scholarly journals First Report of Barley yellow streak mosaic virus-Infected Barley in Alaska

Plant Disease ◽  
2000 ◽  
Vol 84 (5) ◽  
pp. 595-595 ◽  
Author(s):  
N. L. Robertson ◽  
S. K. Brumfield
Keyword(s):  
Electron Microscopy ◽  
Western Blot ◽  
Mosaic Virus ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Virus Disease ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyclonal Antiserum ◽  
Mechanical Transmission

Barley yellow streak mosaic virus (BaYSMV) was first described and reported in Montana and Alberta, Canada, more than 17 years ago (1). Since then, it has been detected in two other locations: Pocatello Valley, ID (3), and across the border in Utah. BaYSMV has now been found in the Alaskan interior. In July 1999, dry-land barley (Hordeum vulgare L.) growing in University of Alaska-Fairbanks experimental plots exhibited symptoms similar to those described for BaYSMV, including parallel chlorotic streaks and leaf banding. Mechanical inoculation of Nicotiana benthamiana with diseased barley sap produced systemic mosaic symptoms. As previously reported for BaYSMV sap-transmission tests (2), parallel inoculations to barley plants yielded no symptoms. Electron microscopy of leaf dips and minipurifications of infected N. benthamiana revealed long filamentous particles that matched the size and shape reported for BaYSMV (1). Ultrathin sections of diseased barley and N. benthamiana leaves displayed characteristic virus particles. BaYSMV was confirmed by immuno-sorbent electron microscopy assays (4) and western blot analysis with polyclonal antiserum. Long filamentous BaYSMV particles appeared only on grids coated with BaYSMV antiserum and exposed to diseased N. benthamiana sap. Total protein extracts from diseased barley tissue and inoculated N. benthamiana, as well as with protein extracted from partially purified preparations, were applied to a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis minigel and stained with Coomassie blue. Diseased samples, but not healthy controls, contained a protein of ≈33 kDa that was within the size range of a previously described protein from partially purified BaYSMV particles (2). Western blot analysis with an Immuno-Blot alkaline phosphatase assay system (Bio-Rad Laboratories, Hercules, CA) confirmed that the protein reacted with polyclonal BaYSMV. This is the first serological documentation of a BaYSMV-specific protein and that the ≈33-kDa protein is the main antigen recognized by the BaYSMV polyclonal antiserum. Based on virus particle shape and size, symptomology, mechanical transmission host range, and serology, we conclude that BaYSMV is associated with the barley disease observed. Barley yellow streak mosaic virus disease outbreaks are associated with recurring drought and are accompanied by infestations of the brown wheat mite vector, Petrobia latens Müller (1), so it is not surprising that this report coincides with abnormally dry conditions occurring throughout the 1990s in the interior of Alaska. References: (1) N. L. Robertson and T. W. Carroll. Science 240:1188, 1988. (2) N. L. Robertson and T. W. Carroll. Plant Dis. 75:839, 1991. (3) J. S. Skaf et al. Plant Dis. 76:861, 1992. (4) J. S. Skaf and T. W. Carroll. Plant Dis. 79:1003, 1995.

scholarly journals Subcellular Localization of the Intracellular Survival-Enhancing Eis Protein of Mycobacterium tuberculosis

2001 ◽  
Vol 69 (7) ◽  
pp. 4295-4302 ◽  
Author(s):  
John L. Dahl ◽  
Jun Wei ◽  
James W. Moulder ◽  
Suman Laal ◽  
Richard L. Friedman
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intracellular Survival ◽  
Terminal Amino Acid ◽  
Terminal Amino

ABSTRACT Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. It is not clear how M. tuberculosis avoids the destructive action of macrophages, but this ability is fundamental in the pathogenicity of tuberculosis. A gene previously identified in M. tuberculosis, designatedeis, was found to enhance intracellular survival ofMycobacterium smegmatis in the human macrophage-like cell line U-937 (J. Wei et al., J. Bacteriol. 182:377–384, 2000). Wheneis was introduced into M. smegmatis on a multicopy vector, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the appearance of a unique 42-kDa protein band corresponding to the predicted molecular weight of the eisgene product. This band was electroeluted from the gel with a purity of >90% and subjected to N-terminal amino acid sequencing, which demonstrated that the 42-kDa band was indeed the protein product ofeis. The Eis protein produced by M. tuberculosis H37Ra had an identical N-terminal amino acid sequence. A synthetic polypeptide corresponding to a carboxyl-terminal region of the deduced eis protein sequence was used to generate affinity-purified rabbit polyclonal antibodies that reacted with the 42-kDa protein in Western blot analysis. Hydropathy profile analysis showed the Eis protein to be predominantly hydrophilic with a potential hydrophobic amino terminus. Phase separation of M. tuberculosis H37Ra lysates by the nonionic detergent Triton X-114 revealed the Eis protein in both the aqueous and detergent phases. After fractionation of M. tuberculosis by differential centrifugation, Eis protein appeared mainly in the cytoplasmic fraction but also in the membrane, cell wall, and culture supernatant fractions as well. Forty percent of the sera from pulmonary tuberculosis patients tested for anti-Eis antibody gave positive reactions in Western blot analysis. Although the function of Eis remains unknown, evidence presented here suggests it associates with the cell surface and is released into the culture medium. It is produced during human tuberculosis infection and therefore may be an important M. tuberculosis immunogen.

