Structure-based design of potent and selective inhibitors of collagenase-3 (MMP-13)

Computer aided drug design led to a new class of spiro-barbiturates (e.g., 4a, MMP-13 K(i)=4.7 nM) that are potent inhibitors of MMP-13.     

A role for epsin N-terminal homology/AP180 N-terminal homology (ENTH/ANTH) domains in tubulin binding

The epsin N-terminal homology (ENTH) domain is a protein module of approximately 150 amino acids found at the N terminus of a variety of proteins identified in yeast, plants, nematode, frog, and mammals. ENTH domains comprise multiple alpha-helices folded upon each other to form a compact globular structure that has been implicated in interactions with lipids and proteins. In characterizing this evolutionarily conserved domain, we isolated and identified tubulin as an ENTH domain-binding partner. The interaction, which is direct and has a dissociation constant of approximately 1 microm, was observed with ENTH domains of proteins present in various species. Tubulin is co-immunoprecipitated from rat brain extracts with the ENTH domain-containing proteins, epsins 1 and 2, and punctate epsin staining is observed along the microtubule cytoskeleton of dissociated cortical neurons. Consistent with a role in microtubule processes, the over-expression of epsin ENTH domain in PC12 cells stimulates neurite outgrowth. These data demonstrate an evolutionarily conserved property of ENTH domains to interact with tubulin and microtubules.     

Statistical alignment based on fragment insertion and deletion models

Motivation: The topic of this paper is the estimation of alignments and mutation rates based on stochastic sequence-evolution models that allow insertions and deletions of subsequences ('fragments') and not just single bases. The model we propose is a variant of a model introduced by Thorne et al., (J. Mol. Evol., 34, 3-16, 1992). The computational tractability of the model depends on certain restrictions in the insertion/deletion process; possible effects we discuss. Results: The process of fragment insertion and deletion in the sequence-evolution model induces a hidden Markov structure at the level of alignments and thus makes possible efficient statistical alignment algorithms. As an example we apply a sampling procedure to assess the variability in alignment and mutation parameter estimates for HVR1 sequences of human and orangutan, improving results of previous work. Simulation studies give evidence that estimation methods based on the proposed model also give satisfactory results when applied to data for which the restrictions in the insertion/deletion process do not hold. Availability: The source code of the software for sampling alignments and mutation rates for a pair of DNA sequences according to the fragment insertion and deletion model is freely available from http://www.math.uni-frankfurt.de/~stoch/software/mcmcsalut under the terms of the GNU public license (GPL, 2000).     

A new missense mutation of the vasopressin-neurophysin II gene in a family with neurohypophyseal diabetes insipidus

OBJECTIVE: Autosomal dominant familial neurohypophyseal diabetes insipidus is a rare disorder characterized by polydipsia and polyuria. We present the results of the molecular analysis of the AVP-NPII gene of a German kindred. METHODS: All three exons of the gene were amplified by polymerase chain reaction and sequenced. RESULTS: In 7 affected individuals a new missense mutation (1770G > T) in exon 2 was found predicting a cysteine to phenylalanine substitution at codon 58 in the neurophysin II domain (NPII). CONCLUSION: As a result of this mutation a cysteine residue is exchanged, which is involved in a disulfide bond with cysteine 44 of the NPII moiety, hypothesizing that the resulting misfolded protein may lead to chronic neurotoxicity by accumulation of these products in the endoplasmatic reticulum.     

The Ews-ERG fusion protein can initiate neoplasia from lineage-committed haematopoietic cells

The EWS-ERG fusion protein is found in human sarcomas with the chromosomal translocation t(21;22)(q22;q12), where the translocation is considered to be an initiating event in sarcoma formation within uncommitted mesenchymal cells, probably long-lived progenitors capable of self renewal. The fusion protein may not therefore have an oncogenic capability beyond these progenitors. To assess whether EWS-ERG can be a tumour initiator in cells other than mesenchymal cells, we have analysed Ews-ERG fusion protein function in a cellular environment not typical of that found in human cancers, namely, committed lymphoid cells. We have used Ews-ERG invertor mice having an inverted ERG cDNA cassette flanked by loxP sites knocked in the Ews intron 8, crossed with mice expressing Cre recombinase under the control of the Rag1 gene to give conditional, lymphoid-specific expression of the fusion protein. Clonal T cell neoplasias arose in these mice. This conditional Ews gene fusion model of tumourigenesis shows that Ews-ERG can cause haematopoietic tumours and the precursor cells are committed cells. Thus, Ews-ERG can function in cells that do not have to be pluripotent progenitors or mesenchymal cells.     

