Evaluation of Porcine Intestinal Epitheliocytes as an In vitro Immunoassay System for the Selection of Probiotic Bifidobacteria to Alleviate Inflammatory Bowel Disease

Probiotics and Antimicrobial Proteins - The use of in vitro systems that allow efficient selection of probiotic candidates with immunomodulatory properties could significantly minimize the use of...     

Functions of beta2-adrenergic receptor in human periodontal ligament cells

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue.     

Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line

Periodontal ligament (PDL) is a specialized connective tissue that influences the lifespan of the tooth. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, but little is known about the effects of TGF-β1 on PDL cells. Our aim has been to demonstrate the expression of TGF-β1 in rat PDL tissues and to evaluate its effects on the proliferation and gene expression in human PDL cells (HPLCs) and a human PDL stem/progenitor cell line, line 1-11, that we have recently developed. The expression of TGF-β1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-β1, TGF-β type I receptor, and TGF-β type II receptor genes. Although exogenous TGF-β1 stimulated the proliferation of HPLCs, it did not upregulate the expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col I), or fibrillin-1 (FBN1) mRNA or of α-SMA protein in HPLCs, whereas expression for these genes was attenuated by an anti-TGF-β1 neutralizing antibody. In contrast, exogenous TGF-β1 reduced the proliferation of cell line 1-11, although it upregulated the expression of α-SMA, Col I, and FBN1 mRNA and of α-SMA protein in this cell line. In addition, interleukin-1 beta stimulation significantly reduced the expression of TGF-β1 mRNA and protein in HPLCs. Thus, TGF-β1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions.     

Mode of antibacterial action by gramicidin S

To elucidate the mode of antibacterial action by gramicidin S (GS), a detailed experiment on GS distribution on bacteria cells was carried out. 14C-Labeled gramicidin S ([14C]GS) was incubated with cells of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli, and the amount of [14C]GS adsorbed on the cells was measured. Adsorption on B. subtilis cells was observed from 1 microgram/ml of [14C]GS. As the concentration of [14C]GS increased, the amount adsorbed on B. subtilis increased discontinuously, producing a curve which had three plateaus. On the other hand, [14C]GS was not easily adsorbed on E. coli cells at lower concentrations, but the amount adsorbed increased above 6 micrograms/ml, and the cells were temporarily saturated with GS at 10 micrograms/ml, which is the minimum inhibitory concentration for E. coli. The amount of [14C]GS adsorbed on the protoplast membrane of B. subtilis was the same as that of natural cells. However, the amount of [14C]GS adsorbed on the cell wall dropped to about 20% of that of natural bacteria. These facts indicate that GS is adsorbed on the cell membrane of bacteria particularly. The uptake of amino acid or glucose in B. subtilis was inhibited by GS. Therefore, it is concluded that GS damages the phospholipid bilayer of the cell membrane by adsorption, and prevents the functioning of the cell membrane. The amount of [14C]GS adsorbed on the spheroplast membrane of E. coli increased remarkably as compared with natural cells, even at a lower concentration of GS. The poor GS adsorption on E. coli cells may be due to the permeability barrier of the E. coli cell wall.     

A multipotent clonal human periodontal ligament cell line with neural crest cell phenotypes promotes neurocytic differentiation, migration, and survival

Repair of injured peripheral nerve is thought to play important roles in tissue homeostasis and regeneration. Recent experiments have demonstrated enhanced functional recovery of damaged neurons by some types of somatic stem cells. It remains unclear, however, if periodontal ligament (PDL) stem cells possess such functions. We recently developed a multipotent clonal human PDL cell line, termed cell line 1-17. Here, we investigated the effects of this cell line on neurocytic differentiation, migration, and survival. This cell line expressed the neural crest cell marker genes Slug, SOX10, Nestin, p75NTR, and CD49d and mesenchymal stem cell-related markers CD13, CD29, CD44, CD71, CD90, CD105, and CD166. Rat adrenal pheochromocytoma cells (PC12 cells) underwent neurocytic differentiation when co-cultured with cell line 1-17 or in conditioned medium from cell line 1-17 (1-17CM). ELISA analysis revealed that 1-17CM contained approximately 50 pg/ml nerve growth factor (NGF). Cell line 1-17-induced migration of PC12 cells, which was inhibited by a neutralizing antibody against NGF. Furthermore, 1-17CM exerted antiapoptotic effects on differentiated PC12 cells as evidenced by inhibition of neurite retraction, reduction in annexin V and caspase-3/7 staining, and induction of Bcl-2 and Bcl-xL mRNA expression. Thus, cell line 1-17 promoted neurocytic differentiation, migration, and survival through secretion of NGF and possibly synergistic factors. PDL stem cells may play a role in peripheral nerve reinnervation during PDL regeneration.     

Detection of hydrolytic activity of trypsin with a fluorescence-chymotryptic peptide on a TLC plate

To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.     

Functional analyses of the activation loop of phototropin2 in Arabidopsis

Phototropins (phot1 and phot2) are autophosphorylating blue-light receptor kinases that mediate blue-light responses such as phototropism, chloroplast accumulation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Only phot2 induces the chloroplast avoidance response under strong blue light. The serine (Ser) residues of the kinase activation loop in phot1 are autophosphorylated by blue light, and autophosphorylation is essential for the phot1-mediated responses. However, the role of autophosphorylation in phot2 remains to be determined. In this study, we substituted the conserved residues of Ser-761 and Ser-763 with alanine (S761A S763A) in the phot2 activation loop and analyzed their function by investigating the phot2-mediated responses after the transformation of phot1 phot2 double mutant with this mutant phot2 gene. Transgenic plants expressing the mutant phot2 protein exhibited impaired responses in chloroplast movement, stomatal opening, phototropic bending, leaf flattening, and plant growth; and those expressing phot2 with S761D S763D mutations showed the normal responses. Substitution of both Ser-761 and Ser-763 with alanine in phot2 did not significantly affect the kinase activity in planta. From these results, we conclude that phosphorylation of Ser-761 and Ser-763 in the activation loop may be a common primary step for phot2-mediated responses.     

Expression and effects of epidermal growth factor on human periodontal ligament cells

Repair of damaged periodontal ligament (PDL) tissue is an essential challenge in tooth preservation. Various researchers have attempted to develop efficient therapies for healing and regenerating PDL tissue based on tissue engineering methods focused on targeting signaling molecules in PDL stem cells and other mesenchymal stem cells. In this context, we investigated the expression of epidermal growth factor (EGF) in normal and surgically wounded PDL tissues and its effect on chemotaxis and expression of osteoinductive and angiogenic factors in human PDL cells (HPDLCs). EGF as well as EGF receptor (EGFR) expression was observed in HPDLCs and entire PDL tissue. In a PDL tissue-injured model of rat, EGF and IL-1β were found to be upregulated in a perilesional pattern. Interleukin-1β induced EGF expression in HPDLCs but not EGFR. It also increased transforming growth factor-α (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) expression. Transwell assays demonstrated the chemotactic activity of EGF on HPDLCs. In addition, EGF treatment significantly induced secretion of bone morphogenetic protein 2 and vascular endothelial growth factor, and gene expression of interleukin-8 (IL-8), and early growth response-1 and -2 (EGR-1/2). Human umbilical vein endothelial cells developed well-formed tube networks when cultured with the supernatant of EGF-treated HPDLCs. These results indicated that EGF upregulated under inflammatory conditions plays roles in the repair of wounded PDL tissue, suggesting its function as a prospective agent to allow the healing and regeneration of this tissue.