Amino Acids 2012, 42:1803–1808 PubMedCrossRef 176 Haff


Amino Acids 2012, 42:1803–1808.PubMedCrossRef 176. Haff

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The level of significance was considered as P < 0 05 Multivariat

The level of significance was considered as P < 0.05. Multivariate logistic regression analysis was used to determine predictor variables that predict the postoperative complications, hospital stay and mortality. Ethical consideration Ethical approval to conduct the study was obtained from the CUHAS-Bugando/BMC joint institutional ethic review committee before the commencement of the study. Patients were required to sign a written informed consent for the study and for HIV testing. Results Socio-demographic data During the study period, a total of 2643 patients were admitted

to our centre and underwent laparotomy for various abdominal conditions. Of these 527 patients underwent laparotomy for bowel obstruction. Out of 527 patients, the underlying cause of SYN-117 obstruction was

intestinal tuberculosis confirmed by histopathology in 129 patients. Of these, 11 patients were excluded from mTOR activity the study due failure to meet the inclusion criteria. Thus, 118 patients representing 22.4% of cases (i.e. 118 out of 527 patients) were enrolled into the study. Seventy-six (64.4%) were males and 42 (35.6%) females, with a male to female ratio of 1.8: 1. The age of patients at presentation ranged from 11 to 67 years with a median age of 26 years. The peak age incidence was in the age group of 21-30 years accounting for 50.0% of cases (Figure 1). Eighty-eight (74.6%) patients were aged 40 years and below. Most of patients, 91 (77.1%) had either primary or no formal education and more than 75% of them were unemployed. The majority of patients, 86 (72.9%) came from the rural areas located a considerable distance from the study area and more than 80% of them had no identifiable health insurance. Figure 1 Distribution of patients according to age group. Clinical presentation among patients with tuberculous bowel obstruction The duration of symptoms prior to admission varied between 4 days to 12 months with a median of 8 months. The majority of our patients, 68 (57.6%) had symptoms of more than 6 months duration at the time of presentation. Out of 118 patients, 87 (73.7%) were considered to have primary intestinal tuberculosis

and the remaining 31 (26.3%) had secondary intestinal tuberculosis (i.e. associated with pulmonary tuberculosis) with 3-mercaptopyruvate sulfurtransferase remarkable chest x-rays, past history of pulmonary tuberculosis was positive in only 28 patients (23.7%). Out of these, eight patients were on treatment with anti-tuberculous drugs while fourteen had already taken a complete course of anti-tubercular drugs. The remaining six patients were defaulters. Sixty two (51.5%) patients presented with acute intestinal obstruction, thirty-four (28.8%) with sub-acute intestinal obstruction, sixteen (13.6%) with signs of peritonism and six (5.1%) with abdominal mass. Abdominal pain was the most common symptom and occurred in all cases (Table 2). In this study, twelve (10.

The bands were detected with EzWest Lumi plus (ATTO, Tokyo, Japan

The bands were detected with EzWest Lumi plus (ATTO, Tokyo, Japan) and ImageQuant LAS 4000mini (GE Healthcare UK Ltd, Little Chalfont, UK). Liquid chromatography (LC)/mass spectrometry (MS) analysis Protein spots in gels were compared and

analyzed by visual inspection. The gel spots were stored in 1% acetic acid and were subjected to LC/MS/MS analysis. Identification of proteins was carried out using Mascot server (Matrix Science) with datasets of rodent and Leptospira proteomes. A protein score of >40 was used to select proteins with significant matching. The difference between the theoretical and experimental mass and pI was also used to determine significant matching. Acknowledgments This study was supported by a grant of the Science and Technology Research Partnership for Sustainable Development (SATREPS) program from Japan Science and Technology Agency (JST) and Japan International Cooperation Agency (JICA). We thank Selleck Ferrostatin-1 Dr. H. Sumimoto and colleagues of the Research Support Center, Graduate

School of Medical Sciences, Kyushu University for their technical support and advice. We also thank Sayaka Akiyoshi, Takayoshi Yamaguchi, Hideko Kameyama, and Naomi Hidaka for their technical cooperation. Electronic supplementary material Additional file 1: Table S1: Amino acid sequence coverage of leptospiral HADH by LC/MS/MS. (DOC 33 KB) References 1. Levett PN: Leptospirosis. PF-01367338 mw Clin Microbiol Rev 2001,14(2):296–326.PubMedCentralPubMedCrossRef

2. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Willig MR, Gotuzzo E, MK-1775 Vinetz JM, Peru-United States Leptospirosis Consortium: Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 2003,3(12):757–771.PubMedCrossRef 3. Picardeau M: Diagnosis and epidemiology of leptospirosis. Med Mal Infect 2013,43(1):1–9.PubMedCrossRef 4. Adler B, de la Pena MA: Leptospira and leptospirosis. Vet Microbiol 2010,140(3–4):287–296.PubMedCrossRef 5. Toyokawa T, Ohnishi N-acetylglucosamine-1-phosphate transferase M, Koizumi N: Diagnosis of acute leptospirosis. Expert Rev Anti Infect Ther 2011,9(1):111–121.PubMedCrossRef 6. Vijayachari P, Sugunan AP, Shriram AN: Leptospirosis: an emerging global public health problem. J Biosci 2008,33(4):557–569.PubMedCrossRef 7. Camargo ED, da Silva MV, Batista L, Vaz AJ, Sakata EE: An evaluation of the ELISA-IgM test in the early diagnosis of human leptospirosis. Rev Inst Med Trop Sao Paulo 1992,34(4):355–357.PubMedCrossRef 8. Fonseca Cde A, Teixeira MM, Romero EC, Tengan FM, Silva MV, Shikanai-Yasuda MA: Leptospira DNA detection for the diagnosis of human leptospirosis. J Infect 2006,52(1):15–22.PubMedCrossRef 9. Balassiano IT, Vital-Brazil JM, Pereira MM: Leptospirosis diagnosis by immunocapture polymerase chain reaction: a new tool for early diagnosis and epidemiologic surveillance. Diagn Microbiol Infect Dis 2012,74(1):11–15.PubMedCrossRef 10.

Conclusion Developing novel approaches for defining oncogene addi

Conclusion Developing novel approaches for defining oncogene addiction networks, coupled with specific combination of molecular targeted agents, will make it possible to achieve more effective and personalized molecular targeted therapy in human gliomas. Author details 1Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, No.6 Tiantan Xili, Dongcheng District, Beijing 100050, China. Acknowledgements This work was supported by grants from National Key Project of Science and Technology Supporting Programs (No. 2007BAI05B08) and National XMU-MP-1 price Natural Science Foundation

of China (No. 30772238 and 30730035). References 1. Mizuarai S, Irie H, Schmatz DM, Kotani H: Integrated genomic and pharmacological

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amplification, PDGFRA oncogene addiction and sunitinib sensitivity. Cancer Biol Ther 2009, 8:2051–2053.PubMedCrossRef 10. Togano T, Sasaki M, Watanabe M, Nakashima M, Tsuruo T, Umezawa K, Higashihara M, Watanabe T, Horie R: Induction of oncogene addiction shift to NF-kappaB by camptothecin in solid tumor cells. Biochem Biophys Res Commun 2009, 390:60–64.PubMedCrossRef 11. Jin Y, Chen Q, Lu Z, Chen B, Pan J: Triptolide abrogates oncogene FIP1L1-PDGFRalpha addiction and induces apoptosis in hypereosinophilic syndrome. Cancer Sci 2009, 100:2210–2217.PubMedCrossRef 12. Calzolari F, Appolloni I, Tutucci E, Caviglia S, Terrile M, Corte G, Malatesta P: Tumor progression and oncogene addiction in a PDGF-B-induced model of gliomagenesis. Neoplasia 2008, 10:1373–1382. following 1382.PubMed 13. Rothenberg SM, Engelman JA, Le S, Riese DJ, Haber DA, Settleman J: Modeling oncogene addiction using RNA interference.

