Marginal and internal fit of 3D printed provisional crowns according to build directions.

PURPOSE: This study aimed to fabricate provisional crowns at varying build directions using the digital light processing (DLP)-based 3D printing and evaluate the marginal and internal fit of the provisional crowns using the silicone replica technique (SRT). MATERIALS AND METHODS: The prepared resin tooth was scanned and a single crown was designed using computer-aided design (CAD) software. Provisional crowns were printed using a DLP-based 3D printer at 6 directions (120°, 135°, 150°, 180°, 210°, 225°) with 10 crowns in each direction. In total, sixty crowns were printed. To measure the marginal and internal fit, a silicone replica was fabricated and the thickness of the silicone impression material was measured using a digital microscope. Sixteen reference points were set and divided into the following 4 groups: marginal gap (MG), cervical gap (CG), axial gap (AG), and occlusal gap (OG). The measurements were statistically analyzed using one-way ANOVA and Dunnett T3. RESULTS: MG, CG, and OG were significantly different by build angle groups (P<.05). The MG and CG were significantly larger in the 120° group than in other groups. OG was the smallest in the 150° and 180° and the largest in the 120° and 135° groups. CONCLUSION: The marginal and internal fit of the 3D-printed provisional crowns can vary depending on the build angle and the best fit was achieved with build angles of 150° and 180°.

Clinical study on screw loosening in dental implant prostheses: a 6-year retrospective study.

OBJECTIVES: In this study, we determined the incidence and pattern of screw loosening in patients who received dental implants. MATERIALS AND METHODS: Patients who received implants between January 2008 and October 2013 and completed their prosthetic rehabilitation were evaluated for the incidence, frequency, and onset of screw loosening using dental charts and radiographs. The association between each factor and screw loosening was analyzed using the chi-square test and a multivariate analysis with binary logistic regression models (P<0.05). RESULTS: Total 1,928 implants were placed in 837 patients (448 males, 389 females), whose follow-up period after loading varied from 0.25 to 70 months (mean period, 31.5 months). Screw loosening occurred in 7.2% of implants. Most cases occurred less than six months after loading. Among those, 22.3% experienced recurrent screw loosening. Screw loosening was most common in the molar region (8.5%) and frequently associated with an implant diameter of ≥5 mm (14.2%). External implant-abutment connections (8.9%) and screw-retained implant prostheses (10.1%) showed higher incidence of problems than internal implant-abutment connections and cement-retained implants, respectively. Screw loosening was most common in implant prostheses with single crowns (14.0%). CONCLUSION: Within the limits of the current study, we conclude that the incidence of screw loosening differs significantly according to the position of implant placement, the type of implant and manufacturer, implant diameter, the type of implant-abutment connection, the type of retention in the implant prosthesis, and the type of implant prosthesis.

Preconditioning with interleukin-1 beta and interferon-gamma enhances the efficacy of human umbilical cord blood-derived mesenchymal stem cells-based therapy via enhancing prostaglandin E2 secretion and indoleamine 2,3-dioxygenase activity in dextran sulfate sodium-induced colitis.

Preconditioning with inflammatory cytokines has improved mesenchymal stem cells characteristics, including differentiation and immunomodulating functions. In this study, we developed a preconditioning combination strategy using interleukin-1beta (IL-1ß) and interferon-gamma (IFN-γ) to enhance the immuneregulatory ability of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). Our results showed that hUCB-MSCs preconditioned with IL-1ß and IFN-γ (primed hUCB-MSCs) created a statistically significant decrease in peripheral blood mononuclear cell proliferation, indicating that their immunosuppressive ability was increased. The secretion of PGE2, cyclooxygenase 2 mRNA expression, and indoleamine 2,3-dioxygenase (IDO) mRNA expression in primed hUCB-MSCs was significantly higher than those in the untreated hUCB-MSCs or the IL-1ß or IFN-γ only treated hUCB-MSCs. When inhibitors of IDO and PGE2 were treated, peripheral blood mononuclear cell proliferation, which is inhibited by primed hUCB-MSCs, was recovered. We found that Th1 T cell differentiation was also inhibited by PGE2 and IDO in the primed hUCB-MSCs, and Tregs differentiation was increased by PGE2 and IDO in the primed hUCB-MSCs. Furthermore, the primed hUCB-MSCs as well as supernatants increase CD4+ T cells migration. We demonstrated the therapeutic effects of primed hUCB-MSCs in dextran sulfate sodium-induced colitis model. In conclusion, we have demonstrated that primed hUCB-MSCs simultaneously enhance PGE2 and IDO and greatly improve the immunoregulatory capacity of MSCs, and we have developed an optimal condition for pretreatment of MSCs for the treatment of immune diseases. Our results raise the possibility that the combination of PGE2 and IDO could be therapeutic mediators for controlling immunosuppression of MSCs.

Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulates rejuvenation of human skin.

Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) play an important role in cutaneous wound healing, and recent studies suggested that MSC-derived exosomes activate several signaling pathways, which are conducive in wound healing and cell growth. In this study, we investigated the roles of exosomes that are derived from USC-CM (USC-CM Exos) in cutaneous collagen synthesis and permeation. We found that USC-CM has various growth factors associated with skin rejuvenation. Our in vitro results showed that USC-CM Exos integrate in Human Dermal Fibroblasts (HDFs) and consequently promote cell migration and collagen synthesis of HDFs. Moreover, we evaluated skin permeation of USC-CM Exos by using human skin tissues. Results showed that Exo-Green labeled USC-CM Exos approached the outermost layer of the epidermis after 3 h and gradually approached the epidermis after 18 h. Moreover, increased expressions of Collagen I and Elastin were found after 3 days of treatment on human skin. The results showed that USC-CM Exos is absorbed into human skin, it promotes Collagen I and Elastin synthesis in the skin, which are essential to skin rejuvenation and shows the potential of USC-CM integration with the cosmetics or therapeutics.

Effect of implant number and distribution on load transfer in implant-supported partial fixed dental prostheses for the anterior maxilla: A photoelastic stress analysis study.

STATEMENT OF PROBLEM: The 4-, 3- or even 2-implant-supported partial fixed dental prosthesis (PFDP) designs have been used to rehabilitate the anterior edentulous maxilla. PURPOSE: The purpose of this in vitro study was to compare the stress distribution in the supporting tissues surrounding implants placed in the anterior maxilla with 5 PFDP designs. MATERIAL AND METHODS: A photoelastic model of the human maxilla with an anterior edentulous region was made with photoelastic resin (PL-2; Vishay Micro-Measurements), and 6 straight implants (OsseoSpeed; Astra Tech AB) were placed in the 6 anterior tooth positions. The 5 design concepts based on implant location were as follows: model 6I: 6 implants; model 2C2CI: 4 implants (2 canines and 2 central incisors); model 2C2LI: 4 implants (2 canines and 2 lateral incisors); model 2C1CI: 3 implants (2 canines and 1 central incisor); and model 2C: 2 canines. A load of 127.4 N was applied on the cingulum of 3 teeth at a 30-degree angle to the long axis of the implant. Stresses that developed in the supporting structure were recorded photographically. RESULTS: The 6-implant-supported PFDP exhibited the most even and lowest distribution of stresses in all loading conditions. When the canine was loaded, the 2- or 3-implant-supported PFDP showed higher stresses around the implant at the canine position than did the 4- or 6-implant-supported PFDP. When the central incisor or lateral incisor was loaded, the two 4-implant-supported PFDPs exhibited similar levels of stresses around the implants and showed lower stresses than did the 2- or 3-implant-supported PFDP. CONCLUSIONS: Implant number and distribution influenced stress distribution around the implants in the anterior maxilla. With a decrease in implant number, the stresses around the implants increased.

DNA methyltransferase inhibition accelerates the immunomodulation and migration of human mesenchymal stem cells.

