Regulatory effects of biomechanical strain on the insulin-like growth factor system in human periodontal cells

During mastication, dental trauma and functional dental habits the tissues that surround and support the teeth, i.e. the periodontium, are subject to complex biomechanical forces. The exact mechanisms mediating the anabolic and catabolic biomechanical effects on the periodontium are yet poorly understood. Therefore, the objective of this in-vitro study was to determine if continuous tensile strain (CTS) regulates the synthesis of components of the insulin-like growth factor (IGF) system in human periodontal ligament (PDL) cells. PDL cells from six donors were phenotyped, seeded on collagen type-I coated silicone membranes, and subjected to CTS of low (3%) or high (20%) magnitudes for 4 and 24 h. The gene expression of IGF1, IGF2, IGF1 receptor (IGF1R), insulin receptor substrate (IRS)1, and IGF-binding proteins (IGFBPs) was detected by real-time PCR. The protein synthesis was determined by immunoblotting. For statistical analysis, ANOVA and the Tukey test (p<0.05) were applied.When cells were subjected to low CTS for 4 h, the IGF1 expression was significantly increased, whereas high CTS or CTS applied for 24 h reduced the constitutive IGF1 synthesis. Although PDL cells also expressed IGF2, IGF1R, and IRS1, no significant differences for these molecules were found between stretched cells and controls. High CTS caused a significant upregulation of IGFBP1 and significant downregulation of IGFBP3 and IGFBP5 at 24 h. In conclusion, this in-vitro study suggests that biomechanical forces may regulate several components of the local IGF system in the human periodontium.     

Substrate binding and the catalytic reactions in cbb3-type oxidases: The lipid membrane modulates ligand binding

Heme–copper oxidases (HCuOs) are the terminal components of the respiratory chain in the mitochondrial membrane or the cell membrane in many bacteria. These enzymes reduce oxygen to water and use the free energy from this reaction to maintain a proton-motive force across the membrane in which they are embedded. The heme–copper oxidases of the cbb₃-type are only found in bacteria, often pathogenic ones since they have a low K_m for O₂, enabling the bacteria to colonize semi-anoxic environments. Cbb₃-type (C) oxidases are highly divergent from the mitochondrial-like aa₃-type (A) oxidases, and within the heme–copper oxidase family, cbb₃ is the closest relative to the most divergent member, the bacterial nitric oxide reductase (NOR). Nitric oxide reductases reduce NO to N₂O without coupling the reaction to the generation of any electrochemical proton gradient. The significant structural differences between A- and C-type heme–copper oxidases are manifested in the lack in cbb₃ of most of the amino acids found to be important for proton pumping in the A-type, as well as in the different binding characteristics of ligands such as CO, O₂ and NO. Investigations of the reasons for these differences at a molecular level have provided insights into the mechanism of O₂ and NO reduction as well as the proton-pumping mechanism in all heme–copper oxidases. In this paper, we discuss results from these studies with the focus on the relationship between proton transfer and ligand binding and reduction. In addition, we present new data, which show that CO binding to one of the c-type hemes of CcoP is modulated by protein–lipid interactions in the membrane. These results show that the heme c-CO binding can be used as a probe of protein–membrane interactions in cbb₃ oxidases, and possible physiological consequences for this behavior are discussed.     

Ligand-induced formation of a transient tryptophan synthase complex with αββ subunit stoichiometry

The prototypical tryptophan synthases form a stable heterotetrameric αββα complex in which the constituting TrpA and TrpB1 subunits activate each other in a bidirectional manner. The hyperthermophilic archaeon Sulfolobus solfataricus does not contain a TrpB1 protein but instead two members of the phylogenetically distinct family of TrpB2 proteins, which are encoded within (sTrpB2i) and outside (sTrpB2a) the tryptophan operon. It has previously been shown that sTrpB2a does not functionally or structurally interact with sTrpA, whereas sTrpB2i substantially activates sTrpA in a unidirectional manner. However, in the absence of catalysis, no physical complex between sTrpB2i and sTrpA could be detected. In order to elucidate the structural requirements for complex formation, we have analyzed the interaction between sTrpA (α-monomer) and sTrpB2i (ββ-dimer) by means of spectroscopy, analytical gel filtration, and analytical ultracentrifugation, as well as isothermal titration calorimetry. In the presence of the TrpA ligand glycerol 3-phosphate (GP) and the TrpB substrate l-serine, sTrpA and sTrpB2i formed a physical complex with a thermodynamic dissociation constant of about 1 μM, indicating that the affinity between the α- and ββ-subunits is weaker by at least 1 order of magnitude than the affinity between the corresponding subunits of prototypical tryptophan synthases. The observed stoichiometry of the complex was 1 subunit of sTrpA per 2 subunits of sTrpB2i, which corresponds to a αββ quaternary structure and testifies to a strong negative cooperativity for the binding of the α-monomers to the ββ-dimer. The analysis of the interaction between sTrpB2i and sTrpA in the presence of several substrate, transition state, and product analogues suggests that the αββ complex remains stable during the whole catalytic cycle and disintegrates into α- and ββ-subunits upon the release of the reaction product tryptophan. The formation of a transient tryptophan synthase complex, together with the observed low affinity of sTrpB2i for l-serine, couples the rate of tryptophan biosynthesis in S. solfataricus to the cytosolic availability of l-serine.     

