RESUMO
The supply and usage of energetic cofactors in metabolism is a central concern for systems metabolic engineering, particularly in case of energy intensive products. One of the most important parameters for systems wide balancing of energetic cofactors is the ATP requirement for biomass formation YATP/Biomass. Despite its fundamental importance, YATP/Biomass values for non-fermentative organisms are still rough estimates deduced from theoretical considerations. For the first time, we present an approach for the experimental determination of YATP/Biomass using comparative 13C metabolic flux analysis (13C MFA) of a wild type strain and an ATP synthase knockout mutant. We show that the energetic profile of a cell can then be deduced from a genome wide stoichiometric model and experimental maintenance data. Particularly, the contributions of substrate level phosphorylation (SLP) and electron transport phosphorylation (ETP) to ATP generation become available which enables the overall energetic efficiency of a cell to be characterized. As a model organism, the industrial platform organism Corynebacterium glutamicum is used. C. glutamicum uses a respiratory type of energy metabolism, implying that ATP can be synthesized either by SLP or by ETP with the membrane-bound F1FO-ATP synthase using the proton motive force (pmf) as driving force. The presence of two terminal oxidases, which differ in their proton translocation efficiency by a factor of three, further complicates energy balancing for this organism. By integration of experimental data and network models, we show that in the wild type SLP and ETP contribute equally to ATP generation. Thus, the role of ETP in respiring bacteria may have been overrated in the past. Remarkably, in the genome wide setting 65% of the pmf is actually not used for ATP synthesis. However, it turns out that, compared to other organisms C. glutamicum still uses its energy budget rather efficiently.
Assuntos
Corynebacterium glutamicum , Trifosfato de Adenosina/metabolismo , Biomassa , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Metabolismo Energético/genética , Engenharia MetabólicaRESUMO
BACKGROUND: Job satisfaction of doctors is an important factor determining quality and performance of a health system. The aim of this study was to assess job satisfaction among doctors of the public and private primary care clinics in Malaysia and evaluate factors that could influence the job satisfaction rating. METHODS: This study was part of the Quality and Costs of Primary Care (QUALICOPC) Malaysia, a cross-sectional survey conducted between August 2015 and June 2016 in Malaysia. Data was collected from doctors recruited from public and private primary care clinics using a standardised questionnaire. Comparisons were made between doctors working in public and private clinics, and logistic regression analysis was used to determine factors influencing the likelihood of job satisfaction outcomes. RESULTS: A total of 221 doctors from the public and 239 doctors from the private sector completed the questionnaire. Compared to private doctors, a higher proportion of public doctors felt they were being overloaded with the administrative task (59.7% vs 36.0%) and part of the work does not make sense (33.9% vs 18.4%). Only 62.9% of public doctors felt that there was a good balance between effort and reward while a significantly higher proportion (85.8%) of private doctors reported the same. Over 80% of doctors in both sectors indicated continued interest in their job and agreed that being a doctor is a well-respected job. Logistic regression analysis showed public-private sector and practice location (urban-rural) to be significantly associated with work satisfaction outcomes. CONCLUSION: A higher proportion of public doctors experienced pressure from administrative tasks and felt that part of their work does not make sense than their colleague in the private sector. At the same time, the majority of private doctors reported positive outcome on effort-and-reward balance compared to only one third of public doctors. The finding suggests that decreasing administrative workload and enhancing work-based supports might be the most effective ways to improve job satisfaction of primary care doctors because these are some of the main aspects of the job that doctors, especially in public clinics, are most unhappy with.
