Essential proteins are inseparable in cell growth and survival. The study of essential proteins is important for understanding cellular functions and biological mechanisms. Therefore, various computable methods have b...Essential proteins are inseparable in cell growth and survival. The study of essential proteins is important for understanding cellular functions and biological mechanisms. Therefore, various computable methods have been proposed to identify essential proteins. Unfortunately, most methods based on network topology only consider the interactions between a protein and its neighboring proteins, and not the interactions with its higher-order distance proteins. In this paper, we propose the DSEP algorithm in which we integrated network topology properties and subcellular localization information in protein–protein interaction(PPI) networks based on four-order distances, and then used random walks to identify the essential proteins. We also propose a method to calculate the finite-order distance of the network, which can greatly reduce the time complexity of our algorithm. We conducted a comprehensive comparison of the DSEP algorithm with 11 existing classical algorithms to identify essential proteins with multiple evaluation methods. The results show that DSEP is superior to these 11 methods.展开更多
BACKGROUND The function of prohibitin 1(Phb1)during liver regeneration(LR)remains relatively unexplored.Our previous research identified downregulation of Phb1 in rat liver mitochondria 24 h after 70%partial hepatecto...BACKGROUND The function of prohibitin 1(Phb1)during liver regeneration(LR)remains relatively unexplored.Our previous research identified downregulation of Phb1 in rat liver mitochondria 24 h after 70%partial hepatectomy(PHx),as determined by subcellular proteomic analysis.AIM To investigate the potential role of Phb1 during LR.METHODS We examined changes in Phb1 mRNA and protein levels,subcellular distribution,and abundance in rat liver during LR following 70%PHx.We also evaluated mitochondrial changes and apoptosis using electron microscopy and flow cytometry.RNA-interference-mediated knockdown of Phb1(PHBi)was performed in BRL-3A cells.RESULTS Compared with sham-operation control groups,Phb1 mRNA and protein levels in 70%PHx test groups were downregulated at 24 h,then upregulated at 72 and 168 h.Phb1 was mainly located in mitochondria,showed a reduced abundance at 24 h,significantly increased at 72 h,and almost recovered to normal at 168 h.Phb1 was also present in nuclei,with continuous increase in abundance observed 72 and 168 h after 70%PHx.The altered ultrastructure and reduced mass of mitochondria during LR had almost completely recovered to normal at 168 h.PHBi in BRL-3A cells resulted in increased S-phase entry,a higher number of apoptotic cells,and disruption of mitochondrial membrane potential.CONCLUSION Phb1 may contribute to maintaining mitochondrial stability and could play a role in regulating cell proliferation and apoptosis of rat liver cells during LR.展开更多
Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression sys...Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.展开更多
Although Platycodon grandiflorum(Jacq.)A.DC.is a renowned medicine food homology plant,reports of excessive cadmium(Cd)levels are common,which affects its safety for clinical use and food consumption.To enable its Cd ...Although Platycodon grandiflorum(Jacq.)A.DC.is a renowned medicine food homology plant,reports of excessive cadmium(Cd)levels are common,which affects its safety for clinical use and food consumption.To enable its Cd levels to be regulated or reduced,it is necessary to first elucidate the mechanism of Cd uptake and accumulation in the plant,in addition to its detoxification mechanisms.This present study used inductively couple plasma-mass-spectrometry to analyze the subcellular distribution and chemical forms of Cd in different tissues of P.grandiflorum.The experimental results showed that Cd was mainly accumulated in the roots[predominantly in the cell wall(50.96%-61.42%)],and it was found primarily in hypomobile and hypotoxic forms.The proportion of Cd in the soluble fraction increased after Cd exposure,and the proportion of insoluble phosphate Cd and oxalate Cd increased in roots and leaves,with a higher increase in oxalate Cd.Therefore,it is likely that root retention mechanisms,cell wall deposition,vacuole sequestration,and the formation of low mobility and low toxicity forms are tolerance strategies for Cd detoxification used by P.grandiflorum.The results of this study provide a theoretical grounding for the study of Cd accumulation and detoxification mechanisms in P.grandiflorum,and they can be used as a reference for developing Cd limits and standards for other medicine food homology plants.展开更多
Infection of plum bark necrosis stem pitting associated virus(PBNSPaV)has been reported in many Prunus species in several countries,causing significant economic losses.The very small proteins encoded by plant viruses ...Infection of plum bark necrosis stem pitting associated virus(PBNSPaV)has been reported in many Prunus species in several countries,causing significant economic losses.The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain significance.However,numerous studies have indicated that they might play important roles in the pathogenesis of virus infection.The role of small hydrophobic protein P6,encoded by the open reading frame 2 of PBNSPaV,has not been well explored.In this study,we amplified the P6 fragment from a PBNSPaV isolate by RT-PCR using specific primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain.Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes to the cytomembrane and nuclear membrane.To further clarify the pathogenicity of P6 proteins,we constructed a PVX-P6 expression vector by inserting the p6 fragment into a potato virus X(PVX)-based vector and transformed it into Agrobacterium tumefaciens GV3101.Infiltration of Nicotiana benthamiana(N.benthamiana)with the PVX vector-transformed A.tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation.Meanwhile,infiltration with the PVX-P6 vector-transformed A.tumefaciens resulted in no significant symptoms.These results demonstrated that heterologous expression of P6 in N.benthamiana could not enhance the pathogenicity of PVX.Our study indicates that P6 may not be a potential pathogenic factor associate with the causing of symptoms,and the mode of action of PBNSPaV-P6 protein remains to be further studied.展开更多
