A region-specific microdissection library for human chromosome 2p23–p25 and the analysis of an interstitial deletion of 2p23.3–p25.1

A region-specific library for human chromosome 2p23–p25 was constructed using microdissection and polymerase chain reaction (PCR)-mediated microcloning techniques. This library is large, comprising 300,000 recombinant microclones. The insert sizes range between 50–600 base pairs (bp) with a mean of 200 bp. About 50%–60% of the clones contain unique or very low copy number sequence inserts as determined by their weak or no hybridization to total human DNA. A subset of 48 microclones that did not hybridize to total human DNA after colony hybridization was analyzed, and 26 (54%) clones were shown to contain single-copy inserts and hybridize to human chromosome 2 DNAs, indicating that they are human chromosome 2 specific. The human genomic fragments identified by these clones after cleavage with HindIII have also been characterized. The single-copy microclones were used to analyze an interstitial deletion in the 2p23.3–p25.1 region — 46,XY, del(2) (pterp25.1::p23.3qter) — previously reported in a patient with severe growth and mental retardation and multiple anomalies. Of the 26 microclones analyzed, 14 clones were mapped to the deletion region. The availability of the 2p23–p25 region-specific library and the probes derived from the library should be valuable for fine structure physical mapping analysis and the cloning of disease-related genes localized to the region. These studies also demonstrate the efficiency with which useful probes can be quickly generated for genome studies and for positional cloning.     

Aptamer–nanoparticle-based chemiluminescence for p53 protein

A simple colorimetric biosensing technique based on the interaction of gold nanoparticles (AuNPs) with the aptamer was developed for detection of p53, a tumor suppressor protein, in the current study. Aggregation of AuNPs was induced by desorption of the p53 binding RNA aptamer from the surface of AuNPs as a result of the aptamer target interaction leading to the color change of AuNPs from red to purple. The detection limit of p53 protein by the colorimetric approach was 0.1 ng/ml after successful optimization of the amount of aptamer, AuNPs, salts, and incubation time. Furthermore, the catalytic activity of the aggregated AuNPs was greatly enhanced by chemiluminescence (CL) reaction, where the detection limit was enhanced to 10 pg/ml with a regression coefficient of R² = 0.9907. Here the sensitivity was increased by 10-fold compared with the AuNP-based colorimetric method. Hence, the sensitivity of detection was increased by employing CL, by using the catalytic activity of aggregated AuNPs, on the luminol–hydrogen peroxide reaction. Thus, the combination of colorimetric and CL-based aptasensor can be of great advantage in increasing the sensitivity of detection for any target analyte.     

Widdrol activates DNA damage checkpoint through the signaling Chk2–p53–Cdc25A–p21–MCM4 pathway in HT29 cells

Widdrol is an odorant compound isolated from Juniperus chinensis. We previously reported that widdrol induces Gap 1 (G1) phase cell cycle arrest and leads to apoptosis in human colon adenocarcinoma HT29 cells. It was also reported that this cell cycle arrest is associated with the induction of checkpoint kinase 2 (Chk2), p53 phosphorylation and cyclin dependent kinase (Cdk) inhibitor p21 expression. In this paper, we investigated the molecular mechanisms of widdrol on the activation of G1 DNA damage checkpoint at early phase when DNA damages occurred in HT29 cells. First of all, we examined that widdrol breaks DNA directly or not. As the results of DNA electrophoresis and formation of phosphorylated histone H2AX (γH2AX) foci in HT29 cells, widdrol generates DNA double-strand breaks directly within 0.5?h both in vitro and in vivo. Based on this result, the change of proteins related in checkpoint pathway was examined over a time course of 0.5-24?h. Treatment of HT29 cells with widdrol elicits the following: (1) phosphorylation of Chk2 and p53, (2) reduction of cell division cycle 25A (Cdc25A) expression, (3) increase of Cdk inhibitor p21 expression, and (4) decrease of the levels of Cdk2 and cyclin E expression in a time-dependent manner. Moreover, only the expression level of mini-chromosome maintenance 4 (MCM4) protein, a subunit of the eukaryotic DNA replicative helicase, is rapidly down-regulated in HT29 cells treated with widdrol over the same time course, but those of the other MCM proteins are unchanged. Overall, our results indicated that widdrol breaks DNA directly in HT29 cells, and this DNA damage results in checkpoint activation via Chk2-p53-Cdc25A-p21-MCM4 pathway and finally cells go to G1-phase cell cycle arrest and apoptosis.     

