BACKGROUND Thrombocytopenia 2,an autosomal dominant inherited disease characterized by moderate thrombocytopenia,predisposition to myeloid malignancies and normal platelet size and function,can be caused by 5’-untran...BACKGROUND Thrombocytopenia 2,an autosomal dominant inherited disease characterized by moderate thrombocytopenia,predisposition to myeloid malignancies and normal platelet size and function,can be caused by 5’-untranslated region(UTR)point mutations in ankyrin repeat domain containing 26(ANKRD26).Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1)have been identified as negative regulators of ANKRD26.However,the positive regulators of ANKRD26 are still unknown.AIM To prove the positive regulatory effect of GATA binding protein 2(GATA2)on ANKRD26 transcription.METHODS Human induced pluripotent stem cells derived from bone marrow(hiPSC-BM)INTRODUCTION Ankyrin repeat domain containing protein 26(ANKRD26)acts as a regulator of adipogenesis and is involved in the regulation of feeding behavior[1-3].The ANKRD26 gene is located on chromosome 10 and shares regions of homology with the primate-specific gene family POTE.According to the Human Protein Atlas database,the ANKRD26 protein is localized to the Golgi apparatus and vesicles,and its expression can be detected in nearly all human tissues[4].Moreover,UniProt annotation revealed that ANKRD26 is localized in the centrosome and contains coiled-coil domains formed by spectrin helices and ankyrin repeats[5,6].The most common disease related to ANKRD26 is thrombocytopenia 2(THC2),which is a rare autosomal dominant inherited disease characterized by lifelong mild-to-moderate thrombocytopenia and mild bleeding[7-9].Caused by the variants in the 5’-untranslated region(UTR)of ANKRD26,THC2 is defined by a decrease in the number of platelets in circulating blood and results in increased bleeding and decreased clotting ability[8,10].Due to the point mutations that occur in the 5’-UTR of ANKRD26,its negative transcription factors(TFs),Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1),lose their repression effect[11].The persistent expression of ANKRD26 increases the activity of the mitogen activated protein kinase and extracellular signal regulated kinase 1/2 signaling pathways,which are potentially involved in the regulation of thrombopoietin-dependent signaling and further impair proplatelet formation by megakaryocytes(MKs)[11].However,the positive regulators of ANKRD26,which might be associated with THC2 pathology,are still unknown.展开更多
Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle ce...Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was~25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from~30 kD to~25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.展开更多
The novel phage lysin PlySs2, is reported to be highly active against various bacteria, including staphylococci, streptococci and Listeria. However, the molecular mechanisms underlying its broad lytic spectrum remain ...The novel phage lysin PlySs2, is reported to be highly active against various bacteria, including staphylococci, streptococci and Listeria. However, the molecular mechanisms underlying its broad lytic spectrum remain to be established. In the present study, the lytic activity of the catalytic domain(CD, PlySc) and binding specificity of the cell wall binding domain(CBD, PlySb) of PlySs2 were examined. Our results showed that PlySc alone maintains very limited lytic activity. Enhanced green fluorescent protein(EGFP)-fused PlySb displayed high binding affinity to the streptococcal strains tested, including S.suis, S.dysgalactiae, and S.agalactiae, but not staphylococci, supporting its utility as a good CBD donor for streptococcal-targeted lysin engineering. EGFP-fused intact PlySs2 similarly displayed high affinity for streptococci, but not staphylococci. Notably, four truncated PlySb fragments showed no binding capacity. These findings collectively indicate that integrity of the PlySc and PlySb domains is an essential determinant of the broad lytic activity of PlySs2.展开更多
Insulin-like growth factor 1 (IGF-I) is a potential nutrient for nerve repair. However, it is impractical as a therapy because of its limited half- life, rapid clearance, and limited target specificity. To achieve t...Insulin-like growth factor 1 (IGF-I) is a potential nutrient for nerve repair. However, it is impractical as a therapy because of its limited half- life, rapid clearance, and limited target specificity. To achieve targeted and long-lasting treatment, we investigated the addition of a binding structure by fusing a collagen-binding domain to IGF- 1. After confirming its affinity for collagen, the biological activity of this construct was examined by measuring cell proliferation after transfection into PC12 and Schwann cells using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-di- phenyl-2-H-tetrazolium bromide assay. Immunofluorescence staining was conducted to detect neurofilament and microtubule-associated protein 2 expression, while real time-polymerase chain reaction was utilized to determine IGF-1 receptor and nerve growth/actor mRNA expression. Our results demonstrate a significant increase in collagen-binding activity of the recombinant protein compared with IGF-1. Moreover, the recombinant protein promoted proliferation of PC12 and Schwann cells, and increased the expression of neurofilament and microtubule-associated protein 2. Importantly, the recombinant protein also stimulated sustained expression of IGF-1 receptor and nerve growth factor mRNA for days. These results show that the recombinant protein achieved the goal of targeting and long-lasting treatment, and thus could become a clinically used factor for promoting nerve regeneration with a prolonged therapeutic effect.展开更多
