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1.
Benjamin S. Johnson Lexie Chafin Daniela Farkas Jessica Adair Ajit Elhance Laszlo Farkas Joseph S. Bednash James D. Londino 《Molecular & cellular proteomics : MCP》2022,21(7):100256
Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions. 相似文献
2.
Nicholas T. Loux David S. Brown Claudia R. Chafin Jerry D. Allison Sayed M. Hassan 《Chemical Speciation and Bioavailability》2013,25(3):111-125
AbstractCoupled geochemical speciation/transport models are being developed to assess potential transport of metal contaminants in the subsurface environment. In a test of the geochemical speciation portion of the effort, MINTEQA2 model predictions are compared with laboratory data concerning the pH dependent partitioning behavior of eight cationic contaminants (Ba, Be, Cd, Cu, Ni, Pb, Tl and Zn) on a sandy aquifer material in an oxidized environment. MINTEQA2 contains provisions for describing potential attenuation due to both mineral phase precipitation processes and adsorption processes resulting from amorphous iron oxides in aquifer materials (MIT Diffuse Layer Model). In the comparison, several trends were discerned. (1) Adsorptive processes tend to better describe the pH-dependent partitioning behavior of transition metals (especially Pb, Zn and Ni). (2) Cd behavior is better described by precipitation as a cadmium carbonate phase. (3) Cu behavior is not reasonably described by the model. (4) Ba and Be comparisons are poor (although presumably their partitioning behavior results from adsorptive and/or pH sensitive solid solution processes). (5) unlike the other elements, the behavior of Tl is relatively insensitive to pH. 相似文献
3.
Nam P. Nguyen Lexie Smith-Raymond Vincent Vinh-Hung Paul Vos Rick Davis Anand Desai Thomas Sroka Dave Abraham Shane P. Krafft Michelle Stevie Homayoun Modarresifar Beng-Hoey Jo Misty Ceizyk 《PloS one》2013,8(3)
Purpose
The study aims to assess the feasibility of Tomotherapy-based image-guided radiotherapy (IGRT) to reduce the aspiration risk in patients with non-laryngeal and non-hypopharyngeal cancer. A retrospective review of 48 patients undergoing radiation for non-laryngeal and non-hypopharyngeal head and neck cancers was conducted. All patients had a modified barium swallow (MBS) prior to treatment, which was repeated one month following radiotherapy. Mean middle and inferior pharyngeal dose was recorded and correlated with the MBS results to determine aspiration risk.Results
Mean pharyngeal dose was 23.2 Gy for the whole group. Two patients (4.2%) developed trace aspiration following radiotherapy which resolved with swallowing therapy. At a median follow-up of 19 months (1–48 months), all patients were able to resume normal oral feeding without aspiration.Conclusion and Clinical Relevance
IGRT may reduce the aspiration risk by decreasing the mean pharyngeal dose in the presence of large cervical lymph nodes. Further prospective studies with IGRT should be performed in patients with non-laryngeal and non-hypopharyngeal head and neck cancers to verify this hypothesis. 相似文献4.
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Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development 总被引:1,自引:0,他引:1
Connolly JP Comerci D Alefantis TG Walz A Quan M Chafin R Grewal P Mujer CV Ugalde RA DelVecchio VG 《Proteomics》2006,6(13):3767-3780
Brucella abortus is the etiologic agent of bovine brucellosis and causes a chronic disease in humans known as undulant fever. In livestock the disease is characterized by abortion and sterility. Live, attenuated vaccines such as S19 and RB51 have been used to control the spread of the disease in animals; however, they are considered unsafe for human use and they induce abortion in pregnant cattle. For the development of a safer and equally efficacious vaccine, immunoproteomics was utilized to identify novel candidate proteins from B. abortus cell envelope (CE). A total of 163 proteins were identified using 2-DE with MALDI-TOF MS and LC-MS/MS. Some of the major protein components include outer-membrane protein (OMP) 25, OMP31, Omp2b porin, and 60 kDa chaperonin GroEL. 2-DE Western blot analyses probed with antiserum from bovine and a human patient infected with Brucella identified several new immunogenic proteins such as fumarate reductase flavoprotein subunit, F0F1-type ATP synthase alpha subunit, and cysteine synthase A. The elucidation of the immunome of B. abortus CE identified a number of candidate proteins for developing vaccines against Brucella infection in bovine and humans. 相似文献
7.
