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Molecular Characterization (molecular + characterization)
Selected AbstractsIdentification and Molecular Characterization of ,Candidatus Phytoplasma mali' Isolates in North-western ItalyJOURNAL OF PHYTOPATHOLOGY, Issue 2 2010Paola Casati Abstract Apple proliferation (AP) is an important disease and is prevalent in several European countries. The causal agent of AP is ,Candidatus Phytoplasma mali' (,Ca. Phytoplasma mali'). In this work, isolates of ,Ca. Phytoplasma mali' were detected and characterized through polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of 16S rRNA gene and non-ribosomal DNA fragment. The presence of three AP subtypes (AT-1, AT-2 and AP-15) was identified in 31 symptomatic apple trees and two samples each constituted by a pool of five insects, collected in north-western Italy, where AT-1 is a dominant subtype. Subsequent nucleotide sequence analysis of the PCR-amplified 1.8 kb (P1/P7) fragment, containing the 16S rDNA, the 16S,23S intergenic ribosomal region and the 5,-end of the 23S rDNA, revealed the presence of at least two phytoplasmal genetic lineages within the AT-1 subtype, designed AT-1a and AT-1b. Moreover, in silico single nucleotide polymorphism (SNP) analysis based on 16S rDNA sequence can differentiate AT-1 subtype from AT-2 and AP-15 subtypes. Our data showed a high degree of genetic diversity among ,Ca. Phytoplasma mali' population in north-western Italy and underlined the possible use of the 16S rDNA analysis for the identification and the geographical origin assignation of isolates of AP phytoplasma. Molecular markers on 16S rDNA, here identified, could be useful for studying the epidemiology of AP disease. [source] Biological and Molecular Characterization of Taiwanese Isolates of Cucumber mosaic virus Associated with Banana Mosaic DiseaseJOURNAL OF PHYTOPATHOLOGY, Issue 2 2009Chun-Nan Chou Abstract Banana mosaic disease (BMD) caused by Cucumber mosaic virus (CMV) has become an important threat to the banana industry. We collected and characterized 10 CMV isolates associated with BMD in Taiwan and compared their biological characteristics and coat protein sequences. The isolates fell into four pathotypes on the basis of the symptoms they induce on banana, Nicotiana glutinosa and Vigna unguiculata (cowpea). Double-stranded RNA analysis revealed that the different pathotypes are not related to the presence of CMV satellite RNA. Phylogenetic analysis of worldwide CMV coat protein sequences revealed that among the currently known CMV subgroups IA, IB and II, subgroup IB is phylogenetically unresolved. Our CMV isolates form a new subgroup, IT, within subgroup I. In addition, we resolved another new CMV subgroup, IS, within subgroup I. The analysis also revealed that isolates within different subgroups can infect the banana. [source] Molecular Characterization of the 3,-Terminal Region of Turnip mosaic virus Isolates from Eastern ChinaJOURNAL OF PHYTOPATHOLOGY, Issue 6 2007Y.-P. Tian Abstract Turnip mosaic virus (TuMV; genus Potyvirus, family Potyviridae) causes great losses to cruciferous crop production worldwide. The 3,-terminal genomic sequences of eight TuMV isolates from eastern China were compared with those of 74 other Chinese TuMV isolates of known host origin in the GenBank and isolated during the past 25 years. The reported sequences of the eight TuMV isolates are 1125 or 1126-nucleotides (nt) long excluding the poly(A) tail. They all contain one partial open reading frame of 912 nt, encoding 304 amino acids, followed by a stop codon and a non-translated region of 209,210 nt. Results of phylogenetic analyses showed that Chinese TuMV isolates clustered into three groups: basal-BR, Asian-BR and world-B. The ratios of non-synonymous and synonymous substitutions and results of amino acid alignment provided evidence for purifying or negative selection in TuMV populations of China. [source] Molecular Characterization and Potential Insect Vector of a Phytoplasma Associated with Garden Beet Witches' Broom in Yazd, IranJOURNAL OF PHYTOPATHOLOGY, Issue 4 2007A. Mirzaie Abstract In 2002, garden beet witches' broom (GBWB) phytoplasma was detected for the first time in garden beet plants (Beta vulgaris L. ssp. esculenta) in Yazd, Iran. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphic (RFLP) analysis of PCR-amplified phytoplasma 16S rDNA were employed for the detection and identification of the phytoplasma associated with garden beet. A phytoplasma belonging to subgroup 16SrII-E, in the peanut witches' broom group (16SrII), was detected in infected plants. Asymptomatic plant samples and the negative control yielded no amplification. The result of analysis of the nucleotide sequence of a 1428 bp fragment of 16S rDNA gene from GBWB phytoplasma (GenBank accession number DQ302722) was basically consistent with the classification based on RFLP analysis, in which GBWB phytoplasma clustered with phytoplasmas of the 16SrII-E subgroup. A search for a natural phytoplasma vector was conducted in Yazd in 2004, in an area where garden beet crops had been affected since 2002. The associated phytoplasma was detected in one leafhopper species, Orosius albicinctus, commonly present in this region. The leafhopper O. albicinctus was used in transmission tests to determine its vector status for the phytoplasma associated with GBWB. Two of eight plants that had been fed on by O. albicinctus, showed mild symptoms of GBWB including stunting and reddening of midveins. A phytoplasma was detected in the two symptomatic test plants by PCR using universal primers and it was identified by RFLP as the GBWB phytoplasma. This finding suggests O. albicinctus is a vector of the GBWB phytoplasma. [source] Molecular Characterization of two Distinct Begomoviruses from Ageratum conyzoides and Malvastrum coromandelianum in ChinaJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2006J. F. Huang Abstract Two weed samples, G52 from Ageratum conyzoides and G87 from Malvastrum