The cellular and molecular mechanisms that mediate interactions between tumour cells and the surrounding bone stroma are to date largely undetermined in prostate cancer (PCa) progression. The purpose of this study was to evaluate the role of alpha 6 and beta 1 integrin subunits in mediating tumour-stromal interactions.in vitro assays we evaluated and compared 1. Monocultures of prostate metastatic PC3, bone stromal derived HS5 and prostate epithelial RWPE-1 cells and 2. Tumour-stromal co-cultures (PC3 + HS5) to ascertain changes in cellular phenotype, function and expression of metastatic markers.Utilising 3D In comparison to 3D monocultures of PC3 or HS5 cells, when cultured together, these cells displayed up-regulated invasive and proliferative qualities, along with altered expression of epithelial-to-mesenchymal and chemokine protein constituents implicated in metastatic dissemination. When co-cultured, HS5 cells were found to re-express N-Cadherin and chemokine receptor CXCR7. Alterations in N-Cadherin expression were found to be mediated by soluble factors secreted by PC3 tumour cells, while chemokine receptor re-expression was dependent on direct cell-cell interactions. We have also shown that integrins beta 1 and alpha 6 play an integral role in maintaining cell homeostasis and mediating expression of E-Cadherin, N-Cadherin and vimentin, in addition to chemokine receptor CXCR7.Collectively our results suggest that both PC3 and HS5 cells provide a “protective” and reciprocal milieu that promotes tumour growth. As such 3D co-cultures may serve as a more complex and valid biological model in the drug discovery pipeline. It is well established that the reciprocal interaction of tumour cells with local bone stroma at the metastatic site plays a critical role in metastatic dissemination in prostate cancer (PCa),2. To-daRecently the evaluation of tumour-stromal cell interactions has been undertaken using a 3D co-culture model. The importance of studying tumours in 3D has been previously described. Using tAn important first step in establishing communication between metastasising cancer cells and surrounding bone stromal cells is the exit of cancer cells from the vasculature once in the bone marrow. Studies suggest that the chemokine, CXCL12, plays a role in trafficking PCa cells to the bone. CXCL12 is expressed by stromal cells in target organs of PCa metastasis , but not in other tissues and its in vitro[in vivo[in vivo[Tumour cells localize to bone regions through integrin-mediated contacts with the extracellular matrix (ECM) and stromal cells. Heavily implicated in PCa bone metastases development and progression is the integrin β1 subunit-13. Exprin vitro and expeo[in vivo. Similaro[in vivo-16. Whilin vitro and in vivo models[in vitro and xenograft experiments primarily in breast and bladder cancer[in vivo prostatic models. To-date one study has confirmed the progressive nature of EMT in prostate cells during xenograft tumour formation and metastasis[Another important factor that allows PCa cells to infiltrate surrounding tissues and metastasise is the induction of EMT. The common feature of EMT is the loss of E-Cadherin and up-regulation of N-Cadherin and vimentin,18. Evido models-21 with r cancer,23. Fromr cancer,25. Surptastasis. Consisttastasis. Thus inin vitro models. In comparison to monocultures, cells in tumour-stromal co-cultures display alterations in morphology, invasion, proliferation and expression of chemokine and EMT markers. Moreover, mediation of EMT and chemokine markers by α6β1 integrins is altered in co-cultures when compared to their monocultured counterparts. Collectively, our results suggest that stromal cells are extremely plastic and together with metastatic cells can co-operate in a reciprocal manner to produce an emergent behaviour that is more malignant. These results may give further insight into the limitations of specific therapeutics that target tumour cells alone.Here we evaluate and compare both monocultures and co-cultures of metastatic PC3 cells and bone stromal derived HS5 cells using 3D To investigate differences in morphological characteristics and cell junction formation between HS5, PC3 and tumour-stromal co-cultures (HS5 + PC3 and HS5 + DU145), we used differential inference contrast (DIC) optics, immunostaining and imaging techniques to reconstruct 3D images from cells grown in 3D cultures. The described 3D model consists of cells grown as 3D spheroids following plating on a bed of extracellular matrix, Matrigel. In order to distinguish HS5, DU145 and PC3 cells in co-culture, we used a bone marrow stromal cell specific marker, STRO-1 to visuain vivo, a central Z-slice of PC3 cells stained for F-actin showed no evidence of polarisation or lumen formation within the centre of the cell mass , there was little difference in the overall phenotypic appearance of these cells Figure A’ when cIn the presence of α6 blocking antibodies, HS5 cells Figure B’ displaWhen cultured together, 3D tumour-stromal cultures displayed disorganised clusters of stellate structures Figure C, with aThese results suggest that β1 integrin can modulate cell-cell contacts and cell-ECM contacts, altering phenotypic morphology in monocultures that are reflective of an epithelial-like reversion. The degree of control exhibited by integrins, however, clearly differs between monocultures and co-cultures as evidenced by the lack of polarisation and acinar formation in HS5 cells in the presence of PC3 cells, suggestive of a more invasive phenotype.Using an Alamar Blue based proliferation assay conducted over a 9 day period, we were able to determine proliferation rates in 3D for both monocultures and tumour-stromal co-cultures.Consistent with previous findings, in compThe ability of cells to metastasise to distal organs is largely mediated by their ability to migrate and invade. Thus we next wanted to ascertain whether there were differences in invasive capacity between monocultures versus tumour-stromal co-cultures and whether α6 and/or β1 integrin may mediate this invasive behaviour. To investigate this we used transwell invasion assays in the presence or absence of α6 and/or β1 function blocking antibodies.In agreement with previous reports, tumour-Inhibition of β1 in HS5 cells saw no significant difference in invasive capacity compared with IgG treated controls Figure G. InhibiWe next wanted to ascertain the relative contribution of invading stromal and tumour cells in co-culture. To investigate this, transwell invasion assays in the presence or absence of α6 and/or β1 function blocking antibodies with FBS and laminin in the lower chamber wells were used. Following invasion, cells were fixed and each cell type was visualised via staining for STRO-1 and cell mask blue. Unlike their monoculture counterparts, when HS5 cells were in the presence of PC3 cells, their invasive capacity was found to equal that of PC3 cells with 52.3% of invaded cells being HS5 positive . Similarly, when co-cultured, HS5 and PC3 cells retained a distribution pattern consistent with a functional IF receptor , hepatoIn addition to soluble factors, there are a plethora of contact mediated variables that could account for the re-expression of CXCR7 in co-cultured HS5 cells. One possibility is the modulation of chemokine receptors through hypoxic conditions, which is known to induce cancer cell expression of c-Met, the bona fide receptor of HGF, and CXCR4, the signalling receptor of the chemokine CXCL12 (SDF1), and further stimulate cancer cell migration and dissemination,52.We report here that both α6 and β1 integrin subunits can influence expression rates of important EMT markers  and chemokine receptor CXCR7 in both monocultured and co-culture assays. Our results regarding integrin mediated changes in these proteins is summarised in Figure Of interest was the clear redistribution of N-Cadherin and vimentin in monocultured PC3 cells when treated with β1 inhibitors. The distribution patterns of these markers were indicative of a reduced junctional and IF protein, respectively. However, the degree to which E-Cadherin in these cells may then activate the Cadherin-catenin complex to mediate the metastatic phenotype needs further clarification. Previous studies have shown that with a decrease in junctional E-Cadherin protein, catenins become localized to the nucleus where they activate the transcription of proto-oncogenes, stimulating mitosis.Using 3D tumour-stromal co-cultures we have shown that the addition of bone derived stromal cells to metastatic PCa cells helps support tumour growth and protects PC3 cells from integrin mediated alterations associated with MET. Reciprocally, we have also found that the addition of PC3 cells results in significant up-regulation of invasive and proliferative behaviour in addition to re-expression of N-Cadherin and CXCR7 on HS5 cells. Further studies now need to evaluate the cross talk that occurs between these two compartments on a systematic, cellular and molecular basis and will likely lead to identification of new targets for therapy.2, 37°C) in cell cultured treated T75 Flasks . Media was replenished every 3 days. Once cells had reached 80-90% confluency, cells were replated (1/10) in T75 flasks. After 10-12 passages, cells were discarded.Cell lines were purchased from ATCC and were passaged for less than 4 weeks during any given assay performed for this paper. ATCC routinely use COI for interspecies identification and STR analysis (DNA profiling) for intra-species identification for all cell-lines. The PCa cell lines , Bone Stromal Cell-line (HS5) and the 3T3 fibroblast cell-line were maintained in RPMI-1640 (Sigma-Aldrich), supplemented with 10% fetal bovine serum  and the prostate epithelial cell-line RWPE-1 was maintained in Keratinocyte Serum Free Media  supplemented with 20 mg/mL bovine pituitary extract (BPE) and 0.2 ng/mL epidermal growth factor (EGF). All cells were propagated in standard cell culture conditions  per well and maintained in standard culture conditions. Media was carefully removed and replenished every 3 days. Cultures were maintained for up to 9 days.For miniaturised 3D cultures, 45 μl phenol red free (PRF) Matrigel™/culture medium (70%: BD Biosciences) was added to 96 well plates and polymerised at 37°C with 5% CO2 for 1 hr. Single cell cultures were then seeded at ~10000 cells per well while co-cultures containing HS5 and PC3 cells were plated at ~5000 cells each (1:1 ratio) per well and media was replenished every three days. After 3, 6 and 9 days in culture, 3D bulk cultures were extracted using Cell Recovery Solution (CRS: BD Biosciences) as per the manufacturer’s instructions. Cell pellets were then lysed and western blotting techniques were carried out.Protein extraction for western blotting was obtained from 3D Matrigel cultures grown in 12-well plates. For 3D cultures, 450 μl PRF Matrigel™/culture medium (70%: BD Biosciences) was added per well and allowed to polymerise at 37°C with 5% COIn order to block α6 or