PRA-Pred: Structure-based prediction of protein-RNA binding affinity.

Understanding crucial factors that affect the binding affinity of protein-RNA complexes is vital for comprehending their recognition mechanisms. This study involved compiling experimentally measured binding affinity (ΔG) values of 217 protein-RNA complexes and extracting numerous structure-based features, considering RNA, protein, and interactions between protein and RNA. Our findings indicate the significance of RNA base-step parameters, interaction energies, number of atomic contacts in the complex, hydrogen bonds, and contact potentials in understanding the binding affinity. Further, we observed that these factors are influenced by the type of RNA strand and the function of the protein in a protein-RNA complex. Multiple regression equations were developed for different classes of complexes to perform the prediction of the binding affinity between the protein and RNA. We evaluated the models using the jack-knife test and achieved an overall correlation 0.77 between the experimental and predicted binding affinities with a mean absolute error of 1.02 kcal/mol. Furthermore, we introduced a web server, PRA-Pred, intended for the prediction of protein-RNA binding affinity, and it is freely accessible through https://web.iitm.ac.in/bioinfo2/prapred/. We propose that our approach could function as a potential resource for investigating protein-RNA recognitions and developing therapeutic strategies.

Molecular dynamics simulations of cognate and non-cognate AspRS-tRNAAsp complexes.

Aspartyl tRNA synthetase (AspRS), one of the 20 aminoacyl-tRNA synthetases, plays an important role in protein synthesis by catalyzing the aminoacylation reaction and synthesises Aspartyl-tRNA (tRNAAsp). A typical three-dimensional structure of AspRS comprises three distinct domains for the recognition of cognate tRNA and catalysis, namely, anti-codon binding domain/N-terminal domain, hinge domain and catalytic domain through their interactions with anti-codon loop, D-stem and acceptor arm of cognate tRNA, respectively. In this work, we have studied the structural characteristics of each domain of AspRS to understand the recognition mechanism of tRNAAsp using molecular dynamics simulations. The dynamics of AspRS-tRNAAsp complexes from E.coli (cognate and non-cognate), S.cerevisiae (cognate) and T.thermophilus (non-cognate) were compared to understand the differences in recognition of cognate and non-cognate tRNAs. Our results explain that the conformational changes associated with the recognition of tRNA occur only in the cognate complexes. Among the cognate complexes, the conformational changes in yeast AspRS are highly controlled during tRNAAsp recognition than that of in the E. coli AspRS. Moreover, the functional motions required for the tRNA recognition are observed only in the cognate complexes, and the conformational changes in AspRS and their recognition of tRNAAsp are organism specific.Communicated by Ramaswamy H. Sarma.

The effects of brain death and ischemia on tolerance induction are organ-specific.

We have previously shown that 12 days of high-dose calcineurin inhibition induced tolerance in MHC inbred miniature swine receiving MHC-mismatched lung, kidney, or co-transplanted heart/kidney allografts. However, if lung grafts were procured from donation after brain death (DBD), and transplanted alone, they were rejected within 19-45 days. Here, we investigated whether donor brain death with or without allograft ischemia would also prevent tolerance induction in kidney or heart/kidney recipients. Four kidney recipients treated with 12 days of calcineurin inhibition received organs from donors rendered brain dead for 4 hours. Six heart/kidney recipients also treated with calcineurin inhibition received organs from donors rendered brain dead for 4 hours, 8 hours, or 4 hours with 4 additional hours of cold storage. In contrast to lung allograft recipients, all isolated kidney or heart/kidney recipients that received organs from DBD donors achieved long-term survival (>100 days) without histologic evidence of rejection. Proinflammatory cytokine gene expression was upregulated in lungs and hearts, but not kidney allografts, after brain death. These data suggest that the deleterious effects of brain death and ischemia on tolerance induction are organ-specific, which has implications for the application of tolerance to clinical transplantation.

Development of the Intestinal Transplantation Model With Major Histocompatibility Complex Inbred CLAWN Miniature Swine.

