OxLDL receptor chromatography from live human U937 cells identifies SYK(L) that regulates phagocytosis of oxLDL.

The binding and activation of macrophages by microscopic aggregates of oxLDL in the intima of the arteries may be an important step towards atherosclerosis leading to heart attack and stroke. Microbeads coated with oxLDL were used to activate, capture and isolate the oxLDL receptor complex from the surface of live cells. Analysis of the resulting tryptic peptides by liquid chromatography and tandem mass spectrometry revealed the Spleen Tyrosine Kinase (SYK), and many of SYK's known interaction network including Fc receptors (FCGR2A, FCER1G and FCGR1A) Toll receptor 4 (TLR4), receptor kinases like EGFRs, as well as RNA binding and metabolism proteins. High-intensity precursor ions (∼9*E3 to 2*E5 counts) were correlated to peptides and specific phosphopeptides from long isoform of SYK (SYK-L) by the SEQUEST, OMSSA and X!TANDEM algorithms. Peptides or phosphopeptides from SYK were observed with the oxLDL-microbeads. Pharmacological inhibitors of SYK activity significantly reduced the engulfment of oxLDL microbeads in the presence of serum factors, but had little effect on IgG phagocytosis. Anti SYK siRNA regulated oxLD engulfment in the context of serum factors and or SYK-L siRNA significantly inhibited engulfment of oxLDL microbeads, but not IgG microbeads.

Identification of protein biomarkers in Dupuytren's contracture using surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS).

BACKGROUND: To study the protein expression profiles associated with Dupuytren's contracture (DC) to identify potential disease protein biomarkers (PBM) using a proteomic technology--Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). METHODS: Normal and disease palmar fascia from DC patients were analyzed using Ciphergen's SELDI-TOF-MS Protein Biological System II (PBSII) ProteinChip reader. Analysis of the resulting SELDI-TOF spectra was carried out using the peak cluster analysis program (BioMarker Wizard, Ciphergen). Common peak clusters were then filtered using a bootstrap algorithm called SAM (Significant Analysis of Microarrays) for increased fidelity in our analysis. RESULTS: Several differentially expressed low molecular weight (<20 kDa) tissue proteins were identified. Spectra generated using both ZipTipC18 aided Au array and WCX2 array based SELDI-TOF-MS were reproducible, with an average peak cluster mass standard deviation for both methods of <1.74 x10(-4). Initial peak cluster analysis of the SELDI spectra identified both up-(14) and down-(3)regulated proteins associated with DC. Further analysis of the peak cluster data using the bootstrap algorithm SAM identified three disease-associated protein features (4600.8 Da, 10254.5 Da, and 11405.1 Da) that were elevated (5.45, 11.7, and 4.28 fold, respectively, with a 0% median false discovery rate). CONCLUSION: SELDI-TOF-MS identified three potential low molecular weight tissue protein markers (p4.6DC, p1ODC, p11.7DC) for DC. The ability of SELDI-TOF-MS to resolve low molecular weight proteins suggests that the method may provide a means of deciphering the biomarker-rich low molecular weight region of the human proteome. Application of such novel technology may help clinicians to focus on specific molecular abnormalities in diseases with no known molecular pathogenesis, and uncover therapeutic and/or diagnostic targets.

A novel mass spectrometry-based assay for GSK-3beta activity.

BACKGROUND: As a component of the progression from genomic to proteomic analysis, there is a need for accurate assessment of protein post-translational modifications such as phosphorylation. Traditional kinase assays rely heavily on the incorporation of gamma-P32 radiolabeled isotopes, monoclonal anti-phospho-protein antibodies, or gel shift analysis of substrate proteins. In addition to the expensive and time consuming nature of these methods, the use of radio-ligands imposes restrictions based on the half-life of the radionucleotides and pose potential health risks to researchers. With the shortcomings of traditional assays in mind, the aim of this study was to develop a high throughput, non-radioactive kinase assay for screening Glycogen Synthase Kinase-3beta (GSK-3beta) activity. RESULTS: Synthetic peptide substrates designed with a GSK-3beta phosphorylation site were assayed with both recombinant enzyme and GSK-3beta immunoprecipitated from NIH 3T3 fibroblasts. A molecular weight shift equal to that of a single phosphate group (80 Da.) was detected by surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) in a GSK-3beta target peptide (2B-Sp). Not only was there a dose-dependent response in molecular weight shift to the amount of recombinant GSK-3beta used in this assay, this shift was also inhibited by lithium chloride (LiCl), in a dose-dependent manner. CONCLUSION: We present here a novel method to sensitively measure peptide phosphorylation by GSK-3beta that, due to the incorporation of substrate controls, is applicable to either purified enzyme or cell extracts. Future studies using this method have the potential to elucidate the activity of GSK-3beta in vivo, and to screen enzyme activity in relation to a variety of GSK-3beta related disorders.

