Osteogenic differentiation and gene expression of dental pulp stem cells under low-level laser irradiation: a good promise for tissue engineering.

The effects of low-level laser therapy (LLLT) has been the focus of recent studies as being assumed responsible for promoting photostimulatory and photobiomodulatory effects in vivo and in vitro, increasing cell metabolism, improving cell regeneration and invoking an anti-inflammatory response. A positive effect of LLLT on the bone proliferation of some cell types has been observed, but little is known about its effect on dental pulp stem cells (DPSCs). Here, we accurately describe the technical procedure to isolate mesenchymal DPSCs, and assay their osteogenic capacity when irradiated with an LLLT source. These preliminary results show that LLLT irradiation influences the in vitro proliferation of DPSCs and increases the expression of essential proteins for bone formation, although it is necessary to carry out further experiments on other cell types and to uniform the methodological designs.

Regulation by the cAMP cascade of oxygen free radical balance in mammalian cells.

A study is presented of the effect of the cAMP cascade on oxygen metabolism in mammalian cell cultures. Serum-starvation of the cell cultures resulted in depression of the forward NADH-ubiquinone oxidoreductase activity of complex I, decreased content of glutathione, and enhancement of the cellular level of H2O2. Depressed transcription of cytosolic Cu/Zn-SOD 1, mitochondrial glutathione peroxidase and catalase was also observed. Activation of the cAMP cascade reversed the depression of the activity of complex I and the accumulation of H2O2. The effect of cAMP involved the cAMP-dependent protein kinase.

Carboxyl residues in the iron-sulfur protein are involved in the proton pumping activity of P. denitrificans bc(1) complex.

A study is presented on chemical modification of the three subunit Paracoccus denitrificans bc(1) complex. N-(Ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) treatment caused a loss of the proton pumping activity of liposome-reconstituted bc(1) complex. A similar effect, which is referred to as the decoupling effect, resulted upon reaction of N,N'-dicyclohexylcarbodiimide (DCCD) with the complex. Direct measurement of the binding of EEDQ to the complex subunits, performed in the presence of the fluorescent hydrophobic nucleophile 4'-[(aminoacetamido)methyl]fluorescein (AMF), showed that the iron-sulfur protein (ISP) and cytochrome c(1) were labeled by EEDQ, whereas cytochrome b was not. Tryptic digestion and sequencing analysis of the fluorescent fragment of the ISP revealed this to consist of a segment with six acidic residues, among which the highly conserved aspartate 160 is present. Analogous experiments on DCCD binding showed that all the three subunits of the complex were labeled. However, DCCD concentration dependence of carboxyl residue modification in the individual subunits and of proton pumping activity showed that the decrease of the H(+)/e(-) ratio correlated only with the modification of the ISP. Tryptic digestion of labeled ISP and sequencing analysis of the fluorescent fragment gave results superimposable upon those obtained with EEDQ. Chymotryptic digestion and sequencing analysis of the single fluorescent fragment of cytochrome b showed that this fragment contained glutamate 174 and aspartate 187. We conclude that, in the P. denitrificans bc(1) complex, carboxyl residues in cytochrome b do not appear to be critically involved in the proton pump mechanism of the complex.

Arachidonic acid causes cytochrome c release from heart mitochondria.

Arachidonic acid interaction with heart mitochondria is known to cause uncoupling as well as inhibition of pyruvate + malate and succinate-supported respiration. Here we present experiments showing that arachidonic acid causes cytochrome c release from Ca(2+)-loaded heart mitochondria. We have also measured mitochondrial matrix swelling and found a fairly good correlation between the two processes, as revealed by the same arachidonic acid concentration dependence and by the same susceptibility toward different free fatty acid species. The effects produced by arachidonic acid are not related to its protonophoric activity since, under the experimental conditions used, saturating concentrations of FCCP did not cause any effect.