DNA methyltransferase (DNMT) inhibitors regulate target gene expression through epigenetic modifications, and these compounds have primarily been studied for cancer therapy or reprogramming. However, the effect of DNMT inhibitors on the immunomodulatory capacity of human mesenchymal stem cells (hMSCs) has not been investigated. In the present study, we treated hMSCs with 5-azacytidine (5-aza), a DNMT inhibitor, and confirmed that the inhibitory effects on mononuclear cell proliferation and cell migration toward activated T cells were increased. To identify the immunomodulatory factors stimulated through 5-aza treatment, we investigated the changes in promoter methylation patterns using methylation arrays and observed that the promoters of immunomodulatory factors, COX2 and PTGES, and migration-related factors, CXCR2 and CXCR4, were hypomethylated after 5-aza treatment. In addition, we observed that the COX2-PGE2 pathway is one of the main pathways for the enhanced immunosuppressive activity of hMSCs through 5-aza treatment. We also determined that the migration of hMSCs toward ligands for CXCR2/CXCR4 was increased after 5-aza treatment. Moreover, using an experimental colitis model, we showed that 5-aza pre-treatment could enhance the therapeutic effect of MSCs against immune-related diseases.

A photoelastic stress analysis of screw- and cement-retained implant prostheses with marginal gaps.

BACKGROUND: The precise fit of an implant prosthesis is considered to be a prerequisite for the success and maintenance of osseointegration. It is unknown how much static stress can be tolerated at the implant-bone interface with ill-fitting prostheses for the two different types of retention (cement vs screw). PURPOSE: The purpose of this study was to evaluate the stress pattern and magnitude in the supporting tissues around ITI (Straumann AG, Waldenburg, Switzerland) implants with screw- or cement-retained prostheses with marginal gaps by photoelastic analysis. MATERIALS AND METHODS: A photoelastic model of a human mandible, partially edentulous distal to the canine, was made of PL-2 resin. Three ITI implants (4.1 × 10 mm, Straumann AG, Waldenburg, Switzerland) were placed in the posterior edentulous region, and screw- or cement-retained three-unit fixed partial dentures (FPDs) were fabricated. Ill-fitting prostheses were made by placing a 100-µm gap between the abutments and the superstructures on the second premolar or the first molar. A static vertical force of 134 N was applied at three loading points on each prosthesis. Photoelastic stress analysis was carried out to measure the fringe order around the implant-supporting structures. RESULTS: Even in the unloaded condition, low-level stresses were generated around the implants after screw tightening or cementing the three-unit FPDs with marginal gaps. Loading on the terminal implants developed high concentrated stresses around the loaded implant, regardless of the types of restorations or the presence of gaps. However, when the middle implant was loaded, moderate stresses were distributed to the anterior and posterior implants. CONCLUSIONS: Screw-retained FPDs with gaps exhibited a wider range of stresses on the interproximal region of adjacent implants than cement-retained FPDs. However, severe misfit in the prosthesis caused the nonaxial stress transfer to the adjacent implants in the cement-retained FPDs with gaps.

Comparison of fracture resistance and fit accuracy of customized zirconia abutments with prefabricated zirconia abutments in internal hexagonal implants.

BACKGROUND: Customized zirconia abutments are increasingly applied for the fabrication of esthetic implant restorations aimed at imitating the natural situation. These abutments are individually shaped according to the anatomical needs of the respective implant site. PURPOSE: This study sought to compare the fracture resistance and fit accuracy of prefabricated and customized zirconia abutments using an internal hexagonal implant system (TSV®, Zimmer, Carlsbad, CA, USA). MATERIALS AND METHODS: Two zirconia abutment groups were tested: prefabricated zirconia abutments (ZirAce, Acucera, Seoul, Korea) and customized zirconia abutments milled by the Zirkonzahn milling system. Twenty zirconia abutments per group were connected to implants on an acrylic resin base with 30-Ncm torque. The fracture resistance of zirconia abutments was measured with an angle of 30° at a crosshead speed of 1 mm/min using the universal testing machine (Z020, Zwick, Ulm, Germany). Marginal and internal gaps between implants and zirconia abutments were measured after sectioning the embedded specimens using a digital microhardness tester (MXT70, Matsuzawa, Tokyo, Japan). RESULTS: The customized abutments were significantly stronger (1,430.2 N) than the prefabricated abutments (1,064.1 N). The mean marginal adaptation of customized abutments revealed a microgap that was increased (11.5 µm) over that in prefabricated abutments (4.3 µm). CONCLUSION: Within the limitations of this study, the customized abutments are significantly stronger than prefabricated abutments, but the fit is less accurate. The strength and fit of both abutments are within clinically acceptable limit.

