A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 col...A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of~29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC_(50) of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M_(21) with a IC_(50) of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.展开更多
From the press-residue of the fresh root tuber of Trichosanthes kirilowii Maxim (Cu-curbitaceae),a new ribosome-inactivating protein (RIP),trichobitacin,was isolated.It has the activity of RNA N-glycosidase and can in...From the press-residue of the fresh root tuber of Trichosanthes kirilowii Maxim (Cu-curbitaceae),a new ribosome-inactivating protein (RIP),trichobitacin,was isolated.It has the activity of RNA N-glycosidase and can inhibit the growth of human placental trophoblastic cells.Its molecular weight is 27,228 Da (ES-MS) and pI 9.6.It is a single chain basic RIP.Its amino acid composition was determined.It is a new RIP.It consists of 0.7~0.9% galactose and may be a glycoprotein.Its A'-and C-terminal amino acid is Asp and Ala,respectively.Its N-terminal preliminary amino acid sequence has been determined.展开更多
Ricin is a highly toxic type 2 ribosome-inactivating protein(RIP)which is extracted from the seeds of castor beans.Ricin is considered a potential bioterror agent and no effective antidote for ricin exists so far.In t...Ricin is a highly toxic type 2 ribosome-inactivating protein(RIP)which is extracted from the seeds of castor beans.Ricin is considered a potential bioterror agent and no effective antidote for ricin exists so far.In this study,by structural modification of a retrograde transport blocker Retro-2cyc1,a series of novel compounds were obtained.The primary screen revealed that compound 27 has an improved antiricin activity compare to positive control.In vitro pre-exposure evaluation in Madin-Darby Canine Kidney(MDCK)cells demonstrated that 27 is a powerful anti-ricin compound with an EC50 of 41.05 nmol/L against one LC(lethal concentration,5.56 ng/mL)of ricin.Further studies surprisingly indicated that 27 confers post-exposure activity against ricin intoxication.An in vivo study showed that 1 h post-exposure administration of 27 can improve the survival rate as well as delay the death of ricin-intoxicated mice.A drug combination of 27 with monoclonal antibody mAb4 C13 rescued mice from one LD(lethal dose)ricin challenge and the survival rate of tested animals is 100%.These results represent,for the first time,indication that small molecule retrograde transport blocker confers both in vitro and in vivo post-exposure protection against ricin and therefore provides a promising candidate for the development of anti-ricin medicines.展开更多
The interaction between ribosome-inactivating proteins (RIPs) and supercoiled DNA was observed with an atomic force microscope (AFM). It was found that RIPs can bind to both supercoiled DNA and the unwound double stra...The interaction between ribosome-inactivating proteins (RIPs) and supercoiled DNA was observed with an atomic force microscope (AFM). It was found that RIPs can bind to both supercoiled DNA and the unwound double stranded loop region in supercoiled DNA. The RIPs hound to the supercoils can induce the conformational change of supercoiled DNA. Furthermore, the supercoiled DNA was relaxed and cleaved into nick or linear form by RIPs. It indicated that RIP seemed to be a supercoil-dependent DNA binding protein and exhibited the activity of su-percoil-dependent DNA endonuclease.展开更多
The crystal structure of trichosanthin (TCS) in space group C2 hasbeen refined by PROLSQ and XPLOR to an R-factor of 0. 204 for 33, 531 reflectionswith F。≥2-σ(F。) between 6 to 1. 9 A resolution. The root mean squa...The crystal structure of trichosanthin (TCS) in space group C2 hasbeen refined by PROLSQ and XPLOR to an R-factor of 0. 204 for 33, 531 reflectionswith F。≥2-σ(F。) between 6 to 1. 9 A resolution. The root mean square(r. m. s. ) devi-ations of bond lengths and bond angles from the standard values are 0. 015 A and 2.77°, respectively. The overall fold of the molecule of TCS is, in general, similar to oth-er RIPs, but there are some differences in secondary structure and in the active sitecleft. An overlay of the two molecules (shown by Mol. A and Mol. B), which are notrelated by symmetry in an asymmetric unit, results in an r. m. s. deviation of 0. 850Afor the main chain atoms. The backbones of the C-terminus of the two molecules arequite different. The structures of the water in two molecules are not completely analo-gous. In the active site cleft of Mol. B, the bonding sites of three water molecules toprotein are similar to those of N atoms of formycin ring in the structure of pokeweedantiviral protein complexed with formycin 5-monophosphate, and the side chains of twoTyr70 in the two crystalline forms of TCS show a similar orientation, which is differentfrom that of the corresponding residue in ricin A-chain.展开更多
