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Both protein kinase C (PKC) and reactive oxygen species (ROS) are well-known signaling messengers cross-talking with each other to activate mitogen-activated protein kinases (MAPKs) for progression of hepatocellular carcinoma (HCC). However, the underlying mechanisms are not well elucidated. Especially, whether mitochondrial ROS (mtROS) is involved and how it triggers MAPK signaling are intriguing. In this study, we found mtROS generation and phosphorylation of MAPKs were mediated by PKCδ in HCCs treated with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Heat shock protein 60 (HSP60), one of the chaperones in mitochondria was the major protein oxidized in TPA-treated HCCs. Moreover, depletion of HSP60 or expression of HSP60 cysteine mutant prevented TPA-induced phosphorylation of MAPKs. To delineate how HSP60 mediated MAPK activation, the role of Raf kinase inhibitor protein (RKIP), a negative regulator of MAPK, was investigated. TPA dissociated RKIP from HSP60 in both mitochondria and cytosol, concurrently with translocation of HSP60 and MAPK from mitochondria to cytosol, which was associated with robust phosphorylation of MAPKs in the cytosol. Moreover, TPA induced opposite phenotypical changes of HCCs, G1 cell cycle arrest, and cell migration, which were prevented by mtROS scavengers and depletion of PKCδ and HSP60. Consistently, TPA increased the migration-related genes, hydrogen peroxide inducible clone5, matrix metalloproteinase-1/3, lamininγ2, and suppressed the cell cycle regulator cyclin E1 (CCNE1) via PKCδ/mtROS/HSP60/MAPK-axis. Finally, c-jun and c-fos were required for TPA-induced expression of the migration-related genes and a novel microRNA, miR-6134, was responsible for TPA-induced suppression of CCNE1. In conclusion, PKCδ cross-talked with mtROS to trigger HSP60 oxidation for release of RKIP to activate MAPK, regulating gene expression for migration, and G1 cell cycle arrest in HCC. Targeted therapy aiming at key players like PKCδ, RKIP, and HSP60 is promising for preventing HCC progression.

SNA is one of the essential EMT transcriptional factors capable of suppressing epithelial maker while upregulating mesenchymal markers. However, the mechanisms for SNA to transactivate mesenchymal markers was not well elucidated. Recently, we demonstrated that SNA collaborates with EGR1 and SP1 to directly upregulate MMP9 and ZEB1. Remarkably, a SNA-binding motif (TCACA) upstream of EGR/SP1 overlapping region on promoters was identified. Herein, we examined whether four other mesenchymal markers, lymphoid enhancer-binding factor (LEF), fibronectin (FN), cyclooxygenase 2 (COX2), and collagen type alpha I (COL1A1) are upregulated by SNA in a similar fashion. Expectedly, SNA is essential for expression of these mesenchymal genes. By deletion mapping and site directed mutagenesis coupled with dual luciferase promoter assay, SNA-binding motif and EGR1/SP1 overlapping region are required for TPA-induced transcription of LEF, FN, COX2 and COL1A1. Consistently, TPA induced binding of SNA and EGR1/SP1 on relevant promoter regions of these mesenchymal genes using ChIP and EMSA. Thus far, we found six of the mesenchymal genes are transcriptionally upregulated by SNA in the same fashion. Moreover, comprehensive screening revealed similar sequence architectures on promoter regions of other SNA-upregulated mesenchymal markers, suggesting that a general model for SNA-upregulated mesenchymal genes can be established.

