Biomedical subjects

Jon B Klein

Publications and source records attributed to Jon B Klein.

60 records · Page 4Linked to original sources

PubMed2002

Proteomic analysis of CA1 and CA3 regions of rat hippocampus and differential susceptibility to intermittent hypoxia.

The CA1 and CA3 regions of the hippocampus markedly differ in their susceptibility to hypoxia in general, and more particularly to the intermittent hypoxia that characterizes sleep apnea. Proteomic approaches were used to identify proteins differentially expressed in the CA1 and CA3 regions of the rat hippocampus and to assess changes in protein expression following a 6-h exposure to intermittent hypoxia (IH). Ninety-nine proteins were identified, and 15 were differentially expressed in the CA1 and the CA3 regions. Following IH, 32 proteins in the CA1 region and only 7 proteins in the more resistant CA3 area were up-regulated. Hypoxia-regulated proteins in the CA1 region included structural proteins, proteins related to apoptosis, primarily chaperone proteins, and proteins involved in cellular metabolic pathways. We conclude that IH-mediated CA1 injury results from complex interactions between pathways involving increased metabolism, induction of stress-induced proteins and apoptosis, and, ultimately, disruption of structural proteins and cell integrity. These findings provide initial insights into mechanisms underlying differences in susceptibility to hypoxia in neural tissue, and may allow for future delineation of interventional strategies aiming to enhance neuronal adaptation to IH.

Animals↗

PubMed2002

Phosphatidylinositol 3-kinase controls antineutrophil cytoplasmic antibodies-induced respiratory burst in human neutrophils.

Antineutrophil cytoplasmic antibodies (ANCA) activate human polymorphonuclear neutrophils (PMN) primed with tumor necrosis factor alpha (TNF-alpha) in vitro. Phosphatidylinositol 3-kinase (PI3-K) and the protein-serine/threonine kinase Akt have been implicated in the control of the phagocyte respiratory burst. The hypothesis that PI3-K controls the ANCA-induced respiratory burst was tested. TNF-alpha-primed PMN were stimulated with a monoclonal antibody to myeloperoxidase (MPO) and with PR3- and MPO-ANCA, respectively. Akt activation was assessed with phospho-specific antibodies. Superoxide release was measured with ferricytochrome. ANCA antigen translocation was assessed by fluorescence-activated cell sorter. The effect of TNF-alpha and MPO-ANCA on Akt signaling was studied with immunoprecipitation and glutathione S-transferase pull-down assays. Western blotting revealed rapid transient Akt phosphorylation during TNF-alpha priming and a second phosphorylation after ANCA. PI3-K inhibition by LY294002 blocked both Akt phosphorylation and superoxide generation. A total of 20 +/- 3 nmol O(2)(-)/0.75 x 10(6) PMN/45 min was released after stimulation with PR3-ANCA. LY294002 (5 microM) decreased this amount to 0.3 +/- 2.6 nmol (n = 10, P < 0.05); the MPO-ANCA values were 23 +/- 3 versus 1.6 +/- 3.6 (n = 10, P < 0.05). p38 MAPK inhibition with 10 microM SB202190 that also decreased ANCA-induced superoxide generation prevented S473 phosphorylation of Akt in response to TNF-alpha and to ANCA. However, SB202190 but not LY294002 abrogated TNF-alpha-mediated ANCA antigen surface translocation, demonstrating that superoxide generation and ANCA antigen translocation proceed by separate mechanisms. Akt, PAK1, and Rac1 existed as cytosolic complex in resting PMN. TNF-alpha stimulation increased association of PAK1 with Akt. An MPO monoclonal antibody did not alter the Akt signaling complex further. The data demonstrate the importance of PI3-K for the ANCA-induced PMN oxidant production.

Antibodies, Antineutrophil Cytoplasmic↗

PubMed2002

Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation.

BACKGROUND: Proteomic techniques have recently become available for large-scale protein analysis. The utility of these techniques in identification of urinary proteins is poorly defined. We constructed a proteome map of normal human urine as a reference protein database by using two differential fractionated techniques to isolate the proteins. METHODS: Proteins were isolated from urine obtained from normal human volunteers by acetone precipitation or ultracentrifugation, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry followed by peptide mass fingerprinting. RESULTS: A total of 67 protein forms of 47 unique proteins were identified, including transporters, adhesion molecules, complement, chaperones, receptors, enzymes, serpins, cell signaling proteins and matrix proteins. Acetone precipitated more acidic and hydrophilic proteins, whereas ultracentrifugation fractionated more basic, hydrophobic, and membrane proteins. Bioinformatic analysis predicted glycosylation to be the most common explanation for multiple forms of the same protein. CONCLUSIONS: Combining two differential isolation techniques magnified protein identification from human urine. Proteomic analysis of urinary proteins is a promising tool to study renal physiology and pathophysiology and to determine biomarkers of renal disease.

