been studied by using a COX-2's PCR fragment of 724 nt spanning 3 introns and a 478nt internal standard for quantitative RT-PCR. The same fragment of 724 nt was used for RNA probe in Northern hybridization. Neonatal estrogenization (10 microg/day of diethylstilbestrol on days 1-5) had no effect on COX-2 expression in prostatic urethra, prostatic lobes, or bladder. Acute estrogen treatment of castrated animals did not induce COX-2 expression, either. In addition the differential expression of basal level of COX-2 in the different lobes of prostate in normal rat was demonstrated. Our results suggest a constant expression of COX-2 gene in prostate and the lower urinary tract of the neonatally estrogenized (neoDES) rats. The present study indicates that the increased expression of COX-2 is probably not essential for the estrogen-driven development of stromal inflammation or hyperplastic and dysplastic alterations in the prostate of neoDES rats.

  4. Expression of Genes Related to Oxidative Stress in Yeast Treated with Ionizing Radiation and N-acetyl -L-cysteine

    Energy Technology Data Exchange (ETDEWEB)

    Park, Ji Young; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)

    2010-10-15

    Ionizing radiation (IR) induces water radiolysis, which generates highly reactive hydroxyl radicals. Reactive oxygen species (ROS) cause apoptosis and cell damage including DNA strand breaks (DSBs), base damage, protein damage and lipid-hydroperoxide. Detoxifying enzymes are immediately triggered for ROS scavenging. Yeast contains two forms of superoxide dismutase (SOD). SOD1 as a cytosolic copper-zinc superoxide dismutase is located in the cytoplasm and cytosol. SOD2 as a manganese containing enzyme is act in mitochondria matrix and mitochondrion. These enzymes scavenge superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen. The hydrogen peroxide formed by superoxide dismutase and by other processes is scavenged by catalase, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. Yeast contains two catalases. Catalase A (CTA1) and Cytosolic catalase T (CTT1) is located in peroxisome and cytoplasm, respectively. Yeast has two glutathione (GSH) peroxidases, which are GPX1 and GPX2. GPX1 and GPX2 are component of cellular component and cytoplasm, respectively. The biochemical function of GSH peroxidase is to reduce lipid-hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. Otherwise, chemicals and materials help ROS detoxification against oxidative damage. N-acetyl-Lcysteine (NAC) having a thiol, a precursor for glutathione (GSH), is known as one of the antioxidants. In this study, we examined the effect of NAC through gene expressions related to protective enzyme against oxidative stress in yeast

  5. Neurospora crassa fmf-1 encodes the homologue of the Schizosaccharomyces pombe Ste11p regulator of sexual development

    Indian Academy of Sciences (India)

    Srividhya V. Iyer; Mukund Ramakrishnan; Durgadas P. Kasbekar

    2009-04-01

    The Neurospora crassa fmf-1 mutation exerts an unusual ‘perithecium-dominant’ developmental arrest; fmf-1 × fmf-1+ cross becomes arrested in perithecial development regardless of whether the mutant participates in the cross as the male or female parent. We localized fmf-1 to the LG IL genome segment between the centromere-proximal breakpoint of the chromosome segment duplication Dp(IL)39311 and the centromere. By mapping crossovers with respect to RFLP markers in this region we further localized fmf-1 to an approximately 34-kb-genome segment. Partial sequencing of this segment revealed a point mutation in the gene NCU 09387.1, a homologue of the Schizosaccharomyces pombe ste11+ regulator of sexual development. The fmf-1 mutation did not complement a NCU 09387.1 deletion mutation, and transformation with wild-type NCU 09387.1 complemented fmf-1. S. pombe Ste11 protein (Ste 11p) is a transcription factor required for sexual differentiation and for the expression of genes required for mating pheromone signalling in matP and matM cells. If FMF-1 also plays a corresponding role in mating pheromone signalling in Neurospora, then protoperithecia in an fmf-1 × fmf-1+ cross would be unable to either send or receive sexual differentiation signals and thus become arrested in development.

  6. Mapping of ionomic traits in Mimulus guttatus reveals Mo and Cd QTLs that colocalize with MOT1 homologues.

    Directory of Open Access Journals (Sweden)

    David B Lowry

    Full Text Available Natural variation in the regulation of the accumulation of mineral nutrients and trace elements in plant tissues is crucial to plant metabolism, development, and survival across different habitats. Studies of the genetic basis of natural variation in nutrient metabolism have been facilitated by the development of ionomics. Ionomics is a functional genomic approach for the identification of the genes and gene networks that regulate the elemental composition, or ionome, of an organism. In this study, we evaluated the genetic basis of divergence in elemental composition between an inland annual and a coastal perennial accession of Mimulus guttatus using a recombinant inbred line (RIL mapping population. Out of 20 elements evaluated, Mo and Cd were the most divergent in accumulation between the two accessions and were highly genetically correlated in the RILs across two replicated experiments. We discovered two major quantitative trait loci (QTL for Mo accumulation, the largest of which consistently colocalized with a QTL for Cd accumulation. Interestingly, both Mo QTLs also colocalized with the two M. guttatus homologues of MOT1, the only known plant transporter to be involved in natural variation in molybdate uptake.

  7. Neurospora crassa fmf-1 encodes the homologue of the Schizosaccharomyces pombe Ste11p regulator of sexual development.

    Science.gov (United States)

    Iyer, Srividhya V; Ramakrishnan, Mukund; Kasbekar, Durgadas P

    2009-04-01

    The Neurospora crassa fmf-1 mutation exerts an unusual 'perithecium-dominant' developmental arrest; fmf-1 x fmf-1+ cross becomes arrested in perithecial development regardless of whether the mutant participates in the cross as the male or female parent. We localized fmf-1 to the LG IL genome segment between the centromere-proximal breakpoint of the chromosome segment duplication Dp(IL)39311 and the centromere. By mapping crossovers with respect to RFLP markers in this region we further localized fmf-1 to an approximately 34-kb-genome segment. Partial sequencing of this segment revealed a point mutation in the gene NCU 09387.1, a homologue of the Schizosaccharomyces pombe ste11+ regulator of sexual development. The fmf-1 mutation did not complement a NCU 09387.1 deletion mutation, and transformation with wild-type NCU 09387.1 complemented fmf-1. S. pombe Ste11 protein (Ste11p) is a transcription factor required for sexual differentiation and for the expression of genes required for mating pheromone signalling in matP and matM cells. If FMF-1 also plays a corresponding role in mating pheromone signalling in Neurospora, then protoperithecia in an fmf-1 x fmf-1+ cross would be unable to either send or receive sexual differentiation signals and thus become arrested in development.

  8. The Gene expression Grade Index: a potential predictor of relapse for endocrine-treated breast cancer patients in the BIG 1–98 trial

    Directory of Open Access Journals (Sweden)

    Haibe-Kains Benjamin

    2009-07-01

    Full Text Available Abstract Background We have previously shown that the Gene expression Grade Index (GGI was able to identify two subtypes of estrogen receptor (ER-positive tumors that were associated with statistically distinct clinical outcomes in both untreated and tamoxifen-treated patients. Here, we aim to investigate the ability of the GGI to predict relapses in postmenopausal women who were treated with tamoxifen (T or letrozole (L within the BIG 1–98 trial. Methods We generated gene expression profiles (Affymetrix and computed the GGI for a matched, case-control sample of patients enrolled in the BIG 1–98 trial from the two hospitals where frozen samples were available. All relapses (cases were identified from patients randomized to receive monotherapy or from the switching treatment arms for whom relapse occurred before the switch. Each case was randomly matched with four controls based upon nodal status and treatment (T or L. The prognostic value of GGI was assessed as a continuous predictor and divided at the median. Predictive accuracy of GGI was estimated using time-dependent area under the curve (AUC of the ROC curves. Results Frozen samples were analyzable for 48 patients (10 cases and 38 controls. Seven of the 10 cases had been assigned to receive L. Cases and controls were comparable with respect to menopausal and nodal status, local and chemotherapy, and HER2 positivity. Cases were slightly older than controls and had a larger proportion of large, poorly differentiated ER+/PgR- tumors. The GGI was significantly and linearly related to risk of relapse: each 10-unit increase in GGI resulted in an increase of approximately 11% in the hazard rate (p = 0.02. Within the subgroups of patients with node-positive disease or who were treated with L, the hazard of relapse was significantly greater for patients with GGI at or above the median. AUC reached a maximum of 78% at 27 months. Conclusion This analysis supports the GGI as a good predictor of

  9. Lack of Association of Multidrug Resistance Gene-1 Polymorphisms with Treatment Outcome in Chronic Myeloid Leukemia Patients Treated with Imatinib

    Directory of Open Access Journals (Sweden)

    Yaya Kassogue

    2015-10-01

    Full Text Available Background: Despite the impressive results obtained with imatinib, inadequate response or resistance are observed in certain patients. It is known that imatinib is a substrate of a multidrug resistance gene (MDR1. Thus, interindividual genetic differences linked to single nucleotide polymorphisms in MDR1 may influence the metabolism of imatinib. The present study has aimed to examine the impact of MDR1 polymorphisms on the hematologic and cytogenetic responses in 70 chronic myeloid leukemia patients who received imatinib. Methods: We used a polymerase chain reaction followed by restriction fragment length polymorphism to identify different profiles of 1236C>T, 2677G>T and 3435C>T in MDR1. Results: The distribution of the three SNPs in responders and poor responders did not show any particular trend (P>0.05. The T allele was slightly higher in responders, but not significantly regardless of the type of SNP (40.3% vs. 33.8% for 1236C>T; 25% vs. 14.7% for 2677G>T and 33.3% vs. 22% for 3435C>T. The dominant model showed a similar trend (P>0.05. Diplotypes composed by the T allele in different exons were frequent in responders. Haplotype analysis showed that 1236C-2677G-3435C was slightly higher in poor responders (60.02% compared to responders (50.42%. However, 1236T-2677T-3435T was frequent in responders (16.98% compared to poor responders (13.1%. Overall, none of the haplotypes were associated with IM response in our cohort (global haplotype association test, P=0.39. Conclusion: The identification of 1236C>T, 2677G>T and 3435C>T polymorphisms may not be advantageous to predict imatinib response for our chronic myeloid leukemia patients.

  10. Epigenetic and genetic variants in the HTR1B gene and clinical improvement in children and adolescents treated with fluoxetine.

    Science.gov (United States)

    Gassó, Patricia; Rodríguez, Natalia; Blázquez, Ana; Monteagudo, Ana; Boloc, Daniel; Plana, Maria Teresa; Lafuente, Amalia; Lázaro, Luisa; Arnaiz, Joan Albert; Mas, Sergi

    2017-04-03

    The serotonin 1B receptor (5-HT1B) is important to both the pathogenesis of major depressive disorder and the antidepressant effects of selective serotonin reuptake inhibitors. Although fluoxetine has been shown to be effective and safe in children and adolescents, not all patients experience a proper clinical response, which has led to further study into the main factors involved in this inter-individual variability. Our aim was to study the effect of epigenetic and genetic factors that could affect 5-hydroxytryptamine receptor 1B (HTR1B) gene expression, and thereby response to fluoxetine. A total of 83 children and adolescents were clinically assessed 12weeks after of initiating an antidepressant treatment with fluoxetine for the first time. We evaluated the influence of single nucleotide polymorphisms (SNPs) specifically located in transcription factor binding sites (TFBSs) on their clinical improvement. A combined genetic analysis considering the significant SNPs together with the functional variant rs130058 previously associated in our population was also performed. Moreover, we assessed, for the first time in the literature, whether methylation levels of the HTR1B promoter region could be associated with the pharmacological response. Two, rs9361233 and rs9361235, were significantly associated with clinical improvement after treatment with fluoxetine. The heterozygous genotype combination analysis showed a negative correlation with clinical improvement. The lowest improvement was experienced by patients who were heterozygous for all three SNPs. Moreover, a negative correlation was found between clinical improvement and the average methylation level of the HTR1B promoter. These results give new evidence for the role of epigenetic and genetic factors which could modulate HTR1B expression in the pharmacological response to antidepressants.

  11. Heme oxygenase-1 is involved in nitric oxide- and cGMP-induced α-Amy2/54 gene expression in GA-treated wheat aleurone layers.

    Science.gov (United States)

    Wu, Mingzhu; Wang, Fangquan; Zhang, Chen; Xie, Yanjie; Han, Bin; Huang, Jingjing; Shen, Wenbiao

    2013-01-01

    Here, α-Amy2/54 gene expression was used as a molecular probe to investigate the interrelationship among nitric oxide (NO), cyclic GMP (cGMP), and heme oxygenase-1 (HO-1) in GA-treated wheat aleurone layers. The inducible expressions of α-Amy2/54 and α-amylase activity were respectively amplified by two NO-releasing compounds, sodium nitroprusside (SNP) and spermine NONOate, in a GA-dependent fashion. Similar responses were observed when an inducer of HO-1, hemin-or one of its catalytic products, carbon monoxide (CO) in aqueous solution-was respectively added. The SNP-induced responses, mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a cGMP derivative, were NO-dependent. This conclusion was supported by the fact that endogenous NO overproduction was rapidly induced by SNP, and thereafter induction of α-Amy2/54 gene expression and increased α-amylase activity were sensitive to the NO scavenger. We further observed that the above induction triggered by SNP and 8-Br-cGMP was partially prevented by zinc protoporphyrin IX (ZnPPIX), an inhibitor of HO-1. These blocking effects were clearly reversed by CO, confirming that the above response was HO-1-specific. Further analyses showed that both SNP and 8-Br-cGMP rapidly up-regulated HO-1 gene expression and increased HO activity, and SNP responses were sensitive to cPTIO and the guanylate cyclase inhibitor 6-anilino-5,8-quinolinedione (LY83583). Molecular evidence confirmed that GA-induced GAMYB and ABA-triggered PKABA1 transcripts were up-regulated or down-regulated by SNP, 8-Br-cGMP or CO cotreated with GA. Contrasting changes were observed when cPTIO, LY83583, or ZnPPIX was added. Together, our results suggested that HO-1 is involved in NO- and cGMP-induced α-Amy2/54 gene expression in GA-treated aleurone layers.

  12. Association between the HTR2C rs1414334 C/G gene polymorphism and the development of the metabolic syndrome in patients treated with atypical antipsychotics

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    José María Rico-Gomis

    2016-07-01

    Full Text Available Few studies have assessed the association between the rs1414334 C/G polymorphism in the HTR2C gene and the development of the metabolic syndrome in patients treated with atypical antipsychotics. To provide further evidence, a cross-sectional study was conducted in Spain between 2012 and 2013 in 166 patients with these characteristics. In these patients, the association between the polymorphism and the presence of the metabolic syndrome was determined by implementing binary logistic regression models adjusted for variables associated with the metabolic syndrome. We did not confirm previous claims that the C allele of the polymorphism was linked to the metabolic syndrome: the association was in the opposite direction and non-significant. This conclusion held after taking gender and lifestyle variables into account.

  13. Cloning of the Zygosaccharomyces bailii GAS1 homologue and effect of cell wall engineering on protein secretory phenotype

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    Dato Laura

    2010-01-01

    Full Text Available Abstract Background Zygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a β-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of Saccharomyces cerevisiae and Pichia pastoris, deleted in the GAS1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the GAS1 homologue of Z. bailii and the evaluation of its deletion on recombinant protein secretion. Results The GAS1 homologue of Z. bailii was cloned by PCR, and when expressed in a S. cerevisiae GAS1 null mutant was able to restore the parental phenotype. The respective Z. bailii Δgas1 deleted strain was obtained by targeted deletion of both alleles of the ZbGAS1 gene with deletion cassettes having flanking regions of ~400 bp. The morphological and physiological characterization of the Z. bailii null mutant resulted very similar to that of the corresponding S. cerevisiae mutant. As for S. cerevisiae, in the Z. bailii Δgas1 the total amount of protein released in the medium was significantly higher. Moreover, three different heterologous proteins were expressed and secreted in said mutant. The amount of enzymatic activity found in the medium was almost doubled in the case of the Candida rugosa lipase CRL1 and of the Yarrowia lipolytica protease XPR2, while for human IL-1β secretion disruption had no relevant effect. Conclusions The data presented confirm that the engineering of the cell wall is an effective way to improve protein secretion in yeast. They also confirmed that Z. bailii is an interesting candidate, despite the knowledge of its genome and the tools for its manipulation still need to be improved. However, as

  14. Influence of methylenetetrahydrofolate reductase gene polymorphisms on the outcome of pediatric patients with non-Hodgkin lymphoma treated with high-dose methotrexate.

    Science.gov (United States)

    D'Angelo, Velia; Ramaglia, Maria; Iannotta, Adriana; Francese, Matteo; Pota, Elvira; Affinita, Maria Carmen; Pecoraro, Giulia; Indolfi, Cristiana; Di Martino, Martina; Di Pinto, Daniela; Buffardi, Salvatore; Poggi, Vincenzo; Indolfi, Paolo; Casale, Fiorina

    2013-12-01

    High-dose methotrexate (MTX) is a key component of most treatment protocols for childhood and adolescent non-Hodgkin lymphoma (NHL). Recent studies have suggested that the toxicity of antifolate drugs, such as MTX, is affected by inherited single nucleotide polymorphisms (SNPs) in folate metabolizing genes. The aim of our study was to investigate the potential influence of the C677T and A1298C genetic variants of the methylenetetrahydrofolate reductase (MTHFR) gene on the clinical toxicity and efficacy of MTX in pediatric patients with NHL (n = 95) treated with therapeutic protocols Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) LNH-97 and EURO LB-02. We demonstrated that patients with the 677T genotype had an approximately six-fold greater risk of developing hematological toxicity compared with wild-type carriers, especially in the 1 g/m(2) treatment group (p = 0.01). Moreover, we identified a correlation between the risk of relapse and the T genotype: T carriers had reduced disease-free survival compared with wild-type patients (67% vs. 100%). Our data suggest a pharmacogenetic influence on the adverse effects of high-dose MTX in the 1 g/m(2) treatment group.

  15. Association between angiotensin-converting enzyme gene polymorphisms and regression of left ventricular hypertrophy in patients treated with angiotensin-converting enzyme inhibitors.

    Science.gov (United States)

    Kohno, M; Yokokawa, K; Minami, M; Kano, H; Yasunari, K; Hanehira, T; Yoshikawa, J

    1999-05-01

    An insertion/deletion (ID) polymorphism of the angiotensin-converting enzyme (ACE) gene is associated with left ventricular hypertrophy. The present study examined polymorphisms of the ACE gene in patients with essential hypertension and left ventricular hypertrophy who were participants in a long-term trial of therapy with an ACE inhibitor. ACE inhibitor therapy was administered for >2 years to 54 patients with hypertension who had moderate or severe left ventricular hypertrophy. Cardiac dimensions were monitored by echocardiography before the initiation of therapy and after 1 and 2 years of treatment. Serum ACE activity and plasma concentrations of brain natriuretic peptide, a marker for left ventricular hypertrophy, were also monitored. Eighteen patients had the II genotype for the angiotensin-converting enzyme gene, 19 had the ID genotype, and 17 had the DD genotype. Baseline (mean +/- SD) serum ACE activity was significantly greater (P <0.05) in the DD (18 +/- 7 IU/L) group than in the II (7 +/- 4 IU/L) or ID (12 +/- 6 IU/L) groups. ACE inhibitor therapy was effective in controlling blood pressure, and it reduced posterior and septal wall thickness, left ventricular mass index, and plasma brain natriuretic peptide concentration in all three groups. Despite similar blood pressure reductions, after 2 years, mean (+/- SD) regression in posterior wall thickness was significantly less (P <0.05) in the DD group (-9% +/- 5%) than in the ID (-21% +/- 7%) and II (-21% +/- 9%) groups. Similar results were seen for the reductions in brain natriuretic peptide levels. The magnitudes of regression of septal wall thickness and left ventricular mass index during therapy were less in the DD group than the II group (P <0.05). Hypertensive patients with the DD genotype are less likely to have regression of left ventricular hypertrophy when treated with ACE inhibitors than are patients with other ACE genotypes.

  16. Changes in Menidia beryllina Gene Expression and In Vitro Hormone-Receptor Activation After Exposure to Estuarine Waters Near Treated Wastewater Outfalls.

    Science.gov (United States)

    Cole, Bryan J; Brander, Susanne M; Jeffries, Ken M; Hasenbein, Simone; He, Guochun; Denison, Michael S; Fangue, Nann A; Connon, Richard E

    2016-08-01

    Fishes in estuarine waters are frequently exposed to treated wastewater effluent, among numerous other sources of contaminants, yet the impacts of these anthropogenic chemicals are not well understood in these dynamic and important waterways. Inland silversides (Menidia beryllina) at an early stage of development [12 days posthatch (dph)] were exposed to waters from two estuarine wastewater-treatment outfall locations in a tidal estuary, the Sacramento/San Joaquin Delta (California, USA) that had varied hydrology and input volumes. The genomic response caused by endocrine-disrupting compounds (EDCs) in these waters was determined using quantitative polymerase chain reaction on a suite of hormonally regulated genes. Relative androgenic and estrogenic activities of the waters were measured using CALUX reporter bioassays. The presence of bifenthrin, a pyrethroid pesticide and known EDC, as well as caffeine and the anti-inflammatory pharmaceutical ibuprofen, which were used as markers of wastewater effluent input, were determined using instrumental analysis. Detectable levels of bifenthrin (2.89 ng L(-1)) were found on one of the sampling dates, and caffeine was found on all sampling dates, in water from the Boynton Slough. Neither compound was detected at the Carquinez Strait site, which has a much smaller effluent discharge input volume relative to the receiving water body size compared with Boynton Slough. Water samples from both sites incubated in the CALUX cell line induced estrogenic and androgenic activity in almost all instances, though the estrogenicity was relatively higher than the androgenicity. Changes in the abundance of mRNA transcripts of endocrine-responsive genes and indicators of general chemical stress were observed after a 96-h exposure to waters from both locations. The relative levels of endocrine response, changes in gene transcript abundance, and contaminant concentrations were greater in water from the Boynton Slough site despite those

  17. Removal of bacterial contaminants and antibiotic resistance genes by conventional wastewater treatment processes in Saudi Arabia: Is the treated wastewater safe to reuse for agricultural irrigation?

    KAUST Repository

    Aljassim, Nada I.

