Glutathione (GSH), the main cellular antioxidant, dynamically influences tumor growth, metastasis, and resistance to therapy in the tumor microenvironment (TME), which comprises cancer cells, immune cells, stromal cells, and non-cellular components, including the extracellular matrix, metabolites, hypoxia, and acidity. Cancer stem cells (CSCs) and T cells are minor but significant cell subsets of the TME. GSH dynamics influences the fate of CSCs and T cells.Here, we explored GSH dynamics in CSCs and T cells within the TME, as well as therapeutic approaches that could target these dynamics.

Glutathione (GSH) is a chief cellular antioxidant, affecting stem cell functions. The cellular GSH level is dynamically altered by the redox buffering system and transcription factors, including NRF2. Additionally, GSH is differentially regulated in each organelle. We previously reported a protocol for monitoring the real-time GSH levels in live stem cells using the reversible GSH sensor FreSHtracer. However, GSH-based stem cell analysis needs be comprehensive and organelle-specific. Hence, in this study, we demonstrate a detailed protocol to measure the GSH regeneration capacity (GRC) in living stem cells by measuring the intensities of the FreSHtracer and the mitochondrial GSH sensor MitoFreSHtracer using a high-content screening confocal microscope. This protocol typically analyses the GRC in approximately 4 h following the seeding of the cells onto plates. This protocol is simple and quantitative. With some minor modifications, it can be employed flexibly to measure the GRC for the whole-cell area or just the mitochondria in all adherent mammalian stem cells.

Lymphomatoid granulomatosis (LYG) is an Epstein-Barr virus (EBV)-associated lymphoproliferative disease. It is considered a rare entity in pediatric patients. An adolescent male with lobar consolidation suspected of having pneumonia was resistant to antibiotics and had persistently abnormal radiographs with chest pain. The patient was diagnosed with pulmonary LYG through video-assisted thoracoscopic surgery (VATS) lung biopsy. He received eight cycles of rituximab, vincristine, cyclophosphamide, and prednisolone (R-CVP) but had progressive disease. As the patient developed hypogammaglobulinemia after eight courses of rituximab, he received intravenous gamma globulin (IVIG) at regular interval. With immune augmentation effect of IVIG and immune modulation treatment with prednisolone, the patient has shown no aggravation of the lung lesions. Considering its rarity, high mortality, and frequent relapses, diagnostic methods investigating the radiologic abnormalities can help in early treatment initiation.

Glutathione (GSH) is a major antioxidant in cells, and plays vital roles in the cellular defense against oxidants and in the regulation of redox signals. In a previous report, we demonstrated that stem cell function is critically affected by heterogeneity and dynamic changes in cellular GSH concentration. Here, we present a detailed protocol for the monitoring of GSH concentration in living stem cells using FreSHtracer, a real-time GSH probe. We describe the steps involved in monitoring GSH concentration in single living stem cells using confocal microscopy and flow cytometry. These methods are simple, rapid, and quantitative, and able to demonstrate intracellular GSH concentration changes in real time. We also describe the application of FreSHtracer to the sorting of stem cells according to their GSH content using flow cytometry. Typically, microscopic or flow cytometric analyses of FreSHtracer and MitoFreSHtracer signals in living stem cells take ~2~3 h, and the fractionation of stem cells into subpopulations on the basis of cellular GSH levels takes 3~4.5 h. This method could be applied to almost every kind of mammalian cell with minor modifications to the protocol described here.
Flow Cytometry ; Fluorescent Dyes ; Glutathione ; Methods ; Microscopy, Confocal ; Oxidants ; Oxidation-Reduction ; Population Characteristics ; Stem Cells

