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Beauveria bassiana, Cordyceps militaris and Ophiocordyceps sinensis (Ascomycotina) are traditional Chinese medicines. Here, mitogenomes of these three Ascomycotina fungi were sequenced and de-novo assembled using single-molecule real-time sequencing. The results showed that their complete mitogenomes were 31,258, 31,854 and 157,584 bp, respectively, with sequencing depth approximately 278,760×, 326,283× and 69,385×. Types of repeat sequences were mainly (AA)n, (AAT)n, (TA)n and (TATT)n. DNA methylation motifs were revealed in DNA modifications of these three fungi. We discovered new models of RNA editing through analysis of transcriptomes from B. bassiana and C. militaris. These data lay a solid foundation for further genetic and biological studies about these three fungi, especially for elucidating the mitogenome evolution and exploring the regulatory mechanism of adapting environment.
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Aclimatación/genética , Beauveria/genética , Cordyceps/genética , Metilación de ADN/genética , Genoma Mitocondrial/genética , Edición de ARN/genética , Aclimatación/fisiología , Perfilación de la Expresión Génica , Variación Genética/genética , Medicina Tradicional China , Transcriptoma/genéticaRESUMEN
Accurate diagnosis of pathogenic Nosema spp. in Antheraea pernyi samples is considered especially useful for reducing economic losses in sericulture and improving food safety by maintaining pathogen-free pupae. However, microscopy and immunologic methods have poor diagnostic sensitivity, while the more sensitive PCR methods remain costly and time-consuming for template preparation. To address this issue, we introduce a sensitive ALMS-qPCR method that combines fast, simple DNA extraction using Alkali Lysis followed by Magnetic bead Separation (ALMS) and quantitative real-time PCR (qPCR). This approach is especially fit for large-scale pathogen molecular screening, because the DNA preparation procedure is fast (<0.94â¯min per sample) and is high-throughput (performs on a 96-well plate). It is cost-effective, since the most expensive materials can be made in the lab and can be recycled, while the automated procedure can help to minimize labor cost. Though the DNA preparation procedure was substantially simplified, common PCR inhibitory factors were not observed. The sensitivity of ALMS-qPCR is high and the limit of detection is 0.045â¯parasites/µL. Large-scale screening of Nosema spp. in 3000 Antheraea pernyi samples confirmed the efficacy of the ALMS-qPCR method. Sensitivity is much higher than clinical microscopy, especially for host groups with low infection prevalence and levels. High-throughput ALMS-qPCR, combining automated DNA preparation and sensitive qPCR, provides an enhanced approach for pébrine screening and epidemiological studies. The application of ALMS-qPCR in the sericulture industry will help to strengthen pébrine control and breed pathogen-free species, which means much safer food provision and better genetic resource conservation.
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Microsporidiosis/diagnóstico , Mariposas Nocturnas/microbiología , Nosema/aislamiento & purificación , Animales , Patología Molecular/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
The complete genome of Cordyceps militaris was sequenced using single-molecule real-time (SMRT) sequencing technology at a coverage over 300×. The genome size was 32.57 Mb, and 14 contigs ranging from 0.35 to 4.58 Mb with an N50 of 2.86 Mb were assembled, including 4 contigs with telomeric sequences on both ends and an additional 8 contigs with telomeric sequences on either the 5' or 3' end. A methylome database of the genome was constructed using SMRT and m4C and m6A methylated nucleotides, and many unknown modification types were identified. The major m6A methylation motif is GA and GGAG, and the major m4C methylation motif is GC or CG/GC. In the C. militaris genome DNA, there were four types of methylated nucleotides that we confirmed using high-resolution LCMS-IT-TOF. Using PacBio Iso-Seq, a total of 31,133 complete cDNA sequences were obtained in the fruiting body. The conserved domains of the nontranscribed regions of the genome include TATA boxes, which are the initial regions of genome replication. There were 406 structural variants between the HN and CM01 strains, and there were 1,114 structural variants between the HN and ATCC strains.