Background Snail (SNA) is responsible for epithelial mesenchymal transition, migration and metastasis of hepatocellular carcinoma. SNA represses the transcription of the essential epithelial marker such as E-cadherin and enhances mesenchymal markers including fibronectin and lymphoid enhancer-binding factor. Our previous studies indicated that SNA, in collaboration with EGR1 and SP1, may directly activate transcription of the mesenchymal markers, matrix degradation enzyme matrix metalloproteinases (MMP9) and zinc finger E-box binding homeobox 1 (ZEB1) in HepG2 cell stimulated by the phorbol ester tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA). Besides, we pinpointed a SNA binding motif (TCACA) upstream of EGR1/SP1 overlapping region on promoters. In this study, we investigated whether LEF and FN are transcriptionally regulated by SNA in a similar fashion. Moreover, a general model for SNA-upregulated mesenchymal markers is proposed.Methods RT/PCR and Western blot were used for analyzing gene expression and shRNA technology for depleting SNA. Dual luciferase assay was used for promoter activation; deletion mapping and mutagenesis were used for confirming the indicated promoter region required for transcription activation. ChIP and EMSA were used for validating the binding of the indicated transcription factor on their putative motifs. Results SNA binding motif and E/S overlapping region are required for TPA-induced transcription of LEF and FN. These were supported by TPA-induced binding of SNA and EGR-1/SP-1 on indicated promoter regions. Moreover, a peroxisome proliferator-activated receptor γ motif upstream of SNA binding motif was found to be a negatively regulatory region in TPA-induced promoter activation of FN, LEF, MMP9 and ZEB1. This was supported by that co-treatment of a PPAR-g inhibitor, GW9662, and mutation of PPAR-g binding motif enhanced TPA-induced promoter activity and expression of the aforementioned genes whereas overexpression of PPAR-g reversed it. Moreover, comprehensive screening of the SNA-upregulated mesenchymal genes revealed similar sequence architecture on the promoter regions of the candidate genes: SNA binding motif (TCACA) coupled with a downstream EGR/SP1 overlapping region and an upstream PPAR-g binding motif. Among them COX2 and COL1A1 were found to potentially exhibit the same transcription mechanisms described above. Conclusions We established a general model for SNA-upregulated mesenchymal gene expressions negatively feed backed by PPAR-g.

Background Hematopoiesis occurs in the bone marrow, producing a complete spectrum of blood cells to maintain homeostasis. In addition to light microscopy, chromosome analysis, and polymerase chain reaction, flow cytometry is a feasible and fast method for quantitatively analyzing hematological diseases. However, because sufficient specific cell markers are scarce, dyserythropoietic diseases are challenging to identify through flow cytometry. Methods Bone marrow samples from C57BL/B6 mice and one healthy donor were analyzed using traditional two-marker (CD71 and glycophorin A) flow cytometry analysis. After cell sorting, the gene expressions of membrane proteins in early and late erythropoiesis precursors and in nonerythroid cells were characterized using microarray analysis. Results Among characterized gene candidates, aquaporin 0 (AQP0) expressed as a surface protein in early- and late-stage erythropoiesis precursors and was not expressed on nonerythroid cells. With the help of AQP0 staining, we could define up to five stages of erythropoiesis in both mouse and human bone marrow using flow cytometry. In addition, because patients with dyserythropoiesis generally exhibited a reduced population of APQ0high cells relative to healthy participants, the analysis results also suggested that the levels of APQ0high cells in early erythropoiesis serve as a novel biomarker that distinguishes normal from dysregulated erythropoiesis. Conclusions AQP0 was successfully demonstrated to be a marker of erythroid differentiation. The expression levels of AQP0 are downregulated in patients with dyserythropoiesis, indicating a critical role of AQP0 in erythropoiesis. Accordingly, the level of AQP0high in early erythroid precursor cells may serve as a reference parameter for diagnosing diseases associated with dyserythropoiesis.

Metabolic regulations play vital roles on maintaining the homeostasis of our body. Evidence have suggested that ATF3 and nuclear factor erythroid 2-related factor 2 (NRF2) are critical for maintaining cell function, metabolism, and inflammation/anti-inflammation regulations when cells are under stress, while the upstream regulators in the stressed cells remain elusive. Recent findings have shown that tricarboxylic acid cycle metabolites such as itaconate and succinate are not just mitochondrial metabolites, but rather important signaling mediators, involving in the regulations of metabolism, immune modulation. Itaconate exerts anti-inflammatory role through regulating ATF3 and NRF2 pathways under stressed conditions. In addition, itaconate inhibits succinate dehydrogenase, succinate oxidation and thus blocking succinate-mediated inflammatory processes. These findings suggest itaconate-ATF3 and itaconate-NRF2 axes are well-coordinated machineries that facilitate the rescue against cellular stress. Here, we review these fascinating discoveries, a research field may help the development of more effective therapeutic approach to manage stress-induced inflammation, tissue damage, and metabolic disorder.