Summary: The role of methyl-CpG binding domain protein 2 (MBD2) in an ApoE-deficient mouse model of age-related macular degeneration (AMD) was investigated. Eight-week-old Mbd2/ApoE double deficient (Mbd2^-/- Ap...Summary: The role of methyl-CpG binding domain protein 2 (MBD2) in an ApoE-deficient mouse model of age-related macular degeneration (AMD) was investigated. Eight-week-old Mbd2/ApoE double deficient (Mbd2^-/- ApoE^-/-) mice (n=12, 24 eyes, experimental group) and MBD2 (wt) ApoE^-/- mice (n=12, 24 eyes, control group) were fed on Western-type diet for 4 months. The mice were sacrificed, and total serum cholesterol levels were analyzed and Bruch's membrane (BM) of the eyes was removed for ultrastructural observation by transmission electron microscopy. Moreover, intercellular adhesion molecule 1 (ICAM-1) immunoreactivities were evaluated by fluorescence microscopy in sections of the eyes in both groups for further understanding the function mechanism of MBD2. There was no significant difference in the total serum cholesterol levels between control group and experimental group (P〉0.05). Transmission electron microscopy revealed that AMD-like lesions, various vacuoles accumulated on BM, notable outer collagenous layer deposits and dilated basal infoldings of retinal pigment epithelium (RPE) were seen in both groups, and the BM in control group was significantly thickened as compared with experimental group (P〈0.05). Fluorescence micrographs exhibited the expression of ICAM-1 in choroid was higher in control group than in experimental group. We are led to conclude that MBD2 gene knockout may lead to accumulation of more deposits on the BM and influence the pathogenesis of AMD via triggering endothelial activation and inflammatory response in choroid, improving microcirculation, and reducing lipid deposition so as to inhibit the development of AMD-like lesions. Our study helps to provide a new therapeutic approach for the clinical treatment of AMD.展开更多
Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, follow...Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.展开更多
AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined...AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined using the TaqMan miRNA assay.Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction,and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay.Mutation analysis was conducted using the QuikChange Site-Directed Mutagenesis kit.The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region(3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.RESULTS:In hilar cholangiocarcinoma,miR-373 decreased and was closely associated with poor cell differentiation,advanced clinical stage,and shorter survival.The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373.MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373.Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island,and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.CONCLUSION:MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.展开更多
Nucleotide-binding oligomerization domain 1(NOD1) is an intracellular innate immune sensor for small molecules derived from bacterial cell components. NOD1 activation by its ligands leads to robust production of pro-i...Nucleotide-binding oligomerization domain 1(NOD1) is an intracellular innate immune sensor for small molecules derived from bacterial cell components. NOD1 activation by its ligands leads to robust production of pro-inflammatory cytokines and chemokines by innate immune cells, thereby mediating mucosal host defense systems against microbes. Chronic gastric infection due to Helicobacter pylori(H. pylori) causes various upper gastrointestinal diseases, including atrophic gastritis, peptic ulcers, and gastric cancer. It is now generally accepted that detection of H. pylori by NOD1 expressed in gastric epithelial cells plays an indispensable role in mucosal host defense systems against this organism. Recent studies have revealed the molecular mechanism by which NOD1 activation caused by H. pylori infection is involved in the development of chronic gastritis and gastric cancer. In this review, we have discussed and summarized how sensing of H. pylori by NOD1 mediates the prevention of chronic gastritis and gastric cancer.展开更多
Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose ...Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomainhelicase-DNA binding protein 5, chromodomain-helicaseDNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation,and should be validated in future studies.展开更多