β1 integrin subunits, well established functional blocking antibodies were diluted (1.5 μg/mL) directly into the 3D matrix as follows: 1. α6: GoH3 (R&D Systems), 2. β1: P5B2 (R&D systems), 3. α6 and β1 and 4. IgG isotope controls (R&D Systems). Cells were then seeded and grown for 9 days in culture in miniaturised 96 well or a bulk 12 well plate format. Functional blocking antibodies and IgG isotope controls were replaced during media changes every 3 days at 1.5 μg/mL concentration. Miniaturised 3D cell cultures were then washed and fixed with 4% paraformaldehyde (PFA) and immunocytochemistry was undertaken. For inhibition assays carried out in a 12 well format, cells were extracted using CRS and western blotting techniques were undertaken.Protein was collected from cells at days 3, 6 and 9 from bulk 3D cultures. Treated cells were lysed in ice-cold RIPA buffer  containing protease inhibitors (Roche), incubated at 4°C for 30 mins prior to centrifugation at 14,100 g for 20 mins to pellet cell debris. The supernatants were then assayed for protein concentration using DC Protein Assay (Bio-Rad), and equal amounts of protein were loaded onto SDS-PAGE gels for electrophoresis. The protein was then transferred to Polyvinylidene Fluoride (PVDF) membranes in transfer buffer  for 30 mins using a Bio-Rad Turbo-Blot system. PVDF membranes were blocked using 5% non-fat milk powder for 1 hr, washed with TBST and primary antibodies were applied in blocking buffer as follows: mouse anti-E-Cadherin , anti-human integrin α6/CD49f , anti-human integrin β1/CD29 , goat anti-human vimentin , rabbit anti-CXCR7  and anti-human N-Cadherin  applied overnight (O/N) at 4°C. Membranes were then washed and HRP-conjugated secondary antibodies (Bio-Rad) applied for 1 hr at 4°C prior to washing and imaging on a Versa Doc (Bio-Rad) imaging station. Membranes were stripped and re-probed for β-actin in the case of vimentin, CXCR7 and CXCR4, whereas E-Cadherin, N-Cadherin and integrin α6, β1 membranes were directly probed for β-actin. Densitometric analysis was performed using Image Lab software and expressed as a fold change in relation to loading controls and normalised against β-actin.Miniaturised 3D cultures of PCa cells  and co-cultures (HS5 + PC3) grown in 384 well format were washed (3 × 5 mins PBS) and fixed with 4% PFA for 20 mins. For immunofluorescence labelling, cells were washed (3 × 5 mins PBS), permeabilised and blocked O/N with 2% BSA, 0.1% Triton-X, 0.05% TWEEN20 at 4°C. Cells were further washed  and the following primary antibodies were applied O/N at 4°C in blocking buffer: mouse anti-E-Cadherin , anti-human Integrin β1/CD29, Anti-human Integrin α6/GoH3, Goat anti-human vimentin, anti-human N-Cadherin, mouse anti-STRO-1  and mouse anti-CXCR7 9C4 . Cells were washed with PBS (3 × 5 mins), incubated with appropriate secondary antibodies , nuclear stain Hoechst , filamentous actin stains: Texas Red Phalloidin or 488 Phalloidin  and Cell Mask Blue  for 4 hrs at room temperature (R/T). Cells were washed then imaged using PerkinElmer Opera™ Confocal Imager and an Olympus IX-81 Scanning Confocal microscope.2 and 95% humidity. Mono culture (HS5 or PC3 cells) were plated at ~800 cells/well and co-cultures (HS5 + PC3 cells) were plated at ~400 cells/well each to make a total of ~800 cells/well in 50 μL complete medium per well and left to adhere O/N at 37°C, 5% CO2 and 95% humidity. A baseline reading was taken 24 hours after plating (Day 0), and readings were obtained on assay days 3, 6 and 9 through application of 5 μl Alamar Blue per well, achieving a final concentration of 10% (v/v). After addition of Alamar Blue cells were further incubated for 4 hrs at 37°C, 5% CO2 and 95% humidity, before plates were read on the Envision Plate Reader (Perkin Elmer) using fluorescence excitation/emission settings of 530 nm/ 595 nm. To investigate the relative contribution of proliferating HS5 and PC3 cells in co-culture, cells were treated with Click-iT EdU HCS 594 kit (Invitrogen) at days 3, 6 and 9 in culture. After incubation with the EdU compound in serum free media , cells were fixed with PFA, washed (3×5 mins PBS) and a 594 fluorescent azide solution  was applied O/N at 4°C in blocking buffer along with STRO-1  antibody. The following day a general cytoplasmic and nuclear stain  and a secondary antibody (5 μg/mL 488 goat anti-mouse) was applied for 4 hrs at R/T. Cells were finally washed (3×5 min PBS) and imaged using an Olympus confocal and results were analysed using Imaris volume and spots.To assess cell proliferation in mono and co-cultured 3D cells, assays were performed in 384-well plates using Alamar Blue reagent (Invitrogen). TC-treated Falcon 384-well plates (BD Biosciences) were applied with 15 μl of 70% Matrigel (diluted in cold SFM) and left to polymerise for 2 hrs at 37°C, 5% C02 and 95% humidity. The following day, cultures were serum starved for 16-24 hours in the presence of integrin function blocking antibodies: 1.5 μg/mL of α6; GoH3, 1.5 μg/mL of β1; P5B2, α6 and β1 and 1.5 μg/mL of mouse IgG isotope controls. On the day of the assay, cells were harvested with accutase and seeded at a density of ~150,000 cells per transwell insert in a volume of 200 μl SFM with the addition of integin inhibitors: 1. 1.5 μg/mL of α6; GoH3, 2. 1.5 μg/mL of β1; P5B2, 3. α6 and β1 or 4. 1.5 μg/mL of mouse IgG isotope controls. Prior to seeding cells, 20 μl of GFR Matrigel (1:5 dilution/SFM) was applied to the transwell insert and polymerised for 1 hr at 37°C, 5% CO2 and 95% humidity. The undersides of the transwell inserts were then coated with 4 μg of laminin (Invitrogen) to encourage attachment of migrated cells. For coating, a 1 mg/mL laminin stock solution was diluted 1/12.5 in warmed PBS, and 50 μl of this solution was dispensed onto each insert and left to evaporate at RT. The inserts were then washed in PBS and equilibrated in SFM for 1 hr at 37°C, 5% CO2 and 95% humidity before cells were seeded onto the prepared transwell inserts. Following addition of cells, 600 μl SFM was added to the lower chamber with or without 10% FBS or 10% FBS + 30 μg/mL of laminin and the plates were incubated at 37°C, 5% CO2 and 95% humidity for 32 hrs to allow for cell invasion to occur.To investigate the role integrin α6 and β1 play in mediating invasive cell behaviour, transwell cell invasion assays  were employed. Two days prior to each invasion assay, PC3 and HS5 cells were seeded in 6 well plates at a density of 500,000 cells/well and co-culture (PC3 + HS5) cells were seeded together at a 1:1 ratio to a total 500, 000 cells/well and left to adhere O/N at 37°C, 5% C0Cell invasion was then quantified through staining with crystal violet. Invaded cells were fixed with 100% Methanol for 10 mins at -20°C, prior to application of crystal violet staining mixture  for 30 mins to allow visualisation of cells. The non-invaded cells on the upper surface of the insert were removed with a cotton swab, the inserts washed in purified water and left to air dry. Cell invasion was quantified using images obtained on the InCell 1000 (GE) and processed by an automated script generated by InCell Developer. Counts were averaged between 3 assay replicates.To further quantify the relative proportion of invading HS5 and PC3 cells in co-culture, experiments were repeated as outlined above and cell invasion was quantified through staining with primary antibody STRO-1  for 2 hrs at R/T followed by a general cytoplasmic and nuclear stain  and a secondary antibody (5 μg/mL 488 goat anti-mouse) application for 2 hrs at R/T. Cells were finally washed (3×5 min PBS), membrane inserts carefully removed from the transwells, placed on a glass slide and imaged using an Olympus confocal and results were analysed using Imaris volume and spots.2, 37°C and 95% humidity). Supernatant from PC3 and 3T3 cells was collected after 48 hrs from T75 flasks and directly transferred to 3D HS5 cells. HS5 cells were plated into 12 well plates on GFR Matrigel and left to adhere O/N in standard culture conditions before addition of PC3 and 3T3 conditioned media. Supernatant was replenished every 2 days. HS5 cells were imaged via Differential Inference Contrast (DIC) optics and processed for western analysis on days 3, 6 and 9 in culture.For these assays, PC3 and 3T3 fibroblast cell-lines were propagated in T75 flasks for a minimum of 48 hrs in RPMI complete media and maintained at 37°C in standard cell culture conditions . 3D reconstructions of z-stacks were created in Imaris x64 Ver. 7 software (Bitplane Scientific Solutions). All live cell imaging was undertaken on the InCell 1000 (GE) Cell Imager using a GE 20/.75 air iris or on an Olympus Cell-R using an Olympus PlanNeo-FLUAR 20/.75 air iris. Images were compiled using Adobe Photoshop CS4 without further nonlinear digital manipulation.Cell invasion by counting the number of migrated cells across 4 fields using ×20 magnification on the InCell 1000 (GE) and processed by an automated script generated by InCell Developer software (GE). Alternatively, quantification of the relative contribution of invaded PC3 and HS5 cells in co-culture was attained across 4 fields using ×40 magnification on an Olympus confocal and processed using Imaris x64 Ver. 7 software (Bitplane Scientific Solutions) volume and surface tool. Counts were averaged between 3 assay replicates. Densitometric analysis was performed using Image Lab software and expressed as a fold change in relation to loading controls and normalised against β-actin. This programme uses volume rendering which is a far more accurate measure of protein concentration as opposed to simple pixel intensity. Proliferation assays were quantified by KC4: Kineticalc for Windows  and counts were averaged between 3 assay replicates. To quantify the relative contribution of proliferating PC3 and HS5 cells in co-culture and relative contribution of β1 and α6 expression, images were attained on the Olympus confocal (n = 10 per variable) and analysed using Imaris x64 Ver. 7 software volume and surface tool. Counts were averaged between 3 assay replicates. Statistical analysis was carried out using Graph-Pad Prism (Version 5) and statistical significance for all given variables was tested using Kruskal-Wallis test and Dunn’s Multiple Comparison test for post-hoc analysis.PCa: Prostate cancer; EMT: Epithelial-to-mesenchymal transition; MET: Mesenchymal-to-epithelial transition; BPE: Bovine pituitary extract; EGF: Epidermal growth factor; ECM: Extracellular matrix; IF: Intermediate filament.There are no potential conflicts of interest or competing interests known to the authors of this paper.LCW carried out the immunoassays, imaging, western blot, proliferation and invasion assays, conceived of the study, performed the statistical analysis, and designed and drafted the manuscript. TTG carried out the immunoassays, imaging, western blot assays and helped to draft the manuscript. VMA conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors have read and approved the final manuscript.