Assuntos
Satisfação no Emprego , Médicos de Atenção Primária/psicologia , Médicos de Atenção Primária/estatística & dados numéricos , Adulto , Estudos Transversais , Feminino , Humanos , Malásia , Masculino , Pessoa de Meia-Idade , Setor Privado , Setor Público , Inquéritos e Questionários , Carga de Trabalho/psicologia , Carga de Trabalho/estatística & dados numéricosRESUMO
Dendritic cells (DCs) are promising therapeutic agents in the field of cancer immunotherapy due to their intrinsic immune-priming capacity. The potency of DCs, however, is readily attenuated immediately after their administration in patients as tumours and various immune cells, including DCs, produce various immunosuppressive factors such as interleukin (IL)-10 and transforming growth factor (TGF)-ß that hamper the function of DCs. In this study, we used small interfering RNA (siRNA) to silence the expression of endogenous molecules in DCs, which can sense immunosuppressive factors. Among the siRNAs targeting various immunosuppressive molecules, we observed that DCs transfected with siRNA targeting IL-10 receptor alpha (siIL-10RA) initiated the strongest antigen-specific CD8(+) T cell immune responses. The potency of siIL-10RA was enhanced further by combining it with siRNA targeting TGF-ß receptor (siTGF-ßR), which was the next best option during the screening of this study, or the previously selected immunoadjuvant siRNA targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or Bcl-2-like protein 11 (BIM). In the midst of sorting out the siRNA cocktails, the cocktail of siIL-10RA and siTGF-ßR generated the strongest antigen-specific CD8(+) T cell immunity. Concordantly, the knock-down of both IL-10RA and TGF-ßR in DCs induced the strongest anti-tumour effects in the TC-1 P0 tumour model, a cervical cancer model expressing the human papillomavirus (HPV)-16 E7 antigen, and even in the immune-resistant TC-1 (P3) tumour model that secretes more IL-10 and TGF-ß than the parental tumour cells (TC-1 P0). These results provide the groundwork for future clinical development of the siRNA cocktail-mediated strategy by co-targeting immunosuppressive molecules to enhance the potency of DC-based vaccines.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , RNA Interferente Pequeno/farmacologia , Receptores de Interleucina-10/genética , Fator de Crescimento Transformador beta/genética , Animais , Antígenos de Neoplasias/imunologia , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Feminino , Papillomavirus Humano 16 , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , PTEN Fosfo-Hidrolase/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/terapia , Neoplasias do Colo do Útero/virologiaRESUMO
BACKGROUND AND OBJECTIVE: Recently it was reported that deferoxamine (DFO), an iron chelator, stimulates bone formation from MG63 and mesenchymal stem cells, but inhibits differentiation in rat calvarial cells; however, the effect of DFO on osteoblastic differentiation in human periodontal ligament cells (hPDLCs) has not been reported. The aim of this study was to investigate the effects and the possible underlying mechanism of DFO on osteoblastic differentiation of hPDLCs. MATERIAL AND METHODS: The effect of DFO on osteoblast differentiation was determined by the staining intensity of calcium deposits with Alizarin red and by RT-PCR analysis of the expression of osteoblastic markers. Signal transduction pathways were analyzed by western blotting. RESULTS: DFO increased osteogenic differentiation in a concentration-dependent manner by expression of the mRNA for differentiation markers and calcium nodule formation. Exposure of hPDLCs to DFO resulted in increases in the production of reactive oxygen species and in the levels of nuclear factor erythroid 2-related factor (Nrf2) protein in nuclear extractions, as well as a dose-dependent increase in the expression of Nrf2 target genes, including glutathione (GSH), glutathione S-transferase, γ-glutamylcysteine lygase, glutathione reductase and glutathione peroxidase. Pretreatment with Nrf2 small interfering RNA, GSH depletion by buthionine sulfoximine and diethyl maleate, and with antioxidants by N-acetylcysteine and vitamin E, blocked DFO-stimulated osteoblastic differentiation. Furthermore, pretreatment with GSH depletion and antioxidants blocked DFO-induced p38 MAPK, ERK, JNK and nuclear factor-kappaB pathways. CONCLUSION: These data indicate, for the first time, that nontoxic DFO promotes osteoblastic differentiation of hPDLCs via modulation of the Nrf2-mediated antioxidant pathway.
