Inhibitory Activity of Cordyceps bassiana Extract on LPS-induced Inflammation in RAW 264.7 Cells by Suppressing NF-κB Activation

Cordyceps bassiana has long been used as an oriental medicine and reported to possess diverse biological activities. The fruiting bodies of Cordyceps bassiana was extracted with ethanol and then further fractionated with n-hexane, ethyl acetate, n-butanol and water. The butanol fraction from Cordyceps bassiana (CBBF) exhibited the most effective in anti-inflammatory activity in RAW 264.7 macrophages and the roles of CBBF on the anti-inflammation cascade in LPS-stimulated RAW 264.7 cells were studied. To investigate the mechanism by which CBBF inhibits NO, iNOS and COX-2, the activation of IκB and MAPKs in LPS-activated macrophage were examined. Our present results demonstrated that CBBF inhibits NO production and iNOS expression in LPS-stimulated RAW 264.7 macrophage cells, and these effects were mediated through the inhibition of IκB-α, JNK and p38 phosphorylation. Also, CBBF suppressed activation of MAPKs including p38 and SAPK/JNK. Furthermore, CBBF significantly suppressed LPS-induced intracellular ROS generation. Its inhibition on iNOS expression, together with its antioxidant activity, may support its anti-inflammatory activity. Thus Cordyceps bassiana can be used as a useful medicinal food or drug for further studies.

(E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone displays suppression of inflammatory responses via inhibition of Src, Syk, and NF-kappaB

(E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone (MPP) is an aldol condensation product resulting from pyrrole-2-carbaldehyde and m- and p- substituted acetophenones. However, its biological activity has not yet been evaluated. Since it has been reported that some propenone-type compounds display anti-inflammatory activity, we investigated whether MPP could negatively modulate inflammatory responses. To do this, we employed lipopolysaccharide (LPS)-stimulated macrophage-like RAW264.7 cells and examined the inhibitory levels of nitric oxide (NO) production and transcriptional activation, as well as the target proteins involved in the inflammatory signaling cascade. Interestingly, MPP was found to reduce the production of NO in LPS-treated RAW264.7 cells, without causing cytotoxicity. Moreover, this compound suppressed the mRNA levels of inflammatory genes, such as inducible NO synthase (iNOS) and tumor necrosis factor (TNF)-alpha. Using luciferase reporter gene assays performed in HEK293 cells and immunoblotting analysis with nuclear protein fractions, we determined that MPP reduced the transcriptional activation of nuclear factor (NF)-kappaB. Furthermore, the activation of a series of upstream signals for NF-kappaB activation, composed of Src, Syk, Akt, and IkappaBalpha, were also blocked by this compound. It was confirmed that MPP was able to suppress autophosphorylation of overexpressed Src and Syk in HEK293 cells. Therefore, these results suggest that MPP can function as an anti-inflammatory drug with NF-kappaB inhibitory properties via the suppression of Src and Syk.

Pyrrole-Derivative of Chalcone, (E)-3-Phenyl-1-(2-Pyrrolyl)-2-Propenone, Inhibits Inflammatory Responses via Inhibition of Src, Syk, and TAK1 Kinase Activities

(E)-3-Phenyl-1-(2-pyrrolyl)-2-propenone (PPP) is a pyrrole derivative of chalcone, in which the B-ring of chalcone linked to β-carbon is replaced by pyrrole group. While pyrrole has been studied for possible Src inhibition activity, chalcone, especially the substituents on the B-ring, has shown pharmaceutical, anti-inflammatory, and anti-oxidant properties via inhibition of NF-κB activity. Our study is aimed to investigate whether this novel synthetic compound retains or enhances the pharmaceutically beneficial activities from the both structures. For this purpose, inflammatory responses of lipopolysaccharide (LPS)-treated RAW264.7 cells were analyzed. Nitric oxide (NO) production, inducible NO synthase (iNOS) and tumor necrosis factor-α (TNF-α) mRNA expression, and the intracellular inflammatory signaling cascade were measured. Interestingly, PPP strongly inhibited NO release in a dose-dependent manner. To further investigate this anti-inflammatory activity, we identified molecular pathways by immunoblot analyses of nuclear fractions and whole cell lysates prepared from LPS-stimulated RAW264.7 cells with or without PPP pretreatment. The nuclear levels of p50, c-Jun, and c-Fos were significantly inhibited when cells were exposed to PPP. Moreover, according to the luciferase reporter gene assay after cotransfection with either TRIF or MyD88 in HEK293 cells, NF-κB-mediated luciferase activity dose-dependently diminished. Additionally, it was confirmed that PPP dampens the upstream signaling cascade of NF-κB and AP-1 activation. Thus, PPP inhibited Syk, Src, and TAK1 activities induced by LPS or induced by overexpression of these genes. Therefore, our results suggest that PPP displays anti-inflammatory activity via inhibition of Syk, Src, and TAK1 activity, which may be developed as a novel anti-inflammatory drug.

