P70S6K and Elf4E dual inhibition is essential to control bladder tumor growth and progression in orthotopic mouse non-muscle invasive bladder tumor model.

We investigated how the dual inhibition of the molecular mechanism of the mammalian target of the rapamycin (mTOR) downstreams, P70S6 kinase (P70S6K) and eukaryotic initiation factor 4E (eIF4E), can lead to a suppression of the proliferation and progression of urothelial carcinoma (UC) in an orthotopic mouse non-muscle invasive bladder tumor (NMIBT) model. A KU-7-luc cell intravesically instilled orthotopic mouse NMIBC model was monitored using bioluminescence imaging (BLI) in vivo by interfering with different molecular components using rapamycin and siRNA technology. We then analyzed the effects on molecular activation status, cell growth, proliferation, and progression. A high concentration of rapamycin (10 µM) blocked both P70S6K and elF4E phosphorylation and inhibited cell proliferation in the KU-7-luc cells. It also reduced cell viability and proliferation more than the transfection of siRNA against p70S6K or elF4E. The groups with dual p70S6K and elF4E siRNA, and rapamycin reduced tumor volume and lamina propria invasion more than the groups with p70S6K or elF4E siRNA instillation, although all groups reduced photon density compared to the control. These findings suggest that both the mTOR pathway downstream of eIF4E and p70S6K can be successfully inhibited by high dose rapamycin only, and p70S6K and Elf4E dual inhibition is essential to control bladder tumor growth and progression.

Establishment of an orthotopic mouse non-muscle invasive bladder cancer model expressing the mammalian target of rapamycin signaling pathway.

We established an orthotopic non-muscle invasive bladder cancer (NMIBC) mouse model expressing the mammalian target of the rapamycin (mTOR) signaling pathway. After intravesical instillation of KU-7-lucs (day 0), animals were subsequently monitored by bioluminescence imaging (BLI) on days 4, 7, 14, and 21, and performed histopathological examination. We also validated the orthotopic mouse model expressing the mTOR signaling pathway immunohistochemically. In vitro BLI photon density was correlated with KU-7-luc cell number (r (2) = 0.97, P < 0.01) and in vivo BLI photon densities increased steadily with time after intravesical instillation. The tumor take rate was 84.2%, formed initially on day 4 and remained NMIBC up to day 21. T1 photon densities were significantly higher than Ta (P < 0.01), and histological tumor volume was positively correlated with BLI photon density (r (2) = 0.87, P < 0.01). The mTOR signaling pathway-related proteins were expressed in the bladder, and were correlated with the western blot results. Our results suggest successful establishment of an orthotopic mouse NMIBC model expressing the mTOR signaling pathway using KU-7-luc cells. This model is expected to be helpful to evaluate preclinical testing of intravesical therapy based on the mTOR signaling pathway against NMIBC.

Human ß-defensin 2 may inhibit internalisation of bacillus Calmette-Guérin (BCG) in bladder cancer cells.

OBJECTIVE: To investigate whether secretion of human ß-defensin 2 (HBD-2) is induced by bacillus Calmette-Guérin (BCG) and to determine whether HBD-2 affects BCG internalisation in bladder cancer cells. MATERIALS AND METHODS: Reverse transcription-polymerase chain reaction analysis was used to determine whether HBD-2 mRNA increases after incubation with BCG. HBD-2 proteins in 5637 and T24 human bladder cancer cell lines were assayed by enzyme-linked immunosorbent assay. The internalisation rate was evaluated by double immunofluorescence assay and confocal microscopy to test the optimal dose of HBD-2 for BCG internalisation. We also investigated the difference in internalisation rates and cell viability between recombinant HBD-2 protein, anti-HBD-2 antibody, and HBD-2 plus anti-HBD-2 antibody pretreatments. RESULTS: BCG induced HBD-2 mRNA expression and HBD-2 production dose and time-dependently in bladder cancer cells and affected BCG internalisation. Pretreatment with recombinant HBD-2 protein lowered internalisation of BCG dose-dependently. Moreover, anti-HBD-2 antibody prevented the effect of HBD-2 on BCG internalisation in bladder cancer cells. The internalisation rate of BCG pretreated with anti-HBD-2 antibody was higher than that in the control in 5637 (P < 0.01) and T24 cells (P < 0.05). The BCG internalisation rate in cells pretreated with anti-HBD-2 antibody plus recombinant HBD-2 protein was higher than that in the control in 5637 (P < 0.01) and T24 cells (P < 0.05). Mycobacterium bovis BCG decreased bladder cancer cell viability, and anti-HBD-2 antibody prevented the inhibitory role of HBD-2 on the anti-proliferative effects of M. bovis BCG in bladder cancer cells CONCLUSION: Bladder cancer cells produce HBD-2 when they are infected by BCG to defend themselves against BCG internalisation, which plays an important role during the initiation and propagation of the immunotherapeutic response in bladder cancer cells.

