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1.
Molecules ; 26(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068164

RESUMO

Astragaloside IV (AS-IV) is one of the major bio-active ingredients of huang qi which is the dried root of Astragalus membranaceus (a traditional Chinese medicinal plant). The pharmacological effects of AS-IV, including anti-oxidative, anti-cancer, and anti-diabetic effects have been actively studied, however, the effects of AS-IV on liver regeneration have not yet been fully described. Thus, the aim of this study was to explore the effects of AS-IV on regenerating liver after 70% partial hepatectomy (PHx) in rats. Differentially expressed mRNAs, proliferative marker and growth factors were analyzed. AS-IV (10 mg/kg) was administrated orally 2 h before surgery. We found 20 core genes showed effects of AS-IV, many of which were involved with functions related to DNA replication during cell division. AS-IV down-regulates MAPK signaling, PI3/Akt signaling, and cell cycle pathway. Hepatocyte growth factor (HGF) and cyclin D1 expression were also decreased by AS-IV administration. Transforming growth factor ß1 (TGFß1, growth regulation signal) was slightly increased. In short, AS-IV down-regulated proliferative signals and genes related to DNA replication. In conclusion, AS-IV showed anti-proliferative activity in regenerating liver tissue after 70% PHx.


Assuntos
Ciclo Celular , Replicação do DNA , Regulação para Baixo , Hepatectomia , Regeneração Hepática/efeitos dos fármacos , Fígado/citologia , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Replicação do DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Fator de Crescimento de Hepatócito/metabolismo , Fígado/efeitos dos fármacos , Fígado/cirurgia , Masculino , Anotação de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Saponinas/química , Análise de Sequência de RNA , Fator de Crescimento Transformador beta1/metabolismo , Triterpenos/química
2.
Int J Biol Macromol ; 166: 45-53, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33068623

RESUMO

Streptococcus mutans is a gram-positive bacterium that causes tooth decay. The exopolyssacharides, mostly glucans synthesized by the bacterium are responsible for establishing pathogenic bio-films associated with dental caries disease. The regulatory immune and inflammatory reactions implicated by the synthesized glucans are still not clearly understood. In this study, a water-soluble exopolyssacharide (WSP) was extracted from culture of Str. mutans. The structural properties of WSP, [α-(1 â†’ 3, 1 â†’ 6)-D-glucan] were confirmed using Fourier-transform infrared spectroscopy and 13C-nuclear magnetic resonance spectroscopy. Furthermore, the effects of WSP on the global gene expression of the macrophage-like RAW 264.7 cells were analyzed using mRNA-seq analysis. Using Gene Ontology analysis, we compiled a total of 24,421 genes that were upregulated or downregulated by more than 5.0-fold and 0.3-fold, respectively. Most of the transcripts were grouped under immune response and inflammation-related gene categories. Among the 802 immunity-related genes analyzed, chemokine ligand 7 (Ccl7), interleukin-1ß (IL-1ß), interleukin-1α (IL-1α) and interleukin-6 (IL-6) were upregulated after WSP exposure. In addition, among a total of 344 genes related to inflammation, Ccl7, IL-1α and IL-6 were upregulated. These results suggest that [α-(1 â†’ 3, 1 â†’ 6)-D-glucan] from Str. mutans produces activates macrophages and may contribute to the immune and inflammatory response to periodontal disease.


Assuntos
Quimiocina CCL7/genética , Glucanos/farmacologia , Interleucinas/genética , Polissacarídeos Bacterianos/farmacologia , Streptococcus mutans/química , Transcriptoma/efeitos dos fármacos , Animais , Quimiocina CCL7/metabolismo , Interleucinas/metabolismo , Ativação de Macrófagos , Camundongos , Células RAW 264.7
3.
Molecules ; 25(13)2020 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-32646056