Assuntos
Elementos de Resposta Antioxidante/efeitos dos fármacos , Desferroxamina/farmacologia , Fator 2 Relacionado a NF-E2/farmacologia , Osteoblastos/efeitos dos fármacos , Ligamento Periodontal/citologia , Sideróforos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Acetilcisteína/farmacologia , Butionina Sulfoximina/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Cálcio/análise , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Glutamato-Cisteína Ligase/análise , Glutamato-Cisteína Ligase/antagonistas & inibidores , Glutationa/análise , Glutationa/antagonistas & inibidores , Glutationa Peroxidase/análise , Glutationa Redutase/análise , Glutationa Transferase/análise , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Maleatos/farmacologia , Fator 2 Relacionado a NF-E2/genética , NF-kappa B/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/análise , Vitamina E/farmacologiaRESUMO
AIM: To determine whether chemokines such as SDF-1 and monocyte chemoattractant protein-1 (MCP-1) are responsible for hydrogen peroxide (H2 O2 )-induced extracellular matrix (ECM) degradation and to identify the underlying mechanism in human dental pulp cells (HDPCs). METHOD: Human dental pulp cells were exposed to 0.4 mmol H2 O2 for 48 h. mRNA expression and protein expression were examined by RT-PCR and Western blot analysis, respectively. The mRNA expression of chemokine (SDF-1 and MCP-1), their receptors (CXCR4 and CXCR2) and extracellular matrix proteins was evaluated by reverse transcriptase-polymerase chain reaction. The production of SDF-1, MCP-1, CXCR4 and CCR2 in the culture medium was determined by enzyme-linked immunosorbent assay. Signal transduction pathway was examined by Western blotting. RESULTS: Hydrogen peroxide provoked the activation of MCP-1 and SDF-1 mRNA and their respective receptors, CXCR4 and CXCR2. H2 O2 treatment concomitantly downregulated the expression of ECM molecules, such as type I collagen, elastin and fibronectin, and upregulated the mRNA expression of matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-8 and MMP-9. Hydrogen peroxide-induced ECM degradation and MMP upregulation were blocked by neutralizing antibodies and siRNAs directed against SDF-1 and MCP-1. Inhibition of SDF-1 and MCP-1 blocked the H2 O2 -induced activation of Akt, p38, ERK and NF-kB. CONCLUSION: Inhibition of SDF and MCP-1 is a potent component of reducing release reactive oxygen species-induced ECM degradation in HDPCs and may play an important role in pulpal and periapical inflammation.
Assuntos
Quimiocina CCL2/metabolismo , Quimiocina CXCL12/metabolismo , Polpa Dentária/citologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Peróxido de Hidrogênio/farmacologia , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Metaloproteinases da Matriz/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Improving dendritic cell (DC) functions is highly promising for therapeutic intervention of diverse diseases, including cancer. Immunosuppressive cytokines such as interleukin (IL)-10 produced by DCs themselves (autocrine) and other regulatory immune cells (paracrine) down-regulate functional profiles of DCs through specific cell surface receptors such as IL-10R. Here, we tried to improve DC functions using small interfering RNA (siRNA) technology to block an IL-10R-mediated immunosuppressive axis. DCs modified with siRNA targeting against IL-10R or IL-10 (DC/siIL-10R or DC/siIL-10) led to up-regulation of major histocompatibility complex (MHC) class II, CD40 co-stimulatory molecule, and IL-12 proinflammatory cytokine after lipopolysacharide (LPS) stimulation compared to DC/siGFP. Notably, the LPS-induced functional profiles of DC/siIL-10R were strongly resistant to the addition of recombinant IL-10, which mimicked paracrine IL-10. In contrast, those of DC/siIL-10 were reversed by adding exogenous IL-10. Consistently, DC/siIL-10R generated more human papilloma virus (HPV) E7-specific CD8(+) T cells and stronger anti-tumour effects against E7-expressing TC-1 tumour cells in vaccinated mice than DC/siGFP, as well as DC/siIL-10. Taken together, these results provide the groundwork for future clinical translation of siRNA-mediated strategy targeting IL-10R to enhance DC-based vaccine potency.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Neoplasias Experimentais/terapia , RNA Interferente Pequeno , Receptores de Interleucina-10/genética , Animais , Antígenos CD40/genética , Antígenos CD40/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/uso terapêutico , Células Dendríticas/metabolismo , Regulação para Baixo , Feminino , Citometria de Fluxo , Genes MHC da Classe II , Tolerância Imunológica , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-12/metabolismo , Lipopolissacarídeos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus/imunologia , Interferência de RNA , Receptores de Interleucina-10/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th1/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
