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BACKGROUND: Microglia, the mononuclear immune cells of the central nervous system (CNS), are essential for the maintenance of CNS homeostasis. BAP31, a resident and ubiquitously expressed protein of the endoplasmic reticulum, serves as a sorting factor for its client proteins, mediating the subsequent export, retention, and degradation or survival. Recently, BAP31 has been defined as a regulatory molecule in the CNS, but the function of BAP31 in microglia has yet to be determined. In the present study, we investigated whether BAP31 is involved in the inflammatory response of microglia. METHODS: This study used the BV2 cell line and BAP31 conditional knockdown mice generated via the Cre/LoxP system. A BAP31 knockdown experiment was performed to elucidate the role of BAP31 in the endogenous inflammatory cytokine production by microglial BV2 cells. A mouse model of lipopolysaccharide (LPS)-induced cognitive impairment was established to evaluate the neuroprotective effect of BAP31 against neuroinflammation-induced memory deficits. Behavioral alterations were assessed with the open field test (OFT), Y maze, and Morris water maze. The activation of microglia in the hippocampus of mice was observed by immunohistochemistry. Western blot, enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, and reverse transcription quantitative real-time polymerase chain reaction (RT-PCR) were used to clarify the mechanisms. RESULTS: BAP31 deficiency upregulates LPS-induced proinflammatory cytokines in BV2 cells and mice by upregulating the protein level of IRAK1, which in turn increases the translocation and transcriptional activity of NF-κB p65 and c-Jun, and moreover, knockdown of IRAK1 or use of an IRAK1 inhibitor reverses these functions. In the cognitive impairment animal model, the BAP31 knockdown mice displayed increased severity in memory deficiency accompanied by an increased expression of proinflammatory factors in the hippocampus. CONCLUSIONS: These findings indicate that BAP31 may modulate inflammatory cytokines and cognitive impairment induced by neuroinflammation through IRAK1, which demonstrates that BAP31 plays an essential role in microglial inflammation and prevention of memory deficits caused by neuroinflammation.
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Encefalitis/metabolismo , Inflamación/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Proteínas de la Membrana/metabolismo , Microglía/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Citocinas/metabolismo , Encefalitis/inducido químicamente , Encefalitis/patología , Inflamación/inducido químicamente , Inflamación/patología , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BLRESUMEN
Our previous study has shown berberine prevents damage to the intestinal mucosal barrier during early phase of sepsis in rat through mechanisms independent of the NOD-like receptors signaling pathway. In this study, we explored the regulatory effects of berberine on Toll-like receptors during the intestinal mucosal damaging process in rats. Male Sprague-Dawlay (SD) rats were treated with berberine for 5 d before undergoing cecal ligation and puncture (CLP) to induce polymicrobial sepsis. The expression of Toll-like receptor 2 (TLR 2), TLR 4, TLR 9, the activity of nuclear factor-kappa B (NF-κB), the levels of selected cytokines and chemokines, percentage of cell death in intestinal epithelial cells, and mucosal permeability were investigated at 0, 2, 6, 12 and 24 h after CLP. Results showed that the tumor necrosis factor-α (TNF-α ) and interleukin-6 (IL-6) level were significantly lower in berberine-treated rats compared to the control animals. Conversely, the expression level of tight junction proteins, percentage of cell death in intestinal epithelial cells and the mucosal permeability were significantly higher in berberine-treated rats. The mRNA expression of TLR 2, TLR 4, and TLR 9 were significantly affected by berberine treatment. Our results indicate that pretreatment with berberine attenuates tissue injury and protects the intestinal mucosal barrier in early phase of sepsis and this may possibly have been mediated through the TLRs pathway.