Probiotics in surgical wound infections: current status.

BACKGROUND: Probiotics--live microorganisms that confer a health benefit when taken in adequate amounts, usually as food supplements--are receiving renewed attention in the medical community. Some have been found to play a role in disease remediation. However, mainstream medicine and science remain divided about the validity of health claims made about them. METHODS: To clarify the potential value of probiotics, we reviewed the scientific data on their role in preventing and treating surgical infections as well as some of our own studies of the effects of certain strains of lactobacilli on surgical implant infections. PRINCIPAL FINDINGS: There is little rigorous evidence that probiotics may be beneficial in the prevention and treatment of wound infections. However, data from 3 clinical trials and from our laboratory indicate that certain strains of probiotic lactobacilli and their byproducts may help reduce infection rates in surgical patients and may ameliorate staphylococcus-related infections of surgical implants. CONCLUSION: Although there is good clinical evidence that certain probiotics may be beneficial in conditions such as diarrheal and inflammatory bowel diseases, more studies are required to apply these concepts to the prevention and treatment of wound and other surgical infections.

Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS): a new proteomic urinary test for patients with urolithiasis.

SELDI-TOF-MS is a highly sensitive protein-analysis tool capable of detecting minute protein profile differences between biological samples. As proteins have been associated with urinary tract calculi, protein-based urinalysis may offer insights into their diagnosis. The purpose of this study was to evaluate SELDI-TOF-MS as a potential method for identifying urinary biomarkers of urolithiasis. Midstream sterile urine samples were obtained from 25 male patients with a confirmed diagnosis of urolithiasis (test group) and 25 male subjects with no known history of the disease (controls). Urinary levels of oxalate, total protein, albumin, and osteopontin were determined. Protein profiles were generated using SELDI-TOF-MS.SELDI-TOF-MS profiling revealed a relationship between protein peak intensities at 67 and 24 kDa that differed between the two groups. The ratio of p67:p24 was found to be less than 1.0 in all of the control samples (mean 0.26), while 18 out of 25 (72%) of the test group samples displayed a ratio greater than 1.0 (total group mean 4.75, P<0.001). Albumin, total protein, and oxalate levels were higher in the test group than the controls. Although SELDI-TOF-MS is not yet in widespread use in hospital and diagnostic laboratories, this system represents a promising new method for rapidly identifying patients with urolithiasis.

Wound healing-associated proteins Hsp47 and fibronectin are elevated in Dupuytren's contracture.

BACKGROUND: Dupuytren's contracture or disease (DD) affects hand function by causing irreversible contraction of the palmar fascia. Histological analysis has shown that DD and wound granulation tissue share many cellular and biochemical characteristics, suggesting that DD may be an exaggerated wound-healing response. The goal of the present study was to examine the possible involvement of two important wound-healing-associated proteins-heat shock protein 47 (Hsp47), fibronectin (Fn), and its oncofetal isoforms-in DD, using clinical tissue samples and primary cell cultures. MATERIALS AND METHODS: We examined the expression of Hsp47, Fn, and an oncofetal isoform of fibronectin (IIICS) in both normal and disease-matched surgical specimens and primary cell cultures using Western blot analysis, and immunocytochemistry (ICC). RESULTS: Our results indicate that Hsp47 and total fibronectin is elevated in DD lesional tissue. In addition, Western and ICC analysis of patient-matched (normal and disease) primary cultures show significantly elevated levels of oncofetal fibronectin (IIICS spliced variant) within disease primary cell cultures. CONCLUSIONS: The high levels of expression of Hsp47 and adult and oncofetal fibronectin in DD suggests that cell-mediated alterations in the extracellular environment may play an important role in the disease process. Furthermore, the involvement of these wound healing-associated proteins in DD supports the notion that this disease may be an exaggerated form of wound healing.

Interventional magnetic resonance image-guided percutaneous cryoablation of renal tumors.

