Sunanta Ratanapo

Interaction of a mulberry leaf lectin with a phytopathogenic bacterium, P. syringae pv mori.

Two N-glycolylneuraminic acid-specific lectins, MLL 1 and 2, from leaves of Morus alba were studied for their anti-bacterial activity against P. syringae pv mori, which was a specific pathogenic bacterium of the mulberry leaf. MLL 1 but not MLL 2 was found to induce the agglutination of P. syringae pv mori. The MLL 1 can induce the agglutination only at the exponential phase of the bacterial growth in a liquid medium and the agglutination was specifically inhibited by N-glycolylneuraminic acid, N-acetylgalactosamine at 12.5 mM and bovine submaxillary mucin at 0.05 &mgr;g/ml.

Monodin-induced agglutination of Vibrio vulnificus, a major infective bacterium in black tiger prawn (Penaeus monodon)

Abstract 1. 1. Monodin, a sialic acid-specific lectin from P. monodon, was found to induce the agglutination of Vibrio vulnificus, a major infective bacterium of the prawn. 2. 2. The monodin-induced agglutination of V.vulnificus was specifically inhibited by N-acetylneuraminic acid and anti-monodin. 3. 3. During growth in a liquid medium, V. vulnificus can be induced to agglutinate only during the early stationary phase. 4. 4. An elevation of the monodin level was found in most of the prawns suffering from bacterial infection.

Sialic acid binding lectins from leaf of mulberry (Morus alba)

Abstract Two new lectins, MLL 1 and MLL 2, were purified from the leaves of mulberry ( Morus alba ) by a procedure consisting of ammonium sulfate precipitation, affinity chromatography on a N -acetylgalactosamine-agarose column, gel filtration on a Sephacryl S-200 column, anion-exchange chromatography using a DEAE-Sephacel column and gel filtration on a Sephadex G-75 column. Both MLL 1 and MLL 2 were found to be glycoproteins with neutral sugar contents of 8.8 and 40%, respectively. They showed the same native molecular weight of 51 000. Each consisted of subunit with molecular weight of 16 500. They were highly specific for N -glycolylneuraminic acid.

Cloning and expression analysis of the Bombyx mori α-amylase gene (Amy) from the indigenous Thai silkworm strain, Nanglai

Abstract &agr;-Amylase is a common enzyme for hydrolyzing starch. In the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), &agr;-amylase is found in both digestive fluid and hemolymph. Here, the complete genomic sequence of the Amy gene encoding &agr;-amylase from a local Thai silkworm, the Nanglai strain, was obtained. This gene was 7981 bp long with 9 exons. The full length Amy cDNA sequence was 1749 bp containing a 1503 bp open reading frame. The ORF encoded 500 amino acid residues. The deduced protein showed 81–54% identity to other insect &agr;-amylases and more than 50% identity to mammalian enzymes. Southern blot analysis revealed that in the Nanglai strain Amy is a single-copy gene. RT- PCR showed that Amy was transcribed only in the foregut. Transgenic B. mori also showed that the Amy promoter activates expression of the transgene only in the foregut.

Chebulin: Terminalia chebula Retz. fruit-derived peptide with angiotensin-I-converting enzyme inhibitory activity.

Angiotensin‐I–converting enzyme (ACE) plays an important role in blood pressure regulation. In this study, an ACE–hexapeptide inhibitor (Asp–Glu–Asn–Ser–Lys–Phe) designated as chebulin was produced from the fruit protein of Terminalia chebula Retz. by pepsin digestion, ultrafiltrated through a 3 KDa cut‐off membrane, a reverse‐phase high‐performance liquid chromatography, and nano‐liquid chromatography tandem mass spectrometry analysis. Chebulin was found to inhibit ACE in a noncompetitive manner, as supported by the structural model. It bounds to ACE by the hydrogen bond, hydrophobic and ionic interactions via the interactions of C‐terminal Phe (Phe‐6), and N‐terminal residues (Asp‐1 and Glu‐2) with the amino acid residues on noncatalytic sites of the ACE. The results showed that chebulin derived from fruits of T. chebula Retz. is a potential ACE–peptide inhibitor that could be used as a functional food additive for the prevention of hypertension and as an alternative to ACE inhibitor drug.

Bioactivities of Jc-SCRIP, a type 1 ribosome-inactivating protein from Jatropha curcas seed coat.

