Angiogenic Properties of Human Dental Pulp Stem Cells

Angiogenesis, the formation of capillaries from pre-existing blood vessels, is a key process in tissue engineering. If blood supply cannot be established rapidly, there is insufficient oxygen and nutrient transport and necrosis of the implanted tissue will occur. Recent studies indicate that the human dental pulp contains precursor cells, named dental pulp stem cells (hDPSC) that show self-renewal and multilineage differentiation capacity. Since these cells can be easily isolated, cultured and cryopreserved, they represent an attractive stem cell source for tissue engineering. Until now, only little is known about the angiogenic abilities and mechanisms of the hDPSC. In this study, the angiogenic profile of both cell lysates and conditioned medium of hDPSC was determined by means of an antibody array. Numerous pro-and anti-angiogenic factors such as vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and endostatin were found both at the mRNA and protein level. hDPSC had no influence on the proliferation of the human microvascular endothelial cells (HMEC-1), but were able to significantly induce HMEC-1 migration in vitro. Addition of the PI3K-inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and the MEK-inhibitor U0126 to the HMEC-1 inhibited this effect, suggesting that both Akt and ERK pathways are involved in hDPSC-mediated HMEC-1 migration. Antibodies against VEGF also abolished the chemotactic actions of hDPSC. Furthermore, in the chicken chorioallantoic membrane (CAM) assay, hDPSC were able to significantly induce blood vessel formation. In conclusion, hDPSC have the ability to induce angiogenesis, meaning that this stem cell population has a great clinical potential, not only for tissue engineering but also for the treatment of chronic wounds, stroke and myocardial infarctions.     

Functional analysis of D-alanylation of lipoteichoic acid in the probiotic strain Lactobacillus rhamnosus GG

Lipoteichoic acid (LTA) is a macroamphiphile molecule which performs several functions in gram-positive bacteria, such as maintenance of cell wall homeostasis. D-alanylation of LTA requires the proteins encoded by the dlt operon, and this process is directly related to the charge properties of this polymer strongly contributing to its function. The insertional inactivation of dltD of the probiotic strain Lactobacillus rhamnosus GG (ATCC 53103) resulted in the complete absence of D-alanyl esters in the LTA as confirmed by nuclear magnetic resonance analysis. This was reflected in modifications of the bacterial cell surface properties. The dltD strain showed 2.4-fold-increased cell length, a low survival capacity in response to gastric juice challenge, an increased sensitivity to human beta-defensin-2, an increased rate of autolysis, an increased capacity to initiate growth in the presence of an anionic detergent, and a decreased capacity to initiate growth in the presence of cationic peptides compared to wild-type results. However, in vitro experiments revealed no major differences for adhesion to human intestinal epithelial cells, biofilm formation, and immunomodulation. These properties are considered to be important for probiotics. The role of the dlt operon in lactobacilli is discussed in view of these results.     

Characterization of mature rat oligodendrocytes: a proteomic approach

Oligodendrocytes are glial cells responsible for the synthesis and maintenance of myelin in the central nervous system (CNS). Oligodendrocytes are vulnerable to damage occurring in a variety of neurological diseases. Understanding oligodendrocyte biology is crucial for the dissemination of de- and remyelination mechanisms. The goal of the present study is the construction of a protein database of mature rat oligodendrocytes. Post-mitotic oligodendrocytes were isolated from mature Wistar rats and subjected to immunocytochemistry. Proteins were extracted and analyzed by means of two-dimensional gel electrophoresis and two-dimensional liquid chromatography, both coupled to mass spectrometry. The combination of the gel-based and gel-free approach resulted in confident identification of a total of 200 proteins. A minority of proteins were identified in both proteomic strategies. The identified proteins represent a variety of functional groups, including novel oligodendrocyte proteins. The results of this study emphasize the power of the applied proteomic strategy to study known or to reveal new proteins and to investigate their regulation in oligodendrocytes in different disease models.     

Genomic analysis of cyclic-di-GMP-related genes in rhizobial type strains and functional analysis in Rhizobium etli

Rhizobia are soil bacteria that can fix nitrogen in symbiosis with leguminous plants or exist free living in the rhizosphere. Crucial to their complex lifestyle is the ability to sense and respond to diverse environmental stimuli, requiring elaborate signaling pathways. In the majority of bacteria, the nucleotide-based second messenger cyclic diguanosine monophosphate (c-di-GMP) is involved in signal transduction. Surprisingly, little is known about the importance of c-di-GMP signaling in rhizobia. We have analyzed the genome sequences of six well-studied type species (Bradyrhizobium japonicum, Mesorhizobium loti, Rhizobium etli, Rhizobium leguminosarum, Sinorhizobium fredii, and Sinorhizobium meliloti) for proteins possibly involved in c-di-GMP signaling based on the presence of four domains: GGDEF (diguanylate cyclase), EAL and HD-GYP (phosphodiesterase), and PilZ (c-di-GMP sensor). We find that rhizobia possess a high number of these proteins. Conservation analysis suggests that c-di-GMP signaling proteins modulate species-specific pathways rather than ancient rhizobia-specific processes. Two hybrid GGDEF-EAL proteins were selected for functional analysis, R. etli RHE_PD00105 (CdgA) and RHE_PD00137 (CdgB). Expression of cdgA and cdgB is repressed by the alarmone (p)ppGpp. cdgB is significantly expressed on plant roots and free living. Mutation of cdgA, cdgB, or both does not affect plant root colonization, nitrogen fixation capacity, biofilm formation, motility, and exopolysaccharide production. However, heterologous expression of the individual GGDEF and EAL domains of each protein in Escherichia coli strongly suggests that CdgA and CdgB are bifunctional proteins, possessing both diguanylate cyclase and phosphodiesterase activities. Taken together, our results provide a platform for future studies of c-di-GMP signaling in rhizobia.     

Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells

Dental pulp stem cells (DPSCs) are an attractive alternative mesenchymal stem cell (MSC) source because of their isolation simplicity compared with the more invasive methods associated with harvesting other MSC sources. However, the isolation method to be favored for obtaining DPSC cultures remains under discussion. This study compares the stem cell properties and multilineage differentiation potential of DPSCs obtained by the two most widely adapted isolation procedures. DPSCs were isolated either by enzymatic digestion of the pulp tissue (DPSC-EZ) or by the explant method (DPSC-OG), while keeping the culture media constant throughout all experiments and in both isolation methods. Assessment of the stem cell properties of DPSC-EZ and DPSC-OG showed no significant differences between the two groups with regard to proliferation rate and colony formation. Phenotype analysis indicated that DPSC-EZ and DPSC-OG were positive for CD29, CD44, CD90, CD105, CD117 and CD146 expression without any significant differences. The multilineage differentiation potential of both stem cell types was confirmed by using standard immuno(histo/cyto)chemical staining together with an in-depth ultrastructural analysis by means of transmission electron microscopy. Our results indicate that both DPSC-EZ and DPSC-OG could be successfully differentiated into adipogenic, chrondrogenic and osteogenic cell types, although the adipogenic differentiation of both stem cell populations was incomplete. The data suggest that both the enzymatic digestion and outgrowth method can be applied to obtain a suitable autologous DPSC resource for tissue replacement therapies of both bone and cartilage.     

The Rhizobium etli opt operon is required for symbiosis and stress resistance

Rhizobium etli is a Gram-negative root-colonizing soil bacterium capable of fixing nitrogen while living in symbiosis with its leguminous host Phaseolus vulgaris. A genome-wide screening for R. etli symbiotic mutants revealed a R. etli operon encoding an oligopeptide ABC-transporter (Opt), two redA homologous genes and one redB gene. Expression analysis showed this opt operon to be transcribed both under free-living and symbiotic conditions and expression levels were demonstrated to be growth-phase-dependent. Plants nodulated by R. etli opt mutants showed a reduced symbiotic nitrogen fixation activity (approximately 50% reduction). Growth experiments with opt mutants in the presence of oligopeptides as the sole nitrogen source confirmed the involvement of the opt genes in oligopeptide uptake. Further phenotypic analysis of the opt mutants revealed them to display an enhanced resistance to the oligopeptide antibiotic bacitracin, an increased susceptibility to the beta-lactam antibiotic ampicillin and a decreased osmotolerance. In conclusion, our results demonstrate that the opt operon plays a crucial role during symbiosis and stress resistance.     

The Highly Autoaggregative and Adhesive Phenotype of the Vaginal Lactobacillus plantarum Strain CMPG5300 Is Sortase Dependent

Lactobacilli are important for the maintenance of a healthy ecosystem in the human vagina. Various mechanisms are postulated but so far are poorly substantiated by molecular studies, such as mutant analysis. Bacterial autoaggregation is an interesting phenomenon that can promote adhesion to host cells and displacement of pathogens. In this study, we report on the identification of a human vaginal isolate, Lactobacillus plantarum strain CMPG5300, which shows high autoaggregative and adhesive capacity. To investigate the importance of sortase-dependent proteins (SDPs) in these phenotypes, a gene deletion mutant was constructed for srtA, the gene encoding the housekeeping sortase that covalently anchors these SDPs to the cell surface. This mutant lost the capacity to autoaggregate, showed a decrease in adhesion to vaginal epithelial cells, and lost biofilm-forming capacity under the conditions tested. These results indicate that the housekeeping sortase SrtA of CMPG5300 is a key determinant of the peculiar surface properties of this vaginal Lactobacillus strain.     

