Finding of bands of higher molecular weight than expected in three proteins in bovine preimplantation embryos

Zygote ◽  
2019 ◽  
Vol 27 (3) ◽  
pp. 187-189
Author(s):  
Veronika Kinterova ◽  
Veronika Petruskova ◽  
Jiri Kanka ◽  
Tereza Toralova
Keyword(s):  
Molecular Weight ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Fibroblast Cells ◽  
Cell Stage

SummaryWe report here the existence of bands of higher molecular weight after western blot analysis in three proteins – Skp1, p27 and IκBα in bovine preimplantation embryos. This finding is specific to preimplantation embryos (from the 2-cell stage to the blastocyst stage) and not differentiated fibroblast cells in which these bands were of expected molecular weight. We suggest that these bands of higher molecular weight represent a complex of proteins that are characteristic of preimplantation embryos.

Immunocharacterization ofTaenia soliumoncosphere and metacestode antigens

1996 ◽  
Vol 70 (4) ◽  
pp. 271-280 ◽  
Author(s):  
C. Garcia-Allan ◽  
N. Martínez ◽  
A. Flisser ◽  
A. Aluja ◽  
J.C. Allan ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Western Blot ◽  
Experimental Infection ◽  
Blot Analysis ◽  
Post Infection ◽  
Western Blot Analysis ◽  
Taenia Solium ◽  
Specific Antibodies ◽  
Specific Antigens ◽  
Related Increase

AbstractA partial immunocharacterization of oncosphere and metacestode antigens ofTaenia soliumwas carried out and compared to antigens from other taeniid species. The results indicated thatT. soliummetacestode antigen contained epitopes cross reactive with rabbit anti-sera to adult and oncospheral stages of the parasite. Oncospheres, however, consisted largely of stage specific antigens. Western blot analysis indicated thatT. soliumandT. pisiformisshared several oncospheral antigens; however, this was not the case withT. soliumandT. hydatigena. Western blot analysis showed a time-related increase in the number of molecules recognized by antibodies toT. soliumoncosphere and metacestode antigens in pigs experimentally infected withT. soliumeggs. Oncosphere specific antibodies were detected in pig sera one month after experimental infection whereas antibodies to cystic stage antigens were not present until the 3rd to 5th month post infection. Sera from neurocysticercotic patients as well as naturally infected cysticercotic pigs recognized high molecular weight antigens in the oncospheres.

Transfusion Of RBCs With Low-Density KEL Induces Tolerance To The KEL Antigen

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1160-1160
Author(s):  
Sean R. Stowell ◽  
Connie M Arthur ◽  
Nicole H. Smith ◽  
Kathryn R. Girard-Pierce ◽  
James C. Zimring ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Antibody Formation ◽  
Research Funding ◽  
Flow Cytometric Analysis ◽  
P Value ◽  
The Impact ◽  
Rbc Transfusion