Heterodimerization of p45–p75 Modulates p75 Signaling: Structural Basis and Mechanism of Action

The p75 neurotrophin receptor, a member of the tumor necrosis factor receptor superfamily, is required as a co-receptor for the Nogo receptor (NgR) to mediate the activity of myelin-associated inhibitors such as Nogo, MAG, and OMgp. p45/NRH2/PLAIDD is a p75 homologue and contains a death domain (DD). Here we report that p45 markedly interferes with the function of p75 as a co-receptor for NgR. P45 forms heterodimers with p75 and thereby blocks RhoA activation and inhibition of neurite outgrowth induced by myelin-associated inhibitors. p45 binds p75 through both its transmembrane (TM) domain and DD. To understand the underlying mechanisms, we have determined the three-dimensional NMR solution structure of the intracellular domain of p45 and characterized its interaction with p75. We have identified the residues involved in such interaction by NMR and co-immunoprecipitation. The DD of p45 binds the DD of p75 by electrostatic interactions. In addition, previous reports suggested that Cys257 in the p75 TM domain is required for signaling. We found that the interaction of the cysteine 58 of p45 with the cysteine 257 of p75 within the TM domain is necessary for p45–p75 heterodimerization. These results suggest a mechanism involving both the TM domain and the DD of p45 to regulate p75-mediated signaling.     

Ctf7p/Eco1p exhibits acetyltransferase activity–but does it matter?

In eukaryotic cells, faithful chromosome segregation depends upon the physical pairing, or cohesion, between sister chromatids. Budding yeast CTF7/ECO1 (herein termed CTF7) encodes an essential protein required to establish cohesion during S-phase and associates with DNA replication factors 1., 2., 3., 4., 5., 6., 7., 8., 9., 10.. However, the molecular mechanism by which Ctf7p establishes cohesion remains unknown. In vitro characterization of Ctf7p as an acetyltransferase led to the model that this activity provides for Ctf7p's essential function [11]. However, in vivo Ctf7p substrates have yet to be documented, nor has an in vivo acetyltransferase activity been demonstrated even when Ctf7p is overexpressed [11] (A. Brands and R.V. Skibbens, unpublished data). In fact, the effects of acetylation-defective Ctf7p (ctf7^ack-) in yeast remain to be rigorously tested, leaving unanswered the critical questions of whether Ctf7p acetyltransferase activity is essential for cell viability and to what extent this activity is required for the establishment of cohesion. Here, we show that yeast strains harboring acetyltransferase-defective alleles [11] as the sole source of Ctf7p function exhibit robust growth and high fidelity chromosome transmission.     

Crucial role of the Rcl1p–Bms1p interaction for yeast pre-ribosomal RNA processing

The essential Rcl1p and Bms1p proteins form a complex required for 40S ribosomal subunit maturation. Bms1p is a GTPase and Rcl1p has been proposed to catalyse the endonucleolytic cleavage at site A₂ separating the pre-40S and pre-60S maturation pathways. We determined the 2.0 Å crystal structure of Bms1p associated with Rcl1p. We demonstrate that Rcl1p nuclear import depends on Bms1p and that the two proteins are loaded into pre-ribosomes at a similar stage of the maturation pathway and remain present within pre-ribosomes after cleavage at A₂. Importantly, GTP binding to Bms1p is not required for the import in the nucleus nor for the incorporation of Rcl1p into pre-ribosomes, but is essential for early pre-rRNA processing. We propose that GTP binding to Bms1p and/or GTP hydrolysis may induce conformational rearrangements within the Bms1p-Rcl1p complex allowing the interaction of Rcl1p with its RNA substrate.     