Contact allergy to dimethacrylate.

Contact allergy to methacrylates is uncommon. We present a 55-year-old woman with a 10-year history of persistent pruritus and burning sensation of the gums every time she wore her dentures. Initially she developed swelling and erythema of the face soon after the dentures were placed on the gums. These symptoms abated after a barrier liner was applied between her gums and the dentures. However, the burning sensation and pruritus of the gums progressively worsened and she started to develop blisters on the gums. The skin allergen patch test was 3+ positive with erythema, edema, papules, ulceration, and pruritus for the denture component dimethacrylate. The diagnosis was supported by the patient's medical history, notably positive patch test, and complete amelioration of the symptoms upon cessation of dimethacrylate denture usage.

Clinical and Laboratory Findings of Middle East Respiratory Syndrome Coronavirus Infection.

There is a paucity of data regarding the differentiating characteristics of patients with laboratory-confirmed and those negative for Middle East respiratory syndrome coronavirus (MERS-CoV) in South Korea. This hospital-based retrospective study compared MERS-CoV-positive and MERS-CoV-negative patients. A total of seven positive patients and 55 negative patients with a median age of 43 years (P = 0.845) were included. No statistical differences were observed with respect to their sex and the presence of comorbidities. At the time of admission, headache (28.6% vs. 3.6%; odds ratio [OR], 10.60; 95% confidence interval [CI], 1.22-92.27), myalgia (57.1% vs. 9.1%; OR, 13.33; 95% CI, 2.30-77.24), and diarrhea (57.1% vs. 14.5%; OR, 7.83; 95% CI, 1.47-41.79) were common among MERS-CoV-positive patients. MERS-CoV-positive patients were more likely to have a low platelet count (164 ± 76.57 vs. 240 ± 79.87) and eosinophil (0.27 ± 0.43 vs. 2.13 ± 2.01; P = 0.003). Chest radiography with diffuse bronchopneumonia was more frequent in MERS-CoV-positive patients than in negative patients (100% vs. 62.5%; P = 0.491). The symptoms of headache, myalgia, and diarrhea, as well as laboratory characteristics, including low platelet counts and eosinophil, and chest X-ray showing diffuse bronchopneumonia might enhance the ability to detect patients in South Korea infected with MERS-CoV.

Histone deacetylase inhibitor scriptaid induces cell cycle arrest and epigenetic change in colon cancer cells.

Histone deacetylase inhibitors (HDACIs) are involved in cell growth, apoptosis and differentiation. This study aimed to investigate the effects of HDACI scriptaid on histone modification, demethylation, cell growth, cell cycle and apoptosis in the RKO colorectal cancer cell line and screening for scriptaid-induced genes. RKO cells were treated with 5-aza-2'-deoxycytidine (5-aza-dC), trichostatin A (TSA) or scriptaid at different concentrations. Histone modification and methylation status of a silenced p16 gene were analyzed using chromatin immunoprecipitation and methylation-specific PCR, respectively. Flow cytometry was performed for the analysis of cell cycle and apoptosis. Scriptaid-induced expression was analyzed using Human OneArray chip. Scriptaid resulted in the demethylation and re-expression of a hypermethylated p16 gene along with 5-aza-dC synergistically in the RKO cells, but not alone. Scriptaid induced modifications of core histone tails important in euchromatin structure: increases in acetyl-H3-K9 and dimethyl-H3-K4 and a decrease in dimethyl-H3-K9. Cell growth was inhibited by scriptaid in a dose-dependent manner. Cell cycle analysis showed that scriptaid induced G1 arrest at 0.5 and 1.0 microM concentrations and G1 and G2/M arrest at 2.0 microM. Scriptaid did not have a significant effect on apoptosis in RKO cells. An altered expression of 278 genes was observed in RKO cells in response to scriptaid treatment. In conclusion, the present study suggests that scriptaid may be effective in growth suppression and cell cycle arrest and in the reversal of repressive chromatin marks at the promoter region of a hypermethylated p16 gene in colorectal cancer.