Laser based spectroscopic methods can be versatile tools in investigating early stage mammalian embryo structure and biochemical processes in live oocytes and embryos. The limiting factor for using the laser methods in embryological studies is the effect of laser irradiation on the ova. The aim of this work is to explore the optimal parameters of the laser exposure in Raman spectroscopic measurements applicable for studying live early embryos in vitro without impacting their developmental capability. Raman spectra from different areas of mouse oocytes and 2-cells embryos were measured and analyzed. The laser power and exposure time were varied and further embryo development was evaluated to select optimal conditions of the measurements. This work demonstrates safe laser irradiation parameters can be selected, which allow acquisition of Raman spectra suitable for further analysis without affecting the early mouse embryo development in vitro up to morphologically normal blastocyst. The estimation of living embryo state is demonstrated via analysis and comparison of the spectra from fertilized embryo with the spectra from unfertilized oocytes or embryos subjected to UV laser irradiation. These results demonstrate the possibility of investigating preimplantation mammalian embryo development and estimating its state/quality. It will have potential in developing prognosis of mammalian embryos in assisted reproductive technologies.

Background Red blood cells are the most abundant cells in the blood that deliver oxygen to the whole body. Erythropoietin (EPO), a positive regulator of erythropoiesis, is currently the major treatment for chronic anemia. Granulocyte colony-stimulating factor (G-CSF) is a multifunctional cytokine and a well-known regulator of hematopoietic stem cell proliferation, differentiation, and mobilization. The use of EPO in combination with G-CSF has been reported to synergistically improve erythroid responses in a group of patients with myelodysplastic syndromes who did not respond to EPO treatment alone; however, the mechanism remains unclear. Methods C57BL/6 J mice injected with G-CSF or EPO were used to compare the erythropoiesis status and the efficiency of erythroid mobilization by flow cytometry. Results In this study, we found that G-CSF induced more orthochromatophilic erythroblast production than did EPO in the bone marrow and spleen. In addition, in contrast to EPO treatments, G-CSF treatments enhanced the efficiency of the mobilization of newly synthesized reticulocytes into peripheral blood. Our results demonstrated that the effects of G-CSF on erythropoiesis and erythrocytic mobilization were independent of EPO secretion and, in contrast to EPO, G-CSF promoted progression of erythropoiesis through transition of early stage R2 (basophilic erythroblasts) to late stage R4 (orthochromatophilic erythroblasts). Conclusions We demonstrate for the first time that G-CSF treatments induce a faster erythropoiesis-enhancing response than that of EPO. These findings suggest an alternative approach to treating acute anemia, especially when patients are experiencing a clinical emergency in remote areas without proper blood bank supplies.

Parkinson's disease (PD), among the most common neurodegenerative diseases worldwide for which there is no cure, is characterized as progressive dopaminergic neuron loss in the substantia nigra through an unknown mechanism. Administering 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) causes neuronal cell death and Parkinsonism in humans. Commonly used in animal models of PD, MPTP can metabolize to 1-methyl-4-phenylpyridinium (MPP(+)); however, the detailed mechanism through which MPP(+) causes neuronal cell death remains undetermined. Previous reports have indicated those knockout mice with Bcl-2 associated protein X (Bax) or caspase-2, two mitochondrial outer membrane permeabilization inducers, are resistant to MPTP administration, suggesting that mitochondria are involved in MPP(+)-triggered apoptosis. Our previous study showed that MPP(+)-triggered apoptosis can be distinguished from spontaneous apoptosis of primary cortical neurons. In the present study, we verified the involvement of mitochondria in MPP(+)-induced and spontaneous apoptosis in cortical neurons through confocal microscope analysis. We demonstrated that caspase-2 activation is specific to MPP(+)-induced apoptosis and occurs before Bax translocation to the mitochondria. Caspase-2 activation is one of the few early molecular events identified in PD models.

