Oyster,as a common aquatic food,play an important role in shellfish allergy.In this study,2 tropomyosin(TM)isoforms TM-αand TM-β(TM-α/-β)in Alectryonella plicatula were identified.The sequences of 852 bp encoding ...Oyster,as a common aquatic food,play an important role in shellfish allergy.In this study,2 tropomyosin(TM)isoforms TM-αand TM-β(TM-α/-β)in Alectryonella plicatula were identified.The sequences of 852 bp encoding 284 amino acids of TM-α/-βand 2 recombinant proteins were obtained,respectively.There were 12 amino acid differences between TM-α/-β.The results of immunological experiments indicated that TM-βhad stronger immunobinding activity and immunoreactivity than those of TM-α.Structural analysis showed that TM-βhad moreα-helix and higher surface hydrophobicity than TM-α.Sequences and epitopes alignment with shellfish TMs revealed that amino acids of TM-βwere more frequently recognized as IgE epitopes in other shellfish TMs than TM-α.Differences in structure and sequence account for the higher immunological activity of TM-βcompared to TM-α.These findings provide a theoretical basis for enriching the understanding of shellfish TM and accurate diagnosis of allergic components.展开更多
In recent years,the allergy rate of oysters has surged,and daily food processing methods make it hard to reduce heat resistance and digestive allergy such as tropomyosin(TM).In this study,the Maillard reaction with xy...In recent years,the allergy rate of oysters has surged,and daily food processing methods make it hard to reduce heat resistance and digestive allergy such as tropomyosin(TM).In this study,the Maillard reaction with xylose significantly reduced the IgE binding capacity of Alectryonella plicatula food matrix(AFM),that reduced by(77.81±2.68)%.The study found the Maillard reaction changes the structure of the AFM,in which the content ofα-helix decreased by(24.64±1.46)%.Structural transformation further explains why the Maillard reaction alters the immunobinding activity of AFM.In addition,the Maillard reaction reduces the digestive stability of the AFM and makes TM in the A.plicatula food matrix Maillard reaction products(AFM-MRPs)more easily digested.Based on the above research,10 amino acids on the 7 IgE epitopes of TM were modified.This result indicates that the Maillard reaction reduces the immunobinding activity of the AFM by changing the structure and modifying the amino acids on the epitope.展开更多
The development and plasticity of central auditory system can be influenced by the change of peripheral neuronal activity. However, the molecular mechanism participating in the process remains elusive. Brain-derived n...The development and plasticity of central auditory system can be influenced by the change of peripheral neuronal activity. However, the molecular mechanism participating in the process remains elusive. Brain-derived neurotrophic factor(BDNF) binding with its functional receptor tropomyosin receptor kinase B(TrkB) has multiple effects on neurons. Here we used a rat model of auditory deprivation by bilateral cochlear ablation, to investigate the changes in expression of BDNF and Trk B in the auditory cortex after auditory deprivation that occurred during the critical period for the development of central auditory system. Reverse transcription-quantitative polymerase chain reaction(RTqPCR) and immunohistochemistry methods were adopted to detect the m RNA and protein expression levels of BDNF and TrkB in the auditory cortex at 2, 4, 6 and 8 weeks after surgery, respectively. The change in the expression of BDNF and TrkB mRNAs and proteins followed similar trend. In the bilateral cochlear ablation groups, the BDNF-TrkB expression level initially decreased at 2 weeks but increased at 4 weeks followed by the reduction at 6 and 8 weeks after cochlear removal, as compared to the age-matched sham control groups. In conclusion, the BDNF-TrkB signaling is involved in the plasticity of auditory cortex in an activity-dependent manner.展开更多
AIM: To investigate the correlation between nerve growth factor-tropomyosin-receptor-kinase (NGF-TrkA) signaling pathway and prognosis in intrahepatic cholangiocarcinoma (IHCC).
