Characterization of anti-Neospora caninum hyperimmune rabbit serum by Western blot analysis and immunoelectron microscopy

1992 ◽  
Vol 78 (8) ◽  
pp. 689-694 ◽  
Author(s):  
J. R. Barta ◽  
J. P. Dubey
Keyword(s):  
Western Blot ◽  
Immunoelectron Microscopy ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Neospora Caninum ◽  
Rabbit Serum ◽  
Hyperimmune Rabbit

scholarly journals Postnatal development and testosterone dependence of a rat epididymal protein identified by neonatal tolerization

Reproduction ◽  
2003 ◽  
pp. 495-507 ◽  
Author(s):  
SA Joshi ◽  
S Shaikh ◽  
S Ranpura ◽  
VV Khole
Keyword(s):  
Western Blot ◽  
Postnatal Development ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyclonal Antiserum ◽  
Cognate Gene ◽  
Specific Proteins ◽  
Androgen Regulation ◽  
Dose Dependent

A rat epididymal protein of 27 kDa was identified using neonatal tolerization. This study reports the production and characterization of a polyclonal antiserum to this protein. ELISA was used to demonstrate that this antiserum reacts strongly with epididymal sperm proteins, but has little or no reactivity with testicular proteins. Western blot analysis revealed that this polyclonal antiserum recognized a 27 kDa protein extracted from the corpus epididymidis as well as from spermatozoa from the corpus and cauda epididymides, and immunostaining revealed the presence of the protein in the corpus to cauda epididymides. Stronger reactivity was observed in the supranuclear region and stereocilla of principal cells of the corpus epididymidis and in the luminal content of the corpus and cauda epididymides. The testicular section showed no reactivity. Treatment with the antiserum resulted in time- and dose-dependent agglutination of rat spermatozoa. By indirect immunofluorescence, the antiserum localized proteins in the mid-piece region of rat spermatozoa. Studies were carried out to determine the age at which the protein first became apparent during postnatal development. The protein was expressed from day 40 onwards, as demonstrated by western blot analysis. The androgen regulation of this protein was ascertained by castration and supplementation studies. Expression of this protein showed a decline starting at day 14 after castration and by day 21 the protein was absent; however, androgen replacement resulted in the reappearance of the protein. The results of these studies indicate that the protein identified is specific to the epididymis, and is regulated by development and androgens. The importance of epididymis-specific proteins that are regulated by androgens in sperm maturation is discussed, and the need to ascertain the sequence of the protein and clone the cognate gene is indicated.

Phenotypic, Genotypic, and Functional Characterization Of AML-Derived Endothelial Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1411-1411
Author(s):  
Russell J Pizzo ◽  
Myra Coppage ◽  
Karen Rosell ◽  
Kimberly Morse ◽  
Jane L. Liesveld
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Leukemia Cell ◽  
Normal Subjects ◽  
Rt Pcr ◽  
Altered Expression ◽  
Normal Subject

