scholarly journals Changes in tear protein profile in dogs with keratoconjunctivitis sicca following topical treatment using cyclosporine A

2021 ◽  
pp. 1711-1717
Author(s):  
Metita Sussadee ◽  
Rucksak Rucksaken ◽  
Phattara-Orn Havanapan ◽  
Onrapak Reamtong ◽  
Aree Thayananuphat
Keyword(s):  
Cyclosporine A ◽  
Protein Identification ◽  
Keratoconjunctivitis Sicca ◽  
Protein Pattern ◽  
Protein Profile ◽  
Expression Patterns ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tear Proteins ◽  
Tear Protein

Background and Aim: Keratoconjunctivitis sicca (KCS) is a chronic inflammatory ocular disease that occurs in many dog breeds worldwide. This study aimed to investigate the tear protein pattern of healthy dogs, KCS dogs, and KCS dogs after treatment with cyclosporine A (CsA). Materials and Methods: Twenty-eight dogs of any breed were enrolled in the study. The subjects were divided into three groups: Healthy, KCS, and CsA-treated dogs. Tear samples were collected using Schirmer strips. Tear proteins extracted from the strips were analyzed using two-dimensional electrophoresis. For the first dimension, total protein from tears was separated by isoelectric focusing. The second dimension was performed using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel images were analyzed and the protein spots of differential expression were manually cut for protein annotation using mass spectrometry. Results: In total, 12 protein spots were excised and subjected to protein identification. Associated with KCS, six protein spots were a downregulated protein, namely, lysozyme. The other six protein spots were upregulated in KCS dogs, consisting of heat shock protein beta-1, protein S100-A12, and keratin type II cytoskeletal 1 and 5. After treatment with CsA for 45 days, the lysozyme protein was still decreasing and the inflammation protein (S100-A12) was not identified. Conclusion: Inflammatory tear proteins and proteins involved in cellular stress were present in KCS dogs and appeared to be reduced in medicated eyes. Treatment with topical CsA in the short term may not improve the activity of antibacterial proteins. Changes in the expression patterns of these four proteins might be useful for disease severity and progression assessment, as well as for exploring a novel method for dry eye management in dogs.

scholarly journals Molecular Characterization and Functional Analysis of PR-1-Like Proteins Identified from the Wheat Head Blight Fungus Fusarium graminearum

2018 ◽  
Vol 108 (4) ◽  
pp. 510-520 ◽  
Author(s):  
Shunwen Lu ◽  
Michael C. Edwards
Keyword(s):  
Functional Analysis ◽  
Fusarium Graminearum ◽  
Expression Patterns ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Transcriptional Analysis ◽  
Fungal Genome ◽  
Dimensional Structure ◽  
Head Blight ◽  
Fungal Virulence

The group 1 pathogenesis-related (PR-1) proteins originally identified from plants and their homologs are also found in other eukaryotic kingdoms. Studies on nonplant PR-1-like (PR-1L) proteins have been pursued widely in humans and animals but rarely in filamentous ascomycetes. Here, we report the characterization of four PR-1L proteins identified from the ascomycete fungus Fusarium graminearum, the primary cause of Fusarium head blight of wheat and barley (designated FgPR-1L). Molecular cloning revealed that the four FgPR-1L proteins are all encoded by small open reading frames (612 to 909 bp) that are often interrupted by introns, in contrast to plant PR-1 genes that lack introns. Sequence analysis indicated that all FgPR-1L proteins contain the PR-1-specific three-dimensional structure, and one of them features a C-terminal transmembrane (TM) domain that has not been reported for any stand-alone PR-1 proteins. Transcriptional analysis revealed that the four FgPR-1L genes are expressed in axenic cultures and in planta with different spatial or temporal expression patterns. Phylogenetic analysis indicated that fungal PR-1L proteins fall into three major groups, one of which harbors FgPR-1L-2-related TM-containing proteins from both phytopathogenic and human-pathogenic ascomycetes. Low-temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteolytic assays indicated that the recombinant FgPR-1L-4 protein exists as a monomer and is resistant to subtilisin of the serine protease family. Functional analysis confirmed that deletion of the FgPR-1L-4 gene from the fungal genome results in significantly reduced virulence on susceptible wheat. This study provides the first example that the F. graminearum–wheat interaction involves a pathogen-derived PR-1L protein that affects fungal virulence on the host.