Protective Effect of Fibroin BF-7 on Neuronal Cell Death in Alzheimer Model using Amyloid beta Peptide / 대한해부학회지

Factors such as senescence,stress and neurodegenerative diseases,including Alzheimer's disease, contribute to the impairment of organs,especially the brain.They also negatively affect normal brain functions such as memory and cognition.In this study,the neuroprotective role of the natural product BF-7 was examined against A beta - induced neurotoxicity in SK-N-SH human neuronal cells.BF-7 significantly attenuated A beta-induced apoptosis as measured by intracellular calcium levels,accumulation of reactive oxygen species,mitochondrial dysfunction,and caspase activity.These results strongly indicate that BF-7 plays an effective and positive role in the improvement of brain functions,including learning and memory,in our model system for Alzheimer's disease.Thus,BF-7 might be useful for developing strategies to protect the nervous system and improve brain function.

Expression of Protoporphyrin IX Induced by Liposome delta-5-aminolevulinic Acid in Cultured Sebocytes and Hair Organs / 대한피부과학회지

BACKGROUND: Photodynamic therapy with topical delta-5-aminolevulinic acid (ALA) has been introduced for effective treatment of facial acne. Topical routes for ALA have been explored via a photosensitizer delivery system. OBJECTIVE: We performed this study to evaluate absorption of liposome ALA (Lipo-ALA) into the sebocytes of cultured sebaceous glands and hair organs. METHODS: We cultured KGM sebocytes that were derived from human sebaceous glands. In addition, we cultured William E media pilosebaceous hair units that were isolated from the occipital scalp. Lipo-ALA (10 microgram/ml) and ALA (10 microgram/ml) were added to the 1 ml KGM containing sebocytes for 24 hours and the 1 ml William E media containing pilosebaceous hair units for 6, 24, 48 and 72 hours, respectively. RESULTS: Absorption of Lipo-ALA into the sebocytes of cultured sebaceous gland and hair organs was higher than that of ALA. CONCLUSION: Lipo-ALA may be considered as an effective photosensitizer in photodynamic therapy for facial acne.

Fatigue life estimation of implant using a finite element method / 대한치과보철학회지

Purpose: The purpose of this study is to use finite element analysis to predict the fatigue life of an implant system subjected to fatigue load by mastication (chewing force). The reliability and the stability of implant system can be defined in terms of the fatigue strength. Not only an implant is expensive but also it is almost impossible to correct after it is inserted. From a bio-engineering standpoint, the fatigue strength of the dental implant system must be evaluated by simulation (FEA). Material and Methods: Finite element analysis and fatigue test are performed to estimate the fatigue strength of the implant system. Mesh of implant is generated with the actual shape and size. In this paper, the fatigue strength of implant system is estimated: U-fit (T.Strong, Korea, internal type). The stress field in implant is calculated by elastic-plastic finite element analysis. The equivalent fatigue stress, considering the contact and preload stretching of a screw by torque for tightening an abutment, is obtained by means of Sine's method. To evaluate the reliability of the calculated fatigue strength, fatigue test is performed. Results: A comparison of the calculated fatigue strength with experimental data showed the validity and accuracy of the proposed method. The initiation points of the fatigue failure in the implant system exist in the region of high equivalent fatigue stress values. Conclusion: The above proposed method for fatigue life estimation can be applied to other configurations of the differently designed and improved implant. In order to prove reliability of prototype implant, fatigue test should be executed. The proposed method is economical for the prediction of fatigue life because fatigue testing, which is time consuming and precision-dependent, is not required.

