Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown.The crude extract of the fruit bearing plant, Herein, we isolated the main pure compound, 4β-Hydroxywithanolide (4βHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug.p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4βHWE in both dose- and time-dependent manners . The half maximal inhibitory concentrations (IC50) of 4βHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 μg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4βHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 μg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 μg/mL for 24 h.It was shown that DNA damage was significantly induced by 1, 5, and 10 μg/mL 4βHWE for 2 h in a dose-dependent manner (In this study, we demonstrated that golden berry-derived 4βHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer. Lung cancer is a leading cause of cancer death in the US  and otheDietary chemoprevention using fruits and foods -7 as a pPhysalis peruviana (golden berry) is a member of the Solanaceae, an edible plant family. The fruit tastes like a sweet tomato and includes high levels of vitamin C, vitamin A, and the vitamin B-complex. The fruit was demonstrated to have both anti-inflammatory and antioxidant properties [P. peruviana extracts were reported to have anti-hepatoma activity due to the induction of apoptosis in a human hepatocellular carcinoma (Hep G2) cell line [operties -18. Moreell line .Tubocapsicum anomalum and reported to be effective against liver, lung, and breast cancer cell lines [P. peruviana [P. peruviana are capable of anticancer activity.Recently, various cytotoxic withanolides  were isolated from eruviana -23; howeeruviana  are stilP. peruviana, this plant was chosen as the basis for our investigations. Using a bioactivity-guided fractionation method, we isolated the major active compound, 4βHWE Although this was first isolated from P. peruviana by other researchers a long time ago [P. peruviana. To test this hypothesis, we used golden berry-derived 4βHWE to investigate the anticancer potential against human lung carcinoma H1299 cells using a new toxicological technique.On the basis of the description of active extracts from time ago , its posThe plant material (10.8 kg) was collected at the Tainan District Agricultural Research and Extension Station, Taiwan, in April 2005. Before the extraction process, we deposited the original plant as a voucher specimen (standard) in the Graduate Institute of Natural Products  to ensure adequate collection data.P. peruviana (10.8 kg) were extracted three times with EtOH at room temperature to obtain a crude extract. The crude extract was partitioned with EtOAc and H2O  to give an EtOAc layer which was then partitioned with n-hexane, MeOH, and H2O  to give an MeOH/H2O layer (65.2 g). The MeOH/H2O layer extract was chromatographed over a silica gel column, using polarity gradient mixtures of n-hexane-EtOAc-MeOH , , , , , , , , , , and  as eluants, and this yielded 24 fractions. The volume for each fraction was 500 ml. Fraction 15 (9154.4 mg) was subjected to Sephadex LH-20 (EtOAc:MeOH 1:4), and the subfraction (fr. 15-3-7) contained crystals which were acetone-washed to provide the main pure compound.The aerial parts (stems and leaves) of 2. A H1299 (human lung adenocarcinoma) cell line was used to test the cytotoxicity against lung cancer; an HGF-1  [Cells were routinely cultured in 1× RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.03% glutamine. All cells were kept at 37°C in a humidified atmosphere containing 5% COroblast)  cell lin6 cells/ml in phosphate-buffered saline; PBS) were mixed with equal volumes of 1.2% low-melting-point agarose  and immediately pipetted onto a slide precoated with 1.2% regular agarose (in distilled water), and the slide was placed on ice to solidify the gel. The third layer (100 μl of 1.2% low-melting agarose gel) was coated onto the solidified second gel, and then the slide was placed on ice to solidify the gel. The slides were immersed in freshly made ice-cold cell lysis solution , 1% N-laurylsarcosine, 1% Triton X-100, and 10% dimethyl sulfoxide (DMSO), stored at 4°C for 2 h, and rinsed with deionized water three times. A 20 μl excision mixture containing 0.6 μg NE, 50 mM Hepes-KOH (pH 7.9), 70 mM KCl, 5 mM MgCl2, 0.4 mM EDTA, 2 mM ATP, 40 mM phosphocreatine, and 2.5 mM creatine phosphokinase was added to each slide for NE digestion. A coverslip was applied, and the slides were then incubated at 37°C for 2 h in a humidified space. The slides were denatured in 0.3 N NaOH and 1 mM EDTA for 20 min followed by electrophoresis at 20 V and 300 mA for 25 min. After being washed with deionized water, the slides were transferred to 0.4 M Tris-HCl (pH 7.5). Then 40 μl propidium iodide  was added. For fluorescence microscopy , the migration of DNA from the nucleus of each cell was measured with a computer program http://tritekcorp.com using the parameter of the percent of tail DNA [The protocol for the comet-NE assay was as described previously ,26. In btail DNA ,28 which50 value was calculated by the slope and intercept according to two concentrations of 4βHWE between the half-maximal proliferative inhibitory concentrations.The proliferation of cells was determined by a Trypan blue dye exclusion assay as described ,29. In b6 cells per 100-mm Petri-dish (at 70%~80% confluency) were treated with the vehicle or 1 μg/mL of 4βHWE for 12 and 24 h. After treatment, cells were collected, washed twice with PBS, and fixed in 70% ethanol overnight. After centrifugation at 700 rpm for 5 min at 4°C, the cell pellet was stained with 10 μg/mL PI  and 10 μg/mL RNase A in PBS for 15 min at room temperature in the dark. The analyses were performed with a FACScan flow cytometer  and Cell-Quest and Modfit software (Becton-Dickinson).The cell cycle histogram was determined as described before . In brie1 population of H1299 cells was determined by flow cytometry as described above. To further confirm apoptosis in 4βHWE-induced H1299 cells, annexin V and PI double-staining  was performed as described before [6 cells per 100-mm Petri-dish (at 70%~80% confluency) were treated with vehicle or 51 μg/mL of 4βHWE for 24 h. Subsequently, cells were labeled with annexin V-FITC (10 μg/mL)/PI (10 μg/mL); stained slides were mounted, and analyzed using an inverted fluorescence microscope . Alternatively, the apoptotic ability of treated cells was analyzed using a flow cytometric analysis as described before [The accumulation of a sub-Gd before . In bried before .t-test was used to test the mean difference between the two groups.All data are presented as the mean ± SD; Student's P. peruviana was 4βHWE according to the extraction method listed in "Materials and Methods". Accordingly, the total collected plant extract was 10.8 kg, and the yield of 4βHWE was 866.5 mg. The chemical structure of 4βHWE is shown in Figure The main pure compound isolated from a, 3.66% ± 2.83%a, 47.89% ± 11.31%b, 62.10% ± 11.78%c, and 67.38% ± 11.58%d, respectively (p < 0.05 between different superscript letters). Accordingly, the DNA damage by 4βHWE had a significant dose-dependent effect. These results suggested that DNA damage was involved in the antiproliferation of 4βHWE towards lung cancer cells.In the comet assay for the H1299 cell line, no "tail" was found in the untreated control  while the "tail" grew after 4βHWE treatment  Figure . Figure 50 values of the 4βHWE-treated H1299 cell line for 24 and 48 h were 0.6 and 0.71 μg/mL, respectively. Growth inhibition of H1299 cells by 4βHWE occurred in a dose-responsive and time-dependent manner .Figure 1, G1, S, and G2/M percentages after drug treatment. It was found that the sub-G1 percentages were 3.5% ± 0.7%, 3.8% ± 0.5%, and 8.1% ± 0.8% (n = 3) for the vehicle, and 12 and 24 h of treatment with 4βHWE, respectively. G1 percentages were dramatically reduced from 69.1% ± 3.3% (vehicle) to 48.5% ± 3.6% and 43.7% ± 5.4% (n = 3) with 12 and 24 h of 4βHWE treatment, respectively. G2/M percentages were 18.6% ± 3.1%, 34.8% ± 1.9%, and 37.8% ± 0.3% (n = 3) for the vehicle or 12 and 24 h of 4βHWE treatment, respectively. Accordingly, moderate sub-G1 accumulation and G2/M arrest were found in a time-dependent manner for the 4βHWE-treated H1299 cell line. All significant differences between the vehicle and drug treatment are shown in Figure Figure 1 phase occurred. To perform a confirmatory study, we enhanced the dosage of 4βHWE to validate the cell cycle disturbance  [50 of the H1299 human lung cancer cell line .At high concentrations, the ethanol extract of function . In this Figures , 5A, dosk Figure , and apoThe conventional alkaline comet assay is sensitive to DNA strand breaks, but is not sensitive to DNA lesions without strand breaks. In contrast, the NE in the comet-NE assay theoretically contains all of the repair enzymes and increases the incision of most kinds of DNA damage. Therefore, the comet-NE assay -36 is ge2/M arrest and apoptosis in time-dependent manners. Our ongoing study further validates the effective cancer-preventive potential of 4βHWE in other cancer cell lines as well as in a xenograft assay. If 4βHWE is indeed proven to exert an effective cancer preventive potential in vivo, golden berry-derived 4βHWE may be a promising ingredient as a cancer-preventive functional food.We demonstrated herein that golden berry-derived 4βHWE had a strong inhibitory effect on the growth of H1299 lung carcinoma cells. This study demonstrated that the DNA damage caused by 4βHWE is the result of a toxicological mechanism. 4βHWE was also demonstrated to have anti-lung cancer activity, along with the potential to produce GThe authors declare that they have no competing interests.C-YY, C-CC, and H-WC participated in the writing and study design. Y-CW instructed F-RC and Y-HL on extracting the pure compound from the fruit. Y-CH, H-LY, and Y-CL provided instruction for the comet assay and its analysis. M-TT, Y-SC, Y-CC, and Y-CK participated in the comet assay experiments. JY-FC, C-CH, AY-LL, and Z-LG performed the apoptosis analysis. H-WC and Y-CW coordinated and oversaw the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/46/prepub


