SLUG is upregulated and induces epithelial mesenchymal transition in canine oral squamous cell carcinoma.

SLUG, encoded by the Snai2 gene, is known to play a role in epithelial-mesenchymal transition (EMT), which contributes to cell invasion and metastasis in some types of human carcinomas. However, the mechanisms and roles of EMT in canine squamous cell carcinoma (SCC) have not yet been elucidated. We have previously established canine oral SCC cell lines, including tonsillar SCC, and in this study, we evaluated the effects of SLUG on the phenotypes regarding EMT of canine SCC cells. First, immunohistochemical analysis revealed that SLUG is upregulated in canine oral SCC tissues compared to that in non-tumoural oral mucosa. Furthermore, gain-of-function and loss-of-function of SLUG revealed that SLUG partly contributed to migration and invasion of cells, as well as the upregulation of EMT markers such as vimentin and SNAIL. Thus, the current study suggests that SLUG promotes cell migration and invasion through EMT induction in canine oral SCC, as well as human cancers.

Initial evaluation of a novel electrocardiography sensor-embedded fabric wear during a full marathon.

Sudden cardiac accident (SCA) during a marathon is a concern due to the popularity of the sport. Preventive strategies, such as cardiac screening and deployment of automated external defibrillators have controversial cost-effectiveness. We investigated the feasibility of use of a new electrocardiography (ECG) sensor-embedded fabric wear (SFW) during a marathon as a novel preventive strategy against SCA. Twenty healthy volunteers participated in a full marathon race. They were equipped with a SFW hitoe® with a transmitter connected via Bluetooth to a standard smartphone for continuous ECG recording. All data were stored in a smartphone and used to analyze the data acquisition rate. The adequate data acquisition rate was > 90% in 13, 30-90% in 3, and < 10% in 4 runners. All of 4 runners with poorly recorded data were female. Inadequate data acquisition was significantly associated with the early phase of the race compared with the mid phase (P = 0.007). Except for 3 runners with poor heart rate data, automated software calculation was significantly associated with manual analysis for both the mean (P < 0.001) and maximum (P = 0.014) heart rate. We tested the feasibility of continuously recording cardiac data during a marathon using a new ECG sensor-embedded wearable device. Although data from 65% of runners were adequately recorded, female runners and the early phase of the race tended to have poor data acquisition. Further improvements in device ergonomics and software are necessary to improve ability to detect abnormal ECGs that may precede SCA.

Establishing cell lines for canine tonsillar and non-tonsillar oral squamous cell carcinoma and identifying characteristics associated with malignancy.

Canine tonsillar squamous cell carcinoma (TSCC) shows a higher metastasis rate than non-tonsillar oral SCC (NTSCC). The mechanisms of metastasis for TSCC have been less studied, because both TSCC and NTSCC cell lines are few. In this study, 6 cloned TSCC (TSCCLN#1-#6), which were from a metastatic lymph node, and 2 cloned NTSCC (oSCC-1 and -4) cell lines, which were from the primary lesion, were established, and their characteristics were evaluated in vitro and in vivo. Results showed that increased expression level of Vimentin in TSCC cell lines and increased expression levels of mesenchymal markers including Vimentin, Snail, and Slug in NTSCC cell lines corelated with the malignant phenotypes such as the cell growth and colony formation abilities in vitro. However, expression levels of mesenchymal markers and in vitro characteristics were unrelated to tumorigenic ability in nude mice. Additionally, the expression levels of E-cadherin and Vimentin were also evaluated by immunohistochemistry using the formalin-fixed paraffin embedded canine oral SCC tissues, and the results show that the expression level of Vimentin in TSCC was higher than in NTSCC. In conclusion, the cell lines established in this study might contribute to elucidating the mechanisms involved in TSCC metastasis.

Nucleolin is a receptor for maleylated-bovine serum albumin on macrophages.