Endothelial barrier integrity is essential for vascular homeostasis and increased vascular permeability and has been implicated in many pathological processes, including diabetic retinopathy. Here, we investigated the effect of Rk1, a ginsenoside extracted from sun ginseng, on regulation of endothelial barrier function. In human retinal endothelial cells, Rk1 strongly inhibited permeability induced by VEGF, advanced glycation end-product, thrombin, or histamine. Furthermore, Rk1 significantly reduced the vessel leakiness of retina in a diabetic mouse model. This anti-permeability activity of Rk1 is correlated with enhanced stability and positioning of tight junction proteins at the boundary between cells. Signaling experiments revealed that Rk1 induces phosphorylation of myosin light chain and cortactin, which are critical regulators for the formation of the cortical actin ring structure and endothelial barrier. These findings raise the possibility that ginsenoside Rk1 could be exploited as a novel prototype compound for the prevention of human diseases that are characterized by vascular leakage. The disruption of endothelial barrier integrity leading to increased vasopermeability contributes to many pathological processes, including various inflammatory diseases, acute lung injury, and diabetic retinopathy ,2. EndotThe reorganization of the actin cytoskeleton into the cortical actin ring and the concomitant redistribution of TJ proteins to the cell periphery is an essential event in endothelial barrier enhancement. Several molecules have been suggested to be important for the formation of the cortical actin ring . PhosphoDiabetic retinopathy (DR) is one of the most common vascular retinopathies and a leading cause of legal blindness in working-age adults . The earPanax. Ginseng, long used in traditional oriental medicine, is known to have a wide range of medicinal effects, including tonic, immunomodulatory, anti-mutagenic, adaptogenic, and anti-aging activities  sucrose  were added to the upper compartment. The amount of radioactivity that diffused into the lower compartment was determined after 30 min with a liquid scintillation counter . Each experimental point was performed at least four times.HRECs were plated onto a Transwell filter . After reaching confluence, HRECs were incubated with M199 containing 1% FBS for 3 h and pretreated for 40 min with or without Rb1, Rg1 or Rk1 (10 µg/ml) prior to stimulation with VEGF (20 ng/ml) , Thrombin (10 units/ml) or Histamine (100 µM)  for 1 h or 5 µM AGE-BSA for 10 h. HRECs were pretreated for 30 min with or without 1C11 (10 µg/ml) prior to stimulation with 5 µM AGE-BSA for 10 h. 50 µl (0.8 µCi [0.0296 MBq]/ml) of , 1 mM Na3VO4, 0.5% Triton X-100) by centrifugation at 15,000 g for 15 min. The proteins in the Triton X-100-insoluble and soluble fractions were analyzed by Western blotting.HRECs plated in 6-well plates were serum starved in M199 containing 1% FBS for 6 h. They were then pretreated for 40 min with or without Rk1 (10µg/ml) prior to stimulation with VEGF (20 ng/ml) for 1 h or pretreated for 30 min with or without 1C11 (10 µg/ml) prior to stimulation with 5 µM AGE-BSA for 10 h, and fractionated in cytoskeleton-stabilizing buffer  prior to stimulation with 20 ng/ml VEGF (1 h) or 5 µM AGE-BSA (10 h). Confluent HRECs were treated with Rk1 (10 µg/ml) for 30min or 1h. HRECs were fixed in 4% paraformaldehyde for 20 min and permeabilized in 0.1% Triton X-100/PBS, then pre-incubated with a blocking solution of PBS containing 5% normal goat serum and 0.05% Tween-20. Cells were then incubated for 2 h in primary antibody . Reactions were visualized by fluorescein-conjugated anti-mouse .Confluent HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with 20 ng/ml VEGF (1 h) or 5 µM AGE-BSA (10 h). Actin filament rearrangement was monitored by Rhodamine Phalloidin staining, which specifically labels F-actin. HRECs monolayers were fixed in 4% paraformaldehyde for 20 min at room temperature, and then washed 3 times with PBS (pH 7.4). Cells were then permeabilized in 0.1% Triton X- 100/PBS and incubated with 0.1 mg/ml of Rhodamine Phalloidin  for 1 h. All samples were observed with a fluorescence microscope .Diabetic mice were injected with 15 µg Rk1 into the vitreous of one eye and with vehicle in the contra lateral eye. At 24 h after injection, the mouse eyes were then enucleated and immediately fixed in 4% PFA for 4h and washed in PBS. The lens were then removed and the eyes were embedded in paraffin wax for sectioning. Sections of mouse eyes were dewaxed and then rehydrated in Tris-buffered saline (TBS). Sections were then washed in TBS and the antigens unmasked in 0.125% trypsin (10 min at 25 °C), washed again in TBS, and blocked in 5% normal goat serum (20 min) in an antibody dilution buffer consisting of TBS containing 0.01% Triton X-100. Sections were then incubated for overnight in primary antibody [rabbit anti-occludin ] at 4 °C. Reactions were visualized by FITC-conjugated anti-rabbit . Nuclei were counterstained with DAPI and are seen in blue. Samples were observed with a fluorescence microscope .Mice were injected with 50 ng VEGF alone or in combination with 15 µg Rk1 into the vitreous of one eye and with vehicle in the contra lateral eye. At each time point after injection, 150µl FITC-Dextran (30 mg/ml in sterile PBS) was injected into the left ventricle. Mice were deeply anaesthetized using ketamine/xylazine and 40 kDa FITC-Dextran  (30 mg/ml in sterile PBS) was injected into the left ventricle. The tracer was allowed to circulate for ~2 min and the mouse eyes were then enucleated and immediately fixed in 4% PFA. The mouse eyes were left overnight in PFA. The following day, the mouse retinas were dissected in a Maltese cross-configuration and flat-mounted onto glass slides. The mouse retinas were then viewed by epifluorescence.−1. All diabetic (blood glucose level after 8 h fasting >150 mg dl−1) and normal mice were maintained with the same diet and water ad libitum for a total of 2 weeks. Each blood sample was taken from the tail vein, and its glucose level was measured with Freestyle Flash Blood Monitoring System .Insulin-deficient diabetes was induced in C57BL/6J mice  by a single i.p. injection of streptozotocin ), which was dissolved in 0.1 M sodium citrate buffer (pH 4.5) just before injection. Age-matched normal control mice received saline alone. Fasting blood glucose level was evaluated 3 days later. A supplemental STZ injection (1.25 mg per BW(g)) i.p., was given if the fasting blood glucose was below 150 mg dlDiabetic mice were injected with Rk1  into the vitreous of one eye and with vehicle in the contra lateral eye. At 24 h after injection, 150 µl FITC-Dextran (30 mg/ml in sterile PBS) was injected into the left ventricle. The retinas were then viewed by fluorescence microscopy.2, 10 mM dithiothreitol, and 1 mM dNTPs at 42 °C for 1 h. The reaction was stopped by heating at 70 °C for 15 min. The cDNA mixture (1 µl) was used directly for enzymatic amplification. The polymerase chain reaction was performed in 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.2 mM dNTPs, 2.5 units of Taq DNA polymerase, and 0.1 µM of primers. Amplification was performed in a DNA thermal cycler  under the following conditions: denaturation at 94 °C for 5 min for the first cycle and for 30 sec thereafter, annealing at 55 °C for 30 sec, and extension at 72 °C for 30 sec for 28 repetitive cycles. Final extension was at 72 °C for 10 min. All results were normalized to GAPDH mRNA. Primers were as follows: ZO-1 5’-CGACCATTTGAACGCAAGTT-3’ (sense) and 5’-TCAAACTCAGGAGGCTGTGC-3’ (antisense); ZO-2 5’-GAGGAGGTGGGAGAGAGCAG-3’ (sense) and 5’-CTGGCTTGTGCGTTTTGATT-3’ (antisense); Occludin 5’-ATTTATGATGAGCAGCCCCC-3’ (sense) and 5’-TGACCTTCCTGCTCTTCCCT-3’ (antisense); VEGF 5’-GTGGACATCTTCCAGGAGTA-3’ (sense) and 5’-GCGAGTCTGTGTTTTTGC-3’ (antisense); GAPDH 5’-CGCCACAGTTTCCCGGAGGG-3’ (sense) and 5’-CCCTCCAAAATCAAGTGGGG-3’ (antisense). Each experimental condition was performed in quadruplicate.HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with VEGF (20 ng/ml) for 6 h or 5 µM AGE-BSA for each time point. Total RNA was obtained from HRECs with a TRIzol reagent kit and 0.5–5 µg RNA was used for RT-PCR. Briefly, target RNA was converted to cDNA by treatment with 200 units of reverse transcriptase and 500 ng of oligo(dT) primer in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgClConfluent HRECs were treated with Rk1  for the 30min. Cellular protein from HRECs was incubated with the anti-cortactin antibody  and protein A or G Sepharose in TEG buffer  at 4°C for 16 h with constant rotation. Sepharose pellets were collected by centrifugation and washed three times with supplemented TEG buffer. Immunoprecipitates were analyzed by SDS-PAGE, followed by Western blot using the indicated antibodies.The mice were housed and maintained under sterile conditions in facilities accredited by the korean Association of Assessment and Accreditation of Laboratory Animal Care. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Yonsei University. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Yonsei. All efforts were made to minimize suffering.All experiments were repeated at least 3 times. Data are presented as the means ± S.E, and statistical comparisons between groups were performed by one-way ANOVA followed by Tukey’s test.in vitro HERCs monolayer permeability assay. As shown in Vascular integrity is required for functional vasculature maintenance and is lost in many pathological states, including DR. Our preliminary results showed that Rk1, a major component of SG135, which is a mixture of ginsenosides extracted from sun ginseng, has a profound effect on the viability of human retina endothelial cells (HRECs) as compared to other derivatives of ginsenosides. Therefore, we decided to investigate whether Rk1 has a protective effect on the integrity of retina vessels using an in vivo. To this end, we co-injected VEGF and Rk1 into the vitreous cavities of mice and performed fluorescein angiography 24 h post injection. To our surprise, we found that in deed Rk1 significantly inhibits VEGF-induced fluorescein leakage in the mouse retina . Unfortunately, the mRNA levels of these proteins were not altered by Rk1 treatment  promoted by hyperglycemia has been suggested to be important in the development of DR by increasing vessel leakiness ,25. Therreatment , 5E, 5F.reatment , 6E, sugin vivo, we created a diabetic mouse (DM) model, in which hyperglycemia is induced with streptozotocin and measured vascular leakage using fluorescein angiography. Streptozotocin-induced hyperglycemia caused fluorescein leakage from retina vessels in the mice, while fluorescein was restricted to vascular lumen in control mice  for the indicated times. Cell lysates were subject to affinity precipitation using PAK-1 (p21-activated kinase) PBD (p21-binding domain) in recombinant protein agarose, which specifically precipitates active Rac (Rac-GTP). “Total Rac” indicates total amount of active and inactive Rac in the HREC. Western blot was performed using an antibody specific for Rac.(TIF)Click here for additional data file.Figure S2(TIF)Click here for additional data file.Figure S3(TIF)Click here for additional data file.


