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1.
Nucleic Acids Res ; 48(5): 2733-2748, 2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32009146

RESUMO

Family with sequence similarity (FAM46) proteins are newly identified metazoan-specific poly(A) polymerases (PAPs). Although predicted as Gld-2-like eukaryotic non-canonical PAPs, the detailed architecture of FAM46 proteins is still unclear. Exact biological functions for most of FAM46 proteins also remain largely unknown. Here, we report the first crystal structure of a FAM46 protein, FAM46B. FAM46B is composed of a prominently larger N-terminal catalytic domain as compared to known eukaryotic PAPs, and a C-terminal helical domain. FAM46B resembles prokaryotic PAP/CCA-adding enzymes in overall folding as well as certain inter-domain connections, which distinguishes FAM46B from other eukaryotic non-canonical PAPs. Biochemical analysis reveals that FAM46B is an active PAP, and prefers adenosine-rich substrate RNAs. FAM46B is uniquely and highly expressed in human pre-implantation embryos and pluripotent stem cells, but sharply down-regulated following differentiation. FAM46B is localized to both cell nucleus and cytosol, and is indispensable for the viability of human embryonic stem cells. Knock-out of FAM46B is lethal. Knock-down of FAM46B induces apoptosis and restricts protein synthesis. The identification of the bacterial-like FAM46B, as a pluripotent stem cell-specific PAP involved in the maintenance of translational efficiency, provides important clues for further functional studies of this PAP in the early embryonic development of high eukaryotes.

2.
Int J Mol Sci ; 21(2)2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936451

RESUMO

To clarify the genetic mechanism underlying grain protein content (GPC) and to improve rice grain qualities, the mapping and cloning of quantitative trait loci (QTLs) controlling the natural variation of GPC are very important. Based on genotyping-by-resequencing, a total of 14 QTLs were detected with the Huanghuazhan/Jizi1560 (HHZ/JZ1560) recombinant inbred line (RIL) population in 2016 and 2017. Seven of the fourteen QTLs were repeatedly identified across two years. Using three residual heterozygote-derived populations, a stably inherited QTL named as qGPC1-1 was validated and delimited to a ~862 kb marker interval JD1006-JD1075 on the short arm of chromosome 1. Comparing the GPC values of the RIL population determined by near infrared reflectance spectroscopy (NIRS) and Kjeldahl nitrogen determination (KND) methods, high correlation coefficients (0.966 and 0.983) were observed in 2016 and 2017. Furthermore, 12 of the 14 QTLs were identically identified with the GPC measured by the two methods. These results indicated that instead of the traditional KND method, the rapid and easy-to-operate NIRS was suitable for analyzing a massive number of samples in mapping and cloning QTLs for GPC. Using the gel-based low-density map consisted of 208 simple sequence repeat (SSR) and insert/deletion (InDel) markers, the same number of QTLs (fourteen) were identified in the same HHZ/JZ1560 RIL population, and three QTLs were repeatedly detected across two years. More stably expressed QTLs were identified based on the genome resequencing, which might be attributed to the high-density map, increasing the detection power of minor QTLs. Our results are helpful in dissecting the genetic basis of GPC and improving rice grain qualities through molecular assisted selection.

3.
Neurosci Bull ; 36(2): 121-133, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31435837

RESUMO

With the shifting role of placebos, there is a need to develop animal models of placebo analgesia and elucidate the mechanisms underlying the effect. In the present study, male Sprague-Dawley rats with chronic inflammatory pain caused by complete Freund's adjuvant (CFA) underwent a series of conditioning procedures, in which morphine was associated with different cues, but they failed to induce placebo analgesia. Then, conditioning with the conditioned place preference apparatus successfully induced analgesic expectancy and placebo analgesia in naïve rats but only induced analgesic expectancy and no analgesic effect in CFA rats. Subsequently, we found enhanced c-fos expression in the nucleus accumbens and reduced expression in the anterior cingulate cortex in naïve rats while c-fos expression in the anterior cingulate cortex in CFA rats was not altered. In summary, the behavioral conditioning model demonstrated the difficulty of establishing a placebo analgesia model in rats with a pathological condition.