RESUMO

The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome has been implicated in a variety of diseases, including atherosclerosis, neurodegenerative diseases, and infectious diseases. Thus, inhibitors of NLRP3 inflammasome have emerged as promising approaches to treat inflammation-related diseases. The aim of this study was to explore the effects of juglone (5-hydroxyl-1,4-naphthoquinone) on NLRP3 inflammasome activation. The inhibitory effects of juglone on nitric oxide (NO) production were assessed in lipopolysaccharide (LPS)-stimulated J774.1 cells by Griess assay, while its effects on reactive oxygen species (ROS) and NLRP3 ATPase activity were assessed. The expression levels of NLRP3, caspase-1, and pro-inflammatory cytokines (IL-1ß, IL-18) and cytotoxicity of juglone in J774.1 cells were also determined. Juglone was non-toxic in J774.1 cells when used at 10 µM (p < 0.01). Juglone treatment inhibited the production of ROS and NO. The levels of NLRP3 and cleaved caspase-1, as well as the secretion of IL-1ß and IL-18, were decreased by treatment with juglone in a concentration-dependent manner. Juglone also inhibited the ATPase activities of NLRP3 in LPS/ATP-stimulated J774.1 macrophages. Our results suggested that juglone could inhibit inflammatory cytokine production and NLRP3 inflammasome activation in macrophages, and should be considered as a therapeutic strategy for inflammation-related diseases.


Assuntos
Lipopolissacarídeos/toxicidade , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Naftoquinonas/farmacologia , Animais , Linhagem Celular , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo
4.
Acta Cir Bras ; 34(6): e201900606, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31432997

RESUMO

PURPOSE: To investigate the effects of pine needle extract (PNE) on the expression of proliferating cell nuclear antigen (PCNA) and Ki-67 during liver regeneration induced by 70% partial hepatectomy (PH) in rat. METHODS: Forty-eight male rats (SD, 7 weeks) had surgery (70% PH). They were randomly divided into two groups. PH + PNE group was only provided PNE diluted in water (10%) for drinking and PH group was provided water from 5 days before surgery to the time of sacrifice. PNE was made by pressing and filtering. Animals were sacrificed at 12h, 24h, 36h, 60h, 84h, 168h after PH, respectively. The expressions of PCNA and Ki-67 were determined as proliferation indices. RESULTS: Immunohistochemistry turned out to increase the expression of PCNA and Ki-67. PCNA expression of PH+PNE group increased up to twice of that of PH group. Western blot also seemed to increase the PCNA expression. These results indicated the promotion of cell proliferation in liver tissue and hepatic regeneration. CONCLUSIONS: Pine needle extract stimulates the expression of some mitotic proteins during liver regeneration induced by 70% PH in rats. It suggests that administration of pine needle extract could accelerate the liver regeneration after partial hepatectomy.


Assuntos
Hepatectomia/métodos , Antígeno Ki-67/efeitos dos fármacos , Regeneração Hepática/efeitos dos fármacos , Pinus/química , Extratos Vegetais/farmacologia , Antígeno Nuclear de Célula em Proliferação/efeitos dos fármacos , Animais , Proliferação de Células , Antígeno Ki-67/metabolismo , Masculino , Índice Mitótico , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
5.
BMC Genomics ; 20(1): 154, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808280