However, the kinetic characteristics of the H2O2 signal and its functional relationships with the insulin receptor during the autophosphorylation process in neurons remain unexplored to date.Insulin receptors are widely distributed in the brain, where they play roles in synaptic function, memory formation, and neuroprotection. Autophosphorylation of the receptor in response to insulin stimulation is a critical step in receptor activation. In neurons, insulin stimulation leads to a rise in mitochondrial H2O2 signal precedes receptor autophosphorylation and represents a single spike with a peak at 5–10 s and duration of less than 30 s. Mitochondrial complexes II and, to a lesser extent, I are involved in generation of the H2O2 signal. The mechanism by which insulin triggers the H2O2 signal involves modulation of succinate dehydrogenase activity. Insulin dose–response for receptor autophosphorylation is well described by hyperbolic function . N-acetylcysteine (NAC), a scavenger of H2O2, dose-dependently inhibited receptor autophosphorylation. The observed dose response is highly sigmoidal , signifying that insulin receptor autophosphorylation is highly ultrasensitive to the H2O2 signal. These results suggest that autophosphorylation occurred as a gradual response to increasing insulin concentrations, only if the H2O2 signal exceeded a certain threshold. Both insulin-stimulated receptor autophosphorylation and H2O2 generation were inhibited by pertussis toxin, suggesting that a pertussis toxin-sensitive G protein may link the insulin receptor to the H2O2-generating system in neurons during the autophosphorylation process.Experiments were carried out in culture of rat cerebellar granule neurons. Kinetic study showed that the insulin-induced H2O2 signal exceeds a certain threshold. This finding provides novel insights into the mechanisms underlying neuronal response to insulin. The neuronal insulin receptor is activated if two conditions are met: 1) insulin binds to the receptor, and 2) the H2O2 signal surpasses a certain threshold, thus, enabling receptor autophosphorylation in all-or-nothing manner. Although the physiological rationale for this control remains to be determined, we propose that malfunction of mitochondrial H2O2 signaling may lead to the development of cerebral insulin resistance.In this study, we demonstrated for the first time that the receptor autophosphorylation occurs only if mitochondrial H Additionally, exogenous H2O2 has been shown to facilitate receptor autophosphorylation in immunoprecipitates of the insulin receptor in the presence of phosphate donors [2O2 also participates in insulin receptor activation. Insulin receptors are widely distributed in the brain, where they play roles in synaptic function, memory formation, and neuroprotection [2O2 in response to insulin stimulation [2O2 is derived from the mitochondrial respiratory chain and plays a role in insulin receptor autophosphorylation. However, the kinetic characteristics of the H2O2 signal and its functional relationships with the insulin receptor during autophosphorylation in neurons remain to be clarified. In the current investigation, these issues have been explored as an extension of our previous study.Insulin receptor is a member of the receptor tyrosine kinase family. Upon insulin binding to the extracellular α-subunits, the receptor undergoes rapid autophosphorylation at three specific tyrosine residues within the activation loop of the cytoplasmic β-subunits ,2, resulmulation ,6. Evidereceptor -9. Accore donors ,11. The otection -14. The otection ,16. Earlmulation . This H250 of 16.3±2.2 nM and Hill coefficient, nH, of 1.1±0.1 (R2=0.99), indicating that this process in neurons is described by a classic hyperbolic function. These results suggest that the insulin dose–response for receptor autophosphorylation in neurons is gradual and not switch-like.We characterized insulin-stimulated receptor autophosphorylation in a primary culture of rat cerebellar granule neurons (CGN). The insulin dose–response curve for the autophosphorylation process is depicted in Figure 2O2 generation during insulin stimulation, we compared the kinetics of insulin-induced autophosphorylation and H2O2 production in CGN. The time-course of receptor autophosphorylation in response to insulin stimulation is depicted in Figure 2O2 signal. H2O2 efflux from neurons into the incubation medium was used as a measure of the signal, given that H2O2 penetrates readily across cellular membranes with an estimated time of gradient formation within 1 s [2O2 spike with a peak at 5–10 s and duration of less than 30 s. We observed a significant difference between the areas under curves (AUCs) calculated for the H2O2 signal in insulin- and vehicle-exposed neurons during the 30 s of insulin stimulation (P<0.001), while no difference was observed at baseline and periods following this time , a scavenger of hydrogen peroxide, on receptor autophosphorylation in neurons stimulated with insulin. NAC dose-dependently and completely (at concentrations ≥4 mM) inhibited receptor autophosphorylation in CGN cultures exposed to 100 nM insulin , signifying that the observed dose response is highly sigmoidal and considerably steeper than a hyperbolic curve (nH=1). Therefore, even a small increase in the H2O2 scavenger dose above the threshold results in complete abrogation of receptor autophosphorylation. Given that a steep dose–response with a Hill coefficient nH>1 is defined as ultrasensitive [2O2 signal generates strong ultrasensitivity in receptor autophosphorylation. Autophosphorylation only occurs when the H2O2 signal has surpassed a certain threshold. Conversely, if the H2O2 signal does not reach this threshold, no autophosphorylation occurs, even in response to the highest insulin dose (100 nM). Therefore, H2O2 appears to function as an enabling signal that permits insulin receptor autophosphorylation in an all-or-nothing manner. This mode of switching between two modes of action is often referred to as a decision-making process. In this context, H2O2 signal above the threshold serves as a decision-making step of the neuron to permit activation of the insulin receptor.To address the specific function of the Hensitive ,20, the 2O2 in neurons [50 of 2.0±0.3 mM and Hill coefficient, nH, of 3.4 (R2=0.92), indicating that the dose response is a highly sigmoidal, and therefore, even a small change in succinate dehydrogenase activity around a certain threshold can have a dramatic effect on insulin receptor autophosphorylation. At concentrations of malonate that completely inhibited receptor autophosphorylation, the insulin-induced H2O2 signal was abolished  were so complete, we conclude that the reducing equivalents for generation of H2O2 do not originate from sources other than the succinate dehydrogenase reaction. The results collectively suggest that activation of succinate dehydrogenase is a possible mechanism underlying insulin-mediated switching of the mitochondrial H2O2 signal critical for receptor autophosphorylation.To extend our previous finding that the mitochondrial electron transport chain (ETC.) provides the source of insulin-induced H neurons , we inve2O2 generation exhibits biphasic behavior, showing a fast initial increase followed by a rapid decrease to the baseline steady-state level while insulin stimulation is sustained. In keeping with common theory, such a response can be generated by an incoherent feed-forward loop (IFFL) in which insulin modulates the H2O2 rate via at least two intermediate pathways with opposite functions of “fast activation” and “delayed inhibition”. We speculate that fast activation and delayed inhibition of succinate dehydrogenase generates the biphasic response. In this scheme, delayed inhibition of succinate dehydrogenase is attributed to the downstream metabolites of succinate oxidation, oxaloacetate [2O2[2O2 signal in response to insulin stimulation.As shown in Figure oacetate  and H2O2tate [2O2. These mtate [2O2 and oxidtate [2O2-29. Givetate [2O2,30 corre2O2 signal, we briefly reviewed current knowledge on succinate-supported H2O2 generation in isolated mitochondria. Succinate promotes the highest rates of H2O2 production among all respiratory substrates [2O2 is produced from superoxide generated by reduction of molecular oxygen in the electron transport chain (ETC.) [m[2O2 production, generation of H2O2 depends on the metabolic state of mitochondria and changes in parallel with succinate dehydrogenase activity. The highest H2O2 rates [2O2 production rates drop rapidly [2O2 production [m and activity of mitochondrial complexes I/II are the candidate factors that influence generation of the insulin-induced H2O2 signal and thus play a role in the control of insulin-stimulated receptor autophosphorylation in neurons.To clarify the factors influencing generation of the insulin-induced Hbstrates -35. Mostn (ETC.) ,37. Althn (ETC.)  and at cn (ETC.) -36, wher(ETC.) [m,39. Nota(ETC.) [m. At succO2 rates ,28 are o rapidly ,39 and s rapidly ,28. Protoduction ,39 and doduction . In summH, of 0.7 (R2=0.83), indicating that the observed dose response is gradual and not switch-like. Our results suggest that ETC. complex I is involved in control of insulin receptor autophosphorylation, presumably as part of the mitochondrial machinery of RET-associated H2O2 generation. However, its role does not appear to be as important as that of ETC. complex II.To investigate whether ETC. complex I functions in the control of insulin-stimulated receptor autophosphorylation, we examined the effects of rotenone on receptor autophosphorylation in neurons stimulated with 100 nM insulin. Rotenone is a selective inhibitor of the ubiquinone reduction site at mitochondrial complex I. Rotenone inhibited insulin-stimulated receptor autophosphorylation, as shown in Figure m, we compared the effects of insulin and other additives on autophosphorylation and ΔΨm in neurons. Rhodamine 123 was used as a measure of mitochondrial ΔΨm[50 of 5.2±0.1 mM and Hill coefficient, nH, of 5.4 (R2=0.99). Comparison of the malonate dose–response curves for autophosphorylation and fluorescence revealed that at a malonate concentration of 2 mM (inducing 50% inhibition of autophosphorylation), mitochondria were depolarized by less than 1%. This finding suggests that ΔΨm in neurons is ultrasensitive to succinate dehydrogenase activity and mitochondrial depolarization is not a causative factor for the inhibitory effect of malonate on receptor autophosphorylation. Figure 50 of 0.030±0.003 (R2=0.99) and IC50 of 0.07±0.02 (R2=0.98), respectively. At a FCCP concentration of 0.07 μM (inducing 50% inhibition of autophosphorylation), >95% mitochondrial depolarization was observed. These data suggest that FCCP-induced mitochondrial depolarization leads to inhibition of insulin-stimulated receptor autophosphorylation. Given that protonophores deactivate succinate dehydrogenase to the lowest observable activity [To address whether receptor autophosphorylation is sensitive to ΔΨdrial ΔΨm. Insulinactivity , we prop2O2 signal to activation of NADPH oxidase (Nox) that is sensitive to inhibition by diphenyleneiodonium (DPI) [2O2 generation and receptor autophosphorylation, the results of the DPI experiment cannot be interpreted to conclude that Nox is a source of the insulin-induced H2O2 signal in neurons. Although the nonspecific flavoprotein inhibitor, DPI, is often claimed to be a specific inhibitor of Nox, compelling evidence shows that DPI inhibits many targets [2O2 generation in rat brain mitochondria [2O2 in neurons. Another argument against a role of Nox in neuronal insulin receptor autophosphorylation is that the kinetics of the insulin-induced H2O2 signal is unusually fast, compared to that previously observed for Nox in non-neuronal cells [2O2 signal is a single spike with a sharp peak at 5–10 s and duration of less than 30 s. In contrast, the insulin-induced oxidant signal in adipocytes reaches a peak at 5 min and begins to dissipate by 10 min [2O2 signals indicates that the source of insulin-induced H2O2 in neurons differs from that in adipocytes and HepG2 cells, which is Nox [2O2 signaling that controls insulin-stimulated receptor autophosphorylation in neurons.A number of previous studies using non-neuronal cells have assigned the insulin-induced Hum (DPI) -43. Acco targets , includi targets ,46 and schondria . In thisal cells ,43. The y 10 min . In HepGh is Nox ,43. Our 2O2 signal is a neuron-specific phenomenon. Although abundant literature is available on insulin-induced H2O2 generation in cells, limited studies have focused on the origin of insulin-induced H2O2 