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.

scholarly journals Investigations on ornithobacterium rhinotracheale in broiler flocks in elazig province located in the east of turkey

2012 ◽  
Vol 49 (No. 8) ◽  
pp. 305-311 ◽  
Author(s):  
G. Ozbey ◽  
H. Ongor ◽  
D. T Balik ◽  
V. Celik ◽  
A. Kilic ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rrna Gene ◽  
Major Band ◽  
Cell Extracts ◽  
Sds Page ◽  
Ornithobacterium Rhinotracheale

In the present study, lung, trachea and serum samples from broiler flocks slaughtered at an abattoir in Elazig province located in the East of Turkey were examined for the presence of Ornithobacterium rhinotracheale using culture and enzyme-linked immunosorbent assay (ELISA). The identity was latter proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis, and polymerase chain reaction (PCR) assays. A total of 324 serum and 250 lung and trachea samples were collected from 10 commercially reared chicken flocks showing respiratory manifestations. The samples were obtained from different flocks. The causative agent (ORT) was isolated from trachea (1.5%) of five chickens and from both lung and trachea (0.4%) of only one chicken in the bacteriological examination of tissues. The presence of antibodies against ORT was detected in 33 (10.2%) of the 324 sera by ELISA. A 784 bp fragment of the 16S rRNA gene was amplified using specific primers in the PCR. All ORT isolates that were positive by culture were also detected to be positive by the PCR. SDS-PAGE protein profiles of whole cell extracts showed a high similarity for all the isolates with a major band of the molecular weight of 33 kDa (kiloDalton). Results of Western blot analysis indicate four antigenic fractions predominantly with molecular weights of 33, 42, 52 and 66 kDa.

Intergeneric distribution and immunolocalization of a putative odorant-binding protein in true bugs (Hemiptera, Heteroptera).

1998 ◽  
Vol 201 (1) ◽  
pp. 33-41
Author(s):  
J C Dickens ◽  
F E Callahan ◽  
W P Wergin ◽  
C A Murphy ◽  
R G Vogt
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cellular Localization ◽  
Polyclonal Antiserum ◽  
Adult Males ◽  
Tarnished Plant Bug ◽  
Olfactory Sensilla ◽  
Dense Granules ◽  
Odorant Binding

Lygus antennal protein (LAP) is an olfactory-related protein of the tarnished plant bug Lygus lineolaris (Hemiptera, Heteroptera: Miridae), a hemimetabolous insect. In previous work, a polyclonal antiserum was generated against the N-terminal sequence of LAP; LAP immunoreactivity was strongest in antennae of adult males, but was also present in antennae of adult females and of nymphs. In the current study, LAP immunoreactivity was examined to determine the species specificity and the tissue and cellular localization of LAP expression. Western blot analysis indicated that LAP immunoreactivity was present in the antennae of the male congeners L. lineolaris and L. hesperous, but was not detectable in male antennae of the more distant relatives Podisus maculiventris or Nezara viridula (Hemiptera, Heteroptera: Pentatomidae). Western blot analysis further confirmed that LAP expression was restricted to antennal tissue. Histological analyses showed that LAP expression within the antennae was specifically associated with chemosensory sensilla on the antenna. Within the sensilla, LAP immunoreactivity was distributed throughout the extracellular lumen and was concentrated in dense granules within the cytoplasm of sensillar support cells. LAP immunoreactivity was restricted to a subset of antennal chemosensory sensilla, specifically the multiporous olfactory sensilla. These findings suggest that LAP has an important olfactory function in Lygus sp., possibly related to that of odorant-binding proteins (OBP) found in other insect orders. If so, LAP would be the first OBP-like protein characterized outside the Endopterygota.