4.
Zhongguo Zhong Yao Za Zhi ; 44(17): 3780-3785, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31602953

RESUMO

The aim of this paper was to investigate the molecular mechanism of Calculus Bovis Sativus( CBS) in alleviating lipid accumulation in vitro by serum pharmacology. The CBS-containing serum of mice was obtained by serum pharmacology method to evaluate its effect on the proliferation of LO2 hepatocytes. The lipid reducing effects of CBS-containing serum through Nrf2 was evaluated by fructose-induced LO2 hepatocyte steatosis model,nuclear factor erythroid 2 related factor 2( Nrf2) agonist oltipraz combined intervention,cell oil red O staining and intracellular triglyceride( TG) content. The effects of CBS-containing serum on lipid peroxidation and hepatocytes apoptosis were evaluated by reactive oxygen species( ROS) and apoptosis assay,respectively. Real-time quantitative polymerase chain reaction( PCR) was used to detect the relative expression of lipid synthesis-related genes and apoptosis-related genes.RESULTS:: showed that CBS drug-containing serum had no significant effect on LO2 hepatocyte proliferation. As compared with the model group,CBS-containing serum could effectively reduce the formation of lipid droplets in fructose-induced LO2 hepatocytes,significantly reduce intracellular TG and ROS levels,and significantly reduce hepatocyte apoptosis rate( P < 0. 05). As compared with the model group,carbohydrate responsive element binding protein( ChREBP),sterol regulatory element binding protein-1 c( SREBP-1 c),fatty acid synthase( FAS),acetyl-CoA carboxylase 1( ACC1),stearoyl-CoA desaturase 1( SCD1),Bax and caspase-3 mRNA levels were significantly reduced in CBS drug-containing serum treatment group( P<0. 05). All of the above effects could be reversed by oltipraz.In conclusion,CBS-containing serum can significantly inhibit the fructose-induced LO2 liver fat deposition,and the mechanism may be related to reducing intracellular ROS level through the Nrf2 pathway and improving intracellular peroxidation state to reduce apoptosis.


Assuntos
Cálculos Biliares/química , Hepatócitos/citologia , Soro/química , Animais , Apoptose , Bovinos , Células Cultivadas , Fígado Gorduroso , Frutose , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Peroxidação de Lipídeos , Fígado , Medicina Tradicional Chinesa , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos
5.
Front Microbiol ; 10: 1354, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31293532

RESUMO

Microbial extracellular proteases play crucial roles in marine protein degradation and nitrogen recycling. Although a large number of marine bacteria are found to produce extracellular proteases, it is still unknown how marine bacteria respond to environmental proteins to activate the expression of genes encoding extracellular proteases. The inducing signal molecule for marine bacterial extracellular proteases has never been identified. In this study, we identified tripeptides as the inducing signal molecules for the extracellular protease MCP-01 of the deep-sea bacterium Pseudoalteromonas sp. SM9913. We found that casein, but not casamino acids, can induce the gene expression and synthesis of MCP-01, suggesting that peptides rather than amino acids derived from casein induce the gene expression and synthesis of MCP-01 in SM9913. Then, casein was hydrolyzed by SM9913 extracellular proteases, and the peptides with inducing effect were isolated and characterized. Finally, four tripeptides, SPP, RYP, RQF and FRQ, were shown to have significant inducing effect on the expression of MCP-01 gene, indicating that they are likely the inducing signal molecules for the expression of protease MCP-01 gene in SM9913. This study sheds light on the induction mechanism for the gene expression and biosynthesis of marine microbial extracellular proteases, which is helpful in better understanding the adaptation of bacteria to deep-sea sedimental environment.

