scholarly journals Cloning of Phospholipase Dα and Lipoxygenase Genes CmPLDa1 and CmLOX1 and Their Expression in Fruit, Floral, and Vegetative Tissues of `Honey Brew' Hybrid Honeydew Melon

2006 ◽  
Vol 131 (4) ◽  
pp. 544-550 ◽  
Author(s):  
Bruce D. Whitaker ◽  
Gene E. Lester
Keyword(s):  
Cucumis Melo ◽  
Critical Role ◽  
Lipid Body ◽  
Nucleotide Identity ◽  
Rt Pcr ◽  
Cucumber Cotyledon ◽  
Coding Regions ◽  
Accelerate Senescence ◽  
Genes Expression ◽  
Honeydew Melon

Increases in phospholipase D [PLD (EC 3.1.4.4)] and lipoxygenase [LOX (EC 1.13.11.12)] activities are thought to play a critical role in senescence of mesocarp tissues in netted and nonnetted muskmelon (Cucumis melo L.) fruits. We have cloned and characterized two full-length cDNAs, CmPLDα1 and CmLOX1, encoding PLDα and LOX proteins in honeydew melon (C. melo Inodorus Group cv. Honey Brew). Relative levels of expression of the corresponding genes were determined by semi-quantitative RT-PCR in developing and mature fruit mesocarp tissues [20-60 d after pollination (DAP)], as well as in roots, leaves, and stems from 4-week-old and flowers from 6- to 7-week-old plants. The coding regions of CmPLDα1 and CmLOX1 cDNAs are, respectively, 2427 and 2634 nucleotides long, encoding proteins 808 and 877 amino acids in length. CmPLDα1 is very similar to PLDα genes from castor bean (Ricinis communis L.), cowpea (Vigna unguiculata L.), strawberry (Fragaria ×ananassa Duch.) and tomato (Lycopersicon esculentum Mill.) (77% nucleotide identity), and is the first PLD gene cloned from a cucurbit species. CmLOX1 has 94% nucleotide identity to a cucumber (Cucumis sativus L.) LOX gene expressed in roots and 80% identity to cucumber cotyledon lipid body LOX. In general, transcript of CmPLDα1 was much more abundant than that of CmLOX1, but relative levels of transcript in the various organs and tissues were similar for the two genes. Expression was highest in roots, flowers, and fruit mesocarp tissues. CmPLDα1 expression in fruit was essentially constitutive throughout development, although maximum levels occurred at 50 and 55 DAP, respectively, in middle and hypodermal mesocarp. CmLOX1 expression was generally higher in middle than in hypodermal mesocarp with maximum transcript levels occurring at 55 and 50 DAP, respectively. Overall, the patterns of expression of CmPLDα1 and CmLOX1 are consistent with a model in which their encoded enzymes act in tandem to promote or accelerate senescence in fruit mesocarp tissues.

scholarly journals (168) Phospholipase Dα and Lipoxygenase Gene Expression in Fruit, Floral, and Vegetative Tissues of `Honey Brew' Hybrid Honeydew Melon

HortScience ◽  
2006 ◽  
Vol 41 (4) ◽  
pp. 1081B-1081
Author(s):  
Bruce D. Whitaker ◽  
Gene E. Lester
Keyword(s):  
Cucumis Melo ◽  
Lipid Body ◽  
Nucleotide Identity ◽  
Rt Pcr ◽  
Cucumber Cotyledon ◽  
Coding Regions ◽  
Accelerate Senescence ◽  
Genes Expression ◽  
Lipoxygenase Gene ◽  
Honeydew Melon

