Abrogation of G2 checkpoint specifically sensitize p53 defective cells to cancer chemotherapeutic agents.

BACKGROUND: Chkl is a checkpoint gene that is activated after DNA damage. It phosphorylates and inactivates Cdc25C at the late G2 phase. The inactivation of Cdc25C and consequently, the inactivation of Cdc2, are required for the G2 arrest induced by DNA damage. METHODS: We treated 184B5 cell line and its E6 transformed cell lines with adriamycin in the presence of staurosporine or UCNO1 and examined G2 arrest and cell death. RESULTS: We found that adriamycin induced a p53 and p21 response as well as a G1 arrest in 184B5 cells, but not in its E6 transformed cells. Staurosporine or UCNO1 abrogated the G2 arrest induced by adriamycin in both cell lines. In addition, staurosporine or UCNO1 specifically sensitized p53 incompetent cells to adriamycin. CONCLUSION: G2/M checkpoint abrogators can potentially enhance the cytotoxic effect of conventional chemotherapeutic reagents specifically to tumor cells.

Structure-directed discovery of potent non-peptidic inhibitors of human urokinase that access a novel binding subsite.

BACKGROUND: Human urokinase-type plasminogen activator has been implicated in the regulation and control of basement membrane and interstitial protein degradation. Because of its role in tissue remodeling, urokinase is a central player in the disease progression of cancer, making it an attractive target for design of an anticancer clinical agent: Few urokinase inhibitors have been described, which suggests that discovery of such a compound is in the early stages. Towards integrating structural data into this process, a new human urokinase crystal form amenable to structure-based drug design has been used to discover potent urokinase inhibitors. RESULTS: On the basis of crystallographic data, 2-naphthamidine was chosen as the lead scaffold for structure-directed optimization. This co-crystal structure shows the compound binding at the primary specificity pocket of the trypsin-like protease and at a novel binding subsite that is accessible from the 8-position of 2-napthamidine. This novel subsite was characterized and used to design two compounds with very different 8-substituents that inhibit urokinase with K(i) values of 30-40 nM. CONCLUSIONS: Utilization of a novel subsite yielded two potent urokinase inhibitors even though this site has not been widely used in inhibitor optimization with other trypsin-like proteases, such as those reported for thrombin or factor Xa. The extensive binding pockets present at the substrate-binding groove of these other proteins are blocked by unique insertion loops in urokinase, thus necessitating the utilization of additional binding subsites. Successful implementation of this strategy and characterization of the novel site provides a significant step towards the discovery of an anticancer clinical agent.

The structure of VanX reveals a novel amino-dipeptidase involved in mediating transposon-based vancomycin resistance.

VanX is a zinc-dependent D-alanyl-D-alanine dipeptidase that is a critical component in a system that mediates transposon-based vancomycin resistance in enterococci. It is also a key drug target in circumventing clinical vancomycin resistance. The structure of VanX from E. faecium has been solved by X-ray crystallography and reveals a Zn(2+)-dipeptidase with a unique overall fold and a well-defined active site confined within a cavity of limited size. The crystal structures of VanX, the VanX:D-alanyl-D-alanine complex, the VanX:D-alanine complex, and VanX in complex with phosphonate and phosphinate transition-state analog inhibitors, are also presented at high resolution. Structural homology searches of known structures revealed that the fold of VanX is similar to those of two proteins: the N-terminal fragment of murine Sonic hedgehog and the Zn(2+)-dependent N-acyl-D-alanyl-D-alanine carboxypeptidase of S. albus G.

Solution structure of an rRNA methyltransferase (ErmAM) that confers macrolide-lincosamide-streptogramin antibiotic resistance.

The Erm family of methyltransferases is responsible for the development of resistance to the macrolide-lincosamide-streptogramin type B (MLS) antibiotics. These enzymes methylate an adenine of 23S ribosomal RNA that prevents the MLS antibiotics from binding to the ribosome and exhibiting their antibacterial activity. Here we describe the three-dimensional structure of an Erm family member, ErmAM, as determined by NMR spectroscopy. The catalytic domain of ErmAM is structurally similar to that found in other methyltransferases and consists of a seven-stranded beta-sheet flanked by alpha-helices and a small two-stranded beta-sheet. In contrast to the catalytic domain, the substrate binding domain is different from other methyltransferases and adopts a novel fold that consists of four alpha-helices.

Behavioral and social correlates of escape from suppression of ovulation in female common marmosets housed with the natal family.