Rhipicephalus (Boophilus) spp. ticks economically impact on cattle production in Africa and other tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The R. microplus Bm86 protective antigen has been produced by recombinant DNA technology and shown to protect cattle against tick infestations.R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) were cloned and characterized from African or Asian tick strains and the recombinant proteins were secreted and purified from P. pastoris. The secretion of recombinant Bm86 ortholog proteins in P. pastoris allowed for a simple purification process rendering a final product with high recovery (35–42%) and purity (80–85%) and likely to result in a more reproducible conformation closely resembling the native protein. Rabbit immunization experiments with recombinant proteins showed immune cross-reactivity between Bm86 ortholog proteins.In this study, the genes for Bm86 (P. pastoris for the control of tick infestations in Africa.These experiments support the development and testing of vaccines containing recombinant Bm86, Ba86 and Bd86 secreted in  Rhipicephalus (Boophilus) spp. ticks are distributed in tropical and subtropical regions of the world with range expansion for some species due to changes in climatic conditions [Rhipicephalus (Boophilus) microplus, economically impact cattle production by reducing weight gain and milk production, and by transmitting pathogens that cause babesiosis (Babesia bovis and B. bigemina) and anaplasmosis  [R. annulatus and R. decoloratus also affect cattle production and vector pathogens in regions of Latin America, Africa or Asia [nditions -3. Infesrginale) . R. annu or Asia .Control of tick infestations has been difficult because ticks have few natural enemies. Integrated tick management strategies include the adaptation of different control methods to a geographic area. A major component of integrated tick control methods is the application of acaricides. However, use of acaricides has had limited efficacy in reducing tick infestations and is often accompanied by serious drawbacks, including the selection of acaricide-resistant ticks, environmental contamination and contamination of milk and meat products with drug residues . FurtherR. microplus Bm86 gut antigen [In the early 1990s, vaccines were developed that induced immunological protection of vertebrate hosts against tick infestations. These vaccines contained the recombinant  antigen . Two vac antigen . These v antigen -14. Thes antigen -15.R. microplus Bm86-containing vaccines also protect against related tick species, R. annulatus and R. decoloratus [R. microplus strain-to-strain variations in the susceptibility to Bm86 vaccination have been reported, which suggests that Bm86 sequence and/or tick physiological differences may influence the efficacy of the vaccine [Controlled immunization trials have shown that oloratus -18. Howe vaccine -22. Ther vaccine .Escherichia coli [Aspergillus nidulans and A. niger [Pichia pastoris [P. pastoris has been shown to be the more efficient for protein secretion [P. pastoris may increase the antigenicity and immunogenicity of the recombinant antigen [P. pastoris is not based on protein secretion but on the expression of the antigen anchored to the yeast membrane, making necessary the purification under denaturing conditions followed by refolding of an antigen with high number of disulfide bonds [R. decoloratus Bm86 orthologs were cloned, expressed in E. coli and partially characterized [R. annulatus and R. decoloratus Bm86 orthologs in P. pastoris have not been reported.The recombinant Bm86 has been expressed in hia coli , AspergiA. niger  and Pichpastoris ,24,25. Oecretion ,27. Furt antigen ,29. Howede bonds . Recentlcterized . HoweverP. pastoris the recombinant R. microplus, R. decoloratus and R. annulatus Bm86 orthologs from African or Asian tick strains and (ii) to simplify the Bm86 production process by secreting recombinant proteins encoded by Bm86 orthologs in P. pastoris.The objectives of this study were (i) to clone and express in R. microplus (Bm86), Israeli R. annulatus (Ba86) and South African R. decoloratus (Bd86) tick strains. Partial sequences were obtained and used to search the NCBI nr database for sequence identity. The first four BLAST hits  showed that cloned Bm86, Bd86 and Ba86 sequences were identical (90–97% identity) to previously reported Bm86 , Bm95  and Bd86-1 and Bd86-2  sequences. The only fragment of 1,107 nucleotides previously reported for Ba86  had 99.9% identity to the Ba86 sequence reported here with a single A × G substitution at position 1,674 (position 1 corresponds to the adenine in the initiation codon of the M29321 reference sequence). The Bm86 sequence of the Mozambique R. microplus strain reported here had a deletion of 66 nucleotides between positions 554 and 619 not found in other Bm86 sequences, which suggested that this region encoding for 22 amino acids may not be important for protein function. The Bd86 sequence of the South African R. decoloratus strain had an 18 nucleotides insertion between positions 1,690 and 1,691, similar to Bd86-2 and three nucleotides longer than in Bd86-1 [The Bm86 orthologs were cloned by RT-PCR from Mozambique n Bd86-1 .Pairwise nucleotide and amino acid sequence alignments were conducted between cloned Bm86, Ba86 and Bd86 sequences and those identified above to have identity to these sequences Table . The resP. pastoris strains GS115, KM71H and X33 for expression of recombinant Bm86, Bd86 and Ba86 proteins. Single colonies of P. pastoris transformants for each gene were grown in an orbital shaker under induction conditions. Culture supernatants were spotted on a nitrocellulose membrane for dot-blot analysis of recombinant proteins. Expression of Bm86 and Bd86 was obtained in GS115 and KM71H strains while Ba86 was expressed in strain X33 only  ended after 20–24 hrs and induction of recombinant protein expression started at the onset of methanol-adoption and utilization phases. As expected, all strains behaved similarly when growing on glycerol as the sole carbon source Table . Cell depastoris ,35.-1 for the strains GS115Moz9-2, KM71HDec8-1 and X33pBaI-3, respectively. However, after 24 hrs growth in methanol, cells stop growing and a steady increase in pO2 levels revealed that a stationary growth phase was achieved. Nevertheless, total protein production continued to increase gradually to 274, 194 and 170 mg·L-1 for the strains GS115Moz9-2, KM71HDec8-1 and X33pBaI-3, respectively  or VWR International Eurolab S.L. . The compositions of the media used in this study were as follows:-1 yeast nitrogen base with ammonium sulphate and without amino acids (YNB); 0.0004 g·L-1biotin; 15 g·L-1 agar and 0.5% methanol.Minimal methanol medium (MM): 13.4 g·L-1 YNB; 0.0004 g·L-1 biotin; 15 g·L-1 agar; 0.04 g·L-1 histidine and 0.5% methanol.Minimal methanol medium + Histidine (MMH): 13.4 g·L-1 YNB; 0.0004 g·L-1 biotin; 15 g·L-1 agar and 20 g·L-1 dextrose.Minimal dextrose medium (MD): 13.4 g·L-1 YNB; 0.0004 g·L-1 biotin; 15 g·L-1 agar; 20 g·L-1 dextrose and 20 g·L-1 dextrose.Minimal dextrose medium + Histidine (MDH): 13.4 g·L-1 yeast extract and 20 g·L-1 peptone.Yeast Extract Peptone medium (YP): 10 g·L-1 yeast extract; 20 g·L-1 peptone and 20 g·L-1 glucose.Yeast Extract Peptone Dextrose medium (YPD): 10 g·L-1yeast extract; 20 g·L-1 peptone; 20 g·L-1 glucose; 182 g·L-1 sorbitol and 20 g·L-1 agar.Yeast Extract Peptone Dextrose Sorbitol medium (YPDS): 10 g·L-1 ZnSO4 × 7H2O; 0.02 g·L-1 CuSO4 × 5H2O; 0.08 g·L-1 KI; 0.3 g·L-1 MnSO4 × H2O; 0.19 g·L-1Na2MoO4 × H2O; 0.02 g·L-1 H3BO3 and 2.9 g·L-1 FeCl3.Trace element solution (TES): 2.0 g·L-1 calcium pantothenate; 0.4 g·L-1 tyamine; 4 g·L-1 myo-inositol; 0.1 g·L-1 nicotinic acid; 0.4 g·L-1 pyridoxine and 0.4 g·L-1 biotin.Vitamin solution (VT): 0.4 g·L-1 KH2PO4; 8.75 g·L-1 (NH4)2SO4; 4.5 g·L-1 MgSO4; 0.5 g·L-1 CaCl2 × 2H2O; 2.5 g·L-1 yeast extract; 5 ml·L-1 TES and 5 ml·L-1 VT.Production medium (PM): 13 g·LR. microplus), Israel (R. annulatus) and South Africa (R. decoloratus).Tick strains were obtained from laboratory colonies maintained at the Utrecht Centre for Tick-borne Diseases, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Originally, tick strains were collected from infested cattle in Mozambique  and following manufacturer's recommendations. Bm86 (R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) coding regions  lacking the signal peptide and GPI anchor sequences were amplified by RT-PCR. The RT-PCR was done using 10 pmol of each primer  in a 50-μl volume  RT/Thermus flavus (Tfl) reaction buffer, 0.2 mM each deoxynucleoside triphosphate (dNTP), 5 u AMV RT, 5 u Tfl DNA polymerase) employing the Access RT-PCR system . Reactions were performed in an automated DNA thermal cycler . RNA was reverse transcribed for 45 min at 45°C prior to PCR consisting of an initial step of 2 min at 94°C followed by 35 cycles of a denaturing step of 30 sec at 94°C and an annealing-extension step of 2 min at 68°C. Control reactions were done using the same procedures, but without RNA added to control contamination of the PCR. PCR products were electrophoresed on 1% agarose gels to check the size of amplified fragments by comparison to a DNA molecular weight marker . The amplicon was resin purified  and cloned into pGEM-T vector (Promega). Partial sequences of cloned Bm86 orthologs were obtained by double-stranded dye-termination cycle sequencing . At least three clones from independent PCR reactions were sequenced for each gene. Multiple sequence alignment was performed using the program AlignX  with an engine based on the Clustal W algorithm [Total RNA was extracted from the viscera of partially fed lgorithm . SearcheR. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) are EU191620–EU191622.The GenBank accession numbers for Bm86  and cloned into P. pastoris expression vector pPICZαA (Invitrogen) digested with Xba I and Xho I. In this way, Bm86 orthologs were cloned under the control of the alcohol oxidase (AOX1) promoter, in frame with the yeast alfa-factor secretion signal but without the C-terminal c-myc/His tag due to a translation termination site introduced by CZABM3 primer during PCR. The expression constructs were sequenced at both ends and selected constructs with correct sequences were named pPAMoz9 (Bm86), pPADec8 (Bd86) and pBaI (Ba86) and used for transformation of P. pastoris.Bm86, Ba86 and Bd86 coding regions were excised from pGEM-T by Sac I and transformed into P. pastoris strains GS115, KM71H and X33 (Invitrogen) by electroporation as described [-1 Zeocin and incubated at 30°C. A functional assay to directly screen for high expression recombinant clones was made by culturing the transformants in an orbital shaker at 250 rpm and 30°C. Single colonies were inoculated in 1 ml YPDS containing 100 μg·ml-1 Zeocin and grown overnight. Cultures were divided into two parts of 500 μl each. Five hundred μl were transferred to 5 ml fresh YP medium with 20 g·L-1 glycerol, grown for 24 hrs and inoculated into 250 ml fresh YP medium supplemented with 20 g·L-1 glycerol. Growth in glycerol was resumed after 24 hrs and then methanol was added daily to 1% (v/v) during the course of induction. After 5 days growing on methanol, supernatants were collected by centrifugation for 15 min at 15,000 × g in a Beckman Allegra™ X-22R centrifuge, rotor F2402H  and dot blots were made to screen for expression of recombinant proteins. The other 500 μl were also transferred to 5 ml fresh YP medium with 20 g·L-1 glycerol, grown for 24 hrs and mixed with glycerol to 250 g·L-1. Long term stocks were prepared as 100 μl aliquots and stored frozen at -80°C.Expression plasmids were linearized by restriction with escribed . Transfo+ or MutS phenotype using the functional assay described in the Invitrogen user's manuals K1710-01 and K1750-01 [S phenotype [-1 Zeocin and incubated at 30°C for 24 hrs. One colony of each transformant was streaked in both MMH and MDH plates for the GS115 and X33 strains. To differentiate Mut+ from MutS, control GS115/Albumin (MutS) and GS115/pPicz/lacZ (Mut+) strains (Invitrogen) were streaked in the MMH and MDH plates. Plates were incubated at 30°C for 3 days and cell growth was observed and compared to controls.The high expression transformants of X33 and GS115 strains were analyzed for MutK1750-01 . The KM7henotype . Briefly-1 glycerol. After 24 hrs, cultures were mixed again and 5 ml were used to inoculate 2 L Erlenmeyer flasks containing 250 ml of YP medium with 20 g·L-1glycerol. Cells were grown to an O.D.600 nm between 15 and 20 and then cultures were inoculated into a 5-L working volume Biostat B bioreactor  containing 3.5 L of PM with 40 g·L-1 glycerol.Pre-inoculums and inoculums for bioreactor cultures were grown in a shaker at 30°C and 250 rpm. Two 100 μl long term stock vials were seeded