6.
Front Pharmacol ; 10: 151, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30890932

RESUMO

Pulmonary fibrosis is common in a variety of inflammatory lung diseases, there is currently no effective clinical drug treatment. It has been reported that the ethanol extract of Eclipta prostrata L. can improve the lung collagen deposition and fibrosis pathology induced by bleomycin (BLM) in mice. In the present study, we studied whether wedelolactone (WEL), a major coumarin ingredient of E. prostrata, provided protection against BLM-induced pulmonary fibrosis. ICR or C57/BL6 strain mice were treated with BLM to establish lung fibrosis model. WEL (2 or 10 mg/kg) was given daily via intragastric administration for 2 weeks starting at 7-day after intratracheal instillation. WEL at 10 mg/kg significantly reduced BLM-induced inflammatory cells infiltration, pro-inflammatory factors expression, and collagen deposition in lung tissues. Additionally, treatment with WEL also impaired BLM-induced increases in fibrotic marker expression (collagen I and α-SMA) and decrease in an anti-fibrotic marker (E-cadherin). Treatment with WEL significantly prevented BLM-induced increase in TGF-ß1 and Smad2/3 phosphorylation in the lungs. WEL administration (10 mg/kg) also significantly promoted AMPK activation compared to model group in BLM-treated mice. Further investigation indicated that activation of AMPK by WEL can suppressed the transdifferentiation of primary lung fibroblasts and the epithelial mesenchymal transition (EMT) of alveolar epithelial cells, the inhibitive effects of WEL was significantly blocked by an AMPK inhibitor (compound C) in vitro. Together, these results suggest that activation of AMPK by WEL followed by reduction in TGFß1/Raf-MAPK signaling pathways may have a therapeutic potential in pulmonary fibrosis.

7.
Curr Med Sci ; 38(1): 167-173, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30074167

RESUMO

Baicalin is one of the main active ingredients of choleretic traditional Chinese medicine drug Radix Scutellariae. The aim of this study was to explore the pharmacokinetic characteristics of baicalin in rats with 17α-ethynylestradiol (EE)-induced intrahepatic cholestasis (IC) based on its choleretic effects. Firstly, rats were subcutaneously injected with EE solution (5 mg/kg, 0.25 mL/100 g) for 5 consecutive days to construct an IC model. Then the bile excretion rate, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bile acid (TBA) and pathological changes of the liver were detected. Secondly, after successfully modeling, the rats were intragastrically given baicalin solution (200 mg/kg) (n=6). Blood samples were collected from the tail vein at different time points after intragastric administration. The protective effects of low- (50 mg/kg), medium- (100 mg/kg) and high-dose (200 mg/kg) baicalin on the liver in IC rats were evaluated. The content of baicalin in plasma was detected by liquid chromatography-mass spectrometry/mass spectrometry and pharmacokinetics parameters were calculated. Pharmacodynamic results showed that low-, medium- and high-dose baicalin all significantly increased the average excretion rate of bile (P<0.05), and significantly decreased serum levels of ALT, AST and ALP and TBA (P<0.05). Meanwhile, HE staining showed that baicalin significantly relieved EE-induced hepatocyte edema and necrosis. Pharmacokinetic results exhibited that the absorption of baicalin in both IC and normal control rats showed bimodal phenomenon. Cmax, AU(0-t) and AUC(0-∞) of baicalin in IC rats were significantly higher than those of the normal control group (P<0.01). T1/2 of plasma baicalin in the model group was significantly extended to (11.09±1.84) h, with clearance dropping to 61.78% of that of the normal control group (P<0.01). The above results suggested that baicalin had protective effects on the liver of IC rats, accompanied by significantly increased in vivo exposure, delayed in vivo clearance and markedly alterative pharmacokinetic characteristics. This study provides a theoretical basis for further development of baicalin as a feasible drug for treating IC.


Assuntos
Anti-Inflamatórios não Esteroides/farmacocinética , Colagogos e Coleréticos/farmacocinética , Colestase Intra-Hepática/tratamento farmacológico , Flavonoides/farmacocinética , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/uso terapêutico , Colagogos e Coleréticos/administração & dosagem , Colagogos e Coleréticos/uso terapêutico , Colestase Intra-Hepática/etiologia , Etinilestradiol/toxicidade , Flavonoides/administração & dosagem , Flavonoides/uso terapêutico , Masculino , Ratos , Ratos Wistar
8.
Nat Commun ; 9(1): 1165, 2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29563550

