Allosteric pluripotency as revealed by protein kinase A.

The functional response of a signaling system to an allosteric stimulus often depends on subcellular conditions, a phenomenon known as pluripotent allostery. For example, a single allosteric modulator, Rp-cAMPS, of the prototypical protein kinase A (PKA) switches from antagonist to agonist depending on MgATP levels. However, the mechanism underlying such pluripotent allostery has remained elusive for decades. Using nuclear magnetic resonance spectroscopy, ensemble models, kinase assays, and molecular dynamics simulations, we show that allosteric pluripotency arises from surprisingly divergent responses of highly homologous tandem domains. The differential responses perturb domain-domain interactions and remodel the free-energy landscape of inhibitory excited states sampled by the regulatory subunit of PKA. The resulting activation threshold values are comparable to the effective free energy of regulatory and catalytic subunit binding, which depends on metabolites, substrates, and mutations, explaining pluripotent allostery and warranting a general redefinition of allosteric targets to include specific subcellular environments.

Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer.

The proinflammatory interleukin-33 (IL-33) binds to its receptor ST2L on the surface of immune cells and stimulates the production of Th2 cytokines; however, the effects of IL-33 on tumour cells are poorly understood. Here we show that ST2 was significantly downregulated in human lung cancer tissues and cells compared with normal lung tissues and cells. IL-33 expression was also inversely correlated with the stages of human lung cancers. In accordance with this finding, low-metastatic cells but not high-metastatic cells derived from Lewis lung carcinoma expressed functional ST2L. IL-33 was abundantly present in the tumours established by the low-metastatic cells compared with those formed by the high-metastatic cells. Although the low-metastatic cells scarcely expressed IL-33 in vitro, these cells did expry 6ess this molecule in vivo, likely due to stimulation by intratumoural IL-1ß and IL-33. Importantly, IL-33 enhanced the cell death of ST2L-positive low-metastatic cells, but not of ST2L-negative high-metastatic cells, under glucose-depleted, glutamine-depleted and hypoxic conditions through p38 MAPK and mTOR activation, and in a mitochondria-dependent manner. The cell death was characterised by cytoplasmic blisters and karyolysis, which are unique morphological features of oncosis. Inevitably, the low-metastatic cells, but not of the high-metastatic cells, grew faster in IL-33(-/-) mice than in wild-type mice. Furthermore, IL-33 selected for the ST2L-positive, oncosis-resistant high-metastatic cells under conditions mimicking the tumour microenvironment. These data suggest that IL-33 enhances lung cancer progression by selecting for more malignant cells in the tumour microenvironment.

Protein tyrosine phosphatase epsilon is a negative regulator of FcepsilonRI-mediated mast cell responses.

Modulation of mast-cell activation may provide novel ways to control allergic diseases. Here, we show that protein tyrosine phosphatase epsilon (PTPepsilon; Ptpre) plays key regulatory roles during mast-cell activation mediated by the high-affinity IgE receptor (FcepsilonRI). Bone marrow-derived mast cells (BMMC) from Ptpre(-/-) mice exhibited enhanced FcepsilonRI-induced Ca(2+) mobilization and mitogen-activated protein kinase (MAPK) (JNK and p38) activation, and showed corresponding enhancement of evoked degranulation and cytokine production, but not leukotriene production. Examination of proteins linking tyrosine kinase activation and Ca(2+) mobilization revealed that the absence of PTPepsilon leads to increased phosphorylation of the linker for activation of T cells and SH2 domain-containing leucocyte phosphoproteins of 76 kDa, but not Grb2-associated binder-2 (Gab2). Because Gab2 is considered to be situated downstream of Fyn kinase, we reasoned that Fyn may not be a target of PTPepsilon. In the event, Syk but not Lyn was hyperphosphorylated in PTPepsilon-deficient BMMC. Thus, PTPepsilon most likely exerts its effects at the level of Syk, inhibiting downstream events including phosphorylation of SLP-76 and linker of activated T cells and mobilization of Ca(2+). Consistent with the in vitro data, antigen- and IgE-mediated passive systemic anaphylactic reactions were augmented in Ptpre(-/-) mice. Given that the number of mast cells is unchanged in these mice, this observation most likely reflects alterations of mast cell-autonomous signalling events. These data suggest that PTPepsilon negatively regulates FcepsilonRI-mediated signalling pathways and thus constitutes a novel target for ameliorating allergic conditions.

