The class Myxosporea encompasses about 2,400 species, most of which are parasites of fish and cause serious damage in aquaculture. Due to the concerns about food safety issues and limited knowledge of Myxozoa life cycle and fish immune system, no chemicals, antibiotics or immune modulators are available to control myxozoa infection. Therefore, little can be done once Myxozoa establishment has occurred.Aeromonas veronii CD3 with significant myxospore shell valve-degrading ability from pond sediment. A 3,057-bp full-length chitinase gene was consequently cloned, and the corresponding mature, recombinant chitinase (ChiCD3) produced by Escherichia coli had substantial chitinase activity. The deduced sequence of ChiCD3 contained one catalytic domain, two chitin-binding domains, and one putative signal peptide. ChiCD3 had an optimal activity at 50°C and pH 6.0, and retained more than 50% of its optimal activity under warm water aquaculture conditions (∼30°C and pH ∼7.0). After incubation with ChiCD3, 38.0±4.8% of the myxospores had damaged shell valves, whereas myxospores incubated with commercially available chitinases remained intact.In this paper we isolated This study reveals a new strategy to control myxozoan disease. ChiCD3 that has capacity to damage the shell valve of myxospores can be supplemented into fish feed and used to control Myxozoa-induced diseases specifically. The phylum Myxozoa is an entirely endoparasitic group of organisms, especially the fish, which produces complex multi- cellular spores Ceratomyxa shastaMyxobolus cerebralisTo control myxozoan disease, there are numerous studies focused on the route of immunization, such as selection of myxozoan-resistant fish species and development of vaccines. Even innate resistance of certain fish species against Myxozoan has been reported, but the underlying mechanisms have not been elucidated in most cases N-acetyl-d-glucosamine (GlcNAc) and is an important element of fungal cell walls. Chitinases have been widely used to control fungal diseases Generally myxospores consist of 2 to 12 shell valves that typically contain substantial amounts of chitin Cyprinus carpio). The spores were pyriform with slight tapering anterior and round posterior ends and measured 18.5±0.5 µm (n = 30) long and 8.9±0.6 µm (n = 30) wide. A single flask-shaped polar capsule lied close to the apex of the spores. Even the mean spore length and width of each species was not identical between species, but the morphological and the scanning light microscopy revealed a smooth spore surface of the sample, which is consistent with that of ThelohanellusThelohanellus kitauei (accession no. GQ396677) and 96% to T. hovorkai (accession no. DQ231155). T. kitauei is a typical gut parasite of Thelohanellus. This site-dependent character differs T. kitauei from other species without morphological identification T. kitauei, Myxobolidae, Myxosporea.The mature myxospores were separated from plasmodia in the gut of a common carp (–1) were detected in the culture supernatant of strain CD3. Comparison of its 16S rDNA sequence with those in GenBank and a phylogenetic analysis classified strain CD3 as Aeromonas veronii . Then the strain CD3 was deposited into the China General Microbiological Culture Collection Center under registration number CGMCC 3169.By using chitinase-screening medium, four bacterial strains were isolated from the sediment of a fish pond which had Myxozoa-induced disease outbreak. Using shell valve of spores as carbon source in culture medium, the reducing sugars   and one catalytic domain (residues Arg318–Asp768) of glycoside hydrolase family 18. The mature protein had a theoretical molecular mass of 110 kDa. The deduced amino acid sequence of ChiCD3 was 90% identical to those of Aeromonas salmonicida subsp. salmonicida A449 and Aeromonas hydrophila subsp. hydrophila ATCC 7966, 53% to the Vibrio mimicus MB-451 chitodextrinase precursor and 52% to the Vibrio sp. RC586 chitodextrinase precursor. Multiple sequence alignment . After induction with 0.1 mM IPTG  at 15°C for 24 h, substantial chitinase activity (2.56 U ml–1) was found in the cell lysate supernatant, and no chitinase activity was detected in the lysate supernatant of an uninduced culture or a culture that harbored an empty pET-30a(+).The gene 2+-NTA metal-chelating affinity chromatography. The purified protein migrated as a single band on SDS–PAGE  followed by barley β-glucan (5.3±0.8 U mg–1) and carboxymethyl cellulose (4.6±1.2 U mg–1). It had no ability to degrade birchwood xylan or locust bean gum. The kinetic constants mK and maxV for the hydrolysis of colloidal chitin were 7.12 mg ml–1 and 19.11 µmol min–1, respectively.ChiCD3 had the greatest activity against colloidal chitin .The myxospores were treated with 19 U of chitinases from Thelohanellus spp. are generally histozoic and highly host-specific parasites Thelohanellus should be paid more attention to be studied. Of them, T. kitauei is the causative agent of the intestinal giant cystic disease of carp, which is a well-known disease in China T. hovorkai causes destruction of capillaries in carp Thelohanellus spores can remain viable in sediment for at least 5 months Thelohanellus infection to the aquaculture industry is substantial and increasing. However, the period in sediment also provides a chance to control Thelohanellus by damaging the spores. Furthermore, this treatment can be carried out either in aquaculture stage or in the interval stage, and is easy for application by comparison with other researches concerning the parasite life-cycle, parasite-host interaction and fish immune system.As the complexity of Myxozoa, it is difficult to control the Myxozoan infection by current available methods. A. veronii strain, named as CD3, with extracellular chitinase activity, from pond sediment. To overcome the initial host defense barriers and provide nutrients for cell proliferation, Aeromonas strains secrete varieties of proteolytic enzymes A. veronii CD3 showed ability to utilize myxospores and its chitinase ChiCD3 produced in E. coli also had the ability to damage myxospores by degrading their shell valves.In this study, we isolated an 453DGVDIDYEY462, which is essential for chitinase activity Comparison of the ChiCD3 catalytic site with that of other bacterial chitinases identified the conserved consensus sequence FIn comparison with homologs that have been characterized, ChiCD3 was more active at a less basic pH and at a lower temperature, i.e., pH 8.0–9.0 and 50°C for the former Thelohanellus is unknown and it cannot be cultured in vitroThe life cycle of The locations for sample collection are not privately-owned or protected in any way and no specific permits are required for the described field studies and sample collection. Also the field studies does not involve endangered or protected species.(aq), collected by centrifugation at 4°C, 500 × g for 5 min, and stored in 1.5 ml microfuge tubes at 4°C. Measurements of spore and capsule dimensions were performed using a Leica DM2500 microscope attached with a digital camera  and ImageProPlus v6 (Media Cybernetics) image analysis system.A Myxozoa-infected common carp that had plasmodia in its gut was taken from an aquaculture pond in Wuqing, Tianjin, China in July 2007. The pond was ∼1.5 m deep, had a temperature of 30°C and a pH of 7.5–8.0 and their surface areas were ∼100 acres. Plasmodia that were filled with mature spores were separated from the fish and ruptured with a needle. Myxospores were washed with sterile 0.7% (w/v) NaCl–1 proteinase K, and homogenized at the maximum speed for 5 min on a mini Beadbeater in the presence of 600 mg of sterile zirconia beads (100 mg of 0.1 mm and 500 mg of 0.5 mm beads). After incubation at 50°C for 30 min, the crude DNA was centrifuged at 16,000 × g, 4°C for 5 min and purified as in E. coli DH5  and sequenced by Sangon (China). The Myxozoa taxon was identified by comparison of its 18S rDNA sequence with those in GenBank.Genomic DNA was extracted and purified g for 20 min, washed repeatedly with sterile H2O until a neutral pH was reached, lyophilized and stored at –20°C. The screening medium contained (per liter) 1 g colloidal chitin (the sole carbon source), 3 g KH2PO4, 2 g K2HPO4, 1 g NH4Cl, 200 mg MgSO4·7H2O, 10 mg CaCl2·2H2O and 2% agar, pH 7.0.The chitinase screening medium was prepared as previously described (aq). The suspension was spread onto chitinase screening agar plates and incubated at 25°C for 3 d. Colonies were streaked onto the same medium to obtain purity.Sediment sample was collected from an aquaculture pond in Tianjin, China that had been the site of a Myxozoa-induced disease outbreak and suspended in sterile 0.7% (w/v) NaClg for 5 min. This step was repeated twice. Then the spores were suspended in sterile 0.7% (w/v) NaCl(aq) and supplemented into chitinase screening medium to replace colloidal chitin as the inducer and the only carbon source The stock myxospores were suspended in 1 ml of 70% ethanol and centrifuged at 1,000 × Genomic DNA of strain CD3 was extracted using DNA Isolation Kit . For identification, 16S rRNA gene fragment was PCR amplified using the primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-CGGYTACCTTGTTACGACTT-3′) and sequenced by Sangon.http://www.cazy.org/GH18.html) and used to touchdown-PCR amplify the chiCD3 core region. The touchdown-PCR program was: 94°C for 5 min; 5 cycles at 94°C for 30 s, 55°C for 30 s that was decreased by 1°C each cycle, and 72°C for 30 s; 30 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 8 min. The PCR product was purified and ligated into a pGEM-T Easy vector for sequencing. To obtain the full-length chitinase gene, the 5′ and 3′ flanking regions were subjected to thermal asymmetrical interlaced (TAIL)-PCR A primer set (CHI-F: 5′-GGIGGITGGACIYTIWSICC-3′ and CHI-R: 5′-ATGCAITAYGAYTTYCAYGG-3′) was designed to include the nucleotide sequences for the two conserved motifs GGWTLSD and SVGAWAD of chitinases belonging to family 18  and SMART (http://smart.embl-heidelberg.de/), respectively. DNA sequences were translated to yield corresponding amino acid sequences with ExPASy (http://au.expasy.org). DNA and protein sequence alignments with known chitinase sequences were performed with blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/), respectively. The key functional residues were predicted at http://pfam.sanger.ac.uk/search. Multiple sequence alignments were performed with Clustal X Sequence assembly was performed using Vector NTI 10.3 software . The signal peptide and functional domains in the deduced amino acid sequence were predicted using SignalP , and the primers pET30a-HindIII (5′-cccAAGCTTGCCAGGCCGCTTATCCCGCC-3′) and pET30a-XhoI (5′-ccgCTCGAGTAAATAGCTACAGGCAGACTT-3′) . The PCR product was purified with TaKaRa Purification Kit, cloned into the HindIII and XhoI sites of pET-30a(+) and then transformed into E. coli BL21(DE3) competent cells. Positive transformants that contained the correct insert were identified by isolating the insert through an agarose gel and sequencing it. These cells were then cultured in 25-ml LB medium containing 100 µM ampicillin at 37°C until the A600 of the culture was 0.6–0.8. Protein expression was induced at 15°C by addition of IPTG  and the culture was incubated for an additional 12 h.The gene fragment encoding ChiCD3  was amplified using the genomic g for 5 min, washed with sterile 0.7% (w/v) NaCl(aq), suspended in 20 mM Tris-HCl (pH 8.0) and lysed with a microfluidizer . Soluble and insoluble fractions were separated by centrifugation at 12,000 × g for 15 min at 4°C. The (His)6-tagged proteins in the supernatant were isolated by Ni2+-NTA metal-chelating affinity chromatography using a His-SelectTM cartridge (Sigma) according to the manufacturer's instructions. SDS–polyacrylamide gel electrophoresis (PAGE) used a 12% running gel Cells were centrifuged at 8,000 × N-acetylglucosamine (reducing sugar) liberated from colloidal chitin g, and the amounts of reducing sugars released into the supernatants were determined by the DNS method Chitinase activity was determined based on the amount of The effect of pH on enzyme activity was determined at 50°C between pH 3.0 and 10.0. The effect of pH on enzyme stability was determined by measuring the chitinase activity under standard conditions after incubation at 50°C in buffers with different pH values for 2 h. The buffers were McIlvaine buffer (pH 3.0–8.0), 0.1 M Tris–HCl (pH 8.0–9.0), and 0.1 M glycine–NaOH (pH 9.0–12.0). The temperature optimum of the enzyme activity was determined at the optimal pH (pH 6.0) for the temperature range of 20 to 70°C. Thermal stability was determined by measuring the residual activity after incubation at 20°C and 50°C, pH 6.0 for 2 h.The substrate specificity of ChiCD3 was determined by measuring the enzyme activity after incubation in 20 mM Tris–HCl containing 0.5% of each substrate  at pH 6.0 and 50°C for 2 h. The amount of reducing sugars produced was estimated using DNS method as described above.mK and maxV for ChiCD3, the enzyme was incubated with colloidal chitin (0.1% to 1.0%) (w/v) and the amounts of reducing sugars were determined using the DNS method. The values for mK and maxV were determined from a Lineweaver-Burk plot.To determine the values of S. griseus chitinase 1  had a specific activity of ∼200 U g–1 against colloidal chitin. S. marcescens chitinase 2  had an activity of 400–1,200 U g–1 against colloidal chitin. Aliquots (100 µl) of the myxospore suspension (∼500 mg wet weight in spores) were treated with 19 U of chitinase 1, chitinase 2, or ChiCD3 in 20 mM Tris–HCl (pH 8.0) or with buffer only (control). After incubation at 37°C for 2 h, each supernatant was collected by centrifugation at 3,000 × g, 4°C for 5 min and subjected to the DNS assay. Each pellet was observed under the scanning electron microscope. As the intact mature spores have smooth shell and without projections, the spores with damaged shell will be counted as the damaged myxospores after treatment with the chitinase. The myxospores treated with 1.9, 19 or 190 U of ChiCD3 were also examined by optical microscope.The shell valve-degrading activities of two commercial chitinases and ChiCD3 were characterized. Thelohanellus kitauei 18S rDNA gene fragment and the chitinase gene chiCD3 were deposited in GenBank under accession numbers GU350406 and FJ561294, respectively.The nucleotide sequences for the


Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ), resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7) when tested at 20 µg/mL.Three members of the δ-endotoxin group of toxins expressed by  Bacillus thuringiensis (Bt), which occurs naturally in several environments in vitro to erythrocytes and other mammalian cells, and lethal to mice after intravenous injection Aedes aegypti gut cell membranes Bacillus thuringiensis subsp. israelensis (Bti) is highly toxic to dipteran insects and is utilized in mosquito control in many countries Culex spp, Aedes spp and Anopheles spp is due to the presence of a megaplasmid, pBtoxis, which encodes at least four Cry and two Cyt toxins: Cry4Aa, Cry4Ba, Cry10, Cry11Aa, Cyt1Aa and Cyt2Ba Crop-damaging insects are responsible for a significant decrease in world crop yields every year in vitro against different cultured cell lines to determine their individual toxic activity and possible synergistic interactions with other toxins under controlled experimental conditions.In our study acrystalliferous Bt strains were engineered to individually express Cry4Aa, Cry11A and Cyt2Ba proteins; the strains could then be tested cyt2Ba gene was identical to the sequence described elsewhere cry11A gene showed 100% nucleotide sequence identity with the cry11A gene described earlier by Berry et al.cry4Aa gene showed 98% nucleotide sequence identity with the first described cry4Aa gene Blast analysis of the sequences from amplified Bti genes showed that the cyt2BaHis, cry11A and cry4Aa coding sequences were first cloned in a PCR cloning vector (not shown), then transferred to the binary B. thuringiensis expression vector pSVP27A under control of the sporulation phase promoter Pcyt . After this additional purification step, SDS-PAGE analysis was carried out, showing a single 27 kDa band as expected  . Despiteexpected .in vivoA. albopictus. In our assays, the Cry4Aa and Cry11A activated toxins showed 10 to 15% of the toxicity of wild type Bti-activated toxins (see below) in A. albopictus C6/36 cells. Furthermore, the mixture of the two recombinant proteins increased toxicity to approximately 36% of the wild type Bti-activated toxins. As reported earlier Choristoneura fumiferana (spruce budworm) showed distinct relative susceptibility when incubated with Bt activated δ-endotoxins. The presence, absence or availability of suitable receptors on the cell surface could explain the diverse levels of susceptibility among different cell lines deriving from one insect species. The increased toxicity of combined Cry4Aa and Cry11A to A. albopictus cells was also observed in previous research A. aegypti than native crystals produced by Bti. Sauka et al. Epinotia aporema (bean shoot borer), suggesting a possible synergism between these Cry toxins. However, no synergistic interaction between 130kDa and 65 kDa Cry proteins was detected in vivo against A. aegypti larvae 50) was estimated  and some antagonism was detected . Aside from this report, it has been demonstrated that binding of Cry11Aa to A. aegypti membranes is greatly enhanced by preincubation of “brush border membrane vesicles” (BBMVs) with Cyt1A et al. in vivoTo verify that Cyt2Ba and Cry toxins (Cry4A and Cry11A) interact cytotoxically, assays were performed using combinations of these toxins. The Cry toxins were used at a concentration of 20 µg/mL, whereas the Cyt2Ba toxin was used at 5 µg/mL, since any increase in the obtained cytotoxicity could be misinterpreted as activity of only Cyt2Ba at its highest concentration .The dataF values  and 2. SS. frugiperda larvae. As previously described, besides the three-domain based structure and the intrinsic amino acid sequence, proteolytic activation is a relevant factor for the toxicity of Cry toxins S. frugiperda midgut proteases resulted in a different SDS-PAGE banding profile than with trypsin activation  BBMVs. In contrast, the Cry3Ba toxin was unable to bind L. decemlineata’s BBMVs using identical proteolytic treatments in vivo against second-instar larvae of A. aegypti. Cry11A and Cry4Aa were toxic to A. aegypti larvae with LC50 values of 53.72 ng/mL and 92.24 ng/mL, respectively  as the source of cyt2Ba, cry4Aa and cry11A genes. Lepidopteran cells from Spodoptera frugiperda, IPLB-SF-21AE  Lymantria dispar, IPLB-LD-652Y (isolated from ovaries of the gypsy moth) Bombyx mori, BM-5 (isolated from ovaries of the silk worm) Aedes albopictus cells C6/36 . In the same way, the complete ORF of the cry11A gene was obtained from the S-1806 strain using specific oligonucleotides . BamHI restriction sites were added to the oligonucleotide sequences (underlined text). The start codon of each gene is shown in italics (ATG) and the 6xHis tag codons fused to the cyt2Ba gene are shown in bold. To amplify the cyt2Ba, cry4Aa and cry11a genes, different PCR programs were performed using the respective three sets of oligonucleotides: cyt2Ba –94°C/5 min and 30 cycles of 94°C/1 min, 52°C/45 s, 72°C/1∶30 min and a final extension of 72°C/7 min; cry4Aa –94°C/5 min and 35 cycles of 94°C/1 min, 53°C/1 min, 72°C/2∶30 min and a final extension of 72°C/7 min; and cry11A –94°C/5 min and 30 cycles of 94°C/1 min, 53°C/1 min, 72°C/1∶30 min and a final extension of 72°C/7 min. Amplified fragments were cloned into the pGem-T®easy (Promega) vector, using Escherichia coli DH5-α® (Invitrogen) as a host, following the manufacturer’s instruction. DNA from recombinant plasmids was isolated BamHI, and then separated by electrophoresis in a 0.8% agarose gel. Fragments of approximately 800, 3,500 and 1,900 bp, corresponding to the cyt2Ba, cry4Aa and cry11 genes, respectively, were purified using the GFX™ PCR DNA and Gel Band Purification Kit . The three genes were then separately cloned into the expression vector pSVP27A, kindly provided by Dr. David Ellar, University of Cambridge, UK E. coli DH5-α®. The positive clones were selected by ampicillin resistance in culture medium (100µg/mL). The recombinant plasmids pSVP27Acyt2BaHis, pSVP27Acry4Aa and pSVP27Acry11A were isolated by alkaline lysis as described in Plasmid DNA from Bti strains S-1989 and S-1806 were extracted by alkaline lysis −1, 200 Ω e 25 µF. After electroporation, the cell suspensions were diluted in 1 mL LB broth and incubated with agitation (200 rpm) for 2 h at 28°C. After cell recovery by centrifugation, the pellets were resuspended and incubated on L-agar with 10 µg mL−1 chloramphenicol g for 20 min and the supernatant was discarded. The pellets were resuspended and washed in 60 mL of a solution containing 0.3 M NaCl, 0.01 M EDTA pH 8.0 and centrifuged at 16,000×g for 20 min. This procedure was repeated twice; at the end of the third centrifugation the supernatant was discarded and the pellets were resuspended in 30 mL 1.0 mM PMSF. Fresh 1.0 mM PMSF solution was used to wash the samples by centrifugation at 16,000×g for 20 min. The sediments were then resuspended in TTN buffer . The samples were centrifuged at 25,000×g for 1 h in a sucrose gradient . The protein bands corresponding to Cry11A, Cry4Aa and Cyt2BaHis, formed in the gradient, were collected and added to a 0.1% Triton X-100 solution. A new centrifugation at 25,000×g for 45 min was performed and the sediments were resuspended in 1,0 mM PMSF. The crystals of Cyt2BaHis and Cry11A were solubilized in 0.1 M Na2CO3, pH 10.5 at 37°C for 1 h. The Cry4Aa crystals were solubilized in the same solution supplemented with the reducing agent 0.2% β-mercapto-ethanol. Any insoluble material was removed by centrifugation. The samples were neutralized with 0.1 M Tris-HCl pH 7.5 2CO3, pH 10.5 solution at 37°C for 1 h and then neutralized with 0.1 M Tris-HCl, pH 7.5. The samples were analysed by electrophoresis in a 12% SDS-PAGE (Mini-protean II – Biorad), following the manufacturer’s instruction.An acrystalliferous Bti strain, 4Q7, kindly provided by Dr. Colin Berry, Cardiff University, UK, was used as a host for the constructed plasmids. The Bti cells were transformed with the recombinant plasmids pSVP27Acyt2BaHis, pSVP27Acry4Aa and pSVP27Acry11A by electroporation as previously described S. frugiperda midguts following the procedure described in The samples containing the solubilized protoxins were quantified by fluorospectrophotometry using the NanoDrop 3300 apparatus together with the Quant-It™ Assay Kit (Invitrogen). The individually purified Cry toxins were activated by two methods. In the first method, the protoxins were incubated with pancreatic bovine trypsin (Sigma) at a 10∶1 ratio (w/w) for 2 h at 37°C. In the second activation method, the protease-containing gastric juice was obtained from ten 2 TPP flasks , were collected and added to 96-well plates (104 cells/well). The culture medium of MCF-7 cells was removed and the cells washed with 1 mL PBS. A trypsin/EDTA (0.05%) solution was added to the cells, incubated at 37°C and 5% CO2 for 4 min and 1 mL of DMEM medium was added to stop trypsinization. The loose cells were centrifuged at 1,000×g for 3 min, resuspended in DMEM medium and added to 96-well plates (104 cells/well). The activated Cyt2Ba, Cry4Aa and Cry11A toxins were added to the wells at predetermined concentrations. Cyt2Ba toxin was tested at two concentrations (5 or 20 µg/mL) separately or in combination with the other toxins. Cry4Aa and Cry11A toxins were used at the concentration of 20 µg/mL, individually or in the presence or absence of Cyt2Ba. The pool of wild type Bti activated toxins were incubated with all cell lines at the concentration of 5 µg/mL. All toxins were also incubated, at the mentioned concentrations, with different culture media in the absence of cells. Trypsin and gastric juice were individually incubated with C6/36 cells. All assays were carried out in triplicate. The time of all incubations with the three toxins as well as with the proteases, was 30 min. Furthermore, the culture media were incubated with CellTiter-Glo® Reagent from the CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega) for 10 min to quantify the amount of ATP released to the media. The luminescence-based measurements were carried out in a Turner TD20/20 luminometer, with the following setup: delay time 5 s, integration time 20 s, sensitivity 50%.The insect  and mammalian (MCF-7) cell lines, grown in 25 cmA. aegypti from the Rockfeller strain were provided by Embrapa Recursos Genéticos e Biotecnologia . The larvae were reared at room temperature (25°C) in autoclaved distilled water and fed on cat biscuit until they reached second instar.Neonate larvae of A. aegypti larvae, and mosquitocidal activities were determined after 24 h A. aegypti larvae were also exposed to a single dose of 20 µg/mL of each GJ-activated toxin (Cry11A and Cry4Aa), following the same method of incubation described above. All bioassays were performed in duplicate.Sucrose-gradient isolated Cry11A and Cry4Aa crystals were added separately to 100 mL sterile tap water in disposable plastic cups containing 20 second-instar 50  of each individual toxin and of the toxin mixtures  was estimated by probit analysis 50, obtained by Tabashnik’s equation 50. When the ratio was >1, the toxin interaction was considered to yield a synergistic effect as the toxicity was higher than the value predicted from individual additive toxicity. A ratio <1 meant that the interaction was antagonistic, whereas a ratio of 1 indicated only additive effects.In order to identify potential synergistic effects, experimental LC