RESUMO

BACKGROUND: Incilaria (= Meghimatium) fruhstorferi is an air-breathing land slug found in restricted habitats of Japan, Taiwan and selected provinces of South Korea (Jeju, Chuncheon, Busan, and Deokjeokdo). The species is on a decline due to depletion of forest cover, predation by natural enemies, and collection. To facilitate the conservation of the species, it is important to decide on a number of traits related to growth, immunity and reproduction addressing fitness advantage of the species. RESULTS: The visceral mass transcriptome of I. fruhstorferi was enabled using the Illumina HiSeq 4000 sequencing platform. According to BUSCO (Benchmarking Universal Single-Copy Orthologs) method, the transcriptome was considered complete with 91.8% of ortholog genes present (Single: 70.7%; Duplicated: 21.1%). A total of 96.79% of the raw read sequences were processed as clean reads. TransDecoder identified 197,271 contigs that contained candidate-coding regions. Of a total of 50,230 unigenes, 34,470 (68.62% of the total unigenes) annotated to homologous proteins in the Protostome database (PANM-DB). The GO term and KEGG pathway analysis indicated genes involved in metabolism, phosphatidylinositol signalling system, aminobenzoate degradation, and T-cell receptor signalling pathway. Many genes associated with molluscan innate immunity were categorized under pathogen recognition receptor, TLR signalling pathway, MyD88 dependent pathway, endogenous ligands, immune effectors, antimicrobial peptides, apoptosis, and adaptation-related. The reproduction-associated unigenes showed homology to protein fem-1, spermatogenesis-associated protein, sperm associated antigen, and testis expressed sequences, among others. In addition, we identified key growth-related genes categorized under somatotrophic axis, muscle growth, chitinases and collagens. A total of 4822 Simple Sequence Repeats (SSRs) were also identified from the unigene sequences of I. fruhstorferi. CONCLUSIONS: This is the first available genomic information for non-model land slug, I. fruhstorferi focusing on genes related to growth, immunity, and reproduction, with additional focus on microsatellites and repeating elements. The transcriptome provides access to greater number of traits of unknown relevance in the species that could be exploited for in-depth analyses of evolutionary plasticity and making informed choices during conservation planning. This would be appropriate for understanding the dynamics of the species on a priority basis considering the ecological, health, and social benefits.


Assuntos
Gastrópodes/genética , Animais , DNA/química , Gastrópodes/crescimento & desenvolvimento , Gastrópodes/imunologia , Gastrópodes/metabolismo , Perfilação da Expressão Gênica , Imunidade/genética , Repetições de Microssatélites , Anotação de Sequência Molecular , Desenvolvimento Muscular/genética , Sequências Repetitivas de Ácido Nucleico , Reprodução/genética , Análise de Sequência de RNA/normas , Homologia de Sequência do Ácido Nucleico , Processos de Determinação Sexual/genética
6.
Acta cir. bras ; 34(6): e201900606, 2019. graf
Artigo em Inglês | LILACS | ID: biblio-1019270

RESUMO

Abstract Purpose To investigate the effects of pine needle extract (PNE) on the expression of proliferating cell nuclear antigen (PCNA) and Ki-67 during liver regeneration induced by 70% partial hepatectomy (PH) in rat. Methods Forty-eight male rats (SD, 7 weeks) had surgery (70% PH). They were randomly divided into two groups. PH + PNE group was only provided PNE diluted in water (10%) for drinking and PH group was provided water from 5 days before surgery to the time of sacrifice. PNE was made by pressing and filtering. Animals were sacrificed at 12h, 24h, 36h, 60h, 84h, 168h after PH, respectively. The expressions of PCNA and Ki-67 were determined as proliferation indices. Results Immunohistochemistry turned out to increase the expression of PCNA and Ki-67. PCNA expression of PH+PNE group increased up to twice of that of PH group. Western blot also seemed to increase the PCNA expression. These results indicated the promotion of cell proliferation in liver tissue and hepatic regeneration. Conclusions Pine needle extract stimulates the expression of some mitotic proteins during liver regeneration induced by 70% PH in rats. It suggests that administration of pine needle extract could accelerate the liver regeneration after partial hepatectomy.