Toll-like receptor 21(TLR21) is a non-mammalian type TLR, and plays an important role in innate immune response in fish. In this paper, the full-length cDNA sequence of TLR21 gene was identified and characterized from...Toll-like receptor 21(TLR21) is a non-mammalian type TLR, and plays an important role in innate immune response in fish. In this paper, the full-length cDNA sequence of TLR21 gene was identified and characterized from large yellow croaker, Larimichthys crocea and was termed as LcTLR21. It consists of 3365 bp, including a 5'-terminal untranslated region(UTR) of 97 bp, a 3'-terminal UTR of 331 bp, and an open reading frame(ORF) of 2937 bp encoding a polypeptide of 978 amino acid residues. The deduced LcTLR21 contains a signal peptide domain at N-terminal, 12 leucine-rich repeats(LRRs) at the extracellular region, a transmembrane domain and a cytoplasmic toll-interleukin-1 receptor(TIR) domain at the C-terminal. Subcellular localization analysis revealed that the LcTLR21-GFP was constitutively expressed in cytoplasm. Tissue expression analysis indicated that LcTLR21 gene broadly expressed in most of the examined tissues, with the most predominant abundance in spleen, followed by head-kidney and liver, while the weakest expression was detected in brain. The expression level of LcTLR21 after LPS, poly I:C and Vibrio parahaemolyticus challenges was investigated in spleen, head-kidney and liver. LcTLR21 gene transcripts increased significantly in all examined tissues after the challenges, and the highest expression level was detected in liver at 24 h after poly I:C stimulation(P <0.05), suggesting that LcTLR21 might play a crucial role in fish resistance to viral and bacterial infections.展开更多
To predict the subcellular location of ascorbate peroxidase 1(APX1)protein in Arabidopsist haliana,aplasmid expressing APX1-enhance green fluorescent protein(EGFP)fusion protein was constructed,transformed into Agroba...To predict the subcellular location of ascorbate peroxidase 1(APX1)protein in Arabidopsist haliana,aplasmid expressing APX1-enhance green fluorescent protein(EGFP)fusion protein was constructed,transformed into Agrobacteriumt umefaciens GV3101,and transiently expressed in tobacco leaves.The localization of APX1 protein in the cells was determined by detecting the distribution of the fusion protein in tobacco epidermal cells.The results showed APX1 protein was mainly distributed in the cytoplasm of the plant.展开更多
Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This co...Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the cell-surface.Methods:Amplify the target gene with PCR from the constructed plasimid SDF-WT-Gly×4-DecPET-30a(+) with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 SDF-1KDEL,pcDNA3.1 SDF-154KDEL,pEGFPSDF-1KDEL and pEGFPSDF-154KDEL eukaryotic expression vectors were constructed and the DNA sequence was accurate,they were transferred into COS-7 cells with liposome . The exogenous expressions were observed, fusion protein SDF-1His and SDF-154His were confirmed by Western blot,and the SDF-1EGFP and SDF-154EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein SDF-1 KDELHis and SDF-1 54 KDELHis expressed in COS-7 cells.Subcelluar localization analysis showed that SDF-1KDELEGFP and SDF-154KDELEGFP were located mainly in endoplasmic reticulum.Conclusion:Four expression vectors pcDNA3.1 SDF-1KDEL, pcDNA3.1SDF-154KDEL, pEGFPSDF-1KDEL and pEGFPSDF-154KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum .展开更多
Plants possess effective mechanisms to respond quickly to the external environment. Rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs after a stimulus. The PLC in Dunaliella salina...Plants possess effective mechanisms to respond quickly to the external environment. Rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs after a stimulus. The PLC in Dunaliella salina plays important roles in growth and stress responses. However, the molecular basis of PLC action in D. salina remains little understood. To gain insight into the potential biological functions of this enzyme, we cloned a phospholipase C gene from D. salina in a previous study, named DsPLC (GenBank No. KF573428). Here, we present the prokaryotic expression, purification, and characterization of the DsPLC gene. The entire coding region of DsPLC was inserted into an expression vector pET32a, and the DsPLC gene was successfully expressed in Escherichia coli. The DsPLC protein was purified and identified using a polyclonal antibody and western blotting. Expressing DsPLC fused with a green fluorescent protein (GFP) in onion showed that DsPLC-GFP was localized to the intracellular membrane. Quantitative real-time PCR analysis revealed that the relative expression of the DsPLC gene was induced significantly by 3.0-mol/L NaCl at 4 h. Our results support the importance of PLC enzymes in plant defense signaling. This study provides a basis for further functional studies of the DsPLC gene and for additional analysis of the potential roles of PLC enzymes in response to abiotic stress.展开更多
The concentrations of 18 elements in subcellular fractions of human liver were determined by combining differential centrifugation and INAA. Samples of human liver were homogenized in a buffer. The homogenate was sepa...The concentrations of 18 elements in subcellular fractions of human liver were determined by combining differential centrifugation and INAA. Samples of human liver were homogenized in a buffer. The homogenate was separated into nuclei, mitochondrial, lysosomal, microsomal and cytosol fractions by successive differential centrifugation. Biological standard reference materials were used to evaluate the accuracy of the INAA method, and the results agree with the certified values. Element levels in subcellular fractions of human liver were discussed.展开更多