Microleakage of different sealing materials in access holes of internal connection implant systems.

STATEMENT OF PROBLEM: Current implant systems cannot completely prevent microleakage from the access holes of screw-retained implant prostheses, which may constitute risks to the clinical success of the implants. PURPOSE: The purpose of this study was to evaluate the levels of microleakage through the access holes of screw-retained implant prostheses sealed with different materials. MATERIAL AND METHODS: An implant with an internal hexagonal configuration was connected to a temporary abutment with an acrylic resin crown. The apical 6.5 mm of the access hole was filled with 1 of the following materials: cotton pellet, silicone sealing material, vinyl polysiloxane, or gutta-percha. The remaining coronal 3 mm was sealed with composite resin. Cyclic loading with 21 N at 1 Hz was applied 16,000 times to the specimens in 0.5% basic fuchsin solution according to the long axis of the tooth. Basic fuchsin dye which penetrated into the internal wall of the abutment through the access hole was dissolved with methyl alcohol. Then the absorbance was measured by a spectrophotometer at 540 nm to evaluate the degree of microleakage. The results were statistically analyzed with 1-way ANOVA and the Tukey HSD test. RESULTS: From greatest to least, the levels of microleakage were in the following order: cotton pellet, silicone sealing material, vinyl polysiloxane, and gutta-percha. The microleakage associated with gutta-percha was not significantly different from that of vinyl polysiloxane. CONCLUSIONS: When sealing the access holes of screw-retained implant prostheses, gutta-percha or vinyl polysiloxane would help reduce microleakage.

Biomechanical comparison of axial and tilted implants for mandibular full-arch fixed prostheses.

PURPOSE: Controversy exists regarding the "All-on-Four" concept for a mandibular full-arch fixed prosthesis. The purpose of this study was to examine photoelastically the effect of the inclination of the two distal implants according to the All-on-Four concept on the stress distribution within the supporting structure. MATERIALS AND METHODS: Two photoelastic models of a human edentulous mandible were fabricated. Each model had four screw-type implants embedded in the interforaminal area. The two distal implants were placed axially in one model and tilted 30 degrees distally in the other model. Two cantilevered acrylic resin prostheses, which used angulated abutments for the distal tilted implants and straight abutments for the axial implants, were fabricated and delivered. Vertical loads of 13 kg were applied at three loading points on the prosthesis: the central fossa of the first molar, the distal fossa of the first premolar, and the distal fossa of the second premolar. Stresses that developed in the supporting structure were monitored photoelastically and recorded photographically. RESULTS: Whereas all cantilever loadings concentrated the stresses at the distal crest of the distal implant sites in both models, the posterior tilting of distal implants splinted in a full-arch fixed prosthesis did not increase the stresses in bone around the distal implants versus the axial-implant model. CONCLUSION: Within the limitations of this photoelastic stress analysis, the use of tilted implants reduced the maximum stress in the distal crestal bone of the distal implant by approximately 17% relative to the axial implants.

Vertically oriented epitaxial germanium nanowires on silicon substrates using thin germanium buffer layers.

We demonstrate a method to realize vertically oriented Ge nanowires on Si(111) substrates. Ge nanowires were grown by chemical vapor deposition using Au nanoparticles to seed nanowire growth via a vapor-liquid-solid growth mechanism. Rapid oxidation of Si during Au nanoparticle application inhibits the growth of vertically oriented Ge nanowires directly on Si. The present method employs thin Ge buffer layers grown at low temperature less than 600 degrees C to circumvent the oxidation problem. By using a thin Ge buffer layer with root-mean-square roughness of approximately 2 nm, the yield of vertically oriented Ge nanowires is as high as 96.3%. This yield is comparable to that of homoepitaxial Ge nanowires. Furthermore, branched Ge nanowires could be successfully grown on these vertically oriented Ge nanowires by a secondary seeding technique. Since the buffer layers are grown under moderate conditions without any high temperature processing steps, this method has a wide process window highly suitable for Si-based microelectronics.

