RESUMEN
The most common genetic risk factor for Parkinson's disease (PD) is heterozygous mutations GBA1, which encodes for the lysosomal enzyme, glucocerebrosidase. Reduced glucocerebrosidase activity associates with an accumulation of abnormal α-synuclein (α-syn) called Lewy pathology, which characterizes PD. PD patients heterozygous for the neuronotypic GBA1L444P mutation (GBA1+/L444P) have a 5.6-fold increased risk of cognitive impairments. In this study, we used GBA1+/L444P mice of either sex to determine its effects on lipid metabolism, expression of synaptic proteins, behavior, and α-syn inclusion formation. At 3 months of age, GBA1+/L444P mice demonstrated impaired contextual fear conditioning, and increased motor activity. Hippocampal levels of vGLUT1 were selectively reduced in GBA1+/L444P mice. We show, using mass spectrometry, that GBA1L444P expression increased levels of glucosylsphingosine, but not glucosylceramide, in the brains and serum of GBA1+/L444P mice. Templated induction of α-syn pathology in mice showed an increase in α-syn inclusion formation in the hippocampus of GBA1+/L444P mice compared with GBA1+/+ mice, but not in the cortex, or substantia nigra pars compacta. Pathologic α-syn reduced SNc dopamine neurons by 50% in both GBA1+/+ and GBA1+/L444P mice. Treatment with a GlcCer synthase inhibitor did not affect abundance of α-syn inclusions in the hippocampus or rescue dopamine neuron loss. Overall, these data suggest the importance of evaluating the contribution of elevated glucosylsphingosine to PD phenotypes. Further, our data suggest that expression of neuronotypic GBA1L444P may cause defects in the hippocampus, which may be a mechanism by which cognitive decline is more prevalent in individuals with GBA1-PD.SIGNIFICANCE STATEMENT Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are both pathologically characterized by abnormal α-synuclein (α-syn). Mutant GBA1 is a risk factor for both PD and DLB. Our data show the expression of neuronotypic GBA1L444P impairs behaviors related to hippocampal function, reduces expression of a hippocampal excitatory synaptic protein, and that the hippocampus is more susceptible to α-syn inclusion formation. Further, our data strengthen support for the importance of evaluating the contribution of glucosylsphingosine to PD phenotypes. These outcomes suggest potential mechanisms by which GBA1L444P contributes to the cognitive symptoms clinically observed in PD and DLB. Our findings also highlight the importance of glucosylsphingosine as a relevant biomarker for future therapeutics.
Asunto(s)
Glucosilceramidasa , Enfermedad de Parkinson , Sinucleinopatías , alfa-Sinucleína , Animales , Ratones , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Hipocampo/metabolismo , Mutación/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Sinucleinopatías/patologíaRESUMEN
BACKGROUND: Bimaxillary protrusion is a clinically common dentofacial deformity, particularly among Chinese patients. This kind of malformation can severely affect facial esthetics and, even in mild cases, is difficult to correct without surgery. Unfortunately, many patients abandon treatment because of fear of surgery. Here, we describe a case of severe skeletal bimaxillary protrusion treated with nonsurgical orthodontic treatments, highlighting an alternative treatment option. CASE SUMMARY: A 31-year-old woman wished to address a severe protrusion profile (approximately 8 mm overbite) and gummy smile. Cephalometric analysis and superimposition showed a severe skeletal class II pattern with a mandibular retrusion, and proclined and protrusive mandibular incisors. Panoramic radiograph showed a missing mandibular right third molar. A diagnosis of severe bimaxillary dentoalveolar protrusion was made. Taking into account the patient's fear of orthognathic surgery, she accepted the proposed alternative treatment using micro-implants and a self-made four-curvature torquing auxiliary. The treatment allowed for maximal en masse anterior tooth retraction, proper relocation of incisors, and alleviation of the skeletal class II pattern. Esthetically, the patient's lip protrusion was significantly decreased as was the overjet (from 10.5 mm to 1.8 mm), and the results remained stable throughout the 2-year follow-up. CONCLUSION: Nonsurgical treatment using micro-implants and a four-curvature torquing auxiliary may benefit severe cases of skeletal bimaxillary protrusion in adults.
