Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one-pot/two-step biocatalytic whole-cell system

The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer–Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L ⁻¹·h ⁻¹) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L ⁻¹·h ⁻¹).     

Reproductive success of a marine teleost was correlated with proactive and reactive stress-coping styles

The present study investigated the relationship between reproductive success and stress-coping styles in gilthead seabream Sparus aurata in captivity. To characterise stress-coping styles, a total of 22 breeders were submitted to three different individual-based tests, one group-based test and post-handling glucocorticoid quantification. To assess spawning participation, a microsatellite analysis was performed on a total of 2698 larvae, which allowed each offspring to be assigned unambiguously to a single parental couple. Overall, S. aurata showed defined proactive and reactive behavioural traits. Proactive breeders exhibited higher levels of activity and risk taking and lower glucocorticoid blood levels than reactive breeders. The stress-coping style traits were consistent over time and context (different tests). Breeders that contributed to a higher number of progeny exhibited proactive behaviours, while those showing low progeny contribution exhibited reactive behaviour. Therefore, breeders with a high proportion of progeny (> 20%) had significantly higher activity and risk taking and lower cortisol than breeders with low progeny contribution (< 20%). In addition, males were more proactive than females and males exhibited significantly higher activity, risk taking and lower cortisol than females. This study is the first to establish in S. aurata breeders: (a) a relationship between stress-coping styles and spawning success; (b) a relationship between stress-coping styles and gender; and (c) the existence of proactive and reactive traits at the adult stage.     

Lamin A and the LINC complex act as potential tumor suppressors in Ewing Sarcoma

Lamin A, a main constituent of the nuclear lamina, is involved in mechanosignaling and cell migration through dynamic interactions with the LINC complex, formed by the nuclear envelope proteins SUN1, SUN2 and the nesprins. Here, we investigated lamin A role in Ewing Sarcoma (EWS), an aggressive bone tumor affecting children and young adults. In patients affected by EWS, we found a significant inverse correlation between LMNA gene expression and tumor aggressiveness. Accordingly, in experimental in vitro models, low lamin A expression correlated with enhanced cell migration and invasiveness and, in vivo, with an increased metastatic load. At the molecular level, this condition was linked to altered expression and anchorage of nuclear envelope proteins and increased nuclear retention of YAP/TAZ, a mechanosignaling effector. Conversely, overexpression of lamin A rescued LINC complex organization, thus reducing YAP/TAZ nuclear recruitment and preventing cell invasiveness. These effects were also obtained through modulation of lamin A maturation by a statin-based pharmacological treatment that further elicited a more differentiated phenotype in EWS cells. These results demonstrate that drugs inducing nuclear envelope remodeling could be exploited to improve therapeutic strategies for EWS.Subject terms: Nuclear organization, Cancer     

D2/D3 dopamine receptor heterodimers exhibit unique functional properties

Evidence for heterodimerization has recently been provided for dopamine D(1) and adenosine A(1) receptors as well as for dopamine D(2) and somatostatin SSTR(5) receptors. In this paper, we have studied the possibility that D(2) and D(3) receptors interact functionally by forming receptor heterodimers. Initially, we split the two receptors at the level of the third cytoplasmic loop into two fragments. The first, containing transmembrane domains (TM) I to V and the N-terminal part of the third cytoplasmic loop, was named D(2trunk) or D(3trunk), and the second, containing the C-terminal part of the third cytoplasmic loop, TMVI and TMVII, and the C-terminal tail, was named D(2tail) or D(3tail). Then we defined the pharmacological profiles of the homologous (D(2trunk)/D(2tail) and D(3trunk)/D(3tail)) as well as of the heterologous (D(2trunk)/D(3tail) and D(3trunk)/D(2tail)) cotransfected receptor fragments. The pharmacological profile of the cross-cotransfected fragments was different from that of the native D(2) or D(3) receptors. In most cases, the D(3trunk)/D(2tail) was the one with the highest affinity for most agonists and antagonists. Moreover, we observed that all of these receptor fragments reduced the expression of the wild type dopamine D(2) and D(3) receptors, suggesting that D(2) and D(3) receptors can form complexes with these fragments and that these complexes bind [(3)H]nemonapride less efficiently or are not correctly targeted to the membrane. In a second set of experiments, we tested the ability of the split and the wild type receptors to inhibit adenylyl cyclase (AC) types V and VI. All of the native and split receptors inhibited AC-V and AC-VI, with the exception of D(3), which was unable to inhibit AC-VI. We therefore studied the ability of D(2) and D(3) to interact functionally with one another to inhibit AC-VI. We found that with D(2) alone, R-(+)-7-hydroxydypropylaminotetralin hydrobromide inhibited AC-VI with an IC(50) of 2.05 +/- 0.15 nm, while in the presence of D(2) and D(3) it inhibited AC-VI with an IC(50) of 0.083 +/- 0.011 nm. Similar results were obtained with a chimeric cyclase made from AC-V and AC-VI. Coimmunoprecipitation experiments indicate that D(2) and D(3) receptors are capable of physical interaction.     

