Joseph Martial

MicroRNA-146a is a therapeutic target and biomarker for peripartum cardiomyopathy

Peripartum cardiomyopathy (PPCM) is a life-threatening pregnancy-associated cardiomyopathy in previously healthy women. Although PPCM is driven in part by the 16-kDa N-terminal prolactin fragment (16K PRL), the underlying molecular mechanisms are poorly understood. We found that 16K PRL induced microRNA-146a (miR-146a) expression in ECs, which attenuated angiogenesis through downregulation of NRAS. 16K PRL stimulated the release of miR-146a-loaded exosomes from ECs. The exosomes were absorbed by cardiomyocytes, increasing miR-146a levels, which resulted in a subsequent decrease in metabolic activity and decreased expression of Erbb4, Notch1, and Irak1. Mice with cardiomyocyte-restricted Stat3 knockout (CKO mice) exhibited a PPCM-like phenotype and displayed increased cardiac miR-146a expression with coincident downregulation of Erbb4, Nras, Notch1, and Irak1. Blocking miR-146a with locked nucleic acids or antago-miRs attenuated PPCM in CKO mice without interrupting full-length prolactin signaling, as indicated by normal nursing activities. Finally, miR-146a was elevated in the plasma and hearts of PPCM patients, but not in patients with dilated cardiomyopathy. These results demonstrate that miR-146a is a downstream-mediator of 16K PRL that could potentially serve as a biomarker and therapeutic target for PPCM.

The Ornithine Decarboxylase Gene Is Essential for Cell Survival during Early Murine Development

ABSTRACT Overexpression and inhibitor studies have suggested that the c-Myc target gene for ornithine decarboxylase (ODC), the enzyme which converts ornithine to putrescine, plays an important role in diverse biological processes, including cell growth, differentiation, transformation, and apoptosis. To explore the physiological function of ODC in mammalian development, we generated mice harboring a disrupted ODC gene.ODC-heterozygous mice were viable, normal, and fertile. Although zygotic ODC is expressed throughout the embryo prior to implantation, loss of ODC did not block normal development to the blastocyst stage. Embryonic day E3.5 ODC-deficient embryos were capable of uterine implantation and induced maternal decidualization yet failed to develop substantially thereafter. Surprisingly, analysis of ODC-deficient blastocysts suggests that loss of ODC does not affect cell growth per se but rather is required for survival of the pluripotent cells of the inner cell mass. Therefore, ODC plays an essential role in murine development, and proper homeostasis of polyamine pools appears to be required for cell survival prior to gastrulation.

MicroRNA-21 Exhibits Antiangiogenic Function by Targeting RhoB Expression in Endothelial Cells

Background MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that regulate gene expression at post-transcriptional level. The recent discovery of the involvement of these RNAs in the control of angiogenesis renders them very attractive in the development of new approaches for restoring the angiogenic balance. Whereas miRNA-21 has been demonstrated to be highly expressed in endothelial cells, the potential function of this miRNA in angiogenesis has never been investigated. Methodology/Principal Findings We first observed in endothelial cells a negative regulation of miR-21 expression by serum and bFGF, two pro-angiogenic factors. Then using in vitro angiogenic assays, we observed that miR-21 acts as a negative modulator of angiogenesis. miR-21 overexpression reduced endothelial cell proliferation, migration and the ability of these cells to form tubes whereas miR-21 inhibition using a LNA-anti-miR led to opposite effects. Expression of miR-21 in endothelial cells also led to a reduction in the organization of actin into stress fibers, which may explain the decrease in cell migration. Further mechanistic studies showed that miR-21 targets RhoB, as revealed by a decrease in RhoB expression and activity in miR-21 overexpressing cells. RhoB silencing impairs endothelial cell migration and tubulogenesis, thus providing a possible mechanism for miR-21 to inhibit angiogenesis. Finally, the therapeutic potential of miR-21 as an angiogenesis inhibitor was demonstrated in vivo in a mouse model of choroidal neovascularization. Conclusions/Significance Our results identify miR-21 as a new angiogenesis inhibitor and suggest that inhibition of cell migration and tubulogenesis is mediated through repression of RhoB.