in 1 ml YP medium, grown for 12 hrs and transferred into 4 × 50 ml tubes containing 5 ml of YP medium with 20 g·L-1 in the culture medium. Upon exhaustion of glycerol, indicated by a sharp increase in dissolved oxygen, methanol induction was made by adding 1% (v/v) methanol to the culture medium and 3 hrs later the fed-batch phase was started by feeding methanol according to the P. pastoris Fermentation Process Guideline [4OH. Prior to methanol induction, pH was adjusted and maintained at 5.5 by adding NH4OH or H3PO3. Throughout the fermentation processes, supplements of 20 ml TES and VT solutions were added to the culture medium every 24 hrs. Additionally, GS115 strain cultures were supplemented with 0.04 g·L-1 L-Histidine every 24 hrs.During the fermentation process, temperature was kept at 30°C and dissolved oxygen was maintained at 30% saturation by regulating agitation and aeration rates. A three-phase cultivation protocol was used in the fermentation. The glycerol growth phase included a 12 to 14 hrs batch stage from the starting point followed by a 10 to 12 hrs glycerol fed-batch stage. A glycerol solution of 50% (v/v) was added to the fermentor for 4 hrs to reach an equivalent total quantity of 60 g·Luideline . The pH 600 nm, either measured as grams of wet weight per litter broth (O.D.600 nm = 1.39 × wet weight (g·L-1) - 27.26), which was obtained by centrifugation of the samples at 15,000 × g for 15 min or measured directly in the culture medium. Total protein concentration in the culture medium was quantified using the Bradford method with BSA as standard [Time-course samples were withdrawn from the fermentor at regular intervals to check growth rate and protein concentration in the supernatant. Cell density was expressed as O.D.standard .® Slice 200 ultrafiltration system having a Hydrosart membrane with a molecular weight cut-off of 50 kDa (Sartorius). Finally, protein solutions were concentrated and diafiltrated against four volumes of phosphate buffer pH 8.3 using a centrifugal concentrator VIVACELL 100  in a Beckman Allegra™ X-22R centrifuge, rotor SX4250 (Beckman-Coulter) at 3,900 × g.Cultures from the 5-L fermentor were centrifuged at 3,900 × g for 15 min in a Beckman Allegra™ X-22R centrifuge, rotor SX4250 (Beckman-Coulter) to separate cells. Supernatants were then collected and filtered through a tandem filtration system with a 20 μm cartridge , 5 μm and 0.45-0.22 μm cartridges  and checked for total and recombinant protein content using the Bradford method with BSA as standard [-1 and filtered through 0.45 and 0.22 μm cartridges (Sartorius AG) under sterile conditions in a laminar flow to obtain a sterile antigen solution. Adjuvation was made by mixing a solution of anhydromannitoletheroctodecenoate  with the recombinant protein solution in batch-by-batch processes using a high-speed mixer Heidolph DIAX 900  at 8,000 rpm and the vaccine was filled manually under sterile conditions in glass bottles of 20 ml . Quality controls were made by testing mechanical and thermal stability of vaccine emulsions as described by Canales et al. [Prior to adjuvation of the vaccine, protein solutions were adjusted to a concentration of 120 μg·mls et al. .Two New Zealand White rabbits per group was each immunized with 3 doses  containing 50 μg/dose of purified recombinant proteins formulated as described above or Gavac  as control. Rabbits were injected subcutaneously with 1 ml/dose using a 1 ml tuberculin syringe and a 27 1/2G needle. Two weeks after the last immunization, blood samples were collected from each rabbit into sterile tubes and maintained at 4°C until arrival at the laboratory. Serum was then separated after centrifugation and stored at -20°C. Rabbits were cared for in accordance with standards specified in the Guide for Care and Use of Laboratory Animals.Protein samples were analyzed by denaturing SDS-PAGE with a 12% PAGEgel-SDS cassette gel  under reducing conditions. Protein bands were visualized by either Coomassie Brilliant Blue R250 or silver staining. Samples were treated with dithiothreitol (DTT) reducer (PAGE-gel Inc.), heated in boiling water for 5 min before loading onto the gel and electrophoresed for 80 min at 90 mA constant current.Electrophoretic transfer of proteins from gels to nitrocellulose membranes  for Western blot analysis was carried out in a Minie-Genie Electroblotter semi-dry transfer unit  according to manufacture's protocol. Protein samples of 2 μl were absorbed onto nitrocellulose membrane by gravity flow to perform the dot blot analysis. A standard curve was constructed with known amounts of recombinant Bm86 extracted from Gavac (Revetmex) and was used for semi-quantitative analysis in dot-blots. The supernatant of the GS115/Albumin strain (Invitrogen) grown under the same conditions was used as a negative control in both dot- and Western-blots. Membranes for dot or Western blots were blocked with 5% skim milk for 1 hr at room temperature, washed three times in TBS  and probed with sera from rabbits immunized with Gavac (Revetmex) (1:1000 dilution) or recombinant proteins (1:5000 dilution) as described above. The antisera were diluted in 3% BSA in TBS. Membranes were then washed three times with TBS and incubated with an anti-rabbit IgG horseradish peroxidase (HRP) conjugate (Sigma-Aldrich) diluted 1:1000 in TBS. After washing the membranes again, color was developed using TMB stabilized substrate for HRP (Promega).MC carried out the expression, fermentation and protein purification and characterization studies. JMPL, VN and AMN carried out the genetic studies and participated in the sequence alignment. MH participated in the design of the study and helped to draft the manuscript. JF did the sequence alignment. FJ and JF conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Complex formation with various metal ions  was investigated by spectrophotometric methods. Only in the case of La3+ and Eu3+ were well defined 1 : 1 complexes formed. On the basis of CD spectroscopy and DFT calculations the configuration at the metal centre of the La3+ complex was determined to show Λ helicity. The coordination compounds [(1a)Ln] presented should be prototypes for further lanthanide(III) complexes with an enterobactine analogue binding situation.A facile synthesis of a highly preorganized tripodal enterobactine-type ligand  The availability of metal ions for biological systems is essential for growth and function. Therefore microorganisms have to develop strategies how to solubilise and take up charged ions through highly non-polar membranes –2.An important class of natural products which is responsible for the uptake of iron are the siderophores –5. The pChelating moieties: Catechol is an efficient chelating moiety, which can form highly stable complexes with a series of first row transition metals. (ii) Chirality: The enterobactin-backbone is based on L-serine units and therefore is chiral . This chiral information gives all three ligand units the same spatial orientation, leading to a bowl-shape structure which is preorganized for the uptake of the metal. Here we present the synthesis of a novel tripodal ligand, which mimics some features of enterobactin, but in contrast is specific for the binding of high coordinated lanthanide(III) ions. The backbone is based on an enantiomerically pure “non-natural” peptide moiety . NMR spectroscopy of the complex did not show significant shift differences between the ligand and the complex. This could be due to the lability of the compound in solution and fast dissociation/association equilibria. However, positive ESI MS in chloroform showed the base peak at m/z = 1600.8 for {K[(1a)La]}+ with correct isotopic pattern (1a-H3)}+.In an orientating coordination study we reacted the ligand  pattern . Due to 1a-H3 with a series of trivalent metal ions we performed UV–vis as well as CD spectroscopic titrations in methanol together with NaOH as base , gallium(III) or iron(III) which are able to form (distorted) octahedral coordination compounds lead to UV–vis spectra which show isosbestic behaviour. However, the titration curves show more or less linear changes of the absorption (in case of iron(III) a kink is observed at 1.5–2.0 equivalents iron(III) salt added). This indicates that not a 1 : 1 but probably polymeric coordination compounds are formed.1a-H3 with lanthanum(III) are shown in Only titrations with lanthanum(III) or europium(III) salts show well defined reliable behaviour . The speKa = 8.3 × 105 M−1) as well as europium(III) (Ka = 7.8 × 105 M−1) for the reaction of deprotonated ligand 1a3− with the Ln3+ ion to form [(1a)Ln] in methanol at room temperature.Analysis of the titration data reveals high binding constants for lanthanum(III) (1a·Ln (R = Et) complexes ab initio calculations were performed for the stereoisomers of complex 1b·La (R = Me)  we describe should be prototypes for further lanthanide(III) complexes with an enterobactine analogue binding situation. In this context the strong complexation of lanthanide(III) ions is of interest, due to the special properties of those compounds as light emitting or magnetic materials +: m/z = 1448.7642; found 1448.7639. HRMS (ESI): calcd. for C81H99N15O9 [M + H]+: m/z = 1426.7823; found 1426.7808. HRMS (ESI): [M + 2Na]++: m/z = 735.8739; found 735.8767.Yield: 81 mg (41 %); mp 107–108 °C. 4  was placed in a Schlenk flask. The rearrangement proceeded at 165 °C under dry inert atmosphere of N2. The dark brown residue was purified by a short silica column  to furnish a bright brown solid.The ether 1H NMR : δ = 8.40 , 8.14 , 8.07 , 7.57 , 7.34 , 4.96 , 4.91 , 4.33 , 4.24 , 3.56 , 3.33 , 2.28 , 1.87 , 1.24 , 1.17 , 0.84 , 0.81 . IR (KBr):  (cm−1) = 3383 (m), 3083 (w), 2964 (m), 2927 (m), 2720 (w), 2283 (w), 2085 (w), 1732 (w), 1629 (vs), 1595 (vs), 1508 (vs), 1453 (vs), 1378 (m), 1321 (m), 1289 (w), 1253 (m), 1204 (s), 1139 (w), 1107 (s), 1021 (m), 992 (w), 909 (m), 848 (s), 809 (w), 774 (m), 723 (m), 662 (w). HRMS (ESI): calcd. for C81H99N15O9 [M + Na]+: m/z = 1448.7619; found 1448.7637.Yield: 0.16 g (90 %); mp 189–196 °C. 3 · 7 H2O  and K2CO3  in MeOH / H2O (4 ml / 1 ml) were added to ligand 2  in MeOH (10 ml). The mixture was stirred at RT for 4 days. The solution was concentrated under reduced pressure and the red coloured residue was washed with water.LaCl81H96N15O9LaK]+, 100), 1464.9 . C81H96N15O9La · 5 H2O: C 58.87, H 6.46, N 12.71; found: C 58.38, H 6.62, N 12.62.Yield: 0.027 g (82%); mp 242–248 °C (dec.). Positive ESI MS (chloroform): m/z (%) = 1600.8 ([C1a-H3 (10−5 M) with NaOH (10−4 M) and a titrant solution containing the metal salt  were prepared. The titrant solution was added in discrete steps to the solution containing ligand 1a-H3. After a mixing time of 2 min the spectra were recorded.To determine the thermodynamic parameters of the metal complexes all titration experiments were accomplished at room temperature by using a Jasco J-815 spectrophotometer connected to an automatic titration unit (Jasco ATS-443). For this purpose a methanolic solution containing ligand The virtual binding constants were evaluated according to The virtual association constants of the complexation systems were calculated by non-linear-square fitting according to the Benesi–Hildebrand equation from the UV absorbtion data set. For a simple 1 : 1 binding model the calculations were carried out with the SigmaPlot program. Best results and lowest error could be observed at 279 nm in the case of lanthanum(III) and at 281 nm in the case of europium(III).