Activator Gcn4p and Cyc8p/Tup1p are interdependent for promoter occupancy at ARG1 in vivo.

The Cyc8p/Tup1p complex mediates repression of diverse genes in Saccharomyces cerevisiae and is recruited by DNA binding proteins specific for the different sets of repressed genes. By screening the yeast deletion library, we identified Cyc8p as a coactivator for Gcn4p, a transcriptional activator of amino acid biosynthetic genes. Deletion of CYC8 confers sensitivity to an inhibitor of isoleucine/valine biosynthesis and impairs activation of Gcn4p-dependent reporters and authentic amino acid biosynthetic target genes. Deletion of TUP1 produces similar but less severe activation defects in vivo. Although expression of Gcn4p is unaffected by deletion of CYC8, chromatin immunoprecipitation assays reveal a strong defect in binding of Gcn4p at the target genes ARG1 and ARG4 in cyc8Delta cells and to a lesser extent in tup1Delta cells. The defects in Gcn4p binding and transcriptional activation in cyc8Delta cells cannot be overcome by Gcn4p overexpression but are partially suppressed in tup1Delta cells. The impairment of Gcn4p binding in cyc8Delta and tup1Delta cells is severe enough to reduce recruitment of SAGA, Srb mediator, TATA binding protein, and RNA polymerase II to the ARG1 and ARG4 promoters, accounting for impaired transcriptional activation of these genes in both mutants. Cyc8p and Tup1p are recruited to the ARG1 and ARG4 promoters, consistent with a direct role for this complex in stimulating Gcn4p occupancy of the upstream activation sequence (UAS). Interestingly, Gcn4p also stimulates binding of Cyc8p/Tup1p at the 3' ends of these genes, raising the possibility that Cyc8p/Tup1p influences transcription elongation. Our findings reveal a novel coactivator function for Cyc8p/Tup1p at the level of activator binding and suggest that Gcn4p may enhance its own binding to the UAS by recruiting Cyc8p/Tup1p.

A multiplicity of coactivators is required by Gcn4p at individual promoters in vivo.

Transcriptional activators interact with multisubunit coactivators that modify chromatin structure or recruit the general transcriptional machinery to their target genes. Budding yeast cells respond to amino acid starvation by inducing an activator of amino acid biosynthetic genes, Gcn4p. We conducted a comprehensive analysis of viable mutants affecting known coactivator subunits from the Saccharomyces Genome Deletion Project for defects in activation by Gcn4p in vivo. The results confirm previous findings that Gcn4p requires SAGA, SWI/SNF, and SRB mediator (SRB/MED) and identify key nonessential subunits of these complexes required for activation. Among the numerous histone acetyltransferases examined, only that present in SAGA, Gcn5p, was required by Gcn4p. We also uncovered a dependence on CCR4-NOT, RSC, and the Paf1 complex. In vitro binding experiments suggest that the Gcn4p activation domain interacts specifically with CCR4-NOT and RSC in addition to SAGA, SWI/SNF, and SRB/MED. Chromatin immunoprecipitation experiments show that Mbf1p, SAGA, SWI/SNF, SRB/MED, RSC, CCR4-NOT, and the Paf1 complex all are recruited by Gcn4p to one of its target genes (ARG1) in vivo. We observed considerable differences in coactivator requirements among several Gcn4p-dependent promoters; thus, only a subset of the array of coactivators that can be recruited by Gcn4p is required at a given target gene in vivo.