Study on the Mitochondrial Dysfunction by p53 Regulation in Ceramide-induced Neuronal Cell Death / 체질인류학회지

Ceramide induces cell death in a dose- and time-dependent manner in neuroblastoma SK-N-SH cells. To investigate the mechanism of SK-N-SH cell death by C2-ceramide, morphological features and Hoechst 33258 staining were analyzed. In these morphlogic study the cell death by ceramide showed typical apoptotic features, nuclear condensation, fragmentation, and membrane blebbing. Ceramide-induced apoptosis was accompanied by nuclear accumulation of p53. Inhibition of p53 expression with p53 antisense oligonucleotides inhibited apoptosis evoked by ceramide. Also, ceramide induced mitochondrial event, collapse of mitochondrial membrane potential (delta psi m) and interestingly, inhibition of p53 attenuated collapse of mitochondrial membrane potential, suggests that ceramide induces mitochondrial dysfunction through upregulation of p53 expression. These results suggest that ceramide-induced apoptosis is dependent upon increase in cellular p53 levels which play a critical role in the regulation of apoptotic cell death and p53 modulates mitochondrial function such as mitochondrial membrane potential level.

FKBP-12 Exhibits an Inhibitory Activity on Calcium Oxalate Crystal Growth in Vitro

Urolithiasis and calcium oxalate crystal deposition diseases are still significant medical problems. In the course of nephrocalcin cDNA cloning, we have identified FKBP-12 as an inhibitory molecule of calcium oxalate crystal growth. lambdagt 11 cDNA libraries were constructed from renal carcinoma tissues and screened for nephrocalcin cDNA clones using anti-nephrocalcin antibody as a probe. Clones expressing recombinant proteins, which appeared to be antigenically cross-reactive to nephrocalcin, were isolated and their DNA sequences and inhibitory activities on the calcium oxalate crystal growth were determined. One of the clone lambdagt 11 #31-1 had a partial fragment (80 bp) of FKBP-12 cDNA as an insert. Therefore, a full-length FKBP-12 cDNA was PCR-cloned from the lambdagt 11 renal carcinoma cDNA library and was subcloned into an expression vector. The resultant recombinant FKBP-12 exhibited an inhibitory activity on the calcium oxalate crystal growth (Kd=10(-7) M). Physiological effect of the extracellular FKBP-12 was investigated in terms of macrophage activation and proinflammatory cytokine gene induction. Extracellular FKBP-12 failed to activate macrophages even at high concentrations. FKBP-12 seems an anti-stone molecule for the oxalate crystal deposition disease and recurrent stone diseases.

Early Detection of Metastasis by Immunohistochemistry in Uterine Cervical Carcinoma

BACKGROUND: Adhesion molecules are important in the maintenance of normal epithelial structure, and altered expression of these molecules may be important in epithelial tumors, particularly in the processes of invasion and metastasis. METHODS: We have examined the expression of E-cadherin, cathepsin-D, CD44, CD44v6, nm23 and transforming growth factor-1 (TGF-1) proteins in the cervical squamous cell carcinoma to evaluate the prognostic significance of these molecules. RESULTS: Immunostain for E-cadherin was highly expressed in the majority of cases of cervical carcinomatous lesions (85.7-100%), but cathepsin-D was very low (7.1-32%). Immunostain for CD44 showed a lower expression in invasive carcinoma with and without metastasis (50.4 and 52.2%) than in carcinoma in situ (68.0%). CD44v6 protein showed some controversy of expression between invasive carcinoma with metastasis (35.7%) without metastasis (56.5%). Staining for nm23 was observed in the high expression of invasive lesions (85.7%). TGF-1 and C-erbB-2 protein were highly expressed, especially in the microinvasive carcinoma (81.8%, 42.8%, respectively). CONCLUSIONS: These results suggest that CD44 and CD44v6 were not highly expressed in the invasive squamous carcinoma of the uterine cervix. However, it is notable that TGF-1 and c-erbB-2 in the microinvasive carcinoma and nm23 in invasive carcinoma were highly expressed compared to these of the other lesions of the uterine cervix.

