A hydrothermal reaction of copper acetate with ammonium molybdate, 4,4-bpy (4,4-bipyridine) and 1,10-phen (1,10-phenanthroline) led to the formation of brown crystals of [Cu2(1,10-phen)2(4,4-bpy)]2 [Mo8O26]4H2O 1. Si...A hydrothermal reaction of copper acetate with ammonium molybdate, 4,4-bpy (4,4-bipyridine) and 1,10-phen (1,10-phenanthroline) led to the formation of brown crystals of [Cu2(1,10-phen)2(4,4-bpy)]2 [Mo8O26]4H2O 1. Single-crystal X-ray analysis has revealed that 1 (C68H56N12O30Cu4Mo8) crystallizes in the triclinic system, space group P with a = 11.270(3), b = 13.113(6), c = 13.906(3) ? = 103.33(4), b = 98.54(2), g = 101.29(2)? V = 1920.1(1) ?3, Mr = 2542.93, Z = 1, Dc = 2.199 g/cm3, m = 2.435 mm-1, F(000) = 1240, the final R = 0.0445, wR = 0.1082 and S = 1.021 for 5052 observed reflections with I >2s(I). It consists of copper (Ⅰ) tetramer units and -[Mo8O26]4- anions, which are further attached into a three-dimensional framework through hydrogen bonding and - stacking interactions.展开更多
A new isopropenyl benzofuran-type tetramer was isolated from the roots of ligularia stenocephala and its structure was established by spectroscopic methods.
Two novel diphoshinoamine ligands have been synthesized. Combination of Cr（Ⅲ） and methylaluminoxane（MAO） generated active catalytic system which can catalyze tetramerization of ethylene with high catalytic activi...Two novel diphoshinoamine ligands have been synthesized. Combination of Cr（Ⅲ） and methylaluminoxane（MAO） generated active catalytic system which can catalyze tetramerization of ethylene with high catalytic activity up to 2.5×10^6 g/mol Cr.h and high selectivity to produce 1-octene （Cs in products is being 89.80 wt%）.展开更多
N-acetylatedα-synuclein(αSyn)has long been established as an intrinsically disordered protein associated with a dysfunctional role in Parkinson’s disease.In recent years,a physiologically relevant,higher order conf...N-acetylatedα-synuclein(αSyn)has long been established as an intrinsically disordered protein associated with a dysfunctional role in Parkinson’s disease.In recent years,a physiologically relevant,higher order conformation has been identified as a helical tetramer that is tailored by buried hydrophobic interactions and is distinctively aggregation resistant.The canonical mechanism by which the tetramer assembles remains elusive.As novel biochemical approaches,computational methods,pioneering purification platforms,and powerful imaging techniques continue to develop,puzzling information that once sparked debate as to the veracity of the tetramer has now shed light upon this new counterpart inαSyn neurobiology.Nuclear magnetic resonance and computational studies on multimericαSyn structure have revealed that the protein folding propensity is controlled by small energy barriers that enable large scale reconfiguration.Alternatively,familial mutations ablate tetramerization and reconfigure polymorphic fibrillization.In this review,we will discuss the dynamic landscape ofαSyn quaternary structure with a focus on the tetrameric conformation.展开更多
Helicobacter pylori 3-Deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the conversion of D-arabinose-5-phosphate (A5P) and phosphoenolpyruvate (PEP)
Chromium acetylacetonate and bis(diphenylphosphino)isopropylamine were coordinated in situ and supported on methylaluminoxane-modified silica. The catalyst structure and effects of reaction temperature, reaction press...Chromium acetylacetonate and bis(diphenylphosphino)isopropylamine were coordinated in situ and supported on methylaluminoxane-modified silica. The catalyst structure and effects of reaction temperature, reaction pressure and Al/Cr molar ratio on ethylene tetramerization were investigated in detail. Chromium was uniformly and firmly immobilized on the support and could not be leached off by methylaluminoxane. The supported catalyst, upon being activated with methylaluminoxane, exhibited catalytic activity of 1.70×107 g/(mol Cr·h) for ethylene tetramerization to form 1-octene at a reaction temperatures of 80 ℃, a pressure of 2.0 MPa and an Al/Cr molar ratio of 300. The supported catalyst presented a good tolerance to high temperature.展开更多
