scholarly journals Proteomic Analysis of Rickettsia parkeri Strain Portsmouth

2009 ◽  
Vol 77 (12) ◽  
pp. 5262-5271 ◽  
Author(s):  
Walairat Pornwiroon ◽  
Apichai Bourchookarn ◽  
Christopher D. Paddock ◽  
Kevin R. Macaluso
Keyword(s):  
Cell Surface ◽  
Vaccine Development ◽  
Spotted Fever ◽  
The United States ◽  
Trna Synthetase ◽  
Surface Proteins ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Two Dimensional ◽  
Rickettsia Parkeri

ABSTRACT Rickettsia parkeri, a recently recognized pathogen of human, is one of several Rickettsia spp. in the United States that causes a spotted fever rickettsiosis. To gain insights into its biology and pathogenesis, we applied the proteomics approach to establish a two-dimensional gel proteome reference map and combined this technique with cell surface biotinylation to identify surface-exposed proteins of a low-passage isolate of R. parkeri obtained from a patient. We identified 91 proteins by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. Of these, 28 were characterized as surface proteins, including virulence-related proteins (e.g., outer membrane protein A [OmpA], OmpB, β-peptide, and RickA). Two-dimensional immunoblotting with serum from the R. parkeri-infected index patient was utilized to identify the immunoreactive proteins as potential targets for diagnosis and vaccine development. In addition to the known rickettsial antigens, OmpA and OmpB, we identified translation initiation factor 2, cell division protein FtsZ, and cysteinyl-tRNA synthetase as immunoreactive proteins. The proteome map with corresponding cell surface protein analysis and antigen detection will facilitate a better understanding of the mechanisms of rickettsial pathogenesis.

scholarly journals Serological evidence of Rickettsia parkeri as the etiological agent of rickettsiosis in Uruguay

2009 ◽  
Vol 51 (6) ◽  
pp. 337-339 ◽  
Author(s):  
Ismael A. Conti-Díaz ◽  
Jonas Moraes-Filho ◽  
Richard C. Pacheco ◽  
Marcelo B. Labruna
Keyword(s):  
Spotted Fever ◽  
Clinical Manifestations ◽  
The United States ◽  
Immunofluorescence Assay ◽  
Etiological Agent ◽  
Serological Evidence ◽  
Rickettsia Parkeri ◽  
Rickettsia Rickettsii ◽  
Antibody Absorption ◽  
Mild Fever

We report three new rickettsiosis human cases in Uruguay. The three clinical cases presented clinical manifestations similar to previous reported cases of Rickettsia parkeri in the United States; that is mild fever (< 40 ºC), malaise, headache, rash, inoculation eschar at the tick bite site, regional lymphadenopathy, and no lethality. Serological antibody-absorption tests with purified antigens of R. parkeri and Rickettsia rickettsii, associated with immunofluorescence assay indicated that the patients in two cases were infected by R. parkeri. Epidemiological and clinical evidences, coupled with our serological analysis, suggest that R. parkeri is the etiological agent of human cases of spotted fever in Uruguay, a disease that has been recognized in that country as cutaneous-ganglionar rickettsiosis.

scholarly journals Identification of phagocytosis-associated surface proteins of macrophages by two-dimensional gel electrophoresis.

1982 ◽  
Vol 92 (2) ◽  
pp. 283-288 ◽  
Author(s):  
F D Howard ◽  
H R Petty ◽  
H M McConnell
Keyword(s):  
Cell Surface ◽  
Gel Electrophoresis ◽  
Protein Composition ◽  
Surface Protein ◽  
Surface Proteins ◽  
Two Dimensional ◽  
Cell Surface Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Reducing Conditions ◽  
Membrane Components