BACKGROUND: Clinical intestinal transplantation (Int-Tx) is associated with some problems such as rejection, infection, graft-versus-host disease, and ischemia-reperfusion injury (IRI). To determine mechanisms of rejection as well as to develop treatment strategies for Int-Tx, this study was designed to establish both heterotopic and orthotropic Int-Tx models using major histocompatibility antigen complex (MHC) inbred CLAWN miniature swine. MATERIALS AND METHODS: Eleven CLAWN miniature swine received MHC matched but minor antigen mismatched allogenic intestinal grafts. Four animals received intestinal grafts heterotopically and kept host intestine intact. The remaining 7 animals received intestinal grafts orthotopically and resected host small intestine. Continuous infusion of tacrolimus was given from day 0 for 12 days. RESULTS: Heterotopically transplanted small intestine were well perfused after revascularization; however, grafts easily underwent ischemic changes during or soon after abdomen closure due to oppression of the grafts in the limited abdominal space. In contrast, all of 7 orthotopically transplanted intestinal grafts in which recipients' small intestine was removed from the jejunum to the ileum had no signs of severe ischemia associated with compartment syndrome. Elevation of the serum concentration of inflammatory cytokines and the progression of lethal acidosis seen in recipients of heterotipic transplantation were markedly less in the case of orthotopic transplantation. Two recipients survived more than 30 days, and 1 long-term survivor showed no evidence of rejection at day 90 despite the fact that tacrolimus was stopped at day 12. CONCLUSIONS: In this study, we demonstrated the establishment of a clinically relevant orthotopic Int-Tx model with long survival in MHC inbred CLAWN miniature swine. We believe that this unique MHC inbred swine Int-Tx model is useful for developing treatment strategies for clinical Int-Tx.

Role of Bone Marrow Maturity, Insulin-Like Growth Factor 1 Receptor, and Forkhead Box Protein N1 in Thymic Involution and Rejuvenation.

Thymic involution is associated with age-related changes of the immune system. Utilizing our innovative technique of transplantation of a thymus as an isolated vascularized graft in MHC-inbred miniature swine, we have previously demonstrated that aged thymi are rejuvenated after transplantation into juvenile swine. Here we have studied the role of insulin-like growth factor (IGF) and forkhead-box protein-N1 (FOXN1) as well as bone marrow (BM) in thymic rejuvenation and involution. We examined thymic rejuvenation and involution by means of histology and flow cytometry. Thymic function was assessed by the ability to induce tolerance of allogeneic kidneys. Aged thymi were rejuvenated in a juvenile environment, and successfully induced organ tolerance, while juvenile thymi in aged recipients involuted and had a limited ability to induce tolerance. However, juvenile BM inhibited the involution process of juvenile thymi in aged recipients. An elevated expression of both FOXN1 and IGF1 receptors (IGF-1R) was observed in juvenile thymi and rejuvenated thymi. Juvenile BM plays a role in promoting the local thymic milieu as indicated by its ability to inhibit thymic involution in aged animals. The expression of FOXN1 and IGF-1R was noted to increase under conditions that stimulated rejuvenation, suggesting that these factors are involved in thymic recovery.

Adoptive Transfer of Renal Allograft Tolerance in a Large Animal Model.

Our recent studies in an inbred swine model demonstrated that both peripheral and intra-graft regulatory cells were required for the adoptive transfer of tolerance to a second, naïve donor-matched kidney. Here, we have asked whether both peripheral and intra-graft regulatory elements are required for adoptive transfer of tolerance when only a long-term tolerant (LTT) kidney is transplanted. Nine highly-inbred swine underwent a tolerance-inducing regimen to prepare LTT kidney grafts which were then transplanted to histocompatible recipients, with or without the peripheral cell populations required for adoptive transfer of tolerance to a naïve kidney. In contrast to our previous studies, tolerance of the LTT kidney transplants alone was achieved without transfer of additional peripheral cells and without strategies to increase the number/potency of regulatory T cells in the donor. This tolerance was systemic, since most subsequent, donor-matched challenge kidney grafts were accepted. These results confirm the presence of a potent tolerance-inducing and/or tolerance-maintaining cell population within LTT renal allografts. They suggest further that additional peripheral tolerance mechanisms, required for adoptive transfer of tolerance to a naïve donor-matched kidney, depend on peripheral cells that, if not transferred with the LTT kidney, require time to develop in the adoptive host.

