Jeffrey C. Howard

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.

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

Dupuytrens 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.

Identification of Collagen-Binding Proteins in Lactobacillus spp. with Surface-Enhanced Laser Desorption/Ionization–Time of Flight ProteinChip Technology

ABSTRACT Biosurfactants produced by Lactobacillus fermentumRC-14, L. rhamnosus GR-1 and 36, and L. caseiShirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)–time of flight mass spectrometry. Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins. Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form). Although biosurfactants isolated from L. casei Shirota and L. rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein. Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions.

Elevated Levels of β-catenin and Fibronectin in Three-dimensional Collagen Cultures of Dupuytren's Disease Cells are Regulated by Tension in Vitro

BackgroundDupuytrens 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 β-catenin may play a role. In this study, collagen matrix cultures of primary disease fibroblasts show enhanced contraction and isometric tension-dependent changes in β-catenin and fibronectin levels.MethodsWestern blots of β-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.ResultsDisease cells exhibited enhanced collagen contraction activity compared to control cells. Alterations in isometric tension of collagen matrices triggered dramatic changes in β-catenin and fibronectin levels, including a transient increase in β-catenin levels within disease cells, while fibronectin levels steadily decreased to levels below those seen in normal cell cultures. In contrast, both fibronectin and β-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.ConclusionThree-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 β-catenin and Fn seen in collagen matrix cultures of disease fibroblasts can be regulated by changes in isometric tension.

Rapid Identification of Probiotic Lactobacillus Biosurfactant Proteins by ProteinChip Tandem Mass Spectrometry Tryptic Peptide Sequencing

ABSTRACT 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.

Enhanced Dupuytren's disease fibroblast populated collagen lattice contraction is independent of endogenous active TGF-β2

BackgroundDupuytrens disease (DD) is a debilitating fibro-proliferative disorder of the hand characterized by the appearance of fibrotic lesions (nodules and cords) leading to flexion contractures of the fingers and loss of hand function. Although the molecular mechanism of DD is unknown, it has been suggested that transforming growth factor-β2 (TGF-β2) may play an important role in the underlying patho-physiology of the disease. The purpose of this study was to further explore this hypothesis by examining the effects of TGF-β2 on primary cell cultures derived from patient-matched disease and normal palmar fascia tissue using a three-dimensional collagen contraction assay.MethodsFibroblast-populated collagen lattice (FPCL) contraction assays using primary cell cultures derived from diseased and control fascia of the same DD patients were studied in response to exogenous TGF-β2 and neutralizing anti-TGF-β2 antibodies.ResultsContraction of the FPCLs occurred significantly faster and to a greater extent in disease cells compared to control cells. The addition of TGF-β2 enhanced the rate and degree of collagen contraction in a dose-dependent fashion for both control and diseased cells. Neutralizing anti-TGF-β2 antibodies abolished exogenous TGF-β2 stimulated collagen contraction, but did not inhibit the enhanced basal collagen contraction activity of disease FPCL cultures.ConclusionsAlthough exogenous TGF-β2 stimulated both disease and control FPCL contraction, neutralizing anti-TGF-β2 antibodies did not affect the elevated basal collagen contraction activity of disease FPCLs, suggesting that the differences in the collagen contraction activity of control and disease FPCL cultures are not due to differences in the levels of endogenous TGF-β2 activity.

A novel mass spectrometry-based assay for GSK-3β activity

BackgroundAs 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 γ-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-3β) activity.ResultsSynthetic peptide substrates designed with a GSK-3β phosphorylation site were assayed with both recombinant enzyme and GSK-3β 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-3β target peptide (2B-Sp). Not only was there a dose-dependent response in molecular weight shift to the amount of recombinant GSK-3β used in this assay, this shift was also inhibited by lithium chloride (LiCl), in a dose-dependent manner.ConclusionWe present here a novel method to sensitively measure peptide phosphorylation by GSK-3β 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-3β in vivo, and to screen enzyme activity in relation to a variety of GSK-3β related disorders.

Bcl-2 expression in F-MuLV-induced erythroleukemias: a role for the anti-apoptotic action of Bcl-2 during tumor progression.

Erythroleukemias induced by various strains of Friend virus are multistage malignancies that result from the accumulation of genetic mutations, including the activation of proto-oncogenes and the inactivation of tumor suppressor genes. In this study, we demonstrate that Bcl-2 expression is activated in the majority of F-MuLV-induced erythroleukemia cell lines. In contrast, Bcl-2 was not expressed in any of the FV-P-induced erythroleukemia cell lines and protein levels were low or negligible in FV-A-induced erythroleukemia cell lines examined. In vivo, Bcl-2 expression levels gradually increased in F-MuLV-induced erythroleukemic cells prior to adaptation to culture. High expression of Bcl-2 in F-MuLV-induced erythroleukemic cells was shown to proceed the emergence of p53 mutation suggesting that Bcl-2 expression may delay p53 mutation in the leukemic cells. This is further supported by the demonstration that the majority of F-MuLV-induced erythroleukemia cell lines established from primary tumors induced in p53 mutant mice express low to negligible levels of Bcl-2. We have shown that the high levels of Bcl-2 expression in FV-P-induced erythroleukemic cells inhibited apoptosis induced by etoposide, low serum and p53 expression. Similarly, ectopic Bcl-2 expression within these cells also provided protection from apoptosis induced by etoposide and growth in low serum. These results suggest that the anti-apoptotic action of Bcl-2 may confer a selective in vivo and in vitro growth advantage to F-MuLV-induced erythroleukemic cells, which is not shared by FV-P/FV-A-induced erythroleukemic cells. The observed induction of Bcl-2 expression in vivo constitutes a novel but late oncogenic event associated with the progression of F-MuLV-induced erythroleukemias.

[32] Understanding urogenital biofilms and potential impact of probiotics

Publisher Summary This chapter discusses the understanding of urogentital biofilms and the potential impact of probiotics. The urogenital tract—namely, vagina, cervix, perineum, and urethra—in females is host to approximately 50 species of bacteria and yeasts. While studies have documented the aerobic and anaerobic composition of the microbiota, little is known about the biofilm dynamics. Organisms such as uropathogenic Escherichia coli, yeast, and Gardnerella can be introduced via sexual intercourse, while other routes of seeding include the skin and intestine. The microbiota and the overall microenvironment can change on an hourly basis, and they are influenced by micturition and urinary components, and by the hormones such as estrogen, exposure to spermicides such as nonoxynol-9, antibiotics, semen, presence of a diaphragm or intrauterine device, menstruation and other factors. The present chapter provides suggestions on methodologies that may be used to examine the impact of probiotic organisms on the microbiota. Basic bacteriology is useful to identify the organisms present, but as many are strict anaerobes and adherent to epithelial cells, special handling (such as avoidance of exposure to oxygen, sonication to remove bacteria from cells) should be undertaken to ensure recovery. Many of the microorganisms in the microbiota cannot be cultivated by standard procedures, molecular techniques that detect nucleic acids, rather than viable cells offer many advantages over classic culture techniques.