Biomedical subjects

Michael J Pabst

Publications and source records attributed to Michael J Pabst.

7 recordsLinked to original sources

PubMed2006

Accumulation of LPS by polyethylene particles decreases bone attachment to implants.

Molecules absorbed on the surface of particulate wear debris may contribute to inflammatory reactions that lead to aseptic loosening of implants. Lipopolysaccharide (LPS), a bacterial endotoxin, can attach to many biomaterials and stimulate macrophages to secrete osteoclast-activating cytokines. We tested the adsorption of LPS by polyethylene particles in vitro and examined the biological effects of LPS absorption on bone remodeling around implants in vivo. Polyethylene particles were incubated in radiolabeled LPS solutions, and adsorption of LPS by the particles was quantified by radioassay. Because polyethylene particles are hydrophobic and less dense than water, they floated and clumped when incubated in a water solution of LPS, resulting in low adsorption of LPS. However, when particles were incubated in an ethanol solution of LPS, most of the LPS was adsorbed by the particles, and was resistant to washing with water. Triton X-100 (10%), however, effectively washed the LPS off the particles. In a rat model, the presence of polyethylene particles around the implant in the femoral canal decreased bone attachment to the implant at 6 weeks. Incubating the particles with LPS before implantation, or intermittent administration of LPS systemically, further decreased bone-implant attachment to similar extents, but had no effect on the bone density of the control side femurs. Our data indicate that polyethylene particles have high affinity for LPS, depending on many factors, especially the solvents of the LPS. Intermittent systemic administration of LPS affects bone remodeling but only occurs in the area containing polyethylene particles and titanium implants, supporting the hypothesis that the presence of polyethylene particles around implants can result in accumulation of LPS from exogenous sources. This may cause local levels of LPS that are high enough to affect bone remodeling around implants.

Adsorption↗

PubMed2005

Local anesthetics inhibit priming of neutrophils by lipopolysaccharide for enhanced release of superoxide: suppression of cytochrome b558 expression by disparate mechanisms.

Local anesthetics have anti-inflammatory effects in vivo and inhibit neutrophil functions in vitro, but how these agents act on neutrophils remains unclear. Phagocytosis and bactericidal activity of neutrophils are enhanced by exposure to bacterial components such as lipopolysaccharide (LPS); this process is termed priming, which for enhanced release of superoxide (O2-) causes mobilization of intracellular granules that contain cytochrome b558, a component of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We studied whether local anesthetics affected LPS priming for enhanced release of O2- in response to triggering by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), and we investigated which element in the LPS signaling pathway might be the target of local anesthetics. Neutrophils were incubated with 10 ng/ml LPS and 1% plasma+/-local anesthetics, washed, and triggered with fMLP. Local anesthetics all inhibited LPS priming, and 50% inhibition was at 0.1 mM tetracaine, 0.5 mM bupivacaine, 3.0 mM lidocaine, or 4.0 mM procaine. Local anesthetics inhibited LPS-induced mobilization of specific granules and secretory vesicles. Local anesthetics inhibited LPS-induced up-regulation of cytochrome b558 but not LPS-induced translocation of p47phox. Inhibition of priming by local anesthetics was reversed by washing and incubating for 5 min. Tetracaine alone, but not the other local anesthetics, inhibited LPS activation of p38 mitogen-activated protein kinase (MAPK) and MAPK kinase 3 (kinases in the LPS signaling pathway). The p38 MAPK inhibitors SB203580 and PD169316 also blocked LPS priming. Thus, tetracaine and the other local anesthetics inhibit by disparate mechanisms, but all the local anesthetics impaired up-regulation of cytochrome b558 and all impaired priming of NADPH oxidase by LPS.

Anesthetics, Local↗

PubMed2005

Functional characterization of two-dimensional gel-separated proteins using sequential staining.

Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications on proteins using MS of peptide fragments is difficult and labor-intensive. Recently, specific staining methods have been developed and merged into the 2-D gel platform so that not only general protein patterns but also patterns of phosphorylated and glycosylated proteins can be obtained. We used the new Pro-Q Diamond phosphoprotein dye technology for the fluorescent detection of phosphoproteins directly in 2-D gels of mouse leukocyte proteins, and Pro-Q Emerald 488 glycoprotein dye to detect glycoproteins. These two fluorescent stains are compatible with general protein stains, such as SYPRO Ruby stain. We devised a sequential procedure using Pro-Q Diamond (phosphoprotein), followed by Pro-Q Emerald 488 (glycoprotein), followed by SYPRO Ruby stain (general protein stain), and finally silver stain for total protein profile. This multiple staining of the proteins in a single gel provided parallel determination of protein expression and preliminary characterization of post-translational modifications of proteins in individual spots on 2-D gels. Although this method does not provide the same degree of certainty as traditional MS methods of characterizing post-translational modifications, it is much simpler, faster, and does not require sophisticated equipment and expertise in MS.