PubMedCrossRef 40 Grimson MJ, Barker

GC: A continuum mod

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GC: A continuum model for the growth of bacterial colonies on a surface. J Phys A: Math Gen 1993, 26:5645–5654.CrossRef 41. Kreft JU, Booth G, Wimpenny JWT: BacSim, a simulator for individual-based modelling of bacterial colony growth. Microbiology 1998, 144:3275–3287.PubMedCrossRef 42. Panikov NS, Belova SE, Dorofeev AG: Nonlinearity in the growth of bacterial colonies: conditions and causes. Microbiology (Mikrobiologiya) 2002, 71:50–56. 43. Sekowska A, Masson JB, Celani A, Danchin A, Vergassola M: Repulsion and metabolic switches in the collective behavior of bacterial colonies. Biophys J 2009, 97:688–698.PubMedCrossRef MLL inhibitor 44. Miyata S, Sasaki T: Asymptotic analysis of a chemotactic model of bacteria colonies. Math Biosci 2006, 201:184–194.PubMedCrossRef 45. Cho HJ, Jönsson H, Campbell K, Melke selleck chemical P, Williams JW, Jedynak B, Stevens AM, Groisman A, Levchenko A: Self-organization in high-density bacterial colonies: efficient crowd control. PLoS Biol 2007, 5:e302.PubMedCrossRef 46. Levine H, Ben-Jacob E: Physical schemata underlying biological pattern formation – examples, issues and strategies. Phys Biol 2004, 1:P14-P22.PubMedCrossRef 47. Pipe L, Grimson MJ: Spatial-temporal modelling of bacterial colony growth on solid media. Mol BioSyst 2008, 4:192–198.PubMedCrossRef 48. Odagiri K, Takatsuka K:

Threshold effect with stochastic fluctuation in bacteria-colony-like proliferation dynamics as analyzed through a comparative study of reaction-diffusion

equations and cellular automata. Phys Rev E 2009, 79:-026202. 49. Ayati BP: A structured-population model of Proteus mirabilis swarm-colony development. J Math Biol 2006, 52:93–114.PubMedCrossRef 50. Grammaticos B, Badoual M, Aubert M: An (almost) solvable model for bacterial pattern formation. Physica D 2007, 234:90–97.CrossRef 51. Arouh S: Analytic model for ring pattern formation by bacterial swarmers. Phys Rev E 2001, 63:031908.CrossRef 52. Python programming language – official website [http://​www.​python.​org] Authors’ contributions JC and IP contributed equally to the designing and performing the experiments and interpreting their results; FC developed the formal model and participated in writing the paper; AB participated in experiments and data interpretation and provided Org 27569 basic technical support; AM participated in study design and data interpretation and drafted the paper. All authors have read and approved the final manuscript.”
“Background Nitrogen is incorporated into glutamate and glutamine which form the major biosynthetic donors for all other nitrogen containing components in a cell. Glutamine is a source of nitrogen for the synthesis of purines, pyrimidines, a number of amino acids, KU55933 supplier glucosamine and ρ-benzoate, whereas glutamate provides nitrogen for most transaminases [1] and is responsible for 85% of nitrogenous compounds in a cell [2]. In most prokaryotes, there are two major routes for ammonium assimilation.

All control groups showed a 100% survival rate In addition to th

All control groups showed a 100% survival rate. In addition to the phage and bacterial host concentrations, the Combretastatin A4 in vitro incubation time was also important

for the bactericidal effect. Approximately 95% of phage particles adsorbed to host cells within 5 min, and nearly 100% were adsorbed by 10 min (Figure 1). Therefore, we selected the 5 and 10 min time points to test the bactericidal effect of ϕAB2 in suspension. At a low phage concentration (103 PFU/ml), an increase in the incubation time from 5 to 10 min resulted in a mean decrease of survival rate of MDRAB between 1.5- and 1,700-fold. In contrast, at higher phage concentrations (105 PFU/ml and 108 PFU/ml) there was a mean reduction of bacterial concentration of 1.4- to 7-fold when the incubation time was increased from 5 to 10 min. Figure 3 Bactericidal effect of JNJ-26481585 purchase different concentrations: (A) 10 3 (B) 10 5 , and (C) 10 8 PFU/ml of ϕAB2 on different concentrations of A. baumannii M3237 in a liquid suspension, at incubation times of 5 and 10 min. The survival rate was calculated as in the Methods section. NF-��B inhibitor These experiments were repeated

three times, and the data shown are the mean ± SEM. *p < 0.05 compared with the respective control group. Bactericidal effect of ϕAB2 on a hard surface The addition of ϕAB2 to a hard glass surface contaminated with A. baumannii M3237 had a bactericidal effect under some conditions (Figure 4). Phage concentrations of 103 and 105 PFU/slide caused a significant reduction (p < 0.05, 40% reduction) of A. baumannii M3237 cells (104 and 105 CFU/slide)

