Transforming growth factor-beta receptor profiles of human and murine embryonic palate mesenchymal cells.

Cell signalling in the developing mammalian palate appears to involve various growth factors and hormones. An important developmental role for the transforming growth factor-beta (TGF-beta) class of growth factors is suggested by the immunolocalization of TGF-beta 1 in the palate during its ontogeny. This study examined the effects of TGF-beta stimulation of, as well as TGF-beta receptor profiles in, murine embryonic palate mesenchymal (MEPM) and human embryonic palate mesenchymal (HEPM) cells. Results showed that TGF-beta 1 (1 ng/ml) stimulated proliferation of HEPM cells and inhibited proliferation of MEPM cells in a dose-dependent manner. The time course of 125I-TGF-beta 1 binding to specific receptors was determined by incubating cells in the presence of 170 pM 125I-TGF-beta 1 for up to 4 h. In both cell types, at 37 degrees C, the binding of 125I-TGF-beta decreased linearly over 4 h, while at 4 degrees C, binding increased with time of incubation. Incubation of both cell types at 4 degrees C for 4 h, with increasing concentrations of 125I-TGF-beta 1, resulted in binding which demonstrated saturation kinetics. Scatchard analyses revealed one class of receptors for HEPM (K 32.3 pM) and MEPM (K 26.3 pM). However, SDS-PAGE analyses of 125I-TGF-beta chemically crosslinked to specific receptor sites revealed that both cell types contained the types I (65,000 Mr) and III (230,000 Mr) TGF-beta receptors while MEPM also contained the type II (86,000 Mr) receptor. Binding studies further demonstrated the ability of platelet-derived growth factor to transmodulate TGF-beta binding. These results indicate that the HEPM cell line and primary cultures of MEPM cells, although obtained from palates at similar developmental stages, are dramatically different in their responsiveness to TGF-beta and have disparate TGF-beta receptor profiles.     

Secalonic acid D blocks embryonic palatal mesenchymal cell-cycle by altering the activity of CDK2 and the expression of p21 and cyclin E

BACKGROUND: The mycotoxin, secalonic acid D (SAD), a known animal and potential human cleft palate (CP)-inducing agent, is produced by Pencillium oxalicum in corn. SAD selectively inhibits proliferation of murine embryonic palatal mesenchymal (MEPM) cells leading to a reduction in cell numbers. These effects can explain the reduction in shelf size and the resulting CP seen in the offspring of SAD-exposed mice. Ability of SAD to inhibit proliferation as well as to block the progression of cells from G1- to S-phase of the cell-cycle were also shown in the human embryonic palatal mesenchymal (HEPM) cells suggesting the potential CP-inducing effect of SAD in human beings METHODS: Gestation day (GD) 12 mouse embryos and HEPM cells were used to test the hypothesis that the cell-cycle block induced by SAD results from a disruption of stage-specific regulatory components both in vivo and in vitro. The effects of SAD on the activity of various cyclin dependent kinases (CDK) and on the levels of various positive (cyclins and CDK) and negative (CDK inhibitors p15, 16, 18, 19, 21, 27, 57) cell-cycle regulators were assessed by performing kinase assays and immunoblots, respectively. RESULTS: In the murine embryonic palates, SAD specifically inhibited G1/S-phase-specific CDK2 activity, reduced the level of cyclin E and tended to increase the level of the CIP/kip CDK inhibitor, p21. In the HEPM cell cultures, exposure to IC50 of SAD significantly affected all of the above targets. In addition, a reduction in the levels/activity of CDK 4/6, a reduction in the levels of cyclins D1, D2, D3, E, A, and all INK4 family proteins, and an increase in the level of the CIP/kip CDK inhibitor, p57, were also seen. CONCLUSIONS: These results suggest that the S-phase-specific cell-cycle proteins CDK2, cyclin E and possibly p21 are the common targets of SAD in murine palatal shelves in vivo and in human embryonic palatal mesenchymal cells in vitro and may be relevant to the pathogenesis of SAD-induced CP.     