RESUMO

Cleavage of transfer (t)RNA and ribosomal (r)RNA are critical and conserved steps of translational control for cells to overcome varied environmental stresses. However, enzymes that are responsible for this event have not been fully identified in high eukaryotes. Here, we report a mammalian tRNA/rRNA-targeting endoribonuclease: SLFN13, a member of the Schlafen family. Structural study reveals a unique pseudo-dimeric U-pillow-shaped architecture of the SLFN13 N'-domain that may clamp base-paired RNAs. SLFN13 is able to digest tRNAs and rRNAs in vitro, and the endonucleolytic cleavage dissevers 11 nucleotides from the 3'-terminus of tRNA at the acceptor stem. The cytoplasmically localised SLFN13 inhibits protein synthesis in 293T cells. Moreover, SLFN13 restricts HIV replication in a nucleolytic activity-dependent manner. According to these observations, we term SLFN13 RNase S13. Our study provides insights into the modulation of translational machinery in high eukaryotes, and sheds light on the functional mechanisms of the Schlafen family.


Assuntos
Endorribonucleases/química , HIV-1/genética , Biossíntese de Proteínas , RNA Ribossômico/química , RNA de Transferência/química , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Citoplasma/química , Citoplasma/enzimologia , Citoplasma/virologia , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos , Células HEK293 , HIV-1/crescimento & desenvolvimento , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Clivagem do RNA , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Replicação Viral
9.
J Neurosci ; 38(7): 1756-1773, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29335353

RESUMO

Potassium voltage-gated channel interacting protein 3 (KChIP3), also termed downstream regulatory element antagonist modulator (DREAM) and calsenilin, is a multifunctional protein belonging to the neuronal calcium sensor (NCS) family. Recent studies revealed the expression of KChIP3 in dorsal root ganglion (DRG) neurons, suggesting the potential role of KChIP3 in peripheral sensory processing. Herein, we show that KChIP3 colocalizes with transient receptor potential ion channel V1 (TRPV1), a critical molecule involved in peripheral sensitization during inflammatory pain. Furthermore, the N-terminal 31-50 fragment of KChIP3 is capable of binding both the intracellular N and C termini of TRPV1, which substantially decreases the surface localization of TRPV1 and the subsequent Ca2+ influx through the channel. Importantly, intrathecal administration of the transmembrane peptide transactivator of transcription (TAT)-31-50 remarkably reduces Ca2+ influx via TRPV1 in DRG neurons and alleviates thermal hyperalgesia and gait alterations in a complete Freund's adjuvant-induced inflammatory pain model in male rats. Moreover, intraplantar injection of TAT-31-50 attenuated the capsaicin-evoked spontaneous pain behavior and thermal hyperalgesia, which further strengthened the regulatory role of TAT-31-50 on TRPV1 channel. In addition, TAT-31-50 could also alleviate inflammatory thermal hyperalgesia in kcnip3-/- rats generated in our study, suggesting that the analgesic effect mediated by TAT-31-50 is independent of endogenous KChIP3. Our study reveals a novel peripheral mechanism for the analgesic function of KChIP3 and provides a potential analgesic agent, TAT-31-50, for the treatment of inflammatory pain.SIGNIFICANCE STATEMENT Inflammatory pain arising from inflamed or injured tissues significantly compromises the quality of life in patients. This study aims to elucidate the role of peripheral potassium channel interacting protein 3 (KChIP3) in inflammatory pain. Direct interaction of the KChIP3 N-terminal 31-50 fragment with transient receptor potential ion channel V1 (TRPV1) was demonstrated. The KChIP3-TRPV1 interaction reduces the surface localization of TRPV1 and thus alleviates heat hyperalgesia and gait alterations induced by peripheral inflammation. Furthermore, the transmembrane transactivator of transcription (TAT)-31-50 peptide showed analgesic effects on inflammatory hyperalgesia independently of endogenous KChIP3. This work reveals a novel mechanism of peripheral KChIP3 in inflammatory hyperalgesia that is distinct from its classical role as a transcriptional repressor in pain modulation.