Intravenous immunoglobulin (IVIg) is an effective treatment against immune thrombocytopenia (ITP). Previous studies suggested that IVIg exerts this ameliorative role through 2 different leukocyte subsets. Dendritic cells (DCs) modulate the immunosuppression in an adoptive cell transfer model, and phagocytes up-regulate their inhibitory IgG Fc receptors (FcγR)IIB expression and thereby ameliorate the inflammatory response and platelet clearance. However, whether or not regulatory mechanisms exist among DCs, phagocytes, and platelets is still largely unknown. In this study we present findings that IVIg-primed splenic CD11c+ DCs (IVIg-DCs) primarily mediate their anti-inflammatory effects at the level of the platelet rather than the phagocyte. IVIg-DCs did not ameliorate ITP in Fcgr2b−/−, Fcgr3−/−, nor P-Selp−/− mice, implicating the potential involvement of these pathways in IVIg action. As platelets are a component of DC regulatory circuits, these findings may suggest an alternative perspective for the use of IVIg treatment.

Snake venom rhodostomin, which belongs to the disintegrin family, binds to mammalian integrins with a high affinity through its Arg-Gly-Asp motif. Soluble rhodostomin served as an integrin inhibitor by competing with the integrin-ligand interaction, whereas the immobilized rhodostomin served as an integrin activator via cross-linking the integrin on cell surfaces. Based on these characteristics, rhodostomin was used either as an antithrombosis agent or, on the other hand, as an integrin agonist to trigger cell activation. Recently, rhodostomin was used to study integrin functions in many aspects, including cell signaling, integrin-mediated endocytosis, and the antithrombosis effects in infectious disease models. Results from these studies indicated that rhodostomin is a powerful tool to dissect the function of integrins.

Established from the calvaria of newborn macrophage colony-stimulating factor (M-CSF)-deficient mice, OP9 is a stromal cell line that used as a feeder layer to support the in vitro differentiation of pluripotent stem cells into various hematopoietic lineage cells, including granulocytes, erythrocytes, lymphocytes, and megakaryocytes. However, as a primary culture cell line, OP9 can be used as stromal cells for only 1 month. Therefore, to obtain functional OP9 cells, numerous M-CSF-deficient newborn mice must be sacrificed. These limitations in some ways restrict the application of OP9 cells in longterm and largescale experiments. In this study, we used human papillomavirus 16 E6 and E7 genes to generate immortalized OP9 stromal cells, designated I-OP9 cells, and then tested their ability to support the megakaryocytic differentiation of pluripotent stem cells in vitro. I-OP9 cells have similar morphology and properties as do parental OP9 cells, and, as expected, have an extended lifespan and can support megakaryocytic differentiation. Our data suggest that the method used in this study, including establishing I-OP9 cells, enables the possibility to enlarge and lengthen the scale of the experiment and, more critically, provides a humanistic approach for preparing stromal cells that support the hematopoietic differentiation of pluripotent stem cells in vitro.

Anthrax lethal toxin (LT), one of the primary virulence factors of Bacillus anthracis, causes anthrax-like symptoms and death in animals. Experiments have indicated that levels of erythrocytopenia and hypoxic stress are associated with disease severity after administering LT. In this study, the granulocyte colony-stimulating factor (G-CSF) was used as a therapeutic agent to ameliorate anthrax-LT- and spore-induced mortality in C57BL/6J mice. We demonstrated that G-CSF promoted the mobilization of mature erythrocytes to peripheral blood, resulting in a significantly faster recovery from erythrocytopenia. In addition, combined treatment using G-CSF and erythropoietin tended to ameliorate B. anthracis-spore-elicited mortality in mice. Although specific treatments against LT-mediated pathogenesis remain elusive, these results may be useful in developing feasible strategies to treat anthrax.