The nuclei and chromosomes were isolated from plasmodia of Physarum polycephalum. The nuclear matrir and chromosome scaffold were obtained after the DNA and most of the proteins were extracted with DNase I and 2 M NaC...The nuclei and chromosomes were isolated from plasmodia of Physarum polycephalum. The nuclear matrir and chromosome scaffold were obtained after the DNA and most of the proteins were extracted with DNase I and 2 M NaCl. SDS-PAGE analyses revealed that the nuclear matrir and chromosome scaffold contained a 37 kD polypeptide which is equivalent to tropomyosin in molecular weight. Immunofluorescence observations upon slide preparations labeled with anti-tropomyosin antibody showed that the nuclear matrix and chromosome scaffold emanated bright fluorescence, suggesting the presence of the antigen in them.Immunodotting results confirmed the presence of tropomyosin in the nuclear matrix and chromosome scaffold. Immunoelectron microscopic obserwtions further demonstrated that tropomyosin was dispersively distributed in the interphase nuclei and metaphase chromosomes.展开更多
The tropomyosin(TM)fractions of crab protems may cause allergic reactions in mdividuals susceptible to allergies;however,efficient and safe methods by which to reduce such allergenicity are not currently available.The...The tropomyosin(TM)fractions of crab protems may cause allergic reactions in mdividuals susceptible to allergies;however,efficient and safe methods by which to reduce such allergenicity are not currently available.Therefore,in this study,the effects of three different processing methods,i.e.,microwave,ultrasound,and high temperature-pressure(HTP)treatments,on the digestion stability of TM from Chinese mitten crab muscle and the allergenicity of TM digestion products were explored.sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that microwaving had little effect on the digestion stability of TM.In contrast,ultrasound and HTP treatments facilitated the degradation of TM.Similarly,Western blotting and inhibition ELISA indicated that the IgE-binding activity of TM was significantly reduced after treatment with ultrasound or HTP.Among the three different proces sing methods,HTP was the most effective method for improving digestibility of TM and reducing immunoreactivity.This finding provides new insights into treatments for crab allergies.展开更多
The neurotrophin-Trk receptor pathway is an intrinsic pathway to relieve damage to the central nervous system. The present study observed the effects of Tongluo Jiunao （TLJN）, which comprises Panax Notoginseng and G...The neurotrophin-Trk receptor pathway is an intrinsic pathway to relieve damage to the central nervous system. The present study observed the effects of Tongluo Jiunao （TLJN）, which comprises Panax Notoginseng and Gardenia Jasminoides, on expression of brain-derived neurotrophic factor （BDNF） and tropomyosin-related kinase B （TrkB） in a rat model of focal cerebral ischemic injury. Xue Sai Tong （XST）, comprising Panax Notoginseng, served as the positive control. Mechanisms of neuroprotection were analyzed following TLJN injection. Following establishment of the middle cerebral artery occlusion models, TLJN and XST were intraperitoneally injected, and 2, 3 5-triphenyltetrazolium chloride staining results revealed that TLJN injection reduced infarct volume, suggesting that TLJN exerted a neuroprotective effect. Enzyme-linked immunosorbent assay results showed that TLJN elevated BDNF and growth associated protein-43 expression in ischemic brain tissues, as well as serum BDNF levels. Reverse-transcription polymerase chain reaction and western blot results showed that TLJN injection did not affect TrkB expression in the ischemic brain tissues of rats. These results suggested that TLJN injection reduced damage to ischemic brain tissues and increased BDNF expression. In addition, TLJN injection resulted in better promoting effects on neurotrophic factor expression compared with XST.展开更多
Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca2+ and regulate striated muscle contraction in vertebrates. TnT’s function has been extensively described in vertebrates, but its role has be...Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca2+ and regulate striated muscle contraction in vertebrates. TnT’s function has been extensively described in vertebrates, but its role has been obscure in molluscan muscles. Our previous work indicated that the TnC and TnI subunits work in adductor phasic muscle, but not in catch muscle. Here, we have characterized TnT from the Japanese bivalve pearl oyster Pinctada fucata to start to explain the function of Tn in molluscan muscle contraction. We determined the primary structure of the full-length TnT protein from the P. fucata adductor muscle (Pifuc-TnT), and found that it is composed of 316 amino acid residues with a predicted molecular mass of 37.4 kDa. Multiple sequence alignment showed that Pifuc-TnT has an extension of >60 residues at the C-terminus that are not present in vertebrate TnTs, including known TnTs from other mollusks. Pifuc-TnT gene structure predictions using Splign alignment of the cDNA generated in this study and genome sequences indicated that Pifuc-TnT consists of 13 exons. Start and stop codons are located in exons 2 and 12, respectively. Quantitative real-time PCR revealed that the Pifuc-TnT gene was predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, slightly in gill, and not at all in mantle and foot. These findings suggest that TnT plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction. Isothermal titration calorimetry revealed that unlike vertebrate TnTs, Pifuc-TnT does not interact with P. fucata tropomyosin-1 nor with tropomyosin-2. These findings in P. fucata imply that Tn functions differently in molluscan muscle than it does in vertebrates.展开更多