BACKGROUND: The risk of outbreaks escalating into pandemics has soared with globalization. Therefore, understanding transmission mechanisms of infectious diseases has become critical to formulating global public health policy. This systematic review assessed evidence in the medical and public health literature for the military as a disease vector. METHODS: We searched 3 electronic databases without temporal restrictions. Two researchers independently extracted study data using a standardized form. Through team discussions, studies were grouped according to their type of transmission mechanism and direct quotes were extracted to generate themes and sub-themes. A content analysis was later performed and frequency distributions for each theme were generated. RESULTS: Of 6477 studies, 210 met our inclusion criteria and provided evidence, spanning over two centuries (1810-2020), for the military as a pathogen transmitter, within itself or between it and civilians. Biological mechanisms driving transmission included person-to-person transmission, contaminated food and water, vector-borne, and airborne routes. Contaminated food and/or water were the most common biological transmission route. Social mechanisms facilitating transmission included crowded living spaces, unhygienic conditions, strenuous working, training conditions, absent or inadequate vaccination programs, pressure from military leadership, poor compliance with public health advice, contractor mismanagement, high-risk behaviours, and occupation-specific freedom of movement. Living conditions were the most common social transmission mechanism, with young, low ranking military personnel repeatedly reported as the most affected group. Selected social mechanisms, such as employment-related freedom of movement, were unique to the military as a social institution. While few studies explicitly studied civilian populations, considerably more contained information that implied that civilians were likely impacted by outbreaks described in the military. CONCLUSIONS: This study identified features of the military that pose a significant threat to global health, especially to civilian health in countries with substantial military presence or underdeveloped health systems. While biological transmission mechanisms are shared by other social groups, selected social transmission mechanisms are unique to the military. As an increasingly interconnected world faces the challenges of COVID-19 and future infectious diseases, the identified features of the military may exacerbate current and similar challenges and impair attempts to implement successful and equitable global public health policies.
Assuntos
COVID-19 , Militares , Surtos de Doenças , Humanos , Pandemias , SARS-CoV-2RESUMO
Gemin5 is a 170-kDa WD-repeat-containing protein that was initially identified as a component of the survival of motor neurons (SMN) complex. We now show that Gemin5 facilitates the activation of apoptosis signal-regulating kinase 1 (ASK1) and downstream signaling. Gemin5 physically interacted with ASK1 as well as with the downstream kinases SEK1 and c-Jun NH(2)-terminal kinase (JNK1), and it potentiated the H(2)O(2)-induced activation of each of these kinases in intact cells. Moreover, Gemin5 promoted the binding of ASK1 to SEK1 and to JNK1, as well as the ASK1-induced activation of JNK1. In comparison, Gemin5 did not physically associate with MKK7, MKK3, MKK6, or p38. Furthermore, depletion of endogenous Gemin5 by RNA interference (RNAi) revealed that Gemin5 contributes to the activation of ASK1 and JNK1, and to apoptosis induced by H(2)O(2) and tumor necrosis factor-alpha (TNFalpha) in HeLa cells. Together, our results suggest that Gemin5 functions as a scaffold protein for the ASK1-JNK1 signaling module and thereby potentiates ASK1-mediated signaling events.
Assuntos
MAP Quinase Quinase Quinase 5/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Sequência de Bases , Linhagem Celular , DNA Complementar/genética , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Técnicas In Vitro , MAP Quinase Quinase Quinase 5/genética , Proteína Quinase 8 Ativada por Mitógeno/genética , Ligação Proteica , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequenas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas do Complexo SMN , Transdução de Sinais , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Barrett's esophagus (BE) with high-grade dysplasia (HGD) or early carcinoma treated with surgery or photodynamic therapy (PDT) is at risk of recurrence. The efficacy of endoscopic ultrasound (EUS) for surveillance after PDT is unknown. Our objective was to determine if EUS is superior to esophagogastroduodenoscopy (EGD) and/or CT scan for surveillance of BE neoplasia after PDT. The study was designed as a retrospective review with the setting as a tertiary referral center. Consecutive patients with BE with HGD or carcinoma in situ treated with PDT were followed with EUS, CT scan and EGD with jumbo biopsies every 1 cm at 3, 4, or 6-month intervals. Exclusion criteria was < 6 months of follow up and/or < 2 EUS procedures. Main outcome measurements were residual or recurrent disease discovered by any method. Results showed that 67/97 patients met the inclusion criteria (56 men and 11 women). Median follow-up was 16 months. Recurrent or residual adenocarcinoma (ACA) was detected in four patients during follow-up. EGD with random biopsies or targeted nodule biopsies detected three patients. EUS with endoscopic mucosal resection of the nodule confirmed T1 recurrence in one of these three. In the fourth patient, CT scan revealed perigastric lymphadenopathy and EUS-FNA (fine needle aspiration) confirmed adenocarcinoma. There were two deaths, one related to disease progression and one unrelated. The rate of recurrent/persistent ACA after PDT was 4/67 = 6%. EUS did not detect disease when EGD and CT were normal. Limitations of this study include non-blinding of results and preferential status of non-invasive imaging (CT) over EUS. Our experience suggests that EUS has little role in the surveillance of these patients, unless discrete abnormalities are found on EGD or cross-sectional imaging.
