Contribution of donor and host mesenchyme to the transplanted tooth germs.

Autologous tooth germ transplantation of immature teeth is an alternative method of tooth replacement that could be used instead of dental implants in younger patients. However, it is paramount that the dental pulp remain vital and that root formation continue in the transplanted location. The goal of this study is to characterize the healing of allogenic tooth grafts in an animal model using GFP-labeled donor or host postnatal mice. In addition, the putative stem cells were labeled before transplantation with a pulse-chase paradigm. Transplanted molars formed cusps and roots and erupted into occlusion by 2 wk postoperatively. Host label-retaining cells (LRCs) were maintained in the center of pulp tissue associating with blood vessels. Dual labeling showed that a proportion of LRCs were incorporated into the odontoblast layer. Host cells, including putative dendritic cells and the endothelium, also immigrated into the pulp tissue but did not contribute to the odontoblast layer. Therefore, LRCs or putative mesenchymal stem cells are retained in the transplanted pulps. Hertwig's epithelial root sheath remains vital, and epithelial LRCs are present in the donor cervical loops. Thus, the dynamic donor-host interaction occurred in the developing transplant, suggesting that these changes affect the characteristics of the dental pulp.

Caffeine and uric acid mediate glutathione synthesis for neuroprotection.

Several lines of epidemiological studies have indicated that caffeine consumption and plasma uric acid (UA) level were negatively correlated with the incidence of some neurodegenerative diseases. We report here a novel mechanism by which these purine derivatives increase neuronal glutathione (GSH) synthesis. Intraperitoneal injection of caffeine or UA into male C57BL/6 mice significantly increased total GSH levels in the hippocampus. Neither SCH58261, an adenosine A2A receptor antagonist, nor rolipram, a phosphodiesterase-4 inhibitor, increased GSH levels. Pretreatment with allopurinol, a drug to inhibit UA production, did not change the GSH level in the caffeine-treated mice. Hippocampal CA1 pyramidal neurons treated with caffeine or UA were resistant to oxidant exposure in the slice culture experiments. In experiments with the SH-SY5Y cell line, cysteine uptake was sodium-dependent and pretreatment with caffeine or UA increased cysteine uptake significantly as compared with the control conditions. Slice culture experiments using the hippocampus also showed increased cysteine and GSH contents after the treatment with caffeine or UA. Immunohistochemical analysis showed increased GSH levels in the hippocampal excitatory amino acid carrier-1 (EAAC1)-positive neurons of mice treated with caffeine or UA. These findings suggest that purine derivatives caffeine and UA induce neuronal GSH synthesis by promoting cysteine uptake, leading to neuroprotection.

[NF-kappa B as a therapeutic drug target].

The transcription factor NF-kappa B has attracted widespread interest based on its unusual regulation, the variety of stimuli that activate it, the diverse genes and biological responses that it controls, the striking evolutionary conservation of structure and function among family members, and its apparent involvement in a variety of human diseases. Recently NF-kappa B has been shown to be the target of new drug discovery. Here, we discuss the so-called NF-kappa B inhibitors and consider the development of new therapeutic agents.

Glufosinate ammonium induces convulsion through N-methyl-D-aspartate receptors in mice.

Glufosinate ammonium, a broad-spectrum herbicide, causes convulsion in rodents and humans. Because of the structural similarities between glufosinate and glutamate, the convulsion induced by glufosinate ammonium may be ascribed to glutamate receptor activation. Three N-methyl-D-asparate (NMDA) receptor antagonists, dizocilpine, LY235959, and Compound 40, and an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist, NBQX, were coadministrated with glufosinate ammonium (80 mg/kg, intraperitoneally) in mice. Statistical analyses showed that the NMDA receptor antagonists markedly inhibited the convulsions, while the AMPA/kainate receptor antagonist had no effect on the convulsion. These results suggest that the convulsion caused by glufosinate ammonium is mediated through NMDA receptors.

Elevated plasma nitrate levels in depressive states.

BACKGROUND: Previous studies have shown that nitric oxide (NO) synthase inhibitors show preclinical antidepressant-like properties, suggesting that NO is involved in the pathogenesis of depression. The purpose of this study is to examine whether or not NO production increases in depressed patients. METHODS: Plasma nitrate concentrations, an index of NO production, were measured by high-performance liquid chromatography in depressed patients (n=17) and compared with patients suffering anxiety (n=6) and with healthy controls (n=12). RESULTS: Plasma nitrate concentrations were significantly higher in depressed patients than in patients with an anxiety disorder (P<0.05) or in controls (P<0.01). LIMITATIONS: The study group was small. The source of the surplus production of NO in patients with major depressive episode remains unclear. CONCLUSIONS: These results suggest that NO production is increased in depression.

