Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.

Prostaglandin A1 inhibits the replication of bovine viral diarrhea virus

ABSTRACT Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5 µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10 µg/mL).

Prostaglandin A1 triggers Mayaro virus inhibition and heat shock protein 70 expression in an epithelial cell model

Abstract INTRODUCTION: The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS: Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6μg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS: PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS: PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.

High levels of interleukin-6 and 8-iso-prostaglandin in the exhaled breath condensate and serum of patients with chronic obstructive pulmonary disease related pulmonary hypertension / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Pulmonary hypertension (PH) is a common complication of chronic obstructive pulmonary disease (COPD). Although alveolar hypoxia is considered as a main cause of PH in COPD, structural and functional changes of pulmonary circulation are apparent at the initial stage of COPD. We hypothesized that an inflammatory response and oxidative stress might contribute to the formation of PH in COPD.</p><p><b>METHODS</b>We measured the levels of interleukin-6 (IL-6) and 8-iso-prostaglandin (8-iso-PSG) in exhaled breath condensate (EBC) and serum in 40 patients with COPD only or in 45 patients with COPD combined with PH. Pulmonary arterial systolic pressure (PASP) was assessed by Doppler echocardiography and defined as PH when the value of systolic pressure was greater than 40 mmHg.</p><p><b>RESULTS</b>Compared with the COPD only group, the level of IL-6 in EBC was significantly increased in all 45 patients with COPD combined with PH ((8.27±2.14) ng/L vs. (4.95±1.19) ng/L, P < 0.01). The level of IL-6 in serum was also elevated in patients with COPD combined with PH compared with the COPD only group ((72.8±21.6) ng/L vs. (43.58±13.38) ng/L, P < 0.01). Similarly, we also observed a significant increase in the level of 8-iso-PSG in both EBC and serum in the COPD with PH group, compared with the COPD only group (EBC: (9.00±2.49) ng/L vs. (5.96±2.31) ng/L, P < 0.01 and serum: (41.87±9.75) ng/L vs. (27.79±11.09) ng/L, P < 0.01). Additionally, the value of PASP in the PH group was confirmed to be positively correlated with the increase in the levels of IL-6 and 8-iso-PSG in both EBC and serum (r = 0.477-0.589, P < 0.05).</p><p><b>CONCLUSION</b>The increase in the levels of IL-6 and 8-iso-PSG in EBC and serum correlates with the pathogenesis of PH in COPD.</p>

The Clinical Efficacy of Mometasone Furoate in Multi-Lamellar Emulsion for Eczema: A Double-blinded Crossover Study

BACKGROUND: Topical application of corticosteroids also has an influence on skin barrier impairment. Physiological lipid mixtures, such as multi-lamellar emulsion (MLE) containing a natural lipid component leads to effective recovery of the barrier function. OBJECTIVE: The purpose of this study was to conduct an evaluation of the therapeutic efficacy and skin barrier protection of topical mometasone furoate in MLE. METHODS: A multi-center randomized, double-blind, controlled study was performed to assess the efficacy and safety of mometasone furoate cream in MLE for Korean patients with eczema. The study group included 175 patients with eczema, who applied either mometasone furoate in MLE cream or methylprednisolone aceponate cream for 2 weeks. Treatment efficacy was evaluated using the physician's global assessment of clinical response (PGA), trans-epidermal water loss (TEWL), and visual analogue scale (VAS) for pruritus. Patients were evaluated using these indices at days 4, 8, and 15. RESULTS: Comparison of PGA score, TEWL, and VAS score at baseline with those at days 4, 8, and 15 of treatment showed a significant improvement in both groups. Patients who applied mometasone furoate in MLE (74.8%) showed better results (p<0.05) than those who applied methylprednisolone aceponate (47.8%). The TEWL improvement ratio was higher in the mometasone furoate in MLE group than that in the methylprednisolone aceponate group, and VAS improvement was also better in the mometasone furoate in MLE group. CONCLUSION: Mometasone furoate in MLE has a better therapeutic efficacy as well as less skin barrier impairment than methylprednisolone aceponate.