We determined the full-length primary structure of the tropomyosin (TM)-1 and -2 proteins from the adductor muscle of the Japanese pearl oyster Pinctada fucata (Pifuc-TM-1 and Pifuc-TM-2), and found that they are each...We determined the full-length primary structure of the tropomyosin (TM)-1 and -2 proteins from the adductor muscle of the Japanese pearl oyster Pinctada fucata (Pifuc-TM-1 and Pifuc-TM-2), and found that they are each composed of 284 amino acid residues. We predicted the gene structure of P. fucata TM (Pifuc-TM) using Splign alignment of our cDNA with genomic sequences and elucidated that Pifuc-TM consists of 10 exons. Exons 1 - 3 and 5 - 10 are used to transcribe Pifuc-TM-1 mRNA, and exons 1 - 4 and 6 - 10 are used to transcribe Pifuc-TM-2 mRNA. Both genes share the same start and stop codons located in exon 1 and exon 10, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TM-1 gene was mainly expressed in adductor phasic muscle, and at a relatively weaker level in adductor catch muscle, whereas the Pifuc-TM-2 gene was expressed equally in both phasic and catch muscles. They were weakly expressed in gill and mantle. Immunoblot analysis using anti-Pifuc-TM-1 and anti-Pifuc-TM-2 antibodies revealed that adductor phasic muscle contained Pifuc-TM-1, while adductor catch muscle contained both Pifuc-TM-1 and Pifuc-TM-2. Differential scanning calorimetry (DSC) analysis was carried out for Pifuc-TM-1 and Pifuc-TM-2 expressed in bacteria, as well as TM purified from P. fucata phasic and catch muscle tissues (phasic-TM and catch-TM). The DSC data indicated that phasic-TM was mainly composed of Pifuc-TM-1, whereas catch-TM contained Pifuc-TM-1 and Pifuc-TM-2. These findings suggest that the distribution of Pifuc-TM-1 and Pifuc-TM-2 in adductor muscle is specific to the muscle fiber type, and reflects the properties of each.展开更多
In vivo imaging of cerebral ischemia/reperfusion injury remains an important challenge.We injected porous Ag/Au@SiO_(2) bimetallic hollow nanoshells carrying anti-tropomyosin 4 as a molecular probe into mice with cere...In vivo imaging of cerebral ischemia/reperfusion injury remains an important challenge.We injected porous Ag/Au@SiO_(2) bimetallic hollow nanoshells carrying anti-tropomyosin 4 as a molecular probe into mice with cerebral ischemia/reperfusion injury and observed microvascular changes in the brain using photoacoustic imaging with ultrasonography.At each measured time point,the total photoacoustic signal was significantly higher on the affected side than on the healthy side.Twelve hours after reperfusion,cerebral perfusion on the affected side increased,cerebrovascular injury worsened,and anti-tropomyosin 4 expression increased.Twenty-four hours after reperfusion and later,perfusion on the affected side declined slowly and stabilized after 1 week;brain injury was also alleviated.Histopathological and immunohistochemical examinations confirmed the brain injury tissue changes.The nanoshell molecular probe carrying anti-tropomyosin 4 has potential for use in early diagnosis of cerebral ischemia/reperfusion injury and evaluating its progression.展开更多
The present study utilized beta amyloid （Aβ）-induced cell apoptosis in PC12 cells as a cell model of Alzheimer＇s disease to investigate the interaction between brain-derived neurotrophic factor （BDNF） and the tr...The present study utilized beta amyloid （Aβ）-induced cell apoptosis in PC12 cells as a cell model of Alzheimer＇s disease to investigate the interaction between brain-derived neurotrophic factor （BDNF） and the tropomyosin-related kinase B receptor. Results showed that Aβ（25-35） can reduce survival of PC12 cells and increase cleaved caspase-3 expression in PC12 cells. However, BDNF inhibited Aβ（25-35）-induced cytotoxicity and cleaved casapase-3 expression. Interestingly, pretreatment with the tropomyosin-related kinase receptor inhibitor K252a for 20 minutes prior to BDNF blocked the neuroprotective effect of BDNF on PC12 cells.展开更多
Background: Tropomyosin 1 alpha chain (Tm1) is an actin-binding protein that regulates the endothelial cell response to oxidative stress following its phosphorylation at Serine 283 (S283). Tm1 is also a major tumor su...Background: Tropomyosin 1 alpha chain (Tm1) is an actin-binding protein that regulates the endothelial cell response to oxidative stress following its phosphorylation at Serine 283 (S283). Tm1 is also a major tumor suppressor in breast cancer. In the present study, we investigated the role of phosphorylation of Tm1 in regulating its tumor suppressor properties. Methods: MDA MB231 breast cancer cells stably overexpressing wild type form of Tm1 or Tm1 mutants (S283A and S283E) were generated. Proliferation and cell viability were assayed by means of the enzymatic cleavage of the tetrazolium salt WST-1 to formazan dye by cellular mitochondrial dehydrogenases. Adhesion assays were performed at various periods of time on cells grown on plastic. Cell migration was evaluated by using the wound-healing assay and by measuring transendothelial migration of cancer cells. Malignant transformation in vitro was determined by using the anchorage-independent growth assay on soft agar. Results: We found that cells expressing the phosphomimetic form of Tm1 S283E/Tm1 are characterized by an increased adhesion to the substratum. Moreover, the migration of MDA-MB231/S283E/Tm1 cells in a wound closure assay is reduced compared to parental cells or those expressing the non-phosphorylatable form of Tm1 (S283A). Similarly, the transendothelial migration of MDA-MB231/S283E/Tm1 cells is also reduced as compared to the other cell lines. Moreover, we found that the cells expressing the S283A mutants form more colonies in soft agar that those expressing the S283E mutants. Conclusion: Phosphorylation of Tm1 at Ser283 contributes to its anti-tumor properties, and this effect results mainly from an increase in cell adhesion associated with a decrease in their migratory and invasive potentials.展开更多