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OBJECTIVE: To investigate the early diagnosis and treatment of acute mesenteric ischemia. METHODS: Forty-two patients with acute mesenteric ischemia from June 2007 to November 2011 were reviewed retrospectively. All patients were diagnosed with DSA and (or) CT and (or) surgery. In this group, there were 32 cases of acute occlusion of meseteric ischemia (AOMI), 9 cases of superior mesenteric venous thrombosis (SMVT) and 1 case of non-occlusive mesenteric ischemia. The patients were treated using comprehensive treatment including early intervention treatment and application of the principle of damage control. The survival of all patients was followed up for 6 months or more in outpatient. RESULTS: (1) Of the 32 AOMI cases, 4 cases healing by systemic anticoagulation; The 19 cases received interventional treatment, including 10 cases received simply interventional treatment, surgery after the failure of intervention in 5 cases, 3 patients died without surgery and postoperative interventional treatment one cases were cured; Eight cases received surgery treatment; One case gave up. (2) Of the 9 SMVT cases, 2 cases healing by systemic anticoagulation; The 6 cases received interventional treatment, including 1 cases received simply interventional treatment, surgery after the failure of intervention in 1 cases, 4 cases to consider intestinal necrosis received interventional treatment again after surgery; One patient died without treatment. (3) Eight cases received delay abdomen close treatment with the principle of damage control surgery. The overall mortality rate of 23.8% (10/42). Interventional treatment of 26 cases, 4 deaths, a mortality rate of 15.3%; The abdomen delayed close of 8 cases, 1 death. CONCLUSIONS: The results show that early diagnosis and treatment is critical to reduce AMI mortality. Comprehensive treatment of early intervention treatment and application of the principle of damage control can significantly reduce the mortality of AMI.
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Isquemia/diagnóstico , Isquemia/terapia , Enfermedades Vasculares/diagnóstico , Enfermedades Vasculares/terapia , Adulto , Anciano , Anciano de 80 o más Años , Diagnóstico Precoz , Femenino , Humanos , Masculino , Isquemia Mesentérica , Persona de Mediana Edad , Estudios Retrospectivos , Tomografía Computarizada por Rayos X , Adulto JovenRESUMEN
Acute lung injury (ALI) and its more severe condition acute respiratory distress syndrome (ARDS) are critical life-threatening disorders characterized by an excessive influx of neutrophils into the alveolar space. Neutrophil infiltration is a multi-step process involving the sequential engagement of adhesion molecules. The adhesion molecule CD11b/CD18 acts as an important role in the recruitment of neutrophils to lung tissues in the ALI model. B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum transmembrane protein, has been reported to regulate the cellular anterograde transport of CD11b/CD18 in human neutrophils. To explore how BAP31 regulates CD11b/CD18 in mouse neutrophils, we constructed myeloid-specific BAP31 knockdown mice in this study. Biological investigations indicated that BAP31 deficiency could significantly alleviated lung injury, as evidenced by the improved histopathological morphology, reduced pulmonary wet/dry weight ratio, inhibited myeloperoxidase level and decreased neutrophil counts in the bronchoalveolar lavage fluid. Further studies clarified that BAP31 deficiency obviously down-regulated the expression of CD11b/CD18 and P-selectin glycoprotein ligand-1 (PSGL-1) by deactivating the nuclear factor kappa B (NF-κB) signaling pathway. Collectively, our results revealed that BAP31 depletion exerted a protective effect on ALI, which was possibly dependent on the attenuation of neutrophil adhesion and infiltration by blocking the expression of adhesion molecules CD11b/CD18 and PSGL-1. These findings implied the potential of BAP31 as an appealing protein to mediate the occurrence of ALI.
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Lesión Pulmonar Aguda , Neutrófilos , Animales , Ratones , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/inmunología , Antígenos CD18/genética , Antígenos CD18/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Neutrófilos/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismoRESUMEN
Reactive oxygen species (ROS) production by activation of microglia is considered to be a major cause of neuronal dysfunction, which can lead to damage and death through direct oxidative damage to neuronal macromolecules or derangement of neuronal redox signaling circuits. BAP31, an integral ER membrane protein, has been defined as a regulatory molecule in the CNS. Our latest studies have found that BAP31 deficiency leads to activation of microglia. In this study, we discovered that BAP31 deficiency upregulated LPS-induced superoxide anion production in BV2 cells and mice by upregulating the expression level of p22phox and by inhibiting the activation of Nrf2-HO-1 signaling. Knockdown of p22phox/keap1 or use of an NADPH oxidase inhibitor (apocynin) reversed the production of superoxide anion and inflammatory cytokines, which then reduced neuronal damage and death in vitro and in vivo. These results suggest that BAP31 deficiency contributes to microglia-related superoxide anion production and neuroinflammation through p22phox and keap1. Furthermore, the excess superoxide anion cooperated with inflammatory cytokines to induce the damage and death of neurons. Thus, we determined that BAP31 is an important regulator in superoxide anion production and neuroinflammation, and the downstream regulators or agonists of BAP31 could therefore be considered as potential therapeutic targets in microglial-related superoxide anion production and neuroinflammation.