Kidney-induced cardiac allograft tolerance in miniature swine is dependent on MHC-matching of donor cardiac and renal parenchyma.

Kidney allografts possess the ability to enable a short course of immunosuppression to induce tolerance of themselves and of cardiac allografts across a full-MHC barrier in miniature swine. However, the renal element(s) responsible for kidney-induced cardiac allograft tolerance (KICAT) are unknown. Here we investigated whether MHC disparities between parenchyma versus hematopoietic-derived "passenger" cells of the heart and kidney allografts affected KICAT. Heart and kidney allografts were co-transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. Group 1 animals (n = 3) received kidney and heart allografts fully MHC-mismatched to each other and to the recipient. Group 2 animals (n = 3) received kidney and heart allografts MHC-matched to each other but MHC-mismatched to the recipient. Group 3 animals (n = 3) received chimeric kidney allografts whose parenchyma was MHC-mismatched to the donor heart. Group 4 animals (n = 3) received chimeric kidney allografts whose passenger leukocytes were MHC-mismatched to the donor heart. Five of six heart allografts in Groups 1 and 3 rejected <40 days. In contrast, heart allografts in Groups 2 and 4 survived >150 days without rejection (p < 0.05). These data demonstrate that KICAT requires MHC-matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.

High incidence of xenogenic bone marrow engraftment in pig-to-baboon intra-bone bone marrow transplantation.

Previous attempts of α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into baboons have demonstrated a loss of macro-chimerism within 24 h in most cases. In order to achieve improved engraftment with persistence of peripheral chimerism, we have developed a new strategy of intra-bone BM (IBBM) Tx. Six baboons received GalTKO BM cells, with one-half of the cells transplanted into the bilateral tibiae directly and the remaining cells injected intravenously (IBBM/BM-Tx) with a conditioning immunosuppressive regimen. In order to assess immune responses induced by the combined IBBM/BM-Tx, three recipients received donor SLA-matched GalTKO kidneys in the peri-operative period of IBBM/BM-Tx (Group 1), and the others received kidneys 2 months after IBBM/BM-Tx (Group 2). Peripheral macro-chimerism was continuously detectable for up to 13 days (mean 7.7 days; range 3-13) post-IBBM/BM-Tx and in three animals, macro-chimerism reappeared at days 10, 14 and 21. Pig CFUs, indicating porcine progenitor cell engraftment, were detected in the host BM in four of six recipients on days 14, 15, 19 and 28. In addition, anti-pig unresponsiveness was observed by in vitro assays. GalTKO/pCMV-kidneys survived for extended periods (47 and 60 days). This strategy may provide a potent adjunct for inducing xenogeneic tolerance through BM-Tx.

Early humoral-mediated graft injuries in ABO-incompatible kidney transplantation in human beings.

INTRODUCTION: Acute humoral rejection is the most important risk factor for early graft loss in ABO-incompatible (ABO-i) renal transplantation (RTx) and is present from the early period after RTx. However, the characteristics of early humoral-mediated graft injury are pathologically uncertain. OBJECTIVE: To analyze tissue from 10 protocol graft biopsies performed in 10 patients within 30 days post-RTx to clarify the pathologic features of early humoral-mediated graft injuries in ABO-i RTx. METHODS: Pathologic findings were examined using light and electron microscopy and immunofluorescence studies for C4d. Protocol biopsies were performed within 30 days after RTx in the absence of an episode of dysfunction (creatinine concentration 1.21-1.81 mg/dL). RESULTS: The immunofluorescence study demonstrated C4d deposition in peritubular and glomerular capillaries. Acute glomerulitis with infiltration of mononuclear cells and neutrophils was observed in 3 patients. Furthermore, glomerulitis was accompanied by endothelial cell injuries, widening of subendothelial spaces with a double-contoured glomerular basement membrane, and mesangiolysis. CONCLUSION: In ABO-i RTx, early humoral-mediated graft injuries were observed in approximately 30% of patients despite normal graft function. They were characterized by C4d deposition and glomerular capillary injury. These findings suggest that renal glomeruli are the first site of graft injury by anti-A or anti-B blood type antibody with complement activation in ABO-i RTx.

