Bioactivity guided isolation of anticancer constituents from leaves of Alnus sieboldiana (Betulaceae).

The leaves of the Japanese Alnus sieboldiana have been extracted with n-hexane and then with methanol. A bioactivity-guided approach based on MTT assay for growth inhibition and quantitative real-time PCR for TNF-α inhibitory activity was taken to identify the active compounds in EtOAc soluble fraction of the methanol extract. From this active fraction, seven compounds have been isolated and four compounds (pinosylvin, galangin, quercetin and methyl gallate) have been examined for their dose-response effect on the viability of A549 cells and on TNF-α inhibitory activity. Based on MTT assay, all of the four examined compounds inhibit growth of human lung cancer cells. Among four tested compounds only galangin (3,5,7-trihydroxyflavone) significantly inhibited TNF-α gene expression in A549 cells (IC50 = 94 µM). Taken together, this finding suggests that galangin may be useful in cancer prevention.

DNA-binding activity of TNF-alpha inducing protein from Helicobacter pylori.

Tumor necrosis factor-alpha (TNF-alpha) inducing protein (Tipalpha) is a carcinogenic factor secreted from Helicobacter pylori (H. pylori), mediated through both enhanced expression of TNF-alpha and chemokine genes and activation of nuclear factor-kappaB. Since Tipalpha enters gastric cancer cells, the Tipalpha binding molecules in the cells should be investigated. The direct DNA-binding activity of Tipalpha was observed by pull down assay using single- and double-stranded genomic DNA cellulose. The surface plasmon resonance assay, indicating an association between Tipalpha and DNA, revealed that the affinity of Tipalpha for (dGdC)10 is 2400 times stronger than that of del-Tipalpha, an inactive Tipalpha. This suggests a strong correlation between DNA-binding activity and carcinogenic activity of Tipalpha. And the DNA-binding activity of Tipalpha was first demonstrated with a molecule secreted from H. pylori.

Purification, crystallization and preliminary X-ray crystallographic analysis of human CCG1-interacting factor B.

A novel human factor CIB (CCG1-interacting factor B) has been isolated using the yeast two-hybrid system. The 22 kDa CIB protein has been expressed in Escherichia coli, purified to homogeneity and crystallized in a form suitable for crystallographic studies. The protein was crystallized in the orthogonal space group P2(1)2(1)2(1), with unit-cell parameters a = 43.60 (2), b = 44.45 (1), c = 110.70 (5) A and one molecule in the asymmetric unit. The crystal diffracted beyond 2.2 A resolution using synchrotron radiation.

Functional interaction of general transcription initiation factor TFIIE with general chromatin factor SPT16/CDC68.

BACKGROUND: Transcriptional initiation of class II genes is one of the major targets for the regulation of gene expression and is carried out by RNA polymerase II and many auxiliary factors, which include general transcription initiation factors (GTFs). TFIIE, one of the GTFs, functions at the later stage of transcription initiation. As recent studies indicated the possibility that TFIIE may have a role in chromatin transcriptional regulation, we isolated TFIIE-interacting factors which have chromatin-related functions. RESULTS: Using the yeast two-hybrid screening system, we isolated the C-terminal part of the human homologue of Saccharomyces cerevisiae (y) Spt16p/Cdc68p, a general chromatin factor. The C-terminal part of human SPT16/CDC68 directly interacts with TFIIE, and ySpt16p/Cdc68p also interacts with yTFIIE (Tfa1p/Tfa2p), thus indicating the existence of an evolutionarily conserved interaction between TFIIE and SPT16/CDC68. Functional interaction of yTFIIE and ySpt16p/Cdc68p was examined using a conditional yTFIIE-alpha mutant strain. Over-expression of ySpt16p/Cdc68p suppressed the phenotype of cold sensitivity of the yTFIIE-alpha-cs mutant strain, and in vitro binding assays revealed that yTFIIE-alpha-cs mutant protein showed diminished binding affinity to ySpt16p/Cdc68p. CONCLUSIONS: These observations indicate that general transcription initiation factor TFIIE functionally interacts with general chromatin factor SPT16/CDC68, a finding which provides new insight into the involvement of TFIIE in chromatin transcription. This may well lead to a breakthrough in relationships between the transcription initiation process and structural changes in chromatin.