Abstract Background Patients requiring repeat transfusion often develop RBC specific alloantibodies that decrease the therapeutic efficacy of transfused cells and limit the availability of compatible RBCs for future transfusion. However, not all RBC antigens possess equal ability to induce alloantibodies. While many factors likely influence this process, several studies suggest that antigen density may independently influence rates of RBC alloimmunization. To directly examine this, we generated transgenic founders with normal or lower levels of the human KEL antigen specifically on RBCs and examined the impact of RBC antigen levels on the development of anti-KEL antibodies following transfusion. Materials and methods Transgenic C57BL/6 founders expressing the human KEL antigen specifically on RBCs were generated using the β-globin promoter and screened for levels of KEL antigen using monoclonal anti-KEL antibodies by flow cytometric analysis. The number of KEL antigens on RBCs isolated from different founders was estimated using QIFIKIT beads. The molecular weight of KEL on RBCs isolated from each founder was assessed by Western blot analysis. C57BL/6 recipients were transfused with RBCs that expressed normal levels of KEL (KEL RBCs) or reduced levels of KEL (KELlo RBCs), followed by harvesting blood on days 3, 5, 7, 14, 21 and 28 following transfusion and analysis of serum for anti-KEL antibodies by indirect immunofluorescence using flow cytometry with KEL and control C57BL/6 RBCs as targets. In addition, C57BL/6 recipients were transfused with KELlo RBCs followed by KEL RBCs and similar analysis for anti-KEL antibody formation on days 3, 5, 7, 14, 21 and 28 following KEL RBC transfusion. All experiments were completed at least three times with 3–5 recipients per group per experiment. Results While KEL RBCs express approximately 1200 antigens per cell, KELlo RBCs express fewer than 200 KEL antigens. However, each KEL transgenic expressed a KEL protein of the predicted molecular weight (83 kD) as assessed by Western blot analysis. Transfusion of KEL RBCs induced IgM anti-KEL antibodies as early as 3 days post transfusion followed by peak IgG anti-KEL antibody levels 14 days following transfusion. In contrast, transfusion of KELlo RBCs failed to induce detectable IgM or IgG anti-KEL antibody formation following transfusion. Similarly, while antibodies could be detected on the surface of KEL RBCs following the development of detectable anti-KEL antibodies in the serum, no antibodies could be detected on KELlo RBCs following transfusion, although anti-KEL generated following KEL RBC transfusion readily bound KELlo RBCs in vitro. Although subsequent KEL RBC exposure following initial KEL RBC transfusion induced considerable increases in anti-KEL antibody formation, KEL RBC transfusion following initial KELlo RBCs transfusion completely failed to induce detectable IgM or IgG anti-KEL antibody formation. (All the above differences achieved a p value of <0.05). Conclusions These results suggest that RBC alloantigen density may significantly impact the immunological outcome of RBC transfusion. KELlo RBC transfusion not only failed to induce anti-KEL antibodies, but also induced an apparent state of tolerance to KEL RBCs following subsequent KEL RBC transfusion. Thus, antigen density may not only influence whether RBC alloimmunization occurs, but may also alter a recipient’s subsequent response to the same antigen. These results also suggest that manipulation of RBCs to express lower levels of RBC antigens may provide a unique tool to tolerize individuals against RBC alloantigens. Disclosures: Zimring: Immucor Inc.: Research Funding; Terumo: Research Funding; Haemonetics: Consultancy; Cerus: Honoraria.

scholarly journals A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis

PLoS ONE ◽  
2016 ◽  
Vol 11 (8) ◽  
pp. e0160418 ◽  
Author(s):  
Wen-Wei Lin ◽  
I-Ju Chen ◽  
Ta-Chun Cheng ◽  
Yi-Ching Tung ◽  
Pei-Yu Chu ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Secondary Antibody ◽  
Rabbit Igg ◽  
Linear Epitopes

scholarly journals 233 EXPRESSION AND LOCALIZATION OF DNMT1 DURING EARLY BOVINE DEVELOPMENT

2005 ◽  
Vol 17 (2) ◽  
pp. 267 ◽  
Author(s):  
D.F. Russell ◽  
D.H. Betts
Keyword(s):  
Dna Methylation ◽  
Embryonic Development ◽  
Western Blot ◽  
Real Time ◽  
Blot Analysis ◽  
Real Time Pcr ◽  
Western Blot Analysis ◽  
Cell Stage ◽  
Rna Levels