Protein kinase Cδ(PKCδ)is a member of the PKC family,and its implications have been reported in various biological and cancerous processes,including cell proliferation,cell death,tumor suppression,and tumor progress...Protein kinase Cδ(PKCδ)is a member of the PKC family,and its implications have been reported in various biological and cancerous processes,including cell proliferation,cell death,tumor suppression,and tumor progression.In liver cancer cells,accumulating reports show the bi-functional regulation of PKCδin cell death and survival.PKCδfunction is defined by various factors,such as phosphorylation,catalytic domain cleavage,and subcellular localization.PKCδhas multiple intracellular distribution patterns,ranging from the cytosol to the nucleus.We recently found a unique extracellular localization of PKCδin liver cancer and its growth factor-like function in liver cancer cells.In this review,we first discuss the structural features of PKCδand then focus on the functional diversity of PKCδbased on its subcellular localization,such as the nucleus,cell surface,and extracellular space.These findings improve our knowledge of PKCδinvolvement in the progression of liver cancer.展开更多
The Dictyostelium discoideum AMP-activated protein kinase (AMPK) snfA subcellular localization was studied in AX2 and stable HPF strains by use of AMPK antipeptide antibody and goat anti-rabbit Alexa-Flour 488-conjuga...The Dictyostelium discoideum AMP-activated protein kinase (AMPK) snfA subcellular localization was studied in AX2 and stable HPF strains by use of AMPK antipeptide antibody and goat anti-rabbit Alexa-Flour 488-conjugated IgG antibody. The AMPK exhibited cytosolic localization patterns and uniform focalised concentrations in wild type and the strains alike. Constitutive activation and attenuation of the α subunit expression did not affect subcellular distribution of AMPK. However, snfA expression was more intense in strains in which AMPK was constitutively active compared with the AX2 but lesser in attenuation strains. The localisation of the snfA reinforced the putative standing that it had a plethora of cytoplasmic functions. Moreover, the oxidative cellular function would require a ubiquitous system and might coordinately regulate responses to metabolic requirements. Furthermore, the developmental phases of the life cycle would support the cytosolic localization;and since organelles were potentially reorganized or removed entirely during the transition from vegetative living to fruiting body morphology. This study provided insight into the subcellular distribution of AMPK in Dictyostelium discoideum. We demonstrated that AMPK localization was steady in AX2 and derived strains whether constitutively active or anti-sense inhibited depicting extreme genetic states.展开更多
Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico clo...Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico cloning and RT-PCR. Bioinformatics analysis showed that deduced amino acid sequences of the six cloned RS genes were highly conserved with a similarity from 95% to 99% when compared to the RS genes which had been deposited at the GenBank. The results of amino acid sequences analysis showed six RS proteins contained the Chal_Sti_Synt_N and ACP_Syn_III_C domains and can be classified to same family but with different evolutionary distance. Expression pattern analysis by QRT-PCR provided evidence indicating that the mRNA of six RS genes were primarily expressed in the peanut shell at different developmental stages with different expression levels, but only lower levels of them were evident in the peanut kernel. The subcellular localization of RS protein in onion epidermal cell was performed by Agrobacterium tumefaciens-mediated transformation and the green fluorescent was monitored by confocal fluorescence microscopy. The results indicated that, RS1 and RS5 were located in the nucleus and plasma membrane respectively, while the RS2, RS3, RS4 and RS6 were located in both nucleus inner membrane and plasma membrane. The data will provide basic information for elucidating the regulatory mechanisms and enzyme kinetics underlying the RS genes in the resveratrol synthase pathway.展开更多
According to the large-scale sequencing of cDNA library from silkworm pupae, the cDNA of a novel gene with blank research background was identified and temporarily named Bm-X. The length of this cDNA is 778 bp. We obt...According to the large-scale sequencing of cDNA library from silkworm pupae, the cDNA of a novel gene with blank research background was identified and temporarily named Bm-X. The length of this cDNA is 778 bp. We obtained its ORF for further study by bioinformatics analysis. It is 444 bp and encodes 147 amino acid residues, with a predicted molecular weight (MW) of 16.4 kD and an isoelectric point (pI) of 3.69. In this study, we successfully constructed a recombinant plasmid pET-28a(+)-Bm-X and expressed it in Escherichia coli. We used the fusion protein rBm-X which purified by Niaffinity chromatography to produce polyclonal antibodies against Bm-X for Western blot analysis. The analysis revealed that Bm-X was expressed in the larval midgut, the epidermis and the silk gland. In addition, the subcellular localization analysis of silkworm ovary epithelial cells (BmN cells) showed that Bm-X protein was located both in cytoplasm and nucleus, and the signal was stronger in cytoplasm than in nucleus. Our findings indicate that Bm-X gene is a novel species-specificity gene and its expression product can be detected in tissues of the fifth silkworm instar larvae and BmN cells.展开更多
Arbuscular mycorrhizal fungi(AMF)can improve plant tolerance to several abiotic stresses,including heavy metals,drought or salinity exposure.However,the role of AMF in alleviation of soil cadmium(Cd)-induced toxicity ...Arbuscular mycorrhizal fungi(AMF)can improve plant tolerance to several abiotic stresses,including heavy metals,drought or salinity exposure.However,the role of AMF in alleviation of soil cadmium(Cd)-induced toxicity to plants is still largely unknown.In this study,Cd speciation in soil and subcellular distribution of Cd were used to characterize the roles of application AM fungi in the alleviation of Cd toxicity in alfalfa plants.Our results showed that the addition of Glomus mosseae in Cd contaminated soil(10 mg/Kg)significantly increased soil pH,cation exchange capacity(CEC)and organic matter in rhizosphere soil with Medicago truncatula L.,and then account for significantly decreased contents of exchangeable and carbonate-bounded Cd speciation in rhizosphere soil,indicating alleviation of plant toxicity by reduction of bioavailable fractions of Cd.Although there is no significant difference found in Cd accumulation by roots and shoots respectively between Cd and AM-Cd treatments,more portion of Cd was recorded compartmentalization in cell wall fraction of both root and shoot in treatment of Cd with AM application,indicating alleviation of Cd toxicity to plant cell.Herein,application of AM fungi in Cd treatments performed to inhibit the appearance of Cd toxicity symptoms,including the improvement of leaf electrolyte leakage,root elongation,seedling growth and biomass.This information provides a clearer understanding of detoxification strategy of AM fungi on Cd behavior with development and stabilization of soil structure and subcellular distribution of plant.展开更多