The enhancement of stability of p53 in MTBP induced p53–MDM2 regulatory network

We have modeled an MTBP-MDM2–p53 regulatory network by integrating p53–MDM2 autoregulatory model (Proctor and Gray, 2008) with the effect of a cellular protein MTBP (MDM2 binding protein) which is allowed to bind with MDM2 (Brady et al., 2005). We study this model to investigate the activation of p53 and MDM2 steady state levels induced by MTBP protein under different stress conditions. Our simulation results in three approaches namely deterministic, Chemical Langevin equation and stochastic simulation of Master equation show a clear transition from damped limit cycle oscillation to fixed point oscillation during a certain time period with constant stress condition in the cell. This transition is the signature of transition of p53 and MDM2 levels from activated state to stabilized steady state levels. We present various phase diagrams to show the transition between unstable and stable states of p53 and MDM2 concentration levels and also their possible relations among critical value of the parameters at which the respective protein level reach stable steady states. In the stochastic approach, the dynamics of the proteins become noise induced process depending on the system size. We found that this noise enhances the stability of the p53 steady state level.     

Inhibition of centriole duplication by centrobin depletion leads to p38–p53 mediated cell-cycle arrest

Previously, we have identified a novel centrosomal protein centrobin that asymmetrically localizes to the daughter centriole. We found that depletion of centrobin expression inhibited the centriole duplication and impaired cytokinesis. However, the biological significance of centrobin in the cell cycle remains unknown. In the current study, we observed that silencing centrobin significantly inhibited the proliferation of lung cancer cell A549 and prevented the cells from G1 to S transition, whereas the growth rate of lung cancer cell line H1299, a p53-null cell line, was not affected. Furthermore, we demonstrated that the G1–S-phase arrest induced by centrobin knockdown in A549 cells is mediated by the upregulation of cell-cycle regulator p53, which is associated with the activation of cellular stress induced p38 pathway instead of DNA damage induced ATM pathway. Inhibition of p38 activity or downregulation of p38 expression could overcome the cell-cycle arrest caused by centrobin depletion. Taken together, our current findings demonstrated that centrobin plays an important role in the progression of cell cycle, and a tight association between the cell-cycle progression and defective centrosomes caused by depletion of centrobin.     

Direct duplication 12p11.21–p13.31 mediated by segmental duplications: a new recurrent rearrangement?

We describe the characterization of an interstitial duplication of 12p, dup(12)(p11.21p13.31), by array-CGH and FISH in a patient with mental retardation and dysmorphic features. The sequence analysis of the breakpoints revealed the presence of homologous low copy repeats (LCRs) flanking the duplication region, thus suggesting that they have mediated the rearrangement. Pip-maker analysis showed that a third cluster of homologous LCRs lie distally to the two mediating the 12p duplication. We hypothesize that this duplication might be a new recurrent rearrangement and that, thanks to the different orientations of the homologous regions lying within each cluster, the three clusters are responsible for at least some of the several 12p aneuploidies reported in the literature such as direct and inverted duplications, deletions and supernumerary analphoid chromosomes. Moreover, we excluded that polymorphic inversions between these three clusters are present in the normal population.Manuela De Gregori, Tiziano Pramparo contributed equally to this paper.     

A Chinese Benign Adult Familial Myoclonic Epilepsy Pedigree Suggesting Linkage to Chromosome 5p15.31–p15.1

Benign adult familial myoclonic epilepsy (BAFME) has been mapped to chromosome 8q23.3–q24.1, 2p11.1–q12.1, 5p15.31–p15.1, and 3q26.32–3q28, in Japanese, Italian, Thai, and French pedigrees, respectively. Recently, we investigated a Chinese BAFME family. Clinical and electrophysiological studies revealed that nine individuals were affected with BAFME. We aimed to establish the causative gene for this pedigree. We genotyped 17 microsatellite markers covering the four previously identified chromosome regions and performed linkage analyses. The linkage analysis data showed that the LOD score was 2.80 for D5S486 at no recombination. This suggested linkage to 5p15.31–p15.1 and excluded linkage to the other three loci (LOD score <0 at no recombination). Our study suggests that the causative gene responsible for BAFME in the Chinese pedigree may be located on chromosome 5p15.31–p15.1.     