CdS/CdSe lateral heterostructure nanobelts by a two-step physical vapor transport method.

The two-dimensional heterostructure nanobelts with a central CdSe region and lateral CdS structures are synthesized by a two-step physical vapor transport method. The large growth rate difference between lateral CdS structures on both +/- (0001) sides of the CdSe region is found. The growth anisotropy is discussed in terms of the polar nature of the side +/- (0001) surfaces of CdSe. High-resolution transmission electron microscopy reveals the CdSe central region covered with non-uniform CdS layer/islands. From micro-photoluminescence measurements, a systematic blueshift of emission energy from the central CdSe region in accordance with the increase of lateral CdS growth temperature is observed. This result indicates that the intermixing rate in the CdSe region with CdS increases with the increase of lateral CdS growth temperature. In conventional CdSSe ternary nanostructures, morphology and emission wavelength were correlated parameters. However, the morphology and emission wavelength are independently controllable in the CdS/CdSe lateral heterostructure nanobelts. This structure is attractive for applications in visible optoelectronic devices.

The growth and optical properties of CdSSe nanosheets.

We have investigated the growth of ternary CdSSe nanostructures by physical vapor transport, specifically aiming at the synthesis of CdSSe nanosheets. CdSSe nanostructures with various S mole fractions are grown at growth temperatures less than 800 degrees C and photoluminescence from these nanostructures covers the entire visible spectral range. Morphological evolution from nanowires to nanosheets is observed as increasing growth temperatures due to the change of relative dominance in the growth mechanism from vapor-liquid-solid to vapor-solid. Nanosheets whose widths extend several tens of micrometers are successfully synthesized. However, nanosheets show a strong suppression effect of S incorporation when the growth is dominated by the vapor-solid mechanism. A micro-photoluminescence study shows high optical quality of individual CdSSe nanostructures with polarization anisotropy ratios rho ~ 0.1. In addition, lateral heterostructure CdS/CdSe nanosheets are successfully synthesized by two step growth. Strong red and green emissions are observed by selective laser excitations focusing at the center and edge parts of these heterostructure nanosheets, respectively. These heterostructure CdS/CdSe nanosheets may be useful for multicolor light emitting devices.

Dioleoyl phosphatidic acid induces morphological changes through an endogenous LPA receptor in C6 glioma cells.

Previously, we suggested that dioleoyl phosphatidic acid (PA) and lysophosphatidic acid (LPA) increased [Ca(2+)](i) through endogenous LPA receptors coupled to pertussis toxin-sensitive G proteins in rat C6 glioma cells. In the present report, we investigated morphological changes and cytotoxicity induced by PA and LPA in C6 glioma cells. Isoproterenol treatment led to changes in the cell morphology of rat C6 glioma cells, which were reverted by the addition of PA and LPA. PA-and LPA-induced morphological reversions were inhibited by treatment with Ki16425, an LPA(1)/LPA(3) receptor antagonist. VPC32183, another LPA(1)/LPA(3) receptor antagonist with a different structure, only inhibited PA-induced morphological reversion but not LPA-induced reversion. However, the reversions were not inhibited by treatment with pertussis toxin, a specific inhibitor of G(i/o) proteins. In addition, cytotoxicity was only induced by LPA but not by PA in C6 glioma cells. Our results suggest that PA may act as a partial agonist at endogenous LPA receptors, which are sensitive to Ki16425 and coupled to PTX-insensitive G proteins, to evoke morphological changes in C6 glioma cells.

Lysophosphatidylserine induces calcium signaling through Ki16425/VPC32183-sensitive GPCR in bone marrow-derived mast cells and in C6 glioma and colon cancer cells.