Abstract Background In addition to participation in homing, egress, and transmigration of hematopoietic cells, marrow endothelium also contributes to regulation of hematopoiesis with effects on cell proliferation and survival. Characteristics of marrow—derived endothelial cells from normal subjects have been described (Blood 1994; 84: 10-19), but characterization of endothelial cells in leukemia states is incomplete. Angiogenesis is known to be increased in AML marrows, and circulating endothelial progenitors are increased and correlate with disease status and response to treatment. Furthermore, cytokines secreted by endothelial cells such as vascular endothelial growth factor (VEGF) have been found to serve as growth factors for leukemia, sometimes in a paracrine or autocrine fashion. Despite these findings, inhibition of VEGF with agents such as bevacizumab has not demonstrated clinical anti-leukemia activity. Since our group and others have shown that endothelial cells from multiple vascular beds (human umbilical vein endothelial cells—HUVECs), human microvascular endothelial cells derived from skin (HMEC-1 cell line), and normal subject—derived endothelial cells are able to prevent spontaneous or therapy-induced apoptosis in AML blasts, it is important to understand the phenotype and characteristics of endothelial cells isolated from AML patients to understand their functional roles and to see if they might have an angiogenic gene expression profile as has been described in multiple myeloma (Clin Cancer Res 2009 15:5369). Methods Endothelial cells were purified from marrow aspirates obtained with consent from normal subjects or from newly diagnosed AML patients. Cells were isolated using anti-CD105-PE (BD Bioscience) followed by anti-PE microbead selection (Miltenyi™) or after disruption of marrow spicules with subsequent selection for endothelial cells in endothelial cell selective medium (EGM-2, Lonza). Cells between 2nd and 4th passage were utilized for analysis. Protein expression was determined by flow cytometry, Western blotting, or RT-PCR. Matrigel™ tubule formation and acetyl-LDL expression were determined as per previously published methods, as were adhesion, CFU-L, and transmigration assays. RNASeq was performed by the Functional Genomics Core at the University of Rochester after extraction of polyadenylated RNA from purified total RNA. Conversion to cDNA occurred with the Illumina TruSeq™ preparation kit, and sequencing was accomplished with the Illumina Genome Analyzer IIx. CASAVA software was utilized for analysis. Results Marrow derived endothelial cells from normal and AML subjects express CD105 (endoglin), CD31(PECAM), CD106 (VCAM), CD146 (MCAM), CD54 (ICAM), and CD34. They do not express CD14 nor CD45, and they demonstrate low level expression of CD144 (VE-cadherin). By RT-PCR, they express Tie-2, VEGF, and eNOS (endothelial nitric oxide synthase). They express acetyl-LDL and form tubular structures in Matrigel™. Phosphorylated components of the mTOR and PI3K/Akt pathways were also expressed by Western blot analysis. Culture of AML cells with endothelial cells from both normal and AML subjects supported adhesion, transmigration, and CFU-L outgrowth, but no significant differences were noted in these functions between normal and AML—derived endothelial cells in vitro assays. RNASeq analysis revealed 130 genes significantly up—or down—regulated in AML derived endothelial cells as compared with those derived from normal marrow. Endothelial cells from both sources had a distinct signature from marrow—derived fibroblasts. The genes differentially expressed (p<0.001) were included in biological function categories involving cancer, cell development, cell growth and proliferation, cell signaling, inflammatory response, and cell death and survival. Further pathway analysis revealed upregulation of c-Fos, and this upregulation in AML vs. normal subject derived endothelial cells was confirmed by Western blot analysis. Genes involved in chemotaxis such as CXCL16 were also upregulated. Conclusions AML—derived endothelial cells exhibit similar phenotype and function as their normal marrow—derived counterparts, but genomic analysis suggests a differential signature with altered expression of genes which could play a role in leukemogenesis or leukemia cell maintenance in the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Partial characterization of 2-β-mercaptoethanol-soluble surface-associated antigens of Onchocerca volvulus

Parasitology ◽  
1991 ◽  
Vol 102 (3) ◽  
pp. 437-444 ◽  
Author(s):  
F. Engelbrecht ◽  
G. Braun ◽  
V. Connor ◽  
M. Downham ◽  
J. A. Whitworth ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Human Infection ◽  
Onchocerca Volvulus ◽  
Rabbit Serum ◽  
Target Antigen ◽  
Molecular Weights ◽  
Igg1 Monoclonal Antibody

Antigens were extracted from the epicuticle/cuticle of intact female Onchocerca volvulus using 2% 2-β-mercaptoethanol and 1% SDS. In Western blot analysis a human infection serum selected for its high antibody titre against whole worm homogenates did not recognize any component solubilized by 1% SDS. However, the same serum did bind at least 7 antigens among the material extracted with 2-β-mercaptoethanol. These antigens have apparent molecular weights (Mr) of: 15 000, 18 000, 28 000, 78 000, 98 000, 120 000 and 200 000. In ELISA using this preparation as target antigen, 151 out of 153 human infection sera gave positive results. An Onchocerca-specific IgG1 monoclonal antibody, designated Cam1, recognized the 28 000 Mr antigen, which is the most prominent antigen detected by Western blot analysis using human infection sera. In ELISA, using material affinity-purified with Cam1 as target antigen, 149 out of 153 human infection sera gave a positive IgG response. From a cDNA library three expressing clones were isolated with a rabbit serum raised against 2-β-mercaptoethanol solubilized material. One of these clones was recognized by the monoclonal antibody Cam1.