Increases in phospholipase D (PLD) and lipoxygenase (LOX) activities are thought to play a key role in senescence of mesocarp tissues in muskmelon fruit. We have cloned and characterized two full-length cDNAs, CmPLDα and CmLOX1, encoding PLDα and LOX proteins in honeydew melon (Cucumis melo L. Inodorus Group). Levels of expression of the corresponding genes were determined by semi-quantitative RT-PCR in developing and mature fruit mesocarp tissues (20–60 d after pollination; DAP), and in roots, leaves, and stems from 4-week-old and flowers from 6-week-old plants. The coding regions of CmPLDα1 and CmLOX1 cDNAs are, respectively, 2427 and 2634 nucleotides long, encoding proteins 808 and 877 amino acids in length. CmPLDα1 is most similar to PLDα genes in castor bean, cowpea, strawberry, and tomato (77% nucleotide identity), and is the first cucurbit PLD gene cloned. CmLOX1 has 94% nucleotide identity to a cucumber LOX gene expressed in roots and 80% identity to cucumber cotyledon lipid body LOX. Transcript of CmPLDα1 was much more abundant than that of CmLOX1, but relative levels of transcript in the various organs and tissues were similar for the two genes. Expression was highest in roots, flowers, and fruit mesocarp tissues. CmPLDα1 expression in fruit was high throughout development, although maximum levels occurred at 50 and 55 DAP, respectively, in middle and hypodermal mesocarp. CmLOX1 expression was generally higher in middle than in hypodermal mesocarp with maximum transcript levels at 55 and 50 DAP, respectively. Overall, the patterns of expression of CmPLDα1 and CmLOX1 are consistent with a model in which their encoded enzymes act in tandem to promote or accelerate senescence in fruit mesocarp tissues.

scholarly journals The potential renal toxicity of silver nanoparticles after repeated oral exposure and its underlying mechanisms

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Hamed Nosrati ◽  
Manijeh Hamzepoor ◽  
Maryam Sohrabi ◽  
Massoud Saidijam ◽  
Mohammad Javad Assari ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Molecular Mechanisms ◽  
Renal Toxicity ◽  
Periodic Acid ◽  
Critical Role ◽  
Oral Exposure ◽  
Control Group ◽  
Rt Pcr ◽  
Periodic Acid Schiff

Abstract Background Silver nanoparticles (AgNPs) can accumulate in various organs after oral exposure. The main objective of the current study is to evaluate the renal toxicity induced by AgNPs after repeated oral exposure and to determine the relevant molecular mechanisms. Methods In this study, 40 male Wistar rats were treated with solutions containing 30, 125, 300, and 700 mg/kg of AgNPs. After 28 days of exposure, histopathological changes were assessed using hematoxylin-eosin (H&E), Masson’s trichrome, and periodic acid-Schiff (PAS) staining. Apoptosis was quantified by TUNEL and immunohistochemistry of caspase-3, and the level of expression of the mRNAs of growth factors was determined using RT-PCR. Results Histopathologic examination revealed degenerative changes in the glomeruli, loss of tubular architecture, loss of brush border, and interrupted tubular basal laminae. These changes were more noticeable in groups treated with 30 and 125 mg/kg. The collagen intensity increased in the group treated with 30 mg/kg in both the cortex and the medulla. Apoptosis was much more evident in middle-dose groups (i.e., 125 and 300 mg/kg). The results of RT-PCR indicated that Bcl-2 and Bax mRNAs upregulated in the treated groups (p < 0.05). Moreover, the data related to EGF, TNF-α, and TGF-β1 revealed that AgNPs induced significant changes in gene expression in the groups treated with 30 and 700 mg/kg compared to the control group. Conclusion Our observations showed that AgNPs played a critical role in in vivo renal toxicity.

scholarly journals Nuevos registros de aislamientos del Cucumber mosaic virus y su RNA satélite en Colima, México

2019 ◽  
Vol 37 (2) ◽  
Author(s):  
Pedro Valadez-Ramírez ◽  
Javier Paz-Román ◽  
Salvador Guzmán-González ◽  
Marco Tulio Buenrostro-Nava ◽  
Daniel Leobardo Ochoa-Martínez
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Solanum Lycopersicum ◽  
Capsicum Annuum ◽  
Catharanthus Roseus ◽  
Cucumis Melo ◽  
Rt Pcr ◽  
Impacto Económico

