Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts

Ascorbic acid (AA) is essential for collagen biosynthesis as a cofactor for prolyl and lysyl hydroxylase and as a stimulus for collagen gene expression. Many studies have evaluated the relationship between AA and collagen expression in short- and long-term effects on cells after a single administration of AA into the culture medium. However, no such study has monitored in detail the stability of AA in medium or the alterations of intracellular AA levels during a protracted interval. Therefore, we examined here intracellular AA levels and stability throughout its exposure to human skin fibroblasts in vitro. Moreover, we determined the effects on type 1 and type 4 collagen and sodium-dependent vitamin C transporter (SVCT) gene expression when medium containing 100 μM AA was replaced every 24 h for 5 days to avoid depletion of AA. Throughout this long-term culture, intracellular AA levels remained constant; the expression of type 1 and type 4 collagens and SVCT2 mRNA was enhanced, and type 1 procollagen synthesis increased. Thus, these results indicate that human skin fibroblasts exposed to AA over time had rising levels of type 1/type 4 collagens and SVCT2 mRNA expression and type 1 procollagen synthesis.     

Energy conversion and changes in physical parameters in chloroplasts and subchloroplast particles II. Energy conversion in chloroplasts and different types of subchloroplast particles

The light-induced absorbance change at 515 nm, light-inducedhydrogen ion uptake and ATP formation were compared in chloroplastsand different types of sonicated subchloroplast particles. Noparallel relationship among the activities for ATP formation,hydrogen ion uptake and the 515-nm change was observed in differenttypes of preparations. NH₄Cl inhibited ATP formation in chloroplastsbut had little effect on subchloroplast particles. In contrast,the light-induced hydrogen ion uptake was inhibited by NH₄Clin a similar manner. Tetraphenylboron (TPB), at 1 µM, inhibited ATP formationby about 30% in both chloroplasts and subchloroplast particles.In the presence of TPB, ATP formation in chloroplasts was stronglyinhibited by NHC₄Cl, but in subchloroplast particles the additionalinhibitory effect of NH₄Cl was small. A synergistic inhibitionof photophosphorylation by valinomycin plus NH₄Cl was much clearer.Although acceleration of the recovery of the 515-nm change byNH₄Cl or valinomycin was moderate, the 515-nm change virtuallydisappeared when NH₄Cl and valinomycin were added simultaneously. Although the membrane potential has a major role as the principaldriving force for ATP formation in subchloroplast particles,the simultaneous abolishment of the pH gradient and membranepotential may be required to uncouple ATP formation. ¹Present address: Fukuoka Women's University, Kasumigaoka, Fukuoka813, Japan. ²Present address: Ryukyu University, Naha, Okinawa 903, Japan. (Received February 5, 1974; )     

Anion-dependent changes of energy transfer in chloroplasts I. Effects of Tris salts on electron flow and phosphorylation

Washing spinach chloroplasts with high-concentration Tris-saltbuffers induced various types of anion-dependent changes inthe electron flow and photophosphorylation in chloroplasts. Tris-HCl buffer caused enhancement of NADP photoreduction andinhibition of phosphorylation. Tris-HNO₃ buffer, on the otherhand, caused inhibition of both electron flow and phosphorylationand decreased trypsin-activated Ca²+-dependent ATPase activity.Tris-H₂SO⁴ and Tris-H₃PO⁴ buffers, however, had no effect onthe rates of electron flow and photophosphorylation. Determination of the presence of the coupling factor (as measuredby ATPase activity) revealed a normal enzyme activity levelin chloroplasts washed with Tris-HCl or Tris-H₂SO⁴ buffer. Removalof the coupling factor by EDTA from chloroplasts washed withTris salts inhibited phosphorylation severely. Phosphorylationactivity could be partially restored by reconstitution withthe coupling factor in die presence of Mg²+. In addition to their different effects on electron flow, Tris-HCland Tris-HNO₃ induced a marked decrease in phosphorylative activityitself. The much decreased rate of phosphorylation can be explainedby the release of the coupling factor and by damage to the high-energystate generating mechanism by Tris-HNO₃-washing and by modificationof the coupling factor in the case of Tris-HCl-washing. ¹Present address: Biology Department, College of Science andEngineering, Ryukyu University, Naha, Okinawa. Japan. (Received June 27, 1972; )     

Anion-dependent changes of energy transfer in chloroplasts III. Evidence for inactivation of light-triggered adenosine triphosphatase in chloroplasts by chloride

