Neuroprotective Effects of Withania somnifera on 4-Hydroxynonenal Induced Cell Death in Human Neuroblastoma SH-SY5Y Cells Through ROS Inhibition and Apoptotic Mitochondrial Pathway

Neurochemical Research - The antioxidant, anti-inflammatory, and anticancer activities of Withania somnifera (WS) are known for a long time. This study was aimed to examine whether WS also...     

Rotenone-induced oxidative stress and apoptosis in human liver HepG2 cells

Rotenone, a commonly used pesticide, is well documented to induce selective degeneration in dopaminergic neurons and motor dysfunction. Such rotenone-induced neurodegenration has been primarily suggested through mitochondria-mediated apoptosis and reactive oxygen species (ROS) generation. But the status of rotenone induced changes in liver, the major metabolic site is poorly investigated. Thus, the present investigation was aimed to study the oxidative stress-induced cytotoxicity and apoptotic cell death in human liver cells-HepG2 receiving experimental exposure of rotenone (12.5–250 μM) for 24 h. Rotenone depicted a dose-dependent cytotoxic response in HepG2 cells. These cytotoxic responses were in concurrence with the markers associated with oxidative stress such as an increase in ROS generation and lipid peroxidation as well as a decrease in the glutathione, catalase, and superoxide dismutase levels. The decrease in mitochondrial membrane potential also confirms the impaired mitochondrial activity. The events of cytotoxicity and oxidative stress were found to be associated with up-regulation in the expressions (mRNA and protein) of pro-apoptotic markers viz., p53, Bax, and caspase-3, and down-regulation of anti-apoptotic marker Bcl-2. The data obtain in this study indicate that rotenone-induced cytotoxicity in HepG2 cells via ROS-induced oxidative stress and mitochondria-mediated apoptosis involving p53, Bax/Bcl-2, and caspase-3.     

Neuropeptide-induced transactivation of a neuronal epidermal growth factor receptor is mediated by metalloprotease-dependent formation of heparin-binding epidermal growth factor

Numerous external stimuli, including G protein-coupled receptor agonists, cytokines, growth factors, and steroids activate mitogen-activated protein kinases (MAPKs) through phosphorylation of the epidermal growth factor receptor (EGF-R). In immortalized hypothalamic neurons (GT1-7 cells), agonist binding to the gonadotropin-releasing hormone receptor (GnRH-R) causes phosphorylation of MAPKs that is mediated by protein kinase C (PKC)-dependent transactivation of the EGF-R. An analysis of the mechanisms involved in this process showed that GnRH stimulation of GT1-7 cells causes release/shedding of the soluble ligand, heparin binding epidermal growth factor (HB-EGF), as a consequence of metalloprotease activation. GnRH-induced phosphorylation of the EGF-R and, subsequently, of Shc, ERK1/2, and its dependent protein, p90RSK-1 (p90 ribosomal S6 kinase 1 or RSK-1), was abolished by metalloprotease inhibition. Similarly, blockade of the effect of HB-EGF with the selective inhibitor CRM197 or a neutralizing antibody attenuated signals generated by GnRH and phorbol 12-myristate 13-acetate, but not those stimulated by EGF. In contrast, phosphorylation of the EGF-R, Shc, and ERK1/2 by EGF and HB-EGF was independent of PKC and metalloprotease activity. The signaling characteristics of HB-EGF closely resembled those of GnRH and EGF in terms of the phosphorylation of EGF-R, Shc, ERK1/2, and RSK-1 as well as the nuclear translocation of RSK-1. However, neither the selective Src kinase inhibitor PP2 nor the overexpression of negative regulatory Src kinase and dominant negative Pyk2 had any effect on HB-EGF-induced responses. In contrast to GT1-7 cells, human embryonic kidney 293 cells expressing the GnRH-R did not exhibit metalloprotease induction and EGF-R transactivation during GnRH stimulation. These data indicate that the GnRH-induced transactivation of the EGF-R and the subsequent ERK1/2 phosphorylation result from ectodomain shedding of HBEGF through PKC-dependent activation of metalloprotease(s) in neuronal GT1-7 cells.     