We investigate the quantum phase transition(QPT) and magnetocaloric effect(MCE) of a tetrameric chain with three-spin interaction using Green's function theory. The magnetization and gap analysis reveals a variety...We investigate the quantum phase transition(QPT) and magnetocaloric effect(MCE) of a tetrameric chain with three-spin interaction using Green's function theory. The magnetization and gap analysis reveals a variety of quantum phases tuned by magnetic field and three-spin interaction, which can open up an energy gap, giving rise to the occurrence of zero magnetization plateau. However, strong three-spin couplings causing strong frustration will destroy the intermediate 1/2 plateau with emergence of a new gapless phase between two cusps. It favors achieving an enhanced MCE at the critical fields, where the minima of isoentropes as well as the valley-peak structure of Gru¨neisen ratio, signaling the accumulation of entropy, lead to cooling via adiabatic(de)magnetization processes. It is also found that the temperature dependence of specific heat combined with Gru¨neisen ratio can testify various quantum phases explicitly.展开更多
A new resveratrol tetramer, sinicin A was isolated from the roots of Ampelopsis sinica, with four known tetramers: vitisin A, cis-vitisin B, ampelopsin H and hopeaphenol. The structure and stereochemistry of sinicin A...A new resveratrol tetramer, sinicin A was isolated from the roots of Ampelopsis sinica, with four known tetramers: vitisin A, cis-vitisin B, ampelopsin H and hopeaphenol. The structure and stereochemistry of sinicin A have been established on the basis of 1D and 2D NMR spectroscopic techniques.展开更多
A new mixed-ligand Zn（II） complex, [Zn（4,4′-bipy）2（H2O）4]-2ANS·6H2O （1, 4,4′- bipy = 4,4′-bipyridine, HANS = 2-aminonaphthalene-l-sulfonic acid）, has been isolated and structurally characterized by sin...A new mixed-ligand Zn（II） complex, [Zn（4,4′-bipy）2（H2O）4]-2ANS·6H2O （1, 4,4′- bipy = 4,4′-bipyridine, HANS = 2-aminonaphthalene-l-sulfonic acid）, has been isolated and structurally characterized by single-crystal X-ray diffwaction, FT-IR spectrum, TG and elemental analysis. It crystallizes in the monoclinic system, space group P2 1/c with a = 12.4852（8）, b = 18.3163（12）, c = 10.9707（7） A, β = 114.2600（10）°, V= 2287.3（3） A3, Dc = 1.455 g/cm3, Mr = 1002.37, Z = 2, F（000） = 1048,μ = 0.704 mm＂1, the final R = 0.0408 and wR = 0.962 for 4029 observed reflections with I 〉 2σ（I）. Interestingly, an unusual one-dimensional （1D） water tape with cyclic tetrameric water clusters can be observed in 1, which are further trapped via Zn-O coordination bonds exhibiting a 2D Zn（Ⅱ）-water layer. These 2D Zn（Ⅱ）-water layers are stacked together into a 3D interdigitated supramolecular architecture via weak π…π interactions, in which free ANS anions are tightly filled by hydrogen-bonding interactions. Thus, π…π and classical hydrogen-bonding interactions are fotmd as main driving forces to stabilize the 2D Zn（Ⅱ）-water layers.展开更多
Analysis of the frequency of antigen-specific cytotoxic T lymphocytes (CTLs) ex vivo is largely dependent on the use of MHC/peptide tetramers. However, the latter reagents have not been widely available, most likely b...Analysis of the frequency of antigen-specific cytotoxic T lymphocytes (CTLs) ex vivo is largely dependent on the use of MHC/peptide tetramers. However, the latter reagents have not been widely available, most likely because of their costly and time-consuming production. In this report we utilized an economic strategy to construct HLA/peptide tetramers with recombinant peptide-linked β2 microglobulin (β2m). The HLA-A2-restricted, melanoma antigen MARTI-derived pep- tide MART127-35( AAGIGILTV) was fused to the N terminus of human β2m through a 15-amino acid (aa)-long linker before being refolded with the recombinant biotinylated HLA-A2 heavy chain ectodomain. The resulted 2-component (2C) monomer was then tetramerized with phycoerythin-labeled streptavidin. The experimental result showed that the 2C HLA-A2/ MART127-35 monomer was shown to bind to the HLA class complex-specific monoclonal antibody W6/32 and the HLA-A2/ MART127-35 complex-specific single chain antibody fragment (scFv) 8.3, suggesting the correctness of its specificity. Fur- thermore, the 2C HLA-A2/MART127-35 tetramer detected a specific CD8+ T cell population in HLA-A2-restricted melanoma infiltrating lymphocytes as the conventional 3C HLA-A2/MART127-35 tetramer. The yield of 2C HLA-A2/MART127-35 monomer was 2. 5 times more than that of the conventional 3C monomer. Taken together, these data indicate that the HLA-A2/ MART127-35 tetramer can be generated conveniently through the use of MART127-35 peptide-β2 m fusion proteins, which can fa- cilitate the monitoring of HLA-A2-restricted, MART1-specific CTL responses in patients with melanoma.展开更多