Mobile phone base station radiation does not affect neoplastic transformation in BALB/3T3 cells.

A large-scale in vitro study focusing on low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields affect malignant transformation or other cellular stress responses. Our group previously reported that DNA strand breaks were not induced in human cells exposed to 2.1425 GHz Wideband Code Division Multiple Access (W-CDMA) radiation up to 800 mW/kg from mobile radio base stations employing the IMT-2000 cellular system. In the current study, BALB/3T3 cells were continuously exposed to 2.1425 GHz W-CDMA RF fields at specific absorption rates (SARs) of 80 and 800 mW/kg for 6 weeks and malignant cell transformation was assessed. In addition, 3-methylcholanthrene (MCA)-treated cells were exposed to RF fields in a similar fashion, to assess for effects on tumor promotion. Finally, the effect of RF fields on tumor co-promotion was assessed in BALB/3T3 cells initiated with MCA and co-exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). At the end of the incubation period, transformation dishes were fixed, stained with Giemsa, and scored for morphologically transformed foci. No significant differences in transformation frequency were observed between the test groups exposed to RF signals and the sham-exposed negative controls in the non-, MCA-, or MCA plus TPA-treated cells. Our studies found no evidence to support the hypothesis that RF fields may affect malignant transformation. Our results suggest that exposure to low-level RF radiation of up to 800 mW/kg does not induce cell transformation, which causes tumor formation.

Mobile phone base station-emitted radiation does not induce phosphorylation of Hsp27.

An in vitro study focusing on the effects of low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields act to induce phosphorylation and overexpression of heat shock protein hsp27. First, we evaluated the responses of human cells to microwave exposure at a specific absorption rate (SAR) of 80 mW/kg, which corresponds to the limit of the average whole-body SAR for general public exposure defined as a basic restriction in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Second, we investigated whether continuous wave (CW) and Wideband Code Division Multiple Access (W-CDMA) modulated signal RF fields at 2.1425 GHz induced activation or gene expression of hsp27 and other heat shock proteins (hsps). Human glioblastoma A172 cells were exposed to W-CDMA radiation at SARs of 80 and 800 mW/kg for 2-48 h, and CW radiation at 80 mW/kg for 24 h. Human IMR-90 fibroblasts from fetal lungs were exposed to W-CDMA at 80 and 800 mW/kg for 2 or 28 h, and CW at 80 mW/kg for 28 h. Under the RF field exposure conditions described above, no significant differences in the expression levels of phosphorylated hsp27 at serine 82 (hsp27[pS82]) were observed between the test groups exposed to W-CDMA or CW signal and the sham-exposed negative controls, as evaluated immediately after the exposure periods by bead-based multiplex assays. Moreover, no noticeable differences in the gene expression of hsps were observed between the test groups and the negative controls by DNA Chip analysis. Our results confirm that exposure to low-level RF field up to 800 mW/kg does not induce phosphorylation of hsp27 or expression of hsp gene family.

Identification of expressed genes characterizing long-term survival in malignant glioma patients.

Better understanding of the underlying biology of malignant gliomas is critical for the development of early detection strategies and new therapeutics. This study aimed to define genes associated with survival. We investigated whether genes coupled with a class prediction model could be used to define subgroups of high-grade gliomas in a more objective manner than standard pathology. RNAs from 29 malignant gliomas were analysed using Agilent microarrays. We identified 21 genes whose expression was most strongly and consistently related to patient survival based on univariate proportional hazards models. In six out of 10 genes, changes in gene expression were validated by quantitative real-time PCR. After adjusting for clinical covariates based on a multivariate analysis, we finally obtained a statistical significance level for DDR1 (discoidin domain receptor family, member 1), DYRK3 (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 3) and KSP37 (Ksp37 protein). In independent samples, it was confirmed that DDR1 protein expression was also correlated to the prognosis of glioma patients detected by immunohistochemical staining. Furthermore, we analysed the efficacy of the short interfering RNA (siRNA)-mediated inhibition of DDR1 mRNA synthesis in glioma cell lines. Cell proliferation and invasion were significantly suppressed by siRNA against DDR1. Thus, DDR1 can be a novel molecular target of therapy as well as an important predictive marker for survival in patients with glioma. Our method was effective at classifying high-grade gliomas objectively, and provided a more accurate predictor of prognosis than histological grading.