Assuntos
Animais , Masculino , Ratos , Extratos Vegetais/farmacologia , Antígeno Nuclear de Célula em Proliferação/efeitos dos fármacos , Antígeno Ki-67/efeitos adversos , Pinus/química , Hepatectomia/métodos , Regeneração Hepática/efeitos dos fármacos , Fatores de Tempo , Ratos Sprague-Dawley , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Ki-67/metabolismo , Proliferação de Células , Índice Mitótico
7.
Int J Med Mushrooms ; 19(6): 521-533, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29199562

RESUMO

The aim of this study was to determine, using murine RAW 264.7 macrophages, the immunomodulatory effect of extracellular ß-glucan isolated from Pleurotus eryngii (PEBG) and its sulfated derivative (PEBG-S) on signaling molecules implicated in host innate immunity. ß-Glucan was extracted and purified from the mycelial culture using optimal medium concentrations. It was then chemically converted to its sulfated form. Monosaccharide composition of ß-glucan was characterized with p-aminobenzoic acid ethyl ester-derivatized sugars through highperformance liquid chromatography analysis. Fourier transform infrared structural analysis showed an S=O bond at 1250 cm-1 and C-S-O binding at 815 cm-1 in PEBG-S. 13C nuclear magnetic resonance analysis showed 1,3-linked α-D-mannopyranosyl and 1,3-ß-D-glucopyranosyl in PEBG-S. A concentration-dependent increase of nitric oxide production was noticed in RAW 264.7 cells treated with PEBG-S or PEBG; those treated with PEBG-S showed less cytotoxicity than those treated with PEBG. Cellular levels of tumor necrosis factor-α, interleukin-1ß, and interleukin-6 were increased by PEBG and PEBG-S treatment, suggesting that they have immunomodulatory activity. Real-time polymerase chain reaction array revealed that the expression levels of nuclear factor-κB and Toll-like receptor signaling genes in cells were upregulated by PEBG and PEBG-S. Moreover, the expression of the ß-glucan receptor dectin-2 was significantly upregulated by PEBG and PEBG-S treatment, reflecting immune activation through the dectin-2-Syk-(CARD9/Bcl-10/MALT1) pathway. Our results suggest that PEBG-S could be used as an effective adjuvant or immune enhancer that can be sustainably produced by recycling the by-product of mycelial culture.


Assuntos
Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Pleurotus/química , Transdução de Sinais/efeitos dos fármacos , beta-Glucanas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Interleucinas/metabolismo , Lectinas Tipo C/efeitos dos fármacos , Camundongos , Óxido Nítrico/metabolismo , Células RAW 264.7 , Sulfatos/farmacologia , Receptores Toll-Like/efeitos dos fármacos , Receptores Toll-Like/metabolismo
8.
Carbohydr Polym ; 137: 561-569, 2016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26686164

RESUMO

Mutan is an extracellular polysaccharide of Streptococcus mutans (S. mutans) that consists of α-(1,3)-linked glucose residues in main chains and α-(1,6) bonds in side chains. In the present study, mutan was isolated from S. mutans, and its structural characteristics were determined using Fourier-transform infrared spectroscopy (FT-IR) and (13)C nuclear magnetic resonance (NMR) spectroscopy. The effects of mutan on RANKL-induced osteoclast differentiation in RAW 264.7 cells were examined. Furthermore, microCT and morphometric analyses were used to determine the contribution of mutan to alveolar bone loss in the maxilla of a rat periodontitis model. Mutan increased (more than 2-fold) RANKL-induced osteoclast differentiation in a dose-dependent manner. Mutan also enhanced the alveolar bone loss in the rat maxilla 2.3-fold. In mutan-treated rats, the bone mineral density, bone volume, trabecular number, and trabecular thickness decreased, whereas trabecular separation significantly increased. In addition, mutan and lipopolysaccharide (LPS) induced similar microarray profiles in RAW 264.7 cells. A total of 43 genes related to osteoclastogenesis were differentially expressed after either mutan or LPS treatment. Five-fold increases in the expression of several genes, including IL-1ß, IL-1α, IL-6, and chemokine ligands, were observed in mutan-treated RAW 264.7 cells. These results suggest a molecular mechanism for the inflammation induced by S. mutans during the establishment of periodontal disease.