El <em>Cucumber mosaic virus</em> (CMV) ocasiona una de las enfermedades virales más importantes a nivel mundial en plantas silvestres y cultivadas. En México son pocos los estudios que se han abordado con este virus, y dada su amplia gama de hospedantes e impacto económico, es necesario contar con mayor información de su presencia y distribución en zonas de importancia agrícola como las del estado de Colima. En este trabajo, se reportan nuevos aislamientos del CMV identificados por RT-PCR, secuenciación de DNA y su análisis filogenético: CMV-Vin en vinca (<em>Catharanthus roseus</em>), CMV-Chi en chile jalapeño (<em>Capsicum annuum</em>) y CMV-Tom en tomate saladette (<em>Solanum lycopersicum</em>). Se confirmó, además, la presencia del CMV en melón cantaloupe (<em>Cucumis melo</em>) (CMV-Mel). Los aislamientos CMV-Vin, CMV-Chi y CMV-Mel agruparon en el subgrupo IB, mientras que CMV-Tom agrupó en el subgrupo IA de CMV. De estos aislamientos, sólo CMV-Vin evidenció la presencia de un RNA satélite (satRNA Vin) sin dominio necrogénico. Este es el primer reporte de la presencia del CMV en vinca, chile y tomate y de un RNA satélite en vinca en Colima, México.

The monocyte transcriptome during pregnancy in multiple sclerosis: prominent expression of the Fc-receptor CD64

2010 ◽  
Vol 17 (4) ◽  
pp. 389-396 ◽  
Author(s):  
Rinze F Neuteboom ◽  
Evert Verbraak ◽  
Annet F Wierenga-Wolf ◽  
Jane SA Voerman ◽  
Marjan van Meurs ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Cell Signalling ◽  
Immune Functions ◽  
Rt Pcr ◽  
Third Trimester ◽  
Fc Gamma Receptor ◽  
The Third ◽  
Gamma Receptor ◽  
Genes Expression ◽  
Biological Interest

Background: During the third trimester of pregnancy multiple sclerosis (MS) disease activity is reduced. It is not fully understood which factors mediate this disease amelioration.Objective: To study alterations of the monocyte transcriptome during pregnancy in MS patients, using a genomewide approach to identify differentially regulated genes.Methods: Women with MS and healthy controls were longitudinally studied, including a visit before pregnancy.Results: RNA-microarray analysis was performed in six patients. We found a significant increase of CD64 (Fc gamma receptor 1a, FcgR1a) during the third trimester compared with baseline, confirmed by RT-PCR in a group of ten patients. Analysis with Ingenuity software was performed using all genes expression of which was altered at least 1.5-fold in at least five out of six patients. Major networks that were altered during MS pregnancy were: cell-to-cell signalling and interaction, immune response, and cell signalling. From the genes selected for Ingenuity analysis, seven additional candidate genes, selected for their biological interest, were tested using RT-PCR in ten patients with MS and nine controls. We found an increased expression of JAK2 and STAT1 directly postpartum in patients with MS and in controls.Conclusion: The increased CD64 expression during pregnancy is indicative of enhanced innate immune functions.

scholarly journals First Report of a Carlavirus in Fuchsia spp. in New Zealand

Plant Disease ◽  
2011 ◽  
Vol 95 (11) ◽  
pp. 1484-1484 ◽  
Author(s):  
Z. Perez-Egusquiza ◽  
L. W. Liefting ◽  
S. Veerakone ◽  
G. R. G. Clover ◽  
M. Ciuffo
Keyword(s):  
Electron Microscopy ◽  
New Zealand ◽  
Coat Protein ◽  
Chenopodium Quinoa ◽  
Plant Diseases ◽  
Nucleotide Identity ◽  
Impatiens Necrotic Spot Virus ◽  
Rt Pcr ◽  
First Report ◽  
Genome Region

