Formation of FADD amyloid fiber and its role in immune signaling in Drosophila melanogaster / 生物工程学报

In this research, we studied the formation of Drosophila melanogaster FADD (Fas-associated death domain-containing protein) amyloid fiber and its influence on signal transduction in IMD (Immune deficiency) signaling pathway to better understand the regulation mechanism of Drosophila innate immune signaling pathway, which will provide reference for the immune regulation in other species. First, we purified dFADD protein expressed in Escherichia coli and performed Sulfur flavin T binding and transmission electron microscopy to identify the dFADD amyloid fibers formed in vitro. Then we investigated the formation of dFADD polymers in S2 cells using SDD-AGE and confocal microscope. We also constructed dFADD mutants to find out which domain is essential to fiber formation and its effect on IMD signal transduction. Our results revealed that dFADD could be polymerized to form amyloid fiber polymers in vitro and inside the cells. Formation of fibers relies on DED (Death-effector domain) domain of dFADD, since DED domain-deleted mutant existed as a monomer. Dual luciferase reporter assay showed that intact DED domain was required for the induction of downstream antimicrobial peptides, indicating that fiber formation was the key to IMD signal transduction. Our study revealed the role of dFADD in mediating the cascade between IMD and Dredd in the IMD signaling pathway by forming amyloid fibers, suggesting an evolutionarily conserved regulatory mechanism of innate immune signaling pathway.

Fluorofenidone inhibits apoptosis of renal tubular epithelial cells in rats with renal interstitial fibrosis

This study aimed to investigate the mechanism of fluorofenidone (AKF-PD) in treating renal interstitial fibrosis in rats with unilateral urinary obstruction (UUO). Thirty-two male Sprague-Dawley rats were randomly divided into sham, UUO, UUO + enalapril, and UUO + AKF-PD groups. All rats, except sham, underwent left urethral obstruction surgery to establish the animal model. Rats were sacrificed 14 days after surgery, and serum was collected for renal function examination. Kidneys were collected to observe pathological changes. Immunohistochemistry was performed to assess collagen I (Col I) protein expression, and terminal deoxynucleotidyl transferase-mediated nick end-labeling staining to observe the apoptosis of renal tubular epithelial cells. The expression of Fas-associated death domain (FADD), apoptotic protease activating factor-1 (Apaf-1), and C/EBP homologous protein (CHOP) proteins was evaluated by immunohistochemistry and western blot analysis. AKF-PD showed no significant effect on renal function in UUO rats. The pathological changes were alleviated significantly after enalapril or AKF-PD treatment, but with no significant differences between the two groups. Col I protein was overexpressed in the UUO group, which was inhibited by both enalapril and AKF-PD. The number of apoptotic renal tubular epithelial cells was much higher in the UUO group, and AKF-PD significantly inhibited epithelial cells apoptosis. The expression of FADD, Apaf-1, and CHOP proteins was significantly upregulated in the UUO group and downregulated by enalapril and AKF-PD. In conclusion, AKF-PD improved renal interstitial fibrosis by inhibiting apoptosis of renal tubular epithelial cells in rats with UUO.

Hepato-protective effect of rutin via IL-6/STAT3 pathway in CCl4-induced hepatotoxicity in rats

BACKGROUND: Carbon tetrachloride (CCl4) induces hepatotoxicity in animal models, including the increased blood flow and cytokine accumulation that are characteristic of tissue inflammation. The present study investigates the hepato-protective effect of rutin on CCl4-induced hepatotoxicity in rats. RESULTS: Forty male Wistar rats were divided into four groups. Group I (control group) received 1 mL/kg of dimethyl sulfoxide intragastrically and 3 mL/kg olive oil intraperitoneally twice a week for 4 weeks. Group II received 70 mg/ kg rutin intragastrically. Groups III and IV received CCl4 (3 mL/kg, 30 % in olive oil) intraperitoneally twice a week for 4 weeks. Group IV received 70 mg/kg rutin intragastrically after 48 h of CCl4 treatment. Liver enzyme levels were determined in all studied groups. Expression of the following genes were monitored with real-time PCR: interleukin-6 (IL-6), dual-specificity protein kinase 5 (MEK5), Fas-associated death domain protein (FADD), epidermal growth factor (EGF), signal transducer and activator of transcription 3 (STAT3), Janus kinase (JAK), B-cell lymphoma 2 (Bcl2) and B-cell lymphoma-extra-large (Bcl-XL). The CCl4 groups showed significant increases in biochemical markers of hepatotoxicity and up-regulation of expression levels of IL-6, Bcl-XL, MEK5, FADD, EGF, STAT3 and JAK compared with the control group. However, CCl4 administration resulted in significant down-regulation of Bcl2 expression compared with the control group. Interestingly, rutin supplementation completely reversed the biochemical markers of hepatotoxicity and the gene expression alterations induced by CCl4. CONCLUSION: CCl4 administration causes alteration in expression of IL-6/STAT3 pathway genes, resulting in hepatotoxicity. Rutin protects against CCl4-induced hepatotoxicity by reversing these expression changes.

