Evaluation of Riproximin Binding Properties Reveals a Novel Mechanism for Cellular Targeting

Riproximin is a cytotoxic type II ribosome-inactivating protein showing high selectivity for tumor cell lines. Its binding to cell surface glycans is crucial for subsequent internalization and cytotoxicity. In this paper, we describe a unique mechanism of interaction and discuss its implications for the cellular targeting and cytotoxicity of riproximin. On a carbohydrate microarray, riproximin specifically bound to two types of asialo-glycans, namely to bi- and triantennary complex N-glycan structures (NA2/NA3) and to repetitive N-acetyl-d-galactosamine (GalNAc), the so-called clustered Tn antigen, a cancer-specific O-glycan on mucins. Two glycoproteins showing high riproximin binding, the NA3-presenting asialofetuin and the clustered Tn-rich asialo-bovine submaxillary mucin, were subsequently chosen as model glycoproteins to mimic the binding interactions of riproximin with the two types of glycans. ELISA analyses were used to relate the two binding specificities of riproximin to its two sugar binding sites. The ability of riproximin to cross-link the two model proteins revealed that binding of the two types of glycoconjugates occurs within different binding sites. The biological implications of these binding properties were analyzed in cellular assays. The cytotoxicity of riproximin was found to depend on its specific and concomitant interaction with the two glycoconjugates as well as on dynamic avidity effects typical for lectins binding to multivalent glycoproteins. The presence of definite, cancer-related structures on the cells to be targeted determines the therapeutic potency of riproximin. Due to its cross-linking ability, riproximin is expected to show a high degree of specificity for cells exposing both NA2/NA3 and clustered Tn structures.     

Riproximin is a recently discovered type II ribosome inactivating protein with potential for treating cancer

The development of new anticancer drugs is a salient problem and the traditional use of plants is a potentially rich source of information for detecting new molecules with antineoplastic activity. Riproximin is a recently detected cytotoxic type II ribosome inactivating protein with high selectivity for certain tumor cell lines. Its activity was recognized as the main component in a plant powder used by African healers for treating cancer. By ribulose bisphosphate carboxylase gene sequencing analysis, the powder was identified to be derived from the plant Ximenia americana. The cDNA sequence of riproximin was identified, the protein was modeled to contain one A- and a B-chain, respectively, and a reliable purification procedure from kernels of X. americana was established. Riproximin displays high but differential antiproliferative activity in a panel of human and rodent cancer cell lines, with concentrations inhibiting cell proliferation by 50% (IC₅₀ values) that diverge by a factor of 100. Consistent antineoplastic activity was detected in colorectal and pancreatic cancer liver metastasis models in rats. The cytotoxic mechanism of action was determined to be based on cellular uptake of riproximin followed by its A-chain prompted depurination of the 28S ribosomal RNA and induction of unfolded protein response. Riproximin's specificity depended on its B-chain connected binding to cell surface glycans, the presence of which is crucial for subsequent internalization into cells and cytotoxicity. These N- and O-glycans include bi- and tri-antennary NA structures (NA2/NA3) as well as Tn3 structures (clustered Tn antigen). Riproximin was found to crosslink proteins with N- and O-glycan structure, thus indicating both types of binding sites on its B chain. Due to this crosslinking ability, riproximin is expected to show prominent cytotoxicity towards cells expressing both, NA2/NA3 and clustered Tn structures. Apart from the properties of riproximin, the plant X. americana has been known for some medical uses in traditional African medicine, including various types of infections.     