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Egg yolk immunoglobulin(IgY),also called egg yolk antibody,is a specific antibody secreted by birds after stimulation with certain antigens.For that it exhibits good resistance to heat,acid and proteinase-mediated degradation,its early-stage products were developed as food or food additives,and they have gained great social and economic benefits in both food safety and ecological agriculture fields.Due to the specific antibodies contained in these products,the products can help to resist pathogen infection.Therefore egg yolk antibody shows great value for development and application in the prevention and treat ment of animal and human diseases,and also shows great potential in human health care area.This review focuses on the development and applications of IgY biologics related with antiviral,antibacterial,antiparasitic and antitumor activities.
RESUMEN
DESIGN: The HIV latent CD4+ T cell reservoir is broadly recognized as a barrier to HIV cure. Induction of HIV expression using protein kinase C (PKC) agonists is one approach under investigation for reactivation of latently infected CD4+ T cells (Beans et al., 2013; Abreu et al., 2014; Jiang et al., 2014; Jiang and Dandekar, 2015). We proposed that an increased understanding of the molecular mechanisms of action of PKC agonists was necessary to inform on biological signaling and pharmacodynamic biomarkers. RNA sequencing (RNA Seq) was applied to identify genes and pathways modulated by PKC agonists. METHODS: Human CD4+ T cells were treated ex vivo with Phorbol 12-myristate 13-acetate, prostatin or ingenol-3-angelate. At 3 h and 24 h post-treatment, cells were harvested and RNA-Seq was performed on RNA isolated from cell lysates. The genes differentially expressed across the PKC agonists were validated by quantitative RT-PCR (qPCR). A subset of genes was evaluated for their role in HIV reactivation using siRNA and CRISPR approaches in the Jurkat latency cell model. RESULTS: Treatment of primary human CD4+ T cells with PKC agonists resulted in alterations in gene expression. qPCR of RNA Seq data confirmed upregulation of 24 genes, including CD69, Egr1, Egr2, Egr3, CSF2, DUSP5, and NR4A1. Gene knockdown of Egr1 and Egr3 resulted in reduced expression and decreased HIV reactivation in response to PKC agonist treatment, indicating a potential role for Egr family members in latency reversal. CONCLUSION: Overall, our results offer new insights into the mechanism of action of PKC agonists, biomarkers of pathway engagement, and the potential role of EGR family in HIV reactivation.
Asunto(s)
VIH-1/fisiología , Proteína Quinasa C/metabolismo , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Biomarcadores , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Diterpenos/química , Diterpenos/farmacología , Agonismo de Drogas , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 3 de la Respuesta de Crecimiento Precoz/genética , Expresión Génica , Infecciones por VIH/virología , Humanos , Células Jurkat , Masculino , Forboles/farmacología , Análisis de Secuencia de ARNRESUMEN
Autoimmune hepatitis (AIH) is a generally progressive, chronic hepatitis of unknown cause that occurs in children and adults of all ages. It is associated with a variety of autoimmune conditions like thyroid disorders (Hashimoto and Graves disease), celiac disease and multiple sclerosis (MS). We report the case of a 61-year-old woman with MS (untreated) and a history of Graves disease who presented with fatigue and right upper quadrant abdominal pain. She was admitted to our hospital for evaluation. Clinical and laboratory workup revealed AIH. She was successfully treated with prednisone and azathioprine, with complete clinical and laboratory improvement. However, to our knowledge there have been only a few reports of a possible association between AIH and untreated MS.