Static pressure regulates connective tissue growth factor expression in human mesangial cells.

Connective tissue growth factor (CTGF) is overexpressed in a variety of fibrotic disorders such as renal fibrosis and atherosclerosis. Fibrosis is a common final pathway of renal diseases of diverse etiology, including inflammation, hemodynamics, and metabolic injury. Mechanical strains such as stretch, shear stress, and static pressure are possible regulatory elements in CTGF expression. In this study, we examined the ability of static pressure to modulate CTGF gene expression in cultured human mesangial cells. Low static pressure (40-80 mm Hg) stimulated cell proliferation via a protein kinase C-dependent pathway. In contrast, high static pressure (100-180 mm Hg) induced apoptosis in human mesangial cells. This effect was reversed by treatment with CTGF antisense oligonucleotide but not with transforming growth factor beta1-neutralizing antibody or protein kinase C inhibitor. High static pressure not only up-regulated the expression of CTGF, but also the expression of extracellular matrix proteins (collagen I and IV, laminin). This up-regulation of extracellular matrix proteins was also reversed by treatment with CTGF antisense oligonucleotide. As judged by mRNA expression of a total of 1100 genes, including apoptosis-associated genes using DNA microarray techniques, recombinant CTGF protein induced apoptosis by down-regulation of a number of anti-apoptotic genes. Overexpression of CTGF in mesangial cells by transient transfection had similar effects. Taken together, these results suggest that high blood pressure up-regulates CTGF expression in mesangial cells. High levels of CTGF in turn enhance extracellular matrix production and induce apoptosis in mesangial cells, and may contribute to remodeling of mesangium and ultimately glomerulosclerosis.

Glufosinate ammonium stimulates nitric oxide production through N-methyl D-aspartate receptors in rat cerebellum.

Glufosinate ammonium, a structural analogue of glutamate, is an active herbicidal ingredient. The neuronal activities of this compound were investigated by use of a microdialysis system that allowed us to measure nitric oxide production in the rat cerebellum in vivo. Kainate (0.3-30 nmol/10 microliter), N-methyl-D-aspartate (NMDA) (3-300 nmol/10 microliter) and glufosinate ammonium (30-3000 nmol/10 microliter), which were administered through the microdialysis probe at a rate of 1 microliter/min for 10 min, stimulated nitric oxide production. The glufosinate ammonium-elicited increase in nitric oxide production was suppressed by an inhibitor of nitric oxide synthase and was antagonized by NMDA receptor antagonists, but not by a kainate/(+/-)-alphaamino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist. These results suggest that glufosinate ammonium stimulates nitric oxide production through NMDA receptors.

Connective tissue growth factor induces apoptosis via caspase 3 in cultured human aortic smooth muscle cells.

Connective tissue growth factor (CTGF) stimulates proliferation of fibroblasts and endothelial cells, but nothing is known about its role in smooth muscle cells. In this study, the effects of recombinant human CTGF (r-hCTGF, 0.5-10 microgram/ml) on cultured human aortic vascular smooth muscle cells were investigated. r-hCTGF significantly reduced cell viability, increased apoptosis, and augmented caspase 3 activity. Moreover, r-hCTGF-induced apoptosis was significantly inhibited by an antibody to CTGF and a caspase-3 inhibitor, Z-Asp(Ome)-Glu-(Ome)Val-Asp(Ome)-FMK. These results suggest that r-hCTGF activates caspase 3 and induces apoptosis.

Connective tissue growth factor induces apoptosis in human breast cancer cell line MCF-7.

Connective tissue growth factor (CTGF) is a member of an emerging CCN gene family that is implicated in various diseases associated with fibro-proliferative disorder including scleroderma and atherosclerosis. The function of CTGF in human cancer is largely unknown. We now show that CTGF induces apoptosis in the human breast cancer cell line MCF-7. CTGF mRNA was completely absent in MCF-7 but strongly induced by treatment with transforming growth factor beta (TGF-beta). TGF-beta by itself induced apoptosis in MCF-7, and this effect was reversed by co-treatment with CTGF antisense oligonucleotide. Overexpression of CTGF gene in transiently transfected MCF-7 cells significantly augmented apoptosis. Moreover, recombinant CTGF protein significantly enhanced apoptosis in MCF-7 cells as evaluated by DNA fragmentation, Tdt-mediated dUTP biotin nick end-labeling staining, flow cytometry analysis, and nuclear staining using Hoechst 33258. Finally, recombinant CTGF showed no effect on Bax protein expression but significantly reduced Bcl2 protein expression. Taken together, these results suggest that CTGF is a major inducer of apoptosis in the human breast cancer cell line MCF-7 and that TGF-beta-induced apoptosis in MCF-7 cells is mediated, in part, by CTGF.