Assuntos
Perda do Osso Alveolar/induzido quimicamente , Diferenciação Celular , Osteoclastos/efeitos dos fármacos , Polissacarídeos Bacterianos/farmacologia , Streptococcus mutans/química , Animais , Linhagem Celular , Citocinas/metabolismo , Glucanos/química , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Osteoclastos/citologia , Osteoclastos/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/toxicidade , Ratos , Ratos Sprague-Dawley
9.
Arch Pharm Res ; 38(6): 973-83, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25586964

RESUMO

Sappanchalcone, a bioactive flavonoid isolated from the heartwood of Caesalpinia sappan L. possesses anti-inflammatory effects. We studied the efficacy of sappanchalcone in attenuating collagen-induced arthritis (CIA) in a mouse model of rheumatoid arthritis. Sappanchalcone was purified to homogeneity from the chloroform fraction of the methanolic extract of C. sappan, and identified using mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. CIA-induced male DBA/1J mice were divided into control, sappanchalcone-treated, and methotrexate-treated groups (n = 10 per group). Paw swelling, arthritis severity, radiographic and histomorphometric changes were assessed to measure the protective role of sappanchalcone against chronic disease progression. Sappanchalcone administration significantly reduced clinical arthritis and inflammatory edema in paws. Bone mineral density and trabecular structure were maintained in CIA mice administered sappanchalcone. The levels of pro-inflammatory cytokines (TNF-α, IL-6, and 1L-1ß) were significantly lower in the serum of sappanchalcone-treated mice as compared with the control group. Our results suggest that sappanchalcone could be used as an anti-inflammatory and bone-protective agent during the treatment of rheumatoid arthritis.


Assuntos
Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/tratamento farmacológico , Caesalpinia/química , Chalconas/isolamento & purificação , Chalconas/farmacologia , Colágeno , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/patologia , Densidade Óssea/efeitos dos fármacos , Osso e Ossos/patologia , Citocinas/metabolismo , Edema/induzido quimicamente , Edema/tratamento farmacológico , Enzimas/metabolismo , Pé/patologia , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos DBA , Extratos Vegetais/química
10.
J Food Sci ; 79(12): M2516-22, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25393163

RESUMO

The Korean traditional seafood jeotgal is consumed directly or as an additive in other foods to improve flavor or fermentation efficiency. Saeujot, made from salted and fermented tiny shrimp (SFS; Acetes japonicus), is the best-selling jeotgal in Korea. In this study, we reveal the microbial diversity and dynamics in naturally fermented shrimp by denaturing gradient gel electrophoresis (DGGE). The population fingerprints of the predominant microbiota and its succession were generated by DGGE analysis of universal V3 16S rDNA polymerase chain reaction (PCR) amplicons. Overall, 17 strains were identified from sequencing of 30 DGGE bands. The DGGE profiles showed diverse bacterial populations in the sample, throughout the fermentation of SFS. Staphylococcus equorum, Halanaerobium saccharolyticum, Salimicrobium luteum, and Halomonas jeotgali were the dominant bacteria, and their levels steadily increased during the fermentation process. Certain other bacteria, such as Psychrobacter jeotgali and Halomonas alimentaria appeared during the early-fermentation process, while Alkalibacterium putridalgicola, Tetragenococcus muriaticus, and Salinicoccus jeotgali appeared during the late-fermentation process. The members of the order Bacillales were found to be predominant during the fermentation of SFS. Furthermore, S. equorum was identified as the dominant bacterial isolate by the traditional method of culturing under aerobic and facultative anaerobic conditions. We expect that this information will facilitate the design of autochthonous starter cultures for the production of SFS with desired characteristic sensory profiles and shorter ripening times.


