Isolation of an N-acetyl-D-glucosamine specific lectin from the rhizomes of Arundo donax with antiproliferative activity

A lectin with antiproliferative activity towards human cancer cell lines and mitogenic towards human peripheral blood mononuclear cells was purified from the rhizomes of Arundo donax (Linn.) by affinity chromatography on N-acetyl-d-glucosamine linked to epoxy-activated sepharose-6B. The pure preparation apparently yielded a single band of approximately 15 kDa on SDS-PAGE, pH 8.3, under both reducing and non-reducing conditions. The molecular mass of native lectin was 32 kDa as determined by gel filtration chromatography. This showed the lectin to be a dimer, with subunits not held together by disulphide linkages. The A. donax lectin (ADL) agglutinated rabbit erythrocytes and the agglutination was inhibited by N-acetyl-d-glucosamine and its di- and trimer. The lectin was thermostable upto 55 degrees C and showed optimum activity in the range of pH 7.0-9.0 and comprised of 2.1% carbohydrate content.     

Steroidal saponins from Smilax china and their anti-inflammatory activities

Steroidal saponins, 1, 2, 3 and 4, were isolated from the BuOH extract of Smilax china L., along with 13 known compounds, 5-17. Their structures were elucidated on the basis of MS, 1D and 2D NMR spectroscopic analyses and chemical evidence. In the bioassay tests, all compounds showed inhibitory effects on cyclooxygenase-2 enzyme (COX-2) activities at final concentration of 10(-5) M, and only compound 5 showed an inhibitory effect on production of TNFalpha (tumor necrosis factor alpha) in murine peritoneal macrophages at the same concentration.     

A novel mitogenic and antiproliferative lectin from a wild cobra lily, Arisaema flavum

A novel lectin having specificity towards a complex glycoprotein asialofetuin was purified from tubers of Arisaema flavum (Schott.) by affinity chromatography on asialofetuin-linked amino-activated silica beads. A. flavum gave a single peak on HPLC size exclusion and a single band on non-denatured PAGE at pH 4.5. The molecular mass of the lectin, as determined by gel filtration chromatography, was 56 kDa. In SDS-PAGE, pH 8.3, the lectin migrated as a single band of 13.5 kDa, under reducing and non-reducing conditions, indicating the homotetrameric nature. A. flavum lectin (AFL) readily agglutinated rabbit, rat, sheep, goat, and guinea pig erythrocytes but not human ABO blood group erythrocytes even after neuraminidase treatment. This lectin is stable up to 55 degrees C and does not require metal ions for its hemagglutination activity. AFL was completely devoid of sulphur containing amino acids and was rich in aspartic acid and glycine. In Oucterlony's double immunodiffusion, the antisera raised against A. flavum lectin showed distinct lines of identity with those of other araceous lectins. AFL showed potent mitogenic activity towards BALB/c splenocytes and human lymphocytes in comparison to Con A, a well-known plant mitogen. AFL also showed significant in vitro antiproliferative activity towards J774 and P388D1 murine cancer cell lines.     

Purification,characterization and immunostimulatory activity of polysaccharide from Cipangopaludina chinensis

In this study, we investigated the purification, preliminary characterization and immunostimulatory activity in vivo of polysaccharide from Cipangopaludina chinensis (CCPS). Firstly, crude CCPS was prepared by hot water extraction. And the crude CCPS was sequentially purified by chromatography of DEAE-52 and Sephadex G-100, resulting in two purified fractions of CCPS-1 and CCPS-2. We found the two fractions were homogeneous heteropolysaccharides mainly composed of rhamnose and glucose with the average molecular weight of 226 and 235 kDa, respectively. CCPS-2 was quite different from CCPS-1. It had much higher content of uronic acid and sulfuric radical. For immunostimulatory activity in vivo, crude CCPS could significantly increase the thymus and spleen indices, enhance the macrophage function, and increase the level of serum hemolysin in cyclophosphamide-treated mice, suggesting CCPS had a potent immunostimulatory activity and could be explored as a potential natural immunomodulatory agent     

Purification and functional characterization of AAV1, a novel P-III metalloproteinase, from Formosan Agkistrodon acutus venom

