New mechanism of BRCA-1 mutation by deletion/insertion at the same nucleotide position in three unrelated French breast/ovarian cancer families

A novel complex mutation consisting of a small deletion/insertion (3958del5ins4) was found in the breast cancer-1 gene (BRCA-1) in three unrelated French breast and/or ovarian cancer families. These mutations occurred at the same nucleotide position of the 3′ end of exon 11. The wild-type sequence, CTCAG, was deleted and replaced by AGGC in the three families. The consequence is the generation of a stop codon, TAG, resulting in a truncated protein. We propose two different mechanisms to explain the generation of this complex mutation: (i) the simultaneous occurrence of a deletion and an insertion in a stem-loop structure and (ii) the abortive integration of a human transposable element (Tigger 1) that deleted 5 nucleotides and inserted a 4-nucleotide “scar”, corresponding to the 5′ extremity of the transposon. Received: 26 November 1997 / Accepted: 6 February 1998     

MRI-based assessment of hip joint translations

To better understand movement limitations and, to some extent, the pathogenesis of osteoarthritis, it is important to quantitatively measure femoroacetabular translations to assess if any joint subluxation occurs. In this paper, we aim at measuring hip joint displacements from magnetic resonance images (MRI) based on a surface registration technique. Because this measurement is related to the location of the hip joint center (HJC), we investigate and compare different HJC estimation approaches based on patient-specific 3D bone models. We estimate the HJC based on a simulated circumduction while minimizing inter-articular distance changes. Measurements of femoroacetabular translations during low amplitude abductions ($80$ samples) and extreme flexions ($60$ samples) in female professional dancers, which is a population potentially exposed to femoroactebaluar impingements, do not show any significant subluxation.     

Evolution in heterogeneous environments: between soft and hard selection

Since their first formulations about half a century ago, the soft and hard selection models have become classical frameworks to study selection in subdivided populations. These models differ in the timing of density regulation and represent two extreme types of selection: density- and frequency-dependent selection (soft) and density- and frequency-independent selection (hard). Yet only few attempts have been made so far to model intermediate scenarios. Here, we design a model where migration may happen twice during the life cycle: before density regulation with probability d(J) (juvenile migration) and after density regulation with probability d(A) (adult migration). In the first step, we analyze the conditions for the coexistence of two specialists. We find that coexistence is possible under a large range of selection types, even when environmental heterogeneity is low. Then, we investigate the different possible outcomes obtained through gradual evolution. We show that polymorphism is more likely to evolve when the trade-off is weak, environmental heterogeneity is high, migration is low, and in particular when juvenile migration is low relative to adult migration, because the timing of migration affects the magnitude of frequency-dependent selection relative to gene flow. This model may provide a more general theoretical framework to experimentally study evolution in heterogeneous environments.     

Half-filter experiments for assignment, structure determination and hydration analysis of unlabelled ligands bound to 13C/15N labelled proteins

A novel variant of the 13C/15N 2 half-filter experiment is reported for studying the hydration of an unlabelled ligand bound to a 15N and 13C uniformly labelled biological macromolecule. This doubly tuned filter experiment represents a powerful tool for obtaining resonance assignments, structure determination and hydration properties of a ligand. Its application to the binary complex formed by the inserted-domain (I-domain) of the leukocyte function-associated antigen-1 (LFA-1) with a ligand reveals the presence of H2O molecules at the binding interface.     

Evaluation of the CFP-substrate-YFP system for protease studies: advantages and limitations

A protease can be defined as an enzyme capable of hydrolyzing peptide bonds. Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities toward these sequences, and determination of kinetic parameters. Biological protease substrates based on fluorescent protein pairs, which allow for use of fluorescence resonance energy transfer (FRET), have been recently developed for in vivo protease activity detection and represent a very interesting alternative to chemical substrates for in vitro protease characterization. Here, we analyze a FRET system consisting of cyan and yellow fluorescent proteins (CFP and YFP, respectively), which are fused by a peptide linker serving as protease substrate. Conditions for CFP-YFP fusion protein production in Escherichia coli and purification of proteins were optimized. FRET between CFP and YFP was found to be optimum at a pH between 5.5 and 10.0, at low concentrations of salt and a temperature superior to 25 degrees C. For efficient FRET to occur, the peptide linker between CFP and YFP can measure up to 25 amino acids. The CFP-substrate-YFP system demonstrated a high degree of resistance to nonspecific proteolysis, making it suitable for enzyme kinetic analysis. As with chemical substrates, substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and kcat/Km values were calculated.     