Isolation and characterization of the human prolactin gene

Prolactin (PRL) and growth hormone (GH) genes derive from a common ancestor and still share some sequence homologies. Their expression in the pituitary gland is regulated in opposite directions by most of the many hormones acting on them. This provides an interesting system to study sequences involved in gene expression. Using a human PRL cDNA clone as a probe, we screened a human genomic DNA library in lambda phage and isolated a single recombinant comprising the whole hPRL gene. It was characterized by restriction endonuclease mapping and cDNA hybridization, by DNA heteroduplex analysis and by nucleotide sequencing. The hPRL gene is present as a single copy per haploid genome, is approximately 10 kb long and contains four introns, three of which interrupt the coding sequence at the same locations as in the known GH and PRL genes. The origin of transcription was determined by S1 mapping on prolactinoma mRNAs. The search for direct and inverted repeats, as well as dyad symmetries was carried out in the 900‐bp sequenced in the 5′‐flanking region. Sequence homologies between hPRL, hGH and rPRL were derived from computer drawn matrices for these upstream regions.

Triose-phosphate isomerase (TIM) of the psychrophilic bacterium Vibrio marinus. Kinetic and structural properties.

The purification and characterization of triose-phosphate isomerase from the psychrophilic bacteriumVibrio marinus (vTIM) is described. Crystal structures of the vTIM-sulfate complex and the vTIM-2-phosphoglycolate complex (at a 2.7-Å resolution) are also presented. The optimal growth temperature of Vibrio marinus is 15 °C. Stability studies show that vTIM is an unstable protein with a half-life of only 10 min at 25 °C. The vTIM sequence is most closely related to the sequence ofEscherichia coli TIM (eTIM) (66% identity), and several unique structural features described for eTIM are also seen in vTIM, but eTIM is considerably more stable. The T d values of vTIM and eTIM, determined by calorimetric studies, are 41 and 54 °C, respectively. Amino acid sequence comparison reveals that vTIM has an alanine in loop 8 (at position 238), whereas all other TIM sequences known to date have a serine. The vTIM mutant A238S was produced and characterized. Compared with wild type, the catalytic efficiency of the A238S mutant is somewhat reduced, and its stability is considerably increased.

The crystal structure of triosephosphate isomerase (TIM) from Thermotoga maritima: a comparative thermostability structural analysis of ten different TIM structures.

The molecular mechanisms that evolution has been employing to adapt to environmental temperatures are poorly understood. To gain some further insight into this subject we solved the crystal structure of triosephosphate isomerase (TIM) from the hyperthermophilic bacterium Thermotoga maritima (TmTIM). The enzyme is a tetramer, assembled as a dimer of dimers, suggesting that the tetrameric wild‐type phosphoglycerate kinase PGK‐TIM fusion protein consists of a core of two TIM dimers covalently linked to 4 PGK units. The crystal structure of TmTIM represents the most thermostable TIM presently known in its 3D‐structure. It adds to a series of nine known TIM structures from a wide variety of organisms, spanning the range from psychrophiles to hyperthermophiles. Several properties believed to be involved in the adaptation to different temperatures were calculated and compared for all ten structures. No sequence preferences, correlated with thermal stability, were apparent from the amino acid composition or from the analysis of the loops and secondary structure elements of the ten TIMs. A common feature for both psychrophilic and T. maritima TIM is the large number of salt bridges compared with the number found in mesophilic TIMs. In the two thermophilic TIMs, the highest amount of accessible hydrophobic surface is buried during the folding and assembly process. Proteins 1999;37:441–453. ©1999 Wiley‐Liss, Inc.

Zebrafish Sox7 and Sox18 function together to control arterial-venous identity.