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Cordyceps/genética , Metilación de ADN , Epigenómica , Genoma Fúngico , Transcriptoma , Biología Computacional/métodos , Secuencia Conservada , Epigénesis Genética , Epigenómica/métodos , Regulación Fúngica de la Expresión Génica , Motivos de Nucleótidos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Regiones no Traducidas , Secuenciación Completa del GenomaRESUMEN
A DNA-binding protein (DBP) [GenBank accession number: M63416] of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been reported to be a regulatory factor in BmNPV, but its detailed functions remain unknown. In order to study the regulatory mechanism of DBP on viral proliferation, genome replication, and gene transcription, a BmNPV dbp gene knockout virus dbp-ko-Bacmid was generated by the means of Red recombination system. In addition, dbp-repaired virus dbp-re-Bacmid was constructed by the means of the Bac to Bac system. Then, the Bacmids were transfected into BmN cells. The results of this viral titer experiment revealed that the TCID50 of the dbp-ko-Bacmid was 0; however, the dbp-re-Bacmid was similar to the wtBacmid (p>0.05), indicating that the dbp-deficient would lead to failure in the assembly of virus particles. In the next step, Real-Time PCR was used to analyze the transcriptional phases of dbp gene in BmN cells, which had been infected with BmNPV. The results of the latter experiment revealed that the transcript of dbp gene was first detected at 3 h post-infection. Furthermore, the replication level of virus genome and the transcriptional level of virus early, late, and very late genes in BmN cells, which had been transfected with 3 kinds of Bacmids, were analyzed by Real-Time PCR. The demonstrating that the replication level of genome was lower than that of wtBacmid and dbp-re-Bacmid (p<0.01). The transcriptional level of dbp-ko-Bacmid early gene lef-3, ie-1, dnapol, late gene vp39 and very late gene p10 were statistically significantly lower than dbp-re-Bacmid and wtBacmid (p<0.01). The results presented are based on Western blot analysis, which indicated that the lack of dbp gene would lead to low expressions of lef3, vp39, and p10. In conclusion, dbp was not only essential for early viral replication, but also a viral gene that has a significant impact on transcription and expression during all periods of baculovirus life cycle.
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Bombyx/virología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/fisiología , Proteínas Virales/genética , Proteínas Virales/fisiología , Animales , Bombyx/ultraestructura , Replicación del ADN/genética , Replicación del ADN/fisiología , Técnicas de Inactivación de Genes , Genes Virales , Microscopía Electrónica de Transmisión , Nucleopoliedrovirus/ultraestructura , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
A new strategy for the convergence of two-dimensional preparative high performance liquid chromatography (HPLC) and real-time cell analysis (RTCA) was developed for rapidly separating and screening anti-tumor components from the ethyl acetate extract of the traditional Chinese medicine (TCM), Euphorbia kansui. Eight compounds: esulone A (1), kansuinin A (2), (3ß,11ß)-3,11-dihydroxylanosta-8,24-dien-7-one (3), kansuinin E (4), kansuinin B (5), isoscopoletin (6), kansuinin D (7) and kansuinin G (8) were efficiently isolated with purity above 97%. Of all the compounds, esulone A has been isolated from this plant for the first time. The structures were identified by NMR spectroscopy and comparisons were made with the data in previous literature. The anti-tumor bioassay was performed on A549 (human lung cancer cells) and Hep-G2 (human liver cancer cells) with a newly developed RTCA system. The result revealed that compounds 1, 3, 7 and 8 almost entirely inhibited the proliferation of A549 cells and that compound 8 was also thought to be the most active compound against Hep-G2 cells. The method provided considerable assistance for the efficient separation of different polar compounds and rapid screening of anti-tumor active compounds.