Isomerization of Peroxidizing Thiadiazolidine Herbicides Is Catalyzed by Glutathione S-Transferase

1996 ◽  
Vol 51 (5-6) ◽  
pp. 342-354 ◽  
Author(s):  
Beate Nicolaus ◽  
Yukiharu Sato ◽  
Ko Wakabayashi ◽  
Peter Böger
Keyword(s):  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Total Activity ◽  
Naphthalic Anhydride ◽  
Molecular Weights ◽  
Isomerase Activity ◽  
Ammonium Sulfate Precipitate ◽  
Catalyzed Reactions

Abstract Thiadiazolidine-converting activity (isomerase), detected in a 45-75% ammonium sulfate precipitate from corn seedlings extracts, was purified by chromatography on hydroxyapatite and by anion exchange on Mono Q Sepharose. Two fractions 1 and 2 with isomerase activity were separated on Mono Q by combination of a stepwise elution and continuous salt gradient; fraction 2 eluting at higher salt concentrations was found the most active. Total activity could be enhanced by treatment of seedlings with naphthalic anhydride. Both fractions containing isomerase activity were further purified by glutathione-(GSH) agarose affinity chromatography and characterized by their specificity for different thiadiazolidines. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration revealed that the isomerase of fraction 2 consists either of a homodimer or a heterodimer of two proteins with apparent molecular weights of 28 and 31 kDa, respectively. The protein pattern as well as the strict dependence of activity on thiol groups (GSH or dithiothreitol) suggested a glutathione Stransferase (GST) catalyzing the thiadiazolidine conversion. Further evidence was obtained by measuring reactions specific for GSTs in both purified fractions, namely the conjugating activity for l-chloro-2,4-dinitrobenzene (CDNB ). atrazine and metazachlor. While no atrazine turnover was found, metazachlor and CDNB conjugation occurred rapidly. Both fractions differed in their activities to several GST substrates with fraction 2 being more effective in metazachlor but less active in C DN B conjugation. Inhibitors specific for GST-catalyzed reactions also inhibited thiadiazolidine conversion confirming that isomerizing activity is attributed to a GST form. We conclude that GST isoforms with different affinities towards thiadiazolidines have been isolated. CDNB activity, molecular weight, the protein pattern on SDS-PAGE as well as the amino acid sequence of one of its polypeptides suggest that fraction 1, less active in thiadiazolidine isomerization, is identical to GST I. The second peptide of this fraction was resistant to Edman degradation probably due to N-terminal blockage. The properties of the high isomerase activity found in fraction 2 are in agreement with characteristics of a GST previously termed as isoform II.

scholarly journals EFFECTS OF HUMAN PLATELET LYSATES WITHOUT ADDITIONAL GROWTH FACTORS ON THE PROTEIN PROFILES OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELL CULTURE MEDIA

2017 ◽  
Vol 10 (17) ◽  
pp. 54
Author(s):  
Hazriani Ra ◽  
Lisa Amir ◽  
Ratna Farida
Keyword(s):  
Culture Media ◽  
Umbilical Vein ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Test Results ◽  
Control Groups ◽  
Cell Culture Media ◽  
Endothelial Cell Culture ◽  
Fetal Bovine

Objectives: The objective of this study is to evaluate the effect of HPLs with no additional GFs on the HUVEC protein profile. Methods: HUVEC cultures were examined in groups as follows: Fetal bovine serum (FBS), 2%-HPL with a GF, and 2%- and 5%-HPL without a GF which were analyzed with a sodium dodecyl sulfate-polyacrylamide gel electrophoresis test. Results: The intensity, thickness, and molecular weight of HUVEC band proteins cultured without a GF were not significantly different compared to the control groups (FBS or HPL with a GF). Conclusions: No difference was found in the HUVEC protein profile after they were cultured with FBS and HPLs, with or without GFs.

scholarly journals Construction and Characterization of anagr Deletion Mutant of Staphylococcus epidermidis

2000 ◽  
Vol 68 (3) ◽  
pp. 1048-1053 ◽  
Author(s):  
Cuong Vuong ◽  
Friedrich Götz ◽  
Michael Otto
Keyword(s):  
Staphylococcus Epidermidis ◽  
Deletion Mutant ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Proteins ◽  
Accessory Gene ◽  
Accessory Gene Regulator ◽  
Sds Page ◽  
The Difference

ABSTRACT The physiological significance of the accessory gene regulator (agr) system of Staphylococcus epidermidis was investigated by construction of an agr deletion mutant via allelic replacement with a spectinomycin resistance cassette. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the protein pattern was strongly altered in the mutant; the amounts of most surface proteins were higher, whereas the amounts of most exoproteins were lower. The agrsystem of S. epidermidis thus appears to have an important impact on growth phase-dependent protein synthesis as has been shown for Staphylococcus aureus. The activity of the exoenzymes lipase and protease, assumed to be involved in staphylococcal pathogenicity, was investigated by agar diffusion assays and SDS-PAGE activity staining. A general reduction of these enzyme activities in the agr mutant was found. The difference in overall lipase activity was small, but zymographic analysis suggested a clear defect in lipase processing in the agr mutant.

