Investigating noise tolerance in an efficient engine for inferring biological regulatory networks.

Biological systems are composed of biomolecules such as genes, proteins, metabolites, and signaling components, which interact in complex networks. To understand complex biological systems, it is important to be capable of inferring regulatory networks from experimental time series data. In previous studies, we developed efficient numerical optimization methods for inferring these networks, but we have yet to test the performance of our methods when considering the error (noise) that is inherent in experimental data. In this study, we investigated the noise tolerance of our proposed inferring engine. We prepared the noise data using the Langevin equation, and compared the performance of our method with that of alternative optimization methods.

Endothelin-1 (ET-1) causes death of retinal neurons through activation of nitric oxide synthase (NOS) and production of superoxide anion.

Endothelin-1 (ET-1) is the most potent and long-acting vasoconstricting peptide presently known. In addition to its vascular effects, endothelin signaling pathway exists in the central nervous system (CNS), which is deeply related to neuronal degeneration. In the present study, we evaluated the effect of ET-1 on death of retinal neurons consisting mainly of amacrine cells, and its interaction with nitric oxide synthase (NOS) and superoxide production. Cultured retinal neurons from fetal rats were exposed to various doses of ET-1 (0.1, 1.0, 10 and 100nM). Neuronal toxicity of ET-1 was assessed by trypan blue exclusion, Hoechst 33,258 staining and TUNEL assay at different times. Intracellular levels of nitric oxide (NO), superoxide and peroxynitrite were determined semiquantitatively by DAF2-DA, hydroethidine and dihydrorhodamine-123, respectively. The effects of adding SOD (100U/ml) and L-NAME with ET-1 on these changes were evaluated. In addition, the receptor mechanisms involved in these reactions were determined by BQ-123 and BQ-788, receptor antagonists for ET A and ET B receptors, respectively. Exposure of cultured retinal neurons to ET-1 reduced the percentage of living cells in a dose- and time-dependent way, and the percentage of living cells was significantly increased by addition of SOD and L-NAME. Fluorometric analyses revealed that ET-1 increased the intracellular NO level in a dose- and time-dependent manner. The intracellular superoxide and peroxynitrite levels were also significantly increased 24h after incubation with 100nM of ET-1, and this elevation was suppressed by SOD and L-NAME. These ET-1-induced alterations were significantly suppressed when both BQ-123 and BQ-788 were added simultaneously with ET-1 to the medium. These results indicate that the neuronal death caused by ET-1 is most likely mediated by the activation of NOS in association with the formation of superoxides and peroxynitrite.

Effect of P2X7 receptor activation on the retinal blood velocity of diabetic rabbits.

OBJECTIVE: To test the effect of activation of P2X7 receptors on retinal blood velocity in diabetic rabbits. METHODS: Immunohistochemical analysis was performed on healthy and diabetic rabbit eyes using P2X7 receptor antibodies. Diabetes was induced using alloxan. Retinal blood velocity was measured with the laser speckle circulation analyzer. Visual function was assessed using electroretinography. RESULTS: Cells in inner retinal layers were positive for anti-P2X7 receptor antibodies in healthy rabbits. The distribution of positive cells extended to outer layers and some small vessels stained in diabetic rabbits. When assayed 24 hours after an intravitreal injection of 150 nmol of benzoylbenzoyl adenosine triphosphate (BzATP), a P2X7 agonist, the retinal blood velocity in healthy rabbits was reduced by approximately 30%; this reduction continued for at least 4 weeks. Only in diabetic rabbits did an injection of 50 nmol of BzATP reduce retinal blood velocity by approximately 30% and the amplitudes of electroretinography a waves, b waves, and oscillatory potentials for at least 4 weeks. CONCLUSIONS: Soon after the onset of alloxan-induced diabetes, retinal blood velocity and function become more vulnerable to reduction initiated through P2X7 receptors. CLINICAL RELEVANCE: Our findings support the hypothesis that the retinal circulation disorder accelerated by activation of P2X7 receptors may be involved in the early changes of diabetic retinopathy.

Endothelin-1 enhances glutamate-induced retinal cell death, possibly through ETA receptors.

PURPOSE: To determine the modification of the glutamate-induced death of retinal neurons by endothelin (ET)-1. METHODS: Cultured retinal neurons from fetal rats were exposed to glutamate (1.0 mM) alone or glutamate with ET-1 (10(-10)-10(-7)M) for 10 minutes. Neuronal death was assessed by the trypan blue exclusion or TUNEL assays at 2, 6, and 24 hours after the exposure. The effects of adding BQ-123 or BQ-788, ET(A), and ET(B) receptor antagonists, respectively, in combination with ET-1 was also assessed. RESULTS: Immunohistochemical analyses showed that the ETs as well as ET(A) and ET(B) receptors were expressed on cultured retinal neurons consisting mainly of amacrine cells. A brief exposure of the cultured retinal neurons to glutamate alone significantly increased the number of dead cells, and the addition of ET-1 with glutamate caused a further significant increase in retinal neuronal death compared with the cells exposed to glutamate alone. A significant increase in neuronal death was detected at doses of 10 nM of ET-1 and higher after a 24-hour exposure (P < 0.05, Dunnett), whereas brief exposure of neurons to up to 1 microM ET-1 alone did not cause delayed cell death of neurons. BQ-123 (10 nM) suppressed the enhancement of retinal toxicity caused by ET-1 (10 nM), whereas BQ-788 had no significant effect. CONCLUSIONS: These results indicate that ET-1 enhances glutamate-induced retinal cell death, possibly through ET(A) receptors. ET-1 may act synergistically with glutamate to damage retinal neurons under hypoxic conditions.

Advanced glycation end products induce death of retinal neurons via activation of nitric oxide synthase.

The purpose of this study was to determine whether advanced glycation end products (AGEs) are neurotoxic for cultured retinal neurons consisting mainly of amacrine cells, and to determine whether endogenous nitric oxide (NO) is involved in the toxicity. Cultured retinal neurons obtained from fetal Wistar rats (gestational age 19 days) were maintained in culture for 10 days, and then exposed to different concentrations of AGEs (0.02, 0.1, and 0.5 mg ml(-1)) in cultured media for different lengths of time. Both trypan blue exclusion and TUNEL assay were used to determine whether AGEs were neurotoxic, and NG-nitro-L-arginine methyl ester (L-NAME, 500 microM), a nitric oxide synthase (NOS) inhibitor, was used to determine whether NO was involved. Immunohistochemical analyses were performed to determine whether specific receptors of AGEs (RAGE) are present on cultured retinal neurons; caspase-3 was activated, and 3-nitrotyrosine was expressed on neurons treated with AGEs. Nitrite levels were measured in the supernatants of the media where neurons were incubated with AGEs. AGEs induced cell death in a time- and dose-dependent manner. TUNEL-positive cells and immunoreactivity to cleaved caspase-3 were enhanced on neurons following exposure to AGEs. L-NAME significantly suppressed the AGEs-induced neurotoxicity as assessed by both trypan blue exclusion and TUNEL assays. Activation of NOS was suggested by enhanced immunoreactivity to 3-nitrotyrosine on neurons and increased nitrite levels in the media incubated with AGEs. These results indicate that AGEs are neurotoxic to retinal neurons in culture through the activation of NOS. Apoptotic pathways may be in part involved in the death of the neurons.