Isolation of a ribosome-inactivating and abortifacient protein from seeds of Luff a acutangula

A glycoprotein with a molecular weight of 28 000 as estimated by SDS-polyacrylamide gel electrophoresis was isolated from seeds of Luffa acutangula using a procedure that involved acetone precipitation, ion exchange chromatography on CM Sepharose CL-6B and gel filtration on Sephadex G-50. In immunodiffusion studies it was found to be immunologically distinct from abortifacient proteins isolated from other members of the Cucurbitaceae family including Momordica charantia, Momordica cochinchinensis, Trichosanthes kirilowii and Trichosanthes cucumeroides. There were some differences in amino acid composition among the proteins although there was a gross similarity. The protein from L. acutangula was capable of inducing mid-term abortion in mice and inhibiting protein synthesis in a cell-free system.     

β-Trichosanthin: a new abortifacient protein from the Chinese drug,Wangua, Trichosanthes cucumeroides

β-Trichosanthin was a new abortifacient protein purified from the Chinese drug, Wangua, root tubers of Trichosanthes cucumeroides (Cucurbitaceae). The purification procedure involved acetone fractionation, ammonium sulfate precipitation, ion-exchange chromatography on CM-Sepharose and preparative agarose electrophoresis. Homogeneity of β-trichosanthin was demonstrated in immunoelectrophoresis, agarose electrophoresis and SDS-polyacrylamide gel electrophoresis. It had a molecular weight of 28 000 and no cysteine in its molecule. It differed from trichosanthin, a known abortifacient protein isolated from a related Chinese drug, Tianhuafen, root tubers of Trichosanthes kirilowii (Cucurbitaceae), in molecular weight, carbohydrate content, charge and amino acid composition. β-Trichosanthin was, however, immunochemically identical to trichosanthin and was about twice as potent as trichosanthin in inducing mid-term abortion in mice.     

Trichosanthin, α-momorcharin and β-momorcharin: identity of abortifacient and ribosome-inactivating proteins

The abortifacient proteins trichosanthin, α-momorcharin and β-momorcharin at nM concentrations inhibit cell-free protein synthesis. The momorcharins and the ribosome-inactivating proteins isolated from Momordica charantia seeds cross-react with the respective antisera. The ribosome-inactivating proteins saporins, pokeweed antiviral protein (PAP) and, to a lesser extent, gelonin have abortifacient activity on pregnant mice.     

Selective killing of choriocarcinoma cells in vitro by trichosanthin, a plant protein purified from root tubers of the Chinese medicinal herb Trichosanthes kirilowii

Trichosanthin is an abortifacient plant protein purified from the Chinese medicinal herb Tian-hua-fen, obtained from root tubers of Trichosanthes kirilowii (Cucurbitaceae). Recently, trichosanthin has also been used in the treatment of trophoblastic tumours, including hydatidiform mole, invasive mole and choriocarcinoma. We have examined the effects of trichosanthin on cultured cells and observed selective cytotoxic effects on choriocarcinoma cells. Mouse melanoma cells were also sensitive to its cytotoxic action. Cytotoxic effects on cultured cells were usually not prominent after 24 hr treatment. Continued incubation for 48 hr in control culture medium after treatment with trichosanthin enhances the cytotoxicity on cells. Our findings suggest that trichosanthin exerts specific cytotoxic activity towards trophoblastic cells.     

Isolation and characterization of an abortifacient protein,momorcochin, from root tubers of Momordica cochinchinensis (Family Cucurbitaceae)

A glycoprotein with a molecular weight of 32 000 as estimated by SDS-polyacrylamide gel electrophoresis, and characterized by an abundance of Asp and Glu residues and an absence of Cys residues in its amino acid analysis, was isolated from fresh root tubers of Momordica cochinchinensis using a procedure that involved acetone precipitation, ammonium sulfate precipitation, ion exchange chromatography on DEAE Sepharose CL-6B and gel filtration on Sephadex G-75. The protein was capable of inducing mid-term abortion in mice. The characteristics of this protein were compared and contrasted with those of the abortifacient proteins isolated from other plants of the Cucurbitaceae family.     