RNA non-enzymatic recombination reactions are of great interest within the hypothesis of the “RNA world”, which argues that at some stage of prebiotic life development proteins were not yet engaged in biochemical reactions and RNA carried out both the information storage task and the full range of catalytic roles necessary in primitive self-replicating systems. Here we report on the study of recombination reaction occuring between two 96 nucleotides (nts) fragments of RNAs under physiological conditions and governed by a short oligodeoxyribonucleotide template, partially complementary to sequences within each of the RNAs. Analysis of recombination products shows that ligation is predominantly template-directed, and occurs within the complementary complex with the template in “butt-to-butt” manner, in 1- or 3- nts bulges or in 2–3 nts internal loops. Minor recombination products formed in the template-independent manner are detected as well. Enzymes: calf intestinal alkaline phosphatase and endonuclease Fok I were purchased from “SibEnzyme”, Russia. T7 RNA polymerase, Taq DNA polymerase, M-MuLV DNA polymerase were produced in this Institute. T4 polynucleotide kinase was from Fermentas. TA cloning was proceeded using “InsTAcloneTM PCR Cloning Kit” (Fermentas). Plasmid DNA was isolated with “GenEluteTM Plasmid Miniprep Kit” (Sigma). BigDye v.1.1 sequencing mixture was from Applied Biosystems. All the chemicals were of analytical or ACS grade, and autoclaved Milli-QTM water was used for all procedures. M2-RNA of influenza virus was synthesized by T7 transcription from plasmid pSVK3M2, this Institute collection. HIV-1 fragment was obtained via transcription of plasmid pHIV-2, kindly provided by Prof. H.J. Gross .-ATP. A reaction system  containing 300 pmol of oligonucleotide, ligation buffer, 10 u of polynucleotide kinase and 0.1 mCi [γ-32P]-ATP was incubated at 37 °C for 1 hour. Then labeled oligonucleotide was purified in 20% denaturing polyacrylamide gel (dPAAG).Primer M3.4.Plasmid pHIV-2 was linearized with Fok I endonuclease according to the manufacturer’s manual. Reaction mixture of a total volume 200 μL, containing 40 μg of plasmid DNA, restriction buffer and 120 u of endonuclease, was incubated at 37 °C for 4 hours. Then mixture was extracted consecutively with solution TE-phenol-chloroform (1:1) and chloroform-isoamyl alcohol (24:1). DNA from the aqueous phase was precipitated with ethanol and dissolved in water.3.5.for and M2–96rev. PCR mixture contained 67 mM Tris-HCl (pH 8.0), 16 mM (NH4)2SO4, 1.5 mM MgCl2, 0.01% Tween-20, 0.2 mM dNTPs, 1.3 μM each PCR primer, 0.5 μg/mL plasmid and 5 u of Taq DNA polymerase in a final volume of 50 μL. PCR was performed in Hybaid PCR Express thermal cycler, starting with 3 min of preincubation at 95 °C, and followed by 30 amplification cycles . Amplification product (121 bp) was purified by phenol extraction (TEphenol : chloroform (1:1), then chloroform only) and precipitated from the aqueous phase with ethanol. Dissolved in water precipitates were used as templates for T7 transcription.M2-DNA fragment was amplified from the plasmid pSVK3M2 using primers M2–963.6.2, 2 mM spermidine, 4 mM NTPs, 5 mM DTT, 0.2 mg/mL BSA, 0.6 mg/mL of DNA and 480 u of T7 polymerase, were incubated at 37 °C for 4 hours. After incubation, RNA was purified by phenol extraction , followed by purification in 8% native PAAG. The visualized band was cut out and RNA was eluted from the gel with 3 mL of elution solution (0.6 M NH4Ac (pH 5.0), 0.1 mM EDTA, 0.1% SDS) on a shaker at 4 °C for 4 hours. Eluted RNA was precipitated with ethanol and dissolved in water.We applied T7 transcription for the synthesis of M2-RNA and HIV-RNA from M2-DNA (obtained by amplification of pSVK3M2 plasmid fragment) and linearized plasmid pHIV-2, respectively. Reaction mixtures of total volume 100 μL, containing 40 mM Tris-HCl (pH 8.1), 16 mM MgCl3.7.A reaction mixture  containing 8 μg of M2-RNA or HIV-RNA, 50 mM bis(tris(oxymethyl)methylamino)propane HCl buffer (pH 8.0), 2% formamide, 0.2% SDS, 1.25 mM DTT and 1.5 u of Calf intestinal alkaline phosphatase (CIP) was incubated at 37 °C for 45 minutes. Then 1.5 u of CIP was added again to the reaction mixture and it was additionally incubated under the same conditions. After incubation, mixture was subjected to twofold extraction with phenolchloroform (1:1), then chloroform-isoamyl alcohol (24:1). RNA was precipitated from the aqueous phase by 95% ethanol, washed twice with 75% ethanol, dissolved in water and stored at −20 °C.3.8.2, were incubated at 37 °C for 3 days. After incubation, EDTA was added to the ligation mixtures in a concentration equal to that of MgCl2. Volumes of all probes were adjusted to 100 μL with water, and RNA was precipitated with ethanol in the presence of 0.3 M NaAc and 10 μg of glycogen. Precipitates were washed with ethanol, dissolved in 15 μL of water and used in reverse transcription reaction.Reaction mixtures  containing bis(tris(oxymethyl)methylamino)propane HCl buffer , 0.5 μM dephosphorylated M2-RNA and HIV-RNA, 1.0 μM of the ONtemplate (in template-directed reactions) and 5 mM MgCl3.9.for and Mrev. In a positive control (to confirm M2-RNA presence in the reaction mixture) primers Mfor and Mrev were used. PCR mixtures contained 67 mM Tris-HCl (pH 8.0), 16 mM (NH4)2SO4, 1.5 mM MgCl2, 0.01% Tween-20, 0.25 mM dNTPs, 0.8 μM each PCR primer, 1 μL of RT-product in dilutions 1:1, 1:10−3, 1:10−6 and 2 u of Taq DNA polymerase in a total volume of 20 μL. PCR was started with 3 min of preincubation at 95 °C and followed by 25 cycles of amplification . Final cycle contained prolonged elongation step  for complete formation of sticky A-ends. PCR products were used directly for TA cloning. In parallel, we run PCR with 5′-32P-labeled Mrev primers for visualization purposes. In this case aliquots of PCR products were mixed with loading buffer and applied on 10% dPAAG electrophoresis. Visualization was accomplished by exposing dried gels on FX Imaging Screen, followed by screen scanning on Molecular Imager FX-PRO Plus (Bio-Rad) and images processing in Quantity One v.4.2.3 Software (Bio-Rad). Amplification products were identified by electrophoresis on 10% dPAAG using DNA ladder, obtained via chemical cleavage of M2–80 radioactive PCR product  at adenine and guanine sites. Reaction mixture of a volume 30 μL, containing 10 μL of 5′-32P-labeled M2–80, and 20 μL of 3% diphenylamine solution in formic acid, was incubated at 37 °C for 5 and 10 minutes. At the end of incubation, 100 μL of water was added and reaction mixtures were subjected to twofold extraction with 300 μL of diethyl ether. DNA from the aqueous phase was precipitated with 10 volumes of 2% LiClO4 solution in acetone, pellets were washed with acetone, dissolved in water and then added to the loading solution.The DNA template for PCR amplification was produced from the ligation mixture of recombinant RNA in reverse transcription reaction. A reaction mixture with an initial volume of 12 μL, containing 1.25 μM of specific reverse primer and 1 μL of mixture of products of the cleavage/ligation reaction (see p. 3.8), was subjected to denaturation at 70 °C for 5 min and then chilled on ice for 3 min. After that NTPs of a final concentration 1 mM and M-MuLV buffer were added, volume was adjusted to 18 μL with water, and mixtures were incubated at 37 °C for 5 min. Finally, 20 u of M-MuLV polymerase were added and the mixture of a final volume 20 μL was incubated at 42 °C for 60 min. The reaction was terminated by heating at 70 °C for 10 min. Obtained cDNA was directly used in PCR. PCR was performed from reverse transcription (RT) products using primers H3.10.TM PCR Cloning Kit” (Fermentas), according to the manual provided by the supplier. Briefly, 0.54 pmol of PCR products, possessing sticky A-ends, was ligated with 0.18 pmol of linear plasmid vector pTZ57R/T, containing sticky ddT-ends, using T4 DNA ligase. The obtained recombinant plasmids were used for transformation of competent E. coli DH5α cells, prepared using the same Kit. Transformed cells were plated at LB agar Petri dishes with ampicillin at concentration of 50 μg/mL. Dishes were incubated at 37 °C for 20 hours and then stored at 4 °C. Plasmids were isolated using “GenEluteTM Plasmid Miniprep Kit” (Sigma) according to the manual of supplier. Briefly, night cultures (OD~0.5) were prepared from corresponding bacterial colonies in 3 mL of LB medium containing ampicillin at a concentration of 50 μg/mL. Cells were harvested by centrifugation, resuspended, lysed and then the solution was neutralized. Cell debris was precipitated by centrifugation, cleared lysate was loaded at microcentrifuge spin columns, and after washing plasmid DNA was eluted from the column with 100 μL of water. Isolated plasmids were used directly for sequencing purposes.The PCR products were cloned using the “InsTAclone3.11.4)2SO4, 1.5 mM MgCl2, 0.01% Tween-20, 0.2 mM dNTPs, 0.3 μM of primers M13/pUC_for and M13/pUC_rev, and 2 u of Taq DNA polymerase. PCR was started with 3 min of preincubation at 95 °C and followed by 27 cycles of amplification . PCR products were analyzed in 8% dPAAG and visualized by staining of gels with ethidium bromide.Fragments of individual bacterial colonies were dissolved in 20 μL of PCR master mix, containing 67 mM Tris-HCl (pH 8.0), 16 mM (NH3.12.via Sanger sequencing reaction with mixture BigDye v.1.1 (Applied Biosystems) followed by further analysis of products at automatic gel analyzer ABI 3130xl (Applied Biosystems). For the sequencing reaction, 5 pmol of M13/pUC_rev primer, 200–400 fmol of plasmid, sequencing buffer and 1.5 μL of BigDye were mixed and adjusted to a final volume of 30 μL by adding water. The reaction was run according to the following program: 10 s preincubation at 96 °C; 3 cycles: 8 s, 96 °C; 4 min, 64 °C; 5 cycles: 8 s, 96 °C; 4 min, 60 °C; 17 cycles: 10 s, 96 °C; 5 s, 50 °C; 4 min, 60 °C; final step denaturation: 3 min, 96 °C. Products of sequencing reaction were purified from the unincorporated BigDye by precipitation with isopropyl alcohol, pellets were dried on SpeedVac evaporator for 3 minutes, dissolved in formaldehyde and subjected to analysis at gene analyzer. Sequences were determined in Sequence Scanner Software v. 1.0 (Applied Biosystems) and aligned with initial RNA sequences using Vector NTI v.10.0.1 Software (Invitrogen).Sequencing of constructs possessing the DNA inserts was performed 4.The non-enzymatic spontaneous recombination reactions discussed in this work were perfomed under very simple conditions, and, presumably, could be easily realized in prebiotic proteinless environment and lead to the formation of a pool of new RNA molecules. Notwithstanding the simplicity of chemical conditions, the processes taking place in the reaction system are very complicated and hardly predicted. It was found that nucleic acid template did not direct, but just facilitated the ligation, providing opportunity for approaching RNA dangling ends of different lengths and nucleotide sequences. RNA molecules appeared not to be prone to ligation within a fully complementary complex with a template, though some clones with this type of recombinant product were detected. Instead, the ligation occurred mostly in the elements of imperfect secondary structure of RNA, namely bulge loops of a size of one or three nucleotides and internal loops of a two or three nucleotide size. In spite of energetic preference of ligation within a duplex with the template, the predominant part of new phosphodiester bonds is formed in the 3 nts RNA bulges that strain spatial structure of RNA due to the double helix bend in the bulge area. However, local conformational preference for formation of this sort of products may be prevalent over thermodynamic stability of newly formed structures, providing an opportunity for occurrence of loop-bearing RNAs with novel sequences. Along with the products of template-governed ligation, we observed a number of products of template-independent ligation. However, formation of this sort of molecules may be attributed to the action of RNA substrates as templates for their self-ligation. If this is the case, proposed non-enzymatic mechanisms seem as a very plausible pathway for the formation, selection and evolution of first informational biopolymers in the prebiotic world.