Immune modulation of glycosaminoglycan derived from P. lewisi in TNF-α stimulated cells.

Poecilocoris lewisi (Korean name: "Kwangdaenolinjae") is a red-striped gold stink bug (insect) which has been used as a crude drug in traditional medicine of East Asia and Korea. In this study, ethanol extract and glycosaminoglycan from P. lewisi (Pl GAG), as an active substance among its components, were investigated for their potential anti-inflammatory actions. They were found to be a potent inducer of nitric oxide (NO) production from calf pulmonary artery endothelial (CPAE) cells and a stimulator of endothelial nitric oxide synthase in a dose-dependent manner. The anti-inflammatory activities were also evaluated by determining the level of adhesion molecules related to atherogenesis and pro-inflammatory cytokines, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), secretory phospholipase A2, and prostaglandin E2, stimulated by tumor necrosis factor (TNF)-α in human umbilical vein endothelial cells (HUVEC). They also showed inhibitory effects on vascular endothelial growth factor (VEGF) production in HUVECs. Matrix metalloproteinases (MMP-2 and 9) were also inhibited by treatment with this extract or glycosaminoglycan. Furthermore, this GAG showed cytotoxicity against CT-26 colon cancer cells whereas having no cytotoxicity in CHO normal cells. Monosaccharide (amino, acidic, neutral monosaccharides) composition of used GAG was characterized by trimethylsilylated GC-MS analysis method.

Modulating the internalization of bacille Calmette-Guérin by cathelicidin in bladder cancer cells.

OBJECTIVE: To confirm the role of cathelicidin (LL-37) in the internalization of bacille Calmette-Guérin (BCG) into bladder cancer cells. METHODS: Enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction analysis evaluated the changes in protein and messenger ribonucleic acid (RNA) expression with BCG incubation after LL-37 pretreatment in 5637 and T24 human bladder cancer cells. The internalization rate was evaluated by a double immunofluorescence assay, and confocal microscopy confirmed the function of LL-37 in BCG internalization. We also investigated the difference in internalization rates and cell viability between LL-37, anti-LL-37 antibody, and LL-37 plus anti-LL-37 antibody. RESULTS: The levels of LL-37 increased after BCG exposure in bladder cancer cells in dose- and time-dependent manners. Increasing LL-37 levels using recombinant LL-37 protein further dose dependently decreased BCG internalization in both cell lines. The internalization rates of BCG after LL-37 instillation were lower compared with the controls, and the internalization rate of BCG after anti-LL-37 antibody instillation was significantly higher compared with the controls in both cell lines (P <.05). Viability of LL-37 plus BCG group was higher compared with the BCG-alone group. The anti-LL-37 antibody plus BCG group had decreased cell viability compared with the BCG-alone group in both cell lines. CONCLUSION: Bladder cancer cells produce cathelicidin when infected with BCG and upregulate cathelicidin to defend against BCG by inhibiting its internalization. Blocking the action of cathelicidin may increase the internalization and effectiveness of BCG in reducing bladder cancer cell proliferation.

Murine ß-defensin-2 may regulate the effect of bacillus Calmette-Guérin (BCG) in normal mouse bladder.