RESUMEN
The current study aimed to investigate the rejection and survival time of grafted skin, and the changes of Treg cells, interleukin 10 (IL-10) and transforming growth factor-ß (TGF-ß) in peripheral blood following skin transplantation with recombinant human interleukin-10 (rhIL-10) or cyclosporin A (CsA), as well as the role of IL-10 in immunological rejection mechanisms. A total of 36 rabbits were divided into two groups. The skin of a donor rabbit was transplanted onto the back of one receptor rabbit. Receptors were randomly divided into six groups, including rhIL-10 low-dose (5 µg/kg/d), rhIL-10 high-dose (10 µg/kg/d), CsA low-dose (5 mg/kg/d), CsA high-dose (10 mg/kg/d), rhIL-10 (5 µg/kg/d) and CsA (5 mg/kg/d) and negative control normal saline (NS; 1 ml/d). All groups received intramuscular drug injection for ten days, beginning one day prior to skin transplantation surgery. Following transplantation, each rabbit's peripheral blood was collected at different times. The changes of CD4+CD25+ regulatory T cells, IL-10 and TGF-ß were determined by flow cytometry and enzyme-linked immunosorbent assay. When compared with the control group, the rejection and survival times of the experimental groups were longer following skin graft. Compared with the two CsA groups and the control group, the proportion of CD4+CD25+ regulatory T cells of rhIL-10 groups was significantly upregulated on the 4th and 7th days following surgery. However, TGF-ß levels were not significantly different. Data suggested that the concentration of IL-10 was positively correlated with the proportion of CD4+CD25+ regulatory T cells. In addition, IL-10 may delay the rejection time of rabbit skin transplantation and prolong the survival time. Thus, the role of IL-10 in inhibited allograft rejection may be associated with CD4+CD25+ regulatory T cells and IL-10, and may be independent of TGF-ß.
Asunto(s)
Interleucina-10/metabolismo , Proteínas Recombinantes/metabolismo , Trasplante de Piel/métodos , Factor de Crecimiento Transformador beta/metabolismo , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Ciclosporina/farmacología , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/inmunología , Humanos , Interleucina-10/administración & dosificación , Interleucina-10/genética , Subunidad alfa del Receptor de Interleucina-2/efectos de los fármacos , Subunidad alfa del Receptor de Interleucina-2/inmunología , Conejos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
A series of macrocyclic compounds containing 2-substituted-quinoline moieties have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K mutant activity while maintaining the high rat liver exposure. Cyclization of the 2-substituted quinoline substituent led to a series of tricyclic P2 compounds which also display superb gt3a potency.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Hepacivirus/enzimología , Compuestos Macrocíclicos/química , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Proteínas Portadoras/metabolismo , Ciclización , Genotipo , Semivida , Hepacivirus/genética , Péptidos y Proteínas de Señalización Intracelular , Cinética , Hígado/metabolismo , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/farmacocinética , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacocinética , Quinolinas/química , Ratas , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
A series of macrocyclic compounds containing a cyclic constraint in the P2-P4 linker region have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K, A156T, A156V, and D168V mutant activity while maintaining high rat liver exposure. The effect of the constraint is most dramatic against gt 1b A156 mutants where ~20-fold improvements in potency are achieved by introduction of a variety of ring systems into the P2-P4 linker.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Hepacivirus/enzimología , Compuestos Macrocíclicos/química , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Sitios de Unión , Proteínas Portadoras/metabolismo , Dominio Catalítico , Ciclización , Genotipo , Semivida , Hepacivirus/genética , Péptidos y Proteínas de Señalización Intracelular , Cinética , Hígado/metabolismo , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/farmacocinética , Simulación del Acoplamiento Molecular , Mutación , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacocinética , Ratas , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
Glassy carbon electrode, which is used to electrochemically determine the content of buformin, is modified with an electropolymerized film of p-aminobenzoic acid in pH 7.0 acetate buffer solution (ABS). The polymer showed an excellent electrocatalytic activity for the reduction of buformin. In pH 7.0 ABS, the cathodic peak current increased linearly over three concentration intervals of buformin, and the detection limit (S/N=3) was 2.0×10-9 g/mL. The method was successfully applied to directly determine buformin in tablets with standard addition recoveries of 95.8-102.5%. The proposed method is simple, cheap and highly efficient.