scholarly journals Characterization of Proteins in the Outer Membrane Preparation of a Murine Pathogen, Helicobacter bilis

2001 ◽  
Vol 69 (5) ◽  
pp. 3502-3506 ◽  
Author(s):  
Zhongming Ge ◽  
Peter Doig ◽  
James G. Fox
Keyword(s):  
Outer Membrane ◽  
Wide Spectrum ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Membrane Preparation ◽  
Laboratory Animals ◽  
Helicobacter Bilis ◽  
H Pylori

ABSTRACT Helicobacter bilis is a bacterial pathogen associated with multifocal hepatitis and inflammatory bowel disease in certain strains of mice. This bacterium colonizes the liver, bile, and lower intestine in mice and has also been isolated from a wide spectrum of laboratory animals. In this study, proteins present in the outer membrane preparation (OMP) of four H. bilis strains isolated from a mouse, a dog, a rat, and a gerbil were characterized and compared with that of Helicobacter pylori, a human gastric pathogen. All four H. bilis strains had similar OMP protein profiles that were distinct from those of H. pylori. Immunoblotting demonstrated that OMP proteins fromH. bilis and H. pylori have little cross-reactivity, except for their flagellins. Nine major immunogenic polypeptides were present in the H. bilis OMPs. By using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five heat-modifiable proteins with molecular masses of 82, 66, 52, 47 and 37 kDa were identified. The N-terminal sequences of the 46- and 47-kDa OMP proteins had no homology with protein sequences available in public databases. These results indicate that H. bilis has a conserved, unique OMP protein profile that is distinct from those of H. pylori.

scholarly journals The population, protein profile and ultrastructure of Ascaridia galli in chicken treated using Areca catechu crude aqueous extract

2019 ◽  
Vol 44 (4) ◽  
pp. 392
Author(s):  
W. W. Mubarokah ◽  
W. Nurcahyo ◽  
J. Prastowo ◽  
K. Kurniasih
Keyword(s):  
Aqueous Extract ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anthelmintic Activity ◽  
Positive Control ◽  
Negative Control ◽  
Ascaridia Galli ◽  
Areca Catechu ◽  
Crude Aqueous Extract

The study aimed at investigating the population, the protein profile and the ultrastructure of adult worms in the intestine of domestic chicken treated using Areca catechu crude aqueous extract. Fifty domestic female chickens of 6 weeks of age were assigned to 5 groups. Group A (negative control) was not given any treatment and any drug. Groups B, C and D were given the treatment at the doses of 26 mg/mL, 53 mg/mL and 79 mg/mL, respectively. Group E (positive control) was given Pyrantel®. Necropsy was conducted to all of the chickens 14 days after the treatment. Adult worms were collected and counted. The worms used in Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Scanning Electron Microscopy (SEM) were those collected from the jejunum of the chickens in the groups A, B and C. The biggest number of the worms was found in the jejunum. The results of electrophoresis showed that the dose 53 mg/mL gave fewer protein bands than the negative control (21:12 ratio), while the results of the SEM showed that there was cuticle damage and anterior labia abrasion at the dose of 53 mg/mL. The Areca catechu crude aqueous extract showed anthelmintic activity potential by reducing the number of the adult worms, lowering their protein profile and damaging the A. galli worms in the intestine.

Oviductal fluid protein patterns in the rhesus monkey (Macaca mulatta) during the menstrual cycle

1992 ◽  
Vol 4 (2) ◽  
pp. 249 ◽  
Author(s):  
A Paliwal ◽  
B Malaviya ◽  
VP Kamboj
Keyword(s):  
Menstrual Cycle ◽  
Protein Profile ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Blue Staining ◽  
Protein Patterns ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Oviductal Fluid

Oviducts were obtained from monkeys on Days 8, 14, 19 and 25 of the menstrual cycle and changes in the pattern of luminal fluid proteins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis after periodic acid Schiff's reagent (PAS) and coomassie blue staining of the gels revealed 85 and 95 kDa proteins only up to Day 14 whereas a 130 kDa glycoprotein persisted up to Day 19 and reached a nadir at mid-menstrual cycle (Day 14). The absence of the 130 kDa glycoprotein in the serum and its presence in cytosolic preparations up to Day 19 suggest that it is of oviductal origin. The 130 kDa glycoprotein is of particular interest since it was present in the oviductal fluid during mid cycle, a period when the oviduct participates in gamete transport, fertilization and embryo development. The conclusion drawn from this study is that the protein profile of monkey oviductal fluid changes during the menstrual cycle.