Objective To investigate the effects of stimulant for nucleotide-binding oligomerization domain 1 (NOD1) on secretion of proinflammatory chemokine/cytokines and insulin-dependent glucose uptake in human differentiated...Objective To investigate the effects of stimulant for nucleotide-binding oligomerization domain 1 (NOD1) on secretion of proinflammatory chemokine/cytokines and insulin-dependent glucose uptake in human differentiated adipocytes. Methods Adipose tissues were obtained from patients undergoing liposuction. Stromal vascular cells were extracted and differentiated into adipocytes. A specific ligand for NOD1, was administered to human adipocytes in culture. Nuclear factor-κB transcriptional activity and proinflammatory chemokine/cytokines production were determined by reporter plasmid assay and enzyme-linked immunosorbent assay, respectively. Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[ 3 H] glucose uptake assay. Furthermore, chemokine/cytokine secretion and glucose uptake in adipocytes transfected with small interfering RNA (siRNA) targeting NOD1展开更多
Pancreatic ductal adenocarcinoma(PDAC) remains a deadly disease with no efficacious treatment options. PDAC incidence is projected to increase, which may be caused at least partially by the obesity epidemic. Significa...Pancreatic ductal adenocarcinoma(PDAC) remains a deadly disease with no efficacious treatment options. PDAC incidence is projected to increase, which may be caused at least partially by the obesity epidemic. Significantly enhanced efforts to prevent or intercept this cancer are clearly warranted. Oncogenic KRAS mutations are recognized initiating events in PDAC development, however, they are not entirely sufficient for the development of fully invasive PDAC.Additional genetic alterations and/or environmental, nutritional, and metabolic signals, as present in obesity, type-2 diabetes mellitus, and inflammation, are required for full PDAC formation. We hypothesize that oncogenic KRAS increases the intensity and duration of the growth-promoting signaling network.Recent exciting studies from different laboratories indicate that the activity of the transcriptional co-activators Yes-associated protein(YAP) and WW-domaincontaining transcriptional co-activator with PDZ-binding motif(TAZ) play a critical role in the promotion and maintenance of PDAC operating as key downstream target of KRAS signaling. While initially thought to be primarily an effector of the tumor-suppressive Hippo pathway, more recent studies revealed that YAP/TAZ subcellular localization and co-transcriptional activity is regulated by multiple upstream signals. Overall, YAP has emerged as a central node of transcriptional convergence in growth-promoting signaling in PDAC cells. Indeed, YAP expression is an independent unfavorable prognostic marker for overall survival of PDAC. In what follows, we will review studies implicating YAP/TAZ in pancreatic cancer development and consider different approaches to target these transcriptional regulators.展开更多
BACKGROUND The regulatory factor X6 (RFX6), a member of regulatory factor X family, is known to play a key role in the development and differentiation of pancreatic beta cells as well as insulin production and secreti...BACKGROUND The regulatory factor X6 (RFX6), a member of regulatory factor X family, is known to play a key role in the development and differentiation of pancreatic beta cells as well as insulin production and secretion. However, the potential role of RFX6 in type 2 diabetes (T2D) is still unclear. AIM Recent studies have indicated that RFX6 binding to DNA could be disrupted in diabetes. Therefore, in this study we investigated whether genetic mutations are present in the DNA binding domain of RFX6 gene that could abrogate its function in T2D. METHODS A cohort of T2D patients was enrolled in this study, and the gene encoding the DNA binding domain of RFX6 was amplified by polymerase chain reaction and then analysed by direct DNA sequencing. RESULTS The DNA sequence analysis revealed the absence of any exonic mutation. However, we have identified a new heterozygous single nucleotide polymorphism (IVS6+31 C>T) in the intronic region of DNA binding domain gene that is present in 9.2% and 8.5% of diabetic and control people, respectively (P = 0.97).CONCLUSION We report the absence of any significant genetic variant that could affect the function of RFX6-DNA binding domain in T2D.展开更多
Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary ce...Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.展开更多
We conducted genome sequence analysis to examine the presence/absence of two types of Z-DNA binding domains in various organisms. We examined 68 organisms from archaea, 914 organisms from bacteria, and 199 organisms f...We conducted genome sequence analysis to examine the presence/absence of two types of Z-DNA binding domains in various organisms. We examined 68 organisms from archaea, 914 organisms from bacteria, and 199 organisms from eukaryotes. RecA protein from Escherichia coli has a Z-DNA binding domain and this protein promotes homologous recombination. All the organisms examined had this domain. This result indicated that this domain is essential for all the organisms. RNA editing enzyme, adenosine deaminase from human has another type of Z-DNA binding domain. This domain was observed in some organisms of archaea, bacteria, and eukaryotes. The presence/absence of Z-DNA binding domain in adenosine deaminase indicated that gain and loss of this domain had occurred in the process of evolution. The implication of presence and absence of this domain is discussed in this study.展开更多