Scavenger receptors have a broad range of functions that include pathogen clearance, and identification of the scavenger receptor family has been of great benefit to the field of physiology. The shuttling-protein nucleolin has recently been shown to possess scavenger receptor-like activity. We therefore investigated whether or not nucleolin is a receptor for maleylated-bovine serum albumin (maleylated-BSA), which is a common ligand for scavenger receptors. Binding and phagocytosis of native control-BSA by thioglycollate-elicited mouse peritoneal macrophages was weak, but that of maleylated-BSA was strong. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with maleylated-BSA but not control-BSA or maleic anhydride. Further, co-treatment of macrophages with anti-nucleolin antibody, but not control-immunoglobulin G, inhibited binding of maleylated-BSA. In addition, antineoplastic guanine rich oligonucleotide (AGRO), a nucleolin-specific oligonucleotide aptamer, inhibited binding of maleylated-BSA. Further, binding of maleylated-BSA to nucleolin-transfected HEK293 cells was higher than that by control HEK cells. These results indicate that nucleolin is a receptor that enables macrophages to recognize maleylated-BSA.

Macrophage recognition of toxic advanced glycosylation end products through the macrophage surface-receptor nucleolin.

Advanced glycosylation end-products (AGEs) are non-enzymatically glycosylated proteins that play an important role in several diseases and aging processes, including angiopathy, renal failure, diabetic complications, and some neurodegenerative diseases. In particular, glyceraldehyde (GCA)- and glycolaldehyde (GOA)-derived AGEs are deemed toxic AGEs, due to their cytotoxicity. Recently, the shuttling-protein nucleolin has been shown to possess scavenger receptor-activity. Here, we investigated whether or not macrophages recognize toxic AGEs through nucleolin receptors expressed on their surface. Free amino acid groups and arginine residues found in bovine serum albumin (BSA) were time-dependently modified by incubation with GCA and GOA. In addition, average molecular size was increased by incubation with GCA and GOA. While GCA-treated BSA (GCA-BSA) and GOA-treated BSA (GOA-BSA) were recognized by thioglycollate-elicited mouse peritoneal macrophages in proportion to their respective aldehyde-modification ratios, aldehyde-untreated control-BSA was not. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with GCA-BSA and GOA-BSA, but not with control-BSA. Further, pretreating macrophages with anti-nucleolin antibody, but not control-Immunoglobulin G, inhibited recognition of GCA-BSA and GOA-BSA by macrophages. Additionally, AGRO, a nucleolin-specific oligonucleotide aptamer, inhibited recognition of GCA-BSA and GOA-BSA. Moreover, nucleolin-transfected HEK293 cells recognized more GCA-BSA and GOA-BSA than control HEK cells did. Binding of nucleolin and GCA-BSA/GOA-BSA was also blocked by anti-nucleolin antibody at molecular level. These results indicate that nucleolin is a receptor that allows macrophages to recognize toxic AGEs.

Shuttling protein nucleolin is a microglia receptor for amyloid beta peptide 1-42.

Amyloid-beta peptide 1-42 (Aß42) plays a key role in the neurotoxicity found in Alzheimer's disease. Mononuclear phagocytes in the brain (microglia), can potentially clear Aß via phagocytosis. Recently, the shuttling-protein nucleolin has been shown to possess scavenger receptor-activity. Here, we investigated whether this receptor interacts specifically with Aß type 1-42 and mediates its phagocytosis by microglia. While monomeric and fibril Aß42 were phagocytosed by mouse microglial EOC2 cells, amyloid ß peptide 1-40 (Aß40) was only weakly phagocytosed. Surface plasmon-resonance analysis revealed that nucleolin strongly associates with Aß42, but only weakly associates with Aß40. Immunofluorescence staining of anti-nucleolin antibody revealed that EOC2 cells and rat primary microglia express nucleolin on their cell surfaces. Further, pretreating EOC2 cells with anti-nucleolin antibody, but not immunoglobulin G (IgG), inhibited phagocytosis of monomeric Aß42 by microglia. Additionally, nucleolin-transfected HEK293 cells phagocytosed monomeric and fibril Aß42 but not monomeric and fibril Aß40. Moreover, AGRO, a nucleolin-specific oligonucleotide aptamer, inhibited phagocytosis of monomeric and fibril Aß42, but not monomeric and fibril Aß40. These results indicate that nucleolin is a receptor that allows microglia to recognize monomeric and fibril Aß42.