超胶AcA_(34)柱层析法分离提纯人血清IgD

本文用超胶AcA_(34)柱层析法从IgD型骨髓瘤病人血清中分离提纯人血清IgD。经聚丙烯酰胺凝胶电泳、SDS聚丙烯酰胺凝胶电泳、免疫电泳和免疫双扩散等方法检查其纯度及活性均较满意。这个方法简单方便,时程短,效果好。此外,还用超薄层胶等电聚焦电泳法得到了IgD的等电聚焦图谱,薄层扫描为五条带,等电点在5.4—6.0。     

Partial purification of superoxide dismutase and peroxidase from ber (Zizyphus mauritiana Lamk.) fruit using anion exchange chromatography

Partial purification of the two enzymes i.e. superoxide dismutase (SOD) and peroxidase (POX) from ber pulp has been obtained by passing the ammonium sulphate fraction through a diethyl amino ethyl-cellulose (DEAE-Cellulose) column. The fractions showing SOD and POX activity were pooled separately and passed through a Sephadex G100 column for further purification. SOD was purified 12.2 fold with 12.6% yield while POX was purified 15.6 fold with 19.3% yield. Approximate molecular mass for SOD and POX, as judged by gel filtration method was 35.6 and 81.5 kDa, respectively.Key words: Ber fruit, superoxide dismutase, peroxidase, DEAE-cellulose chromatography, fold purification, yield, Zizyphus mauritiana Lamk     

PURIFICATION AND PROPERTIES OF THE PROTHORACICOTROPIC HORMONE OF THE SILKWORM, BOMBYX MORI

A simple and highly reproducible procedure for partial purification of prothoracicotropic hormone (PTTH) was established starting with 96,000 male adult Bombyx heads. Approximately 28,500-fold purification of PTTH was accomplished with a yield of about 50% and 6 ng of the most purified preparation ("highly purified PTTH") caused adult development in a brainless Samia pupa. The peptidal nature of PTTH was reconfirmed through the effects of various enzymatic and chemical treatments on the biological activity of "highly purified PTTH". Gel-filtration indicated the molecular weight of PTTH to be 4,400.     

Purification of potato lectin (Solanum tuberosum agglutinin) from tubers or fruits using chromatofocusing

The lectin from potato tubers (Solanum tuberosum agglutinin) has been purified to homogeneity by a procedure involving chromatofocusing followed by gel filtration. By subjecting tuber and fruit extracts from an individual plant to this purification scheme, it was demonstrated that the lectins from those two tissues, though similar, are not identical.     

Purification and characterization of osteopontin from human milk

Osteopontin (OPN) is expressed in many organs and tissues and has different biological properties related to different molecular forms in respect to size and posttranslational modifications. However, a purification procedure for authentic intact OPN as well as fragments of OPN from an accessible biological source is missing. A four-step procedure was used to purify OPN from human milk, based on its crystal growth inhibitory activity, including anion exchange chromatography, the elimination of casein, hydroxyapatite chromatography, and negative affinity chromatography. Purified OPN was further separated into its different molecular forms by means of a two-step procedure, involving size exclusion chromatography and reverse phase chromatography. A rabbit polyclonal antibody was raised to purified intact OPN and high M(r) OPN components; the immunoreactivity of both forms was almost equal when investigated by enzyme immunoassay (EIA). The procedures facilitate the purification of intact OPN and OPN fragments for purposes of standardization, preparation of monospecific antibodies, and functional studies.     

Design,production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins

To expand the application of the streptavidin-biotin technology for reversible affinity purification of biotinylated proteins, a novel form of monomeric streptavidin was engineered and produced using Bacillus subtilis as the expression host. By changing as little as two amino acid residues (T90 and D128) to alanine, the resulting mutant streptavidin designated DM3 was produced 100% in the monomeric form as a soluble functional protein via secretion. It remained in the monomeric state in the presence or absence of biotin. Interaction of purified monomeric streptavidin with biotin was studied by surface plasmon resonance-based BIAcore biosensor. Its on-rate is comparable to that of monomeric avidin while its off-rate is seven times lower. The dissociation constant was determined to be 1.3 x 10(-8)M. These properties make it an attractive agent for affinity purification of biotinylated proteins. An affinity matrix with immobilized DM3 mutein was prepared and applied to purify biotinylated cytochrome c from a crude extract. Biotinylated cytochrome c could be purified to homogeneity in one step and was shown to retain full biological activity. Advantages of using DM3 mutein over other traditional methods in the purification of biotinylated proteins are discussed.     