Nicotinamide adenine dinucleotide(NAD^(+))kinase(NADK)phosphorylates NAD^(+)to generate NADP^(+),which plays a crucial role in maintaining NAD^(+)/NADp^(+)homeostasis,cellular redox balance,and metabolism.However,how ...Nicotinamide adenine dinucleotide(NAD^(+))kinase(NADK)phosphorylates NAD^(+)to generate NADP^(+),which plays a crucial role in maintaining NAD^(+)/NADp^(+)homeostasis,cellular redox balance,and metabolism.However,how human NADK activity is regulated,and how dysregulation or mutation of NADK is linked to human diseases,such as cancers,are still not fully understood.Here,we present a cryo-EM structure of human tetrameric NADK and elaborate on the necessity of the NADK tetramer for its activity.The N-terminal region of human NADK,which does not exist in bacterial NADKs,modulates tetramer conformation,thereby regulating its activity.A methylation-deficient mutant,R45H,within the N-terminal region results in increased NADK activity and confers cancer chemotherapy resistance.Conversely,mutations in NADK identified among cancer patients alter the tetramer conformation,resulting in NADK inactivation and increasing the sensitivity of lung cancer cells to chemotherapy.Our findings partially unveil the structural basis for NADK regulation,offering insights into the cancer etiology of patients carrying NADK mutations.展开更多
The current single-cell analysis technologies such as fluorescence-activated cell sorting(FACS)and fluorescence-activated droplet sorting(FADS)could decipher the cellular heterogeneity but were constrained by low sort...The current single-cell analysis technologies such as fluorescence-activated cell sorting(FACS)and fluorescence-activated droplet sorting(FADS)could decipher the cellular heterogeneity but were constrained by low sorting performance and cell viability.Here,an ultra-sensitive single-cell sorting platform has been developed by integrating the FADS technology with Tetramer-HCR-EvaGreen(THE)fluorescence signal amplification.The THE system produced much higher fluorescence signal than that of the single Tetramer or Tetramer-HCR signal amplification.Upon application to target MCF-7 cells,the platform exhibited high efficacy and selectivity while maintaining more than 95%cell viability.The THE-FADS achieved sorting efficiencies of 55.5%and 50.3%with purities of 91%and 85%for MCF-7 cells in PBS solutions and simulated serum samples,respectively.The sorted MCF-7 cells showed similar proliferation together with CK19 and EGFR mRNA expression compared with the control cells.The established THE-FADS showed the promising prospects to cellular heterogeneity understanding and personalized medicine.展开更多
Chinese-descent rhesus macaques have become more prevalent for HIV infection and vaccine investigation than Indian-origin macaques. Most of the currently available data and reagents such as major histocompatibility co...Chinese-descent rhesus macaques have become more prevalent for HIV infection and vaccine investigation than Indian-origin macaques. Most of the currently available data and reagents such as major histocompatibility complex （MHC） class I tetramers, however, were derived from Indian-origin macaques due to the dominant use of these animals in history. Although there are significant differences in the immunogenetic background between the two macaque populations, they share a few of common MHC class I alleles. We reported in this study the procedure for preparation of a soluble Mamu-B＊1703 （a MHC class I molecule of Chinese macaques） monomer and tetramer loaded with a dominant simian immunodeficiency virus （SIV） epitope IW9 （IRYPKTFGW） that was identified to be Mamu-B＊1701-restricted in Indian macaques. The DNA fragment encoding the Mamu-B＊1703 extracellular domain fused with a BirA substrate peptide （BSP） was amplified from a previously cloned cDNA and inserted into a prokaratic expression vector. In the presence of the antigenic peptide IW9 and light chain β2-microglobulin, the expressed heavy chain was refolded into a soluble monomer. After biotinylation, four monomers were polymerized as a tetramer by phycoerythrin-conjugated streptavidin. The tetramer, having been confirmed to have the right conformation, was a potential tool for investigation of antigen-specific CD8^＋ T-lymphocytes in SIV vaccine models of Chinese macaques. And our results also suggested that some antigenic peptides reported in Indian-origin macaques could be directly recruited as ligands for construction of Chinese macaque MHC tetramers.展开更多