Essential role for ADAM19 in cardiovascular morphogenesis

Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19(-/-) animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects.     

Development of a gene-based radiation hybrid map of chicken Chromosome 7 and comparison to human and mouse

To validate the ChickRH6 whole-genome radiation hybrid (WGRH) panel, we constructed a map of chicken Chromosome 7 based on 19 microsatellite markers from the genetic map and 76 ESTs (expressed sequence tags), whose efficient targeted development was made possible by using the ICCARE software. This high-density radiation hybrid (RH) map of a chicken macrochromosome gives us indications on characteristics of ChickRH6. The potential resolution of the panel is 325 kb and the practical resolution of our framework map is 1.3 Mb. Based on these results, a complete framework map of the chicken genome would comprise 1000 markers. The marker order is in good agreement with the genetic map and comparison with the human and mouse sequence maps revealed a number of internal rearrangements.     

Mu opioid receptor: a gateway to drug addiction

Mu opioid receptors mediate positive reinforcement following direct (morphine) or indirect (alcohol, cannabinoids, nicotine) activation, and our understanding of mu receptor function is central to the development of addiction therapies. Recent data obtained in native neurons confirm that mu receptor signaling and regulation are strongly agonist-dependent. Current functional mapping reveals morphine-activated neurons in the extended amygdala and early genomic approaches have identified novel mu receptor-associated proteins. A classification of about 30 genes either promoting or counteracting the addictive properties of morphine is proposed from the analysis of knockout mice data. The targeting of effectors or regulatory proteins, beyond the mu receptor itself, might provide valuable strategies to treat addictive disorders.     

A polyclonal antibody directed against syringylpropane epitopes of native lignins

With a view to visualizing the ultrastructural distribution of syringyl lignins in secondary plant cell walls, a polyclonal antibody raised from a synthetic DHP polymer consisting only of syringyl propane units was prepared. To test the reactivity of the antiserum, a mini-dot-blot immunoassay reducing the amounts of substrates and antiserum was developed. A characteristic attribute of the S-antiserum appears to be its specific recognition of sequences of three or more consecutive syringyl units. On ultra-thin sections of model plants of Arabidopsis thaliana, Populus and tobacco, the antiserum allowed us to demonstrate a higher concentration of syringyl epitopes in fibres than in vessels. Variations in the distribution pattern of these epitopes between the three plants examined suggest that the synthesis of syringyl lignins in angiosperms depends on the species.     

In vivo analysis of cell division, cell growth, and differentiation at the shoot apical meristem in Arabidopsis

The aerial parts of the plant are generated by groups of rapidly dividing cells called shoot apical meristems. To analyze cell behavior in these structures, we developed a technique to visualize living shoot apical meristems using the confocal microscope. This method, combined with green fluorescent protein marker lines and vital stains, allows us to follow the dynamics of cell proliferation, cell expansion, and cell differentiation at the shoot apex. Using this approach, the effects of several mitotic drugs on meristem development were studied. Oryzalin (depolymerizing microtubules) very rapidly caused cell division arrest. Nevertheless, both cell expansion and cell differentiation proceeded in the treated meristems. Interestingly, DNA synthesis was not blocked, and the meristematic cells went through several rounds of endoreduplication in the presence of the drug. We next treated the meristems with two inhibitors of DNA synthesis, aphidicolin and hydroxyurea. In this case, cell growth and, later, cell differentiation were inhibited, suggesting an important role for DNA synthesis in growth and patterning.