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Antineoplásicos/aislamiento & purificación , Bioensayo/métodos , Cromatografía Líquida de Alta Presión , Euphorbia/química , Extractos Vegetales/aislamiento & purificación , Células A549 , Antineoplásicos/química , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Células Hep G2 , Humanos , Estructura Molecular , Neoplasias/tratamiento farmacológico , Extractos Vegetales/farmacologíaRESUMEN
An in vitro three-dimensional (3D) cell culture system that can mimic organ and tissue structure and function in vivo will be of great benefit for drug discovery and toxicity testing. In this study, the neuroprotective properties of the three most prevalent flavonoid monomers extracted from EGb 761 (isorharmnetin, kaempferol, and quercetin) were investigated using the developed 3D stem cell-derived neural co-culture model. Rat neural stem cells were differentiated into co-culture of both neurons and astrocytes at an equal ratio in the developed 3D model and standard two-dimensional (2D) model using a two-step differentiation protocol for 14 days. The level of neuroprotective effect offered by each flavonoid was found to be aligned with its effect as an antioxidant and its ability to inhibit Caspase-3 activity in a dose-dependent manner. Cell exposure to quercetin (100 µM) following oxidative insult provided the highest levels of neuroprotection in both 2D and 3D models, comparable with exposure to 100 µM of Vitamin E, whilst exposure to isorhamnetin and kaempferol provided a reduced level of neuroprotection in both 2D and 3D models. At lower dosages (10 µM flavonoid concentration), the 3D model was more representative of results previously reported in vivo. The co-cultures of stem cell derived neurons and astrocytes in 3D hydrogel scaffolds as an in vitro neural model closely replicates in vivo results for routine neural drug toxicity and efficacy testing. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:735-744, 2016.
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Flavonoides/farmacología , Modelos Biológicos , Células-Madre Neurales/efectos de los fármacos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Animales , Células Cultivadas , Técnicas de Cocultivo , Flavonoides/química , Flavonoides/aislamiento & purificación , Ginkgo biloba , Células-Madre Neurales/citología , Neuronas/citología , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/aislamiento & purificación , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , RatasRESUMEN
FK506 binding proteins (FKBPs) are intracellular receptors of the immunosuppressant FK506 and play important roles in the correct folding of new proteins and the self-assembly of biological macromolecules. FKBP12 is a member of the FKBP family that is widely expressed and highly conserved in many species. In this study, we identified the complete cDNA sequence encoding the FKBP12 ortholog in Bombyx mori, named Bm-FKBP12B (GenBank accession no. DQ443423). Multiple-sequence alignment among different species revealed a high similarity among FKBP12 paralogs and orthologs. Bioinformatics analysis of the Bm-FKBP12B gene showed that it is located on chromosome 20 and consists of three exons and two introns. We cloned, expressed, and purified the Bm-FKBP12B protein in Escherichia coli and generated a specific polyclonal antibody against Bm-FKBP12B. The real-time quantitative reverse-transcription (qRT) PCR and Western blotting results showed that Bm-FKBP12B was present throughout all of the development stages, but it was abundant in the adult and embryo stages. Bm-FKBP12B expression was higher in the silk gland and gut, suggesting that it might play important roles in regulating gene expression in the silk gland and during silk fiber formation. Bm-FKBP12B protein was distributed in the cytoplasm, nucleus, and nuclear membrane.
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Bombyx/metabolismo , Filogenia , Proteína 1A de Unión a Tacrolimus/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bombyx/genética , Biología Computacional , Expresión Génica , Proteínas de Insectos/genética , Proteínas de Insectos/aislamiento & purificación , Proteínas de Insectos/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Interferencia de ARN , Homología de Secuencia de Aminoácido , Proteína 1A de Unión a Tacrolimus/genética , Proteína 1A de Unión a Tacrolimus/aislamiento & purificaciónRESUMEN
In this study, we identified a heat-resistant protein from the chrysalis stage of the silkworm which we named sex-specific storage protein 2 (SSP2). This protein was stable even at 80 °C, and has an amino acid sequence that is 90.65 % homologous to SP2. We utilized the heat-resistant characteristics of SSP2 to purify the protein and maintain its biological activity. In addition, using flow cytometry and the MTT assay, we found that SSP2 had anti-apoptotic effects on BmN cells, and that SSP2 could also inhibit cell apoptosis induced by chemical factors. These results suggest that SSP2 has a cell-protective function, and provides a basis for future work on the function of storage proteins in silkworm.