Short-term plasticity plays a key role in synaptic transmission and has been extensively investigated for excitatory synapses. Much less is known about inhibitory synapses. Here we analyze the performance of glycinergic connections between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) in the auditory brainstem, where high spike rates as well as fast and precise neurotransmission are hallmarks. Analysis was performed in acute mouse slices shortly after hearing onset 11) and 8 days later (P19). Stimulation was done at 37°C with 1–400 Hz for 40 s. Moreover, in a novel approach named marathon experiments, a very prolonged stimulation protocol was employed, comprising 10 trials of 1-min challenge and 1-min recovery periods at 50 and 1 Hz, respectively, thus lasting up to 20 min and amounting to >30,000 stimulus pulses. IPSC peak amplitudes displayed short-term depression (STD) and synaptic attenuation in a frequency-dependent manner. No facilitation was observed. STD in the MNTB-LSO connections was less pronounced than reported in the upstream calyx of Held-MNTB connections. At P11, the STD level and the failure rate were slightly lower within the ms-to-s range than at P19. During prolonged stimulation periods lasting 40 s, P19 connections sustained virtually failure-free transmission up to frequencies of 100 Hz, whereas P11 connections did so only up to 50 Hz. In marathon experiments, P11 synapses recuperated reproducibly from synaptic attenuation during all recovery periods, demonstrating a robust synaptic machinery at hearing onset. At 26°C, transmission was severely impaired and comprised abnormally high amplitudes after minutes of silence, indicative of imprecisely regulated vesicle pools. Our study takes a fresh look at synaptic plasticity and stability by extending conventional stimulus periods in the ms-to-s range to minutes. It also provides a framework for future analyses of synaptic plasticity. Fast and reliable synaptic transmission at high frequencies is one of the hallmarks of the auditory system. In particular, it is a key feature for processing interaural time and intensity differences, the two major features for computation of sound localization  at frequencies between 50 and 500 Hz  and the tens of seconds-to-minute range, for which we use the term “synaptic attenuation” as the counterpart to the term synaptic augmentation , two temperatures (37 and 26°C), and various frequencies (1–400 Hz). Moreover, “marathon experiments” involved very prolonged stimulation (20 min) comprising up to 30,600 stimuli, many-fold more than routinely used 11 ± 1 or P19 ± 1. They were in accordance with the German law for conducting animal experiments and followed the NIH guide for the care and use of laboratory animals.3 25; NaH2PO4 1.25; Na-pyruvate 2; D-glucose 10; CaCl2 2; MgCl2 1; myo-inositol 3; ascorbic acid 0.44; pH = 7.3 when bubbled with 95% O2-5% CO2) before being transferred into a recording chamber in which they were continually superfused with ACSF (1–2 ml/min). Recordings were performed at near physiological temperature  or at room temperature (26 ± 2°C). The recording chamber was mounted on an upright microscope  equipped with differential interference contrast optics  and an infrared video camera system . LSO principal neurons were identified by their location in the center of the nucleus, orientation, size, spindle-shaped somata, the presence of hyperpolarization-activated inward currents . The Cl− concentration of 2 mM was slightly lower than the native 5 mM previously determined for P9–11 via perforated-patch clamp recordings -8P, Science Products) with a horizontal puller . They were connected to an Axopatch-1D patch-clamp amplifier (Molecular Devices) and had resistances of 3–7 MΩ when filled with intracellular solution , a theta glass electrode  with a tip diameter of 10–20 μm was filled with ACSF and placed lateral to the MNTB Figure . MonopolFor normalization, 40 control stimuli were presented at 1 Hz during a baseline period and the peak amplitudes of the 40 IPSCs were averaged . All drugs were added to the ACSF. In control experiments, the residual current could be completely abolished with strychnine (0.3 – 1 μM), thus verifying their glycinergic nature.All chemicals were purchased from Sigma-Aldrich unless stated otherwise. Glycine-mediated receptor currents were isolated pharmacologically. To do so, N-methyl-D-aspartic acid (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor-mediated currents were blocked by kynurenic acid (5 mM). Furthermore, GABAzine (10 μM) and CGP55845 (3 μM) were used for blocking GABA1, IPSC2, IPSC10), amplitudes after the 1st and the 40th s , and averaged amplitudes over several 10-s periods, namely 30–40 s, 50–60 s, and 110–120 s . When individual IPSC peak amplitudes were ≤5% of the control value, the event was considered a failure. A failure was also attributed if the peak amplitude was <15 pA, which equaled the noise level. For such failures, the peak amplitude was set to 0 pA. For kinetic measurements, the decay time constant τ (decay to 37% of peak amplitude) was determined by fitting a single exponential to the decay phase of the IPSC.Data analysis was performed with Mini Analysis 6.0.3 (Synaptosoft). At the beginning of each experiment, the control value of glycinergic IPSC peak amplitudes was obtained for each neuron (see above). The following peak amplitudes underwent statistical analysis: amplitudes of the 1st, 2nd, and 10th IPSC . Outliers (more than four times standard deviation above/below mean) were excluded. Such outliers occurred in the range of 5.3–11.8% . In absolute numbers, ngross = 19 became nnet = 18 in the best case, and ngross = 17 became nnet = 15 in the worst case. Samples with a Gaussian distribution (Kolmogorov–Smirnov) were compared using a paired or an unpaired two-tailed Student's t-test. A Wilcoxon-signed rank test was used for unequal variances. Equality of variances in unpaired samples Student's t-tests was assessed with an F-test. Values in bar charts are presented as mean ± s.e.m. Significance values were as follows: *p < 0.05, **p < 0.01, ***p < 0.001.Statistical analysis was performed on data sets if in vivo at P11–12 and thereafter  had the highest peak amplitude . For example, at 1, 10, and 100 Hz, IPSC2/IPSC1 ratios amounted to 0.84, 0.85, and 0.80, respectively  to high frequency stimulation, even during the first 40 pulses Table 1–A8. Vir1 to IPSC40 were evoked reliably, even at a stimulus frequency of 200 Hz  revealed similarities as well as differences in STD behavior compared to 37°C  was more pronounced at 26°C than at 37°C (IPSC1 s/IPSC2 ratio: 0.24 vs. 0.39). The greater reduction with time at lower temperature was corroborated by the IPSC40 s/IPSC1 s ratios. For example at 50 Hz, the ratio was 0.07 at 26°C, yet 0.61 at 37°C , STD was consistently observed, progressing up to 200 Hz . At P11/37°C, the corresponding ratio was 0.85. A significant decline between IPSC2 and IPSC10 did not occur systematically. If there was one, it was moderate . Noticeably, the absolute amplitudes should be treated carefully. First, they may have been reduced by the usage of kynurenic acid  and a 1.5-fold shorter decay time . Analysis of age-dependency (P19/37°C vs. P11/37°C) revealed significant differences for all three parameters tested , a 25% shorter rise time  and a 2-fold shorter decay time .The time course of the IPSCs was significantly shorter at 37°C, as evidenced by a 2-fold shorter rise time  and 26°C (n = 17). Exemplary results from a neuron at 26°C and another at 37°C are illustrated in Figures 60 s) had completely collapsed to 0.0 ± 0.0% at 26°C, yet they remained at 20.6 ± 3.3% at 37°C , corroborating that 37°C responses are quite stable in the second-to-minute range. In contrast, IPSCs declined further between 40 and 60 s at 26°C . Together, this demonstrates that glycinergic MNTB-LSO neurotransmission is sustained at a steady-state level in the minute range only at physiological temperature. The better performance in the minute range at 37°C was also evident in drastically higher peak amplitudes during the last 10 s of the challenge period: at 37°C, the mean IPSC50−60 s value was 23.3 ± 3.5%, but it was almost 40-fold lower at 26°C  and then stimulated uniformly with 50 Hz, now extending the stimulus train to 60 s Figure 2. Immedi Figures 2,B2. Thep = 0.016). In comparison to recovery data obtained from IPSCs in other neuron types, the recovery of MNTB-LSO synapses was similarly fast , albeit considerably fewer at 37°C . Likewise, the IPSC1 amplitudes declined significantly, albeit only moderately, from trial 1 to trial 10 , yet almost 10% in trial 10 . At the end of the recovery period, however, the situation was more diverse: at 37°C, IPSC110−120 s values were significantly lower than those obtained at 26°C in trial 1, yet they were higher in trial 10. No difference occurred in trial 5 .At both 37 and 26°C, the course of recovery from synaptic attenuation was quite variable across neurons, particularly in later trials Figures , 11A. Ases Table , 11B. At0 Figure , Table 3d Figure . At the The temperature dependency of STD and amplitude recovery across trials was reflected by the time course of the failure rates. During the first 40 pulses at 37°C, failure rates of about 10% occurred in trial 10 only, whereas this level was reached already in trial 5 at 26°C Table . At the In a final series of experiments, we addressed the question whether possible AP conduction problems  and failures was quite similar to that obtained via orthodromic stimulation Figures . The perMature LSO neurons receive powerful inhibitory, glycinergic input from MNTB neurons, which are able to respond reliably to high frequency stimulation supplied via the excitatory calyx of Held, up to 800 Hz for brief trains lasting ca. 20 ms  were similar to estimated time constants described previously for rodent LSO principal neurons , whereas we have stimulated over a period of 60 s and in 10 trials, resulting in 30,600 stimuli total (marathon experiments). We did so because the IPSC peak amplitudes had not reached a steady state after 20 pulses. Rather, they further declined considerably, e.g., reaching values of <30% after 40 s at 50Hz/P11/37°C  did not reveal any effect on the time course of synaptic depression and recovery. Thus, we exclude a considerable role of GABAB receptor signaling from our experiments.As shown in gerbils, LSO neurons release GABA during spiking activity which acts as a retrograde transmitter at presynaptic GABAA receptors . This low value does not explain the 64% failures in IPSCs  comes close to the observed IPSC failure rate of 71%. Finally, also the course of recovery cannot be explained by spike failures.Our results from antidromic stimulation experiments imply that AP conduction failures, which occurred only at stimulus frequencies ≥100 Hz, do not at all contribute to impaired transmission at frequencies ≤50 Hz. Rather, the presynaptic release machinery cannot faithfully follow such frequencies, resulting in failures. Furthermore, as the fidelity of AP conduction at 100 Hz was 45% Figure 2, and asIn sum, although glycinergic MNTB-LSO connections undergo STD and synaptic attenuation, they can sustain high frequency transmission at hearing onset over long periods (minutes) and thousands of stimuli if stimulus frequency does not exceed 50 Hz. They also recover robustly (τ = 6.3 s). At P11, connections appear to function quite reliably, yet sustained transmission at 100 Hz is reached only by P19. If the physiological temperature is reduced by 11°C, the performance worsens drastically, in that 10 Hz is now the upper frequency for reliable transmission. Depletion of transmitter release is the most likely cause of the depression behavior. It is important to assess the impact of glycine recycling in replenishing the transmitter pool. One aspect is neuronal and glial re-uptake from the synaptic cleft, which is mediated by glycine transporters 1 and 2, respectively. The role of these transporters may be addressed in pharmacological (Jeong et al., Eckhard Friauf, Florian Kramer, and Désirée Griesemer designed the study; Florian Kramer, Dennis Bakker, Sina Brill, and Erik Frotscher performed the experiments; Eckhard Friauf, Désirée Griesemer, Jürgen Franke, and Florian Kramer analyzed data and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of our study was to evaluate the feasibility of 4D phase contrast (PC) flow imaging for visualisation and analysis of cerebrospinal fluid (CSF) flow in Chiari I malformation. CSF flow was compared between healthy volunteers and patients with Chiari I malformation with and without syringomyelia. Moreover, the effect of a craniocervical decompression on CSF flow was studied in Chiari I patients with syringomyelia.20 patients with Chiari I malformation and 10 healthy volunteers were examined using 4D PC flow imaging. 11 patients had syringomyelia and 1 patient presented with a presyrinx. Of these, 9 patients underwent craniocervical decompression and flow data was assessed pre- and postoperatively. Flow images were analyzed by 2 blinded readers. Quantitative flow parameters including peak flow velocities, maximum flow volume and timing of systole were assessed at the craniocervical junction and the cervical spinal canal.Qualitative analysis showed superior interreader agreement for 4D PC flow images compared to single-slice 2D axial (C1) and sagittal (mid-plane) images (κ = 0.67 vs. κ = 0.57 vs. κ = 0.02). Flow alterations were found in 17 patients consisting of anterolateral flow jet (n=14) or flow vortex (n=5) formation and flow inhomogeneity (n=3). In patients peak velocities at the craniocervical junction were found to be increased  with maximal velocities of up to 37.9 cm/sec. Of the 9 patients who underwent craniocervical decompression 4 showed a decrease in syrinx size postoperatively. Postoperatively, maximum flow volumes increased significantly at C2 and the lower levels of the cervical spine. In the group with regredient syrinx size peak flow velocities decreased (-19.4 ± 14.4 vs. -10.5 ± 3.8 p=0.07) while patients without change in syrinx size showed increased peak flow velocities postoperatively (-19.4 ± 14.4 vs. -25.7 ± 17.3 p=0.08).Analysis of cerebrospinal fluid dynamics using 4D PC flow imaging is superior to a 2D single slice approach. It allows for a comprehensive characterization of complex flow phenomena, which might be missed by 2D imaging. CSF flow is considerably altered in patients with Chiari I malformation. Analysis of CSF flow pre- and postoperatively suggests a correlation between reduction in peak flow velocity and change in syrinx size.