Assuntos
Hiperalgesia/fisiopatologia , Inflamação/fisiopatologia , Proteínas Interatuantes com Canais de Kv/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Sinalização do Cálcio , Repressão Epigenética , Adjuvante de Freund , Marcha , Gânglios Espinais/efeitos dos fármacos , Técnicas de Inativação de Genes , Hiperalgesia/induzido quimicamente , Inflamação/induzido quimicamente , Injeções Espinhais , Proteínas Interatuantes com Canais de Kv/genética , Masculino , Medição da Dor/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Ratos , Canais de Cátion TRPV/efeitos dos fármacos
10.
Life Sci ; 186: 125-132, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28802904

RESUMO

AIMS: To investigate the therapeutic effect of baicalin treatment in chronic ulcerative colitis (UC), and explore the potential anti-inflammation mechanism(s) via IL-33 pathway. MAIN METHODS: UC model were established by giving three cycles of 5-day 2% dextran sodium sulfate (DSS) with two intervals of 14-day recovery in mice, totaling 43days. At the 13th day of the UC modeling, mice received baicalin at doses of 50, 100, or 150mg/kg, respectively. Disease activity index (DAI) assessment as well as HE and PAS staining were performed. Serum levels of TNF-α, IL-1ß and IL-6 were determined by ELISA. Myeloperoxidase (MPO) activity and nitric oxide (NO) contents in colon were measured. The expressions of IL-33 and Ly6/G were examined by immunochemistry. And contents of IL-33 protein and NF-κB-related proteins were tested by Western blot. KEY FINDINGS: Morphological and histological analyses revealed that baicalin administration had a significant effect on reducing the severity of DSS-induced UC in mice. Besides, baicalin treatment significantly reduced the levels of MPO and NO. Moreover, increased levels of inflammatory cytokines, such as TNF-α, IL-1ß, and IL-6, have been identified in damaged colon tissue, which was noticeably reduced by baicalin treatment. Our data demonstrated that protein levels of IL-33 and NF-κB p65 were elevated in colon tissues of chronic UC mice. Baicalin treatment significantly suppressed levels of IL-33 and NF-κB p65, whereas levels of IκB-α were increased. SIGNIFICANCE: Baicalin treatment effectively alleviated DSS-induced chronic UC, and the protective mechanisms may involve inhibition of IL-33 expression and subsequent NF-κB activation.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Colo/efeitos dos fármacos , Flavonoides/uso terapêutico , Interleucina-33/genética , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Doença Crônica , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Colo/imunologia , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Flavonoides/administração & dosagem , Interleucina-33/imunologia , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia , NF-kappa B/metabolismo
11.
Environ Sci Technol ; 51(11): 6044-6052, 2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28462990

RESUMO

Sediment samples from the East China and Yellow seas collected adjacent to continental China were found to have lower δ15N values (expressed as δ15N = [15N:14Nsample/15N:14Nair - 1] × 1000‰; the sediment 15N:14N ratio relative to the air nitrogen 15N:14N ratio). In contrast, the Arctic sediments from the Chukchi Sea, the sampling region furthest from China, showed higher δ15N values (2-3‰ higher than those representing the East China and the Yellow sea sediments). Across the sites sampled, the levels of sediment δ15N increased with increasing distance from China, which is broadly consistent with the decreasing influence of anthropogenic nitrogen (NANTH) resulting from fossil fuel combustion and fertilizer use. We concluded that, of several processes, the input of NANTH appears to be emerging as a new driver of change in the sediment δ15N value in marginal seas adjacent to China. The present results indicate that the effect of NANTH has extended beyond the ocean water column into the deep sedimentary environment, presumably via biological assimilation of NANTH followed by deposition. Further, the findings indicate that NANTH is taking over from the conventional paradigm of nitrate flux from nitrate-rich deep water as the primary driver of biological export production in this region of the Pacific Ocean.