OBJECTIVE: To perform cloning of the gene encoding Chinese Schistosoma japonicum tropomyosin (SjcTM) and its expression in Escherichia coli. METHODS: SjcTM cDNA fragment, except for 14 amino acids at the amino terminu...OBJECTIVE: To perform cloning of the gene encoding Chinese Schistosoma japonicum tropomyosin (SjcTM) and its expression in Escherichia coli. METHODS: SjcTM cDNA fragment, except for 14 amino acids at the amino terminus, was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from adult worms of S. japonicum. The RT-PCR product was cloned into T vector and sequenced. The SjcTM cDNA, derived from the constructed TA clone pGEM-SjcTM, was then subcloned into the expressing vector pBV220. After characterization by agarose gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for expression under the temperature-dependent condition. RESULTS: The RT-PCR product, cloned into a T vector, was sequenced and shown to be 96.5% identical at the nuclei acid level and 98.1% identical in deduced amino acid sequence to that of S. mansoni tropomyosin. The target DNA fragment was then subcloned into a prokaryotic vector pBV220. Induced expression in E. coli DH5alpha cells resulted in a constant level of recombinant protein production. The results of SDS-PAGE and Western blot revealed that the molecular weight of non-fusion recombinant protein (rSjcTM) was approximately 32 kDa and could be recognized specifically by a polyclonal antiserum specific for native S. japonicum tropomyosin (SjcTM). CONCLUSION: The engineering of the cDNA encoding S. japonicum tropomyosin and its bacterial expression was successfully made.展开更多
The one-bead-one-compound （OBOC） combinatorial peptide library is a powerful tool to identify ligand and receptor interactions. Here, we applied the OBOC library technology to identify mimotopes specific to the immu...The one-bead-one-compound （OBOC） combinatorial peptide library is a powerful tool to identify ligand and receptor interactions. Here, we applied the OBOC library technology to identify mimotopes specific to the immunoglobulin E （IgE） epitopes of the major shellfish allergen tropomyosin. OBOC peptide libraries with 8-12 amino acid residues were screened with serum samples from patients with shellfish allergy for IgE mimotopes of tropomyosin. Twenty-five mimotopes were identified from the screening and their binding reactivity to tropomyosin-specific IgE was confirmed by peptide ELISA. These mimotopes could be divided into seven clusters based on sequence homology, and epitope mapping by EpiSearch of the clustered mimotopes was performed to characterize and confirm the validity of mimotopes. Five out of six of the predicted epitopes were found to overlap with previously identified epitopes of tropomyosin. To further confirm the mimicry potential of mimotopes, BALB/c mice were immunized with mimotopes conjugated to keyhole limpet hemocyanin and assayed for their capacity to induce tropomyosin-specific antibodies. BALB/c mice that received mimotope immunization were found to have an elevated level of tropomyosin-specific immunoglobulin G, but not mice that received an irrelevant mimotope. This study pioneers the successful application of the OBOC libraries using whole sera to screen and identify multiple shrimp allergen mimotopes and validates their mimicry potential using in vitro, in vivo, and in silico methods.展开更多
Tropomyosin receptor kinase A,B and C(TRKA,TRKB and TRKC),which are well-known members of the cell surface receptor tyrosine kinase(RTK)family,are encoded by the neurotrophic receptor tyrosine kinase 1,2 and 3(NTRK1,N...Tropomyosin receptor kinase A,B and C(TRKA,TRKB and TRKC),which are well-known members of the cell surface receptor tyrosine kinase(RTK)family,are encoded by the neurotrophic receptor tyrosine kinase 1,2 and 3(NTRK1,NTRK2 and NTRK3)genes,respectively.TRKs can regulate cell proliferation,differentiation and even apoptosis through the RAS/MAPKs,PI3 K/AKT and PLCγtyrosine kinase fusions;Small-molecule inhibitor;NTRK fusion cancer pathways.Gene fusions involving NTRK act as oncogenic drivers of a broad diversity of adult and pediatric tumors,and TRKs have become promising antitumor targets.Therefore,achieving a comprehensive understanding of TRKs and relevant TRK inhibitors should be urgently pursued for the further development of novel TRK inhibitors for potential clinical applications.This review focuses on summarizing the biological functions of TRKs and NTRK fusion proteins,the development of small-molecule TRK inhibitors with different chemotypes and their activity and selectivity,and the potential therapeutic applications of these inhibitors for future cancer drug discovery efforts.展开更多
基金supported by the National Natural Scientific Foundation of China(32072336,32472449).