Immunogenicity of an eight amino acid domain shared by Fas (CD95/Apo-I) and HIV-1 gp120. I. Structural and antigenic analysis.

UNLABELLED: Previous studies have demonstrated that immunoglobulin G (IgG) antibodies to VEINCTR-N, a domain shared by Fas (CD95/Apo-I) and gp120, contribute to T-cell apoptosis during human immunodeficiency virus-type 1 (HIV-1) infection as a result of the agonist cross-linking of Fas. The present work was designed to determine whether these molecules are elicited primarily to HIV-1 or the cell receptor. MATERIALS AND METHODS: Sera from 439 HIV-1-infected patients were screened by ELISA for their reactivity to VEINCTR-N. Subjects with significant serum elevations of IgG anti-VEINCTR-N were further investigated. Immunologic parameters, including CD4+ and CD8+ lymphocyte count, extent of T-cell apoptosis, occurrence of both anti-Fas antibodies and circulating soluble Fas titers, and reactivity to the 8-mer peptides resembling the flank-regions of VEINCTR-N on both gp120 V3 loop and Fas were examined. In addition, the antigenicity of these domains was assessed by biochemical and computerized analyses. RESULTS: 21 patients with significant levels of IgG to VEINCTR-N showed both an increased extent of peripheral T-cell apoptosis and binding to full-length Fas. A weak, though positive correlation of the anti-VEINCTR-N activity with its antecedent peptide on Fas was also found. Charge and structural analysis revealed that, although the extended 26-amino acid (a.a.) regions on both proteins were hydrophilic, the Fas peptide adjacent to VEINCTR-N expressed a short beta-conformed a.a. sequence in contiguity with a portion of the shared epitope, also in beta-sheet conformation. Patterns of antigenicity confirmed an apparent immunodominance of the full VEINCTR-N, based on its homology with the consensus sequence of other members of the tumor necrosis factor (TNF) receptor family. The hypothesis that the high immunogenicity of this region of Fas, rather than gp120, can drive the production of anti-VEINCTR-N antibodies also was supported by the concurrent significant elevations of soluble Fas in almost all of the sera studied. CONCLUSIONS: Our results indicate that a high release of the soluble form of Fas by T cells during the chronic immune activation of HIV-1 infection primes a humoral response against this epitope of Fas as a result of its high antigenicity. This is similar to the antibodies to tumor necrosis factor alpha (TNFalpha) receptor (R) (TNFalpha-R) that occur in response to increased levels of the soluble receptor for TNF during autoimmunity.

Plasma 5alpha-androstane-3alpha,17betadiol, an endogenous steroid that positively modulates GABA(A) receptor function, and anxiety: a study in menopausal women.

We tested the hypothesis that changes in endogenous neuroactive steroids acting as positive allosteric modulators of gamma-aminobutyric acid (GABA)(A) receptors may be related to the menopause-associated mood alterations. The study sample consisted of twenty five drug-free menopausal women, 1-3 years since the onset of menopause, homogeneous for age and body mass index (BMI) and without personal history of psychiatric, metabolic or endocrine disorders. Depression and anxiety-related symptoms were assessed with the Zung Self-administered Depression Scale (ZSDS) and the Cornell's Dysthymia Rating Scale (CDRS). The cut-off value predicted by the ZSDS index defined two groups of women (asymptomatic [35.5+/-4.6, n=12] and symptomatic [60.8+/-7.9, n=13]), that were also significantly different according to the CDRS scores (10.6+/-3.4 and 31.5+/-12, respectively, P<0.05). Upon evaluation of the scores relative to the anxiety factor of the CDRS (items 11-15) the symptomatic, but not the asymptomatic, group showed a moderate level of anxiety. The plasma concentrations of several neuroactive steroids were measured, after extraction and HPLC purification, by radioimmunoassay with specific antisera. Only dehydroepiandrosterone and its metabolite 5alpha-androstane-3alpha,17betadiol (3alpha-ADIOL), a positive allosteric modulator of GABA(A) receptors, were significantly (P<0.05 and P<0.005) higher (+110% and +64%, respectively) in the asymptomatic group. A highly significant and negative correlation (r=-0.672, P=0.003) was found between the plasma 3alpha-ADIOL concentrations and the scores of the anxiety factor of the CDRS. These data suggest that endogenous 3alpha-ADIOL modulates the central GABAergic tone and that higher 3alpha-ADIOL concentrations could have a role in preventing the expression of anxiety in the asymptomatic women.