The three-dimensional structure of trichosanthin at 2.7A resolution has been improved further, by refitting one of the C-terminal tails, adjusting 16 residues in the molecular surface regions, discarding some water mo...The three-dimensional structure of trichosanthin at 2.7A resolution has been improved further, by refitting one of the C-terminal tails, adjusting 16 residues in the molecular surface regions, discarding some water molecules with high B values, and adjusting weights during the further refinement. The R-factor has been reduced to 18.5% and the r.m.s deviations from ideal geometry are also improved. The structures of the two molecules in the monoclinic asymmetric unit and the only molecule in the orthorhombic asymmetric unit are compared with one another. The main-chain structures for most of the residues in the three molecules are substantially the same. However, the courses of the three C-terminal tails are completely different, and the intermolecular interactions resulting from the particular packing of the molecules in the crystals account for the differences. The strand Be-2 and the preceding B-turn in small domain show large r.m.s. deviations among the three molecules and they are also involved in intermolecular interactions.展开更多
The effect of the administration of large amounts of green tea polyphenols is a matter of controversy. We explored whether a polyphenol mixture from a concentrated green tea extract (Polyphenon 60) could alter the eff...The effect of the administration of large amounts of green tea polyphenols is a matter of controversy. We explored whether a polyphenol mixture from a concentrated green tea extract (Polyphenon 60) could alter the effects on mice of the type 2 (two chains) ribosome-inactivating protein nigrin b isolated from Sambucus nigra L. Nigrin b triggers specific reversible toxic effects on the mouse intestines featured by apoptosis of mice Lieberkühn crypt cells upon parenteral administration of sub-lethal amounts. Independent administration to mice of 30 mg/kg body weight of Polyphenon 60 by oral gavage or 10 mg/kg body weight of nigrin b administered via the intraperitoneal route (i.p.) did not affect survival. In contrast, the simultaneous treatment greatly enhanced nigrin b toxicity leading to the death of some animals. The histological analysis revealed that the most serious injury was inflicted on the small intestine crypts, which disappeared, and on the liver, which evidenced hepatotoxicity showing haemorrhagic areas. These findings raise concerns about the abuse of high concentrations of green tea polyphenols especially when the intestinal mucosa is damaged, for instance by toxins or therapeutic drugs.展开更多
The structure of the N-linked oligosaccharide chain of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae), was determined. A glycopeptide liberated by pronase d... The structure of the N-linked oligosaccharide chain of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae), was determined. A glycopeptide liberated by pronase digestion of the glycoprotein was subjected to amino acid and neutral carbohydrate analysis to establish the composition of amino acid and sugar residues. The sequences and glycosylation hnkages of the sugar and amino acid residues in the glycopeptide were determined as Manαl-6(XyIβ1-2)-Manβ1-4GlcNAcβ1-4(Fucαl-3)-GlcNAc-Asn-Leu by 2D-NMR spectroscopy and FAB-MS data.展开更多
Chemically based rodent models are used to assess the positive effects promoted by foods and gut microbiota on gut health. Lectins with enzymatic activity, such as type 2 ribosome-inactivating proteins, might also pro...Chemically based rodent models are used to assess the positive effects promoted by foods and gut microbiota on gut health. Lectins with enzymatic activity, such as type 2 ribosome-inactivating proteins, might also prove useful for exploring these issues. Sub-lethal doses of the lectin nigrin from Sambucus nigra L. to mice promoted reversible derangement of gut epithelium by induction of apoptosis of transit amplifying cells of the small intestine crypts in a time-dependent course. The present work seeks to study vitamin B6 accumulation in plasma from an oral bolus in a mouse nigrin model. 24 h after sub-lethal nigrin b treatment, there was clear body weight reduction associated to a notable increase in Evan’s blue stain accumulation in excised small intestine, an increase in myeloperoxidase activity, and a near 50% reduction in plasma accumulation of vitamin B6. Histological analysis of small intestine sections of nigrin b-treated animals also revealed significant derangement of intestinal crypts. Seventy two hours after nigrin b treatment, stain uptake decreased and vitamin B6 accumulation was almost restored despite villi derangement. Large intestine crypts were scarcely or not at all affected. Eight days after nigrin b treatment, vitamin B6 uptake and intestinal crypt structure had fully recovered. The nigrin b mice model supports the view that, under these conditions, the carrier-mediated vitamin B6 uptake component of the small intestine crypts is probably the most active when the vitamin is administered orally as a bolus. The findings provide insights into the suitability of the present mice model for nutritional or drug absorption studies in conditions of partially altered or injured intestinal mucosa.展开更多
