Structural analysis of urinary and serum IgG in normal individuals and patients by Western blotting in relation to stability of urinary IgG and total IgG value.

Although mycobacterial proteins in exosomes from peripheral serum of patients with tuberculosis (TB) have been identified, other exact compositions of exosomes remain unknown. In the present study, a comprehensive proteomics analysis of serum exosomes derived from patients with active TB (ATB) was performed. Exosomes from patients with ATB were characterized using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blotting analysis. Then identified protein components were quantified by label-free proteomics and were determined via bioinformatics analysis. A total of 123 differential proteins were identified in ATB serum exosomes and analyzed with Gene Ontology (GO) analysis. Among these proteins heat shock protein70 (HSP70), CD81, major histocompatibility complex-I (MHC-I ) and tumor susceptibility gene101 (TSG101) were present in exosomes of ATB and normal individuals confirmed via western blotting. In addition, among identified exosomal proteins lipopolysaccharide binding protein (LBP) increased significantly, but CD36 and MHC-I decreased significantly in ATB exosomes. Meanwhile, MHC-I was down-expressed in serum and peripheral blood mononuclear cells (PBMCs) of ATB, but interestingly CD36 was down-regulated in serum and up-expressed in PBMCs of ATB patients validated with ELISA and flow cytometry. CD36 was up-regulated by M. tuberculosis H37Ra infection in macrophages and suppressed in exosomes from H37Ra infected macrophages detected by western blotting. This study provided a comprehensive description of the exosome proteome in the serum of patients with ATB and revealed certain potential biomarkers associated with TB infection.

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Radioimmunoassay for immunologlobulin G in serum and urine.

Clinical Chemistry ◽

10.1093/clinchem/25.12.2015 ◽

1979 ◽

Vol 25 (12) ◽

pp. 2015-2019 ◽

Cited By ~ 2

Author(s):

J Woo ◽

M Floyd ◽

M A Longley ◽

D C Cannon

Keyword(s):

Dose Response ◽

Allograft Rejection ◽

Response Curve ◽

Renal Allograft ◽

Response Curves ◽

Assay Sensitivity ◽

Renal Allografts ◽

Serum Igg ◽

Normal Individuals ◽

Serum And Urine

Abstract We describe a radioimmunoassay for immunoglobulin G (IgG) in serum and urine. Aliquots of diluted samples and 125I-labeled IgG were incubated in antibody-coated tubes at 37 degrees C for 24 h, the supernates were decanted, and the radioactivity in tubes containing the bound fraction was counted. The dose-response curve in the range of 0.4--500 mg/L of urine or 640--40 000 mg/L of serum was linear on logit-log transformation and iterative weighted regression. Assay sensitivity was 10 ng of IgG. Validation studies included testing for precision, accuracy, antibody specificity, and parallelism of the dose-response curves for standard and unknown. In a study of 14 apparently normal individuals, serum IgG = 4.0-10.9 gL, urine IgG = 1.1-4.8 mg/24 h, and IgG clearance = 0.2 X 10(-4) to 4.8 X 10(-4) mL/min. In 20 patients with renal allografts, serum IgG = 15.8-66 g/L, urine IgG - 9.6-626 mg/24 h, and IgG clearance = 9 X 10(-4) to 1.99 X 10(-1) mL/min. IgG values correlated well with severity of renal allograft rejection.

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Elevated Phosphate Levels Trigger Autophagy-Mediated Cellular Apoptosis in H9c2 Cardiomyoblasts

Cardiorenal Medicine ◽

10.1159/000479010 ◽

2017 ◽

Vol 8 (1) ◽

pp. 31-40 ◽

Cited By ~ 2

Author(s):

Yao-Lung Liu ◽

Kuan-Ho Lin ◽

Shanmugam Tamilselvi ◽

Wei-Kung Chen ◽

Chia-Yao Shen ◽

...

Keyword(s):

Cytochrome C ◽

Western Blotting ◽

Annexin V ◽

Western Blotting Analysis ◽

Blotting Analysis ◽

Normal Individuals ◽

Associated Proteins ◽

Cellular Apoptosis ◽

H9c2 Cardiomyoblasts ◽

Related Proteins

Background/Aim: In chronic kidney disease (CKD), kidneys fail to maintain phosphorus homeostasis in serum. Elevated phosphorus levels in serum have been associated with cardiovascular diseases in CKD patients and in normal individuals. In this study, we evaluated the level of autophagy- and apoptosis-related markers under different concentrations of hyperphosphate in myocardial cells. Methods: Modulation inflicted on the levels of various survival-, autophagy-, and apoptosis-related markers were determined by Western blotting analysis using total protein extract. FITC-annexin V staining was performed to quantify the apoptotic cells in all groups. Results: Hyperphosphate treatments showed to induce autophagy-related proteins beclin-1, ATG7, and LC3 II through the pAMPK-ULK1 pathway in Western blotting analysis. Further, apoptosis-associated proteins such as Bax, Bid, cytochrome c, and c-caspase-9 were also upregulated with hyperphosphate treatment. 3-Methyladenine, an autophagy inhibitor, inhibited apoptosis significantly in FITC-annexin V staining, and the inhibition of Bax, cytochrome c, and c-caspase-3 was shown by Western blotting. Conclusion: The results suggest that hyperphosphate in H9c2 cardiomyoblasts would lead to cellular apoptosis via autophagy, which is mediated by the pAMPK signaling pathway. Our findings revealed the possible mechanism responsible for the heart damage under hyperphosphatemia.