The genus Fuchsia has 110 known species and numerous hybrids. These ornamental plants with brightly colored flowers originate from Central and South America, New Zealand, and Tahiti, but a wider variety are now grown all over the world. Few viruses have been reported in Fuchsia spp.: a carlavirus, Fuchsia latent virus (FLV) (1–3), a cucumovirus, Cucumber mosaic virus (CMV) (3), and two tospoviruses, Impatiens necrotic spot virus (INSV) and Tomato spotted wilt virus (TSWV) (4). In August 2009, five plants, each representing a different cultivar of Fuchsia hybrid, from home gardens in the Auckland and Southland regions of New Zealand, displayed variable symptoms including mild chlorosis, mild mottle, or purple spots on leaves. Plants tested negative for CMV, INSV, and TSWV using commercial ImmunoStrips (Agdia Inc., Elkhart, IN); however, flexuous particles of ~650 to 700 nm were found by electron microscopy in all samples. Local lesions were also observed on Chenopodium quinoa plants 4 weeks after sap inoculation. Total RNA was extracted from all plants with a RNeasy Plant Mini Kit (Qiagen Inc., Doncaster, Australia) and tested by reverse transcription (RT)-PCR using two generic sets of primers (R. van der Vlugt, personal communication) designed to amplify fragments of ~730 and 550 bp of the replicase and coat protein genes of carlaviruses, respectively. Amplicons of the expected size were obtained for all samples, cloned, and at least three clones per sample were sequenced. No differences within clones from the same samples were observed (GenBank Accession Nos. HQ197672 to HQ197681). A BLASTn search of the viral replicase fragment showed the highest nucleotide identity (76%) to Potato rough dwarf virus (PRDV) (EU020009), whereas the coat protein fragment had maximum nucleotide identity (70 to 72%) to PRDV (EU020009 and DQ640311) and Potato virus P (DQ516055). Sequences obtained were also pairwise aligned using the MegAlign program (DNASTAR, Inc., Madison, WI) and results showed that the isolates had 83 to 97% identity to each other within each genome region. Further sequences (HQ197925 and HQ197926) were obtained from a Fuchsia plant originating from Belgium, a BLASTn analysis showed high nucleotide identity (84 to 99%) to the New Zealand isolates. The low genetic identity to other Carlavirus members suggests that these isolates belong to a different species from those previously sequenced. On the basis of electron microscopy and herbaceous indexing, the isolates had similar characteristics to a carlavirus reported from Fuchsia in Italy (1) and FLV reported in Canada (2). The Italian carlavirus isolate was obtained and tested with the same primers by RT-PCR. Pairwise analysis of the Italian sequences (HQ197927 and HQ197928) with the New Zealand and Belgian sequences showed between 84 and 95% similarity within each genome region. These results suggest that the carlavirus infecting these plants is the same virus, possibly FLV. To our knowledge, this is the first report of this carlavirus infecting Fuchsia spp. in New Zealand, but the virus has probably been present for some time in this country and is likely to be distributed worldwide. References: (1) G. Dellavalle et al. Acta Hortic. 432:332, 1996. (2) L. J. John et al. Acta Hortic. 110:195, 1980. (3) P. Roggero et al. Plant Pathol. 49:802, 2000. (4) R. Wick and B. Dicklow. Diseases in Fuchsia. Common Names of Plant Diseases. Online publication. The American Phytopathological Society, St. Paul, MN, 1999.

Application of Novel Laser Capture Microdissection and RT-PCR to a Functional Analysis of Osteopontin in Mouse Growth Plate Cartilage

2001 ◽  
Vol 7 (S2) ◽  
pp. 44-45
Author(s):  
R. Jacquet ◽  
J. Hillyer ◽  
J. Zhang ◽  
W. J. Landis
Keyword(s):  
Growth Plate ◽  
Laser Capture Microdissection ◽  
Critical Role ◽  
Rna Extraction ◽  
Normal Growth ◽  
Alternative Possibilities ◽  
Rt Pcr ◽  
Hypertrophic Growth ◽  
Growth Plate Cartilage ◽  
Laser Capture