Platelets are an ideal model for studying a rapid morphological change in response to various signal transduction systems. Morphological changes via the activation of integrin alphaIIbbeta3 in platelets have been investigated intensively. In contrast, activation via integrin alpha6beta1 is less well studied. Here, we provide the first biochemical evidence that integrins alpha6beta1 and alphaIIbbeta3 of platelets are associated with different membrane proteins. We also demonstrate that platelets activated by integrin alpha6beta1 show dynamic change by actively forming filopodia and never fully spreading over a period of more than an hour. In addition, platelets activated by integrin alpha6beta1 are different from those activated by integrin alphaIIbbeta3 in terms of cell-substrate contact and in their distribution pattern of actin, Arp2/3 and various phosphotyrosine proteins. The morphological appearance of platelets produced through integrin alpha6beta1 activation is highly dependent on PI3 kinase (PI3K) but less dependent on Src kinase. Suppression of PI3K activity in integrin alpha6beta1 activated platelets induces an increase in Cdc42 activity and more filopodium formation. However, both Cdc42 and PI3K activity are higher in platelets activated by integrin alpha6beta1 than in those activated by integrin alphaIIbbeta3. Taken together, this study demonstrates that the signals induced by integrin alpha6beta1 modulate at the level of PI3K and Cdc42 activity to allow platelets to actively form filopodia.

Cell-adhesion and spread are important for cell survival. Although extensive studies have suggested several potential mechanisms of action, it is not yet clear how important cell-morphological change per se contributes to the cell-surviving signal. We employed a non-integrin-mediated cell-adhesion system to explore this question. BHK-Japanese encephalitis virus (JEV) cells (BHK21 cells that are persistently infected with JEV) express a large amount of JEV-envelope protein (JEV E) on their surfaces, and can attach and form pseudopodia on the anti-JEV E antibody-coated substrates. However, cells that adhered on the antibody substrate underwent a caspase-3-mediated apoptosis together with a down-regulation of mitogen-activated protein kinase activity within 20 h after adhesion, which indicates that viral-protein-mediated cell-adhesion and cell-spread are not sufficient for supporting cell survival. This provides a different perspective for the study of the relationships between the cell-morphological change and the cell-survival signal.

MPTP (1-methyl-1,2,3,6-tetrahydropyridine), a chemical contaminant of synthetic heroin, induces neuropathological changes with clinical features similar to idiopathic Parkinson's disease. The mechanism by which MPTP and its metabolite MPP + (1-methyl-4-phenylpyridinium) induces neuronal cell death remains unclear. We employed primary cortical/telencephalon neuronal cultures to investigate the potential role of caspase and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) pathways in MPP + -induced neuronal death. DNA fragmentation and caspase-3 activity analysis showed that cortical neuronal cells underwent apoptosis after MPP + treatment. However, a basal level of apoptotic cells was also observed in untreated cultures. Interestingly, JNK activity increased in untreated cultures over time, whereas it was down-regulated after MPP + treatment. This indicates that the JNK pathways could be differentially regulated in different apoptotic processes.

Rhodostomin (RHO), a disintegrin isolated from snake venom, has been demonstrated to inhibit platelet aggregation through interaction with integrin αIIbβ3, but there is a lack of direct evidence for RHO–integrin αIIbβ3 binding. In addition, no study on the length of Arg49-Gly50-Asp51 (RGD) loop of RHO influencing on its binding to integrin αIIbβ3 has been reported. In the present study we have developed a highly sensitive dot-blot and glutathione S-transferase–RHO pull-down assays; the latter was coupled with a biotin–avidin–horseradish peroxidase enhanced-chemiluminescence detection system. These were able to demonstrate the direct binding of RHO to integrin αIIbβ3. The pull-down assay further showed that four alanine-insertion mutants upstream of the RGD motif and three insertions downstream of the RGD were able to decrease integrin αIIbβ3 binding activity to only a limited extent. By contrast, two insertions immediately next to RGD and one insertion in front of the Cys57 caused almost complete loss of binding activity to αIIbβ3. The results of the platelet-aggregation-inhibition assay and platelet-adhesion assay for the insertion mutants were consistent with results of the pull-down assay. It is thus concluded that, although an insertion of a single alanine residue in many positions of the RGD loop has only minor effects on RHO binding to integrin αIIbβ3, the specific position of Pro53 residue adjacent to the RGD sequence is important for RHO binding to platelet integrin αIIbβ3.