Copper impact on heat shock protein 70 expression and apoptosis in rainbow trout hepatocytes

The mechanism underlying copper hepatotoxicity was investigated in primary cultures of rainbow trout hepatocytes maintained in Leibovitz-15 media. CuSO4 treatment (0, 25, 50, 100 and 200 microM) resulted in a dose-dependent elevation in heat shock protein 70 (hsp70) expression at 24 and 48 h post-exposure. There was no effect of copper (200 microM CuSO4) on hepatotoxicity at 24 h, whereas longer exposures (48 h) resulted in increased lactate dehydrogenase (LDH) leakage and apoptosis, demonstrated by fluorescence nuclear staining and DNA fragmentation. Vitamin C (1 mM), a free radical scavenger, inhibited this copper-induced apoptosis implying a role for reactive oxygen species in copper toxicity. However, no parallel inhibition of either LDH leakage or hsp70 protein expression was observed with vitamin C suggesting that at least two independent mechanisms are involved in the cellular response to copper. Also, copper exposed (24 h) cells were unable to mount an hsp70 response to a standardized heat shock (+15 degrees C for 1 h), even in the presence of vitamin C. Together, these results suggest that hepatotoxicity of copper includes impairment of hsp70 response to subsequent stressors and/or signals, which is crucial for protecting cells from proteotoxicity.     

Oxidation affects the regulation of hepatic lipid synthesis by chylomicron remnants

The effects of native and oxidized chylomicron remnants on lipid synthesis in normal and oxidatively stressed liver cells were investigated using MET murine hepatocytes (MMH cells), a nontransformed mouse hepatocyte cell line that maintains a highly differentiated hepatic phenotype in culture. Lipid synthesis was determined by measuring the incorporation of [(3)H]oleate into cholesteryl ester, triacylglycerol, and phospholipid by the cells. The formation of cholesteryl ester and phospholipid was decreased by chylomicron remnants in a dose-dependent manner, while triacylglycerol synthesis was increased. Exposure of MMH cells to mild oxidative stress by incubation with CuSO(4) (2.5 microM) for 24 h led to significantly increased incorporation of [(3)H]oleate into triacylglycerol and phospholipid, but not cholesteryl ester, in the absence of chylomicron remnants. In the presence of the lipoproteins, however, similar effects to those found in untreated cells were observed. Oxidatively modified chylomicron remnants prepared by incubation with CuSO(4) (10 microM, 18 h, 37 degrees C) did not influence cholesteryl ester or phospholipid synthesis in MMH cells, but had a similar effect to that found with native remnants on triacylglycerol synthesis. These findings show that hepatic lipid metabolism is altered by exposure to mild oxidative stress and by lipids from the diet delivered to the liver in chylomicron remnants, and these effects may play a role in the development of atherosclerosis.     

Folate supplementation reduces serum hsp70 levels in patients with type 2 diabetes

Type 2 diabetes patients are subject to oxidative stress as a result of hyperglycemia. The aim of this study was to determine whether administration of the antioxidant folic acid, previously shown to reduce homocysteine levels, would reduce circulating levels of Hsp70 while improving the condition of type 2 diabetes patients with microalbuminuria. Plasma homocysteine fell from pretreatment values of 12.9 to 10.3 microM (P < 0.0001). The urine albumin-creatinine ratio fell from 12.4 to 10.4 mg/mM (P = 0.38). Pretreatment Hsp70 levels were higher in patients not taking insulin (5.32 ng/mL) compared with those on insulin (2.44 ng/mL) (P = 0.012). Folic acid supplementation resulted in a significant fall in Hsp70 (5.32 to 2.05 ng/mL) (P = 0.004). There was no change in Hsp70 in those receiving insulin. Folic acid supplementation in non-insulin-treated type 2 diabetes patients, therefore, resulted in a fall in Hsp70, reflecting an improvement in oxidative stress. The data shows that improvement in homocysteine status can lead to a reduction in Hsp70, indicating the possibility of its use as a marker for severity of disease.     

Folate deficiency and in vitro palatogenesis II: Effects of methotrexate on rabbit palate fusion, folate pools, and dihydrofolate reductase activity

Folate deficiency during pregnancy induced by dietary deprivation or folate antagonists is teratogenic. Methotrexate is a commonly used antifolate drug that produces congenital defects, with the craniofacial complex being especially vulnerable. The molecular action of this compound, therefore, was examined in vitro using fetal rabbit palates in an organ culture system. Dihydrofolate reductase activity was measured in these palates on the 4 days preceding fusion. There was no significant fluctuation in activity with gestational age. Methotrexate treatment of the palates in vitro resulted in significant reduction of enzyme activity and also decreased total folate concentration. However, the in vitro fusion ability of these palates was not altered by exposure to methotrexate. These results suggest that since methotrexate is a potent inhibitor of DNA synthesis and has its maximal effect on rapidly dividing systems, the peak in cellular proliferative activity in the fetal rabbit palate occurs very early in its development.     