Phosphorylation and gene expression of p53 are not affected in human cells exposed to 2.1425 GHz band CW or W-CDMA modulated radiation allocated to mobile radio base stations.

A large-scale in vitro study focusing on low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields induce apoptosis or other cellular stress response that activate p53 or the p53-signaling pathway. First, we evaluated the response of human cells to microwave exposure at a specific absorption rate (SAR) of 80 mW/kg, which corresponds to the limit of the average whole-body SAR for general public exposure defined as a basic restriction by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Second, we investigated whether continuous wave (CW) and wideband code division multiple access (W-CDMA) modulated signal RF fields at 2.1425 GHz induced apoptosis or any signs of stress. Human glioblastoma A172 cells were exposed to W-CDMA radiation at SARs of 80, 250, and 800 mW/kg, and CW radiation at 80 mW/kg for 24 or 48 h. Human IMR-90 fibroblasts from fetal lungs were exposed to both W-CDMA and CW radiation at a SAR of 80 mW/kg for 28 h. Under the RF field exposure conditions described above, no significant differences in the percentage of apoptotic cells were observed between the test groups exposed to RF signals and the sham-exposed negative controls, as evaluated by the Annexin V affinity assay. No significant differences in expression levels of phosphorylated p53 at serine 15 or total p53 were observed between the test groups and the negative controls by the bead-based multiplex assay. Moreover, microarray hybridization and real-time RT-PCR analysis showed no noticeable differences in gene expression of the subsequent downstream targets of p53 signaling involved in apoptosis between the test groups and the negative controls. Our results confirm that exposure to low-level RF signals up to 800 mW/kg does not induce p53-dependent apoptosis, DNA damage, or other stress response in human cells.

DNA strand breaks are not induced in human cells exposed to 2.1425 GHz band CW and W-CDMA modulated radiofrequency fields allocated to mobile radio base stations.

We conducted a large-scale in vitro study focused on the effects of low level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system in order to test the hypothesis that modulated RF fields may act as a DNA damaging agent. First, we evaluated the responses of human cells to microwave exposure at a specific absorption rate (SAR) of 80 mW/kg, which corresponds to the limit of the average whole body SAR for general public exposure defined as a basic restriction in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Second, we investigated whether continuous wave (CW) and Wideband Code Division Multiple Access (W-CDMA) modulated signal RF fields at 2.1425 GHz induced different levels of DNA damage. Human glioblastoma A172 cells and normal human IMR-90 fibroblasts from fetal lungs were exposed to mobile communication frequency radiation to investigate whether such exposure produced DNA strand breaks in cell culture. A172 cells were exposed to W-CDMA radiation at SARs of 80, 250, and 800 mW/kg and CW radiation at 80 mW/kg for 2 and 24 h, while IMR-90 cells were exposed to both W-CDMA and CW radiations at a SAR of 80 mW/kg for the same time periods. Under the same RF field exposure conditions, no significant differences in the DNA strand breaks were observed between the test groups exposed to W-CDMA or CW radiation and the sham exposed negative controls, as evaluated immediately after the exposure periods by alkaline comet assays. Our results confirm that low level exposures do not act as a genotoxicant up to a SAR of 800 mW/kg.

Comparative study of clinical outcome in kidney transplantation between early steroid withdrawal protocol using basiliximab, calcineurin inhibitor, and mycophenolate mofetil and triple regimen consisting of calcineurin inhibitor, mycophenolate mofetil, and steroid.