Assuntos
Adenocarcinoma/diagnóstico , Adenocarcinoma/cirurgia , Esôfago de Barrett/diagnóstico , Esôfago de Barrett/terapia , Endossonografia , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/terapia , Esofagoscopia , Fotoquimioterapia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Vigilância da População , Estudos RetrospectivosRESUMO
Foreign body aspiration is commonly described in infants and children. However, recently, a new high-risk group was identified among young women, especially those from the Muslim population who wear the traditional hair scarf. This is due to the habit of holding the scarf pin in between the lips to free hands to adjust the scarf more easily. Talking, laughing, or coughing while fixing the scarf may result in inadvertent inhalation of the pin into the tracheobronchial tree. We present a case of scarf pin inhalation and the challenges encountered in managing this patient during the successful removal of the pin via flexible bronchoscopy under fluoroscopy guidance. This particular case was technically challenging for us as the sharp tip of the needle was pointing upward and piercing the bronchial mucosa.
RESUMO
Anti-vascular endothelial growth factor (VEGF) therapy has demonstrated efficacy in treating human metastatic cancers, but therapeutic resistance is a practical limitation and most tumors eventually become unresponsive. To identify microenvironmental factors underlying the resistance of cancer to antiangiogenesis therapy, we conducted genomic analyses of intraperitoneal ovarian tumors in which adaptive resistance to anti-VEGF therapy (B20 antibody) developed. We found that expression of the microseminoprotein, prostate-associated (MSMP) gene was substantially upregulated in resistant compared with control tumors. MSMP secretion from cancer cells was induced by hypoxia, triggering MAPK signaling in endothelial cells to promote tube formation in vitro. Recruitment of the transcriptional repressor CCCTC-binding factor (CTCF) to the MSMP enhancer region was decreased by histone acetylation under hypoxic conditions in cancer cells. MSMP siRNA, delivered in vivo using the DOPC nanoliposomes, restored tumor sensitivity to anti-VEGF therapy. In ovarian cancer patients treated with bevacizumab, serum MSMP concentration increased significantly only in non-responders. These findings imply that MSMP inhibition combined with the use of antiangiogenesis drugs may be a new strategy to overcome resistance to antiangiogenesis therapy.
Assuntos
Bevacizumab/farmacologia , Carcinoma Epitelial do Ovário/patologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias das Tubas Uterinas/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias Peritoneais/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Apoptose , Biomarcadores Tumorais , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/metabolismo , Hipóxia Celular , Proliferação de Células , Neoplasias das Tubas Uterinas/tratamento farmacológico , Neoplasias das Tubas Uterinas/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Neovascularização Patológica , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/metabolismo , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immune selection drives the evolution of tumor cells toward an immune-resistant and cancer stem cell (CSC)-like phenotype. We reported that apoptosis inhibitor-5 (API5) acts as an immune escape factor, which has a significant role in controlling immune resistance to antigen-specific T cells, but its functional association with CSC-like properties remains largely unknown. In this study, we demonstrated for the first time that API5 confers CSC-like properties, including NANOG expression, the frequency of CD44-positive cells and sphere-forming capacity. Critically, these CSC-like properties mediated by API5 are dependent on FGFR1 signaling, which is triggered by E2F1-dependent FGF2 expression. Furthermore, we uncovered the FGF2-NANOG molecular axis as a downstream component of API5 signaling that is conserved in cervical cancer patients. Finally, we found that the blockade of FGFR signaling is an effective strategy to control API5high human cancer. Thus, our findings reveal a crucial role of API5 in linking immune resistance and CSC-like properties, and provide the rationale for its therapeutic application for the treatment of API5+ refractory tumors.