Previously, we developed a TraT display system to express snake venom rhodostomin (RHO), a disintegrin, on the external surface of Escherichia coli [J Biomed Sci 6:64-70;1999]. To show a new potential use of the TraT display system, we employed a biotin labeling technique coupled with SDS-PAGE and flow cytometry analyses to further demonstrate and confirm the expression of TraT-RHO on the E. coli surface. We also showed that the expression of TraT-RHO on the cell surface not only facilitated the bacteria adhesion to BHK-21 cells but also induced bacterial internalization into BHK-21 cells. This feature allowed us to enrich the TraT-RHO expression bacteria about 10,000-fold starting with a mixture of TraT-RHO bacteria with beta-galactosidase-positive bacteria in a ratio of 10(2):10(7) through four cycles of BHK-21 cell endocytosis and replating of engulfed bacteria on agar plates. We therefore suggest that the TraT display system can be applied to select out bacteria expressing a specific peptide sequence from a large population of display library through the process of receptor-mediated endocytosis and reamplification cycles.

We have previously reported that non-activated platelets can be induced by morphological changes from the recombinant fusion protein of GST-rhodostomin [GST-RHO(RGD)], a member of disintegrin with an arginine-glycine-aspartic acid (RGD) motif. In this study, we further characterized the factors involved in platelet shape changes induced by rhodostomin. From less to full-spreading, four cell spreading indexes, p1, p2, s1 and s2, were designated to the platelet shape based on the scanning electron micrographs. Results of peptide competition and antibody blocking confirmed that interaction between the RGD of rhodostomin and the alpha(IIb)beta3 integrins of platelets was required for induction of a higher percentage of s2 cells. When platelets were pretreated with calphostin C, herbimycin A and cytochalasin B, respectively, the percentage of p1 and p2 cells on rhodostomin-coated plates was increased and, concomitantly, the percentage of s1 and s2 cells was decreased. Biochemical analyses indicated that the focal adhesion kinase (FAK or pp125FAK) in platelets that adhered to GST-RHO(RGD) was phosphorylated in contrast to little or no phosphorylation of FAK in cells adhered to fibrinogen or non-activated cells. Furthermore, the degree of FAK phosphorylation was consistently correlated with morphological changes in platelets treated with various drugs. Taking all the results together, we suggested that rhodostomin could directly bind to integrins of platelets and then trigger signal transduction leading to FAK phosphorylation and actin polymerization and finally resulting in platelet full-spreading.

Rhodostomin (RHO) from Agkistrodon rhodostoma venom, consisting of 68 amino acids with an arginine-glycine-aspartic acid (RGD) sequence and 12 cysteine residues, is a potent inhibitor of platelet aggregation. We previously demonstrated that cell culture plates coated with the bacterially produced fusion protein of glutathione S-transferase-RHO [GST-RHO(RGD)] can facilitate human hepatoma cell attachment via intergrin interaction within 15 min. In this study, we further characterized the effect of RHO fusion protein on platelet cells by creating two other related fusion proteins, GST-RHO(RGE) and GST-(PS)RHO. The former was a single amino acid-substituted mutant, in which the aspartic acid residue of RGD was replaced by glutamic acid, and the latter was an insertion mutant, in which a pentapeptide of protein kinase A phosphorylation site was inserted between GST and RHO. These two mutant proteins together with a wild-type of GST-RHO(RGD) and native form of RHO were used to study effects on the inhibition of ADP-induced platelet aggregation. Results indicated that GST-RHO(RGD) inhibited platelet aggregation as potently as the native RHO, while the two other mutants were inactive. Furthermore, when unactivated platelet cells attached on the GST-RHO(RGD)-coated plate, they became a flattened pancake shape. From the results of facilitation of cell attachment on fusion protein-coated plates, we concluded that: (1) the GST-RHO(RGD) fusion protein is equally functional in inhibition of platelet aggregation and facilitation of cell attachment, which is through the interaction of RGD and integrins on the cell membrane; (2) the GST-RHO(RGE) mutant protein is unable to bind with integrins and results in loss of function; (3) the insertion mutant of GST-(PS)RHO may disrupt a proper conformation of RHO and also results in loss of function; (4) the bacterially produced fusion protein GST-RHO(RGD) can be properly used as an antithrombotic agent and an extracellular matrix.