Chaperone-assisted translocation

We investigate the translocation of a stiff polymer through a nanopore in a membrane, in the presence of binding particles (chaperones) that bind reversibly to the polymer on both sides of the membrane. A bound chaperone covers one (univalent binding) or many (multivalent binding) binding sites. Assuming that the diffusion of the chaperones is fast compared to the rate of translocation we describe the process by a one-dimensional master equation. We expand previous models by a detailed study of the effective force in the master equation, which is obtained by the appropriate statistical mechanical average over the chaperone states. The dependence of the force on the degree of valency (the number of binding sites occupied by a chaperone) is studied in detail. We obtain finite size corrections (to the thermodynamical expression for the force), which, for univalent binding, can be expressed analytically. We finally investigate the mean velocity for translocation as a function of chaperone binding strength and size. For both univalent and multivalent binding simple results are obtained for the case of a sufficiently long translocating polymer.     

Lesion bacterial communities in American lobsters with diet-induced shell disease

In southern New England, USA, shell disease affects the profitability of the American lobster Homarus americanus fishery. In laboratory trials using juvenile lobsters, exclusive feeding of herring Clupea harengus induces shell disease typified initially by small melanized spots that progress into distinct lesions. Amongst a cohabitated, but segregated, cohort of 11 juvenile lobsters fed exclusively herring, bacterial communities colonizing spots and lesions were investigated by denaturing gradient gel electrophoresis of 16S rDNA amplified using 1 group-specific and 2 universal primer sets. The Bacteroidetes and Proteobacteria predominated in both spots and lesions and included members of the orders Flavobacteriales (Bacteriodetes), Rhodobacterales, Rhodospirillales and Rhizobiales (Alphaproteobacteria), Xanthomonadales (Gammaproteobacteria) and unclassified Gammaproteobacteria. Bacterial communities in spot lesions displayed more diversity than communities with larger (older) lesions, indicating that the lesion communities stabilize over time. At least 8 bacterial types persisted as lesions developed from spots. Aquimarina 'homaria', a species commonly cultured from lesions present on wild lobsters with epizootic shell disease, was found ubiquitously in spots and lesions, as was the 'Candidatus Kopriimonas aquarianus', implicating putative roles of these species in diet-induced shell disease of captive lobsters.     

Large amplicon droplet digital PCR for DNA‐based monitoring of pediatric chronic myeloid leukaemia

Quantification of tumour‐specific molecular markers at the RNA and DNA level for treatment response monitoring is crucial for risk‐adapted stratification and guidance of individualized therapy in leukaemia and other malignancies. Most pediatric leukaemias and solid tumours of mesenchymal origin are characterized by a relatively low mutation burden at the single nucleotide level and the presence of recurrent chromosomal translocations. The genomic fusion sites resulting from translocations are stable molecular tumour markers; however, repeat‐rich DNA sequences flanking intronic breakpoints limit the design of high sensitivity PCR assays for minimal residual disease (MRD) monitoring. Here, we quantitatively evaluated the impact of repeat elements on assay selection and the feasibility of using extended amplicons (≤1330 bp) amplified by droplet digital PCR to monitor pediatric chronic myeloid leukaemia (CML). Molecular characterization of 178 genomic BCR‐ABL1 fusion sites showed that 64% were located within sequence repeat elements, impeding optimal primer/probe design. Comparative quantification of DNA and RNA BCR‐ABL1 copy numbers in 687 specimens from 55 pediatric patients revealed that their levels were highly correlated. The combination of droplet digital PCR, double quenched probes and extended amplicons represents a valuable tool for sensitive MRD assessment in CML and may be adapted to other translocation‐positive tumours.