scholarly journals Proteomic profile of pulmonary and extra pulmonary TB samples

2018 ◽  
Vol 16 (2) ◽  
pp. 21-27
Author(s):  
J. Kumar ◽  
C. Cheekavolu ◽  
V.L. Ashalatha ◽  
P. Leela ◽  
H.F. Daginawala
Keyword(s):  
Early Diagnosis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Secretory Proteins ◽  
Healthy Individuals ◽  
Serum Samples ◽  
Major Health Problem ◽  
Major Health ◽  
Therapeutic Drugs ◽  
Proteomic Approach

Introduction: Tuberculosis (TB) is a major health problem in India, so early diagnosis and treatment of Mycobacterium tuberculosis (M.tb.) infection can prevent deaths from this pathogen. The secretion of proteins by M.tb. is important in diagnostic purposes for generation of therapeutic drugs and vaccines candidates for TB. The objective of this study was to identify the protein expression (biomarkers) in TB and Tuberculosis meningitis (TBM) using proteomic approach. Methods: In this study, using Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), we analyzed the secretory proteins of M.tb. in serum, cerebrospinal fluid (CSF) samples. The identified proteins were determined by Total Lab- 100 Quantity One densitometry software. Results: Our study showed that protein bands expressed in CSF samples reveals the presence of 72kD, 70kD, 44kD, 40kD & 16kD predominantly in TBM patients compared to healthy individuals. The electrophoretogram identified 97kD, 72kD, 44kD, 38kD, 29kD & 16kD predominant proteins in serum samples of TB patients. Conclusion: The detection of secretory proteins in serum and CSF samples of M.tb. in TB and TBM patients gives reliable and early diagnosis of TB and TBM. The secretory proteins can be useful as immunodiagnostic and vaccine targets which can serve as important biomarkers

Radioimmunoassay for the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen: a new serum marker of bone collagen degradation

1993 ◽  
Vol 39 (4) ◽  
pp. 635-640 ◽  
Author(s):  
J Risteli ◽  
I Elomaa ◽  
S Niemi ◽  
A Novamo ◽  
L Risteli
Keyword(s):  
Type I Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bone Collagen ◽  
Collagen Molecule ◽  
Type I ◽  
Serum Samples ◽  
Amino Terminal ◽  
Wide Range ◽  
Carboxy Terminal

Abstract We developed a radioimmunoassay (RIA) for the carboxy-terminal telopeptides of type I collagen (ICTP), cross-linked with the helical domain of another type I collagen molecule, after isolation from human femoral bone. The cross-linked peptide was liberated by digesting insoluble, denatured bone collagen either with bacterial collagenase or with trypsin, and purified by two successive reversed-phase separations on HPLC, with monitoring of pyridinoline-specific fluorescence. The purity of the peptide was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its origin in the type I collagen fibers was determined by amino-terminal amino acid sequencing. Polyclonal antibodies and a separation reagent containing second antibody and polyethylene glycol are used in the RIA. An immunologically identical, somewhat larger antigen is present in human serum; its concentration increases in multiple myeloma and in rheumatoid arthritis. The ICTP antigen seems to be cleared from the circulation by the kidneys, because glomerular filtration rates that are two-thirds of normal or less are associated with increased circulating ICTP concentrations. The CVs of the method are between 3% and 8% for a wide range of concentrations. The analysis of 40 serum samples can be completed in 4 h.

Apoptotic cell thrombospondin-1 and heparin-binding domain lead to dendritic-cell phagocytic and tolerizing states

Blood ◽  
2006 ◽  
Vol 108 (10) ◽  
pp. 3580-3589 ◽  
Author(s):  
Alon Krispin ◽  
Yaniv Bledi ◽  
Mizhir Atallah ◽  
Uriel Trahtemberg ◽  
Inna Verbovetski ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Apoptotic Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Domain ◽  
Apoptotic Cells ◽  
Heparin Binding ◽  
Proteomic Approach ◽  
Thrombospondin 1 ◽  
Heparin Binding Domain

Abstract Apoptotic cells were shown to induce dendritic cell immune tolerance. We applied a proteomic approach to identify molecules that are secreted from apoptotic monocytes, and thus may mediate engulfment and immune suppression. Supernatants of monocytes undergoing apoptosis were collected and compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and differentially expressed proteins were identified using tandem mass spectrometry. Thrombospondin-1 (TSP-1) and its cleaved 26-kDa heparin-binding domain (HBD) were identified. We show that TSP-1 is expressed upon induction of monocyte apoptosis in a caspase-dependent pattern and the HBD is cleaved by chymotrypsin-like serine protease. We further show that CD29, CD36, CD47, CD51, and CD91 simultaneously participate in engulfment induction and generation of an immature dendritic cell (iDC) tolerogenic and phagocytic state. We conclude that apoptotic cell TSP-1, and notably its HBD, creates a signalosome in iDCs to improve engulfment and to tolerate engulfed material prior to the interaction with apoptotic cells.