We describe the first two cases of percutaneous cryoablation under magnetic resonance imaging guidance. To date, this minimally invasive procedure has been used for the treatment of renal cell tumors in patients who cannot tolerate or refuse surgical nephrectomy. The two patients described showed no evidence of recurrence or complications 35 and 36 months after the procedure.

Elevated levels of beta-catenin and fibronectin in three-dimensional collagen cultures of Dupuytren's disease cells are regulated by tension in vitro.

BACKGROUND: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in the development of scar-like, collagen-rich disease cords within specific palmar fascia bands. Although the molecular pathology of DD is unknown, recent evidence suggests that beta-catenin may play a role. In this study, collagen matrix cultures of primary disease fibroblasts show enhanced contraction and isometric tension-dependent changes in beta-catenin and fibronectin levels. METHODS: Western blots of beta-catenin and fibronectin levels were determined for control and disease primary cell cultures grown within stressed- and attached-collagen matrices. Collagen contraction was quantified, and immunocytochemistry analysis of filamentous actin performed. RESULTS: Disease cells exhibited enhanced collagen contraction activity compared to control cells. Alterations in isometric tension of collagen matrices triggered dramatic changes in beta-catenin and fibronectin levels, including a transient increase in beta-catenin levels within disease cells, while fibronectin levels steadily decreased to levels below those seen in normal cell cultures. In contrast, both fibronectin and beta-catenin levels increased in attached collagen-matrix cultures of disease cells, while control cultures showed only increases in fibronectin levels. Immunocytochemistry analysis also revealed extensive filamentous actin networks in disease cells, and enhanced attachment and spreading of disease cell in collagen matrices. CONCLUSION: Three-dimensional collagen matrix cultures of primary disease cell lines are more contractile and express a more extensive filamentous actin network than patient-matched control cultures. The elevated levels of beta-catenin and Fn seen in collagen matrix cultures of disease fibroblasts can be regulated by changes in isometric tension.

Beta-catenin expression in Dupuytren's disease: potential role for cell-matrix interactions in modulating beta-catenin levels in vivo and in vitro.

Dupuytren's disease (DD) is a superficial fibromatosis of the hand. Although the molecular mechanisms responsible for this disease are unknown, recent studies suggest that beta-catenin may be a key factor involved in fibromatosis. In this study, we analysed the in vivo and in vitro expression levels of beta-catenin in DD, using surgical specimens and primary cell lines. Although no somatic mutations (exon 3) of beta-catenin were detected, Western blot analysis revealed high levels of beta-catenin in diseased palmar fascia, and low to undetectable levels of beta-catenin in patient-matched normal palmar fascia. Immunohistochemistry analysis showed high levels of beta-catenin expression within the disease fascia, as well as cytoplasmic and nuclear accumulations of the protein. Immunoprecipitation of beta-catenin from seven patient lesions showed the protein to be tyrosine phosphorylated. Lastly, Western analysis of three patient-matched (disease and normal fascia) primary cell cultures showed significantly elevated levels of beta-catenin in disease cells cultured in three-dimensional collagen lattices. This is the first extensive in vivo and in vitro characterization of beta-catenin in DD, and the first to suggest that the extracellular matrix may play an important role in modulating beta-catenin stability in DD.

Lactobacillus fermentum RC-14 inhibits Staphylococcus aureus infection of surgical implants in rats.

Staphylococcus aureus is a common cause of community and hospital-acquired infections. Moreover, the clinical impact of S. aureus is on the rise because of the global increase in the incidence of multidrug-resistant strains and its growing prevalence as a major cause of surgical infections. As a result, there is a pressing need to identify new antistaphylococcal agents and preventative strategies that will help in the management of these types of infections. This report describes the successful use of a probiotic, Lactobacillus fermentum RC-14, and its secreted biosurfactant to inhibit surgical implant infections caused by S. aureus. L. fermentum RC-14 and its secreted biosurfactant both significantly inhibited S. aureus infection and bacteria adherence to surgical implants.

Rapid identification of probiotic lactobacillus biosurfactant proteins by ProteinChip tandem mass spectrometry tryptic peptide sequencing.

A novel ProteinChip-interfaced tandem mass spectrometer was employed to identify collagen binding proteins from biosurfactant produced by Lactobacillus fermentum RC-14. On-chip tryptic digestion of the captured collagen binding proteins resulted in rapid sequence identification of five novel tryptic peptide sequences via collision-induced dissociation tandem mass spectrometry.