Preparation of a Homogeneous Tomato Fruit Peroxidase

Tomato fruit (Lycopersicon esculentum Mill cv. Walters) peroxidase was purified to apparent homogeneity by a three step procedure: hydrophobic chromatography, DEAE Sephacel chromatography and semi-preparative electrophoresis. A purification of 71 fold and a yield of 52% relative to crude extract were obtained. The pure enzyme was brown in color and showed a molecular weight of 45,000 as estimated from SDS disc gel electrophoresis and gel filtration on Ultrogel AcA 34. The pH optimum of tomato peroxidase varied with substrate dyes used and the enzyme may have some hydrophobic properties near its active site. The optimum temperature was 35 C for this enzyme, and IAA oxidase activity was evident in the presence of 2,4-dichloro-phenol and manganese. The apparent K_M for IAA was measured to be 0.24 mM.     

Purification and Properties of In Vitro–produced Anthrax Toxin Components

The three components of the toxin of Bacillus anthracis, edema factor (EF), protective antigen (PA), and lethal factor (LF), were purified 197-, 156-, and 1,025- fold, with 38, 78, and 11% recovery, respectively. Each purified component was serologically active, distinct, and free from the other components. The purified EF produced edema when mixed with PA, and the purified PA was an active immunogen. The components did not appear to be simple proteins by spectrophotometric analysis. As they were purified, the pH range in which they were most stable narrowed, centering between pH 7.4 and 7.8. Heat readily destroyed the biological activity of the components but not their serological activity. The rat lethality test showed that, with a constant amount of LF and an increasing amount of PA, the time to death reached a minimum and then was extended. When an increasing amount of LF was added to a constant amount of PA, the time to death became shorter as more LF was added. The biological, immunological, and serological properties of the components were shown to vary independently with storage and extent of purification so that serological activity was not always directly correlated with biological activity. Evidence is presented that the components can exist in different molecular configurations or as aggregates, and that this property is influenced by the state of component purity and by the environment.     

γ-Glutamyl transpeptidase of the ackee plant

The enzyme γ-glutamyl transpeptidase was purified from seeds of immature ackee fruit (Blighia sapida; Sapindaceae) by salt fractionation and gel filtration on Biogel P-10 and P-200. The procedure, which differs from an earlier one applied to kidney bean fruit, achieves 9.8% yield and 577-fold purification. The enzyme is also present in other parts of the fruit and in leaves. A MW of 12 500 was found by SDS-polyacrylamide gel electrophoresis, a value much lower that that reported for the enzyme from kidney bean fruit. Neutral or amino sugar accounts for 10% of the dry weight. In vitro, the enzyme catalysed synthesis of an unusual γ-glutamyl dipeptide which occurs in ackee seeds, using glutathione as glutamyl group donor. The enzyme mechanism was of the double displacement (ping-pong) type.     

Multiple forms of ADP‐glucose pyrophosphorylase from tomato leaf

ADP‐glucose pyrophosphorylase (AGP, EC 2.7.7.27) was partially purified from tomato ( Lycopersicon esculentum Mill. cv. Laura) leaf by a procedure previously used for purification of AGP from tomato pericarp. SDS‐PAGE and western blot analysis of the final preparation indicated that the leaf enzyme is composed of two subunits of 50 and 54 kDa. Two‐dimensional PAGE and western blot analysis of the same preparation, however, revealed at least six isoforms of the large subunit and two isoforms of the small subunit. The leaf AGP is very sensitive to regulation by 3‐phosphoglycerate and inorganic phosphate. Its properties are compared to those of AGP from tomato fruit.     