Sox7 and Sox18 are members of the F-subgroup of Sox transcription factors family and are mostly expressed in endothelial compartments. In humans, dominant mutations in Sox18 are the underlying cause of the severe hypotrichosis-lymphedema-telangiectasia disorder characterized by vascular defects. However little is known about which vasculogenic processes Sox7 and Sox18 regulate in vivo. We cloned the orthologs of Sox7 and Sox18 in zebrafish, analysed their expression pattern and performed functional analyses. Both genes are expressed in the lateral plate mesoderm during somitogenesis. At later stages, Sox18 is expressed in all axial vessels whereas Sox7 expression is mainly restricted to the dorsal aorta. Knockdown of Sox7 or Sox18 alone failed to reveal any phenotype. In contrast, blocking the two genes simultaneously led to embryos displaying dysmorphogenesis of the proximal aorta and arteriovenous shunts, all of which can account for the lack of circulation observed in the trunk and tail. Gene expression analyses performed with general endothelial markers on double morphants revealed that Sox7 and Sox18 are dispensable for the initial specification and positioning of the major trunk vessels. However, morphants display ectopic expression of the venous Flt4 marker in the dorsal aorta and a concomitant reduction of the artery-specific markers EphrinB2a and Gridlock. The striking similarities between the phenotype of Sox7/Sox18 morphants and Gridlock mutants strongly suggest that Sox7 and Sox18 control arterial-venous identity by regulating Gridlock expression.

Synthesis of growth hormone by bacteria

A hybrid gene was constructed between the β-lactamase gene of plasmid pBR322 and the cloned coding sequence for rat growth hormone. This gene is expressed in bacteria and growth hormone sequences are detectable by immunological methods.

De novo Backbone and Sequence Design of an Idealized α/β-barrel Protein: Evidence of Stable Tertiary Structure

We have designed, synthesized, and characterized a 216 amino acid residue sequence encoding a putative idealized α/β-barrel protein. The design was elaborated in two steps. First, the idealized backbone was defined with geometric parameters representing our target fold: a central eight parallel-stranded β-sheet surrounded by eight parallel α-helices, connected together with short structural turns on both sides of the barrel. An automated sequence selection algorithm, based on the dead-end elimination theorem, was used to find the optimal amino acid sequence fitting the target structure. A synthetic gene coding for the designed sequence was constructed and the recombinant artificial protein was expressed in bacteria, purified and characterized. Far-UV CD spectra with prominent bands at 222 nm and 208 nm revealed the presence of α-helix secondary structures (50%) in fairly good agreement with the model. A pronounced absorption band in the near-UV CD region, arising from immobilized aromatic side-chains, showed that the artificial protein is folded in solution. Chemical unfolding monitored by tryptophan fluorescence revealed a conformational stability (ΔGH_2O) of 35 kJ/mol. Thermal unfolding monitored by near-UV CD revealed a cooperative transition with an apparent T_m of 65 °C. Moreover, the artificial protein did not exhibit any affinity for the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS), providing additional evidence that the artificial barrel is not in the molten globule state, contrary to previously designed artificial a/ b-barrels. Finally, ^1H NMR spectra of the folded and unfolded proteins provided evidence for specific interactions in the folded protein. Taken together, the results indicate that the de novo designed α/β-barrel protein adopts a stable three-dimensional structure in solution. These encouraging results show that de novo design of an idealized protein structure of more than 200 amino acid residues is now possible, from construction of a particular backbone conformation to determination of an amino acid sequence with an automated sequence selection algorithm.

A recombinant viral haemorrhagic septicaemia virus glycoprotein expressed in insect cells induces protective immunity in rainbow trout

Viral haemorrhagic septicaemia (VHS) is a fish rhabdovirus infection of world-wide importance. Control policies have been established but the disease still causes heavy losses in fish farming. The development of a recombinant subunit vaccine was initiated to produce a safe and effective vaccine to protect fish against VHS. The VHS virus (VHSV) glycoprotein, which induces neutralizing antibodies in rainbow trout, was chosen for expression in insect cells using a baculovirus vector. The M(r) of the recombinant protein estimated by SDS-PAGE was slightly lower than that of the native viral protein. The recombinant protein displayed different degrees of glycosylation and was recognized in ELISA by neutralizing antibodies. It was transported to the plasma membrane of insect cells where its ability to induce membrane fusion was preserved. The efficacy of the recombinant protein as a vaccine was compared with those of an inactivated and an attenuated vaccine. When injected intraperitoneally into rainbow trout, the baculovirus-encoded protein was shown (i) to induce the synthesis of VHSV-neutralizing antibodies and (ii) to confer protection against virus challenge. Immunization performed by immersion failed. This is the first report of a recombinant vaccine that protects fish against VHSV.