AIMS: Effect of early steroid withdrawal protocol using basiliximab in kidney transplantation (KTx) on the clinical outcomes was investigated as compared with triple regimen. METHODS: Kidney transplant patients in group 1 (n = 62) were treated with 8 mg/kg of cyclosporine (CsA), 2000 mg of MMF, two bolus IV injections of 20 mg of basiliximab and 500 mg of methylprednisolone (MP) rapidly tapered and withdrawn at 14 postoperative days (POD). Group 2 (n = 56) was treated with same dose of CsA and MMF, and 250 mg of MP tapered and continued. Acute rejection (AR) episodes were treated with MP pulse therapy followed by muromonab CD3 (OKT3) in case of steroid-resistant rejection. RESULTS: In 46 of 62 cases (74.2%) in group 1, steroid was successfully withdrawn at 13.7 +/- 1.7 POD. Graft survival at 3, 6, and 12 months in group 1 was 100%, 100%, and 98.4% (one death with functioning graft), and 100%, 98.2%, and 96.4% in group 2, respectively. The incidence of AR was 12.9% for group 1 and 42.9% for group 2, among which 21 cases in group 2 were treated with ALG or OKT3; no patient needed ALG or OKT3 in group 1. Fifteen cases in group 1 and 13 cases in group 2 developed CMV antigenemia, among which febrile episode was exhibited in 3 cases (4.8%) in group 1 and 5 cases (8.9%) in group 2. CONCLUSIONS: Early steroid withdrawal protocol using basiliximab is promising for reducing the incidence of AR (especially steroid-resistant rejection), CMV diseases, and steroid-related complications.

Cyclosporine-sparing effect of basiliximab in renal transplant recipients with mycophenolate mofetil.

UNLABELLED: Basiliximab added to a maintenance regimen consisting of cyclosporine microemulsion and mycophenolate mofetil was studied for its effectiveness in allowing early steroid withdrawal in renal transplantation. Furthermore, the cyclosporine-sparing effects between groups with and without basiliximab induction therapy were compared. PATIENTS: Between September 2001 and June 2003, 90 patients underwent renal transplants with cyclosporine-based immunosuppression, namely, cyclosporine, mycophenolate mofetil, and methylprednisolone, (group 1; n = 25). During the latter half of the study basiliximab was administered during the induction phase (group 2; n = 65). In group 2, steroids were completely withdrawn on postoperative day 14 in 57 patients. RESULTS: The incidence of acute rejection was significantly higher among group 1 patients (P = .005). The incidence of steroid-resistant rejection in group 1 patients was significantly higher (P = .025). At each time point cyclosporine levels in group 1 patients were significantly higher (P < .01). The incidence of infection was comparable between the groups. Patient and graft survival rates in group 1 were 100% and 100%; in group 2, they were 99% and 99%, respectively. In group 2, steroids were discontinued in 57 patients with permanent withdrawal achieved in 32 patients (56%). CONCLUSION: The use of basiliximab, together with mycophenolate mofetil allowed for a significant reduction in the cyclosporine dose without increasing the risk of acute rejection. Although further follow-up is necessary to confirm the effect, this regimen may attenuate cyclosporine nephrotoxicity thereby affecting the long-term outcomes of renal transplantation.

An intraoperative fluorescent imaging system in organ transplantation.

UNLABELLED: An intraoperative fluorescent imaging system (SPY system; Novadaq Technologies, Inc, Concord, Ontario, Canada) that enables vascular surgeons to confirm the location and states of the reconstructed vessels during surgery, has been developed in the field of open heart surgery. In this paper, we evaluated the usefulness of the SPY system in kidney and liver transplantation. PATIENTS AND METHODS: SPY system visualizes arteries and grafts intraoperatively, using indocyanine green (ICG) with a portable imaging device. The modality was evaluated in 15 patients undergoing kidney (n = 13) or liver (n = 2) transplantation with respect to safety, feasibility of use, and image quality. Images were generated and acquired with a portable laser diode/infrared camera device after injection of 10 mL of ICG (2.5 mg/mL) intravenously. RESULT: There was no complication associated with ICG injection or the imaging device. The SPY system was easily used during transplant surgery and adequately demonstrated reconstructed arteries and patency in all patients. CONCLUSION: The intraoperative imaging system enables the surgeon to view, record, and replay real-time images of the reconstructed arteries during surgery. The system may provide useful information during surgery such as solid organ transplantation that requires vascular reconstruction.

Enhancement of glutathione S-transferase placental-form positive liver cell foci development by microcystin-LR in aflatoxin B1-initiated rats.

