scholarly journals Cloning, Sequencing, and Expression of the Leukotoxin Gene from Fusobacterium necrophorum†

2001 ◽  
Vol 69 (9) ◽  
pp. 5447-5455 ◽  
Author(s):  
Sanjeev Kumar Narayanan ◽  
T. G. Nagaraja ◽  
M. M. Chengappa ◽  
George C. Stewart
Keyword(s):  
Molecular Weight ◽  
Western Blot ◽  
Sequence Similarity ◽  
Blot Hybridization ◽  
Flow Cytometric Analysis ◽  
Southern Blot Hybridization ◽  
Fusobacterium Necrophorum ◽  
Inverse Pcr ◽  
Western Blot Assay ◽  
Polyclonal Antisera

ABSTRACT Fusobacterium necrophorum is a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF; lktA) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the wholelktA ORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin fromF. necrophorum culture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed in Escherichia coli. Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against the F. necrophorum leukotoxin. Southern blot hybridization analysis revealed different patterns of lktAhybridizing bands between isolates of the two subspecies ofF. necrophorum.

scholarly journals Comparison of Diagnostic Techniques for Detecting Tomato Infectious Chlorosis Virus

Plant Disease ◽  
1998 ◽  
Vol 82 (1) ◽  
pp. 84-88 ◽  
Author(s):  
R. H. Li ◽  
G. C. Wisler ◽  
H.-Y. Liu ◽  
J. E. Duffus
Keyword(s):  
Western Blot ◽  
Blot Hybridization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Dot Blot ◽  
Tomato Plants ◽  
Dot Blot Hybridization ◽  
Field Samples ◽  
Infected Tomato ◽  
Tomato Infectious Chlorosis Virus

A polyclonal antiserum prepared against purified virions of tomato infectious chlorosis virus (TICV) was used to evaluate serological tests for its detection, to determine its distribution in infected plants, to study relationships among isolates of this virus, and to detect it in field samples. A cRNA probe representing TICV RNA 1 and RNA 2 was used in dot blot hybridization tests. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was also developed for detection of TICV isolates. The comparative study of these four techniques indicated that RT-PCR was 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA), Western blot, and dot blot hybridization assays for TICV detection. TICV was detected in leaf, stem, flower, and root tissues of the infected tomato plants. However, the virus was not uniformly distributed throughout the infected tomato plants, and the highest viral concentration was observed in fully developed young tomato leaves at the onset of yellowing symptoms. The virus was detected by indirect ELISA, Western blot, dot blot hybridization, and RT-PCR assays in laboratory-infected tomato, tomatillo, potato, and Nicotiana clevelandii and in naturally infected tomato, petunia, and Ranunculus sp. plants obtained from commercial sources. These tests indicate that there are apparently no detectable serological or nucleic acid differences among four TICV isolates obtained from Orange and Yolo Counties of California or from North Carolina or Italy.

scholarly journals The induction of the synthesis of interferon, CD4 and CD8 t lymphocytes in blood and spleen of mice after oral vaccination with the “early” protein HPV16 E2

2019 ◽  
Vol 488 (3) ◽  
pp. 333-337
Author(s):  
R. K. Salyaev ◽  
N. I. Rekoslavskaya ◽  
A. S. Stolbikov
Keyword(s):  
T Lymphocytes ◽  
Mononuclear Cells ◽  
Blot Hybridization ◽  
Oral Vaccination ◽  
Enzyme Immunoassays ◽  
Early Protein ◽  
Cd8 T Lymphocytes ◽  
Western Blot Hybridization ◽  
Characteristic Features ◽  
Very High

Very high contents of the interferon, CD4 and CD8 T lymphocytes were found in blood and spleen cells after the oral vaccination of mice with the “early” protein of high-risk oncogenic papillomavirus HPV16 E2 by using immunoassays analyses: ELISPOT for the detection of the content of interferon, Western-blot hybridization and enzyme immunoassays for analyses of СD4 and CD8 T lymphocytes. Both blood cells and splenocytes showed high content of alive mononuclear cells during ELISA and ELISPOT analyses that had very characteristic features for T lymphocytes. The ratio of CD4/CD8 was very close to 1 after the quanttitive determination by the enzyme immunoassays with appropriate antibodies. It was concluded that the “early” protein HPV16 E2 participated in the activation of the immune system during pathogenesis and neoplasia.

scholarly journals Genome-wide identification and expression profiling of duplicated flavonoid 3'-hydroxylase gene family in Carthamus tinctorius L.

