Hernandezine(Her),a bisbenzylisoquinoline alkaloid extracted from Thalictrum flavum,is recognized for its range of biological activities inherent to this herbal medicine.Despite its notable properties,the anti-cancer ...Hernandezine(Her),a bisbenzylisoquinoline alkaloid extracted from Thalictrum flavum,is recognized for its range of biological activities inherent to this herbal medicine.Despite its notable properties,the anti-cancer effects of Her have remained largely unexplored.In this study,we elucidated that Her significantly induced cytotoxicity in cancer cells through the activation of apoptosis and necroptosis mechanisms.Furthermore,Her triggered autophagosome formation by activating the AMPK and ATG5 conjugation systems,leading to LC3 lipidation.Our findings revealed that Her caused damage to the mitochondrial membrane,with the damaged mitochondria undergoing mitophagy,as evidenced by the elevated expression of mitophagy markers.Conversely,Her disrupted autophagic flux,demonstrated by the upregulation of p62 and accumulation of autolysosomes,as observed in the RFP-GFP-LC3 reporter assay.Initially,we determined that Her did not prevent the fusion of autophagosomes and lysosomes.However,it inhibited the maturation of cathepsin D and increased lysosomal pH,indicating an impairment of lysosomal function.The use of the early-stage autophagy inhibitor,3-methyladenine(3-MA),did not suppress LC3II,suggesting that Her also induces noncanonical autophagy in autophagosome formation.The application of Bafilomycin A1,an inhibitor of noncanonical autophagy,diminished the recruitment of ATG16L1 and the accumulation of LC3II by Her,thereby augmenting Her-induced cell death.These observations imply that while autophagy initially plays a protective role,the disruption of the autophagic process by Her promotes programmed cell death.This study provides the first evidence of Her’s dual role in inducing apoptosis and necroptosis while also initiating and subsequently impairing autophagy to promote apoptotic cell death.These insights contribute to a deeper understanding of the mechanisms underlying programmed cell death,offering potential avenues for enhancing cancer prevention and therapeutic strategies.展开更多
Both Helicobacter pylori(H. pylori) infection and liver diseases, including nonalcoholic fatty liver disease(NAFLD), viral hepatitis, and hepatocellular carcinoma(HCC), have high prevalences worldwide, and the relatio...Both Helicobacter pylori(H. pylori) infection and liver diseases, including nonalcoholic fatty liver disease(NAFLD), viral hepatitis, and hepatocellular carcinoma(HCC), have high prevalences worldwide, and the relationship between H. pylori infection and liver disease has been discussed for many years. Although positive correlations between H. pylori and NAFLD have been identified in some clinical and experimental studies, nega-tive correlations have also been obtained in high-quality clinical studies. Associations between H. pylori and the pathogenesis of chronic viral hepatitis, mainly disease progression with fibrosis, have also been suggested in some clinical studies. Concerning HCC, a possible role for H. pylori in hepatocarcinogenesis has been identified since H. pylori genes have frequently been detected in resected HCC specimens. However, no study hasrevealed the direct involvement of H. pylori in promoting the development of HCC. Although findings regarding the correlations between H. pylori and liver disease pathogenesis have been accumulating, the existing data do not completely lead to an unequivocal conclusion. Further high-quality clinical and experimental analyses are necessary to evaluate the efficacy of H. pylori eradication in ameliorating the histopathological changes observed in each liver disease.展开更多
The global increase in the prevalence of drug-resistant bacteria has necessitated the development of alternative treatments that do not rely on conventional antimicrobial agents.Using bacteriophage-derived lytic enzym...The global increase in the prevalence of drug-resistant bacteria has necessitated the development of alternative treatments that do not rely on conventional antimicrobial agents.Using bacteriophage-derived lytic enzymes in antibacterial therapy shows promise;however,a thorough comparison and evaluation of their bactericidal efficacy are lacking.This study aimed to compare and investigate the bactericidal activity and spectrum of such lytic enzymes,with the goal of harnessing them for antibacterial therapy.First,we examined the bactericidal activity of spanins,endolysins,and holins derived from 2 Escherichia coli model phages,T1 and T7.Among these,T1-spanin exhibited the highest bactericidal activity against E.coli.Subsequently,we expressed T1-spanin within bacterial cells and assessed its bactericidal activity.T1-spanin showed potent bactericidal activity against all clinical isolates tested,including bacterial strains of 111 E.coli,2 Acinetobacter spp.,3 Klebsiella spp.,and 3 Pseudomonas aeruginosa.In contrast,T1 phage-derived endolysin showed bactericidal activity against E.coli and P.aeruginosa,yet its efficacy against other bacteria was inferior to that of T1-spanin.Finally,we developed a phage-based technology to introduce the T1-spanin gene into target bacteria.The synthesized non-proliferative phage exhibited strong antibacterial activity against the targeted bacteria.The potent bactericidal activity exhibited by spanins,combined with the novel phage synthetic technology,holds promise for the development of innovative antimicrobial agents.展开更多