Assuntos
Bacillaceae/isolamento & purificação , Clostridium/isolamento & purificação , Halomonas/isolamento & purificação , Alimentos Marinhos/microbiologia , Staphylococcus/isolamento & purificação , Animais , Bacillaceae/classificação , Biomassa , Fenômenos Químicos , Clostridium/classificação , DNA Bacteriano/genética , Decápodes/microbiologia , Eletroforese em Gel de Gradiente Desnaturante , Fermentação , Microbiologia de Alimentos , Halomonas/classificação , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Cloreto de Sódio , Staphylococcus/classificação
11.
Arch Pharm Res ; 36(7): 897-904, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23585332

RESUMO

tert-Butylhydroquinone (tBHQ) is a commonly used antioxidant additive that is approved for human use by both the Food and Agriculture Organization and the World Health Organization (FAO/WHO). In this study, we examined the effect of tBHQ on body weight gain and found that food supplementation with 0.001 % (w/w) tBHQ inhibited 61.4 % (P < 0.01) of body weight gain in high-fat diet (HFD)-induced C57BL/6 mice, and the oral administration of tBHQ (1.5 mg/kg) reduced 47.5 % (P < 0.05) of body weight gain in normal diet fed db/db mice. The HFD increased lipid deposit in adipocytes, but these were reduced significantly by tBHQ treatment in C57BL/6 mice. tBHQ supplementation significantly lowered the plasma triglyceride and total cholesterol, with reduced size of accumulated fat mass. The rate limiting enzyme of beta-oxidation (ACOX1) was significantly over-expressed in the liver with tBHQ treatment. These results indicate that tBHQ suppresses body weight gain in mice, possibly at least related to the up-regulation of ACOX1 gene expression.


Assuntos
Peso Corporal/efeitos dos fármacos , Dieta Hiperlipídica , Hidroquinonas/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Aumento de Peso/efeitos dos fármacos , Animais , Antioxidantes/efeitos adversos , Antioxidantes/farmacologia , Peso Corporal/fisiologia , Dieta Hiperlipídica/métodos , Hidroquinonas/efeitos adversos , Metabolismo dos Lipídeos/fisiologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/diagnóstico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distribuição Aleatória , Aumento de Peso/fisiologia
12.
Arch Virol ; 158(8): 1701-18, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23512574

RESUMO

The chitinase gene of baculoviruses is expressed in the late phase of virus replication in insects and possesses high exo- and endochitinase activity, which can hydrolyze chitin in the body of the insect, thus promoting terminal host liquefaction. Alphabaculovirus viral chitinases (vChitA) have been well analyzed, but information regarding viral chitinases from betabaculoviruses is limited. Whole-genome sequencing of a Korean isolate of Pieris rapae GV (PiraGV-K) predicted a putative chitinase gene corresponding to ORF10. The PiraGV-K chitinase gene had a coding sequence of 1,761 bp, encoding a protein of 586 amino acid (aa) residues, including an 18-aa putative signal peptide. Time course induction pattern observed by SDS-PAGE and subsequent Western blot with anti-PiraGV-K chitinase antibody revealed the cleavage of the signal peptide from the intact chitinase. Edman sequencing analysis was further conducted to confirm the exact nature of the mature chitinase, and the N-terminal amino acid sequence (KPGAP) exactly matched the sequence following the signal peptide sequence. The transcriptomics of PiraGV-K chitinase in infected P. rapae larvae, examined by real-time PCR, revealed a significant 75-fold increase after four days of feeding with PiraGV-K-treated leaves, with a subsequent decline at the later stages of infection. Confocal microscopic analysis showed that PiraGV-K chitinase possibly exists as a secreted protein, with strong chitinase-specific signals in fat body cells and integument at four days postinfection. Furthermore, immunogold labeling and electron microscopy studies localized the PiraGV-K chitinase in the cytoplasm and sparsely within vacuolar structures in the fat body apart from the extensive aggregation in the cuticular lining of the integument.