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Bombyx/química , Proteínas de Insectos/química , Proteínas de Insectos/aislamiento & purificación , Pupa/química , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Calor , Proteínas de Insectos/farmacología , Datos de Secuencia Molecular , Estabilidad Proteica , Alineación de SecuenciaRESUMEN
Lysine acetylation in proteins is a dynamic and reversible PTM and plays an important role in diverse cellular processes. In this study, using lysine-acetylation (Kac) peptide enrichment coupled with nano HPLC/MS/MS, we initially identified the acetylome in the silkworms. Overall, a total of 342 acetylated proteins with 667 Kac sites were identified in silkworm. Sequence motifs analysis around Kac sites revealed an enrichment of Y, F, and H in the +1 position, and F was also enriched in the +2 and -2 positions, indicating the presences of preferred amino acids around Kac sites in the silkworm. Functional analysis showed the acetylated proteins were primarily involved in some specific biological processes. Furthermore, lots of nutrient-storage proteins, such as apolipophorin, vitellogenin, storage proteins, and 30 K proteins, were highly acetylated, indicating lysine acetylation may represent a common regulatory mechanism of nutrient utilization in the silkworm. Interestingly, Ser2 proteins, the coating proteins of larval silk, were found to contain many Kac sites, suggesting lysine acetylation may be involved in the regulation of larval silk synthesis. This study is the first to identify the acetylome in a lepidoptera insect, and expands greatly the catalog of lysine acetylation substrates and sites in insects.
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Bombyx/metabolismo , Proteínas de Insectos/química , Lisina/química , Proteoma/química , Acetilación , Animales , Cromatografía Líquida de Alta Presión , Proteínas de Insectos/análisis , Proteínas de Insectos/metabolismo , Lisina/análisis , Lisina/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Proteómica , Espectrometría de Masas en TándemRESUMEN
AIM: To explore the association between COX-2 polymorphisms and non-small cell lung cancer (NSCLC) susceptibility. METHODS: We collected fasting peripheral venous blood from 60 cases with NSCLC and 62 healthy controls through physical examinations, and applied PCR-RFLP to analyze COX-2 polymorphisms of two groups. RESULTS: With respect to detecting COX-2 rs689466 and rs5275 polymorphisms, the distribution frequency of mutant genotype AA of COX-2 rs689466 in case group was higher than that in control group, which possessed significant difference between two groups (P<0.05). Carriers with AA genotype were 4.05 times at risk of NSCLC than those with GG genotype (P = 0.04, OR=4.05, 95% CI=1.14-14.43). The distribution of mutant genotype CC of COX-2 rs5275 was different between two groups, and carriers with genotype CC were at 5.70 times higher risk of NSCLC than those with genotype TT. After corrected by sex, gender, smoking and drinking factors, AA genotype of COX-2 rs689466 and CC genotype of COX-2 rs5275 still contributed to increased risk of NSCLC (OR=4.22, 95% CI=1.10-16.17, OR=6.95, 95% CI=1.27-38.11). After analyzed of linkage disequilibrium (LD) and haplotypes of alleles in two SNPs, the distribution frequency of A-C haplotype in case group was higher than that in control group, with significant difference between two groups (P<0.05). After corrected by sex, gender, smoking and drinking factors, statistical difference was still found in the total distribution of A-C haplotype between two groups (P=0.03, OR=6.11, 95% CI=1.16-32.2). CONCLUSIONS: COX-2 rs689466 and rs5275 polymorphisms may be related to NSCLC susceptibility. And A-C haplotype might be a susceptibility haplotype for NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Ciclooxigenasa 2/genética , Predisposición Genética a la Enfermedad/genética , Neoplasias Pulmonares/genética , Anciano , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido SimpleRESUMEN