scholarly journals Initiation and processing in vitro of the primary translation products of guinea-pig caseins

1979 ◽  
Vol 184 (2) ◽  
pp. 261-267 ◽  
Author(s):  
R K Craig ◽  
P A J Perera ◽  
A Mellor ◽  
A E Smith
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Secretory Proteins ◽  
Post Translational Modifications ◽  
Microsomal Membranes

1. Guinea-pig caseins synthesized in a mRNA-directed wheat-germ cell-free protein-synthesizing system represent the primary translation products, even though they appear to be of lower molecular weight when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in parallel with caseins isolated from guinea-pig milk. 2. Identification of the N-terminal dipeptide of the primary translational product of caseins A, B and C and alpha-lactalbumin showed that all shared a common sequence, which was identified as either Met-Arg or Met-Lys. 3. Procedures utilizing methionyl-tRNAfMet or methionyl-tRNAMet in the presence or absence of microsomal membranes during translation provide a rapid method of distinguishing between N-terminal processing of peptides synthesized in vitro and other post-translational modifications (glycosylation, phosphorylation), which also result in a change in mobility of peptides when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The results demonstrate that guinea-pig caseins, in common with most other secretory proteins, are synthesized with transient N-terminal ‘signal’-peptide extensions, which are cleaved during synthesis in the presence of microsomal membranes.

scholarly journals Secretory Immune Response to Membrane Antigens during Giardia lamblia Infection in Humans

1998 ◽  
Vol 66 (2) ◽  
pp. 756-759 ◽  
Author(s):  
Disney M. Rosales-Borjas ◽  
Juan Díaz-Rivadeneyra ◽  
Antonio Doña-Leyva ◽  
Sergio A. Zambrano-Villa ◽  
Carmen Mascaró ◽  
...  
Keyword(s):  
Immune Response ◽  
Sodium Dodecyl Sulfate ◽  
Giardia Lamblia ◽  
Gel Electrophoresis ◽  
Membrane Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Healthy Individuals ◽  
Future Studies ◽  
Membrane Antigens

ABSTRACT The secretory immune response in humans infected with Giardia lamblia was studied by using saliva samples and a membrane-rich protein fraction. The membrane fraction, studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed 24 antigen bands, ranging from 170 to 14 kDa. Saliva samples from giardiasis patients showed a heterogeneous response against the membrane fraction when they were assayed by immunoblotting. Among the antigens recognized by patient saliva samples, those of 170, 105, 92, 66, 32, 29, and 14 kDa stood out. These antigens were not recognized by saliva samples from healthy individuals. They may be of importance in future studies of protection from or diagnosis of G. lamblia infections.

Quantitative analysis of serum proteins separated by capillary electrophoresis

1993 ◽  
Vol 39 (4) ◽  
pp. 689-692 ◽  
Author(s):  
J W Kim ◽  
J H Park ◽  
J W Park ◽  
H J Doh ◽  
G S Heo ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Gel Electrophoresis ◽  
Serum Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Serum Samples ◽  
Polyclonal Gammopathy ◽  
Quantitative Analyses

Abstract The possibility of open tubular capillary electrophoresis for clinical diagnostic use is examined. Capillary electrophoresis was performed in an untreated 50 microns (i.d.) x 100 cm (65 cm to detector) capillary with detection of absorbance at 200 nm. Conditions for the separation of serum proteins without adsorption to the capillary surface were established. Quantitative analyses of serum samples from 38 patients with liver cirrhosis, nephrotic syndrome, or polyclonal gammopathy by capillary electrophoresis were done and the results were compared with those by conventional agarose gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All samples were analyzed in duplicate. We evaluated linearity of response, within-run CV, and the correlation between capillary electrophoresis and agarose gel electrophoresis.

scholarly journals Detection of Serum Thermolabile β-2 Macroglycoprotein (Hakata Antigen) by Enzyme-Linked Immunosorbent Assay Using Polysaccharide Produced by Aerococcus viridans

2001 ◽  
Vol 8 (2) ◽  
pp. 454-459 ◽  
Author(s):  
Mitsushi Tsujimura ◽  
Chuzo Ishida ◽  
Yasuko Sagara ◽  
Takashi Miyazaki ◽  
Koichi Murakami ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gas Chromatography Mass Spectrometry ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Serum Samples ◽  
Aerococcus Viridans ◽  
Sds Page