Helicobacter bilis infection in experimental mice populations are well recognized, but the role of this bacterium in human diseases is less known. Limited data are available on virulence determinants of this species. In Helicobacter pylori, γ-glutamyltranspeptidase (γGT) contributes to the colonization of the gastric mucosa and to the pathogenesis of peptic ulcer. The role of γGT in H. bilis infections remains unknown. The annotated genome sequence of H. bilis revealed two putative ggt genes and our aim was to characterize these H. bilis γGT paralogues. We performed a phylogenetic analysis to understand the evolution of Helicobacter γGTs and to predict functional activities of these two genes. In addition, both copies of H. bilis γGTs were expressed as recombinant proteins and their biochemical characteristics were analysed. Functional complementation of Esherichia coli deficient in γGT activity and deletion of γGT in H. bilis were performed. Finally, the inhibitory effect of T-cell and gastric cell proliferation by H. bilis γGT was assessed. Our results indicated that one gene is responsible for γGT activity, while the other showed no γGT activity due to lack of autoprocessing. Although both H. bilis and H. pylori γGTs exhibited a similar affinity to L-Glutamine and γ-Glutamyl-p-nitroanilide, the H. bilis γGT was significantly less active. Nevertheless, H. bilis γGT inhibited T-cell proliferation at a similar level to that observed for H. pylori. Finally, we showed a similar suppressive influence of both H. bilis and H. pylori γGTs on AGS cell proliferation mediated by an apoptosis-independent mechanism. Our data suggest a conserved function of γGT in the Helicobacter genus. Since γGT is present only in a few enterohepatic Helicobacter species, its expression appears not to be essential for colonization of the lower gastrointestinal tract, but it could provide metabolic advantages in colonization capability of different niches.The confounding consequences of  However, among the 20 validly published enterohepatic Helicobacter species (EHS), only H. aurati, H. bilis, H. canis, H. muridarum and H. trogontum express this enzyme H. pylori, γGT represents an important virulence factor, playing a role in colonization in vivo and in vitroH. pylori–mediated H2O2 generation Helicobacter genus H bilis, an endemic EHS in most experimental mouse colonies, induces disease in susceptible animals and may substantially confound interpretations of some research studies H. bilis infection in inbred H. bilis has been used experimentally to induce inflammatory bowel disease (IBD) in mdr−/− and IL-10−/− knock-out mice H. bilis is able to infect and cause diseases in different animal hosts, showing one of the broadest host spectrums in the Helicobacter genus H. bilis has been also isolated from human patients with chronic diarrhoea H. bilisH. bilis γGT (Hb-γGT).γGT is found in all gastric Helicobacter spp., the genome sequence of H. bilis ATCC 43879 revealed the presence of two ggt copies. In this study, we used a phylogenetic and a functional approach to analyse both H. bilis γGT paralogues. Although both genes were phylogenetically related to other Helicobacter γGTs, analysis of the recombinant proteins, western blot using specific antibodies, complementation of E. coli Δggt and mutation in H. bilis clearly showed that only one gene was responsible for H. bilis γGT activity. The γGT of H. bilis exhibited a similar affinity as H. pylori to γ-Glutamyl-p-nitroanilide and to L-Glutamine; however, it was significantly less active. Nevertheless, H. bilis γGT inhibited T-cell and gastric cell proliferation at a similar level to that observed for H. pylori γGT. The inhibition observed was mediated by an apoptosis-independent mechanism and suggested a conserved function of γGT in Helicobacter genus.In contrast to observations in gastric ggt paralogues HRAG_01341 and HRAG_01828 of the human-associated H. bilis strain ATCC 43879 genome (NCBI ACDN0000000), were named bgh1 (bilis ggtH. homologue 1) and bgh2 (bilis ggtH. homologue 2), respectively. Nucleotide and amino acid similarity between the two H. bilis γGT paralogues, and between each homologue and H. pylori γGT  were analysed by pairwise global alignment. The two H. bilis γGT paralogues showed 65.8% and 62.0% of nucleotide and amino acid identity, respectively. Moreover, in comparison with Hp-γGT, Bgh2 showed 65.2% of amino acid identity, while Bgh1 showed 53.4%. To detect amino acid positions potentially involved in functional change, conserved sites of both Bgh1 and Bgh2 genes were evaluated on the basis of γGT structural data available for H. pyloriE. coliHelicobacter γGT sequences, other bacterial γGTs and class IV cephalosporin acylase (CA) of Pseudomonas sp. strain SE82, was constructed .The structed . In Bgh2H. bilis γGT paralogues, the sequences were submitted to SignalP 3.0, using both Neural networks and Hidden Markov Models (HMM) methods sec signal peptide sequence with a potential cleavage site between positions 30 and 31 (VFA-AS). The same prediction was obtained for all γGTs of Helicobacter spp. and C. jejuni. No twin-arginine signal peptide cleavage site was detected in any γGTs of Helicobacter spp. and C. jejuni.To predict the localization of both Pseudomonas spp. CA were analysed  was evaluated by sliding window analysis (window size 50 bp) using SWAKK bgh1 and bgh2 pairs of H. trogontum and H. bilis genomospecies FL56 (data not shown).To examine the phylogenetic relationships among d motifs . A fragm. jejuni . Phylogeuridarum  clusterebgh1 and bgh2 in H. bilis, 33 H. bilis strains from our collection H. bilis type strain ATCC 51630T, H. bilis ATCC 49314 and H. bilis ATCC 49320, were subjected to a specific BclI RFLP-PCR for bgh1 and a specific PCR for bgh2. All strains were positive for the presence of both genes. Furthermore, bgh1 orthologues of H. bilis type strain ATCC 51630T and three canine strains To determine the frequency of bgh1 and bgh2 was evaluated in H. bilis CCUG 23435 at different time points. Bacterial growth was monitored by measuring OD600 up to 24 h, corresponding approximately to the end of the exponential phase. At 8, 12 and 24 h both genes were transcribed (data not shown).The transcription of both H. bilis γGT paralogues, we expressed recombinant His-tagged Bgh1 and Bgh2 proteins in E. coli. Bgh2 was expressed without the signal peptide, while for Bgh1 the entire sequence was used. Recombinant expression of Bgh2 resulted in a soluble protein that was directly purified by Ni-affinity chromatography. By contrast, Bgh1 was expressed as an insoluble protein of ∼70 KDa and was purified after refolding. The purity of the proteins on SDS-PAGE was >90% . The western blot performed on H. bilis whole lysate using the antiserum against Bgh1 clearly showed a single band without indication of autoprocessing  were introduced in E. coli CY128 E. coli DH5α grown in LB overnight at 25°C was used as positive control in γGT assay. Only Bgh2 was found to successfully complement E. coli CY128, while Bgh1 was unable to restore γGT activity (data not shown). The induction of pMRg3 in E. coli CY128 resulted in a clear inhibition of growth. This finding was consistent with formation of inclusion bodies, as a consequence of the overexpression of Bgh1. However, when the protein was expressed overnight at a lower temperature (15°C) with low amount of inducer (0.002% instead of 0.2%), there was no inhibition of the growth rate of E. coli, suggesting a low accumulation of insoluble proteins. The successful expression of Bgh1 under improved conditions was confirmed by SDS-PAGE (data not shown). Nevertheless, Bgh1 was not able to complement E. coli Δggt, while Bgh2, expressed under the same conditions, restored γGT activity in the mutant strain.To evaluate the capability of H. bilis, a mutant of bgh2 was constructed in CCUG 23435 strain. The bgh2 gene was disrupted by insertion of a chloramphenicol cassette between positions 1182 and 1246, in order to delete the threonine-threonine catalytic dyad. The successful insertion of the choramphenicol resistance cassette and the deletion of bgh2 were confirmed by PCR. RT-PCR and western blot analysis showed the expression of bgh1 in both wild-type and mutant strains (data not shown), indicating that the replacement of bgh2 had no effect on the expression of the bgh1 paralogue gene. Since complementation approaches are currently unavailable for H. bilis, another independent Δbgh2 mutant was used as a control for secondary mutations. Neither of the H. bilis mutants were able to hydrolyse the substrate analogue gGpNA. Moreover, in the supernatant of H. bilis wild-type γGT activity was determined with a gGpNA turnover of 0.48 µM/min at a concentration of 50 µg/mL of total protein. No activity was detected in the supernatant of the mutant strain MR9 . The mutants grew normally in vitro, as described for H. pylori γGT-deficient strains H. pylori and H. bilis in vitro.To confirm that Bgh2 is responsible of the γGT activity in rain MR9 . These rM was determined by titration of the gGpNA concentrations between 1 and 1000 µM. The processed Hb-γGT had a KM of 7.7±1.2 µM and a kcat of 1.12±0.03•102/sec. Compared with KM and kcat values calculated for Hp-γGT (KM = 9.8±1.5 µM and kcat = 17.7±0.5•102/sec), the apparent KM value for Hb-γGT was 20% lower, while the kcat value was >10-fold reduced  were measured. The initial experiments were performed at pH 8.0. The K reduced . The pH  reduced . The pH ggt strains confirmed the results obtained with the recombinant proteins. The supernatants of H. bilis wild type reduced T-cell proliferation up to 60%, while the supernatant of Δggt strain had no significant inhibitory activity. When Jurkat cells were infected with acivicin pre-treated supernatant of H. bilis wild type, no significant reduction in proliferation of T-cells was observed, indicating that only Hb-γGT is responsible for the inhibition of T-cell proliferation in our system  compared to that able to inhibit the proliferation of human T-cells . No statThe ability of recombinant Hp-γGT