coromandelianum, showing leaf curling and vein thickening symptoms were collected in Nanning, Guangxi Province, China. The complete nucleotide sequences of DNA-A-like molecules of G52 and G87 were determined to be 2735 and 2745 nucleotides respectively. Both DNA-A molecules have a genomic organization typical of begomoviruses and share 73.4% sequence identity with each other. Sequence comparisons showed that the DNA-A of G52 and G87 were most closely related to those of Ageratum yellow vein virus (AYVV; 85% sequence identity) and Tobacco leaf curl Yunnanvirus (75.7% sequence identity) respectively. Further sequence comparisons showed that G52 has arisen by recombination among viruses related to AYVV, Papaya leaf curl China virus and an unidentified Begomovirus species. The molecular data suggest that G52 and G87 are two distinct begomoviruses, for which the names Ageratum leaf curl virus for G52 and Malvastrum leaf curl virus for G87 are proposed. The satellite DNA, molecule was only found to be associated with G87. G87 DNA, consists of 1354 nucleotides, and shares the highest nucleotide sequence identity (68.9%) with that associated with Sida yellow vein China virus. A defective DNA, molecule was also found to be associated with G87. [source] Biological and Molecular Characterization of Melon-Infecting Kyuri Green Mottle Mosaic Virus in IndonesiaJOURNAL OF PHYTOPATHOLOGY, Issue 10 2005B. S. Daryono Abstract Melon (Cucumis melo L.) plants showing fruit deformation and mosaic symptoms were found in Java, Indonesia, in 2001. Leaf dips of the symptomatic melon tissue revealed rod-shaped viral particles 300 × 18 nm in size. Biological and serological data described in this study indicate that the virus belonged to the genus tobamovirus and was related to the kyuri green mottle mosaic virus (KGMMV). The genome of the virus has been completely sequenced, consisting of 6512 nucleotides and was compared in detail with KGMMV-C1 and KGMMV-Y. The sequence of their 5,- and 3,- non-coding regions (NCRs) were 91% and 94% identical to KGMMV-C1, and only 82% and 95% identical to KGMMV-Y respectively. The amino acid sequence of the shorter and longer RNA replicase components, movement protein and coat protein were 94%, 91%, 95% and 94% identical to KGMMV-C1 and 93%, 89%, 91% and 85% identical of KGMMV-Y respectively. The results from phylogenetic analysis of the coding regions revealed that KGMMV-YM is a new strain of KGMMV. This is the first report of the complete nucleotide sequence and analysis of genome organization for KGMMV isolated in anywhere in South-East Asia. [source] Molecular Characterization of a Distinct Begomovirus and its Associated Satellite DNA Molecule Infecting Sida acuta in China,JOURNAL OF PHYTOPATHOLOGY, Issue 5 2005Q. Xiong Abstract Three viral isolates Hn8, Hn40 and Hn41 were obtained from Sida acuta showing yellow mosaic symptom in the Hainan province, China. Comparison of partial DNA-A sequences amplified with degenerate primers confirmed the existence of single type of Begomovirus. The complete nucleotide sequence of the DNA-A-like molecule of Hn8 was determined to be 2749 nucleotides, having a typical genetic organization of a Begomovirus. Hn8 DNA-A had the highest sequence identity (78%) with that of Ageratum yellow vein China virus-[G13] (AJ558120), and had less sequence identity with other begomoviruses. Based on the above molecular data, Hn8 was thus considered as a new Begomovirus species, for which the name Sida yellow mosaic China virus (SiYMCNV) is proposed. Satellite DNA- , molecules (Hn8- ,, Hn40- , and Hn41- ,) were found to be associated with Hn8, Hn40 and Hn41 and their complete nucleotide sequences were determined. Sequence analysis showed that Hn8- ,, Hn40- , and Hn41- , shared more than 84% nucleotide sequence identity, and they were different from other characterized DNA- ,, sharing the highest nucleotide sequence identity (47.8%) with DNA- , of Ageratum yellow vein virus. [source] Identification and Molecular Characterization of Viruses Infecting Cucurbits in PakistanJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2004A. Ali Abstract Cucurbits are grown throughout the North-West Frontier Province of Pakistan as summer and winter crops. Plants having mosaic, mottling, chlorosis and leaf distortion symptoms were frequently found in most of the cucurbit fields during the survey. Using dot immunobinding assay, Cucumber green mottle mosaic virus (CGMMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV) and Papaya ringspot virus (PRSV) were found infecting cucurbits. CGMMV was widespread, infecting 46.9% of the samples tested followed by ZYMV (14.8%), WMV (12.5%) and PRSV (7.8%). Multiple infections were common with 42% of the samples being infected with two viruses and 8% with three viruses. The nucleotide sequences of the coat protein (CP) genes of these four viruses were determined and deduced amino acid sequence comparisons revealed 88.3,99% similarity of the ZYMV-Pak isolate with other isolates of ZYMV reported worldwide. The amino acid sequence identity of Pakistani isolates of WMV, CGMMV and PRSV ranged from 96.8 to 98.4%, 98.1 to 99.4% and 79.3 to 84.2%, respectively, with other isolates reported elsewhere. Little variability was observed in the sequences of WMV and CGMMV. ZYMV-Pak was very close to the USA isolate, and the PRSV-Pak isolate was close to Indian isolates of PRSV possibly reflecting the geographical relationship between these isolates. [source] Molecular Characterization of a Strain of Squash Leaf Curl China Virus from the PhilippinesJOURNAL OF PHYTOPATHOLOGY, Issue 10 2003T. Kon Abstract The complete nucleotide sequence of infectious cloned DNA components (A and B) of the causal agent of squash leaf curl disease in the Philippines was determined. DNA-A and DNA-B comprise 2739 and 2705 nucleotides, respectively; the common region is 174 bases in length. Five ORFs were found in DNA-A and two in DNA-B. Partial dimeric clones containing DNA-A and DNA-B, constructed in a binary vector and transformed