PURPOSE: We investigated whether bacillus Calmette-Guérin (BCG)-induced secretion of murine ß-defensin-2 (mBD2) and determined whether mBD2 regulated BCG effects in the normal mouse bladder. MATERIALS AND METHODS: A total of 140 C57BL/6 female mice were divided into 28 groups, and the experiment was performed over 3 steps. In the first step (20 groups), mice bladders were stimulated with different doses of BCG (multiplicity of infection [MOI] 0, 1, 10, 30, and 100) and histological analysis was conducted in bladder specimens isolated at different times (0, 4, 8, and 24h after instillation) to determine optimal dose and time point of BCG internalization and urine mBD2 and cytokine concentration. In the second step (4 groups), BCG internalization and urine cytokine levels were measured after pretreatment of different recombinant mBD2 (rmBD2) (0, 1, 2.5, and 5 ng/ml) at optimal dose and time point. In the third step (4 groups), BCG internalization and urine cytokine levels were compared between pretreatment conditions (control, rmBD2, anti-mBD2 Ab, and rmBD2+anti-mBD2 Ab). Urine was collected for estimating mBD2 levels and a multiplex analysis for 9 cytokines. Real-time polymerase chain reaction assay was used for estimating the relative BCG cell number in mice bladder tissue. RESULTS: Bladder edema was induced by BCG (MOI 30 and 100), which progressed to an inflammatory infiltrate composed primarily of neutrophils and increased mBD2 secretion at 4 hours after instillation. Relative BCG cell number and urinary cytokine levels (interferon-γ and interleukins [IL]-2, -4, -6, and -10) response pattern was characterized by a peak at 4 hours after instillation followed by rapid decline. The levels of interferon-γ, and IL-1ß, -2, -4, -6, and -10 and relative BCG cell numbers decreased in a dose-dependent manner according to pretreatment with rmBD2 protein, and the responses were potentiated in the anti-mBD2 pretreatment group at 4 hours after BCG (MOI 30) instillation. CONCLUSION: The present results suggest that the mouse urothelium produces mBD2 in response to intravesicular BCG as a defense mechanism against BCG, and blocking mBD2 by an anti-mBD2 antibody increased the effectiveness of BCG.

Transesterification of plant oils using Staphylococcus haemolyticus L62 lipase displayed on Escherichia coli cell surface using the OmpA signal peptide and EstAß8 anchoring motif.

Staphylococcus haemolyticus L62 (SHL62) lipase was displayed on the outer membrane of Escherichia coli using the OmpA signal peptide and the autotransporter EstAß8 protein. Localization of SHL62 lipase on the outer membrane of E. coli was confirmed using immunofluorescence microscopy and flow cytometry analysis. Lipase activity of the displayed SHL62 lipase was also measured using spectrophotometry and pH titration. SHL62 lipase activity of whole cells reached 2.0U/ml culture (OD600nm of 10) when it was measured by the p-nitrophenyl caprylate assay after being induced with 1mM IPTG for 24h. The optimum temperature and pH for the lipase was 45°C and 10, respectively. Furthermore, it maintained more than 90% of maximum lipase activity at up to 50°C and in a pH range of 5-9. The hydrolytic activity assay conduted with various substrates confirmed that p-nitrophenyl caprylate and corn oil were preferred substrates among various synthetic and natural substrates, respectively. The displayed SHL62 lipase produced fatty acid esters from various alcohols and plant oils through transesterification.

Dual inhibition by S6K1 and Elf4E is essential for controlling cellular growth and invasion in bladder cancer.

PURPOSE: We investigated how dual inhibition of the molecular mechanism of mammalian target of rapamycin (mTOR) downstream of S6K1 and the eukaryotic initiation factor 4E (eIF4E) can lead to suppression of proliferation and progression of urothelial carcinoma. MATERIALS AND METHODS: We characterized the molecular mechanism of the mTOR pathway in the T24 and 5637 urothelial carcinoma cell lines by interfering with different molecular components using rapamycin and short interfering (siRNA) technology (S6K1 or elF4E) and analyzed the effects on molecular activation status, cell growth, proliferation, and invasion. RESULTS: A high concentration of rapamycin (10 µM) blocked both S6K1 and elF4E phosphorylation and inhibited cell proliferation in T24 and 5637 cells. The inhibition of both S6K1 and elF4E phosphorylation by rapamycin reduced cell viability and proliferation more than transfection of siRNA against S6K1 or elF4E in 5637 and T24 cells. Cells silenced for S6K1 or elF4E expression exhibited significantly reduced cell migration and invasion compared with those of the control but inhibition of both S6K1 and elF4E phosphorylation by rapamycin reduced cell migration and invasion more than siRNA transfection against S6K1 or elF4E in 5637 and T24 cells. CONCLUSION: These findings suggest that both the mTOR pathway downstream of eukaryotic initiation factor 4E and S6K1 can be successfully inhibited, therefore, the recurrence of bladder cancer can be prevented by high-dose rapamycin only, suggesting that 4E-BP1 might be still under mTORC1.

