The Efficacy of Molecular Markers Analysis with Integration of Sensory Methods in Detection of Aroma in Rice

Allele Specific Amplification with four primers (External Antisense Primer, External Sense Primer, Internal Nonfragrant Sense Primer, and Internal Fragrant Antisense Primer) and sensory evaluation with leaves and grains were executed to identify aromatic rice genotypes and their F1individuals derived from different crosses of 2 Malaysian varieties with 4 popular land races and 3 advance lines. Homozygous aromatic (fgr/fgr) F1individuals demonstrated better aroma scores compared to both heterozygous nonaromatic (FGR/fgr) and homozygous nonaromatic (FGR/FGR) individuals, while, some F1individuals expressed aroma in both leaf and grain aromatic tests without possessing thefgrallele. Genotypic analysis of F1individuals for thefgrgene represented homozygous aromatic, heterozygous nonaromatic and homozygous nonaromatic genotypes in the ratio 20 : 19 : 3. Genotypic and phenotypic analysis revealed that aroma in F1individuals was successfully inherited from the parents, but either molecular analysis or sensory evaluation alone could not determine aromatic condition completely. The integration of molecular analysis with sensory methods was observed as rapid and reliable for the screening of aromatic genotypes because molecular analysis could distinguish aromatic homozygous, nonaromatic homozygous and nonaromatic heterozygous individuals, whilst the sensory method facilitated the evaluation of aroma emitted from leaf and grain during flowering to maturity stages.

Expression of Kindlins in Acute Myeloid Leukemia

Abstract 4910 The Kindlin family of intracellular proteins has recently emerged as key regulators of cellular functions and cell-matrix interactions. They comprise of three evolutionarily conserved members, kindlin-1, kindlin-2 and kindlin-3, they share considerable sequence and structural similarities. A few of study revealed that Kindlin-2 influences solid tumor cell invasion and resistance. With regard to AML, the influence of Kindlins is still unknown. To evaluate the clinical significance of Kindlin-2 in acute myeloid leukemia (AML), we investigated the expression of Kindlin-2, kindling-3 in AML cells. 1. Materials and methods K562, KG-1a, HL60, U937, Jurkat cell lines were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS, GIBCO) at 37°C in a humidified atmosphere of 5% CO2. Bone marrow (BM) samples were obtained from 88 patients with de novo AML from Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC). Samples of 9 normal donors and ITP were used as the control group. Bone marrow mononuclear cells (BMMCs) were prepared by Ficoll-Hypaque density gradient centrifugation. Expressions of Kindlin-2, Kindlin-3 were detected by RQ-PCR. The following primers for real-time PCR were used: (a) Kindlin-2 sense primer, 5'-CCGCTCGAGCTATGCGTATCCCCGTAG-3'; (b) Kindlin-2 antisense primer, 5'-CGACGCGTCTAGCGAGGGGTTGTC-3'; (c) Kindlin-3 sense primer, 5'-CCGCTCGAGCTATGCGTATCCCCGTAG-3'; (d) Kindlin-3 antisense primer, 5'-CGACGCGTCTAGCGAGGGGTTGTC-3'; (e) GAPDH sense primer, 5'-GAAGGTGAAGGTCGGAGTC-3'; (f) GAPDH antisense primer, 5'-GAAGATGGTGATGGGATTTC-3'. Analysis was performed using ABI 7500 Sequence Detection software (Applied Biosystems). The expression of Kindlin-2 and Kindlin-3 were showed as RQ value calculated through ΔΔCt method [ΔΔCt = (CtKindlin □ CtGAPDH)sample □ (CtKindlin □ CtGAPDH)calibrator]. The ΔCt (CtKindlin □ CtGAPDH) of K562 was defined as calibrator, and the RQ of calibrator was 1.000. Relationships between Kindlin-2, Kindlin-3 and the patients' clinical data were analyzed. 2. Results Expression of Kindlins in newly diagnosis AML The level of Kindlin-2 in AML (0.163±1.665) was significantly lower than that in non-AML (1.683±1.395) controls (p=0.010). No significant difference was found between the AML and controls in levels of Kindlin-3 (p=0.216). Out of the 79 patients who accepted treatment, 61 patients achieved complete remission (CR) and 18 patients were NR. Patients with higher expression of Kindlin-2 had a higher CR rate (86.8% vs 68.3%) (p=0.050). Expression of kindling 3 was unrelated to CR rate. Both of kindling-2 and kindling-3 increased after CR. This finding implicates Kindlin-2 as a potential prognostic factor of AML. Disclosures: No relevant conflicts of interest to declare.