Quantification of Fas protein in CSF of patients with neurocysticercosis / Quantificação da proteína Fas no LCR de pacientes com neurocisticercose

Neurocysticercosis is a parasitic disease that affects the central nervous system. The objective of this study was to investigate the correlation between neuronal death evaluated by the quantification of Fas apoptotic factor and the different evolutive forms of neurocysticercosis accompanied or not by epileptic seizures. METHODS: Cerebrospinal fluid samples from 36 patients with a diagnosis of neurocysticercosis divided into the following groups: active cystic form (n=15), 9 patients with and 6 without seizures, and calcified form (=21), 9 with and 12 without seizures. Fourteen patients comprised the control group. Fas protein concentrations were determined by ELISA. RESULTS: Only the group of patients with calcified cysts without seizures presented cerebrospinal fluid levels of Fas similar to those of the control group. Higher levels were observed for the other groups. CONCLUSIONS: The present finding suggests high cerebrospinal fluid levels of soluble Fas protein, except for patients with calcified cysts without seizures. Significant differences were observed for the group with calcified cysts and seizures, suggesting greater neuronal damage in these patients. Replacement of the term inactive cyst with reactive inactive cyst is suggested.

Neurocisticercose é uma doença parasitária que afeta o sistema nervoso central. O objetivo deste estudo foi investigar a correlação entre morte neuronal por meio da quantificação do fator apoptótico Fas e a presença de neurocisticercose nas suas diferentes fases evolutivas, acompanhadas ou não de crises epilépticas. MÉTODOS: Foram analisadas amostras de líquido cefalorraquidiano em 36 pacientes com diagnóstico de neurocisticercose, determinando-se as concentrações da proteína Fas pelo método ELISA. Foram considerados os seguintes grupos: forma cística ativa n=15 (9 com crises, 6 sem crises), forma calcificada n=21 (9 com crises, 12 sem crises) e 14 pacientes (grupo controle). RESULTADOS: Apenas o grupo com calcificações sem crises apresentou níveis de Fas semelhantes ao controle. Maiores níveis foram observados nos outros grupos. CONCLUSÕES: As formas ativa e calcificada apresentam níveis elevados da proteína Fas, exceto para as formas calcificadas sem crises. No grupo de calcificações com crise, observamos diferenças mais expressivas, sugerindo maior dano neuronal. Sugerimos a substituição da denominação "cisto inativo" por "cisto inativo reagente".

Effect of schizandrin B on H(2)O(2)-induced apoptosis of human hepatocytes in vitro: role of Fas pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of Fas pathway in H(2)O(2)-induced apoptosis of L02 human hepatocytes and the effect of schisandrin B on Fas pathway.</p><p><b>METHODS</b>Real-time quantitative PCR was used to detect the expressions of FAS, fas associated death domain protein (FADD) and caspase-8 mRNA in L02 cells exposed to H(2)O(2). Flow cytometry was employed to assess the cell apoptosis. ELISA, Western blotting and spectrophotometric assay were performed to determine the expressions of FAS protein, FADD protein and caspase-8 activity.</p><p><b>RESULTS</b>Within the dose range of 5-15 mol/L, schisandrin B dose-dependently inhibited FAS and FADD expressions and caspase-8 activation.</p><p><b>CONCLUSION</b>Schisandrin B can partially inhibit H(2)O(2)-induced L02 cell apoptosis possibly by affecting the FAS-FADD-caspase-8 pathway.</p>