into Agrobacterium tumefaciens, induced systemic infection in agro-inoculated pumpkin plants (Cucurbita moschata). The total DNA-A sequence was most closely related to that of Squash leaf curl China virus (SLCCNV) (88% identity), although the existence of B component of SLCCNV has not been reported. The deduced coat protein was like that of SLCCNV (98% amino acid sequence identity) and the Philippines virus has low sequence identity to Squash leaf curl virus (SLCV) and Squash mild leaf curl virus (SMLCV) (63 and 64% total nucleotide sequence identities, respectively). From these results, we propose that the Philippines virus be designated Squash leaf curl China virus -[Philippines] (SLCCNV-[PH]). [source] Molecular Characterization of Gregarines from Sand Flies (Diptera: Psychodidae) and Description of Psychodiella n. g. (Apicomplexa: Gregarinida)THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2009JAN VOTİPKA ABSTRACT. Sand fly and mosquito gregarines have been lumped for a long time in the single genus Ascogregarina and on the basis of their morphological characters and the lack of merogony been placed into the eugregarine family Lecudinidae. Phylogenetic analyses performed in this study clearly demonstrated paraphyly of the current genus Ascogregarina and revealed disparate phylogenetic positions of gregarines parasitizing mosquitoes and gregarines retrieved from sand flies. Therefore, we reclassified the genus Ascogregarina and created a new genus Psychodiella to accommodate gregarines from sand flies. The genus Psychodiella is distinguished from all other related gregarine genera by the characteristic localization of oocysts in accessory glands of female hosts, distinctive nucleotide sequences of the small subunit rDNA, and host specificity to flies belonging to the subfamily Phlebotominae. The genus comprises three described species: the type species for the new genus,Psychodiella chagasi (Adler and Mayrink 1961) n. comb., Psychodiella mackiei (Shortt and Swaminath 1927) n. comb., and Psychodiella saraviae (Ostrovska, Warburg, and Montoya-Lerma 1990) n. comb. Its creation is additionally supported by sequencing data from other gregarine species originating from the sand fly Phlebotomus sergenti. In the evolutionary context, both genera of gregarines from mosquitoes (Ascogregarina) and sand flies (Psychodiella) have a close relationship to neogregarines; the genera represent clades distinct from the other previously sequenced gregarines. [source] Morphological and Molecular Characterization of a New Protist Family, Sandmanniellidae n. fam. (Ciliophora, Colpodea), with Description of Sandmanniella terricola n. g., n. sp. from the Chobe Floodplain in BotswanaTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 5 2009WILHELM FOISSNER ABSTRACT. Sandmanniella terricola n. g., n. sp. was discovered in soil from the Chobe floodplain, Botswana, southern Africa. Its morphology and 18S rDNA gene sequence were studied with standard methods. Sandmanniella terricola is very likely an adversity strategist because it reaches peak abundances 6,12 h after rewetting the soil and maintains trophic food vacuoles with undigested bacteria in the resting cyst, a highly specific feature suggested as an indicator for an adversity life strategy. Possibly, the energy of the stored food vacuoles is used for reproduction and support of the cyst wall. Morphologically, Sandmanniella terricola is inconspicuous, having a size of only 50 × 40 ,m and a simple, ellipsoidal shape. The main characteristics of the genus are a colpodid silverline pattern; a perioral cilia condensation; a flat, dish-shaped oral cavity, in the centre of which originates a long, conical oral basket resembling that of certain nassulid ciliates; and a vertically oriented left oral polykinetid composed of brick-shaped adoral organelles. This unique mixture of features and the gene sequence trees, where Sandmanniella shows an isolated position, suggest establishing a new family, the Sandmanniellidae n. fam., possibly related to the families Colpodidae or Bryophryidae. The curious oral basket provides some support for the hypothesis of a common ancestor of colpodid and nassulid ciliates. [source] Establishment of Liebermannia dichroplusae n. comb. on the Basis of Molecular Characterization of Perezia dichroplusae Lange, 1987 (Microsporidia)THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2007YULIYA Y. SOKOLOVA ABSTRACT. Perezia dichroplusae Lange, 1987 is a parasite of the Malpighian tubules of an Argentine grasshopper, Dichroplus elongatus (Orthoptera, Acrididae, Melanoplinae). In order to determine relationships of this microsporidium with Perezia nelsoni and with other microsporidia, we sequenced its small subunit ribosomal RNA gene (SSU rDNA) (GenBank Accession No. EF016249) and performed phylogenetic analysis of the novel sequence against 17 microsporidian SSU rDNA sequences from GenBank, using neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) methods. This analysis revealed the highest similarity (96%) of the new sequence to Liebermannia patagonica, a parasite of gut epithelium cells of another grasshopper from Argentina, versus only 65% similarity to P. nelsoni, a parasite of muscles of paenaeid shrimps. In phylogenetic trees inferred from SSU rDNA sequences, the microsporidium from D. elongatus is sister taxon to L. patagonica and both cluster with Orthosomella operophterae. At the higher hierarchical level, the Liebermania,Orthosomella branch forms a clade with the Endoreticulatus,Cystosporogenus,Vittaforma group and with Enterocytozoon bieneusi. Perezia nelsoni falls into another large clade together with Nosema and Ameson species. We propose transferring P. dichroplusae to the genus Liebermannia and creating a new combination Liebermannia dichroplusae n. comb., based both on SSU rDNA sequence analysis and on common characters between P. dichroplusae and L. patagonica, which include the presence of elongated multinuclear sporonts, sporoblastogenesis by a similar process of