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme that catalyzes crosslinking, polyamination or deamidation of glutamine residues in proteins. It has been reported that TG2 is involved in the pathogenesis of various inflammatory diseases including celiac disease, pulmonary fibrosis, cystic fibrosis, multiple sclerosis and sepsis. Recently, using a mouse model of bleomycin-induced lung fibrosis, we showed that TG2 is required to trigger inflammation via the induction of T helper type 17 (Th17) cell differentiation in response to tissue damage. However, the role of TG2 in inflammatory bowel disease (IBD), which is thought to be a Th17 cell-associated disease, has remained elusive. In this study, we investigated the role of TG2 in dextran sulfate sodium (DSS)-induced colitis, the most widely used mouse model for IBD. Age- and sex-matched wild-type and TG2(−/−) mice were fed 2% DSS for 7 days or 3.5% DSS for 5 days in drinking water. An in situ TG activity assay revealed that DSS treatment activates TG2 in various colon cell types, including columnar absorptive cells and goblet cells. DSS-treated TG2(−/−) mice showed lower interleukin (IL)-6, but higher IL-17A and RORγt (retinoic acid receptor-related orphan receptor-γt) expression levels in the colon tissues than that in the wild-type mice. Moreover, TG2(−/−) mice showed higher mortality than the wild-type mice because of DSS treatment. Nevertheless, we found no significant differences in changes of body weight, colon length, morphology, immune cell infiltration and in vivo intestinal permeability between DSS-treated wild-type and TG2(−/−) mice. These results indicate that TG2-mediated Th17 cell differentiation is not required for the pathogenesis of DSS-induced acute colitis.
Animals ; Body Weight ; Celiac Disease ; Cell Differentiation ; Child ; Child, Orphaned ; Colitis* ; Colon ; Cystic Fibrosis ; Dextran Sulfate* ; Dextrans* ; Drinking Water ; Fibrosis ; Glutamine ; Goblet Cells ; Humans ; Inflammation ; Inflammatory Bowel Diseases ; Interleukin-17 ; Interleukins ; Lung ; Mice* ; Mortality ; Multiple Sclerosis ; Permeability ; Pulmonary Fibrosis ; Sepsis ; Th17 Cells

Arterial restenosis frequently develops after open or endovascular surgery due to intimal hyperplasia. Since tissue transglutaminase (TG2) is known to involve in fibrosis, wound healing, and extracellular matrix remodeling, we examined the role of TG2 in the process of intimal hyperplasia using TG2-null mice. The neointimal formation was compared between TG2-null and wild-type (C57BL/6) mice by two different injury models; carotid ligation and carotid loop injury. In ligation model, there was no difference in intimal thickness between two groups. In loop injury model, intimal hyperplasia developed in both groups and the intimal/medial area ratio was significantly reduced in TG2-null mice (P = 0.007). TG2 was intensely stained in neointimal cells in 2 weeks. In situ activity of TG2 in the injured arteries steadily increased until 4 weeks compared to uninjured arteries. Taken together, intimal hyperplasia was significantly reduced in TG2-null mice, indicating that TG2 has an important role in the development of intimal hyperplasia. This suggests that TG2 may be a novel target to prevent the arterial restenosis after vascular surgery.
Animals ; Carotid Arteries/pathology/*surgery ; Disease Models, Animal ; GTP-Binding Proteins/deficiency/genetics/*metabolism ; Hyperplasia ; Mice ; Mice, Inbred C57BL ; Transglutaminases/deficiency/genetics/*metabolism ; Tunica Intima/*pathology

An abrupt increase of intracellular Ca2+ is observed in cells under hypoxic or oxidatively stressed conditions. The dysregulated increase of cytosolic Ca2+ triggers apoptotic cell death through mitochondrial swelling and activation of Ca2+-dependent enzymes. Transglutaminase 2 (TG2) is a Ca2+-dependent enzyme that catalyzes transamidation reaction producing cross-linked and polyaminated proteins. TG2 activity is known to be involved in the apoptotic process. However, the pro-apoptotic role of TG2 is still controversial. In this study, we investigate the role of TG2 in apoptosis induced by Ca2+-overload. Overexpression of TG2 inhibited the A23187-induced apoptosis through suppression of caspase-3 and -9 activities, cytochrome c release into cytosol, and mitochondria membrane depolarization. Conversely, down-regulation of TG2 caused the increases of cell death, caspase-3 activity and cytochrome c in cytosol in response to Ca2+-overload. Western blot analysis of Bcl-2 family proteins showed that TG2 reduced the expression level of Bax protein. Moreover, overexpression of Bax abrogated the anti-apoptotic effect of TG2, indicating that TG2-mediated suppression of Bax is responsible for inhibiting cell death under Ca2+-overloaded conditions. Our findings revealed a novel anti-apoptotic pathway involving TG2, and suggested the induction of TG2 as a novel strategy for promoting cell survival in diseases such as ischemia and neurodegeneration.
*Apoptosis ; Apoptosis Regulatory Proteins/metabolism ; Calcimycin/pharmacology ; Calcium/*metabolism ; Caspases/metabolism ; Cell Death ; Cell Survival ; Cytochromes c/metabolism ; Down-Regulation ; GTP-Binding Proteins/*metabolism ; HEK293 Cells ; Hela Cells ; Humans ; Ionophores/pharmacology ; Mitochondria/metabolism ; Transglutaminases/*metabolism ; bcl-2-Associated X Protein/genetics/*metabolism