    2015-04-01

    This study aims to assess the removal efficiency of microbial contaminants in a local wastewater treatment plant over the duration of one year, and to assess the microbial risk associated with reusing treated wastewater in agricultural irrigation. The treatment process achieved 3.5 logs removal of heterotrophic bacteria and up to 3.5 logs removal of fecal coliforms. The final chlorinated effluent had 1.8×102 MPN/100mL of fecal coliforms and fulfils the required quality for restricted irrigation. 16S rRNA gene-based high-throughput sequencing showed that several genera associated with opportunistic pathogens (e.g. Acinetobacter, Aeromonas, Arcobacter, Legionella, Mycobacterium, Neisseria, Pseudomonas and Streptococcus) were detected at relative abundance ranging from 0.014 to 21 % of the total microbial community in the influent. Among them, Pseudomonas spp. had the highest approximated cell number in the influent but decreased to less than 30 cells/100mL in both types of effluent. A culture-based approach further revealed that Pseudomonas aeruginosa was mainly found in the influent and non-chlorinated effluent but was replaced by other Pseudomonas spp. in the chlorinated effluent. Aeromonas hydrophila could still be recovered in the chlorinated effluent. Quantitative microbial risk assessment (QMRA) determined that only chlorinated effluent should be permitted for use in agricultural irrigation as it achieved an acceptable annual microbial risk lower than 10-4 arising from both P. aeruginosa and A. hydrophila. However, the proportion of bacterial isolates resistant to 6 types of antibiotics increased from 3.8% in the influent to 6.9% in the chlorinated effluent. Examples of these antibiotic-resistant isolates in the chlorinated effluent include Enterococcus and Enterobacter spp. Besides the presence of antibiotic-resistant bacterial isolates, tetracycline resistance genes tetO, tetQ, tetW, tetH, tetZ were also present at an average 2.5×102, 1.6×102, 4.4×102, 1

  18. Resistance mutations in protease gene at baseline are not related to virological failure in patients treated with darunavir/ritonavir monotherapy

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    Angela Gutierrez-Liarte

    2014-11-01

    Full Text Available Introduction: Monotherapy with darunavir plus ritonavir (DRV/r is a good maintenance strategy for suppressed HIV-infected patients. The clinical trials designed to prove the efficacy of PI/r do not include patients with resistance mutation in protease gene [1,2]. Sometimes in routine practice, basically to avoid NRTIs toxicity, monotherapy with DRV/r is used despite PI resistance mutations. The aim of this study is to know the effect of previous protease resistance mutation on DRV/r monotherapy efficacy. Materials and Methods: We designed an observational cohort study of adults in treatment with DRV/r monotherapy in a tertiary Spanish hospital since 2011 to 2014. Demographic data and clinical outcomes were described. The analysis of efficacy was done according to the snapshot algorithm (defining virological failure as viral load >50 copies/mL, ITTe, at 48 and 96 weeks. We analyzed the difference of efficacy between patients with and without baseline resistance mutations at 48 and 96 weeks by using the χ2 test; and during the follow-up by using the Kaplan–Meier test. The statistical analysis was done with SPSS 17.0. Results: Eighty-nine patients were included in the cohort but 14 were excluded because they had not reached more than six months with monotherapy. The cohort was composed mainly by men (78%, the medium age was 51 years (SD±10, 35% were MSM and 19% were former IDU. Twenty-four patients (35% had a previous diagnosis of AIDS. The mean time taking NRTIs was 10.5 years (SD±5.4. Sixty-four patients (85% had been treated with PI in the past. Previous failure with PI had been reported in 15 (20%. A resistance mutation test had been done at baseline in 45 patients (51%. Twenty-two patients (29% had some mutations in protease gene, 10 patients (13% had major mutations and 1 patient had some mutations of resistance for darunavir (I64V. At 48 weeks, 93% (CI 95% 86–98% had VL<50 copies/mL, and 79% (CI 95% 67–89% at 96 weeks. There were

  19. Comparison of the regulation, metabolic functions, and roles in virulence of the glyceraldehyde-3-phosphate dehydrogenase homologues gapA and gapB in Staphylococcus aureus.

    Science.gov (United States)

    Purves, Joanne; Cockayne, Alan; Moody, Peter C E; Morrissey, Julie A

    2010-12-01

    The Gram-positive bacterium Staphylococcus aureus contains two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologues known as GapA and GapB. GapA has been characterized as a functional GAPDH protein, but currently there is no biological evidence for the role of GapB in metabolism in S. aureus. In this study we show through a number of complementary methods that S. aureus GapA is essential for glycolysis while GapB is essential in gluconeogenesis. These proteins are reciprocally regulated in response to glucose concentrations, and both are influenced by the glycolysis regulator protein GapR, which is the first demonstration of the role of this regulator in S. aureus and the first indication that GapR homologues control genes other than those within the glycolytic operon. Furthermore, we show that both GapA and GapB are important in the pathogenesis of S. aureus in a Galleria mellonella model of infection, showing for the first time in any bacteria that both glycolysis and gluconeogenesis have important roles in virulence.

  20. Mycosphaerella graminicola LysM effector-mediated stealth pathogenesis subverts recognition through both CERK1 and CEBiP homologues in wheat.

    Science.gov (United States)

    Lee, Wing-Sham; Rudd, Jason J; Hammond-Kosack, Kim E; Kanyuka, Kostya

    2014-03-01

    Fungal cell-wall chitin is a well-recognized pathogen-associated molecular pattern. Recognition of chitin in plants by pattern recognition receptors activates pathogen-triggered immunity (PTI). In Arabidopsis, this process is mediated by a plasma membrane receptor kinase, CERK1, whereas in rice, a receptor-like protein, CEBiP, in addition to CERK1 is required. Secreted chitin-binding lysin motif (LysM) containing fungal effector proteins, such as Ecp6 from the biotrophic fungus Cladosporium fulvum, have been reported to interfere with PTI. Here, we identified wheat homologues of CERK1 and CEBiP and investigated their role in the interaction with the nonbiotrophic pathogen of wheat Mycosphaerella graminicola (synonym Zymoseptoria tritici). We show that silencing of either CERK1 or CEBiP in wheat, using Barley stripe mosaic virus-mediated virus-induced gene silencing, is sufficient in allowing leaf colonization by the normally nonpathogenic M. graminicola Mg3LysM (homologue of Ecp6) deletion mutant, while the Mg1LysM deletion mutant was fully pathogenic toward both silenced and wild-type wheat leaves. These data indicate that Mg3LysM is important for fungal evasion of PTI in wheat leaf tissue and that both CERK1 and CEBiP are required for activation of chitin-induced defenses, a feature conserved between rice and wheat, and perhaps, also in other cereal species.

  1. Comparison of the Regulation, Metabolic Functions, and Roles in Virulence of the Glyceraldehyde-3-Phosphate Dehydrogenase Homologues gapA and gapB in Staphylococcus aureus▿

    Science.gov (United States)

    Purves, Joanne; Cockayne, Alan; Moody, Peter C. E.; Morrissey, Julie A.

    2010-01-01

    The Gram-positive bacterium Staphylococcus aureus contains two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologues known as GapA and GapB. GapA has been characterized as a functional GAPDH protein, but currently there is no biological evidence for the role of GapB in metabolism in S. aureus. In this study we show through a number of complementary methods that S. aureus GapA is essential for glycolysis while GapB is essential in gluconeogenesis. These proteins are reciprocally regulated in response to glucose concentrations, and both are influenced by the glycolysis regulator protein GapR, which is the first demonstration of the role of this regulator in S. aureus and the first indication that GapR homologues control genes other than those within the glycolytic operon. Furthermore, we show that both GapA and GapB are important in the pathogenesis of S. aureus in a Galleria mellonella model of infection, showing for the first time in any bacteria that both glycolysis and gluconeogenesis have important roles in virulence. PMID:20876289

  2. Rabbit muscle creatine phosphokinase. CDNA cloning, primary structure and detection of human homologues.

    Science.gov (United States)

    Putney, S; Herlihy, W; Royal, N; Pang, H; Aposhian, H V; Pickering, L; Belagaje, R; Biemann, K; Page, D; Kuby, S

    1984-12-10

    A cDNA library was constructed from rabbit muscle poly(A) RNA. Limited amino acid sequence information was obtained on rabbit muscle creatine phosphokinase and this was the basis for design and synthesis of two oligonucleotide probes complementary to a creatine kinase cDNA sequence which encodes a pentapeptide. Colony hybridizations with the probes and subsequent steps led to isolation of two clones, whose cDNA segments partially overlap and which together encode the entire protein. The primary structure was established from the sequence of two cDNA clones and from independently determined sequences of scattered portions of the polypeptide. The reactive cysteine has been located to position 282 within the 380 amino acid polypeptide. The rabbit cDNA hybridizes to digests of human chromosomal DNA. This reveals a restriction fragment length polymorphism associated with the human homologue(s) which hybridizes to the rabbit cDNA.

  3. Formation of Benzyl Carbanion in Collision-Induced Dissociation of Deprotonated Phenylalanine Homologues.

    Science.gov (United States)

    Sekimoto, Kanako; Matsuda, Natsuki; Takayama, Mitsuo

    2014-01-01

    The fragmentation behavior of deprotonated L-phenylalanine (Phe) and its homologues including L-homophenylalanine (HPA) and L-phenylglycine (PG) was investigated using collision-induced dissociation mass spectrometry coupled with a negative ion atmospheric pressure corona discharge ionization (APCDI) technique. The deprotonated molecules [M-H](-) fragmented to lose unique neutral species, e.g., the loss of NH3, CO2, toluene and iminoglycine for [Phe-H](-); styrene and ethenamine/CO2 for [HPA-H](-); and CO2 for [PG-H](-). All of the fragmentations observed are attributable to the formation of intermediates and/or product ions which include benzyl carbanions having resonance-stabilized structures. The carbanions are formed via proton rearrangement through a transition state or via a simple dissociation reaction. These results suggest that the principal factor governing the fragmentation behavior of deprotonated Phe homologues is the stability of the intermediate and/or product ion structures.

  4. A spinach O-acetylserine(thiollyase homologue, SoCSaseLP, suppresses cysteine biosynthesis catalysed by other enzyme isoforms

    Directory of Open Access Journals (Sweden)

    Miki Noda

    2016-06-01

    Full Text Available An enzyme, O-acetylserine(thiollyase (OASTL, also known as O-acetylserine sulfhydrylase or cysteine synthase (CSase, catalyses the incorporation of sulfide into O-acetylserine and produces cysteine. We previously identified a cDNA encoding an OASTL-like protein from Spinacia oleracea, (SoCSaseLP, but a recombinant SoCSaseLP produced in Escherichia coli did not show OASTL activity. The exon-intron structure of the SoCSaseLP gene shared conserved structures with other spinach OASTL genes. The SoCSaseLP and a Beta vulgaris homologue protein, KMT13462, comprise a unique clade in the phylogenetic tree of the OASTL family. Interestingly, when the SoCSaseLP gene was expressed in tobacco plants, total OASTL activity in tobacco leaves was reduced. This reduction in total OASTL activity was most likely caused by interference by SoCSaseLP with cytosolic OASTL. To investigate the possible interaction of SoCSaseLP with a spinach cytosolic OASTL isoform SoCSaseA, a pull-down assay was carried out. The recombinant glutathione S-transferase (GST-SoCSaseLP fusion protein was expressed in E. coli together with the histidine-tagged SoCSaseA protein, and the protein extract was subjected to glutathione affinity chromatography. The histidine-tagged SoCSaseA was co-purified with the GST-SoCSaseLP fusion protein, indicating the binding of SoCSaseLP to SoCSaseA. Consistent with this interaction, the OASTL activity of the co-purified SoCSaseA was reduced compared with the activity of SoCSaseA that was purified on its own. These results strongly suggest that SoCSaseLP negatively regulates the activity of other cytosolic OASTL family members by direct interaction.

  5. [Homologue pairing: initiation sites and effects on crossing over and chromosome disjunction in Drosophila melanogaster].

    Science.gov (United States)

    Chubykin, V L

    1996-01-01

    The role of homologue pairing and chromocentral association of chromosomes in recombination and segregation during cell division is discussed. Peculiarities of mitotic and meiotic chromosome pairing in Drosophila males and females are considered. On the basis of our own and published data, the presence and localization of sites of homologue pairing initiation in euchromatin are substantiated. The effects of transfer of initiation sites along a chromosome (exemplified by inversions) on chromosome pairing (asynapsis), crossing over (intrachromosomal, interchromosomal, and centromeric effects), and segregation are discussed. To record the effects of pairing sites on crossing over, a method of comparing crossing-over frequencies in an inverted region with those in a region of the same size and position with regard to the centromere on cytological maps was proposed. Chromosomes orient toward opposite division poles during paracentromeric heterochromatin pairing. This occurs after successful euchromatin pairing, during which the chromocentral circular structure is reorganized. If heterochromatin pairing is disrupted because of structural or locus mutations, nonexchange bivalents segregate randomly. In this case, chromosome coordination may occur due to proximal chiasmata or chromocentral associations between homologues.

  6. Homologue of mammalian apolipoprotein A-Ⅱ in non-mammalian vertebrates

    Institute of Scientific and Technical Information of China (English)

    Malay Choudhury; Shoji Yamada; Masaharu Komatsu; Hideki Kishimura; Seiichi Ando

    2009-01-01

    Although apolipoprotein with molecular weight 14 kDa (apo-14 kDa) is associated with fish plasma highdensity lipoproteins(HDLs),it remains to be determined whether apo-14 kDa is the homologue of mammalian apoA-Ⅱ.We have obtained the full cDNA sequences that encode Japanese eel and rainbow trout apo-14 kDa.Homologues of Japanese eel apo-14 kDa sequence could be found in 14 fish species deposited in the DDBJ/EMBL/GenBank or TGI database.Fish apo14 kDa lacks propeptide and contains more internal repeats than mammalian apoA-Ⅱ.Nevertheless,phylogenetic analysis allowed fish apo-14 kDa to be the homologue of mammalian apoA-Ⅱ.In addition,in silico cloning of the TGI,Ensembl,or NCBI database revealed apoA-Ⅱs in dog,chicken,green anole lizard,and African clawed frog whose sequences had not so far been available,suggesting both apoA-Ⅰ and apoA-Ⅱas fundamental constituents of vertebrate HDLs.

  7. Obesity-linked homologues TfAP-2 and Twz establish meal frequency in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Michael J Williams

    2014-09-01

    Full Text Available In all animals managing the size of individual meals and frequency of feeding is crucial for metabolic homeostasis. In the current study we demonstrate that the noradrenalin analogue octopamine and the cholecystokinin (CCK homologue Drosulfakinin (Dsk function downstream of TfAP-2 and Tiwaz (Twz to control the number of meals in adult flies. Loss of TfAP-2 or Twz in octopaminergic neurons increased the size of individual meals, while overexpression of TfAP-2 significantly decreased meal size and increased feeding frequency. Of note, our study reveals that TfAP-2 and Twz regulate octopamine signaling to initiate feeding; then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior. Intriguingly, we found that the mouse TfAP-2 and Twz homologues, AP-2β and Kctd15, co-localize in areas of the brain known to regulate feeding behavior and reward, and a proximity ligation assay (PLA demonstrated that AP-2β and Kctd15 interact directly in a mouse hypothalamus-derived cell line. Finally, we show that in this mouse hypothalamic cell line AP-2β and Kctd15 directly interact with Ube2i, a mouse sumoylation enzyme, and that AP-2β may itself be sumoylated. Our study reveals how two obesity-linked homologues regulate metabolic homeostasis by modulating consummatory behavior.

  8. Deletion of the fission yeast homologue of human insulinase reveals a TORC1-dependent pathway mediating resistance to proteotoxic stress.

    Directory of Open Access Journals (Sweden)

    Clémentine Beuzelin

    Full Text Available Insulin Degrading Enzyme (IDE is a protease conserved through evolution with a role in diabetes and Alzheimer's disease. The reason underlying its ubiquitous expression including cells lacking identified IDE substrates remains unknown. Here we show that the fission yeast IDE homologue (Iph1 modulates cellular sensitivity to endoplasmic reticulum (ER stress in a manner dependent on TORC1 (Target of Rapamycin Complex 1. Reduced sensitivity to tunicamycin was associated with a smaller number of cells undergoing apoptosis. Wild type levels of tunicamycin sensitivity were restored in iph1 null cells when the TORC1 complex was inhibited by rapamycin or by heat inactivation of the Tor2 kinase. Although Iph1 cleaved hallmark IDE substrates including insulin efficiently, its role in the ER stress response was independent of its catalytic activity since expression of inactive Iph1 restored normal sensitivity. Importantly, wild type as well as inactive human IDE complemented gene-invalidated yeast cells when expressed at the genomic locus under the control of iph1(+ promoter. These results suggest that IDE has a previously unknown function unrelated to substrate cleavage, which links sensitivity to ER stress to a pro-survival role of the TORC1 pathway.

  9. Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway.

    Science.gov (United States)

    Shimamura, Mai; Kyotani, Akane; Azuma, Yumiko; Yoshida, Hideki; Binh Nguyen, Thanh; Mizuta, Ikuko; Yoshida, Tomokatsu; Mizuno, Toshiki; Nakagawa, Masanori; Tokuda, Takahiko; Yamaguchi, Masamitsu

    2014-08-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila.

  10. The Caenorhabditis elegans p120 catenin homologue, JAC-1, modulates cadherin-catenin function during epidermal morphogenesis.

    Science.gov (United States)

    Pettitt, Jonathan; Cox, Elisabeth A; Broadbent, Ian D; Flett, Aileen; Hardin, Jeff

    2003-07-07

    The cadherin-catenin complex is essential for tissue morphogenesis during animal development. In cultured mammalian cells, p120 catenin (p120ctn) is an important regulator of cadherin-catenin complex function. However, information on the role of p120ctn family members in cadherin-dependent events in vivo is limited. We have examined the role of the single Caenorhabditis elegans p120ctn homologue JAC-1 (juxtamembrane domain [JMD]-associated catenin) during epidermal morphogenesis. Similar to other p120ctn family members, JAC-1 binds the JMD of the classical cadherin HMR-1, and GFP-tagged JAC-1 localizes to adherens junctions in an HMR-1-dependent manner. Surprisingly, depleting JAC-1 expression using RNA interference (RNAi) does not result in any obvious defects in embryonic or postembryonic development. However, jac-1(RNAi) does increase the severity and penetrance of morphogenetic defects caused by a hypomorphic mutation in the hmp-1/alpha-catenin gene. In these hmp-1 mutants, jac-1 depletion causes failure of the embryo to elongate into a worm-like shape, a process that involves contraction of the epidermis. Associated with failed elongation is the detachment of actin bundles from epidermal adherens junctions and failure to maintain cadherin in adherens junctions. These results suggest that JAC-1 acts as a positive modulator of cadherin function in C. elegans.

  11. Mammalian mitochondrial intermediate peptidase: Structure/function analysis of a new homologue from Schizophyllum commune and relationship to thimet oligopeptidases

    Energy Technology Data Exchange (ETDEWEB)

    Isaya, G.; Sakati, W.R.; Rollins, R.A. [Yale Univ. School of Medicine, New Haven, CT (United States)] [and others

    1995-08-10

    Mitochondrial intermediate peptidase (MIP) is a component of the mitochondrial protein import machinery required for maturation of nuclear-encoded precursor proteins targeted to the mitochondrial matrix or inner membrane. We previously characterized this enzyme in rat (RMIP) and Saccharomyces cerevisiae (YMIP) and showed that MIP activity is essential for mitochondrial function in yeast. We have now defined the structure of a new MIP homologue (SMIP) from the basidiomycete fungus Schizophyllum commune. SMIP includes 4 exons of 523, 486, 660, and 629 bp separated by 3 short introns. The predicted SMIP, YMIP, and RMIP sequences share 31-37% identity and 54-57% similarity over 700 amino acids. When SMIP and RMIP were expressed in a yeast mip1{Delta} mutant, they were both able to rescue the respiratory-deficient phenotype caused by genetic inactivation of YMIP, indicating that the function of this enzyme is conserved in eukaryotes. Moreover, the MIP sequences show 20-24% identity and 40-47% similarity to a family of oligopeptidases from bacteria, yeast, and mammals. MIP and these proteins are characterized by a highly conserved motif, F-H-E-X-G-H-(X){sub 12}-G-(X){sub 5}-D-(X){sub 2}-E-X-P-S-(X){sub 3}-E-X, centered around a zinc-binding site and appear to represent a new family of genes associated with proteolytic processing in the mitochondrial and cytosolic compartments. 48 refs., 8 figs.

  12. Mammalian mitochondrial intermediate peptidase: structure/function analysis of a new homologue from Schizophyllum commune and relationship to thimet oligopeptidases.

    Science.gov (United States)

    Isaya, G; Sakati, W R; Rollins, R A; Shen, G P; Hanson, L C; Ullrich, R C; Novotny, C P

    1995-08-10

    Mitochondrial intermediate peptidase (MIP) is a component of the mitochondrial protein import machinery required for maturation of nuclear-encoded precursor proteins targeted to the mitochondrial matrix or inner membrane. We previously characterized this enzyme in rat (RMIP) and Saccharomyces cerevisiae (YMIP) and showed that MIP activity is essential for mitochondrial function in yeast. We have now defined the structure of a new MIP homologue (SMIP) from the basidiomycete fungus Schizophyllum commune. SMIP includes 4 exons of 523, 486, 660, and 629 bp separated by 3 short introns. The predicted SMIP, YMIP, and RMIP sequences share 31-37% identity and 54-57% similarity over 700 amino acids. When SMIP and RMIP were expressed in a yeast mip1 delta mutant, they were both able to rescue the respiratory-deficient phenotype caused by genetic inactivation of YMIP, indicating that the function of this enzyme is conserved in eukaryotes. Moreover, the MIP sequences show 20-24% identity and 40-47% similarity to a family of oligopeptidases from bacteria, yeast, and mammals. MIP and these proteins are characterized by a highly conserved motif, F-H-E-X-G-H-(X)2-H-(X)12-G-(X)5-D-(X)2-E-X-P-S-(X)3-E-X, centered around a zinc-binding site and appear to represent a new family of genes associated with proteolytic processing in the mitochondrial and cytosolic compartments.

  13. Ectopic expression of Cvh (Chicken Vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate.

    Science.gov (United States)

    Lavial, Fabrice; Acloque, Hervé; Bachelard, Elodie; Nieto, M Angela; Samarut, Jacques; Pain, Bertrand

    2009-06-01

    When they are derived from blastodermal cells of the pre-primitive streak in vitro, the pluripotency of Chicken Embryonic Stem Cells (cESC) can be controlled by the cPouV and Nanog genes. These cESC can differentiate into derivatives of the three germ layers both in vitro and in vivo, but they only weakly colonize the gonads of host embryos. By contrast, non-cultured blastodermal cells and long-term cultured chicken primordial germ cells maintain full germline competence. This restriction in the germline potential of the cESC may result from either early germline determination in the donor embryos or it may occur as a result of in vitro culture. We are interested in understanding the genetic determinants of germline programming. The RNA binding protein Cvh (Chicken Vasa Homologue) is considered as one such determinant, although its role in germ cell physiology is still unclear. Here we show that the exogenous expression of Cvh, combined with appropriate culture conditions, induces cESC reprogramming towards a germ cell fate. Indeed, these cells express the Dazl, Tudor and Sycp3 germline markers, and they display improved germline colonization and adopt a germ cell fate when injected into recipient embryos. Thus, our results demonstrate that Vasa can drive ES cell differentiation towards the germ cell lineage, both in vitro and in vivo.