after 10 min (Figure 4A and B). When a phage concentration of 108 PFU/slide was used, the number of A. baumannii ADP ribosylation factor M3237 was significantly reduced (p < 0.05, >90% reduction) after 5 or 10 min for all concentrations of bacteria tested (Figure 4C). However, the bactericidal effect of ϕAB2 at 108 PFU/slide was significantly lower for A. baumannii M3237 at 104 and 105 CFU/slide than at 106 CFU/slide (p < 0.05). To date, there is no standard method for evaluating phage biocontrol efficiency on a hard surface. Incubation times of 5 and 10 min were chosen for surface tests on the basis of ϕAB2 adsorption data (Figure 1) and a previous study by Abuladze et al. [26]. Extending the incubation time from 5 to 10 min increased the mean bactericidal effect on A. baumannii M3237 1.3-fold under all test conditions. Figure 4 Bactericidal effect of different concentrations: (A) 10 3 (B) 10 5 , and (C) 10 8 PFU/slide of ϕAB2 on different concentrations of A. baumannii M3237 on a glass surface following incubation times of 5 and 10 min. The survival rate was calculated as in the Methods section. These experiments were repeated three times, and the data shown are the mean ± SEM. *p < 0.05 compared with respective control group. Use of ϕAB2 as a hand sanitizer in a paraffin oil-based lotion The stability of ϕAB2 in a lotion and its ability to kill A.

Using this criterion we constructed GlnJ

Using this criterion we constructed GlnJ variants with the following substitutions: R17K, Q42H, N54D, K85R, V100M and E109G (in each position the residue in GlnJ was replaced by the corresponding one in GlnB). These variants were expressed and purified as N-terminal AUY-922 mouse histidine tagged fusions. Figure 1 Alignment of the amino acid sequence of the R. rubrum GlnB and GlnJ proteins, constructed using ClustalW (http://​www.​ebi.​ac.​uk/​Tools/​clustalw2/​index.​html).

The loop regions are highlighted and the positions of the amino acid substitutions used in this study are marked with a star. Although not all the residues selected are located in regions of the PII protein that have previously been shown to be involved in metabolite binding, we decided to analyze amino acids occurring in areas of high conservation as, due to the considerable flexibility of the PII structure, they may also play a role in this response to divalent cations. An example of this high flexibility comes from the recent structure of S. elongatus mTOR inhibitor GlnB, where the

very C-terminal portion of the protein displays a large conformational change upon binding of the ligands to the T-loop region [9]. Uridylylation of GlnJ variants in the presence of Mn2+ and Mg2+ Using purified GlnD and GlnJ variants we analysed the uridylylation profile in the conditions that were previously determined [11] and described in the Materials and methods, with either Mg2+ or Mn2+ present in the assays. As shown in Figure 2, GlnJ is only extensively modified in the presence of Mn2+ (A) while GlnB is modified with both Mn2+ and Mg2+ (B), as analyzed by native PAGE, with a selleckchem slower migrating band

converted to a faster migrating band (all 3 subunits modified). The identity (and uridylylation status) of the two forms was also confirmed by mass spectrometry (results not shown). The GlnJ variants R17K, V100I and E109G showed the same pattern as GlnJ (Figure 2A). The GlnJN54D variant can still be modified in the presence of Mn2+ albeit to a lower extent, but there was also no modification in the presence of Mg2+. The variants GlnJQ42H and GlnJK85R show normal uridylylation in the presence of Mn2+ but enhanced with Mg2+(Figure 2A). Given the fact that only the GlnJQ42H and GlnJK85R substitutions 6-phosphogluconolactonase supported modification with Mg2+, we combined them and constructed the GlnJQ42HK85R variant. In this case, the modification in the presence of Mn2+ was identical to GlnJ, but substantially improved with Mg2+ (Figure 2A). Figure 2 Uridylylation of GlnJ (A) and GlnB (B) variants. The reactions were performed as described in the Materials and methods in the presence of Mn2+, Mg2+ or without either divalent cation (control – C), and the uridylylation status analyzed by native PAGE. U – unmodified, M3- modified (fully modified trimmers).

