Multiple Functions of Nm23-H1 Are Regulated by Oxido-Reduction System

Nucleoside diphosphate kinase (NDPK, Nm23), a housekeeping enzyme, is known to be a multifunctional protein, acting as a metastasis suppressor, transactivation activity on c-myc, and regulating endocytosis. The cellular mechanisms regulating Nm23 functions are poorly understood. In this study, we identified the modifications and interacting proteins of Nm23-H1 in response to oxidative stress. We found that Cys109 in Nm23-H1 is oxidized to various oxidation states including intra- and inter-disulfide crosslinks, glutathionylation, and sulfonic acid formation in response to H2O2 treatment both in vivo and in vitro. The cross-linking sites and modifications of oxidized Nm23-H1 were identified by peptide sequencing using UPLC-ESI-q-TOF tandem MS. Glutathionylation and oxidation of Cys109 inhibited the NDPK enzymatic activity of Nm23-H1. We also found that thioredoxin reductase 1 (TrxR1) is an interacting protein of Nm23-H1, and it binds specifically to oxidized Nm23-H1. Oxidized Nm23 is a substrate of NADPH-TrxR1-thioredoxin shuttle system, and the disulfide crosslinking is reversibly reduced and the enzymatic activity is recovered by this system. Oxidation of Cys109 in Nm23-H1 inhibited its metastatic suppressor activity as well as the enzymatic activities. The mutant, Nm23-H1 C109A, retained both the enzymatic and metastasis suppressor activities under oxidative stress. This suggests that key enzymatic and metastasis suppressor functions of Nm23-H1 are regulated by oxido-reduction of its Cys109

Subarachnoid hemorrhage and Terson's syndrome: a prospective study

PURPOSE: To analyze the incidence, clinical course, ophthalmic findings, and prognosis of the patients with intracranial bleeding and Terson's syndrome. METHODS: A prospective consecutive study of patients admitted to the emergency room with the diagnosis of acute subarachnoid hemorrhage. Neurological and funduscopic examinations were performed upon admission and at days 3, 7, 30 and 60 after the diagnosis. In all cases the clinical condition was graded according to the Hunt and Hess classification. RESULTS: Seventeen patients were enrolled in this study from July to October, 2000. Terson's syndrome was diagnosed in 5 of the patients (29.4%). Fifteen cases were associated with ruptured cerebral aneurysms, and the remaining two were caused by head trauma. There was no gender preponderance (9F:8M) and the median age was 48 years (range 22-80 years). Four of the five patients (80%) with Terson's syndrome died from intracranial complications, whereas 11 patients with intracranial bleeding and no ocular involvement survived. The leading cause of mortality was rebleeding. The ophthalmoscopic findings included intraretinal bleeding (3 cases), intraretinal and with subhyaloid hemorrhage (1 case) and intraretinal hemorrhage with cotton wool spots (1 case). CONCLUSIONS: In our study, the incidence of Terson's syndrome was 29.4% and it was associated with a high mortality rate (80%).OBJETIVO: Analisar a incidência, evolução clínica, alteração oftalmológica e prognóstico de vida de pacientes com hemorragia subaracnóidea e síndrome de Terson. MÉTODOS: Estudo prospectivo e consecutivo de pacientes admitidos no pronto socorro de neurocirurgia da Universidade Federal de São Paulo (UNIFESP) com diagnóstico de hemorragia subaracnóidea aguda. Após exame neurológico, o mapeamento de retina foi realizado em todos os pacientes na admissão e no 3º, 7º, 30º e 60º dia. Em todos os casos foi realizada a correlação entre a escala de Hunt e Hess e a presença de hemorragia intra-ocular. RESULTADOS: Dezessete pacientes foram examinados durante julho a outubro de 2000. A síndrome de Terson foi observada em 5 casos (29,4%). Em 15 pacientes a etiologia da hemorragia foi ruptura de aneurisma cerebral e em 2 casos a causa foi relacionada a traumatismo crânio-encefálico. Não houve predominância significante de sexo (9F e 8M) e a idade mediana foi de 48 anos (22 a 80 anos). Houve 4 óbitos de pacientes com síndrome de Terson e apenas 1 no grupo de pacientes sem alteração ocular. Não houve nenhuma correlação entre a gravidade do quadro clínico e a presença da síndrome de Terson. CONCLUSÃO: Neste estudo, a incidência da síndrome de Terson foi de 29,4% e sua presença indicou alto risco de mortalidade (80% dos casos com a síndrome de Terson).Universidade Federal de São Paulo (UNIFESP) Setor de Retina e VítreoUniversidade Federal de São Paulo (UNIFESP)UNIFESP, Setor de Retina e VítreoUNIFESPSciEL