Identification of polysaccharides from pericarp tissues of litchi (Litchi chinensis Sonn.) fruit in relation to their antioxidant activities

A large number of polysaccharides are present in the pericarp tissues of harvested litchi fruits. A DEAE Sepharose fast-flow anion-exchange column and a Sephadex G-50 gel-permeation column were used to isolate and purify the major polysaccharides from litchi fruit pericarp tissues. Antioxidant activities of these major polysaccharide components were also evaluated. An aqueous extract of the polysaccharides from litchi fruit pericarp tissues was chromatographed on a DEAE anion-exchange column to yield two fractions. The largest amount of the polysaccharide fraction was subjected to further purification by gel filtration on Sephadex G-50. The purified product was a neutral polysaccharide, with a molecular weight of 14 kDa, comprised mainly of 65.6% mannose, 33.0% galactose and 1.4% arabinose. Analysis by Smith degradation indicated that there were 8.7% of (1-->2)-glycosidic linkages, 83.3% of (1-->3)-glycosidic linkages and 8.0% of (1-->6)-glycosidic linkages in the polysaccharide. Furthermore, different polysaccharide fractions extracted and purified from litchi fruit pericarp tissues exhibited strong antioxidant activities. Among these fractions, the purified polysaccharide had the highest antioxidant activity and should be explored as a novel potential antioxidant.     

Purification and characterization of luteinizing hormone-releasing hormone from codfish brain

We have purified luteinizing hormone-releasing hormone (LH-RH) from codfish brain and have demonstrated its identity with salmon LH-RH (sLH-RH). An antiserum raised against sLH-RH was used in a specific radioimmunoassay (RIA) to monitor purification and to manufacture an immunoaffinity chromatography column for the initial purification step. The cross-reactivity of the sLH-RH RIA with mammalian LH-RH was 0.1%. Acid extracts of codfish brains were sequentially purified by immunoaffinity chromatography, gel-filtration chromatography, and three steps of reverse-phase HPLC. The purified material and synthetic sLH-RH coeluted on reverse-phase HPLC and exhibited similar biological activity in a dispersed pituitary cell bioassay. Furthermore, the amino acid composition of the purified material was identical to salmon LH-RH. These results suggest that there is structural conservation of LH-RH between these species of teleost fish.     

Biological and biophysical characterization of recombinant soluble human E-selectin purified at large scale by reversed-phase high-performance liquid chromatography

A first step in the development of a high-throughput screening assay for antagonists of human E-selectin is the purification and characterization of the selectin. In the present paper we describe a single-step, rapid, reversed-phase HPLC purification protocol for the recombinant, soluble from of human E-selectin (rshE-selectin) produced in Chinese hamster ovary cells. The procedure resulted in high protein yields with recoveries of greater than 98%. Characterization of the reversed-phase purified rshE-selectin showed this product to be analogous to rshE-selectin purified using conventional chromatographic techniques with respect to biological activity and molecular shape. However, the carbohydrate composition of reversed-phase purified rshE-selectin, which had been variable with conventionally purified material, was found to be constant across several isolations. The protocol described herein eliminated the high mannose component associated with previously purified rshE-selectin and provided a uniform carbohydrate composition for addtitional experimental studies, such as NMR. This fact, coupled with the high yield and simplicity of the present purification scheme are distinct advantages over those previously published. It is expected that other mammalian selectins, such as P-selectin and L-selectin, would also be amenable to reversed-phase HPLC purification.     

Prostaglandin E2 9-keto reductase from bovine term placenta.