Effects of homocysteine on the binding of extracellular-superoxide dismutase to the endothelial cell surface

Homocysteine is known to be a risk factor for several vascular diseases. Previously, we found a significant association between plasma homocysteine and plasma extracellular-superoxide dismutase (EC-SOD) levels. The binding of EC-SOD to human and bovine aortic endothelial cell cultures showed significant decreases after incubation with 10 microM homocysteine, whereas the expression of EC-SOD in fibroblast cell cultures was inhibited with a high concentration (1 mM) of homocysteine. Furthermore, binding of EC-SOD to heparin immobilized on plates was decreased with homocysteine. These observations suggested that homocysteine decreases the binding of EC-SOD to vascular endothelial cell surfaces by degradation of endothelial heparan sulfate proteoglycan, which results in a loss of the ability to protect endothelial cell surfaces from oxidative stress.     

Differences in Growth Generate the Diverse Palate Shapes of New World Leaf-Nosed Bats (Order Chiroptera,Family Phyllostomidae)

New World leaf-nosed bats (Family Phyllostomidae) display incredible craniofacial diversity that is associated with their broad range of dietary preferences. The short and broad palates of highly frugivorous bats are functionally linked to high bite forces, and the long and narrow palates of nectarivorous bats to flower feeding. Although the functional correlates and evolutionary history of shape variation in phyllostomid palates are beginning to be understood, the specific developmental processes that govern palate diversification remain unknown. To begin to resolve this issue, this study quantified palate morphology in seven phyllostomid species from a range of developmental stages and in adults. This sample includes species with short and broad, long and narrow, and intermediate palate shapes, and thereby covers the range of palate shapes displayed by phyllostomids. Results indicate that while initial palate shape (i.e., width vs. length) varies among species, the pattern of this variation does not match that observed in adults. In contrast, the relative growth of palate width and length in developing phyllostomids and the ratio of these axes in adults are significantly correlated. These and other results suggest that evolutionary alterations in patterns of palate growth have governed the diversification of palate shapes in adult phyllostomids. This implies that the diverse palate shapes of phyllostomids are the result of relatively subtle evolutionary changes in later rather than earlier development events.     

Endosulfan-induced apoptosis and glutathione depletion in human peripheral blood mononuclear cells: Attenuation by N-acetylcysteine

Present study investigated whether endosulfan, an organochlorine pesticide is able to deplete glutathione (GSH) and induce apoptosis in human peripheral blood mononuclear cells (PBMC) in vitro. The role of oxidative stress in the induction of apoptosis was also evaluated by the measurement of the GSH level in cell lysate. The protective role of N-acetylcysteine (NAC) on endosulfan-induced apoptosis was also studied. Isolated human PBMC were exposed to increasing concentrations (0-100 microM) of endosulfan (alpha/beta at 70:30 mixture) alone and in combination with NAC (20 microM) up to 24 h. Apoptotic cell death was determined by Annexin-V Cy3.18 binding and DNA fragmentation assays. Cellular GSH level was measured using dithionitrobenzene. Endosulfan at low concentrations, i.e., 5 and 10 microM, did not cause significant death during 6 h/12 h incubation, whereas a concentration-dependent cell death was observed at 24 h. DNA fragmentation analysis revealed no appreciable difference between control cells and 5 microM/10 microM endosulfan treated cells, where only high molecular weight DNA band was observed. Significant ladder formation was observed at higher concentration, which is indicative of apoptotic cell death. Intracellular GSH levels decreased significantly in endosulfan-treated cells in a dose-dependent manner, showing a close correlation between oxidative stress and degree of apoptosis of PBMC. Cotreatment with NAC attenuated GSH depletion as well as apoptosis. Our results provide experimental evidence of involvement of oxidative stress in endosulfan-mediated apoptosis in human PBMC in vitro.     