Allelopathy  has been largely studied as an autecological process, often assuming simplistic associations between pairs of isolated species. The growth inhibition of a species in filter paper bioassay enriched with a single chemical is commonly interpreted as evidence of an allelopathic interaction, but for some of these putative examples of allelopathy, the results have not been verifiable in more natural settings with plants growing in soil.Festuca rubra ssp. commutata. We re-examined the allelopathic effects of m-tyrosine to understand its dynamics in soil environment. Allelopathic potential of m-tyrosine with filter paper and soil (non-sterile or sterile) bioassays was studied using Lactuca sativa, Phalaris minor and Bambusa arundinacea as assay species. Experimental application of m-tyrosine to non-sterile and sterile soil revealed the impact of soil microbial communities in determining the soil concentration of m-tyrosine and growth responses.On the basis of filter paper bioassay, a recent study established allelopathic effects of m-tyrosine, a component of root exudates of F. rubra ssp. commutata rhizosphere. We hope that our study will motivate researchers to integrate the role of soil microbial communities in bioassays in allelopathic research so that its importance in plant-plant competitive interactions can be thoroughly evaluated.Here, we show that the allelopathic effects of m-tyrosine, which could be seen in sterilized soil with particular plant species were significantly diminished when non-sterile soil was used, which points to an important role for rhizosphere-specific and bulk soil microbial activity in determining the outcome of this allelopathic interaction. Our data show that the amounts of m-tyrosine required for root growth inhibition were higher than what would normally be found in  Since 8-hydroxyquinoline has affinity for cations such as aluminum, iron or magnesium, the addition of EDTA (disodium ethylenediaminetetraacetate) to the extraction solution improved the recovery of 8-hydroxyquinoline probably due to dissociation of 8-hydroxyquinoline-metal cation complex C. diffusa-invaded soil and since the influence of microbial communities were excluded from this work, it remains an open question whether 8-hydroxyquinoline is allelopathic in soil.Phytotoxicity of 8-hydroxyquinoline, a root-exuded metabolite from Festuca rubra ssp. commutata. These results were extrapolated to the development of a biorational approach for weed control On the basis of filter paper and agar bioassays, Bertin and colleagues Lactuca sativa), littleseed canarygrass  and bamboo (Bambusa arundinacea) using filter paper and soil (non-sterile or sterile) bioassays. Since m-tyrosine was suggested to be a potent allelopathic and herbicidal compound We tested the phytotoxic effects of m-tyrosine on lettuce  or 30°C (for bamboo). Three seeds of lettuce, littleseed canarygrass or bamboo were sown in 10 g non-sterile soil and inoculated at 22 (lettuce and littleseed canarygrass) or 30°C (bamboo). After 8 d of seed germination, the non-sterile soil was treated with 0, 4.25, 8.5, 17, 34, 68 or 136 µg m-tyrosine/g soil and incubated under same conditions for 24 h. In order to investigate the recovery of m-tyrosine in soil devoid of microbes, sterile and non-sterile soils without assay species were treated with 0, 4.25, 8.5, 17, 34, 68 or 136 µg m-tyrosine/g soil and incubated at 22 or 30°C for 24 h. Each m-tyrosine treatment was replicated thrice.After an incubation period of 24 h, soil samples were extracted in 5 ml water, and briefly mixed by vigorous vortexing. After centrifugation for 10 min at 4500 rpm, supernatant was passed through 0.2 µm filters (Sartorius) and placed in sample vials for high-pressure liquid chromatography (HPLC) analysis. m-Tyrosine recovery was measured by HPLC with an Agilent-HPLC, 1100 series equipped with an ODS inertsil C-18 column  protected by a Phenomenex Security Guard C18 pre-column. The mobile phases were (A) water: 0.05% formic acid and (B) acetonitrile: 0.05% formic acid. The elution system was as follows: 0–3 min, 3% of B; 3–12 min, 3–50% of B; 12–15 min, 50% of B. The flow rate was 1 mL/min, the injection volume was 10 µL, and the column oven was set at 24°C, and the elutent was monitored at 280 nm. m-Tyrosine concentration among or between treatments was analyzed by means of one-way ANOVAs or t-tests, respectively F. rubra ssp. commutata were sown in 10 g soil at 22°C. Eight days after the germination of seeds, seedlings were removed, and soil was extracted with 10 mL water. Soil extracts were then subjected to HPLC as described above.In order to examine the root exudation of m-tyrosine in non-sterile soil, seeds  of 2 respiration by chemical titration is an effective method to compare microbial activity in different soils 2. Three replicates were used. Soil was incubated at 22°C or 30°C, respectively for 24 h, and was terminated by adding 1 mL of 0.1 N BaCl2 to NaOH. A 10 mL of NaOH taken from blank, control or treatment was titrated against 0.1 N HCl, and amount of CO2 released was calculated Measuring soil COlettuce = 56.65, df = 6, 284; P<0.001; Flittleseed canarygrass = 301.1; df = 6, 318; P<0.001; Fbamboo = 16.97, df = 6, 230, P<0.001) or in sterile soil treated with m-tyrosine   and lettuce  was inhibited in soils amended with 68 or 136 µg/g tyrosine  . No significant difference in m-tyrosine recovery was observed among three assay species at 34 or 68 µg m-tyrosine/g soil . HoweverWe also examined the recovery of m-tyrosine in sterile and non-sterile soil without assay species when treated with 0, 4.25, 8.5, 17, 34, 68 or 136 µg m-tyrosine/g soil and incubated at 22 or 30°C. A higher availability of m-tyrosine was observed in non-sterile soil, incubated at 22°C, treated with 17 (12.1%), 34 (22.1%), 68 (29.7%) or 136 (40.3%) µg m-tyrosine/g non-sterile soil compared to non-sterile soil, incubated at 30°C, treated with 17 (0%), 34 (13.1%), 68 (18.5%) or 136 (21.8%) µg m-tyrosine/g non-sterile soil . The rec2 release  = 0.035) compared to untreated soil incubated at 22°C (2 release in treated soil incubated at 30°C was significantly (P<0.0001) higher compared to untreated soil incubated at 30°C. No significant differences (except 4.25 or 8.5 µg m-tyrosine/g soil) were observed in the CO2 released from m-tyrosine-treated soil incubated at 22°C compared to untreated soil incubated at 22°C. A significantly higher CO2 release from soil treated with 17 <0.0001), 34 <0.0001), 68 <0.0001) or 136 <0.0001) µg m-tyrosine/g soil was observed at 30°C compared to respective soils incubated at 22°C  in non-sterile soil with bamboo compared to non-sterile soil with lettuce (31.1%) or littleseed canarygrass (34.1%) . The low2 efflux is a reliable technique for comparing microbial activity in different soils 2 release in untreated and m-tyrosine-treated soil incubated at 22 or 30°C. We observed a higher CO2 release in the untreated soil incubated at 30°C (4±0.2 µg CO2 released/g soil/h) compared to 22°C (2.6±0.6 µg CO2 released/g soil/h). A higher m-tyrosine recovery of 12.1, 22.1, 29.7 and 40.3% was observed in non-sterile soil treated with 17, 34, 68 or 136 µg m-tyrosine/g soil at 22°C compared to 0, 13.1, 18.5 or 21.8%, respectively in m-tyrosine treated soil at 30°C. The lower recovery was associated with higher CO2 efflux suggesting higher microbial activity in soil treated with 17 (47.4%), 34 (59.1%), 68 (81.1%) or 136 (61.4%) µg m-tyrosine/g soil at 30°C compared to respective soils incubated at 22°C (2 release (except from the soil treated with 8.5 µg m-tyrosine/g soil) compared to soil treated with 17, 34, 68 or 136 µg m-tyrosine/g soil at 22°C. No differences in the CO2 release were observed among soils treated with 17, 34, 68 or 136 µg m-tyrosine/g soil at 22°C  reflect the amount actually released from the plant that could interact with competing plants. Moreover, it is difficult to collect root surface washings in a system without causing physical damage to the roots, which is unlikely to occur in the artificial soil system. In natural conditions, biotic stresses such as herbivory, however, might cause a local release of m-tyrosine. Ecologists have often criticized the extraction procedures used in past experiments claiming the demonstration of allelopathic effects. Harper Almost all species can, by appropriate digestion, extraction and concentration, be persuaded to yield a product that is toxic to one species or another”. These comments hold for most of the recent claims of allelopathic function F. rubra ssp. commutata could have some ecological function, which yet remains to be discovered.We did not detect m-tyrosine in non-sterile soils with 3, 6 or 9 seedlings of While m-tyrosine may exert allelopathic effects at high concentrations, our research suggests that the quantities of m-tyrosine released from the plant are unlikely to persist in soil to exert phytotoxic effects. Soil microbial communities form a major sink that influences accumulation of a metabolite to phytotoxic levels . Microbiin vitro demonstrations of allelopathic activity of a chemical can rarely be applied to field situation, the most recent studies published in high profile journals in vitro experiments. It is our hope that this study will motivate researchers to integrate the role of soil microbial communities in bioassays in allelopathic research so that its importance in plant-plant competitive interactions can be thoroughly evaluated.Bioassays for allelopathy should investigate rhizosphere-specific and bulk soil microbial activity; and also temperature-driven microbial activity. The differences between species that are ‘sensitive’ or ‘resistant’ to a putative allelochemical, as is commonly invoked in invasion ecology, may reflect differences in microbial communities harbored by different plant species. Although it is well known that