sequentially splitting off sporoblasts, ovocylindrical spores of variable size, tissue tropism limited to epithelial cells, Orthoptera as hosts, and geographical distribution of hosts in the southern temperate region of Argentina. We argue that the condition of the nuclei in spores (i.e. diplokaryotic in L. patagonica or monokaryotic in L. dichroplusae) cannot be used to distinguish genera. Therefore, we remove the statement about the presence of diplokaryotic spores from the revised diagnosis of the genus Liebermannia. [source] Molecular Characterization of the Obligate Endosymbiont "Caedibacter macronucleorum"Fokin and Görtz, 1993 and of its Host Paramecium duboscqui Strain Ku4-8THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2006MARTINA SCHRALLHAMMER ABSTRACT. Bacterial endosymbionts of protozoa were often described as new species by protozoologists mainly on the basis of few morphological characters and partly by host specificity. Many of these species have never been validated by prokaryotic microbiologists whose taxonomic rules are quite different from those of protozoologists, who use the Zoological Code of Nomenclature. "Caedibacter macronucleorum"Fokin and Görtz 1993, an endosymbiont of Paramecium duboscqui, belongs to this category. Here we provide the molecular characterization of this organism and of its host P. duboscqui strain Ku4-8. Bacterial 16S rRNA gene sequence analysis proved that "C. macronucleorum" belongs to the Alphaproteobacteria. It is closely related to Caedibacter caryophilus but not to Caedibacter taeniospiralis, which belongs to the Gammaproteobacteria. "Caedibacter macronucleorum" and C. caryophilus 16S rRNA genes show a similarity value of 99%. This high 16S rRNA sequence similarity and the lack of a specific oligonucleotide probe for distinguishing the two endosymbionts do not allow validating "C. macronucleorum" as a provisional taxon (Candidatus). Nevertheless, "C. macronucleorum" and C. caryophilus can be easily discriminated on the basis of a highly variable stretch of nucleotides that interrupts the 16S rRNA genes of both organisms. [source] Morphological, Biochemical and Molecular Characterization of Herpetomonas samuelpessoai camargoi n. subsp., a Trypanosomatid Isolated from the Flower of the Squash Cucurbita moschataTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 1 2001JOAO E. FIORINI ABSTRACT. We report the morphological, biochemical and molecular characteristics of a trypanosomatid isolated from the flower of Cucurbita moschata. Although the trypanosomatid was isolated from a plant, the lack of recognition of Phytomonas-spccific molecular markers based on spliced-leader and ribosomal genes as well as by monoclonal antibodies specific for Phytomonas argues against assigning it to this genus. Because the isolate displayed typical opisthomastigote forms in culture, it is assigned to the genus Herpetomonas. Analysis of randomly amplified polymorphic DNA (RAPD) patterns and characterization of ribosomal SSU and ITS markers suggest that it is more closely related to H. samuelpessoai than to any other species. However, the presence of spined flagellates in culture (displaying lateral expansions of the plasma membrane originating near the flagellar pocket) and isolate-specific RAPD fingerprints argue strongly that the trypanosomatid belongs to a new subspecies, for which the name Herpetomonas samuelpessoai camargoi n. subsp. is proposed. [source] Drug Transporters: Molecular Characterization and Role in Drug Disposition.BIOTECHNOLOGY JOURNAL, Issue 8 2008By Guofeng You, Marilyn E. Morris (Eds.) No abstract is available for this article. [source] Molecular Characterization of Anaerobic Microbial Communities from Benzene-Degrading Sediments under Methanogenic ConditionsBIOTECHNOLOGY PROGRESS, Issue 6 2005Wook Chang Anaerobic benzene degradation was confirmed in microbial communities enriched from Baltimore Harbor (Baltimore, MD) sediments under methanogenic conditions. Molecular characterization based on 16S rDNA gene sequences revealed that the strains in the communities were diversely affiliated with such phylogenetic branches as the Bacteroidetes, Euryarchaeota, Firmicutes, and Thermotogaephyla. Of interest was that the majority of the microbial populations detected in these cultures were closely related to the members of dechlorinating microbial communities. Further, some of those species were previously found in naphthalene- or phenanthrene-degrading methanogenic communities. Finally, this result could be used to design targeted isolation strategies for anaerobic benzene-degrading strains under methanogenic conditions. [source] Production and Molecular Characterization of Clinical Phase I Anti-Melanoma Mouse IgG3 Monoclonal Antibody R24BIOTECHNOLOGY PROGRESS, Issue 5 2001Sven E. Kemminer R24 is a mouse IgG3 monoclonal antibody (mab) that reacts with the ganglioside GD3 expressed by cells of neuroectodermal origin. The anti-tumor activity of R24 has been demonstrated in initial phase I and pilot trials in patients suffering from metastatic melanoma. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important antibody. Growth, metabolism, and IgG production of R24 secreting hybridoma cells were analyzed on 1 L bioreactor bench scale using repeated-batch mode. The amount of 57 mg of pure mab was obtained from 1.6 L crude supernatant by protein A chromatography. Western blot binding assays with sugar-specific lectins revealed glycosylation of the heavy chains, whereas no carbohydrates were detectable on the light chains. Because glycosylation is essential for antibody effector functions in vivo (such as complement fixation or binding to macrophage Fc receptors), mab R24 was subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Released glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Six major biantennary chains of the complex glycosylation phenotype were found with variations in galactosylation and core fucosylation. The predominant N-linked structure, indicating the high degree of