The objective of this study was to elucidate whether microcystin-LR (MC-LR), a hepatotoxic blue-green algal toxin in drinking water, is carcinogenic or possesses the ability to modulate aflatoxin B1 (AFB1)-induced hepatocarcinogenicity. In a medium-term liver bioassay, male Fischer 344 rats were given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg) followed by an i.p. injection of MC-LR for 6 weeks after 2 weeks of DEN treatment. To study the synergism between AFB1 and MC-LR, DEN-treated rats were given an i.p. injection of AFB1 (0.5 mg/kg) dissolved in dimethyl sulfoxide (DMSO) followed by MC-LR at 2 weeks after the treatment. In a separate experiment, the rats were first given AFB1 (0.5 mg/kg) and 2 weeks later an i.p. injection of 1 or 10 microg/kg of MC-LR twice a week for 6 weeks. Most rats were subjected to a two-thirds partial hepatectomy (PH) at week 3 and were killed under anesthesia at week 8. Liver sections were analyzed for glutathione S-transferase placental form (GST-P) expression, and subjected to histopathological examination for phenotypic alteration of hepatocellular foci. In rats that did not receive DEN, MC-LR did not cause a significant increase in the numbers of GST-P-positive foci, whereas AFB1 induced a slight increase in GST-P-positive foci development. In rats given DEN, MC-LR enhanced the expression of GST-P-positive foci, as did AFB1 but no synergism was observed. Histopathological analysis revealed that the area of eosinophilic foci, a biomarker for preneoplastic liver lesion, markedly increased because of MC-LR. In rats given AFB1 as an initiator, treatment with MC-LR resulted in a synergistic increase in the development of GST-P-positive foci. These results suggest that the hepatocarcinogenicities of MC-LR and AFB1 can be predicted in experimental animals with a medium-term bioassay. Furthermore, tumor promoting activity of MC-LR was demonstrated in rats treated with AFB1.

Immunohistochemical localization of microcystin-LR in the liver of mice: a study on the pathogenesis of microcystin-LR-induced hepatotoxicity.

The relationship between the intralobular sites of hepatotoxic injury and the distribution of microcystin-LR (MCLR), an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), was examined using an immunohistochemical method with a monoclonal antibody specific to MCLR on the livers of mice receiving a single i.p. injection of the MCLR. Immunoblotting and high-performance liquid chromatography analyses of liver extracts were also performed to determine the binding form of MCLR to PP1 and PP2A (MCLR-PP1/PP2A adducts) and free MCLR. Immunohistochemistry revealed a discernible intensity of staining in the centrilobular regions where hemorrhage and apoptosis occurred. In these regions, immunopositivity was evident in the cytoplasm and nuclei of the hepatocytes; some apoptotic cells were also immunopositive. In contrast, coagulative necrosis, which was mainly evident in the midlobular regions, was completely negative. Analysis of liver extracts demonstrated MCLR-PP1/PP2A adducts, but free MCLR was below detection limit. These results suggest that the immunohistochemical localization of MCLR in centrilobular hepatocytes is closely associated with the onset of hemorrhage and apoptosis and is related to adduct formation. The occurrence of coagulative necrosis however might also be related to other factors such as ischemia/hypoxia.

Acute oral toxicity of microcystin-LR, a cyanobacterial hepatotoxin, in mice.

Microcystin-LR (MCLR) is a hepatotoxic peptide produced by Microcystis aeruginosa, an alga found worldwide in reservoirs for drinking supply; however, acute oral toxicity of purified MCLR remains unknown. Therefore, a single dose of MCLR (more than 95% purity) ranging from 8.0 to 20.0 mg/kg body weight was orally given to female 6-week old BALB/c mice, and lethality and pathological changes were observed. Median lethal dose (LD50) of the orally given MCLR estimated by the up and down method was 10.9 mg/kg, which was 167 times higher than the i.p. LD50 value (65.4 microgram/kg by moving average method). Orally administrated toxin caused primarily hepatocellular injuries with characteristics of hemorrhage and necrosis. In situ end-labeling as well as electron microscopic observation revealed an induction of apoptotic cell death to hepatocytes. These results indicate the lethality of MCLR was much lower in oral dosage than by i.p. administration, but toxic effects are similar. In addition, apoptosis is considered one of major components in MCLR-induced hepatotoxicity.