Transforming growth factor-beta(1) induces apoptosis via connective tissue growth factor in human aortic smooth muscle cells.

We examined the possible involvement of connective tissue growth factor (CTGF) in the apoptosis induced by transforming growth factor-beta(1) (TGF-beta(1)) in human aortic vascular smooth muscle cells (HASC). In quiescent HASC, TGF-beta(1) induced the mRNA and protein of CTGF. A CTGF antisense oligonucleotide inhibited this induction. TGF-beta(1) significantly reduced cell viability and induced DNA fragmentation, and the CTGF antisense oligonucleotide reversed these effects. Moreover, TGF-beta(1) activated caspase 3 in HASC, and the CTGF antisense oligonucleotide reduced this activation. These findings show that CTGF plays a key role in the TGF-beta(1)-induced apoptosis in HASC.

Overexpression of connective tissue growth factor gene induces apoptosis in human aortic smooth muscle cells.

BACKGROUND: Connective tissue growth factor (CTGF) is expressed at very high levels particularly in the shoulder of human atherosclerotic lesions but not in normal blood vessels. Thus, CTGF may be important in the regulation of vascular smooth muscle cell function in atherosclerosis, but its precise role remains elusive. METHODS AND RESULTS: Full-length CTGF cDNA driven by a cytomegalovirus promoter was transiently transfected into cultured human aortic smooth muscle cells (HASCs). Northern and Western analysis demonstrated that CTGF was overexpressed in these cells 48 hours after transfection. The effects of CTGF overexpression on cell proliferation were evaluated by [(3)H]thymidine uptake and cell count in quiescent HASCs or those stimulated with platelet-derived growth factor (PDGF). Although mock transfection showed no effect, CTGF overexpression significantly inhibited cell proliferation in cells stimulated by PDGF. Moreover, CTGF overexpression, but not mock transfection, significantly increased apoptosis as assessed by DNA fragmentation associated with histone, TdT-mediated dUTP biotin nick end-labeling, and appearance of hypodiploid cells by flow cytometry. CONCLUSIONS: Our results for the first time demonstrate that CTGF can also act as a growth inhibitor in human aortic smooth muscle cells at least in part by inducing apoptosis. This may be important for the formation and composition of lesions and plaque stability in atherosclerosis.

Nitration modifying function of proteins, hormones and neurotransmitters.

Several lines of evidence have been accumulated for occurrence of nitration in vivo. In this brief review, we summarized nitration studies on functional changes of proteins, hormones and neurotransmitters, before as well as after the discovery of peroxynitrite. Most of nitrated molecules exhibit less active properties than the parental compounds. It is still unknown whether nitration is merely a footprint of oxidative stress, an important pathway of nitric oxide metabolisms or a part of integral processes for maintaining cellular homeostasis.

Endothelin ET(A) receptor antagonist reverses the inhibitory effect of platelet-derived growth factor on cytokine-induced nitric oxide production.

Cytokines and cytokine-induced nitric oxide (NO) play important roles in inflammatory glomerular diseases, and both platelet-derived growth factor and transforming growth factor-beta inhibit cytokine-induced NO production. In this study, we demonstrated that a selective endothelin ET(A) receptor antagonist, BQ-485 (Hexahydro-1H-azepinylcarbonyl-Leu-D-Trp-D-Trp-OH), reversed the inhibitory effect of platelet-derived growth factor on cytokine-induced NO production, but not that of transforming growth factor-beta. Our findings suggest a difference between the inhibitory mechanisms of platelet-derived growth factor and transforming growth factor-beta on cytokine-induced NO production.

Endothelium-independent and -dependent vasoactivity of 6-nitronorepinephrine.

