Phenotypic characterization of epiphycan-deficient and epiphycan/biglycan double-deficient mice.

Objective: To characterize the in vivo role epiphycan (Epn) has in cartilage development and/or maintenance.

Methods: Epn-deficient mice were generated by disrupting the Epn gene in mouse embryonic stem cells. Epn/biglycan (Bgn) double-deficient mice were produced by crossing Epn-deficient mice with Bgn-deficient mice.

The Enterococcus faecalis MSCRAMM ACE binds its ligand by the Collagen Hug model.

We have determined the crystal structure of the ligand binding segment of the Enterococcus faecalis collagen binding MSCRAMM ACE (microbial surface components recognizing adhesive matrix molecules adhesin of collagen from enterococci). This segment is composed of two subdomains, N(1) and N(2), each adopting an IgG-like fold and forming a putative collagen binding surface at the interface between the two subdomains. This structure is very similar to that recently reported for CNA, the collagen binding MSCRAMM of Staphylococcus aureus, for which a unique ligand binding mechanism called the Collagen Hug was proposed.

Multicomponent Lyme vaccine: three is not a crowd.

Lyme disease is caused by the spirochete Borrelia burgdorferi and it is the most common vector-borne disease in the United States. Disseminated spirochetes can persist in various tissues and can result in a variety of different disease manifestations. Vaccination trials testing various lipoprotein candidates have yielded mixed results despite the generation of robust antibody titers.

Identification and biochemical characterization of two novel collagen binding MSCRAMMs of Bacillus anthracis.

Cell wall-anchored proteins play critical roles in the pathogenesis of infections caused by Gram-positive bacteria. Through the analysis of the genome of Bacillus anthracis Ames strain, we identified two novel putative cell wall-anchored proteins, BA0871 and BA5258, which have sequence homology to CNA, a cell wall-anchored collagen adhesin of Staphylococcus aureus. The two proteins have similar domain organization to that of CNA, with typical signal peptide sequences, a non-repetitive A region followed by repeats, and a characteristic cell wall-anchoring region.

Identification and characterization of the C3 binding domain of the Staphylococcus aureus extracellular fibrinogen-binding protein (Efb).

The secreted Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) is a virulence factor that binds to both the complement component C3b and fibrinogen. Our laboratory previously reported that by binding to C3b, Efb inhibited complement activation and blocked opsonophagocytosis. We have now located the Efb binding domain in C3b to the C3d fragment and determined a disassociation constant (Kd) of 0.

BBK32, a fibronectin binding MSCRAMM from Borrelia burgdorferi, contains a disordered region that undergoes a conformational change on ligand binding.

BBK32 is a fibronectin-binding lipoprotein on Borrelia burgdorferi, the causative agent of Lyme disease. Analysis using secondary structure prediction programs suggested that BBK32 is composed of two domains, an N-terminal segment lacking well defined secondary structure and a C-terminal segment composed largely of alpha-helices. Analysis of purified recombinant forms of the two domains by circular dichroism spectroscopy, gel permeation chromatography, and intrinsic viscosity determination were consistent with an N-terminal-extended, unstructured segment and a C-terminal globular domain in BBK32.

Inhibition of complement activation by a secreted Staphylococcus aureus protein.

Staphylococcus aureus can cause a variety of acute and chronic diseases. The ability of S. aureus to cause persistent infections has been linked to its ability to evade or inactivate host immune responses.

Decorin-binding sites in the adhesin DbpA from Borrelia burgdorferi: a synthetic peptide approach.

Lyme disease is caused by the spirochete Borrelia burgdorferi following transmission from infected Ixodes ticks to human hosts. Following colonization of the skin, spirochetes can disseminate throughout the body, resulting in complications that can include ocular, cardiac, neural, and skeletal disease. We have previously shown that B.

The Staphylococcus aureus Map protein is an immunomodulator that interferes with T cell-mediated responses.

Staphylococcus aureus (SA) is an opportunistic pathogen that affects a variety of organ systems and is responsible for many diseases worldwide. SA express an MHC class II analog protein (Map), which may potentiate SA survival by modulating host immunity. We tested this hypothesis in mice by generating Map-deficient SA (Map(-)SA) and comparing disease outcome to wild-type Map(+)SA-infected mice.