RESUMEN
Next generation NNRTIs are sought which possess both broad spectrum antiviral activity against key mutant strains and a high genetic barrier to the selection of new mutant viral strains. Pyridones were evaluated as an acyclic conformational constraint to replace the aryl ether core of MK-4965 (1) and the more rigid indazole constraint of MK-6186 (2). The resulting pyridone compounds are potent inhibitors of HIV RT and have antiviral activity in cell culture that is superior to other next generation NNRTI's.
Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Piridonas/química , Inhibidores de la Transcriptasa Inversa/síntesis química , Sitios de Unión , Línea Celular , Simulación por Computador , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , VIH/enzimología , Transcriptasa Inversa del VIH/metabolismo , Humanos , Estructura Terciaria de Proteína , Pirazoles/química , Piridinas/química , Piridonas/síntesis química , Piridonas/farmacología , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacologíaRESUMEN
Highly active antiretroviral therapy (HAART) significantly reduces human immunodeficiency virus (HIV) viral load and has led to a dramatic decrease in acquired immunodeficiency syndrome (AIDS) related mortality. Despite this success, there remains a critical need for new HIV therapies to address the emergence of drug resistant viral strains. Next generation NNRTIs are sought that are effective against these mutant forms of the HIV virus. The bound conformations of our lead inhibitors, MK-1107 (1) and MK-4965 (2), were divergent about the oxymethylene linker, and each of these conformations was rigidified using two isomeric cyclic constraints. The constraint derived from the bioactive conformation of 2provided novel, highly potent NNRTIs that possess broad spectrum antiviral activity and good pharmacokinetic profiles. Systematic SAR led to the identification of indazole as the optimal conformational constraint to provide MK-6186 (3) and MK-7445 (6). Despite their reduced flexibility, these compounds had potency comparable to that of the corresponding acyclic ethers in both recombinant enzyme and cell based assays against both the wild-type and the clinically relevant mutant strains.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Imidazoles/síntesis química , Indazoles/síntesis química , Pirazoles/síntesis química , Inhibidores de la Transcriptasa Inversa/síntesis química , Triazoles/síntesis química , Animales , Fármacos Anti-VIH/farmacocinética , Fármacos Anti-VIH/farmacología , Células Cultivadas , Perros , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Imidazoles/farmacocinética , Imidazoles/farmacología , Indazoles/farmacocinética , Indazoles/farmacología , Modelos Moleculares , Conformación Molecular , Mutación , Nitrilos/síntesis química , Nitrilos/farmacocinética , Nitrilos/farmacología , Nitrobencenos/síntesis química , Nitrobencenos/farmacocinética , Nitrobencenos/farmacología , Pirazoles/farmacocinética , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Inhibidores de la Transcriptasa Inversa/farmacocinética , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Termodinámica , Triazoles/farmacocinética , Triazoles/farmacologíaRESUMEN
The discovery of MK-1220 is reported along with the development of a series of HCV NS3/4A protease inhibitors containing a P2 to P4 macrocyclic constraint with improved preclinical pharmacokinetics. Optimization of the P2 heterocycle substitution pattern as well as the P3 amino acid led to compounds with greatly improved plasma exposure following oral dosing in both rats and dogs while maintaining excellent enzyme potency and cellular activity. These studies led to the identification of MK-1220.
RESUMEN
Biaryl ethers were recently reported as potent NNRTIs. Herein, we disclose a detailed effort to modify the previously reported compound 1. We have designed and synthesized a series of novel pyrazole derivatives as a surrogate for pyrazolopyridine motif that were potent inhibitors of HIV-1 RT with nanomolar intrinsic activity on the WT and key mutant enzymes and potent antiviral activity in infected cells.