Nucleoside Phosphorylation: A Feasible Step in the Prebiotic Pathway to RNA

Plausible prebiotic conditions for the phosphorylation of nucleosides by inorganic phosphate were reported by Lohrmann and Orgel in 1971. This reaction was carried out on heated dry films and promoted by urea. The major products formed were nucleoside-2:3 cyclicPs; 5-NMPs and other derivatives were also formed. Minor modifications of the Lohrmann and Orgel system have resulted in the preferential formation of 5-NMPs. In this modified system a 2-fold preference for phosphorylation of the 5-OH group over the 3(2)-OH group was observed and the formation of other derivatives was minimized. The small amounts of bis compounds that were formed in this system could be quantitatively removed by selective binding to the mineral hydroxylapatite at moderate ionic strengths. It was also discovered that under hydrolytic conditions there was a 3:1 preference for removal of phosphates attached to the 3-OH group over the 5-OH group. A recycling procedure for obtaining additional 5-NMPs from bis compounds and 3-NMPs is proposed.     

Antigenic epitopes fused to cationic peptide bound to oligonucleotides facilitate Toll-like receptor 9-dependent, but CD4+ T cell help-independent, priming of CD8+ T cells

A priority in current vaccine research is the development of adjuvants that support the efficient priming of long-lasting, CD4(+) T cell help-independent CD8(+) T cell immunity. Oligodeoxynucleotides (ODN) with immune-stimulating sequences (ISS) containing CpG motifs facilitate the priming of MHC class I-restricted CD8(+) T cell responses to proteins or peptides. We show that the adjuvant effect of ISS(+) ODN on CD8(+) T cell priming to large, recombinant Ag is enhanced by binding them to short, cationic (arginine-rich) peptides that themselves have no adjuvant activity in CD8(+) T cell priming. Fusing antigenic epitopes to cationic (8- to 10-mer) peptides bound to immune-stimulating ISS(+) ODN or nonstimulating NSS(+) ODN (without CpG-containing sequences) generated immunogens that efficiently primed long-lasting, specific CD8(+) T cell immunity of high magnitude. Different MHC class I-binding epitopes fused to short cationic peptides of different origins showed this adjuvant activity. Quantitative ODN binding to cationic peptides strikingly reduced the toxicity of the latter, suggesting that it improves the safety profile of the adjuvant. CD8(+) T cell priming supported by this adjuvant was Toll-like receptor 9 dependent, but required no CD4(+) T cell help. ODN (with or without CpG-containing sequences) are thus potent Th1-promoting adjuvants when bound to cationic peptides covalently linked to antigenic epitopes, a mode of Ag delivery prevailing in many viral nucleocapsids.     

Activating immunity in the liver. II. IFN-beta attenuates NK cell-dependent liver injury triggered by liver NKT cell activation

Dendritic cell (DC)-dependent activation of liver NKT cells triggered by a single i.v. injection of a low dose (10-100 ng/mouse) of alpha-galactosyl ceramide (alphaGalCer) into mice induces liver injury. This response is particularly evident in HBs-tg B6 mice that express a transgene-encoded hepatitis B surface Ag in the liver. Liver injury following alphaGalCer injection is suppressed in mice depleted of NK cells, indicating that NK cells play a role in NK T cell-initiated liver injury. In vitro, liver NKT cells provide a CD80/86-dependent signal to alphaGalCer-pulsed liver DC to release IL-12 p70 that stimulates the IFN-gamma response of NKT and NK cells. Adoptive transfer of NKT cell-activated liver DC into the liver of nontreated, normal (immunocompetent), or immunodeficient (RAG(-/-) or HBs-tg/RAG(-/-)) hosts via the portal vein elicited IFN-gamma responses of liver NK cells in situ. IFN-beta down-regulates the pathogenic IL-12/IFN-gamma cytokine cascade triggered by NKT cell/DC/NK cell interactions in the liver. Pretreating liver DC in vitro with IFN-beta suppressed their IL-12 (but not IL-10) release in response to CD40 ligation or specific (alphaGalCer-dependent) interaction with liver NKT cells and down-regulated the IFN-gamma response of the specifically activated liver NKT cells. In vivo, IFN-beta attenuated the NKT cell-triggered induction of liver immunopathology. This study identifies interacting subsets of the hepatic innate immune system (and cytokines that up- and down-regulate these interactions) activated early in immune-mediated liver pathology.     