Two-dimensional PAGE (P. Z. O'Farrell, H. M. Goodman, and P. H. O'Farrell. 1977. Cell. 12:1133-1142) has been employed to assess the effects of antibody-dependent phagocytosis on the cell surface protein composition of RAW264 macrophages. Unilamellar phospholipid vesicles containing 1% dinitrophenyl-aminocaproyl-phosphatidylethanolamine (DNP-cap-PE) were used as the target particle. Macrophages were exposed to anti-DNP antibody alone, vesicles alone, or vesicles in the presence of antibody for 1 h at 37 degrees C. Cell surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination at 4 degrees C. After detergent solubilization, membrane proteins were analyzed by two-dimensional gel electrophoresis. The resulting pattern of spots was compared to that of standard proteins. We have identified several surface proteins, not apparently associated with the phagocytic process, which are present either in a multichain structure or in several discretely charged forms. After phagocytosis, we have observed the appearance of two proteins of 45 and 50 kdaltons in nonreducing gels. In addition, we have noted the disappearance of a 140-kdalton protein in gels run under reducing conditions. These alterations would not be detected in the conventional one-dimensional gel electrophoresis. This evidence shows that phagocytosis leads to a modification of cell surface protein composition. Our results support the concept of specific enrichment and depletion of membrane components during antibody-dependent phagocytosis.

scholarly journals Identification of Adrenomedullin-Induced S-Nitrosylated Proteins in JEG-3 Placental Cells

2021 ◽  
Author(s):  
Yingting Li ◽  
Liuying Zhong ◽  
Cheuk-Lun Lee ◽  
Philip C.N. Chiu ◽  
Min Chen
Keyword(s):  
Cell Surface ◽  
Cell Invasion ◽  
Surface Expression ◽  
Translation Initiation Factor ◽  
Cell Surface Receptor ◽  
Initiation Factor ◽  
Trophoblast Invasion ◽  
Spectrometric Analysis ◽  
Nitric Oxide Signaling ◽  
Placental Cells

AbstractExtravillous cytotrophoblast (EVCT) is responsible for trophoblast invasion, which is important during placentation. Dysregulation of the process leads to pregnancy complications. S-nitrosylation of proteins is associated with cell invasion in many cell types. Adrenomedullin (ADM), a polypeptide expressed abundantly in the first-trimester placentas, induces EVCT invasion by upregulation of protein S-nitrosylation. This study aimed to identify the S-nitrosylated proteins induced by ADM in the JEG-3 placental cells. By using affinity chromatography followed by mass spectrometric analysis, tubulin, enolase, eukaryotic translation initiation factor 4A1, actin, annexin II (ANX II), and glyceraldehyde 3-phosphate dehydrogenaseprotein-1 were found to be S-nitrosylated by ADM. In vitro treatment with ADM or S-Nitrosoglutathione (GSNO) significantly increased the ANX II surface expression, but not its total expression in the JEG-3 cells. Translocation of ANX II to cell surface has been reported to act as a cell surface receptor to plasmin, plasminogen, and tissue plasminogen activator (tPA), thereby stimulating cell invasion and migration. However, in this study, ADM-induced surface expression of ANX II in the JEG-3 cells was not associated with changes in the secretory and membrane-bound tPA activities. Future studies are required to understand the roles of surface expression of S-nitrosylated ANX II on trophoblast functions. To conclude, this study provided evidences that ADM regulated the nitric oxide signaling pathway and modulated trophoblast invasion.

Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases

1992 ◽  
Vol 12 (12) ◽  
pp. 5801-5815
Author(s):  
M Ramirez ◽  
R C Wek ◽  
C R Vazquez de Aldana ◽  
B M Jackson ◽  
B Freeman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Translation Initiation ◽  
Trna Synthetase ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Atp Binding ◽  
Sequence Motif ◽  
Trna Synthetases

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN2c alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2c alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2 alpha kinase GCN2.

scholarly journals Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases.