  14. LC2, the Chlamydomonas Homologue of the t Complex-encoded Protein Tctex2, Is Essential for Outer Dynein Arm Assembly

    Science.gov (United States)

    Pazour, Gregory J.; Koutoulis, Anthony; Benashski, Sharon E.; Dickert, Bethany L.; Sheng, Hong; Patel-King, Ramila S.; King, Stephen M.; Witman, George B.

    1999-01-01

    Tctex2 is thought to be one of the distorter genes of the mouse t haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain of Chlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonas insertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules of t-bearing or wild-type sperm, with resulting differences in their motility. PMID:10512883

  15. Achaete-scute complex homologue-1 promotes development of laryngocarcinoma via facilitating the epithelial-mesenchymal transformation.

    Science.gov (United States)

    Ma, Huaci; Du, Xiaodong; Zhang, Shu; Wang, Qiang; Yin, Yong; Qiu, Xiaoxia; Da, Peng; Yue, Huijun; Wu, Hao; Xu, Fenglei

    2017-06-01

    Laryngeal cancer is one of the most common fatal cancers among head and neck carcinomas, whose mechanism, however, remains unclear. The proneural basic-helix-loop-helix protein achaete-scute complex homologue-1, a member of the basic helix-loop-helix family, plays a very important role in many cancers. This study aims to explore the clinical value and mechanism of achaete-scute complex homologue-1 in laryngeal cancer. Methods including Cell Counting Kit-8, flow cytometry, Transwell invasion assays, and scratch assay were adopted to further explore the bio-function of achaete-scute complex homologue-1, whose expression was examined in fresh and paraffin chip of laryngeal carcinoma tissues by means of western blot and immunohistochemistry, after the interference of achaete-scute complex homologue-1; achaete-scute complex homologue-1, an overexpression in laryngeal carcinoma whose carcinogenicity potential was confirmed via western blot, was correlative with T classification (p = 0.002), histological differentiation (p = 0.000), lymph node metastasis (p = 0.000), and poor survival (p = 0.000). Multivariate analysis shows that achaete-scute complex homologue-1 overexpression is an independent prognostic factor unfavorable to laryngeal carcinoma patients (p = 0.000). Moreover, knocking down achaete-scute complex homologue-1 expression could significantly suppress the proliferation, migration, and invasion of laryngeal carcinoma cell in vitro and disorder epithelial-mesenchymal transformation-associated protein expression. Achaete-scute complex homologue-1 plays an important role in the genesis and progression of laryngeal carcinoma and may act as a potential biomarker for therapeutic target and prognostic prediction.

  16. Mapping of a macular drusen susceptibility locus in rhesus macaques to the homologue of human chromosome 6q14-15.

    Science.gov (United States)

    Singh, Krishna K; Ristau, Steven; Dawson, William W; Krawczak, Michael; Schmidtke, Jörg

    2005-10-01

    Rhesus macaques (Macaca mulatta) are a natural model for retinal drusen formation. The present study aimed at clarifying whether chromosomal regions homologous to candidate genes for drusen formation and progression in humans are also associated with a drusen phenotype in rhesus macaques. Some 42 genetic markers from seven chromosomal regions implicated in macular degeneration syndromes in humans were tested for whether they identified homologous, polymorphic sequences in rhesus DNA. This was found to be the case for seven markers, all of which were subsequently screened for the presence of potentially disease-predisposing alleles in 52 randomly chosen adult animals from the Cayo Santiago population of rhesus macaques (Caribbean Primate Research Center, PR, USA). The high drusen prevalence expected in the Cayo Santiago colony was confirmed in our sample in that 38 animals were found to have drusen (73%). Logistic regression analysis revealed that some alleles of the rhesus homologue of anonymous human marker D6S1036 were consistently over-represented among affected animals. Of two candidate genes located in the respective region, allelic variation in one (IMPG1) showed strong association with drusen formation. We conclude that one or more genes located at the rhesus homologue of human 6q14-15 are likely to play a role in retinal drusen formation, a finding that represents a first step towards the identification of genetic factors implicated in macular drusen formation in rhesus macaques. This is an important tool for the separation of genetic and environmental factors which must occur before satisfactory management methods can be developed.

  17. Deficiency in frataxin homologue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation.

    Science.gov (United States)

    Pynyaha, Yuriy V; Boretsky, Yuriy R; Fedorovych, Daria V; Fayura, Lubov R; Levkiv, Andriy I; Ubiyvovk, Vira M; Protchenko, Olha V; Philpott, Caroline C; Sibirny, Andriy A

    2009-12-01

    Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B2) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Δyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-L-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Δyfh1 mutant was ~3-3.5 times higher as compared to the parental strain. It produced 50-70 times more riboflavin in iron sufficient synthetic media relative to the parental wildtype strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Δyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.

  18. Regulation of demethylation and re-expression of RASSF1A gene in hepatocellular carcinoma cell lines treated with NCTD in vitro

    Directory of Open Access Journals (Sweden)

    Yong Wang

    2015-01-01

    Full Text Available Background: Hepatocellular carcinoma, a lethal malignant neoplasm with poor prognosis, has dismal results of surgical resection and chemoradiotherapy. Norcantharidin (NCTD, the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of cancer. However, the detailed mechanisms underlying this process are generally unclear. Purpose: The aim of this study was to investigate the mechanism of NCTD-induced apoptosis in HepG2 cells. Materials and Methods: Human HepG2 cell lines were treated with NCTD at different concentrations (2.50, 5.00, 10.00, 20.00, 40.00 μg/mL for 24 hours. Cell proliferation was evaluated by measurement of cellular 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT. The methylation levels of RASSF1A (Ras-association domain family 1 A in HepG2 cells were detected by methylation-specific PCR (MSP. The mRNA levels of RASSF1A in HepG2 cells were detected by real-time fluorescent quantitative PCR (RT-PCR. The levels of RASSF1A protein expression of HepG2 cells were detected by Western blotting assay. Results: The inhibition of cell proliferation was observed when treated with NCTD at concentrations (2.5 μg/mL, and as concentration increased, the proliferation of HepG2 cells was markedly inhibited by NCTD in dose-dependent manners. The levels of methylation of RASSF1A decreased at the increasing concentration of 10, 20 and 40 μg/mL. The levels of RASSF1A mRNA and protein were decreased when treated with NCTD at the concentrations of 10, 20 and 40 μg/mL, which were also in a dose-dependent manner. Conclusion: NCTD can reverse the methylation state of RASSF1A gene and induce its re-expression, which will provide the theoretical basis for the clinical practice.

  19. Gene-expression profiling and not immunophenotypic algorithms predicts prognosis in patients with diffuse large B-cell lymphoma treated with immunochemotherapy.

    Science.gov (United States)

    Gutiérrez-García, Gonzalo; Cardesa-Salzmann, Teresa; Climent, Fina; González-Barca, Eva; Mercadal, Santiago; Mate, José L; Sancho, Juan M; Arenillas, Leonor; Serrano, Sergi; Escoda, Lourdes; Martínez, Salomé; Valera, Alexandra; Martínez, Antonio; Jares, Pedro; Pinyol, Magdalena; García-Herrera, Adriana; Martínez-Trillos, Alejandra; Giné, Eva; Villamor, Neus; Campo, Elías; Colomo, Luis; López-Guillermo, Armando

    2011-05-05

    Diffuse large B-cell lymphomas (DLBCLs) can be divided into germinal-center B cell-like (GCB) and activated-B cell-like (ABC) subtypes by gene-expression profiling (GEP), with the latter showing a poorer outcome. Although this classification can be mimicked by different immunostaining algorithms, their reliability is the object of controversy. We constructed tissue microarrays with samples of 157 DLBCL patients homogeneously treated with immunochemotherapy to apply the following algorithms: Colomo (MUM1/IRF4, CD10, and BCL6 antigens), Hans (CD10, BCL6, and MUM1/IRF4), Muris (CD10 and MUM1/IRF4 plus BCL2), Choi (GCET1, MUM1/IRF4, CD10, FOXP1, and BCL6), and Tally (CD10, GCET1, MUM1/IRF4, FOXP1, and LMO2). GEP information was available in 62 cases. The proportion of misclassified cases by immunohistochemistry compared with GEP was higher when defining the GCB subset: 41%, 48%, 30%, 60%, and 40% for Colomo, Hans, Muris, Choi, and Tally, respectively. Whereas the GEP groups showed significantly different 5-year progression-free survival (76% vs 31% for GCB and activated DLBCL) and overall survival (80% vs 45%), none of the immunostaining algorithms was able to retain the prognostic impact of the groups (GCB vs non-GCB). In conclusion, stratification based on immunostaining algorithms should be used with caution in guiding therapy, even in clinical trials.

  20. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  1. Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Shimamura, Mai; Kyotani, Akane [Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Azuma, Yumiko [Department of Neurology, Kyoto Prefectural University of Medicine, 465 Kajii-cho,Kamigyo-ku, Kyoto 602-8566 (Japan); Yoshida, Hideki; Binh Nguyen, Thanh [Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Mizuta, Ikuko; Yoshida, Tomokatsu; Mizuno, Toshiki [Department of Neurology, Kyoto Prefectural University of Medicine, 465 Kajii-cho,Kamigyo-ku, Kyoto 602-8566 (Japan); Nakagawa, Masanori [North Medical Center, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566 (Japan); Tokuda, Takahiko, E-mail: ttokuda@koto.kpu-m.ac.jp [Department of Neurology, Kyoto Prefectural University of Medicine, 465 Kajii-cho,Kamigyo-ku, Kyoto 602-8566 (Japan); Department of Molecular Pathobiology of Brain Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566 (Japan); Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp [Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan)

    2014-08-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila. - Highlights: • Knockdown of Cabeza induced rough eye phenotype. • Knockdown of Cabeza induced fusion of cone cells in pupal retinae. • Knockdown of Cabeza induced apoptosis in pupal retinae. • Mutation in EGFR pathway-related genes suppressed the rough eye phenotype. • Cabeza may negatively regulate the EGFR pathway.

  2. Atlantic cod (Gadus morhua L.) possesses three homologues of ISG15 with different expression kinetics and conjugation properties.

    Science.gov (United States)

    Furnes, Clemens; Kileng, Øyvind; Rinaldo, Christine Hanssen; Seppola, Marit; Jensen, Ingvill; Robertsen, Børre

    2009-12-01

    Two new interferon stimulated gene 15 (ISG15) family members were identified in a subtractive cDNA library constructed from a mixture of head kidney and spleen of Atlantic cod (Gadus morhua) stimulated with polyinosinic:polycytidylic acid (poly I:C). Two full-length Atlantic cod (Ac) ISG15-2 and AcISG15-3 cDNAs were cloned with rapid amplification of cDNA ends (RACE). The cDNA sequence of AcISG15-2 encodes a 16.9kDa protein and AcISG15-3 encodes a 18.4kDa protein, both of which possess the characteristic structural features of two tandem ubiquitin-like domains and the LRGG motif necessary for conjugation. Furthermore, the AcISG15-3 protein is expressed with a C-terminal extension in common with the human ISG15 protein. Gene expression analysis using quantitative reverse transcriptase PCR (RT-qPCR) showed that AcISG15-1, AcISG15-2, and AcISG15-3 transcripts were up-regulated in head kidney after poly I:C stimulation, suggesting that these proteins may be involved in the cod immune response. However, transient expression of myc-tagged AcISG15 proteins revealed differences in their abilities to form conjugates in vitro. We show that AcISG15-2 forms covalent conjugates to a range of cellular protein as a response to poly I:C, recombinant Atlantic salmon IFNa1 (rSasaIFNa1) and infectious pancreatic necrosis virus (IPNV), whereas conjugation was absent for AcISG15-1 and AcISG15-3. Thus, these results suggest there are three ISG15 homologues in Atlantic cod and that the three proteins may play different roles in innate immunity.

  3. Identification and expression analysis of an IL-18 homologue and its alternatively spliced form in rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Zou, Jun; Bird, Steve; Truckle, Jonathan; Bols, Niels; Horne, Mike; Secombes, Chris

    2004-05-01

    A homologue of interleukin 18 has been identified from rainbow trout, Oncorhynchus mykiss. The trout IL-18 gene spans 3.7 kb and consists of six exons and five introns, sharing the same gene organization with its human counterpart. The putative translated protein is 199 amino acids in length with no predicted signal peptide. Analysis of the multiple sequence alignment reveals a conserved ICE cut site, resulting in a mature peptide of 162 amino acids. The trout IL-18 shares 41-45% similarity with known IL-18 molecules and contains an IL-1 family signature motif. It is constitutively expressed in a wide range of tissues including brain, gill, gut, heart, kidney, liver, muscle, skin and spleen. Transcription is not modulated by lipopolysaccharide, poly(I:C) or trout recombinant IL-1beta in primary head kidney leucocyte cultures and RTS-11 cells, a macrophage cell line. However, expression is downregulated by lipopolysaccharide and rIL-1beta in RTG-2 cells, a fibroblast-like cell line. An alternatively spliced form of IL-18 mRNA has also been found and translates into a 182 amino acid protein with a 17 amino acid deletion in the precursor region of the authentic form. This alternatively spliced form is also widely expressed although much lower than the authentic form. Interestingly, its expression is upregulated by lipopolysaccharide and poly(I:C), but is not affected by rIL-1beta in RTG-2 cells. The present study suggests that alternative splicing may play an important role in regulating IL-18 activities in rainbow trout.

  4. Sex-dependent modulation of longevity by two Drosophila homologues of human Apolipoprotein D, GLaz and NLaz.

    Science.gov (United States)

    Ruiz, Mario; Sanchez, Diego; Canal, Inmaculada; Acebes, Angel; Ganfornina, Maria D

    2011-07-01

    Apolipoprotein D (ApoD), a member of the Lipocalin family, is the gene most up-regulated with age in the mammalian brain. Its expression strongly correlates with aging-associated neurodegenerative and metabolic diseases. Two homologues of ApoD expressed in the Drosophila brain, Glial Lazarillo (GLaz) and Neural Lazarillo (NLaz), are known to alter longevity in male flies. However, sex differences in the aging process have not been explored so far for these genes. Here we demonstrate that NLaz alters lifespan in both sexes, but unexpectedly the lack of GLaz influences longevity in a sex-specific way, reducing longevity in males but not in females. While NLaz has metabolic functions similar to ApoD, the regulation of GLaz expression upon aging is the closest to ApoD in the aging brain. A multivariate analysis of physiological parameters relevant to lifespan modulation uncovers both common and specialized functions for the two Lipocalins, and reveals that changes in protein homeostasis account for the observed sex-specific patterns of longevity. The response to oxidative stress and accumulation of lipid peroxides are among their common functions, while the transcriptional and behavioral response to starvation, the pattern of daily locomotor activity, storage of fat along aging, fertility, and courtship behavior differentiate NLaz from GLaz mutants. We also demonstrate that food composition is an important environmental parameter influencing stress resistance and reproductive phenotypes of both Lipocalin mutants. Since ApoD shares many properties with the common ancestor of invertebrate Lipocalins, we must benefit from this global comparison with both GLaz and NLaz to understand the complex functions of ApoD in mammalian aging and neurodegeneration.

  5. Cloning and characterization of three apple MADS box genes isolated from vegetative tissue

    NARCIS (Netherlands)

    Linden, van der C.G.; Vosman, B.J.; Smulders, M.J.M.

    2002-01-01

    With the aim of finding genes involved in the floral transition of woody species four MADS box genes containing cDNAs from apple (Malus domestica) have been isolated. Three genes were isolated from vegetative tissue of apple, but were homologues of known genes that specify floral organ identity. MdM

  6. Cloning and characterization of three apple MADS box genes isolated from vegetative tissue

    NARCIS (Netherlands)

    Linden, van der C.G.; Vosman, B.J.; Smulders, M.J.M.

    2002-01-01

    With the aim of finding genes involved in the floral transition of woody species four MADS box genes containing cDNAs from apple (Malus domestica) have been isolated. Three genes were isolated from vegetative tissue of apple, but were homologues of known genes that specify floral organ identity.

  7. The tomato Orion locus comprises a unique class of Hcr9 genes

    NARCIS (Netherlands)

    Kock, de M.J.D.; Brandwagt, B.F.; Bonnema, A.B.; Wit, de P.J.G.M.; Lindhout, P.

    2005-01-01

    Resistance against the tomato fungal pathogen Cladosporium fulvum is often conferred by Hcr9 genes (Homologues of the C. fulvum resistance gene Cf-9) that are located in the Milky Way cluster on the short arm of chromosome 1. These Hcr9 genes mediate recognition of fungal avirulence gene products. I

  8. Altered expression of GFLO, the Gesneriaceae homologue of FLORICAULA/LEAFY, is associated with the transition to bulbil formation in Titanotrichum oldhamii.

    Science.gov (United States)

    Wang, Chun-Neng; Möller, Michael; Cronk, Quentin C B

    2004-03-01

    Titanotrichum oldhamii inflorescences switch from flower to bulbil production at the end of the flowering season. The structure of the bulbiliferous shoots resembles the abnormal meristematic organization of the Antirrhinum mutant, floricaula. Gesneriaceae- FLORICAULA (GFLO) is thus a candidate gene in the regulation of bulbil formation. To investigate this hypothesis, part of the GFLO gene (between the second and third exon) was isolated using degenerate primers designed in regions conserved between Antirrhinum, Nicotiana and Arabidopsis, followed by genome walking to obtain the complete gene and flanking sequences. RT-PCR results showed that the GFLO homologue is strongly expressed in inflorescence apical meristems and young flowers. However, in meristems that had switched to bulbil formation, GFLO transcription was greatly reduced. The down-regulation of GFLO in bulbil primordia indicates that this gene is connected to, or part of, the bulbil-flower regulatory pathway. Phylogenetic analysis confirms the orthology of GFLO and FLO, and indicates that the gene may be useful for phylogenetic reconstruction at the genus or family level.

  9. Tsp66E, the Drosophila KAI1 homologue, and Tsp74F function to regulate ovarian follicle cell and wing development by stabilizing integrin localization.

    Science.gov (United States)

    Han, Seung Yeop; Lee, Minjung; Hong, Yoon Ki; Hwang, Soojin; Choi, Gahee; Suh, Yoon Seok; Park, Seung Hwan; Lee, Soojin; Lee, Sang-Hee; Chung, Jongkyeong; Baek, Sung Hee; Cho, Kyoung Sang

    2012-11-16

    The metastasis suppressor KAI1/CD82 has been implicated in various cellular processes; however, its function in development is not fully understood. Here, we generated and characterized mutants of Tsp66E and Tsp74F, which are Drosophila homologues of KAI1/CD82 and Tspan11, respectively. These mutants exhibited egg elongation defects along with disturbed integrin localization and actin polarity. Moreover, the defects were enhanced by mutation of inflated, an αPS2 integrin gene. Mutant ovaries had elevated αPS2 integrin levels and reduced endocytic trafficking. These results suggest that Drosophila KAI1/CD82 affects the polarized localization and the level of integrin, which may contribute to epithelial cell polarity.

  10. Single-nucleotide polymorphism in the 5-alpha-reductase gene (SRD5A2) is associated with increased prevalence of metabolic syndrome in chemotherapy-treated testicular cancer survivors

    NARCIS (Netherlands)

    Boer, Hink; Westerink, Nico-Derk L.; Altena, Renske; Nuver, Janine; Dijck-Brouwer, D. A. Janneke; van Faassen, Martijn; Klont, Frank; Kema, Ido P.; Lefrandt, Joop D.; Zwart, Nynke; Boezen, H. Marike; Smit, Andries J.; Meijer, Coby; Gietema, Jourik A.

    2016-01-01

    Purpose: Chemotherapy-treated testicular cancer survivors are at risk for development of the metabolic syndrome, especially in case of decreased androgen levels. Polymorphisms in the gene encoding steroid 5-alpha-reductase type II (SRD5A2) are involved in altered androgen metabolism. We investigated

  11. Single-nucleotide polymorphism in the 5-α-reductase gene (SRD5A2) is associated with increased prevalence of metabolic syndrome in chemotherapy-treated testicular cancer survivors

    NARCIS (Netherlands)

    Boer, Hink; Westerink, Nico-Derk L; Altena, Renske; Nuver, Janine; Dijck-Brouwer, D A Janneke; van Faassen, Martijn; Klont, Frank; Kema, Ido P; Lefrandt, Joop D; Zwart, Nynke; Boezen, H Marike; Smit, Andries J; Meijer, Coby; Gietema, Jourik A

    2016-01-01

    PURPOSE: Chemotherapy-treated testicular cancer survivors are at risk for development of the metabolic syndrome, especially in case of decreased androgen levels. Polymorphisms in the gene encoding steroid 5-α-reductase type II (SRD5A2) are involved in altered androgen metabolism. We investigated whe

  12. New Orthogonal Transcriptional Switches Derived from Tet Repressor Homologues for Saccharomyces cerevisiae Regulated by 2,4-Diacetylphloroglucinol and Other Ligands.

    Science.gov (United States)

    Ikushima, Shigehito; Boeke, Jef D

    2017-03-17

    Here we describe the development of tightly regulated expression switches in yeast, by engineering distant homologues of Escherichia coli TetR, including the transcriptional regulator PhlF from Pseudomonas and others. Previous studies demonstrated that the PhlF protein bound its operator sequence (phlO) in the absence of 2,4-diacetylphloroglucinol (DAPG) but dissociated from phlO in the presence of DAPG. Thus, we developed a DAPG-Off system in which expression of a gene preceded by the phlO-embedded promoter was activated by a fusion of PhlF to a multimerized viral activator protein (VP16) domain in a DAPG-free environment but repressed when DAPG was added to growth medium. In addition, we constructed a DAPG-On system with the opposite behavior of the DAPG-Off system; i.e., DAPG triggers the expression of a reporter gene. Exposure of DAPG to yeast cells did not cause any serious deleterious effect on yeast physiology in terms of growth. Efforts to engineer additional Tet repressor homologues were partially successful and a known mammalian switch, the p-cumate switch based on CymR from Pseudomonas, was found to function in yeast. Orthogonality between the TetR (doxycycline), CamR (d-camphor), PhlF (DAPG), and CymR (p-cumate)-based Off switches was demonstrated by evaluating all 4 ligands against suitably engineered yeast strains. This study expands the toolbox of "On" and "Off" switches for yeast biotechnology.