agalactosyl glycoforms, was the agalacto biantennary chain with a relative percentage of 57% (51% core-fucosylated, 6% nonfucosylated). The second most abundant oligosaccharide was the monogalacto biantennary chain amounting to 30% (26% core- and 4% nonfucosylated). The antibody contained 0.46 ,g sialic acid per mg protein, which splits into 0.243 ,g Neu5Gc and 0.217 ,g Neu5Ac, corresponding to a Neu5Ac:Neu5Gc ratio of 1:1.06. Furthermore, the antigen specificity of R24 was determined by immunodetection of GD3 on thin-layer chromatograms, and real time GD3-antibody binding interactions were measured with an optical biosensor (BIAcore). From the structural data obtained in this study it is concluded that glycosylation of the antibody may be important in the clinical outcome of targeted anti-cancer immunotherapy. [source] Chordoid Glioma: A Case Report and Molecular Characterization of Five CasesBRAIN PATHOLOGY, Issue 3 2009Craig Horbinski MD Abstract Chordoid gliomas are rare, slow-growing neoplasms of the anterior third ventricle. We reported a case of chordoid glioma in a 41-year-old man with obstructive hydrocephalus. Histologically, the tumor consisted of polygonal epithelioid cells admixed with elongated cells in a myxoid stroma. A prominent lymphoplasmacytic infiltrate was present. The tumor cells expressed glial fibrillary acidic protein (GFAP), epithelial membrane antigen (EMA), vimentin, CD31, CD34, epidermal growth factor receptor (EGFR) and S100 but were negative for pankeratin and E-cadherin. The percentage of Ki67 positive cells was approximately 3%. Weak p53 immunoreactivity was seen in less than 10% of the cells. Array comparative genomic hybridization performed on this case, as well as on four other archived cases, showed losses at several loci. Fluorescence in situ hybridization (FISH) confirmed consistent genetic alterations at 9p21 and 11q13. These are the fifth through ninth reported cases of chordoid gliomas with molecular characterization suggesting a distinct genetic origin from other gliomas. [source] Molecular Characterization of the NCoA-1,STAT,6 InteractionCHEMBIOCHEM, Issue 8 2008Markus Seitz Abstract Many protein,protein interactions involved in cell signalling, cell adhesion and regulation of transcription are mediated by short ,-helical recognition motifs with the sequence Leu-Xaa-Xaa-Leu-Leu (LXXLL, where Xaa is any amino acid). Originally observed in cofactors that interact with hormone-activated nuclear receptors, LXXLL motifs are now known to occur in many transcription factors, including the STAT family, which transmit signals from activated cytokine receptors at the cell surface to target genes in the nucleus. STAT,6 becomes activated in response to IL-4 and IL-13, which regulate immune and anti-inflammatory responses. Structural studies have revealed how an LXXLL motif located in 2.5 turns of an ,-helical peptide derived from STAT,6 provide contacts through the leucine side chains to the coactivator of transcription, NCoA-1. However, since many protein,protein interactions are mediated by LXXLL motifs, it is important to understand how specificity is achieved in this and other signalling pathways. Here, we show that energetically important contacts between STAT,6 and NCoA-1 are made in residues that flank the LXXLL motif, including the underlined residues in the sequence LLPPTEQDLTKLL. We also demonstrate how the affinity for NCoA-1 of peptides derived from this region of STAT,6 can be significantly improved by optimising knobs-into-holes contacts on the surface of the protein. The results provide important new insights into the origins of binding specificity, and might be of practical value in the design of novel small-molecule inhibitors of this important protein,protein interaction. [source] Molecular characterization of regenerated cardiomyocytes derived from adult mesenchymal stem cellsCONGENITAL ANOMALIES, Issue 1 2002Keiichi Fukuda ABSTRACT, We recently isolated a cardiomyogenic (CMG) cell line from murine bone marrow stroma, and in this paper characterize regenerated cardiomyocytes derived from adult mesenchymal stem cells at the molecular level. Stromal cells were immortalized, exposed to 5-azacytidine, and repeatedly screened for spontaneously beating cells. CMG cells began to beat spontaneously after 2 weeks, and beat synchronously after 3 weeks. They exhibited sinus-node-like or ventricular-cell-like action potentials. Analysis of the isoforms of contractile protein genes, such as of myosin and ,-actin, indicated that their phenotype was similar to that of fetal ventricular cardiomyocytes. The cells expressed Nkx2.5, GATA4, TEF-1, and MEF2-C mRNA before 5-azacytidine exposure, and MEF2-A and MEF2-D after exposure. CMG cells expressed ,1A, ,1B, and ,1D -adrenergic receptor mRNA prior to differentiation, and ,1, ,2 -adrenergic and M1, M2 -muscarinic receptors after acquiring the cardiomyocyte phenotype. Phenylephrine induced phosphorylation of ERK1/ 2, and the phosphorylation was inhibited by prazosin. Isoproterenol increased the cAMP level 38-fold and beating rate, cell motion, % shortening, and contractile velocity by 48%, 38%, 27%, and 51%, respectively, and the increases were blocked by CGP20712A (,1 -selective blocker). Car-bachol increased IP3 32-fold, and the increase was inhibited by AFDX116 (M2 -selective blocker). These findings demonstrated that the regenerated cardiomyocytes were capable of responding to adrenergic and muscarinic stimulation. This new cell line provides a model for the study of cardiomyocyte transplantation. [source] Molecular characterization of the effects of Y-27632CYTOSKELETON, Issue 2 2007Hassina Darenfed Abstract Many key cellular functions, such as cell motility and cellular differentiation are mediated by Rho-associated protein kinases (ROCKs). Numerous studies have been conducted to examine the ROCK signal transduction pathways involved in these motile and contractile events with the aid of pharmacological inhibitors such as Y-27632. However the molecular mechanism