Ceramide interaction with the respiratory chain of heart mitochondria.

A study is presented on the interaction of ceramide with the respiratory chain of rat heart mitochondria, and a comparison is made between the effects elicited by short- and long-chain ceramides. N-Acetylsphingosine (C(2)-ceramide) and N-palmitoylsphingosine (C(16)-ceramide) inhibited to the same extent the pyruvate+malate-dependent oxygen consumption. Succinate-supported respiration was also inhibited by ceramides, but this activity was substantially restored upon the addition of cytochrome c, which, on the contrary, was ineffective toward the ceramide-inhibited NADH-linked substrate oxidation. Direct measurements showed that short- and long-chain ceramides caused a large release of cytochrome c from mitochondria. The ceramide-dependent inhibition of pyruvate+malate and succinate oxidation caused reactive oxygen species to be produced at the level of either complex I or complex III. The activity of the cytochrome c oxidase, measured as ascorbate/TMPD oxidase activity, was significantly stimulated and inhibited by C(2)- and C(16)-ceramide, respectively. Similar effects were observed on the activity of the individual respiratory complexes isolated from bovine heart. Short- and long-chain ceramides had definitely different effects on the mitochondrial membrane potential. C(2)-ceramide caused an almost complete collapse of the respiration-dependent membrane potential, whereas C(16)-ceramide had a negligible effect. Similar results were obtained when the potential was generated in liposome-reconstituted complex III respiring at the steady-state. Furthermore, C(2)-ceramide caused a drop of the membrane potential generated by ATP hydrolysis instead of respiration, whereas C(16)-ceramide did not. Finally, only short-chain ceramides inhibited markedly the reactive oxygen species generation associated with membrane potential-dependent reverse electron flow from succinate to complex I. The emerging indication is that the short-chain ceramide-dependent collapse of membrane potential is a consequence of their ability to perturb the membrane structure, leading to an unspecific increase of its permeability.

Arachidonic acid interaction with the mitochondrial electron transport chain promotes reactive oxygen species generation.

A study has been carried out on the interaction of arachidonic acid and other long chain free fatty acids with bovine heart mitochondria. It is shown that arachidonic acid causes an uncoupling effect under state 4 respiration of intact mitochondria as well as a marked inhibition of uncoupled respiration. While, under our conditions, the uncoupling effect is independent of the fatty acid species considered, the inhibition is stronger for unsaturated acids. Experiments carried out with mitochondrial particles indicated that the arachidonic acid dependent decrease of the respiratory activity is caused by a selective inhibition of Complex I and III. It is also shown that arachidonic acid causes a remarkable increase of hydrogen peroxide production when added to mitochondria respiring with either pyruvate+malate or succinate as substrate. The production of reactive oxygen species (ROS) at the coupling site II was almost double than that at site I. The results obtained are discussed with regard to the impairment of the mitochondrial respiratory activity as occurring during the heart ischemia/reperfusion process.

Localization of acidic residues involved in the proton pumping activity of the bovine heart mitochondrial bc1 complex.

Chemical modification of carboxyl residues in polypeptide subunits of the mitochondrial bc1 complex causes a decoupling effect, that is inhibition of the proton pumping activity, without affecting the rate of electron transfer to ferricytochrome c. The study presented here is aimed at localizing and identifying the residues whose modification results in decoupling of the complex. Glutamate-53 in subunit IX (the DCCD-binding protein) and aspartate-166 in the Rieske iron-sulfur protein are the residues modified by N,N'-dicyclohexylcarbodiimide (DCCD) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), respectively. The results obtained also suggest that the carboxy-terminal sequence of the Core protein II, which is fairly rich in acidic residues, may also play a role in the vectorial proton translocation activity of the complex.