Washing chloroplasts with a high concentration of Tris-Cl^- buffercaused Cl^- dependent inhibition of photophosphorylation, light-inducedpH rise and light-triggered Mg²⁺-dependent ATPase activity.The inhibition of these activities was largely prevented bythe presence of 10^–4 M ADP or ATP during Tris washing,especially that of Mg²⁺-ATPase activity. The results were interpretedas suggesting that the inactivation of light-triggered ATPaseactivity in chloroplasts by chloride is one of the causes ofthe uncoupling of chloroplasts with Tris washing. (Received April 30, 1974; )     

Generation and function of reactive oxygen species in dendritic cells during antigen presentation

Although reactive oxygen species (ROS) have long been considered to play pathogenic roles in various disorders, this classic view is now being challenged by the recent discovery of their physiological roles in cellular signaling. To determine the immunological consequence of pharmacological disruption of endogenous redox regulation, we used a selenium-containing antioxidant compound ebselen known to modulate both thioredoxin and glutaredoxin pathways. Ebselen at 5-20 micro M inhibited Con A-induced proliferation and cytokine production by the HDK-1 T cell line as well as the LPS-triggered cytokine production by XS52 dendritic cell (DC) line. Working with the in vitro-reconstituted Ag presentation system composed of bone marrow-derived DC, CD4(+) T cells purified from DO11.10 TCR-transgenic mice and OVA peptide (serving as Ag), we observed that 1) both T cells and DC elevate intracellular oxidation states upon Ag-specific interaction; 2) ebselen significantly inhibits ROS production in both populations; and 3) ebselen at 5-20 micro M inhibits DC-induced proliferation and cytokine production by T cells as well as T cell-induced cytokine production by DC. Thus, Ag-specific, bidirectional DC-T cell communication can be blocked by interfering with the redox regulation pathways. Allergic contact hypersensitivity responses in BALB/c mice to oxazolone, but not irritant contact hypersensitivity responses to croton oil, were suppressed significantly by postchallenge treatment with oral administrations of ebselen (100 mg/kg per day). These results provide both conceptual and technical frameworks for studying ROS-dependent regulation of DC-T cell communication during Ag presentation and for testing the potential utility of antioxidants for the treatment of immunological disease.     

CD39 is the dominant Langerhans cell-associated ecto-NTPDase: modulatory roles in inflammation and immune responsiveness

CD39, the endothelial ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), regulates vascular inflammation and thrombosis by hydrolyzing ATP and ADP. Although ecto-NTPDase activities have been used as a marker of epidermal dendritic cells (DCs) known as Langerhans cells, the identity and function of these activities remain unknown. Here we report that Langerhans cells in CD39-/- mice express no detectable ecto-NTPDase activity. Irritant chemicals triggered rapid ATP and ADP release from keratinocytes and caused exacerbated skin inflammation in CD39-/- mice. Paradoxically, T cell-mediated allergic contact hypersensitivity was severely attenuated in CD39-/- mice. As to mechanisms, T cells increased pericellular ATP concentrations upon activation, and CD39-/- DCs showed ATP unresponsiveness (secondary to P2-receptor desensitization) and impaired antigen-presenting capacity. Our results show opposing outcomes of CD39 deficiency in irritant versus allergic contact dermatitis, reflecting its diverse roles in regulating extracellular nucleotide-mediated signaling in inflammatory responses to environmental insults and DC-T cell communication in antigen presentation.     

Anion-dependent changes of energy transfer in chloroplasts II. Relationships of the coupling factor to light-induced proton transport and absorbance change at 515 nm

Roles of the coupling factor in light-induced proton transportand 515-nm absorption change were investigated in chloroplastswashed with high concentrations of Tris salts (pH 7.2). Washingthe chloroplasts with Tris-HCl and Tris-HNO₃ buffers diminishedboth the light-induced pH rise and absorbance change at 515-nm,while Tris-H₂SO₄ buffer was much less effective. Inhibited activitiescould be restored by replacement of the coupling factor afterextraction with EDTA. N,N'-dicyclohexylcarbodiimide also restoredboth activities. Effects of various anions on the proton pumpand 515-nm shift were also investigated. The order of effectivenesswas NO₃^–>Cl^–>SO₄^2–. The role of thecoupling factor and its mode of action; the action mechanismsof Tris and anionsn energy transducing processes in chloroplasts,photophosphorylation, proton transport and absorbance changeat 515 nm, are discussed. ¹Present address: Biology Department, College of Science andEngineering, Ryukyu University, Naha, Okinawa, Japan. (Received June 27, 1972; )