AAV1, an alkaline glycoprotein (GP), was purified from Agkistrodon acutus venom by two chromatographic steps on successive DEAE-Sephadex A-50 and Superdex 75 FPLC columns. AAV1 on SDS-PAGE under non-reducing conditions migrated as a monomeric and a polymeric forms with apparent molecular mass of 57 and 180 kDa, respectively. Upon reduction, it appeared as a single broad band with a mass of 50.3 kDa corresponding to the size of a typical P-III metalloproteinase acurhagin. The N-terminal sequence of an autoproteolytical 30 kDa-fragment of AAV1 showed a high homology to that of venom proteins with Metalloproteinase, Disintegrin-like, and Cysteine-rich (MDC) domains. Although it was devoid of cleaving activity toward gelatin, fibronectin and prothrombin, AAV1 preferentially digested the Aalpha chain of fibrinogen and followed by the Bbeta chain, leading to the inhibition of fibrinogen-induced platelet aggregation in elastase-treated human platelets. However, the proteolytic activity of AAV1 was completely inactivated by the chelating agent but not serine proteinase inhibitor. Furthermore, AAV1 could concentration-dependently inhibit platelet aggregation and suppress tyrosine phosphorylation of intracellular proteins in collagen- and convulxin-stimulated platelets, respectively. The interaction of MDC domains in AAV1 molecule with platelet GPVI was responsible for the inhibitory effect of AAV1 on collagen- and convulxin-induced platelet aggregation. Taken together, these pieces of evidence suggest that AAV1 from Formosan viper venom belongs to a new member of high-molecular mass metalloproteinase family and functions as a GPVI antagonist.     

Tay1 protein, a novel telomere binding factor from Yarrowia lipolytica

Inspection of the complete genome of the yeast Yarrowia lipolytica for the presence of genes encoding homologues of known telomere-binding proteins surprisingly revealed no counterparts of typical yeast Myb domain-containing telomeric factors including Rap1 or Taz1. Instead, we identified a gene, YALIOD10923g, encoding a protein containing two Myb domains, exhibiting a high degree of similarity to the Myb domain of human telomeric proteins TRF1 and TRF2 and homologous to an essential fission yeast protein Mug152 whose expression is elevated during meiosis. The protein, which we named Tay1p (telomere-associated in Yarrowia lipolytica 1), was purified for biochemical studies. Using a model Y. lipolytica telomere, we demonstrate that the protein preferentially binds to Y. lipolytica telomeric tracts. Tay1p binds along the telomeric tract as dimers and larger oligomers, and it is able to remodel the telomeric DNA into both looped structures and synaptic complexes of two model telomere DNAs. The ability of Tay1p to induce dimerization of telomeres in vitro goes in line with its oligomeric nature, where each oligomer can employ several Myb domains to form intermolecular telomere clusters. We also provide experimental evidence that Tay1p may be associated with Y. lipolytica telomeres in vivo. Together with its homologues from Schizosaccharomyces pombe and several basidiomycetous fungi (Sánchez-Alonso, P., and Guzman, P. (2008) Fungal Genet. Biol. 45, S54-S62), Tay1p constitutes a novel family of putative telomeric factors whose analysis may be instrumental in understanding the function and evolution of double-stranded DNA telomeric proteins.     

Synthesis of antiproliferative flavones from calycopterin, major flavonoid of Calycopteris floribunda Lamk

Eighteen new analogues of 5,3′-dihydroxy-3,6,7,8,4′-pentamethoxy-flavone, a potent natural cytotoxic and antimitotic flavone, were synthesized from calycopterin, the major flavonoid of Calycopteris floribunda Lamk., a traditional Asian medicinal plant. One of them, the 3′-amino substituted analogue, displayed almost the same activity as the reference compound. Pharmacomodulation at C-3′ on the B-ring, and at C-5,6,7 and 8 on the A-ring allowed to refine structure-activity relationships within the cytotoxic flavones series.     

A novel approach for over-expression, characterization, and isotopic enrichment of a homogeneous species of acyl carrier protein from Plasmodium falciparum

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. Holo-ACP is the obligatory substrate for the synthesis of acyl-ACPs which act as the carrier and donor for various metabolic reactions. Despite its interactions with numerous proteins in the cell, its mode of interaction is poorly understood. Here, we report the over-expression of PfACP in minimal medium solely in its holo form and in high yield. Expression in minimal media provides a means to isotopically label PfACP for high resolution multi-nuclear and multi-dimensional NMR studies. Indeed, the proton-nitrogen correlated NMR spectrum exhibits very high chemical shift dispersion and resolution. We also show that holo-PfACP thus expressed is amenable to acylation reactions using Escherichia coli acyl-ACP synthetase as well as by standard chemical methods.