A human homologue of yeast anti-silencing factor has histone chaperone activity.

BACKGROUND: Structural changes in chromatin play essential roles in regulating eukaryotic gene expression. Silencing, potent repression of transcription in Saccharomyces cerevisiae, occurs near telomeres and at the silent mating-type loci, as well as at rDNA loci. This type of repression relates to the condensation of chromatin that occurs in the heterochromatin of multicellular organisms. Anti-silencing is a reaction by which silenced loci are de-repressed. Genetic studies revealed that several factors participate in the anti-silencing reaction. However, actions of factors and molecular mechanisms underlying anti-silencing remain unknown. RESULTS: Here we report the functional activity of a highly evolutionarily conserved human factor termed CIA (CCG1-interacting factor A), whose budding yeast homologue ASF1 has anti-silencing activity. Using yeast two-hybrid screening, we isolated histone H3 as an interacting factor of CIA. We also showed that CIA binds to histones H3/H4 in vitro, and that the interacting region of histone H3 is located in the C-terminal helices. Considering the functional role of CIA as a histone-interacting protein, we found that CIA forms a nucleosome-like structure with DNA and histones. CONCLUSIONS: These results show that human CIA, whose yeast homologue ASF1 is an anti-silencing factor, possesses histone chaperone activity. This leads to a better understanding of the relationship between chromatin structural changes and anti-silencing processes.

Relationship between the subcellular localization and structures of catalytic domains of FKBP-type PPIases.

The Schizosaccharomyces pombe gene, fkp39(+), encoding a homolog of FKBP(FK506 binding protein)-type peptidyl prolyl cis-trans isomerase (PPIase), was isolated and the primary structure was determined. This gene product (SpFkbp39p) showed PPIase enzymatic activity in a chymotrypsin-dependent enzyme assay involving recombinant SpFkbp39p. Comparison of the primary structures of the catalytic domains of FKBPs, including SpFkbp39p, revealed that FKBPs could be classified into four groups. This categorization corresponding to the known subcellular localization of the FKBPs, makes the prediction of the subcellular localization of FKBPs based on their primary structures feasible. SpFkbp39p was considered to be a member of the nuclear-type FKBP group from this relationship between primary structure and subcellular localization. An immunofluorescence assay against HA-epitope-tagged SpFkbp39p revealed that SpFkbp39p is localized to the nucleus, as predicted. Residues conserved in a "group-specific" manner in the catalytic domain were mapped to their corresponding three-dimensional positions; these "group-specific" residues were located in close proximity in distinct regions mostly on the protein surface, which implies the presence of "group-specific" regulatory functional regions. We also found that nuclear-type FKBPs, including SpFkbp39p, have two highly conserved domains other than catalytic ones, with further basic and acidic charged regions, especially in the case of nuclear-type FKBPs. This is the first report indicating that there is a rule for the relationship between the subcellular localization and structure of the catalytic domain of a FKBP.

Isolation and characterization of a cDNA encoding a human TFIID subunit containing a variety of putative structural motifs including direct repeats.

A cDNA encoding human TFIID subunit p30 beta (hp30 beta) was isolated and sequenced. Comparison of the deduced 211 amino acid (aa) sequence with that of Drosophila melanogaster p30 beta (dp30 beta) showed 45% identity in overall length, and subunits with highly conserved C-terminal (81% identity) and less conserved N-terminal (21% identity) regions. Three acidic and basic regions were alternately placed. Potent target sites for protein kinases and a putative nuclear localization signal (NLS) were located in the N-terminal region. Interestingly, analysis of the aa sequence of p30 beta led us to find several interesting structural motifs/regions including 1) direct repeats, 2) a region similar to histone H4 between the two direct repeats and 3) acidic and hydrophobic aa surfaces in the potent helical region. Northern blot analysis showed ubiquitous expression of mRNA corresponding to hp 30 beta.