Assuntos
Quitinases/análise , DNA Viral/química , DNA Viral/genética , Genoma Viral , Granulovirus/enzimologia , Lepidópteros/virologia , Estruturas Animais/enzimologia , Estruturas Animais/virologia , Animais , Western Blotting , Quitinases/genética , Quitinases/imunologia , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Granulovirus/genética , Granulovirus/imunologia , Microscopia Confocal , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Análise de Sequência de Proteína
13.
J Invertebr Pathol ; 113(1): 7-17, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23295682

RESUMO

Pieris rapae granulovirus (PiraGV) is highly pathogenic to the cabbage butterfly (P. rapae), an important pest of cultivated cabbages and mustard crops. It therefore holds significant promise towards exploitation as a potent bio-control agent in the field controlling the pest population. Whole-genome elucidation of the Korean isolate of the granulovirus (PiraGV-K), reported the presence of a granulin gene corresponding to ORF 1 in its genome. Comprehensive studies towards functional characterization of the gene, established that it is composed of 744 nucleotides and encodes a peptide of 247 amino acid residues. It possessed significant homology with AoGV and ClanGV with 87% identity at amino acid level. Multiple alignment data suggests that the C-terminus region of the gene had three different conserved regions. Time-course studies conducted in PiraGV-K infected P. rapae larvae revealed a significant upsurge of the transcript (134-fold) at 4 days post infection followed by a significant decline at the most advanced stages of infection. Anti-PiraGV-K granulin antibody was produced and western blot conducted with the infected larvae further confirmed the induction pattern with a protein of 30 kDa. Immunofluorescent staining showed a granulin-specific signal in fat body and integument of the infected larvae. Granulin-specific signals were noticed 2 days post infection with the eventual systemic spread of infection to the associated tracheal matrix witnessed at 4 days post infection. Immunogold labeling and electron microscopic studies further proved the cytopathological effects as the presence of numerous membrane-bound vesicles with nucleocapsids and abruption of intercellular junctions in fat body and hypertrophied cells in the integument.


Assuntos
Genoma Viral , Granulovirus/genética , Proteínas Estruturais Virais , Animais , Borboletas/virologia , Corpo Adiposo/virologia , Granulovirus/isolamento & purificação , Imuno-Histoquímica , Larva/virologia , Dados de Sequência Molecular , Proteínas de Matriz de Corpos de Inclusão , Alinhamento de Sequência , Análise de Sequência de DNA , Análise de Sequência de Proteína
14.
Mol Hum Reprod ; 19(2): 93-101, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23112252

RESUMO

Endocannabinoid signaling plays various roles in directing reproductive processes. Mouse embryos are shown to express high levels of CB1 receptor (CB1R). Low concentrations of anandamide stimulate embryo growth and implantation but at higher concentrations it adversely affects implantation. We tested the hypothesis that high levels of endocannabinoids cause autophagic activation and cell death in preimplantation mouse embryos. We used methanandamide (METH), a selective CB1R agonist, to examine the effect of heightened endocannabinoid signaling on autophagy in mouse embryos. Western blotting, immunofluorescence staining, transmission electron microscopy and TUNEL analysis were performed. We observed that METH treatment in vitro or in vivo up-regulated autophagic response in preimplantation mouse embryos. In blastocysts, apoptosis was also increased after METH injections. At 28 nM, which is considered a high physiological dose to embryonic cells, METH up-regulated autophagic activation in trophoblast stem cells. This work demonstrates for the first time that blastocysts respond to higher than normal levels of endocannabinoid by increasing autophagic activation and apoptosis.


Assuntos
Blastocisto/metabolismo , Endocanabinoides/metabolismo , Animais , Ácidos Araquidônicos/farmacologia , Blastocisto/efeitos dos fármacos , Blastocisto/ultraestrutura , Endocanabinoides/farmacologia , Feminino , Imunofluorescência , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Transmissão , Alcamidas Poli-Insaturadas/farmacologia , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
15.
J Environ Biol ; 34(2): 283-7, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24620593