In the present work, computational analyses were applied to study the subcellular localiza-tion and posttranslational modifications of hu-man prion proteins (PrPs). The tentative location of prion protein was determin...In the present work, computational analyses were applied to study the subcellular localiza-tion and posttranslational modifications of hu-man prion proteins (PrPs). The tentative location of prion protein was determined to be in the nu-cleolus inside the nucleus by the following bio-informatics tools: Hum-PLoc, Euk-PLoc and Nuc-PLoc. Based on our results signal peptides with average of 22 base pairs in N-terminal were identified in human PrPs. This theoretical study demonstrates that PrP is post-translationally modified by: 1) attachment of two N-linked complex carbohydrate moieties (N181 and N197), 2) attachmet of glycosylphosphatidylinositol (GPI) at serine 230 and 3) formation of two di-sulfide bonds between “6–22” and “179–214” cysteines. Furthermore, ten protein kinase phosphorylation sites were predicted in human PrP. The above-noted phosphorylation was car-ried out by PKC and CK2. By using bioinfor-matics tools, we have shown that computation-ally human PrPs locate particularly into the nu-cleolus.展开更多
Apoptosis proteins have a central role in the development and homeostasis of an organism. These proteins are very important for the understanding the mechanism of programmed cell death, and their function is related t...Apoptosis proteins have a central role in the development and homeostasis of an organism. These proteins are very important for the understanding the mechanism of programmed cell death, and their function is related to their types. The apoptosis proteins are categorized into the following four types: (1) Cytoplasmic protein;(2) Plasma membrane-bound protein;(3) Mitochondrial inner and outer proteins;(4) Other proteins. A novel method, the Hilbert-Huang transform, is applied for predicting the type of a given apoptosis protein with support vector machine. High success rates were obtained by the re-substitute test (98/98=100%), jackknife test (91/98 = 92.9%).展开更多
The leukaemia cells HL60, incubated in 10 mM/ml ALA (5-aminolevulinic) for 4 hours, were carried an experimental research with fluorescent probes in photodynamic therapy (PDT) based on ALA by using PDT reaction room (...The leukaemia cells HL60, incubated in 10 mM/ml ALA (5-aminolevulinic) for 4 hours, were carried an experimental research with fluorescent probes in photodynamic therapy (PDT) based on ALA by using PDT reaction room (The average fluence rate of the 412 nm source was 5 mW/cm2). Cells viability were determined using a Cell Counting Kit-8 (CCK-8) assay, and PpIX Photobleaching of subcellular distributed sites of HL60 cells in vitro were investigated by fluorescence spectra acquired during treatment. The results showed that the fluorescence intensity of mitochondria, lysosomes, endoplasmic reticulum had decreased by 81.5%, 52.3% and 21.0%, respectively, compared with their initial values after a 45-minute light treatment. The rate of PpIX photobleaching in mitochondria was significantly higher than others. Addi-tionally, the change of the activity of HL60 cells was basically characterized by the change fluorescence intensity in mitochondria, which suggest that mitochondria is one of main therapeutic targets of photodynamic therapy.展开更多
As one of the essential topics in proteomics and molecular biology, protein subcellular localization has been extensively studied in previous decades. However, most of the methods are limited to the prediction of sing...As one of the essential topics in proteomics and molecular biology, protein subcellular localization has been extensively studied in previous decades. However, most of the methods are limited to the prediction of single-location proteins. In many studies, multi-location proteins are either not considered or assumed not existing. This paper proposes a novel multi-label subcellular-localization predictor based on the semantic similarity between Gene Ontology (GO) terms. Given a protein, the accession numbers of its homologs are obtained via BLAST search. Then, the homologous accession numbers of the protein are used as keys to search against the gene ontology annotation database to obtain a set of GO terms. The semantic similarity between GO terms is used to formulate semantic similarity vectors for classification. A support vector machine (SVM) classifier with a new decision scheme is proposed to classify the multi-label GO semantic similarity vectors. Experimental results show that the proposed multi-label predictor significantly outperforms the state-of-the-art predictors such as iLoc-Plant and Plant-mPLoc.展开更多
基金Project supported by the Gansu Province Industrial Support Plan (Grant No.2023CYZC-25)the Natural Science Foundation of Gansu Province (Grant No.23JRRA770)the National Natural Science Foundation of China (Grant No.62162040)。
文摘Essential proteins are inseparable in cell growth and survival. The study of essential proteins is important for understanding cellular functions and biological mechanisms. Therefore, various computable methods have been proposed to identify essential proteins. Unfortunately, most methods based on network topology only consider the interactions between a protein and its neighboring proteins, and not the interactions with its higher-order distance proteins. In this paper, we propose the DSEP algorithm in which we integrated network topology properties and subcellular localization information in protein–protein interaction(PPI) networks based on four-order distances, and then used random walks to identify the essential proteins. We also propose a method to calculate the finite-order distance of the network, which can greatly reduce the time complexity of our algorithm. We conducted a comprehensive comparison of the DSEP algorithm with 11 existing classical algorithms to identify essential proteins with multiple evaluation methods. The results show that DSEP is superior to these 11 methods.