The sequence of a Xenopus laevis TFIID subunit reveals remarkable conservation among vertebrates.

A cDNA clone encoding a Xenopus laevis (Xl) homologue of human transcription factor IID (TFIID) subunit p80 was isolated and sequenced. The deduced 618-amino-acid (aa) sequence was compared to the homologous from human, mouse, rat and Drosophila melanogaster (Dm). A highly conserved region exists in the central region among these species. In contrast, the C-terminal region has significant homology among vertebrates, whereas the corresponding region of the Dm homologue shows poor homology.

Reduction in tumor formation on polyethylene by collagen immobilization.

After surface modification with collagen immobilization through covalent binding, porous polyethylene pieces were implanted subcutaneously into the back of rats for 1 year and the tumorigenesis-reducing effect was examined. In the virgin pieces without collagen immobilization, tumors were observed in 11 out of 24 pieces implanted (45.8%). On the other hand, in the collagen immobilized pieces a tumor was found only in one of 24 implanted pieces (4.2%). These results suggest that immobilization of collagen on the surface of an artificial material through covalent binding is very effective for a reduction of tumor formation.

Reduction in tumour formation on porous polyethylene by collagen immobilization.

After surface modification with collagen immobilization through covalent bonding, porous polyethylene pieces with an average pore size of 400 microns were implanted subcutaneously into the back of rats for 1 yr. It was found that connective tissues with abundant blood vessels were formed clearly, filling more than 90% of the pore volume and bound firmly to the pore walls. A tumour was found in only one of 24 implanted pieces (4.2%). On the other hand, the virgin porous polyethylene pieces without collagen immobilization exhibited inflammatory reactions within the pores and the connective tissues produced filled only 15% of the pore volume. Formation of a malignant histiocytoma was observed in 11 of the 24 pieces which had been implanted (45.8%). Thus, immobilization of collagen on the surface of an artificial material through covalent bonding proved to be very effective not only for firm bonding with soft connective tissues but also for a reduction of tumour formation.

Soft tissue reaction to collagen-immobilized porous polyethylene: subcutaneous implantation in rats for 20 wk.

Collagen-immobilized porous polyethylene, in which the immobilization was through covalent bonding, and virgin porous polyethylene were implanted subcutaneously in rats from 1 to 20 wk. The results were the ingrowth of the connective tissue into collagen-immobilized porous polyethylene was rich and contained a low level of inflammatory cellular infiltration compared with that of virgin porous polyethylene. The material-tissue interface showed that the living body-originated collagen fibres were firmly anchored into the immobilized collagen layer. These results suggested that covalent immobilization of collagen on to the biomaterial surface is useful in promoting the ingrowth of soft tissue and the tissue adhesion.

Heterogeneity and tissue-specific expression of eukaryotic transcription factor S-II-related protein mRNA.

Two new cDNA clones for S-II-related proteins were isolated from a mouse liver cDNA library. Analyses of these clones suggested that S-II proteins are a family of proteins with relatively conserved C-terminal regions but variable N-terminal regions, and that some members of this family are expressed in a tissue-specific manner.

1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin.

The solution conformation of an antibacterial protein sapecin has been determined by 1H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4-12), one helix (residues 15-23), and two extended strands (residues 24-31 and 34-40). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP-5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three-dimensional structure determination has revealed that a basic-residue rich region and the hydrophobic surface face each other on the surface of sapecin.

Determination of the disulfide array in sapecin, an antibacterial peptide of Sarcophaga peregrina (flesh fly).

Sapecin is a 40-residue peptide containing 6 half-cystine residues. The disulfide structure of sapecin was determined by sequencing cystine-containing peptides obtained by digesting sapecin with thermolysin. Results showed that sapecin has a vortical structure fixed by 3 disulfide bonds between cysteine residues 3 and 30, 16 and 36, and 20 and 38, respectively, and that these disulfide bonds are essential for its antibacterial activity.