Soluble Fas in pemphigus vulgaris

The Fas/Fas ligand [FasL] system has been recognized as an important pathway for apoptosis induction in cells and tissues. It has recently been shown that skin lesions of pemphigus vulgaris are associated with Fas mediated apoptosis. The aim of this study was to evaluated the level of serum soluble Fas of ten newly diagnosed patients with pemphigus vulgaris. Sera were collected from ten patients with pemphigus vulgaris. Commercial sandwich enzyme-linked immunosorbent assay [ELISA] for quantitative detection of soluble Fas was applied. Patients with mucosal skin involvement had higher median values in contrast to patients with cutaneous involvement. Elevation of soluble Fas in our study may give insights for the pathogenesis of pemphigus vulgaris. Suppression of this underlying mechanism may be an important target for novel therapies and relapse prevention

Changes of death receptor regulator expression in the articular cartilage of patients with Kashin-Beck disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the changes in the expressions of Fas-associated death domain protein (FADD) and cellular-FLICE inhibitory protein (c-FLIP) in the articular cartilage of patients with Kashin-Beck disease (KBD) and the role of these proteins in the pathogenesis of KBD.</p><p><b>METHODS</b>The cartilage samples were collected from patients with established diagnosis of KBD and osteoarthritis and from healthy control subjects undergoing amputation due to traffic accidents. The expressions of Fas-associated death domain protein (FADD) and cellular-FLICE inhibitory protein (c-FLIP) in the cartilage were detected by immunohistochemistry, and the positive chondrocytes were counted in different layers of the articular cartilage under microscope.</p><p><b>RESULTS</b>The positivity rates of FADD in the middle layer of articular cartilage from patients with KBD [(28.68∓2.19)%] and osteoarthritis [(35.40∓2.34)%] were significantly higher than that in normal cartilage [(10.51∓5.02)%, F=16.245, P=0.000], but the rates in the upper and deeper layers were comparable among the 3 groups (P=0.206-0.761). In KBD cartilage, FADD expression was the highest in the middle layer [(28.68∓5.38)%] followed by the deeper layer [(17.94∓8.38)%]. Compared with the healthy controls, KBD and osteoarthritis patients showed significantly higher FLIP expression in the upper layer of the cartilage (F=5.929, P=0.018) but similar expressions in middle and deeper layers.</p><p><b>CONCLUSIONS</b>KBD patients have significant increased FADD expression in the middle layer but decreased FLIP expression in the upper layer of the cartilage, suggesting that the death receptor pathway and its regulators play important roles in the pathogenesis of KBD.</p>

effects of resveratrol and tannic acid on apoptosis in colon adenocarcinoma cell line

To investigate the effects of resveratrol and tannic acid on apoptosis, and Bcl-2 homologous antagonist/killer [Bak] and fas associated death domain [FADD] proteins in the CaCo-2 cell line. In the present study, resveratrol and tannic acid were administrated in the CaCo-2 cell line at doses of 25, 50, and 100 uM. The CaCo-2 cells were grown and cultured in the Medical Biology Department, Eskisehir Osmangazi University, Eskisehir, Turkey in 2007. The effects of these agents on apoptotic index were determined by Apop Taq peroxidase kit and their effects on the ratios of Bak and FADD proteins by the immunohistochemical staining method at 24, 48, and 72 hours. Stained and non-stained cells in 30 separate areas of the 3 separate chamber slides, prepared for each group, were counted. The percentage of apoptosis, and Bak and FADD proteins was calculated with the control. Mean +/- standard error values were calculated for the 3 experiments. Apoptotic index, Bak protein percentage ratio, and FADD protein percentage ratio values in all groups that received tannic acid and resveratrol increased when compared within the groups. This increase was found to be time and dose independent in all parameters. Cells undergo apoptosis in 2 pathways [mitochondrial and death receptor] in resveratrol and tannic acid induced CaCo-2 cells