HLA-A~*2402 is one of the most frequent HLA-A allele in Asian population.To construct HLA-A~*2402-peptide tetramers,the transmembrane and intracellular segments of HLA-A~*2402 cDNA were replaced with BSP sequence to f...HLA-A~*2402 is one of the most frequent HLA-A allele in Asian population.To construct HLA-A~*2402-peptide tetramers,the transmembrane and intracellular segments of HLA-A~*2402 cDNA were replaced with BSP sequence to form a fusion gene of sHLA-A~*2402-BSP.The sHLA-A~*2402-BSP fusion protein and β2m were high-level expressed as insoluble aggregates in E.coli,and refoided to form an HLA-A~*2402-peptide monomeric complex by dilution method in the presence of an antigenic peptide.The HLA-A~*2402-peptide monomeric complex was biotinated and tetramized to prepare HLA-A~*2402-peptide tetramer.Then using the HLA-A~*2402-peptide tetramers to detect antigen-specific cytotoxic T lymphocyte(CTL)induced by artificial antigen presenting cell (aAPC)in vitro.The results showed that HLA-A~*2402-peptide tetramer was prepared correctly,and functional in detecting antigen-specific CTL in vitro,HLA-A~*2402-peptide monomeric and its multimeric complexes are expected to provide a powerful tool for studying mechanisms of immune-related diseases in Asian populations. Cellular & Molecular Immunology.2005;2(2):145-149.展开更多
Major histocompatibility complex （MHC） class I tetramer technology has become the central technique for analyzing antigen-specific CD8^＋ T cell responses and it has been widely used to explore the differentiation a...Major histocompatibility complex （MHC） class I tetramer technology has become the central technique for analyzing antigen-specific CD8^＋ T cell responses and it has been widely used to explore the differentiation and formation of memory CD8^＋ T cells. Previously, a simplified and efficient procedure for preparing high quality HLA-A＊0201 tetramers has been established in our lab and the tetramers loaded with HCMV peptide pp6549s.50a has been successfully applied to investigate HCMV-specific CD8^＋ T cells in Chinese populations. Using similar procedure we reported here the construction of HLA-A＊0201 tetramer loaded with another dominant epitope derived from immediate early （IE）-1 316.324 （VLEETSVML, VLE） of HCMV （A2-VLE） and characterization of this tetramer. After A2-VLE monomer was prepared and purified, its tetramer was then formed at a yield of 83%. The optimized amount of A2-VLE tetramer for staining 100 μl whole blood was 0.5 μg with incubation at 4℃ for 1 h. Furthermore, the dissociation constant of the tetramer binding to the specific CD8^＋ T cells of one HLA-A2^＋ donor was estimated to be 32.7 nmol/L, which is markedly higher than that of MHC monomer. The construction of A2-VLE tetramer provides an alternative choice for investigating HCMV-specific CD8^＋ T cell responses and will deepen our understanding of the differentiation and formation of HCMV-specific memory CD8^＋ T cells. Cellular ＆ Molecular Immunology.展开更多
Major histocompatibility complex （MHC） tetramer technology is critical for characterization of antigen-specific T cells. In the present study we reported the successful generation of HLA-A＊0203 tetramer loaded with...Major histocompatibility complex （MHC） tetramer technology is critical for characterization of antigen-specific T cells. In the present study we reported the successful generation of HLA-A＊0203 tetramer loaded with Epstein- Barr virus EBNA3596-604 peptide （SVRDRLARL, SVR）. Prokaryotic expression vector for the ectodomain of the heavy chain of HLA-A＊0203 fused with a BirA substrate peptide （HLA-A＊0203-BSP） was constructed and the expression conditions of the fusion protein in Escherichia coli （E. coli） were optimized. The fusion protein was highly expressed in inclusion bodies within E. coil It was then refolded in the presence of 132-microglobulin and SVR peptide to form a soluble HLA-A＊0203-SVR monomer. After biotinylation with BirA, the monomer was purified by anion-exchange chromatography and its purity was up to 95%. The tetramer was then formulated by mixing the biotinylated monomer with streptavidin-PE at a ratio of 4：1. Flow cytometry showed that this tetramer could specifically react with antigen-specific CD8^＋ T cells, indicating that it was biologically functional. These results provide a foundation for further characterization of antigen-specific CD8^＋ T cells from HLA-A＊0203 subjects.展开更多
