Haploinsufficiency of Mdm2 and Mdm4 in tumorigenesis and development

The tumor suppressor p53 is inactivated by multiple mechanisms that include mutations of the p53 gene itself and increased levels of the p53 inhibitors MDM2 and MDM4. Mice lacking Mdm2 or Mdm4 exhibit embryo-lethal phenotypes that are completely rescued by concomitant deletion of p53. Here we show that Mdm2 and Mdm4 haploinsufficiency leads to increased p53 activity, exhibited as increased sensitivity to DNA damage and decreased transformation potential. Moreover, in in vivo tumor development, Emu-myc Mdm4+/- mice show a delayed onset of B-cell lymphomas compared to Emu-myc mice. Additionally, Mdm2+/- Mdm4+/- double-heterozygous mice are not viable and exhibit defects in hematopoiesis and cerebellar development. The defects in Mdm2+/- Mdm4+/- mice are corrected by deletion of a single p53 allele. These findings highlight the exquisite sensitivity of p53 to Mdm2 and Mdm4 levels and suggest that some cell types may be more sensitive to therapeutic drugs that inhibit the Mdm-p53 interaction.     

Polyhead formation in phage P22 pinpoints a region in coat protein required for conformational switching

Eighteen single amino acid substitutions in phage P22 coat protein cause temperature-sensitive folding defects (tsf). Three intragenic global suppressor (su) substitutions (D163G, T166I and F170L), localized to a flexible loop, rescue the folding of several tsf coat proteins. Here we investigate the su substitutions in the absence of the original tsf substitutions. None of the su variant coat proteins displayed protein folding defects. Individual su substitutions had little effect on phage production in vivo; yet double and triple combinations resulted in a cold-sensitive (cs) phenotype, consistent with a defect in assembly. During virus assembly and maturation, conformational switching of capsid subunits is required when chemically identical capsid subunits form an icosahedron. Analysis of double- and triple-su phage-infected cell lysates by negative-stain electron microscopy reveals an increase in aberrant structures at the cs temperature. In vitro assembly of F170L coat protein causes production of polyheads, never seen before in phage P22. Purified procapsids composed of all of the su coat proteins showed defects in expansion, which mimics maturation in vitro. Our results suggest that a previously identified surface-exposed loop in coat protein is critical in conformational switching of subunits during both procapsid assembly and maturation.     

Study of feline oral squamous cell carcinoma: potential target for cyclooxygenase inhibitor treatment

Oral squamous cell carcinoma (OSCC) is associated with high morbidity and mortality. A potential target for OSCC treatment is cyclooxygenase-2 (cox-2). Pet cats with naturally occurring OSCC may offer the opportunity to study anticancer activity of cox inhibitors. Cox-2 expression in feline OSCC was determined by immunohistochemistry. High intensity cox-2 immunoreactivity was detected in 6 of 34 (18%) feline OSCC samples. Weak immunoreactivity was noted in 22 OSCCs and in epithelial cells from oral mucosa of clinically normal cats. Pharmacokinetics of a cox inhibitor (piroxicam, 0.3 mg/kg) were studied in carcinoma-bearing cats to confirm a dose for follow-up trials. The average peak serum piroxicam concentration (948 ng/ml, which inhibited cox-2 activity) and serum half-life (15.9 h) were similar to that in normal cats. These results provide information (cox-2 expression as an inclusion criteria, 0.3 mg/kg daily piroxicam) for the design of follow-up trials of cox inhibitor treatment in pet cats with OSCC.     

Single-cell-based sensors and synchrotron FTIR spectroscopy: a hybrid system towards bacterial detection