implicated in insulin receptor autophosphorylation. In experiments with 3T3-L1 adipocytes [2O2 was assigned to activated Nox on the basis of data obtained with DPI only. Strong evidence from experiments with dominant-negative Nox4 constructs and Nox4 siRNA further suggested that Nox4 is the source of the insulin-induced H2O2 signal in adipocytes [2O2 in neurons. In summary, owing to limited data, it is not currently possible to address whether mitochondria represent the only neuronal source of insulin-induced H2O2 implicated in receptor activation.Data from the present study raise the issue of whether the mitochondrial origin of the insulin-induced Hipocytes ,42 and Hipocytes , the souipocytes . The resipocytes , indicat2O2 signal in non-neuronal cells [2O2 in neurons, we examined the effects of pertussis toxin (PTX), an inhibitor of Gi/0 protein-receptor coupling, on H2O2 generation and receptor autophosphorylation in CGN exposed to 100 nM insulin. PTX treatment completely inhibited insulin-stimulated receptor autophosphorylation at the highest dose of 2 mg/L, and this suppression was dose-dependent , implying that insulin-induced receptor autophosphorylation is ultrasensitive to PTX. PTX (2 mg/L) completely eliminated the insulin-induced H2O2 signal , receptor autophosphorylation was abolished, as shown in Figure 2O2-generating system in neurons during generation of the insulin-induced H2O2 signal critical for insulin receptor autophosphorylation.A pertussis toxin-sensitive G protein links the insulin receptor to the insulin-induced Hal cells . To detet Figure A. Approxl Figure B. Thus, 2O2 generation [Although our data show for the first time that a PTX-sensitive G protein is possibly involved in activation of the insulin receptor in CNS, the involvement of a PTX-sensitive G protein in insulin receptor activation in peripheral tissues is widely documented. In mice, Gαi2 deficiency results in impaired tyrosine phosphorylation of the insulin receptor substrate, IRS1, and frank insulin resistance in peripheral tissues , whereasneration  and reguneration . Althougneration . Gαi2 opneration -56. Our 2O2 signal, which plays a permissive role in activation of the insulin receptor. Autophosphorylation only occurs once the H2O2 signal has surpassed a certain threshold. Under conditions where the H2O2 signal does not reach this threshold, no autophosphorylation occurs, even in response to the highest insulin dose. In this context, H2O2 signal above the threshold serves as the neuron’s decision to activate the insulin receptor. The insulin-induced H2O2 signal is derived from mitochondria. Succinate dehydrogenase in complex II plays a key role in control of H2O2 generation. An unknown pertussis toxin-sensitive G protein links the insulin receptor to the mitochondrial H2O2-generating system during H2O2 signal activation.It is possible to draw some tentative conclusions regarding the functional relationship between the insulin receptor and mitochondria during receptor activation in neurons Figure . Insulin2O2 signal exceeds a certain threshold, enabling receptor autophosphorylation. Generally, this mode of control means that activation of the neuronal insulin receptor is conditional on mitochondrial functioning. Moreover, receptor activation may be conditional on neural activity. Neural activity evokes insulin release from synaptosomes within nerve endings into the synaptic cleft [The current study provides novel insights into the mechanisms underlying neuronal response to insulin. The insulin receptor is activated if two conditions are met: 1) insulin binds to the receptor, and 2) the Hic cleft ,58 and iic cleft , which a2O2 signal in autophosphorylation of the neuronal insulin receptor, factors that disrupt H2O2 signaling may generate unresponsiveness or resistance to insulin. In line with the above findings, mitochondrial dysfunction that affects generation of the H2O2 signal may induce central insulin resistance. Moreover, insulin resistance may be induced by pathologically elevated activity of antioxidant enzymes that metabolize H2O2, e.g., arising as a compensatory response to oxidative stress. Accumulating evidence from animal and human studies supports this hypothesis. Glutathione peroxidase (Gpx1) and peroxiredoxins (Prx) are primary H2O2 scavengers capable of metabolizing the insulin-induced H2O2 signal, according to kinetic estimations based on published second-order rate constants of ~107 M-1 s-1 and abundance data [2O2 scavenger, NAC, renders them more insulin-resistant [2O2 signal and consequently induces central insulin resistance.Given the importance of the insulin-induced Hnce data -62. Micence data . In contesistant . Brain iesistant , and sigesistant ,67 and pesistant -71. Elevesistant ,73. From2O2 signal exceeds a certain threshold. Our findings provide a novel insight into the mechanisms underlying neuronal response to insulin. The neuronal insulin receptor is activated if two conditions are met: 1) insulin binds to the receptor, and 2) the H2O2 signal surpasses a certain threshold, thus, enabling receptor autophosphorylation in all-or-nothing manner. Although the physiological rationale for this control remains to be determined, we propose that malfunction of insulin-induced H2O2 signaling may lead to cerebral insulin resistance, in view of the critical significance of the H2O2 signal for insulin receptor activation in neurons.In summary, we have demonstrated for the first time that the receptor autophosphorylation occurs only if mitochondrial HPhosphoDetect™ Insulin Receptor (pTyr1162/1163) ELISA kit and Insulin Receptor (β-Subunit) ELISA Kit were from Calbiochem. Other materials were purchased from Sigma, ICN, Gibco, Biosource, Invitrogen and Acros.2+/Mg2+-free Hanks’ buffered salt solution (HBSS) without Phenol Red (Gibco). After mincing with fine scissors, tissue was placed in Ca2+/Mg2+-free HBSS with Phenol Red and 0.1% trypsin for 15 min at 37°C. Trypsin was inactivated by washing with normal HBSS. Cells were dissociated via trituration and pelleted in HBSS. Next, cells were resuspended in Neurobasal Medium (Gibco) supplemented with serum-free B-27 Supplement , 20 mmol/L KCl, GlutaMax  and antibiotic-antimycotic , and plated at a density of 5×106 cells/ml onto 35X10 mm sterile cell culture dishes or 6×24 plates (Corning) previously coated with polyethyleneimine (10 mkg/ml). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air, and fed with supplemented Neurobasal Medium. Cultures were treated on day 3 with 5 μM cytosine arabinoside (Sigma) for 24 h to prevent glial proliferation, and the medium subsequently changed. Neurons on days 7 to 9 were used for experiments.Cerebellar granule neurons were prepared from 7- to 8-day-old Wistar rats, as described earlier . Cerebel2O2 efflux from CGN cultures was measured using fluorimetry employing the cell-impermeable Amplex Red dye in the presence of horseradish peroxidase . CGN cultures were pre-incubated for 120 min in Hepes-buffered salt solution composed of 156 mM NaCl, 3 mM KCl, 2 mM MgSO4, 1.25 mM KH2PO4, 2 mM CaCl2, 0.5 mM glucose, 20 mM HEPES, and the pH adjusted to 7.4 with NaOH. Cultures were subsequently exposed to Hepes-buffered salt solution supplemented with 50 μM Amplex Red and 0.1 U/ml horseradish peroxidase (incubation medium), and H2O2 efflux recorded every 5 s for 2 min. The incubation medium was rapidly exchanged with fresh medium containing no insulin (control) or 100 nM insulin, and H2O2 efflux recorded every 5 s for 2 min. Where indicated, PTX (2 μg/ml) was added 120 min before and during insulin stimulation. Malonate (6 mM) was added 5 min before and during insulin stimulation. Fluorescence was measured with an epifluorescent inverted microscope Axiovert 200  equipped with a 20X fluorite objective, using excitation at 550±10 nm and fluorescence detection at 610±30 nm. All imaging data were collected and analyzed using Metafluor 6.1 software . Standard curves of H2O2 concentrations were linear up to 1500 nM and used to convert fluorescence values to H2O2 values. The calculated detection limit of the assay was 7 nM. Data were normalized to total amounts of cell protein, and expressed as H2O2 nM μg-1.H2, 1 mM MgCl2, 20 mM HEPES, and 0.5 mM glucose) at pH 7.4 for 30 min, followed by exposure to insulin  or no insulin (control) in the presence or absence of other additives (preincubation for 5–30 min before insulin). The experiment was terminated by removing the medium, washing with ice-cold PBS, and adding 200 μL/dish cell lysis buffer (Biosource) supplemented with 1 mM PMSF, 50 mM protease inhibitor set III (Sigma), and 2 mM sodium orthovanadate as the tyrosine phosphatase inhibitor on ice for 10 min. Lysates were centrifuged at 12,000 rpm at 4°C for 12 min. In each CGN lysate, amounts of autophosphorylated receptors were measured as described by the manufacturer. For each experiment, values were normalized to the total amounts of insulin receptor β-subunit (IR) measured using the insulin receptor (β-subunit) ELISA kit  and normalized to scale between 0 and 1 using Equation 1:Amounts of the autophosphorylated β-subunit of the insulin receptor were measured with the PhosphoDetect™ insulin receptor (pTyr1162/1163) ELISA kit  suitable for studies with the rat insulin receptor. CGN cultures were incubated in Hepes-buffered salt solution  the normalized autophosphorylation value, YCTR the mean autophosphorylation value in neurons exposed to vehicle, and YINS the mean autophosphorylation value in neurons exposed to 100 nM insulin.where 0 ratio of the fluorescence signal measured after exposure of neurons to additives to that measured at baseline.Mitochondrial depolarization changes in individual neurons within CGN cultures in response to additives were measured via fluorescence of Rhodamine 123, as described earlier . CGN culExperimental data were fitted by non-linear regression to the Hill equation to provide the parameter values in Equation 2:Y is a parameter value, X a variable, K the concentration of the variable producing half the effect, and nH the Hill coefficient.where P<0.05.Data were analyzed for statistical significance with one-way analysis of variance (ANOVA). Values are presented as means ± SEM. Differences were considered significant at CGN: Cerebellar granule neurons; DPI: Diphenyleneiodonium; FCCP: Carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone; HBSS: Hanks’ buffered salt solution; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; NAC: N-acetylcysteine; PBS: Phosphate-buffered saline; PMSF: Phenylmethylsulfonyl fluoride; PTX: Pertussis toxin; PTPs: Protein tyrosine phosphatases; SEM: Standard error of mean.The authors declare that they have no competing interests.in vitro studies with CGN cultures and data analysis. TPS carried out the in vitro studies with CGN cultures and data analysis. YES carried out the in vitro studies with CGN cultures and data analysis. LRG carried out the DPI experiments in CGN cultures and data analysis; VGP participated in the design of the in vitro studies with CGN cultures, and manuscript evaluation/critique. IAP conceived, designed and coordinated the study, and drafted the manuscript. All authors read and approved the final manuscript.NAP carried out the