Rh, Cl and one P atom lie on a mirror plane, as does the solvent molecule. There are two intermolecular hydrogen bonds, one between the methanol O atom and an aryl H atom (2.51 Å), and one between the Cl atom and the hydroxy H atom of methanol [2.34 (3) Å]. The complex precipitates in trace amounts from a reaction between RhCl(cod)(thp)  and P(p-FC6H4)3 under argon in CD3OD.  Two C6H4-F units are disordered over two positions; for one the site occupancy factors are ca. 0.53 and 0.47, for the other the values are ca. 0.64 and 0.36. The methyl H atoms of the solvent molecule are disordered across the mirror plane. In the title compound, [RhCl{P( DOI: 10.1107/S1600536808005485/rk2075Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs.Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including 50 1–7 µg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus  at concentrations close to IC50. The candidate compounds suppressed resuscitation of dormant (“non-culturable”) cells of M. smegmatis at 1 µg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 µg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations.Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with ICNPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins. Mycobacterium tuberculosis has been suggested to persist in a dormant state in approximately 2 billion of people Dormancy is a reversible physiological state of the bacteria characterized by low metabolic activity, in which cells can persist for extended periods without division). Due to reasons that are not fully clear, such latent infection can reactivate at any time causing active tuberculosis Latency is a clinical state of asymptomatic, chronic infection). Importantly, dormant mycobacteria are less susceptible to antibiotics which are normally used for treatment of active tuberculosis. Therefore, finding of new compounds with potential activity against latent forms of tuberculosis is an urgent task for the scientific community. Ideally, such compounds should sterilize dormant M. tuberculosis in the host environment or block its reactivation.Tuberculosis (TB) still kills millions of people around the Globe. Moreover, M. tuberculosis persistent organisms associated with latent infections. M. tuberculosis contains five rpf-like genes which products, RpfA-E expressed as recombinant proteins in E. coli stimulated replication and resuscitation of mycobacterial cells tuberculosis cells in vitro and for growth of viable cells in vivo was also confirmed in the study of the Rpf knockout mutants M. tuberculosis. Such drugs will have a great potential for prophylaxis of latent TB reactivation, provoked by the application of anti-TNF antibodies therapy The discovery of Rpfs (resuscitation promoting factor), a family of proteins involved in the control of dormancy and “non-culturability” of mycobacteria and related organisms, has provided a promising opportunity to explore new strategies on targeting Despite the progress in the Rpf research, the mechanisms of its biological activity remain unclear. Recent findings suggest that Rpfs are probably involved in the remodelling of bacterial cell envelope 1H NMR are reported in ppm downfield from TMS (σ). Mass spectra were obtained using a Finnigan SSQ-700 (USA) instrument with direct inject. Silicagel 60 F254 aluminium sheets  were used for all analytical thin layer chromatography (TLC). All chemicals were purchased from Alfa Chemicals  including starting otho-chloronitroaryles for synthesis target compounds (III), (IV), (V), (VI), (VIII), (IX), (X).Melting points were determined according to the BP procedure and are uncorrected . If analyses are indicated only by the symbols of the elements, analytical results are within ± 0.3% of the theoretical values . NMR spectra were determined with a Varian Unity Plus 400 (USA). Shifts for 4-chloro-6-methoxy-5-nitropyrimidine was synthesised according to A suspension of 2-chloronitroaryles (4.00 mmol) and 1,5-dichloroantraquinone  in ethanol (25 ml) was treated by 10 ml ethanol solution of KSCN  and heated at 50°C for 2–6 h. After cooling the reaction mixture was dissolved in 100 ml of cold water and light yellow precipitate was collected by filtration. The solid residue was twice crystallised from suitable solvent to obtain 2-thiocyanatonitroaryles with >96% purity (yield 35–55%).+. 212. 1H NMR (DMSO) σ 9.25  and 4.17 . Anal. (C6H4N4O3S), C,H,N,S.Suspension of 1.94 g (1.0 mmol) of 4,6-dichloro-5-nitropyrimidine in 20 ml methanol was treated by methanol solution of MeONa (prepared in advance from 0.23 g (1.0 mmol) of natrium and 10 ml of methanol) at 0°C. Reaction mixture was stored for 2 h at room temperature and mixed with 100 ml of cold water. White crystalline solid of 4-chloro-6-methoxy-5-nitropyrimidine was filtered off and crystallised from ethanol (mp. 54–56°C). Follow reaction according “General procedure for 2-thiocyanatonitroaryles synthesis” leads to the target compound (I). Mp 128–31°C (ethanol). m/z+. 239. 1H NMR (DMSO) σ 8.43 , 4.86 2) and 1.37 . Anal. (C8H9N5O2S), C,H,N,S.Solution of 1.94 g (1.0 mmol) of 4,6-dichloro-5-nitropyrimidine in 20 ml dioxane was treated by water solution of isopropylamine acetate (prepared in advance from 1.20 g (2.0 mmol) of isopropylamine and excess of acetic acid in 5 ml of water). Reaction mixture was stored for 2 h at room temperature and mixed with 100 ml of cold water. Light yellow crystalline solid of 4-chloro-6-isopropylamine-5-nitropyrimidine was filtered out and crystallised from ethanol (mp. 96–98°C). Follow reaction according “General procedure for 2-thiocyanatonitroaryles synthesis” leads to the target compound (II). Mp 143–5°C (ethanol). m/z+. 282. 1H NMR (DMSO) σ 9.66  and 8.61 . Anal. (C8H2N4O4S3), C,H,N,S.Usage of 1,5-dinitro-2,4-dichlorobenzene according “General procedure for 2-thiocyanatonitroaryles synthesis” leads to the target compound (III). Mp 78-80°C (DMF/water). m/z+. 248. 1H NMR (DMSO) σ 8.51 , 8.29  and 7.84 . Anal. (C8H3F3N2O2S), C,H,N,S.Usage 4-chloro-3-nitrobenzotrifluoride according “General procedure for 2-thiocyanatonitroaryles synthesis” leads to the target compound (IV). Mp 78–81°C (ethanol). m/z+. 205. 1H NMR (DMSO) σ 8.40 , 8.26  and 7.90 . Anal. (C8H3N3O2S), C,H,N,S.Usage 4-chloro-3-nitrobenzonitrile according “General procedure for 2-thiocyanatonitroaryles synthesis” leads to the target compound (V). Mp 143-5°C (ethanol). m/z+. 222. 1H NMR (DMSO) σ 8.69 , 8.09 , 7.85  and 2.57 . Anal. (C9H6N2O3S), C,H,N,S.Usage 4′-chloro-3′-nitroacetophenone according “General procedure for 2-thiocyanatonitroaryles synthesis” leads to the target compound (VI). Mp 96-8°C (ethanol). m/z+. 284. 1H NMR (DMSO) σ 8.58 , 8.11 , 7.93 , 7.67 and 7.41 . Anal. (C14H8N2O3S), C,H,N,S.Solution of 4.35 g (2.0 mmol) 4-chlorobenzophenone in 6 ml of sulfuric acid was treated by mixture of 2 ml of nitric acid and 2 ml of sulfuric acid at 0°C. Reaction mixture was warmed up to the room temperature, heated at 65°C for 2 h and put on 100 g of crashed ice. White solid of 4-chloro-3-nitrobenzophenone was collected, dried and crystallized from hexane (mp 104–6°C). Follow reaction according “General procedure for 2-thiocyanatonitroaryles synthesis” leads to the target compound (VII). Mp 132–5°C (ethyl acetate). m/z+. 224. 1H NMR (DMSO) σ 8.56 , 8.18  and 7.87 . Anal. (C8H4N2O4S), C,H,N,S.Usage 4-chloro-3-nitrobenzoic acid according “General procedure for 2-thiocyanatonitroaryles synthesis” leads to the target compound (VIII). mp 220-4°C (ethanol/water). m/z3-Nitro-4-thiocyanatobenzoic acid methyl ester (IX) and 3-nitro-4-thiocyanatobenzoic acid ethyl ester (X) were synthesized according Micrococcus luteus, Fleming strain 2665, NCIMB13267 was grown in Nutrient Broth (Hi Media) on an orbital shaker at 30°C. Mycobacterium smegmatis mc2155 was grown in Sauton's medium on an orbital shaker at 37°C. M. tuberculosis H37Rv was grown under agitation at 37°C (200 rpm), in Sauton's medium supplemented by ADC with 0.05% w/v Tween 80 M. luteus were grown to the stationary phase in the rich medium  for 48 h. Cultures were washed twice by centrifugation and diluted to 105 cells/ml by the same medium. Diluted cells were distributed on 96 well plates (250 µl per well) and incubated in the Multiskan Analyzer  with 620 nm filter at 30°C for 24 h with shaking. Prior the incubation, the inhibitors, freshly dissolved in DMSO (1 mg/ml), were added in 0.5–10 µg/ml concentration range to the appropriate wells in triplicates. The culture of M. luteus without inhibitors served as a control. Bacterial growth was followed by measurement of optical density at 620 nm. Cells of M. smegmatis grew in NB medium to the stationary phase for 24–26 h and were diluted to 105 cells/ml by the same medium containing 0.05% w/v Tween-80. Cell growth with and without inhibitors was detected at 37°C in the Multiskan Analyzer, similarly to M. luteus experimental conditions. M. tuberculosis grew in Sauton's medium supplemented by ADC in the presence of 0.05% Tween-80 for 8 days. Cells were inoculated in 2 ml of fresh medium at ca 106 cells/ml in test tubes. Inhibitors were added at 1-25 µg/ml in triplicates. Test tubes were incubated without shaking at 37°C OD600 was measured periodically, using Eppendopf Biophotometer for 14 days.Cells of M. smegmatis mc2155 harbouring the pAGR plasmid with the rpf gene was used to produce “non-culturable” (NC) cells in the stationary phase as described previously M. tuberculosis “non-culturable” cells + for 50 days M. smegmatis were performed in 48-well plastic plates (Corning) containing 1 ml of Sauton's medium diluted 1∶1 with 0,6% (w/v) glycerol in water. Some wells contained inhibitors at 0.1–25 µg/ml. All wells were supplemented with 0.05%(w/v) yeast extract (LabM). Appropriate serial dilutions of the starved M. smegmatis cells (100 µl) were added to each well. Plates were incubated at 37°C with agitation at 150 rpm for 10–14 days. Wells with visible bacterial growth were counted as positive, and MPN values were calculated using standard statistical methods Resuscitation and most probable number (MPN) assays of M. tuberculosis cells, save that the diluted Sauton's medium was supplemented by 10% v/v ADC, and cell dilutions were prepared in screw capped test tubes which were kept standing at 37°C for 2 months.Similar procedure was applied for resuscitation of M.luteus Rpf (RpfSm) was used for the analysis. The truncated protein contained conserved Rpf domain and additional 20 amino acids of the gene product, corresponding to the following amino acid sequence:The truncated form of ATVDTWDRLAECESNGTWDINTGNGFYGGVQFTLSSWQAVGGEGYPHQASKAEQIKRAEILQDLQGWGAWPLCSQKLGLTQADADAGDVDATE. The truncated gene was amplified by PCR from the pET-19b-Rpf described before CGCGAAATTAATACGACTCACTAT and a reverse primer: GGATCCTCACTCGGTGGCGTCACGTCGAC (BamH1 restriction cite is underlined). After denaturation for 5 min at 94°C, samples were subjected to 2 cycles of: 30 s at 94°C, 30 s at 58°C, 60 s at 72°C, followed by 2 cycles of: 30 s at 94°C, 30 s at 56°C, 60 s at 72°C and 25 cycles of: 30 s at 94°C, 30 s at 54°C, 60 s at 72°C, followed by 5 min at 72°C. The purified PCR product was digested with XbaI and BamH1, purified, ligated into pET19b vector and established in E. coli DH5α. The construct containing the truncated rpf gene (RpfSm) was sequenced and transformed in E. coli HSM174 (DE3). RpfSm was purified from 350 ml cultures of the E. coli producer strain, grown at 37°C in the rich medium (Hi Media) containing 100 µg/ml ampicillin to optical density OD600 nm 0.65–0.8. After induction with 1 mM IPTG, growth continued for 2 h at room temperature. Cells were collected by centrifugation at 3000 g for 15 min and frozen in the binding buffer (BB) . After thawing out, RNAase and DNAase, both at concentrations of 10 µg/ml were added in the presence of 10 MM MgSO4, followed by addition of 8 M urea. After sonication, the crude extract was centrifuged at 6000 g for 30 min to remove cell debris, and supernatant was applied to the Ni2+-chelation column (V = 2 ml) , equilibrated with BB. The Biological LP system («Biorad») was employed for the elution and refolding of the protein on the Ni-NTA column: first, the series of washing steps, with the BB contained 8 M urea, were applied. The second (refolding step) was the application of BB with urea concentration declining linearly from 8 M to zero. The protein was eluted by linear gradient of imidazole from 5 mM to 500 mM. Protein was collected in the range of imidazole concentrations of ∼250 mM in a total volume of 4–5 ml. Dialysis of Rpf contained fraction (30–50 µg/ml) was performed against 50 mM citric acid-sodium citrate buffer, pH 6.0. The protein was stored at +4°C for one week without significant loss of the activity.