and Hb-γGT to induce apoptosis in AGS cells was evaluated using AnnexinV-Propidium Iodide double staining and FACS analysis. After 24 h of incubation, concentrations of up to 5 µg/mL of both recombinant proteins did not induce apoptosis in AGS cells . The aveHelicobacter γGT genes, identified in the shotgun genome sequence of the human strain H. bilis ATCC 43879 on the basis of sequence homology, were subjected to functional analysis to confirm their annotations. Although the two genes bgh1 and bgh2 (bilis ggtH. homologue 1 and 2) showed significant homology and both are classified as T03.01 peptidases in MEROPS, the evaluation of conserved amino acid residues indicated a marked difference between these two paralogues, especially in the substrate-binding pocket. In fact, all critical functional residues are present in Bgh2, as well as in all the other Helicobacter γGTs, but not in Bgh1. The amino acid substitutions observed in Bgh1 could potentially affect the enzyme maturation, catalytic activity and substrate specificity, suggesting different biochemical properties. Moreover, the absence of a signal peptide in Bgh1, in contrast to observations for Bgh2 and for the other Helicobacter γGTs, suggests a different sub-cellular localization of this protein.Two Helicobacter γGTs in relation with T03 proteases of other bacterial species. The Minimum Evolution tree clearly showed that both H. bilis T03.01 peptidase paralogues were phylogenetically associated with other ε-proteobacteria γGTs. Nevertheless, Bgh1 formed an independent branch, indicating a different evolution. Moreover, among Helicobacter species, only H. trogontum showed two distinct γGT paralogues, as observed in H. bilis. In addition, the data suggested that both genes are not restricted to H. bilis ATCC 43879, but are common features among H. bilis genomospecies and universally shared by all members of the taxon.We first evaluated the phylogeny of H. bilis in the same branch as other enterohepatic Helicobacter spp. H. bilis and related species  from others enterohepatic Helicobacter species. On the basis of our phylogenetic analysis we can hypothesise that differing phylogeny of the subunits reflects a possible divergence of substrate specificity between the γGTs of H. bilis and related species and the other Helicobacter or Campylobacter γGTs. More studies are needed to verify this hypothesis.A more detailed phylogenetic analysis further revealed that the two subunits of γGTs have followed different evolutionary paths. The tree based on the C-terminal part resembled the topology of the tree of 16S rRNA and other housekeeping genes and includes bgh1 indicated that the paralogues potentially evolved by gene duplication from a common ancestral sequence probably before the speciation event of H. bilis and related species. The hypothesis of gene duplication is well supported also by the low Ka∶Ks ratio between the two paralogues , indicating that the two genes are under strong purifying selection In the trees based on the sequences of both subunits, the position of the clade including the orthologues of H. bilis T03.01 peptidases paralogues. To compare their biochemical properties, both genes were overexpressed in E. coli and the recombinant proteins were purified by affinity chromatography. The overexpression of Bgh1 resulted in formation of inclusion bodies due to the accumulation of an insoluble protein of ∼70 KDa. After solubilisation and refolding, recombinant Bgh1 showed no significant maturation into two subunits and, subsequently, no γGT activity. In contrast, recombinant Bgh2 was expressed in soluble form as a pro-enzyme of ∼60 KDa, which undergoes autocatalytic processing to generate two subunits of ∼40 and ∼20 KDa and showed γGT activity. No clear molecular evidence explains the absence of an autocatalytic process for Bgh1 since all of the amino acid residues critical for Hp-γGT in vitro. However, the CD spectrum of purified Bgh1 showed that the protein has a defined set of secondary structures indicating a non-random folding of the recombinant protein. Moreover, Bgh1 was unable to restore γGT activity after complementation of E. coli Δggt mutant. Furthermore, the H. bilis strain in which bgh2 was disrupted but still able to transcribe bgh1 resulted in deficient γGT activity. This data clearly indicate bgh1 does not encode a functional H. bilis γGT. As reported previously, the single amino acid substitution D433N converts Ec-γGT to a class IV cephalosporin acylase H. bilis CCUG 23435, which expresses both γGT paralogues, as previously described for E. coliH. bilis did not show any cephalosporin acylase activity (data not shown), indicating that glutaryl-7-aminocephalosporanic acid is unlikely to be a substrate for Bgh1. To elucidate whether the lack of enzyme activity of Bgh1 is caused by the absence of autocatalytic maturation, western blot analysis on H. bilis whole lysate using specific antisera for both γGT paralogues were performed. The results showed that H. bilis expresses Bgh1 which does not undergo autocatalytic processing, confirming the results obtained with the recombinant protein. However, further studies are needed to determinate potential substrates for Bgh1 and to investigate its role in the metabolism of H. bilis.Both sequence and phylogenetic analyses reveal clear differences between the two H. bilis whole lysate, complementation of E. coli Δggt and deletion of the gene in H. bilis CCUG 23435 clearly showed that only bgh2 encodes the functional H. bilis γGT. Hb-γGT exhibited similar affinity to the substrate analogue gGpNA at pH 8.0 compared with that of Hp-γGT; however, it was significantly less proficient, having more than 10-fold less kcat. Moreover, in contrast to observations for Hp-γGT H. pylori and other bacterial γGTs H. bilis γGT lost its ability to interfere the host redox system. However, the enzyme enables the bacterial cells to use extracellular glutamine as the source of glutamate. This hypothesis is supported by the presence in the H. bilis genome of a putative glutamate transporter gene  showing ∼60% of amino acid identity with H. pylori GltS homologue gltS as well as ggt genes are missing in the EHS H. hepaticus genomes H. hepaticus and H. bilis are urease-positive Helicobacter spp., commensal bacterial species and/or opportunistic pathogens of mice, sharing the same niches in the lower gastrointestinal tract and causing similar diseases H. bilis, but not H. hepaticus, was also isolated from the stomach of different hosts Helicobacter spp. does not appear to be essential for colonization of the lower gastrointestinal tract of mice, but it could provide metabolic advantages in colonization capability of other tissues or in the adaptation of different hosts.Analysis of the recombinant protein, western blot on H. pylori and the host by inhibiting T-cell proliferation H. pylori–mediated H2O2 generation H. bilis γGT inhibited T-cell proliferation similar to what was observed for H. pyloriH. bilis. Our data demonstrate that this activity is not limited to H. pylori but is conserved among the genus, confirming the potential immune suppressive role of secreted Helicobacter γGTs. The conserved function of this enzyme among Helicobacter spp. with different niches and host specificity opens a new scenario on the role of γGT in the pathogenesis of Helicobacter associated diseases.Although the importance of γGT for physiology and pathobiology of mammalian tissues has been known for many years, its function in prokaryotic metabolism and its role in interaction with the host have been studied for only a few bacterial species We detected AGS cell proliferation inhibition after treatment with both recombinant γGTs, as observed for T-cells, but only when a high concentration was used (2.5 µg/mL). However, in contrast to what was previously described Helicobacter strains were routinely grown on HP medium  containing 5% bovine blood at 37°C in a microaerobic atmosphere  . E. coli strains were cultivated on Luria-Bertani (LB) agar or broth supplemented with 100 mg/L of ampicillin, 15 mg/L of kanamycin or 20 mg/L of chloramphenicol when needed. Jurkat T-cells  were cultured in RPMI 1640  with 10% of foetal bovine serum . AGS cells  were grown in Dulbecco's Modified Eagle's growth medium  supplemented with 10% FBS and 200 U/mL of penicillin and 0.2 mg/mL of streptomycin . Cell lines were incubated at 37°C with 5% CO2. Bacterial DNA extraction was performed as described earlier The bacterial strains and plasmids used in this study are listed in Helicobacter and C. jejuni γGTs. Standard PCR was done in a 50 µL reaction mixture containing 50 ng of DNA template, 25 pmol of each primer and 1.25 U of DyNAzyme™ . The 5′ consensus clamp region contains the sequencing primers used for direct sequencing of the PCR products. If secondary peaks or multiple sequences were present, the PCR products were inserted into pGEM®-T and sequenced on both strands using M13 primers. The area corresponding to nucleotides 702–1569 (amino acids 234–523) of bgh1 was used to design specific primers for RFLP-PCR protocol. The PCR was done in a 50 µL reaction mixture, as described above, and then the products were digested by BclI . BclI specifically cuts the PCR fragment in two pieces of 606 and 260 bp. EMBL accession numbers are listed in Consensus DEgenerate Hybrid Oligonucleotide Primers iCODEHOP  were desggt sequences of Helicobacter spp and C. jejuni was built in MEGA4 on the basis of the amino acid sequence alignment. Maximum likelihood (ML) trees were constructed using PHYML http://www.hiv.lanl.gov/). Trees were visualized by iTOL Phylogenetic analyses of γGT amino acid sequences were conducted in MEGA4 bgh1 and bgh2 genes using Phusion® High-Fidelity DNA Polymerase (Finnzymes). Bgh1 and Bgh2 were expressed as 6×His-tagged proteins by inserting the genes respectively in NdeI and HindIII or NheI and HindIII restriction sites of pet28b+ vector . The 6×His-tag was fused in the N-terminal part of the proteins. The resulting expression constructs pMRg1 and pMRg2 were sequenced and confirmed to be identical