Photodynamic therapy with talaporfin sodium induces dose-dependent apoptotic cell death in human glioma cell lines.

OBJECTIVE: To investigate the kinetics of cell death in human glioma cell lines induced by photodynamic therapy (PDT) with the second-generation photosensitizer talaporfin sodium (TS) and a 664-nm diode laser. MATERIALS AND METHODS: Three human glioma cell lines (T98G, A172, U251) were studied. After incubation of the cell lines with various concentrations of TS for 4 h, PDT using diode laser irradiation at 33 mW/cm² and 10 J/cm² was performed. Cell viability and changes in cell morphology were examined by the Cell Counting Kit-8 assay and phase-contrast microscopy, respectively. In addition, to evaluate the pathology of cell death, changes in cell viability after treatment with a caspase activation inhibitor and an autophagy inhibitor were also examined. RESULTS: In all 3 human glioma cell lines, TS induced dose-dependent cell death. However, the 50% lethal dose of TS varied among these cell lines. The main morphological feature of cell death was shrinkage of the cell body, and the number of cells with this morphological change increased in a time-dependent manner, resulting in cell death. In addition, a dose-dependent improvement in cell viability by the caspase inhibitor Z-VAD-fmk was observed. CONCLUSION: PDT with TS induces dose-dependent apoptosis in human glioma cell lines. However, the sensitivity to PDT varied among the cell lines, indicating a possible difference in the intracellular content of TS, or a difference in the susceptibility to the intracellular oxidative stress caused by PDT.

Macrophage recognition of cells with elevated calcium is mediated by carbohydrate chains of CD43.

Macrophages remove deteriorating cells (those undergoing apoptosis and oxidation) via poly-N-acetyllactosaminyl chains on CD43 caps, a major cell-surface glycoprotein. Unusually high intracellular calcium levels are also deteriorating for cells and tissue. Here we artificially elevated calcium levels in cells and examined the mechanism by which this elevation was resolved by macrophages. Results showed that treatment with the calcium ionophore A23187 and ionomycin induces capping of CD43 on Jurkat cells, which are subsequently recognized and phagocytosed by macrophages, indicating that macrophages regard cells with elevated calcium as targets for removal. Further tests showed that A23187- and ionomycin-treated Jurkat cells did not induce apoptotic changes such as DNA fragmentation or phosphatidylserine expression, indicating that these cells were removed despite still being viable. Jurkat cells pretreated with anti-CD43 antibody or those with poly-N-acetyllactosaminyl chains containing oligosaccharides inhibited macrophage binding, indicating that macrophages recognize the poly-N-acetyllactosaminyl chains on CD43. Binding was also inhibited by treating macrophages with anti-nucleolin antibody, indicating that recognition occurs through nucleolin, a cell-surface receptor. Further, nucleolin-transfected HEK293 cells bound A23187-treated cells, and this binding was inhibited by in the presence of oligosaccharides. Taken together, these results show that elevated calcium levels induce CD43 capping, and macrophages remove the cells if their nucleolin receptors can bind to the poly-N-acetyllactosaminyl chains of capped CD43.

Photodynamic therapy in combination with talaporfin sodium induces mitochondrial apoptotic cell death accompanied with necrosis in glioma cells.