A comparative molecular dynamics (MD) simulation study was performed on the p53 oncoprotein to investigate the effect of the Arg273His (R273H) mutation on the p53→DNA Binding Domain (DBD). The two p53 dimer structure...A comparative molecular dynamics (MD) simulation study was performed on the p53 oncoprotein to investigate the effect of the Arg273His (R273H) mutation on the p53→DNA Binding Domain (DBD). The two p53 dimer structures of the wild-type and mutant Arg273His (R273H) were simulated with the same thermodynamic and environmental parameters. The obtained results demonstrate that the induced Arg273His mutation has a considerable effect on the p53→DNA close contact interaction and changes the picture of hydrogen formation. The Arg273His mutation, in some cases, destroys the existing native hydrogen bond, but, in other cases, forms a strong p53→DNA hydrogen bond, which is not proper for the native protein. The MD simulation results illustrate some molecular mechanism of the conformational changes of the Arg273His key amino acid residue in the p53→DNA binding domain, which might be important for the understanding of the physiological functioning of the p53 protein and the origin of cancer.展开更多
Objective To investigate the potential role of nucleotide-binding oligomerization domain 1(NOD1),a component of the innate immune system,in mediating lipid-induced insulin resistance in adipocytes.Methods Adipocytes f...Objective To investigate the potential role of nucleotide-binding oligomerization domain 1(NOD1),a component of the innate immune system,in mediating lipid-induced insulin resistance in adipocytes.Methods Adipocytes from Toll-like receptor 4 deficiency mice were used for stimulation experiments.The effect of oleate/palmitate mixture on nuclear factor-κB(NF-κB)activation was analyzed by reporter plasmid assay.The release of proinflammatory chemokine/cytokines production was determined by using real-time PCR.Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[3H]glucose uptake assay.Chemokine/cytokine expression and glucose uptake in adipocytes transfected with small interfering RNA(siRNA)targeting NOD1 upon fatty acids treatment were analyzed.Results Oleate/palmitate mixture activated the NF-κB pathway and induced interleukin-6,tumor necrosis factor-α,and monocyte chemoattractant protein-1 mRNA expressions in adipocytes from mice deficient in Toll-like receptor 4,and these effects were blocked by siRNA targeting NOD1.Furthermore,saturated fatty acids decreased the ability of insulin-stimulated glucose uptake.Importantly,siRNA targeting NOD1 partially reversed saturated fatty acid-induced suppression of insulin-induced glucose uptake.Conclusion NOD1 might play an important role in saturated fatty acid-induced insulin resistance in adipocytes,suggesting a mechanism by which reduced NOD1 activity confers beneficial effects on insulin action.展开更多
AIM To uncover the roles of tumor-promoting gene ZEB1 in aerobic glycolysis regulation and shed light on the underlying molecular mechanism.METHODS Endogenous zinc finger E-box binding homeobox-1(ZEB1) was silenced us...AIM To uncover the roles of tumor-promoting gene ZEB1 in aerobic glycolysis regulation and shed light on the underlying molecular mechanism.METHODS Endogenous zinc finger E-box binding homeobox-1(ZEB1) was silenced using a lentivirus-mediated method, and the impact of ZEB1 and methyl-CpG binding domain protein 1(MBD1) on aerobic glycolysis was measured using seahorse cellular flux analyzers, reactive oxygen species quantification, and mitochondrial membrane potential measurement. The interaction between ZEB1 and MBD1 was assessed by co-immunoprecipitation and immunofluorescence assays. The impact of ZEB1 and MBD1 interaction on sirtuin 3(SIRT3) expression was confirmed by quantitative polymerase chain reaction, western blotting, and dual-luciferase and chromatinimmunoprecipitation assays.RESULTS ZEB1 was a positive regulator of aerobic glycolysis in pancreatic cancer. ZEB1 transcriptionally silenced expression of SIRT3, a mitochondrial-localized tumor suppressor, through interaction with MBD1. CONCLUSION ZEB1 silenced SIRT3 expression via interaction with MBD1 to promote aerobic glycolysis in pancreatic cancer.展开更多
Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1 Ai-h-loop 2, in contrast to the very low to...Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1 Ai-h-loop 2, in contrast to the very low toxicity of Cry1Ai, is closely associated with the eleven residues in the loop 2 region. To characterize the key sites of loop 2 in Cry1Ai-h-loop 2, alaninesubstituted mutants were generated. The toxicity of these mutants against H. armigera indicated that dual-mutant on Gly373 and Asn375 caused a significant decrease in toxic activity. ELISA binding and competition binding assays demonstrated that the reduction of toxicity in the mutant of interest was correlated with decreased binding affinity.展开更多
基金Supported by General Program of National Natural Science Foundation of China,No.81770197Scientific and Technological Research Major Program of Chongqing Municipal Education Commission,No.KJZD-M202312802+1 种基金Chongqing Natural Science Foundation of China,No.CSTB2022NSCQ-MSX0190,No.CSTB2022NSCQ-MSX0176,and No.cstc2020jcyj-msxmX0051Xinqiao Young Postdoc Talent Incubation Program,No.2022YQB098.