Serial I-123-FP-CIT SPECT Image Findings of Parkinson's Disease Patients With Levodopa-Induced Dyskinesia

Background: Levodopa-induced dyskinesia (LID) is a major complication of dopamine replacement drug usage in Parkinson's disease (PD) patients. Since the mechanism of LID is yet unclear, we analyzed serial [I-123] N-ω-fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) nortropane (I-123 FP-CIT) single photon emission computed tomography (SPECT) images. We investigated the changes of dopaminergic innervation during the progression of PD in relation to the development of LID.Methods: Data were obtained from the Parkinson's Progression Markers Initiative (PPMI) database. Two hundred and ninety PD dopamine replacement drug-naïve patients (age 61.0 ± 9.7, M: F = 195: 95) were enrolled. I-123 FP-CIT SPECT images from baseline, 12, 24, and 48 months were analyzed among with clinical factors. specific binding ratios (SBRs) of the striatal regions from I-123 FP-CIT SPECT images were analyzed. We used independent tests and logistic regression for analysis of LID risk association.Results: Among 290 patients, 36 patients developed LID after 48 months follow-up. Baseline MDS-UPDRS Part II and III scores were significantly higher in the PD patients with LID, compared with the PD patients without LID. Striatal SBRs were significantly lower in the PD patients with LID at baseline, 24 and 48 months (p < 0.001). Multivariate analysis revealed MDS-UPDRS Part II and putaminal SBRs at baseline and 24 months to be significantly associated with the development of LID (p < 0.001). Also, patients who developed LID at 48 months had a higher decrease rate of putaminal SBR at the 24 months (p < 0.05), and 48 months (p < 0.01) period.Conclusion: In this study, we demonstrated the serial changes of the nigrostriatal dopaminergic innervation in relationship to LID development for the first time. The deterioration rate of dopaminergic innervation was significantly higher in the PD patients who developed LID, compared with the PD patients who did not develop LID. Serial follow up I-123 FP-CIT SPECT acquisition during the course of PD could be helpful in predicting the development of LID

Potential role and mechanism of IFN-gamma inducible protein-10 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in rheumatoid arthritis

Introduction IFN-gamma inducible protein-10 (CXCL10), a member of the CXC chemokine family, and its receptor CXCR3 contribute to the recruitment of T cells from the blood stream into the inflamed joints and have a crucial role in perpetuating inflammation in rheumatoid arthritis (RA) synovial joints. Recently we showed the role of CXCL10 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in an animal model of RA and suggested the contribution to osteoclastogenesis. We tested the effects of CXCL10 on the expression of RANKL in RA synoviocytes and T cells, and we investigated which subunit of CXCR3 contributes to RANKL expression by CXCL10. Methods Synoviocytes derived from RA patients were kept in culture for 24 hours in the presence or absence of TNF-α. CXCL10 expression was measured by reverse transcriptase polymerase chain reaction (RT-PCR) of cultured synoviocytes. Expression of RANKL was measured by RT-PCR and western blot in cultured synoviocytes with or without CXCL10 and also measured in Jurkat/Hut 78 T cells and CD4+ T cells in the presence of CXCL10 or dexamethasone. CXCL10 induced RANKL expression in Jurkat T cells was tested upon the pertussis toxin (PTX), an inhibitor of Gi subunit of G protein coupled receptor (GPCR). The synthetic siRNA for Gαi2 was used to knock down gene expression of respective proteins. Results CXCL10 expression in RA synoviocytes was increased by TNF-α. CXCL10 slightly increased RANKL expression in RA synoviocytes, but markedly increased RANKL expression in Jurkat/Hut 78 T cell or CD4+ T cell. CXCL10 augmented the expression of RANKL by 62.6%, and PTX inhibited both basal level of RANKL (from 37.4 ± 16.0 to 18.9 ± 13.0%) and CXCL10-induced RANKL expression in Jurkat T cells (from 100% to 48.6 ± 27.3%). Knock down of Gαi2 by siRNA transfection, which suppressed the basal level of RANKL (from 61.8 ± 17.9% to 31.1 ± 15.9%) and CXCL10-induced RANKL expression (from 100% to 53.1 ± 27.1%) in Jurkat T cells, is consistent with PTX, which inhibited RANKL expression. Conclusions CXCL10 increased RANKL expression in CD4+ T cells and it was mediated by Gαi subunits of CXCR3. These results indicate that CXCL10 may have a potential role in osteoclastogenesis of RA synovial tissue and subsequent joint erosion