RESUMO
Zinc overload may be a key mechanism of neuronal death in acute brain injury. We have demonstrated previously that zinc overload neurotoxicity involves protein kinase C (PKC)-dependent rises in intracellular levels of reactive oxygen species (ROS). However, the cascade linking PKC activation to ROS generation in cultured cortical neurons has been unknown. A recent study has demonstrated that ROS-generating NADPH oxidase is present in sympathetic neurons and contributes to NGF deprivation-induced cell death. Because NADPH oxidase is activated by PKC, in the present study, we examined the possibility that NADPH oxidase is the effector for oxidative stress in zinc-overloaded cortical cells. Reverse transcription-PCR and Western blot analyses revealed that naive cultured cortical cells express subunits of NADPH oxidase at low levels. Exposure to zinc substantially increased levels of NADPH oxidase subunits in both neurons and astrocytes. In addition, zinc exposure induced translocation of the p47(PHOX) and p67(PHOX) subunits to the membrane, a signature event for NADPH oxidase activation. Addition of a selective PKC inhibitor, GF109203X, blocked both the induction and the membrane translocation of NADPH oxidase by zinc. Supporting the role for NADPH oxidase in zinc-triggered oxidative injury, NADPH oxidase inhibitors attenuated ROS production and cortical neuronal death induced by zinc. In addition, Cu/Zn-superoxide dismutase and catalase attenuated zinc-induced cortical neuronal death. Our results have demonstrated that zinc overload induces and activates NADPH oxidase in cortical neurons and astrocytes in a PKC-dependent manner. Thus, NADPH oxidase may be an enzyme contributing to ROS generation in zinc-overloaded cortical neurons and astrocytes.
Assuntos
Astrócitos/enzimologia , Córtex Cerebral/enzimologia , NADPH Oxidases/metabolismo , Neurônios/enzimologia , Zinco/farmacologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Catalase/metabolismo , Catalase/farmacologia , Morte Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Técnicas de Cocultura , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Camundongos , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/química , Neurônios/citologia , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosfoproteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Subunidades Proteicas , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase/farmacologiaRESUMO
The extended single-reaction multiplex PCR (esr-mPCR) developed in this study to detect staphylococcal enterotoxins (SEs), including SEA, SEB, SEC, SED, SEE, SEH, SEI, and SEJ, requires fewer sets of primers than other conventional multiplex PCRs and can be used to detect newly identified staphylococcal enterotoxins SEs more readily. Esr-mPCR analysis of 141 isolates of Staphylococcus aureus obtained from abattoir and livestock product samples revealed that 27 of the S. aureus isolates were toxigenic, and two were 2 multitoxigenic isolates. The most prevalent SE type was SEI followed by SEA and SEH. In addition, we investigated the clonal relatedness of toxigenic S. aureus isolates by arbitrarily primed PCR (AP-PCR). AP-PCR analysis of toxigenic S. aureus isolates revealed that the discriminatory power of AP-PCR was 9 (D=0.81), 8 (D=0.77), and 10 types (D=0.83) with primers AP1, ERIC2, and AP7, respectively. The combination of three each AP-PCR result could rearrange toxigenic S. aureus isolates into 10 types and five subtypes, with the D-value of 0.92. Interestingly, our data showed that toxigenic S. aureus isolates from different sources had different fingerprinting patterns although some of them carried the same types of SE genes. These data suggest that combinations of esr-mPCR and AP-PCR can provide a powerful approach for epidemiological investigation of toxigenic S. aureus isolates.