文摘BACKGROUND Thrombocytopenia 2,an autosomal dominant inherited disease characterized by moderate thrombocytopenia,predisposition to myeloid malignancies and normal platelet size and function,can be caused by 5’-untranslated region(UTR)point mutations in ankyrin repeat domain containing 26(ANKRD26).Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1)have been identified as negative regulators of ANKRD26.However,the positive regulators of ANKRD26 are still unknown.AIM To prove the positive regulatory effect of GATA binding protein 2(GATA2)on ANKRD26 transcription.METHODS Human induced pluripotent stem cells derived from bone marrow(hiPSC-BM)INTRODUCTION Ankyrin repeat domain containing protein 26(ANKRD26)acts as a regulator of adipogenesis and is involved in the regulation of feeding behavior[1-3].The ANKRD26 gene is located on chromosome 10 and shares regions of homology with the primate-specific gene family POTE.According to the Human Protein Atlas database,the ANKRD26 protein is localized to the Golgi apparatus and vesicles,and its expression can be detected in nearly all human tissues[4].Moreover,UniProt annotation revealed that ANKRD26 is localized in the centrosome and contains coiled-coil domains formed by spectrin helices and ankyrin repeats[5,6].The most common disease related to ANKRD26 is thrombocytopenia 2(THC2),which is a rare autosomal dominant inherited disease characterized by lifelong mild-to-moderate thrombocytopenia and mild bleeding[7-9].Caused by the variants in the 5’-untranslated region(UTR)of ANKRD26,THC2 is defined by a decrease in the number of platelets in circulating blood and results in increased bleeding and decreased clotting ability[8,10].Due to the point mutations that occur in the 5’-UTR of ANKRD26,its negative transcription factors(TFs),Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1),lose their repression effect[11].The persistent expression of ANKRD26 increases the activity of the mitogen activated protein kinase and extracellular signal regulated kinase 1/2 signaling pathways,which are potentially involved in the regulation of thrombopoietin-dependent signaling and further impair proplatelet formation by megakaryocytes(MKs)[11].However,the positive regulators of ANKRD26,which might be associated with THC2 pathology,are still unknown.
文摘Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was~25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from~30 kD to~25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.
基金supported by the National Natural Science Foundation of China (No. 31400126)Basic Research Program of the Ministry of Science and Technology of China (No. 2012CB721102)+2 种基金Chinese Academy of Sciences (No. KJZD-EW-L02)Open Research Fund Program of the State Key Laboratory of Virology of China (No. 2014IOV002)Key Laboratory of Emerging Infectious Diseases and Biosafety, Wuhan
文摘The novel phage lysin PlySs2, is reported to be highly active against various bacteria, including staphylococci, streptococci and Listeria. However, the molecular mechanisms underlying its broad lytic spectrum remain to be established. In the present study, the lytic activity of the catalytic domain(CD, PlySc) and binding specificity of the cell wall binding domain(CBD, PlySb) of PlySs2 were examined. Our results showed that PlySc alone maintains very limited lytic activity. Enhanced green fluorescent protein(EGFP)-fused PlySb displayed high binding affinity to the streptococcal strains tested, including S.suis, S.dysgalactiae, and S.agalactiae, but not staphylococci, supporting its utility as a good CBD donor for streptococcal-targeted lysin engineering. EGFP-fused intact PlySs2 similarly displayed high affinity for streptococci, but not staphylococci. Notably, four truncated PlySb fragments showed no binding capacity. These findings collectively indicate that integrity of the PlySc and PlySb domains is an essential determinant of the broad lytic activity of PlySs2.
基金supported by the National Natural Science Foundation of China,No.81350013a grant from the Jilin Provincial Science and Technology Plan of China,No.20160101027JC&SC201502001the Graduate Innovation Fund of Jilin University in China,No.2017031&2017176
文摘Insulin-like growth factor 1 (IGF-I) is a potential nutrient for nerve repair. However, it is impractical as a therapy because of its limited half- life, rapid clearance, and limited target specificity. To achieve targeted and long-lasting treatment, we investigated the addition of a binding structure by fusing a collagen-binding domain to IGF- 1. After confirming its affinity for collagen, the biological activity of this construct was examined by measuring cell proliferation after transfection into PC12 and Schwann cells using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-di- phenyl-2-H-tetrazolium bromide assay. Immunofluorescence staining was conducted to detect neurofilament and microtubule-associated protein 2 expression, while real time-polymerase chain reaction was utilized to determine IGF-1 receptor and nerve growth/actor mRNA expression. Our results demonstrate a significant increase in collagen-binding activity of the recombinant protein compared with IGF-1. Moreover, the recombinant protein promoted proliferation of PC12 and Schwann cells, and increased the expression of neurofilament and microtubule-associated protein 2. Importantly, the recombinant protein also stimulated sustained expression of IGF-1 receptor and nerve growth factor mRNA for days. These results show that the recombinant protein achieved the goal of targeting and long-lasting treatment, and thus could become a clinically used factor for promoting nerve regeneration with a prolonged therapeutic effect.