Fibronectin binding protein A of Staphylococcus aureus can mediate human T lymphocyte adhesion and coactivation.

The extracellular matrix protein fibronectin (FN) mediates the adhesion of bacteria as well as T lymphocytes. Mammalian cells express integrins alpha(4)beta(1) and alpha(5)beta(1) as the major FN-binding cell surface receptors. Bacteria such as Staphylococcus aureus, also express FN-binding receptors that are important for adherence to host tissue and initiation of infection.

Resistance to Lyme disease in decorin-deficient mice.

Microbial adhesion to the host tissue represents an early, critical step in the pathogenesis of most infectious diseases. BORRELIA: burgdorferi, the causative agent of Lyme disease (LD), expresses two surface-exposed decorin-binding adhesins, DbpA and DbpB. A decorin-deficient (Dcn(-/-)) mouse was recently developed and found to have a relatively mild phenotype.

Prolactin receptor and uterine milk protein expression in the ovine endometrium during the estrous cycle and pregnancy.

Lactogenic hormones regulate epithelial proliferation, differentiation, and function in a variety of epitheliomesenchymal organs. During pregnancy, the ovine uterus is a potential site for endocrine and paracrine actions of lactogenic hormones in the form of pituitary prolactin (PRL) and placental lactogen (PL). These studies determined temporal and spatial alterations in PRL receptor (PRL-R) and expression of uterine milk proteins (UTMP), a marker of endometrial secretory activity, in the ovine endometrium during the estrous cycle and pregnancy.

Ovine uterine gland knock-out model: effects of gland ablation on the estrous cycle.

Ovine endometrial gland development is a postnatal event that can be inhibited epigenetically by chronic exposure of ewe lambs to a synthetic progestin from birth to puberty. As adults, these neonatally progestin-treated ewes lack endometrial glands and display a uterine gland knockout (UGKO) phenotype that is useful as a model for study of endometrial function. Here, objectives were to determine: 1) length of progestin exposure necessary from birth to produce the UGKO phenotype in ewes; 2) if UGKO ewes display normal estrous cycles; and 3) if UGKO ewes could establish and/or maintain pregnancy.

Expression and localization of PG-Lb/epiphycan during mouse development.

We have examined the expression pattern of the PG-Lb/epiphycan gene that encodes a small leucine-rich repeat proteoglycan during mouse embryonic development. PG-Lb/epiphycan mRNA transcripts were first detected at E12.5 days postcoitus (dpc) at high levels in structures that were developing cartilage elements.

Development and characterization of immortalized ovine endometrial cell lines.

The objective of this study was to generate immortalized endometrial epithelial and stromal cell lines from the ovine uterus. Luminal (LE) and glandular epithelial (GE) cells and stromal (ST) cells were enzymatically isolated from the uterus of a Day 5 cyclic ewe (estrus on Day 0), and primary cultures were immortalized by transduction with a retroviral vector (LXSN-16E6E7) packaged by the amphotropic fibroblast line PA-317. Cells having integrated the vector were selected by resistance to the neomycin analogue G418 (0.

Matrix-binding proteins of Staphylococcus aureus: functional analysis of mutant and hybrid molecules.

The fibrinogen-binding protein ClfA and the collagen-binding protein Cna are surface-associated adhesins of Staphylococcus aureus. ClfA has a dipeptide repeat region R composed mainly of serine and aspartate residues, more than 40 of which are required along with the 28-residue region W, the LPXTG motif and region M to display the ligand-binding region A on the cell surface in a functional form. Cna has a 61-residue region W and at least one 187-residue region B linking the collagen-binding region A to peptidoglycan.

Adherence of Borrelia burgdorferi. Identification of critical lysine residues in DbpA required for decorin binding.

Borrelia burgdorferi, the causative agent of Lyme disease, expresses on its surface two decorin binding adhesins, DbpA and DbpB. Previous studies have demonstrated that vaccination of mice with DbpA provided protection against challenge with heterologous Borrelia strains despite considerable sequence variability among DbpA in these strains. We have now examined the importance of individual amino acid residues in DbpA for decorin binding.

Role of fibronectin-binding MSCRAMMs in bacterial adherence and entry into mammalian cells.