Asunto(s)
Fármacos Anti-VIH/química , Éteres/química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Pirazoles/química , Piridinas/química , Inhibidores de la Transcriptasa Inversa/química , Regulación Alostérica , Animales , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacocinética , Perros , Éteres/síntesis química , Éteres/farmacocinética , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Humanos , Mutación , Pirazoles/síntesis química , Pirazoles/farmacocinética , Piridinas/síntesis química , Piridinas/farmacocinética , Ratas , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/farmacocinética , Relación Estructura-ActividadRESUMEN
A new, efficient synthesis of sphingofungin F has been accomplished with 10.4% overall yield in 15 steps. The salient features of the synthesis are the utilization of methyl tricyclic iminolactone 3 for the asymmetric aldol reaction as a key step to introduce two desired stereogenic centers, one-pot reaction for the synthesis of omega-hydroxyketone from epsilon-caprolactone, and an effective one-step deprotection strategy to remove all protecting groups using 0.2 N HCl.
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Compuestos Heterocíclicos con 3 Anillos/química , Lactonas/química , Aminoácidos/síntesis química , Aminoácidos/química , Ácidos Grasos Insaturados/síntesis química , Ácidos Grasos Insaturados/química , Hidrólisis , EstereoisomerismoRESUMEN
A new class of HCV NS3/4a protease inhibitors which contain a P2 to P4 macrocyclic constraint was designed using a molecular-modeling derived strategy. Exploration of the P2 heterocyclic region, the P2 to P4 linker, and the P1 side chain of this class of compounds via a modular synthetic strategy allowed for the optimization of enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 35b (vaniprevir, MK-7009), which is active against both the genotype 1 and genotype 2 NS3/4a protease enzymes and has good plasma exposure and excellent liver exposure in multiple species.
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Hepacivirus/enzimología , Indoles/farmacología , Inhibidores de Serina Proteinasa/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Área Bajo la Curva , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Ciclopropanos , Perros , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Indoles/química , Indoles/farmacocinética , Concentración 50 Inhibidora , Péptidos y Proteínas de Señalización Intracelular , Isoindoles , Lactamas Macrocíclicas , Leucina/análogos & derivados , Hígado/metabolismo , Macaca mulatta , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacocinética , Compuestos Macrocíclicos/farmacología , Tasa de Depuración Metabólica , Modelos Químicos , Estructura Molecular , Pan troglodytes , Prolina/análogos & derivados , Ratas , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacocinética , Relación Estructura-Actividad , Sulfonamidas , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/metabolismoRESUMEN
Biaryl ethers were recently reported as potent NNRTIs. Herein we disclose a detailed SAR study that led to the biaryl ether 6. This compound possessed excellent potency against WT RT and key clinically observed RT mutants and had an excellent pharmacokinetic profile in rats, dogs, and rhesus macaques. The compound also exhibited a clean safety profile in preclinical safety studies.
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Éteres/química , Éteres/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Mutación , Animales , Línea Celular , Perros , Éteres/síntesis química , Éteres/farmacocinética , VIH-1/enzimología , Humanos , Macaca mulatta , Nucleósidos/química , Ratas , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacocinética , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-ActividadRESUMEN
OBJECTIVE: To evaluate the biological characteristics of Campylobacter jejuni (CJ) cultured on different culture media and their expression abundance of outer membrane proteins (OMPs). METHODS: CJ was cultured on the improved Bull's medium yolk agar, improved Bull's blood agar or improved Bull's agar for 48 h. The biological characteristics of the bacteria, including the colony feature, morphology, motility, biochemistry, and results of indirect fluorescence test were observed and compared. OMP of the cultured CJ was extracted using 0.2 mol/L and glycine-hydrochloride buffered solution (pH 2.2) and identified by SDS-PAGE to compare the expression abundance of the OMPs with molecular weight of 28-31 kD. RESULTS: CJ exhibited typical biological characteristics with larger cell body and more rapid growth on improved Bull's medium yolk agar than those on improved Bull's blood agar and improved Bull's agar. The bacteria grown on improved Bull's medium yolk agar showed also greater expression abundance of the OMPs with molecule mass between 28 kD and 31 kD. CONCLUSION: Improved Bull's medium yolk agar allows rapid growth of CJ with typical biological characteristics and enhanced expression of the OMPs with molecular weight of 28 -31 kD, and can be widely used in CJ subunit vaccine development, CJ epidemiological survey, CJ food safety examination, and CJ quarantine.
