Regulation of periodontal ligament cell functions by interleukin-1beta

Periodontal ligament (PDL) cells maintain the attachment of the tooth to alveolar bone. These cells reside at a site in which they are challenged frequently by bacterial products and proinflammatory cytokines, such as interleukin-1beta (IL-1beta), during infections. In our initial studies we observed that IL-1beta down-regulates the osteoblast-like characteristics of PDL cells in vitro. Therefore, we examined the functional significance of the loss of the PDL cell's osteoblast-like characteristics during inflammation. In this report we show that, during inflammation, IL-1beta can modulate the phenotypic characteristics of PDL cells to a more functionally significant lipopolysaccharide (LPS)-responsive phenotype. In a healthy periodontium PDL cells exhibit an osteoblast-like phenotype and are unresponsive to gram-negative bacterial LPS. Treatment of PDL cells with IL-1beta inhibits the expression of their osteoblast-like characteristics, as assessed by the failure to express transforming growth factor beta1 (TGF-beta1) and proteins associated with mineralization, such as alkaline phosphatase and osteocalcin. As a consequence of this IL-1beta-induced phenotypic change, PDL cells become responsive to LPS and synthesize proinflammatory cytokines. The IL-1beta-induced phenotypic changes in PDL cells were transient, as removal of IL-1beta from PDL cell cultures resulted in reacquisition of their osteoblast-like characteristics and lack of LPS responsiveness. The IL-1beta-induced phenotypic changes occurred at concentrations that are frequently observed in tissue exudates during periodontal inflammation (0.05 to 5 ng/ml). The results suggest that, during inflammation in vivo, IL-1beta may modulate PDL cell functions, allowing PDL cells to participate directly in the disease process by assuming LPS responsiveness at the expense of their normal structural properties and functions.     

Protein thermostability above 100 degreesC: a key role for ionic interactions

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100 degreesC. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100 degreesC and 88 degreesC, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104 degreesC. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104 degreesC over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.     

Stochastic kinetic analysis of developmental pathway bifurcation in phage lambda-infected Escherichia coli cells

Fluctuations in rates of gene expression can produce highly erratic time patterns of protein production in individual cells and wide diversity in instantaneous protein concentrations across cell populations. When two independently produced regulatory proteins acting at low cellular concentrations competitively control a switch point in a pathway, stochastic variations in their concentrations can produce probabilistic pathway selection, so that an initially homogeneous cell population partitions into distinct phenotypic subpopulations. Many pathogenic organisms, for example, use this mechanism to randomly switch surface features to evade host responses. This coupling between molecular-level fluctuations and macroscopic phenotype selection is analyzed using the phage lambda lysis-lysogeny decision circuit as a model system. The fraction of infected cells selecting the lysogenic pathway at different phage:cell ratios, predicted using a molecular-level stochastic kinetic model of the genetic regulatory circuit, is consistent with experimental observations. The kinetic model of the decision circuit uses the stochastic formulation of chemical kinetics, stochastic mechanisms of gene expression, and a statistical-thermodynamic model of promoter regulation. Conventional deterministic kinetics cannot be used to predict statistics of regulatory systems that produce probabilistic outcomes. Rather, a stochastic kinetic analysis must be used to predict statistics of regulatory outcomes for such stochastically regulated systems.     

Assessment of tissue viability using near-infrared spectroscopy

Both anti-CD40 antibodies and anti-immunoglobulin (Ig) coupled to Sepharose induced proliferation of resting B cells and suppressed lipopolysaccharide (LPS)-induced B-cell differentiation to immunoglobulin secretion at comparable levels determined with the plaque-forming assay and Ig RNA steady state levels. Anti-CD40 antibodies also increased the proliferation of B cells stimulated by T helper cells in vitro while suppressing their differentiation to Ig secretion. Further, B cells preactivated by anti-Ig, anti-CD40 or a combination of the two mitogens could be restimulated by anti-CD40 but not by anti-Ig antibodies. Phenotypic divergence of Ig and CD40 signals regarding surface expression of activation markers was observed. Restimulation of anti-Ig- or anti-CD40-prestimulated cells with anti-Ig induced apoptosis whereas apoptosis could be inhibited when cells were recultivated with anti-CD40.     

Influence of cell polarity on retrovirus-mediated gene transfer to differentiated human airway epithelia

Gene transfer with recombinant murine leukemia viruses (MuLV) provides the potential to permanently correct inherited lung diseases, such as cystic fibrosis (CF). Several problems prevent the application of MuLV-based recombinant retroviruses to lung gene therapy: (i) the lack of cell proliferation in mature pulmonary epithelia, (ii) inefficient gene transfer with a vector applied to the apical surface, and (iii) low titers of many retroviral preparations. We found that keratinocyte growth factor (KGF) stimulated proliferation of differentiated human tracheal and bronchial epithelia. Approximately 50% of epithelia divided in response to KGF as assessed by bromodeoxyuridine histochemistry. In airway epithelia stimulated to divide with KGF, high-titer ampho- and xenotropic enveloped vectors preferentially infected cells from the basal side. However, treatment with hypotonic shock or EGTA transiently increased transepithelial permeability, enhancing gene transfer with the vector applied to the mucosal surfaces of KGF-stimulated epithelia. Up to 35% of cells expressed the transgene after gene transfer. By using this approach, cells throughout the epithelial sheet, including basal cells, were targeted. Moreover, the Cl- transport defect in differentiated CF airway epithelia was corrected. These findings suggest that barriers to apical infection with MuLV can be overcome.