文摘BACKGROUND The function of prohibitin 1(Phb1)during liver regeneration(LR)remains relatively unexplored.Our previous research identified downregulation of Phb1 in rat liver mitochondria 24 h after 70%partial hepatectomy(PHx),as determined by subcellular proteomic analysis.AIM To investigate the potential role of Phb1 during LR.METHODS We examined changes in Phb1 mRNA and protein levels,subcellular distribution,and abundance in rat liver during LR following 70%PHx.We also evaluated mitochondrial changes and apoptosis using electron microscopy and flow cytometry.RNA-interference-mediated knockdown of Phb1(PHBi)was performed in BRL-3A cells.RESULTS Compared with sham-operation control groups,Phb1 mRNA and protein levels in 70%PHx test groups were downregulated at 24 h,then upregulated at 72 and 168 h.Phb1 was mainly located in mitochondria,showed a reduced abundance at 24 h,significantly increased at 72 h,and almost recovered to normal at 168 h.Phb1 was also present in nuclei,with continuous increase in abundance observed 72 and 168 h after 70%PHx.The altered ultrastructure and reduced mass of mitochondria during LR had almost completely recovered to normal at 168 h.PHBi in BRL-3A cells resulted in increased S-phase entry,a higher number of apoptotic cells,and disruption of mitochondrial membrane potential.CONCLUSION Phb1 may contribute to maintaining mitochondrial stability and could play a role in regulating cell proliferation and apoptosis of rat liver cells during LR.
基金supported by the National Natural Science Foundation of ChinaChina (Grant Nos. 31872051, 32072528)the Foundation of Hubei Hongshan Laboratory (Grant No.2021hszd009)。
文摘Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.
基金This work was supported by the Major Science and Technology Projects in Inner Mongolia Autonomous Region(No.2019ZD005)the National Natural Science Foundation of China(No.81903751)+1 种基金by the Natural Science Basic Research Project of Shaanxi Science and Technology Department(No.2019JQ-877)by the Scientific Research Project of Shaanxi Administration of Traditional Chinese Medicine(No.2019-ZZ-ZY018).
文摘Although Platycodon grandiflorum(Jacq.)A.DC.is a renowned medicine food homology plant,reports of excessive cadmium(Cd)levels are common,which affects its safety for clinical use and food consumption.To enable its Cd levels to be regulated or reduced,it is necessary to first elucidate the mechanism of Cd uptake and accumulation in the plant,in addition to its detoxification mechanisms.This present study used inductively couple plasma-mass-spectrometry to analyze the subcellular distribution and chemical forms of Cd in different tissues of P.grandiflorum.The experimental results showed that Cd was mainly accumulated in the roots[predominantly in the cell wall(50.96%-61.42%)],and it was found primarily in hypomobile and hypotoxic forms.The proportion of Cd in the soluble fraction increased after Cd exposure,and the proportion of insoluble phosphate Cd and oxalate Cd increased in roots and leaves,with a higher increase in oxalate Cd.Therefore,it is likely that root retention mechanisms,cell wall deposition,vacuole sequestration,and the formation of low mobility and low toxicity forms are tolerance strategies for Cd detoxification used by P.grandiflorum.The results of this study provide a theoretical grounding for the study of Cd accumulation and detoxification mechanisms in P.grandiflorum,and they can be used as a reference for developing Cd limits and standards for other medicine food homology plants.
基金funded by the National Natural Science Foundation of China(32102143)Shandong Province Natural Sciences Foundation of China(ZR2019PC011 and ZR2020QC122)+1 种基金Scientific Research Foundation for Ph.D.Programs of Zaozhuang University(2018BS040 and 2018BS042)Science and Technology Program of Zaozhuang(2019NS03).