Effect of infosheet for topical tacrolimus 0.1% and its efficacy and compliance in the treatment of atopic dermatitis

PURPOSE: Topical calcineurin inhibitor is recently developed topical immunomodulator, and preliminary studies showed its effectiveness in the treatment of atopic dermatitis (AD). However, some side effects including transient irritation can influence the patient compliance. So, there are some needs to improve the patient compliance. The purpose of this study was to evaluate the efficacy, safety and patient compliance with using topical tacrolimus 0.1% to treat AD when the correct information about topical tacrolimus are properly given to patients. METHODS: We examined the medical recordings, clinical severity scoring of total 194 AD patients at 9 general hospitals in Seoul, Korea from September 2010 to August 2011. We offered an infosheet of topical tacrolimus 0.1% and the patients applied it twice a day for 2 weeks. And we measured the efficacy of the topical tacrolimus 0.1% with SCORing atopic dermatitis (SCORAD) index, patient's global assessment (PGA), and investigator's global assessment (IGA). RESULTS: Topical tacrolimus 0.1% effectively controlled AD with a reduction of the SCORAD index from baseline 31.9 to 20.2 at 2 weeks of application. In IGA results showed 98% got improvement and in PGA, results showed 96% got improvement after treatment. Although 42.3% of the patients complained of adverse effects, these were all transient. The effect of information on topical tacrolimus 0.1% showed 34% patients could predict the side effect, 35% patients could feel safety to use, and 18% patients experienced side effect but could maintain topical calcineurin inhibitor. CONCLUSION: Topical tacrolimus 0.1% may be an effective treatment modality for AD when patients show good compliance for applying the ointment. And properly given, the correct information may increase the patient compliance.

Evaluation of tensile strength of surgical synthetic absorbable suture materials: an in vitro study

PURPOSE: The purpose of this study was to evaluate the tensile strength of surgical synthetic absorbable sutures over a period of 14 days under simulated oral conditions. METHODS: Three suture materials (polyglycolic acid [PGA], polyglactin [PG] 910, and poly (glycolide-co-small je, Ukrainian-caprolactone) [PGC]) were used in 4-0 and 5-0 gauges. 210 suture samples (35 of each material and gauge) were used. All of the samples were tested preimmersion and 1 hour and 1, 3, 7, 10, and 14 days postimmersion. The tensile strength of each suture material and gauge was assessed. The point of breakage and the resorption pattern of the sutures were also assessed. RESULTS: During the first 24 hours of immersion, all 4-0 and 5-0 samples of PGA, PG 910, and PGC maintained their initial tensile strength. At baseline (preimmersion), there was a statistically significant (P<0.001) difference in the tensile strengths between the 4-0 and 5-0 gauge of PGA, PG 910, and PGC. PGA 4-0 showed the highest tensile strength until day 10. At 7 days, all the 4-0 sutures of the three materials had maintained their tensile strength with PGA 4-0 having significantly greater (P=0.003) tensile strength compared to PG. CONCLUSIONS: 4-0 sutures are stronger and have greater tensile strength than 5-0 sutures. The PGA 4-0 suture showed the highest tensile strength at the end of day 10.

Comparison of Severity Scoring Systems of Atopic Dermatitis / 대한피부과학회지

BACKGROUND: There are no serologic markers that accurately reflect the severity of atopic dermatitis (AD), many different scoring systems used in clinical practice and for research purposes have been developed for assessing the severity of AD. OBJECTIVE: The goal of the present study was to evaluate the correlations between severity scoring systems of AD and clinical assessments of physicians and patients. METHODS: We graded the severity of 418 AD patients, applying the SCORing of Atopic Dermtaitis (SCORAD), Objective SCORAD (oSCORAD), Eczema Area and Severity Index (EASI), Three Item Severity score, Rajka and Langeland score. Also, we evaluated the Physician's Global Assessment (PGA) and Visual Analog Scale (VAS) of pruritus and sleep loss. RESULTS: here was significant positive correlation between oSCORAD and EASI and oSCORAD showed a good correlation with the other severity scoring systems. oSCORAD was highly correlated with PGA and VAS of pruritus and sleep loss. oSCORAD demonstrated the greatest correlation with body surface area, showing a linear relation. CONCLUSION: oSCORAD can assess the severity of AD with reflecting clinical assessments of physicians and patients and disease extent.