PD-1/PD-L1 expression in human T-cell leukemia virus type 1 carriers and adult T-cell leukemia/lymphoma patients.

Adult T-cell leukemia/lymphoma (ATLL) develops after infection with human T-cell leukemia virus-1 (HTLV-1) after a long latency period. The negative regulatory programmed death-1/programmed death-1 ligand 1 (PD-1/PD-L1) pathway has been implicated in the induction of cytotoxic T-lymphocyte (CTL) exhaustion during chronic viral infection along with tumor escape from host immunity. To determine whether the PD-1/PD-L1 pathway could be involved in the establishment of persistent HTLV-1 infections and immune evasion of ATLL cells in patients, we examined PD-1/PD-L1 expression on cells from 27 asymptomatic HTLV-1 carriers (ACs) and 27 ATLL patients in comparison with cells from 18 healthy donors. PD-1 expression on HTLV-1-specific CTLs from ACs and ATLL patients was dramatically elevated. In addition, PD-1 expression was significantly higher on CD8+ T cells along with cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-specific CTLs in ATLL patients compared with ACs and control individuals. Primary ATLL cells in 21.7% of ATLL patients expressed PD-L1, whereas elevated expression was not observed in cells from ACs. Finally, in functional studies, we observed that an anti-PD-L1 antagonistic antibody upregulated HTLV-1-specific CD8+T-cell response. These observations suggest that the PD-1/PD-L1 pathway plays a role in fostering persistent HTLV-1 infections, which may further ATLL development and facilitate immune evasion by ATLL cells.

Gene expression profiles in differentiating leukemia cells induced by methyl jasmonate are similar to those of cytokinins and methyl jasmonate analogs induce the differentiation of human leukemia cells in primary culture.

Jasmonates are potent lipid regulators in plants that play pivotal roles in their biological activities. Methyl jasmonate (MJ) is very effective at inducing the myelomonocytic differentiation of human myeloid leukemia HL-60 cells. We examined the gene expression profiles associated with exposure to MJ using cDNA microarrays, and compared the results with those obtained with other inducers of differentiation, such as all-trans retinoic acid (ATRA), 1alpha,25-dihydroxyvitamin D(3) (VD(3)), isopentenyladenine (IPA) and cotylenin A (CN-A). Many genes were upregulated, and only a small fraction was downregulated, upon exposure to the inducers. MJ, IPA and CN-A, but not ATRA or VD(3), immediately induced the expression of mRNA for the calcium-binding protein S100P. The gene expression profile induced by MJ resembled that induced by IPA, suggesting that these inducers share many common signal transduction systems for inducing the differentiation of leukemia cells. Methyl 4,5-didehydrojasmonate was about 30 times more potent than MJ and the natural form of the stereoisomer was more effective than the unnatural isomer. It significantly stimulated both the functional and morphological differentiation of leukemia cells that had been freshly isolated from patients with hematological malignancies. Jasmonate derivatives may be promising therapeutic agents for differentiation therapy of leukemia.

Outcomes of 23- and 25-gauge transconjunctival sutureless vitrectomies for idiopathic macular holes.

BACKGROUND/AIMS: To assess the outcomes of 23-gauge sutureless transconjunctival vitrectomies (TSV), as compared with 25-gauge TSV in macular hole surgeries. METHODS: A retrospective, consecutive, interventional case series of 47 eyes with idiopathic macular holes treated by 23- or 25-gauge TSV were analysed. RESULTS: The operative time was 37.2 (SD 8.9) min with 23-gauge TSV and 34.2 (8.7) min with 25-gauge TSV (p = 0.388). The anatomical success rate was 96% with 23-gauge TSV and 92% with 25-gauge TSV (p>0.999). The logarithm of the minimum angle of resolution of best-corrected visual acuity (BCVA) at the sixth postoperative month was 0.19 (0.16) with 23-gauge TSV and 0.19 (0.25) with 25-gauge TSV (p = 0.521). Postoperative improvement in BCVA was comparable between the two TSVs. IOP on postoperative day 1 was lower with 25-gauge TSV (12.3 (4.9) mm Hg) than with 23-gauge TSV (17.4 (5.8) mm Hg) (p = 0.036). Complications included retinal break, intraoperative bleeding and slippage of the infusion cannula with 23-gauge TSV, while retinal detachment and postoperative hypotony occurred in the 25-gauge TSV group (p = 0.570). CONCLUSION: 23-gauge TSV appears to be as safe and effective as 25-gauge TSV in macular hole surgery.