Photodynamic therapy (PDT) induces selective cell death of neoplastic tissue and connecting vasculature by combining photosensitizers with light. Here we clarified the types of cell death induced by PDT in combination with the photosensitizer talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6) in order to evaluate the potential of this therapy as a treatment for glioma. PDT with NPe6 (NPe6-PDT) induces dose-dependent cell death in human glioblastoma T98G cells. Specifically, cell death modalities were observed in NPe6-PDT treated T98G cells, including signs of apoptosis (activation of caspase-3, expression of phosphatidylserine, and DNA fragmentation) and necrosis (stainability of propidium iodide). In addition, high doses of NPe6-PDT decreased the proportion of apoptotic cell death, while increasing necrosis. Closer examination of apoptotic characteristics revealed release of cytochrome-c from mitochondria as well as activation of both caspse-9 and caspase-3 in cells treated with low doses of NPe6-PDT. Benziloxycarbonyl-Leu-Gln(OMe)-His-Asp(OMe)-fluoromethyl-ketone (Z-LEHD-fmk), a caspase-9 specific inhibitor, and benziloxycarbonyl-Asp(OMe)-Gln-Met-Asp(OMe)-fluoromethyl-ketone (Z-DQMD-fmk), a caspase-3 specific inhibitor, showed dose-dependent prevention of cell death in NPe6-PDT treated cells, indicating that mitochondrial apoptotic pathway was a factor in the observed cell death. Further, the cell morphology was observed after PDT. Time- and NPe6-dose dependent necrotic features were increased in NPe6-PDT treated cells. These results suggest that NPe6-PDT could be an effective treatment for glioma if used in mild doses to avoid the increased necrosis that may induce undesirable obstacles.

Macrophage recognition of thiol-group oxidized cells: recognition of carbohydrate chains by macrophage surface nucleolin as apoptotic cells.

The mechanism was investigated for macrophage recognition of cells oxidized by diamide, a thiol group-specific oxidizing reagent. Jurkat cells exposed to various concentrations of diamide were recognized by macrophages, the cells exposed to 25 µM diamide being best recognized. CD43, a major glycoprotein on the Jurkat cell surface, tended to form clusters upon diamide oxidization, and pretreating Jurkat cells with the anti-CD43 antibody inhibited macrophage binding. This indicates that macrophages appeared to recognize CD43. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and a Western blot analysis of CD43 of the diamide-oxidized cells showed no increase in the amount of cross-linked CD43 compared with control cells, indicating that cross-linking of CD43 by a disulphide bond was not involved in the clustering. Both CD43 clustering and binding of the oxidized cells to macrophages was prevented by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), suggesting that the oxidized and macrophage-bound cells were undergoing apoptosis. A closer examination revealed that the caspase-3 activity, chromatin condensation, and DNA fragmentation in Jurkat cells were all increased by oxidation. The macrophage receptor involved in the binding appeared to be the cell-surface protein, nucleolin; an anti-nucleolin antibody treatment inhibited the binding. These results suggest that thiol group-oxidized cells underwent early apoptosis and were recognized by nucleolin on macrophages as early apoptotic cells.

Clearance of CD43-capped cells by macrophages: capping alone leads to phagocytosis.

Apoptotic cells must be recognized early for phagocytosis to ensure that their toxic contents do not damage neighboring cells. In some cases this is achieved via CD43-capped membrane glycoproteins, the sialylpolylactosaminyl chains of which serve as ligands for phagocytosis by macrophages. However, because many additional changes occur during apoptosis, determining exactly which events are responsible for signaling macrophages to initiate phagocytosis remains a challenge. Here, we examined one clearance mechanism in detail and determined that capping of CD43 alone is sufficient to initiate phagocytosis. We induced macrophage-mediated phagocytosis by using cytochalasin B to artificially cap CD43 on healthy (non-apoptotic) Jurkat cells. Additional experiments confirmed that sialylpolylactosaminyl chains formed through this capping method are a prerequisite for removal, and that nucleolin is the macrophage receptor responsible for their detection. These findings strongly suggest that capping of CD43 presents a sufficient signal for phagocytosis without any additional membrane changes.

Nucleolin on the cell surface as a new molecular target for gastric cancer treatment.