A rapid method of species identification of wild chironomids (Diptera: Chironomidae) via electrophoresis of hemoglobin proteins in sodium dodecyl sulfate polyacrylamide gel (SDS–PAGE)

2014 ◽  
Vol 104 (5) ◽  
pp. 639-651 ◽  
Author(s):  
J.T. Oh ◽  
J.H. Epler ◽  
C.S. Bentivegna
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Head Capsule ◽  
Species Level ◽  
Polyacrylamide Gel ◽  
Taxonomic Identification ◽  
Sds Page ◽  
Dodecyl Sulfate

AbstractStudying aquatic benthic macroinvertebrates (BMIs) in the field requires accurate taxonomic identification, which can be difficult and time consuming. Conventionally, head capsule morphology has been used to identify wild larvae of Chironomidae. However, due to the number of species and possible damage and/or deformity of their head capsules, another supporting approach for identification is needed. Here, we provide hemoglobin (Hb) protein in hemolymph of chironomids as a new biomarker that may help resolve some of the ambiguities and difficulties encountered during taxonomic identification. Chironomids collected from two locations in Maine and New Jersey, USA were identified to the genus level and in some cases to the species-level using head capsule and body morphologies. The head capsule for a particular individual was then associated with a corresponding Hb protein profile generated from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Distinct Hb profiles were observed from one group (Thienemannimyia) and four genera (Chironomus, Cricotopus, Dicrotendipes, and Glyptotendipes) of chironomids. Several species were polymorphic, having more than one Hb profile and/or having bands of the same size as those of other species. However, major bands and the combination of bands could distinguish individuals at the genus and sometimes species-level. Overall, this study showed that Hb profiles can be used in combination with head capsule morphology to identify wild chironomids.

Whole-cell protein and ITS rDNA profiles as diagnostic tools to discriminate Fusarium avenaceum intraspecific variability and associated virulence

2009 ◽  
Vol 55 (2) ◽  
pp. 117-125 ◽  
Author(s):  
V. Vujanovic ◽  
S. Vidovic ◽  
M. R. Fernandez ◽  
P. Daida ◽  
D. Korber
Keyword(s):  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intraspecific Variability ◽  
Fusarium Avenaceum ◽  
Cell Protein ◽  
Diagnostic Tools ◽  
Group Method ◽  
Head Blight ◽  
Sds Page

A total of 91 isolates of Fusarium avenaceum were regrouped into 15 phenotypes and 10 vegetative compatibility groups showing specific one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1-D SDS–PAGE) protein profiles and less-specific internal transcribed spacer rDNA profiles. Each isolate possessed reproducible signature protein bands. Indeed, the unweighted pair group method with arithmetic averages clustering revealed that the protein profile of each group of isolates correlated with fungus virulence. The use of SDS–PAGE offers a simple and sensitive technique for routine differentiation between pathogenic and nonpathogenic isolates within unknown F. avenaceum populations. The discovery has significant implications for risk assessment of cereal yield to ensure food and feed safety. This low-cost approach has the potential to be optimized and extended to a broad spectrum of Fusarium head blight pathogens.

scholarly journals Comparison of protein and peptide fractionation approaches in protein identification and quantification from Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Liting Deng ◽  
David C. L. Handler ◽  
Dylan Multari ◽  
Paul A. Haynes
Keyword(s):  
Sample Preparation ◽  
Mass Spectrometer ◽  
Protein Identification ◽  
Ion Trap ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Similar Amount ◽  
Linear Ion Trap ◽  
Sds Page ◽  
Q Exactive

ABSTRACTProteomics, as a high-throughput technology, has been developed with the aim of investigating the maximum number of proteins in cells. However, protein discovery and data generation vary in depth and coverage when different technical strategies are used. In this study, four different sample preparation, and peptide or protein fractionation, methods were applied to identify and quantify proteins from log-phase yeast lysate: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); gas phase fractionation (GPF); filter-aided sample preparation (FASP)- GPF; and FASP-high pH reversed phase fractionation (HpH). Fractionated samples were initially analyzed and compared using nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS) employing data dependent acquisition on a linear ion trap instrument. The number of fractions and replicates was adjusted so that each experiment used a similar amount of mass spectrometric instrument time, approximately 16 hours. A second set of experiments was performed using a Q Exactive Orbitrap instrument, comparing FASP-GPF, SDS-PAGE and FASP-HpH. Compared with results from the linear ion trap mass spectrometer, the use of a Q Exactive Orbitrap mass spectrometer enabled a small increase in protein identifications using SDS-PAGE and FASP-GPF methods, and a large increase using FASP-HpH. A big advantage of using the higher resolution instrument found in this study was the substantially increased peptide identifications which enhance the proteome coverage. A total of 1035, 1357 and 2134 proteins were separately identified by FASP-GPF, SDS-PAGE and FASP-HpH. Combining results from the Orbitrap experiments, there were a total of 2269 proteins found, with 94% of them identified using the FASP-HpH method. Therefore, the FASP-HpH method is the optimal choice among these approaches when using a high resolution spectrometer, when applied to this type of sample.

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