Despite the high mutation rate of HIV-1,the amino acid sequences of the membrane-spanning domain(MSD)of HIV-1 gp41 are well conserved.Arginine residues are rarely found in single membrane-spanning domains,yet an argin...Despite the high mutation rate of HIV-1,the amino acid sequences of the membrane-spanning domain(MSD)of HIV-1 gp41 are well conserved.Arginine residues are rarely found in single membrane-spanning domains,yet an arginine residue,R696(the numbering is based on that of HXB2),is highly conserved in HIV-1 gp41.To examine the role of R696,it was mutated to K,A,I,L,D,E,N,and Q.Most of these substitutions did not affect the expression,processing or surface distribution of the envelope protein(Env).However,a syncytia formation assay showed that the substitution of R696 with amino acid residues other than K,a naturally observed mutation in the gp41 MSD,decreased fusion activity.Substitution with hydrophobic amino acid residues(A,I,and L)resulted in a modest decrease,while substitution with D or E,potentially negatively-charged residues,almost abolished the syncytia formation.All the fusion-defective mutants showed slower kinetics with the cell-based dual split protein(DSP)assay that scores the degree of membrane fusion based on pore formation between fusing cells.Interestingly,the D and E substitutions did show some fusion activity in the DSP assays,suggesting that proteins containing D or E substitutions retained some fusion pore-forming capability.However,nascent pores failed to develop,due probably to impaired activity in the pore enlargement process.Our data show the importance of this conserved arginine residue for efficient membrane fusion.展开更多
Acquired immunodeficiency syndrome(AIDS)was reported in 1981((CDC),1981).Human immunodeficiency virus type-1(HIV-1),the virus causing AIDS,was isolated in 1983(Barré-Sinoussi et al.,1983).Since AIDS patients were...Acquired immunodeficiency syndrome(AIDS)was reported in 1981((CDC),1981).Human immunodeficiency virus type-1(HIV-1),the virus causing AIDS,was isolated in 1983(Barré-Sinoussi et al.,1983).Since AIDS patients were characterized by a profound decrease in CD4-positive T cell count(Gottlieb et al.,1981),it was a logical consequence that CD4 would be the most probable receptor for the virus.Actually,it was confirmed that CD4 is the primary receptor for HIV-1 in 1986(Maddon et al.,1986).However,it was speculated that HIV-1 requires another factor to infect a cell,since CD4 alone could not support HIV-1 infection to nonhuman cells(Weiner et al.,1991).展开更多
基金This work was supported by JSPS KAKENHI(Nos.20K10449 and 23K09645)。
文摘Hernandezine(Her),a bisbenzylisoquinoline alkaloid extracted from Thalictrum flavum,is recognized for its range of biological activities inherent to this herbal medicine.Despite its notable properties,the anti-cancer effects of Her have remained largely unexplored.In this study,we elucidated that Her significantly induced cytotoxicity in cancer cells through the activation of apoptosis and necroptosis mechanisms.Furthermore,Her triggered autophagosome formation by activating the AMPK and ATG5 conjugation systems,leading to LC3 lipidation.Our findings revealed that Her caused damage to the mitochondrial membrane,with the damaged mitochondria undergoing mitophagy,as evidenced by the elevated expression of mitophagy markers.Conversely,Her disrupted autophagic flux,demonstrated by the upregulation of p62 and accumulation of autolysosomes,as observed in the RFP-GFP-LC3 reporter assay.Initially,we determined that Her did not prevent the fusion of autophagosomes and lysosomes.However,it inhibited the maturation of cathepsin D and increased lysosomal pH,indicating an impairment of lysosomal function.The use of the early-stage autophagy inhibitor,3-methyladenine(3-MA),did not suppress LC3II,suggesting that Her also induces noncanonical autophagy in autophagosome formation.The application of Bafilomycin A1,an inhibitor of noncanonical autophagy,diminished the recruitment of ATG16L1 and the accumulation of LC3II by Her,thereby augmenting Her-induced cell death.These observations imply that while autophagy initially plays a protective role,the disruption of the autophagic process by Her promotes programmed cell death.This study provides the first evidence of Her’s dual role in inducing apoptosis and necroptosis while also initiating and subsequently impairing autophagy to promote apoptotic cell death.These insights contribute to a deeper understanding of the mechanisms underlying programmed cell death,offering potential avenues for enhancing cancer prevention and therapeutic strategies.