The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of pepti... The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptides obtained by enzymatic digestion with trypsin, chymotrypsin and S. aureus V8 protease, as well as by chemical cleavage with BNPS-skatole. The protein consists of 249 amino acid residues containing one asparagine - linked sugar group attached to the site of Asn 5 1 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of trichosanthin and other ribosome-inactivating proteins from different species of plants shows a significant homology with each other. Regarding the similarity of their biological properties, an active domain of these proteins has been predicted here.展开更多
Cucurmosin 2, a type 1 ribosome-inactivating protein （RIP） isolated from sarcocarp of Cucurbita moschata, has been crystallized by the vapor-diffusion method using PEG6000 as the precipitant. The crystals belong to ...Cucurmosin 2, a type 1 ribosome-inactivating protein （RIP） isolated from sarcocarp of Cucurbita moschata, has been crystallized by the vapor-diffusion method using PEG6000 as the precipitant. The crystals belong to the orthorhombic space group P212121, with unit cell parameters a = 55.853, b = 65.507, c = 91.754 А, and have one molecule per asymmetric unit. X-ray data have been collected to 1.8А, using a synchrotron source.展开更多
Luffaculin 1 purified from the seeds of Luffa acutangula belongs to the type I ribosome-inactivating proteins （RIPs）. It has been crystallized by the vapor-diffusion method using polyethylene glycol as a precipitant...Luffaculin 1 purified from the seeds of Luffa acutangula belongs to the type I ribosome-inactivating proteins （RIPs）. It has been crystallized by the vapor-diffusion method using polyethylene glycol as a precipitant. The crystal is of space group P1 with a = 39.135, b = 46.813, c = 83.571 A, α = 891068,β = 80.009 and y = 72.143°, and has two molecules per asymmetric unit. X-ray data have been collected to be 1.4 A using a synchrotron source.展开更多
β-Trichosanthin, a type 1 ribosome-inactivating protein （RIP） isolated from the root tuber of Trichosanthe kirilowii Maxim, is an isoform of trichosanthin. Here we report its crystallization in two crystal forms us...β-Trichosanthin, a type 1 ribosome-inactivating protein （RIP） isolated from the root tuber of Trichosanthe kirilowii Maxim, is an isoform of trichosanthin. Here we report its crystallization in two crystal forms using the hanging-drop vapor-diffusion method. The form A and form B crystals belong to the orthorhombic space group P212121 and monoclinic space group P21, respectively. X-ray data have been collected to 1.6 and 1.2 A resolution for form A and form B crystals, respectively, using a synchrotron source.展开更多
文摘A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of~29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC_(50) of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M_(21) with a IC_(50) of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.
基金Project supported by grants from the High Technology Development Program of China,from the Key Project of Chinese Academy 8.5 and from the National Natural Science Foundation of China.
文摘From the press-residue of the fresh root tuber of Trichosanthes kirilowii Maxim (Cu-curbitaceae),a new ribosome-inactivating protein (RIP),trichobitacin,was isolated.It has the activity of RNA N-glycosidase and can inhibit the growth of human placental trophoblastic cells.Its molecular weight is 27,228 Da (ES-MS) and pI 9.6.It is a single chain basic RIP.Its amino acid composition was determined.It is a new RIP.It consists of 0.7~0.9% galactose and may be a glycoprotein.Its A'-and C-terminal amino acid is Asp and Ala,respectively.Its N-terminal preliminary amino acid sequence has been determined.
基金the financial supports of the National Science and Technology Major Projects for"Major New Drugs Innovation and Development"(2018ZX09711003-001-001)of China.
文摘Ricin is a highly toxic type 2 ribosome-inactivating protein(RIP)which is extracted from the seeds of castor beans.Ricin is considered a potential bioterror agent and no effective antidote for ricin exists so far.In this study,by structural modification of a retrograde transport blocker Retro-2cyc1,a series of novel compounds were obtained.The primary screen revealed that compound 27 has an improved antiricin activity compare to positive control.In vitro pre-exposure evaluation in Madin-Darby Canine Kidney(MDCK)cells demonstrated that 27 is a powerful anti-ricin compound with an EC50 of 41.05 nmol/L against one LC(lethal concentration,5.56 ng/mL)of ricin.Further studies surprisingly indicated that 27 confers post-exposure activity against ricin intoxication.An in vivo study showed that 1 h post-exposure administration of 27 can improve the survival rate as well as delay the death of ricin-intoxicated mice.A drug combination of 27 with monoclonal antibody mAb4 C13 rescued mice from one LD(lethal dose)ricin challenge and the survival rate of tested animals is 100%.These results represent,for the first time,indication that small molecule retrograde transport blocker confers both in vitro and in vivo post-exposure protection against ricin and therefore provides a promising candidate for the development of anti-ricin medicines.