Identifying pathogenic genetic background of simplex or multiplex retinitis pigmentosa patients: a large scale mutation screening study.

BACKGROUND AND PURPOSE: More than half of the retinitis pigmentosa (RP) cases are genetically simplex or multiplex. To date, 37 causative genes of RP have been identified; however, the elucidation of gene defects in simplex or multiplex RP patients/families remains problematic. The aim of our study was to identify the genetic causes of RP in patients with unknown or non-Mendelian inheritance. METHODS AND RESULTS: Since 2003, 52 simplex RP patients, 151 patients from 141 multiplex RP families, and six sporadic patients with retinal degeneration were studied. A total of 108 exons of 30 RP-causing genes that harboured the reported mutations were screened by an efficient denaturing high performance liquid chromatography (dHPLC) based assay. Aberrant fragments were subsequently analysed by automatic sequencing. Twenty-six mutations, including two frameshift mutations, one single amino acid deletion, and 23 missense mutations, were identified in 28 probands (14.07%). Eighteen mutations have not been reported to date. Three pairs of combined mutations in different genes were identified in two sporadic cases and one multiplex family, indicating the possibility of novel digenic patterns. Of the 23 missense mutations, 21 were predicted as deleterious mutations by computational methods using PolyPhen, SIFT, PANTHER, and PMut programs. CONCLUSION: We elucidated the mutation spectrum in Japanese RP patients and demonstrated the validity of the mutation detection system using dHPLC sequencing for genetic diagnosis in RP patients independent of familial incidence, which may provide a model strategy for identifying genetic causes in other diseases linked to a wide range of genes.

Retinal repair by transplantation of photoreceptor precursors.

Photoreceptor loss causes irreversible blindness in many retinal diseases. Repair of such damage by cell transplantation is one of the most feasible types of central nervous system repair; photoreceptor degeneration initially leaves the inner retinal circuitry intact and new photoreceptors need only make single, short synaptic connections to contribute to the retinotopic map. So far, brain- and retina-derived stem cells transplanted into adult retina have shown little evidence of being able to integrate into the outer nuclear layer and differentiate into new photoreceptors. Furthermore, there has been no demonstration that transplanted cells form functional synaptic connections with other neurons in the recipient retina or restore visual function. This might be because the mature mammalian retina lacks the ability to accept and incorporate stem cells or to promote photoreceptor differentiation. We hypothesized that committed progenitor or precursor cells at later ontogenetic stages might have a higher probability of success upon transplantation. Here we show that donor cells can integrate into the adult or degenerating retina if they are taken from the developing retina at a time coincident with the peak of rod genesis. These transplanted cells integrate, differentiate into rod photoreceptors, form synaptic connections and improve visual function. Furthermore, we use genetically tagged post-mitotic rod precursors expressing the transcription factor Nrl (ref. 6) (neural retina leucine zipper) to show that successfully integrated rod photoreceptors are derived only from immature post-mitotic rod precursors and not from proliferating progenitor or stem cells. These findings define the ontogenetic stage of donor cells for successful rod photoreceptor transplantation.

Hepatocyte growth factor as an enhancer of nmda currents and synaptic plasticity in the hippocampus.

Hepatocyte growth factor (HGF) promotes the survival and migration of immature neurons, but its role in the mature brain has remained elusive. In the hippocampus of juvenile rats, we found that the HGF receptor c-Met was expressed in neurons. Furthermore, it was highly Tyr-phosphorylated, more so than in the liver under normal conditions, suggesting that the receptor is activated and that HGF may act continuously in the intact brain. Exogenously applied HGF enhanced synaptic long-term potentiation (LTP) in the CA1 region of hippocampus, but did not affect long-term depression. We further found that HGF augmented N-methyl-D-aspartate receptor-mediated currents in both slices and dissociated neurons. This augmentation is likely to underlie the enhancement of LTP. Considering that the expression of both HGF and c-Met are known to be induced by ischemic stimuli, this modulation would provide a novel understanding of a neuronal regulatory systems shared with pathogenic ischemic states.

Cyclooxygenase-2 expression and relationship to tumour progression in human renal cell carcinoma.