to bgh1 and bgh2, respectively. Both expression constructs were used to transform E. coli Rosetta 2 (Novagen®). Expression of recombinant protein was induced with 0.5 mM IPTG for 4 h at 27°C. After centrifugation, pellets were solubilized in ice-cold binding buffer  containing protease inhibitors and then lysed by subsequent sonication (5 times for 30 s) on ice. The supernatant was loaded on the HisTrap™ column  at 1 mL/min at 4°C, and bound protein was eluted with an imidazole-gradient (0–100 mM imidazole) using the elution buffer . Eluates were collected and then tested for purity by SDS-PAGE. Since purification of a soluble form of Bgh1 was not possible, the sonicated cells of E. coli (4 times for 1 min) were homogenized, solubilized in 6 M Gua-HCl, 1 mM DTT, 10% Glucose pH 8.5 and stirred for 1 h at 4°C. Following centrifugation, the supernatant containing the unfolded protein was loaded on a HisTrapHP™ column  at 4°C. The bound protein was refolded with a gradient of 100 mM HEPES, 5% Glucose, 250 mM NaCl, 10 mM MgCl2, 1 mM DTT pH 8.5 (0–100% of HEPES buffer). After the refolding, the column was washed extensively with 5 column volumes of HEPES buffer, and bound protein was eluted with an imidazole-gradient (0–1000 mM) using HEPES buffer with 1000 mM imidazole. Eluate was collected and then tested for purity by SDS-PAGE. For further purification, fractions with the recombinant proteins from Ni-Sepharose affinity chromatography were pooled, dialysed overnight against 100 nM HEPES, 140 mM NaCl, 2.5% Sucrose pH 7.0, at 4°C and processed to the second and final purification step. The dialysed samples were loaded on a Superdex™75 column  and equilibrated with 100 nM HEPES, 140 mM NaCl, 2.5% Sucrose pH 7.0. All fractions collected were analysed by SDS-PAGE for the presence of recombinant proteins. Fractions containing the protein were pooled, aliquoted and stored at −80°C until further use.Primers pairs were designed to amplify complete sequences of 2PO4 pH 7.5, 50 mM K2SO4. All CD measurements were corrected by subtracting the buffer spectrum. The following parameters were used: data pitch, 0.1 nm; continuous scanning mode at 100 nm/min; response, 4 sec; band width, 1 nm; accumulation, 16. Smoothing was made by the method Means-Movement. Data were collected using Spectra manager v1.54.03 (Jasco).To estimate the secondary structure of Bgh1 a CD spectrum of the recombinant protein was calculated. The measurements were performed on a JASCO J-810 spectropolarimeter equipped  using a 0.2-mm-pathlength quartz-glass cuvette 1-mm. CD spectra were recorded with 50 µl of the sample at final concentration of 15 µM in 20 mM KHBalb/c mice (3 mice per group) were immunized i.p. with 30 µg of antigen and 10 µg Cholera toxin (CT) (Sigma-Aldrich) as adjuvant at days 0, 7 and 14 (control group with CT only). Sera were taken 21 days after the last immunization and stored at −20°C. Animal experiments were performed according to the guidelines of the Bavarian Ministry of Animal Wealth Fare. For evaluation of Bgh-specificity, ELISA plates were coated with 2 µg/mL recombinant Bgh1 and Bgh2 proteins in PBS pH 7.4 overnight at 4°C. After washing (4×0.01% Tween-20 in PBS) and blocking (1% BSA in PBS), different dilutions of sera (in blocking buffer) were incubated for 1 h at 37°C. HRP-conjugated anti-mouse IgG antibody (1∶3000)  was incubated (1 h at 37°C) and detected with ELISA Opt EIATM  according to the manufacturer's instructions.H. bilis overnight culture was resuspended in 1 mL BHI (BD). Following lysis (sonication 4×30 sec), the protein-concentration in cleared lysates was determined by BCA-Assay and adjusted to 1 mg/mL. Per lane 5 µg of total protein were loaded. After blocking with 5% BSA in Tris-buffered saline (TBS), the Whatman Protran® Nitrocellulose membranes  were incubated for 1 h at RT with the sera of the immunized mice (1∶800 diluted in TBS). Following washing, HRP-conjugated secondary anti-mouse IgG antibody (Promega) (diluted 1∶3000 in TBS) was added and detected by the acridan-based ECL-system (Thermo Fisher Scientific).For the western blot, bgh1 and bgh2 were amplified as described above and were inserted between KpnI and HindIII restriction sites of pBAD24 expression vector E. coli CY128 Δggt. The recombinant strains were grown in LB medium with ampicillin at 37°C to OD600≈0.4, and the expression of the corresponding gene was induced with 0.2% L-arabinose for 4 h at 25°C. The basal expression of pMRg3 and pMRg5 vectors was inhibited by using 0.2% of glucose instead of L-arabinose. Expressions were verified by SDS-PAGE electrophoresis and Coomassie Blue stain. A total of 109 cells were harvested by centrifugation and stored at −70°C before the γGT assay.bgh2 was performed in H. bilis CCUG 23435. Deletion was introduced by allelic exchange using vector pUC119 in which ∼1100 bp of the 5′ –end and ∼500 bp of the 3′ –end of the target gene and the chloramphenicol resistance gene from pUOA14 E. coli DH5α and used as a suicide plasmid in H. bilis CCUG 23435. H. bilis mutant was obtained by electroporation as described for H. hepaticusH. bilis MR9 (bgh2::cam), was selected on an HP medium containing 5% bovine blood supplemented with chloramphenicol (20 mg/mL). The site of recombination was verified by PCR.Chromosomal inactivation of bgh1 and bgh2 during growth, H. bilis wild-type CCUG 23435 was cultivated in Brain Heart Infusion  containing 20% FBS and Vitox supplement  at 37°C microaerobically by continuous shaking at 150 rpm. OD600 was measured at 8, 16 and 24 h and the same amount of cells was treated with RNAprotect Bacteria Reagent . RNA was extracted using RNeasy Mini Kit (Qiagen) and treated with TURBO DNase™ . Moreover, H. bilis CCUG 23435 and the mutant MR9 were cultivated in YT×2 medium  containing 10% FBS at 37°C in a microaerobic atmosphere. After 24 h RNA was extracted as described above. cDNAs were synthesized from 1 µg of total RNA using SuperScript® III Reverse Transcriptase (Invitrogen) using random hexamers (Finnzymes). RT-PCR reactions were carried out in a total volume of 50 µL as described above, using 1 µL of cDNA and 1 µL of RNA as negative control and 20 pmol of specific primers for bgh1 and bgh2. Thermocycling conditions were 94°C for 2 min followed 40 cycles of 94°C for 45 s, 58°C for 45 s and 72°C for 1 min. The PCR products were sequenced using the same primers.To analyse the expression of both H. bilis γGT on T-cell proliferation, Jurkat cells were seeded at 5×103 per well in a complete culture medium in 96-well microtiter plates and a variable amount of protein (0.001 to 10 µg/mL) or sterile-filtrated culture supernatant  was added in a total volume of 100 µl. As a control for the inhibition of T-cell proliferation, the recombinant γGT from H. pylori 26695 (HP-γGT) was used. The HP-γGT was produced as previously described For measurement of the effect of H. bilis and H. pylori γGT on proliferation of AGS cells, 5×103 cells per well were seeded in culture medium in a 96-well plate and treated with 0.5–5 µg/mL of protein in a total volume of 100 µl. Cells were incubated at 37°C for 48 h and analysed for proliferation as described above.For the measurement of the effect of recombinant H. pylori and H. bilis in DMEM with 10% of FBS. Untreated cells were used as a negative control and cells treated with 10 µM of Staurosporine  as a positive control for apoptosis. Cells were harvested with 0.25% Trypsin-EDTA (Invitrogen) and 2×105 cells were washed once with PBS. The pellets were resuspended in 10 µg/mL of AnnexinV-FITC antibody  in FACS buffer and incubated for 20 minutes in dark at room temperature. Cell suspensions were then diluted with FACS binding buffer and 10 µl of 50 µg/mL of Propidium Iodide  was added before acquiring samples. Cell debris was excluded by scatter gating (forward vs. side scatter). Approximately 50,000 events were collected per sample using CyAn ADP FACS cytometer  and data was analysed with FloJo software . The percentage of apoptotic cells is expressed as the double positive cell population (AnnexinV+PI) and pre-apoptotic cells stained only for AnnexinV.Apoptosis in gastric epithelial cells was determined by Fluorescence-activated cell sorting (FACS). AGS cells were seeded in a 6-well plate till they reached a confluence of 60–70%, and treated for 24 hours with 0.5–5 µg/mL of purified recombinant γGT proteins from 9 bacteria were suspended in 400 µL of reaction buffer containing 1 mM of L-γ-glutamyl-p-nitroanilide (gGpNA) as described previously For determination of γGT activity, ∼10Figure S1CD spectrum and photomultiplier voltage for recombinant Bgh1. (A) CD spectrum for recombinant Bgh1 (black line), the predicted spectrum for Bgh1 obtained by K2D3 (red line) and lysozyme (blue line). The spectrum for lysozyme was taken from the Protein Circular Dichroism Data Bank (http://pcddb.cryst.bbk.ac.uk/). (B) Photomultiplier voltage of the spectrum for recombinant Bgh1.(TIF)Click here for additional data file.Figure S2Rate of gGpNA turnover of culture supernatant  of Helicobacter bilis wild-type CCUG 23435 (WT) and H. bilis Δggt MR9 (Δggt). The white bars show the results for untreated culture supernatants, while the black bars show the results after treatment of culture supernatants with 50 µM of Acivicin.(TIF)Click here for additional data file.Figure S3γGT-activity competition with glutathione, L-/D-Glutamine and L-Glutamic Acid. Error bars in the graphs were calculated as SEM. The analysis was performed using Prism4 v4.03 (GraphPad Software). Inhibitory effect with various amounts of substrate analogues on (A) Helicobacter bilis γGT (Hb-γGT) and (B) Helicobacter pylori γGT (Hp-γGT).(TIF)Click here for additional data file.Figure S4Apoptosis analysis of AGS cells after 24 hour treatment with γGT recombinant proteins. AnnexinV and PI staining of AGS cells analysed by Flow cytometer treated with 0.5, 0.25 and 5 µg/mL of recombinant proteins from Helicobacter bilis (Hb-γGT), and Helicobacter pylori γGT (Hp-γGT), compared to untreated cells and cells treated with staurosporin.(TIF)Click here for additional data file.