Phosphorylated p70S6K in noninvasive low-grade urothelial carcinoma of the bladder: correlation with tumor recurrence.

We investigated whether inhibiting phosphorylated p70S6K (p-p70S6K) suppresses the proliferation and growth of noninvasive low-grade urothelial carcinoma (LG-URCa) in vitroand whether p-p70S6K can serve as a predictive biomarker for the recurrence of noninvasive LG-URCa of the bladder in patients. We constructed a tissue microarray (TMA) for 95 LG-URCa and 35 benign urothelium samples and performed immunohistochemical staining for p-p70S6K and p-4E-BP1. A Cox regression model was used to investigate the predictive factors for recurrence of LG-URCa. We investigated the dose-dependent antiproliferative effect of rapamycin, its antiproliferative effect and the growth-inhibition effect of p70S6K siRNA transfection in RT4 and 253J cell lines. The pT1 staged group (P < 0.05; hazard ratio (HR), 2.415) and the high p-p70S6K staining group (P < 0.05; HR, 2.249) were independent factors for predicting recurrence. Rapamycin inhibited RT4 and 253J cell proliferation in a dose-dependent manner (r = -0.850, P< 0.001 in RT4 cells; r = -0.835, P< 0.001 in 253J cells). RT4 and 253J cell proliferation and growth were inhibited by the transfection of p70S6K siRNA and rapamycin, respectively (P < 0.05). Transfection of p70S6K siRNA resulted in inhibitory effects on cell proliferation and growth that were similar to those of rapamycin. Our results suggest that inhibiting p70S6K phosphorylation is important to prevent recurrence and that p70S6K phosphorylation can be used as a molecular biomarker to predict recurrence of certain LG-URCa of the bladder.

Immune Modulation of B. terrestris Worker (a Type of Bumblebee), Extract on CFA-induced Paw Edema in Rats.

To develop a composition for enhancing immunity, based on alcohol extracts of the bumblebee as an active ingredient, bumblebee ethanol extracts were evaluated for their protective effect in chronic models of inflammation, adjuvant induced rat arthritis. B. terrestris worker extract (SDIEX) and, B. hypocrita sapporoensis lava an pupa extract (SPDYBEX), significantly decreased paw edema in arthritic rats, at a dose 100 mg/kg, respectively. The cytokine levels related inflammation of COX-2, sPLA2, VEGF, and TNF-α, were decreased, compared to positive control, indomethacin (5 mg/kg). Histopathological data demonstrated decreases inflammatory activity, hind paw edema, and repaired hyaline articular cartilage in DRG over a 2 wk administration. HPLC and GC-MS analysis of SDIEX and SPDYBEX revealed the presence of cantharidin.

Isolation and biochemical characterization of Bacillus pumilus lipases from the Antarctic.

Lipase-producing bacterial strains were isolated from Antarctic soil samples using the tricaprylin agar plate method. Seven strains with relatively strong lipase activities were selected. All of them turned out to be Bacillus pumilus strains by the 16S rRNA gene sequence analysis. Their corresponding lipase genes were cloned, sequenced, and compared. Finally, three different Bacillus pumilus lipases (BPL1, BPL2, and BPL3) were chosen. Their amino acid sequence identities were in the range of 92-98% with the previous Bacillus pumilus lipases. Their optimum temperatures and pHs were measured to be 40 degrees C and pH 9. Lipase BPL1 and lipase BPL2 were stable up to 30 degrees C, whereas lipase BPL3 was stable up to 20 degrees C. Lipase BPL2 was stable within a pH range of 6-10, whereas lipase BPL1 and lipase BPL3 were stable within a pH range of 5-11, showing strong alkaline tolerance. All these lipases exhibited high hydrolytic activity toward pnitrophenyl caprylate (C8). In addition, lipase BPL1 showed high hydrolytic activity toward tributyrin, whereas lipase BPL2 and lipase BPL3 hydrolyzed tricaprylin and castor oil preferentially. These results demonstrated that the three Antarctic Bacillus lipases were alkaliphilic and had a substrate preference toward short- and mediumchain triglycerides. These Antarctic Bacillus lipases might be used in detergent and food industries.