To test the ability of klotho to modulate IGF-1-induced prolifera

To test the ability of klotho to modulate IGF-1-induced proliferation and survival, A549 cells were transiently transfected with either pCMV6 or SN-38 ic50 pCMV6-MYC-KL and grown in 0.5% serum with either IGF-1 or a control vehicle for 24-96 hr. Klotho transfection obviously

inhibited cell proliferation in the untreated cells, and this inhibition was only mildly restored following addition of IGF-1 to Akt inhibitor review the cells. Thus, whereas IGF-1 increased cell proliferation by up to 33% in control pCMV6-transfected cells, cell proliferation in the pCMV6-MYC-KL-transfected cells increased by only 11% (Figure 3B). Klotho inhibits the activation of the IGF-1/insulin pathways and is directly associated with IGF-1R in lung cancer cells We studied the effect of klotho on IGF-1 pathway activation in A549 lung cancer cells, which check details express high levels of IGF-1R and show an enhanced proliferation following IGF-1 treatment. A549 cells were transfected with either pCMV6-MYC-KL or pCMV6, starved for 24 hr, treated with IGF-1 (10 min, 25 nM) and analysed using western blotting for the expression and phosphorylation of IGF-1R. Klotho overexpression in A549 cells was associated with reduced phosphorylation of IGF-1R (P < 0.01). The effects of overexpression of klotho on the insulin

(10 min, 100 nM) pathway were also examined, and similar to IGF-1 activation, klotho overexpression in A549 cells was associated with reduced phosphorylation of insulin receptor (IR, P < 0.01), indicating that klotho also inhibited the activation of the insulin pathway in A549 cells. We further studied the effect of klotho knockdown in Miconazole A549 cells using sh-2, and found a significant increase in IGF-1R/IR phosphorylation following IGF-1/insulin stimulation in sh-2-transfected cells

compared with siRNAc-transfected cells. The results were shown in Figure 4. Figure 4 Downregulation of the IGF-1/insulin pathways by klotho in lung cancer cell line A549. A549 cells were transfected with either MYC-KL or control vector pCMV6. After 24 hr, cells were serum-starved for 24 hr and treated with IGF-1 (10 min, 25 nM) or insulin (10 min, 100 nM). Following treatment, cells were harvested and proteins were resolved and immunoblotted using antibodies either directed against phospho (P) and total (T) IGF-1R or phospho (P) and total (T) insulin-R (IR). Similar treatment was done when silenced the klotho of the cells using sh-2 or control shRNAc. Data shown are the mean results ± SD of a representative experiment performed in triplicate (n = 3), *indicates p < 0.01. Klotho-induced apoptosis of A549 cells To determine the effects of overexpression or downregulation of klotho on the klotho-induced apoptosis in A549 cells, the rate of apoptosis was evaluated by flow cytometry analysis. As shown in Figure 5, the effects of klotho-induced apoptosis were investigated in pCMV6 cells as well as cells transfected with pCMV6-MYC-KL, sh-2 or shRNAc.

Ea1189-3(pBlueKS acrD), expressing acrD under control of Plac, ex

Ea1189-3(pBlueKS.acrD), expressing acrD under control of Plac, exhibited elevated resistance to clotrimazole (2-fold), fusidic acid (2-fold), novobiocin (4-fold), hygromycin B (2-fold), cadmium acetate (2-fold), zinc sulfate (2-fold), bile salt (2-fold), deoxycholate (4-fold), and SDS (2-fold). The expression of acrD under control of its native promoter in Ea1189-3 showed an increase in resistance similar to that of Plac-controlled acrD expression (data not shown). When acrD was under control of both promoters, Plac and PacrD, it conferred elevated resistance. Compared to the control, Ea1189-3(pBlueKS.acrD-ext) displayed increased resistance

to clotrimazole (4-fold), fusidic acid (8-fold), novobiocin (16-fold), hygromycin B (2-fold), cadmium acetate (2-fold), zinc this website sulfate (2-fold), bile salt (8-fold), deoxycholate (8-fold), SDS (2-fold), luteolin (8-fold) and ethidium bromide (2-fold) (Table 1). RND-type efflux pump expression during cellular growth To monitor the expression levels of the RND-type efflux pumps AcrAB and AcrD at different ACY-1215 purchase growth states, total RNA was isolated at distinct optical densities and expression levels analyzed by quantitative RT-PCR. The expression values were normalized to the highest expression of the acrA and acrD transcript, respectively