Lysophosphatidylserine (LPS) can be generated following phosphatidylserine-specific phospholipase A2 activation. The effects of LPS on cellular activities and the identities of its target molecules, however, have not been fully elucidated. In this study, we observed that LPS stimulated intracellular calcium increased in mouse bone marrow-derived mast cells (BMMC), and rat C6 glioma and human HCT116 colon cancer cells and compared the LPS-induced Ca2+ increases with the response by lysophosphatidic acid (LPA), a structurally related bioactive lysolipid. In order to test involvement of signaling molecules in the LPS-induced Ca2+ signaling, we used pertussis toxin (PTX), U73122, and 2-APB, which are specific inhibitors for G proteins, phospholipase C (PLC), and IP3 receptors, respectively. The increases due to LPS and LPA were inhibited by PTX, U-73122 and 2-APB, suggesting that both lipids stimulate calcium signaling via G proteins (Gi/o types), PLC activation, and subsequent IP3 production, although the sensitivity to pharmacological inhibitors varied from complete inhibition to partial inhibition depending on cell type and lysolipid. Furthermore, we observed that Ki16425 completely inhibited an LPS-induced Ca2+ response in three cell types, but that the effect of VPC32183 varied from complete inhibition in BMMC and C6 glioma cells to partial inhibition in HCT116 cells. Therefore, we conclude that LPS increases [Ca2+]i through Ki16425/VPC32183-sensitive G protein-coupled receptors (GPCR), G protein, PLC, and IP3 in mouse BMMC, rat C6, and human HCT116 cells.

Effect of direct albumin binding to sphingosylphosphorylcholine in Jurkat T cells.

We investigated the effects of serum on lysophospholipid-induced cytotoxicity in Jurkat T cells. We found that sphingosylphosphorylcholine (SPC, also known as lysosphingomyelin) induced cytotoxicity and that albumin in serum could protect cells by binding directly to SPC. Furthermore, we also found that SPC induced ROS generation, increased [Ca(2+)](i), and decreased MMP. However, those effects were only observed at concentrations higher than 10 microM and were only induced in albumin-free media. Therefore, SPC may be trapped by albumin in plasma and unable to exert its effects under normal conditions, although at high concentrations, SPC could induce several responses such as ROS generation, increased [Ca(2+)](i), and decreased MMP in Jurkat T cells.

Sphingosine 1-phosphate (S1P) induces shape change in rat C6 glioma cells through the S1P2 receptor: development of an agonist for S1P receptors.

Treatment with isoprenaline led to a change in the cell morphology of rat C6 glioma cells. This morphological change was reverted by the addition of sphingosine 1-phosphate (S1P). Using this morphological change as a response marker we determined that DS-SG-44 ((2S,3R)-2-amino-3-hydroxy-4-(4-octylphenyl)butyl phosphoric acid) was an agonist of S1P receptors. The DS-SG-44-induced morphological reversion was not observed with such structurally related molecules as DS-SG-45 ((2S,3R)-2-amino-3-hydroxy-4-(3-octylphenyl)butyl phosphoric acid) and DS-SG-12 ((2S,3R)-2-amino-4-(4-octylphenyl)butane-1,3-diol). The S1P- and DS-SG-44-induced shape changes were neither reproduced with the S1P1/S1P3 receptor agonist VPC24191 nor inhibited by the S1P1/S1P3 receptor antagonist, VPC23019. Transfection with small interfering RNA (siRNA) for the S1P2 receptor greatly inhibited the DS-SG-44-induced shape change, and in part an S1P-induced response. In the presence of VPC23019, siRNA transfection for the S1P2 receptor almost completely blocked the S1P- and DS-SG-44-induced shape changes. Our results suggested that DS-SG-44, a newly-synthesized S1P analogue, acted as an S1P receptor agonist and that the S1P-induced shape change in rat C6 glioma cells was mediated mainly through the S1P2 receptor, and cooperatively through the S1P1/S1P3 receptors.

Dioleoyl phosphatidic acid increases intracellular Ca2+ through endogenous LPA receptors in C6 glioma and L2071 fibroblasts.