Production of IL-15 and lts Functional Study in Mouse Splenocyte Activation / 대한면역학회지

After the synthesis of IL-15 cDNA from the total RNA of mouse spleen, it was inserted into the prokaryotic expression vector, pRseta, and eukaryotic expression vector, pcDNA3.0, respectively. Subsequently, the insertion of gene and open reading frame were confirmed by sequencing of each plasmid, respectively. Using pRseta- IL-15 plasmid, the recombinant IL-15 protein was induced by IPTG under BL21 (DE 3) host cells and recombinant IL-15 was expressed at 14.5 KDa with time. Then, IL- 15 was separated by His-tag affinity chromatography and analyzed by SDS-PAGE to yield soluble IL-15 at 14.5 KDa as monomer and 29.0 KDa as dimer. In order to inspect the function and contribution of IL-15, the in vitro experiment was established using mononuclear cells separated from the mouse spleen. After 48h exposure of PHA to mouse splenocyte and 24h treatment with recombinant IL-15, the effects of cytokine inductions inspected against IL-2, IL-6, IL-10, IL-12, IFN-r, and GM-CSF. The results showed that comparing with the control, IL-6 increased, IL-2, IL-12 and IFN-r increased and similar, and GM-CSF decreased. In addition, the direct injection of pcDNA3.0-IL-15 plasmid into mice gave the similar results to in vitro studies. Namely, IL-6 and IL-12 increased, and IL-2, IFN-r and GM-CSF were similar or decreased. IL-10 was not induced in in vitro and in vivo experiments. These results suggested that the IL-15 induce the splenocyte activation and can be an important factor in proliferation and fuction recovery of weakened T-cell.

Detection of Chemokine Gene Expression Induced by IL-12/IL-2 in Renca Tumor / 대한면역학회지

In order to evaluate antitumor rnechanisms of interleukin (IL)-12/IL-2 that has been shown significant tumor suppressive activity on established primary and metastatic Renca tumor, we studied chemokine gene expression induced by direct action of IL- 12/IL-2 or cytokine cascade. IL-12/IL-2 induced gene expression of interferon gamma (IFN-r) and granulocyte monocyte-colony stimulating factor (GM-CSF) in splenocytes, and it induced gene expression of monokine induced by IFN-r (Mig), interferon inducible protein 10 (IP- 10), SDF-1, macrophage inflammatory protein (MIP)-1a, MIP-1B, MIP-2, monocyte chemotactic protein (MCP)-1, and Rantes in tumor mass. However IL-12/IL-2 could not induce these chemokines in tumor mass of GKO mice and Renca cell in vitro. IL- 12 also did not increased chemokine gene expression in Renca cell in vitro, but IFN-r induced gene expression of Mig, IP-10, MCP-1 in Renca cell in vitro. In the chemotaxis assay, culture supernatant of Renca cell stimulated with IFN-r increased splenocyte migration in vitro. All these data suggest IL-12/IL-2 can induce IFN-r-chemokine cascade in tumor mass, and Mig, IP-10, MCP-1 produced from tumor cell may play an important role for initial immune cell migration into tumor mass.

The Effect of GTP Cyclohydrolase I on the Activity and Expression of Tyrosine Hydroxylase and Cell Growth

BACKGROUND: Parkinson's Disease (PD) is a progressive neurodegenerative disorder characterized by resting tremor, rigidity, and bradykinesia. L-3,4-dihydroxyphenylalanine (L-dopa) has been used for over last 3 decades to treat this disorder, however, its usage is limited due to the reducing effectiveness on time and severe side effects. The best strategy for treating this disorder without serious side effects would be to keep a constant level of dopamine in the brain. This could be achieved by gene or cell therapy using gene(S) involved in dopamine biosynthesis or cells from other individual. For Parkinson's gene therapy, however, there still are controversies on which gene In what combination will yield the best result. In this report, we propose a biochemical background for using GTP cyc]ohydrolase I (GTPCH I) in addition to TH for higher and/or more stable expression of TH. METHODS: TH and GTPCH I cDNA were subcloned into retroviral vectos and resulting recombinant retrovirus packaged in BOSC 23 cells were used to infect NIH-3T3. Confirming successful infections by westers blot analysls, the new cell lines were used to examine steady state TH expression level and TH activity. Furthermore, the effect of ectopic expression of BH4 to the proliferation of these cells were studied. RESULTS: NIH-3T3 cells expressing both TH and GTPCH I showed approximately 10 fold higher expression of TH protein than the cells expressing TH alone. The activity of KNTH2GC6 was approximately 4-6 fold higher than that of striatal tissue and 60 fold higher than KNTH2. Furthermore, growth rate of KNTH2GC6 was strikingly reduced by inhibiting the biosynthesis of BH4. CONCLUSIONS: We showed that the use of GTPCH I in addition to TH not only increased the stability and/or expression of TH protein but also the activity of the enzyme. These improved characteristics of TH protein are very likely due to the expression of BH4 and should be very seriously considered for Parkinson's gene therapy.