  13. An Evolutionary Network of Genes Present in the Eukaryote Common Ancestor Polls Genomes on Eukaryotic and Mitochondrial Origin

    OpenAIRE

    Thiergart, T.; Landan, G; Schenk, M.; Dagan, T.; Martin, W F

    2012-01-01

    To test the predictions of competing and mutually exclusive hypotheses for the origin of eukaryotes, we identified from a sample of 27 sequenced eukaryotic and 994 sequenced prokaryotic genomes 571 genes that were present in the eukaryote common ancestor and that have homologues among eubacterial and archaebacterial genomes. Maximum-likelihood trees identified the prokaryotic genomes that most frequently contained genes branching as the sister to the eukaryotic nuclear homologues. Among the a...

  14. Vaccination against Bm86 Homologues in Rabbits Does Not Impair Ixodes ricinus Feeding or Oviposition.

    Directory of Open Access Journals (Sweden)

    Jeroen Coumou

    Full Text Available Human tick-borne diseases that are transmitted by Ixodes ricinus, such as Lyme borreliosis and tick borne encephalitis, are on the rise in Europe. Diminishing I. ricinus populations in nature can reduce tick exposure to humans, and one way to do so is by developing an anti-vector vaccine against tick antigens. Currently, there is only one anti-vector vaccine available against ticks, which is a veterinary vaccine based on the tick antigen Bm86 in the gut of Rhipicephalus microplus. Bm86 vaccine formulations cause a reduction in the number of Rhipicephalus microplus ticks that successfully feed, i.e. lower engorgement weights and a decrease in the number of oviposited eggs. Furthermore, Bm86 vaccines reduce transmission of bovine Babesia spp. Previously two conserved Bm86 homologues in I. ricinus ticks, designated as Ir86-1 and Ir86-2, were described. Here we investigated the effect of a vaccine against recombinant Ir86-1, Ir86-2 or a combination of both on Ixodes ricinus feeding. Recombinant Ixodes ricinus Bm86 homologues were expressed in a Drosophila expression system and rabbits were immunized with rIr86-1, rIr86-2, a combination of both or ovalbumin as a control. Each animal was infested with 50 female adults and 50 male adults Ixodes ricinus and tick mortality, engorgement weights and egg mass were analyzed. Although serum IgG titers against rIr86 proteins were elicited, no effect was found on tick feeding between the rIr86 vaccinated animals and ovalbumin vaccinated animals. We conclude that vaccination against Bm86 homologues in Ixodes ricinus is not an effective approach to control Ixodes ricinus populations, despite the clear effects of Bm86 vaccination against Rhipicephalus microplus.

  15. Vaccination against Bm86 Homologues in Rabbits Does Not Impair Ixodes ricinus Feeding or Oviposition

    Science.gov (United States)

    Coumou, Jeroen; Wagemakers, Alex; Trentelman, Jos J.; Nijhof, Ard M.; Hovius, Joppe W.

    2015-01-01

    Human tick-borne diseases that are transmitted by Ixodes ricinus, such as Lyme borreliosis and tick borne encephalitis, are on the rise in Europe. Diminishing I. ricinus populations in nature can reduce tick exposure to humans, and one way to do so is by developing an anti-vector vaccine against tick antigens. Currently, there is only one anti-vector vaccine available against ticks, which is a veterinary vaccine based on the tick antigen Bm86 in the gut of Rhipicephalus microplus. Bm86 vaccine formulations cause a reduction in the number of Rhipicephalus microplus ticks that successfully feed, i.e. lower engorgement weights and a decrease in the number of oviposited eggs. Furthermore, Bm86 vaccines reduce transmission of bovine Babesia spp. Previously two conserved Bm86 homologues in I. ricinus ticks, designated as Ir86-1 and Ir86-2, were described. Here we investigated the effect of a vaccine against recombinant Ir86-1, Ir86-2 or a combination of both on Ixodes ricinus feeding. Recombinant Ixodes ricinus Bm86 homologues were expressed in a Drosophila expression system and rabbits were immunized with rIr86-1, rIr86-2, a combination of both or ovalbumin as a control. Each animal was infested with 50 female adults and 50 male adults Ixodes ricinus and tick mortality, engorgement weights and egg mass were analyzed. Although serum IgG titers against rIr86 proteins were elicited, no effect was found on tick feeding between the rIr86 vaccinated animals and ovalbumin vaccinated animals. We conclude that vaccination against Bm86 homologues in Ixodes ricinus is not an effective approach to control Ixodes ricinus populations, despite the clear effects of Bm86 vaccination against Rhipicephalus microplus. PMID:25919587

  16. Polymorphism of CYP3A4 and ABCB1 genes increase the risk of neuropathy in breast cancer patients treated with paclitaxel and docetaxel

    Directory of Open Access Journals (Sweden)

    Kus T

    2016-08-01

    Full Text Available Tulay Kus,1 Gokmen Aktas,1 Mehmet Emin Kalender,1 Abdullah Tuncay Demiryurek,2 Mustafa Ulasli,1 Serdar Oztuzcu,3 Alper Sevinc,1 Seval Kul,4 Celaletdin Camci1 1Department of Internal Medicine, Division of Medical Oncology, University of Gaziantep, Gaziantep Oncology Hospital, Gaziantep, Turkey; 2Department of Medical Pharmacology, 3Department of Medical Biology, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey; 4Department of Biostatistics, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey Background: Interindividual variability of pharmacogenetics may account for unpredictable neurotoxicities of taxanes. Methods: From March 2011 to June 2015, female patients with operable breast cancer who had received docetaxel- or paclitaxel-containing adjuvant chemotherapy were included in this study. All patients were treated with single-agent paclitaxel intravenously (IV 175 mg/m2 every 3 weeks for four cycles, or IV 80 mg/m2 weekly for 12 cycles, and IV 100 mg/m2 docetaxel for four cycles as adjuvant treatment. We evaluated the relationship between neurotoxicity of taxanes and single-nucleotide polymorphisms of ABCB1, CYP3A4, ERCC1, ERCC2, FGFR4, TP53, ERBB2, and CYP2C8 genes. Taxane-induced neurotoxicity during the treatment was evaluated according to the National Cancer Institute Common Toxicity Criteria version 4.03 prior to each cycle. Chi-squared tests were used to compare the two groups, and multivariate binary logistic regression models were used for determining possible risk factors of neuropathy. Results: Pharmacogenetic analysis was performed in 219 females. ABCB1 3435 TT genotype had significantly higher risk for grade ≥2 neurotoxicity (odds ratio [OR]: 2.759, 95% confidence interval [CI]: 1.172–6.493, P: 0.017 compared to TC and CC genotype, and also CYP3A4 392 AA and AG genotype had significantly higher risk for grade ≥2 neurotoxicity (OR: 2.259, 95% CI: 1.033–4.941, P: 0.038 compared to GG genotype. For

  17. Polymorphism of CYP3A4 and ABCB1 genes increase the risk of neuropathy in breast cancer patients treated with paclitaxel and docetaxel

    Science.gov (United States)

    Kus, Tulay; Aktas, Gokmen; Kalender, Mehmet Emin; Demiryurek, Abdullah Tuncay; Ulasli, Mustafa; Oztuzcu, Serdar; Sevinc, Alper; Kul, Seval; Camci, Celaletdin

    2016-01-01

    Background Interindividual variability of pharmacogenetics may account for unpredictable neurotoxicities of taxanes. Methods From March 2011 to June 2015, female patients with operable breast cancer who had received docetaxel- or paclitaxel-containing adjuvant chemotherapy were included in this study. All patients were treated with single-agent paclitaxel intravenously (IV) 175 mg/m2 every 3 weeks for four cycles, or IV 80 mg/m2 weekly for 12 cycles, and IV 100 mg/m2 docetaxel for four cycles as adjuvant treatment. We evaluated the relationship between neurotoxicity of taxanes and single-nucleotide polymorphisms of ABCB1, CYP3A4, ERCC1, ERCC2, FGFR4, TP53, ERBB2, and CYP2C8 genes. Taxane-induced neurotoxicity during the treatment was evaluated according to the National Cancer Institute Common Toxicity Criteria version 4.03 prior to each cycle. Chi-squared tests were used to compare the two groups, and multivariate binary logistic regression models were used for determining possible risk factors of neuropathy. Results Pharmacogenetic analysis was performed in 219 females. ABCB1 3435 TT genotype had significantly higher risk for grade ≥2 neurotoxicity (odds ratio [OR]: 2.759, 95% confidence interval [CI]: 1.172–6.493, P: 0.017) compared to TC and CC genotype, and also CYP3A4 392 AA and AG genotype had significantly higher risk for grade ≥2 neurotoxicity (OR: 2.259, 95% CI: 1.033–4.941, P: 0.038) compared to GG genotype. For FDGF4 gene with AG and GG genotype, OR was 1.879 (95% CI: 1.001–3.525, P: 0.048) compared to AA genotype with regard to any grade of neuropathy risk. We could not find any other association of other genotypes with neurotoxicity grades. Conclusion ABCB1 3435 TT genotype and CYP3A4 392 AA/AG genotypes may be used as predictors of neurotoxicity during taxane chemotherapy. PMID:27574448

  18. Evaluation of the ability of adjuvant tamoxifen-benefit gene signatures to predict outcome of hormone-naive estrogen receptor-positive breast cancer patients treated with tamoxifen in the advanced setting

    DEFF Research Database (Denmark)

    Sieuwerts, Anieta M; Lyng, Maria Bibi; Meijer-van Gelder, Marion E

    2014-01-01

    To identify molecular markers indicative of response to tamoxifen and easily implemented in the routine setting, we recently reported three gene signatures that could stratify post-menopausal tamoxifen-treated, estrogen receptor-positive (ER+) patients according to outcome in the adjuvant setting...... exhibit predictive promise regarding the efficacy of tamoxifen treatment in recurrent disease, in addition to the previously shown favorable outcome in the adjuvant setting........ Here, we evaluated the predictive potential of the total of 14 genes included in the 3 gene signatures using 2 hormone-naïve Dutch ER+ cohorts of a total of 285 recurrent breast cancer patients treated with first-line tamoxifen. mRNA levels were measured by reverse transcriptase quantitative PCR (RT...

  19. The primary structure of ammodytin L, a myotoxic phospholipase A2 homologue from Vipera ammodytes venom.

    Science.gov (United States)

    Krizaj, I; Bieber, A L; Ritonja, A; Gubensek, F

    1991-12-18

    A new myotoxic phospholipase A2 homologue, having a serine residue in position 49 instead of highly conserved aspartic acid, was found in the venom of Vipera ammodytes. The primary structure revealed additional mutations in the positions important for enzymatic activity. Tyr28 is exchanged for a histidine and Gly33 for asparagine. These changes render earlier-reported weak enzymatic activity unlikely. The role of this rather abundant venom fraction is apparently in myotoxicity, which was confirmed in the muscle-cell culture from neonatal rats. The muscle-cell culture proved to be a good tool to investigate the effects of various myotoxins on muscle cells.

  20. Partial functional complementation between human and mouse cytomegalovirus chemokine receptor homologues

    DEFF Research Database (Denmark)

    Farrell, Helen E; Abraham, Alexander M; Cardin, Rhonda D

    2011-01-01

    The human cytomegalovirus (CMV) proteins US28 and UL33 are homologous to chemokine receptors (CKRs). Knockout of the mouse CMV M33 protein (UL33 homologue) results in substantial attenuation of salivary gland infection/replication and reduced efficiency of reactivation from tissue explants. M33......-mediated G protein-coupled signaling is critical for the salivary gland phenotype. In this report, we demonstrate that US28 and (to a lesser degree) UL33 restore reactivation from tissue explants and partially restore replication in salivary glands (compared to a signaling-deficient M33 mutant...

  1. Mutations in the Plasmodium falciparum cytochrome b gene are associated with delayed parasite recrudescence in malaria patients treated with atovaquone-proguanil

    Directory of Open Access Journals (Sweden)

    Fivelman Quinton L

    2008-11-01

    Full Text Available Abstract Background Fixed-dose combination antimalarial drugs have played an increasingly important role in the treatment and chemoprophylaxis of falciparum malaria since the worldwide failure of monotherapy with chloroquine. Atovaquone-proguanil is one such combination drug used both for prophylaxis in travellers, and for treatment of acute malaria cases in European hospitals and clinics. Methods A series of eight atovaquone-proguanil treatment failures and two prophylaxis breakthroughs from four UK hospitals from 2004–2008 were analysed for evidence of mutations in the pfcyt-b gene, previously found to be associated with failure of the atovaquone component. Results Parasites carrying pfcyt-b mutations were found in five falciparum malaria patients with recrudescent parasitaemia occurring weeks after apparently successful treatment of a primary infection with atovaquone-proguanil. Four of these cases carried parasites with the Tyr268Cys mutation in pfcyt-b, previously reported in two French patients with malaria. In contrast, mutations in pfcyt-b were not found in three patients treated with atovaquone-proguanil who exhibited delayed clearance of the primary infection, nor in two returning travellers with malaria who had used the combination for prophylaxis. Using current and previously published data, mean time to recrudescence of parasites carrying pfcytb codon 268 mutations was estimated as 28.0 days after treatment (95% C.I. 23.0 – 33.0 days, whereas treatment failures without codon 268 mutations received rescue treatment an average of 4.71 days after initial AP treatment (95% C.I. 1.76 – 7.67 days. Conclusion Genetically-determined parasite resistance to atovaquone is associated with delayed recrudescence of resistant parasites three weeks or more after initial clearance of parasitaemia by atovaquone/proguanil therapy. The 268-Cys allele of pfcyt-b may have been overlooked in previous studies of atovaquone-proguanil treatment failure

  2. The POU-er of gene nomenclature

    DEFF Research Database (Denmark)

    Frankenberg, Stephen R; Frank, Dale; Harland, Richard

    2014-01-01

    this family provides insight into the evolution of early embryonic potency. Here, we seek to clarify the relationship between POU5F1 homologues in the vertebrate lineage, both phylogenetically and functionally. We resolve the confusion over the identity of the zebrafish gene, which was originally named pou2...

  3. Initial characterization of a bolA homologue from Pseudomonas fluorescens indicates different roles for BolA-like proteins in P. fluorescens and Escherichia coli.

    Science.gov (United States)

    Koch, Birgit; Nybroe, Ole

    2006-09-01

    The RpoS-regulated bolA gene in Escherichia coli is important for the decrease in cell size during stationary phase or sudden carbon starvation. A Pseudomonas fluorescens strain mutated in a gene with homology to bolA reduced its cell size upon carbon starvation, and RpoS had little effect on bolA expression. The mutant grew slower than the wild-type strain in minimal medium with L-serine as the sole nitrogen source, while growth rates were similar on a mixture of L-serine and L-cysteine. Reverse transcriptase polymerase chain reaction analysis indicated that the bolA homologue is the second gene in an operon where the two next ORFs encode putative proteins with homology to sulphurtransferases and protein disulphide isomerases. Complementation of the mutant phenotypes was only obtained by plasmids encoding BolA as well as the above two proteins. Growth phenotypes and gene homologies suggest that BolA-like proteins have different functions in E. coli and Pseudomonas.

  4. Fission yeast Mog1p homologue, which interacts with the small GTPase Ran, is required for mitosis-to-interphase transition and poly(A)(+) RNA metabolism.

    Science.gov (United States)

    Tatebayashi, K; Tani, T; Ikeda, H

    2001-04-01

    We have cloned and characterized the Schizosaccharomyces pombe gene mog1(+), which encodes a protein with homology to the Saccharomyces cerevisiae Mog1p participating in the Ran-GTPase system. The S. pombe Mog1p is predominantly localized in the nucleus. In contrast to the S. cerevisiae MOG1 gene, the S. pombe mog1(+) gene is essential for cell viability. mog1(+) is required for the mitosis-to-interphase transition, as the mog1-1 mutant arrests at restrictive temperatures as septated, binucleated cells with highly condensed chromosomes and an aberrant nuclear envelope. FACS analysis showed that these cells do not undergo a subsequent round of DNA replication. Surprisingly, also unlike the Delta mog1 mutation in S. cerevisiae, the mog1-1 mutation causes nucleolar accumulation of poly(A)(+) RNA at the restrictive temperature in S. pombe, but the signals do not overlap with the fibrillarin-rich region of the nucleolus. Thus, we found that mog1(+) is required for the mitosis-to-interphase transition and a class of RNA metabolism. In our attempt to identify suppressors of mog1-1, we isolated the spi1(+) gene, which encodes the fission yeast homologue of Ran. We found that overexpression of Spi1p rescues the S. pombe Delta mog1 cells from death. On the basis of these results, we conclude that mog1(+) is involved in the Ran-GTPase system.

  5. Wax crystal-sparse leaf2, a rice homologue of WAX2/GL1, is involved in synthesis of leaf cuticular wax.

    Science.gov (United States)

    Mao, Bigang; Cheng, Zhijun; Lei, Cailin; Xu, Fenghua; Gao, Suwei; Ren, Yulong; Wang, Jiulin; Zhang, Xin; Wang, Jie; Wu, Fuqing; Guo, Xiuping; Liu, Xiaolu; Wu, Chuanyin; Wang, Haiyang; Wan, Jianmin

    2012-01-01

    Epicuticular wax in plants limits non-stomatal water loss, inhibits postgenital organ fusion, protects plants against damage from UV radiation and imposes a physical barrier against pathogen infection. Here, we give a detailed description of the genetic, physiological and morphological consequences of a mutation in the rice gene WSL2, based on a comparison between the wild-type and an EMS mutant. The mutant's leaf cuticle membrane is thicker and less organized than that of the wild type, and its total wax content is diminished by ~80%. The mutant is also more sensitive to drought stress. WSL2 was isolated by positional cloning, and was shown to encode a homologue of the Arabidopsis thaliana genes CER3/WAX2/YRE/FLP1 and the maize gene GL1. It is expressed throughout the plant, except in the root. A transient assay carried out in both A. thaliana and rice protoplasts showed that the gene product is deposited in the endoplasmic reticulum. An analysis of the overall composition of the wax revealed that the mutant produces a substantially reduced quantity of C22-C32 fatty acids, which suggests that the function of WSL2 is associated with the elongation of very long-chain fatty acids.

  6. Strboh A homologue of NADPH oxidase regulates wound-induced oxidative burst and facilitates wound-healing in potato tubers.

    Science.gov (United States)

    Kumar, G N Mohan; Iyer, Suresh; Knowles, N Richard

    2007-12-01

    During 30-months of storage at 4 degrees C, potato (Solanum tuberosum L.) tubers progressively lose the ability to produce superoxide in response to wounding, resist microbial infection, and develop a suberized wound periderm. Using differentially aged tubers, we demonstrate that Strboh A is responsible for the wound-induced oxidative burst in potato and aging attenuates its expression. In vivo superoxide production and NADPH oxidase (NOX) activity from 1-month-old tubers increased to a maximum 18-24 h after wounding and then decreased to barely detectable levels by 72 h. Wounding also induced a 68% increase in microsomal protein within 18 h. These wound-induced responses were lost over a 25- to 30-month storage period. Superoxide production and NOX activity were inhibited by diphenylene iodonium chloride, a specific inhibitor of NOX, which in turn effectively inhibited wound-healing and increased susceptibility to microbial infection and decay in 1-month-old tubers. Wound-induced superoxide production was also inhibited by EGTA-mediated destabilization of membranes. The ability to restore superoxide production to EGTA-treated tissue with Ca(+2) declined with advancing tuber age, likely a consequence of age-related changes in membrane architecture. Of the five homologues of NOX (Strboh A-D and F), wounding induced the expression of Strboh A in 6-month-old tubers but this response was absent in tubers stored for 25-30 months. Strboh A thus mediates the initial burst of superoxide in response to wounding of potato tubers; loss of its expression increases the susceptibility to microbial infection and contributes to the age-induced loss of wound-healing ability.

  7. Gibberellins regulate the transcription of the continuous flowering regulator, RoKSN, a rose TFL1 homologue.

    Science.gov (United States)

    Randoux, Marie; Jeauffre, Julien; Thouroude, Tatiana; Vasseur, François; Hamama, Latifa; Juchaux, Marjorie; Sakr, Soulaiman; Foucher, Fabrice

    2012-11-01

    The role of gibberellins (GAs) during floral induction has been widely studied in the annual plant Arabidopsis thaliana. Less is known about this control in perennials. It is thought that GA is a major regulator of flowering in rose. In spring, low GA content may be necessary for floral initiation. GA inhibited flowering in once-flowering roses, whereas GA did not block blooming in continuous-flowering roses. Recently, RoKSN, a homologue of TFL1, was shown to control continuous flowering. The loss of RoKSN function led to continuous flowering behaviour. The objective of this study was to understand the molecular control of flowering by GA and the involvement of RoKSN in this inhibition. In once-flowering rose, the exogenous application of GA(3) in spring inhibited floral initiation. Application of GA(3) during a short period of 1 month, corresponding to the floral transition, was sufficient to inhibit flowering. At the molecular level, RoKSN transcripts were accumulated after GA(3) treatment. In spring, this accumulation is correlated with floral inhibition. Other floral genes such as RoFT, RoSOC1, and RoAP1 were repressed in a RoKSN-dependent pathway, whereas RoLFY and RoFD repression was RoKSN independent. The RoKSN promoter contained GA-responsive cis-elements, whose deletion suppressed the response to GA in a heterologous system. In summer, once-flowering roses did not flower even after exogenous application of a GA synthesis inhibitor that failed to repress RoKSN. A model is presented for the GA inhibition of flowering in spring mediated by the induction of RoKSN. In summer, factors other than GA may control RoKSN.

  8. Characterization of a Plasmodium falciparum macrophage-migration inhibitory factor homologue.

    Science.gov (United States)

    Cordery, Damien V; Kishore, Uday; Kyes, Sue; Shafi, Mohammed J; Watkins, Katherine R; Williams, Thomas N; Marsh, Kevin; Urban, Britta C

    2007-03-15

    Macrophage-migration inhibitory factor (MIF), one of the first cytokines described, has a broad range of proinflammatory properties. The genome sequencing project of Plasmodium falciparum identified a parasite homologue of MIF. The protein is expressed during the asexual blood stages of the parasite life cycle that cause malarial disease. The identification of a parasite homologue of MIF raised the question of whether it affects monocyte function in a manner similar to its human counterpart. Recombinant P. falciparum MIF (PfMIF) was generated and used in vitro to assess its influence on monocyte function. Antibodies generated against PfMIF were used to determine the expression profile and localization of the protein in blood-stage parasites. Antibody responses to PfMIF were determined in Kenyan children with acute malaria and in control subjects. PfMIF protein was expressed in asexual blood-stage parasites, localized to the Maurer's cleft. In vitro treatment of monocytes with PfMIF inhibited random migration and reduced the surface expression of Toll-like receptor (TLR) 2, TLR4, and CD86. These results indicate that PfMIF is released during blood-stage malaria and potentially modulates the function of monocytes during acute P. falciparum infection.