Synthesis, conjugation and evaluation of some novel polymers and their micro particles for sustained release drug formulations

To prepare and evaluate three novels functionalized polymers [PGA, PGA-co-caprolactone and PGA-copentadecalactone] for the development of nanoparticles which were further used in the development of a novel polymeric prodrug using Ibuprofen as a model drug. The Ibuprofen-polymer prodrug was developed by coupling the drug to one of the three prepared polyester polymers via ester linkage. A hydrolytic enzyme was used to prepare two polymer monomers, glycerol and polyvinyl adipate, which are non toxic, ester linked biological monomers. The polymers and their prodrug were characterized using NMR, GPC, UV-spectrophotometer and DSC. In vitro drug release study of Ibuprofen-polymer conjugate was performed in phosphate buffer PH 7.4 using a roller [Stuart STR 1] placed in an incubator [Stuart SI 60] and the temperature was kept constant at 37 +/- 1°C. Among the three polymers, glycerol-adipateco-pentadecalactone was observed to give a burst release following slow release in the medium. These characteristics suggest that these polymers can be successfully used in sustained release drug formulations

Comparison The Safety and The Efficacy between the Group of using Pelubiprofen Tab. and the Group of using Aceclofenac Tab. on Back Pain Patients: Multi Institution, Double Blind, Random Sample

STUDY DESIGN: Multi institution, double blind, random sample. OBJECTIVES: We conducted a comparative study with Aceclofenac Tab, which is widely used in the clinical field in order to observe the Pelubiprofen Tab's clinical efficiency in patients with back pain. SUMMARY OF LITERATURE REVIEW: Among the numerous literatures regarding the chronic back pain, there is are few studies with Pelubiprofen Tab's clinical efficiency. MATERIALS AND METHODS: We computed an experimental model through a case control study, practiced from January, 2010 to January, 2011, and thereby, 298 back pain patients were selected. This study was conducted through a multi institution, double blind, and random sample. We compared the experimental and control groups' clinical efficiency that was estimated by VAS after 28 days of medication. Also, we compared the treatment efficiency of both drugs by using a variation of Oswestry Disability Index (ODI) and Physician's Global assessment, with a total usage of relief medicine. Also, the side effect and clinical pathologic result were tested. Statistical analysis was done with three different methods, Safety method, ITT (Intent-To-Treat), and PP (Per Protocol). Logistic regression model was used, and this result was compared by a Chi-square or Fisher's Exact test. RESULTS: Comparing the VAS of both groups, VAS decreased with statistical significance. Both groups didn't show a significant difference in VAS (p=0.6764). As the decrement of the total dosage of relief medicine, the decrease in the control group was rather higher, but the difference didn't show any statistical significance (p=0.9955). The experimental group was not inferior than that of the control group in ODI and PGA variation. Analyzing the side effect, both groups didn't show any significant difference (p=0.9843). CONCLUSIONS: As a result of the clinical trial, Pelubiprofen Tab. applied to back pain patients was not inferior to that of aceclofenac Tab., in terms of efficiency, and didn't show any significant difference in safety.

Production of poly-gama-glutamate (PGA) biopolymer by batch and semicontinuous cultures of immobilized Bacilluslicheniformis strain-R

Production of Polyglutamate (PGA) biopolymer by immobilized Bacilluslicheniformis strain-R was intensively investigated. Preliminary experiments were carried out to address the most suitable immobilization methodology. Entrapment of Bacillus cells in alginate-agar led optimal PGA production (36.75 g/l), with 1.32- and 2.18-fold increase in comparison with alginate- or K-carrageenan-immobilized cells, respectively. During semicontinuous cultivation of agar-alginate gel-cell mixture, production of PGA by 10 ml mixture was increased from 2nd to 3rd run whereas, increased till the 4th run using 15ml mixture. Adsorption was the most suitable immobilization technique for production of PGA and the sponge cubes was the preferred matrix recording 43.2 g/l of PGA with the highest cell adsorption. Furthermore, no PGA was detected when B. licheniformis cells were adsorbed on wood and pumice. Although luffa pulp-adsorbed cells recorded the highest PGA production (50.4 g/l), cell adsorption was the lowest. Semicontinuous cultivation of B. licheniformis cells adsorbed on sponge led to increase of PGA production till the 3rd run and reached 55.5 g/l then slightly decreased in the 4th run. The successful use of fixed-bed bioreactor for semicontinuous cultivation of B. licheniformis cells held on sponge cubes (3 runs, 96 hours/run) provides insight for the potential biotechnological production of PGA by immobilized cells.