Chemical and biological characterization of the galactose binding lectins from Trichosanthes kirilowii root tubers

Three galactose binding isolectins have been isolated from Trichosanthes kirilowii root tubers. Two of the isolectins, TK-I and TK-II, are similar in many aspects including molecular weight, amino acid composition, NH₂-terminal amino acid residue, blood group and carbohydrate specificities, immunodiffusion and immunoelectrophoretic behavior, hemagglutinating and insulinomimetic activities, and in possessing subunits with different molecular weights. Compared to TK-I and TK-II, lectin TK-III has a larger molecular weight, subunits with the same molecular weight, a single and distinctive NH₂-terminal amino acid residue, a different isoelectric point and lower hemagglutinating activity. The three lectins share common antigenic determinants in their structures. β-Linked terminal oligosaccharides containing D-galactose inhibit hemagglutination induced by the lectins with a higher potency than α-linked oligosaccharides. The lectins are non-mitogenic and did not inhibit the concanavalin-A induced mitogenic response of lymphocytes.     

Cochinin B, a novel ribosome-inactivating protein from the seeds of Momordica cochinchinensis

Cochinin B, a novel ribosome-inactivating protein (RIP) with a molecular weight of 28 kDa, was purified from the seeds of Momordica cochinchinensis (Cucurbitaceae). The isolation procedure entailed ammonium sulfate precipitation, cation-exchange chromatography on SP Sepharose column and size-exclusion chromatography on Superdex 75 column with a fast protein liquid chromatography (FPLC) system. The first twenty N-terminal amino acid residues of Cochinin B showed homology to type I RIPs from other Momordica species. The purified Cochinin B displayed a strong inhibitory activity on protein synthesis in the cell-free rabbit reticulocyte lysate system with IC50 of 0.36 nM. Furthermore, it exhibited N-glycosidase activity and cytotoxicity against Vero cell line with IC50 higher than 1540 nM. Interestingly, Cochinin B manifested strong anti-tumor activities on human cervical epithelial carcinoma (HeLa), human embryonic kidney (HEK293) and human small cell lung cancer (NCI-H187) cell lines with IC50 of 16.9, 114 and 574 nM, respectively.     

Isolation of a putative ribosome inactivating protein from dried roots of Trichosanthes kirilowii used in Traditional Chinese Medicine

A putative ribosome inactivating protein (RIP), named T 33, was isolated from dried roots of Trichosanthes kirilowii Maximovicz which are used in Traditional Chinese Medicine. After chromatographic enrichment a protein band with molecular mass of 33 kDa was found by SDS-PAGE. Partial amino acid sequences of this protein consisting of 8 - 33 amino acids reveal homology to highly conserved regions of different RIPs, but also indicate T 33 to be a novel type 2 RIP. Future heterologous expression of this protein will allow investigation of its biological activities in detail.     

The immunosuppressive activities of two abortifacient proteins isolated from the seeds of bitter melon (Momordica charantia)

Two abortifacient proteins, alpha- and beta-momorcharin, have been purified from the seeds of the bitter melon (Momordica charantia). It was found that non-cytotoxic concentrations of these plant proteins can significantly inhibit the mitogenic responses of mouse splenocytes to concanavalin A, phytohaemagglutinin and lipopolysaccharide in a dose-dependent manner. In addition, the alloantigen-induced lymphoproliferation and the in vitro generation of a primary cytotoxic lymphocyte response were severely suppressed in the presence of these proteins. In contrast, the cytolytic activity of cytotoxic lymphocytes and natural killer cells was unimpaired by in vitro exposure to momorcharin. On the other hand, a clear decrease in the functional capacity of macrophages, such as the cytostatic and phagocytic activities, was observed under similar conditions. In vivo studies have shown that single injections of nontoxic microgram amounts of momorcharin into mice resulted in a significant depression of the delayed-type hypersensitivity response as well as the humoral antibody formation to sheep red blood cells. Similarly, the thioglycollate-induced in vivo migration of macrophages was also suppressed. Interestingly, the in vivo activation of natural killer cells was not appreciably affected. Our data suggests that the observed potent immunosuppressive effect of alpha- and beta-momorcharin is unlikely to be due to direct lymphocytotoxicity or due to a shift in the kinetic parameter of the immune response.     