Genome-Wide Linkage Study Suggests a Susceptibility Locus for Isolated Bilateral Microtia on 4p15.32–4p16.2

Microtia is a congenital deformity where the external ear is underdeveloped. Genetic investigations have identified many susceptibility genes of microtia-related syndromes. However, no causal genes were reported for isolated microtia, the main form of microtia. We conducted a genome-wide linkage analysis on a 5-generation Chinese pedigree with isolated bilateral microtia. We identified a suggestive linkage locus on 4p15.32–4p16.2 with parametric LOD score of 2.70 and nonparametric linkage score (Zmean) of 12.28 (simulated occurrence per genome scan equal to 0.46 and 0.47, respectively). Haplotype reconstruction analysis of the 4p15.32–4p16.2 region further confined the linkage signal to a 10-Mb segment located between rs12505562 and rs12649803 (9.65–30.24 cM; 5.54–15.58 Mb). Various human organ developmental genes reside in this 10-Mb susceptibility region, such as EVC, EVC2, SLC2A9, NKX3-2, and HMX1. The coding regions of three genes, EVC known for cartilage development and NKX3-2, HMX1 involved in microtia, were selected for sequencing with 5 individuals from the pedigree. Of the 38 identified sequence variants, none segregates along with the disease phenotype. Other genes or DNA sequences of the 10-Mb region warrant for further investigation. In conclusion, we report a susceptibility locus of isolated microtia, and this finding will encourage future studies on the genetic basis of ear deformity.     

Peptide, Peptidomimetic, and Small-molecule Antagonists of the p53–HDM2 Protein–Protein Interaction

Modulation of intracellular protein–protein interactions has been – and remains – a challenging goal for the discovery and development of small-molecule therapeutic agents. Progress in the pharmacological targeting and understanding at the molecular level of one such interaction that is relevant to cancer drug research, viz. that between the tumour suppressor protein p53 and its negative regulator HDM2, is reviewed here. The first X-ray crystal structure of a complex between a small peptide from the trans-activation domain of p53 and the N-terminal domain of HDM2 was reported almost 10 years ago. The nature of this interaction, which involves just three residue side chains in the p53 peptide ligand and a compact hydrophobic binding pocket in the HDM2 receptor, together with the attractive concept of reactivating the anti-proliferative functions of p53 in tumour cells, has spurned a great deal of effort aimed at finding drug-like antagonists of this interaction. A variety of approaches, including both structure-guided peptidomimetic and de novo design, as well as high through-put screening campaigns, have provided a wealth of leads that might be turned into actual drugs. There is still some way to go as far as optimisation and preclinical development of such leads is concerned, but it is clear already now that antagonists of the p53–HDM2 protein–protein interaction have a good chance of ultimately being successful in providing a new anti-cancer therapy modality, both in monotherapy and to potentiate the effectiveness of existing chemotherapies.     

6p21.2–p12.3 deletion detected by aCGH in an 8-year-old girl with cleidocranial dysplasia and developmental delay

We present an 8-year-old girl with cleidocranial dysplasia, psychomotor developmental delay, poor wound healing and a 6p21.2–p12.3 deletion detected by aCGH. The patient was previously found to have a normal karyotype on conventional cytogenetic analysis and no RUNX2 mutation on sequence analysis. We discuss the genotype–phenotype correlation and the consequence of haploinsufficiency of CUL7, VEGFA, NFKBIE and RUNX2 in this case.     

Investigation of the inhibitory mechanism of apomorphine against MDM2–p53 interaction

Mirror-image screening using d-proteins is a powerful approach to provide mirror-image structures of chiral natural products for drug screening. During the course of our screening study for novel MDM2–p53 interaction inhibitors, we identified that NPD6878 (R-(?)-apomorphine) inhibited both the native l-MDM2–l-p53 interaction and the mirror-image d-MDM2–d-p53 interaction at equipotent doses. In addition, both enantiomers of apomorphine showed potent inhibitory activity against the native MDM2–p53 interaction. In this study, we investigated the inhibitory mechanism of both enantiomers of apomorphine against the MDM2–p53 interaction. Achiral oxoapomorphine, which was converted from chiral apomorphines under aerobic conditions, served as the reactive species to form a covalent bond at Cys77 of MDM2, leading to the inhibitory effect against the binding to p53.