Epigenetic mechanisms are involved in regulating chromatin structure and gene expression through repression. In this study, we show that histone deacetylase inhibitors (DAIs) that alter the acetylation of histones in chromatin enhance the expression of several genes on tumor cells including: MHC class I, II, and the costimulatory molecule CD40. Enhanced transcription results in a significant increase in protein expression on the tumor cell surface, and expression can be elicited on some tumors that are unresponsive to IFN-γ. The magnitude of induction of these genes cannot be explained by the effect of DAIs on the cell cycle or enhanced apoptosis. Induction of class II genes by DAIs was accompanied by activation of a repressed class II transactivator gene in a plasma cell tumor but, in several other tumor cell lines, class II was induced in the apparent absence of class II transactivator transcripts. These findings also suggest that the abnormalities observed in some tumors in the expression of genes critical to tumor immunity may result from epigenetic alterations in chromatin and gene regulation in addition to well-established mutational mechanisms.

Background: All known ligands of EGF receptor (EGFR) are characterized by the EGF motif and generated from transmembrane precursors. Results: Prolidase, a cytosolic dipeptidase devoid of EGF motif, binds and activates EGFR independent of its dipeptidase activity when present outside of cell. Conclusion: Prolidase is a novel EGFR ligand.Significance: This shows a new function of prolidase and new mechanism of EGFR activation.

Allyl isothiocyanate (AITC) occurs in many commonly consumed cruciferous vegetables and exhibits significant anti-cancer activities. Available data suggest that it is particularly promising for bladder cancer prevention and/or treatment. Here, we show that AITC arrests human bladder cancer cells in mitosis and also induces apoptosis. Mitotic arrest by AITC was associated with increased ubiquitination and degradation of ␣-and ␤-tubulin. AITC directly binds to multiple cysteine residues of the tubulins. AITC induced mitochondrion-mediated apoptosis, as shown by cytochrome c release from mitochondria to cytoplasm, activation of caspase-9 and caspase-3, and formation of TUNEL-positive cells. Inhibition of caspase-9 blocked AITCinduced apoptosis. Moreover, we found that apoptosis induction by AITC depended entirely on mitotic arrest and was mediated via Bcl-2 phosphorylation at Ser-70. Pre-arresting cells in G 1 phase by hydroxyurea abrogated both AITC-induced mitotic arrest and Bcl-2 phosphorylation. Overexpression of a Bcl-2 mutant prevented AITC from inducing apoptosis. We further showed that AITC-induced Bcl-2 phosphorylation was caused by c-Jun N-terminal kinase (JNK), and AITC activates JNK. Taken together, this study has revealed a novel anticancer mechanism of a phytochemical that is commonly present in human diet.Allyl isothiocyanate (AITC) 2 is a naturally occurring compound that possesses both antimicrobial and anticancer activities. Many commonly consumed cruciferous vegetables are rich sources of AITC, such as mustard, horseradish, wasabi, and cabbage. Its bactericidal and fungicidal activities were demonstrated against a variety of pathogens, and its anticancer activities were shown in both cultured cancer cell lines and animal tumor models (1). Bioavailability of AITC is extremely high; nearly 90% of orally administered AITC is absorbed (1). Although available evidence indicates that the anticancer activity of AITC is neither cell-nor tissue-specific, we have recently shown that AITC is selectively delivered to bladder tissue through urinary excretion and potently inhibits cancer development and muscle invasion in an orthotopic rat bladder cancer model (2). Moreover, an AITC-rich mustard seed powder also strongly inhibited bladder cancer development and muscle invasion in vivo (3). Thus, AITC is highly promising for bladder cancer prevention and/or treatment. These results are also consistent with epidemiological studies showing an inverse association between consumption of cruciferous vegetables and bladder cancer risk (4, 5). In light of these findings, this study focuses on human bladder cancer cells.Previous studies have shown that AITC causes cell cycle arrest and apoptosis in cancer cell lines of different tissue origins in vitro and in several tumor xenograft models in vivo, and it modulates many genes and proteins involved in cancer cell survival and proliferation (1). In our recent studies, both AITC and the AITC-rich mustard seed powder mentioned above arrested bladder cancer cells in G 2 /M ...

A comprehensive synthetic approach for the determination of continuous antigenic sites of proteins is presented. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire primary structure of the protein under study. Its application to the alpha-chain of human haemoglobin afforded, for the first time, a full profile of immunochemically active alpha-chain peptides and enabled the localization of all the major continuous antigenic sites of this haemoglobin subunit.