(Figure 1A). While the expression levels of acrA changed during the cell cycle, indicating a growth phase-dependent transcription Mannose-binding protein-associated serine protease with the highest expression in the early exponential phase, acrD showed constant expression

during growth. Additionally, the constant expression of acrD was also connected to a low expression level as determined by Ct values (data not shown). Figure 1 Promoter activities of acrA and acrD from Erwinia amylovora. The activity was determined in the course of growth in LB broth, OD600, optical density at 600 nm. (A) Relative mRNA transcript VE-822 concentration abundance of acrA and acrD during cellular growth of Ea1189 as determined by quantitative RT-PCR. The relative mRNA level was related to the highest average value determined for a gene, which was defined as 100%. (B) Expression of acrA and acrD as determined by transcriptional fusions with the reporter gene egfp. E. amylovora wild type was transformed with pBBR.acrA-Pro.egfp and pBBR.acrD-Pro.egfp, respectively. Experiments were performed in triplicates with similar results. Furthermore, we studied the effect of temperature on activation of the RND-type efflux pump AcrD using qRT-PCR. Bacteria were cultured in LB broth at 18°C and 28°C, respectively, where 28°C represents the optimal growth temperature and 18°C represents the temperature at which several genes involved in pathogenicity showed induction in E. amylovora[30, 31]. However, no temperature dependence of the acrD expression was observed in vitro (data not shown). Promoter activity of acrAB and acrD in vitro In order to monitor promoter activities of the RND-type efflux pumps AcrAB and AcrD in E.

Motility was determined using sulfide-indole-motility medium Fat

Motility was determined using sulfide-indole-motility medium. Fatty acid methyl esters were extracted and analyzed by the Sherlock Microbial Identification system (MIDI, Newark, DE) according to the manufacturer’s instructions. All assays were selleck products performed in triplicate. The 16S rRNA gene of strain B7 was amplified by PCR with the universal MGCD0103 in vitro primers 27F and 1541R and sequenced [16]. Phylogenetic trees were constructed using the neighbor-joining and maximum-parsimony algorithm within MEGA4 [17]. The DNA-DNA hybridization between B7

and Paenibacillus ehimensis IFO 15659T was performed using the thermal denaturation method [14]. Production and purification of active compounds Strain B7 maintained on nutrient agar slants was inoculated into 50 mL of nutrient broth and cultivated at 30°C for 24 h. The seed culture of strain B7 was transferred

to a 2L Erlenmeyer flask that contained 500 mL of the KL medium. The culture was incubated on a rotary shaker (200 rpm) at 30°C for 3 d. After centrifugation at 4500 g for 30 min at 4°C, the cell-free LY2109761 molecular weight supernatant was loaded onto a column packed with Amberlite XAD-16 resin (Sigma, St. Louis, MO). The column was washed with distilled water prior to elution with stepwise gradients of aqueous methanol (30, 60, and 100%, v/v). Each fraction was concentrated and assessed for activity using the paper disc method. The Branched chain aminotransferase active fraction was evaporated and dried before being redissolved in acetonitrile. The concentrated solution was then applied to a C18 SPE column (Hardwee, Germany). The column was washed with five bed volumes of distilled water, followed by five bed volumes of an acetonitrile/water mixture (20:80, v/v). The fraction that contained the active

compounds was eluted from the column by washing with three bed volumes of an acetonitrile/water mixture (68:32, v/v). Further purification was performed using a preparative HPLC system (Dalian Elite, Dalian, China) that was equipped with an YMC-pack DOS-A C18 (5 μm, 250 × 20 mm) column. The mobile phase consisted of Milli-Q water that contained 0.02% trifluoroacetic acid and acetonitrile. A linear gradient of 15% to 55% acetonitrile (40 min) was used for elution at a flow rate of 10 mL/min. UV detection was performed at a wavelength of 210 nm. Fractions from multiple runs were collected and combined for the subsequent antimicrobial activity assays. The active fractions were passed through the HPLC column two consecutive times. Amino acid analysis Approximately 300 μg of the purified compound in 0.4 ml of 6 M HCl with 0.1% phenol was hydrolyzed at 110°C for 16 h. Amino acid analyses was performed using ion-exchange chromatography with a Hitachi L-8900 amino acid analyzer (Tokyo, Japan) according to the method described by Qian et al. [18].