RESUMO

The aim of the study was to examine the liver tissue damage induced by nanosized-TiO2 in mouse. The biochemical parameters of liver, namely glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and alkaline phosphatase were enhanced approximately 18%, 35% and 69% by exposure to nanosized-TiO2, respectively. The nanosized-TiO2 accumulated in the periphery of sinusoid in liver when the ultrastructure was examined through transmission electron microscopy. Enzymes, such as superoxide dismutase, catalase and aldehyde dehydrogenase were significantly inhibited by 22%, 38% and 15%, respectively, whereas glutathione peroxidase was constant following exposure to nanosized-TiO2.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Nanopartículas , Titânio/toxicidade , Animais , Masculino , Camundongos , Camundongos Endogâmicos ICR , Titânio/química
16.
Int J Biochem Cell Biol ; 44(6): 989-97, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22465708

RESUMO

Microtubule-associated protein 1 light chain-3 (LC3) plays a critical role in autophagosome formation during autophagy; however, its potential alternative functions remain largely unexplored. Here we demonstrate a discrete role for LC3 in osteoclast, a specialized bone-resorbing cell that requires a dynamic microtubule network for its activity. We found that an increase in the conversion of soluble LC3-I to lipid-bound LC3-II in mature osteoclast was correlated with osteoclast activity, but not with autophagic activity. Knockdown of LC3 using small interfering RNA did not affect TRAP-positive multinucleated cell formation, but suppressed actin ring formation, cathepsin K release, and the subsequent bone-resorbing capacity of osteoclasts. LC3 mediated this function by associating with microtubules and regulating Cdc42 activity. More importantly, LC3-II protein levels were reduced by the Atg5 knockdown, and this knockdown led to decrease in Cdc42 activity, indicating that LC3-II is critical for Cdc42 activity. Overexpression of a constitutively active form of Cdc42 partially rescued the phenotype induced by LC3 knockdown. Our results demonstrate that LC3 contributes to the regulatory link between the microtubule and Cdc42 involved in bone-resorbing activity, providing evidence for a role for LC3 in mediating diverse cellular functions beyond its role as an autophagy protein.


Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Osteoclastos/metabolismo , Proteína cdc42 de Ligação ao GTP/fisiologia , Animais , Catepsina K/metabolismo , Diferenciação Celular , Técnicas de Silenciamento de Genes , Humanos , Microscopia Confocal , Osteoclastos/citologia , RNA Interferente Pequeno
17.
Aquat Toxicol ; 72(4): 383-96, 2005 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15848257

RESUMO

The ultrastructure of testicular cells of adult male African clawed frogs (Xenopus laevis) exposed to either estradiol (0.1 microg/L) or 2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine (atrazine; 10 or 100 microg/L) was examined by electron microscopy and compared to plasma concentrations of the steroid hormones, testosterone (T) and estradiol (E2), testicular aromatase activity and gonad growth expressed as the gonado-somatic index (GSI). Exposure to E2 caused significant changes both at the sub-cellular and biochemical levels. Exposure to E2 resulted in significantly fewer sperm cells, inhibition of meiotic division of germ cells, more lipid droplets that are storage compartments for the sex steroid hormone precursor cholesterol, and lesser plasma T concentrations. Although not statistically significant, frogs exposed to E2 had slightly smaller GSI values. These results may be indicative of an inhibition of gonad growth and disrupted germ cell development by E2. Concentrations of E2 in plasma were greater in frogs exposed to E2 in water. Exposure to neither concentration of atrazine caused effects on germ cell development, testicular aromatase activity or plasma hormone concentrations. These results suggest that atrazine does not affect testicular function. In contrast, exposure of male X. laevis to E2 led to sub-cellular events that are indicative of disruption of testicular development, and demasculinization processes (decrease of androgen hormone titers). These results indicate that atrazine does not cause responses that are similar to those caused by exposure to E2.


Assuntos
Aromatase/metabolismo , Estradiol/sangue , Testículo/ultraestrutura , Testosterona/sangue , Xenopus laevis/sangue , Análise de Variância , Animais , Atrazina/toxicidade , Estradiol/toxicidade , Masculino , Microscopia Eletrônica , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/metabolismo , Xenopus laevis/anatomia & histologia
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