ΔDUFRpfB was expressed and purified under native conditions according to 4 in total volume of 0.4 ml. A number of experiments were performed in the presence of NPT compounds, in concentration range of 0.5–10 µg/ml. After 3 hours of incubation at 37°C, 2 µl of 10 M NaOH was added and the samples were frozen and kept at −20°C. The kinetic analysis of Rpf activity and inhibition was performed using the range of concentrations of the substrate from 3 to 24 µM and the inhibitor (VII) from 0.5 to10 µg/ml. The intensity of fluorescence was detected by the RF-5301PC fluorimeter  using excitation wavelength of 364 nm and a read-out of 448 nm.To determine a muralytic activity of the recombinant Rpfs', 4-methylumbelliferyl-β-D-N, N′, N″-triacetylchitotrioside (4-MUF-3-NAG)  was applied. 6 µM of 4-MUF-3-NAG was mixed with the proteins (1–10 µg/ml) in 100 mM citric acid-sodium citrate buffer, pH 6.0 with 0.5 mM MgSOCD analysis of Rpf proteins was performed in a buffer −20 mM phosphate, 50 mM sodium chloride, pH 7.0. The traces of imidazole in the protein were removed by extensive dialysis prior to the CD spectra recording. Measurements were performed using Jasco – 720 automatic recording spectrophotometer; 1 mm path length quvette was used; slit width 1.5 nm; time constant 3 sec. Experiment was performed at 20°C. Inhibitors were used as acetonitrile solution (1 mg/ml).ΔDUFRpf B were recorded at room temperature using the RF-5301PC fluorimeter  in 0.3×1 cm path length quartz quvette with slit width 1.5 nm for excitation and 3 nm for emission channels. Proteins (40–50 µg/ml) were dissolved in 50 mM phosphate buffer, pH 7.5. The fluorescence emission spectra were measured under excitation wavelength at 280 nm. Fluorescence quenching measurements were analysed using the Stern-Volmer equation 0F and F are the relative fluorescence intensities in the absence and presence of quencher, respectively, [Q]- concentration of quencher and qK is the Stern – Volmer quenching constant.The steady-state fluorescence excitation and emission spectra of M. luteusM. luteus Rpf lacking the last 89 amino acid residues of the C-terminal part (Rpf Sm) and secondly, recently described truncated form of Rpf B (ΔDUF RpfB) ΔDUFRpfB correspondingly. We found that four out of five of the initially tested compounds (I)-(IV) and (IX) inhibited muralytic activities of both proteins in the 1–7 µg/ml concentration range , was used for synthesis of derivative compounds . Apart of negatively charged compound (VIII), the derivative compounds displayed inhibitory activity on the Rpf- mediated MUF-3-NAG hydrolysis (VIII) (Hydrolytic enzymes play a key role for spore germination in many microorganisms on range . The moss (VIII) . The Lins (VIII) .To exclude possible alterations of the Rpf secondary structure caused by NPT, CD measurement studies were undertaken using both Rpfs. ΔDUFRpfB resulted in significant quenching of its auto-fluorescence (excitation 280 and emission 340) (af is an accessible fraction of fluorochromes. From equation (2), the accessible fraction of ΔDUFRpfB tryptophanes for compound (VII) (af) was close to 65%. These results were compared with quenching of ΔDUFRpfB by iodide which interacts with the exposed fluorochromes only ΔDUFRpfB molecule contains 5 tryptophan residues (the major contributors in an auto-fluorescence under excitation used), 65% of accessibility to (VII) corresponds to three tryptophanes. 40% accessibility for iodide corresponds two exposed tryptophan residues localized on the molecule surface, indicating that the substance (VII) interacted with at least two exposed and one buried tryptophan. Note, that in case of inactive compound (VIII) the accessible fraction was similar to iodide (not shown).The existence of several tryptophanes and phenylalanines residues in the Rpf molecule enables one to analyse an interaction between NPTs and Rpf by fluorescence quenching. The addition of the highly efficient inhibitor of the enzymatic activity of Rpf, the compound (VII), to ion 340) . ApplicaM. luteus, M. smegmatis and M. tuberculosis. M. luteus, containing the single rpf gene essential for growth M. luteus growth in the NB liquid medium (MICs were between 1-10 µg/ml). Moreover, there was clear correlation between inhibition of the enzymatic activity of Rpf and bacteriostatic effects displayed by the compounds. For example, the compounds (II) and (VIII) did not influence the Rpf enzymatic activity and had no effect on M. luteus growth . In the second model “non-culturable” cells of M.tuberculosis resuscitate spontaneously, nevertheless this process is Rpf-dependent since the Rpf mutants were unable to resuscitate M.smegmatis (III) practically did not influence resuscitation of M.tuberculosis. Compounds  had no influence on the resuscitation kinetics (not shown). None of the compounds tested suppressed growth of active cells of M. tuberculosis at the same concentration (not shown).In the first model the majority of ependent , and theThe structural studies of the conserved domain of Rpf could be considered as a “compact hybrid” of c-type lysozyme and lytic transglycosylases Through our efforts related to the search of new antimicrobial compounds we observed that derivatives of ortho-nitrodialkyldithiocarbamate have potent antimycobacterial activity We found that some of these compounds strongly suppressed germination of fungal spores (unpublished). Structural similarity between Rpf and cell wall lytic enzymes involved in spore germination (see above) allowed us to suggest that NPTs may influence on the activity of Rpf.ΔDUFRpfB M. luteus is in a good accordance of Rpf essentiality in this bacterium. Remarkably, we found a good correlation between the effectiveness of different NPTs to inhibit the enzymatic activity of Rpf and to arrest growth of M. luteus cells (with the exception of compound (I)). The inhibitory concentration range for these compounds was also similar for both processes.We first showed that most of candidate compounds indeed inhibit muralytic activity of Rpf in the non-competitive mode . The mearpf genes did not significantly impair the growth of the axenic cultures of M. tuberculosis, but exhibited a vital effect on their resuscitation in the liquid medium in vitroin vivoM. smegmatis and M.tuberculosis but to the less extent against the growth of viable cells, providing additional evidence for collective non-essentiality of rpf genes in M.tuberculosisIt was stated that Rpf proteins stimulate growth of cells at suboptimal or stressful conditions, but not of actively multiplying cells M. luteus and resuscitation of M. smegmatis, most likely could be explained by presence the negative charge on the molecule. Indeed, in published crystal structure of ΔDUFRpfB, a negatively charged region in the catalytic site has been established Availability of derivatives of NPT with different effects against Rpf activities provides some insight in the mechanism of their action. So, the failure of the compound (VIII) to inhibit 4-MUF-3-NAG hydrolyzing activity, growth of Despite a rather good correlation between anti-enzymatic and anti-resuscitation activities observed in this study, more experiments are required to confirm that Rpf proteins are, indeed, the targets for NPT in cells. The unique ability of NPT to suppress or at least delay resuscitation of “non-culturable” mycobacteria makes them attractive agents for specific target - non-replicating mycobacteria.Because Rpf proteins are likely important for the establishing of chronic Figure S1VII). 4-MUF-3-NAG hydrolysis by RpfSm (30 µg/ml) was performed according to “VII): - 0.5 µg/ml; ▴ - 1 µg/ml; × - 5 µg/ml; *- 10 µg/ml. The Lineweaver-Burk plotInhibition of 4-MUF-3-NAG hydrolysis by RpfSm in the presence of ((4.81 MB TIF)Click here for additional data file.Figure S2Influence of compound (VII) on the secondary structure of Rpfs. CD spectra of ΔDUFRpf B (A) and Rpf Sm (B) in absence and presence of (VII) were obtained as described in the “(5.81 MB TIF)Click here for additional data file.Table S1M. tuberculosis “non-culturable” cells obtained in vitro. Confidence limits (95%) for MPN are shown. These numbers present logarithmic transformation of cell concentration (cells/ml) estimated by MPN assay. The “lower “ and “upper” values are logarithmic transformation of cell concentrations representing 95% confidence limits of the assay.Influence of NPT compounds on resuscitation of (0.04 MB DOC)Click here for additional data file.Table S21/2- time is needed to achieve a 50% level of substrate conversion. Alcoholdehydrohenase(1.1.1.1) activity: ADH was dissolved in 50 mM phospahe buffer pH 7 at the concentration 10 µg/ml. The rate of the reaction was judged by transformation of NAD+ (1 mM) into NADH+H+. As the substrate ethanol was used. Activity was measured by OD340. Laccase(1.10.3.2) activity: Laccase was dissolved in the 50 mM citrate-phosphate buffer, pH4.5 at concentration 9 µg/ml. Pyrocatechin was used at concentration 11 µg/ml. Activity was measured by OD410. Trypsin(3.4.21.4) activity: The enzyme at concentration 8.0 µg/ml was dissolved in 50 mM Tris-HCl buffer pH 8.0. Concentration of the substrate (N-CBz-Gly-Gly-Arg-β-naphthylamide - Sigma-Aldrich)- 11 µg/ml. Registration of the kinetic curves was made fluorimetrically - excitation wavelength - 330 nm; emission - 420 nm.Influence of compounds (III) and (VII) on activity of some enzymes τ(0.02 MB DOC)Click here for additional data file.