文摘Oyster,as a common aquatic food,play an important role in shellfish allergy.In this study,2 tropomyosin(TM)isoforms TM-αand TM-β(TM-α/-β)in Alectryonella plicatula were identified.The sequences of 852 bp encoding 284 amino acids of TM-α/-βand 2 recombinant proteins were obtained,respectively.There were 12 amino acid differences between TM-α/-β.The results of immunological experiments indicated that TM-βhad stronger immunobinding activity and immunoreactivity than those of TM-α.Structural analysis showed that TM-βhad moreα-helix and higher surface hydrophobicity than TM-α.Sequences and epitopes alignment with shellfish TMs revealed that amino acids of TM-βwere more frequently recognized as IgE epitopes in other shellfish TMs than TM-α.Differences in structure and sequence account for the higher immunological activity of TM-βcompared to TM-α.These findings provide a theoretical basis for enriching the understanding of shellfish TM and accurate diagnosis of allergic components.
基金supported by the National Natural Scientific Foundation of China(32072336,31871720)the National Key R&D Program of China(2019YFD0901703).
文摘In recent years,the allergy rate of oysters has surged,and daily food processing methods make it hard to reduce heat resistance and digestive allergy such as tropomyosin(TM).In this study,the Maillard reaction with xylose significantly reduced the IgE binding capacity of Alectryonella plicatula food matrix(AFM),that reduced by(77.81±2.68)%.The study found the Maillard reaction changes the structure of the AFM,in which the content ofα-helix decreased by(24.64±1.46)%.Structural transformation further explains why the Maillard reaction alters the immunobinding activity of AFM.In addition,the Maillard reaction reduces the digestive stability of the AFM and makes TM in the A.plicatula food matrix Maillard reaction products(AFM-MRPs)more easily digested.Based on the above research,10 amino acids on the 7 IgE epitopes of TM were modified.This result indicates that the Maillard reaction reduces the immunobinding activity of the AFM by changing the structure and modifying the amino acids on the epitope.
基金supported by National Science Foundation of China (Grant 30600125)National Science Foundation Hebei Province (Grant C2011206036)
文摘The development and plasticity of central auditory system can be influenced by the change of peripheral neuronal activity. However, the molecular mechanism participating in the process remains elusive. Brain-derived neurotrophic factor(BDNF) binding with its functional receptor tropomyosin receptor kinase B(TrkB) has multiple effects on neurons. Here we used a rat model of auditory deprivation by bilateral cochlear ablation, to investigate the changes in expression of BDNF and Trk B in the auditory cortex after auditory deprivation that occurred during the critical period for the development of central auditory system. Reverse transcription-quantitative polymerase chain reaction(RTqPCR) and immunohistochemistry methods were adopted to detect the m RNA and protein expression levels of BDNF and TrkB in the auditory cortex at 2, 4, 6 and 8 weeks after surgery, respectively. The change in the expression of BDNF and TrkB mRNAs and proteins followed similar trend. In the bilateral cochlear ablation groups, the BDNF-TrkB expression level initially decreased at 2 weeks but increased at 4 weeks followed by the reduction at 6 and 8 weeks after cochlear removal, as compared to the age-matched sham control groups. In conclusion, the BDNF-TrkB signaling is involved in the plasticity of auditory cortex in an activity-dependent manner.
基金Supported by National Natural Science Foundation of China,No.81172044
文摘AIM: To investigate the correlation between nerve growth factor-tropomyosin-receptor-kinase (NGF-TrkA) signaling pathway and prognosis in intrahepatic cholangiocarcinoma (IHCC).