基金Project supported by the National Natural Science Foundation of China, Chinese Academy of Sciences and Shanghai Center of Life Sciences.
文摘The interaction between ribosome-inactivating proteins (RIPs) and supercoiled DNA was observed with an atomic force microscope (AFM). It was found that RIPs can bind to both supercoiled DNA and the unwound double stranded loop region in supercoiled DNA. The RIPs hound to the supercoils can induce the conformational change of supercoiled DNA. Furthermore, the supercoiled DNA was relaxed and cleaved into nick or linear form by RIPs. It indicated that RIP seemed to be a supercoil-dependent DNA binding protein and exhibited the activity of su-percoil-dependent DNA endonuclease.
文摘The crystal structure of trichosanthin (TCS) in space group C2 hasbeen refined by PROLSQ and XPLOR to an R-factor of 0. 204 for 33, 531 reflectionswith F。≥2-σ(F。) between 6 to 1. 9 A resolution. The root mean square(r. m. s. ) devi-ations of bond lengths and bond angles from the standard values are 0. 015 A and 2.77°, respectively. The overall fold of the molecule of TCS is, in general, similar to oth-er RIPs, but there are some differences in secondary structure and in the active sitecleft. An overlay of the two molecules (shown by Mol. A and Mol. B), which are notrelated by symmetry in an asymmetric unit, results in an r. m. s. deviation of 0. 850Afor the main chain atoms. The backbones of the C-terminus of the two molecules arequite different. The structures of the water in two molecules are not completely analo-gous. In the active site cleft of Mol. B, the bonding sites of three water molecules toprotein are similar to those of N atoms of formycin ring in the structure of pokeweedantiviral protein complexed with formycin 5-monophosphate, and the side chains of twoTyr70 in the two crystalline forms of TCS show a similar orientation, which is differentfrom that of the corresponding residue in ricin A-chain.
文摘The three-dimensional structure of trichosanthin at 2.7A resolution has been improved further, by refitting one of the C-terminal tails, adjusting 16 residues in the molecular surface regions, discarding some water molecules with high B values, and adjusting weights during the further refinement. The R-factor has been reduced to 18.5% and the r.m.s deviations from ideal geometry are also improved. The structures of the two molecules in the monoclinic asymmetric unit and the only molecule in the orthorhombic asymmetric unit are compared with one another. The main-chain structures for most of the residues in the three molecules are substantially the same. However, the courses of the three C-terminal tails are completely different, and the intermolecular interactions resulting from the particular packing of the molecules in the crystals account for the differences. The strand Be-2 and the preceding B-turn in small domain show large r.m.s. deviations among the three molecules and they are also involved in intermolecular interactions.
文摘The effect of the administration of large amounts of green tea polyphenols is a matter of controversy. We explored whether a polyphenol mixture from a concentrated green tea extract (Polyphenon 60) could alter the effects on mice of the type 2 (two chains) ribosome-inactivating protein nigrin b isolated from Sambucus nigra L. Nigrin b triggers specific reversible toxic effects on the mouse intestines featured by apoptosis of mice Lieberkühn crypt cells upon parenteral administration of sub-lethal amounts. Independent administration to mice of 30 mg/kg body weight of Polyphenon 60 by oral gavage or 10 mg/kg body weight of nigrin b administered via the intraperitoneal route (i.p.) did not affect survival. In contrast, the simultaneous treatment greatly enhanced nigrin b toxicity leading to the death of some animals. The histological analysis revealed that the most serious injury was inflicted on the small intestine crypts, which disappeared, and on the liver, which evidenced hepatotoxicity showing haemorrhagic areas. These findings raise concerns about the abuse of high concentrations of green tea polyphenols especially when the intestinal mucosa is damaged, for instance by toxins or therapeutic drugs.
基金National Ministry of Science and Technology (No. 970211006)Chinese Academy of Sciences (No.KJ951-A1-504-01).