Prostaglandin A1 inhibits the replication of Sindbis virus in monkey kidney and mosquito cells

The present study reports the effect of prostaglandin A1 (PGA1) on the replication of Sindbis virus in monkey kidney and mosquito cells. In PGA1 treated cells we observed a severe reduction of virus yield. In both cells lines the highest nontoxic concentration of PGA1 (10 ìg/mL) decreased virus replication, dose dependently, by more than 90 por cento. SDS-PAGE analysis of [35S] methionine labeled proteins showed that viral proteins (E1/E2 and C) were normally synthesized in PGA1 treated Vero cells, and induction of stress proteins (HSP70 and HSP90 ) was detected in uninfected and infected cells. In Vero cells the inhibition of virus replication was accompanied by a decrease in [3H] glucosamine incorporation into the virus glycoproteins.

Development of Artificial Vessels with Autologous Bone Marrow Cells and Polymers / 대한흉부외과학회지

BACKGROUND: To treat anastomosis site stenosis and occlusion of the artificial vessels used in vascular surgery, tissue-engineered artificial vessels using autologous cells have been constructed. We developed artificial vessels using a polymer scaffold and autologous bone marrow cells and performed an in vivo evaluation. MATERIAL AND METHOD: We manufactured a vascular scaffold using biodegradable PLCL (poly lactide-co-epsilon-caprolactone) and PGA (poly glycolic acid) fibers. Then we seeded autologous bone marrow cells onto the scaffold. After implantation of the artificial vessel into the abdominal aorta, we performed an angiography 3 weeks after surgery. After the dogs were euthanized we retrieved the artificial vessels and performed histological analysis. RESULT: Among the six dogs, 2 dogs died of massive bleeding due to a crack in the vascular scaffold 10 days after the operation. The remaining four dogs lived for 3 weeks after the operation. In these dogs, the angiography revealed no stenosis or occlusion at 3 weeks after the operation. Gross examination revealed small thrombi on the inner surface of the vessels and the histological analysis showed three layers of vessel structure similar to the native vessel. Immunohistochemical analysis demonstrated regeneration of the endothelial and smooth muscle cell layers. CONCLUSION: A tissue engineered vascular graft was manufactured using a polymer scaffold and autologous bone marrow cells that had a structure similar to that of the native artery. Further research is needed to determine how to accommodate the aortic pressure.

Prostaglandin A2 Induces Caspase-independent Apoptosis in Hepatocellular Carcinoma Cells / 대한간학회지

BACKGROUND/AIMS: Prostaglandin (PG) A2 has been reported to inhibit the growth of hepatocellular carcinoma cells via activation of apoptosis, although the molecular mechanisms involved have not been clarified, yet. To investigate the mechanism of the PGA2-induced apoptosis, we analyzed the activation of caspases during the apoptosis of hepatoma cell lines. METHODS: Induction of apoptosis by PGA2 in hepatoma cell lines, Hep 3B and Hep G2, was assessed by DAPI staining of nuclei and agarose gel electrophoresis of genomic DNA. The involvement of caspases was analyzed by immunoblot analysis of poly ADP-ribose polymerase (PARP) and by checking the effect of caspase inhibitors on PGA2-induced apoptosis. RESULTS: PGA2 inhibited the growth of Hep 3B and Hep G2 cells, accompanying nuclear condensation and fragmentation, and genomic DNA laddering, which are the hallmarks of apoptosis. The PARP was not cleaved during the apoptosis of Hep 3B and Hep G2 cells and caspase inhibitors such as z-VAD-Fmk and z-DEVD-Fmk exerted no effect on the PGA2-induced apoptosis. CONCLUSIONS: These results suggest that PGA2 induces apoptosis in Hep 3B and Hep G2 cells via caspase-independent pathway.