Effects of the synthetic coprisin analog peptide, CopA3 in pathogenic microorganisms and mammalian cancer cells.

A synthetic coprisin analog peptide, 9-mer dimer CopA3 (CopA3) was designed based on a defensin-like peptide, Coprisin, isolated from the bacteria-immunized dung beetle Copris tripartitus. Here, CopA3 was investigated for its antimicrobial activity and cancer cell growth inhibition. CopA3 showed antimicrobial activities against various pathogenic bacteria and yeast fungus with MIC values in 2~32 µM ranges, and inhibited the cell viabilities of pancreatic and hepatocellular cancer cells, except MIAPaca2, Hep3B, and HepG2 cells, in a dose-dependent manner. The average IC(50) values of CopA3 against pancreatic and hepatocellular cancer cells were 61.7 µM and 67.8 µM, respectively. The results indicate that CopA3 has potential in the treatments of pancreatic and hepatocellular cancers as well as microorganism infection disease.

Synthetic Coprisin analog peptide, D-CopA3 has antimicrobial activity and pro-apoptotic effects in human leukemia cells.

Recently, we reported that the synthetic Coprisin analog peptide 9-mer dimer CopA3 (consisted of all-L amino acid sequence) was designed based on a defensin-like peptide, Coprisin isolated from Copris tripartitus. The 9-mer dimer CopA3 (L-CopA3) had antibacterial activity and induced apoptosis in human leukemia cells via a caspase-independent pathway. In this study, all of amino acid sequences of L-CopA3 were modified to all D-form amino acids (D-CopA3) to develop a more effective antimicrobial peptide. We investigated whether D-CopA3 had antimicrobial activities against pathogenic microorganisms and proapoptotic effects in human leukemia cells (U937, Jurkat, and AML-2). The synthetic peptide D-CopA3 had antimicrobial activities against various pathogenic bacteria and yeast fungus with MIC values in the 4~64 microM range. Moreover, D-CopA3 caused cell growth inhibition, and increased the chromosomal DNA fragmentation and the expression of inflammatory cytokines, TNF-alpha and IL1-beta, transcripts in human leukemia cells. The all-D amino acid peptide D-CopA3 proved as effective as the L-CopA3 reported previously. These results provide the basis for developing D-CopA3 as a new antibiotic peptide.

Thirteen-Week Oral Dose Toxicity Study of G. bimaculatus in Sprague-Dawley Rats.

Gryllus bimaculatus (Gb) was orally administered at doses of 0, 0.04, 0.2, 1 and 5 g/kg bw/day for 13 consecutive weeks. There were no observed clinical signs or deaths related to treatment in all the groups tested. Therefore, the approximate lethal oral dose of G. bimaculatus was considered to be higher than 5 g/kg in rats. Throughout the administration period, no significant changes in diet consumption, ophthalmologic findings, organ weight, clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis) or gross pathology were detected. Minor changes were found in hematological parameters for the 5 g/kg Gb-treated group (triglyceride reduction of 35.8%), but all changes were within normal physiological ranges. Microscopic examination did not identify any treatment-related histopathologic changes in the organs of Gb-treated rats in the high dose group. From these results, one can conclude that the no-observed adverse effect level (NOAEL) of G. bimaculatus is higher than 5 g/kg bw/day in rats.

Antibacterial activity of recombinant hCAP18/LL37 protein secreted from Pichia pastoris.

Human antimicrobial peptide CAP18/LL37 (hCAP18/LL37) was expressed in Pichia pastoris and its antibacterial activity was tested against pathogenic bacteria. The full length ORF of hCAP18/LL37 was cloned into the pPICZaA vector followed by integration into the genomic AOX1 gene of P. pastoris. Agar diffusion assay demonstrated that the different hCAP18/LL37 transformants showed various antibacterial activities against Staphylococcus aureus, Micrococcus luteus, and Salmonella gastroenteritis. The secreted form of hCAP18/LL37 exhibited its maximum activity after 72 h incubation with 2% methanol in MM media, not in BMM. This result suggests that the yeast secreted expression system can be used as a production tool of antimicrobial peptides for industrial or pharmaceutical application.