The long bones of vertebrates, such as the tibiae and femurs of humans, extend in length by means of genotypic and phenotypic changes orchestrated by the chondrocytes comprising growth plate cartilage. Among the constituents synthesized by these cartilage cells, osteopontin (OPN), a phosphorylated glycoprotein, is thought to play a critical role in events leading to normal growth plate function and ultimate mineralization of the deeper zones of this cartilage region. The precise role of OPN, however, is uncertain with regard to mineralization, and present evidence supports the alternative possibilities that the protein may be either facilitative or inhibitory to mineral deposition. in order to investigate OPN function in a model growth plate, cartilage from normal 1-11 day old postnatal mice was examined by the novel techique of laser capture microdissection (LCM) followed by RT-PCR to obtain a measure of OPN gene expression by chondrocytes of known age and specific location in the plate. LCM permits identification of individual or clusters of cells within a tissue section and subsequent unique isolation (“capture”) of such cells for a variety of molecular analyses.In this study, mouse tibiae were dissected, placed in RNAlater (Ambion, Austin, TX) to preserve message, and stored at −20°C. Sections (5 μm thick) of fresh frozen developing epiphyseal growth plates were obtained in a cryostat maintained at −20°C, fixed briefly in 70% ethanol, and stained with eosin. Sections were examined in a Pixcell LCM system (Arcturus Engineering, Mountain View, CA) where chondrocytes were attached to the surface of polymer film substrates and lifted free of sections. in separate experiments, ∼200-1200 cells were captured and analyzed. Substrates were transferred to Eppendorf tubes containing RNA extraction buffer. RNA was extracted from cells by microisolation (Stratagene, La Jolla, CA), DNAse-treated, reverse-transcribed, and then subjected to PCR (40 cycles) with AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA). Ethidium bromide agarose gels revealed OPN mRNA from groups of chondrocytes isolated from whole cartilage and from resting, proliferating, and hypertrophic growth plate zones from the mouse tibiae. Brain cells captured by LCM from the same mouse sections served as positive controls and reactions containing no reverse transcriptase were negative controls. 18S rRNA was used as a marker for semiquantitation and standardization of expressed message from captured cells.

scholarly journals Molecular Identification and Characterization of Tomato zonate spot virus in Tobacco in Guangxi, China

Plant Disease ◽  
2011 ◽  
Vol 95 (11) ◽  
pp. 1483-1483 ◽  
Author(s):  
J.-H. Cai ◽  
B.-X. Qin ◽  
X.-P. Wei ◽  
J. Huang ◽  
W.-L. Zhou ◽  
...  
Keyword(s):  
Amino Acids ◽  
Southwest China ◽  
Nucleotide Identity ◽  
Dead Leaf ◽  
Guangxi Province ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Spot Virus ◽  
Tobacco Plants ◽  
Partial Rdrp