Phosphatidic acid (PA) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in C6 rat glioma and L2071 mouse fibroblast cells. Dioleoyl PA (PA, 18:1) was the most efficacious, followed by dipalmitoyl PA (16:0 PA) and dimyristoyl PA (14:0 PA). Lysophosphatidic acid (LPA) also increased the [Ca(2+)](i) in the both cells. PA desensitized LPA-induced Ca(2+) response completely in C6 cells, but partly in L2071 cells. Treatment of pertussis toxin (PTX), a specific inhibitor of G(i/o)-type G proteins, completely ameliorated LPA- and PA-induced Ca(2+) response in C6 cells. However, in L2071 cells, PTX inhibited PA-induced Ca(2+) increase by 80% and LPA-induced one by 20%. Ki16425, a specific inhibitor of LPA(1)/LPA(3) receptors, completely inhibited both LPA- and PA-induced Ca(2+) responses in C6 cells. On the other hand, in L2071 cells, Ki16425 completely inhibited PA-induced Ca(2+) response, but partly LPA-induced one. VPC32183, another specific inhibitor of LPA(1)/LPA(3) receptors, completely inhibited LPA- and PA-induced Ca(2+) responses in both C6 and L2071 cells. Therefore, PA and LPA appear to increase [Ca(2+)](i) through Ki16425/VPC32183-sensitive LPA receptor coupled to PTX-sensitive G proteins in C6 cells. In L2071 cells, however, LPA increases [Ca(2+)](i) through Ki16425-insensitive LPA receptor coupled to PTX-insensitive G proteins and Ki16425-sensitive LPA receptor coupled to PTX-sensitive G protein, whereas PA utilized only the latter pathway. Our results suggest that PA acts as a partial agonist on endogenous LPA receptors, which are sensitive to Ki16425 and coupled to PTX-sensitive G protein, but not on LPA receptors, which are not sensitive to Ki16425 and coupled to PTX-insensitive G protein.

Multiple actions of dimethylsphingosine in 1321N1 astrocytes.

N,N-dimethyl-D-erythro-sphingosine (DMS) is an N-methyl derivative of sphingosine and an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In the present study, we examined the effects of DMS on intracellular Ca2+ concentration, pH, and glutamate uptake in human 1321N1 astrocytes. DMS increased intracellular Ca2+ concentration and cytosolic pH in a concentration-dependent manner. Pretreatment of the cells with the Gi/o protein inhibitor PTX and the PLC inhibitor U73122 had no obvious effect. However, removal of extracellular Ca2+ with the Ca2+ chelator EGTA or depletion of intracellular Ca2+ stores with thapsigargin impeded the DMS-induced increase of intracellular Ca2+ concentration. Pretreatment of cells with NH4Cl or monensin reduced the DMS-induced Ca2+ increase. However, inhibition of the DMS-induced Ca2+ increase with BAPTA did not influence the DMS-induced pH increase. DMS also inhibited glutamate uptake by the 1321N1 astrocytes in a concentration-dependent manner. It also increased intracellular Ca2+ and pH in PC12 neuronal cells. Our observations on the effects of DMS on 1321N1 astrocytes and PC12 neuronal cells point to a physiological role of DMS in the brain.

Albumin inhibits cytotoxic activity of lysophosphatidylcholine by direct binding.

Fetal bovine serum (FBS) was found to protect Jurkat T cells from LPC-induced cytotoxicity. Twenty micromolar LPC-induced cytotoxicity of 80-90% of the cells in media without FBS for 3 h, whereas 50-70% in media with 0.5% FBS. However, LPC-induced cytotoxicity was not observed in the presence of 5% FBS in media. The cytotoxicity was specific for LPC among lysophospholipids tested and significantly observed with palmitoyl (C16:0) LPC, stearoyl (C18:0) LPC, and oleoyl (C18:1) LPC among 11 synthetic LPCs. Furthermore, the cytoprotective effect of FBS was observed only when it was added before the treatment, but not after the treatment of LPC, and premixing of FBS and LPC before addition to the cells ameliorated LPC-induced cytotoxicity. Finally, albumin, a major constituent of FBS, prevented completely LPC-induced cytotoxicity even at as low as 3 microM concentration. We also found that five molecules of LPC could sequentially bind to one BSA using isothermal titration calorimetry. The above results suggest that the cytotoxic activity of LPC could be attenuated by albumin in blood. Finally, it should be cautioned that, when experiments are conducted with LPC dissolved in assay buffers containing albumin, the albumin in the buffer could influence the results.