AIMS: Cyclooxygenase (COX), which catalyses the synthesis of prostaglandins from arachidonic acid, has two isoforms; COX-1 and COX-2. There is ample evidence to suggest an important role for COX-2 in cancer. The aim of this study was to evaluate the clinical significance of COX-2 expression and its localization in the development and progression of human renal cell carcinoma (RCC). METHODS AND RESULTS: The expression and localization of COX-2 were evaluated in human RCC tissues from 75 patients by immunohistochemistry. Immunoreactive COX-2 protein was observed in all cases of RCC, and the levels of COX-2 expression were correlated with tumour grade and pathological stage. Expression of COX-2 was higher in the granular cell subtype than in the clear cell subtype of RCC. Immunoelectron microscopy revealed that COX-2 was expressed in the nuclear membrane, rough endoplasmic reticulum, Golgi complex and mitochondrial membrane of RCC cells. CONCLUSION: COX-2 overexpression within these intracellular organelles in RCC may be associated with renal cell carcinogenesis and COX-2 may be a useful biomarker in RCC.

Comparison of stepwise and simultaneous estimations of population pharmacokinetics and pharmacodynamics of TS-943.

The prediction performances of population pharmacokinetic-pharmacodynamic analysis of the two methods (a stepwise and a simultaneous estimations) were evaluated with respect to their accuracies and precisions. A study was designed to investigate the safety and efficacy of TS-943 by a 4 hours constant infusion in 36 healthy male subjects. Population analysis was performed using pharmacokinetic and pharmacodynamic models with NONMEM. The mean of the prediction error (MPE) and the root mean squared error (RMSE) served as a measure of accuracy and precision. In addition, a bootstrap validation was also performed. The results indicate that those population pharmacokinetic-pharmacodynamic parameters for the two methods were comparable. The results of simultaneous estimations are similar to those obtained using a stepwise estimation. The mean parameter estimates obtained with the additional 200 bootstrap replicates of data were within 15% of those obtained with the final model in both methods. The present results demonstrated that the accuracy of pharmacodynamic evaluations using a stepwise end a simultaneous estimations was comparable.

S-oxidation of S-methyl-esonarimod by flavin-containing monooxygenases in human liver microsomes.

1. Studies using human liver microsomes and recombinant human cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) were performed to identify the enzymes responsible for the metabolism of S-methyl-esonarimod (M2), an active metabolite of esonarimod (KE-298, a novel antirheumatic drug). 2. S-oxidative activities of M2 significantly correlated with those of methyl p-tolyl sulfide, a specific substrate of FMOs, as tested using 10 different human liver microsomes (r(2) = 0.539, p<0.05). Thermal treatment of microsomes reduced the S-oxidative activity in the absence of the NADPH-generating system at 45 degrees C for 5 min. However, methimazole, a known competitive substrate of FMOs, was a weak inhibitor of the S-oxidation in liver microsomes. 3. Recombinant human FMO1 and FMO5 produced M3 in greater quantities than recombinant human FMO3. The S-oxidation of M2 by recombinant human FMO5 was not appreciably inhibited in the presence of methimazole. In contrast, methimazole was effective in suppressing the catalytic activity of recombinant human FMO1 and FMO3. 4. The apparent K(m) (K(m app)) for the S-oxidation of M2 in human recombinant FMO5 (2.71 microM) was similar to that obtained using human liver microsomes (2.43 microM). 5. The present results suggest that the S-oxidation of S-methyl esonarimod reflects FMO5 activity in the human liver because the recombinant FMO5 data match well with the human liver microsomal experiments.

Pharmacokinetic and pharmacodynamic analysis of TS-943, a selective non-peptide platelet glycoprotein-IIb/IIIa (GPIIb/IIIa) receptor antagonist, using a nonlinear mixed effect model in dogs.