基金This work was supported by the National Natural Science Foundation of China (20073048) NSF of Fujian and the Chinese Academy of Sciences
文摘A hydrothermal reaction of copper acetate with ammonium molybdate, 4,4-bpy (4,4-bipyridine) and 1,10-phen (1,10-phenanthroline) led to the formation of brown crystals of [Cu2(1,10-phen)2(4,4-bpy)]2 [Mo8O26]4H2O 1. Single-crystal X-ray analysis has revealed that 1 (C68H56N12O30Cu4Mo8) crystallizes in the triclinic system, space group P with a = 11.270(3), b = 13.113(6), c = 13.906(3) ? = 103.33(4), b = 98.54(2), g = 101.29(2)? V = 1920.1(1) ?3, Mr = 2542.93, Z = 1, Dc = 2.199 g/cm3, m = 2.435 mm-1, F(000) = 1240, the final R = 0.0445, wR = 0.1082 and S = 1.021 for 5052 observed reflections with I >2s(I). It consists of copper (Ⅰ) tetramer units and -[Mo8O26]4- anions, which are further attached into a three-dimensional framework through hydrogen bonding and - stacking interactions.
基金supoaed by the National Natural Science Foundation of China(No.29972017)
文摘A new isopropenyl benzofuran-type tetramer was isolated from the roots of ligularia stenocephala and its structure was established by spectroscopic methods.
文摘Two novel diphoshinoamine ligands have been synthesized. Combination of Cr（Ⅲ） and methylaluminoxane（MAO） generated active catalytic system which can catalyze tetramerization of ethylene with high catalytic activity up to 2.5×10^6 g/mol Cr.h and high selectivity to produce 1-octene （Cs in products is being 89.80 wt%）.
基金supported in part by Award No.18-7(to HRL)from the Commonwealth of Virginia’s Alzheimer’s and Related Diseases Research Award Fund,administered by the Virginia Center on Aging
文摘N-acetylatedα-synuclein(αSyn)has long been established as an intrinsically disordered protein associated with a dysfunctional role in Parkinson’s disease.In recent years,a physiologically relevant,higher order conformation has been identified as a helical tetramer that is tailored by buried hydrophobic interactions and is distinctively aggregation resistant.The canonical mechanism by which the tetramer assembles remains elusive.As novel biochemical approaches,computational methods,pioneering purification platforms,and powerful imaging techniques continue to develop,puzzling information that once sparked debate as to the veracity of the tetramer has now shed light upon this new counterpart inαSyn neurobiology.Nuclear magnetic resonance and computational studies on multimericαSyn structure have revealed that the protein folding propensity is controlled by small energy barriers that enable large scale reconfiguration.Alternatively,familial mutations ablate tetramerization and reconfigure polymorphic fibrillization.In this review,we will discuss the dynamic landscape ofαSyn quaternary structure with a focus on the tetrameric conformation.
文摘Helicobacter pylori 3-Deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the conversion of D-arabinose-5-phosphate (A5P) and phosphoenolpyruvate (PEP)
基金supported by the National Natural Science Foundation of China (U1162114)the PetroChina Innovation Foundation (2012D-5006-0501)+2 种基金the Tianjin Municipal Education Commission of China (20110505)the Natural Science Foundation of Tianjin (12JCQNJC06000)the Program for New Century Excellent Talents in University (NCET-07-0142)
文摘Chromium acetylacetonate and bis(diphenylphosphino)isopropylamine were coordinated in situ and supported on methylaluminoxane-modified silica. The catalyst structure and effects of reaction temperature, reaction pressure and Al/Cr molar ratio on ethylene tetramerization were investigated in detail. Chromium was uniformly and firmly immobilized on the support and could not be leached off by methylaluminoxane. The supported catalyst, upon being activated with methylaluminoxane, exhibited catalytic activity of 1.70×107 g/(mol Cr·h) for ethylene tetramerization to form 1-octene at a reaction temperatures of 80 ℃, a pressure of 2.0 MPa and an Al/Cr molar ratio of 300. The supported catalyst presented a good tolerance to high temperature.
