Jisnuson Svasti

Antiviral isoflavonoid sulfate and steroidal glycosides from the fruits of Solanum torvum.

The C-4 sulfated isoflavonoid, torvanol A (1), and the steroidal glycoside, torvoside H (3), together with the known glycoside, torvoside A (2), were isolated from a MeOH extract of Solanum torvum fruits. Upon enzymatic hydrolysis with beta-glucosidase, torvoside A (2) and torvoside H (3) yielded the corresponding acetal derivatives 4 and 5, respectively. Torvanol A (1), torvoside H (3) and compound 5 exhibited antiviral activity (herpes simplex virus type 1) with IC(50) values of 9.6, 23.2 and 17.4 microg/ml, respectively. Compounds 1-5 showed no cytotoxicity (at 50 microg/ml) against BC, KB and Vero cell lines.

Vanillin suppresses metastatic potential of human cancer cells through PI3K inhibition and decreases angiogenesis in vivo.

Vanillin, a food flavoring agent, has been shown to suppress cancer cell migration and metastasis in a mouse model, but its mechanism of action is unknown. In this report, we have examined the antimetastatic potential of vanillin and its structurally related compounds, vanillic acid, vanillyl alcohol, and apocynin on hepatocyte growth factor (HGF)-induced migration of human lung cancer cells by the Transwell assay. Vanillin and apocynin could inhibit cell migration, and both compounds selectively inhibited Akt phosphorylation of HGF signaling, without affecting phosphorylation of Met and Erk. Vanillin and apocynin could inhibit the enzymatic activity of phosphoinositide 3-kinase (PI3K), as revealed by an in vitro lipid kinase assay, suggesting that inhibition of PI3K activity was a mechanism underlying the inhibitory effect on cancer cell migration, and the presence of an aldehyde or ketone group in the vanillin structure was important for this inhibition. Vanillin and apocynin also inhibited angiogenesis, determined by the chick chorioallantoic membrane assay.

Mucoadhesive curcumin nanospheres: Biological activity, adhesion to stomach mucosa and release of curcumin into the circulation

Although mucoadhesive drug carriers for the gastro-intestinal tract (GIT) have been reported, the mucoadhesive property and drug release characteristics have never been evaluated separately, whilst the adherence of the carriers to the surface of GIT has not been directly visualized. Here, a monopolymeric carrier made from ethylcellulose (EC) and a dipolymeric carrier made from a blend of methylcellulose (MC) and EC (ECMC) were easily fabricated through a self-assembling process and yielded the highest reported curcumin loading of ~48-49%. Both curcumin loaded ECMC (C-ECMC) and curcumin loaded EC (C-EC) particles showed an in vitro free radical scavenging activity and a dose-dependent in vitro cytotoxic effect towards MCF-7 human breast adenocarcinoma and HepG2 hepatoblastoma cells in tissue culture. The in vivo evaluation of their adherence to stomach mucosa and their ability to release curcumin into the circulation were carried out through quantification of curcumin levels in the stomach tissue and in blood of mice orally administered with the two spheres. Direct evidence of the adherence of the C-EC and C-ECMC particles along the mucosal epithelia of the stomach is also presented for the first time through SEM images. The mucoadhesive property of self-assembled C-EC nanoparticles is discussed.

Detection of cathepsin B up-regulation in neoplastic thyroid tissues by proteomic analysis

Nodular or multinodular goiter is the most common non‐neoplastic thyroid disease and may be difficult to distinguish from true neoplastic thyroid diseases using microscopic criteria. We have used two‐dimensional gel electrophoresis to study the protein patterns of thyroid tissues including normal thyroid, multinodular goiter, diffuse hyperplasia, follicular adenoma, follicular carcinoma and papillary carcinoma. Specific proteins, in the region of molecular mass 15–30 kDa and isoelectric point 4.5–6.5, were identified by electrospray tandem mass spectrometry and protein sequencing. The most distinctive protein found is cathepsin B, which could be detected as four spots, with differential expression in different thyroid diseases. In particular, two of these cathepsin B spots CB2 and CB3 are strongly up‐regulated in neoplastic diseases, compared to non‐neoplastic diseases. In addition, overexpression of ATP synthase D chain and prohibitin were observed in papillary carcinoma, which should allow it to be differentiated from follicular carcinoma. Changes in expression of other proteins were also observed in disease states compared to normal tissues, namely translationally controlled tumor protein, thioredoxin peroxidase 1, glutathione‐S‐transferase P, DJ‐1 protein, superoxide dismutase (Cu, Zn), and heat shock protein 27, but these changes are less characteristic, so they do not allow the differentiation between neoplastic and non‐neoplastic tissues. Thus, the proteomic approach is a useful diagnostic tool for studying diseases involving the thyroid nodule.

Structural Insights into Rice BGlu1 β-Glucosidase Oligosaccharide Hydrolysis and Transglycosylation

The structures of rice BGlu1 beta-glucosidase, a plant beta-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 A and 1.55 A resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) beta-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long beta-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa beta-glucosidase B, which hydrolyzes similar oligosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-beta-glucanase/beta-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/beta-II beta-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, I179, N190 and N245 did appear to interact with the substrates.

Proteomic studies of cholangiocarcinoma and hepatocellular carcinoma cell secretomes.

Cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) occur with relatively high incidence in Thailand. The secretome, proteins secreted from cancer cells, are potentially useful as biomarkers of the diseases. Proteomic analysis was performed on the secreted proteins of cholangiocarcinoma (HuCCA-1) and hepatocellular carcinoma (HCC-S102, HepG2, SK-Hep-1, and Alexander) cell lines. The secretomes of the five cancer cell lines were analyzed by SDS-PAGE combined with LC/MS/MS. Sixty-eight proteins were found to be expressed only in HuCCA-1. Examples include neutrophil gelatinase-associated lipocalin (lipocalin 2), laminin 5 beta 3, cathepsin D precursor, desmoplakin, annexin IV variant, and annexin A5. Immunoblotting was used to confirm the presence of lipocalin 2 in conditioned media and cell lysate of 5 cell lines. The results showed that lipocalin 2 was a secreted protein which is expressed only in the conditioned media of the cholangiocarcinoma cell line. Study of lipocalin 2 expression in different types of cancer and normal tissues from cholangiocarcinoma patients showed that lipocalin 2 was expressed only in the cancer tissues. We suggest that lipocalin 2 may be a potential biomarker for cholangiocarcinoma.

Characterization of a rice β-glucosidase highly expressed in flower and germinating shoot

The cDNAs for two β-glucosidase (E.C. 3.2.1.21) isozymes from rice (Oryza sativa L.), designated bglu1 and bglu2, were cloned and sequenced. The cDNA sequences for bglu1 and bglu2 included open reading frames encoding 504 and 500 amino acid precursor proteins, respectively. Both of these enzymes appeared to enter the secretory pathway, as judged by their N-terminal signal sequences. Southern blots using gene-specific probes indicated that bglu1 and bglu2 were single copy genes. The bglu1 and bglu2 mRNAs were highly expressed in the shoot during germination, with a similar time course. However, differences were seen in expression in mature plants, where bglu1 was highly expressed in flowers, but bglu2 was not. A recombinant thioredoxin fusion protein produced from the bglu1 cDNA in redox-deficient Escherichia coli (BGlu1) hydrolyzed p-nitrophenol β-d-glucoside, β-d-fucoside, and other p-nitrophenol β-d-glycosides, and was strongly inhibited by glucono-1,5-lactone. It also hydrolyzed some natural glucosides, including the rice-derived pyridoxine-5′-O-β-d-glucoside, and hydrolyzed and transglycosylated short β-(1→3) and β-(1→4) linked gluco-oligosaccharides. Based on the results, possible functions of BGlu1 include hydrolysis and recycling of oligosaccharides generated from rapid cell wall expansion during seed germination and flower expansion, and release of the coenzyme pyridoxine from its glucose-conjugated storage form.

Proteomic analysis and abrogated expression of O-GlcNAcylated proteins associated with primary breast cancer

O‐GlcNAcylation is a dynamic PTM of nuclear and cytoplasmic proteins, regulated by O‐GlcNAc transferase (OGT) and O‐GlcNAcase, which catalyze the addition and removal of O‐GlcNAc, respectively. This modification is associated with glucose metabolism, which plays important roles in many diseases including cancer. Although emerging evidence reveals that some tumor‐associated proteins are O‐GlcNAc modified, the total O‐GlcNAcylation in cancer is still largely unexplored. Here, we demonstrate that O‐GlcNAcylation was increased in primary breast malignant tumors, not in benign tumors and that this augmentation was associated with increased expression of OGT level. Using 2D O‐GlcNAc immnoblotting and LC‐MS/MS analysis, we successfully identified 29 proteins, with seven being uniquely O‐GlcNAcylated or associated with O‐GlcNAcylation in cancer. Of these identified proteins, some were related to the Warburg effect, including metabolic enzymes, proteins involved in stress responses and biosynthesis. In addition, proteins associated with RNA metabolism, gene expression, and cytoskeleton were highly O‐GlcNAcylated or associated with O‐GlcNAcylation. Moreover, OGT knockdown showed that decreasing O‐GlcNAcylation was related to inhibition of the anchorage‐independent growth in vitro. These data indicate that aberrant protein O‐GlcNAcylation is associated with breast cancer. Abnormal modification of these O‐GlcNAc‐modified proteins might be one of the vital malignant characteristics of cancer.

Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts.

Four novel antibacterial peptides, Leucrocin I-IV from Siamese crocodile white blood cell extracts were purified by reverse phase high performance liquid chromatography (RP-HPLC). Leucrocins exhibit strong antibacterial activity towards Staphylococcus epidermidis, Salmonella typhi and Vibrio cholerae. The peptides were 7-10 residues in length with different primary structure. The amino acid sequence of Leucrocin I is NGVQPKY with molecular mass around 806.99 Da and Leucrocin II is NAGSLLSGWG with molecular mass around 956.3 Da. Further, the interaction between peptides and bacterial membranes as part of their killing mechanism was studied by fluorescence and electron microscopy. The outer membrane and cytoplasmic membrane was the target of action of Leucrocins as assayed in model membrane by release of β-galactosidase due to the membrane permeabilization. Finally, the hemolytic effect was tested against human red blood cell. Leucrocin I, III and IV showed less toxicity against human red blood cells than Leucrocin II.

The heterogeneity of the protamines from human spermatozoa

Nuclear basic protein from ejaculated human spermatozoa were labelled with iodo[14C1]acetic acid and fractionated by ion-exchange chromatography into several pools (named A-K). Gel electrophoresis indicated that the minor protamine components, were present in pool D and that, of the major protamine components, component 1 (pools E, F, G, H) was well separated from the unresolved mixture of component 2 and component 3 (pools I, J, K). Pools G and J were free of other contaminants. Pools D, G, and J produced different radioactive peptides on digestion with trypsin and with thermolysin, and also had quite distinct amino acid compositions. This suggests that the heterogeneity of human protamines is caused by differences in amino acid sequence. Major component 1 also seems to be heterogenous, since it was found in two distinct peaks (pools E and G), but post-translational modification as a cause of the two types of component 1 has not been ruled out. Although all the human protamine components are similar to other mammalian protamines in containing half-cysteine and tyrosine, they also have unique common features such as high histidine and high glutamic acid contents.