Characterization of anti-BRCA2 antibodies in cell lines by Western blot analysis

2001 ◽  
Author(s):  
D. Bernard-Gallon ◽  
L. Cravello ◽  
C. Vissac ◽  
Y.-J. Bignon
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis

Human cyritestin genes (CYRN1 and CYRN2) are non-functional

2001 ◽  
Vol 357 (2) ◽  
pp. 551-556 ◽  
Author(s):  
Pawel GRZMIL ◽  
Youngmin KIM ◽  
Rahman SHAMSADIN ◽  
Jürgen NEESEN ◽  
Ibrahim M. ADHAM ◽  
...  
Keyword(s):  
Western Blot ◽  
Gene Family ◽  
Zona Pellucida ◽  
Blot Analysis ◽  
Male Fertility ◽  
Western Blot Analysis ◽  
Normal Development ◽  
Sperm Protein ◽  
Deficient Mice

The mouse cyritestin gene is a member of the ADAM (adisintegrin and metalloprotease) gene family and codes for a membrane-anchored sperm protein. Recently, it was shown that cyritestin is critical for male fertility in the mouse. Spermatozoa of cyritestin-deficient mice are not able to bind to the zona pellucida of the oocyte and therefore unable to fertilize the egg. However, zona-free oocytes can be fertilized and the resulting embryos show normal development. In contrast to the mouse, where only one gene for cyritestin (Cyrn) is reported, two cyritestin genes (CYRN1 and CYRN2) are known in humans. The human CYRN1 and CYRN2 genes are located on chromosomes 8 and 16, respectively. We report that 27% of fertile men are deficient for the CYRN1 gene but that all have a CYRN2 gene, suggesting that the CYRN2 gene is the orthologous mouse cyritestin gene in humans and might be involved in sperm–egg interactions. However, the characterization of CYRN2 transcripts from testicular RNA of CYRN1-deficient men demonstrated many termination codons in the synthesized cyritestin cDNA. Furthermore, Western-blot analysis with human testicular protein extracts using an anti-cyritestin antibody failed to detect any cyritestin protein. These results demonstrate clearly that both cyritestin genes are non-functional in humans.

scholarly journals Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice

2006 ◽  
Vol 290 (4) ◽  
pp. H1713-H1720 ◽  
Author(s):  
Bunyen Teng ◽  
Habib R. Ansari ◽  
Peter J. Oldenburg ◽  
J. Schnermann ◽  
S. Jamal Mustafa
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Western Blot ◽  
Smooth Muscle Cells ◽  
Knockout Mice ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Muscle Cells ◽  
Positive Staining

Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A1 receptor (A1AR)-knockout (A1KO) mice and their wild-type (A1WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiotics) through the aortic arch. The efflux was collected at 30-, 60-, and 90-min intervals. The cells were centrifuged, and the pellets were mixed with medium [medium 199-F-12 medium with 10% FBS and 2% antibiotics (for endothelial cells) and advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax, and 2% antibiotics (for smooth muscle cells)] and plated. Endothelial cells were characterized by a cobblestone appearance and positive staining with acetylated LDL labeled with 1,1′-dioctadecyl-3,3,3′,3-tetramethylindocarbocyanine perchlorate. Smooth muscle cells were characterized by positive staining of smooth muscle α-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was ∼91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A1WT and A1KO mice. Furthermore, the A1AR was characterized by Western blot analysis using an A1AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.

scholarly journals Identification and Characterization of Epp, the Secreted Processing Protease for the Vibrio anguillarum EmpA Metalloprotease

2008 ◽  
Vol 190 (20) ◽  
pp. 6589-6597 ◽  
Author(s):  
Maureen Varina ◽  
Steven M. Denkin ◽  
Andrew M. Staroscik ◽  
David R. Nelson
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Protease Activity ◽  
Western Blot Analysis ◽  
Culture Supernatant ◽  
Vibrio Anguillarum ◽  
Wild Type ◽  
Processing Protease ◽  
Culture Supernatants

ABSTRACT The zinc metalloprotease EmpA is a virulence factor for the fish pathogen Vibrio anguillarum. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an ∼10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn10Km) clone bank of V. anguillarum was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn10Km was cloned, sequenced, and found to contain epp, an open reading frame encoding a putative protease. Further characterization of epp was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when empA protease activity was induced by salmon gastrointestinal mucus. When the epp mutation was complemented with a wild-type copy of epp (M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the empA mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease.

Sign in / Sign up

Export Citation Format

Share Document