2021 ◽  
Vol 49 (4) ◽  
pp. 12509
Author(s):  
Nguyen Q. V. HOANG ◽  
Kong JIE ◽  
Naveed AHMAD ◽  
Ma XINTONG ◽  
Zhang XINYUE ◽  
...  
Keyword(s):  
Plant Species ◽  
Blot Hybridization ◽  
Carthamus Tinctorius ◽  
Fusion Construct ◽  
Protein Motifs ◽  
Functional Studies ◽  
Genome Wide ◽  
Western Blot Hybridization ◽  
Onion Epidermal Cells ◽  
Encoding Genes

Flavonoid 3′-hydroxylase (F3’H) enzyme is essential in determining the flavonoids B-ring hydroxylation pattern. It is mainly implicated in the biosynthetic pathway of cyaniding-based anthocyanins, flavonols, and flavan-3-ols. However, the evolution and regulatory mechanism of these important flavonoid hydroxylases have not been systematically investigated in safflower (Carthamus tinctorius L.). In this study, we identified 22 duplicatedCtF3'H-encoding genes from safflower through genome-wide prediction and conservation analysis. Phylogenetic analysis revealed the pattern of conservation and divergence of CtF3'Hs encoding proteins and their homologs from different plant species. The distribution of conserved protein motifs and cis-regulatory units suggested several structural components that could be crucial in deciphering the final function of CtF3'H proteins. Furthermore, the results of RNA-seq and qRT-PCR assay in different flowering tissues suggested differential expression level of CtF3’H genes during flower development. Based on the unique homology of CtF3’H5 with flavonoid 3’ hydroxylases from other plant species, further validation of CtF3’H5 was carried out. The transient expression of CtF3’H5 in onion epidermal cells implied that the subcellular localization of the fusion construct containing CtF3’H5 and GFP was predominantly detected in the plasma membrane. Similarly, the prokaryotic expression and western blot hybridization of CtF3’H5 demonstrated the detection of a stable 50.3kD target protein. However, more efforts are needed to further extend the functional validation of CtF3’H5 in safflower. This study provides a fundamental gateway for future functional studies and understanding the genetic evolution of F3'Hs in plants.

Vorkommen von Antikörpern gegen das bovine Immunschwächevirus bei Rindern in Bayern

2003 ◽  
Vol 31 (05) ◽  
pp. 248-253
Author(s):  
Maren Bartels ◽  
Katrin Hartmann ◽  
L. Scobie ◽  
O. Jarrett ◽  
W. Klee
Keyword(s):  
Western Blot

ZusammenfassungIm Rahmen einer epidemiologischen Untersuchung zur Infektion mit dem bovinen Immunschwächevirus (BIV) bei Rindern in Oberbayern erfolgten zwei Studien, in denen Serum mittels indirektem ELISA auf BIV-Antikörper untersucht wurde. Die ELISA-Ergebnisse der BIV-positiven Tiere der Studie I wurden mittels Western Blot bestätigt. In Studie I wurde Blut von 173 ungezielt ausgewählten Rinderpatienten der II. Medizinischen Tierklinik der Ludwig-Maximilians-Universität München untersucht. Von diesen waren acht Tiere BIV-infiziert. Das entspricht einer Prävalenz von 4,6%. Alle positiven Tiere waren über zwei Monate alt. In Studie II wurden 550 Kühe aus 11 oberbayerischen landwirtschaftlichen Betrieben untersucht. Hiervon waren 11 Tiere BIVAntikör-perpositiv. Dies entspricht einer Prävalenz von 2,0%. Die positiven Tiere stammten aus fünf Betrieben mit Boxenlaufstallhaltung. Kein Tier aus Betrieben mit Anbindehaltung war positiv. In Studie II lag das Durchschnittsalter der Kühe aus den Betrieben ohne BIV-infizierte Tiere signifikant höher als in den Betrieben mit BIV-infizierten Tieren. Die Prävalenz von BIV-Antikörpern war zwar unter den kranken Probanden aus Studie I signifikant höher als bei den klinisch unauffälligen Rindern der Studie II, die pathogene Bedeutung des BIV erscheint jedoch fraglich.

Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

1989 ◽  
Vol 61 (03) ◽  
pp. 437-441 ◽  
Author(s):  
Cindra Condra ◽  
Elka Nutt ◽  
Christopher J Petroski ◽  
Ellen Simpson ◽  
P A Friedman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Salivary Gland ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Potent Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Sds Page ◽  
Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

Western Blot Analyses of Prekallikrein and its Activation Products in Human Plasma

1991 ◽  
Vol 65 (04) ◽  
pp. 382-388 ◽  
Author(s):  
Dulce Veloso ◽  
Robert W Colman
Keyword(s):  
Complex Formation ◽  
Western Blot ◽  
Human Plasma ◽  
Contact System ◽  
Plasma Activation ◽  
Normal Plasma ◽  
Sds Page ◽  
Activation Products ◽  
Major Antigen ◽  
Formation Of Complexes