Assuntos
Monitoramento Ambiental , Sedimentos Geológicos , Nitrogênio , Regiões Árticas , China , Oceanos e Mares , Oceano Pacífico
12.
Nature ; 542(7641): 372-376, 2017 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-28114303

RESUMO

Mitochondria are double-membraned organelles with variable shapes influenced by metabolic conditions, developmental stage, and environmental stimuli. Their dynamic morphology is a result of regulated and balanced fusion and fission processes. Fusion is crucial for the health and physiological functions of mitochondria, including complementation of damaged mitochondrial DNAs and the maintenance of membrane potential. Mitofusins are dynamin-related GTPases that are essential for mitochondrial fusion. They are embedded in the mitochondrial outer membrane and thought to fuse adjacent mitochondria via combined oligomerization and GTP hydrolysis. However, the molecular mechanisms of this process remain unknown. Here we present crystal structures of engineered human MFN1 containing the GTPase domain and a helical domain during different stages of GTP hydrolysis. The helical domain is composed of elements from widely dispersed sequence regions of MFN1 and resembles the 'neck' of the bacterial dynamin-like protein. The structures reveal unique features of its catalytic machinery and explain how GTP binding induces conformational changes to promote GTPase domain dimerization in the transition state. Disruption of GTPase domain dimerization abolishes the fusogenic activity of MFN1. Moreover, a conserved aspartate residue trigger was found to affect mitochondrial elongation in MFN1, probably through a GTP-loading-dependent domain rearrangement. Thus, we propose a mechanistic model for MFN1-mediated mitochondrial tethering, and our results shed light on the molecular basis of mitochondrial fusion and mitofusin-related human neuromuscular disorders.


Assuntos
GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sequência de Aminoácidos , Biocatálise , Cristalografia por Raios X , GTP Fosfo-Hidrolases/genética , Humanos , Hidrólise , Fusão de Membrana , Potenciais da Membrana , Proteínas de Transporte da Membrana Mitocondrial/genética , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Triptofano/metabolismo
13.
ACS Chem Biol ; 11(9): 2414-9, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27409357

RESUMO

Four kinds of 2',3'-cNMPs have been identified in animals and plants, and their physiological roles are also suggested. However, in prokaryotes, while 2',3'-cCMP and 2',3'-cUMP are reported, 2',3'-cGMP or 2',3'-cAMP have never been identified from bacteria or archaea. In addition, there has been no biological method to prepare these cyclic nucleotides. During the study of a novel gene LfliZ from deep-sea bacterium Pseudoalteromonas sp. SM9913, we found that the recombinant LfliZ oligomers contained endogenous substrates, which were then identified to be 2',3'-cCMP, 2',3'-cUMP, 2',3'-cGMP, and 2',3'-cAMP, showing the first evidence for the biological existence of four kinds of 2',3'-cNMPs in prokaryotes. In addition, we further developed a biological method to simultaneously prepare four kinds of 2',3'-cNMPs from Escherichia coli through expressing LfliZ.


Assuntos
Nucleotídeos Cíclicos/química , Animais , Cromatografia em Gel , Escherichia coli/genética , Genes Bacterianos , Espectrometria de Massas , Nucleotídeos Cíclicos/classificação , Plantas/química , Pseudoalteromonas/química , Pseudoalteromonas/genética , Espectrofotometria Ultravioleta
14.
Front Microbiol ; 6: 1498, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26779153

RESUMO

Bacterial extracellular peptidases are important for bacterial nutrition and organic nitrogen degradation in the ocean. While many peptidases of the M13 family from terrestrial animals and bacteria are studied, there has been no report on M13 peptidases from marine bacteria. Here, we characterized an M13 peptidase, PepS, from the deep-sea sedimentary strain Shewanella sp. E525-6, and investigated its substrate specificity and catalytic mechanism. The gene pepS cloned from strain E525-6 contains 2085 bp and encodes an M13 metallopeptidase. PepS was expressed in Escherichia coli and purified. Among the characterized M13 peptidases, PepS shares the highest sequence identity (47%) with Zmp1 from Mycobacterium tuberculosis, indicating that PepS is a new member of the M13 family. PepS had the highest activity at 30°C and pH 8.0. It retained 15% activity at 0°C. Its half life at 40°C was only 4 min. These properties indicate that PepS is a cold-adapted enzyme. The smallest substrate for PepS is pentapeptide, and it is probably unable to cleave peptides of more than 30 residues. PepS prefers to hydrolyze peptide bonds with P1' hydrophobic residues. Structural and mutational analyses suggested that His531, His535 and Glu592 coordinate the catalytic zinc ion in PepS, Glu532 acts as a nucleophile, and His654 is probably involved in the transition state stabilization. Asp538 and Asp596 can stablize the orientations of His531 and His535, and Arg660 can stablize the orientation of Asp596. These results help in understanding marine bacterial peptidases and organic nitrogen degradation.