Recent studies have demonstrated that recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) produced by the silkworm pupae bioreactor is absorbed into blood through oral administration and functions as an active cytokine. The aim of this study was to further examine and identify synergetic protein factors in silkworm pupae that improve rhGM-CSF absorption via an oral route. The concentrations of rhGM-CSF in serum were evaluated in mice after oral administration of rhGM-CSF using different chemical compositions of silkworm pupae as pharmaceutical excipients. The experimental data revealed that the supernatant lyophilized powder (SLP) of a homogenized slurry of silkworm pupae caused a significant increase in the rhGM-CSF level in blood when rhGM-CSF was orally administered with SLP, suggesting that synergetic protein factors that improve the oral absorption of rhGM-CSF primarily exist in SLP. As shown by scanning electron microscopy, microspheres were formed when rhGM-CSF was coated with SLP. Animal experimental data showed that the absorption of orally administered rhGM-CSF through the gastrointestinal (GI) tract primarily resulted from protein factors present in the SLP retentate obtained after 10 kDa ultrafiltration. Surface plasmon resonance spectroscopy analysis demonstrated that several protein factors present in the SLP retentate obtained after 10 kDa ultrafiltration were bound to rhGM-CSF. Proteins bound to rhGM-CSF by liquid chromatography-mass spectrometry were identified as chymotrypsin inhibitor SCI-II precursor, cationic peptide CP8 precursor, Kazal-type proteinase inhibitor, and chymotrypsin inhibitor SCI-I. These findings indicate that these proteinase inhibitors play an important role in improving rhGM-CSF absorption in the GI tract.
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Bombyx/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Pupa/metabolismo , Administración Oral , Animales , Reactores Biológicos , Cromatografía Liquida , Femenino , Tracto Gastrointestinal/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Microscopía Electrónica de Rastreo , Microesferas , Resonancia por Plasmón de Superficie , UltrafiltraciónRESUMEN
Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the ß-trefoil superfamily, with ß domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people's interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.
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Bombyx/química , Hormona de Crecimiento Humana/química , Proteínas de Insectos/química , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Hormona de Crecimiento Humana/aislamiento & purificación , Hormona de Crecimiento Humana/fisiología , Humanos , Proteínas de Insectos/aislamiento & purificación , Proteínas de Insectos/fisiología , Células K562 , Modelos Moleculares , Datos de Secuencia Molecular , Pupa/química , Homología Estructural de ProteínaRESUMEN
Recent studies have demonstrated that Notch-1 expression is increased in the hippocampus of Alzheimer's disease patients. We speculate that Notch-1 signaling may be involved in PC12 cell apoptosis induced by amyloid beta-peptide (25-35) (Aß25-35). In the present study, PC12 cells were cultured with different doses (0, 0.1, 1.0, 10 and 100 nmol/L) of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, a Notch-1 signaling pathway inhibitor, for 30 minutes. Then cultured cells were induced with Aß25-35 for 48 hours. Pretreatment of PC12 cells with high doses of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (> 10 nmol/L) prolonged the survival of PC12 cells after Aß25-35 induction, decreased the expression of apoptosis-related proteins caspase-3, -8, -9, increased the activity of oxidative stress-related superoxide dismutase and catalase, inhibited the production of active oxygen, and reduced nuclear factor kappa B expression. This study indicates that the Notch-1 signaling pathway plays a pivotal role in Aß25-35-induced PC12 apoptosis.