基金Supported by the National Natural Science Foundation of China under Grant Nos.11204157,11174179,11247020the China Three Gorges University Project KJ2011B068
文摘We investigate the quantum phase transition(QPT) and magnetocaloric effect(MCE) of a tetrameric chain with three-spin interaction using Green's function theory. The magnetization and gap analysis reveals a variety of quantum phases tuned by magnetic field and three-spin interaction, which can open up an energy gap, giving rise to the occurrence of zero magnetization plateau. However, strong three-spin couplings causing strong frustration will destroy the intermediate 1/2 plateau with emergence of a new gapless phase between two cusps. It favors achieving an enhanced MCE at the critical fields, where the minima of isoentropes as well as the valley-peak structure of Gru¨neisen ratio, signaling the accumulation of entropy, lead to cooling via adiabatic(de)magnetization processes. It is also found that the temperature dependence of specific heat combined with Gru¨neisen ratio can testify various quantum phases explicitly.
基金This research program was supported by the National Natural Science Foundation of China (No. 30070+2 种基金889). The authors thank the Department of Instrumental Analysis Institute of Materia Medica Chinese Academy of Medical Sciences & Peking Union Col
文摘A new resveratrol tetramer, sinicin A was isolated from the roots of Ampelopsis sinica, with four known tetramers: vitisin A, cis-vitisin B, ampelopsin H and hopeaphenol. The structure and stereochemistry of sinicin A have been established on the basis of 1D and 2D NMR spectroscopic techniques.
基金Supported by the Advanced Project for Young Teachers in Tianjin Normal University
文摘A new mixed-ligand Zn（II） complex, [Zn（4,4′-bipy）2（H2O）4]-2ANS·6H2O （1, 4,4′- bipy = 4,4′-bipyridine, HANS = 2-aminonaphthalene-l-sulfonic acid）, has been isolated and structurally characterized by single-crystal X-ray diffwaction, FT-IR spectrum, TG and elemental analysis. It crystallizes in the monoclinic system, space group P2 1/c with a = 12.4852（8）, b = 18.3163（12）, c = 10.9707（7） A, β = 114.2600（10）°, V= 2287.3（3） A3, Dc = 1.455 g/cm3, Mr = 1002.37, Z = 2, F（000） = 1048,μ = 0.704 mm＂1, the final R = 0.0408 and wR = 0.962 for 4029 observed reflections with I 〉 2σ（I）. Interestingly, an unusual one-dimensional （1D） water tape with cyclic tetrameric water clusters can be observed in 1, which are further trapped via Zn-O coordination bonds exhibiting a 2D Zn（Ⅱ）-water layer. These 2D Zn（Ⅱ）-water layers are stacked together into a 3D interdigitated supramolecular architecture via weak π…π interactions, in which free ANS anions are tightly filled by hydrogen-bonding interactions. Thus, π…π and classical hydrogen-bonding interactions are fotmd as main driving forces to stabilize the 2D Zn（Ⅱ）-water layers.
文摘Analysis of the frequency of antigen-specific cytotoxic T lymphocytes (CTLs) ex vivo is largely dependent on the use of MHC/peptide tetramers. However, the latter reagents have not been widely available, most likely because of their costly and time-consuming production. In this report we utilized an economic strategy to construct HLA/peptide tetramers with recombinant peptide-linked β2 microglobulin (β2m). The HLA-A2-restricted, melanoma antigen MARTI-derived pep- tide MART127-35( AAGIGILTV) was fused to the N terminus of human β2m through a 15-amino acid (aa)-long linker before being refolded with the recombinant biotinylated HLA-A2 heavy chain ectodomain. The resulted 2-component (2C) monomer was then tetramerized with phycoerythin-labeled streptavidin. The experimental result showed that the 2C HLA-A2/ MART127-35 monomer was shown to bind to the HLA class complex-specific monoclonal antibody W6/32 and the HLA-A2/ MART127-35 complex-specific single chain antibody fragment (scFv) 8.3, suggesting the correctness of its specificity. Fur- thermore, the 2C HLA-A2/MART127-35 tetramer detected a specific CD8+ T cell population in HLA-A2-restricted melanoma infiltrating lymphocytes as the conventional 3C HLA-A2/MART127-35 tetramer. The yield of 2C HLA-A2/MART127-35 monomer was 2. 5 times more than that of the conventional 3C monomer. Taken together, these data indicate that the HLA-A2/ MART127-35 tetramer can be generated conveniently through the use of MART127-35 peptide-β2 m fusion proteins, which can fa- cilitate the monitoring of HLA-A2-restricted, MART1-specific CTL responses in patients with melanoma.