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Hemo-Oxigenasa 1/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteínas de la Membrana/metabolismo , Microglía/metabolismo , NADPH Oxidasas/metabolismo , Transducción de Señal , Superóxidos/metabolismo , Línea Celular Tumoral , Hemo-Oxigenasa 1/genética , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteínas de la Membrana/genética , Microglía/patología , NADPH Oxidasas/genéticaRESUMEN
OBJECTIVES: Our aim was to investigate the prevalence and predictive variables of sarcopenia. METHODS: We recruited participants from the Peking Union Medical College Hospital Multicenter Prospective Longitudinal Sarcopenia Study (PPLSS). Muscle mass was quantified using bioimpedance, and muscle function was quantified using grip strength and gait speed. Logistic regression revealed the relationships between sarcopenia and nutritional, lifestyle, disease, psychosocial and physical variables. RESULTS: The prevalence of sarcopenia and sarcopenic obesity was 9.2%-16.2% and 0.26%-9.1%, respectively. Old age, single status, undernourishment, higher income, smoking, low physical activity, poor appetite and low protein diets were significantly associated with sarcopenia. Multiple logistic regression analysis showed that age was a risk factor for all stages of sarcopenia, and participants above 80 years were greater than fivefold more susceptible to sarcopenia, while lower physical activity was an independent risk factor. The optimal cut-off value for age was 71 years, which departs from the commonly accepted cut-off of 60 years. Female participants were greater than twofold less susceptible to sarcopenia than male participants. The sterol derivative 25-hydroxyvitamin D was associated with fourfold lower odds of sarcopenia in male participants. Several protein intake variables were also correlated with sarcopenia. Based on these parameters, we defined a highly predictive index for sarcopenia. CONCLUSIONS: Our findings support a predictive index of sarcopenia, which agglomerates the complex influences that sterol metabolism and nutrition exert on male vs female participants.
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Proteínas/metabolismo , Sarcopenia/patología , Esteroles/metabolismo , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Calcifediol/metabolismo , China/epidemiología , Ejercicio Físico , Femenino , Humanos , Modelos Logísticos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Curva ROC , Factores de Riesgo , Sarcopenia/epidemiología , Factores Sexuales , Testosterona/análisisRESUMEN
BACKGROUND: Ageing, chronic diseases, prolonged inactivity, and inadequate nutrition pose a severe threat to skeletal muscle health and function. To date, experimental evidence suggests that ageing-related subclinical inflammation could be an important causative factor in sarcopenia. Although inflammatory signalling has been implicated in the pathogenesis of experimental animal models of sarcopenia, few studies have surveyed the clinical association between circulating factors and muscle mass in patients before and after lifestyle interventions. In this study, we evaluated whether proinflammatory cytokines are associated with the onset of sarcopenia, which circulating factors are associated with the severity of sarcopenia, and how these factors change after lifestyle interventions in sarcopenic elderly persons. METHODS: A total of 56 elderly subjects (age ≥ 60 years) with sarcopenia and 56 elderly non-sarcopenic subjects, who met entry criteria and had given informed consent, were selected from the Peking Union Medical College Hospital multicentre prospective longitudinal sarcopenia study for testing relevant circulating factors. Thirty-two elderly subjects from the sarcopenic cohort completed a 12 week intensive lifestyle intervention programme with whey supplements (30 g/day) and a personalized resistance training regimen. The levels of proinflammatory cytokines and metabolic hormones, pre-intensive and post-intensive lifestyle interventions, were measured. RESULTS: The sarcopenic group was significantly older (72.05 ± 6.54 years; P < 0.001), more likely to be inactive and female (57.1% of all sarcopenic patients), and had a higher prevalence of type 2 diabetes (16% higher risk). Compared with non-sarcopenic subjects, serum interleukin (IL)-6, IL-18, tumour necrosis factor-α (TNF-α), TNF-like weak inducer of apoptosis (TWEAK), and leptin were significantly higher, while insulin growth factor 1, insulin, and adiponectin were significantly lower in sarcopenic patients (all P < 0.05). Logistic regression analyses revealed that high levels of TNF-α (>11.15 pg/mL) and TWEAK (>1276.48 pg/mL) were associated with a 7.6-fold and 14.3-fold increased risk of sarcopenia, respectively. After adjustment for confounding variables, high levels of TWEAK were still associated with a 13.4-fold increased risk of sarcopenia. Intensive lifestyle interventions led to significant improvements in sarcopenic patients' muscle mass and serum profiles of TWEAK, TNF-α, IL-18, insulin, and adiponectin (all P < 0.05). CONCLUSIONS: High levels of the inflammatory cytokines TWEAK and TNF-α are associated with an increased risk of sarcopenia, while the metabolic hormones insulin growth factor 1, insulin, and adiponectin are associated with a decreased risk of sarcopenia in our Chinese patient cohort. Intensive lifestyle interventions could significantly improve muscle mass, reduce inflammation, and restore metabolic hormone levels in sarcopenic patients. This trial was registered at clinicaltrials.gov as NCT02873676.