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Detection of Antibodies to BrucellaCytoplasmic Proteins in the Cerebrospinal Fluid of Patients with Neurobrucellosis

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.6.5.756-759.1999 ◽

1999 ◽

Vol 6 (5) ◽

pp. 756-759 ◽

Cited By ~ 29

Author(s):

Pablo C. Baldi ◽

George F. Araj ◽

Graciela C. Racaro ◽

Jorge C. Wallach ◽

Carlos A. Fossati

Keyword(s):

Cerebrospinal Fluid ◽

Western Blotting ◽

Immunosorbent Assay ◽

Immunoglobulin G ◽

Serological Diagnosis ◽

Neurological Involvement ◽

Enzyme Linked Immunosorbent Assay ◽

Specific Diagnosis ◽

Serum Igg ◽

Cytoplasmic Proteins

ABSTRACT The diagnosis of human neurobrucellosis usually relies on the detection of antibodies to Brucella lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) by agglutination tests or enzyme-linked immunosorbent assay (ELISA). Here we describe the detection of immunoglobulin G (IgG) to cytoplasmic proteins (CP) ofBrucella spp. by ELISA and Western blotting in seven CSF samples from five patients with neurobrucellosis. While IgG to CP (titers of 200 to 12,800) and IgG to LPS (800 to 6,400) were found in the CSF of these patients, these antibodies were not detected in CSF samples from two patients who had systemic brucellosis without neurological involvement. The latter, however, had serum IgG and IgM to both LPS and CP. No reactivity to these antigens was found in CSF samples from 14 and 20 patients suffering from nonbrucellar meningitis and noninfectious diseases, respectively. These findings suggest that, in addition to its usefulness in the serological diagnosis of human systemic brucellosis, the ELISA with CP antigen can be used for the specific diagnosis of human neurobrucellosis.

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Pseudomonas aeruginosa 1244 Pilin Glycosylation: Glycan Substrate Recognition

Journal of Bacteriology ◽

10.1128/jb.00273-06 ◽

2006 ◽

Vol 188 (12) ◽

pp. 4244-4252 ◽

Cited By ~ 29

Author(s):

Joseph Horzempa ◽

Charles R. Dean ◽

Joanna B. Goldberg ◽

Peter Castric

Keyword(s):

Mass Spectrometry ◽

Pseudomonas Aeruginosa ◽

Structural Analysis ◽

Western Blotting ◽

Biosynthetic Pathway ◽

Substrate Recognition ◽

O Antigen ◽

Recognition Elements ◽

Glycosylation Reaction

ABSTRACT The pilin of Pseudomonas aeruginosa 1244 is glycosylated with an oligosaccharide that is structurally identical to the O-antigen repeating unit of this organism. Concordantly, the metabolic source of the pilin glycan is the O-antigen biosynthetic pathway. The present study was conducted to investigate glycan substrate recognition in the 1244 pilin glycosylation reaction. Comparative structural analysis of O subunits that had been previously shown to be compatible with the 1244 glycosylation machinery revealed similarities among sugars at the presumed reducing termini of these oligosaccharides. We therefore hypothesized that the glycosylation substrate was within the sugar at the reducing end of the glycan precursor. Since much is known of PA103 O-antigen genetics and because the sugars at the reducing termini of the O7 (strain 1244) and O11 (strain PA103) are identical (β-N-acetyl fucosamine), we utilized PA103 and strains that express lipopolysaccharide (LPS) with a truncated O-antigen subunit to test our hypothesis. LPS from a strain mutated in the wbjE gene produced an incomplete O subunit, consisting only of the monosaccharide at the reducing end (β-d-N-acetyl fucosamine), indicating that this moiety contained substrate recognition elements for WaaL. Expression of pilAO 1244 in PA103 wbjE::aacC1, followed by Western blotting of extracts of these cells, indicated that pilin produced has been modified by the addition of material consistent with a single N-acetyl fucosamine. This was confirmed by analyzing endopeptidase-treated pilin by mass spectrometry. These data suggest that the pilin glycosylation substrate recognition features lie within the reducing-end moiety of the O repeat and that structures of the remaining sugars are irrelevant.