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Animal medicine is an important part in traditional Tibetan medicine. However, information about the chemical composition of animal medicine is very limited, and there is a lack of comprehensive chromatographic purification methods. In the present work, animal medicine Osteon Myospalacem Baileyi was taken as an example and a novel two-dimensional preparative chromatographic method was established for the preparation of single compounds with high purity from the extract of Osteon Myospalacem Baileyi. The first-dimension preparation was carried on a DAISO Silica prep column, and ten fractions were obtained from the 112.3 g crude sample within 12 injections. A diol prep column used in nonaqueous mobile phase was selected for the second-dimension preparation. The purity of the compounds isolated from the crude extract was >98%, which indicated that the method built in this work was efficient to manufacture single compounds of high purity from the extract of Osteon Myospalacem Baileyi. Additionally, this method showed great potential in the purification of weakly polar chemicals and it could act as a good example in the purification of other traditional animal medicines.
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Osteón/química , Roedores , Animales , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Medicina Tradicional China , TibetRESUMEN
Bombyx mori nucleopolyhedrovirus (BmNPV) bm47 gene is found in all sequenced lepidopteran nucleopolyhedroviruses (NPVs). It is one of the core genes of NPVs. However, the role of bm47 in the biological cycle of NPV remains unknown. In this study, the Red recombination system was used to knock out bm47 from BmNPV to construct bm47-ko-Bacmid in E. coli BW25113 system. Then bm47 gene was introduced back to the viral genome using the Bac-to-Bac system to create the repair virus bm47-re-Bacmid. TCID50 assay and real-time PCR (qPCR) were used to evaluate the effects of bm47 deletion on viral DNA replication, gene transcription, and protein expression. qPCR results showed that bm47 knock-out had no significant effect on viral DNA replication. However, the qPCR results showed that bm47-ko-Bacmid significantly decreased the transcription levels of early gene lef-3, late gene vp39, and very late gene p10 at 48 h and 72 h after viral transfection of BmN cells (P < 0.05). This work will provide a foundation for further studies on the biological function of BmNPV bm47 in viral replication and transcription.
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Eliminación de Gen , Nucleopoliedrovirus/genética , Transcripción Genética , Proteínas Virales/genética , Replicación Viral , Animales , Bombyx/virología , Regulación Viral de la Expresión Génica , Nucleopoliedrovirus/fisiología , Proteínas Virales/metabolismoRESUMEN
The E(spl)mγ gene in Drosophila is a regulatory target gene downstream of the Notch pathway. BmE(spl)mγ (Bombyx mori, E(spl)mγ) is an ortholog of the Drosophila E(spl)mγ gene, and the gene encodes a protein with 248 amino acid residues. This gene was cloned and overexpressed in Escherichia coli BL21(DE3). The recombinant protein was purified and subsequently used to generate a rabbit polyclonal antibody. Western blotting analyses showed that BmE(spl)mγ expression is high in pupa and egg, and low in larva and moth. In the fifth instar larva, the protein levels are high in head, epidermis, sexual gland, trachea, and the fatbody and low in the Malpighian tubule, hemolymph, gut, and silk gland. The further immunohistochemical analyses also showed higher BmE(spl)mγ expression in the head of fifth instar larva and pupa. Of the four moth parts studied, the thorax had the highest expression level. Thus, BmE(spl)mγ might be associated with neurogenesis in silkworm. Furthermore, DAPT (a γ-secretase inhibitor and an indirect inhibitor of Notch) blocking experiments showed that higher concentrations of the blocking agent and a longer processing time reduce the transcription levels of the BmE(spl)mγ gene, demonstrating that the silkworm BmE(spl)mγ gene is associated with the Notch signal pathway. These findings suggest that the function of BmE(spl)mγ may be similar to that of its Drosophila homolog.