ABSTRACT Although a serum thermolabile β-2 macroglycoprotein (TMG) may play a role in host defense as a lectin, little is known of its related physiological functions, mainly due to a lack of appropriate methods for tracing the functions of TMG. We identified a polysaccharide fromAerococcus viridans, PSA, which reacts with TMG, and based on this finding, we developed an enzyme-linked immunosorbent assay to trace the functions of TMG. Using ethanol precipitation and DEAE-Sepharose and Sephacryl S-400 column chromatographies, we isolated PSA from cultured medium of A. viridans, and it exhibited specific binding against TMG in blood samples. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated PSA showed ladder bands that implied the existence of repeating units composed of d-glucose,N-acetyl-d-glucosamine, d-mannose, and d-xylose, as confirmed by gas chromatography-mass spectrometry. SDS-PAGE and immunochemical analysis, using rabbit anti-TMG antibody, showed that PSA specifically binds solely to intact serum TMG but not to TMG heated at 56°C for 30 min, a condition under which antigenicity is lost. TMG in serum samples bound to PSA in a dose-dependent manner, and this binding was clearly suppressed by addition of PSA. These observations indicate that PSA is a useful adsorbent to TMG and can be used to develop appropriate methods for tracing the functions of TMG.

scholarly journals Identification and Characterization of Immunogenic Proteins of Mycoplasma genitalium

2006 ◽  
Vol 13 (8) ◽  
pp. 913-922 ◽  
Author(s):  
Helle Friis Svenstrup ◽  
Jørgen Skov Jensen ◽  
Kris Gevaert ◽  
Svend Birkelund ◽  
Gunna Christiansen
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mycoplasma Genitalium ◽  
Cross Reactivity ◽  
Rabbit Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Immunogenic Proteins ◽  
Recombinant Fragments

ABSTRACT Mycoplasma genitalium causes nonchlamydial nongonococcal urethritis. M. genitalium was detected by PCR in 17 urethral swabs obtained from 99 men with and without urethritis (J. S. Jensen, R. Orsum, B. Dohn, S. Uldum, A. M. Worm, and K. Lind, Genitourin. Med. 69:265-269, 1993), and later, four M. genitalium strains were isolated (J. S. Jensen, H. T. Hansen, and K. Lind, J. Clin. Microbiol. 34:286-291, 1996). The objective of this study was to characterize immunogenic proteins of M. genitalium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting by using a hyperimmune rabbit serum against M. genitalium G37, determine their identity by mass spectrometry, and develop an M. genitalium-specific enzyme-linked immunosorbent assay (ELISA) free from cross-reactivity with M. pneumoniae antibodies. Using recombinant fragments of the C-terminal part of MgPa (rMgPa), we developed a specific ELISA for detection of M. genitalium antibodies. This antigen did not bind M. pneumoniae antibodies. Using serum samples from the 99 men with and without urethritis, we found that 26 had immunoglobulin G (IgG) antibodies to M. genitalium. There was a strong statistically significant correlation between PCR and IgG antibodies to M. genitalium (odds ratio [OR], 5.9; 95% confidence interval [CI], 2.3 to 21.5; P = 0.002). Furthermore, men with recurrent urethritis were more likely to have antibodies to M. genitalium than were those without recurrent urethritis (OR, 4.0; 95% CI, 1.1 to 14.5; P = 0.0383) and they had significantly higher antibody titers. By use of the rMgPa ELISA, this study further substantiates the importance of M. genitalium as a cause of male urethritis.

scholarly journals Phase Variation among Major Surface Antigens ofMycoplasma penetrans

2001 ◽  
Vol 69 (12) ◽  
pp. 7642-7651 ◽  
Author(s):  
Kerstin Röske ◽  
Alain Blanchard ◽  
Isabelle Chambaud ◽  
Christine Citti ◽  
Jürgen H. Helbig ◽  
...  
Keyword(s):  
Infected Patient ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Phase Variation ◽  
Immunoblot Analysis ◽  
Phase Variable ◽  
Serum Samples ◽  
Independent Manner ◽  
Major Surface