In Guangxi Province of southwest China, diseases caused by Tospoviruses (family Bunyaviridae) pose a serious threat to tobacco (Nicotiana tobacum) production. During surveys conducted annually at Xinrong Village in Jingxi County from 2008 to 2010, more than 130 ha of fields were found to have 10 to 50% of plants exhibiting symptoms similar to spotted wilt caused by Tomato spotted wilt virus (TSWV). During this period, disease symptoms at similar prevalence and incidence were also found at Fushan, Debao County in most of the cultivars produced in these areas, including Yunyan 85, 87, 92, 97, and K326. Symptoms on tobacco varied but commonly included dwarfing, midrib browning, distorted apical buds, and concentric ringspots that coalesced to form large areas of dead leaf tissue. Mechanical inoculation from diseased tobacco leaves with concentric ringspots back to tobacco cv. Yunyan 85 or 87, resulted in 12 of 16 plants with symptoms similar to those observed in the field. No symptoms on plants developed following inoculation with buffer only. Symptoms found in the field resembled those caused by TSWV. However, testing using TSWV-specific antiserum was shown to be negative by double-antibody sandwich-ELISA (Agdia, Elkhart, IN). Total RNA was extracted from 27 diseased tobacco plants collected from different regions in Guangxi using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA extracts were amplified by reverse transcription (RT)-PCR using the degenerate primers T2740 (ATGGGDATNTTTGATTTCATG) and T3920c (TCATGCTCATSAGRTAAATYTCTCT) designed to target the partial RNA-dependent RNA polymerase (RdRp) sequence of members in the genus Tospovirus (3). Amplification was performed at 42°C for 60 min, followed by 35 cycles of PCR (30 s denaturation at 94°C, 45 s annealing at 55°C, and 30 s extension at 72°C) and a 7-min final extension at 72°C. A PCR product of approximately 1.2 kb was amplified from 21 diseased plants. RT-PCR amplicons were cloned into the pUC19-T Simple Vector (TaKaRa, Dalian, China) and sequenced in both directions. Sequences were assembled and analyzed by DNAStar 5.01 (DNASTAR, Madison, WI). Sequences of representative isolates were deposited in GenBank (Accession Nos. JN020022 to JN020027). The 1.2-kb partial RdRp sequences of these isolates were shown to have 94.4 to 95.3% nucleotide identity and 96.5 to 97.5% amino acids identity to Tomato zonate spot virus (TZSV) (GenBank Accession No. NC_010491) (1). Among these TZSV isolates from Guangxi, the partial RdRp sequences have 98.0 to 99.4% nucleotide identity and 98.8 to 100% amino acids identity with each other. The presence of TZSV was further confirmed in diseased tobacco plants by indirect ELISA using antiserum of TZSV (provided by Prof. Zhongkai Zhang, Agricultural Academy of Yunnan, China). TZSV has been characterized as a novel tospovirus on various hosts including tobacco in Yunnan province (1,2). To our knowledge, this is the first report of TZSV-associated disease on tobacco in Guangxi Province, southwest China. Further work is necessary to study the epidemiology and management of the disease. References: (1) J. Dong et al. Arch. Virol. 153:855, 2008. (2) J. Dong et al. J. Insect Sci. 10:166, 2010. (3) Y. Lin. Master Thesis. National Chung Hsing University, Taichung, Taiwan, Republic of China, 2007.

Transcription Factors Regulating T-Cells: Implications for Graft vs. Host Disease.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3929-3929
Author(s):  
R. P. Weitzel ◽  
Y. Huang ◽  
L. Fanning ◽  
M. Kozik ◽  
P. Leahy ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factors ◽  
T Cell ◽  
Lower Incidence ◽  
Time Course ◽  
Critical Role ◽  
Cell Protein ◽  
Rt Pcr ◽  
Host Disease ◽  
Cyclophilin B