Nucleolin is an abundant non-ribosomal protein found in nucleolus and a major component of silver-stained nucleolar organizer region (AgNOR), a histopathological marker of cancer which is highly elevated in cancer cells. We recently reported that nucleolin on the cell surface of mouse gastric cancer cells acts as a receptor for tumor necrosis factor-alpha-inducing protein (Tipalpha), a new carcinogenic factor of Helicobacter pylori. In this study, we first examined the localization of nucleolin on cell surface of five gastric cancer cell lines by cell fractionation and flow cytometry: We found that large amounts of nucleolin were present on surface of MKN-45, KATOIII, MKN-74, and AGS cells, with smaller amounts on surface of MKN-1 cells. The membrane fraction of normal epithelial cells of mouse glandular stomach did not contain much nucleolin, suggesting that translocation of nucleolin to the cell surface occurs during carcinogenesis, making for easier binding with Tipalpha. AS1411, a nucleolin targeted DNA aptamer, inhibited growth of gastric cancer cell lines in this order of potency: MKN-45>KATOIII>AGS>MKN-74=MKN-1, associated with induction of S-phase cell cycle arrest. Fluorescein isothiocyanate (FITC)-AS1411 was more rapidly incorporated into MKN-45 and AGS than into MKN-1 cells, based on varying amounts of cell surface nucleolin. We think that AS1411 first binds to nucleolin on the cell surface and that the binding complex is then incorporated into the cells. All results indicate that nucleolin on the cell surface is a new and promising therapeutic target for treatment of gastric cancer.

Nucleolin as cell surface receptor for tumor necrosis factor-alpha inducing protein: a carcinogenic factor of Helicobacter pylori.

PURPOSE: Tumor necrosis factor-alpha inducing protein (Tipalpha) is a unique carcinogenic factor released from Helicobacter pylori (H. pylori). Tipalpha specifically binds to cells and is incorporated into cytosol and nucleus, where it strongly induces expression of TNF-alpha and chemokine genes mediated through NF-kappaB activation, resulting in tumor development. To elucidate mechanism of action of Tipalpha, we studied a binding protein of Tipalpha in gastric epithelial cells. METHODS: Tipalpha binding protein was found in cell lysates of mouse gastric cancer cell line MGT-40 by FLAG-pull down assay and identified to be cell surface nucleolin by flow cytometry using anti-nucleolin antibody. Incorporation of Tipalpha into the cells was determined by Western blotting and expression of TNF-alpha gene was quantified by RT-PCR. RESULTS: Nucleolin was co-precipitated with Tipalpha-FLAG, but not with del-Tipalpha-FLAG (an inactive mutant). After treatment with Tipalpha-FLAG, incorporated Tipalpha was co-immunoprecipitated with endogenous nucleolin using anti-nucleolin antibody. The direct binding of Tipalpha to recombinant His-tagged nucleolin fragment (284-710) was also confirmed. Although nucleolin is an abundant non-ribosomal protein of the nucleolus, we found that nucleolin is present on the cell surface of MGT-40 cells. Pretreatment with anti-nucleolin antibody enhanced Tipalpha-incorporation into the cells through nucleolin internalization. In addition, pretreatment with tunicamycin, an inhibitor of N-glycosylation, decreased the amounts of cell surface nucleolin and inhibited both internalization of Tipalpha and expression of TNF-alpha gene. CONCLUSIONS: All the results indicate that nucleolin acts as a receptor for Tipalpha and shuttles Tipalpha from cell surface to cytosol and nuclei. These findings provide a new mechanistic insight into gastric cancer development with Tipalpha.

Clearance of oxidatively damaged cells by macrophages: recognition of glycoprotein clusters by macrophage-surface nucleolin as early apoptotic cells.

The mechanism of macrophage recognition of oxidatively damaged cells was investigated. Jurkat T cells exposed to various concentrations of H(2)O(2) were bound and phagocytosed by macrophages. The cells exposed to 0.1 mM H(2)O(2) were best bound. The cell-surface ligands recognized by macrophages were suggested to be sialylpolylactosaminyl sugar chains of a major sialoglycoprotein CD43 because 1) the cell binding was inhibited by oligosaccharides containing sialylpolylactosaminyl chains, and their inhibitory activity was destroyed by a polylactosamine-cleaving enzyme endo-beta-galactosidase, and by neuraminidase; 2) the oxidized Jurkat cells pretreated with either glycosidase or with anti-CD43 antibody were not bound. The macrophage receptor involved in the binding was suggested to be cell-surface nucleolin because 1) anti-nucleolin antibody inhibited the binding; 2) nucleolin-transfected HEK293 cells bound the oxidized cells; and 3) this binding was inhibited by anti-nucleolin antibody and by anti-CD43 antibody. CD43 on oxidized Jurkat cells tended to form clusters in good accordance with their susceptibility to the macrophage binding. CD43 clustering and the oxidized-cell binding to macrophages were prevented by a caspase inhibitor Z-VAD-fmk, suggesting that the oxidized and bound cells were undergoing apoptosis. Indeed, caspase-3 activity of Jurkat cells increased by the oxidation. These results suggest that moderately oxidized cells undergo apoptosis and are recognized by macrophages as early apoptotic cells.