Impact of luxS and Suppressor Mutations on the Gastrointestinal Transit of Lactobacillus rhamnosus GG

It is generally believed that probiotic bacteria need to survive gastrointestinal transit to exert a health-promoting effect. In this study, a genuine luxS mutant and a luxS mutant containing unknown suppressor mutations of the probiotic strain Lactobacillus rhamnosus GG were compared to the wild type for survival and persistence in the murine gastrointestinal tract. The LuxS enzyme, catalyzing the production of the autoinducer-2 signaling molecule, also forms an integral part of the activated methyl cycle and the metabolism of methionine and cysteine. The genuine luxS mutant CMPG5412 showed drastically reduced persistence in mice, which was related to less survival in simulated gastric juice, indicating that LuxS metabolism is crucial for the gastric stress resistance of L. rhamnosus GG. The suppressor mutations in the other luxS mutant, CMPG5413, appear to compensate for the metabolic defects of the luxS mutation and to restore the resistance to gastric juice but cause a defect in adherence, biofilm formation, and exopolysaccharide production. The shorter residence time of this suppressor mutant in the murine gastrointestinal tract indicates a role for biofilm formation and exopolysaccharides in the persistence capacity of L. rhamnosus GG.     

Identification of a Gene Cluster for the Biosynthesis of a Long,Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase

Cell surface polysaccharides have an established role as virulence factors in human bacterial pathogens. Less documented are the biosynthesis and biological functions of surface polysaccharides in beneficial bacteria. We identified a gene cluster that encodes the enzymes and regulatory and transporter proteins for the different steps in the biosynthesis of extracellular polysaccharides (EPS) of the well-documented probiotic strain Lactobacillus rhamnosus GG. Subsequent mutation of the welE gene, encoding the priming glycosyltransferase within this cluster, and comparative phenotypic analyses of wild-type versus mutant strains confirmed the specific function of this gene cluster in the biosynthesis of high-molecular-weight, galactose-rich heteropolymeric EPS molecules. The phenotypic analyses included monomer composition determination, estimation of the polymer length of the isolated EPS molecules, and single-molecule force spectroscopy of the surface polysaccharides. Further characterization of the welE mutant also showed that deprivation of these long, galactose-rich EPS molecules results in an increased adherence and biofilm formation capacity of L. rhamnosus GG, possibly because of less shielding of adhesins such as fimbria-like structures.Bacterial surface polysaccharides are considered to be key macromolecules in determining microbe-host interactions, as they display a high degree of variety and diversity among bacterial species in terms of composition, monomer linkages, branching degree, polymer size, production level, etc. (24, 46). Since most bacteria contain more than one type of surface polysaccharides, such as lipopolysaccharides (O antigens), capsular polysaccharides (CPS), exopolysaccharides (EPS), and/or glycan chains as part of glycoproteins, the elucidation of their exact role is complex. Nevertheless, surface polysaccharides are now known to exert important functions at several stages during pathogenesis, including tissue adherence, biofilm formation, and evasion of host defenses such as phagocytosis (9, 24, 33). In addition to their role in pathogens, an important biological role for CPS and glycoproteins has also recently been shown in colonization of the gut by bacteria of the genus Bacteroides (10, 34).Conversely, the role of surface polysaccharides in probiotic-host interactions has not yet been studied in great detail. A probiotic bacterium is defined as “a live microorganism that, when administered or ingested in adequate amounts, confers a health benefit on the host” (18). Members of the genus Lactobacillus are commonly studied for their health-promoting capacities (26, 31, 37). As polysaccharides display a high diversity among lactobacilli, they are thought to be involved in determining strain-specific properties important for probiotic action, such as adhesion, stress resistance, and interactions with specific receptors and effectors of the host defense system (13, 56). Moreover, these EPS molecules are of interest in the dairy industry for conferring textural and rheological properties to fermented products such as yogurt and soft cheese (56). Nevertheless, detailed genetic and functional studies of EPS molecules of lactobacilli are currently limited (26, 56).Lactobacillus rhamnosus GG (ATCC 53103) is one of the probiotic strains with the largest number of proven health benefits (15). Several clinical trials have reported that L. rhamnosus GG can prevent and relieve certain types of diarrhea (22) and atopic disease (25) and reduce inflammation in some milder states of inflammatory bowel diseases (60). However, the cell surface factors or specific characteristics of L. rhamnosus GG that underlie these health benefits are largely unknown.We recently showed by single-molecule force spectroscopy (SMFS) with specific lectin tips that the cell surface of L. rhamnosus GG wild-type cells contains two major types of cell wall-associated polysaccharides (CW-PS) (21). The longest and most abundant polysaccharides are galactose-rich and seem to correspond with the EPS molecules of L. rhamnosus GG, which were previously structurally identified by Landersjö et al. (27) using nuclear magnetic resonance spectroscopy. Additionally, shorter, yet-uncharacterized glucose-rich polysaccharides are present on the L. rhamnosus GG surface (21). In the current study, we describe the identification and annotation of the L. rhamnosus GG gene cluster that encodes the enzymes and transporter and regulatory proteins involved in the biosynthesis of long, galactose-rich EPS molecules. This was experimentally confirmed by the construction of a knockout mutant of the corresponding priming glycosyltransferase and subsequent characterization of the surface polysaccharides of wild-type and mutant strains. We also studied the specific role of these EPS molecules in adherence to mucus and gut epithelial cells and in biofilm formation by L. rhamnosus GG.