Abstract Background. Graft versus host disease (GVHD) after allogeneic stem cell transplantation is associated with significant morbidity and mortality and targeted therapies are needed. The transcription factor nuclear factor of activated T-cells 1 (NFAT1) is known to interact with various other proteins, including AP-1 (fos/jun heterodimer), regulating both active immune responses and T-cell anergy. We have previously demonstrated lower expression of NFAT-associated transcripts via microarray, including IFN-γ, TNF-α, M-CSF, IL-3, 4, 5, and 13, IL-2R-α, CD40L, MIP1-α, as well as related transcription factors such as JunB, FOSL1, STAT4, T-bet, and c-maf in umbilical cord blood (UCB) CD4+ cells following primary stimulation when compared to cells obtained from adult blood (AB). We hypothesize a critical role for these NFAT1-dependent factors in the increased proliferation, decreased cytokine production seen in UCB, and lower incidence of GVHD following UCB transplantation. Here we present data more closely exploring the behavior of NFAT1 and associated genes in both UCB and AB CD4+ T-cells following primary stimulation. Methods. siRNA targeting NFAT1 mRNA was utilized to study the effects of lowered NFAT1 in primary human T-cells. CD14neg/4+ T-cells were isolated from adult peripheral blood by ficoll density gradient and selection by MACS. These cells were then immediately transfected via Nucleofector electroporation (Amaxa Biosystems, Gaithersburg, MD) with siRNA duplexes targeting NFAT1 and Cyclophilin B as well as an eGFP-encoding plasmid along with appropriate controls. Cultures were stimulated by anti-CD3/CD28 for 16h. 24h following transfection. Whole cell protein was harvested from a portion of each culture, and the remaining cells washed with PBS and replaced in non-stimulating media. Protein was extracted from the remaining cells 24h later. Protein lysate was analyzed by western blot for NFAT1, Cyclophilin-B, and β-Actin. Separately, expression of 7 transcripts (FOS, JUN, JUNB, FOSL1, BACH2, NFAM, and NFAT1) was analyzed via real-time PCR (RT-PCR). mRNA was obtained from 4 UCB and 4 AB samples at baseline (0h) (n=2) and 16h (n=2) of stimulation, using β-2-microglobulin as an endogenous control. Results. Transfection efficiency was measured to be approximately 70% via fluorescence microscopy of the eGFP-transfected culture. Our data indicate marginal reduction of NFAT1 protein at 24h. Notably after 48h approximately 65% knockdown is evident. Time course studies to determine the stability of siRNA-mediated NFAT1 knockdown are ongoing. No significant increase in the level of NFAT1 mRNA as measured by RT-PCR was detected. This suggests that the increase in NFAT1 protein levels post stimulation may not be a result of transcriptional regulation. Both UCB and AB samples exhibited a strong (15 fold) increase in FOSL1 transcription, and confirmed the enhanced up-regulation (about 3 fold) in the AB sample seen in microarray. Conclusions. Our data elucidate differences in the transcriptional program between UCB and AB T-cells, suggesting a crucial role for post-transcriptional events in the regulation of NFAT1 and its associated genes. Our findings identify differing regulation and expression of transcription factors in UCB vs. AB T-cells that may underlie the lower incidence and severity of GVHD seen in patients infused with UCB-derived grafts, and implicate additional elements which may modulate UCB T-cell alloreactivity. Studies to analyze the direct effects of decreased NFAT1 expression on relevant cytokines and other signaling molecules are currently ongoing.

Frequent Dysregulation of Fc Gamma Receptor IIb (Fc γRIIb) and Immunoreceptor Tyrosine-Based Inhibitory Motif (ITIM) Signaling Occurs in Multiple Myeloma (MM) Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2917-2917
Author(s):  
Jennifer Li ◽  
Andrew Leu ◽  
Mingjie Li ◽  
Ethan D Hobel ◽  
Kevin Delijani ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Critical Role ◽  
Rt Pcr ◽  
Binding Ability ◽  
Downstream Signaling

Abstract Abstract 2917 The inhibitory Fc receptor, Fc γRIIb, is expressed on plasma cells, controls their persistence in the bone marrow (BM) and their ability to produce serum Ig. Activation of Fc γRIIb leads to the phosphorylation of ITIM and recruitment of SH2-containing inositol 5'-phosphatase (SHIP) in plasma cells. Immunoreceptor tyrosine-based activation motif (ITAM) and ITIM provide the basis for two opposing signaling modules that duel for control of plasma cell activation. Fc γRIIb-mediated SHIP phosphorylation activates downstream ITAM or ITIM signaling. To determine whether multiple myeloma (MM) cells express Fc γRIIb, we performed immunohistochemical staining on bone marrow mononuclear cells from MM patients and controls. We found that not only CD20+ B cells expressed Fc γRIIb but more importantly CD138+ cells from MM patients also showed expression of this receptor. Next, we examined whether Fc γRIIb was present and expressed in CD138+ primary MM cells purified from fresh MM BM and the MM cell lines MM1s, RPMI8226, and U266 using PCR and RT-PCR on DNA and mRNA, respectively. We focused on the transmembrane domain of the Fc γRIIb gene with four primers from different parts of this domain since this portion plays a critical role in this receptor's function. The MM cell lines expressed different amounts of Fc γRIIb. Notably, we found that 17% (5/30) of MM patients showed absence of Fc γRIIb both using RT-PCR for mRNA and PCR for DNA. Moreover, use of these same primers on nonmalignant PBMCs from the MM patients also showed absence of this gene in the same five patients. As a result of these findings, we are currently sequencing Fc γRIIb in MM patients to determine if additional patients show mutational changes that affect the function of this receptor. We also further determined SHIP-1 phosphorylation using Western blot analysis since this protein mediates downstream signaling of Fc γRIIb. Following stimulation with Fc complexes, phosphorylation of SHIP-1 was markedly reduced in MM tumor cells compared to normal CD20+ B cells. Interestingly, the patients with missing Fc γRIIb expressed higher levels of SHIP-1 gene expression compared to patients with normal Fc γRIIb expression. We investigated the IgG-binding ability of MM patients (n=33) and normal donors (n=33) to Fc γRIIb. Each serum sample was incubated with cells from MHC1, a cell line that specifically expresses Fc γRIIb but not Fc γRI and Fc γRIIa. The results showed MM patients' serum IgG have much lower Fc γRIIb-binding ability than normal human IgG (P<0.05) by using both flow cytometric and immunofluorescence assays. Our findings suggest that the monoclonal protein produced by MM patients has a very low Fc γRIIb-binding ability and is incapable of signaling through the inhibitory ITIM pathway. Germline loss of Fc γRIIb in MM patients with variation in its expression level and its downstream signaling molecule SHIP and its phosphorylation as well as the inability of MM IgG to bind cells containing this receptor is a potential new mechanism that contributes to the uncontrolled growth of MM. Disclosures: Berenson: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Medtronic: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck: Research Funding; Genentech: Research Funding.