Expression of TNF-alpha signaling adapter proteins in peripheral blood mononuclear cells in lupus nephritis patients of different TCM asthenia syndromes / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the mRNA expressions of the TNF adapter proteins, including TNF receptor-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor-interacting protein 1 (RIP-1) and TNF receptor-associated factor-2 (TRAF-2) in peripheral blood mononuclear cells (PBMCs) of lupus nephritis (LN) patients of various TCM asthenia syndromes. Methods Fifty-one inpatients with LN were differentiated according to TCM syndrome differentiation, 13 cases of yin-deficiency with inner heat syndrome (A); 26 cases of both qi-yin deficiency syndrome (B), 12 cases of Pi-Shen yang-deficiency syndrome (C). Peripheral venous blood samples from the 51 LN patients and 17 healthy subjects were collected to separate PBMCs. The mRNA expressions of TNF adapter molecules (TRADD, FADD, RIP-1 and TRAF-2), as well as Caspase-3 and interleukin-1beta (IL-1beta) were analyzed by quantitative real-time PCR and the differences among them were compared.</p><p><b>RESULTS</b>(1) As compared with the healthy subjects, expression of TRADD mRNA in patients of syndrome A, B and C was lowered to 0.54, 0.32, and 0.38-fold, respectively (P < 0.05, P < 0.01), showing insignificant difference among the three syndromes; (2) FADD mRNA lowered to 0.79, 0.62, and 0.72-fold respectively, only with significance shown in syndrome B (P < 0.05); (3) RIP-1 mRNA lowered to 0.79, 0.50, and 0.60-fold respectively with significance shown in syndrome B and C (P < 0.01, P < 0.05), and insignificant difference was shown among the three syndromes; (4) TRAF-2 lowered to 0.70, 0.52, and 0.50-fold respectively (P < 0.01, P < 0.01, P = 0.07), significance shown in syndrome B and C (P < 0.01), but with insignificant difference among the three; (5) Caspase-3 elevated in all patients of the three syndromes (all P < 0.01); (6) IL-1beta in syndrome A was apparently lower ed to the normal range and also lower than that in the other two syndromes (both P < 0.05).</p><p><b>CONCLUSIONS</b>Expressions of TRADD, FADD, RIP-1 and TRAF-2 mRNA decreased in all the patients of various TCM asthenia syndromes, the decrement in patients of syndrome B and C was lesser than that in syndrome A. These abnormal low expressions of signal proteins might be the substantial bases for asthenia syndromes of LN patients, and the apoptotic signal mediated by them may involve in the formation of asthenia syndrome in LN.</p>

Construction of mammalian cell expression vector for pAcGFP-bFADD fusion protein and its expression in CHO-K1 cell / 生物工程学报

Fas-associated death domain (FADD) is a signal connection protein in Fas/FasL apoptotic path which might play a key role on apoptosis by transferring apoptotic signal. To reveal the intracellular signal transduction molecules involved in the procedure of follicular development in bovine ovary, we cloned FADD gene in bovine ovary tissue with RT-PCR, deleted the termination codon in its cDNA and directionally cloned the amplified FADD gene into eukaryotic expression vector pAcGFP-N1 including AcGFP, successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme Bgl II/EcoR I and sequencing, transfected pAcGFP-bFADD into CHO-K1 cell mediated by Lipofectamine 2000, observed the expression of AcGFP and detected the transcription and expression of FADD by RT-PCR and Western blotting. The results showed that the cattle FADD was successfully cloned, the pAcGFP-bFADD fusion protein recombinant plasmid was successfully constructed by introducing Bgl II, EcoR I cloning site at two ends of FADD open reading frame and inserting a Kozak sequence before start codon. AcGFP expression was detected as early as 24 h after transfection. The percentage of AcGFP positive cells reached about 65% after 24 h. A 654 bp transcription was amplified by RT-PCR, and 51.4 kD target protein was detected by Western blotting. Construction of pAcGFP-bFADD recombinant plasmid should be helpful for further understanding the mechanism of regulation of FADD on bovine oocytes formation and development.

Immunohistochemical Analysis of Fas-associated Death Domain Protein Expression in Stomach Cancers

PURPOSE: Evidence exists that dysregulation of apoptosis is involved in the pathogenesis of cancer development. Fas- associated death domain (FADD) protein, an adaptor protein of death receptors, is a critical regulatory component of the extrinsic cell- death pathway that exerts its pro-apoptotic effect upon binding with death receptors. Expression of the FADD protein has not been reported in stomach cancer. The aim of this study was to explore the expression status of the FADD protein in stomach cancers. MATERIALS AND METHODS: In the current study, we analyzed the expression of the FADD protein in 60 advanced stomach cancer by using immunohistochemistry and a tissue microarray approach. RESULTS: Immunopositivity (defined as > or =30%) was observed for the FADD protein in 23 (38%) of the 60 cancers. Normal gastric mucosal cells showed expression of the FADD protein. CONCLUSION: Taken together, these results indicate that decreased expression of the FADD protein is a frequent event in stomach cancers and suggest that to avoid apoptosis, stomach cancer cells in vivo may need loss of FADD expression, which might contribute to tumor development.