of action of Y-27632 has not been fully defined. To assess the relative contribution of these Rho effectors to the effects of Y-27632, we compared the cytoskeletal phenotype, wound healing and neurite outgrowth in cells treated with Y-27632 or subjected to knockdown with ROCK-I, ROCK-II or PRK-2- specific siRNAs. Reduction of ROCK-I enhances the formation of thin actin-rich membrane extensions, a phenotype that closely resembles the effect of Y-27632. Knockdown of ROCK II or PRK-2, leads to the formation of disc-like extenstions and thick actin bundles, respectively. The effect of ROCK-I knockdown also mimicked the effect of Y-27632 on wound closer rates. ROCK-I knockdown and Y-27632 enhanced wound closure rates, while ROCK-II and PRK-2 were not appreciably different from control cells. In neurite outgrowth assays, knockdown of ROCK-I, ROCK-II or PRK-2 enhances neurite lengths, however no individual knockdown stimulated neurite outgrowth as robustly as Y-27632. We conclude that several kinases contribute to the global effect of Y-27632 on cellular responses. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Molecular characterization of Ciona sperm outer arm dynein reveals multiple components related to outer arm docking complex protein 2CYTOSKELETON, Issue 10 2006Akiko Hozumi Abstract Using proteomic and immunochemical techniques, we have identified the light and intermediate chains (IC) of outer arm dynein from sperm axonemes of the ascidian Cionaintestinalis. Ciona outer arm dynein contains six light chains (LC) including a leucine-rich repeat protein, Tctex1- and Tctex2-related proteins, a protein similar to Drosophila roadblock and two components related to Chlamydomonas LC8. No LC with thioredoxin domains is included in Ciona outer arm dynein. Among the five ICs in Ciona, three are orthologs of those in sea urchin dynein: two are WD-repeat proteins and the third one, unique to metazoan sperm flagella, contains both thioredoxin and nucleoside diphosphate kinase modules. The remaining two Ciona ICs have extensive coiled coil structure and show sequence similarity to outer arm dynein docking complex protein 2 (DC2) that was first identified in Chlamydomonas flagella. We recently identified a third DC2-like protein with coiled coil structure, Ci-Axp66.0 that is also associated in substoichiometric amounts with Ciona outer arm dynein. In addition, Oda5p, a component of an additional complex required for assembly of outer arm dynein in Chlamydomonas flagella, also groups with this family of DC2-like proteins. Thus, the assembly of outer arm dynein onto doublet microtubules involves multiple coiled-coil proteins related to DC2. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Molecular characterization of conditionally immortalized cell lines derived from mouse early embryonic inner earDEVELOPMENTAL DYNAMICS, Issue 4 2004John A. Germiller Abstract Inner ear sensory hair cells (HCs), supporting cells (SCs), and sensory neurons (SNs) are hypothesized to develop from common progenitors in the early embryonic otocyst. Because little is known about the molecular signals that control this lineage specification, we derived a model system of early otic development: conditionally immortalized otocyst (IMO) cell lines from the embryonic day 9.5 Immortomouse. This age is the earliest stage at which the otocyst can easily be separated from surrounding mesenchymal, nervous system, and epithelial cells. At 9.5 days post coitum, there are still pluripotent cells in the otocyst, allowing for the eventual identification of both SN and HC precursors,and possibly an elusive inner ear stem cell. Cell lines derived from primitive precursor cells can also be used as blank canvases for transfections of genes that can affect lineage decisions as the cells differentiate. It is important, therefore, to characterize the "baseline state" of these cell lines in as much detail as possible. We characterized seven representative "precursor-like" IMO cell populations and the uncloned IMO cells, before cell sorting, at the molecular level by polymerase chain reaction (PCR) and immunocytochemistry (IHC), and one line (IMO-2B1) in detail by real-time quantitative PCR and IHC. Many of the phenotypic markers characteristic of differentiated HCs or SCs were detected in IMO-2B1 proliferating cells, as well as during differentiation for up to 30 days in culture. These IMO cell lines represent a unique model system for studying early stages of inner ear development and determining the consequences of affecting key molecular events in their differentiation. Developmental Dynamics 231:815,827, 2004. © 2004 Wiley-Liss, Inc. [source] Genetic dissection reveals two separate pathways for rod and cone regeneration in the teleost retinaDEVELOPMENTAL NEUROBIOLOGY, Issue 5 2008Ann C. Morris Abstract Development of therapies to treat visual system dystrophies resulting from the degeneration of rod and cone photoreceptors may directly benefit from studies of animal models, such as the zebrafish, that display continuous retinal neurogenesis and the capacity for injury-induced regeneration. Previous studies of retinal regeneration in fish have been conducted on adult animals and have relied on methods that cause acute damage to both rods and cones, as well as other retinal cell types. We report here the use of a genetic approach to study progenitor cell responses to photoreceptor degeneration in the larval and adult zebrafish retina. We have compared the responses to selective rod or cone degeneration using, respectively, the XOPS-mCFP transgenic line and zebrafish with a null mutation in the pde6c gene. Notably, rod degeneration induces increased proliferation of progenitors in the outer nuclear layer (ONL) and is not associated with proliferation or reactive gliosis in the inner nuclear layer (INL). Molecular characterization of the rod progenitor cells demonstrated that they are committed to the rod photoreceptor fate while they are still mitotic. In contrast, cone degeneration induces both Müller cell proliferation and reactive gliosis, with little change in proliferation in the ONL. We found that in both lines, proliferative responses to photoreceptor degeneration can be observed as 7 days post fertilization (dpf). These two genetic models therefore offer new opportunities for investigating the molecular mechanisms of selective degeneration and regeneration of rods and cones. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008. [source] Molecular characterization of sulfate-reducing bacteria in a New England salt marshENVIRONMENTAL MICROBIOLOGY, Issue 8 2005Michele Bahr Summary Sulfate reduction, mediated by sulfate-reducing bacteria (SRB), is the dominant remineralization pathway in sediments of New England salt marshes. High sulfate reduction rates are associated with the rhizosphere of Spartina alterniflora when plants elongate aboveground. The growth process concurrently produces significant amounts of new rhizome material belowground and the plants leak dissolved organic compounds. This study investigated the diversity of SRB in a salt marsh over an annual growth cycle of S. alterniflora by exploring the diversity of a functional gene, dissimilatory sulfite reductase (dsrAB). Because the dsrAB gene is a key gene in the anaerobic sulfate-respiration pathway, it allows the identification of microorganisms responsible for sulfate reduction. Conserved dsrAB primers in polymerase chain reaction (PCR) generated full-length dsrAB amplicons for cloning and DNA sequence analysis. Nearly 80% of 380 clone sequences were similar to genes from Desulfosarcina and Desulfobacterium species within Desulfobacteraceae. This reinforces the hypothesis that complete oxidizers with high substrate versatility dominate the marsh. However, the phylotypes formed several clades that were distinct from cultured representatives, indicating a greater diversity of SRB than previously appreciated. Several dsrAB sequences were related to homologues from Gram-positive, thermophilic and non-thermophilic Desulfotomaculum species. One dsrAB lineage formed a sister group to cultured members of the delta-proteobacterial group Syntrophobacteraceae. A deeply branching dsrAB lineage was not affiliated with genes from any cultured SRB. The sequence data from this study will allow for the design of probes or primers that can quantitatively assess the diverse range of sulfate reducers present in the environment. [source] Molecular characterization of artemin and ferritin from Artemia franciscanaFEBS JOURNAL, Issue 1 2003Tao Chen Embryos of the brine shrimp, Artemia franciscana, exhibit remarkable resistance to physiological stress, which is temporally correlated with the presence of two proteins, one a small heat shock/,-crystallin protein termed p26 and the other called artemin, of unknown function. Artemin was sequenced previously by Edman degradation, and its relationship to ferritin, an iron storage protein, established. The isolation from an Artemia expressed sequence tag library of artemin and ferritin cDNAs extends this work. Artemin cDNA was found to contain an ORF of 693 nucleotides, and its deduced amino-acid sequence, except for the initiator methionine, was identical with that determined previously. Ferritin cDNA is 725 bp in length with an ORF of 516 nucleotides. Artemin amino-acid residues 32,185 are most similar to ferritin, but artemin is enriched in cysteines. The abundance of cysteines and their intramolecular spatial distribution suggest that artemin protects embryos against oxidative damage and/or that its function is redox regulated. The conserved regions in artemin and ferritin monomers are structurally similar to one another and both proteins assemble into oligomers. However, modeling of the quaternary structure indicated that artemin multimers lack the central space used for metal storage that characterizes ferritin oligomers, implying different roles for this protein. Probing of Northern blots revealed two artemin transcripts, one of 3.5 kb and another of 2.2 kb. These transcripts decreased in parallel and had almost disappeared by 16 h of development. The ferritin transcript of 0.8 kb increased slightly during reinitiation of development, then declined, and was almost completely gone by 16 h. Clearly, the loss of artemin and ferritin during embryo development is due to transcriptional regulation and proteolytic degradation of the proteins. [source] Molecular characterization of a human scavenger receptor, human MARCOFEBS JOURNAL, Issue 3 2000Nabil A. Elshourbagy Murine MARCO has been identified recently in subsets of macrophages located in the peritoneum, marginal zone of the spleen, and the medullary cord of lymph nodes, where it has been proposed that it serves as a bacteria-binding receptor. A scavenger receptor family member with an extended collagenous domain, murine MARCO has also been demonstrated in atherosclerotic lesions of susceptible mice. We report here the identification, tissue and chromosomal localization, and pharmacological characterization of human (h)MARCO. hMARCO was identified from a macrophage cDNA library by electronic screening with the murine MARCO sequence. Nucleotide sequence analysis confirmed that the full-length hMARCO clone encoded a 519-amino acid protein sharing 68.5% identity with murine MARCO. RNA blot analysis indicated that the hMARCO transcript is 2.0 kb in length and is predominantly expressed in human lung, liver, and lymph nodes. Radiation hybrid mapping localized hMARCO to chromosome 2q14. Ligand-binding studies of COS cells expressing hMARCO demonstrated significant specific binding of both Escherichia coli and Staphylococcus aureus. In contrast, the hMARCO receptor expressed in COS cells did not specifically bind the scavenger receptor ligand acetylated low-density lipoprotein (LDL), despite its similarity to the elongated collagen-like binding domain of the macrophage scavenger receptor. In addition, acetylated (Ac)LDL and oxidized (Ox)LDL did not inhibit E. coli binding to hMARCO. These data