Although female common marmosets typically do not breed while housed with their natal families, up to half ovulate at least once while housed with the intact natal family, and a similar proportion conceive if an unrelated adult male is present in the group. In this study, we investigated the behavioral and social correlates of escape from suppression of ovulation by daughters housed in intact natal families or in families in which the father had been replaced by an unrelated adult male. Focal-animal behavioral data were collected from daughters that were (N = 7) or were not (N = 10) undergoing ovulatory cycles while housed with the natal family and from daughters that were (N = 5) or were not (N = 3) cycling or pregnant in families containing an unrelated male. Additionally, four cyclic and six acyclic females housed in intact natal families underwent simulated "prospecting" tests. Cyclic and acyclic daughters in intact natal families did not engage in sexual interactions with the father and showed few differences from one another in their interactions with the parents. Moreover, cyclic and acyclic daughters did not differ in their willingness to leave the family for short periods or to investigate an unfamiliar family in "prospecting" tests. However, daughters that underwent ovarian cycles in the presence of an unrelated male showed numerous behavioral differences from those in intact natal families, including frequent courtship and sexual behaviors with the male, reduced affiliative interactions with the mother, and elevated frequencies of aggressive display behavior. Moreover, these females were less likely to behave submissively towards the mother or the adult male. These findings suggest that both suppression of ovulation and inhibition of sexual behavior normally contribute to reproductive failure in female marmosets living with their natal families, and that the two components of suppression may become dissociated under specific social conditions.

M13-102: a vector for facilitating construction and improving quality of M13 shotgun libraries.

A modified vector, M13-102, is described which utilizes the previously reported M13-100 direct selection strategy for shotgun cloning [Guilfoyle and Smith, Nucleic Acids Res. 22 (1994) 100-107]. In these vectors, direct selection replaces the need for phosphatase treatment of vector DNA and is achieved by insertional inactivation of M13 gene X. When not inactivated, the engineered overproduction of the M13 gene X product mediates phage replication repression. M13-102 contains two new additions: (1) a sequence enabling triple-helix-mediated affinity capture (TAC) for purification of linearized vector DNA, and (2) universal primer sequences for wider compatibility with commercial instruments that support fluorescence-based sequencing. Using a biotinylated homopyrimidine oligodeoxyribonucleotide as third-strand probe, TAC is performed on streptavidin-coated magnetic beads [Ji et al., Genetics Analysis: Techniques and Applications 11 (1994) 43-47], and serves as a rapid and efficient alternative to gel purification. To reduce tandem insertions, phosphatase treatment of insert DNA was easily invoked without sacrificing cloning efficiency. The combined capabilities of direct selection, TAC purification and phosphatase treatment of inserts should facilitate library construction and improve overall library quality.

High-level expression of recombinant human FK-binding protein from a fusion precursor.

The human peptidyl-prolyl isomerase FK-binding protein (FKBP) was cloned as a fusion partner with CMP-KDO synthetase (CKS), and the resultant construct was characterized as an improved high-expression source for FKBP. The CKS-FKBP fusion was expressed as a soluble protein at levels approaching 1 gm/L in Escherichia coli fermentations. The fusion protein was purified to near homogeneity by a one-step ammonium sulfate fractionation of whole cell lysate. After selective cleavage, the fusion precursor produced yields approaching 300 mg of purified FKBP per liter of harvested culture, a approximately 30 to 60-fold increase over that observed for a nonfusion construct. Selective cleavage of the fusion partners was accomplished using either hydroxylamine or specific, limited proteolysis. Once separated from the CKS fusion partner, the FKBP was isolated in a single step by either reversed-phase HPLC or chromatography on Q-Sepharose. For comparison of physical and chemical properties, a nonfusion construct of recombinant human FKBP was expressed in E. coli and isolated. The purified FKBPs exhibited expected SDS-PAGE molecular weights and N-terminal sequences. The proteins had similar proton NMR spectra and binding to [3H]FK-506. The fusion construct, CKS-FKBP, was also found to bind [3H]FK-506. These data indicate that FKBP fused to the C-terminus of CKS folds independently of the fusion partner and suggests the fused FKBP adopts a conformation resembling that of the native protein.

Stimulation of fibroblast proliferation and prostaglandin production by purified recombinant murine interleukin 1.

Recombinant murine interleukin 1 (IL-1) obtained from a clone of Escherichia coli containing an IL-1 expression plasmid was purified to homogeneity using a sequential extraction procedure and gel filtration chromatography. The purified recombinant IL-1 exhibited a pI of approximately 5.2 and a sp act of 6 X 10(6) units/mg. These values are in agreement with those obtained with natural murine IL-1. The purified recombinant IL-1 enhanced the proliferation of human HEL and WI-38 fibroblasts in a serum-free medium. In addition, IL-1 stimulated fibroblast PGE2 5- to 30-fold over a 24-hr period. The effects of IL-1 on fibroblast activation were obtained with the same concentrations of IL-1 that are effective in the mouse thymocyte assay. These results unequivocally establish the ability of IL-1 to modulate fibroblast proliferation and function.