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Bombyx/genética , Clonación Molecular , Proteínas de Insectos/genética , Receptores Notch/metabolismo , Proteínas Represoras/genética , Secuencia de Aminoácidos , Animales , Bombyx/química , Bombyx/crecimiento & desarrollo , Bombyx/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Larva/química , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Masculino , Datos de Secuencia Molecular , Transporte de Proteínas , Pupa/genética , Pupa/crecimiento & desarrollo , Pupa/metabolismo , Conejos , Receptores Notch/genética , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Alineación de Secuencia , Transducción de SeñalRESUMEN
To better understand the molecular mechanism underlying of diapause in Antheraea pernyi (A. pernyi), we cloned a novel diapause-associated protein 3 (DAP3) gene from A. pernyi by reverse transcription-polymerase chain reaction (RT-PCR) and studied the biological functions. Sequence analysis revealed that this gene encodes 171 amino acids and has a conserved domain of Copper/Zinc superoxide dismutase (Cu/Zn-SOD). Western blot and qRT-PCR results showed that DAP3 was mainly expressed in the pupal stage, and gradually decreased as diapause development. DAP3 was also expressed in 1st and 5th instar larvae of A. pernyi. In tissues of 5th instar larvae of A. pernyi, DAP3 was mainly expressed in the epidermis, followed by the head, hemolymph and fat body. To identify the SOD activity of DAP3, we constructed a prokaryotic expression vector by inserting the coding region sequence into plasmid pET-28a (+) and obtained the purified rHIS-DAP3 fusion protein by Ni-NTA affinitive column. Importantly, we found the SOD activity of DAP3 fusion protein was approximately 0.6674 U/µg. To further confirm the SOD activity of DAP3 in vivo, we induced the oxidative stress model of pupae by UV irradiation. The results showed that both the mRNA and protein level of DAP3 significantly increased by UV irradiation. Furthermore, the SOD activity of the total lysate of pupae increased significantly at 10 min post UV irradiation and transiently returned to normal level afterwards. These results suggested that DAP3 might be a novel protein with SOD activity getting involved in regulation of diapause in A. pernyi.
Asunto(s)
Diapausa de Insecto , Proteínas de Insectos/metabolismo , Mariposas Nocturnas/enzimología , Superóxido Dismutasa/metabolismo , Secuencia de Aminoácidos , Animales , Bombyx , China , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genes de Insecto , Proteínas de Insectos/genética , Larva/enzimología , Larva/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mariposas Nocturnas/genética , Estrés Oxidativo , Plásmidos/metabolismo , Pupa/enzimología , Pupa/genética , Quercus , Proteínas Recombinantes/biosíntesis , Alineación de Secuencia , Análisis de Secuencia de Proteína , Superóxido Dismutasa/genética , Transcripción GenéticaRESUMEN
To determine whether cholera toxin B subunit and active peptide from shark liver (CTB-APSL) fusion protein plays a role in treatment of type 2 diabetic mice, the CTB-APSL gene was cloned and expressed in silkworm (Bombyx mori) baculovirus expression vector system (BEVS), then the fusion protein was orally administrated at a dose of 100 mg/kg for five weeks in diabetic mice. The results demonstrated that the oral administration of CTB-APSL fusion protein can effectively reduce the levels of both fasting blood glucose (FBG) and glycosylated hemoglobin (GHb), promote insulin secretion and improve insulin resistance, significantly improve lipid metabolism, reduce triglycerides (TG), total cholesterol (TC) and low density lipoprotein (LDL) levels and increase high density lipoprotein (HDL) levels, as well as effectively improve the inflammatory response of type 2 diabetic mice through the reduction of the levels of inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Histopathology shows that the fusion protein can significantly repair damaged pancreatic tissue in type 2 diabetic mice, significantly improve hepatic steatosis and hepatic cell cloudy swelling, reduce the content of lipid droplets in type 2 diabetic mice, effectively inhibit renal interstitial inflammatory cells invasion and improve renal tubular epithelial cell nucleus pyknosis, thus providing an experimental basis for the development of a new type of oral therapy for type 2 diabetes.