基金supported by grants from Natural Science Foundation of Hubei Province(No:2012FFB02304)Scientific Research Foundation of Ministry of Education(No:2013-1792),China
文摘Summary: The role of methyl-CpG binding domain protein 2 (MBD2) in an ApoE-deficient mouse model of age-related macular degeneration (AMD) was investigated. Eight-week-old Mbd2/ApoE double deficient (Mbd2^-/- ApoE^-/-) mice (n=12, 24 eyes, experimental group) and MBD2 (wt) ApoE^-/- mice (n=12, 24 eyes, control group) were fed on Western-type diet for 4 months. The mice were sacrificed, and total serum cholesterol levels were analyzed and Bruch's membrane (BM) of the eyes was removed for ultrastructural observation by transmission electron microscopy. Moreover, intercellular adhesion molecule 1 (ICAM-1) immunoreactivities were evaluated by fluorescence microscopy in sections of the eyes in both groups for further understanding the function mechanism of MBD2. There was no significant difference in the total serum cholesterol levels between control group and experimental group (P〉0.05). Transmission electron microscopy revealed that AMD-like lesions, various vacuoles accumulated on BM, notable outer collagenous layer deposits and dilated basal infoldings of retinal pigment epithelium (RPE) were seen in both groups, and the BM in control group was significantly thickened as compared with experimental group (P〈0.05). Fluorescence micrographs exhibited the expression of ICAM-1 in choroid was higher in control group than in experimental group. We are led to conclude that MBD2 gene knockout may lead to accumulation of more deposits on the BM and influence the pathogenesis of AMD via triggering endothelial activation and inflammatory response in choroid, improving microcirculation, and reducing lipid deposition so as to inhibit the development of AMD-like lesions. Our study helps to provide a new therapeutic approach for the clinical treatment of AMD.
基金National Institutes of Health Grant (AI059570 and AI077767)
文摘Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.
基金Supported by National Natural Science Foundation of China,No. 81071998Hubei Natural Science Foundation,No.2008CDB159Specialized Research Fund for the Doctoral Program of Higher Education,No. 20070487114
文摘AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined using the TaqMan miRNA assay.Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction,and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay.Mutation analysis was conducted using the QuikChange Site-Directed Mutagenesis kit.The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region(3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.RESULTS:In hilar cholangiocarcinoma,miR-373 decreased and was closely associated with poor cell differentiation,advanced clinical stage,and shorter survival.The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373.MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373.Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island,and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.CONCLUSION:MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.
文摘Nucleotide-binding oligomerization domain 1(NOD1) is an intracellular innate immune sensor for small molecules derived from bacterial cell components. NOD1 activation by its ligands leads to robust production of pro-inflammatory cytokines and chemokines by innate immune cells, thereby mediating mucosal host defense systems against microbes. Chronic gastric infection due to Helicobacter pylori(H. pylori) causes various upper gastrointestinal diseases, including atrophic gastritis, peptic ulcers, and gastric cancer. It is now generally accepted that detection of H. pylori by NOD1 expressed in gastric epithelial cells plays an indispensable role in mucosal host defense systems against this organism. Recent studies have revealed the molecular mechanism by which NOD1 activation caused by H. pylori infection is involved in the development of chronic gastritis and gastric cancer. In this review, we have discussed and summarized how sensing of H. pylori by NOD1 mediates the prevention of chronic gastritis and gastric cancer.
基金Supported by The National Center for Advancing Translational Sciences of the National Institutes of Health under award numbers ULl TR000454 previously awarded to Dr.Colbert and Dr.Fisher and TLlT R000456 to Dr.ColbertPancreatic Cancer Action Network(Pan-CAN)&sol American Association for Cancer Research(AACR)award 16982+1 种基金Department of Defense(DOD)/Peer Reviewed Cancer Research Program(PRCRP)award CA110535Georgia Cancer Coalition award 11072,all to Dr.Yu
文摘Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomainhelicase-DNA binding protein 5, chromodomain-helicaseDNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation,and should be validated in future studies.