DNA methylation of CG motifs is an important mechanism of transcriptional regulation. During embryonic development DNA methyltransferase 1 (DNMT1) has been implicated in the maintainance of gametic and embryonic epigenetic patterns. Here we report the characterization of DNMT1 expression patterns within in vitro-produced (IVP) bovine embryos. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries and either denuded (germinal vesicle, GV) or matured in vitro for 24 h (metaphase II, MII). Embryos at the 1-, 2-, 4-, 8-, 16-cell stage, and morula (Mo, Day 6 post-insemination, p.i.) and blastocyst (Days 7, 8, and 9 p.i.) stages were produced by in vitro fertilization and cultured in SOFm for appropriate times under a 5% O2, 5% CO2, and 90% N2 atmosphere. Oocytes/embryos were either snap frozen (−80°C) for DNMT1 mRNA and protein expression profiles analysis or wholemount fixed and permeabilized for DNMT1 immunostaining followed by laser confocal microscopy. DNMT1 RNA, determined by real-time PCR analysis, was present throughout bovine embryonic development (replications = 3, n = 10 embryos/pool and normalized by Histone H2A expression). Growing oocytes accumulated DNMT1 transcripts until the MII stage (20-fold increase), whereas after fertilization DNMT1 RNA levels decreased and remained constant until the 16-cell stage when DNMT1 RNA levels decreased further and then remains constant until the blastocyst (Day 8 p.i.) stage. Confocal analysis of DNMT1 immunostained oocytes/embryos (n = 20/stage) revealed that DNMT1 is localized in the cytoplasm of the oocyte and pre-implantation embryo with the exception of the 16-cell stage, when the enzyme is translocated to nucleus (confirmed by Hoechst co-localization). Moreover, a punctuate staining pattern was observed for DNMT1, which could be due to its association with the mitochondria or endoplasmic reticulum. Interestingly, in GV oocytes DNMT1 was present in localized areas of the nucleus, suggestive of nucleoli association. DNMT1 was observed in the majority of the nuclei in early blastocysts, while after expansion, DNMT1 accumulated in the cytoplasm of the trophectoderm and was localized in both the cytoplasm and the nucleus of the majority of cells within the inner cell mass. Western blot analysis revealed low levels of DNMT1 protein in oocytes and pre-implantation embryos with the exception of the 16-cell embryos and Mo stages during which a significant (P < 0.05) increase in the levels of DNMT1 protein was observed. Specificity of primers and conditions for the real-time PCR assay were confirmed by cDNA sequencing whereas specificity of the antibody used for immunofluorescence and western blot analysis was confirmed by amino acid sequencing. These results suggest the participation of DNMT1 in the bovine embryonic genome activation process, supporting a passive DNA demethylation process during the early cleavage stages in the bovine. The low expression profile of DNMT1 after morula stage indicates that other methylases are required for the maintenance of DNA methylation during blastocyst formation and expansion. This work was funded by CFIA, NSERC, CIHR, and OMAF.

241 GENE EXPRESSION OF DNMT1 ISOFORMS IN PORCINE OOCYTES, EMBRYOS, AND SOMATIC CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 278
Author(s):  
A. M. Giraldo ◽  
M. V. Mendicino ◽  
A. M. Vance ◽  
D. L. Ayares
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Early Embryos ◽  
Genomic Methylation