文摘The nuclei and chromosomes were isolated from plasmodia of Physarum polycephalum. The nuclear matrir and chromosome scaffold were obtained after the DNA and most of the proteins were extracted with DNase I and 2 M NaCl. SDS-PAGE analyses revealed that the nuclear matrir and chromosome scaffold contained a 37 kD polypeptide which is equivalent to tropomyosin in molecular weight. Immunofluorescence observations upon slide preparations labeled with anti-tropomyosin antibody showed that the nuclear matrix and chromosome scaffold emanated bright fluorescence, suggesting the presence of the antigen in them.Immunodotting results confirmed the presence of tropomyosin in the nuclear matrix and chromosome scaffold. Immunoelectron microscopic obserwtions further demonstrated that tropomyosin was dispersively distributed in the interphase nuclei and metaphase chromosomes.
基金supported by the earmarked fund for the Anhui Provincial Modern Agri-industry Technology Research System(AHCYJSTX-08)the China Agriculture Research System of MOF and MARA(CARS-48)。
文摘The tropomyosin(TM)fractions of crab protems may cause allergic reactions in mdividuals susceptible to allergies;however,efficient and safe methods by which to reduce such allergenicity are not currently available.Therefore,in this study,the effects of three different processing methods,i.e.,microwave,ultrasound,and high temperature-pressure(HTP)treatments,on the digestion stability of TM from Chinese mitten crab muscle and the allergenicity of TM digestion products were explored.sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that microwaving had little effect on the digestion stability of TM.In contrast,ultrasound and HTP treatments facilitated the degradation of TM.Similarly,Western blotting and inhibition ELISA indicated that the IgE-binding activity of TM was significantly reduced after treatment with ultrasound or HTP.Among the three different proces sing methods,HTP was the most effective method for improving digestibility of TM and reducing immunoreactivity.This finding provides new insights into treatments for crab allergies.
文摘The neurotrophin-Trk receptor pathway is an intrinsic pathway to relieve damage to the central nervous system. The present study observed the effects of Tongluo Jiunao （TLJN）, which comprises Panax Notoginseng and Gardenia Jasminoides, on expression of brain-derived neurotrophic factor （BDNF） and tropomyosin-related kinase B （TrkB） in a rat model of focal cerebral ischemic injury. Xue Sai Tong （XST）, comprising Panax Notoginseng, served as the positive control. Mechanisms of neuroprotection were analyzed following TLJN injection. Following establishment of the middle cerebral artery occlusion models, TLJN and XST were intraperitoneally injected, and 2, 3 5-triphenyltetrazolium chloride staining results revealed that TLJN injection reduced infarct volume, suggesting that TLJN exerted a neuroprotective effect. Enzyme-linked immunosorbent assay results showed that TLJN elevated BDNF and growth associated protein-43 expression in ischemic brain tissues, as well as serum BDNF levels. Reverse-transcription polymerase chain reaction and western blot results showed that TLJN injection did not affect TrkB expression in the ischemic brain tissues of rats. These results suggested that TLJN injection reduced damage to ischemic brain tissues and increased BDNF expression. In addition, TLJN injection resulted in better promoting effects on neurotrophic factor expression compared with XST.
文摘Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca2+ and regulate striated muscle contraction in vertebrates. TnT’s function has been extensively described in vertebrates, but its role has been obscure in molluscan muscles. Our previous work indicated that the TnC and TnI subunits work in adductor phasic muscle, but not in catch muscle. Here, we have characterized TnT from the Japanese bivalve pearl oyster Pinctada fucata to start to explain the function of Tn in molluscan muscle contraction. We determined the primary structure of the full-length TnT protein from the P. fucata adductor muscle (Pifuc-TnT), and found that it is composed of 316 amino acid residues with a predicted molecular mass of 37.4 kDa. Multiple sequence alignment showed that Pifuc-TnT has an extension of >60 residues at the C-terminus that are not present in vertebrate TnTs, including known TnTs from other mollusks. Pifuc-TnT gene structure predictions using Splign alignment of the cDNA generated in this study and genome sequences indicated that Pifuc-TnT consists of 13 exons. Start and stop codons are located in exons 2 and 12, respectively. Quantitative real-time PCR revealed that the Pifuc-TnT gene was predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, slightly in gill, and not at all in mantle and foot. These findings suggest that TnT plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction. Isothermal titration calorimetry revealed that unlike vertebrate TnTs, Pifuc-TnT does not interact with P. fucata tropomyosin-1 nor with tropomyosin-2. These findings in P. fucata imply that Tn functions differently in molluscan muscle than it does in vertebrates.