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Envejecimiento/inmunología , Mediadores de Inflamación/sangre , Inflamación/rehabilitación , Sarcopenia/inmunología , Anciano , Envejecimiento/sangre , Composición Corporal , China , Estudios Transversales , Citocina TWEAK/sangre , Citocina TWEAK/inmunología , Femenino , Estilo de Vida Saludable , Humanos , Inflamación/sangre , Inflamación/diagnóstico , Inflamación/inmunología , Mediadores de Inflamación/inmunología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Músculo Esquelético/inmunología , Estudios Prospectivos , Entrenamiento de Fuerza , Sarcopenia/sangre , Sarcopenia/diagnóstico , Sarcopenia/rehabilitación , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
OBJECTIVE: To explore the changes of mitogen activated protein kinase (MAPK) cascade pathway and anti-stress response of hepatocytes after liver transplantation. METHODS: Ten normal liver specimens and 18 punctured donor liver specimens were divided into 3 groups: A (control:10), B (no rejection:10) and C (acute rejection:8). MAPK, Ras, Jun and heat shock protein 70 (HSP70) were tested immunohistochemically while Ras and HSP70 were tested by in situ hybridization. All sections were subjected to image analysis. RESULTS: Protein expressions of MAPK, Ras and Jun were increased by an ascending order of groups A, B and C. The protein expression of HSP70 was the highest in group B but lower in group C. Expressions of Ras and HSP70 mRNA were consistent with those of protein. CONCLUSIONS: The changes of the MAPK cascade pathway and anti-stress response of hepatocytes after liver transplantation are one of regulation mechanisms for protecting the hepatocytes from damage after liver transplantation. This mechanism is active to support individual survival
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Rechazo de Injerto/fisiopatología , Trasplante de Hígado , Sistema de Señalización de MAP Quinasas/fisiología , Estrés Fisiológico/fisiopatología , Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Inmunohistoquímica , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
OBJECTIVES: To explorer the change of several signal pathway and their signal after liver transplantation. METHODS: Classified 34 punctured donor liver samples and 10 normal liver samples as A (no rejection) groups, B (mild/moderate acute rejection) groups, C (serious acute rejection) groups, D (chronic rejection/fibrosis) groups and E (control) groups, MAPK, Ras and p53 were performed immunohistochemistry analysis and image analysis. MAPK and Ras were performed in situ hybridizition. Then image analysis was performed. RESULTS: The protein expression of MAPK, Ras, increase by turns of A, B and C groups (1.42+/-0.28, 3.88+/-0.87, 6.68+/-0.57 in MAPK; 1.27+/-0.12, 2.80+/-0.30, 3.93+/-0.20 in Ras; corresponding), and decrease by turns of D and E groups (1.49+/-0.37, 0.88+/-0.20 in MAPK; 1.47+/-0.21, 1.01+/-0.12 in Ras; corresponding, F=178.39 in MAPK and 320.59 in Ras, groups B, C vs groups A, D, E, P<0.001 in MAPK and Ras), The protein expression of p53 is higher in treated groups (The results of groups A to E are 2.09+/-0.13, 2.39+/-0.11, 2.03+/-0.19, 2.26+/-0.18 and 0.35+/-0.08, corresponding, F=360.08, groups E vs groups A, B, C, D, P<0.001). Expression of MAPK, Ras mRNA is as same as that of protein. CONCLUSION: The MAPKs pathway has role in rejection response after liver transplantation. And it seemed that the MAPKs and p53 are one regulation mechanism for protecting the hepatocyte from damage after liver transplantation.