In sinoatrial-node cells (SANC) the SR generates periodic local, subsarcolemmal Ca2+ releases (LCRs) that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na+-Ca2+ exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP).Mitochondria dynamically buffer cytosolic Ca2+ (Ca2+m), cytosolic Ca2+ (Ca2+c)-SR-Ca2+ crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.To determine if mitochondrial Ca2+ influx into (Ru360) or Ca2+ efflux from (CGP-37157) decreased [Ca2+]m to 80±8% control or increased [Ca2+]m to 119±7% control, respectively. Concurrent with inhibition of mitochondrial Ca2+ influx or efflux, the SR Ca2+ load, and LCR size, duration, amplitude and period  significantly increased or decreased, respectively. Changes in total ensemble LCR Ca2+ signal were highly correlated with the change in the SR Ca2+ load (r2 = 0.97). Changes in the spontaneous AP cycle length  in response to changes in [Ca2+]m were predicted by concurrent changes in LCR period (r2 = 0.84).Inhibition of mitochondrial Ca2+m flux translates into a change in the AP firing rate by effecting changes in Ca2+c and SR Ca2+ loading, which affects the characteristics of spontaneous SR Ca2+ release.A change in SANC Ca Unlike VM, even in the absence of β-adrenergic stimulation in SANC, high levels of basal cAMP and cAMP-mediated protein kinase A-dependent (PKA) and Ca2+/calmodulin-dependent protein kinase II phosphorylation of Ca2+ cycling-proteins of both clocks enables the SR (Ca2+ clock) to generate spontaneous rhythmic local, subsarcolemmal Ca2+releases (LCRs) via SR ryanodine receptors. LCRs activate an inward Na+-Ca2+ exchange current that accelerates the rate of diastolic depolarization, prompting the membrane clock to generate the next action potential (AP).Sinoatrial nodal cells (SANC) initiate each heart beat by generating spontaneous APs that emanate from the sinoatrial node. Numerous studies over the last decade indicate a crucial role for SR cycling in SANC normal automaticity, giving rise to a 2+ flux into and out of mitochondria in SANC will directly affect SR Ca2+ loading, and thus indirectly affect SR Ca2+ release, and therefore that a change in mitochondrial Ca2+ (Ca2+m) cycling will modulate the normal automaticity of the coupled-clock system.We hypothesized that changes in Ca2+ cycling. But, this method can not be explored in SANC, which fire spontaneous AP’s. Therefore, to test our hypothesis that mitochondrial-SR Ca2+ crosstalk exists and links to SANC AP firing, we measured [Ca2+]c, [Ca2+]m, SR Ca2+ load and Ca2+ releases, and the spontaneous AP firing rate in intact, single, isolated SANC in response to specific inhibitors of Ca2+ flux into (Ru360) and Ca2+ flux from (CGP-37157) mitochondria.Changing the external electrical stimulation rate in VM is a convenient approach to induce changes in cross-talk between mitochondrial and SR Ca2+m was indexed by selective Mn2+ quenching of the cytosolic fluorescence of the Ca2+ probe, Indo-1 2 to Indo-1-AM loaded SANC selectively quenched the cytosolic Indo-1 fluorescence ratio 410/490 in a time dependent manner (11±2 min), as evidenced by the disappearance of the AP-induced cytosolic Ca2+ transient . Saponin (25 µg/ml) reduced the fluorescence to 64±5% (n = 5), i.e. to the same level as permeabilizing of sarcolemma with the lower concentration of digitonin. Thus, when the SANC plasma membrane was selectively permeablized, the Indo fluorescence decreased to the same level as it did in cells with an intact surface membrane in the presence of Mn2+. In contrast, a higher concentration of digitonin, which also permeabilizes mitochondrial membranes, decreased the fluorescence to 10±4% control . These results provide evidence that the Mn2+-resistant fluorescence of Indo-1 loaded cells is derived from Indo-1 within the mitochondrial compartment. Note, that in the time-control experiment protocol without Mn2+ application, Indo-1 fluorescence was only slightly quenched (−7±3%).To define the compartmental Indo-quenching, we quenched Indo-1 fluorescence using varying concentrations of digitonin: a low concentration of digitonin (5 µM) or saponin (25 µg/ml), which permeabilizes only the sarcolemma ; and a h2+]m. 2+]m to 80±8% of control (from 210±19 to 171±19 nM) of the Mn2+ quenched control; conversely, CGP-37157 (1 µM) increased [Ca2+]m to 119±7% of control (from 210±19 to 270±13 nM)  caused, over a 10-min period, a progressive increase in the spontaneous SANC AP firing rate to 111±1% control (from 2.7±0.25 to 3±0.3 Hz). Conversely, inhibition of Ca2+ efflux from mitochondria by CGP-37157 (1 µM) progressively reduced the spontaneous AP firing rate to 89±2% control (from 2.65±0.2 to 2.3±0.2 Hz) over a 10-min period. Note that both effects evolved and saturated with roughly the same time course. These drug effects were not reversible even after 10-min wash. The changes in other AP parameters are summarized in The main question of our study is whether a change in Ca2+ buffering by the mitochondria affects [Ca2+]c we loaded cells with Indo-1AM. Changes in spontaneous AP firing rate by inhibition of mitochondrial Ca2+ influx or efflux were determined in Indo-1/AM loaded cells (see representative examples in 2+c transients measured by Indo-1 and 2+c transients in the presence of Ru360 or CGP-37157. Inhibition of Ca2+m influx significantly increased peak systolic Ca2+ from 380±23 to 415±28 nM (p = 0.001) and decreased the 90% decay time of cytosolic [Ca2+] (T-90c) from 302±13 to 285±11 ms (p = 0.04). The diastolic Indo fluorescence (p = 0.6), time to peak Ca2+ , and 50% decay time of cytosolic Ca2+  were not significantly altered. Conversely, inhibition of Ca2+m efflux significantly decreased peak systolic [Ca2+] from 369±13 to 342±13 nM (p = 0.02) and increased T-90c from 301±14 to 325±12 ms (p = 0.04). The diastolic Indo fluorescence (p = 0.12), T-Pc (p = 0.3), and T-50c (p = 0.2) were not significantly altered. Therefore, the effects of inhibition of [Ca2+]m influx or efflux on [Ca2+]c in SANC are similar to their effects in prior studies in guinea pig ventricular cardiac myocytes To determine how Ca2+ cycling are determined, in part, by the SR Ca2+ load. Perturbations of mitochondrial Ca2+ flux might affect SR Ca2+ loading if these two organelles were located substantially close to each other. To determine the proximity of mitochondria to SR we employed dual immunolabeling to SANC with anti-SERCA2 antibody and Mitotracker orange to visualize the SR and mitochondrial membrane, respectively. SANC of varying size exhibit robust uniform SERCA2 immunolabeling  onto the cell following drug application to perturb Ca2+m flux. Representative examples and average data are presented in 2+ influx into mitochondria increased the SR Ca2+ load (assessed by change in amplitude of the Ca2+ transient induced by a brief rapid application of caffeine) by 17±5% of control ; conversely inhibition of Ca2+ efflux from mitochondria reduced the SR Ca2+ load by 13±2% of control  , these effects were not statistically significant , and the LCR duration from 35±0.8 to 41±0.8ms (p<0.001). In contrast to Ru360, CGP-37157 decreased the average LCR size from 4.3±0.1 to 2.6±0.1 µm (p<0.001), the average LCR amplitude from 1.22±0.02 to 1.14±0.01F/Fo (p = 0.02), and the average LCR duration from 35±0.5 to 29±0.6ms (p<0.001).Prior studies have shown that changes in SR Caexamples  show tha2+ of the LCR ensemble shifted to lower values in response to CGP-37157, and to higher values in response to Ru360 (2+ from 38±6 to 61±7 ms*µm*F/F0 (p<0.001) and CGP-37157 decreased it from 35±3 to 17±2 ms*µm*F/F0 (p = 0.003). The Ru360-induced increase, and the CGP-37157-induced decrease in the total LCR ensemble Ca2+ paralleled the effects of Ru360 and CGP-37157 on the amplitude of caffeine induced Ca2+ release into the cytosol  to an LCR onset . The relationship between the change in LCR period and AP cycle length by perturbations of Ca2+m flux is quantitatively strikingly similar to that which occurs in response to other interventions which affect the LCR period and AP cycle length of SANC m, the spontaneous AP firing rate, nor LCR characteristics  (5 µM) 2+m decreased  decreased the spontaneous AP firing rate by 15±3%  and reduced the SR Ca2+ load by 17±3% . This decrease in SR load by CPA is comparable to decreases in both spontaneous AP firing rate and SR Ca2+ load effected by CGP-37157. These results are consistent with our interpretation that the changes in AP cycle length due to manipulation of [Ca2+]m content are linked to changes in the SR Ca2+ load.To prove that a change in SR load is a necessary component in the feedback mechanism between m, under basal conditions, and when mitochondrial Ca2+ flux is inhibited (2+ influx by Ru360 decreased the maximum diastolic [Ca2+]m (from 89.8 to 60 nM) and the systolic [Ca2+]m (from 222.7 to 160 nM). Inhibition of mitochondrial Ca2+ efflux by CGP-37157, however, increased both diastolic [Ca2+]m (to 137.2 nM) and systolic [Ca2+]m (to 308 nM). Note, that manipulation of Ca2+m affected the cycle length without affecting If kinetics, which is also consistent with our experimental result. Moreover, the model predicts that sarcolemmal Na+-Ca2+ exchanger current kinetics are altered indirectly due to change in LCR characteristics  to measure the kinetics of the changes in intracellular Caing rate . Simulat2+ cycling proteins and surface membrane electrogenic molecules that regulates SANC normal automaticity includes SR-mitochondrial Ca2+ cross-talk.In summary, we conclude that the coupled clock system of intracellular Ca2+ cross talk, previously demonstrated in ventricular myocytes 2+ cycling into and out of mitochondria in SANC, effected by using Ru360 and CGP-37157, modulate basal coupled-clock system automaticity via an impact on Ca2+c, SR loading and indirectly on Ca2+ release characteristics: blocking mitochondrial Ca2+ efflux increases [Ca2+]m, decreases the SR load, decreases the amplitude of the AP triggered cytosolic Ca2+ transient and total Ca2+ released spontaneously by the LCR ensemble, and reduces the AP firing rate. Conversely, reducing mitochondrial Ca2+ influx decreases [Ca2+]m, increases SR Ca2+ loading, increases the amplitude of the AP triggered cytosolic Ca2+ transient and total Ca2+ released spontaneously by the LCR ensemble, and increases the AP firing spontaneous rate . External stimulation in SANC is not feasible, however, because SANC beat spontaneously, and it is challenging to over-drive the spontaneous SANC AP firing rate, while maintaining a normal rhythm . Beat-to-beat Ca2+m flux into or out of the mitochondria of SANC in the present study can be roughly estimated, however assuming that Ru360 or CGP-37157 reduces the influx or efflux by 30%, respectively , and assuming a linear time-course of drug effects to inhibit their targets. Given the experimentally determined time course of the drug-induced changes in [Ca2+]m, and the average number of spontaneous AP cycles over this time, a rough estimation of drug-induced changes in Ca2+ flux from or into the mitochondria per AP cycle is on the order of 0.5 nM/beat.Whether mitochondrial Ca2+m does not substantially affect [Ca2+]m itself, or affect other functions within SANC that are related to the AP firing rate. Because our major goal was to explore whether perturbations of mitochondrial Ca2+ cycling modulates the basal AP firing rate of SANC, neither cell permeabilization 2+]m that requires acute rupture of a membrane patch, often creating a small leak current, that notably affects the balance of ionic currents during diastolic depolarization and spontaneous AP frequency of SANC 2+ quench approach to assess [Ca2+]m in SANC because measurements of [Ca2+]m in VM by cell permeabilization and Mn2+ quench are in close agreement 2+ to selectively quench Ca2+c fluorescence, without causing marked changes in Ca2+m, is based on its slower quenching of the mitochondrial fluorescence 2+ that rapidly quenches cytosolic Ca2+but has minimal effects on sarcolemmal or mitochondrial Ca2+ ion transport be employed and 2) that during Ca2+c quenching the temperature be maintained at a near physiological level. We employed 50 µM Mn2+ to quench Ca2+c fluorescence, which at this concentration has no effect on our major functional endpoint, the spontaneous AP firing rate. Specifically, since we previously have shown 2+ and AP firing rate, the lack of a sustained effect of Mn2+ on the SANC spontaneous AP firing rate strongly suggests that there is no functionally significant inhibition of sarcolemmal Ca2+ channels (i.e. L-type channels) by 50 µM Mn2+. Another concern about the Mn2+ quench technique is that Mn2+ can inhibit oxidative phosphorylation. The lack of a sustained effect of Mn2+ on the basal spontaneous AP firing rate again suggests that Mn2+ does not perturb the matching of ATP supply to demand, since we have previously shown