Synthesis,characterization, and in vitro antimicrobial activities of 5-alkenyl/hydroxyalkenyl-2-phenylamine-1,3,4-oxadiazoles and thiadiazoles

The long-chain alkenoic acid hydrazides (1a–d) on reaction with phenylisocyanate and phenylthiocyanate gave their corresponding semicarbazides (2a–d) and thiosemicarbazides (4a–d), which on further refluxing with POCl₃ and Ac₂O yielded corresponding 1,3,4-oxadiazoles (3a–d) and thiadiazoles (5a–d), respectively.The structure elucidation of synthesized compounds is based on the elemental analysis and spectral data (IR, ¹H NMR, ¹³C NMR and MS). The synthesized oxadiazoles and thiadiazoles have been screened for antibacterial and antifungal activities. The investigation of antimicrobial screening revealed that compounds 3c, 3d, 5c, 5d and compounds 3b, 5b, showed good antibacterial and antifungal activities, respectively.     

Redistribution and Phosphorylation of Occludin During Opening and Resealing of Tight Junctions in Cultured Epithelial Cells

We studied the expression, distribution, and phosphorylation of the tight junction (TJ) protein occludin in confluent MDCK cell monolayers following three procedures for opening and resealing of TJs. When Ca²⁺ is transiently removed from the culture medium, the TJs open and the cells separate from each other, but the occludin band around each cell is retained. When Ca²⁺ is reintroduced, the TJs reseal. When the monolayers are exposed to prolonged Ca²⁺ starvation the cells maintain contact, but occludin disappears from the cell borders and can be detected only in a cytoplasmic compartment. When Ca²⁺ is reintroduced, new TJs are assembled and the transepithelial electrical resistance (TER) is reestablished in about 20 hr. Monolayers treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) show a different pattern of TJ opening: the cell-cell contact is maintained but the TJ strand network, as seen in freeze-fracture replicas, becomes discontinuous. Occludin is still localized at the cell periphery, but in a pattern of distribution that matches the discontinuous TJ. These TJs do not reseal even 24 hr after removal of the TPA. Western blot analysis showed that the 62–65 kD double band of occludin did not change with these treatments. However, in vivo phosphorylation analysis showed that the TPA treatment reduced the phosphorylation levels of occludin, while the prolonged Ca²⁺ starvation completely dephosphorylated the two occludin bands. In addition, a highly phosphorylated 71 kD band that immunoprecipitates with occludin is not present when TJ is opened by the Ca²⁺ removal. Phosphoaminoacid analysis showed that the 62–65 kD occludin bands are phosphorylated on serine and threonine, while the 71 kD band was phosphorylated exclusively on serine. Our results provide further evidence that phosphorylation of occludin is an important step in regulating TJ formation and permeability. Received: 28 December 1998/Revised: 8 April 1999     

Chemical composition and antimicrobial,antioxidant, and anti-inflammatory activities of Lepidium sativum seed oil

Lepidium sativum (garden cress) seed oil was examined for its antimicrobial, antioxidant, and anti-inflammatory activities. The oil was obtained by hydrodistillation, where gas chromatography coupled with mass spectrometry that utilized to study its chemical composition. Microdilution method was used to test the antimicrobial effect of oil against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella pneumoniae, and Candida albicans. The antioxidant activity was assessed by radical scavenging activity assay using 2,2-diphenyl-1-picrylhydrazyl radical. The major constituents found in the oil were 7,10-hexadecadienoic acid, 11-octadecenoic acid, 7,10,13-hexadecatrienoic acid, and behenic acid. The minimum inhibitory concentration (MIC) against all pathogens was 47.5 mg/ml, except for Salmonella enterica, which showed MIC of 90 mg/ml. The oil demonstrated antioxidant activity in a dose dependent pattern, with a half maximal inhibitory concentration (IC₅₀) value of 40 mg/ml, and exerted anti-inflammatory activity, wherein 21% protection was shown at a concentration of 300 μg/ml. Thus, L. sativum seed oil shows antimicrobial, antioxidant, and anti-inflammatory properties.     

Small Synthetic Peptides Homologous to Segments of the First External Loop of Occludin Impair Tight Junction Resealing

This study shows that resealing of opened tight junctions (TJs) is impaired by interaction with oligopeptides homologous to the external domain of chick occludin. The experiments were carried out with confluent A6 cell monolayers grown on collagen supports under stable transepithelial electrical resistance (TER). The monolayers were bathed on the apical side with a 75 mm KCl solution and on the basolateral side by NaCl-Ringer's solution. TJ opening was induced by basolateral Ca²⁺ removal and was characterized by a marked drop of TER. The reintroduction of Ca²⁺ triggered junction resealing as indicated by an elevation of TER to control values. Custom-made peptides SNYYGSGLSY (corresponding to the residues 100 to 109) and SNYYGSGLS (residues 100 to 108), homologous to segments of the first external loop of chick occludin molecule, impaired junction resealing when the peptides were included in the apical bathing fluid (concentrations in the range of 0.5 to 1.5 mg/ml). Peptide removal from the apical solution usually triggered a slow recovery of TER, indicating a slow recovery of the TJ seal. Changes in localization of ZO-1, a cytoplasmic protein that underlies the membrane at the TJs, were evaluated immunocytochemically following Ca²⁺ removal and reintroduction. The presence or absence of the oligopeptides showed no influence on the pattern of change of ZO-1 localization. These observations support the hypothesis that the TJ seal results from the interaction of specific homologous segments of occludin on the surface of adjacent cells. Additionally, our results show that small peptides homologous to segments of the occludin first external loop can be used as specific reagents to manipulate the permeability of tight junctions. Received: 4 December 1998/Revised: 22 January 1999     

Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation

The duration as well as the magnitude of mitogen-activated protein kinase activation has been proposed to regulate gene expression and other specific intracellular responses in individual cell types. Activation of ERK1/2 by the hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) is relatively sustained in alpha T3-1 pituitary gonadotropes and HEK293 cells but is transient in immortalized GT1-7 neurons. Each of these cell types expresses the epidermal growth factor receptor (EGFR) and responds to EGF stimulation with significant but transient ERK1/2 phosphorylation. However, GnRH-induced ERK1/2 phosphorylation caused by EGFR transactivation was confined to GT1-7 cells and was attenuated by EGFR kinase inhibition. Neither EGF nor GnRH receptor activation caused translocation of phospho-ERK1/2 into the nucleus in GT1-7 cells. In contrast, agonist stimulation of GnRH receptors expressed in HEK293 cells caused sustained phosphorylation and nuclear translocation of ERK1/2 by a protein kinase C-dependent but EGFR-independent pathway. GnRH-induced activation of ERK1/2 was attenuated by the selective Src kinase inhibitor PP2 and the negative regulatory C-terminal Src kinase in GT1-7 cells but not in HEK293 cells. In GT1-7 cells, GnRH stimulated phosphorylation and nuclear translocation of the ERK1/2-dependent protein, p90RSK-1 (RSK-1). These results indicate that the duration of ERK1/2 activation depends on the signaling pathways utilized by GnRH in specific target cells. Whereas activation of the Gq/protein kinase C pathway in HEK293 cells causes sustained phosphorylation and translocation of ERK1/2 to the nucleus, transactivation of the EGFR by GnRH in GT1-7 cells elicits transient ERK1/2 signals without nuclear accumulation. These findings suggest that transactivation of the tightly regulated EGFR can account for the transient ERK1/2 responses that are elicited by stimulation of certain G protein-coupled receptors.     

Activation and nuclear translocation of PKCdelta,Pyk2 and ERK1/2 by gonadotropin releasing hormone in HEK293 cells

The mechanism of agonist-induced activation of Pyk2 and its relationship with ERK1/2 phosphorylation was analyzed in HEK293 cells stably expressing the gonadotropin releasing hormone (GnRH) receptor. GnRH stimulation caused rapid and sustained phosphorylation of ERK1/2 and Pyk2 that was accompanied by their nuclear translocation. Pyk2 was also localized on cell membranes and at focal adhesions. Dominant negative Pyk2 (PKM) had no effect on GnRH-induced ERK1/2 phosphorylation and c-fos expression. These actions of GnRH on ERK1/2 and Pyk2 were mimicked by activation of protein kinase C (PKC) and were abolished by its inhibition. GnRH caused translocation of PKC and δ, but not of , ι and λ, to the cell membrane, as well as phosphorylation of Raf at Ser338, a major site in the activation of MEK/ERK1/2. Stimulation of HEK293 cells by EGF caused marked ERK1/2 phosphorylation that was attenuated by the selective EGFR receptor (EGF-R) kinase inhibitor, AG1478. However, GnRH-induced ERK1/2 activation was independent of EGF-R activation. These results indicate that activation of PKC is responsible for GnRH-induced phosphorylation of both ERK1/2 and Pyk2, and that Pyk2 activation does not contribute to GnRH signaling. Moreover, GnRH-induced phosphorylation of ERK1/2 and expression of c-fos in HEK293 cells is independent of Src and EGF-R transactivation, and is mediated through the PKC/Raf/MEK cascade.     

Petroselinum sativum protects HepG2 cells from cytotoxicity and oxidative stress induced by hydrogen peroxide

Molecular Biology Reports - A number of liver diseases are known to be caused by oxidative stress. Petroselinum sativum (P. sativum; parsley) is popular for its anti-inflammatory, antimicrobial,...