文摘Nicotinamide adenine dinucleotide(NAD^(+))kinase(NADK)phosphorylates NAD^(+)to generate NADP^(+),which plays a crucial role in maintaining NAD^(+)/NADp^(+)homeostasis,cellular redox balance,and metabolism.However,how human NADK activity is regulated,and how dysregulation or mutation of NADK is linked to human diseases,such as cancers,are still not fully understood.Here,we present a cryo-EM structure of human tetrameric NADK and elaborate on the necessity of the NADK tetramer for its activity.The N-terminal region of human NADK,which does not exist in bacterial NADKs,modulates tetramer conformation,thereby regulating its activity.A methylation-deficient mutant,R45H,within the N-terminal region results in increased NADK activity and confers cancer chemotherapy resistance.Conversely,mutations in NADK identified among cancer patients alter the tetramer conformation,resulting in NADK inactivation and increasing the sensitivity of lung cancer cells to chemotherapy.Our findings partially unveil the structural basis for NADK regulation,offering insights into the cancer etiology of patients carrying NADK mutations.
基金funded by National Key Research and Development Program of China(grant number 2022YFC3502002)CAS-NSTDA Joint Research Project(2024)(grant number 101GJHZ2024017MI).
文摘The current single-cell analysis technologies such as fluorescence-activated cell sorting(FACS)and fluorescence-activated droplet sorting(FADS)could decipher the cellular heterogeneity but were constrained by low sorting performance and cell viability.Here,an ultra-sensitive single-cell sorting platform has been developed by integrating the FADS technology with Tetramer-HCR-EvaGreen(THE)fluorescence signal amplification.The THE system produced much higher fluorescence signal than that of the single Tetramer or Tetramer-HCR signal amplification.Upon application to target MCF-7 cells,the platform exhibited high efficacy and selectivity while maintaining more than 95%cell viability.The THE-FADS achieved sorting efficiencies of 55.5%and 50.3%with purities of 91%and 85%for MCF-7 cells in PBS solutions and simulated serum samples,respectively.The sorted MCF-7 cells showed similar proliferation together with CK19 and EGFR mRNA expression compared with the control cells.The established THE-FADS showed the promising prospects to cellular heterogeneity understanding and personalized medicine.
基金supported by Natural Science Fund of Guangdong Province (No.8451063201000340)the Talented Man Initiation Fund of Jinan University (No.51208004, No.51208017) to Dr. DY Ouyang +1 种基金grants from the National Natural Science Foundation of China (No.30572199, No.30230350 and No.30371651) to Prof. XH Hethe Biochemistry and Molecular Biology Key Discipline of Guangdong Province.
文摘Chinese-descent rhesus macaques have become more prevalent for HIV infection and vaccine investigation than Indian-origin macaques. Most of the currently available data and reagents such as major histocompatibility complex （MHC） class I tetramers, however, were derived from Indian-origin macaques due to the dominant use of these animals in history. Although there are significant differences in the immunogenetic background between the two macaque populations, they share a few of common MHC class I alleles. We reported in this study the procedure for preparation of a soluble Mamu-B＊1703 （a MHC class I molecule of Chinese macaques） monomer and tetramer loaded with a dominant simian immunodeficiency virus （SIV） epitope IW9 （IRYPKTFGW） that was identified to be Mamu-B＊1701-restricted in Indian macaques. The DNA fragment encoding the Mamu-B＊1703 extracellular domain fused with a BirA substrate peptide （BSP） was amplified from a previously cloned cDNA and inserted into a prokaratic expression vector. In the presence of the antigenic peptide IW9 and light chain β2-microglobulin, the expressed heavy chain was refolded into a soluble monomer. After biotinylation, four monomers were polymerized as a tetramer by phycoerythrin-conjugated streptavidin. The tetramer, having been confirmed to have the right conformation, was a potential tool for investigation of antigen-specific CD8^＋ T-lymphocytes in SIV vaccine models of Chinese macaques. And our results also suggested that some antigenic peptides reported in Indian-origin macaques could be directly recruited as ligands for construction of Chinese macaque MHC tetramers.