In this study, a type 1 RIP, designated as Jc‐SCRIP, was first isolated from the seed coat of Jatropha curcas Linn. It was purified by ammonium sulfate precipitation and chromatography on DEAE‐Sephacel™ and CM‐cellulose columns. Purification fold of Jc‐SCRIP increased 113.8 times, and the yield was 1.13% of the total protein in the final step. It was shown to be a monomeric glycoprotein with a molecular mass of 38 938 Da, as determined by MALDI‐TOF/MS. It exhibited hemagglutination activity and possessed strong N‐glycosidase activity. The antimicrobial activity of Jc‐SCRIP was tested against nine human pathogenic bacteria and one fungus; the most potent inhibitory activity was against Staphylococcus epidermidis ATCC 12228, with minimum inhibitory concentration value of 0.20 μm. Jc‐SCRIP demonstrated in vitro cytotoxicity against human breast adenocarcinoma cell line (MCF‐7), a colon adenocarcinoma (SW620), and a liver carcinoma cell line (HepG2), with IC50 values of 0.15, 0.25, and 0.40 mm, respectively. The results suggested that Jc‐SCRIP may be a potential natural antimicrobial and anticancer agent in medical applications.

Brucin, an antibacterial peptide derived from fruit protein of Fructus Bruceae, Brucea javanica (L.) Merr

A novel antibacterial peptide specific to Streptococcus pyogenes was produced from dried fruit protein of Brucea javanica (L.) Merr. A mixture of active peptides from the fruit protein was produced in vitro by pepsin hydrolysis. The hydrolysate was purified by reverse‐phase HPLC, and antimicrobial peptides active against Gram‐negative and Gram‐positive bacteria were analysed using SDS‐PAGE and nanoLC‐MS/MS. Here, four possible peptides were obtained and chemically synthesized for comparative study of the growth inhibition of Strep. pyogenes. One chemically synthesized peptide with a molecular mass of 1168·31 Da, His‐Thr‐Leu‐Cys‐Met‐Asp‐Gly‐Gly‐Ala‐Thr‐Tyr, showed the most potent antibacterial activity against Strep. pyogenes. This 11‐amino acid peptide was named Brucin. Its bacterial inhibitory activity was 16‐fold and 12·5‐fold higher than penicillin G and chloramphenicol, respectively, with a MIC value of 20 μmol l−1. The results suggest that Brucin, a potent antibiotic peptide, may be developed as an alternative drug for the treatment of the disease caused by Strep. pyogenes.

A Novel Anti-cancer Peptide Extracted from Gynura pseudochina Rhizome: Cytotoxicity Dependent on Disulfide Bond Formation

As current anticancer drugs have limitations, researchers have sought novel anti-cancer agents with high specificity and fewer side effects to normal cells. Bioactive peptides isolated from plants are of interest for their therapeutic potential and pharmaceutical development. In this study, a novel anticancer peptide, Gynurin (monomeric sequence: LNCCNLLL) was isolated and identified for the first time from the protein hydrolysate of Gynura pseudochina rhizomes. The presence of homodimeric Gynurin drastically inhibited human gastric cancer cells (KATO-III) with a greater inhibitory effect than 5-flurouracil without significant cytotoxicity of normal cells (Vero). By performing structural analysis using circular dichroism, Gynurin could form a random coil conformation with a partial helical structure in a mimic membrane model environment of 50% trifluoroethanol. However, in the presence of a reducing agent (DTT), treated Gynurin completely lost its cytotoxicity against KATO-III cells and adopted a random coil structure. Taken altogether, this study suggested that disulfide bond formation and peptide dimerization were crucial parameters for the anti-cancer activity of Gynurin. Consequently, dimeric Gynurin peptide could potentially be an effective therapeutic component for pharmaceutical development in the treatment of gastric cancer.

Partially Purified Gloriosa superba Peptides Inhibit Colon Cancer Cell Viability by Inducing Apoptosis Through p53 Upregulation

Background: Colon cancer is a major health problem worldwide. Available treatments such as surgery, chemotherapy, radiation and anticancer drugs are limited due to stage of cancer, side effects and altered biodistribution. The use of peptides extracted from natural products has appeared as a potential therapy. Gloriosa superba is known to contain colchicine and other alkaloids with anticancer activity. However, these peptides contained within the extracts have not been studied. This study, therefore, focuses on an investigation of anti–colon cancer activity from a partially purified protein hydrolysate of G superba rhizome. Methods: Dried G superba rhizome was extracted using 0.5% sodium dodecyl sulfate and digested with pepsin. The protein hydrolysates with molecular weight lesser than 3 kDa were collected and subjected for cell viability assay. Then, the partial purification of the protein hydrolysate was performed using reverse‐phase high‐performance liquid chromatography. Fractions containing anticancer peptides were investigated, and their effects on apoptosis and protein expression using apoptosis test and Western blot, respectively. Results: Partially purified peptides of G superba rhizome demonstrated anticolon activity in SW620 cells by inducing apoptosis through upregulation of p53 and downregulation of nuclear factor kappa B (NF‐&kgr;B). Conclusions: Consequently, G superba peptides showed high potential for further purification and development of anticolon therapeutics.