First Report of Little Cherry Disease from Sweet Cherry (Prunus avium) and Sour Cherry (P. cerasus) in the Czech Republic

Little cherry disease (LChD), a virus disease of sweet (Prunus avium) and sour cherries (P. cerasus), is caused by members of the Closteroviridae family. Symptoms are especially visible on fruits and leaves. Leaves become red or bronze in late summer and fall. Fruit are small, angular, and pointed. Fruits are unmarketable due to a characteristic bitter flavor. LChD also causes reduction of yield (1). Sweet and sour cherries are the second (after apples) most often grown fruit species in the Czech Republic. Since LChD occurred in Germany (1) and Poland (2) in 2007 and 2008, sweet and sour cherry trees with LChD symptoms were surveyed in orchards in the East Bohemia Region of the Czech Republic. The presence of LChD was determined by reverse transcription (RT)-PCR and woody indicator plants, as recommended by the European and Mediterranean Plant Protection Organization (EPPO). Different parts of plants were taken from trees with suspicious symptoms to observe the dynamics of virus infection during the 2009 growing season. Total RNA was isolated from young leaves, blossoms, fruits, and fully developed leaves with a CONCERT Plant RNA Purification Reagent (Invitrogen, Carlsbad, CA) (3). RT-PCR was performed with a QIAGEN OneStep RT-PCR Kit (Qiagen, Hilden, Germany) and oligonucleotides previously described (4). Oligonucleotide LCV3EC (5′-GCTCTAGAGGCACCTTTTATTTTTTATATATGC-3′), complementary to position 16910 to 16934 (GenBankAccession No. Y10237) (with the addition of eight nonviral nucleotides to introduce an XbaI site), was used as a negative-sense primer in RT reactions and PCR. Oligonucleotide LCV16659 (5′-GTTATAGAATTCACTGCAAGTG-3′) was used as a positive-sense primer for PCR amplification. The program used for cDNA synthesis was 50°C for 30 min, followed by denaturation for 10 min at 95°C, 35 cycles of 45 s at 94°C, 45 s at 58°C, and 45 s at 72°C. A final incubation was at 72°C for 5 min (1). The finished PCR products (430 bp) were analyzed on 1% agarose gels (stained with SYBR green). According to the preliminary results, young leaves from buds (67% of samples of selected trees with LChD were positive), blossoms (67% positive), and leaves taken in autumn (67% positive) were optimal for the detection of LChD by RT-PCR. The trial with woody indicator plant species was established in the field. Indicators P. avium cv. Sam and P. avium cvs. Bing, F12/1, and Canindex (4) were inoculated with buds from LChD-infected trees and observed for 2 years. Woody indicators remained symptomless throughout the first year of observation, but the indicators showed red coloration of leaves in late summer of the second year. P. avium cv. Canindex seems to be the best woody indicator for testing of LChD in the climatic conditions of the Czech Republic. To our knowledge, this is the first report of LChD in the Czech Republic. References: (1) W. Jelkmann et al. Acta Hortic. 781:321, 2008. (2) B. Komorowska and M. Cieślińska. Plant Dis. 92:1366, 2008. (3) J. Matoušek et al. Biol.Chem. 388:1, 2007. (4) M. Vitushkina et al. Eur. J. Plant Pathol. 103:803, 1997.

First Report of Pepper yellow leaf curl Indonesia virus in Ageratum conyzoides in Indonesia