Sox-4 is a positive regulator of Hep3B and HepG2 cells' apoptosis induced by prostaglandin (PG)A2 and 12-PGJ2

We reported earlier that expression of Sox-4 was found to be elevated during prostaglandin (PG) A2 and delta(12)-PGJ(12) induced apoptosis in human hepatocarcinoma Hep3B cells. In this study, the role of Sox-4 was examined using human Hep3B and HepG2 cell lines. Sox-4 induction by several apoptotic inducer such as A23187 (Ca(2+) ionophore) and etoposide (topoisomerase II inhibitor) and Sox-4 transfection into the cells were able to induce apoptosis as observed by the cellular DNA fragmentation. Antisense oligonucleotide of Sox-4 inhibited the induction of Sox-4 expression and blocked the formation of DNA fragmentation by PGA(2) and delta(12)-PGJ(12) in Hep3B and HepG2 cells. Sox-4-induced apoptosis was accompanied with caspase-1 activation indicating that caspase cascade was involved in this apoptotic pathway. These results indicate that Sox-4 is involved in Hep3B and HepG2 cells apoptosis as an important apoptotic mediator.

Prostaglandin A1, inhibits replication of Classical Swine Fever Virus

Prostaglandins (Pgs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report the effect of prostaglandin (PgA1) on the multiplication of a positive strand RNA virus, Classical Swine Fever Virus (CSFV) in PK15 cells. PgA1 was found to inhibit the multiplication of CSFV. At a concentration of 5 µg/ml, which was nontoxic to the cell, PgA1 inhibits virus production in 99 per cent. In PgA1 treated cells the size and number of characteristic Classical Swine Fever focus decreased in amount.

Inhibition of Mayaro virus replication by prostagandin A1 in Aedes albopictus cells

Prostaglandin Al (PGA1) inhibits Mayaro virus replication in Aedes albopictus cells at nontoxic doses to uninfected cells . At 10 ug/ml, PGA1 decreases virus production by 90 (per cent). The presence of PGA1 during virus adsorption, with no treatment after infection, reduces virus yield by 41 (per cent). Antiviral activity is observed even when treatment starts at one or two hours post-infection. However, in cells pre-treated with PGA1 during 24 hours, virus replication is not impaired. Thus, events ocurring during initial stages of infection and after virus adsorption and penetration must be the target of PGA1 action. SDS-PAGE analysis of 35S-methionine labelled proteins shows that PGA1 inhibits the synthesis of viral proteins and induces the synthesis of polypeptides with molecular weight of 70 kDa, 57 kDa and 23 kDa. In cells pre-treated with actinomycin D the induction of those proteins is suppressed. In addition, actinomycin D treatment prevents PGA1 antiviral activity, indicating that PGA1-induced stress proteins are probably involved in this mechanism

Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells

The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 µg/ml PGB2 inhibited virus yield by 60 percent, at the same dose PGA1 suppressed virus replication by more than 90 percent. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2

Effect of prostaglandin A1 in the induction of stress proteins in Aedes albopictus cells

Prostaglandin are natural fatty acid derivatives with diverse physiological effects, including immune function and the control of cell growth. While the action of prostaglandins in the induction of stress proteins in vertebrate cells in well documented, their functions in invertebrate cells have been poorly investigated. The purpose of the present study was to investigate the effect of prostaglandin A1 (PGA1; 0.25, 1.25 and 12.5 mug/ml) on protein synthesis during the growth of Aedes albopictus cells. We found that PGA1 stimulates the synthesis of several polypeptides with molecular masses of 87,80,70,57,29,27 and 23 kDa in Aedes albopictus cells. When the protein induced by PGA1 and those induced by heat treatment were compared by polyacrylamide gel electrophoresis, PGA1 was found to induce the stress proteins. The HSP70 family and the low-molecular weight polypeptides (29 and 27 kDa, respectively) were induced by PGA1 in the lag phase. We lso observed that PGA1 is able to induce a 23-kDa polypeptide independently of the growth phase of the cell.