that this matching is closely linked to the AP firing rate The validity of our interpretations of the present results assumes that the method used to measure Ca2+m flux is another important issue that merits consideration in interpreting the present results. We employed Ru360 at concentration of 2 µM to selectively block Ca2+ influx into the mitochondria, i.e. a concentration similar to that used in prior studies in VM 2+ influx to the mitochondria and to decrease [Ca2+]m. In rat VM, in which Ca2+c is not substantially affected by changes in Ca2+m, this concentration of Ru360 does not affect SR Ca2+ uptake or L-type current 2+m is higher during ouabain application than during control conditions The selectivity of drugs employed to perturb Ca+-Ca2+ exchanger (IC50 = 0.5 µM) 2+]m, but has only minor effects on sarolemmal Na+-Ca2+ exchanger (IC50 = 13 µM), or Ca2+ uptake by SR (IC50 = 10 µM) or on ryanodine receptor open probability (IC50 = 9.5 µM) 2+ current is a crucial element of SANC automaticity 50 = 0.3 µM) We employed CGP-37157 at concentration of 1 µM because it selectively blocks the mitochondrial Na2+ flux affect the spontaneous AP firing rate of SANC via changes in SR Ca2+ loading and release. Furthermore, while our experimental method to measure [Ca2+]m does not permit evaluation of systolic and diastolic [Ca2+]m, our model predicts that under basal conditions both diastolic and systolic [Ca2+]m are lower than the diastolic and systolic [Ca2+]c, respectively. However, the systolic [Ca2+]m is higher than diastolic [Ca2+]c.Our novel numerical model simulations reproduce our experimental findings and validate our interpretation of these findings: changes in mitochondrial Ca2+ cycling into and out of the mitochondria acts as a dynamic buffer of cytosolic Ca2+, thus affecting Ca2+ availability for SR Ca2+ loading and release. When mitochondrial Ca2+ influx or Ca2+ efflux is selectively blocked, SR Ca2+ cycling and Ca2+ load are altered. This leads to changes in spontaneous AP triggered SR Ca2+ release that produce changes in both global AP induced cytosolic Ca2+ transients and to spontaneous, localized submembrane diastolic SR Ca2+ release. Therefore, under basal conditions mitochondrial Ca2+ fluxes modulate SR Ca2+ cycling via an effect on SR Ca2+ loading, and on this basis mitochondrial Ca2+ flux is indirectly linked to the SANC spontaneous AP firing rate.In summary, the present study shows, for the first time, that SANC mitochondria and SR are closely associated, i.e., that Ca2+ cycling were studied in Tyrode solution at 35±0.5°C, with the following composition (in mM): 140 NaCl, 5.4 KCl, 2 MgCl2, 5 HEPES, 1.8 CaCl2, and 5.5 Glucose, and was titrated to pH 7.4 with NaOH. Cells were imaged with an LSM-510 inverted confocal microscope using a 63×/1.4 N.A. oil immersion lens (Carl Zeiss). Linescan images , were recorded with a scan line oriented along the short axis of the cell to quantify the spontaneous AP induced contraction rate. Cell contraction measurements were recorded for 15 min under control conditions and following drug application for times specified. Spontaneous action potentials were measured via a perforated patch-clamp technique with 35 µM β-escin (Sigma) added to the pipette solution that contained (in mM): 120 K-gluconate, 5 NaCl, 5 MgATP, 5 NaATP, 5 HEPES and 20 KCl, pH 7.2 with KOH. Action potentials were recorded using an Axopatch-200 B patch-clamp amplifier . For both cell contraction and electrophysiological recordings cells from at least 5 rabbits were used.Single, spontaneously beating SANC were isolated from New Zealand White rabbit hearts as previously described 2+ was measured with calibrated Indo-1 fluorescence to assess cytosolic Ca2+ signals. SANC were placed in a chamber on the inverted fluorescence microscope stage (Zeiss IM-35) and were loaded with 14 µM Indo-1 AM (Molecular Probes) for 15-min at room temperature, and subsequently superfused with Tyrode’s solution for 20-min to remove excess indicator and allow full de-esterification as the bath temperature slowly was increased to 35±0.5°C. The apparatus to detect Indo-1 fluorescence is as described previously 2+]i was calculated according to the equation [Ca2+]i = βxKdx(R-Rmin)/(Rmax-R), using a Kd of 844 nM. The average Rmin , Rmax , and β  for the fluorescence system were determined by sequential exposure of SANC to a high potassium, zero- Ca2+ solution  containing metabolic inhibitors  (2), the same solution with 1 mM EGTA and 20 µM ionmycin (for Rmin). Measurements were taken when the fluorescence at both wavelengths reached stable values, and (3) high Ca2+ Tyrode’s solution (5 mM Ca2+ instead of EGTA) was used for determining Rmax. Average Rmax, Rmin and β were 1.9±0.16, 0.9±0.01, and 2.1±0.6, respectively (n = 10). The method limitation derived from: 1) a change in pH from the calibrated solution (pH = 7.4 in bath solution compare to 7.2 see above), 2) imprecision of Kd of Indo-1 and 3) partial quenching of mitochondrial Ca2+ by Mn2+ affect the calculated [Ca2+]m. For Ca2+ recordings cells from at least 5 rabbits were used.Intracellular Ca2+ cycling into and out of the cytosol was measured with Flou-4 AM (Molecular Probes) to assess AP triggered Ca2+ release, spontaneous LCRs, and caffeine induced SR Ca2+ release. SANC were loaded with 5 µM Flou-4 AM for 20 min at room temperature, and subsequently superfused with Tyrode’s solution at 35±0.5°C. The Ca2+ fluorescence was imaged by a LSM510 confocal microscope (see above) using a 40x/1.3 N.A. oil immersion lens. Cells were excited with the 488 nm laser line of an Ar laser, and fluorescence emission was collected with LP 505 nm, with the pinhole set to obtain no more than 5 µm optical slice (512×1 pixels at 14.9 pixel/µm and 2 ms/line). All images were recorded with a scan line oriented along the long axis of the cell, close to the sarcolemmal membrane, and processed with IDL software or Matlab.Ca2+ transient was expressed as a peak value (F) normalized to minimal fluorescence (F0). The amplitude of individual LCRs was also expressed as a peak value (F) normalized to minimal fluorescence (F0). LCR spatial size (FWHM) was indexed as the full width at half-maximum amplitude. LCR duration (FDHM) was characterized as the full duration at half-maximum amplitude. The number of LCRs was normalized per 100 µm of the linescan image and during a 1s time interval. The integral of Ca2+ of an individual LCR was estimated as follows: FWHMxFDHMx(F/F0-1)/2. The total Ca2+ released by LCR ensemble was calculated as the sum of the signal masses of diastolic LCRs that occurred between AP-induced Ca2+ transients. The SR Ca2+ content was estimated by the amplitude of the Ca2+ transient induced by a brief rapid application of caffeine  onto the cell by pressure-ejection via a nearby pipette, as described previously 2+ recordings cells from at least 5 rabbits were used.The AP induced Ca2+] ∼1 mM), 20 HEPES, 0.5 EGTA, 10 phosphocreatine, and 5 U/ml creatine phosphokinase; pH 7.2 with KOH.SANC were permeabilized with 5 or 25 µM digitonin at 35±0.5°C with solution containing (in mM): 100 K aspartate, 25 KCl, 10 NaCl, 3 MgATP, 0.8 MgCl2,  at 4°C overnight. This was followed by labeling with a Cy5-conjugated secondary antibody  incubation. The fluorescence was imaged by a LSM510 confocal microscope (see above) using a 40x/1.3 N.A oil immersion lens. The Cy5 and MT fluorophores were excited with 633 and 543 nm He-Ne lasers, respectively.SANC were incubated with 500 nM Mitotracker Orange , for 1 h at 37°C, fixed with 4% formaldehyde in phosphate buffer (PBS) and permeabilized with 1% Triton X-100/PBS. Nonspecific cross-reactivity was blocked by incubating the samples overnight in solution containing: 2% BSA/PBS, 5% donkey serum, 0.01% NaN3VO4. Total protein concentration was determined using a standard, commercially available protein quant kit . The accuracy of protein quantification was verified on the 4–12% Bis-Tris polyacrylamide SDS-PAGE gel (Invitrogen) labeled with the fluorescent Sypro Ruby gel stain (Invitrogen). The total integral of fluorescence intensities for each protein lane was calculated using ImageQuant5 software . 5-15 µg of proteins from each aliquot were mixed with the NuPage sample buffer and DTT , and resolved on 4–12% Bis-Tris polyacrylamide SDS-PAGE gel using standard MES/SDS running buffer. Proteins were transferred for 25 min to the activated PVDF membrane  using standard semidry transfer unit (Bio-Rad) in NuPage transfer buffer . PVDF membranes with transferred proteins were blocked overnight in 5% nonfat milk (Bio-Rad), and resuspended in TBS-T buffer .SANC suspensions were divided equally into 4 aliquots: the first aliquot was treated with 2 µM Ru360, the second with 1 µM CGP-37157, the third with 1 µM isoproterenol; and the forth was used as a control. Cells were incubated at 36°C for 10 min. After the treatment, samples were centrifuged and the pellets were frozen in liquid nitrogen. Pelleted cells were resuspended in RIPA lysis buffer  that also included a standard mammalian protease inhibitor cocktail (Sigma) and phosphatase inhibitors 1 mM NaF, 2 mM NaTo detect total phospholamban the first membrane was incubated over-night with a total anti-PLB monoclonal phospholamban antibody  and incubated 1 h at room temperature with goat anti-mouse conjugated antibodies . To detect phospholamban phosphorylation, the second and the third membranes were incubated over-night with anti-P-Ser-16 rabbit polyclonal antibody  or the anti- P-Thr-17 rabbit antibody , and incubated for 1 h at room temperature with polyclonal goat anti-rabbit secondary antibody . To detect actin, the membranes were incubated over-night with anti-actin goat polyclonal antibodies , and incubated for 1 h at room temperature with donkey anti-goat-IgG-HRP secondary antibody . Membranes were developed for 5 min using SuperSignal West Pico (or Dura) Chemiluminescent Substrate (Thermo Fisher Scientific) and visualized on the HyBlot CL autoradiography film using a series of sequential exposure times (Denville Scientific Inc.) to obtain an optimum signal intensity.+-Ca2+ exchanger inhibitor, and Ru360, an inhibitor of Ca2+ flux into the mitochondria, were purchased from EMD Chemicals; Isoproterenol, digitonin, saponin, ionmycin, cesium chloride, cyclopiazonic acid, caffeine and MnCl2 were purchased from Sigma.CGP-37157, a mitochondrial NaP<0.05 was taken to indicate statistical significance.Data are presented as mean±SEM. When the means of paired samples were to be compared, a paired t-test was employed. For multiple pharmacologic treatments, a linear mixed-effects model with Dunnett’s method to adjust p-values was used. This model accounts for repeated measurements on the same preparation while allowing testing for differences among pharmacological perturbations. Figure S1Validation of compartmentatal indo-quenching. (A–B) Low concentration of digitonin (5 µmol/L) does not permeabilize mitochondrial membrane, however high concentration of digitonin (25 µmol/L) does permeabilize mitochondrial membrane (C–D) visualized by 125 nmol/L tetramethylrhodamine methyl ester (TMRM).(TIF)Click here for additional data file.Figure S2Representative examples of Indo-1 fluorescence. Mn2+ (50 µmol/L) quenching of the cytosolic indo-1 fluorescence ratio 410/490 in the presence of Ru360 (upper panel) and CGP-37157 (lower panel).(TIF)Click here for additional data file.Figure S3Co-immunoelabeling of SERCA2 and part of the mitochondrial mass in SANC, visualized by anti-SERCA2 antibody and Mitotracker orange (MT) staining, respectively.(TIF)Click here for additional data file.Figure S4Western blots of phospholamban phosphorylation. (A) Representative examples of phospholamban phosphorylated at serine-16 site (PKA site) and total phospholamban in the basal state and following Ru360 (2 µmol/L), CGP-37157 (1 µmol/L) or isoproterenol (1 µmol/L). Actin is used as a protein loading control. (B) Fluorescent Sypro Ruby gel stain to validate the accuracy of protein loading. (C) Average phosphorylation ratio (n = 5) of phospholamban phosphorylated at serine-16 site to protein loading. *p<0.05 vs. control.(TIF)Click here for additional data file.Figure S5Average time-dependent change in the rate of AP induced contractions in the presence of CsCl and CGP-37157 (n = 7) or CsCl and Ru360 (n = 7).(TIF)Click here for additional data file.Figure S6Mitochondrial-SR numerical model. The extended coupled clock numerical model simulations of the effect of specific inhibition of Ca influx into or efflux from mitochondria in intact SANC on (A) Ca2+ in network SR, (B) Ca2+ in junctional SR, (C) Ca2+ release flux from the SR, and (D) Ca2+ in the sub membrane space.(TIF)Click here for additional data file.Table S1(DOC)Click here for additional data file.Table S2(DOC)Click here for additional data file.Text S1(DOC)Click here for additional data file.