We report that P450cam catalysis is controlled by oxygen levels: at high O2 concentration, P450cam catalyzes the known oxidation reaction, whereas at low O2 concentration the enzyme catalyzes the reduction of camphor to borneol. We confirmed, using 17O and 2H NMR, that the hydrogen atom added to camphor comes from water, which is oxidized to hydrogen peroxide (H2O2). This is the first time a cytochrome P450 has been observed to catalyze oxidation of water to H2O2, a difficult reaction to catalyze due to its high barrier. The reduction of camphor and simultaneous oxidation of water are likely catalyzed by the iron-oxo intermediate of P450cam, and we present a plausible mechanism that accounts for the 1∶1 borneol:H2O2 stoichiometry we observed. This reaction has an adaptive value to bacteria that express this camphor catabolism pathway, which requires O2, for two reasons: 1) the borneol and H2O2 mixture generated is toxic to other bacteria and 2) borneol down-regulates the expression of P450cam and its electron transfer partners. Since the reaction described here only occurs under low O2 conditions, the down-regulation only occurs when O2 is scarce.P450 Enzyme-substrate complex 2 accepts two electrons from the nicotinamide cofactor (NADH) via two redox partner proteins: an iron-sulfur protein, putidaredoxin (PdX), and a flavoprotein, putidaredoxin reductase (PdR) 2, in a stepwise manner, via intermediates 3 and 45, which is protonated to give hydroperoxo complex 6. Protonation of the distal oxygen of 6 and elimination of water gives rise to a high valent iron-oxo complex 7 known as compound I (Cpd I) 7 is then inserted into a C-H bond of the substrate, giving an alcohol product complexed to the iron, 8. The catalytic cycle is complete when water displaces the product.Cytochrome P450 enzymes (P450s or CYPs) belong to a family of heme-thiolate enzymes that couple the reduction of oxygen to the oxidation of non-activated hydrocarbons 2, P450 enzymes can be shunted to Cpd I by using oxidants such as cumene hydroperoxide or meta-chloroperbenzoic acid (m-CPBA) (i”) 2 and water. This reductive pathway is known as four-electron uncoupling because it requires two NADH equivalents (ii”) 2O2 (iii”) from the ferric hydroperoxo species 6. Third, superoxide can dissociate from superoxo complex 4 (iv”) Instead of proceeding through the complete reduction and splitting of Ocam (CYP101A1) enables a strain of Pseudomonas putida (a soil bacterium) to use (1R)-(+)-camphor 9 , protease inhibitors leupeptin, aprotinin, and 4-(2-aminoethyl)-benzenesulfonyl fluoride, butylated hydroxytoluene (BHT), cytochrome P450 CYP3A4 (C-4982), superoxide dismutase (S5639), catalase (C-1345), glucose oxidase (G-2133) were purchased from Sigma. Ethylenediaminetetraacetic acid (EDTA) was purchased from Fisher Scientific. Ferrous sulphate (FeSO4) was purchased from Allied Chemical, Canada. Gas chromatography/mass spectrometry (GC-MS) was carried out on a Varian Saturn 2000 MS equipped with a 30-m SPB-5 column  and the column was programmed as follows: 45°C (0.5 min), 7°C min−1 to 120°C (1 min), 50°C min−1 to 260°C (3 min). Electron impact (EI) spectra were obtained at an emission current of 30 µA, scanning from 50 to 365 amu, with ion storage (SIS mode) 49–375, trap temperature 170°C and transfer line 250°C. UV/Vis spectra were obtained on a Cary 300 Bio UV-visible double beam instrument. NADH utilization rates and hydrogen peroxide formation were measured on a thermostatted Hach DR/4000 U spectrophotometer. Activity assays were carried out at 22°C. Electrophoresis was performed on polyacrylamide gels  with 0.5% SDS (SDS-PAGE). The samples were reduced by treating with 1 µL of DTT stock (31 mg/mL) before loading on gels. Gels were stained with Coomassie Brilliant Blue R (Sigma). Sonication was done using a Branson Ultrasonic sonicator. Centrifugations were carried out with a Beckmann Avanti J-26 XPI centrifuge, equipped with a JLA 8.1000 rotor.All solvents were distilled prior to use. Nicotine adenine dinucleotide, reduced (NADH), dithiothreitol (DTT), lysozyme, DNase, RNase, vitamin B+), pH 7.4 with 1 mM camphor; T-100 ; T-400 . Buffers for nickel columns were: rinse buffer ; low imidazole buffer ; strip buffer (0.1 M Ethylenediaminetetraacetic acid (EDTA)), 0.5 M NaCl, pH 8.0). For P450cam purifications, all buffers contained 1 mM camphor. Substrate-free P450 was prepared by passing the substrate bound enzyme over a Sephadex G-10 column equilibrated with 100 mM 3-(N-morpholino) propane sulfonic acid .The buffers used were: lysis (20 mM phosphate buffer (K2H) NMR spectra were recorded on a Bruker AVANCE II 600 MHz spectrometer (operating at 92.124 MHz). A Bruker 5 mm TCI cryoprobe was used with samples maintained at a temperature of 298 K. 2H field-locking and field sweep were turned off. Samples were contained in 3 mm diameter MATCH nmr tubes filled to 40 mm (volume ca. 185 µL). Acquisition details: 10,240 transients summed, spectral width 15 ppm, transmitter offset 6.5 ppm, 11054 complex points acquired, 15 degree pulse with recycle delay of 1 s between transients, no decoupling of 1H during FID acquisition. Acquisition time was 14.2 h per spectrum.Deuterium (17O NMR spectra were run on a Bruker AVANCE III 500 MHz NMR spectrometer (operating at 67.808 MHz) equipped with a Bruker 5 mm TBO probe and samples were maintained at a temperature of 298 K. Samples were contained in 5 mm diameter nmr tubes filled to 50 mm (volume ca. 600 µL). Acquisition details: 1,000 or 25,000 transients summed, spectral width 503 ppm, 17O transmitter offset 50 ppm, 1H transmitter offset 4.78 ppm, 32768 complex points acquired, 90 degree pulse with recycle delay of 1 s between transients, and inverse-gated WALTZ-16 composite pulse decoupling of 1H during FID acquisition. Acquisition time was 12 min per spectrum or 300 min . The chemical shifts (δ) for all compounds are listed in parts per million using the NMR solvent as an internal reference (0 ppm for 17O or 4.78 ppm for 2H).The E. coli strain BL-21 (DE 3) (Novagen) containing the appropriate plasmid 600 = 0.9–1.0 −1) and trace additives were added. The cultures, except for PdR, were grown for 12 h at 27°C (PdR was grown for 6 h). The cells were harvested by centrifugation  and stored at −85°C until lysis. Additives were: FeCl2 (0.1 µM), 5-aminolevulinic acid (1 mM), Vitamin B1 (10 µM) for P450; FeCl2 (0.1 µM) and Na2S.9H2O (0.1 µM) for redoxin; riboflavin (1 mM) for reductase.P. putida−1. The fractions with high absorbances at λ392 (in the case of P450), λ454 (in the case of PdR) were checked with SDS-PAGE. The collected fractions were pooled and concentrated using an Amicon ultrafiltration cell equipped with a YM-10 membrane and the concentrated protein was individually loaded onto a S-100 column, eluted with T-100 buffer, 1 mM sucrose, 1 mM camphor (P450 only) at 1 mL min−1. SDS-polyacrylamide gel electrophoresis showed a single band for P450 and PdR.The lysis steps of P450 and PdR remained the same as described for −1), DNase , and RNase  were added and the solution was stirred for 30 min at 4°C. The lysate was sonicated with 50% duty cycle for 10 minutes, stirred for 10 min at 4°C, and homogenized with a pestle. The homogenized cells were then harvested by centrifugation  and dialysed with frequent changes of lysis buffer followed by further purification by ammonium sulphate precipitation. The dialysed lysate was subjected to a 20% ammonium sulphate cut to remove the cell debris. The 20% supernatant was then carried forward to 90% ammonium sulphate saturation overnight to isolate the protein. The 20–90% pellet was resuspended in 5 mL of rinse buffer , dialysed against this buffer for 3 h and harvested at 5000 rpm for 5 min. The dialysed supernatant was loaded on a ∼5 cm Ni2+-His bind column and eluted with strip buffer (10 mL×3), low imidazole buffer (10 mL×2), high imidazole buffer (10 mL×4). The fractions with A280/A325<5.0 were pooled, dialysed with 100 mM Tris, 100 mM KCl, pH 7.4 and the concentrated PdX protein was frozen to −85°C. The concentrations of ferric P450 (with camphor), PdR and PdX were determined by their extinction coefficients .In the case of PdX, cells from 2 L of culture were lysed in lysis buffer . Lysozyme  are included in Material S1.cam when camphor is present and the O2 concentration is low . In vivo, this occurs when cultures are poorly aerated in vitro, this occurs when the buffer is sparged with argon in an open vial. In contrast, the known oxidation products 10 and 11 form at high O2 concentrations (∼9 mg/L = 284 µM). In vivo, this occurs when cultures are well aerated in vitro, this occurs when pure O2 is bubbled into the buffer , isolated from expression in E. coli , with NADH and camphor. Our extinction coefficient values were used for the calculation of the enzyme concentration (2 bubbled into the buffer), we observed 5-exo-hydroxy camphor as a major product .We have observed that borneol forms as a major product of P450e buffer . To map  E. coli . Assays ntration . Under h product , entry 1 product  entry 2.cam shunted with m-CPBA (i”) reduced camphor to borneol . Using recombinant proteins , under mid-range oxygenated conditions (with air), we detected the enzymatic conversion of camphor to 2-D-borneol 12D  also yielded borneol that was deuterated at C-2 (Hexo)  . All the2O2 along with borneol, approximately in a 1∶1 stoichiometric ratio when P450cam was shunted with m-CPBA (217O m-CPBA and recombinant P450cam (0.1 µM) in 17O phosphate buffer  for 12 h to detect the formation of H217O2. To this assay mixture, P450cam (0.02 µM) and m-CPBA (0.2 mM) were added at 2 h intervals, to form detectable amounts of H217O2. A new resonance was observed at 178 ppm in the 17O NMR spectrum,  were run simultaneously, and this resonance was not detected  was added to the reaction mixture, the resonance at 178 ppm disappeared  of ∼50 (1H/2H) for hydrogen peroxide formation are much smaller, suggesting that this product does not form at the rate-limiting step  of ∼50  suggestsing step .KM = 699±88 µM and kcat = 426±20 min−1 for camphor  . Simultaneous transfer of the hydrogen atom from step III to the carbonyl group of camphor forms a borneol radical  H°∼−79±8 kJ/mol, Material S1, section 2.9). Finally, the transfer of a hydrogen atom from protonated Cpd II to the borneol radical forms borneol and Cpd I , completing the “borneol cycle”. The net reaction is endothermic, with ΔH° ≅ 305±8 kJ/mol  . The forJ/mol 13 , which iJ/mol 13 . Three wStep IV,. A non-s2O2 formation has been proposed previously for electrolytic catalysts that oxidize water to O2 (via an intermediate peroxide) 2O2 during electrolysis x catalyst stops the water oxidation process at H2O2, because the peroxide is solvated and stabilized by hydrogen bonding to ethylamine and/or water in the electrolyte. cam active site is essential for stabilization of the various reactive intermediates and of the H2O2 formed. The turnover numbers with regard to H2O2 formation we have observed are ∼7, whereas the electrocatalytic systems give turnover numbers of 20–1500 for complete water oxidation to O2. cam only has access to thermal energy to perform this “uphill” reaction, whereas the electrocatalytic systems are run at overpotentials. The involvement of OH-radicals in H2 levels: under high oxygenation, Cpd I sequentially hydroxylates camphor to 10, or 10 to 11 . A BLAST search against the P450cam sequence revealed many other bacterial cytochromes P450 that show sequence identities for the three residues that hold a set of water molecules above the porphyrin . Superposition of P450cam  under shunt conditions, 5-exo-hydroxy camphor formed as a major product. There were no detectable amounts of borneol, suggesting that the reduction cycle is specific to P450ediates.  The onlyediates. . Dockingediates. . This moet. al. have stated that mammalian P450s can reduce 4-hydroxynonenal to 1,4-dihydroxynonenal under low oxygen conditions, et. al. have stated that P450BM-3 from Bacillus megaterium can reduce p-methoxy-benzaldehyde to methoxy-benzalcohol Amunom In vitro assays with P450 under shunt conditions were performed with a free radical quencher (BHT), a free metal chelator (EDTA), catalase and superoxide dismutase, to determine whether free reactive oxygen species are involved in borneol formation   , entry 3ormation , entry 4ormation  can difformation , entry 5ormation , entry 6ocamphor , entry 7cam . The binding sites are good candidates for O2 binding because they are hydrophobic and distinct from the substrate access route 2 molecule bound near the heme could affect the reactivity of Cpd I.We have found a hydrophobic tunnel in P4502 binding site in P450cam is closer to the porphyrin than the equivalent site in CYP3A4, and the O2 binding site is lined by different residues  than that for borneol formation under low O2 concentration. This suggests that camphor binding and possibly positioning might be affected by O2 concentrations. Surprisingly, the kcat is the same for both reactions, even though there appears to be a larger barrier in the borneol cycle than in the normal oxidation reaction. This larger-than-expected kcat suggests that, consistent with the observed KIE, H-atom tunneling is occurring in the borneol cycle. Under high O2 concentrations using D2O as the solvent, 5-ketocamphor  was detected for borneol formation    and S9. cam, PdX, and PdR might be advantageous to P. putida during periods of low soil aeration. Because the camphor degradation pathway requires four O2/camphor , and the P450cam-catalyzed oxidation is the first committed step cam convert borneol back to camphor The borneol down-regulation of P450cam, a cycle that occurs at low O2 concentration. The cycle connects to the known catalytic cycle via Cpd I which is regulated by O2 levels: at low O2 concentration, Cpd I oxidises water, whereas at high O2 concentration, Cpd I oxidises camphor. Under low O2 concentrations, the slower formation of compound I and high energy barrier with tunneling could account for lower formation of borneol.We describe the borneol cycle of P450cam proposed here (2 reduction or shunt). The reaction occurs both in vitro (this paper) and in vivocam couples the oxidation of water to H2O2 and the reduction of camphor to borneol. We have presented evidence that: i) water is the source of the 2-H in the borneol; ii) water is oxidized to form H2O2 when camphor is reduced; and iii) the transfer of an H atom from water to C-2 of camphor occurs at a rate-limiting step in the borneol cycle. We propose that the reactivity of Cpd I is regulated by O2 concentration, and we have located a potential access channel where O2 might bind to P450cam to exert its allosteric control.The Cpd I-catalyzed reaction of P450sed here  is indep2O2 formed serve several ecological functions. First, borneol and H2O2 are not very toxic to P. putida, whereas the combination is lethal to bacteria such as E. coli which do not contain any P450 P. putida an advantage in bacterial communities. Secondly camphor induces the expression of P450, PdX, and PdR, whereas borneol decreases the expression of these gene products in P. putida. These features of the P450cam may protect the bacteria from excessive exposure to borneol and reactive oxygen species during prolonged periods of low oxygen concentration.The borneol and HFigure S117O NMR spectra of H217O2 (obtained by electrolysis of H217O) buffered at a) pH 10, b) pH 3, and c) pH 9.