Animals↗

PubMed2004

Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting.

We compared trysin-digested protein samples desalted by ZipTip(C18) reverse-phase microcolumns with on-plate washing of peptides deposited either on paraffin-coated plates (PCP), Teflon-based AnchorChip plates, or stainless steel plates, before analysis by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Trypsinized bovine serum albumin and ovalbumin and 16 protein spots extracted from silver-stained two-dimensional gels of murine C(2)C(12) myoblasts or human leukocytes, prepared by the above two methods, were subjected to MALDI on PCP, AnchorChip plates, or uncoated stainless steel plates. Although most peptide mass peaks were identical regardless of the method of desalting and concentrating of protein samples, samples washed and concentrated by the PCP-based method had peptide peaks that were not seen in the samples prepared using the ZipTip(C18) columns. The mass spectra of peptides desalted and washed on uncoated stainless steel MALDI plates were consistently inferior due to loss of peptides. Some peptides of large molecular masses were apparently lost from samples desalted by ZipTip(C18) microcolumns, thus diminishing the quality of the fingerprint needed for protein identification. We demonstrate that the method of washing of protein samples on paraffin-coated plates provides an easy, reproducible, inexpensive, and high-throughput alternative to ZipTip(C18)-based purification of protein prior to MALDI-TOF-MS analysis.

Animals↗

PubMed2004

Invasive M1T1 group A Streptococcus undergoes a phase-shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB.

A globally disseminated strain of M1T1 group A Streptococcus (GAS) has been associated with severe infections in humans including necrotizing fasciitis and toxic shock syndrome. Recent clinicoepidemiologic data showed a striking inverse relationship between disease severity and the degree to which M1T1 GAS express the streptococcal cysteine protease, SpeB. Electrophoretic 2-D gel analysis of the secreted M1T1 proteome, coupled with MALDI-TOF mass spectroscopy, revealed that expression of active SpeB caused the degradation of the vast majority of secreted GAS proteins, including several known virulence factors. Injection of a SpeB+/SpeA- M1T1 GAS strain into a murine subcutanous chamber model of infection selected for a stable phase-shift to a SpeB-/SpeA+ phenotype that expressed a full repertoire of secreted proteins and possessed enhanced lymphocyte-stimulating capacity. The proteome of the SpeB-in vivo phase-shift form closely matched the proteome of an isogenic speB gene deletion mutant of the original M1T1 isolate. The absence or the inactivation of SpeB allowed proteomic identification of proteins in this M1T1 clone that are not present in the previously sequenced M1 genome including SpeA and another bacteriophage-encoded novel streptodornase allele. Further proteomic analysis of the M1T1 SpeB+ and SpeB- phase-shift forms in the presence of a cysteine protease inhibitor demonstrated differences in the expression of several proteins, including the in vivo upregulation of SpeA, which occurred independently of SpeB inactivation.

Animals↗

PubMed2003

Proteome of monocytes primed with lipopolysaccharide: analysis of the abundant proteins.

We performed a proteomic analysis of monocytes primed by lipopolysaccharide (LPS) in vitro, using two-dimensional gels stained with Coomassie blue. We found 16 proteins of approximately 500 detected that either increased or decreased in abundance as a result of priming by LPS (14 with P </= 0.05). The proteins were identified by comparing the masses of their tryptic peptides with those of all known proteins, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the SWISS-PROT database. Identities were confirmed by matching the sequence of several tryptic peptides, using liquid chromatography electrospray-ionization quadrupole ion trap mass spectrometry. There were increases in the protective enzymes superoxide dismutase and catalase, in four calgranulins, in the cytokine pre-B cell enhancing factor, and in annexin 2, macrophage capping protein, transketolase, pyruvate kinase, and serine/threonine protein kinase 10. Proteins that decreased in abundance were integrin alpha-IIB, protein disulfide isomerase, and platelet-activating factor acetylhydrolase. Many of these altered proteins have interesting functions in inflammation.

Amino Acid Sequence↗

PubMed2002

Preliminary analysis of the mouse cerebellum proteome.

This paper reports on the initial analysis of protein expression in the mouse cerebellum with the proteomics approach. Proteins from cerebellar tissue homogenates were separated by two-dimensional gel electrophoresis, and the proteins were stained with colloidal Coomassie Blue to produce a high-resolution map of the cerebellum proteome. Selected proteins from this map were digested with trypsin, and the resulting tryptic peptides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography-electrospray quadrupole ion trap mass spectrometry. The mass spectrometric data were used to identify the proteins through searches of the SWISSPROT protein sequence database. To date, 30 prominent proteins with various functional characteristics were identified. These data will be used for future studies of differential protein expression in mouse models of neurological disorders.

Animals↗