scholarly journals Detection of immunogenic protein from salivary gland of Aedes albopictus

2021 ◽  
Vol 40 (3) ◽  
pp. 234-242
Author(s):  
Rike Oktarianti ◽  
Rochmatul Nuryu Khasanah ◽  
Syubbanul Wathon ◽  
Kartika Senjarini
Keyword(s):  
Salivary Gland ◽  
Western Blot ◽  
Salivary Glands ◽  
Aedes Albopictus ◽  
Blot Analysis ◽  
Gel Electrophoresis ◽  
Western Blot Analysis ◽  
Sodium Dodecyl ◽  
Immunogenic Proteins ◽  
Healthy Persons

BackgroundDengue virus is transmitted by several species of Aedes mosquitoes, with Aedes albopictus as secondary vector. During blood feeding, these vectors inject saliva into the vertebrate hosts. The saliva contains anticoagulant, anti-inflammatory and immunogenic factors. The objective of this research was to detect immunogenic proteins from Ae.albopictus salivary glands reacting with sera of people living in dengue endemic areas. MethodsThe identification of immunogenic proteins of Ae. albopictus salivary gland used one-dimensional gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and western blot analysis, respectively. To determine the immunogenic nature of the candidate proteins, the antigens from the salivary gland of Ae. albopictus were reacted with sera from healthy persons, dengue hemorrhagic fever (DHF) patients, and neonates, each of the groups comprising 10 samples. ResultsThe protein profiles of Ae. albopictus salivary glands showed 13 bands with molecular weights from 16 kDa up to 97 kDa, i.e. 16, 17, 26, 28, 31, 32, 45, 55, 60, 67, 73, 76, and 97 kDa. According to western blot analysis result, the 31 kDa proteins were recognized in all endemic population sera, both in DHF patients and healthy persons. In contrast, protein bands of 47 and 67 kDa were only recognized by the sera of DHF patients. ConclusionThree immunogenic proteins of 31, 47 and 67 kDa were detected from Ae. albopictus salivary glands. These immunogenic proteins may be developed as candidate biomarkers for bite exposure to Ae. albopictus and as vector-based DHF vaccines.

Isolation of an aspartic proteinase precursor from the egg of a hard tick, Boophilus microplus

Parasitology ◽  
1998 ◽  
Vol 116 (6) ◽  
pp. 525-532 ◽  
Author(s):  
C. LOGULLO ◽  
I. DA SILVA VAZ ◽  
M. H. F. SORGINE ◽  
G. O. PAIVA-SILVA ◽  
F. S. FARIA ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Fat Body ◽  
Sodium Dodecyl ◽  
Aspartic Proteinase ◽  
Boophilus Microplus ◽  
Proteolytic Processing ◽  
Protein Sequencing ◽  
Hard Tick

An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS–PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S] methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3·5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.

Localization of a phosphatidylglycerol/ phosphatidylinositol transfer protein in Aspergillus oryzae

1998 ◽  
Vol 44 (10) ◽  
pp. 945-953 ◽  
Author(s):  
Eric Record ◽  
Michèle Asther ◽  
Serge Moukha ◽  
Didier Marion ◽  
Vincent Burlat ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Western Blot ◽  
Aspergillus Oryzae ◽  
Filamentous Fungus ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Phospholipid Transfer Protein ◽  
Transfer Protein ◽  
Phospholipid Transfer ◽  
Phosphatidylinositol Transfer

The subcellular localization of the phosphatidylglycerol/phosphatidylinositol transfer protein (PG/PI-TP) of Aspergillus oryzae was investigated using Western blot analysis of the cell protein extracts, a cellular membrane fractionation technique, and transmission electron microscopy. The PG/PI-TP, as detected by Western blot analysis with a specific immune serum, was found to be mainly cytoplasmic and partly associated with intracellular membranes. A fractionation experiment was conducted after homogenization of the filamentous fungus mycelium. The endoplasmic reticulum, Golgi-like vesicles, and the plasma membrane were separated by isopycnic ultracentrifugation on a sucrose gradient, and our data revealed that the immunodetected PG/PI-TP was only associated with the Golgi-like apparatus. All these results were documented by electron microscopy and indicate here for the first time that there exists a specific phospholipid transfer protein in a filamentous fungus that is localized in the cytoplasm and associated with Golgi-like vesicles.Key words: phospholipid transfer protein, subcellular fractionation, ultrastructural localization, filamentous fungus, Aspergillus oryzae.