Mitochondrial oxidative alterations following partial hepatectomy.

Mitochondria, isolated from rat livers during the early phase of liver regeneration (7-24 h after partial hepatectomy), show: (i) decrease in the rate of ATP synthesis; (ii) increase of malondialdehyde and of oxidized protein production; (iii) decrease of the content of intramitochondrial glutathione and of protein thiols on mitochondrial proteins; (iv) increase of the glutathione bound to mitochondrial proteins by disulfide bonds. These observations suggest an increase of production of oxygen radicals in liver mitochondria, following partial hepatectomy, which can alter the function of the enzymes involved in the oxidative phosphorylation. Blue-native gel electrophoresis of rat liver mitochondria, isolated after partial hepatectomy, shows, during the early phase of liver regeneration (0-24 h after partial hepatectomy), a progressive decrease of the content of F0F1-ATP synthase complex. The amount of glutathione bound to the F0F1-ATP synthase, electroeluted from the blue-native gels, progressively increased during the early phase of liver regeneration. It is concluded that partial hepatectomy causes mitochondrial oxidative stress that, in turn, modifies proteins (such as F0F1-ATP synthase) involved in the mitochondrial oxidative phosphorylation.

Chemical modification of the bovine mitochondrial bc1 complex reveals critical acidic residues involved in the proton pumping activity.

Bovine heart ubiquinol-cytochrome c reductase (bc1 complex) was modified with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), which is a selective reagent for buried carboxyl groups. EEDQ treatment caused a loss of the proton pumping activity of liposome-reconstituted bc1 complex, without effect on the passive proton conductivity of the proteoliposomes. Although the decoupling effect produced on proton translocation was similar to that elicited by N,N'-dicyclohexylcarbodiimide (DCCD) modification of cytochrome b and subunit IX, EEDQ modified different subunits, namely the Core protein II and the iron-sulfur protein (ISP). A time-dependent increase of the labeling of both subunits was observed which was kinetically comparable with the decrease of the H+/e- ratio. Trypsin treatment of the complex showed that the EEDQ-modified carboxyl group in the ISP belongs to the protruding moiety of the protein, holding the Fe/S cluster. The results obtained show that critical acidic residues, located in different subunits of the bc1 complex, at both sides of the membrane, contribute to its proton pumping activity.

Steady-state proton translocation in bovine heart mitochondrial bc1 complex reconstituted into liposomes.

The effect of different anions on the steady-state proton translocation in bovine bc1 complex reconstituted in liposomes was studied. The H+/e- ratio for vectorial proton translocation is at the steady state definitely lower than that measured at level flow, (0.3 vs. 1.0). The presence of azide or arachidonate at micro- and submicromolar concentrations, respectively, gave a substantial reactivation of the proton pumping activity at the steady state, without any appreciable effect on respiration-dependent transmembrane pH difference. Addition of azide to turning-over bc1 vesicles also caused a transition of b cytochromes toward oxidation. The results are discussed in terms of possible involvement of an acidic residue in the protonation of the semiquinone/quinol couple at the N side of the membrane.

Identification of functional domains and critical residues in the adenosinetriphosphatase inhibitor protein of mitochondrial F0F1 ATP synthase.