Time-resolved pathogenic gene expression analysis of the plant pathogen Xanthomonas oryzae pv. oryzae

Virulence of wild-type and mutant Xoo strains on rice. (DOCX 16 kb

Protective Effect of Heme Oxygenase-1 on High Glucose-Induced Pancreatic β-Cell Injury

BackgroundGlucose toxicity that is caused by chronic exposure to a high glucose concentration leads to islet dysfunction and induces apoptosis in pancreatic β-cells. Heme oxygenase-1 (HO-1) has been identified as an anti-apoptotic and cytoprotective gene. The purpose of this study is to investigate whether HO-1 up-regulation when using metalloprotophyrin (cobalt protoporphyrin, CoPP) could protect pancreatic β-cells from high glucose-induced apoptosis.MethodsReverse transcription-polymerase chain reaction was performed to analyze the CoPP-induced mRNA expression of HO-1. Cell viability of INS-1 cells cultured in the presence of CoPP was examined by acridine orange/propidium iodide staining. The generation of intracellular reactive oxygen species (ROS) was measured using flow cytometry. Glucose stimulated insulin secretion (GSIS) was determined following incubation with CoPP in different glucose concentrations.ResultsCoPP increased HO-1 mRNA expression in both a dose- and time-dependent manner. Overexpression of HO-1 inhibited caspase-3, and the number of dead cells in the presence of CoPP was significantly decreased when exposed to high glucose conditions (HG). CoPP also decreased the generation of intracellular ROS by 50% during 72 hours of culture with HG. However, decreased GSIS was not recovered even in the presence of CoPP.ConclusionOur data suggest that CoPP-induced HO-1 up-regulation results in protection from high glucose-induced apoptosis in INS-1 cells; however, glucose stimulated insulin secretion is not restored

Signal Transduction Mechanisms Underlying Group I mGluR-mediated Increase in Frequency and Amplitude of Spontaneous EPSCs in the Spinal Trigeminal Subnucleus Oralis of the Rat

Group I mGluRs (mGluR1 and 5) pre- and/or postsynaptically regulate synaptic transmission at glutamatergic synapses. By recording spontaneous EPSCs (sEPSCs) in the spinal trigeminal subnucleus oralis (Vo), we here investigated the regulation of glutamatergic transmission through the activation of group I mGluRs. Bath-applied DHPG (10 μM/5 min), activating the group I mGluRs, increased sEPSCs both in frequency and amplitude; particularly, the increased amplitude was long-lasting. The DHPG-induced increases of sEPSC frequency and amplitude were not NMDA receptor-dependent. The DHPG-induced increase in the frequency of sEPSCs, the presynaptic effect being further confirmed by the DHPG effect on paired-pulse ratio of trigeminal tract-evoked EPSCs, an index of presynaptic modulation, was significantly but partially reduced by blockades of voltage-dependent sodium channel, mGluR1 or mGluR5. Interestingly, PKC inhibition markedly enhanced the DHPG-induced increase of sEPSC frequency, which was mainly accomplished through mGluR1, indicating an inhibitory role of PKC. In contrast, the DHPG-induced increase of sEPSC amplitude was not affected by mGluR1 or mGluR5 antagonists although the long-lasting property of the increase was disappeared; however, the increase was completely inhibited by blocking both mGluR1 and mGluR5. Further study of signal transduction mechanisms revealed that PLC and CaMKII mediated the increases of sEPSC in both frequency and amplitude by DHPG, while IP3 receptor, NO and ERK only that of amplitude during DHPG application. Altogether, these results indicate that the activation of group I mGluRs and their signal transduction pathways differentially regulate glutamate release and synaptic responses in Vo, thereby contributing to the processing of somatosensory signals from orofacial region