NP Nguyen A Chi M Betz F Almeida P Vos R Davis B Slane M Ceizyk D Abraham L Smith-Raymond M Stevie S Jang S Gelumbauskas V Vinh-Hung 《PloS one》2012,7(8):e42729
Purpose
The study aims to assess the feasibility of intensity-modulated and image-guided radiotherapy (IMRT, and IGRT, respectively) for functional preservation in locally advanced laryngeal cancer. A retrospective review of 27 patients undergoing concurrent chemoradiation for locally advanced laryngeal cancers (8 IMRT, 19 IGRT) was undertaken. In addition to regular clinical examinations, all patients had PET imaging at 4 months and 10 months after radiotherapy, then yearly. Loco-regional control, speech quality and feeding-tube dependency were assessed during follow-up visits.Results
At a median follow-up of 20 months (range 6–57 months), four out of 27 patients (14.8%) developed local recurrence and underwent salvage total laryngectomy. One patient developed distant metastases following salvage surgery. Among the 23 patients who conserved their larynx with no sign of recurrence at last follow-up, 22 (95%) reported normal or near normal voice quality, allowing them to communicate adequately. Four patients (14.8%) had long-term tube feeding-dependency because of severe dysphagia (2 patients) and chronic aspiration (2 patients, with ensuing death from aspiration pneumonia in one patient).Conclusions and Clinical Relevance
Functional laryngeal preservation is feasible with IMRT and IGRT for locally advanced laryngeal cancer. However, dysphagia and aspiration remain serious complications, due most likely to high radiation dose delivery to the pharyngeal musculatures. 相似文献8.
Several proteins of viral and cellular origins are myristoylated on an amino-terminal glycine residue during biosynthesis. The enzyme responsible for this modification, myristoyl CoA:protein N-myristoyltransferase (NMT), can be measured in cell-free systems by following the transfer of [3H]myristate from [3H]myristoyl CoA to a synthetic peptide substrate. We report here a procedure for the analysis of NMT activity using ion-exchange chromatography on CM-Sepharose to separate [3H]myristoyl peptide from radiolabeled reactants. This technique provides a convenient method for assaying multiple samples that is much more rapid and sensitive than procedures that rely on reversed-phase HPLC for the separation of reaction components. Characterization of this assay indicates that it is suitable for the kinetic analysis of NMT activity and for the rapid analysis of column fractions generated during the purification of NMT. 相似文献
9.
Siew Ping Han Lexie R. Friend John H. Carson George Korza Elisa Barbarese Michael Maggipinto Jodie T. Hatfield Joseph A. Rothnagel Ross Smith 《Traffic (Copenhagen, Denmark)》2010,11(7):886-898
Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans‐acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform‐specific antibodies and isoform‐specific green fluorescent protein (GFP)‐fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform‐specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic‐trafficking functions. These findings highlight the importance of considering isoform‐specific functions for alternatively spliced proteins. 相似文献
10.
The Bacillus subtilis DinR Binding Site: Redefinition of the Consensus Sequence 总被引:4,自引:0,他引:4 下载免费PDF全文
Kevin W. Winterling David Chafin Jeffery J. Hayes Ji Sun Arthur S. Levine Ronald E. Yasbin Roger Woodgate 《Journal of bacteriology》1998,180(8):2201-2211
Recently, the DinR protein was established as the cellular repressor of the SOS response in the bacterium Bacillus subtilis. It is believed that DinR functions as the repressor by binding to a consensus sequence located in the promoter region of each SOS gene. The binding site for DinR is believed to be synonymous with the formerly identified Cheo box, a region of 12 bp displaying dyad symmetry (GAAC-N4-GTTC). Electrophoretic mobility shift assays revealed that highly purified DinR does bind to such sites located upstream of the dinA, dinB, dinC, and dinR genes. Furthermore, detailed mutational analysis of the B. subtilis recA operator indicates that some nucleotides are more important than others for maintaining efficient DinR binding. For example, nucleotide substitutions immediately 5′ and 3′ of the Cheo box as well as those in the N4 region appear to affect DinR binding. This data, combined with computational analyses of potential binding sites in other gram-positive organisms, yields a new consensus (DinR box) of 5′-CGAACRNRYGTTYC-3′. DNA footprint analysis of the B. subtilis dinR and recA DinR boxes revealed that the DinR box is centrally located within a DNA region of 31 bp that is protected from hydroxyl radical cleavage in the presence of DinR. Furthermore, while DinR is predominantly monomeric in solution, it apparently binds to the DinR box in a dimeric state. 相似文献