Microarrays of single macrophage cell-based sensors were developed and demonstrated for potential real-time bacterium detection by synchrotron FTIR microscopy. The cells were patterned on gold electrodes of silicon oxide substrates by a surface engineering technique, in which the gold electrodes were immobilized with fibronectin to mediate cell adhesion and the silicon oxide background was passivated with polyethylene glycol (PEG) to resist protein adsorption and cell adhesion. Cell morphology and IR spectra of single, double, and triple cells on gold electrodes exposed to lipopolysaccharide (LPS) of different concentrations were compared to reveal the detection capability of this cell-based sensing platform. The single-cell-based system was found to generate the most significant and consistent IR spectrum shifts upon exposure to LPS, thus providing the highest detection sensitivity. Changes in cell morphology and IR shifts upon cell exposure to LPS were found to be dependent on the LPS concentration and exposure time, which established a method for the identification of LPS concentration and infected cell population. Possibility of using this single-cell system with conventional IR spectroscopy as well as its limitation was investigated by comparing IR spectra of single-cell arrays with gold electrode surface areas of 25, 100, and 400 microm2 using both synchrotron and conventional FTIR spectromicroscopes. This cell-based platform may potentially provide real-time, label-free, and rapid bacterial detection, and allow for high-throughput statistical analyses, and portability.     

A high-throughput assay for O-GlcNAc transferase detects primary sequence preferences in peptide substrates

O-GlcNAc transferase (OGT) catalyzes the addition of N-acetylglucosamine (O-GlcNAc) onto a diverse array of intracellular proteins. Although hundreds of proteins are known to be modified by O-GlcNAc, a strict amino acid consensus sequence for OGT has not been identified. In this study, we describe the development of a high-throughput assay for OGT and use it to profile the specificity of the enzyme among a panel of peptide substrates.     

Synthesis and evaluation of substituted benzoisoquinolinones as potent inhibitors of Chk1 kinase

From HTS lead 1, a novel benzoisoquinolinone class of ATP-competitive Chk1 inhibitors was devised and synthesized via a photochemical route. Using X-ray crystallography as a guide, potency was rapidly enhanced through the installation of a tethered basic amine designed to interact with an acidic residue (Glu91) in the enzyme pocket. Further SAR was explored at the solvent front and near to the H1 pocket and resulted in the discovery of low MW, sub-nanomolar inhibitors of Chk1.     

The discovery of 2-amino-3,5-diarylbenzamide inhibitors of IKK-alpha and IKK-beta kinases

A potent and selective series of 2-amino-3,5-diarylbenzamide inhibitors of IKK-alpha and IKK-beta is described. The most potent compounds are 8h, 8r and 8v, with IKK-beta inhibitory potencies of pIC(50) 7.0, 6.8 and 6.8, respectively. The series has excellent selectivity, both within the IKK family over IKK-epsilon, and across a wide variety of kinase assays. The potency of 8h in the IKK-beta enzyme assay translates to significant cellular activity (pIC(50) 5.7-6.1) in assays of functional and mechanistic relevance.     

Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor

Unlike processive cellular motors such as myosin V, whose structure has recently been determined in a "rigor-like" conformation, myosin II from contracting muscle filaments necessarily spends most of its time detached from actin. By using squid and sea scallop sources, however, we have now obtained similar rigor-like atomic structures for muscle myosin heads (S1). The significance of the hallmark closed actin-binding cleft in these crystal structures is supported here by actin/S1-binding studies. These structures reveal how different duty ratios, and hence cellular functions, of the myosin isoforms may be accounted for, in part, on the basis of detailed differences in interdomain contacts. Moreover, the rigor-like position of switch II turns out to be unique for myosin V. The overall arrangements of subdomains in the motor are relatively conserved in each of the known contractile states, and we explore qualitatively the energetics of these states.     

A comprehensive analysis of common copy-number variations in the human genome

Segmental copy-number variations (CNVs) in the human genome are associated with developmental disorders and susceptibility to diseases. More importantly, CNVs may represent a major genetic component of our phenotypic diversity. In this study, using a whole-genome array comparative genomic hybridization assay, we identified 3,654 autosomal segmental CNVs, 800 of which appeared at a frequency of at least 3%. Of these frequent CNVs, 77% are novel. In the 95 individuals analyzed, the two most diverse genomes differed by at least 9 Mb in size or varied by at least 266 loci in content. Approximately 68% of the 800 polymorphic regions overlap with genes, which may reflect human diversity in senses (smell, hearing, taste, and sight), rhesus phenotype, metabolism, and disease susceptibility. Intriguingly, 14 polymorphic regions harbor 21 of the known human microRNAs, raising the possibility of the contribution of microRNAs to phenotypic diversity in humans. This in-depth survey of CNVs across the human genome provides a valuable baseline for studies involving human genetics.