Correlation between individual differences in intraocular pressure reduction and outflow facility due to latanoprost in normal-tension glaucoma patients.

PURPOSE: The amount of intraocular pressure (IOP) reduction achieved by the use of latanoprost eyedrops varies among patients, and there are even nonresponders. This report examines whether there is any correlation between the amount of individual variability in IOP reduction and the uveoscleral outflow facility after latanoprost eyedrop instillation in normal-tension glaucoma patients. METHODS: Sixteen normal-tension glaucoma patients (mean age, 56.4 years) were enrolled in the study to investigate the relationship between the amount of IOP reduction and outflow facility. Before treatment, subjects underwent circadian IOP measurement and then tonography, and the outflow facility was calculated. Subsequently, patients began treatment once daily with latanoprost instillation in one eye. After 4 weeks of daily latanoprost treatment, circadian IOP was measured again. RESULTS: Mean pretreatment outflow facility was 0.23 +/- 0.05 microl/min per mmHg. On average, latanoprost instillation decreased IOP by 2.8 mmHg, but the reduction varied among individuals from -0.3 mmHg to 5.8 mmHg. No significant correlation was noted between the outflow facility and the IOP decline associated with latanoprost. CONCLUSION: Because there was no significant correlation between individual IOP reduction by latanoprost and outflow facility, the differences in substantial change in uveoscleral outflow after latanoprost administration may be one explanation for the individual variation in IOP reduction after treatment with this drug.

Garcinone B reduces prostaglandin E2 release and NF-kappaB-mediated transcription in C6 rat glioma cells.

In the course of our survey of natural compounds inhibiting prostaglandin E2 release and/or lipopolysaccharide (LPS)-induced transcriptional stimulation via NF-kappaB, a central regulator of inflammatory genes, from natural resources, we found garcinone B, a xanthone from callus tissue culture of Hypericum patulum, as a compound with such pharmacological activities, that is a derivative of gamma-mangostin which potently inhibits COX-1 and COX-2 activities to reduce PGE2 release from C6 rat glioma cells, and inhibits IKK activity to prevent NF-kappaB-dependent COX-2 gene transcription. Garcinone B, to a lesser extent, reduced A23187-induced increase in prostaglandin E2 release than gamma-mangostin and its structurally related compound, patulone, in C6 cells. This compound also prevented LPS-induced stimulation of NF-kappaB-dependent transcription. These results suggest that garcinone B becomes a unique pharmacological tool to investigate intracellular signaling pathways involved in inflammation.

A calcium binding protein, S100A4, mediates T cell dependent cytotoxicity as a transformation-associated antigen.

The nature of the target molecule of TCR gamma delta T cell-mediated lysis remains to be determined. As we previously reported, #067 monoclonal antibody (mAb) recognizes one of the transformation-associated antigens, designated as #067 antigen. This antigen is expressed on the cell surface of rat fibrosarcoma W31 cells, which are established by transformation of fetal fibroblastic WFB cells with H-ras oncogene. It has been suggested that the #067 antigen is a target molecule for TCR gamma delta T cells since #067 mAb inhibited TCR gamma delta T cell-mediated lysis against #067 positive cells. In this study we attempted to identify the protein sequence of the #067 antigen. By using molecular cloning techniques, we demonstrated that a calcium binding protein, S100A4, was possibly one and the same molecule as the #067 antigen. It was shown that the expression of S100A4 was higher in W31 cells than in WFB cells at transcription and protein level. Flow cytometry and immunocytochemical studies showed that #067 antigen partially co-localized with S100A4 on the cell surface as well as the cytoplasm of W31 cells. Moreover, rabbit anti-S100A4 polyclonal antibodies (pAb) inhibited TCR gamma delta T cell-mediated lysis against #067 positive cells. Our results indicated that S100A4 may play a role as a possible target molecule for TCR gamma delta T cell-mediated lysis although how S100A4 is involved in TCR gamma delta T cell-mediated lysis remains to be determined.

gamma-Mangostin inhibits inhibitor-kappaB kinase activity and decreases lipopolysaccharide-induced cyclooxygenase-2 gene expression in C6 rat glioma cells.

We investigated the effect of gamma-mangostin purified from the fruit hull of the medicinal plant Garcinia mangostana on spontaneous prostaglandin E(2) (PGE(2)) genase release and inducible cyclooxy-2 (COX-2) gene expression in C6 rat glioma cells. An 18-h treatment with gamma-mangostin potently inhibited spontaneous PGE(2) release in a concentration-dependent manner with the IC(50) value of approximately 2 microM, without affecting the cell viability even at 30 microM. By immunoblotting and reverse-transcription polymerase chain reaction, we showed that gamma-mangostin concentration-dependently inhibited lipopolysaccharide (LPS)-induced expression of COX-2 protein and its mRNA, but not those of constitutive COX-1 cyclooxygenase. Because LPS is known to stimulate inhibitor kappaB (IkappaB) kinase (IKK)-mediated phosphorylation of IkappaB followed by its degradation, which in turn induces nuclear factor (NF)-kappaB nuclear translocation leading to transcriptional activation of COX-2 gene, the effect of gamma-mangostin on the IKK/IkappaB cascade controlling the NF-kappaB activation was examined. An in vitro IKK assay using IKK protein immunoprecipitated from C6 cell extract showed that this compound inhibited IKK activity in a concentration-dependent manner, with the IC(50) value of approximately 10 microM. Consistently gamma-mangostin was also observed to decrease the LPS-induced IkappaB degradation and phosphorylation in a concentration-dependent manner, as assayed by immunoblotting. Furthermore, luciferase reporter assays showed that gamma-mangostin reduced the LPS-inducible activation of NF-kappaB-and human COX-2 gene promoter region-dependent transcription. gamma-Mangostin also inhibited rat carrageenan-induced paw edema. These results suggest that gamma-mangostin directly inhibits IKK activity and thereby prevents COX-2 gene transcription, an NF-kappaB target gene, probably to decrease the inflammatory agent-stimulated PGE(2) production in vivo, and is a new useful lead compound for anti-inflammatory drug development.