  9. Protease inhibitor homologues from mamba venoms: facilitation of acetylcholine release and interactions with prejunctional blocking toxins.

    Science.gov (United States)

    Harvey, A L; Karlsson, E

    1982-09-01

    1 Five polypeptides, which were isolated from elapid snake venoms and which are structurally related to protease inhibitors, were tested for action on isolated biventer cervicis nerve-muscle preparations of the chick. 2 Dendrotoxin from the Eastern green mamba (Dendroaspis angusticeps) and toxins K and I from the black mamba (Dendroaspis polylepis polylepis) increased to indirect stimulation without affecting responses to exogenous acetylcholine, carbachol of KCl. 3 The two other protease inhibitor homologues, HHV-II from Ringhals cobra (Hemachatus haemachatus) and NNV-II from Cape cobra (Naja nivea) did not increase responses to nerve stimulation. Trypsin inhibitor from bovine pancreas also had no facilitatory effects on neuromuscular transmission. 4 The facilitatory toxins from mamba venoms interacted with the prejunctional blocking toxins, beta-bungarotoxin, crotoxin and notexin, but not with taipoxin. The blocking effects of beta-bungarotoxin were reduced by pretreatment with the mamba toxins, whereas the blocking actions of crotoxin and notexin were enhanced. 5 The results indicate that protease inhibitor homologues from mamba venoms form a new class of neurotoxin, which acts to increase the release of acetylcholine in response to motor nerve stimulation. 6 From the interaction studies it is concluded that the facilitatory toxins bind to motor nerve terminals at sites related to those occupied by the prejunctional blocking toxins. However, differences in interactions with individual toxins suggest that there must be several related binding sites on the nerve terminals.

  10. Cloning of rat sp56, the homologue of mouse sperm ZP3 receptor-sp56

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Mouse sp56 is considered as one of the candidates for mouse zona pellucida 3 (mZP3) receptor. Up to date, its homologue has only been cloned from guinea pig, namely AM67. Based on the cDNA sequence of mouse sp56, we designed a pair of primer to amplify its homologue from rat testis cDNA. Using RT-PCR,two fragments of 743 bp and 938 bp were amplified. The PCR products show very high homology to mouse sp56. However, the 743 bp product completely lacks one of the seven Sushi domains of mouse sp56. Using the 743 bp product as the probe to detect the expression profile of sp56 in rat tissues, Northern blot shows that a ~2.0 kb mRNA expresses specifically in testis. Employed the RACE method, two full cDNA sequences of rat sp56 were obtained. A Mr ~42 KD band was detected in denatured and non-reducing protein sample of rat testis and sperm with anti-mouse sp56 monoclonal antibody by Western blot method. Rat sp56was localized on rat sperm head by the indirect immunofiuorescence method. Rat sp56 immunoreactivitywas detected from the early pachytene spermatocytes and throughout the spermatogenesis. Its cloning willfurther our understanding of the mechanism of the sperm-egg recognition and binding.

  11. Heavy Atom Effect on the First Hyperpolarizabilities of Squaric Acid Homologues Studied by Ab Initio and DFT Methods

    Institute of Scientific and Technical Information of China (English)

    FU Wei-Wei; ZHOU Li-Xin; WAN Hua-Ping

    2004-01-01

    We have calculated the first hyperpolarizabilities of four squaric acid homologue molecules: 3,4-dithiohydroxy-3-cyclobutene-1,2-dione (OSSQ), 3,4-dithiohydroxy-3-cyclobutene-1, 2-dithione (SSSQ), 3,4-dithiohydroxy-3-cyclobutene-1,2-diselenone (SeSSQ) and 3,4-dithiohydroxy- 3-cyclobutene-1,2-ditellurone (TeSSQ). The correlation effect was investigated at the second-order Mφller-Plesset (MP2) perturbation and density functional theory (DFT) levels. The frequency disper- sion and solvent effect were considered to compare the theoretical values with the experimental observations. Based on all of these studies, it is worthy to point out that the heavy atom effect dis- covered for furan homologues is an influence on the first hyperpolarizabilities of squaric acid homologues.

  12. Validation of reference genes for RT-qPCR in marine bivalve ecotoxicology: Systematic review and case study using copper treated primary Ruditapes philippinarum hemocytes.

    Science.gov (United States)

    Volland, Moritz; Blasco, Julián; Hampel, Miriam

    2017-04-01

    The appropriate selection of reference genes for the normalization of non-biological variance in reverse transcription real-time quantitative PCR (RT-qPCR) is essential for the accurate interpretation of the collected data. The use of multiple validated reference genes has been shown to substantially increase the robustness of the normalization. It is therefore considered good practice to validate putative genes under specific conditions, determine the optimal number of genes to be employed, and report the method or methods used. Under this premise, we assessed the current state of reference gene based normalization in RT-qPCR bivalve ecotoxicology studies (post 2011), employing a systematic quantitative literature review. A total of 52 papers met our criteria and were analysed for genes used, the use of multiple reference genes, as well as the validation method employed. We further critically discuss methods for reference gene validation based on a case study using copper exposed primary hemocytes from the marine bivalve Ruditapes philippinarum; including the established algorithms geNorm, NormFinder and BestKeeper, as well as the popular online tool RefFinder. We identified that RT-qPCR normalization is largely performed using single reference genes, while less than 40% of the studies attempted to experimentally validate the expression stability of the genes used. 18s rRNA and β-Actin were the most popular genes, yet their un-validated use did introduce artefactual variance that altered the interpretation of the resulting data. Our findings further suggest that combining the results from multiple individual algorithms and calculating the overall best-ranked gene, as computed by the RefFinder tool, does not by default lead to the identification of the most suitable reference genes. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Pharmacogenetic Study in Rectal Cancer Patients Treated With Preoperative Chemoradiotherapy: Polymorphisms in Thymidylate Synthase, Epidermal Growth Factor Receptor, GSTP1, and DNA Repair Genes

    Energy Technology Data Exchange (ETDEWEB)

    Paez, David, E-mail: dpaez@santpau.cat [Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Salazar, Juliana; Pare, Laia [Centre for Biomedical Network Research on Rare Diseases, Barcelona (Spain); Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Pertriz, Lourdes [Department of Radiotherapy, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Targarona, Eduardo [Department of Surgery, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Rio, Elisabeth del [Centre for Biomedical Network Research on Rare Diseases, Barcelona (Spain); Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Barnadas, Agusti; Marcuello, Eugenio [Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain); Baiget, Montserrat [Centre for Biomedical Network Research on Rare Diseases, Barcelona (Spain); Department of Genetics, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona (Spain)

    2011-12-01

    Purpose: Several studies have been performed to evaluate the usefulness of neoadjuvant treatment using oxaliplatin and fluoropyrimidines for locally advanced rectal cancer. However, preoperative biomarkers of outcome are lacking. We studied the polymorphisms in thymidylate synthase, epidermal growth factor receptor, glutathione S-transferase pi 1 (GSTP1), and several DNA repair genes to evaluate their usefulness as pharmacogenetic markers in a cohort of 128 rectal cancer patients treated with preoperative chemoradiotherapy. Methods and Materials: Blood samples were obtained from 128 patients with Stage II-III rectal cancer. DNA was extracted from the peripheral blood nucleated cells, and the genotypes were analyzed by polymerase chain reaction amplification and automated sequencing techniques or using a 48.48 dynamic array on the BioMark system. The germline polymorphisms studied were thymidylate synthase, (VNTR/5 Prime UTR, 2R G>C single nucleotide polymorphism [SNP], 3R G>C SNP), epidermal growth factor receptor (Arg497Lys), GSTP1 (Ile105val), excision repair cross-complementing 1 (Asn118Asn, 8092C>A, 19716G>C), X-ray repair cross-complementing group 1 (XRCC1) (Arg194Trp, Arg280His, Arg399Gln), and xeroderma pigmentosum group D (Lys751Gln). The pathologic response, pathologic regression, progression-free survival, and overall survival were evaluated according to each genotype. Results: The Asterisk-Operator 3/ Asterisk-Operator 3 thymidylate synthase genotype was associated with a greater response rate (pathologic complete remission and microfoci residual tumor, 59% in Asterisk-Operator 3/ Asterisk-Operator 3 vs. 35% in Asterisk-Operator 2/ Asterisk-Operator 2 and Asterisk-Operator 2/ Asterisk-Operator 3; p = .013). For the thymidylate synthase genotype, the median progression-free survival was 103 months for the Asterisk-Operator 3/ Asterisk-Operator 3 patients and 84 months for the Asterisk-Operator 2/ Asterisk-Operator 2 and Asterisk-Operator 2/ Asterisk

  14. Sequence analysis of β-subunit genes of the 20S proteasome in patients with relapsed multiple myeloma treated with bortezomib or dexamethasone

    NARCIS (Netherlands)

    D.I. Lichter (David); H. Danaee (Hadi); M.D. Pickard (Michael); O. Tayber (Olga); M. Sintchak (Michael); H. Shi (Hongliang); P.G. Richardson (Paul Gerard); J. Cavenagh (Jamie); J. Bladé (Joan); T. Facon (Thierry); R. Niesvizky; M. Alsina (Melissa); W. Dalton (William); P. Sonneveld (Pieter); S. Lonial (Sagar); H. van de Velde (Helgi); D. Ricci (Deborah); D.-L. Esseltine (Dixie-Lee); W.L. Trepicchio (William); G. Mulligan (George); K.C. Anderson (Kenneth Carl)

    2012-01-01

    textabstractVariations within proteasome β (PSMB) genes, which encode the β subunits of the 20S proteasome, may affect proteasome function, assembly, and/or binding of proteasome inhibitors. To investigate the potential association between PSMB gene variants and treatment-emergent resistance to bort

  15. Treating Meningitis

    Science.gov (United States)

    ... David C. Spencer, MD Steven Karceski, MD Treating meningitis Steven Karceski, MD WHAT DID THE AUTHORS STUDY? ... study, “ Dexamethasone and long-term survival in bacterial meningitis, ” Dr. Fritz and his colleagues carefully evaluated 2 ...

  16. Treating Vomiting

    Science.gov (United States)

    ... hours. Your pediatrician usually won’t prescribe a drug to treat the vomiting, but some doctors will prescribe antinausea medications to children. If your child also has diarrhea, ask your pediatrician for instructions on giving liquids ...

  17. AMPKα, C/EBPβ, CPT1β, GPR43, PPARγ, and SCD Gene Expression in Single- and Co-cultured Bovine Satellite Cells and Intramuscular Preadipocytes Treated with Palmitic, Stearic, Oleic, and Linoleic Acid.

    Science.gov (United States)

    Choi, S H; Park, S K; Johnson, B J; Chung, K Y; Choi, C W; Kim, K H; Kim, W Y; Smith, B

    2015-03-01

    We previously demonstrated that bovine subcutaneous preadipocytes promote adipogenic gene expression in muscle satellite cells in a co-culture system. Herein we hypothesize that saturated fatty acids would promote adipogenic/lipogenic gene expression, whereas mono- and polyunsaturated fatty acids would have the opposite effect. Bovine semimembranosus satellite cells (BSC) and intramuscular preadipocytes (IPA) were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS)/Dulbecco's Modified Eagle Medium (DMEM) and 1% antibiotics during the 3-d proliferation period. After proliferation, cells were treated for 3 d with 3% horse serum/DMEM (BSC) or 5% FBS/DMEM (IPA) with antibiotics. Media also contained 10 μg/mL insulin and 10 μg/mL pioglitazone. Subsequently, differentiating BSC and IPA were cultured in their respective media with 40 μM palmitic, stearic, oleic, or linoleic acid for 4 d. Finally, BSC and IPA were single- or co-cultured for an additional 2 h. All fatty acid treatments increased (p = 0.001) carnitine palmitoyltransferase-1 beta (CPT1β) gene expression, but the increase in CPT1β gene expression was especially pronounced in IPA incubated with palmitic and stearic acid (6- to 17- fold increases). Oleic and linoleic acid decreased (p = 0.001) stearoyl-CoA desaturase (SCD) gene expression over 80% in both BSC and IPA. Conversely, palmitic and stearic acid increased SCD gene expression three fold in co-cultured in IPA, and stearic acid increased AMPKα gene expression in single- and co-cultured BSC and IPA. Consistent with our hypothesis, saturated fatty acids, especially stearic acid, promoted adipogenic and lipogenic gene expression, whereas unsaturated fatty acids decreased expression of those genes associated with fatty acid metabolism.

  18. Synthesis and Characterization of a New Five and Six Membered Selenoheterocyclic Compounds Homologues of Ebselen

    Directory of Open Access Journals (Sweden)

    Mouslim Messali

    2011-01-01

    Full Text Available The discovery of the antioxidant activity of selenoenzyme glutathione peroxidase (GPx has attracted growing attention in the biochemistry of selenium. Among molecules which mimic the structure of the active site of the enzyme, N-phenyl-1,2-benzisoselenazolin-3-one 1, Ebselen, exhibited useful anti-inflammatory properties. It has been extensively investigated and has undergone clinical trials as an anti-inflammatory agent. Unfortunately, Ebselen exhibits relatively poor catalytic activity, prompting attempts to design more efficacious GPx mimetics that would retain his low toxicity while manifesting improved catalytic properties. In this context, novel 1,2-benzoselenazine and 1,2-benzoselenazols, which are five and six membered homologues of Ebselen were synthesized and characterized. One structure has been proven by single crystal X-ray crystallography.

  19. Molecular investigations of pathogenesis-related Bet v 1 homologues in Passiflora (Passifloraceae).

    Science.gov (United States)

    Finkler, Carla; Giacomet, Carolina; Muschner, Valéria C; Salzano, Francisco M; Freitas, Loreta B

    2005-07-01

    The major birch pollen allergen, Bet v 1, responsible for allergic reactions in many areas of the world, is homologous to a large number of pathogenesis-related proteins (PRs), identified as PR10. As part of a long-range investigation of these types of proteins and of evolution in Passiflora, DNA sequences from eight Bet v 1 homologue isoforms were obtained from five species of this genus in Brazil, and their sequences compared among themselves and with 30 others from 8 different species, classified in different taxonomic units. The objective was a first characterization of these PRs in wild passionflowers, and their use for evolutionary and applied investigations. High interspecific, but low intraspecific variability was observed, as expected from multigenic families subjected to concerted evolution. The relationships obtained both within Passiflora and between it and seven other genera probably best reflect functional similarities than evolutionary history.

  20. Homologue Structure of the SLAC1 Anion Channel for Closing Stomata in Leaves

    Energy Technology Data Exchange (ETDEWEB)

    Y Chen; L Hu; M Punta; R Bruni; B Hillerich; B Kloss; B Rost; J Love; S Siegelbaum; W Hendrickson

    2011-12-31

    The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 {angstrom} resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.

  1. A Salmonella Typhi homologue of bacteriophage muramidases controls typhoid toxin secretion.

    Science.gov (United States)

    Hodak, Hélène; Galán, Jorge E

    2013-01-01

    Unlike other Salmonella, which can infect a broad range of hosts causing self-limiting infection, Salmonella Typhi is an exclusively human pathogen that causes typhoid fever, a life-threatening systemic disease. Typhoid toxin is a unique virulence factor of Salmonella Typhi, which is expressed when the bacteria are within mammalian cells. Here, we report that an N-acetyl-β-D-muramidase similar to phage endolysins encoded within the same pathogenicity islet as the toxin is required for typhoid toxin secretion. Genetic and functional analysis of TtsA revealed unique amino acids at its predicted peptidoglycan-binding domain that are essential for protein secretion and that distinguishes this protein from other homologues. We propose that TtsA defines a new protein secretion mechanism recently evolved from the machine that mediates phage release.

  2. Iron uptake by melanoma cells from the soluble form of the transferrin homologue, melanotransferrin.

    Science.gov (United States)

    Food, Michael R; Des Richardson, R

    2002-01-01

    Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue that can also exist in a soluble form (sMTf). Considering the high homology of MTf to Tf, it is possible to suggest that sMTf could bind to the high affinity transferrin receptor 1 (TfR1) or lower affinity TfR2. We have used sMTf labelled with 59Fe to examine its ability to donate Fe to cells. Our experiments demonstrate that sMTf is far less effective than Tf at donating Fe to cells and this does not occur via specific receptors. Indeed, the uptake of sMTf by cells occurred via a non-specific process (e.g. adsorptive pinocytosis).

  3. Identification of an AfsA homologue (BarX) from Streptomyces virginiae as a pleiotropic regulator controlling autoregulator biosynthesis, virginiamycin biosynthesis and virginiamycin M1 resistance.

    Science.gov (United States)

    Kawachi, R; Akashi, T; Kamitani, Y; Sy, A; Wangchaisoonthorn, U; Nihira, T; Yamada, Y

    2000-04-01

    Virginiae butanolide (VB)-BarA of Streptomyces virginiae is one of the newly discovered pairs of a gamma-butyrolactone autoregulator and the corresponding receptor protein of the Streptomyces species, and has been shown to regulate the production of antibiotic virginiamycin (VM) in S. virginiae. A divergently transcribed barX gene is situated 259 bp upstream of the barA gene, and the BarX protein has been shown to be highly homologous (39.8% identity, 74. 6% similarity) to S. griseus AfsA. Although AfsA is thought to be a biosynthetic enzyme for A-factor, another member of the family of gamma-butyrolactone autoregulators, the in vivo function of S. virginiae BarX was investigated in this study by phenotypic and transcriptional comparison between wild-type S. virginiae and a barX deletion mutant. With the same growth rate as wild-type S. virginiae on both solid and liquid media, the barX mutant showed no apparent changes in its morphological behaviour, indicating that barX does not participate in morphological control in S. virginiae. However, the barX mutant became more sensitive to virginiamycin M1 than did the wild-type strain (minimum inhibitory concentration, 50 microgram ml-1 compared with > 200 microgram ml-1) and exhibited reduced VB and VM production. The VM production was not restored by exogenous addition of VB, suggesting that BarX per se is not a biosynthetic enzyme of VBs but a pleiotropic regulatory protein controlling VB biosynthesis. DNA sequencing of a 5.6 kbp downstream region of barX revealed the presence of five open reading frames (ORFs): barZ, encoding a BarB-like regulatory protein; orf2, encoding a Streptomyces coelicolor RedD-like pathway specific regulator; varM, encoding a homologue of ATP-dependent transporters for macrolide antibiotics; orf4, encoding a homologue of beta-ketoacyl ACP/CoA reductase; and orf5, encoding a homologue of dNDP-glucose dehydratase. Reverse transcription polymerase chain reaction (RT-PCR) analyses of the

  4. Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A. (UNL)

    2012-08-21

    DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

  5. The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics.

    Directory of Open Access Journals (Sweden)

    Marcel Sluijter

    Full Text Available The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn and RuvA(Mge, respectively were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn and RuvA(Mge (68.8% identity, substantial differences were found between these proteins in their activities. First, RuvA(Mge was found to preferentially bind to HJs, whereas RuvA(Mpn displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn is able to form two distinct complexes with HJs, RuvA(Mge only produced a single HJ complex. Third, RuvA(Mge stimulated the DNA helicase and ATPase activities of RuvB(Mge, whereas RuvA(Mpn did not augment RuvB activity. Finally, while both RuvA(Mge and RecU(Mge efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge.

  6. The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics.

    Science.gov (United States)

    Sluijter, Marcel; Estevão, Silvia; Hoogenboezem, Theo; Hartwig, Nico G; van Rossum, Annemarie M C; Vink, Cornelis

    2012-01-01

    The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).

  7. Gene expression profiling identifies FYN as an important molecule in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy

    DEFF Research Database (Denmark)

    Elias, D; (Hansen) Vever, Henriette; Lænkholm, A-V;

    2015-01-01

    To elucidate the molecular mechanisms of tamoxifen resistance in breast cancer, we performed gene array analyses and identified 366 genes with altered expression in four unique tamoxifen-resistant (TamR) cell lines vs the parental tamoxifen-sensitive MCF-7/S0.5 cell line. Most of these genes were...... an important role in tamoxifen resistance, and its subcellular localization in breast tumor cells may be an important novel biomarker of response to endocrine therapy in breast cancer.Oncogene advance online publication, 2 June 2014; doi:10.1038/onc.2014.138.......To elucidate the molecular mechanisms of tamoxifen resistance in breast cancer, we performed gene array analyses and identified 366 genes with altered expression in four unique tamoxifen-resistant (TamR) cell lines vs the parental tamoxifen-sensitive MCF-7/S0.5 cell line. Most of these genes were...... functionally linked to cell proliferation, death and control of gene expression, and include FYN, PRKCA, ITPR1, DPYD, DACH1, LYN, GBP1 and PRLR. Treatment with FYN-specific small interfering RNA or a SRC family kinase inhibitor reduced cell growth of TamR cell lines while exerting no significant effect on MCF...

  8. Transcriptome analysis of H2O2-treated wheat seedlings reveals a H2O2-responsive fatty acid desaturase gene participating in powdery mildew resistance.

    Directory of Open Access Journals (Sweden)

    Aili Li

    Full Text Available Hydrogen peroxide (H(2O(2 plays important roles in plant biotic and abiotic stress responses. However, the effect of H(2O(2 stress on the bread wheat transcriptome is still lacking. To investigate the cellular and metabolic responses triggered by H(2O(2, we performed an mRNA tag analysis of wheat seedlings under 10 mM H(2O(2 treatment for 6 hour in one powdery mildew (PM resistant (PmA and two susceptible (Cha and Han lines. In total, 6,156, 6,875 and 3,276 transcripts were found to be differentially expressed in PmA, Han and Cha respectively. Among them, 260 genes exhibited consistent expression patterns in all three wheat lines and may represent a subset of basal H(2O(2 responsive genes that were associated with cell defense, signal transduction, photosynthesis, carbohydrate metabolism, lipid metabolism, redox homeostasis, and transport. Among genes specific to PmA, 'transport' activity was significantly enriched in Gene Ontology analysis. MapMan classification showed that, while both up- and down- regulations were observed for auxin, abscisic acid, and brassinolides signaling genes, the jasmonic acid and ethylene signaling pathway genes were all up-regulated, suggesting H(2O(2-enhanced JA/Et functions in PmA. To further study whether any of these genes were involved in wheat PM response, 19 H(2O(2-responsive putative defense related genes were assayed in wheat seedlings infected with Blumeria graminis f. sp. tritici (Bgt. Eight of these genes were found to be co-regulated by H(2O(2 and Bgt, among which a fatty acid desaturase gene TaFAD was then confirmed by virus induced gene silencing (VIGS to be required for the PM resistance. Together, our data presents the first global picture of the wheat transcriptome under H(2O(2 stress and uncovers potential links between H(2O(2 and Bgt responses, hence providing important candidate genes for the PM resistance in wheat.