文摘Both Helicobacter pylori(H. pylori) infection and liver diseases, including nonalcoholic fatty liver disease(NAFLD), viral hepatitis, and hepatocellular carcinoma(HCC), have high prevalences worldwide, and the relationship between H. pylori infection and liver disease has been discussed for many years. Although positive correlations between H. pylori and NAFLD have been identified in some clinical and experimental studies, nega-tive correlations have also been obtained in high-quality clinical studies. Associations between H. pylori and the pathogenesis of chronic viral hepatitis, mainly disease progression with fibrosis, have also been suggested in some clinical studies. Concerning HCC, a possible role for H. pylori in hepatocarcinogenesis has been identified since H. pylori genes have frequently been detected in resected HCC specimens. However, no study hasrevealed the direct involvement of H. pylori in promoting the development of HCC. Although findings regarding the correlations between H. pylori and liver disease pathogenesis have been accumulating, the existing data do not completely lead to an unequivocal conclusion. Further high-quality clinical and experimental analyses are necessary to evaluate the efficacy of H. pylori eradication in ameliorating the histopathological changes observed in each liver disease.
基金supported by the Japan Agency for Medical Research and Development under grant numbers JP23wm0325065,JP22fk0108532,JP21fk0108496,and JP21 wm0325022 to K.K.grant number JP21gm1610002 to L.C.and K.K.JSPS KAKENHI grants numbers 21H02110 and 21K19666 to K.K.
文摘The global increase in the prevalence of drug-resistant bacteria has necessitated the development of alternative treatments that do not rely on conventional antimicrobial agents.Using bacteriophage-derived lytic enzymes in antibacterial therapy shows promise;however,a thorough comparison and evaluation of their bactericidal efficacy are lacking.This study aimed to compare and investigate the bactericidal activity and spectrum of such lytic enzymes,with the goal of harnessing them for antibacterial therapy.First,we examined the bactericidal activity of spanins,endolysins,and holins derived from 2 Escherichia coli model phages,T1 and T7.Among these,T1-spanin exhibited the highest bactericidal activity against E.coli.Subsequently,we expressed T1-spanin within bacterial cells and assessed its bactericidal activity.T1-spanin showed potent bactericidal activity against all clinical isolates tested,including bacterial strains of 111 E.coli,2 Acinetobacter spp.,3 Klebsiella spp.,and 3 Pseudomonas aeruginosa.In contrast,T1 phage-derived endolysin showed bactericidal activity against E.coli and P.aeruginosa,yet its efficacy against other bacteria was inferior to that of T1-spanin.Finally,we developed a phage-based technology to introduce the T1-spanin gene into target bacteria.The synthesized non-proliferative phage exhibited strong antibacterial activity against the targeted bacteria.The potent bactericidal activity exhibited by spanins,combined with the novel phage synthetic technology,holds promise for the development of innovative antimicrobial agents.
基金supported by the Ministry of Education,Culture,Sports,Science and Technology of Japan,Japan Initiative for Global Research Network on Infectious Diseases(J-GRID).
文摘Despite the high mutation rate of HIV-1,the amino acid sequences of the membrane-spanning domain(MSD)of HIV-1 gp41 are well conserved.Arginine residues are rarely found in single membrane-spanning domains,yet an arginine residue,R696(the numbering is based on that of HXB2),is highly conserved in HIV-1 gp41.To examine the role of R696,it was mutated to K,A,I,L,D,E,N,and Q.Most of these substitutions did not affect the expression,processing or surface distribution of the envelope protein(Env).However,a syncytia formation assay showed that the substitution of R696 with amino acid residues other than K,a naturally observed mutation in the gp41 MSD,decreased fusion activity.Substitution with hydrophobic amino acid residues(A,I,and L)resulted in a modest decrease,while substitution with D or E,potentially negatively-charged residues,almost abolished the syncytia formation.All the fusion-defective mutants showed slower kinetics with the cell-based dual split protein(DSP)assay that scores the degree of membrane fusion based on pore formation between fusing cells.Interestingly,the D and E substitutions did show some fusion activity in the DSP assays,suggesting that proteins containing D or E substitutions retained some fusion pore-forming capability.However,nascent pores failed to develop,due probably to impaired activity in the pore enlargement process.Our data show the importance of this conserved arginine residue for efficient membrane fusion.
文摘Acquired immunodeficiency syndrome(AIDS)was reported in 1981((CDC),1981).Human immunodeficiency virus type-1(HIV-1),the virus causing AIDS,was isolated in 1983(Barré-Sinoussi et al.,1983).Since AIDS patients were characterized by a profound decrease in CD4-positive T cell count(Gottlieb et al.,1981),it was a logical consequence that CD4 would be the most probable receptor for the virus.Actually,it was confirmed that CD4 is the primary receptor for HIV-1 in 1986(Maddon et al.,1986).However,it was speculated that HIV-1 requires another factor to infect a cell,since CD4 alone could not support HIV-1 infection to nonhuman cells(Weiner et al.,1991).