Peptide segments of the inhibitor protein (IF1) of the F0F1 ATP synthase complex from bovine-heart mitochondria have been constructed by chemical synthesis. The IF1-(42-58)-peptide was equally effective as IF1 in inhibiting the ATPase activity of both the F0F1 complex in the mitochondrial membrane deprived of IF1 (SMP) and soluble F1. The IF1-(22-46)-peptide inhibited the ATPase activity in the soluble F1 but had no effect on either the ATPase activity or H+ conduction in SMP. Substitution of the His or Lys residues with Ala in the IF1-(42-58)-peptide decreased the inhibition of ATP hydrolysis. The inhibition exerted by the IF1-(42-58)-peptide on ATP hydrolysis in SMP exhibited a pH dependence, similar to that observed with IF1, which was lost upon replacement of His or Lys with Ala. In soluble F1, inhibition of ATP hydrolysis by IF1, the IF1-(42-58)-peptide and the IF1-(22-46)-peptide was pH dependent when F1 was first incubated with ATP. The IF1-(42-58)-peptide also caused inhibition of passive H+ conduction in SMP. This activity of the synthetic peptide was weaker, as compared to that of IF1, and practically unaffected by substitution of His or Lys with Ala. An antibody against the IF1-(42-58)-synthetic peptide stimulated ATP hydrolysis in the membrane-bound F0F1 complex with associated IF1 but was without effect on H+ conduction. An antibody against IF1 stimulated both processes.

The nuclear-encoded 18 kDa (IP) AQDQ subunit of bovine heart complex I is phosphorylated by the mitochondrial cAMP-dependent protein kinase.

In bovine heart mitochondria a protein of M(r) 18 kDa, phosphorylated by mtPKA, is associated to the NADH-ubiquinone oxidoreductase in the inner membrane and is present in purified preparation of this complex. The 18 kDa phosphoprotein has now been isolated and sequenced. It is identified as the 18 kDa (IP) AQDQ subunit of complex I, a protein of 133 amino acids with a phosphorylation consensus site RVS at position 129-131.

Correlation between rat liver regeneration and mitochondrial energy metabolism.

The time course of changes in mitochondrial energy metabolism during liver regeneration, following partial hepatectomy, is analyzed. For 24 h after surgical operation, a lag phase in the time course of the growth of liver is observed. In this period mitochondria showed a decrease of: (1) the respiratory control index; (2) the rate of oxidative phosphorylation; (3) the amount of immunodetected beta-F1 and F01-PVP subunits of F0F1-ATP synthase. No decrease, but instead a small increase in the content of mRNA for beta-F1 was observed in this phase. After this lag phase the growth of liver started, the content of mRNA for beta F1, as well as the level of immunodetected mitochondrial beta-F1 and F01-PVP subunits, increased and oxidative phosphorylation recovered. Analysis of the relative beta F1 protein/mRNA ratio indicates a decrease of beta F1 translational efficiency which remained low up to 72 h after partial hepatectomy and reached the same ratio of control at 96 h. It is concluded that the regenerating capability of rat liver is correlated with the efficiency of oxidative phosphorylation.

Proton/electron stoichiometry of mitochondrial bc1 complex. Influence of pH and transmembrane delta pH.

The effect of pH and transmembrane delta pH on the efficiency of the proton pump of the mitochondrial bc1 complex both in situ and in the reconstituted state was studied. In both cases the H+/e- ratio for vectorial proton translocation by the bc1 complex respiring at the steady state, under conditions in which the transmembrane pH difference (delta pH) represents the only component of the proton motive force (delta p), was significantly lower than that measured under level flow conditions. The latter amounts, at neutral pH, to 1 (2 including the scalar H+ release). In the reconstituted system steady-state delta pH was modulated by changing the intravesicular buffer as well as the intra/extra-liposomal pH. Under these conditions the H+/e- ratio varied inversely with the delta pH. The data presented show that delta pH exerts a critical control on the proton pump of the bc1 complex. Increasing the external pH above neutrality caused a decrease of the level flow H+/e- ratio. This effect is explained in terms of proton/electron linkage in b cytochromes.

Characteristics of energy-linked proton translocation in liposome reconstituted bovine cytochrome bc1 complex. Influence of the protonmotive force on the H+/e- stoichiometry.