文摘Infection of plum bark necrosis stem pitting associated virus(PBNSPaV)has been reported in many Prunus species in several countries,causing significant economic losses.The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain significance.However,numerous studies have indicated that they might play important roles in the pathogenesis of virus infection.The role of small hydrophobic protein P6,encoded by the open reading frame 2 of PBNSPaV,has not been well explored.In this study,we amplified the P6 fragment from a PBNSPaV isolate by RT-PCR using specific primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain.Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes to the cytomembrane and nuclear membrane.To further clarify the pathogenicity of P6 proteins,we constructed a PVX-P6 expression vector by inserting the p6 fragment into a potato virus X(PVX)-based vector and transformed it into Agrobacterium tumefaciens GV3101.Infiltration of Nicotiana benthamiana(N.benthamiana)with the PVX vector-transformed A.tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation.Meanwhile,infiltration with the PVX-P6 vector-transformed A.tumefaciens resulted in no significant symptoms.These results demonstrated that heterologous expression of P6 in N.benthamiana could not enhance the pathogenicity of PVX.Our study indicates that P6 may not be a potential pathogenic factor associate with the causing of symptoms,and the mode of action of PBNSPaV-P6 protein remains to be further studied.
基金funded by the Qingdao National Laboratory for Marine Science and Technology to Cuiluan Yaothe Natural Science Foundation of Fujian (No.2018J01454)the National Natural Science Foundation of China (Nos.31101882 and 41276178)
文摘Toll-like receptor 21(TLR21) is a non-mammalian type TLR, and plays an important role in innate immune response in fish. In this paper, the full-length cDNA sequence of TLR21 gene was identified and characterized from large yellow croaker, Larimichthys crocea and was termed as LcTLR21. It consists of 3365 bp, including a 5'-terminal untranslated region(UTR) of 97 bp, a 3'-terminal UTR of 331 bp, and an open reading frame(ORF) of 2937 bp encoding a polypeptide of 978 amino acid residues. The deduced LcTLR21 contains a signal peptide domain at N-terminal, 12 leucine-rich repeats(LRRs) at the extracellular region, a transmembrane domain and a cytoplasmic toll-interleukin-1 receptor(TIR) domain at the C-terminal. Subcellular localization analysis revealed that the LcTLR21-GFP was constitutively expressed in cytoplasm. Tissue expression analysis indicated that LcTLR21 gene broadly expressed in most of the examined tissues, with the most predominant abundance in spleen, followed by head-kidney and liver, while the weakest expression was detected in brain. The expression level of LcTLR21 after LPS, poly I:C and Vibrio parahaemolyticus challenges was investigated in spleen, head-kidney and liver. LcTLR21 gene transcripts increased significantly in all examined tissues after the challenges, and the highest expression level was detected in liver at 24 h after poly I:C stimulation(P <0.05), suggesting that LcTLR21 might play a crucial role in fish resistance to viral and bacterial infections.
基金Supported by the Undergraduate Innovation and Entrepreneurship Training Program of Anhui Province(2016CXCYS099)
文摘To predict the subcellular location of ascorbate peroxidase 1(APX1)protein in Arabidopsist haliana,aplasmid expressing APX1-enhance green fluorescent protein(EGFP)fusion protein was constructed,transformed into Agrobacteriumt umefaciens GV3101,and transiently expressed in tobacco leaves.The localization of APX1 protein in the cells was determined by detecting the distribution of the fusion protein in tobacco epidermal cells.The results showed APX1 protein was mainly distributed in the cytoplasm of the plant.
基金This work was supported by grants from the National Natural Science Foundation of China(30170270)
文摘Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the cell-surface.Methods:Amplify the target gene with PCR from the constructed plasimid SDF-WT-Gly×4-DecPET-30a(+) with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 SDF-1KDEL,pcDNA3.1 SDF-154KDEL,pEGFPSDF-1KDEL and pEGFPSDF-154KDEL eukaryotic expression vectors were constructed and the DNA sequence was accurate,they were transferred into COS-7 cells with liposome . The exogenous expressions were observed, fusion protein SDF-1His and SDF-154His were confirmed by Western blot,and the SDF-1EGFP and SDF-154EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein SDF-1 KDELHis and SDF-1 54 KDELHis expressed in COS-7 cells.Subcelluar localization analysis showed that SDF-1KDELEGFP and SDF-154KDELEGFP were located mainly in endoplasmic reticulum.Conclusion:Four expression vectors pcDNA3.1 SDF-1KDEL, pcDNA3.1SDF-154KDEL, pEGFPSDF-1KDEL and pEGFPSDF-154KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum .
基金Supported by the National Natural Science Foundation of China(No.31472260)the Key Laboratory of Hydrobiology in Liaoning Province,College of Fisheries and Life Science,Dalian Ocean University
文摘Plants possess effective mechanisms to respond quickly to the external environment. Rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs after a stimulus. The PLC in Dunaliella salina plays important roles in growth and stress responses. However, the molecular basis of PLC action in D. salina remains little understood. To gain insight into the potential biological functions of this enzyme, we cloned a phospholipase C gene from D. salina in a previous study, named DsPLC (GenBank No. KF573428). Here, we present the prokaryotic expression, purification, and characterization of the DsPLC gene. The entire coding region of DsPLC was inserted into an expression vector pET32a, and the DsPLC gene was successfully expressed in Escherichia coli. The DsPLC protein was purified and identified using a polyclonal antibody and western blotting. Expressing DsPLC fused with a green fluorescent protein (GFP) in onion showed that DsPLC-GFP was localized to the intracellular membrane. Quantitative real-time PCR analysis revealed that the relative expression of the DsPLC gene was induced significantly by 3.0-mol/L NaCl at 4 h. Our results support the importance of PLC enzymes in plant defense signaling. This study provides a basis for further functional studies of the DsPLC gene and for additional analysis of the potential roles of PLC enzymes in response to abiotic stress.