文摘We determined the full-length primary structure of the tropomyosin (TM)-1 and -2 proteins from the adductor muscle of the Japanese pearl oyster Pinctada fucata (Pifuc-TM-1 and Pifuc-TM-2), and found that they are each composed of 284 amino acid residues. We predicted the gene structure of P. fucata TM (Pifuc-TM) using Splign alignment of our cDNA with genomic sequences and elucidated that Pifuc-TM consists of 10 exons. Exons 1 - 3 and 5 - 10 are used to transcribe Pifuc-TM-1 mRNA, and exons 1 - 4 and 6 - 10 are used to transcribe Pifuc-TM-2 mRNA. Both genes share the same start and stop codons located in exon 1 and exon 10, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TM-1 gene was mainly expressed in adductor phasic muscle, and at a relatively weaker level in adductor catch muscle, whereas the Pifuc-TM-2 gene was expressed equally in both phasic and catch muscles. They were weakly expressed in gill and mantle. Immunoblot analysis using anti-Pifuc-TM-1 and anti-Pifuc-TM-2 antibodies revealed that adductor phasic muscle contained Pifuc-TM-1, while adductor catch muscle contained both Pifuc-TM-1 and Pifuc-TM-2. Differential scanning calorimetry (DSC) analysis was carried out for Pifuc-TM-1 and Pifuc-TM-2 expressed in bacteria, as well as TM purified from P. fucata phasic and catch muscle tissues (phasic-TM and catch-TM). The DSC data indicated that phasic-TM was mainly composed of Pifuc-TM-1, whereas catch-TM contained Pifuc-TM-1 and Pifuc-TM-2. These findings suggest that the distribution of Pifuc-TM-1 and Pifuc-TM-2 in adductor muscle is specific to the muscle fiber type, and reflects the properties of each.
基金supported by the National Natural Science Foundation of China,No.81730050(to WH).
文摘In vivo imaging of cerebral ischemia/reperfusion injury remains an important challenge.We injected porous Ag/Au@SiO_(2) bimetallic hollow nanoshells carrying anti-tropomyosin 4 as a molecular probe into mice with cerebral ischemia/reperfusion injury and observed microvascular changes in the brain using photoacoustic imaging with ultrasonography.At each measured time point,the total photoacoustic signal was significantly higher on the affected side than on the healthy side.Twelve hours after reperfusion,cerebral perfusion on the affected side increased,cerebrovascular injury worsened,and anti-tropomyosin 4 expression increased.Twenty-four hours after reperfusion and later,perfusion on the affected side declined slowly and stabilized after 1 week;brain injury was also alleviated.Histopathological and immunohistochemical examinations confirmed the brain injury tissue changes.The nanoshell molecular probe carrying anti-tropomyosin 4 has potential for use in early diagnosis of cerebral ischemia/reperfusion injury and evaluating its progression.
文摘The present study utilized beta amyloid （Aβ）-induced cell apoptosis in PC12 cells as a cell model of Alzheimer＇s disease to investigate the interaction between brain-derived neurotrophic factor （BDNF） and the tropomyosin-related kinase B receptor. Results showed that Aβ（25-35） can reduce survival of PC12 cells and increase cleaved caspase-3 expression in PC12 cells. However, BDNF inhibited Aβ（25-35）-induced cytotoxicity and cleaved casapase-3 expression. Interestingly, pretreatment with the tropomyosin-related kinase receptor inhibitor K252a for 20 minutes prior to BDNF blocked the neuroprotective effect of BDNF on PC12 cells.
基金supported by the Canadian Institutes of Health Research.
文摘Background: Tropomyosin 1 alpha chain (Tm1) is an actin-binding protein that regulates the endothelial cell response to oxidative stress following its phosphorylation at Serine 283 (S283). Tm1 is also a major tumor suppressor in breast cancer. In the present study, we investigated the role of phosphorylation of Tm1 in regulating its tumor suppressor properties. Methods: MDA MB231 breast cancer cells stably overexpressing wild type form of Tm1 or Tm1 mutants (S283A and S283E) were generated. Proliferation and cell viability were assayed by means of the enzymatic cleavage of the tetrazolium salt WST-1 to formazan dye by cellular mitochondrial dehydrogenases. Adhesion assays were performed at various periods of time on cells grown on plastic. Cell migration was evaluated by using the wound-healing assay and by measuring transendothelial migration of cancer cells. Malignant transformation in vitro was determined by using the anchorage-independent growth assay on soft agar. Results: We found that cells expressing the phosphomimetic form of Tm1 S283E/Tm1 are characterized by an increased adhesion to the substratum. Moreover, the migration of MDA-MB231/S283E/Tm1 cells in a wound closure assay is reduced compared to parental cells or those expressing the non-phosphorylatable form of Tm1 (S283A). Similarly, the transendothelial migration of MDA-MB231/S283E/Tm1 cells is also reduced as compared to the other cell lines. Moreover, we found that the cells expressing the S283A mutants form more colonies in soft agar that those expressing the S283E mutants. Conclusion: Phosphorylation of Tm1 at Ser283 contributes to its anti-tumor properties, and this effect results mainly from an increase in cell adhesion associated with a decrease in their migratory and invasive potentials.