Vasoactivities of 6-nitronorepinephrine were investigated using rat aorta. 6-Nitronorepinephrine (> 100 microM) caused dose-dependent contraction in both endothelium-intact and -denuded aorta, although the latter showed greater contraction than the former. Prazosin (> 3 nM), an alpha1-adrenoceptor antagonist, attenuated significantly the 6-nitronorepinephrine-induced contractions, thereby suggesting the alpha1-adrenoceptor involvement. Aortic rings prepared from reserpine-pretreated rats showed the 6-nitronorepinephrine-induced a contraction to the extent similar to those from untreated rats, suggesting that endogenous norepinephrine does not play a role in the 6-nitronorepinephrine-induced contraction. 6-Nitronorepinephrine (> 10 microM) potentiated norepinephrine-induced contraction only in the presence of endothelium. The augmentation was attenuated by catalase (1200 U/ml). H2O2 (10-300 microM) augmented the norepinephrine-induced contraction only in the endothelium-intact rat aortic rings. 6-Nitronorepinephrine attenuated significantly acetylcholine-induced relaxation. Catalase prevented the 6-nitronorepinephrine-induced inhibition of the acetylcholine-induced relaxation. These results suggest that 6-nitronorepinephrine has a weak alpha1-adrenoceptor agonistic property and that the endothelium-dependent potentiation by 6-nitronorepinephrine of the norepinephrine-induced contraction is mediated through production of H2O2.

[Biological significance of nitration, nitrosation and nitrosylation in vivo].

Nitric oxide is an exceptionally stable molecule as a radical species. The smallest signal molecule is formed from oxygen and L-arginine by well-defined enzymes. Nitric oxide plays roles not only in physiological regulation such as blood pressure, platelet aggregation and neuronal function, but also in pathophysiological states. The reactions of nitric oxide with target molecules involve electron transfer. This mechanism differs essentially from those of neurotransmitters and hormones identified so far. It is reasonable to assume that distinct signal transduction pathways are involved in this redox signal molecule. In this review, we briefly summarize nitration, nitrosation and nitrosylation of target molecules in vivo by nitric oxide.

Immobilization stress increases mRNA levels of interleukin-1 receptor antagonist in various rat brain regions.

1. Interleukin-1 receptor antagonist (IL-1Ra), as well as the interleukin-1 beta (IL-1 beta) gene response to immobilization stress (IMS), was examined in the rat brain. The reverse transcription-polymerase chain reaction was employed to determine mRNA levels. 3. IL-1 beta and IL-1Ra mRNA levels peaked at approximately 0.5 and 2-4 hr, respectively. The maximum mRNA levels of IL-1 beta were 15-fold higher than pre-IMS levels, whereas those of IL-1Ra were 250-fold higher in the hypothalamus. 3. After the biosynthesis of IL-1 beta has peaked, IL-1Ra may contribute to attenuation of the IL-1 activity which has been enhanced by IMS.

The effects of transmural pressure on prostacyclin release from porcine endocardial endothelial cells--comparison with vascular endothelial cells.

We assessed the effect of pressure on the release of prostacyclin (PGI2) from cultured endocardial endothelial cells (EECs) and vascular endothelial cells (VECs). EECs were harvested from the right ventricle (RV) and left ventricle (LV) of porcine hearts, and VECs from pulmonary artery (PA), aorta (Ao) and coronary artery (CA). Confluent EECs and VECs were incubated for 30 min under various pressures (0, 50, 100, 150 mmHg) and PGI2 release from each cell was measured. Pressure-induced PGI2 release from LV-EECs was larger than that from RV-EECs. Pressure also increased PGI2 release from both PA- and Ao-VECs, but not from CA-VECs. These findings suggest that endocardium can produce PGI2 in response to pressure and PGI2 released into the coronary blood from the ventricle may play an important role in the prevention of myocardial ischemia.

Dexamethasone and nitric oxide synthase gene expression in brain.

Systemic administration of lipopolysaccharide (LPS), which causes endotoxemia and systemic inflammation, has been reported to induce expression of the gene for type II inducible nitric oxide synthase (iNOS) in peripheral organs. This study was carried out to examine whether intraperitoneally injected LPS elicits the expression of iNOS messenger ribonucleic acid (mRNA) in the rat brain. We also investigated whether intraperitoneal treatment with dexamethasone (DEX) prevents this induction. To determine levels of iNOS mRNA, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was employed. Treatment with LPS induced the expression of iNOS mRNA in various brain regions, accounting for approximately 1 x 10(5) to 4 x 10(5) molecules per micrograms of poly A+ RNA, and these inductions were markedly suppressed by DEX. The results suggest that, during systemic inflammation, iNOS mRNA induction occurs in brain through a DEX-sensitive mechanism.