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Structural analysis of the surface-layer protein of spirillum serpens by high-resolution electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077268 ◽

1983 ◽

Vol 41 ◽

pp. 738-739

Author(s):

W. H. Wu ◽

R. M. Glaeser

Keyword(s):

Electron Microscopy ◽

Surface Layer ◽

Structural Analysis ◽

High Resolution ◽

Low Dose ◽

High Resolution Electron Microscopy ◽

Optical Diffraction ◽

Surface Layer Protein ◽

Morphological Unit ◽

Resolution Electron

Spirillum serpens possesses a surface layer protein which exhibits a regular hexagonal packing of the morphological subunits. A morphological model of the structure of the protein has been proposed at a resolution of about 25 Å, in which the morphological unit might be described as having the appearance of a flared-out, hollow cylinder with six ÅspokesÅ at the flared end. In order to understand the detailed association of the macromolecules, it is necessary to do a high resolution structural analysis. Large, single layered arrays of the surface layer protein have been obtained for this purpose by means of extensive heating in high CaCl2, a procedure derived from that of Buckmire and Murray. Low dose, low temperature electron microscopy has been applied to the large arrays.As a first step, the samples were negatively stained with neutralized phosphotungstic acid, and the specimens were imaged at 40,000 magnification by use of a high resolution cold stage on a JE0L 100B. Low dose images were recorded with exposures of 7-9 electrons/Å2. The micrographs obtained (Fig. 1) were examined by use of optical diffraction (Fig. 2) to tell what areas were especially well ordered.

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RNA polymerase: Preparation and structural analysis of helical polymers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010011091x ◽

1984 ◽

Vol 42 ◽

pp. 152-153

Author(s):

E. Loren Buhle ◽

Pamela Rew ◽

Ueli Aebi

Keyword(s):

Structural Analysis ◽

Rna Polymerase ◽

Molecular Level ◽

X Ray Diffraction ◽

Helical Polymers ◽

X Ray ◽

E Coli ◽

The Past ◽

Ordered Arrays ◽

Low Ionic Strength

While DNA-dependent RNA polymerase represents one of the key enzymes involved in transcription and ultimately in gene expression in procaryotic and eucaryotic cells, little progress has been made towards elucidation of its 3-D structure at the molecular level over the past few years. This is mainly because to date no 3-D crystals suitable for X-ray diffraction analysis have been obtained with this rather large (MW ~500 kd) multi-subunit (α2ββ'ζ). As an alternative, we have been trying to form ordered arrays of RNA polymerase from E. coli suitable for structural analysis in the electron microscope combined with image processing. Here we report about helical polymers induced from holoenzyme (α2ββ'ζ) at low ionic strength with 5-7 mM MnCl2 (see Fig. 1a). The presence of the ζ-subunit (MW 86 kd) is required to form these polymers, since the core enzyme (α2ββ') does fail to assemble into such structures under these conditions.

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Interpreting HVEM of muscle-cell impulse networks

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010012103x ◽

1992 ◽

Vol 50 (1) ◽

pp. 126-127

Author(s):

Paul DeCosta ◽

Kyugon Cho ◽

Stephen Shemlon ◽

Heesung Jun ◽

Stanley M. Dunn

Keyword(s):

Structural Analysis ◽

Muscle Cell ◽

Electron Micrographs ◽

Relative Size ◽

Automatic Classification ◽

Nerve Fibers ◽

Analysis Algorithm ◽

Structural Networks

Introduction: The analysis and interpretation of electron micrographs of cells and tissues, often requires the accurate extraction of structural networks, which either provide immediate 2D or 3D information, or from which the desired information can be inferred. The images of these structures contain lines and/or curves whose orientation, lengths, and intersections characterize the overall network.Some examples exist of studies that have been done in the analysis of networks of natural structures. In, Sebok and Roemer determine the complexity of nerve structures in an EM formed slide. Here the number of nodes that exist in the image describes how dense nerve fibers are in a particular region of the skin. Hildith proposes a network structural analysis algorithm for the automatic classification of chromosome spreads (type, relative size and orientation).

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Auditory Selective Attention in Cerebral-Palsied Individuals

Language Speech and Hearing Services in Schools ◽

10.1044/0161-1461.1604.260 ◽

1985 ◽

Vol 16 (4) ◽

pp. 260-266 ◽

Cited By ~ 4

Author(s):

Lee Ann Laraway

Keyword(s):

Selective Attention ◽

White Noise ◽

Normal Subjects ◽

Auditory Selective Attention ◽

Normal Individuals ◽

Significant Difference

The purpose of this study was to determine whether there is a statistically significant difference between the auditory selective attention abilities of normal and cerebral-palsied individuals. Twenty-three cerebral-palsied and 23 normal subjects between the ages of 5 and 21 were asked to repeat a series of 30 items consisting of from 2 to 4 digits in the presence of intermittent white noise. Results of the study indicate that cerebral-palsied individuals perform significantly poorer than normal individuals when the stimulus is accompanied by noise. Noise was not a significant factor in the performance of the normal subjects regardless of age.

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