A study is presented on the H+/e- stoichiometry for proton translocation by the isolated cytochrome bc1 complex under level-flow and steady-state conditions. An experimental procedure was used which allows the determination of pure vectorial proton translocation in both conditions in a single experiment. The results obtained indicate an H+/e- ratio of 1 at level-flow and 0.3 at steady-state. The ratios appear to be independent of the rate of electron transfer through the complex. Making use of pyranine-entrapped bc1 vesicles, a respiration-dependent steady-state delta pH value of 0.4 was determined in the presence of valinomycin. This value could be either decreased by subsaturating concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or increased by introducing bovine serum albumin in the assay mixture. The steady-state H+/e- ratio appeared to be in linear inverse correlation with the delta pH. This indicates that delta pH exerts a control on the proton pump of the bc1 complex at the steady state. The effect of valinomycin-mediated potassium-diffusion potential on electron-transfer and proton-translocation activities is also shown. The experiments presented show that the H+/e- ratio is unaffected, both at level flow and steady state, by an imposed diffusion potential up to around 100 mV. At higher potential values the level-flow H+/e- ratio slightly decreased. Measurements as a function of imposed membrane potential of the rate of electron transfer at level flow and of the rate of the pre-steady-state reduction of b and c1 cytochromes in the complex indicate activation of electron transfer at potential values of 40-50 mV. This activation appears, however, to involve a rate-limiting step which remains normally coupled to proton translocation.

Interaction of Zn2+ with the bovine-heart mitochondrial bc1 complex.

A study is presented of the effect of Zn2+ on the enzymatic properties of the bovine-heart cytochrome-bc1 complex. Micromolar concentrations of Zn2+ reversibly inhibit the cytochrome-c reductase activity of either the cholate-solubilized or liposome-reconstituted complex. Kinetic analysis of the redox reactions of the cytochromes indicate that Zn2+ affects the activity of the complex at the quinol oxidation site. The following have been determined: (a) Zn2+ inhibits the pre-steady-state reduction of cytochrome c1 by duroquinol either in the absence or in the presence of antimycin, (b) it does not inhibit the reduction of b cytochromes in the absence of antimycin or in the presence of myxothiazol, (c) it inhibits cytochrome-b reduction in the presence of antimycin. Furthermore Zn2+ inhibits the antimycin-promoted oxidant-induced extrareduction of b cytochromes. Addition of Zn2+ to reduced bc1 complex causes a red shift in the absorption spectrum of cytochrome b566 and a substantial decrease in the signal intensity of the EPR spectrum of the Fe-S protein. This is interpreted as an interaction of Zn2+ with the 2Fe-2S-cluster region of the Fe-S protein, thus giving rise to inhibition of the reductase activity and of the antimycin-insensitive reduction route of b cytochromes. A Scatchard-plot of 65Zn2+ binding to the native isolated complex gave a straight line from which a value of three binding sites and a single dissociation constant of 3 x 10(-6) M can be calculated, which is practically equal to the concentration causing 50% inhibition of electron flow.

Structural and functional characteristics of polypeptide subunits of the bovine heart ubiquinol--cytochrome-c reductase complex.

Structural and functional characteristics of subunits of bovine heart cytochrome-c reductase have been investigated by controlled digestion of soluble and membrane-reconstituted purified bc1 complex and direct amino acid sequencing of native and digested protein subunits. The results obtained show that the N-terminal segments of core protein II and the 14-kDa protein extend at the periphery of the complex, protruding into the inner matrix space. The Fe-S protein, located at the outer C-periphery of the complex, is shown to be anchored to other subunits of the complex by the amphipathic N-terminal region. Proteolytic cleavage of 7-11 residues from the N-terminal segment of the 14-kDa protein is apparently associated with decoupling of redox-linked proton pumping. Partial digestion of core protein II, the 6.4-kDa protein, and the C-terminal region of the 9.2-kDa protein, is without effect on the redox and proton-motive activity of the complex.