Liposomes as Acitivators of Lipophilic Platinum (2 ) Complexes / 대한암학회지

PURPOSE: The goal of this study is to understand the activation processes that take place within the liposomal formulation of lipophilic diaminocyclohexane platinum (DACH-Pt) complexes, to identify the activated species of this class of compounds, and to use that information to develop a reproducible liposomal formulation of DACH-Pt complexes. MATERIALS AND METHODS: Liposomal DACH-Pt complexes were prepared by lyophilization-rehydration method using PC, PG and PA. Their intraliposomal stability and biological activity were determined by HPLC and in vitro/in vivo experiments. RESULTS: DACH-Pt complexes in a liposomal formulation have shown significant promise in preclinical studies and clinical phase I, II trials. Interestingly, they are prodrugs which converts into one or more undetennined activated platinum species within the liposomes ex vivo. Our studies have shown that the stability of liposomal DACH-Pt complexes is inversely related with the antitumor activity of those complexes. The configuratian of leaving group in the complexes and pH of the liposome suspension affect significantly the degradation/activation process that takes place within the liposomes. DACH-Pt complexes with linear (L10) leaving groups are more stable than complexes with branched ones (B10 and NDDP), but also significantly less potent. The presence of PG and PA in the liposome is a prerequisite for the degradation/activation process of DACH-Pt complexes. As PG and PA formulation gave more dramatic changes of the original complexes than PC alone due to lower pH, the cytotoxicity and antitumor activity at those fonnulations increased against PC alone. DACH-Pt complexes are very stable in liposomes containing PC alone but inactive in vitro/in vivo experiments. CONCLUSION: These results also support that the active species produced within the liposomal DACH-Pt complexes is DACH-Pt-Cl2.

Study on the Production and Function of Macrophage Migration Inhibitory Factor ( MIF ) : Effect of Recombinant MIF and MIF cDNA on the Induction of Cytokine mRNA / 대한면역학회지

In order to study the functions of migration inhibitory factor (MIF) as macrophage activating cytokine and to investigate the possibility of MIF cDNA as gene therapeutic agent or adjuvant, we produced recombinant MIF (rMIF), anti-MIF antibody and pcDNA I plasmid containing mMIF cDNA (mMIF plasmid). We have investigated the effects of recombinant mMIF or mMIF plasmid on the expression of immune response-related gene in the mouse peritoneal macrophage or splenocyte. Recombinant mMIF produced by Baculovirus expression system was biologically active; it increased mRNA expression of tumor necrosis factor (TNF)-a, Interleukin (IL)-1, IL-6, granulocyte monocyte-colony stimulating factor (GM-CSF), nitric oxide synthase (NOS), Fas and Bcl-x when applied to the cultures of mouse peritoneal macrophage. Anti-mMIF antibody blocked these effects of mMIF on macrophage. Plasmid DNA carrying MIF cDNA inoculated into mouse peritoneal cavity also increased mRNA transcriptions from TNF, IL-1, IL-6, IL-12, GM-CSF, NOS genes of peritoneal macrophage. It enhanced proliferation of splenocyte stimulated with phorbol myristate acetate and IL-2 mRNA expression of splenocytes. Frorn these results, we conclude that rMIF is a strong macrophage activating factor and especially MIF plasmid can be used as an immune potentiating DNA drug in gene therapy for cancer or DNA adjuvant in vaccination in future.