Bacterial production of biologically active canine interleukin-1beta by seamless SUMO tagging and removal.

Interleukin 1beta (IL-1beta) is a potent stimulator of extracellular matrix degradation in models of osteoarthritis (OA). In contrast to bovine explant models which effectively respond to recombinant human IL-1beta, canine models are relatively refractory to human IL-1beta stimulation. Canine IL-1beta cDNA was cloned in order to produce a fully potent species matched preparation of IL-1beta for use specifically in canine models of OA. Established methods for the production of various orthologous IL-1beta proteins from different species are problematic due to the exquisite sensitivity of the mature IL-1beta product to N-terminal variations and the intrinsic technical challenges associated with producing an unmodified product. We have applied a seamless method of SUMO tagging and removal in order to produce a homogeneous unmodified preparation of canine IL-1beta from Escherichia coli which was found to be a potent inducer of aggrecanase activity in isolated canine articular chondrocytes. This method combines highly efficient aspects of seamless plasmid engineering, protein purification, and precise tag removal.

A second p53 binding site in the central domain of Mdm2 is essential for p53 ubiquitination.

Mdm2 negatively regulates p53 by inhibiting its transcriptional activity and promoting its degradation by functioning as an E3 ubiquitin ligase. The primary p53 binding site on mdm2 is located in its N-terminal domain. Through binding to p53 at its N-terminal transactivation domain, mdm2 directly blocks the transcriptional activation function of p53. We discovered that truncated mdm2 protein constructs without the N-terminal p53 binding domain are at least as active as full-length mdm2 in catalyzing p53 ubiquitination. Furthermore, the deletion of the central acidic domain significantly reduces the E3 ligase activity of mdm2 toward p53. We have also performed GST pull-down experiments to probe the direct binding of various mdm2 domain constructs toward full length p53 and found that mdm2 constructs without the N-terminal p53 binding domain retain the ability to bind to p53. Our kinetic and binding data localize the second p53 binding site between amino acids 211 and 361, including the acidic domain and the zinc finger region. Our work, consistent with other reports, suggests that the p53 tetramer interacts with at least two sites on mdm2. Although the interaction between the N-termini of mdm2 and p53 blocks the transactivation activity of p53, the interaction between the central domain of mdm2 and the core domain of p53 is critical for the ubiquitination and degradation of p53. This second mdm2-p53 interaction site represents an alternative target for small molecule modulators of the mdm2-p53 pathway.

A biochemical rationale for the anticancer effects of Hsp90 inhibitors: slow, tight binding inhibition by geldanamycin and its analogues.

Heat shock protein (Hsp)90 is emerging as an important therapeutic target for the treatment of cancer. Two analogues of the Hsp90 inhibitor geldanamycin are currently in clinical trials. Geldanamycin (GA) and its analogues have been reported to bind purified Hsp90 with low micromolar potency, in stark contrast to their low nanomolar antiproliferative activity in cell culture and their potent antitumor activity in animal models. Several models have been proposed to account for the approximately 100-fold-greater potency in cell culture, including that GA analogues bind with greater affinity to a five-protein Hsp90 complex than to Hsp90 alone. We have determined that GA and the fluorescent analogue BODIPY-GA (BDGA) both demonstrate slow, tight binding to purified Hsp90. BDGA, used to characterize the kinetics of ligand-Hsp90 interactions, was found to bind Hsp90alpha with k(off) = 2.5 x 10(-3) min(-1), t(1/2) = 4.6 h, and Ki* = 10 nM. It was found that BDGA binds to a functional multiprotein Hsp90 complex with kinetics and affinity identical to that of Hsp90 alone. Also, BDGA binds to Hsp90 from multiple cell lysates in a time-dependent manner with similar kinetics. Therefore, our results indicate that the high potency of GA in cell culture and in vivo can be accounted for by its time-dependent, tight binding to Hsp90 alone. In the broader context, these studies highlight the essentiality of detailed biochemical characterization of drug-target interactions for the effective translation of in vitro pharmacology to cellular and in vivo efficacy.

Caries experience associated with attention-deficit/hyperactivity disorder.

UNLABELLED: The purpose of this cross-sectional study was to examine whether children with attention-deficit/hyperactivity disorder (ADHD) had a total caries experience that was equivalent to children without the disorder. METHODS: The test sample included children ages 6 to 10 years old diagnosed with ADHD by physicians at Duke University Medical Center. The control group also included healthy children 6 to 10 years old without the diagnosis of ADHD. A visual dental exam for caries was performed and a sample of whole, unstimulated saliva was collected. The parent/guardian of each participant completed a health/medication history and a questionnaire concerning the child's oral health and habits, daily routine, and demographic information. RESULTS: Wilcoxon and chi-square tests showed that children with ADHD have significantly more enamel caries in the primary and permanent dentitions and a significantly higher prevalence of total caries experience when compared to controls. There was no significant difference in whole saliva production between the ADHD children and the controls. CONCLUSIONS: Dentists should be aware that children with ADHD are at higher risk for caries.

Crystal structure and substrate specificity of the beta-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus.

beta-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 A resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.