However, the model is complicated, and it is not clear which specific DNA sequence properties are most important for intrinsic nucleosome-forming preferences.The relative preference of nucleosomes to form on individual DNA sequences plays a major role in genome packaging. A wide variety of DNA sequence features are believed to influence nucleosome formation, including periodic dinucleotide signals, poly-A stretches and other short motifs, and sequence properties that influence DNA structure, including base content. It was recently shown by Kaplan et al. that a probabilistic model using composition of all 5-mers within a nucleosome-sized tiling window accurately predicts intrinsic nucleosome occupancy across an entire genome in vitro and in vivo in a manner comparable to the Kaplan model. G+C content and frequency of AAAA are the most important features. G+C content is dominant, alone explaining ~50% of the variation in nucleosome occupancy in vitro.We find that a simple linear combination of only 14 simple DNA sequence attributes  explains nucleosome occupancy Our findings provide a dramatically simplified means to predict and understand intrinsic nucleosome occupancy. G+C content may dominate because it both reduces frequency of poly-A-like stretches and correlates with many other DNA structural characteristics. Since G+C content is enriched or depleted at many types of features in diverse eukaryotic genomes, our results suggest that variation in nucleotide composition may have a widespread and direct influence on chromatin structure. In the in vivo,4. Activin vivo-7, presuS. cerevisiae, studies examining the relative incorporation of yeast genomic DNA into nucleosomes in vitro have demonstrated that nucleosome depletion at promoters is to a large extent programmed into the DNA sequence[in vivo. Therefore these results also indicate that the sequence preferences of nucleosomes are likely to be broadly conserved across eukarya.In  sequence,9. These sequence or humanin vivo and in vitro experiments[in vivo[in vivo correlates with and can be predicted by base composition (G+C content)[in vivo nucleosome occupancy, and did not directly demonstrate intrinsic nucleosome sequence preference.To fully understand the function and evolution of gene regulation and genome packaging, it will be essential to understand the sequence preferences of nucleosomes. A variety of sequence cues have been shown to influence nucleosome sequence preference. These include nucleosome positioning,11 and eperiments,21 and ts[in vivo,23. Howes[in vivo,24-27, as[in vivo. Some of content),28 and o content), many ofin vitro. The Kaplan model should inherently capture the effects of both base composition and aspects of large-scale structural properties which are thought to depend primarily on dinucleotide content[Kaplan et al. showed re content. Howeverin vitro data, it performs comparably or better than the best previous models on in vivo data in both yeast and C. elegans. The 14 feature model is heavily dependent on G+C and poly-A content, with G+C having the highest independent correlation with measured nucleosome occupancy. We suggest possible explanations and implications of the strong association between G+C content and intrinsic nucleosome occupancy.Here we used Lasso, a lineain vitro nucleosome data of Kaplan et al.[Table S1 , including representatives of all categories (a-d) above . In each case, Lasso assigned nonzero weights to a similar set of features [-308). The Kaplan model has a correlation coefficient of 0.74 over the same test data. Thus, the 14 feature model encapsulates the vast majority of the information in the Kaplan model. Indeed, the correlation between the 14 feature model and the Kaplan model over the entire yeast genome is 0.88 , independently of all other features . We note that the Kaplan model weights for individual 5-mers also scale highly with G+C content  and that the scores assigned by the Kaplan model to 147-base windows across the yeast genome correlate highly with G+C content , both correlate well with G+C content when calculated as an average over a tiling window using dinucleotide tables  is a major component. G+C content may be a dominant feature because it correlates with many structural properties of DNA, and also reduces the frequency of poly-A-like stretches. Since local variations in G+C content occur at many types of features in diverse eukaryotic genomes, our findings suggest that nucleotide composition may have a widespread and direct influence on chromatin structure.in vitro and in vivo)[2 scale. We also used the in vivo nucleosome occupancy measurements from a tiling array study in yeast[in vivo map of nucleosome occupancy in C. elegans[2 ratio between the basepair's total occupancy and the mean genomic average occupancy. Then, we set the genomic average to zero by subtracting the new genome-wide mean from each basepair.We converted the average nucleosome occupancy measurements from yeast ( in vivo) to log2 . elegans using bo. elegans. For thi2 in vitro nucleosome occupancy data), Lasso generates a linear model ŷ = β x1 + β x2 + ... β xn, where the output ŷ is the nucleosome occupancy prediction for a given base position, and β are the weights for each feature (x1..n), calculated at that position. The Lasso algorithm imposes a constraint on the sum of the weights, such that only the most important features are given non-zero weights. Input features are listed in Table 2H42 tetramer of the histone octamer[in vitro[We downloaded a MATLAB version of the Lasso algorithm,40. Give[in vitro. The frein vitro occupancy, and used the presence or absence of the sequence feature to define positive and negative instances. For the base composition and dinucleotide feature models, we calculated the Pearson correlation to the measured in vitro nucleosome occupancy, and retained those with correlation > |0.10|. We then ran Lasso on the selected sequence features, training on 1,000,000 randomly selected data points from chromosomes 1-9 (or other sets of chromosomes as indicated) which had been standardized to have mean zero and unit variance  and selected the optimal weights by means of 10-fold internal cross validation. The 14 feature linear model is as follows  resulted in more stable results. We therefore selected input features as follows: for 4-mer frequency and nucleosome excluding/positioning motifs, the AUROC (area under the receiver operating curve) ≤0.45 and AUROC > 0.54. To calculate the AUROC for each sequence motif, we first sorted each 150-base sequence by Intrinsic sequence preference = 1.67175 × G+C content + 0.145742 × propeller twist + 1.31928 × slide - 0.10549 × freqAAAA - 0.07628 × freqAAAT - 0.03006 × freqAAGT - 0.05055 × freqAATA - 0.02564 × freqAATT - 0.02154 × freqAGAA - 0.03949 × freqATAA - 0.02354 × freqATAT - 0.03214 × freqATTA - 0.03314 × freqGAAA - 0.0334 × freqTATA + 1.788022Where 4-mer occurrence is calculated in 150 bp windows, and G+C content, propeller twist and slide are calculated in 75 bp windows as described above. Propeller twist and slide were calculated as averages over all dinucleotides from tables found in:S(i) is the structural feature  score for the dinucleotide at position i.Where http://srs6.bionet.nsc.ru/srs6bin/cgi-bin/wgetz?-page+LibInfo+-newId+-lib+PROPERTY:For propeller twist and slide, the full equations are (from the PROPERTY databasehttp://srAverage propeller twist = (-17.3 × freqAA - 6.7 × freqAC - 14.3 × freqAG - 16.9 × freqAT - 8.6 × freqCA - 12.8 × freqCC - 11.2 × freqCG - 14.3 × freqCT - 15.1 × freqGA - 11.7 × freqGC - 12.8 × freqGG - 6.7 × freqGT - 11.1 × freqTA - 15.1 × freqTC - 8.6 × freqTG - 17.3 × freqTT)/75Average slide = (-0.03 × freqAA - 0.13 × freqAC + 0.47 × freqAG - 0.37 × freqAT + 1.46 × freqCA + 0.6 × freqCC + 0.63 × freqCG + 0.47 × freqCT - 0.07 × freqGA + 0.29 × freqGC + 0.6 × freqGG - 0.13 × freqGT + 0.74 × freqTA - 0.07 × freqTC + 1.46 × freqTG - 0.03 × freqTT)/75C. elegans genomes using the model scored on 150-base windows surrounding each data point in both in vitro and in vivo nucleosome maps [We predicted nucleosome occupancy in yeast and ome maps ,34 at 1 http://genie.weizmann.ac.il/software/nucleo_exe.html[occ or "average occupancy" measure. For other models[C. elegans chrIII, and synthetic 150-mer oligonucleotides (both microarray and sequencing data sets)[We obtained nucleosome prediction software from the authors' website _exe.html,23,24, aer models,29,31,32er models was obtaer models on all ser models, we predata sets), using data sets), which oDT performed the analyses. TRH coordinated the study. TRH and DT contributed to the preparation of the manuscript.Supplementary data and figures. Contains Table S1, and Figures S1-3.Click here for file


Upon the addition of a series of tetrabutylammonium salts to receptor 1 in aqueous CH3CN, only when the counter ion was F− was a significant color change (from light violet to dark orange) observed.A novel indolocarbazole-based chemosensor  In particular, the selective sensing of fluoride has gained attention due to its significant role in clinical treatments e.g. dental care −.Deprotonation of receptor ceptor 1 . The binceptor 1  with flu1 +F−, the signal for phenolic OH protons of 1 underwent large downfield shift (Δδ = 1.34 ppm) and the proton signal was broadened. These observations further indicated that the first added F− establishes an H-bond interaction with the OH subunit of 1, while an excess of F− induces the deprotonation of the catechol moieties and NH proton, which brings electron density onto the π-conjugated framework through bond propagation, thus causing a shielding effect and inducing upfield shift of aromatic protons. The above mentioned results indicate that receptor 1 exhibits selective sensing for F− (F− > AcO− >> other anions) in an appropriate solvent.The interaction of receptor 3OH. The addition of the polar protic solvent methanol results in a gradual decrease of absorbance in UV–vis studies. This is presumably because the presence of a relatively high amount of protic solvent disfavors the formation of the deprotonated receptor. However, in the water–acetonitrile system, no color changes were observed upon addition of organic bases such as triethylamine whilst the addition of excess [Bu4N]+OH− can deprotonate receptor 1 and induce a color change.The fluoride- and acetate-induced deprotonation process is reversible as evidenced from the addition of CH1 based on indolocarbazole was synthesized in high yield, which can form 1:2 complex with anions by multiple hydrogen-bonding interactions. Among the anions, only receptor 1 has higher selectivity for F− and leads to a distinct color change that can be observed by the naked eye. The binding results with a series of anions suggest that receptor 1 efficiently binds F− as established by UV–vis, fluorescence and 1H NMR spectroscopic methods. As a colorimetric anion sensor, the indolocarbazole-based receptor 1 displayed highly selective coloration for F− ion even in the presence of other anions.In conclusion, a new colorimetric receptor 1H NMR spectra were recorded on a Bruker AM-500 spectrometer. The 1H NMR chemical shift values are expressed in ppm (δ). UV–visible and fluorescence spectra measurements were performed on a JASCO V530 and a PerkinElmer LS-55 spectrofluorimeter, respectively. Receptor 1 and guest anions were dissolved in UV-grade acetonitrile and water (4:1 v/v). The corresponding absorbance values for receptor 1 were noted during titration and used for the determination of binding constant values. Binding constants were determined by using the expression A0/A − A0 = [εM/(εM − εC)](Ka−1Cg−1 + 1), where εM and εC are molar extinction coefficients for receptor and the hydrogen-bonding complex, respectively, at selected wavelengths, A0 denotes the absorbance of the free receptors at the specific wavelength, and Cg is the concentration of [Bu4N]+X− . The measured absorbance A0/A − A0 as a function of the inverse of the guest anion concentration fits a linear relationship, indicating a 1:2 complexation of the receptor and anions. The ratio of the intercepts to the slope was used to determine the binding constant Ka.All reactions were carried out under a nitrogen atmosphere. Solvents were dried before use. Solvents for spectroscopic measurements were of spectroscopic or HPLC grade. The 1 at the AM1 level was carried out using the minimal valence basis as STO 3G in ArgusLab 4.0.1 software suite. We have refrained from citing calculated total energy value, the calculation being for molecule only in the gas phase.Geometric optimization of their stable conformation of receptor 2, 0.5 g, 1.41 mmol) in a round-bottom flask containing dry acetonitrile (5 mL), I2 (2 mol %) was added and the mixture refluxed for 45 min. The solid obtained was filtered, dried and recrystallized from a mixture of DMF–CHCl3. Yield 82%, mp 258 °C; 1H NMR : δ (ppm) 10.53 , 8.63 , 7.24 , 7.12 , 6.94 , 6.80 , 6.68 , 6.48 , 6.43 ; 13C NMR : δ (ppm) 145.1, 143.9, 137.1 (for two carbon), 135.1 (for two carbon), 125.9, 120.4, 119.4, 118.59, 118.0, 115.4, 115.1, 11.0, 110.1; FTIR : 3472, 3430, 1521, 1457, 1262, 1224; C30H20N2O4 (473.1496); Anal. Calcd C, 76.26; H, 4.27; N, 5.93; O, 13.54; found C, 76.35; H, 4.19; N, 5.73; O, 13.60; HRMS (MH+ + 2): 475.21.3,3′-Bis(indolyl)-3,4-dihydroxyphenylmethane .