Proteins in saliva are needed for preprocessing food in the mouth, maintenance of tooth mineralization, and protection from microbial pathogens. Novel insights into human lineage-specific functions of salivary proteins and clues to their involvement in human disease can be gained through evolutionary studies, as recently shown for salivary amylase AMY1 and salivary agglutinin DMBT1/gp340. However, the entirety of proteins in saliva, the salivary proteome, has not yet been investigated from an evolutionary perspective. Here, we compared the proteomes of human saliva and the saliva of our closest extant evolutionary relatives, chimpanzees and gorillas, using macaques as an outgroup, with the aim to uncover features in saliva protein composition that are unique to each species. We found that humans produce a waterier saliva, containing less than half total protein than great apes and Old World monkeys. For all major salivary proteins in humans, we could identify counterparts in chimpanzee and gorilla saliva. However, we discovered unique protein profiles in saliva of humans that were distinct from those of nonhuman primates. These findings open up the possibility that dietary differences and pathogenic pressures may have shaped a distinct salivary proteome in the human lineage.

The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major 'continuous' antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of 'structurally inherent antigenic sites', namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.

Expression of extrachromosomal tet genes increased the susceptibility of gram-negative bacteria to specific aminoglycoside antibiotics. The magnitude of the increase in susceptibility was dependent on the amount and the class of the tet gene product (designated Tet) and the bacterial species in which the tet gene was expressed. Truncated Tet proteins that contained more than the first 33, but no more than the first 97, N-terminal amino acids of Tet also increased the susceptibility to aminoglycosides and complemented the potassium uptake defects in Escherichia coli. The primary structure of this N-terminal Tet fragment has the hydropathic characteristics of a multimeric, transmembrane structure and is highly conserved in three different classes of Tet proteins.Four classes of plasmid-encoded tet genes (A, B, C, and D) in gram-negative bacteria have been described (25). These four classes encode related inner membrane Tet proteins that are distinguished by differences in amino acid sequence and the relative levels of resistance to tetracycline and tetracycline analogs which they confer (18,23,26). Expression of tet genes has several pleiotropic effects. Tet proteins mediate tetracycline efflux (18,25), complement certain defects in potassium uptake (10), and confer increased susceptibility to heavy metals (14) and specific organic acids (2,15,20). Overexpression of tet can also reduce viability (18,27,28). We report here that tet expression also increases the susceptibility of gram-negative bacteria to specific aminoglycoside antibiotics. Defining the physiological or structural differences between tetracyclineresistant (Tcr) and tetracycline-sensitive (Tcs) ColEl origin of plasmid DNA replication (32). The regulation of the pCC42 tet structural gene by the tetR repressor was verified by comparing the growth rates of HB101, HB101(pBR322), and HB101(pCC42) in L broth containing 20 ,ug of tetracycline per ml, a concentration that is inhibitory for Tcs HB101, with and without prior derepression (induction) of the tetR repressor with 0.5 ,ug of acid-inactivated tetracycline per ml. Acid-inactivated tetracycline was prepared by refluxing tetracycline in 0.2 N H2S04 for 20 h.The pH was then adjusted to 6.0 with NaOH, and contaminating 5a,6-anhydrotetracycline was removed by precipitation in the cold. Chromatographic analysis on octadecylsilane columns with 30% methanol-70% 1 mM EDTA indicated that the final product contained less than 1.0% unreacted tetracycline. Accordingly, acid-inactivated tetracycline prepared in this manner had no antibacterial activity at 150 ,ug/ml against E. coli. Acid-inactivated tetracycline was approximately 10-fold more effective than tetracycline at inducing tet gene expression (data not shown).Plasmid pCC100 was recovered from mutagenized HB101(pBR322) by selecting for Tcr at 75 p.g/ml. Preliminary data indicate that the segment of the pCC100 plasmid that is responsible for the increased Tcr is not located within the tet gene itself, but lies within the PstI-PvuII fragment that contains...

Previous studies indicated that human and bovine factor VII exhibit 71% amino acid sequence identity. In the present study, competition binding experiments revealed that the interaction of human factor VII with cell-surface human tissue factor was not inhibited by 100-fold molar excess of bovine factor VII. This rinding indicated that bovine and human factor VII are not structurally homologous in the region(s) where human factor VII interacts with human tissue factor. On this premise, we synthesized three peptides corresponding to regions of human factor VII that exhibited marked structural dissimilarity to bovine factor VII; these regions of dissimilarity included residues [195][196][197][198][199][200][201][202][203][204][205][206][263][264][265][266][267][268][269][270][271][272][273][274][314][315][316][317][318][319][320][321][322][323][324][325][326] inhibited the interaction of factor VII with cell-surface tissue factor and the activation of factor X by a complex of factor VIla and tissue factor half-maximally at concentrations of 1-2 mM. A structurally rearranged form of peptide 195-206 containing an aspartimide residue inhibited these reactions half-maximally at concentrations of 250-300 uiM. In contrast, neither peptide 263-274 nor peptide 314-326, at 2 mM concentration, significantly affected either factor Vila interaction with tissue factor or factor Vila-mediated activatiop of factor X. Our data provide presumptive evidence that residues 195-206 of human factor VII are involved in the interaction of human factor VII with the extracellular domain of human tissue factor apoprotein.