Assuntos
DNA Bacteriano/genética , Enterotoxinas/genética , Reação em Cadeia da Polimerase/métodos , Staphylococcus aureus/metabolismo , Matadouros , Técnicas de Tipagem Bacteriana , Impressões Digitais de DNA , Primers do DNA , DNA Bacteriano/isolamento & purificação , Enterotoxinas/isolamento & purificação , Microbiologia de Alimentos , Intoxicação Alimentar Estafilocócica/prevenção & controle , Staphylococcus aureus/classificação , Staphylococcus aureus/isolamento & purificaçãoRESUMO
An efficacy test of GC-100X, a noncorrosive alkaline ionic fluid (pH 12) composed of free radicals and supplemented with xylitol, was carried out against six major foodborne pathogens-Staphylococcus aureus FRI 913, Salmonella enterica serovar Enteritidis ATCC 13076, S. enterica serovar Typhimurium DT104 Korean isolate, Vibrio parahaemolyticus ATCC 17803, Escherichia coli O157:H7 ATCC 43894, and Pseudomonas aeruginosa KCTC 1637-at three different temperatures (4, 25, and 36 degrees C) with or without organic load (2% yeast extract). Results revealed a more than 4-log10 (CFU/ml) reduction (1.0 x 10(4) CFU/ml reduction) against all pathogens reacted at 37 degrees C for 3 h in the absence of organic material. GC-100X solution diluted with an equal volume of distilled or standard hard water (300 ppm CaCO3) showed effective bactericidal activity, particularly against gram-negative bacteria. Washing efficacy of GC-100X solution was compared against E. coli O157:H7 on cherry tomato surfaces with those of a commercially used detergent and chlorine water (100 ppm). Viable cell counts of E. coli O157:H7 that had penetrated to the cores of tomatoes after sanitizing treatment revealed that GC-100X stock and its 5% diluted solutions had similar washing effects to 100-ppm chlorine water and were more effective than the other kitchen detergent. These results indicate that GC-100X has good bactericidal and sanitizing activities and is useful as a new sanitizer for food safety and kitchen hygiene.
Assuntos
Desinfetantes/farmacologia , Escherichia coli O157/efeitos dos fármacos , Solanum lycopersicum/microbiologia , Cloro/farmacologia , Contagem de Colônia Microbiana , Escherichia coli O157/crescimento & desenvolvimento , Microbiologia de Alimentos , Concentração de Íons de Hidrogênio , Temperatura , Fatores de Tempo , Resultado do Tratamento , Xilitol/farmacologiaRESUMO
From a nationwide survey of otitis media in Korea, 44.52% of the population were found to have some type of otitis media or its sequelae. A high prevalence rate was seen in the age group over 41 years. This finding suggests a strong relationship between socioeconomic status and incidence of otitis media. From a clinical study of surgical cases of otitis media seen in the past 10 years, we have found that the prevalence of chronic otitis media is decreasing every year. However, severity and pathological findings of otitis media were reflected remarkably in a decreased incidence of acute purulent otitis media and an increased incidence of middle ear effusion in children. In recent years our efforts to control chronic otitis media in children have focused on the treatment of chronic middle ear effusion. To prevent the latter condition, it is strongly emphasized that pediatricians and primary care physicians should be competent in diagnosing otitis media as early as possible, and that they should refer appropriate patients to otolaryngologists for further evaluation and management.
Assuntos
Otite Média/epidemiologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Colesteatoma/epidemiologia , Estudos Transversais , Feminino , Humanos , Coreia (Geográfico) , Masculino , Pessoa de Meia-Idade , Otite Média/cirurgia , Fatores SocioeconômicosRESUMO
The nucleotide sequence of the nucleocapsid protein (N) coding region of a Hantaan virus strain (CFC94-2) isolated from a Korean Hemorrhagic Fever (KHF) patient was determined by sequencing a series of deletion mutants. Comparison of the N coding sequence of CFC94-2 to the sequence of the prototype of Hantaan virus, strain 76-118 reveals a 4.97% difference in the nucleotide sequence and a 1.6% difference in the deduced amino acid sequence. The rate of amino acid sequence variation in N protein of different Hantaan viruses (1.6%) is quite similar to that in G1 and G2 envelope proteins (1.7%). These results suggest that N protein may be under a similar selection pressure to G1 and G2 envelope proteins against host immune system.