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Proteínas de la Membrana Bacteriana Externa/metabolismo , Campylobacter jejuni/crecimiento & desarrollo , Campylobacter jejuni/metabolismo , Medios de Cultivo , Proteínas de la Membrana Bacteriana Externa/análisisRESUMEN
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are key elements of multidrug regimens, called HAART (Highly Active Antiretroviral Therapy), that are used to treat HIV-1 infections. Elucidation of the structure-activity relationships of the thiocarbamate moiety of the previous published lead compound 2 provided a series of novel tetrahydroquinoline derivatives as potent inhibitors of HIV-1 RT with nanomolar intrinsic activity on the WT and key mutant enzymes and potent antiviral activity in infected cells. The SAR optimization, mutation profiles, preparation of compounds, and pharmacokinetic profile of compounds are described.
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Fármacos Anti-VIH/química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Quinolinas/química , Inhibidores de la Transcriptasa Inversa/química , Sitio Alostérico , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Sitios de Unión , Cristalografía por Rayos X , Transcriptasa Inversa del VIH/metabolismo , Conformación Molecular , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/metabolismo , Quinolinas/síntesis química , Quinolinas/farmacología , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Tiocarbamatos/química , Tiocarbamatos/farmacologíaRESUMEN
<p><b>OBJECTIVE</b>To evaluate the biological characteristics of Campylobacter jejuni (CJ) cultured on different culture media and their expression abundance of outer membrane proteins (OMPs).</p><p><b>METHODS</b>CJ was cultured on the improved Bull's medium yolk agar, improved Bull's blood agar or improved Bull's agar for 48 h. The biological characteristics of the bacteria, including the colony feature, morphology, motility, biochemistry, and results of indirect fluorescence test were observed and compared. OMP of the cultured CJ was extracted using 0.2 mol/L and glycine-hydrochloride buffered solution (pH 2.2) and identified by SDS-PAGE to compare the expression abundance of the OMPs with molecular weight of 28-31 kD.</p><p><b>RESULTS</b>CJ exhibited typical biological characteristics with larger cell body and more rapid growth on improved Bull's medium yolk agar than those on improved Bull's blood agar and improved Bull's agar. The bacteria grown on improved Bull's medium yolk agar showed also greater expression abundance of the OMPs with molecule mass between 28 kD and 31 kD.</p><p><b>CONCLUSION</b>Improved Bull's medium yolk agar allows rapid growth of CJ with typical biological characteristics and enhanced expression of the OMPs with molecular weight of 28 -31 kD, and can be widely used in CJ subunit vaccine development, CJ epidemiological survey, CJ food safety examination, and CJ quarantine.</p>
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Proteínas de la Membrana Bacteriana Externa , Metabolismo , Campylobacter jejuni , Metabolismo , Medios de CultivoRESUMEN
AIM: To construct a prokaryotic expression vector carrying Campylobacter jejuni peb1A gene and express it in Escherichia coli. Immunoreactivity and antigenicity of rPEB1 were evaluated. The ability of rPEB1 to induce antibody responses and protective efficacy was identified. METHODS: peb1A gene was amplified by PCR, target gene and prokaryotic expression plasmid pET28a (+) was digested with BamHI and XhoI, respectively. DNA was ligated with T4 DNA ligase to construct recombinant plasmid pET28a(+)-peb1A. The rPEB1 was expressed in E. coli BL21 (DE3) and identified by SDS-PAGE. BALB/c mice were immunized with rPEB1. ELISA was used to detect the specific antibody titer and MTT method was used to measure the stimulation index of spleen lymphocyte transformation. RESULTS: The recombinant plasmid pET28a (+)-peb1A was correctly constructed. The expression output of PEB1 protein in pET28a (+)-peb1A system was approximately 33% of total proteins in E. coli. The specific IgG antibody was detected in serum of BALB/c mice immunized with rPEB1 protein. Effective immunological protection with a lower sickness incidence and mortality was seen in the mice suffering from massive C. jejuni infection. CONCLUSION: rPEB1 protein is a valuable candidate for C. jejuni subunit vaccine.