scholarly journals Reduced connexin-43 expression in the aorta of pre-hypertensive rats

2008 ◽  
pp. S23-S29
Author(s):  
K Dlugošová ◽  
M Mitašíková ◽  
I Bernátová ◽  
P Weismann ◽  
Ľ Okruhlicová
Keyword(s):  
Electron Microscopy ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Connexin 43 ◽  
Cell Communication ◽  
Hypertensive Rats ◽  
Cx43 Expression ◽  
Aortic Endothelium ◽  
Junction Protein

Genetic component represents an important factor in the development of hypertension, which is known to be associated with changes in expression of vascular gap junction protein connexin 43 (Cx43). The aim of the study was to examine the distribution and expression of Cx43 in the aortic endothelium of adult normotensive Wistar rats (W), borderline hypertensive rats (BHR) and spontaneously hypertensive rats (SHR). Rings of the thoracic aorta were processed for immunofluorescence and Western blot analysis of endothelial Cx43 and for electron microscopy. Both, BHR and SHR exhibited significantly increased blood pressure vs. W (132±2 mm Hg and 185±3 mm Hg vs. 110±2 mm Hg). Reduced Cx43 immunofluorescence was observed in the endothelium of BHR and these alterations were more pronounced in SHR. Western blot analysis showed significant suppression of Cx43 expression in the aorta of both BHR (p<0.05) and SHR (p<0.001) vs. W. Electron microscopy revealed local subcellular alterations of interendothelial connections in BHR including extended tight junctions. These alterations were more frequent and marked in SHR. The results indicate that connexin 43 expression is reduced in the aortic endothelium already in prehypertensive period, which may affect cell-to-cell communication and thus participate in acceleration of hypertensive disease.

Cytoskeletons of X-Linked GATA-1, G208S Macrothrombocytes Are Deficient in Talin.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1829-1829
Author(s):  
James G. White ◽  
Steven M. Burris ◽  
Angela Thomas
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Single Cells ◽  
Sodium Dodecyl ◽  
Normal Subjects ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Integrin Activation ◽  
Normal Platelet

Abstract Germline mutations in the X-linked transcription factor, GATA-1, cause several hematopoietic disorders resulting in anemia and/or macrothrombocytopenia. Features of the platelet ultrastructural pathology, including hypo- and agranular megathrombocytes (Mtc) in all mutations, tubular membrane sheets often in parallel association in megakaryocytes and Mtc of patients with the V205M and G208S variations, platelets sequestered in Mtc and platelets in platelets in Mtc (as many as five in one Mtc) in the V205M, G208S and R216Q disorders, together with platelets attached to platelets attached to platelets forming large Mtc in the V205M and G208S mutations have helped to explain how Mtc are formed and why they fail to separate from proplatelets of megakaryocytes and enter the circulation as single cells. However, the findings do not explain why patients with GATA-1 mutations often have serious bleeding problems. Major receptors for hemostasis and thrombosis are present on GATA-1 Mtc, although, in some cases, slightly reduced in frequency. Yet, while the cells bind to and spread fully on glass and plastic, they adhere poorly to vWF or collagen coated glass slides under flow conditions, aggregate less well than normal platelets when stirred with collagen or ristocetin in an aggregometer and fail to retract clots. The findings suggest that internal activation mechanisms for the surface receptors are defective. The present study has solubilized GATA-1 Mtc from two males with the G208S mutation and normal platelets in sodium dodecyl sulfate/ EDTA in the presence of protease inhibitors and analyzed their cytoskeletal proteins on reduced, one dimensional polyacrylamide gels using a Mini-Slab Gel Apparatus. Gels were electrophoresed by the method of Laemmli. Proteins were stained with colloidal Coomassie G250 blue. GATA-1 Mtc cytoskeletal proteins were the same as those from the normal subjects, except for the absence of Talin. Western blot analysis to prove the missing protein is Talin was carried out. Normal platelet and GATA-1 Mtc cytoskeletal proteins were transferred to nitrocellulose sheets. Identification and localization of the missing protein was accomplished using a mouse monoclonal antibody against human Talin and a secondary anti-mouse IgG peroxidase conjugate. The Western blot analysis confirmed that the missing Mtc protein is Talin. Recent studies have shown that binding of the 235–245 kD actin-binding protein Talin to the β subunit cytoplasmic tails of integrins is the final step of integrin activation. The absence of Talin in GATA-1 Mtc may result in failure of full integrin activation accounting for functional failure in hemostasis.

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