文摘The concentrations of 18 elements in subcellular fractions of human liver were determined by combining differential centrifugation and INAA. Samples of human liver were homogenized in a buffer. The homogenate was separated into nuclei, mitochondrial, lysosomal, microsomal and cytosol fractions by successive differential centrifugation. Biological standard reference materials were used to evaluate the accuracy of the INAA method, and the results agree with the certified values. Element levels in subcellular fractions of human liver were discussed.
基金Supported by the Japan Society for the Promotion of Science,KAKENHI Grant,No.19H03519,16K18434,and 18K15253 to Yamada K,No.17H03584,18K19484,and 20H03519 to Yoshida KAMED under Grant No.A326TS to Yamada K+1 种基金The Jikei University Graduate Research Fund to Yamada Kand the Science Research Promotion Fund to Yoshida K.
文摘Protein kinase Cδ(PKCδ)is a member of the PKC family,and its implications have been reported in various biological and cancerous processes,including cell proliferation,cell death,tumor suppression,and tumor progression.In liver cancer cells,accumulating reports show the bi-functional regulation of PKCδin cell death and survival.PKCδfunction is defined by various factors,such as phosphorylation,catalytic domain cleavage,and subcellular localization.PKCδhas multiple intracellular distribution patterns,ranging from the cytosol to the nucleus.We recently found a unique extracellular localization of PKCδin liver cancer and its growth factor-like function in liver cancer cells.In this review,we first discuss the structural features of PKCδand then focus on the functional diversity of PKCδbased on its subcellular localization,such as the nucleus,cell surface,and extracellular space.These findings improve our knowledge of PKCδinvolvement in the progression of liver cancer.
文摘The Dictyostelium discoideum AMP-activated protein kinase (AMPK) snfA subcellular localization was studied in AX2 and stable HPF strains by use of AMPK antipeptide antibody and goat anti-rabbit Alexa-Flour 488-conjugated IgG antibody. The AMPK exhibited cytosolic localization patterns and uniform focalised concentrations in wild type and the strains alike. Constitutive activation and attenuation of the α subunit expression did not affect subcellular distribution of AMPK. However, snfA expression was more intense in strains in which AMPK was constitutively active compared with the AX2 but lesser in attenuation strains. The localisation of the snfA reinforced the putative standing that it had a plethora of cytoplasmic functions. Moreover, the oxidative cellular function would require a ubiquitous system and might coordinately regulate responses to metabolic requirements. Furthermore, the developmental phases of the life cycle would support the cytosolic localization;and since organelles were potentially reorganized or removed entirely during the transition from vegetative living to fruiting body morphology. This study provided insight into the subcellular distribution of AMPK in Dictyostelium discoideum. We demonstrated that AMPK localization was steady in AX2 and derived strains whether constitutively active or anti-sense inhibited depicting extreme genetic states.
文摘Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico cloning and RT-PCR. Bioinformatics analysis showed that deduced amino acid sequences of the six cloned RS genes were highly conserved with a similarity from 95% to 99% when compared to the RS genes which had been deposited at the GenBank. The results of amino acid sequences analysis showed six RS proteins contained the Chal_Sti_Synt_N and ACP_Syn_III_C domains and can be classified to same family but with different evolutionary distance. Expression pattern analysis by QRT-PCR provided evidence indicating that the mRNA of six RS genes were primarily expressed in the peanut shell at different developmental stages with different expression levels, but only lower levels of them were evident in the peanut kernel. The subcellular localization of RS protein in onion epidermal cell was performed by Agrobacterium tumefaciens-mediated transformation and the green fluorescent was monitored by confocal fluorescence microscopy. The results indicated that, RS1 and RS5 were located in the nucleus and plasma membrane respectively, while the RS2, RS3, RS4 and RS6 were located in both nucleus inner membrane and plasma membrane. The data will provide basic information for elucidating the regulatory mechanisms and enzyme kinetics underlying the RS genes in the resveratrol synthase pathway.
文摘According to the large-scale sequencing of cDNA library from silkworm pupae, the cDNA of a novel gene with blank research background was identified and temporarily named Bm-X. The length of this cDNA is 778 bp. We obtained its ORF for further study by bioinformatics analysis. It is 444 bp and encodes 147 amino acid residues, with a predicted molecular weight (MW) of 16.4 kD and an isoelectric point (pI) of 3.69. In this study, we successfully constructed a recombinant plasmid pET-28a(+)-Bm-X and expressed it in Escherichia coli. We used the fusion protein rBm-X which purified by Niaffinity chromatography to produce polyclonal antibodies against Bm-X for Western blot analysis. The analysis revealed that Bm-X was expressed in the larval midgut, the epidermis and the silk gland. In addition, the subcellular localization analysis of silkworm ovary epithelial cells (BmN cells) showed that Bm-X protein was located both in cytoplasm and nucleus, and the signal was stronger in cytoplasm than in nucleus. Our findings indicate that Bm-X gene is a novel species-specificity gene and its expression product can be detected in tissues of the fifth silkworm instar larvae and BmN cells.