15.
Zhonghua Wai Ke Za Zhi ; 48(15): 1137-40, 2010 Aug 01.
Artigo em Chinês | MEDLINE | ID: mdl-21055005

RESUMO

OBJECTIVE: To explore the relationship between systemic inflammatory response syndrome(SIRS) and severity of acute pancreatitis combined with plateau erythrocythemia in the high altitude. METHODS: A retrospective analysis on the clinical data which involved acute pancreatitis combined with plateau erythrocythemia (n = 40) and without plateau erythrocythemia (n = 40) admitted from September 2006 to September 2009 was conducted. According to the unified standards, these cases were divided into plateau erythrocythemia group and no plateau erythrocythemia group. The patients in plateau erythrocythemia group were further divided into severe group and mild group according to scores of APACHEII. The data was analyzed according to the patient with (or without) SIRS, SIRS's standard indicators, diagnostic parameter and relation of severity and duration of SIRS in acute pancreatitis combined with plateau erythrocythemia. RESULTS: There was significantly discrepancy between plateau erythrocythemia group and no plateau erythrocythemia group not only in the incidence of patients who developed SIRS, but also in two items of patients fulfilling or not fulfilling diagnostic criteria of SIRS (P < 0.05). There was significant statistical difference in three items of diagnostic parameter of SIRS between plateau erythrocythemia group and no plateau erythrocythemia group (P < 0.05). Significant difference in two and three diagnostic parameter was found on severity of SIRS in acute pancreatitis combined with plateau erythrocythemia (P < 0.05). The more severity acute pancreatitis combined with plateau erythrocythemia was, the longer duration of SIRS was. CONCLUSION: SIRS is highly correlated with the severity of SIRS in acute pancreatitis combined with plateau erythrocythemia in the high altitude.


Assuntos
Altitude , Pancreatite/complicações , Policitemia/complicações , Síndrome de Resposta Inflamatória Sistêmica/etiologia , APACHE , Doença Aguda , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
16.
Mol Biol Rep ; 37(4): 2099-103, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19680788

RESUMO

Isolation of high quality RNA from ramie (Boehmeria nivea L. Gaud.) is difficult due to its high levels of polyphenols, polysaccharides, pectin, fat, wax and other secondary metabolites. A modified procedure based on guanidinium isothiocyanate for RNA preparation of ramie was developed in this study. High concentrations (5%, v/v) of guanidinium isothiocyanate, PVP-4000, sodium citrate and sodium lauryl sarcosinate and beta-mercaptoethanol were used in the extraction buffer, together with a low pH sodium acetate (pH 4.0) added to improve the RNA quality. The average yield was about 400 microg RNAg(-1) fresh leaves. One SSH library which was induced by ramie anthracnose was constructed by utilizing the RNA extracted through the present method. These results showed that our protocol was applicable for RNA isolation from recalcitrant ramie tissues.


Assuntos
Boehmeria/genética , Biblioteca Gênica , Hibridização de Ácido Nucleico/métodos , RNA de Plantas/isolamento & purificação , RNA de Plantas/normas , Northern Blotting , Boehmeria/microbiologia , Células Clonais , Colletotrichum/fisiologia , Eletroforese em Gel de Ágar , Espectrofotometria
17.
Zhonghua Wai Ke Za Zhi ; 44(23): 1624-5, 2006 Dec 01.
Artigo em Chinês | MEDLINE | ID: mdl-17359694