Clearance of oxidized erythrocytes by macrophages: involvement of caspases in the generation of clearance signal at band 3 glycoprotein.

Human erythrocytes exposed to appropriate concentrations of H(2)O(2) for 1h became susceptible to the binding and phagocytosis by macrophages. The binding was inhibited by anti-band 3 serum and prevented by pretreatment of erythrocytes with a polylactosamine-cleaving enzyme endo-beta-galactosidase, indicating that polylactosaminyl sugar chains of band 3 are recognized by macrophages. The macrophage receptor involved was suggested to be nucleolin, a recently identified macrophage surface protein recognizing sialylpolylactosaminyl-chain clusters on early apoptotic cells, because anti-nucleolin antibody and a soluble form of recombinant nucleolin blocked the recognition. Treatment of erythrocytes with caspase inhibitors Z-VAD-fmk or Z-DQMD-fmk (caspase 3 selective) before the oxidation resulted in lowered binding of the oxidized erythrocytes to macrophages, suggesting that actions of caspases, particularly those of caspase 3, are prerequisite for the membrane changes leading to band 3 aggregation. Moreover, the cytosolic caspase 3 was found to be activated by H(2)O(2), and the extent of the activation correlated well with the susceptibility of the oxidized erythrocytes to the macrophage recognition. These results suggest that oxidative stress renders the erythrocytes susceptible to clearance by macrophages through activation of caspases leading to band 3 aggregation.

Plasma KL-6 predicts the development and outcome of bronchopulmonary dysplasia.

Circulating KL-6 is a specific indicator of pulmonary injury affecting the alveolar epithelium and interstitium. Our preliminary study suggested the usefulness of plasma KL-6 as a marker of bronchopulmonary dysplasia (BPD). To confirm the diagnostic value of KL-6 for BPD as well as to determine the reference range, we conducted a larger prospective study in 135 preterm infants <32 wk GA. Among the infants without oxygen dependence at a postconceptional age of 36 wk, the plasma KL-6 level showed no significant association with GA at any time. Among 42 infants <28 wk GA, plasma KL-6 levels were significantly higher in those with moderate/severe BPD compared with those with no/mild BPD. A plasma level of 199 U/mL at 1 wk or 232 U/mL at 2 wk was an excellent predictor of moderate/severe BPD <28 wk GA (positive predictive value of 83% and 80%, respectively). Unlike nonspecific markers of inflammation or fibrosis, KL-6 objectively reflects the severity of pulmonary injury irrespective of the treatment or the radiographic changes. Therefore, not only as a good marker, measurement of KL-6 may also help to provide new insights into the pathogenesis of BPD.

A multifunctional shuttling protein nucleolin is a macrophage receptor for apoptotic cells.

Early apoptotic Jurkat T cells undergo capping of CD43, and its polylactosaminyl saccharide chains serve as ligands for phagocytosis by macrophages. This suggests the presence of a polylactosaminoglycan-binding receptor on macrophages. Here we show that this receptor is nucleolin, a multifunctional shuttling protein present in nucleus, cytoplasm, and on the surface of some types of cells. Nucleolin was detected at the surface of macrophages, and anti-nucleolin antibody inhibited the binding of the early apoptotic cells to macrophages. Nucleolin-transfected HEK293 cells expressed nucleolin on the cell surface and bound the early apoptotic cells but not phosphatidylserine-exposing late apoptotic cells. This binding was inhibited by anti-nucleolin antibody, by polylactosamine-containing oligosaccharides, and by anti-CD43 antibody. Deletion of the antibody binding region of nucleolin resulted in loss of the apoptotic cell-binding ability. Moreover, truncated recombinant nucleolin in solution containing this region blocked the apoptotic cell binding to macrophages, and the blocking effect was cancelled by the oligosaccharides. These results indicate that nucleolin is a macrophage receptor for apoptotic cells.