Transglutaminase 4 is a member of enzyme family that catalyzes calcium-dependent posttranslational modification of proteins. Although transglutaminase 4 has been shown to have prostate-restricted expression pattern, little is known about the biological function of transglutaminase 4 in human. To gain insight into its role in prostate, we analyzed the expression status of human transglutaminase 4 in benign prostate hyperplasia (BPH) and prostate cancer (PCa). Unexpectedly, RT-PCR and nucleotide sequence analysis showed four alternative splicing variants of transglutaminase 4: transglutaminase 4-L, -M (-M1 and -M2) and -S. The difference between transglutaminase 4-M1 and -M2 is attributed to splicing sites, but not nucleotide size. The deduced amino acid sequences showed that transglutaminase 4-L, -M1 and -M2 have correct open reading frames, whereas transglutaminase 4-S has a truncated reading frame. RT-PCR analysis of clinical samples revealed that transglutaminase 4-M and -S were detected in all tested prostate tissue (80 BPH and 48 PCa). Interestingly, transglutaminase 4-L was found in 56% of BPH (45 out of 80) and only in 15% of PCa (7 out of 48). However, transglutaminase 4-L expression did not correlate with serum prostate-specific antigen (PSA) level, prostate volumes or PSA densities. These results will provide a clue to future investigation aiming at delineating physiological and pathological roles of human transglutaminase 4.

This erratum is being published to correct the printing error on page 286 of the article entitled 'Panton-Valentine leukocidin positive Staphylococcus aureus isolated from blood in Korea' by Kim JS, Park JS, Song W, Kim HS, Cho HC, Lee KM, Kim EC in Korean J Lab Med 2007;27:286-91. DOI 10.3343/kjlm. 2007.27.4.286 as follows. The heading of the right column of the Table 1 was misprinted as methicillin-resistant, so it should be corrected to methicillin-susceptible.
Adult ; Amino Acid Substitution ; Brain Neoplasms/radiotherapy/surgery ; Breast Neoplasms/diagnosis/radiotherapy/surgery ; Female ; *Genetic Counseling ; Genetic Predisposition to Disease ; *Germ-Line Mutation ; Humans ; Li-Fraumeni Syndrome/*diagnosis/genetics/therapy ; Mutation, Missense ; Pedigree ; Tumor Suppressor Protein p53/*genetics

BACKGROUND: Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by some Staphylococcus aureus strains and associated with skin and soft tissue infections; these strains are epidemiologically associated with current outbreaks of community-acquired methicillin-resistant S. aureus (MRSA) and with necrotizing pneumonia in healthy adults in USA and Europe. This study was performed to investigate the presence of PVL-positive S. aureus and the significant infections known to be caused by this organism. METHODS: A total of 573 strains of S. aureus blood isolates at university-affiliated hospital during 2002 to 2005 were selected. The presence of PVL was investigated using PCR. Additional 12 staphylococcal toxin genes were also examined in PVL-positive S. aureus strains, and MRSA isolates were typed for the staphylococcal cassette chromosome mec (SCCmec). RESULTS: PVL genes were detected in 5 (0.9%) of 573 S. aureus strains, including 1 MRSA and 4 MSSA. The PVL-positive MRSA isolate was SCCmec type IV, and no other staphylococcal toxins were detected. The median age of the patients infected with PVL-positive S. aureus was 36 yr. Three cases of bacteremia were preceded by skin and soft-tissue infections. CONCLUSIONS: Bacteremia caused by PVL-positive S. aureus strain were detected in 5 patients in Korea, and some of the patients were associated with severe skin and soft-tissue infections. In addition, the PVL-positive MRSA strain of SCCmec type IV, a characteristic of community-acquired MRSA isolates in USA and Europe, also exists in Korea, and can cause the severe infections known to be associated with this organism.
Adult ; Bacteremia/*microbiology ; Bacterial Proteins/genetics ; Bacterial Toxins/*blood ; Exotoxins/*blood ; Female ; Humans ; Korea ; Leukocidins/*blood ; Male ; Methicillin/pharmacology ; Methicillin Resistance/drug effects ; Middle Aged ; Polymerase Chain Reaction/methods ; Staphylococcal Infections/*microbiology ; Staphylococcus aureus/genetics/*isolation & purification