SummaryPrekallikrein (PK), a zymogen of the contact system, and its activation products, kallikrein (KAL), KAl-inhibitor complexes and fragments containing KAL epitope(s) have been detected in human plasma by immunoblotting with a monoclonal anti-human plasma PK antibody, MAb 13G1L. Detection of antigen-MAb 13G11 complexes with peroxidase-conjugated anti-IgG showed that the two variants of PK (85- and 88-kDa) are the only major antigen species in normal, non-activated plasma. Upon plasma activation with kaolin, the intensity of the PK bands decreased with formation of complexes of KAL with CL inhibitor (C1 INH) and α2-rrtzcroglobulin (α2M) identical to those formed by the purified proteins. Immunoblots of normal plasma showed good correlation between the PK detected and the amount of plasma assayed. Increasing amounts of KAL incubated with a constant volume of PK-deficient plasma showed increasing amounts of KAL and of KAL-C1 INH and KAL-α2M complexes. Complexes of KALantithrombin III (ATIII) and the ratio of KALα2M/ KAL-CL INH were higher in activated CL INH-deficient plasmas than in activated normal plasmas. Protein resolution by 3-12% gradient SDS-PAGE and epitope detection with [125I]MAb 13G11 showed four KALα2M species and a 45-kDa fragment(s) in both surface-activated normal plasma and complexes formed by purified KAL and α2M. Immunoblots of activated plasma also showed bands at the position of KALCL INH and KALATIII complexes. When α1-antitrypsin Pittsburgh (cα1-AT, Pitts) was added to plasma before activation, KAL-α1-ALPitts was the main complex. The non-activated normal plasma revealed only an overloaded PK band. This is the first report of an antibody that recognizes KAL epitope(s) in KAL-α2M, KALATIII and KALa1-α1Pitts complexes and in the 45-kDa fragment(s). Therefore, MAb 13G11 should be useful for studying the structure of these complexes as well as the mechanism of complex formation. In addition, immunoblotting with MAb 13G11 would allow detection of KAl-inhibitor complexes in patient plasmas as indicators of activation of the contact system.

Differences in the Interactions of Lupus Anticoagulant IgG with Human Prothrombin and Bovine Prothrombin

1995 ◽  
Vol 73 (04) ◽  
pp. 668-674 ◽  
Author(s):  
L Vijaya Mohan Rao ◽  
An D Hoang ◽  
Samuel I Rapaport
Keyword(s):  
Western Blot ◽  
Lupus Anticoagulant ◽  
Protein A ◽  
Phospholipid Complex ◽  
Experimental Conditions ◽  
Bovine Plasma ◽  
Activation System ◽  
Rate Limiting ◽  
Substantial Extent ◽  
Prothrombin Activation

SummaryLupus anticoagulant (LA) IgGs have been reported to inhibit more effectively and consistently the Xa/Va/phospholipid complex-catalyzed activation of human prothrombin than the Xa/Va/phospholipid complex-catalyzed activation of bovine prothrombin. This led us to carry out studies to determine whether the ability to inhibit the activation of prothrombin of LA IgGs, separated from the plasma of 15 patients by protein A affinity chromatography, could be related to the ability of the LA IgGs to bind to prothrombin under various experimental conditions. Of 14 LA IgG preparations tested all prolonged to a variable but substantial extent the dilute Russell’s viper venom time (dRVVT) of human plasma but only minimally prolonged the dRVVT of bovine plasma. In a purified prothrombin activation system with a rate limiting concentration of phospholipid, all 15 LA IgG preparations inhibited the activation of human prothrombin with the majority showing >50% of inhibition. In contrast, only one LA IgG markedly inhibited (>50%) the activation of bovine prothrombin and five others moderately inhibited (25-40%) the activation of bovine prothrombin. Nevertheless, the majority of LA IgG preparations bound to immobilized bovine prothrombin on a Western blot and also to immobilized bovine prothrombin on a microtiter well. In an ELISA in which phosphatidylserine (PS) was immobilized on microtiter wells, bovine prothrombin supported the binding of 10 of 15 LA IgG preparations to PS. However, the extent of binding was lower than that observed with human prothrombin. In experiments with 125I-human prothrombin or 125I-bovine prothrombin in a solution containing Ca2+, the addition of PS/PC vesicles enhanced the binding of both human and bovine prothrombin to some LA IgG preparations. The enhanced binding was particularly evident for bovine prothrombin. Although seemingly related for some preparations, the ability of a LA IgG to bind to bovine prothrombin, either in the presence or absence of PS, and the ability of that LA IgG to inhibit the activation of bovine prothrombin was not consistently related for all preparations.

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