Expression profiles and functional implications of p53-like transcription factors in thymic epithelial cell subtypes.

In this study, we investigated the localization and functional significance of p53 tumor suppressor-like molecules, p63 and p73, in human thymic epithelial cells (TECs). Immunohistochemical studies showed particular distribution profiles of p63 and p73 in thymic epithelium, in which cortical TECs preferentially expressed p63 in their nuclei whereas subcapsular and medullary TECs expressed both p63 and p73 in their nuclei. The wide distribution of p63 in TECs was further suggested by studies using TECs of primary culture. In vitro studies using two human TEC lines demonstrated that p63 was capable of up-regulating intercellular adhesion molecule-1 (ICAM-1) and enhancing the production of IL-6 and IL-8. Moreover, in vitro studies also indicated that p73, but not p63, had the capacity to induce granulocyte macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) in the TEC lines. These findings suggest that p63 would regulate the cell adhesive property through ICAM-1/LFA-1 interaction and the production of IL-6 and IL-8, probably in all TEC subtypes. p73 in subcapslar and medullary TECs was suggested to play a role in the regulation of the production of GM-CSF and G-CSF, which might stimulate other stromal cells such as dendritic cells, macrophages and endothelial cells around these regions.

Histone H2AX sensitizes glioma cells to genotoxic stimuli by recruiting DNA double-strand break repair proteins.

Glioblastoma is a life-threatening tumor in the human brain despite the fact that radio-chemotherapy inducing DNA damage has been improved in the last decade. Various studies focusing on the enhancement of the susceptibility of glioblastoma cells to DNA damage have been reported, which are aimed at more efficient treatment for the tumor. In this study, we show that radioresistant T98G glioblastoma cells can develop sensitivity to DNA damage induced by irradiation and etoposide as a result of the introduction of a DNA repair-associated histone, H2AX. Interestingly, when H2AX-transformed T98G cells were irradiated, Brca1 and Nbs1 were readily recruited in DNA double-strand break (DSB) foci and showed the G2/M-phase arrest of the cell cycle. Moreover, up-regulation of Brca1 was observed in H2AX-T98G cells after exposure to irradiation. Together with the evidence that H2AX transfection does not affect growth activities of non-tumor cells under genotoxic stimuli, this suggests that H2AX gene transfer would provide a new modality for radio-chemotherapy for glioblastomas, probably through overcoming the instability of the genome, and that Brca1 and Nbs1 might be crucial in this methodology.

T-cell receptor variable gamma chain gene expression in the interaction between rat gammadelta-type T cells and heat-shock protein 70-like molecule.

We previously reported that rat T-cell receptor (TCR) Vdelta6 of T-cell hybridomas was preferentially involved in recognition of the cell surface-expressed 70 kDa rat heat-shock cognate (hsc70, a constitutively expressed member of the hsp 70 family) protein-like molecule (#067 molecule). In the present study, we analyzed usage of the TCR Vgamma family of #067-restricted T-cell hybridomas. Our data indicated that most of these hybridomas expressed transcripts of TCR Vgamma1 and/or Vgamma2. However, some of the Vgamma2 transcripts were out-of-frame, suggesting that the TCR Vgamma1 family may be important for the recognition of #067-defined molecules. TCR Vgamma1 transcripts were detected in not only #067-restricted T-cell hybridomas, but #067-non restricted ones as well. However, V-J nucleotide sequences of #067-restricted and #067-non restricted T-cell hybridomas suggested that #067-restricted T-cell hybridomas showed limited insertion of nucleotide stretch as compared with #067-non restricted ones. In terms of amino acids, only one amino acid was added in #067-restricted T-cell hybridomas, whereas two or three amino acids were added in #067-non restricted ones. These data suggest that the heterodimer of the TCR relatively short stretch form of Vgamma1 molecule and TCR Vdelta6 may participate in recognition of the #067 molecule.

p73 is expressed in human thymic epithelial cells.

The thymus is a heterogeneous immune organ in which immature T-cells develop and eventually specialize to make certain immune responses of their own. Among various types of stromal cells in the thymus, thymic epithelial cells (TECs) have a crucially important function for presenting self-antigens and secreting cytokines to thymocytes for their maturation into T-cells. In this study we show that the p73 gene, a homologue of the tumor suppressor gene p53, was expressed in the nucleus of the human TEC in vivo and in TEC lines in vitro. Because p73 has the capacity to be a transactivator like p53, it may contribute to T-cell development in the context of TEC biology as regulated in the cell cycle and apoptosis.