文摘OBJECTIVE: To perform cloning of the gene encoding Chinese Schistosoma japonicum tropomyosin (SjcTM) and its expression in Escherichia coli. METHODS: SjcTM cDNA fragment, except for 14 amino acids at the amino terminus, was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from adult worms of S. japonicum. The RT-PCR product was cloned into T vector and sequenced. The SjcTM cDNA, derived from the constructed TA clone pGEM-SjcTM, was then subcloned into the expressing vector pBV220. After characterization by agarose gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for expression under the temperature-dependent condition. RESULTS: The RT-PCR product, cloned into a T vector, was sequenced and shown to be 96.5% identical at the nuclei acid level and 98.1% identical in deduced amino acid sequence to that of S. mansoni tropomyosin. The target DNA fragment was then subcloned into a prokaryotic vector pBV220. Induced expression in E. coli DH5alpha cells resulted in a constant level of recombinant protein production. The results of SDS-PAGE and Western blot revealed that the molecular weight of non-fusion recombinant protein (rSjcTM) was approximately 32 kDa and could be recognized specifically by a polyclonal antiserum specific for native S. japonicum tropomyosin (SjcTM). CONCLUSION: The engineering of the cDNA encoding S. japonicum tropomyosin and its bacterial expression was successfully made.
文摘The one-bead-one-compound （OBOC） combinatorial peptide library is a powerful tool to identify ligand and receptor interactions. Here, we applied the OBOC library technology to identify mimotopes specific to the immunoglobulin E （IgE） epitopes of the major shellfish allergen tropomyosin. OBOC peptide libraries with 8-12 amino acid residues were screened with serum samples from patients with shellfish allergy for IgE mimotopes of tropomyosin. Twenty-five mimotopes were identified from the screening and their binding reactivity to tropomyosin-specific IgE was confirmed by peptide ELISA. These mimotopes could be divided into seven clusters based on sequence homology, and epitope mapping by EpiSearch of the clustered mimotopes was performed to characterize and confirm the validity of mimotopes. Five out of six of the predicted epitopes were found to overlap with previously identified epitopes of tropomyosin. To further confirm the mimicry potential of mimotopes, BALB/c mice were immunized with mimotopes conjugated to keyhole limpet hemocyanin and assayed for their capacity to induce tropomyosin-specific antibodies. BALB/c mice that received mimotope immunization were found to have an elevated level of tropomyosin-specific immunoglobulin G, but not mice that received an irrelevant mimotope. This study pioneers the successful application of the OBOC libraries using whole sera to screen and identify multiple shrimp allergen mimotopes and validates their mimicry potential using in vitro, in vivo, and in silico methods.
基金supported by grants from National Natural Science Foundation of China(Grants 81922064,81874290,81803755,and 91853109)Sichuan Science and Technology Program(Grants 2019YFSY0038 and 2019JDRC0091,China)
文摘Tropomyosin receptor kinase A,B and C(TRKA,TRKB and TRKC),which are well-known members of the cell surface receptor tyrosine kinase(RTK)family,are encoded by the neurotrophic receptor tyrosine kinase 1,2 and 3(NTRK1,NTRK2 and NTRK3)genes,respectively.TRKs can regulate cell proliferation,differentiation and even apoptosis through the RAS/MAPKs,PI3 K/AKT and PLCγtyrosine kinase fusions;Small-molecule inhibitor;NTRK fusion cancer pathways.Gene fusions involving NTRK act as oncogenic drivers of a broad diversity of adult and pediatric tumors,and TRKs have become promising antitumor targets.Therefore,achieving a comprehensive understanding of TRKs and relevant TRK inhibitors should be urgently pursued for the further development of novel TRK inhibitors for potential clinical applications.This review focuses on summarizing the biological functions of TRKs and NTRK fusion proteins,the development of small-molecule TRK inhibitors with different chemotypes and their activity and selectivity,and the potential therapeutic applications of these inhibitors for future cancer drug discovery efforts.