(TIF)Click here for additional data file.Figure S217O NMR spectra of the incubation mixture under shunt conditions using 1 mM m17O phosphate buffer  containing 1 mM substrate camphor and recombinant P450cam.-CPBA in : a) before and b) after addition of catalase (1 unit). The peak at 178 ppm corresponds to H217O2 and that at 0 ppm is due to H217O.(TIF)Click here for additional data file.Figure S3GC-MS traces of camphor incubated under shunt conditions using mcam, and b) with unreduced P450cam.-CPBA with: a) reduced P450 1-indanone was used as an internal standard.(TIF)Click here for additional data file.Figure S4a) Summary of the borneol cycle steps and of the net reaction. b) Possible routes by which the borneol cycle could end.(TIF)Click here for additional data file.Figure S5cam (upper portion) and of vertebrate class II P450s, also against P450cam (lower portion).Alignment of microbial cytochromes P450 against P450Microbial sequences used: gamma prot 1 = marine gamma proteobacterium HTCC2207 (ZP_01225512), Novo ar CYP = Novosphingbium aromaticivorans CYP 101D2 (PDB 3NV6), Sphingo echi = Sphingomonas echinoides ATCC14820 (ZP_10341012), Novo CYP 101D1 = a camphor hydroxylase from Novosphingobium aromaticivorans DSM 12444 (PDB 3LXI), Sphing chlor = Sphingomonas chlorophenolicum camphor hydroxylase (ZP_10341012), Azospir B510 = Azospirillium sp. B510 (YP_003451823), Azospir = (BAI74843), P450 Burk H160 = Burkholderia sp. H160 (ZP_03264429), P450 Burk MCO-3 Burkholderia cenocepacia MC0-3 = (YP_001774494), Sping Witt R = Sphingomonas wittichii RW1 (YP_001262244), Citromicrobi = Citromicrobium bathyomarinum JL354 (ZP_06860768), Novo CYP 101 = Novosphingobium aromaticivorans DSM12444 CYP 101C1 (PDB 3OFT_C), Sping E 14820 = Sphingomonas echinoides ATCC 14820 (ZP_10339023), gamma prot 2 = marine gamma proteobacterium NOR51-B (ZP_04956740), Sphingomonas = Sphingomonas sp. KC8 (ZP_09138048), Sphing chl L = Sphingobium chlorophenolicum L-1 (YP_004553185), P450 nor = Cytochrome P450nor from Fusarium oxysporum (BAA03390). Vertebrate P450s: Cyp lan deme = lanosterol 14-α demethylase isoform 1 precursor Homo sapiens (NP_000777), CYP 2C9 = human liver limonene hydroxylase (P11712), CYP 4A11 Homo sapiens (NP_000769), CYP 4F12 = fatty acyl Ω-hydroxylase Homo sapiens (NP_076433), CYP 4F2 = leukotriene-B(4) omega-hydroxylase 1 precursor Homo sapiens (NP_001073), CYP 3A5 form 1 = CYP 3A5 isoform 1 Homo sapiens (NP_000768), CYP 3A4 = CYP 3A4 isoform 1 Homo sapiens (NP_059488), CYP26B1 = retinoic acid hydroxylase Homo sapiens (NP_063938).(TIF)Click here for additional data file.Figure S650 determination of a) H2O2 and b) of a 1∶1 (molar) mixture of borneol and H2O2 against ICE. coli, a species of bacterium that lacks cytochrome P450.(TIF)Click here for additional data file.Figure S7Effect of 16 h incubation of stationary E. coli (a) and P. putida2O2 (1∶1), borneol, or H2O2 (1 mM). (b) cultures with borneol: H(TIF)Click here for additional data file.Figure S8Expression profile of PdX in P. putida, in the presence and absence of camphor or borneol . Points represent the average ± S. E. of three replicates.(TIF)Click here for additional data file.Figure S9Expression profile of PdR in P. putida, in the presence and absence of camphor or borneol . Points represent the average ± S. E. of three replicates.(TIF)Click here for additional data file.Table S1cam extinction coefficients at selected wavelengths.Calculated and literature values of P450(DOC)Click here for additional data file.Table S22O buffer, with the full P450cam system and with the shunted P450cam.Formation of 2-D-borneol and 5-ketocamphor in D(DOC)Click here for additional data file.Table S3Assays with recombinant proteins at selected temperatures. Formation of borneol, D-borneol, under shunt conditions with the addition of m-CPBA.(DOC)Click here for additional data file.Table S4cam, shunted with Assays with recombinant P450m2O and D2O at selected temperatures. Formation of H2O2 or D2O2.-CPBA in H(DOC)Click here for additional data file.Table S5Formation of borneol and hydrogen peroxide from the P450 catalytic cycle using several shunt agents.(DOC)Click here for additional data file.Material S1Supporting information for this paper. This file contains detailed descriptions of various assays, calculations, sequence alignments and P450cam system induction experiments. In addition, there are 9 supplemental Figures and 5 supplemental Tables.(DOC)Click here for additional data file.


Bacillus from chitin-containing wastes, production of a thermostable and alkaline chitinasese, and enzyme characterization. Bacillus thuringiensis subsp. kurstaki HBK-51 was isolated from soil and was identified. Chitinase was obtained from supernatant of B. thuringiensis HBK-51 strain and showed its optimum activity at 110°C and at pH 9.0. Following 3 hours of incubation period, the enzyme showed a high level of activity at 110°C (96% remaining activity) and between pH 9.0 and 12.0 (98% remaining activity). Considering these characteristics, the enzyme was described as hyperthermophile-thermostable and highly alkaline. Two bands of the enzyme weighing 50 and 125 kDa were obtained following 12% SDS-PAGE analyses. Among the metal ions and chemicals used, Ni2+ (32%), K+ (44%), and Cu2+ (56%) increased the enzyme activity while EDTA (7%), SDS (7%), Hg2+ (11%), and ethyl-acetimidate (20%) decreased the activity of the enzyme. Bacillus thuringiensis subsp. kurstaki HBK-51 is an important strain which can be used in several biotechnological applications as a chitinase producer.This paper reports the isolation and identification of chitinase-producing  Chitinase plays a variety of important roles in these organisms ranging from nutrition to defense and control of ecdysis in insects. The importance of chitinases in many biological processes makes their inhibitors important targets for potential antifungal and insecticidal agents as well as antimalarial agents [Chitin, a linear β-,4-linkedIt is one of the most abundant naturally occurring polysaccharides and has attracted tremendous attention in the fields of agriculture, pharmacology, and biotechnology. Chitin is the second most abundant component of biomass on earth . This liChitin-Azure was (Sigma-Aldrich), PMFS, Protein Marker (BSA), CBB R-250, DMSO and the others were purchased from Merck (Germany).−6 − 10−7 fold with freshly prepared LB, then streaked on N1 agar plate. After incubation at 37°C overnight, single colonies were selected  and stock cultures prepared [Crabs, campus soil, and compost were mixed and held to approximately 30 days outside . After this period, sterile distilled water was added to material and homogenized. 10 mL supernatant wae transferred to glass tube and incubated at 80°C for 10 min to eliminate vegetative forms of bacteria. 1 mL sample inoculated to 10 mL LB and incubated at 37°C for 6 hours and then sample was diluted 10Preparation of colloidal chitin was performed from commercial chitin  according to Wen et al.  with min4, 1.5 KH2PO4, 0.5 NH4Cl, 0.12 MgSO4, 0.005 CaCl2, 0.25 NaCl, pH = 6.5 [All strains  were selected from soil including chitin wastes, tested one by one for chitinase activity on the CHDA (chitinase-detection agar). Chitinase producer strains were determined after 3–5 days at 37°C by visualizing the clear zone formed surrounding of the colonies on the CHDA plate. CHDA agar, used first detection of chitinase positive strains and preparation of stock culture, by adding 10% g of colloidal chitin and 20 g of agar in M9 medium containing (g/L); 0.65 NaHPOpH = 6.5 .Bacillus thuringiensis subsp. kurstaki) was used as chitinase producer. 1 mL of HBK-51 strain fresh culture was inoculated into 10 mL of LB medium  and incubated overnight at 37°C with shaking (150 rev min−1). The culture was transferred into a 2 litres glass bottle containing 500 mL M9-Medium which was supplemented with 1% colloidal chitin and incubated at 37°C on a rotary shaker at 150 rev min−1 for 3 days [HBK-51 strain  at −33°C overnight. Et-OH  was added 70% of the original volume of supernatant. The precipitate was recovered by centrifugation  for 30 min.), dissolved in a small amount of 0.1 M Na-phosphate buffer (pH 7.0), and stored at 4°C , 11.Chitinase activity was measured with chitin-azure as a substrate . Enzyme The optimum pH and temperature were determined by incubation in a buffer at different pH values (3.0–10.0) and temperatures (30–120°C) under standard assay conditions using chitin-azure as the substrate. The buffers used were 50 mM sodium acetate buffer (pH: 3.0–6.0), 50 mM Tris-HCl buffer (pH: 7.0–9.0), and 50 mM Glycine-NaOH buffer (pH: 10.0–12.0). The optimum temperature for enzyme activity was measured under standard assay in the range of 30–120°C with intervals of 10°C . The effect of the temperature on the stability of chitinolytic enzyme was determined by exposure of the enzyme solution in 50 mM Tris-HCl buffer, pH: 9.0 (optimum pH) to different temperatures (30–110°C) for 3 h. The residual activity was then assayed under standard conditions using chitin-azure as the substrate , 15.For pH stability assay, the enzyme solution was preincubated for 3 h at room temperature in the buffers of various pH values (pH: 5.0–12.0) and then the residual activity was determined under standard conditions , 15.All bacteria which produced chitinase were tested with EtBr for plasmid elimination according to Hardy . Bacillus strain HBK-51 were based on morphological [Criteria used for classification and identification of ological , physiolological , fatty aological , 18, 19. 27F 5′-AGAGTTTGATCMTGGCTCAG-3′ (8–27 position),  530F 5′-GTGCCAGAGCMGCCGCGGTAA-3′ (515–533 position) and reverse primers:  1525R 5′-AAGGAGGTGWTCCRCC-3′ (1541–1525 position) 1100R 5′-AGGGTTGCGCTCGTTG-3′ (1115–1100 position). The amplified PCR product was sequenced by the Beckman Coulter-CQ 8800 model sequence analyzer with their methods. The 16S rDNA sequence was aligned with other 16S rDNA bacterial sequence obtained from GenBank by basic local alignment search tool (BLAST) program [The 16S rRNA gene was amplified by polymerase chain reaction (PCR) with forward primers: program .2SO3, NaCl, CaCl2, ZnCl2, MgCl2, BaCl2, FeCl3, MnCl2, CuCl2, CoCl2, NiCl2, KCl, HgCl2, and 1–5% SDS and DMSO for 30 min at room temperature after which the residual activity was measured with standard assay [The chemicals were used in two different concentrations . These crd assay . The enzyme molecular weight was determined with sodium dodecyl sulphate polyacrylamide gel electrophoresis (PAGE) . The gelChitinase producing bacteria were isolated from chitin wastes on CHDA  confirmed the isolate as being Bacillus genus and according to BLAST confirmation, it was Bacillus thuringiensis subsp. kurstaki. Theamplified16S rRNA sequence was submitted to GenBank for possible identification. The result showed 99.00% identity with Bacillus thuringiensis subsp. kurstaki. The accession number is EU153549. Chitinolytic activity of HBK-51 on CHDA is shown in The HBK-51 strain was identified according to morphological and physiological characteristics  with an optimum value 0f 8.0 and 40°C, respectively. Li et al. [Bacillus cereus strain CH2 had optimum activity at pH 7.1 and temperature at 40°C. According to these results, Bacillus thuringiensis subsp. kurstaki HBK-51 chitinase is a thermotolerant and alkaline enzyme. Guo et al. [Thermomyces lanuginosus SY2 chitinase exhibited optimum catalytic activity at pH 4.5, 55°C and enzyme was stable at 50°C and its half-life time at 65°C was 25 min. On the other hand, they reported that the thermostable chitinase had major advantages over industrial catalysis for its high activity at high temperature. Thermostable chitinase could be useful for the chitin industry and biotechnological applications.The pH effect on the chitinase activity was determined using three buffer systems at various pH values. The HBK-51 chitinase was active at broad range of pH 3.0–10.0) but had optimum activity at pH 9.0  enzyme activity lost by 95%. Yuli et al. [2, ethyl=acetimidate the enzyme activity was partially inhibited by 7%, 7%, 11%, 20%, respectively, but  and insects (bio-insecticide friend with environment) and bioconversion of chitin waste to single-cell protein, Et-OH and fertilizer .The thermostable chitinase had major advantages over industrial catalysis for its high activity at high temperature . The pro