suggest that hMARCO may play an important role in host defense, but it has no obvious role in the accumulation of modified lipoproteins during atherogenesis. [source] Molecular characterization of microbial community in nitrate-removing activated sludgeFEMS MICROBIOLOGY ECOLOGY, Issue 2 2002Han-Woong Lee Abstract The microbial community composition and dominant denitrifying populations in high-nitrate-removing (CR-I) and low-nitrate-removing (CR-II) activated sludge from continuous bioreactors were investigated with most probable number (MPN) enumeration, fluorescence in situ hybridization (FISH) and 16S rDNA characterization. MPNs of nitrate-reducing bacteria of sludge CR-I and sludge CR-II were 2.82×107 and 2.69×104 colony-forming units ml,1, respectively. Eight denitrifying bacteria and two nitrate-reducing bacteria were isolated from sludge CR-I, and four denitrifying bacteria and three nitrate-reducing bacteria from sludge CR-II. Small subunit rDNA characterization of the isolates showed that the majority belonged to the genus Pseudomonas. By using FISH up to 76% (CR-I) and 52% (CR-II) of total 4,6-diamidino-2-phenylindole cell counts hybridized to the bacterial probe EUB338. Members of ,-Proteobacteria were the most abundant proteobacterial group in both sludges, accounting for up to 41.6% and 37.1% of those detected by EUB338, respectively, whereas a higher number of Cytophaga,Flexibacter cluster members were observed in CR-I sludge compared to CR-II sludge. In contrast with culture-based results, the numbers of rRNA group I Pseudomonads accounted for less than 0.01% of those detected by EUB338 in both sludges. Ribosomal DNA clone library analysis showed that the ,-Proteobacteria were also dominant in both sludges. In CR-I sludge, they were related to Zooglorea ramigera, Alcaligenes defragrans, denitrifying Fe-oxidizing bacteria and Dechlorimonas sp., whereas in CR-II sludge, they were related to Nitrosomonas sp. and Dechlorimonas agitatus. When this reactor was operated under anaerobic and anoxic conditions, nitrifying bacteria could adapt to the anoxic environment. We inferred that anaerobic ammonium oxidation and nitrite oxidation may occur in low-nitrate-removing sludge CR-II and inhibit denitrification. [source] Molecular characterization and antifungal activity of a family 46 chitosanase from Amycolatopsis sp.FEMS MICROBIOLOGY LETTERS, Issue 1 2009CsO- Abstract An actinomycete strain, Amycolatopsis sp. CsO-2, produces a 27-kDa chitosanase. To reveal the molecular characteristics of the enzyme, its corresponding gene ctoA was cloned by a reverse genetic technique, based on the N-terminal amino acid sequence of the protein. The encoded CtoA protein was deduced to be composed of 286 amino acids, including a putative signal peptide (1,48), and exhibited 83% identity in the amino acid sequence with the family 46 chitosanases from Streptomyces sp. N174 or Nocardioides sp. N106. The active recombinant CtoA protein was successfully overproduced in Escherichia coli. The mutant protein E22Q, in which the glutamic acid residue 22 was replaced with glutamine, abolished the chitosanase activity, showing that the Glu22 residue is required for the enzymatic activity. CtoA exhibited antifungal activity against Rhizopus oryzae, which is known to produce chitosan probably as a cell wall component. In contrast, E22Q did not inhibit the growth of the fungus, suggesting that chitosan-hydrolyzing activity is essential for the antifungal activity. It is noteworthy that the antifungal effect of CtoA against R. oryzae was drastically enhanced by the simultaneous addition of the family 19 chitinase ChiC from Streptomyces griseus. [source] Molecular characterization of the spectrum of genomic deletions in the mismatch repair genes MSH2, MLH1, MSH6, and PMS2 responsible for hereditary nonpolyposis colorectal cancer (HNPCC)GENES, CHROMOSOMES AND CANCER, Issue 2 2005Heleen van der Klift A systematic search by Southern blot analysis in a cohort of 439 hereditary nonpolyposis colorectal cancer (HNPCC) families for genomic rearrangements in the main mismatch repair (MMR) genes, namely, MSH2, MLH1, MSH6, and PMS2, identified 48 genomic rearrangements causative of this inherited predisposition to colorectal cancer in 68 unrelated kindreds. Twenty-nine of the 48 rearrangements were found in MSH2, 13 in MLH1, 2 in MSH6, and 4 in PMS2. The vast majority were deletions, although one previously described large inversion, an intronic insertion, and a more complex rearrangement also were found. Twenty-four deletion breakpoints have been identified and sequenced in order to determine the underlying recombination mechanisms. Most fall within repetitive sequences, mainly Alu repeats, in agreement with the differential distribution of deletions between the MSH2 and MLH1 genes: the higher number and density of Alu repeats in MSH2 corresponded with a higher incidence of genomic rearrangement at this disease locus when compared with other MMR genes. Long interspersed nuclear element (LINE) repeats, relatively abundant in, for example, MLH1, did not seem to contribute to the genesis of the deletions, presumably because of their older evolutionary age and divergence among individual repeat units when compared with short interspersed nuclear element (SINE) repeats, including Alu repeats. Moreover, Southern blot analysis of the introns and the genomic regions flanking the MMR genes allowed us to detect 6 novel genomic rearrangements that left the coding region of the disease-causing gene intact. These rearrangements comprised 4 deletions upstream of the coding region of MSH2 (3 cases) and MSH6 (1 case), a 2-kb insertion in intron 7 of PMS2, and a small (459-bp) deletion in intron 13 of MLH1. The characterization of these genomic rearrangements underlines the importance of genomic deletions in the etiology of HNPCC and will facilitate the development of PCR-based tests for their detection in diagnostic laboratories. © 2005 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||||||||