1,3-Propanediol formation with product-tolerant mutants of Clostridium butyricum DSM 5431 in continuous culture: productivity, carbon and electron flow

Glycerol fermentation and product formation of two product-tolerant mutants of Clostridium butyricum DSM 5431 were investigated in continuous culture at increasing glycerol feed concentrations. Under conditions of glycerol excess (above 55 g l⁻¹ at D = 0·15 h⁻¹), the mutants maintained a constant level of glycerol consumption and product formation, whereas the parent strain exhibited a substantial decrease in substrate conversion, 1,3-propanediol and butyrate formation, and an increase in acetate formation. The activities of the glycerol dehydrogenase, the glycerol dehydratase and the 1,3-propanediol dehydrogenase showed only slight changes with glycerol concentrations in the mutants, but dropped markedly at high concentrations in the wild type. Intracellular concentrations of NADH, NAD ⁺ and acetyl-CoA remained at a relatively constant level in the mutants, but increased sharply with the wild type strain. The NADH content was always higher than the NAD ⁺ content in the mutants as well as in the wild type.     

Direct cytotoxicity test applied to patients in stage I and II of malignant skin melanoma

Summary Using the direct cytotoxicity test described by Takasugi and Klein (1970), 59 patients suffering from localized malignant skin melanoma (stages I and II) were investigated for cell-mediated immunity. On an average, peripheral lymphocytes obtained preoperatively from 52 patients showed a higher cytotoxicity against one of two established melanoma cell lines (RPMI 7931) as compared to lymphocytes from control persons. No significant differences were found between lymphocytotoxicity of the patients and the controls against the control cell lines.In a prospective study, 17 patients were investigated. The results obtained so far suggest a correlation between a positive reaction postoperatively and recurrence of melanoma.It is clear that the control group exerted a certain degree of nonspecific cytotoxicity depending on the target cell used (established cell lines versus short-term cultures) and the lymphocyte/target cell ratio. Furthermore, there was a day-to-day variation in the nonspecific cytotoxicity exerted by lymphocytes derived from the same control person.It is concluded that considerable refinements have to be made before the microcytotoxicity assay becomes of clinical use in the evaluation of the postoperative status and course in melanoma patients.With the technical assistance of Marianne Barfod and Vibeke AhrenkielSponsored by the Danish Cancer Society.     

Early molecular events during the interaction of enveloped riboviruses with cells

A kinetic model was constructed and partly solved to describe the migration of the fluorescence label 1,6-diphenylhexatriene (DPH) in both directions when enveloped viruses, labelled with DPH in their envelopes are in contact with unlabelled cells or cell labelled in their membranes are in contact with unlabelled enveloped viruses. The central assumption is that two types of receptor sites exist on the cell surface, i.e., physical adsorption sites (P-sites), available to all the viruses studied in these papers and binding sites (B-sites) available only to the viruses which penetrate into the specific cells.The differential equations for the label migration, for different values of the ratio number of viruses number of sites were numerically solved, assuming different fractions of P- and B-sites.The equations also describe, appropriately the mechanism of rapid label migration in the system and substantiate the magnitude time of residence of the nonpenetrating viruses adsorbed on the cell surface. The resulting curves match satisfactorily those for the label release by the viruses and account well for the steady state values of the kinetics of label migration in the virus-cell system.     

Malformations, anomalies and variations in patients with severe iron deficiency]

In severe iron deficiency which frequently occurs in the population of Turkey, malformations, anomalies and variations are often observed. In 190 patients with severe iron deficiency of long duration such abnormalities could be found in 107 cases. The abnormal changes were of different character and occured in various parts of the body. In the majority multiple changes, ranging from 2 to 7 and more could be registered. 100 persons showing no iron deficiency and no anemia presented a much lower incidence of the same changes; in a second group of 54 patients suffering from a severe anemia without iron deficiency the incidence was still lower. These observations suggest that the occurrence of the abnormalities is closely connected with the iron deficiency. The character of the abnormal changes which are not susceptible to iron treatment are pointing to a prenatal origin. The diversity of the changes, the occurrence in various parts of the body and skeleton as well as the multiplicity of incidence are showing that they are due to impairment of the process of development in the embryonic organism. This view is supported by the results of the examination of the chromosomes. A distinct relation could be established between the incidence of the malformations and the occurrence of the chromosomal aberrations. As the iron deficiency in the Turkish population is mainly caused by an insufficient supply of iron with the food it is likely that by sufficient iron supply in pregnant women the incidence of malformations and anomalies caused by the iron deficiency can be prevented and by a general amelioration of the nutrition their occurrence in the population markedly reduced.