A simultaneous analysis of the pharmacokinetics and pharmacodynamics of TS-943, a selective nonpeptide platelet glycoprotein-IIb/IIIa (GPIIb/IIIa) receptor antagonist, was made in dogs using a nonlinear mixed effect model. Plasma concentrations of TS-943 were determined after bolus intravenous injection, constant infusion and bolus plus constant infusion. Pharmacokinetic/pharmacodynamic data were fitted using NONMEM software. The pharmacokinetics of TS-943 fitted a two-compartment open model with first-order elimination. The pharmacodynamic model that best fitted platelet aggregation was an inhibitory sigmoid Emax model. The final estimates for E0 (baseline effect), Emax (maximum effect), IC50 (50% inhibitory concentration) and gamma (Hill coefficient) were 66.3%, 64.3%, 104 ng mL(-1) and 1.37, respectively. Correlations between TS-943 plasma concentration and extension of template bleeding time were examined by fitting with an exponential model. The TS-943 plasma concentration necessary to double bleeding time (C2-BTE) was approximately 209 ng mL(-1). The model estimated that the C2-BTE/IC50 (inhibition of platelet aggregation) ratio was approximately 2.0-fold in dogs. Our results suggest that the ratio values for dogs and man are comparable. A nonlinear mixed effect model was a useful tool for exploring the concentration-effect relationship for both efficacy and safety of TS-943 in dogs and man. In this study, the dog was found to be a useful model for screening of efficacy and safety of TS-943 in man.

A new delivery system for 5-fluorouracil using prodrug and converting enzyme.

AIMS: To evaluate a new delivery system of 5-fluorouracil (5-FU) using 5-fluorocytosine (5-FC) as a prodrug and cytosine deaminase induced in vitro and in vivo. METHODS: Fibroblastic cells from rabbit Tenon's capsule were cultured. The cells were exposed to 5-FU and 5-FC with or without cytosine deaminase induced by recombinant adenovirus. In the in vitro study, cell proliferation and DNA synthesis were assessed by MTS, BrdU assay. The effect of 5-FC removal after the treatment of 5-FC and cytosine deaminase induction was also assayed. In the in vivo study cells with or without cytosine deaminase induction were transplanted into the subconjunctival space of mice, followed by eye drops of 1000 microg/ml of 5-FC three times a day. The mice were sacrificed at days 1, 5, and 10, then the cells transplanted were evaluated. RESULTS: Cell proliferation was inhibited by exposure to 5-FU in a dose dependent manner; however, up to 1000 microg/ml of 5-FC did not affect cell proliferation. Cell proliferation was inhibited by exposure to 5-FC in a time dependent manner with induction of cytosine deaminase following infection of recombinant adenovirus. When 5-FC was removed 3 or 6 days after the treatment, the cells grew again. The effect was reproduced in the in vivo model of subconjunctival cellular proliferation although 5-FC was administrated as eye drops. There were no cases with corneal erosion. CONCLUSION: Cell proliferation was inhibited by co-exposure of 5-FC and cytosine deaminase. This new delivery system may merit controlled delivery of 5-FU after filtering surgery.

[Evaluation of endoscopic papillary balloon dilation (EPBD) using one-step catheter for removal of common bile duct stones].

We compared the results of EPBD in 18 patients using one-step catheter (one-step group) with those in 46 patients using usual two-step catheter (two-step group). The frequency of endoscopy and bile duct clearance were the equal in both groups. Pancreatitis occurred in only one patient of two-step group. Elevations of the amylase values were the equal in both groups. Amylase values after EPBD tended to decrease in 11 patients whose amylase values were above 500 IU/l before EPBD. Using one-step catheter in EPBD was useful for removing common bile duct stones easily and safety.

Electromyography of male rat perineal musculature during copulatory behavior.

Electromyogram (EMG) recordings were made from the bulbospongiosus (BS) and ischiocavernosus (IC) muscles of the awake male rat in order to evaluate the roles played by these muscles in the implementation of penile intromission during a copulatory encounter. Male rats with bipolar wire electrodes implanted chronically in the BS and IC muscles were mated with sexually receptive females. Concurrent videotape recordings established a relationship between EMG and components of male rat copulatory behavior, i.e. mount, intromission and ejaculation. On EMGs recorded from the IC muscle, the initial low-amplitude phase at the initiation of each behavioral component was distinguished from the late high-amplitude phase whenever the male accomplished mounting or intromission. In the BS muscle, the late high-amplitude phase was observed only when the male accomplished intromission. Power spectrum analysis by fast Fourier transformation revealed that in the IC muscle the two phases differed at an overall range of 100-1,000 Hz. In the BS muscle, however, the spectra of the two phases differed at a specific range of 400-600 Hz. Recruitment of additional motor units would culminate in such a characteristic rise in the power spectrum. The results suggest that somatosensory inputs contingent on the intromission cause such recruitment in the BS to regulate penile cups and other movements.