In human, mouse, and some marsupials, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (DNMT1o) enzyme. The identification and characterization of a similar oocyte transcript variant in farm animals would greatly contribute to the understanding of the methylation processes that occur during nuclear remodeling of in vivo embryos as well as in cloned embryos. The objectives of this study were to identify the alternative splicing variants of DNMT1 in porcine oocytes and determine the gene expression pattern of the different DNMT1 isoforms during embryo development. Total RNA was isolated from a pool of 50 zona free mature porcine oocytes using the Trizol method. A RACE System (Invitrogen, Carlsbad, CA) was used to amplify the 5′ cDNA end of DNMT1. Polymerase chain reaction products were separated by electrophoresis, recovered from the agarose gel, subcloned into a cloning vector, and sequenced. The location of exons and introns of every transcript variant was determined by aligning the 5′RACE-derived sequences to the Sus scrofa DNMT1 genomic sequence located on chromosome 2 (CU462940). RNA levels of the DNMT1 isoforms were analyzed in porcine oocytes and in vivo embryos, as well and in cells from ovary, lung, spleen, liver, heart, and skin by quantitative PCR using the AACT method. DNMT1 protein levels of porcine oocytes and different somatic cells types were analyzed by Western blot. Two new DNMT1o RNA isoforms were identified (EU908730 and EU908731). The previously reported DNMT1s isoform (DQ060156) was expressed at low but constant levels in oocytes, as well as in embryos from the 2-cell to the blastocyst stage. Abundant RNA levels of EU908730 and EU908731 were detected in oocytes and embryos from the 2-cell to the 8- to 16-cell stage. Levels of these DNMT1o transcripts were low at the morula and blastocyst stage. Although DQ060156 was present in all the somatic cell types analyzed, EU908730 and EU908731 were not detected in any somatic tissues. As predicted by the RNA sequence and verified by Western blot analysis, EU908730 and EU908731 RNAs translate one DNMT1o enzyme (ˆ170 KDa). Western blot analysis confirmed that both the oocyte and the somatic forms of DNMT1 protein are present in porcine oocytes and early embryos, while somatic cells produce only DNMT1s protein. In conclusion, this study demonstrates that porcine oocytes express a DNMT1o RNA in addition to the DNMT1s somatic transcript. The 2 newly identified RNA isoforms are produced by alternative splicing of the DNMT1 gene and translate a DNMT1o protein that is unique to oocytes and early embryos. Analysis of the oocyte and somatic DNMT1 isoforms in pre-implantation embryos will determine the expression pattern of this transcript during genomic methylation and its involvement during nuclear reprogramming and cellular differentiation.

scholarly journals Spliced mRNA Encoding the Murine Cytomegalovirus Chemokine Homolog Predicts a β Chemokine of Novel Structure

1999 ◽  
Vol 73 (5) ◽  
pp. 3682-3691 ◽  
Author(s):  
Margaret R. MacDonald ◽  
Mary W. Burney ◽  
Stuart B. Resnick ◽  
Herbert W. Virgin
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blot ◽  
Molecular Mass ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Stop Codon ◽  
Murine Cytomegalovirus ◽  
Fibroblast Cells ◽  
Cos Cells ◽  
Amino Terminal

ABSTRACT A viral mRNA of the late kinetic class expressed by murine cytomegalovirus (MCMV) contains an open reading frame (ORF) whose predicted protein, designated MCK-1, has homology to β chemokines (M. R. MacDonald, X.-Y. Li, and H. W. Virgin IV, J. Virol. 71:1671–1678, 1997). The present study analyzed further the structure of the transcript in infected fibroblast cells. A splicing event removed the MCK-1 stop codon, bringing a downstream ORF into frame with the chemokine homolog and demonstrating that the MCK-1 ORF was an exon of a larger gene. The predicted 31.4-kDa protein, designated MCK-2, contains a putative amino-terminal signal sequence and a β chemokine domain, followed by a carboxyl-terminal domain without significant homology to known proteins. Quantitative analysis of mRNA forms in MCMV-infected fibroblast cells at late times after infection indicated that the viral chemokine RNA was predominantly spliced. There was no evidence for expression of RNA encoding either MCK-1 or MCK-2 at immediate early or early times after infection with MCMV. Monoclonal antibodies generated against bacterially expressed MCK-2 recognized multiple proteins in the range of ∼30 to ∼45 kDa in Western blot analysis of MCK-2 expressed in transfected COS cells. The monoclonal antibodies immunoprecipitated a similar group of proteins in transfected COS cells metabolically labeled with radioactive cysteine. Radiolabelled protein of apparent higher molecular mass was immunoprecipitated from culture medium overlying the transfected cells, suggesting that posttranslationally modified MCK-2 can be secreted. Two proteins with apparent molecular mass suggestive of posttranslational modification were detected by Western blot analysis of cells harvested at late times after infection with MCMV. These studies show that MCMV encodes and expresses a β chemokine homolog with a novel predicted structure.

Sign in / Sign up

Export Citation Format

Share Document