基金Supported by Grant from Department of Education of Liaoning Province(2008810)
文摘Objective To investigate the effects of stimulant for nucleotide-binding oligomerization domain 1 (NOD1) on secretion of proinflammatory chemokine/cytokines and insulin-dependent glucose uptake in human differentiated adipocytes. Methods Adipose tissues were obtained from patients undergoing liposuction. Stromal vascular cells were extracted and differentiated into adipocytes. A specific ligand for NOD1, was administered to human adipocytes in culture. Nuclear factor-κB transcriptional activity and proinflammatory chemokine/cytokines production were determined by reporter plasmid assay and enzyme-linked immunosorbent assay, respectively. Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[ 3 H] glucose uptake assay. Furthermore, chemokine/cytokine secretion and glucose uptake in adipocytes transfected with small interfering RNA (siRNA) targeting NOD1
文摘Pancreatic ductal adenocarcinoma(PDAC) remains a deadly disease with no efficacious treatment options. PDAC incidence is projected to increase, which may be caused at least partially by the obesity epidemic. Significantly enhanced efforts to prevent or intercept this cancer are clearly warranted. Oncogenic KRAS mutations are recognized initiating events in PDAC development, however, they are not entirely sufficient for the development of fully invasive PDAC.Additional genetic alterations and/or environmental, nutritional, and metabolic signals, as present in obesity, type-2 diabetes mellitus, and inflammation, are required for full PDAC formation. We hypothesize that oncogenic KRAS increases the intensity and duration of the growth-promoting signaling network.Recent exciting studies from different laboratories indicate that the activity of the transcriptional co-activators Yes-associated protein(YAP) and WW-domaincontaining transcriptional co-activator with PDZ-binding motif(TAZ) play a critical role in the promotion and maintenance of PDAC operating as key downstream target of KRAS signaling. While initially thought to be primarily an effector of the tumor-suppressive Hippo pathway, more recent studies revealed that YAP/TAZ subcellular localization and co-transcriptional activity is regulated by multiple upstream signals. Overall, YAP has emerged as a central node of transcriptional convergence in growth-promoting signaling in PDAC cells. Indeed, YAP expression is an independent unfavorable prognostic marker for overall survival of PDAC. In what follows, we will review studies implicating YAP/TAZ in pancreatic cancer development and consider different approaches to target these transcriptional regulators.
文摘BACKGROUND The regulatory factor X6 (RFX6), a member of regulatory factor X family, is known to play a key role in the development and differentiation of pancreatic beta cells as well as insulin production and secretion. However, the potential role of RFX6 in type 2 diabetes (T2D) is still unclear. AIM Recent studies have indicated that RFX6 binding to DNA could be disrupted in diabetes. Therefore, in this study we investigated whether genetic mutations are present in the DNA binding domain of RFX6 gene that could abrogate its function in T2D. METHODS A cohort of T2D patients was enrolled in this study, and the gene encoding the DNA binding domain of RFX6 was amplified by polymerase chain reaction and then analysed by direct DNA sequencing. RESULTS The DNA sequence analysis revealed the absence of any exonic mutation. However, we have identified a new heterozygous single nucleotide polymorphism (IVS6+31 C>T) in the intronic region of DNA binding domain gene that is present in 9.2% and 8.5% of diabetic and control people, respectively (P = 0.97).CONCLUSION We report the absence of any significant genetic variant that could affect the function of RFX6-DNA binding domain in T2D.
文摘Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.
文摘We conducted genome sequence analysis to examine the presence/absence of two types of Z-DNA binding domains in various organisms. We examined 68 organisms from archaea, 914 organisms from bacteria, and 199 organisms from eukaryotes. RecA protein from Escherichia coli has a Z-DNA binding domain and this protein promotes homologous recombination. All the organisms examined had this domain. This result indicated that this domain is essential for all the organisms. RNA editing enzyme, adenosine deaminase from human has another type of Z-DNA binding domain. This domain was observed in some organisms of archaea, bacteria, and eukaryotes. The presence/absence of Z-DNA binding domain in adenosine deaminase indicated that gain and loss of this domain had occurred in the process of evolution. The implication of presence and absence of this domain is discussed in this study.