Alpha and beta momorcharins

Two abortifacient proteins designated α and β momorcharins were isolated from seeds of the bitter gourd Momordica charantia by a procedure involving acetone fractionation, CM-Sepharose chromatography and gel filtration on Sephadex G-100. The two proteins were approximately equipotent in inducing midterm abortion in mice. Hemagglutinating (lectin) activity was lacking. Homogeneity of the preparations was attested by a single band in polyacrylamide gel electrophoresis and immunoelectrophoresis. Although the two proteins had similar molecular weights as judged by SDS-polyacrylamide gel electrophoresis, they were immunochemically distinct.     

Peptides with antilipolytic and lipogenic activities from seeds of the bitter gourd Momordica charantia (Family Cucurbitaceae)

• 1.1. Decorticated Momordica charantia seeds were extracted with acidic ethanol. The pH of the extract was adjusted to 8 and the resulting precipitate was chromatographed on CM Sepharose CL-6B and then desalted on Sephadex G-10.
• 2.2. A number of factors which exhibited antilipolytic and lipogenic activities in rat adipocytes were isolated.
• 3.3. Two of the factors were demonstrated to be peptides with similar amino acid compositions and a molecular weight of approximately 8000. However the peptides greatly differed in their chromatographic behavior on CM-Sepharose CL-6B and in their mobilities in agarose and polyacrylamide gel electrophoresis.

     

双边栝蒌种子油含量及其脂肪酸的测定

目的：测定双边栝蒌种子油的含量及其脂肪酸组成。方法：用重量法和GC-MS法测定。结果：双边栝蒌种子油含量为31.24%，脂肪酸组成以亚油酸和油酸为主，不饱和脂肪酸占脂肪酸总量的90.48%,饱和脂肪酸以棕榈酸和硬脂酸为主。结论：双边栝篓种子油属优质植物油，可作为油脂资源开发利用。     

Comparative study of two medicinal Cucurbitaceae

Comparative study of the seeds of two Cucurbitaceae Momordica charantia L. and Herpetospermum caudigerum Wall. shows and important difference for their metabolism concerning particularly aspartic acid. Momordica can produce vicin, while for Herpetospermum, this amino acid would give asparagin. In the other hand, between the two species, Herpetospermum is the one which is able to accumulate lignoids.     

N-terminal sequence of some ribosome-inactivating proteins

The N-terminal portion of some type 1 ribosome-inactivating proteins (RIPs) isolated from the seeds of Gelonium multifiorum, Momordica charantia, Bryonia dioica, Saponaria oficinalis and from the leaves of Saponaria oficinalis are reported in the present paper. Their relationship with other RIPS is discussed.     

Purification and characterization of hemorrhagic components from Agkistrodon acutus (hundred pace snake) venom

Three toxic components, designated Aa-hemorrhagin I,II,III, were isolated from the venom of Agkistrodon acutus by DEAE-Sephadex A-50 chromatography and were further purified by gel filtration on Sephadex G-75, and by column chromatography on DE52 cellulose and CM-Sephadex C-25. All of these components show single bands on acrylamide gel electrophoresis and one precipitin line on immunoelectrophoresis. They are distinct from each other immunochemically. Aa-hemorrhagin I and II are acidic proteins, while III is basic. These hemorrhagins have similar mol. wts of about 22,000. In addition to hemorrhagic and lethal activities, these hemorrhagins possess caseinolytic activities. Their hemorrhagic and caseinolytic activities are inhibited by EDTA, cysteine and the serum of homologous snakes. The amino acid composition of the Aa-hemorrhagin I was determined.     

Isolation and characterization of a galactose binding lectin with insulinomimetic activities

A galactose binding lectin was isolated from the seeds of the bitter gourd Momordica charantia by delipidation with petroleum ether, extraction with phosphate buffered saline, ammonium sulfate precipitation and affinity chromatography on lactogel. The lectin had a molecular weight of 124,000 and approximately 5% carbohydrate content. The molecular weights of the individual subunits were 37,000, 35,000 and 33,000. The lectin exhibited potent hemag-glutinating activity. In addition, it demonstrated antilipolytic and lipogenic activities in isolated rat adipocytes although it did not possess intrinsic lipolytic activity. The antilipolytic activity was susceptible to destruction by heat, trypsin, chymotrypsin, glutathione and galactose, indicating that the integrity of the protein moiety, the disulfide linkages, and galactose, which is the sugar specifically bound by the lectin, all play an important role in interaction with the adipocyte leading to an expression of this insulin-like activity.     