ABSTRACT The pathogenicity and prevalence of Mycoplasma penetrans, a Mycoplasma species recently isolated from humans, are still debated. A major P35 antigen, which is used as target epitope in serological assays, was shown to be a phase-variable lipid-associated membrane protein (LAMP). In this study, we performed a comparative analysis of the LAMP patterns from fiveM. penetrans clinical isolates and from the type strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and immunoblots with sera serially collected from an M. penetrans-infected patient indicated that these strains expressed different LAMP repertoires. Furthermore, the intraclonal variation in the expression of LAMPs (P34A, P34B, P35, and P38) was monitored by immunoblot analysis with three specific monoclonal antibodies (MAbs) developed in this study and MAb 7 to P35. The phase variation of these LAMPs occurs in an independent manner, with frequencies of variation ranging from 10−2 to 10−4 per cell per generation. Consistent with their amphipathic nature, the P34B and P38 antigens were found exposed at the cell surface. The DNA sequence encoding the P38 antigen was defined and found to be related to those of the P35 gene and other putative LAMP-encoding genes, suggesting that these variable antigens are encoded by a family of related genes. Finally, the serum samples from an M. penetrans-infected patient contained antibodies that reacted with a P36 antigen expressed in different M. penetrans strains but not in the isolate recovered from this patient. This result suggested that in vivo phase variation of P36 occurred, which would support a role for these LAMP variations in avoiding the host's immune vigilance.

Expression, purification and structural analysis of recombinant rBdh-2His6, a spermadhesin from buck (Capra hircus) seminal plasma

2012 ◽  
Vol 24 (4) ◽  
pp. 580 ◽  
Author(s):  
Antônia Sâmia F. Nascimento ◽  
João B. Cajazeiras ◽  
Kyria S. Nascimento ◽  
Sara Monalisa S. Nogueira ◽  
Bruno L. Sousa ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Structural Changes ◽  
Polymerase Chain Reaction Amplification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Capra Hircus ◽  
Secretory Proteins ◽  
Reaction Amplification ◽  
Polymerase Chain

Spermadhesins, a family of secretory proteins from the male genital tract of ungulate species, belong to the group of animal lectins. Spermadhesins have a prominent role in different aspects of fertilisation, such as spermatozoid capacitation, acrosomal stabilisation, sperm–oviduct interaction and during sperm–oocyte fusion. Proteins (spermadhesins) in buck seminal plasma were described. In the present study, bodhesin Bdh-2 cDNA present in buck seminal plasma was subcloned with the expression plasmid pTrcHis TOPO used to transform Escherichia coli Top10 One shot cells. The recombinant clones were selected by growth in 50 µg mL–1 ampicillin-containing LB broth and polymerase chain reaction amplification. Recombinant rBdh-2His6 synthesis was monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and followed by immunoblotting using monoclonal anti-His antibody. Production of rBdh-2 using low temperatures was not satisfactory. Greater production of rBdh-2 occurred with 1.5 mM isopropyl β-d-thiogalactoside after 2 h of induction. The method used to purify rBdh-2 was affinity chromatography on a His-Trap column following ion-exchange chromatography on a DEAE-Sephacel column. The secondary structure of the rBdh-2His6 was evaluated by spectral profile circular dichroism (CD). The prevalence of secondary structures like β-sheets, with fewer unfolded structures and α-helices, was confirmed. The structure of rBdh-2His6 remained stable up to 35°C. However, significant structural changes were observed at temperatures higher than 40°C related to a distortion of the CD spectrum.

scholarly journals Human T Cell and Antibody-Mediated Responses to theMycobacterium tuberculosisRecombinant 85A, 85B, and ESAT-6 Antigens

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Gilson C. Macedo ◽  
Adriana Bozzi ◽  
Helena Rachel Weinreich ◽  
Andre Bafica ◽  
Henrique C. Teixeira ◽  
...  
Keyword(s):  
Healthy Individuals ◽  
Major Health Problem ◽  
Major Health ◽  
Tuberculosis Patients ◽  
Cellular Immune Responses ◽  
Human T Cell ◽  
Tnf Α ◽  
Ifn Γ ◽  
Cellular Immune ◽  
Control And Eradication

Tuberculosis remains a major health problem throughout the world causing large number of deaths. Effective disease control and eradication programs require the identification of major antigens recognized by the protective responses againstM. tuberculosis. In this study, we have investigated humoral and cellular immune responses toM. tuberculosis-specific Ag85A, Ag85B, and ESAT-6 antigens in Brazilian patients with pulmonary (P,n=13) or extrapulmonary (EP,n=12) tuberculosis, patients undergoing chemotherapy (PT,n=23), and noninfected healthy individuals (NI,n=7). Compared to NI, we observed increased levels of IgG1 responses to Ag85B and ESAT-6 in P and PT groups. Regarding cellular immunity, Ag85A and ESAT-6 were able to discriminate P, PT, and EP patients from healthy individuals by IFN-γ production and P and PT groups from EP individuals by production of TNF-α. In summary, these findings demonstrate the ability of Ag85A, Ag85B, and ESAT-6 to differentiate TB patients from controls by IgG1, IFN-γ and TNF-α production.

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