Rhodostomin (Rho) from snake venom, a potent inhibitor of platelet aggregation, contains 68 amino acids having an ROD sequence and 12 cysteine residues. A chemically synthesized Rho gene was cloned and expressed in Escherichia coli . The expression of Rho gene fused with the glutathione S-transferase (GST) gene was about 10-30% of total cell proteins. The Rho-fusion protein could be recognized by antibodies raised against either a native Rho peptide or a synthetic peptide. The purified GST-Rho coated on culture plates facilitated the attachment of human hepatoma cells, which was inhibitable by co-incubation with a synthetic hexapeptide GRGDSP but not with a related peptide of GRGESP, suggesting that the E. coli -expressed Rho-fusion protein was properly folded and biologically functional.

Phosphatidylinositol 3-kinase (PI3K) pathway is important for platelet activation. Recent studies showed that PI3K and oscillative calcium could cross talk to each other and positively regulate integrin alpha (IIb)beta3-mediated outside-in signaling. However, the mechanism of this feedback regulation remains to be further characterized. Here we found that treatments of both PI3K inhibitor wortmannin and P2Y1 inhibitor A3P5P could inhibit granular secretion in platelets. Additionally, when RGD-substrate adherent platelets were treated with the ADP scavenger apyrase to deplete the granular-released ADP, their attachments in engaging with substrates became looser and the frequency of calcium oscillation decreased. Since it is known that ADP stimulates the PI3K and calcium signal primarily through P2Y12 and P2Y1 receptors respectively, our data indicated that integrin alpha(IIb)beta3 downstream PI3K and calcium activation might be not completely coupled to integrin associated signaling complex, but in part through feedback stimulation by granular released ADP. Our data indicates the important roles of PI3K and granular released ADP in coordinating the feedback regulations in integrin alpha(IIb)beta3-mediated platelet activation.

The frequency of calcium oscillation reveals the platelet activation status, however, the biological significance of the periodic calcium responses and methods of communication with other integrin-mediated signals are not clear. RGD-containing disintegrin rhodostomin coated substrates were employed to enhance platelet spreading and calcium oscillation through direct binding and clustering of the receptor integrin alpha(IIb)beta3. The results showed that the activation of phosphatidylinositol 3-kinase (PI3-K) and internal calcium pathways were crucial for alpha(IIb)beta3 outside-in signaling. PI3-K antagonists wortmannin and LY294002 inhibited disintegrin substrates and induced platelet spreading and calcium oscillation. At the same time, pretreatment of platelets with the microsomal calcium-ATPase inhibitor thapsigargin to deplete internal calcium stores severely impaired the calcium oscillation as well as PI3-K activation and spreading on disintegrin substrates. Because inhibition of one pathway could inhibit the other, our data indicates that PI3-K and calcium oscillation are synergistically operated and form a positive-feedback regulation in integrin alpha(IIb)beta3-mediated outside-in signaling.

Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igkappa leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins.

Platelet activation has been a focus of numerous studies in normal and abnormal states. Morphological changes and calcium signals found with activated platelets in vitro have been well characterized. However, the rate of cell spreading on substrates and the frequency of calcium oscillation within individual platelets upon activation have not yet been reported. In this study, we first examined the ability of a recombinant fusion protein of rhodostomin (GST-rhodostomin), a snake disintegrin containing an Arg-Gly-Asp (RGD) motif, to activate platelets when GST-rhodostomin served as a substrate. Four aspects of platelet activities induced by immobilized GST-rhodostomin and fibrinogen were analyzed in parallel. Examinations of (1) translocation of P-selectin from intracellular compartments to the plasma membrane, (2) platelet adhesion to and spreading on substrates, (3) platelet contact pattern on substrates, and (4) the degree of phosphorylation of focal adhesion kinase in platelets indicated that GST-rhodostomin was a better substrate for platelet activation than fibrinogen. Analysis of the rate of platelet spreading on GST-rhodostomin was examined by time-lapsed video microscopy. The spreading rate averaged 0.43 micrometer/minute, while cell spreading averaged 0.22 micrometer/minute when platelets were plated on fibrinogen and treated with thrombin. A newly developed method, using time-lapsed microscopy and the Metamorph program, was used to analyze calcium signals within platelets. We found that platelets on GST-rhodostomin evoked calcium oscillation at a frequency of 4.77 spike/cell/minute vs 2.76 spike/cell/minute on fibrinogen. The results of cell spreading and calcium oscillation were consistent with the results of microscopic and biochemical assays. We therefore conclude that the determination of the rate of platelet spreading and the frequency of calcium oscillation within platelets performed in this study provides more quantitative parameters for measuring platelet activities. Our results also suggest that GST-rhodostomin might potentially be used as a probe to dissect the molecular mechanisms underlying the kinetic processes of platelet activation.