基金This study was supported by National Key Basic Research Program of China(No.CB 510008)National Natural Science Foundation of China(No.30271201).
文摘HLA-A~*2402 is one of the most frequent HLA-A allele in Asian population.To construct HLA-A~*2402-peptide tetramers,the transmembrane and intracellular segments of HLA-A~*2402 cDNA were replaced with BSP sequence to form a fusion gene of sHLA-A~*2402-BSP.The sHLA-A~*2402-BSP fusion protein and β2m were high-level expressed as insoluble aggregates in E.coli,and refoided to form an HLA-A~*2402-peptide monomeric complex by dilution method in the presence of an antigenic peptide.The HLA-A~*2402-peptide monomeric complex was biotinated and tetramized to prepare HLA-A~*2402-peptide tetramer.Then using the HLA-A~*2402-peptide tetramers to detect antigen-specific cytotoxic T lymphocyte(CTL)induced by artificial antigen presenting cell (aAPC)in vitro.The results showed that HLA-A~*2402-peptide tetramer was prepared correctly,and functional in detecting antigen-specific CTL in vitro,HLA-A~*2402-peptide monomeric and its multimeric complexes are expected to provide a powerful tool for studying mechanisms of immune-related diseases in Asian populations. Cellular & Molecular Immunology.2005;2(2):145-149.
基金This work was supported by Key Project of the National Natural Science Foundation of China （No.30230350）.
文摘Major histocompatibility complex （MHC） class I tetramer technology has become the central technique for analyzing antigen-specific CD8^＋ T cell responses and it has been widely used to explore the differentiation and formation of memory CD8^＋ T cells. Previously, a simplified and efficient procedure for preparing high quality HLA-A＊0201 tetramers has been established in our lab and the tetramers loaded with HCMV peptide pp6549s.50a has been successfully applied to investigate HCMV-specific CD8^＋ T cells in Chinese populations. Using similar procedure we reported here the construction of HLA-A＊0201 tetramer loaded with another dominant epitope derived from immediate early （IE）-1 316.324 （VLEETSVML, VLE） of HCMV （A2-VLE） and characterization of this tetramer. After A2-VLE monomer was prepared and purified, its tetramer was then formed at a yield of 83%. The optimized amount of A2-VLE tetramer for staining 100 μl whole blood was 0.5 μg with incubation at 4℃ for 1 h. Furthermore, the dissociation constant of the tetramer binding to the specific CD8^＋ T cells of one HLA-A2^＋ donor was estimated to be 32.7 nmol/L, which is markedly higher than that of MHC monomer. The construction of A2-VLE tetramer provides an alternative choice for investigating HCMV-specific CD8^＋ T cell responses and will deepen our understanding of the differentiation and formation of HCMV-specific memory CD8^＋ T cells. Cellular ＆ Molecular Immunology.
文摘Major histocompatibility complex （MHC） tetramer technology is critical for characterization of antigen-specific T cells. In the present study we reported the successful generation of HLA-A＊0203 tetramer loaded with Epstein- Barr virus EBNA3596-604 peptide （SVRDRLARL, SVR）. Prokaryotic expression vector for the ectodomain of the heavy chain of HLA-A＊0203 fused with a BirA substrate peptide （HLA-A＊0203-BSP） was constructed and the expression conditions of the fusion protein in Escherichia coli （E. coli） were optimized. The fusion protein was highly expressed in inclusion bodies within E. coil It was then refolded in the presence of 132-microglobulin and SVR peptide to form a soluble HLA-A＊0203-SVR monomer. After biotinylation with BirA, the monomer was purified by anion-exchange chromatography and its purity was up to 95%. The tetramer was then formulated by mixing the biotinylated monomer with streptavidin-PE at a ratio of 4：1. Flow cytometry showed that this tetramer could specifically react with antigen-specific CD8^＋ T cells, indicating that it was biologically functional. These results provide a foundation for further characterization of antigen-specific CD8^＋ T cells from HLA-A＊0203 subjects.