Ageratum conyzoides L. plants affected with yellow vein disease were collected from Magelang, Bandung, and Purwokerto locations in Indonesia during 2001. A. conyzoides is a naturally occurring weed that is found in and around fields of cultivated pepper (Capsicum annuum L.) and tomato (Lycopersicon esculentum L.). It is frequently found with symptoms of yellow vein disease and the abundance of whiteflies on the affected plants suggested the possible involvement of a geminivirus. Total nucleic acids were extracted from nine samples collected from these locations of A. conyzoides-affected plants exhibiting yellow vein disease and amplified using PCR with geminivirus DNA-A-specific designed primers (virion-sense primer 5′-GAGCTCTTAGCCGCCTGAATGTTC-3′; complementary-sense primer 5′-GAGCTCGTCAGATGTTAAGACCTAC-3′) (1). A PCR-amplified product of approximately 2.7 kbp was obtained from each sample. Five independent sequences were cloned and sequenced from each sample. Sequence analysis showed that five of nine samples were Ageratum yellow vein virus (one each from Bandung and Purwokerto and three from Magelang) and the remaining four samples (two samples each from Bandung and Purwokerto) were a strain of Pepper yellow leaf curl Indonesia virus (PepYLCIDV). Full-length DNA-A of PepYLCIDV from systemic A. coniziodes was amplified using PCR with additional primers designed at only one restriction site (BamHI) (5′-GGATCCGCTTGTTCATCCTTTTCCAG-3′/5′-GGATCCCACATCTTTGGTTAGTGGAGGGTG-3′) and cloned. Three independent clones obtained were sequenced and analyzed. The sequence of a full-length DNA-A component was determined (2,760 bases, GenBank Accession No. AB267838). PCR using degenerate primers (DNABLC1: 5′-GTVAATGGRGTDCACTTCTG-3′; DNABLC2: 5′-RGTDCACTTCTGYARGATGC-3′, DNABLV2: 5′-GAGTAGTAGTGBAKGTTGCA-3′) of begomovirus DNA-B component (2), five independent clones were obtained and sequenced. Primers designed to amplify a full-length B component were constructed around a unique restriction site (BamHI) (5′-GGATCCCCTCATTCCTTTTGCGGAG-3′/5′-GGATCCACAGAGGAAAACTCGCAAGGC-3′). A PCR product was obtained from A. conyzoides samples and three independent clones were sequenced and analyzed. A full-length sequence of a begomovirus B component was determined (2,746 bases, GenBank Accession No. AB267839). Five open reading frames (ORF) were found in DNA-A and two in DNA-B. The DNA-A and DNA-B had a common region (CR) (74% nucleotide sequence identity) that comprised approximately 160 nucleotides. The DNA-A and DNA-B had an identical 31-base stem loop region in the CR. In addition, DNA-A and DNA-B had the highest nucleotide sequence identity (93%) with those of PepYLCIDV (GenBank Accession Nos. AB267834 and AB267835), suggesting it is a strain of PepYLCIDV, which is widely prevalent in Indonesia. To our knowledge, this is the first report of PepYLCIDV isolated from A. conyzoides plants affected with yellow vein disease. References: (1) R. W. Briddon and P. G. Markham. Mol. Biotechnol. 1:202, 1994. (2) S. K. Green et al. Plant Dis. 85:1286, 2001.

A modified GLGI method for identification of novel porcine genes from long serial analysis of gene expression tags

Combining the long serial analysis of gene expression (LongSAGE) and the generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification (GLGI) technique, a new strategy called modified GLGI (M-GLGI) was developed to isolate unknown 3′ expressed sequence tags (ESTs) and discover novel genes. A 17 bp LongSAGE tag was used as sense primer instead of a 10-base SAGE tag; PCR reaction was performed under an appropriate annealing temperature for each tag; universal DNA polymerase was used in PCR amplification instead of Pfu enzyme; a common cloning strategy using pMD-18T vector and Escherichia coli DH5α cells were used instead of a special vector and competent cells. Moreover, ESTs isolated by M-GLGI had 3′ ends with the polyadenylation signals and poly(dA) tails. This method is more sensitive for identifying genes expressed in low abundance than conventional EST sequencing.