The critical role of surgical pathology in personalized medicine: The impact of biopsy cavities in breast cancer samples on recurrence risk when assessed by quantitative RT-PCR

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e22016-e22016
Author(s):  
F. L. Baehner ◽  
J. Anderson ◽  
C. Millward ◽  
C. Sangli ◽  
C. Quale ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Expression Profiles ◽  
Critical Role ◽  
Recurrence Score ◽  
Pcr Analysis ◽  
Breast Cancers ◽  
Rt Pcr ◽  
Poorly Differentiated ◽  
The Impact

e22016 Background: Tumor gene expression analysis using the Recurrence Score (RS) assay is frequently used in ER+ breast cancer. Manual microdissection is performed in cases where biopsy cavities (BxC) are present in the submitted specimen. The objective of this was to characterize by quantitative RT-PCR the impact of BxC on 21 gene expression profiles and the RS. Methods: 48 (15 well, 18 moderate, and 15 poorly differentiated) breast cancers were evaluated for gene expression differences between whole sections (WS; containing BxC) and enriched tumor (ET; BxC excluded). Standardized quantitative RT-PCR analysis for the 21 Oncotype DX genes was performed; reference normalized gene expression measurements ranged from 0 to 15, where each 1-unit reflects an approximate 2-fold change in RNA. Analyses of individual genes and the RS were performed on the entire sample set and stratified by tumor grade. Correlation analyses used Pearson's R, concordance analysis used Lin's sample concordance and paired t- tests to characterize differences. Results: There were statistically significant differences in reference normalized gene expression between ET and WS in 6 genes: BAG1 (ET-WS: 0.13 units, p=0.0025), CD68 (ET-WS: -0.64 units, p<0.0001), ER (ET-WS: 0.29 units, p=0.0012), GSTM1 (ET-WS: 0.18 units p=0.0025), STK15 (ET-WS: -0.18 units, p=0.0041) and STMY3 (ET-WS: 0.62 units, p<0.0001). Expression of the macrophage marker CD68 was higher and expression of ER was lower in WS containing BxC. The correlation (0.95) and concordance (0.92) were generally high between WS and ET for RS overall however among moderately differentially tumors, there was a statistically significant mean increase in RS for WS of 3.3 units (p = 0.0012) while among poorly differentiated tumors there was a trend toward a statistically significant decrease in RS for WS of 2.2 units (p=0.0569). Conclusions: Histologic identification of invasive carcinoma and exclusion of BxC is essential for precise RS assessment. Inclusion of BxC in breast cancer specimens is associated with significant changes in the expression of individual genes and impacts the RS. Removal of BxC from breast cancer specimens assessed for gene expression levels is warranted. [Table: see text]

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