文摘A comparative molecular dynamics (MD) simulation study was performed on the p53 oncoprotein to investigate the effect of the Arg273His (R273H) mutation on the p53→DNA Binding Domain (DBD). The two p53 dimer structures of the wild-type and mutant Arg273His (R273H) were simulated with the same thermodynamic and environmental parameters. The obtained results demonstrate that the induced Arg273His mutation has a considerable effect on the p53→DNA close contact interaction and changes the picture of hydrogen formation. The Arg273His mutation, in some cases, destroys the existing native hydrogen bond, but, in other cases, forms a strong p53→DNA hydrogen bond, which is not proper for the native protein. The MD simulation results illustrate some molecular mechanism of the conformational changes of the Arg273His key amino acid residue in the p53→DNA binding domain, which might be important for the understanding of the physiological functioning of the p53 protein and the origin of cancer.
基金Supported by the Grant from the Educational Department of Liaoning Province(2008810)
文摘Objective To investigate the potential role of nucleotide-binding oligomerization domain 1(NOD1),a component of the innate immune system,in mediating lipid-induced insulin resistance in adipocytes.Methods Adipocytes from Toll-like receptor 4 deficiency mice were used for stimulation experiments.The effect of oleate/palmitate mixture on nuclear factor-κB(NF-κB)activation was analyzed by reporter plasmid assay.The release of proinflammatory chemokine/cytokines production was determined by using real-time PCR.Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[3H]glucose uptake assay.Chemokine/cytokine expression and glucose uptake in adipocytes transfected with small interfering RNA(siRNA)targeting NOD1 upon fatty acids treatment were analyzed.Results Oleate/palmitate mixture activated the NF-κB pathway and induced interleukin-6,tumor necrosis factor-α,and monocyte chemoattractant protein-1 mRNA expressions in adipocytes from mice deficient in Toll-like receptor 4,and these effects were blocked by siRNA targeting NOD1.Furthermore,saturated fatty acids decreased the ability of insulin-stimulated glucose uptake.Importantly,siRNA targeting NOD1 partially reversed saturated fatty acid-induced suppression of insulin-induced glucose uptake.Conclusion NOD1 might play an important role in saturated fatty acid-induced insulin resistance in adipocytes,suggesting a mechanism by which reduced NOD1 activity confers beneficial effects on insulin action.
基金Acknowledgments We thank Dr Lena Claesson-Welsh (Uppsala University), and PIs of Model Animal Research Center (MARC, Nanjing University) for the helpful discussion about the work, and Yanlan Cao, Wenting Shi and all the staff in the MARC Animal facility of Nanjing University for excellent technical assistance. This work wasfinancially supported by grants from the National Natural Science Foundation of China (30771069, 30671038, and 30930028), the Ministry of Science and Technology of China (2006CB943500), and the Ministry of Education of China (NCET: Program for New Century Excellent Talents in University).
基金the National Science Fund for Distinguished Young Scholars of China,No.81625016the National Science Foundation of China,No.81502031 and No.81772555+1 种基金Shanghai Municipal Commission of Health and Family Planning Grant,No.20154Y0090Youth Research Foundation of Shanghai Municipal Commission of Health and Family Planning,No.Z0124Y074
文摘AIM To uncover the roles of tumor-promoting gene ZEB1 in aerobic glycolysis regulation and shed light on the underlying molecular mechanism.METHODS Endogenous zinc finger E-box binding homeobox-1(ZEB1) was silenced using a lentivirus-mediated method, and the impact of ZEB1 and methyl-CpG binding domain protein 1(MBD1) on aerobic glycolysis was measured using seahorse cellular flux analyzers, reactive oxygen species quantification, and mitochondrial membrane potential measurement. The interaction between ZEB1 and MBD1 was assessed by co-immunoprecipitation and immunofluorescence assays. The impact of ZEB1 and MBD1 interaction on sirtuin 3(SIRT3) expression was confirmed by quantitative polymerase chain reaction, western blotting, and dual-luciferase and chromatinimmunoprecipitation assays.RESULTS ZEB1 was a positive regulator of aerobic glycolysis in pancreatic cancer. ZEB1 transcriptionally silenced expression of SIRT3, a mitochondrial-localized tumor suppressor, through interaction with MBD1. CONCLUSION ZEB1 silenced SIRT3 expression via interaction with MBD1 to promote aerobic glycolysis in pancreatic cancer.
基金supported by the National Key R&D Program of China(2017YFD0200400)the National Natural Science Foundation of China(31272115)
文摘Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1 Ai-h-loop 2, in contrast to the very low toxicity of Cry1Ai, is closely associated with the eleven residues in the loop 2 region. To characterize the key sites of loop 2 in Cry1Ai-h-loop 2, alaninesubstituted mutants were generated. The toxicity of these mutants against H. armigera indicated that dual-mutant on Gly373 and Asn375 caused a significant decrease in toxic activity. ELISA binding and competition binding assays demonstrated that the reduction of toxicity in the mutant of interest was correlated with decreased binding affinity.