Sprouty2 controls proliferation of palate mesenchymal cells via fibroblast growth factor signaling

Cleft palate is one of the most common craniofacial deformities. The fibroblast growth factor (FGF) plays a central role in reciprocal interactions between adjacent tissues during palatal development, and the FGF signaling pathway has been shown to be inhibited by members of the Sprouty protein family. In this study, we report the incidence of cleft palate, possibly caused by failure of palatal shelf elevation, in Sprouty2-deficient (KO) mice. Sprouty2-deficient palates fused completely in palatal organ culture. However, palate mesenchymal cell proliferation estimated by Ki-67 staining was increased in Sprouty2 KO mice compared with WT mice. Sprouty2-null palates expressed higher levels of FGF target genes, such as Msx1, Etv5, and Ptx1 than WT controls. Furthermore, proliferation and the extracellular signal-regulated kinase (Erk) activation in response to FGF was enhanced in palate mesenchymal cells transfected with Sprouty2 small interfering RNA. These results suggest that Sprouty2 regulates palate mesenchymal cell proliferation via FGF signaling and is involved in palatal shelf elevation.     

Effect of copper intoxication on rat liver proteasome activity: relationship with oxidative stress

Copper toxicity is associated with formation of reactive oxygen species, which are capable to oxidize proteins. The selective removal of the latter by the 20S proteasome is considered an essential part of the cell antioxidant defense system. The aim of the present study was to investigate whether peptidase activities of rat liver proteasomes were affected by chronic (40 mg CuSO(4)/rat/daily with the drinking water for 2 weeks) and acute (20 mg/kg CuSO(4), s.c.) copper treatment. To evaluate the role of proteasome, its inhibitor MG132 was also used. The degree of copper-induced oxidative stress (OS), established by measuring lipid peroxidation, protein oxidation, and cellular glutathione level, as well as activities of antioxidant enzymes--catalase, superoxide dismutase, and gultathionine peroxidase, depended on the mode of copper administration. Chronic copper administration (mild oxidative stress) did not affect proteasome activities, whereas acute copper treatment (severe oxidative stress) caused a decline in chymotryptic- and tryptic-like activities. The treatment of copper-loaded animals with MG132 did not change copper-induced alterations in the tested indices, except an additional increase in protein oxidation and inhibition of glutathionine peroxidase activity. The results suggested that the in vivo copper-induced oxidative stress was associated with changes in the catalytic activity of proteasome.     

Dopaminergic modulation of oxidative stress and apoptosis in human peripheral blood lymphocytes: evidence for a D1-like receptor-dependent protective effect

Dopamine (DA) is a neurotransmitter in the central and peripheral nervous system, which can be either cytotoxic or cytoprotective under selected conditions. Such effects involve oxidative mechanisms and are likely to play a role in neurodegenerative disorders. Because increasing evidence points to peripheral blood lymphocytes (PBL) as a feasible model for studying DA-related mechanisms of cell death and survival, we have explored in these cells the effects of DA on oxidative metabolism and apoptosis. Our results show that, whereas DA 100-500 microM resulted in increased intracellular reactive oxygen species (ROS) levels and apoptotic cell death through oxidative stress, DA 0.1-5 microM decreased ROS levels and apoptosis. DA (both 1 and 500 microM) partially counteracted the decrease in Cu/Zn superoxide dismutase levels observed in untreated PBL. However, whereas the effect of the low dose lasted for the whole incubation period (24 h), the effect of DA 500 microM was transient. DA-dependent reduction of ROS levels and apoptosis was prevented by D1-like (but not D2-like) receptor antagonism. The present findings add knowledge about the sensitivity of PBL to DA and strengthen the rationale for exploiting these cells as an easily accessible peripheral model for the ex vivo investigation of oxidative stress-related dopaminergic mechanisms underlying human neurodegenerative diseases.     