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Trasplante de Hígado , Transducción de Señal/fisiología , Humanos , Inmunohistoquímica , Hibridación in Situ , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/análisis , Proteína p53 Supresora de Tumor/análisis , Proteínas ras/análisisRESUMEN
NOD-like receptors play a crucial role in host defense against intestinal infection. We explored the regulatory effects of berberine on NLRs during the intestinal mucosal damaging process in rats. Male Sprague-Dawlay (SD) rats were treated with berberine for 5d before undergoing cecal ligation and puncture (CLP) to induce polymicrobiol sepsis. The expression of nucleotide-binding oligomerization domain 2 (NOD2), NLR family-pyrin domain containing 3 (NLRP3), the activity of nuclear factor-kappa B (NF-κB), the levels of selected cytokines and chemokines, percentage of cell death in intestinal epithelial cells, and mucosal permeability were investigated at 0h, 2h, 6h, 12h and 24h after CLP. Results showed that the Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) level in were significantly lower in berberine treated rats compared to the control animals. The tight junction proteins level, percentage of cell death in intestinal epithelial cells and the mucosal permeability were, on the other hand, significantly elevated in berberine treated rats. The expression of NOD and NLRP3, however, were not significantly affected by berberine treatment. Our results indicate that Pretreatment with berberine attenuates tissue injury and protects the intestinal mucosal barrier in early phase of sepsis but it is likely that the mechanisms of this preventive effect do not involve the NLR pathway.
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Berberina/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Sepsis/metabolismo , Sepsis/patología , Transducción de Señal/efectos de los fármacos , Animales , Proteínas Portadoras , Muerte Celular/efectos de los fármacos , Claudina-4/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-6/sangre , Mucosa Intestinal/metabolismo , Masculino , Proteína con Dominio Pirina 3 de la Familia NLR , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/metabolismo , Permeabilidad/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
OBJECTIVE: To initially investigate the expressing regularity and effect of enterocyte NOD like receptors on gut mucosal barrier during early phase of acute intra-abdominal infection. METHODS: Sprague-Dawley (SD) rats were randomly allocated into control group (n=6) and experimental group (n=24). Acute intra-abdominal infection model was induced by cecal ligation and puncture (CLP). The level of NOD2 and NOD like receptor 3 (NLRP3) mRNA expression in gut mucosa was determined using fluorescent polymerase chain reaction (PCR); the expression of caspase-1 and tight junction protein was determined by Western blotting; the activity of nuclear factor-ΚB (NF-ΚB) was determined by electrophoretic mobility shift assay (EMSA); the level of serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was determined by enzyme linked immunosorbent assay (ELISA). The dead cell percentage of enterocyte was observed by terminal deoxynucleotidyl transferase mediated nick end labeling, and the gut mucosal permeability using an in situ loop preparation of gut with fluorescence isothiocyanate-conjugated dextran was determined. RESULTS: NOD2 mRNA expression was quickly increased to a very high apex at 2 hours after operation, compared with the control group, the difference was statistically significant (75.50±13.03 vs. 1.00±0.00, P<0.01), and quickly descended at 6 hours, and then slowing descended. The expression of NLRP3 mRNA was decreased at 2 hours after the operation, then increased gradually, and peaked at 12 hours, which was significantly higher than that in control group (4.03±0.71 vs. 1.00±0.00,P<0.05). The level of caspase-1 was significantly higher than that in control group at 2 hours (3.56±0.14 vs. 2.10±0.11,P<0.01) and then gradually increased. The levels of Occludin, ZO-1 and Claudin-4 were obviously lowered than that in control group at 2-6 hours (2 hours Occludin: 7.24±1.13 vs. 12.72±1.34, 6 hours ZO-1: 0.47±0.09 vs. 1.57±0.17, 2 hours Claudin-4: 1.63±0.28 vs. 3.40±0.34, P<0.05 or P<0.01), and then all slowly decreased. The activity of NF-ΚB was quickly increased at 2 hours, obviously higher than that in control group (24.85±0.57 vs. 12.42±0.73, P<0.01), and then slowly decreased at a state of high expression. The expression of IL-6 in experimental group had a peak at 6 hours (compared with the control group, 3088.07±330.03 vs. 26.19±7.58,P<0.01), and then slowly decreased. The level of TNF-α was significantly higher than that in control group at 2 hours (110.75±19.18 vs. 7.86±3.58,P<0.01), and then gradually increased. The percentage of dead enterocyte was higher than that in control group with infection progress (0.12±0.02 vs. 0.03±0.01,P<0.05), and then gradually increased, so mucosal permeability was gradually increased too. Compared with the control group, the difference was statistically significant through 2 hours [glucosans: (35.75±4.66)% vs. (2.84±0.35)%, P<0.01]. The relevance analysis showed that NLRP3 have a little higher correlation with mucosal permeability and caspase-1 protein expression than other targets. Caspase-1 had a strong correlation with the percentage of dead cell, TNF-α and gut mucosal permeability. Gut mucosal permeability had highest correlation with the expression of caspase-1 protein. CONCLUSIONS: The data of our study suggested that NOD2 and NLRP3 take role in early phase of intra-abdominal infection, the huge wave of the expression level of NOD2 hinted that it was feed backed by some accurate mechanism in case of its express was too strong or too weak. The correlation of NLRP3, caspase-1, and percentage of dead cell imply they maybe have some extent of causation, and the percentage of dead cell in gut mucosa was as important as tight junction protein in maintaining the function of intestinal mucosal barrier.