Influenza viruses continuously undergo antigenic changes with gradual accumulation of mutations in hemagglutinin (HA) that is a major determinant in subtype specificity. The identification of conserved epitopes within specific HA subtypes gives an important clue for developing new vaccines and diagnostics. We produced and characterized nine monoclonal antibodies that showed significant neutralizing activities against H1 subtype influenza viruses, and determined the complex structure of HA derived from a 2009 pandemic virus A/Korea/01/2009 (KR01) and the Fab fragment from H1-specific monoclonal antibody GC0587. The overall structure of the complex was essentially identical to the previously determined KR01 HA-Fab0757 complex structure. Both Fab0587 and Fab0757 recognize readily accessible head regions of HA, revealing broadly shared and conserved antigenic determinants among H1 subtypes. The β-strands constituted by Ser110-Glu115 and Lys169-Lys170 form H1 epitopes with distinct conformations from those of H1 and H3 HA sites. In particular, Glu112, Glu115, Lys169, and Lys171 that are highly conserved among H1 subtype HAs have close contacts with HCDR3 and LCDR3. The differences between Fab0587 and Fab0757 complexes reside mainly in HCDR3 and LCDR3, providing distinct antigenic determinants specific for 1918 pdm influenza strain. Our results demonstrate a potential key neutralizing epitope important for H1 subtype specificity in influenza virus. Influenza is a viral infectious disease of the respiratory tract that affects millions of people annually Orthomyxoviridae have three antigenically distinct types of virus, A, B, and C Influenza viruses which belong to the family of The 2009 pdm viruses were derived from a reassortment of six gene segments from triple reassortant swine virus and two gene segments from Eurasian influenza A (H1N1) swine virus lineage HA is synthesized as a precursor, HA0, that trimerizes in the endoplasmic reticulum and is transferred through the Golgi apparatus to the cell surface During the production of H1-specific monoclonal antibodies against 2009 pdm H1N1 strains, we isolated and characterized nine H1-specific monoclonal antibodies which neutralized a broad range of H1 subtype influenza viruses. Among them, we determined the structure of the HA protein from a 2009 pandemic virus A/Korea/01/2009 (KR01) in complex with the Fab fragment from GC0587 and compared with the KR01 HA-Fab757 complex structure. In addition to GC0587, GC0757 exhibits additional activity against A/Brevig Mission/1/1918. The structural features of the complexes provide a understanding of how antibodies with subtype specificity can distinguish antigenic determinants.In our initial immunogenicity experiment, A/California/07/2009 (CA07) was used to immunize groups of BALB/c mice and antibody-producing cells were screened by ELISA for secretion of antibodies. Nine monoclonal antibodies were then tested for neutralization against a panel of 14 isolates including H1, 2009 pdm H1, H2, H3, H5, and H7 . Among td 2.5–8.9 nM). The dissociation constants were within the range typically required for effective neutralization, comparable to stem binding antibodies (Kd ∼10 nM) Examination based on ELISA assays for the antigenicity against KR01 HA revealed that GC0346, GC0587, GC0757, and GC1517 had significantly high affinity to HA , whereas2. A possible orientation of the HA2 domain can be derived from this partial model. When the KR01 HA structure was superimposed to that of the HA-Fab0587 complex, the head region of KR01 HA was found to be rotated clockwise by ∼20° relative to the stem region  of KR01 HA-Fab0587 complex was determined at 3.1 Å resolution . The asym region . We obsem region . The conm region .2, of which 48% arise from binding to the H-chain and 52% from the L-chain. In the Fab0757 complex, it is 993.2 Å2, with 50% from the H- and L-chains each. The buried surface areas are in the increasing order of LCDR1, HCDR2, LCDR3, and HCDR3, ranging from 230 to 300 Å2. LCDR3 and HCDR3 have the largest number of interactions in the HA-Fab0587 complex, whereas LCDR3 and HCDR2 have the largest in the HA-Fab0587 complex  and Vero cells (Korean Cell Line Bank 10081) were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 1% glutamine, and 1% penicillin-streptomycin  under 5% CO2 atmosphere at 37°C for 2–3 days. Virus titer was determined by using plaque assay (PFU/ml), TCID50, and hemagglutination assay and virus stocks were stored in deep freezer (−77°C) until experiments.H1N1 A/Thailand/CU44/2006 (CU44), A/Brisbane/59/2007 (BR59) and 2009 pdm KR01, and H3N2 A/Gyeongnam/684/2006 (Gy684) were propagated in MDCK or Vero cells with DMEM supplemented with 0.2% BSA, 25 mM HEPES, 1% glutamine, 1% penicillin-streptomycin, and 2 µg/ml of L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin  under 5% COFour-week old female Balb/c mice were immunized by intraperitoneal injection of 100 µg of 2009 pdm H1N1 A/California/04/2009 (CA04) vaccine dissolved in 100 µl PBS plus complete Freund’s adjuvant . Boost immunizations were given at 3 weeks post-vaccination. Animals were maintained and treated according to the Green Cross guidelines. All experiments in mice were approved by the Institutional Animal Care and Use Committee (IACUC) of the Green Cross.Mouse spleen cells collected at 4 days after the 5th immunization boost with vaccine were fused to the sp2/0 myeloma cells. Fused cells were distributed over 96 well tissue culture plates at 2,000 cells per well in complete DMEM medium containing 100 µM hypoxanthine, 0.4 µM aminopterin, and 16 µM thymidine (HAT). With weekly replacement of medium, antibody-producing cells were screened by ELISA and subcloned by limiting dilution. Ferret anti-BR59 whole serum was used as 1/50 dilution and monoclonal antibody was used at a final concentration of 80 µg/ml. Positive clones were checked for isotype by using Iso-Gold™ rapid mouse-monoclonal isotyping test kit  as described in the manufacturer’s protocol.Microtiter plates were coated with 100 µl of vaccine or its equivalent HA (5 µg/ml) diluted in PBS per well at 4°C overnight, and blocked with PBS containing 1% BSA and 0.05% Tween 20 at room temperature for 1 hr. One hundred µl of hybridoma supernatant or purified antibodies per well was transferred into the ELISA plates. Binding reaction was carried out at room temperature for 2 hrs. Plates were subsequently washed four times with PBS containing 0.05% Tween 20, and 100 µl of HRP-conjugated goat anti-human Fab  diluted 1∶10,000 in PBS was added, and reactions were carried out at room temperature for 1 hr. Plates were washed four times, and 100 µl of tetramethyl benzidine substrate  per well was added. The absorbance was determined at 490 nm.E. coli strain DH5α and then used to co-transfect Sf9 cells along with linearized baculovirus chromosomal DNA  by the calcium transfection method. The virus was harvested after 5 days, and transfection efficiency was confirmed by PCR after extraction of DNA from 400 µl of virus (cell supernatant). This original virus stock was amplified through repeated rounds of infection.HA gene were cloned downstream of the gp67 secretion signal sequence of the transfer vector pAcGP67A . Based on H3 numbering, the corresponding residues were 11–329 (1–327) (HA1) and 330–506 (1–176) (HA2), with a thrombin cleavage site, foldon region, and 6xHis-tag downstream of the HA gene sequence Baculovirus containing HA gene was used to infect suspension cultures of Hi5 cells. After 3∼4 days at 28°C, the culture medium was harvested and applied to a Ni-NTA column equilibrated with working buffer . The column was washed with buffer containing 50 mM imidazole and precursor HA protein was eluted in an imidazole gradient, which was dialysed against 10 mM Tris-HCl (pH 8.0) and 50 mM NaCl, and hydrolysed by thrombin for 12 hrs at 4°C for removal of the foldon region and 6xHis-tag. The reaction was halted by the addition of 1 mM phenylmethylsulphonyl fluoride, and the active form of HA was purified by Mono Q ion-exchange chromatography and Superdex 200HR size exclusion chromatography.). Each was then treated with papain at a ratio of 1∶50 (w/w) for 2 hrs in PBS buffer  with 5 mM EDTA and 5 mM L-cysteine, and the reaction was stopped with 30 mM crystalline iodoacetamide. The solution containing Fab fragments was dialyzed against the PBS buffer and applied to protein A column. The unbound fraction was purified by Superdex 200HR gel filtration chromatography. It was then mixed with HA at a 1∶1.5 molar ratio for 12 hr incubation, and the complex was further purified by Superdex 200HR gel filtration chromatography.The neutralizing antibodies with activity against H1 subtype influenza viruses, GC0346, GC0587, GC0757, and GC1517, were purified using protein A columns . The crystals of HA-Fab0587 diffracted to 2.8 Å resolution, which belong to space group P21 with unit cell dimensions a = 72.8 Å, b = 237.8 Å, c = 94.2 Å, α = 90°, β = 110.3°, γ = 90° (The complex of HA and Fab0587 was screened by hanging drop vapour diffusion method. 1 µl of the 10 mg/ml complex in 20 mM Tri-HCl (pH 8.0) and 50 mM NaCl was mixed with 1 µl of screening solution of 20% PEG3350 and 200 mM potassium iodide and incubated at 4°C. After one week, small rhombic crystals were obtained. Diffraction data were collected with the crystals flash-cooled at 100 K in a stream of liquid N γ = 90° . All dato-Fc map peaks above 3.0σ, if the B factors were below 50 Å2 after refinement. The Rfree value was used as an indicator to validate the water picking and refinement procedure and to guard against possible overfitting of the data The crystal structure of KR01 HA-Fab0587 was solved by molecular replacement using the KR01 HA-Fab0757 structure (PDB ID 4F15) as a template using CCP4 www.rcsb.org) with PDB ID code 4LVH for H1N1 HA-Fab0587 structure.Data deposition−The atomic coordinates and structure factors have been deposited in the Protein Data Bank  of HA is shown in red color and Fab in blue in one complex, and others are in green. The stem regions (HA2) of HA could not be modeled due to weak electron density, but there are empty space for them. A partial model for HA2 is shown in the lower panel. Figure S2, Sequence alignments of HAs. Epitopes in H1 HAs are highlighted in red, and more conserved residues are highlighted in more red. Residues in other HA those are same with that of epitope in H1 HAs are indicated by red boxes. Potential glycosylation sites are highlighted in green. Figure S3, LCDR1 at Fab binding site. Residues involved in interactions between HA and Fab0587 are represented as a ball-and-stick model and hydrophilic interactions as dotted line. Residues of HA in complex with Fab0587 are colored in orange and H-chains of Fab0587 and Fab0757 are in dark and light blue, respectively. Figure S4, HCDR2 at Fab binding site. Residues involved in interactions between HA and Fab0587 are represented as a ball-and-stick model and hydrophilic interactions as dotted line. Residues of HA in complex with Fab0587 are colored in orange and H-chains of Fab0587 and Fab0757 are in dark and light blue, respectively. Figure S5, Comparison of novel H1 specific epitope regions among different subtypes. Superposition of the novel epitope conformations of H1 (blue), H2 (gray), H3 (dark gray), and H5 (green) HA. RMSD was calculated based on secondary structure matching of head domain of H1 with that of other subtypes, to show 1.19, 1.68 and 1.34 Å for H2, H3 and H5, respectively.(DOCX)Click here for additional data file.