Retardation of removal of radiation-induced apoptotic cells in developing neural tubes in macrophage galactose-type C-type lectin-1-deficient mouse embryos.

MGL1/CD301a is a C-type lectin that recognizes galactose and N-acetylgalactosamine as monosaccharides and is expressed on limited populations of macrophages and dendritic cells at least in adult mice. In this study, pregnant mice with Mgl1+/- genotype were mated with Mgl1+/- or Mgl1-/- genotype males, and the embryos were used to assess a hypothesis that this molecule plays an important role in the clearance of apoptotic cells. After X-ray irradiation at 1 Gy of developing embryos at 10.5 days post coitus (d.p.c.), the number of Mgl1-/- pups was significantly reduced as compared with Mgl1+/+ pups. Distributions of MGL1-positive cells, MGL2-positive cells, and apoptotic cells were histologically examined in irradiated Mgl1+/+ embryos. MGL1-positive cells were detected in the neural tube in which many cells undergo apoptosis, whereas MGL2-positive cells were not observed. Biotinylated recombinant MGL1 bound a significant portion of the apoptotic cells. When Mgl1+/+ and Mgl1-/- embryos were examined for the presence of apoptotic cells, similar numbers of apoptotic cells gave rise, but the clearance of these cells was slower in Mgl1-/- embryos than in Mgl1+/+ embryos. These results strongly suggest that MGL1/CD301a is involved in the clearance of apoptotic cells. This process should be essential in the repair and normal development of X-ray-irradiated embryos.

Regulation of apoptosis by glutathione redox state in PC12 cells exposed simultaneously to iron and ascorbic acid.

We previously reported that the levels of non-protein-bound iron (NPBI) and ascorbic acid (AA) are markedly increased in the cerebrospinal fluid of infants with perinatal asphyxia. The present study showed that FeSO4 and AA synergistically induced apoptosis of PC12 cells, which was prevented by alpha-tocopherol and glutathione (GSH) ethyl ester. Markers of free radical damage, such as ortho-tyrosine, meta-tyrosine, and F(2alpha)-isoprostane, showed a gradual increase. AA and ferrous NPBI disappeared rapidly from the culture medium, but exposure for only a few hours was sufficient to trigger apoptosis. Intracellular GSH decreased progressively along with a concomitant increase of glutathione disulfide (GSSG). The baseline half-cell reduction potential (Ehc) for GSSG, 2H+/2GSH couple was -246 mV and an Ehc of -200 mV was the critical level to switch on apoptosis, although some cells escaped this fate by transient increase of intracellular GSH. Once Ehc reached around -165 mV (81 mV oxidation from the baseline), all cells lost the ability to maintain an adequate intracellular GSH level and subsequently underwent apoptosis. These findings at least partly explain the mechanism of Fe-AA cytotoxicity, in that ferrous iron catalyzes hydroxyl radical generation and induces lipid peroxidation, after which subsequent depletion of GSH raises Ehc to the critical level for triggering or potentiating the apoptotic cascade.

Identification of apo- and holo-forms of ceruloplasmin in patients with Wilson's disease using native polyacrylamide gel electrophoresis.

OBJECTIVES: To determine the serum ratio of holo-ceruloplasmin to total ceruloplasmin in patients with Wilson's disease (WD), we identified apo- and holo-forms of serum ceruloplasmin with native-PAGE electrophoresis. DESIGN AND METHODS: Serum obtained from nine patients with Wilson's disease was subjected to native-PAGE analysis. We also determined the ceruloplasmin level and ferroxidase activity in all of the samples. RESULTS: Among the nine patients, three had both forms of ceruloplasmin and six had only apo-ceruloplasmin. In the patients who had only apo-ceruloplasmin, the serum ceruloplasmin level was significantly lower than in the patients with both forms. CONCLUSIONS: Differences in the ability to incorporate copper into ceruloplasmin may have a role in ceruloplasmin concentrations in patients with Wilson's disease.