  9. Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF

    DEFF Research Database (Denmark)

    Tratwal, Josefine; Follin, Bjarke; Ekblond, Annette

    2014-01-01

    on reference genes (RGs) for the normalization of qPCR data. RESULTS: BMSCs and ASCs were stimulated with vascular endothelial growth factor A-165 (VEGF) for one week, and compared with un-stimulated cells from the same donor. The stability of nine RGs through VEGF treatment as well as the donor variation...... gene, TBP, was found to be the most stable standalone gene, while TBP and YWHAZ were found to be the best two-RG combination for qPCR analyses for both BMSCs and ASCs through the VEGF stimulation. The presented stepwise elimination procedure was validated, while we found the final normalization...

  10. Predominant recognition of species-specific determinants of the GroES homologues from Mycobacterium leprae and M. tuberculosis

    NARCIS (Netherlands)

    Chua-Intra, B.; Ivanyi, J.; Hills, A.; Thole, J.; Moreno, C.; Vordermeier, H.M.

    1998-01-01

    The Mycobacterium leprae and M. tuberculosis 10000 MW heat-shock protein homologues of GroES have previously been identified as major immunogens for human T cells. We used synthetic peptides to characterize the determinants recognized by murine T cells. The findings suggest that, despite 90% sequenc

  11. GTP analogue inhibits polymerization and GTPase activity of the bacterial protein FtsZ without affecting its eukaryotic homologue tubulin.

    NARCIS (Netherlands)

    Läppchen, T.; Hartog, A.F.; Pinas, V.; Koomen, G.J.; den Blaauwen, T.

    2005-01-01

    The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubul

  12. 4-Oxalocrotonate tautomerase, its homologue YwhB, and active vinylpyruvate hydratase : Synthesis and evaluation of 2-fluoro substrate analogues

    NARCIS (Netherlands)

    Johnson, William H; Wang, Susan C; Stanley, Thanuja M; Czerwinski, Robert M; Almrud, Jeffrey J; Poelarends, Gerrit J; Murzin, Alexey G; Whitman, Christian P

    2004-01-01

    A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized and examined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvate hydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found in B

  13. Secretory pathway antagonism by calicivirus homologues of Norwalk virus nonstructural protein p22 is restricted to noroviruses

    Directory of Open Access Journals (Sweden)

    Sharp Tyler M

    2012-09-01

    Full Text Available Abstract Background Our previous report that the Norwalk virus nonstructural protein p22 is an antagonist of the cellular secretory pathway suggests a new aspect of norovirus/host interaction. To explore conservation of function of this highly divergent calicivirus protein, we examined the effects of p22 homologues from four human and two murine noroviruses, and feline calicivirus on the secretory pathway. Findings All human noroviruses examined induced Golgi disruption and inhibited protein secretion, with the genogroup II.4 Houston virus being the most potent antagonist. Genogroup II.6 viruses have a conserved mutation in the mimic of an Endoplasmic Reticulum export signal (MERES motif that is highly conserved in human norovirus homologues of p22 and is critical for secretory pathway antagonism, and these viruses had reduced levels of Golgi disruption and inhibition of protein secretion. p22 homologues from both persistent and nonpersistent strains of murine norovirus induced Golgi disruption, but only mildly inhibited cellular protein secretion. Feline calicivirus p30 did not induce Golgi disruption or inhibit cellular protein secretion. Conclusions These differences confirm a norovirus-specific effect on host cell secretory pathway antagonism by homologues of p22, which may affect viral replication and/or cellular pathogenesis.

  14. Secretory pathway antagonism by calicivirus homologues of Norwalk virus nonstructural protein p22 is restricted to noroviruses.

    Science.gov (United States)

    Sharp, Tyler M; Crawford, Sue E; Ajami, Nadim J; Neill, Frederick H; Atmar, Robert L; Katayama, Kazuhiko; Utama, Budi; Estes, Mary K

    2012-09-03

    Our previous report that the Norwalk virus nonstructural protein p22 is an antagonist of the cellular secretory pathway suggests a new aspect of norovirus/host interaction. To explore conservation of function of this highly divergent calicivirus protein, we examined the effects of p22 homologues from four human and two murine noroviruses, and feline calicivirus on the secretory pathway. All human noroviruses examined induced Golgi disruption and inhibited protein secretion, with the genogroup II.4 Houston virus being the most potent antagonist. Genogroup II.6 viruses have a conserved mutation in the mimic of an Endoplasmic Reticulum export signal (MERES) motif that is highly conserved in human norovirus homologues of p22 and is critical for secretory pathway antagonism, and these viruses had reduced levels of Golgi disruption and inhibition of protein secretion. p22 homologues from both persistent and nonpersistent strains of murine norovirus induced Golgi disruption, but only mildly inhibited cellular protein secretion. Feline calicivirus p30 did not induce Golgi disruption or inhibit cellular protein secretion. These differences confirm a norovirus-specific effect on host cell secretory pathway antagonism by homologues of p22, which may affect viral replication and/or cellular pathogenesis.

  15. 4-Oxalocrotonate tautomerase, its homologue YwhB, and active vinylpyruvate hydratase : Synthesis and evaluation of 2-fluoro substrate analogues

    NARCIS (Netherlands)

    Johnson, William H; Wang, Susan C; Stanley, Thanuja M; Czerwinski, Robert M; Almrud, Jeffrey J; Poelarends, Gerrit J; Murzin, Alexey G; Whitman, Christian P

    2004-01-01

    A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized and examined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvate hydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found in B

  16. Inflammatory gene expression in monocytes of patients with schizophrenia : overlap and difference with bipolar disorder. A study in naturalistically treated patients

    NARCIS (Netherlands)

    Drexhage, Roosmarijn C.; van der Heul-Nieuwenhuijsen, Leonie; Padmos, Roos C.; van Beveren, Nico; Cohen, Dan; Versnel, Marjan A.; Nolen, Willem A.; Drexhage, Hemmo A.

    2010-01-01

    Accumulating evidence indicates an activated inflammatory response system as a vulnerability factor for schizophrenia (SZ) and bipolar disorder (BD). We aimed to detect a specific inflammatory monocyte gene expression signature in SZ and compare such signature with our recently described

  17. Coupling Neurogenetics (GARS™) and a Nutrigenomic Based Dopaminergic Agonist to Treat Reward Deficiency Syndrome (RDS): Targeting Polymorphic Reward Genes for Carbohydrate Addiction Algorithms

    OpenAIRE

    Kenneth Blum; Thomas Simpatico; Badgaiyan, Rajendra D.; Zsolt Demetrovics; James Fratantonio; Gozde Agan; Marcelo Febo; Gold, Mark S.

    2015-01-01

    Earlier work from our laboratory, showing anti-addiction activity of a nutraceutical consisting of amino-acid precursors and enkephalinase inhibition properties and our discovery of the first polymorphic gene (Dopamine D2 Receptor Gene [DRD2]) to associate with severe alcoholism serves as a blue-print for the development of “Personalized Medicine” in addiction. Prior to the later genetic finding, we developed the concept of Brain Reward Cascade, which continues to act as an important componen...

  18. Transcriptome analysis of acetic-acid-treated yeast cells identifies a large set of genes whose overexpression or deletion enhances acetic acid tolerance.

    Science.gov (United States)

    Lee, Yeji; Nasution, Olviyani; Choi, Eunyong; Choi, In-Geol; Kim, Wankee; Choi, Wonja

    2015-08-01

    Acetic acid inhibits the metabolic activities of Saccharomyces cerevisiae. Therefore, a better understanding of how S. cerevisiae cells acquire the tolerance to acetic acid is of importance to develop robust yeast strains to be used in industry. To do this, we examined the transcriptional changes that occur at 12 h post-exposure to acetic acid, revealing that 56 and 58 genes were upregulated and downregulated, respectively. Functional categorization of them revealed that 22 protein synthesis genes and 14 stress response genes constituted the largest portion of the upregulated and downregulated genes, respectively. To evaluate the association of the regulated genes with acetic acid tolerance, 3 upregulated genes (DBP2, ASC1, and GND1) were selected among 34 non-protein synthesis genes, and 54 viable mutants individually deleted for the downregulated genes were retrieved from the non-essential haploid deletion library. Strains overexpressing ASC1 and GND1 displayed enhanced tolerance to acetic acid, whereas a strain overexpressing DBP2 was sensitive. Fifty of 54 deletion mutants displayed enhanced acetic acid tolerance. Three chosen deletion mutants (hsps82Δ, ato2Δ, and ssa3Δ) were also tolerant to benzoic acid but not propionic and sorbic acids. Moreover, all those five (two overexpressing and three deleted) strains were more efficient in proton efflux and lower in membrane permeability and internal hydrogen peroxide content than controls. Individually or in combination, those physiological changes are likely to contribute at least in part to enhanced acetic acid tolerance. Overall, information of our transcriptional profile was very useful to identify molecular factors associated with acetic acid tolerance.

  19. Coupling Neurogenetics (GARS™) and a Nutrigenomic Based Dopaminergic Agonist to Treat Reward Deficiency Syndrome (RDS): Targeting Polymorphic Reward Genes for Carbohydrate Addiction Algorithms

    OpenAIRE

    Kenneth Blum; Thomas Simpatico; Badgaiyan, Rajendra D.; Zsolt Demetrovics; James Fratantonio; Gozde Agan; Marcelo Febo; Mark S. Gold

    2015-01-01

    Earlier work from our laboratory, showing anti-addiction activity of a nutraceutical consisting of amino-acid precursors and enkephalinase inhibition properties and our discovery of the first polymorphic gene (Dopamine D2 Receptor Gene [DRD2]) to associate with severe alcoholism serves as a blue-print for the development of “Personalized Medicine” in addiction. Prior to the later genetic finding, we developed the concept of Brain Reward Cascade, which continues to act as an important componen...

  20. Gene expression predicts overall survival in paraffin-embedded tissues of diffuse large B-cell lymphoma treated with R-CHOP

    Science.gov (United States)

    LeBlanc, Michael L.; Unger, Joseph M.; Miller, Thomas P.; Grogan, Thomas M.; Persky, Daniel O.; Martel, Ralph R.; Sabalos, Constantine M.; Seligmann, Bruce; Braziel, Rita M.; Campo, Elias; Rosenwald, Andreas; Connors, Joseph M.; Sehn, Laurie H.; Johnson, Nathalie; Gascoyne, Randy D.

    2008-01-01

    Gene expression profiling (GEP) on frozen tissues has identified genes predicting outcome in patients with diffuse large B-cell lymphoma (DLBCL). Confirmation of results in current patients is limited by availability of frozen samples and addition of monoclonal antibodies to treatment regimens. We used a quantitative nuclease protection assay (qNPA) to analyze formalin-fixed, paraffin-embedded tissue blocks for 36 previously identified genes (N = 209, 93 chemotherapy; 116 rituximab + chemotherapy). By qNPA, 208 cases were successfully analyzed (99.5%). In addition, 15 of 36 and 11 of 36 genes, representing each functional group previously identified by GEP, were associated with survival (P 80%) as independent indicators of survival, together distinguishing cases with the worst prognosis. Our results solve a clinical research problem by demonstrating that prognostic genes can be meaningfully quantified using qNPA technology on formalin-fixed, paraffin-embedded tissues; previous GEP findings in DLBCL are relevant with current treatments; and 2 genes, representing immune escape and proliferation, are the common features of the most aggressive DLBCL. PMID:18544678

  1. Tricky Treats

    Centers for Disease Control (CDC) Podcasts

    2008-08-04

    The Eagle Books are a series of four books that are brought to life by wise animal characters - Mr. Eagle, Miss Rabbit, and Coyote - who engage Rain That Dances and his young friends in the joy of physical activity, eating healthy foods, and learning from their elders about health and diabetes prevention. Tricky Treats shows children the difference between healthy snacks and sweet treats.  Created: 8/4/2008 by National Center for Chronic Disease Prevention and Health Promotion (NCCDPHP).   Date Released: 8/5/2008.

  2. Robust identification of orthologues and paralogues for microbial pan-genomics using GET_HOMOLOGUES: a case study of pIncA/C plasmids.

    Science.gov (United States)

    Vinuesa, Pablo; Contreras-Moreira, Bruno

    2015-01-01

    GET_HOMOLOGUES is an open-source software package written in Perl and R to define robust core- and pan-genomes by computing consensus clusters of orthologous gene families from whole-genome sequences using the bidirectional best-hit, COGtriangles, and OrthoMCL clustering algorithms. The granularity of the clusters can be fine-tuned by a user-configurable filtering strategy based on a combination of blastp pairwise alignment parameters, hmmscan-based scanning of Pfam domain composition of the proteins in each cluster, and a partial synteny criterion. We present detailed protocols to fit exponential and binomial mixture models to estimate core- and pan-genome sizes, compute pan-genome trees from the pan-genome matrix using a parsimony criterion, analyze and graphically represent the pan-genome structure, and identify lineage-specific gene families for the 12 complete pIncA/C plasmids currently available in NCBI's RefSeq. The software package, license, and detailed user manual can be downloaded for free for academic use from two mirrors: http://www.eead.csic.es/compbio/soft/gethoms.php and http://maya.ccg.unam.mx/soft/gethoms.php.

  3. Treating Infertility

    Science.gov (United States)

    ... by a number of factors. Both male and female factors can contribute to infertility. What treatment options are available for infertility? Treatment ... problems. A common problem that leads to male infertility, varicocele , sometimes can be ... are hormonal problems treated in women? Abnormal levels of hormones ...

  4. Association between the insulin-induced gene 2 (INSIG2) and weight gain in a German sample of antipsychotic-treated schizophrenic patients: perturbation of SREBP-controlled lipogenesis in drug-related metabolic adverse effects?

    Science.gov (United States)

    Le Hellard, S; Theisen, F M; Haberhausen, M; Raeder, M B; Fernø, J; Gebhardt, S; Hinney, A; Remschmidt, H; Krieg, J C; Mehler-Wex, C; Nöthen, M M; Hebebrand, J; Steen, V M

    2009-03-01

    Atypical antipsychotics are nowadays the most widely used drugs to treat schizophrenia and other psychosis. Unfortunately, some of them can cause major metabolic adverse effects, such as weight gain, dyslipidemia and type 2 diabetes. The underlying lipogenic mechanisms of the antipsychotic drugs are not known, but several studies have focused on a central effect in the hypothalamic control of appetite regulation and energy expenditure. In a functional convergent genomic approach we recently used a cellular model and demonstrated that orexigenic antipsychotics that induce weight gain activate the expression of lipid biosynthesis genes controlled by the sterol regulatory element-binding protein (SREBP) transcription factors. We therefore hypothesized that the major genes involved in the SREBP activation of fatty acids and cholesterol production (SREBF1, SREBF2, SCAP, INSIG1 and INSIG2) would be strong candidate genes for interindividual variation in drug-induced weight gain. We genotyped a total of 44 HapMap-selected tagging single nucleotide polymorphisms in a sample of 160 German patients with schizophrenia that had been monitored with respect to changes in body mass index during antipsychotic drug treatment. We found a strong association (P=0.0003-0.00007) between three markers localized within or near the INSIG2 gene (rs17587100, rs10490624 and rs17047764) and antipsychotic-related weight gain. Our finding is supported by the recent involvement of the INSIG2 gene in obesity in the general population and implicates SREBP-controlled lipogenesis in drug-induced metabolic adverse effects.

  5. MGMT Gene Promoter Methylation as a Potent Prognostic Factor in Glioblastoma Treated With Temozolomide-Based Chemoradiotherapy: A Single-Institution Study

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young Suk [Department of Radiation Oncology, Yonsei University College of Medicine, Yonsei University Health System, Seoul (Korea, Republic of); Kim, Se Hoon [Department of Pathology, Yonsei University College of Medicine, Yonsei University Health System, Seoul (Korea, Republic of); Cho, Jaeho; Kim, Jun Won [Department of Radiation Oncology, Yonsei University College of Medicine, Yonsei University Health System, Seoul (Korea, Republic of); Chang, Jong Hee; Kim, Dong Suk; Lee, Kyu Sung [Department of Neurosurgery, Yonsei University College of Medicine, Yonsei University Health System, Seoul (Korea, Republic of); Suh, Chang-Ok, E-mail: cosuh317@yuhs.ac [Department of Radiation Oncology, Yonsei University College of Medicine, Yonsei University Health System, Seoul (Korea, Republic of)

    2012-11-01

    Purpose: Recently, cells deficient in O{sup 6}-methylguanine-DNA methyltransferase (MGMT) were found to show increased sensitivity to temozolomide (TMZ). We evaluated whether hypermethylation of MGMT was associated with survival in patients with glioblastoma multiforme (GBM). Methods and Materials: We retrospectively analyzed 93 patients with histologically confirmed GBM who received involved-field radiotherapy with TMZ from 2001 to 2008. The median age was 58 years (range, 24-78 years). Surgical resection was total in 39 patients (42%), subtotal in 30 patients (32%), and partial in 17 patients (18%); only a biopsy was performed in 7 patients (8%). Postoperative radiotherapy began within 3 weeks of surgery in 87% of the patients. Radiotherapy doses ranged from 50 to 74 Gy (median, 70 Gy). MGMT gene methylation was determined in 78 patients; MGMT was unmethylated in 43 patients (55%) and methylated in 35 patients (45%). The median follow-up period was 22 months (range, 3-88 months) for all patients. Results: The median overall survival (OS) was 22 months, and progression-free survival (PFS) was 11 months. MGMT gene methylation was an independently significant prognostic factor for both OS (p = 0.002) and PFS (p = 0.008) in multivariate analysis. The median OS was 29 months for the methylated group and 20 months for the unmethylated group. In 35 patients with methylated MGMT genes, the 2-year and 5-year OS rates were 54% and 31%, respectively. Six patients with combined prognostic factors of methylated MGMT genes, age {<=}50 years, and total/subtotal resections are all alive 38 to 77 months after operation, whereas the median OS in 8 patients with unmethylated MGMT genes, age >50 years, and less than subtotal resection was 13.2 months. Conclusion: We confirmed that MGMT gene methylation is a potent prognostic factor in patients with GBM. Our results suggest that early postoperative radiotherapy and a high total/subtotal resection rate might further improve the

  6. Integration of retroviral vectors induces minor changes in the transcriptional activity of T cells from ADA-SCID patients treated with gene therapy.

    Science.gov (United States)

    Cassani, Barbara; Montini, Eugenio; Maruggi, Giulietta; Ambrosi, Alessandro; Mirolo, Massimiliano; Selleri, Silvia; Biral, Erika; Frugnoli, Ilaria; Hernandez-Trujillo, Vivian; Di Serio, Clelia; Roncarolo, Maria Grazia; Naldini, Luigi; Mavilio, Fulvio; Aiuti, Alessandro

    2009-10-22

    Gene transfer into hematopoietic stem cells by gamma-retroviral vectors (RVs) is an effective treatment for inherited blood disorders, although potentially limited by the risk of insertional mutagenesis. We evaluated the genomic impact of RV integration in T lymphocytes from adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) patients 10 to 30 months after infusion of autologous, genetically corrected CD34(+) cells. Expression profiling on ex vivo T-cell bulk population revealed no difference with respect to healthy controls. To assess the effect of vector integration on gene expression at the single-cell level, primary T-cell clones were isolated from 2 patients. T-cell clones harbored either 1 (89.8%) or 2 (10.2%) vector copies per cell and displayed partial to full correction of ADA expression, purine metabolism, and T-cell receptor-driven functions. Analysis of RV integration sites indicated a high diversity in T-cell origin, consistently with the polyclonal T-cell receptor-Vbeta repertoire. Quantitative transcript analysis of 120 genes within a 200-kb window around RV integration sites showed modest (2.8- to 5.2-fold) dysregulation of 5.8% genes in 18.6% of the T-cell clones compared with controls. Nonetheless, affected clones maintained a stable phenotype and normal in vitro functions. These results confirm that RV-mediated gene transfer for ADA-SCID is safe, and provide crucial information for the development of future gene therapy protocols. The trials described herein have been registered at http://www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

  7. Heterologous expression of a plant RelA-SpoT homologue results in increased stress tolerance in Saccharomyces cerevisiae by accumulation of the bacterial alarmone ppGpp.

    Science.gov (United States)

    Ochi, Kozo; Nishizawa, Tomoyasu; Inaoka, Takashi; Yamada, Akiyo; Hashimoto, Kohsuke; Hosaka, Takeshi; Okamoto, Susumu; Ozeki, Yoshihiro

    2012-08-01

    The bacterial alarmone ppGpp is present only in bacteria and the chloroplasts of plants, but not in mammalian cells or eukaryotic micro-organisms such as yeasts and fungi. The importance of the ppGpp signalling system in eukaryotes has therefore been largely overlooked. Here, we demonstrated that heterologous expression of a relA-spoT homologue (Sj-RSH) isolated from the halophilic plant Suaeda japonica in the yeast Saccharomyces cerevisiae results in accumulation of ppGpp, accompanied by enhancement of tolerance against various stress stimuli, such as osmotic stress, ethanol, hydrogen peroxide, high temperature and freezing. Unlike bacterial ppGpp accumulation, ppGpp was accumulated in the early growth phase but not in the late growth phase. Moreover, nutritional downshift resulted in a decrease in ppGpp level, suggesting that the observed Sj-RSH activity to synthesize ppGpp is not starvation-dependent, contrary to our expectations based on bacteria. Accumulated ppGpp was found to be present solely in the cytosolic fraction and not in the mitochondrial fraction, perhaps reflecting the ribosome-independent ppGpp synthesis in S. cerevisiae cells. Unlike bacterial inosine monophosphate (IMP) dehydrogenases, the IMP dehydrogenase of S. cerevisiae was insensitive to ppGpp. Microarray analysis showed that ppGpp accumulation gave rise to marked changes in gene expression, with both upregulation and downregulation, including changes in mitochondrial gene expression. The most prominent upregulation (38-fold) was detected in the hypothetical gene YBR072C-A of unknown function, followed by many other known stress-responsive genes. S. cerevisiae may provide new opportunities to uncover and analyse the ppGpp signalling system in eukaryotic cells.