Asunto(s)
Toxina del Cólera/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacología , Proteínas Virales de Fusión/farmacología , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Glucemia/metabolismo , Peso Corporal , Bombyx/virología , Toxina del Cólera/química , ADN Viral/genética , Diabetes Mellitus Experimental/tratamiento farmacológico , Escherichia coli/genética , Escherichia coli/metabolismo , Gangliósido G(M1)/metabolismo , Vectores Genéticos , Hemoglobina Glucada/análisis , Hipolipemiantes/farmacología , Insulina/metabolismo , Resistencia a la Insulina , Riñón/patología , Lípidos/sangre , Masculino , Ratones , Ratones Endogámicos ICR , Tiburones , Bazo/patología , Proteínas Virales de Fusión/químicaRESUMEN
Dysregulation of miR-452 has been observed in many tumors, but its biological function in hepatocellular carcinoma (HCC) is still unknown. Our results showed that miR-452 expression is significantly increased in HCC tissues and HCC cell lines. We also found that overexpression of miR-452 dramatically accelerated proliferation, induced cell cycle from G1 to S transition, and blocked apoptosis of HCC cells. Migration and matrigel invasion assays indicated that miR-452 significantly promotes HepG2 and QGY-7703 cells migration and invasion in vitro. Further studies showed that miR-452 directly targets the 3'-untranslated region of cyclin-dependent kinase inhibitor 1B (CDKN1B), ectopic miR-452 expression suppressed CDKN1B expression on mRNA and protein level. Silencing CDKN1B by small interfering RNA resembled the phenotype resulting from ectopic miR-452 expression. This study provides new insights into the potential molecular mechanisms that miRNA-452 contributed to HCC.
Asunto(s)
Carcinogénesis/genética , Carcinoma Hepatocelular/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Neoplasias Hepáticas/genética , MicroARNs/genética , Regiones no Traducidas 3'/genética , Apoptosis/genética , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Regulación hacia Abajo/genética , Fase G1/genética , Células HEK293 , Células Hep G2 , Humanos , ARN Mensajero/genética , Fase S/genética , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Small non-coding RNAs (ncRNAs) are important regulators of gene expression in eukaryotes. Previously, only microRNAs (miRNAs) and piRNAs have been identified in the silkworm, Bombyx mori. Furthermore, only ncRNAs (50-500nt) of intermediate size have been systematically identified in the silkworm. RESULTS: Here, we performed a systematic identification and analysis of small RNAs (18-50nt) associated with the Bombyx mori argonaute2 (BmAgo2) protein. Using RIP-seq, we identified various types of small ncRNAs associated with BmAGO2. These ncRNAs showed a multimodal length distribution, with three peaks at ~20nt, ~27nt and ~33nt, which included tRNA-, transposable element (TE)-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. The tRNA-derived fragments (tRFs) were found at an extremely high abundance and accounted for 69.90% of the BmAgo2-associated small RNAs. Northern blotting confirmed that many tRFs were expressed or up-regulated only in the BmNPV-infected cells, implying that the tRFs play a prominent role by binding to BmAgo2 during BmNPV infection. Additional evidence suggested that there are potential cleavage sites on the D, anti-codon and TψC loops of the tRNAs. TE-derived small RNAs and piRNAs also accounted for a significant proportion of the BmAgo2-associated small RNAs, suggesting that BmAgo2 could be involved in the maintenance of genome stability by suppressing the activities of transposons guided by these small RNAs. Finally, Northern blotting was also used to confirm the Bombyx 5.8 s rRNA-derived small RNAs, demonstrating that various novel small RNAs exist in the silkworm. CONCLUSIONS: Using an RIP-seq method in combination with Northern blotting, we identified various types of small RNAs associated with the BmAgo2 protein, including tRNA-, TE-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. Our findings provide new clues for future functional studies of the role of small RNAs in insect development and evolution.