RESUMO

OBJECTIVE: To evaluate the application of total-cystectomy in the hepatic cystic echinococcosis (HCE). METHODS: An retrospective analysis was made in 223 cases of HCE. The observation targets included the days post operative, blood loss during operation, complications of residual cavity and local recurrence. The patients were divided into two groups: cyst-resection group and cyst ectomy group. The cyst-resection group was divided into two sub-groups, per cystectomy group and liver resection group. RESULTS: The incidences of residual cavity and local recurrence in the cyst-resection group were lower than that in the cystectomy group (P < 0.01). The incidences of blood loss and average days in ward in the per cystectomy group were lower than that in partial liver resection group (P < 0.01). CONCLUSIONS: HCE can decrease the complication of liver residual cavity and local recurrence effectively, total cystectomy should be selected as first procedures for treatment of hepatic cystic echinococcosis.


Assuntos
Cistectomia/métodos , Equinococose Hepática/cirurgia , Adolescente , Adulto , Idoso , Criança , Feminino , Seguimentos , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do Tratamento
18.
Sheng Wu Gong Cheng Xue Bao ; 21(2): 211-5, 2005 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-16013477

RESUMO

Based on the genomic sequence of SARS-CoV strain BJ101, antigenic immunodominant genes coding for the structure proteins of SARS-CoV were predicted by bio-informatics methods, and two chimeric genes A and B with multi-immunodominants lined up by Gly-Pro-Gly linker were synthesized. The chimeric genes were cloned into plasmid pGEX-6p-1 and expressed in E. coli with IPGT inducing. BALB/c mice were immunized with the purified recombinant fusion protein. The specificity of monoclonal antibodies were tested with a commercial ELISA kit for detecting antibody against SARS-CoV. The results showed that two peptides with molecular weights of 34kD and 35kD expressed by the two chimeric genes could be recognized by SARS patient convalescent serum in Western blot. Six positive hybridoma cell lines stably secreting monoclonal antibodies were selected. The subtype of monoclonal antibody D3C5 is IgG2a, and subtypes of all other five monoclonal antibodies are IgG1. Light chains of all monoclonal antibodies are kappa. With a commercial SARS-CoV antibodies detection ELISA kit, five out of six monoclonal antibodies were positively recognized. In western blot analysis with inactived virus cultures, D3D1 specifically recognized a band of about 180 kD. To further analyse the epitopes corresponding to the monoclonal antibodies, six oligoes (S1-S6) from S gene were synthesized and expressed. The results showed that the monoclonal antibodies D3D1 and D3C5 specifically recognized expression product of S2 and S5 oligoes, respectively. The S2 and S5 oligoes are corresponding to 447-458aa and 789-799aa of SARS-CoV S protein respectively.


Assuntos
Anticorpos Monoclonais/biossíntese , Epitopos , Glicoproteínas de Membrana/imunologia , Vírus da SARS/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Epitopos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Humanos , Hibridomas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Glicoproteína da Espícula de Coronavírus
19.
Virus Genes ; 31(1): 81-7, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15965612

RESUMO

A phage display peptide library targeting the nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) strain CH-1a was generated and used for epitope mapping. After 3 rounds of biopanning with the monoclonal antibody (MAb) N3H2 directed against the N protein, 3 positive phages were screened and sequenced. These phages share a consensus sequence, IQTAFNQGA, which corresponds to the amino acid (AA) 79-87 segment of the CH-1a N protein. A small DNA fragment coding for IQTAFNQGA was expressed as a fusion product, and reacted to N3H2 in Western blots and indirect ELISA. Four truncated peptides (IQTAFNQG, IQTAFNQ, QTAFNQGA, and TAFNQGA) expressed as GST fusion products failed to react with N3H2. The sequences around the N3H2-binding site among the N proteins of 57 PRRSV strains were compared. Our results indicate that the IQTAFNQGA motif is highly conserved among North American and European isolates. We concluded that the precisely defined nona-peptide epitope is a novel conserved Linear B cell epitope on the N protein of PRRSV.


Assuntos
Epitopos de Linfócito B/genética , Proteínas do Nucleocapsídeo/imunologia , Biblioteca de Peptídeos , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Sequência de Aminoácidos , Animais , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/química , Alinhamento de Sequência
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