Low doses of reactive oxygen species protect endothelial cells from apoptosis by increasing thioredoxin-1 expression

The redox regulator thioredoxin-1 (Trx-1) is required for the redox potential of the cell and exerts important functions in cell growth and apoptosis. Severe oxidative stress has been implicated in the oxidation of proteins and cell death. However, the role of low doses of reactive oxygen species (ROS) is poorly understood. Here, we show that 10 and 50 microM H2O2 and short-term exposure to shear stress significantly increased Trx-1 mRNA and protein levels in endothelial cells. Since it is known that Trx-1 exerts anti-apoptotic functions, we next investigated whether low doses of ROS can inhibit basal and serum-depletion induced endothelial cell apoptosis. Indeed, treatment of endothelial cells with 10 and 50 microM H2O2 significantly reduced apoptosis induction. Reduction of Trx-1 expression using an antisense oligonucleotide approach resulted in the induction of apoptosis and abolished the inhibitory effect of low doses of H2O2. Taken together, our results demonstrate that low doses of ROS act as signaling molecules and exert anti-apoptotic functions in endothelial cells via upregulation of the redox-regulator Trx-1.     

Immunohistochemical localization of TGF-B1 during morphogenetic movements of the developing mouse palate.

Transforming growth factor-beta (TGF-B1) may play an important role in developmentally active tissues in which it is found in high concentrations. We localized TGF-B1 in the developing fetal mouse palate immunohistochemically using a polyclonal antibody. Mouse fetal palates at 12-17 days (inclusive) of gestation were examined and specific focal concentrations of TGF-B1 identified regions undergoing active morphogenesis. The association of TGF-B1 with aggregates of mesenchymal cells in the palate and chondroblasts, rhabdomyocytes, and epithelia of the craniofacial complex strongly implicates its role in proliferation and differentiation in the developing mouse palate. We believe these findings have important bearing on the normal development of the palate as well as cleft anomalies.     

Effects of oxidizing and reducing agents on ovine pulmonary artery responses to nitric oxide donors, sodium nitroprusside and 3-morpholino-sydnonimine

Nitrovasodilators-sodium nitroprusside (SNP; 10(-9)-10(-4) M) and 3-morpholino-sydnonimine (SIN-1; 10(-9)-10(-4) M) produced concentration-dependent relaxation of the fourth generation sheep pulmonary artery, preconstricted with 5-hydroxytryptamine (1 microM). Oxidizing agents [oxidized glutathione (GSSG, 1 mM) and CuSO4 (5 and 20 microM)] and reducing agents [dithiothreitol (DTT, 0.1 mM), ascorbic acid (1 mM) and reduced glutathione (GSH, 1 mM)] caused opposite effects on nitric oxide (NO)-induced vasodilation in the artery. Ascorbic acid and GSH potentiated the NO responses, while GSSG and CuSO4 inhibited relaxation caused by the nitrovasodilators. DTT, however, reduced the relaxant potency and efficacy of SNP and SIN-1. Pretreatment of the pulmonary artery strips with DTT (0.1 mM) inhibited SNP (10 microM)-induced Na(+)-K(+)-ATPase activity, while ascorbic acid (1 mM) and GSH (1 mM) had no effect either on basal or SNP (10 microM)-stimulated 86Rb uptake, an index of Na(+)-K(+)-ATPase activity, in ovine pulmonary artery. The results suggest that reducing agents like ascorbic acid may have beneficial effect in improving the vascular function under oxidative stress.     

Mechanisms underlying reductant-induced reactive oxygen species formation by anticancer copper(II) compounds

Intracellular generation of reactive oxygen species (ROS) via thiol-mediated reduction of copper(II) to copper(I) has been assumed as the major mechanism underlying the anticancer activity of copper(II) complexes. The aim of this study was to compare the anticancer potential of copper(II) complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone; currently in phase II clinical trials) and its terminally dimethylated derivative with that of 2-formylpyridine thiosemicarbazone and that of 2,2′-bipyridyl-6-carbothioamide. Experiments on generation of oxidative stress and the influence of biologically relevant reductants (glutathione, ascorbic acid) on the anticancer activity of the copper complexes revealed that reductant-dependent redox cycling occurred mainly outside the cells, leading to generation and dismutation of superoxide radicals resulting in cytotoxic amounts of H₂O₂. However, without extracellular reductants only weak intracellular ROS generation was observed at IC₅₀ levels, suggesting that cellular thiols are not involved in copper-complex-induced oxidative stress. Taken together, thiol-induced intracellular ROS generation might contribute to the anticancer activity of copper thiosemicarbazone complexes but is not the determining factor.