A Ribosome-Inactivating Protein Principle from Hairy Roots and Seeds of Luffa cylindrica (L) Roem and its Cytotoxicity on Melanotic and Amelanotic Melanoma Cell Lines

Single-chain ribosome-inactivating proteins (sc-RIP) are plant proteins that deadenylate ribosomal RNA at a specific site corresponding to A4324 of 28S r-RNA; they are therefore highly specific RNA N-glycosidases. RIPs have been studied as potential tumor cytotoxic agents, both in their native form and after conjugation with monoclonal antibodies. This study tested the sc-RIP extracts from the seeds and hairy root tissue cultures of Luffa cylindrica (established by transformation with Agrobacterium rhizogenes strain 1855) for inhibitory effects on the growth of in vitro melanotic and amelanotic human melanoma cell lines. Crude extracts from hairy roots and seeds inhibited protein synthesis in a reaction mixture containing an in vitro translation system based on rabbit reticulocyte lysate. Inhibitory activity increased during the hairy root culture period, reaching a maximum value in the stationary phase. Growth was inhibited in melanotic and amelanotic melanoma cells exposed to crude extracts from hairy roots and seeds of Luffa cylindrica for 24, 48 and 72 h.     

Purification and characterization of anticoagulation factors from the venom of Agkistrodon acutus.

Two anticoagulants from five-pace snake (Agkistrodon acutus) venom, anticoagulation factor I (ACF I) and anticoagulation factor II (ACF II), have been purified by a multiple-step chromatography procedure of anion-exchange chromatography, gel permeation chromatography and cation-exchange chromatography. Each of them is shown to be homogeneous as judged by PAGE, SDS-PAGE and mass spectrometry. In vitro, both proteins show equivalent anticoagulant activity, and are devoid of proteolytic, esterolytic, L-amino acid oxidase, phospholipase A, thrombin-like, fibrinolytic, hemorrhagic and lethal activities. They have similar amino acid compositions with similar absorption coeffecients (A(1%)(280)) (30.5 for ACF I and 30.0 for ACF II). Both are disulfide-linked consisting of two 14.7 kD chains for ACF I and two 14.6 kD chains for ACFII. ACF I has a molecular mass of 29,604+/-8 atomic mass units (amu) compared to 29,468+/-6 amu for ACF II, determined by mass spectrometry. The isoelectric points of ACF I and ACF II are 5.7 and 7.0, respectively. We conclude that the two isoforms possess equivalent biological activities with similar amino acid compositions and molecular masses, but different isoelectric points.     

基于性别差异的栝楼药用部位氨基酸含量比较研究

目的：比较雌雄株栝楼根氨基酸含量差异。方法：蛋白经盐酸水解得氨基酸,柱前衍生反相高效液相色谱法检测其含量。结果：雌、雄株栝楼根氨基酸含量为7.35%、7.04%。结论：柱前衍生高效液相色谱法可用于栝楼根氨基酸含量测定。总氨基酸含量雄株略低于雌株;碱性氨基酸含量雄株高于雌株,组氨酸含量约为雌株两倍;必需氨基酸含量无显著差异。     

苦瓜籽核糖体失活蛋白的分离纯化及抗氧化活性的研究

目的改进苦瓜籽核糖体失活蛋白的提取工艺 ,并对其抗氧化活性进行研究。方法苦瓜籽经粉碎 ,50mmol/L乙酸抽提 ,硫酸铵沉淀 ,经阳离子交换及凝胶柱色谱等步骤进一步纯化后 ,测定所得两种核糖体失活蛋白的理化性质。结果纯化后得到一种分子量为 2 9.6kD的蛋白质和一种小分子量蛋白质 ,两者在无细胞系统中抑制50 %蛋白质合成浓度分别为 4 .3× 1 0 - 6 、1 .4× 1 0 - 6 mol/L ,含糖量分别为 4 .83 %、1 .82 % ,类超氧化物歧化酶 (SOD)活性分别为 79.0 5、1 7.2 5u/mg。阳性对照纯品SOD活性为 1 2 67u/mg。结论从苦瓜籽中分离纯化出两种不同分子量的核糖体失活蛋白均为糖蛋白质 ,且具有一定的抗氧化活性