基金This work was financially supported by Grants of Science and Technology Department of Sichuan Province(2019YFS0469,2020YFS0344)Educational Department of Sichuan province(17ZB0438).
文摘Arbuscular mycorrhizal fungi(AMF)can improve plant tolerance to several abiotic stresses,including heavy metals,drought or salinity exposure.However,the role of AMF in alleviation of soil cadmium(Cd)-induced toxicity to plants is still largely unknown.In this study,Cd speciation in soil and subcellular distribution of Cd were used to characterize the roles of application AM fungi in the alleviation of Cd toxicity in alfalfa plants.Our results showed that the addition of Glomus mosseae in Cd contaminated soil(10 mg/Kg)significantly increased soil pH,cation exchange capacity(CEC)and organic matter in rhizosphere soil with Medicago truncatula L.,and then account for significantly decreased contents of exchangeable and carbonate-bounded Cd speciation in rhizosphere soil,indicating alleviation of plant toxicity by reduction of bioavailable fractions of Cd.Although there is no significant difference found in Cd accumulation by roots and shoots respectively between Cd and AM-Cd treatments,more portion of Cd was recorded compartmentalization in cell wall fraction of both root and shoot in treatment of Cd with AM application,indicating alleviation of Cd toxicity to plant cell.Herein,application of AM fungi in Cd treatments performed to inhibit the appearance of Cd toxicity symptoms,including the improvement of leaf electrolyte leakage,root elongation,seedling growth and biomass.This information provides a clearer understanding of detoxification strategy of AM fungi on Cd behavior with development and stabilization of soil structure and subcellular distribution of plant.
文摘In the present work, computational analyses were applied to study the subcellular localiza-tion and posttranslational modifications of hu-man prion proteins (PrPs). The tentative location of prion protein was determined to be in the nu-cleolus inside the nucleus by the following bio-informatics tools: Hum-PLoc, Euk-PLoc and Nuc-PLoc. Based on our results signal peptides with average of 22 base pairs in N-terminal were identified in human PrPs. This theoretical study demonstrates that PrP is post-translationally modified by: 1) attachment of two N-linked complex carbohydrate moieties (N181 and N197), 2) attachmet of glycosylphosphatidylinositol (GPI) at serine 230 and 3) formation of two di-sulfide bonds between “6–22” and “179–214” cysteines. Furthermore, ten protein kinase phosphorylation sites were predicted in human PrP. The above-noted phosphorylation was car-ried out by PKC and CK2. By using bioinfor-matics tools, we have shown that computation-ally human PrPs locate particularly into the nu-cleolus.
文摘Apoptosis proteins have a central role in the development and homeostasis of an organism. These proteins are very important for the understanding the mechanism of programmed cell death, and their function is related to their types. The apoptosis proteins are categorized into the following four types: (1) Cytoplasmic protein;(2) Plasma membrane-bound protein;(3) Mitochondrial inner and outer proteins;(4) Other proteins. A novel method, the Hilbert-Huang transform, is applied for predicting the type of a given apoptosis protein with support vector machine. High success rates were obtained by the re-substitute test (98/98=100%), jackknife test (91/98 = 92.9%).
文摘The leukaemia cells HL60, incubated in 10 mM/ml ALA (5-aminolevulinic) for 4 hours, were carried an experimental research with fluorescent probes in photodynamic therapy (PDT) based on ALA by using PDT reaction room (The average fluence rate of the 412 nm source was 5 mW/cm2). Cells viability were determined using a Cell Counting Kit-8 (CCK-8) assay, and PpIX Photobleaching of subcellular distributed sites of HL60 cells in vitro were investigated by fluorescence spectra acquired during treatment. The results showed that the fluorescence intensity of mitochondria, lysosomes, endoplasmic reticulum had decreased by 81.5%, 52.3% and 21.0%, respectively, compared with their initial values after a 45-minute light treatment. The rate of PpIX photobleaching in mitochondria was significantly higher than others. Addi-tionally, the change of the activity of HL60 cells was basically characterized by the change fluorescence intensity in mitochondria, which suggest that mitochondria is one of main therapeutic targets of photodynamic therapy.
文摘As one of the essential topics in proteomics and molecular biology, protein subcellular localization has been extensively studied in previous decades. However, most of the methods are limited to the prediction of single-location proteins. In many studies, multi-location proteins are either not considered or assumed not existing. This paper proposes a novel multi-label subcellular-localization predictor based on the semantic similarity between Gene Ontology (GO) terms. Given a protein, the accession numbers of its homologs are obtained via BLAST search. Then, the homologous accession numbers of the protein are used as keys to search against the gene ontology annotation database to obtain a set of GO terms. The semantic similarity between GO terms is used to formulate semantic similarity vectors for classification. A support vector machine (SVM) classifier with a new decision scheme is proposed to classify the multi-label GO semantic similarity vectors. Experimental results show that the proposed multi-label predictor significantly outperforms the state-of-the-art predictors such as iLoc-Plant and Plant-mPLoc.