Developmental regulation of prostaglandin E2 synthase in porcine ductus arteriosus

The synthesis of PGE(2), the major vasodilator prostanoid of the ductus arteriosus (DA), is catalyzed by PGE(2) synthases (PGES). The factors implicated in increased PGE(2) synthesis in the perinatal DA are not known. We studied the developmental changes of PGES along with that of cyclooxygenase (COX)-2 and cytosolic phospholipase A(2) (cPLA(2)) in the DA of fetal (75-90% gestation) and immediately postnatal newborn (NB) piglets. Levels of microsomal PGES (mPGES), COX-2, and PGE(2) in the DA of NB were approximately 7-fold higher than in fetus; activities of cytosolic PGES (cPGES) and cPLA(2) in DA of the fetus and NB did not differ. Because platelet-activating factor (PAF) could regulate COX-2 expression, the former was measured and found to be more abundant in the DA of the NB than of fetus. PAF elicited an increase in mPGES, COX-2, and PGE(2) in fetal DA to levels approaching those of the NB; cPGES, cPLA(2), and COX-1 were unaffected. In perinatal NB DA, PAF receptor antagonists BN-52021 and THG-315 reduced mPGES, COX-2, and PGE(2) levels and were associated with increased DA tone. It is concluded that PAF contributes in regulating DA tone by governing mPGES, COX-2, and ensuing PGE(2) levels in the perinate.     

Living recombinant Saccharomyces cerevisiae secreting proteins or peptides as a new drug delivery system in the gut

New strategies to prevent or treat diseases have been focusing on innovative approaches, such as the oral administration of living recombinant micro-organisms delivering active compounds in the digestive environment. The survival rate and the ability of two recombinant Saccharomyces cerevisiae strains (WppV(5)H(6) and WppGSTV(5)H(6)) to initiate the synthesis and secrete either a model peptide (peptide-V(5)H(6), MW: 5.6 kDa) or a model protein (glutathione-S-transferase-V(5)H(6), MW: 31.5 kDa) were studied in a gastric-small intestinal system simulating human digestive conditions. The WppV(5)H(6) and WppGSTV(5)H(6) strains respectively showed 83.1%+/-9.6 (n=3) and 95.3%+/-22.7 (n=4) survival rates in the model upper digestive tract after 270 min of digestion. The secretion products were detected as early as 90 min after the yeast intake/gene induction in each compartment of the in vitro system, but mostly in the jejunum and ileum. The GST-V(5)H(6) concentrations in the digestive medium reached 15 ng ml(-1), close to values measured in batch cultures. These results open up new opportunities for the set up of drug delivery systems based on engineered yeasts secreting compounds directly in the digestive tract. The main potential medical applications include the development of oral vaccines, the correction of metabolic disorders and the in situ production of biological mediators.     

Alpha- and beta- cleavages of the amino-terminus of the cellular prion protein

It is commonly assumed that the physiological isoform of prion protein, PrP(C), is cleaved during its normal processing between residues 111/112, whereas the pathogenic isoform, PrP(Sc), is cleaved at an alternate site in the octapeptide repeat region around position 90. Here we demonstrated both in cultured cells and in vivo, that PrP(C) is subject to a complex set of post-translational processing with the molecule being cleaved upstream of position 111/112, in the octapeptide repeat region or at position 96. PrP has therefore two main cleavage sites that we decided to name alpha and beta. Cleavage of PrP(C) at these sites leads us to re-evaluate the function of both N- and C-terminus fragments thus generated.     

Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.