Assuntos
Proteínas do Capsídeo , Capsídeo/genética , Vírus Hantaan/química , Febre Hemorrágica com Síndrome Renal/virologia , Proteínas do Core Viral/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Variação Genética , Humanos , Coreia (Geográfico) , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
We used the IL088 Otodynamic Analyzer system to study click-evoked otoacoustic emissions (CEOAEs) in 30 healthy guinea pigs. The animals were anesthetized and patterns of the CEOAEs were evaluated before manipution, after the tympanic bulla was opened, and after formation of a microfistula on the basal turn of the cochlea. The animals then were divided into three pressure loading groups (10, 20, and 30 cm H2O). CEOAEs were recorded with a capillary manometer at pretest, 5, 10, 20, 30, 45, and 60 min after perilymphatic pressure loading to the basal turn of cochlea, and 10 and 20 min after pressure unloading. As perilymphatic pressure increased, all three pressure groups showed maximum decreases in both echo response and reproducibility 5 min after pressure loading. In the 10 cm H2O pressure group, emissions recovered 10 min after pressure loading, and this tendency continued. However, in the 20 and 30 cm H2O pressure groups, no recovery of emissions was seen throughout the 60 min observation period, except for emissions after pressure unloading. The results suggest that the echo response and reproducibility may be sensitive indicators of cochlear function and perilymphatic pressure regulation capacity.
Assuntos
Estimulação Acústica , Cóclea/fisiologia , Cobaias , Perilinfa/fisiologia , AnimaisRESUMO
Antibiotic resistance in Salmonella enteritidis and S. typhimurium, one of most frequent etiologic pathogens of food-borne bacterial gastroenteritidis in humans, is a serious health problem worldwide. Fifteen and 22 each of S. enteritidis and S. typhimurium were isolated from animals from 1983 to 1999 in Korea and tested for their antibiotic resistance patterns and phage types. S. enteritides isolates were highly resistant to sulfonamides (86.7%) and four of them (26.6%) showed multiple antibiotic resistance. The most frequent phage type (PT) of S. enteritids was PT1 (33.3%) even though none of them had multiple antibiotic resistance. S. typhimurium isolates were highly resistant to streptomycin, sulfonamides, and tetracycline, 100%, 95.5%, and 86.4% respectively. The incidence of multiple antibiotic resistance of S. typhimurium isolates was extremely high (100%) comparing to S. enteritidis isolates (26.7%). Two of the five ACSSuT type S. typhimurium isolates, resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline, were phage type DT104. All S. typhimurium isolates were sensitive to florfenicol. For the rapid detection of multiple antibiotic resistant S. enteritidis and S. typhimurium isolates, particularly ACSSuT type S. typhimurium DT104, antibiotic resistance genes, cmlA/tetR, PSE-1, and TEM, and Salmonella spp. Specific gene, SipB/C, were amplified using four pairs of primers in hot-started multiplex polymerase chain reaction. Two Korean isolates of S. typhimurium DT104 showed TEM amplicons instead of PSE-1 for the ampicillin resistance. The multiplex PCR used in this study was useful in rapid detection of ACSSuT type S. typhimurium and identification of b-lactamase gene distribution among Salmonella isolates.
Assuntos
Antibacterianos/farmacologia , Salmonelose Animal/microbiologia , Salmonella enteritidis/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacos , Animais , Tipagem de Bacteriófagos , Sequência de Bases , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana Múltipla/genética , Amplificação de Genes , Humanos , Testes de Sensibilidade Microbiana/veterinária , Fenótipo , Reação em Cadeia da Polimerase/veterinária , Salmonelose Animal/tratamento farmacológico , Salmonella enteritidis/classificação , Salmonella enteritidis/genética , Salmonella typhimurium/classificação , Salmonella typhimurium/genéticaRESUMO
This study was conducted to identify attention deficit hyperactivity disorder (ADHD) in Korean juvenile delinquents. Intelligence tests (KEDI-WISC, K-WAIS), the Test of Variables of Attention (TOVA), the Teacher Report Form (TRF), the Youth Self-Report (YSR), and the Rosenberg Self-Esteem Scale were administered to 98 incarcerated Korean adolescents (the delinquent group) and 84 adolescent nondelinquents (the control group). The groups were compared, and significant differences were found for ADHD; 42.4% of the adolescents in the delinquent group were identified as having ADHD, in comparison to 11.9% of the adolescents in the control group. Delinquent adolescents and adolescents with ADHD were found to have lower IQ scores, poorer TOVA performance, more severe problem behaviors, and lower self-esteem than nondelinquent adolescents and adolescents without ADHD. Delinquent adolescents with ADHD consistently fared the worst on assessments of intelligence, TOVA performance, problem behaviors, and self-esteem.