The aim of the following study was to determine the activity and physico-chemical properties of prostaglandin E2 9-keto reductase from bovine placenta. Placental tissues obtained immediately after parturition were subjected to purification procedure consisting of homogenization, affinity chromatography, gel filtration and allowed to electrophoresis. The activity of enzyme was measured spectrophotometrically. The purification procedures receive 135-fold purified enzyme preparate of the molecular weight of 45 kDa with the following kinetic values: Michaelis constant for PGE2, 117 microM and max velocity 183 pmol/min. The activity of enzyme was also detected with 20 alpha-hydroxypregn-4en-3-one and with 9,10-phenanthrenquinone (Michaelis constant 22 microM and 6 microM, respectively). The determination of physico-chemical properties of prostaglandin E2 9-keto reductase, performed for the first time in bovine placenta, should aid the understanding of the metabolism of prostaglandins and their biological importance in physiological and pathological conditions in cattle.     

Isolation and determination of the amino acid sequence of chicken calcitonin I from chicken ultimobranchial glands

A radioimmunoassay for chicken calcitonin in chicken ultimobranchial glands was established utilizing a rabbit antiserum against eel calcitonin. This assay method, which is about 100 times as sensitive as the usual bioassay for hypocalcemic activity, was used for monitoring chicken calcitonin during its purification. The immunoreactivity in chicken ultimobranchial extract was separated by SP-Sephadex C-25 chromatography into two fractions. Chicken calcitonin I, which was occurred in the major immunoreactive fraction, was further purified to homogeneity as shown by reverse phase HPLC. In the end, 39 nmol of chicken calcitonin I was obtained from 3,384 chickens following a 12,000-fold purification. The complete amino acid sequence of purified chicken calcitonin I was determined to be H-Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Ly s-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-NH2 and confirmed by synthesis. The specific biological activity of chicken calcitonin I (4,500 MRCU/mg) was identical to that of eel calcitonin, which has the highest specific biological activity among the calcitonins so far isolated. Chicken calcitonin I resembled the calcitonins from the ultimobranchial glands both of salmon and eel in sequence, biological activity, and immunological property.     

Simple and rapid procedure for the purification of lipoprotein(a)

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein-like particle displaying strong athero-thrombotic properties. Highly purified Lp(a) is increasingly requested for standardization of Lp(a) measurements and for biological studies. Several procedures have been described for Lp(a) separation and purification but none of them appear completely suitable. We present here a procedure for Lp(a) purification based on sequential elutions after lysine-Sepharose affinity chromatography. We were able to identify four distinct subspecies of Lp(a) showing different affinity to ε-amino groups of lysine-Sepharose, simply by modifying molarity and pH of the eluents; the fraction obtained in highly purified state represented the major form and could be eluted with 0.5 M sodium phosphate buffer (pH 4.4). Advantages of this procedure are represented by simplicity, rapidity and final yield.     

Applications of bromelain from pineapple waste towards acne

Bromelain is a proteolytic mixture obtained from pineapple (Ananas comosus (L. Merr)). It has diversified clinical properties and is used in alleviation of cancer, inflammation and oxidative stress. The current study focuses on extraction of bromelain from different parts of pineapple such as core, crown, fruit, peel and stem. The extracted enzyme was precipitated using ammonium sulphate at 40% saturation followed by dialysis. The fold of purification obtained for peel, crown, core, fruit and stem were found to be 1.948, 1.536, 1,027, 1.989, and 1.232 respectively. Bromelain activity was estimated using Azocasein assay, the highest activity was seen in peel at 3.417 U/μg. Antimicrobial activity and MIC of the bromelain purified and crude fractions was studied against the test organisms. Peel crude and purified extract exhibited highest inhibitory effect towards S. aureus followed by P. acne. The antioxidant activity was evaluated using DPPH antioxidant assay. IC50 values peel, fruit, stem and crown are found to be 13.158 μg/ml, 24.13 μg/ml and 23.33 μg/ml and 113.79 μg/ml respectively. The purified bromelain from peel, stem and crown was used to create a facewash formulation towards pathogens frequently associated with skin infections. Common skin pathogens like S. aureus and P. acne were found highly sensitive to its action. The aim of this study was to evaluate the potential of bromelain isolated from waste parts of pineapple in alleviation of acne due to its diverse antimicrobial properties.