Nuclear factor kappaB (NF-kappaB) is a transcription factor, which plays an important role in inflammation, cell proliferation, apoptosis, and immunity in eukaryotes. In cancer cells, NF-kappaB induces resistance to anticancer chemotherapeutic agents by increasing cell proliferation and inhibiting apoptosis. Therefore, inhibition of NF-kappaB activation in cancer cells is advantageous in cancer therapy by lowing the resistance to chemotherapy. Several phytochemicals from fruits and vegetables have been reported to inhibit NF-kappaB activation, but the mechanisms of how the phytochemicals work have not been fully understood. The present study examines the effects of selected phytochemicals and apple extracts on TNF-alpha-induced NF-kappaB activation in human breast cancer MCF-7 cells. Apple extracts significantly inhibited the TNF-alpha-induced NF-kappaB activation at a dose of 5 mg/mL (p < 0.05). Curcumin also significantly blocked the TNF-alpha-induced NF-kappaB activation at doses of 10 and 20 microM (p < 0.05). Neither apple extracts nor curcumin affected phosphorylation of inhibitor of NF-kappaB-alpha (IkappaB-alpha); both significantly inhibited proteasomal activity of MCF-7 cells at doses of 2.5 and 5 mg/mL of apple extracts and 20 microM of curcumin (p < 0.05). These results suggest that apple extracts and curcumin have the capabilities of inhibiting TNF-alpha-induced NF-kappaB activation of MCF-7 cells by inhibiting the proteasomal activities instead of IkappaB kinase (IKK) activation.

Cdk4-mediated phosphorylation of Rb protein is inhibited by p16, a product of a possible tumor suppressor gene. We examined the expression of p16 and Rb protein by means of immunohistochemistry in 61 non-small cell lung cancers and have demonstrated an inverse relationship between the expression of p16 and Rb protein: 28/30 specimens that did not stain for p16 stained for Rb and 21/31 p16-positive specimens did not stain for Rb. Only 1 of the p16-negative specimens had a mutation of exon 2 of the CDKN2 gene. Our results indirectly support the theory that p16 expression is negatively regulated by the functional Rb protein.

A gene encoding a maltogenic amylase of Bacillus stearothermophilus ET1 was cloned and expressed in Escherichia coli. DNA sequence analysis indicated that the gene could encode a 69627-Da protein containing 590 amino acids. The predicted amino acid sequence of the enzyme shared 47Ϫ70% identity with the sequences of maltogenic amylase from Bacillus licheniformis, neopullulanase from B. stearothermophilus, and cyclodextrin hydrolase (CDase) I-5 from an alkalophilic Bacillus I-5 strain. In addition to starch, pullulan and cyclodextrin, B. stearothermophilus could hydrolyze isopanose, but not panose, to glucose and maltose. Maltogenic amylase hydrolyzed acarbose, a competitive inhibitor of amylases, to glucose and a trisaccharide. When acarbose was incubated with 10% glucose, isoacarbose, containing an A-1,6-glucosidic linkage was produced as an acceptor reaction product. B. stearothermophilus maltogenic amylase shared four highly similar regions of amino acids with several amylolytic enzymes. The β-cyclodextrinϪhydrolyzing activity of maltogenic amylase was enhanced to a level equivalent to the activity of CDase when its amino acid sequence between the third and the fourth conserved regions was made more hydrophobic by site-directed mutagenesis. Enhanced transglycosylation activity was observed in most of the mutants. This result suggested that the members of a subfamily of amylolytic enzymes, including maltogenic amylase and CDase, could share similar substrate specificities, enzymatic mechanisms and structure/function relationships.Keywords : Bacillus stearothermophilus; maltogenic amylase ; acarbose ; transglycosylation; site-directed mutagenesis.Many types of amylases with unique properties have been megaterium [7], and A-amylase of Thermoactinomyces vulgaris [8] have been reported to hydrolyze the A-1,4 linkages of pulluisolated and characterized for various applications in the starch industry [1,2]. These proteins share many structural and mecha-lan to produce panose. Amylolytic enzymes, such as cyclodextrin glucanotransferases (CGTase) and CDase exhibit their nistic characteristics. However, amylases can be divided into several groups according to substrate specificities, patterns of highest levels of activity on cyclomaltodextrins [9Ϫ11]. Some starch cleavage, transglycosylation or cyclization activities, and amylolytic enzymes, including debranching enzymes and structural features. Classical A-amylases (e.g. 1,4-A-D-glucan CGTase, catalyze transglycosylation by forming A-1,4 or A-1,6 glucanohydrolase) catalyze hydrolysis of A-1,4-glucosidic link-linkages. ages in starch, and different amylases give rise to oligosacchaJespersen et al.[12] used sequence alignments and structurerides with specific lengths of glucose as major product [2]. De-prediction models to predict the presence of A-amylase-type branching enzymes are capable of hydrolyzing A-1,6-glucosidic (β/A) 8 -barrel domains and the positions of the β-strands and Alinkages in starch and/or pullulan [1, 3Ϫ5] to produce maltotri-helices found in 47 amy...

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There have been few reports on genetic alterations in thymomas. To investigate the expression of p16 INK4A , RB, p53 and cyclin D1 in thymomas, we first examined 36 thymomas (non-invasive type, 16 cases; invasive type, 20 cases) and 3 thymic carcinomas, using immunohistochemistry. Abnormal expression of p16 INK4A , RB, p53 and cyclin D1 was observed in 18, 8, 10 and 7 cases, respectively. Only a subgroup of invasive thymomas and thymic carcinomas showed an inverse correlation between p16 INK4A and RB expression. Subsequently, we examined the 36 thymomas and 4 thymic carcinomas for mutations in p53 and CDKN2 genes, using PCR-SSCP and direct-sequencing analyses. No mutation of these genes was detected in the thymomas and thymic carcinomas examined. A polymorphism in the 3Ј untranslated region of exon 3 of CDKN2 was detected in 5 cases of thymoma. We searched for hypermethylation in the promoter region of CDKN2, observing it in 4 thymomas and 1 thymic carcinoma. Our data suggest that, unlike other more common cancers, alteration of the p53 gene may not play a significant role in the tumorigenesis of thymoma. However, inactivation of p16 INK4A and RB may play a role in the progression of thymoma and thymic carcinoma. Int.