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Mucosa Intestinal/metabolismo , Infecciones Intraabdominales/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Proteínas Portadoras , Caspasa 1/metabolismo , Masculino , Proteína con Dominio Pirina 3 de la Familia NLR , Proteína Adaptadora de Señalización NOD2/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells. They have a distinct karyotype, membrane lipid composition, morphology and growth kinetics from the parental cell lines. In this paper, we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility. Adaptation of cell lines sf-9, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1) to 33°C and 35°C was carried out by shifting the culture temperature between 28°C and higher temperatures by a gradual stepwise increase in temperature. The process of adaption to a higher culture temperature was accomplished over a period of 2 months. The cell lines with the temperature adaption were designated as sf9-ht33, sf9-ht35, High5-ht33, High5-ht35, MG1-ht33, MG1-ht35. These cell lines have been subcultured over 70 passages. Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines. The population doubling time of heat adapted cell lines were 1-4 h less than these of parental cell lines. Cell shapes did not show obvious change, however, the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption. When the cell lines were infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) at 28°C, 33°C, 35°C and 37°C, production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.
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Baculoviridae/fisiología , Línea Celular/fisiología , Lepidópteros/citología , Animales , Técnicas de Cultivo de Célula , Línea Celular/química , Línea Celular/virología , Calor , Cinética , Lepidópteros/químicaRESUMEN
It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines. But the characteristics of the cell line do not always remain stable and may change upon continuous passage. Recently an alphanodavirus, named Tn5 Cell Line Virus (or TNCL Virus), was identified in High Five cells in particular. Therefore, we established a new cell line, QB-Tn9-4s, from Trichoplusia ni, which was determined to be free of TNCL virus by RT-PCR analysis. In this paper, we describe the development of a novel cell clone, QB-CL-B, from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV, and the level of recombinant protein production. This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate; although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies, there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells.
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Baculoviridae/crecimiento & desarrollo , Línea Celular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Animales , Técnicas de Cultivo de Célula , Vectores Genéticos , LepidópterosRESUMEN
The full length cry2Ab gene was cloned by PCR-RFLP method from Bt strain B-Pr-88, which was isolated in China with high toxicity to the Lepidopteran insect pests. Nucleic acid sequence analysis showed that this gene was 1902 base pairs encoding 633 amino acids. This cry gene was named cry2Ab4 as a novel gene by Bacillus thuringiensis Delta Endotoxin Nomenclature Committee. The full open reading frame sequence of the cry2Ab4 gene was amplified with a pair of PCR primers L2ab5/L2ab3 designed according to its DNA sequence,and inserted into the BamH I /EcoR I sites of E. coli expression vector pET21b to obtain the recombinant plasmid pET-2Ab4. The result of SDS-PAGE proved that Cry2Ab4 could be expressed as a 60 kD protein in E. coli BL21 (DE3)strain induced by IPTG. Bioassay of the expressed product of the cry2Ab4 gene showed that Cry2Ab4 was highly toxic to the larvae of Helicoverpa armigera and Leguminivora glycinivorella, moderately active to the larvae of Plutella xylostella and Chilo suppressalis, but not insecticidal to the larvae of Spodotera exigua and Ostrinia furnacalis. Our result indicated that cry2Ab4 gene could be used as a novel gene for generation of transgenic plants and engineered microorganism.