Acetone↗

PubMed2002

Differential expression of proteins in renal cortex and medulla: a proteomic approach.

BACKGROUND: Western blotting has previously been used to identify changes in protein expression in renal tissue. However, only a few proteins can be studied in each experiment by Western blot. We have used proteomic tools to construct protein maps of rat kidney cortex and medulla. METHODS: Expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were excised and digested with trypsin. Peptide masses were identified by MALDI-TOF mass spectrometry. The Mascot search engine was used to analyze the peptide masses and identify the proteins. RESULTS: Seventy-two proteins were identified (54 unique proteins) out of approximately 1000 spots visualized on each gel. Most of the spots were expressed both in cortex and medulla. Of the identified proteins, three were expressed only in medulla and one only in cortex. Nine proteins were expressed in both regions but to a greater extent in cortex and three proteins were expressed more in medulla. Differential expression was confirmed for three proteins by Western blot. CONCLUSIONS: A large group of proteins and their relative expression levels from cortical and medullary portions of rat kidneys were found. Sixteen proteins are differentially expressed. Proteomics can be used to identify differential expression of proteins in the kidney on a large scale. Proteomics should be useful to detect changes in renal protein expression in response to a large range of physiological and pathophysiological stimuli.

Animals↗

PubMed2002

Proteomic analysis of differentially expressed Chlamydia pneumoniae genes during persistent infection of HEp-2 cells.

Recent data have shown that the respiratory pathogen Chlamydia pneumoniae expresses an altered gene transcription profile during gamma interferon (IFN-gamma)-induced persistent infection in vitro. In the present study, we examined, by proteomics, expression of C. pneumoniae proteins labeled intracellularly with [(35)S]methionine/cysteine under normal conditions or IFN-gamma-mediated persistence. The identity of differentially expressed proteins during persistent infection was determined by matching spots to those of proteins identified in C. pneumoniae elementary bodies by matrix-assisted laser desorption ionization mass spectrometry. Upon treatment with 50 U of IFN-gamma per ml, a marked upregulation of major outer membrane protein (MOMP), heat shock protein 60 (Hsp-60/GroEL), and proteins with functions in DNA replication (GyrA), transcription (RpoA, PnP), translation (Rrf), glycolysis (PgK, GlgP), and type III secretion (SctN) was observed at 24 h of infection. In contrast, no significant decreases in bacterial protein expression were found in C. pneumoniae-infected cells due to IFN-gamma treatment. Upregulation of C. pneumoniae proteins involved in diverse functions during persistent infection may allow the organism to resist the inhibitory effects of IFN-gamma while retaining basic functions. Future studies should examine the differential expression of chlamydial proteins during the developmental cycle under IFN-gamma pressure to obtain a finer representation of the gene products involved in establishing persistence.

Bacterial Proteins↗

PubMed2002

Molecular conformation dictates signaling module formation: example of PKCepsilon and Src tyrosine kinase.

Our laboratory has conducted multiple functional proteomic analyses to characterize the components of protein kinase C (PKC)epsilon cardioprotective signaling complexes and found that activation of PKCepsilon induces dynamic modulation of these complexes. In addition, it is known that signal transduction within a complex involves the formation of modules, one of which has been shown to include PKCepsilon and Src tyrosine kinase in the rabbit heart. However, the cellular mechanisms that define the assembly of PKCepsilon modules remain largely unknown. To address this issue, the interactions between PKCepsilon and Src were studied. We used recombinant proteins of wild-type PKCepsilon (PKCepsilon-WT) and open conformation mutants of the kinase (PKCepsilon-AE5 and PKCepsilon-AN59), the regulatory and catalytic domains of PKCepsilon, along with glutathione-S-transferase (GST) fusion proteins of Src (GST-Src) and two domains of Src (GST-SH2 and GST-SH3). GST pulldown assays demonstrated that Src and PKCepsilon are binding partners and that the interaction between PKCepsilon and Src appears to involve multiple sites. This finding was supported for endogenous PKCepsilon and Src in the murine heart using immunofluorescence-based confocal microscopy and coimmunoprecipitation. Furthermore, PKCepsilon-WT and GST-Src interactions were significantly enhanced in the presence of phosphatidyl-L-serine, an activator of PKC, indicating that Src favors interaction with activated PKCepsilon. This finding was confirmed when the PKCepsilon-WT was replaced with PKCepsilon-AE5 or PKCepsilon-AN59, demonstrating that the conformation of PKCepsilon is a critical determinant of its interactions with Src. Together, these results illustrate that formation of a signaling module between PKCepsilon and Src involves specific domains within the two molecules and is governed by the molecular conformation of PKCepsilon.

Animals↗