First Report of Citrus leaf blotch virus on Kumquat in Italy

During the spring of 2006, nurserymen reported observations of the bud union disorder of ‘Nagami’ kumquat scions propagated on Troyer citrange rootstock to the CRA–Istituto Sperimentale per l'Agrumicoltura. These plants showed reduced canopy volume and new shoots below graft points 6 months after propagation; the bud union was brittle and broke down easily after 1 year. After tests excluded common citrus viruses and viroids that might cause the incompatibility (e.g., Citrus tristeza virus, Citrus psorosis virus, Citrus exocortis viroid, and Hop stunt viroid), we tested for Citrus leaf blotch virus (CLBV), a virus previously associated with a bud union crease in kumquat (2). Leaves were collected from 100 2-year-old kumquat plants from a nursery near Messina (Sicily [Italy]); 50 were grafted on sour orange rootstock (asymptomatic) and 50 were grafted on Troyer citrange rootstock (symptomatic). Total RNA was extracted using Qiagen RNeasy Plant Mini Kit (Qiagen S.P.A. Milan, Italy). Primers previously reported (1,2) and designed from a published CLBV sequence (Genbank Accession No. AJ318061) were used in reverse transcription (RT)-PCR assays to amplify the RNA-dependent RNA polymerase gene (sense primer KU 27, 5′-GATGCAAGCCAGGATGAATAC-3′, genomic positions 5321–5340 and anti-sense primer KU 15, 5′-CAGACACTCCAAGACCTTTCC-3′, genomic positions 5776–5756) and the coat protein gene (sense primer KU18, 5′-TTAAGATTACAGACACGAAGG-3′ genomic positions 7686–7706 and anti-sense primer KU 19 5′-CTGTTTTTGAATTTTGCTCG-3′, genomic positions 8123–8104). All kumquat samples yielded amplicons of the expected size (456 and 438 bp). No amplicons were obtained from healthy plants. Amplicons for each gene were cloned into the pGEM-T Easy Vector (Promega Italy, Milan), and four clones for each plasmid DNA were sequenced in both directions. Consensus sequences of the two genes (Genbank Accession Nos. EF203229 and EF203230) had 96 and 97% nucleotide sequence identity, respectively, and both had 99% amino acid identity with the previously reported CLBV sequence (Genbank Accession No. AJ318061). Approximately 400,000 ornamental kumquats are produced annually in Italy. CLBV infection can cause serious production losses because of the decline associated with bud union disorders grafted onto trifoliate orange and trifoliate-derived rootstocks. References: (1) L. Galipienso et al. Eur. J. Plant Pathol. 110:175, 2004. (2) M. C. Vives et al. Virology 287:225, 2001.

KONSTRUKSI MUTASI DAERAH RESISTEN RIFAMPIN (GEN rpoB) DARI Mycobacterium leprae PADA PENDERITA LEPRA DI SURABAYA MELALUI ANALISIS GENOM HASIL PCR

Rifampin is a key component in the chemoterapeutic regimens used to combat both leprosy and tuberculosis. Owing to exquisite rifampin susceptibility of Mycobacterium leprae, this drug is the backbone of the multidrug therapy currently recommended by WHO for the treatment of leprosy. Resistant mutant are known to arise in leprosy patients receiving rifampin (RIF) monotherapy. The aim of this study was to elucidation of the sequence of the M. leprae rpoB gene permitted identification of mutations associated with rifampin resistance of leprosy patients in Surabaya by genome analysis. M. leprae was detected by nested PCR. In brief, PCR was run with the sense primer rpoBF and anti sense primer rpoBR for 45 cycles. Amplified DNA was analyzed by 3 percent agarose electrophoresis and the 342 base pairs product was visualized by UV fluorescence after staining with ethidium bromide. PCR product will be purified by phenolchloroform methods and then sequencing directly by ABI PRISM 310. After that sequence data from samples will be analyzed by Genetic Mac ver. 8.0, and comparing with reference data from Gen bank. The result show that only six of 10 samples could be analyzed construct of mutations by Genetic Mac ver 8.0. They have construct no mutation or 100 percent homology with reference (Z14314 or GI:44382).

Derivative (1) t(1;1) (p36; q21) Produces the Fusion Protein with Phosphatase 10 and MEL1 (MDS1/EVI1-Like Gene 1) in a AML (M5a) Case.

Objective: A 25 years-old Japanese male was diagnosed as AML (M5a) with 46, XY, der (1) t(1; 1) (p36; q21). To determine the leukemogenesis in this case, we analyzed the fusion product of this chromosomal translocation. Method: After obtaining informed consent, RNA was extracted from his bone marrow cells (blastic cells were occupied in almost 100%) with GTC method, and poly-A rich RNA was separated with oligo-dT latex particles. 1st strand cDNA was synthesized with oligo-dT primer. Oligo-dA stretch was added with TdT to make 5′ RACE. 1st PCR was performed with the synthetic antisense-1 primers from several candidates including p73, and MEL1, and with QT primer (5′-TGAGCCAGAGTGACGAGGACTCGAGCTCAAGCT17-3′) as a sense primer. 2nd PCR was performed with internal antisense-2 primers from several candidates, and Q0 primer (5′-CCAGTGAGCAGAGTGACG-3′) as the sense primer. The reaction of DNA sequencing was performed with BigDye Terminator Cycle Sequencing kit (Applied Biosystems), and analyzed with ABI Prism 3700 DNA analyzer. Result and Discussion: The fusion cDNA product was obtained, which was consisted of MEL1 and Phosphatase 10. The breakpoint of MEL1 was exon 4, and PR domain of MEL1 was disrupted. This domain is reported to be the key function of MEL1, and when MEL1 without PR domain is expressed, myeloid differentiation is blocked. In this case the fusion of Phosphatase 10 and MEL1 is seemed to be the main cause of leukemogenesis.