Most bacterial infections are initiated by the adherence of microorganisms to host tissues. This process involves the interaction of specific bacterial surface structures, called adhesins, with host components. In this review, we discuss a group of microbial adhesins known as Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) which recognize and bind FN.

Differential effects of intrauterine and subcutaneous administration of recombinant ovine interferon tau on the endometrium of cyclic ewes.

Interferon tau (IFNtau) is the antiluteolytic signal produced by the conceptus of ruminants. Intrauterine administration of recombinant ovine IFNtau suppresses expression of endometrial estrogen receptor (ER) and oxytocin receptor (OTR) in the luminal and superficial glandular epithelia to abrogate the production of luteolytic prostaglandin F(2alpha) (PGF(2alpha)) pulses. Subcutaneous (s.

Decorin-binding adhesins from Borrelia burgdorferi.

Lyme disease is a tick-transmitted infection caused by the spirochete Borrelia burgdorferi. Ticks deposit B. burgdorferi into the dermis of the host, where they eventually become associated with collagen fibres.

Identification and characterization of glycosylation-dependent cell adhesion molecule 1-like protein expression in the ovine uterus.

Glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) is an endothelial glycoprotein secreted in lymph nodes that serves as a ligand for leukocyte cell surface selectin and mediates lymphocyte extravasation. In the present studies, rabbit anti-rat GlyCAM-1 IgG was used in immunochemical analyses of GlyCAM-1-like protein in the ovine uterus. In cyclic ewes, GlyCAM-1 expression increased in the endometrial luminal epithelium (LE) and shallow glandular epithelium (cGE) between Days 1 and 5 and then decreased between Days 11 and 15.

Ligand-dependent and -independent interactions with the transforming growth factor type II and I receptor subunits reside in the aminoterminal portion of the ectodomain of the type III subunit.

The type III receptor for transforming growth factor beta (TGFbeta), which exhibits no kinase activity, binds TGFbeta1 and TGFbeta2 and is involved in assembly and activity of the multi-subunit TGFbeta signal transduction complex. Recently we showed that TGFbeta receptor type III (TbetaRIII) can participate in a complex composed of the dimeric TGFbeta ligand and a type III, II, and I receptor subunit. The interaction of the TbetaRIII subunit with TbetaRII is TGFbeta-dependent, whereas interaction with TbetaRI is TGFbeta-independent.

Inhibition of growth of malignant rat prostate tumor cells by restoration of fibroblast growth factor receptor 2.

A loss of expression of fibroblast growth factor (FGF) receptor 2 IIIb (FGFR2IIIb), which responds to stroma-derived FGF, accompanies progression of premalignant androgen-responsive rat prostate tumor epithelial cells to the malignant phenotype. Concurrently, the level of FGFR2 gene expression is reduced and lost altogether in over 30% of cells, whereas all malignant cells abnormally express FGFR1, which is normally confined to stromal cells (S. Feng et al.

Heparan sulfate is required for interaction and activation of the epithelial cell fibroblast growth factor receptor-2IIIb with stromal-derived fibroblast growth factor-7.

Fibroblast growth factor-7 (FGF-7) and a specific splice variant of the FGF tyrosine kinase receptor family (FGFR2IIIb) constitute a paracrine signaling system from stroma to epithelium. Different effects of the manipulation of cellular heparan sulfates and heparin on activities of FGF-7 relative to FGF-1 in epithelial cells suggest that pericellular heparan sulfates may regulate the activity of FGF-7 by a different mechanism than other FGFs. In this report, we employ the heparan sulfate-binding protein, protamine sulfate, to reversibly block cellular heparan sulfates.

The heparan sulfate-fibroblast growth factor family: diversity of structure and function.

The fibroblast growth factor (FGF) receptor complex is a ubiquitous regulator of development and adult tissue homeostasis that bridges the peri-cellular matrix and the intracellular environment. Diverse members of the FGF polypeptide family, the FGF receptor tyrosine kinase (FGFRTK) family and the FGF receptor heparan sulfate proteoglycan (FGFRHS) family combine to result in active and specific FGFR signal transduction complexes. Regulated alternate splicing and combination of variant subdomains give rise to diversity of FGFRTK monomers.