文摘 The structure of the N-linked oligosaccharide chain of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae), was determined. A glycopeptide liberated by pronase digestion of the glycoprotein was subjected to amino acid and neutral carbohydrate analysis to establish the composition of amino acid and sugar residues. The sequences and glycosylation hnkages of the sugar and amino acid residues in the glycopeptide were determined as Manαl-6(XyIβ1-2)-Manβ1-4GlcNAcβ1-4(Fucαl-3)-GlcNAc-Asn-Leu by 2D-NMR spectroscopy and FAB-MS data.
文摘Chemically based rodent models are used to assess the positive effects promoted by foods and gut microbiota on gut health. Lectins with enzymatic activity, such as type 2 ribosome-inactivating proteins, might also prove useful for exploring these issues. Sub-lethal doses of the lectin nigrin from Sambucus nigra L. to mice promoted reversible derangement of gut epithelium by induction of apoptosis of transit amplifying cells of the small intestine crypts in a time-dependent course. The present work seeks to study vitamin B6 accumulation in plasma from an oral bolus in a mouse nigrin model. 24 h after sub-lethal nigrin b treatment, there was clear body weight reduction associated to a notable increase in Evan’s blue stain accumulation in excised small intestine, an increase in myeloperoxidase activity, and a near 50% reduction in plasma accumulation of vitamin B6. Histological analysis of small intestine sections of nigrin b-treated animals also revealed significant derangement of intestinal crypts. Seventy two hours after nigrin b treatment, stain uptake decreased and vitamin B6 accumulation was almost restored despite villi derangement. Large intestine crypts were scarcely or not at all affected. Eight days after nigrin b treatment, vitamin B6 uptake and intestinal crypt structure had fully recovered. The nigrin b mice model supports the view that, under these conditions, the carrier-mediated vitamin B6 uptake component of the small intestine crypts is probably the most active when the vitamin is administered orally as a bolus. The findings provide insights into the suitability of the present mice model for nutritional or drug absorption studies in conditions of partially altered or injured intestinal mucosa.
基金Project supported by Grants from the High Technology Development Program of China from the National Natural Science Foundation of China(No. 29272074)
文摘 The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptides obtained by enzymatic digestion with trypsin, chymotrypsin and S. aureus V8 protease, as well as by chemical cleavage with BNPS-skatole. The protein consists of 249 amino acid residues containing one asparagine - linked sugar group attached to the site of Asn 5 1 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of trichosanthin and other ribosome-inactivating proteins from different species of plants shows a significant homology with each other. Regarding the similarity of their biological properties, an active domain of these proteins has been predicted here.
基金supported by the National Natural Science Foundation of China (No. 39970872, 30772587)the Natural Science Foundation of Fujian Province (C97052,C0510012)+1 种基金Special Fund of Fujian Development and Reform Commission (2006-34)supported by the National Science Foundation of USA
文摘Cucurmosin 2, a type 1 ribosome-inactivating protein （RIP） isolated from sarcocarp of Cucurbita moschata, has been crystallized by the vapor-diffusion method using PEG6000 as the precipitant. The crystals belong to the orthorhombic space group P212121, with unit cell parameters a = 55.853, b = 65.507, c = 91.754 А, and have one molecule per asymmetric unit. X-ray data have been collected to 1.8А, using a synchrotron source.
基金This work was supported by the National Natural Science Foundation of China (No. 39970872) and Natural Science Foundation of Fujian Province (C97052)
文摘Luffaculin 1 purified from the seeds of Luffa acutangula belongs to the type I ribosome-inactivating proteins （RIPs）. It has been crystallized by the vapor-diffusion method using polyethylene glycol as a precipitant. The crystal is of space group P1 with a = 39.135, b = 46.813, c = 83.571 A, α = 891068,β = 80.009 and y = 72.143°, and has two molecules per asymmetric unit. X-ray data have been collected to be 1.4 A using a synchrotron source.
基金the NNSF of China (No.39970872,No.30772587)the Natural Science Foundation of Fujian Province (C97052,C0510012)+1 种基金Special Fund of Fujian Development and Reform Commissionthe National Science Foundation of USA
文摘β-Trichosanthin, a type 1 ribosome-inactivating protein （RIP） isolated from the root tuber of Trichosanthe kirilowii Maxim, is an isoform of trichosanthin. Here we report its crystallization in two crystal forms using the hanging-drop vapor-diffusion method. The form A and form B crystals belong to the orthorhombic space group P212121 and monoclinic space group P21, respectively. X-ray data have been collected to 1.6 and 1.2 A resolution for form A and form B crystals, respectively, using a synchrotron source.