1992 ◽  
Vol 12 (12) ◽  
pp. 5801-5815 ◽  
Author(s):  
M Ramirez ◽  
R C Wek ◽  
C R Vazquez de Aldana ◽  
B M Jackson ◽  
B Freeman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Translation Initiation ◽  
Trna Synthetase ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Atp Binding ◽  
Sequence Motif ◽  
Trna Synthetases

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN2c alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2c alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2 alpha kinase GCN2.

scholarly journals Curation of a Multiple Myeloma Cell Surface Database for Immunotherapy Development

2020 ◽  
Vol 3 ◽  
Author(s):  
Emily Adaniya ◽  
Christina Yu ◽  
Fabiana Perna
Keyword(s):  
Multiple Myeloma ◽  
Cell Surface ◽  
Plasma Cells ◽  
Expression Profiles ◽  
The United States ◽  
Myeloma Cell ◽  
Surface Proteins ◽  
Computational Tool ◽  
Genomic Databases ◽  
Manual Curation

Background/Objective: Every year in the United States, 32,000 individuals are diagnosed with Multiple Myeloma (MM) and 13,000 die from the disease. MM is a cancer of the plasma cells and while treatment exists, MM remains incurable. Our overall goal is to identify biologically and therapeutically relevant cell surface targets to develop MM immunotherapy.      Methods: Given the genomic heterogeneity of MM, we performed surface analysis of seven MM cell lines by Mass-Spectrometry (MS), generating a pool of candidate proteins. Lacking specific tools for studying cell surface proteins, we developed an integrated computational tool with five distinct databases, and scored the likelihood of cell surface location for each molecule. One point was given for each database a protein appeared in. A protein receiving a score of three or more was considered a cell surface protein with high confidence. However, about one third of the candidates were not detected by the computational tool, thus requiring manual curation. We used UniProt and GeneCards to determine the subcellular locations of the unannotated IDs, and further confirmed their location with the computational tool.    Results: Of the 5,454 UniProt IDs produced by MS, 2,026 were not annotated. Causes of an unannotated ID included having an obsolete ID, being an isoform, and/or not being located on the cell surface. Through manual annotation, 8 unique cell surface IDs were added to the existing 448. The 456 targets were further analyzed based on their expression profiles in both MM and normal tissues resulting in 94 of the most promising targets, some of which would have been missed without manual curation.     Conclusion/Potential Impact: Public genomic databases are often incomplete and may contain errors making manual curation a necessary step in error reduction. Left unchecked, promising surface targets could have been overlooked hindering the creation of potentially curative immunotherapies. 

scholarly journals Avian Migrants Facilitate Invasions of Neotropical Ticks and Tick-Borne Pathogens into the United States

2015 ◽  
Vol 81 (24) ◽  
pp. 8366-8378 ◽  
Author(s):  
Emily B. Cohen ◽  
Lisa D. Auckland ◽  
Peter P. Marra ◽  
Sarah A. Hamer
Keyword(s):  
United States ◽  
Migratory Birds ◽  
Spotted Fever ◽  
Animal Health ◽  
The United States ◽  
Etiologic Agent ◽  
Rickettsia Parkeri ◽  
Blood Samples ◽  
Content Type ◽  
Amblyomma Triste

ABSTRACTMigratory birds have the potential to transport exotic vectors and pathogens of human and animal health importance across vast distances. We systematically examined birds that recently migrated to the United States from the Neotropics for ticks. We screened both ticks and birds for tick-borne pathogens, includingRickettsiaspecies andBorrelia burgdorferi. Over two spring seasons (2013 and 2014), 3.56% of birds (n= 3,844) representing 42.35% of the species examined (n= 85) were infested by ticks. Ground-foraging birds with reduced fuel stores were most commonly infested. Eight tick species were identified, including seven in the genusAmblyomma, of which onlyAmblyomma maculatum/Amblyomma tristeis known to be established in the United States. Most ticks on birds (67%) were neotropical species with ranges in Central and South America. Additionally, a singleIxodesgenus tick was detected. A total of 29% of the ticks (n= 137) and no avian blood samples (n= 100) were positive for infection withRickettsiaspecies, includingRickettsia parkeri, an emerging cause of spotted fever in humans in the southern United States, a species in the group ofRickettsiamonacensis, and uncharacterized species and endosymbionts of unknown pathogenicity. No avian tick or blood samples tested positive forB. burgdorferi, the etiologic agent of Lyme disease. An extrapolation of our findings suggests that anywhere from 4 to 39 million exotic neotropical ticks are transported to the United States annually on migratory songbirds, with uncertain consequences for human and animal health if the current barriers to their establishment and spread are overcome.

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