  8. Gene expression profiles of inducible nitric oxide synthase and cytokines in Leishmania major-infected macrophage-like RAW 264.7 cells treated with gallic acid

    NARCIS (Netherlands)

    Radtke, O.A.; Kiderlen, A.F.; Kayser, Oliver; Kolodziej, H

    2004-01-01

    The effects of gallic acid on the gene expressions of inducible nitric oxide synthase (iNOS) and the cytokines interleukin (IL)-1, IL-10, IL-12, IL-18, TNF-alpha, and interferon (IFN)-gamma were investigated by reverse-transcription polymerase chain reaction (RT-PCR). The experiments were performed

  9. Inflammatory gene expression in monocytes of patients with schizophrenia : overlap and difference with bipolar disorder. A study in naturalistically treated patients

    NARCIS (Netherlands)

    Drexhage, Roosmarijn C.; van der Heul-Nieuwenhuijsen, Leonie; Padmos, Roos C.; van Beveren, Nico; Cohen, Dan; Versnel, Marjan A.; Nolen, Willem A.; Drexhage, Hemmo A.

    2010-01-01

    Accumulating evidence indicates an activated inflammatory response system as a vulnerability factor for schizophrenia (SZ) and bipolar disorder (BD). We aimed to detect a specific inflammatory monocyte gene expression signature in SZ and compare such signature with our recently described inflammator

  10. Expression of the outcome predictor in acute leukemia 1 (OPAL1) gene is not an independent prognostic factor in patients treated according to COALL or St Jude protocols

    NARCIS (Netherlands)

    A. Holleman (Amy); C.H. Pui (Ching-Hon); W.E. Evans (William); R. Pieters (Rob); M.L. den Boer (Monique); M.H. Cheok (Meyling); K.M. Kazemier (Karin); D. Pei (Deqing); J.R. Downing (James); G.E. Janka-Schaub (Gritta); U. Göbel (Ulrich); U. Graubner (Ulrike)

    2006-01-01

    textabstractNew prognostic factors may result in better risk classification and improved treatment of children with acute lymphoblastic leukemia (ALL). Recently, high expression of a gene named OPAL1 (outcome predictor in acute leukemia) was reported to be associated with favorable prognosis in ALL.

  11. Diversity of nifH gene pools in the rhizosphere of two cultivars of sorghum (Sorghum bicolor) treated with contrasting levels of nitrogen fertilizer

    NARCIS (Netherlands)

    Coelho, M.R.R.; Vos, de M.; Carneiro, N.P.; Marriel, I.E.; Paiva, E.; Seldin, L.

    2008-01-01

    The diversity of nitrogen-fixing bacteria was assessed in the rhizospheres of two cultivars of sorghum (IS 5322-C and IPA 1011) sown in Cerrado soil amended with two levels of nitrogen fertilizer (12 and 120 kg ha(-1)). The nifH gene was amplified directly from DNA extracted from the rhizospheres, a

  12. Expression Changes of Serotonin Receptor Gene Subtype 5HT3a in Peripheral Blood Mononuclear Cells from Schizophrenic Patients Treated with Haloperidol and Olanzapin

    Directory of Open Access Journals (Sweden)

    Gholam Reza Shariati

    2009-09-01

    Full Text Available Serotonin receptors are involved in pathophysiology of schizophrenia and may mediate other neurotransmitter effects. We investigated serotonin receptors gene expression in peripheral blood mononuclear cells (PBMC of naïve schizophrenic patients, before and after treatment. Also serotonin receptor gene expression was compared in two treatment groups including Haloperidol and Olanzapine. The PBMC was separated from whole blood by Ficoll-hypaque. The total cellular RNA was extracted and the cDNA was synthesized. This process was followed by real-time PCR using primer pairs specific for 5HT3a serotonin receptor mRNA and beta-actin as internal control. The results showed the presence of subtype of serotonin receptor in lymphocytes. Serotonin gene expression showed significant changes in Olanzapine treatment group which correlated with Clinical Global Impression (CGI score improvement. In conclusion, the present study has shown that human PBMC express serotonin receptors 5HT3a. Moreover, clinical symptom improvement of Olanzapin may be demonstrated by a change in serotonin receptor gene expression.

  13. Diversity of nifH gene pools in the rhizosphere of two cultivars of sorghum (Sorghum bicolor) treated with contrasting levels of nitrogen fertilizer

    NARCIS (Netherlands)

    Coelho, M.R.R.; Vos, de M.; Carneiro, N.P.; Marriel, I.E.; Paiva, E.; Seldin, L.

    2008-01-01

    The diversity of nitrogen-fixing bacteria was assessed in the rhizospheres of two cultivars of sorghum (IS 5322-C and IPA 1011) sown in Cerrado soil amended with two levels of nitrogen fertilizer (12 and 120 kg ha(-1)). The nifH gene was amplified directly from DNA extracted from the rhizospheres,

  14. Gene expression profiling and pathway analysis in MCF-7 and MDA-MB-231 human breast cancer cell lines treated with dioscin

    Science.gov (United States)

    The long-term goal of our study is to understand the genetic and epigenetic mechanisms of breast cancer metastasis in human and to discover new possible genetic markers for use in clinical practice. We have used microarray technology (Human OneArray microarray, phylanxbiotech.com) to compare gene ex...

  15. Gene expression signatures that predict outcome of tamoxifen-treated estrogen receptor-positive, high-risk, primary breast cancer patients

    DEFF Research Database (Denmark)

    Lyng, Maria Bibi; Lænkholm, Anne-Vibeke; Tan, Qihua;

    2013-01-01

    Tamoxifen significantly improves outcome for estrogen receptor-positive (ER+) breast cancer, but the 15-year recurrence rate remains 30%. The aim of this study was to identify gene profiles that accurately predicted the outcome of ER+ breast cancer patients who received adjuvant Tamoxifen mono-therapy....

  16. Gene regulation in response to protein disulphide isomerase deficiency

    DEFF Research Database (Denmark)

    Nørgaard, Per; Tachibana, Christine; Bruun, Anette W

    2003-01-01

    We have examined the activities of promoters of a number of yeast genes encoding resident endoplasmic reticulum proteins, and found increased expression in a strain with severe protein disulphide isomerase deficiency. Serial deletion in the promoter of the MPD1 gene, which encodes a PDI1-homologue...... element. The sequence (GACACG) does not resemble the unfolded protein response element. It is present in the upstream regions of the MPD1, MPD2, KAR2, PDI1 and ERO1 genes....

  17. Endometrial gene expression in the early luteal phase is impacted by mode of triggering final oocyte maturation in recFSH stimulated and GnRH antagonist co-treated IVF cycles

    DEFF Research Database (Denmark)

    Humaidan, P; Van Vaerenbergh, I; Bourgain, C

    2012-01-01

    Do differences in endometrial gene expression exist after ovarian stimulation with four different regimens of triggering final oocyte maturation and luteal phase support in the same patient? SUMMARY ANSWER: Significant differences in the expression of genes involved in receptivity and early...... implantation were seen between the four protocols. WHAT IS KNOWN ALREADY: GnRH agonist triggering is an alternative to hCG triggering in GnRH antagonist co-treated cycles, resulting in an elimination of early ovarian hyperstimulation syndrome. Whereas previous studies have revealed a low ongoing clinical...... pregnancy rate after GnRH agonist trigger due to a high early pregnancy loss rate, despite supplementation with the standard luteal phase support, more recent studies, employing a 'modified' luteal phase support including a bolus of 1500 IU hCG on the day of oocyte aspiration, have reported ongoing...

  18. PAI-1 4G/5G gene polymorphism is associated with angiographic patency in ST-elevation myocardial infarction patients treated with thrombolytic therapy.

    Science.gov (United States)

    Ozkan, Bugra; Cagliyan, Caglar E; Elbasan, Zafer; Uysal, Onur K; Kalkan, Gulhan Y; Bozkurt, Mehmet; Tekin, Kamuran; Bozdogan, Sevcan T; Ozalp, Ozge; Duran, Mustafa; Sahin, Durmus Y; Cayli, Murat

    2012-09-01

    In this study, we examined the relationship between PAI-1 4G/5G polymorphism and patency of the infarct-related artery after thrombolysis in patients with ST-elevation myocardial infarction (STEMI). Acute STEMI patients who received thrombolytic therapy within first 12 h were included in our study. The PAI-1 4G/5G promoter region insertion/deletion polymorphism was studied from venous blood samples. Patients with the PAI-1 4G/5G gene polymorphism were included in group 1 and the others were included in group 2. Coronary angiography was performed in all patients in the first 24 h after receiving thrombolytic therapy. Thrombolysis in myocardial infarction (TIMI) 0-1 flow in the infarct-related artery was considered as 'no flow', TIMI 2 flow as 'slow flow', and TIMI 3 flow as 'normal flow'. A total of 61 patients were included in our study. Thirty patients (49.2%) were positive for the PAI-1 4G/5G gene polymorphism, whereas 31 of them (50.8%) were in the control group. There were significantly more patients with 'no flow' (14 vs. 6; P=0.02) and less patients with 'normal flow' (8 vs. 19; P=0.02) in group 1. In addition, time to thrombolytic therapy (TTT) was maximum in the 'no flow' group and minimum in the 'normal flow' group (P=0.005). In the logistic regression analysis, TTT (odds ratio: 0.9898; 95% confidence interval: 0.982-0.997; P=0.004) and the PAI-1 4G/5G gene polymorphism (odds ratio: 4.621; 95% confidence interval: 1.399-15.268; P5G gene polymorphism and TTT are associated independently with 'no flow' after thrombolysis in patients with STEMI.

  19. Interleukin-10.rs1800896 and Interleukin-18.rs1946518 gene polymorphisms could not predict the outcome of hepatitis C virus infection in Egyptian patients treated with pegylated interferon plus ribavirin.

    Science.gov (United States)

    Abdelraheem, Wedad M; Hassuna, Noha A; Abuloyoun, Sahar M; Abdel Ghany, Hend M; Rizk, Hazem A; Abdelwahab, Sayed F

    2016-09-01

    A single-nucleotide polymorphism (SNP) in the interleukin (IL)-28B gene was used as a major predictor of the response to treatment in patients with hepatitis C virus (HCV) infection. Data examining the role of IL-10 and IL-18 gene polymorphisms among HCV genotype 4 (G4)-infected Egyptians in response to pegylated interferon (PEG-IFN) plus ribavirin (RBV) therapy are limited. This study investigated the impact of SNP at IL-10.rs1800896 (at position -1082) and IL-18.rs1946518 genes (at position -607) on the response to PEG-IFN/RBV therapy in HCV-infected Egyptians. This study was carried out on 100 HCV patients treated with PEG-IFN plus RBV and 100 healthy controls. The HCV patients included 50 treatment non-responders (NR) and 50 subjects with sustained virologic response (SVR). Genomic DNA from venous blood of subjects was extracted and IL-10.rs1800896 and IL-18.rs1946518 genotypes were determined using allele-specific amplification and SYBR Green real-time PCR. Linkage disequilibrium between the two SNPs was estimated using Haploview software. The frequency of the IL-10.rs1800896 AA, AG and GG genotypes among non-responders were 16 %, 70 % and 14 % while among SVR subjects, the frequency was 34 %, 60 % and 6 %, respectively (p=0.073). On the other hand, the frequency of the IL-18.rs1946518 AA, AC and CC genotypes among non-responders was 14 %, 50 % and 36 %, respectively, while among responders, these frequencies were 28 %, 44 % and 28 %, (p = 0.220). Both markers were in linkage equilibrium (D' = 0.23; r (2) = 0.052). SNPs in the IL-10.rs1800896 and IL-18.rs1946518 genes could not predict the outcome of HCV infection in Egyptians treated with PEG-IFN/RBV.

  20. Studies of the Cataluminescence of Benzene Homologues onNanosized γ–Al2O3/Eu2O3 and the Development of a Gas Sensorfor Benzene Homologue Vapors

    Directory of Open Access Journals (Sweden)

    Cuiqin Wu

    2006-12-01

    Full Text Available The cataluminescence (CTL of benzene and the benzene homologues tolueneand xylene on nanosized γ–Al2O3 doped with Eu2O3 (γ–Al2O3/Eu2O3 was studied and asensor of determining these gases was designed. The proposed sensor showed highsensitivity and selectivity at an optimal temperature of 432 ºC, a wavelength of 425 nm anda flow rate of 400 mL/min. Quantitative analysis was performed at the optimal conditions.The linear ranges of CTL intensity versus concentration of the benzene homologues were asfollows: benzene 2.4~5000 mL/m3, toluene 4.0~5000 mL/m3 and xylene 6.8~5000 mL/m3,with detection limits (3σ of 1.8 mL/m3, 3.0 mL/m3 and 3.4 mL/m3 for each one,respectively. The response time of this system was less than 3 s. The coexistence of othergases, such as SO2, CO and NH3, caused interference at levels around 11.7%, 5.8% and8.9% respectively. The technique is a convenient and fast way of determining the vapors ofbenzene homologues in air.

  1. The Drosophila homologue of the amyloid precursor protein is a conserved modulator of Wnt PCP signaling.

    Directory of Open Access Journals (Sweden)

    Alessia Soldano

    Full Text Available Wnt Planar Cell Polarity (PCP signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs are intensely investigated because of their link to Alzheimer's disease (AD. APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh by Abelson kinase (Abl. Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.

  2. A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells.

    Science.gov (United States)

    Lindskog, Eva; Svensson, Ingrid; Häggström, Lena

    2006-07-01

    Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.

  3. The nicotinic acetylcholine receptor and its prokaryotic homologues: Structure, conformational transitions & allosteric modulation.

    Science.gov (United States)

    Cecchini, Marco; Changeux, Jean-Pierre

    2015-09-01

    Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger - a neurotransmitter - into an ion flux through the postsynaptic membrane. Here, we present an overview of the most recent advances on the signal transduction mechanism boosted by X-ray crystallography of both prokaryotic and eukaryotic homologues of the nicotinic acetylcholine receptor (nAChR) in conjunction with time-resolved analyses based on single-channel electrophysiology and Molecular Dynamics simulations. The available data consistently point to a global mechanism of gating that involves a large reorganization of the receptor mediated by two distinct quaternary transitions: a global twisting and a radial expansion/contraction of the extracellular domain. These transitions profoundly modify the organization of the interface between subunits, which host several sites for orthosteric and allosteric modulatory ligands. The same mechanism may thus mediate both positive and negative allosteric modulations of pLGICs ligand binding at topographically distinct sites. The emerging picture of signal transduction is expected to pave the way to new pharmacological strategies for the development of allosteric modulators of nAChR and pLGICs in general. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. NRMT2 is an N-terminal monomethylase that primes for its homologue NRMT1.

    Science.gov (United States)

    Petkowski, Janusz J; Bonsignore, Lindsay A; Tooley, John G; Wilkey, Daniel W; Merchant, Michael L; Macara, Ian G; Schaner Tooley, Christine E

    2013-12-15

    NRMT (N-terminal regulator of chromatin condensation 1 methyltransferase) was the first eukaryotic methyltransferase identified to specifically methylate the free α-amino group of proteins. Since the discovery of this N-terminal methyltransferase, many new substrates have been identified and the modification itself has been shown to regulate DNA-protein interactions. Sequence analysis predicts one close human homologue of NRMT, METTL11B (methyltransferase-like protein 11B, now renamed NRMT2). We show in the present paper for the first time that NRMT2 also has N-terminal methylation activity and recognizes the same N-terminal consensus sequences as NRMT (now NRMT1). Both enzymes have similar tissue expression and cellular localization patterns. However, enzyme assays and MS experiments indicate that they differ in their specific catalytic functions. Although NRMT1 is a distributive methyltransferase that can mono-, di- and tri-methylate its substrates, NRMT2 is primarily a monomethylase. Concurrent expression of NRMT1 and NRMT2 accelerates the production of trimethylation, and we propose that NRMT2 activates NRMT1 by priming its substrates for trimethylation.

  5. A caseian point for the evolution of a diaphragm homologue among the earliest synapsids.

    Science.gov (United States)

    Lambertz, Markus; Shelton, Christen D; Spindler, Frederik; Perry, Steven F

    2016-12-01

    The origin of the diaphragm remains a poorly understood yet crucial step in the evolution of terrestrial vertebrates, as this unique structure serves as the main respiratory motor for mammals. Here, we analyze the paleobiology and the respiratory apparatus of one of the oldest lineages of mammal-like reptiles: the Caseidae. Combining quantitative bone histology and functional morphological and physiological modeling approaches, we deduce a scenario in which an auxiliary ventilatory structure was present in these early synapsids. Crucial to this hypothesis are indications that at least the phylogenetically advanced caseids might not have been primarily terrestrial but rather were bound to a predominantly aquatic life. Such a lifestyle would have resulted in severe constraints on their ventilatory system, which consequently would have had to cope with diving-related problems. Our modeling of breathing parameters revealed that these caseids were capable of only limited costal breathing and, if aquatic, must have employed some auxiliary ventilatory mechanism to quickly meet their oxygen demand upon surfacing. Given caseids' phylogenetic position at the base of Synapsida and under this aquatic scenario, it would be most parsimonious to assume that a homologue of the mammalian diaphragm had already evolved about 50 Ma earlier than previously assumed.

  6. The Drosophila homologue of the amyloid precursor protein is a conserved modulator of Wnt PCP signaling.

    Science.gov (United States)

    Soldano, Alessia; Okray, Zeynep; Janovska, Pavlina; Tmejová, Kateřina; Reynaud, Elodie; Claeys, Annelies; Yan, Jiekun; Atak, Zeynep Kalender; De Strooper, Bart; Dura, Jean-Maurice; Bryja, Vítězslav; Hassan, Bassem A

    2013-01-01

    Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.

  7. DNA Damage Induced MutS Homologue hMSH4 Acetylation

    Directory of Open Access Journals (Sweden)

    Chengtao Her

    2013-10-01

    Full Text Available Acetylation of non-histone proteins is increasingly recognized as an important post-translational modification for controlling the actions of various cellular processes including DNA repair and damage response. Here, we report that the human MutS homologue hMSH4 undergoes acetylation following DNA damage induced by ionizing radiation (IR. To determine which acetyltransferases are responsible for hMSH4 acetylation in response to DNA damage, potential interactions of hMSH4 with hTip60, hGCN5, and hMof were analyzed. The results of these experiments indicate that only hMof interacts with hMSH4 in a DNA damage-dependent manner. Intriguingly, the interplay between hMSH4 and hMof manipulates the outcomes of nonhomologous end joining (NHEJ-mediated DNA double strand break (DSB repair and thereby controls cell survival in response to IR. This study also shows that hMSH4 interacts with HDAC3, by which HDAC3 negatively regulates the levels of hMSH4 acetylation. Interestingly, elevated levels of HDAC3 correlate with increased NHEJ-mediated DSB repair, suggesting that hMSH4 acetylation per se may not directly affect the role of hMSH4 in DSB repair.

  8. TRBP and eIF6 homologue in Marsupenaeus japonicus play crucial roles in antiviral response.

    Directory of Open Access Journals (Sweden)

    Shuai Wang

    Full Text Available Plants and invertebrates can suppress viral infection through RNA silencing, mediated by RNA-induced silencing complex (RISC. Trans-activation response RNA-binding protein (TRBP, consisting of three double-stranded RNA-binding domains, is a component of the RISC. In our previous paper, a TRBP homologue in Fenneropenaeus chinensis (Fc-TRBP was reported to directly bind to eukaryotic initiation factor 6 (Fc-eIF6. In this study, we further characterized the function of TRBP and the involvement of TRBP and eIF6 in antiviral RNA interference (RNAi pathway of shrimp. The double-stranded RNA binding domains (dsRBDs B and C of the TRBP from Marsupenaeus japonicus (Mj-TRBP were found to mediate the interaction of TRBP and eIF6. Gel-shift assays revealed that the N-terminal of Mj-TRBP dsRBD strongly binds to double-stranded RNA (dsRNA and that the homodimer of the TRBP mediated by the C-terminal dsRBD increases the affinity to dsRNA. RNAi against either Mj-TRBP or Mj-eIF6 impairs the dsRNA-induced sequence-specific RNAi pathway and facilitates the proliferation of white spot syndrome virus (WSSV. These results further proved the important roles of TRBP and eIF6 in the antiviral response of shrimp.

  9. Crystal Structure of the Heterotrimer Core of Saccharomyces cerevisiae AMPK Homologue SNF1

    Energy Technology Data Exchange (ETDEWEB)

    Amodeo,G.; Rudolph, M.; Tong, L.

    2007-01-01

    AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals and is an attractive target for drug discovery against diabetes, obesity and other diseases. The AMPK homologue in Saccharomyces cerevisiae, known as SNF1, is essential for responses to glucose starvation as well as for other cellular processes, although SNF1 seems to be activated by a ligand other than AMP. Here we report the crystal structure at 2.6 resolution of the heterotrimer core of SNF1. The ligand-binding site in the {gamma}-subunit (Snf4) has clear structural differences from that of the Schizosaccharomyces pombe enzyme, although our crystallographic data indicate that AMP can also bind to Snf4. The glycogen-binding domain in the {beta}-subunit (Sip2) interacts with Snf4 in the heterotrimer but should still be able to bind carbohydrates. Our structure is supported by a large body of biochemical and genetic data on this complex. Most significantly, the structure reveals that part of the regulatory sequence in the {alpha}-subunit (Snf1) is sequestered by Snf4, demonstrating a direct interaction between the {alpha}- and {gamma}-subunits and indicating that our structure may represent the heterotrimer core of SNF1 in its activated state.

  10. Crystallization and preliminary diffraction analysis of a DsbA homologue from Wolbachia pipientis

    Energy Technology Data Exchange (ETDEWEB)

    Kurz, M. [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, QLD 4072 (Australia); Iturbe-Ormaetxe, I. [School of Integrative Biology, The University of Queensland, St Lucia, QLD 4072 (Australia); Jarrott, R. [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, QLD 4072 (Australia); O’Neill, S. L. [School of Integrative Biology, The University of Queensland, St Lucia, QLD 4072 (Australia); Byriel, K. A.; Martin, J. L., E-mail: j.martin@imb.uq.edu.au; Heras, B., E-mail: j.martin@imb.uq.edu.au [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, QLD 4072 (Australia)

    2008-02-01

    The first crystallization of a W. pipientis protein, α-DsbA1, was achieved using hanging-drop and sitting-drop vapour diffusion. α-DsbA1 is one of two DsbA homologues encoded by the Gram-negative α-proteobacterium Wolbachia pipientis, an endosymbiont that can behave as a reproductive parasite in insects and as a mutualist in medically important filarial nematodes. The α-DsbA1 protein is thought to be important for the folding and secretion of Wolbachia proteins involved in the induction of reproductive distortions. Crystals of native and SeMet α-DsbA1 were grown by vapour diffusion and belong to the monoclinic space group C2, with unit-cell parameters a = 71.4, b = 49.5, c = 69.3 Å, β = 107.0° and one molecule in the asymmetric unit (44% solvent content). X-ray data were recorded from native crystals to a resolution of 2.01 Å using a copper anode and data from SeMet α-DsbA1 crystals were recorded to 2.45 Å resolution using a chromium anode.