The traT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between an Escherichia coli and its surroundings. This protein was altered so as to permit the expression of foreign proteins on the outer membrane of E. coli in this study. A 729-bp DNA fragment, including the leader and entire structural gene sequence of traT, was amplified and obtained by PCR. This sequence was then subcloned downstream of the tac promoter of pDR540, resulting in a TraT expression vector, pT2. Here, we report that the expression of TraT protein, fused either with a partial pre-S antigen of hepatitis B virus (60 and 98 amino acids, respectively) or with the snake venom rhodostomin (72 amino acids), was successfully achieved on the outer membrane of E. coli, using the pT2 plasmid. This result was demonstrated using dot blot and immunofluorescence analysis. This finding supports the notion that the pT2 plasmid can be used as an E. coli display system. This system can detect a foreign peptide of about 100 amino acid residues in length on the bacterial surface.

Rhodostomin from venom of Agkistrodon rhodostoma (also called Calloselasma rhodostoma) contains 68 amino acid residues including 6 pairs of disulfide bonds and an arginine-glycine-aspartic acid (RGD) sequence at positions 49-51. It has been known as one of the strongest antagonists to platelet aggregation among the family termed disintegrin. In this review paper, in addition to introducing the characteristics of disintegrin and its related molecules, the advantages of using recombinant DNA technology to produce rhodostomin are described. The recombinant rhodostomin has been demonstrated to facilitate cell adhesion via interaction between the RGD motif of rhodostomin and integrins on the cell surface. This property allowed us to use the recombinant rhodostomin as an extracellular matrix to study cell adhesion and to distinguish attachment efficiency between two melanoma cell lines B16-F1 and B16-F10, the former is a low metastasis cell while the latter is a high metastasis cell. Furthermore, by using the recombinant rhodostomin as a substrate, osteoprogenitor-like cells are able to be selected and enriched within 3 days from rat bone marrow which contains a heterogeneous cell population. Finally, we show that the recombinant rhodostomin can be immobilized on beads and which serve as an affinity column to dissect cell-surface protein(s) binding to the RGD motif of rhodostomin.

Background Lethal toxin (LT) is a major virulence factor of Bacillus anthracis. Sprague Dawley rats manifest pronounced lung edema and shock after LT treatments, resulting in high mortality. The heart failure that is induced by LT has been suggested to be a principal mechanism of lung edema and mortality in rodents. Since LT-induced death occurs more rapidly in rats than in mice, suggesting that other mechanisms in addition to the heart dysfunction may be contributed to the fast progression of LT-induced pathogenesis in rats. Coagulopathy may contribute to circulatory failure and lung injury. However, the effect of LT on coagulation-induced lung dysfunction is unclear. Methods To investigate the involvement of coagulopathy in LT-mediated pathogenesis, the mortality, lung histology and coagulant levels of LT-treated rats were examined. The effects of activated protein C (aPC) on LT-mediated pathogenesis were also evaluated. Results Fibrin depositions were detected in the lungs of LT-treated rats, indicating that coagulation was activated. Increased levels of plasma D-dimer and thrombomodulin, and the ameliorative effect of aPC further suggested that the activation of coagulation-fibrinolysis pathways plays a role in LT-mediated pathogenesis in rats. Reduced mortality was associated with decreased plasma levels of D-dimer and thrombomodulin following aPC treatments in rats with LT-mediated pathogenesis. Conclusions These findings suggest that the activation of coagulation in lung tissue contributes to mortality in LT-mediated pathogenesis in rats. In addition, anticoagulant aPC may help to develop a feasible therapeutic strategy.