Parvovirus B19 and Aplastic Anemia: Case Control Study: Preliminary Report from the ITESM Hematology Study Group.

Aplastic anemia (AA) is a classic bone marrow failure syndrome simply defined as peripheral blood pancytopenia and a hypocelular bone marrow, yet the diagnosis must be made by excluding other causes of bone marrow failure. The incidence rate of AA reported by the International Aplastic Anemia and Agranulocytosis Study (IAAAS) in the 1980s was 2 cases per 1 million people. This disease is known to be caused by exposure to radiation, chemotherapy and some viral agents, yet most of the cases are idiopathic. Epstein Barr virus and non-A, non-B or non-C Hepatitis virus have classically been related to the development of some AA cases. Recently there have been some reports of AA following Parvovirus B19 (PvB19) infection. This virus, the only parvoviridae virus capable of infecting humans, attacks erythrocyte precursors attaching to the P antigen in their surface and requiring Beta1 integrin for viral entry. Although PvB19 seems to infect only erytroid precursors, it is widely recognized that the infection with this virus can cause not only anemia, but neutropenia and thrombocytopenia as well, producing aplastic crisis of varying intensity. A correlation has recently been found between PvB19 DNA in peripheral blood and AA in children. We pretend to corroborate this observation and include adult patients in order to improve our understanding of the relationship between PvB19 and AA. So far we have taken peripheral blood samples from 9 AA patients and 9 controls paired by age, sex and community; we plan to include 100 AA patients and their controls from several hospitals around Mexico. DNA was extracted using the PUREGENE DNA extraction kit (Gentra, Minneapolis MN). Nested PCR was performed using the sense primer (P1) 5-AATACACTGTGGTTTTATGGGCCG-3, antisense (P2) 5-CCATTGCTGGTTATAACCACAGGT-3 for the first round and the sense primer (P3) 5-AATGAAAACTTTCCATTTAATGATGTAG-3 and antisense primer (P4) 5-CTAAAATGGCTTTTGCAGCTTCTAC-3for the second round. A DNA sample from a patient with active infectious mononucleosis with positive IgG and IgM against PvB19 in serum was used as positive control. Two samples from the AA group (22%) and 1 from the control group (11%) have turned positive for PvB19 DNA. The reported incidence for the presence of this virusDNA in the peripheral blood of the population is 3%. We expect that, as the number of patients grows, the percentage of positive samples in the control group will decrease, while the percentage of positive samples in the AA group will rise or be sustained. Our partial results point towards a possible relationship between AA and the presence of PvB19 DNA in the peripheral blood cells. It is possible that this virus is one of many factors capable of precipitating the development of AA by limiting the bone marrows capacity to produce blood cells. We are in the process of gathering more samples to prove if a relationship really exists and, if so, future studies will likely shed light upon the mechanism by which PvB19 contributes to the development of AA and other marrow failure syndromes.

Mouse Amelogenin Exons 8 and 9: Sequence Analysis and Protein Distribution

Amelogenin is the major protein of the developing enamel. Two additional exons, termed 8 and 9, have been characterized in the rat. Our aim was: to identify the mouse amelogenin exons 8/9 sequences; to investigate the potential presence of the alternative spliced isoforms of amelogenin exons 8/9; and to immunolocalize proteins containing sequences encoded by exons 8/9 during odontogenesis. RT-PCR analysis with exon 9 anti-sense primer generated 2 major amplicons with the use of a mouse tooth cDNA library and dental cell lines. DNA sequence analysis showed 93% identify with the rat exons 8/9 sequence. Alternative splicing of exon 3 was also found, but only in cDNAs lacking exons 8 and 9. Immunohistochemistry localized exons 8/9-encoded proteins in ameloblasts, young odontoblasts, and stratum intermedium cells. Analysis of our data supports the hypothesis that: (1) AMELX contains 2 additional exons; (2) ameloblasts and odontoblasts synthesize amelogenin 8/9; and (3) amelogenin splice variants may have unique functions during tooth formation.