  11. Homologues of sox8 and sox10 in the orange-spotted grouper Epinephelus coioides: sequences, expression patterns, and their effects on cyp19a1a promoter activities in vitro.

    Science.gov (United States)

    Liu, Qiongyou; Lu, Huijie; Zhang, Lihong; Xie, Jun; Shen, Wenying; Zhang, Weimin

    2012-09-01

    Sox8 and Sox10 are members of group E Sox proteins involved in a wide range of developmental processes including sex determination and neurogenesis in vertebrates. The orange-spotted grouper sox8a and sox10a homologues were isolated and characterized in the present study. Both sox8a and sox10a genes contain three exons and two introns, and encode putative proteins with typical structures of group E Sox. Sox8a was expressed in diverse tissues including the central nervous system and some peripheral tissues. In contrast, sox10a mRNA was detected primarily in the central nervous system. During embryogenesis, sox8a mRNA seemed to be de novo synthesized in the embryos from otic vesicle stage. However, sox10a mRNA was only detectable in juvenile fish 35 days post hatching and thereafter. The mRNA levels of sox8a in the gonads were not significantly different among ovarian developmental stages but increased in the testis. In vitro transfection assays showed that the Sox10a but not Sox8a up-regulated cyp19a1a promoter activities. Taken together, these results suggested that the sox8a may play roles in diverse tissues and during embryogenesis, whereas sox10a may be mainly involved in the neural regulation of juvenile and adult fish, and that certain Sox homologues may regulate the orange-spotted grouper cyp19a1a promoter.

  12. Coupling Neurogenetics (GARS™ and a Nutrigenomic Based Dopaminergic Agonist to Treat Reward Deficiency Syndrome (RDS: Targeting Polymorphic Reward Genes for Carbohydrate Addiction Algorithms

    Directory of Open Access Journals (Sweden)

    Kenneth Blum

    2015-06-01

    Full Text Available Earlier work from our laboratory, showing anti-addiction activity of a nutraceutical consisting of amino-acid precursors and enkephalinase inhibition properties and our discovery of the first polymorphic gene (Dopamine D2 Receptor Gene [DRD2] to associate with severe alcoholism serves as a blue-print for the development of "Personalized Medicine" in addiction. Prior to the later genetic finding, we developed the concept of Brain Reward Cascade, which continues to act as an important component for stratification of addiction risk through neurogenetics. In 1996 our laboratory also coined the term "Reward Deficiency Syndrome (RDS" to define a common genetic rubric for both substance and non-substance related addictive behaviors. Following many reiterations we utilized polymorphic targets of a number of reward genes (serotonergic, Opioidergic, GABAergic and Dopaminergic to customize KB220 [Neuroadaptogen- amino-acid therapy (NAAT] by specific algorithms. Identifying 1,000 obese subjects in the Netherlands a subsequent small subset was administered various KB220Z formulae customized according to respective DNA polymorphisms individualized that translated to significant decreases in both Body Mass Index (BMI and weight in pounds. Following these experiments, we have been successfully developing a panel of genes known as "Genetic Addiction Risk Score" (GARSpDX™. Selection of 10 genes with appropriate variants, a statistically significant association between the ASIMedia Version-alcohol and drug severity scores and GARSpDx was found A variant of KB220Z in abstinent heroin addicts increased resting state functional connectivity in a putative network including: dorsal anterior cingulate, medial frontal gyrus, nucleus accumbens, posterior cingulate, occipital cortical areas, and cerebellum. In addition, we show that KB220Z significantly activates, above placebo, seed regions of interest including the left nucleus accumbens, cingulate gyrus, anterior

  13. Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

    Science.gov (United States)

    Drozd, Ewa; Krzysztoń-Russjan, Jolanta; Marczewska, Jadwiga; Drozd, Janina; Bubko, Irena; Bielak, Magda; Lubelska, Katarzyna; Wiktorska, Katarzyna; Chilmonczyk, Zdzisław; Anuszewska, Elżbieta; Gruber-Bzura, Beata

    2016-10-01

    Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely

  14. Coupling Neurogenetics (GARS™) and a Nutrigenomic Based Dopaminergic Agonist to Treat Reward Deficiency Syndrome (RDS): Targeting Polymorphic Reward Genes for Carbohydrate Addiction Algorithms.

    Science.gov (United States)

    Blum, Kenneth; Simpatico, Thomas; Badgaiyan, Rajendra D; Demetrovics, Zsolt; Fratantonio, James; Agan, Gozde; Febo, Marcelo; Gold, Mark S

    Earlier work from our laboratory, showing anti-addiction activity of a nutraceutical consisting of amino-acid precursors and enkephalinase inhibition properties and our discovery of the first polymorphic gene (Dopamine D2 Receptor Gene [DRD2]) to associate with severe alcoholism serves as a blue-print for the development of "Personalized Medicine" in addiction. Prior to the later genetic finding, we developed the concept of Brain Reward Cascade, which continues to act as an important component for stratification of addiction risk through neurogenetics. In 1996 our laboratory also coined the term "Reward Deficiency Syndrome (RDS)" to define a common genetic rubric for both substance and non-substance related addictive behaviors. Following many reiterations we utilized polymorphic targets of a number of reward genes (serotonergic, Opioidergic, GABAergic and Dopaminergic) to customize KB220 [Neuroadaptogen- amino-acid therapy (NAAT)] by specific algorithms. Identifying 1,000 obese subjects in the Netherlands a subsequent small subset was administered various KB220Z formulae customized according to respective DNA polymorphisms individualized that translated to significant decreases in both Body Mass Index (BMI) and weight in pounds. Following these experiments, we have been successfully developing a panel of genes known as "Genetic Addiction Risk Score" (GARSpDX)™. Selection of 10 genes with appropriate variants, a statistically significant association between the ASI-Media Version-alcohol and drug severity scores and GARSpDx was found A variant of KB220Z in abstinent heroin addicts increased resting state functional connectivity in a putative network including: dorsal anterior cingulate, medial frontal gyrus, nucleus accumbens, posterior cingulate, occipital cortical areas, and cerebellum. In addition, we show that KB220Z significantly activates, above placebo, seed regions of interest including the left nucleus accumbens, cingulate gyrus, anterior thalamic nuclei

  15. Characterization of the POU5F1 Homologue in Nile Tilapia: From Expression Pattern to Biological Activity.

    Science.gov (United States)

    Xiaohuan, Huang; Yang, Zhao; Linyan, Liu; Zhenhua, Fan; Linyan, Zhou; Zhijian, Wang; Ling, Wei; Deshou, Wang; Jing, Wei

    2016-09-15

    POU5F1 (OCT4) is a crucial transcription factor for induction and maintenance of cellular pluripotency, as well as survival of germ cells in mammals. However, the homologues of POU5F1 in teleost fish, including zebrafish and medaka, now named Pou5f3, exhibit considerable differences in expression pattern and pluripotency-maintaining activity. To what extent the POU5F1 homologues are conserved in vertebrates has been unclear. In this study, we report that the POU5F1 homologue from the Nile tilapia (Oreochromis niloticus), OnPou5f3, displays an expression pattern and biological activity somewhat different from those in zebrafish or medaka. The expression of Onpou5f3 at both mRNA and protein levels was abundant in early development embryos until blastula stages, barely detectable as proceeding, and then displayed a transiently strong expression domain in the brain region during neurula stages similar to zebrafish but not medaka. Afterward, OnPou5f3 appeared as germline-restricted (including primordial germ cells and female and male gonad germ cells) expression just like medaka. Notably, OnPou5f3 depletion through morpholino oligos caused blastula blockage or lethality and failure of survival and proliferation of blastula cell-derived cells. These findings indicate that equivalent POU5F1-like expression and activity of Pou5f3 might be conserved accompanying with species-specific expression pattern during evolution. Our study provides insight into the evolutionary conservation of the POU5F1 homologues across vertebrates.

  16. A new MOF-5 homologue for selective separation of methane from C2 hydrocarbons at room temperature

    Directory of Open Access Journals (Sweden)

    Yabing He

    2014-12-01

    Full Text Available A new MOF-5 homologue compound UTSA-10 has been obtained under solvothermal conditions from a mixture of Zn(NO32⋅6H2O and commercially available linker, 2-methylfumaric acid, in N,N-dimethylformamide. The moderate surface area and suitable pore sizes enable the activated UTSA-10a to separate methane from C2 hydrocarbons at room temperature.

  17. Shrimp Serine Proteinase Homologues PmMasSPH-1 and -2 Play a Role in the Activation of the Prophenoloxidase System

    Science.gov (United States)

    Jearaphunt, Miti; Amparyup, Piti; Sangsuriya, Pakkakul; Charoensapsri, Walaiporn; Senapin, Saengchan; Tassanakajon, Anchalee

    2015-01-01

    Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune response used by invertebrates against intruding pathogens. Several masquerade-like and serine proteinase homologues (SPHs) have been demonstrated to play an essential role in proPO activation in insects and crustaceans. In a previous study, we characterized the masquerade-like SPH, PmMasSPH1, in the black tiger shrimp Penaeus monodon as a multifunctional immune protein based on its recognition and antimicrobial activity against the Gram-negative bacteria Vibrio harveyi. In the present study, we identify a novel SPH, known as PmMasSPH2, composed of an N-terminal clip domain and a C-terminal SP-like domain that share high similarity to those of other insect and crustacean SPHs. We demonstrate that gene silencing of PmMasSPH1 and PmMasSPH2 significantly reduces PO activity, resulting in a high number of V. harveyi in the hemolymph. Interestingly, knockdown of PmMasSPH1 suppressed not only its gene transcript but also other immune-related genes in the proPO system (e.g., PmPPAE2) and antimicrobial peptides (e.g., PenmonPEN3, PenmonPEN5, crustinPm1 and Crus-likePm). The PmMasSPH1 and PmMasSPH2 also show binding activity to peptidoglycan (PGN) of Gram-positive bacteria. Using a yeast two-hybrid analysis and co-immunoprecipitation, we demonstrate that PmMasSPH1 specifically interacted with the final proteinase of the proPO cascade, PmPPAE2. Furthermore, the presence of both PmMasSPH1 and PmPPAE2 enhances PGN-induced PO activity in vitro. Taken together, these results suggest the importance of PmMasSPHs in the activation of the shrimp proPO system. PMID:25803442

  18. Pseudo-response regulator (PRR) homologues of the moss Physcomitrella patens: insights into the evolution of the PRR family in land plants.

    Science.gov (United States)

    Satbhai, Santosh B; Yamashino, Takafumi; Okada, Ryo; Nomoto, Yuji; Mizuno, Takeshi; Tezuka, Yuki; Itoh, Tomonori; Tomita, Mitsuru; Otsuki, Susumu; Aoki, Setsuyuki

    2011-02-01

    The pseudo-response regulators (PRRs) are the circadian clock component proteins in the model dicot Arabidopsis thaliana. They contain a receiver-like domain (RLD) similar to the receiver domains of the RRs in the His-Asp phosphorelay system, but the RLDs lack the phosphoacceptor aspartic acid residue invariably conserved in the receiver domains. To study the evolution of PRR genes in plants, here we characterize their homologue genes, PpPRR1, PpPRR2, PpPRR3 and PpPRR4, from the moss Physcomitrella patens. In the phylogenetic analysis, PpPRRs cluster together, sister to an angiosperm PRR gene subfamily, illustrating their close relationships with the angiosperm PRRs. However, distinct from the angiosperm sequences, the RLDs of PpPRR2/3/4 exhibit a potential phosphoacceptor aspartic acid-aspartic acid-lysine (DDK) motif. Consistently, the PpPRR2 RLD had phosphotransfer ability in vitro, suggesting that PpPRR2 functions as an RR. The PpPRR1 RLD, on the other hand, shows a partially diverged DDK motif, and it did not show phosphotransfer ability. All PpPRRs were expressed in a circadian and light-dependent manner, with differential regulation between PpPRR2/4 and PpPRR1/3. Altogether, our results illustrate that PRRs originated from an RR(s) and that there are intraspecific divergences among PpPRRs. Finally, we offer scenarios for the evolution of the PRR family in land plants.

  19. Species-specific flight styles of flies are reflected in the response dynamics of a homologue motion sensitive neuron

    Directory of Open Access Journals (Sweden)

    Bart eGeurten

    2012-03-01

    Full Text Available Hoverflies and blowflies have distinctly different flight styles. Yet, both species have been shown to structure their flight behaviour in a way that facilitates extraction of 3D information from the image flow on the retina (optic flow. Neuronal candidates to analyse the optic flow are the tangential cells in the third optical ganglion – the lobula complex. These neurons are directionally selective and integrate the optic flow over large parts of the visual field. Homologue tangential cells in hoverflies and blowflies have a similar morphology. Because blowflies and hoverflies have similar neuronal layout but distinctly different flight behaviours, they are an ideal substrate to pinpoint potential neuronal adaptations to the different flight styles.In this article we describe the relationship between locomotion behaviour and motion vision on three different levels:1.We compare the different flight styles based on the categorisation of flight behaviour into prototypical movements.2.We measure the species specific dynamics of the optic flow under naturalistic flight conditions. We found the translational optic flow of both species to be very different.3.We describe possible adaptations of a homologue motion sensitive neuron. We stimulate this cell in blowflies (Calliphora and hoverflies (Eristalis with naturalistic optic flow generated by both species during free flight. The characterized hoverfly tangential cell responds faster to transient changes in the optic flow than its blowfly homologue. It is discussed whether and how the different dynamical response properties aid optic flow analysis.

  20. Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling.

    Science.gov (United States)

    Pazman, C; Mayes, C A; Fanto, M; Haynes, S R; Mlodzik, M

    2000-04-01

    The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.

  1. Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p.

    Science.gov (United States)

    Jaspersen, Sue L; Giddings, Thomas H; Winey, Mark

    2002-12-23

    Accurate duplication of the Saccharomyces cerevisiae spindle pole body (SPB) is required for formation of a bipolar mitotic spindle. We identified mutants in SPB assembly by screening a temperature-sensitive collection of yeast for defects in SPB incorporation of a fluorescently marked integral SPB component, Spc42p. One SPB assembly mutant contained a mutation in a previously uncharacterized open reading frame that we call MPS3 (for monopolar spindle). mps3-1 mutants arrest in mitosis with monopolar spindles at the nonpermissive temperature, suggesting a defect in SPB duplication. Execution point experiments revealed that MPS3 function is required for the first step of SPB duplication in G1. Like cells containing mutations in two other genes required for this step of SPB duplication (CDC31 and KAR1), mps3-1 mutants arrest with a single unduplicated SPB that lacks an associated half-bridge. MPS3 encodes an essential integral membrane protein that localizes to the SPB half-bridge. Genetic interactions between MPS3 and CDC31 and binding of Cdc31p to Mps3p in vitro, as well as the fact that Cdc31p localization to the SPB is partially dependent on Mps3p function, suggest that one function for Mps3p during SPB duplication is to recruit Cdc31p, the yeast centrin homologue, to the half-bridge.

  2. Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue.

    Science.gov (United States)

    Goncharov, I; Palfi, Z; Bindereif, A; Michaeli, S

    1999-04-30

    Trans-splicing in trypanosomes involves the addition of a common spliced leader (SL) sequence, which is derived from a small RNA, the SL RNA, to all mRNA precursors. The SL RNA is present in the cell in the form of a ribonucleoprotein, the SL RNP. Using conventional chromatography and affinity selection with 2'-O-methylated RNA oligonucleotides at high ionic strength, five proteins of 70, 16, 13, 12, and 8 kDa were co-selected with the SL RNA from Leptomonas collosoma, representing the SL RNP core particle. Under conditions of lower ionic strength, additional proteins of 28 and 20 kDa were revealed. On the basis of peptide sequences, the gene coding for a protein with a predicted molecular weight of 11.9 kDa was cloned and identified as homologue of the cis-spliceosomal SmE. The protein carries the Sm motifs 1 and 2 characteristic of Sm antigens that bind to all known cis-spliceosomal uridylic acid-rich small nuclear RNAs (U snRNAs), suggesting the existence of Sm proteins in trypanosomes. This finding is of special interest because trypanosome snRNPs are the only snRNPs examined to date that are not recognized by anti-Sm antibodies. Because of the early divergence of trypanosomes from the eukaryotic lineage, the trypanosome SmE protein represents one of the primordial Sm proteins in nature.

  3. Small cell lung cancer, an epithelial to mesenchymal transition (EMT)-like cancer: significance of inactive Notch signaling and expression of achaete-scute complex homologue 1.

    Science.gov (United States)

    Ito, Takaaki; Kudoh, Shinji; Ichimura, Takaya; Fujino, Kosuke; Hassan, Wael Ahmed Maher Abdo; Udaka, Naoko

    2017-01-01

    Small cell lung cancer (SCLC) is one of the most malignant neoplasms in common human cancers. The tumor is composed of small immature-looking cells with a round or fusiform shape, which possesses weak adhesion features among them, suggesting that SCLC shows the morphological characteristics of epithelial to mesenchymal transition (EMT). SCLC is characterized by high metastatic and recurrent rates, sensitivity to the initial chemotherapy, and easy acquirement of chemoresistance afterwards. These characters may be related to the EMT phenotype of SCLC. Notch signaling is an important signaling pathway, and could have roles in regulating neuroendocrine differentiation, proliferation, cell adhesion, EMT, and chemoresistance. Notch1 is usually absent in SCLC in vivo, but could appear after chemotherapy. Notch1 can enhance cell adhesion by induction of E-cadherin in SCLC, which indicates mesenchymal to epithelial transition. On the other hand, achaete-scute complex homologue 1 (ASCL1), negatively regulated by Notch signaling, is a lineage-specific gene of SCLC, and functions to promote neuroendocrine differentiation as well as EMT. ASCL1-transfected adenocarcinoma cell lines induced neuroendocrine phenotypes and lost epithelial cell features. SCLC is characterized by neuroendocrine differentiation and EMT-like features, which could be produced by inactive Notch signaling and ASCL1 expression. In addition, chemical and radiation treatments can activate Notch signaling, which suppress neuroendocrine differentiation and induces chemoradioresistance, accompanied by secession from EMT. Thus, the status of Notch signaling and ASCL1 expression may determine the cell behaviors of SCLC partly through modifying EMT phenotypes.

  4. Cloning of interleukin-10 from African clawed frog (Xenopus tropicalis), with the Finding of IL-19/20 homologue in the IL-10 locus.

    Science.gov (United States)

    Qi, Zhitao; Zhang, Qihuan; Wang, Zisheng; Zhao, Weihong; Gao, Qian

    2015-01-01

    Interleukin-10 (IL-10) is a pleiotropic cytokine that plays an important role in immune system. In the present study, the IL-10 gene of African clawed frog (Xenopus tropicalis) was first cloned, and its expression pattern and 3D structure were also analyzed. The frog IL-10 mRNA encoded 172 amino acids which possessed several conserved features found in IL-10s from other species, including five-exon/four-intron genomic structure, conserved four cysteine residues, IL-10 family motif, and six α-helices. Real-time PCR showed that frog IL-10 mRNA was ubiquitous expressed in all examined tissues, highly in some immune related tissues including kidney, spleen, and intestine and lowly in heart, stomach, and liver. The frog IL-10 mRNA was upregulated at 24 h after LPS stimulation, indicating that it plays a part in the host immune response to bacterial infection. Another IL, termed as IL-20, was identified from the frog IL-10 locus, which might be the homologue of mammalian IL-19/20 according to the analysis results of the phylogenetic tree and the sequence identities.

  5. Gene Therapy

    Science.gov (United States)

    ... or improve your body's ability to fight disease. Gene therapy holds promise for treating a wide range of diseases, such as cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS. Researchers are still studying how and ...

  6. Genetic variations in DNA repair genes, radiosensitivity to cancer and susceptibility to acute tissue reactions in radiotherapy-treated cancer patients

    Energy Technology Data Exchange (ETDEWEB)

    Chistiakov, Dimitry A. (Dept. of Pathology, Univ. of Pittsburgh, Pittsburgh (US)); Voronova, Natalia V. (Dept. of Molecular Diagnostics, National Research Center GosNIIgenetika, Moscow (RU)); Chistiakov, Pavel A. (Dept. of Radiology, Cancer Research Center, Moscow (RU))

    2008-06-15

    Ionizing radiation is a well established carcinogen for human cells. At low doses, radiation exposure mainly results in generation of double strand breaks (DSBs). Radiation-related DSBs could be directly linked to the formation of chromosomal rearrangements as has been proven for radiation-induced thyroid tumors. Repair of DSBs presumably involves two main pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). A number of known inherited syndromes, such as ataxia telangiectasia, ataxia-telangiectasia like-disorder, radiosensitive severe combined immunodeficiency, Nijmegen breakage syndrome, and LIG4 deficiency are associated with increased radiosensitivity and/or cancer risk. Many of them are caused by mutations in DNA repair genes. Recent studies also suggest that variations in the DNA repair capacity in the general population may influence cancer susceptibility. In this paper, we summarize the current status of DNA repair proteins as potential targets for radiation-induced cancer risk. We will focus on genetic alterations in genes involved in HR- and NHEJ-mediated repair of DSBs, which could influence predisposition to radiation-related cancer and thereby explain interindividual differences in radiosensitivity or radioresistance in a general population

  7. Analysis of HIV-1 protease gene reveals frequent multiple infections followed by recombination among drug treated individuals living in Sao Paulo and Santos, Brazil.

    Directory of Open Access Journals (Sweden)

    Edsel Renata De Morais Nunes

    Full Text Available The present study investigated the prevalence of HIV-1 multiple infections in a population composed by 47 patients under HAART failure and enrolled at the National DST/AIDS, Program, Ministry of Health, Brazil.Detection of multiple infections was done using a previously published RFLP assay for the HIV-1 protease gene, which is able of distinguishing between infections caused by a single or multiple HIV-1 subtypes. Samples with multiple infections were cloned, and sequence data submitted to phylogenetic analysis. We were able to identify 17 HIV-1 multiple infections out of 47 samples. Multiple infections were mostly composed by a mixture of recombinant viruses (94%, with only one case in which protease gene pure subtypes B and F were recovered. This is the first study that reports the prevalence of multiple infections and intersubtype recombinants in a population undergoing HAART in Brazil. Based on the data there was a steep increase of multiple infections after the introduction of the combined antiretroviral therapy in Brazil. Cases of multiple infections may be associated with HIV-1 genetic diversity through recombination allowing for the generation of viruses showing a combination of resistance mutations.