Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type–specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan-neural marker class III β-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron-specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration.     

Tapirus balkanicus nov. sp., nouveau tapir (Perissodactyla, Mammalia) du Turolien de Bulgarie

A new species of Tapiridae, Tapirus balkanicus, is etablished for new material of the Bulgarian Upper Miocene. The comparison with other Neogene tapirids leads us to consider the upper jaw as more characteristic than the lower one for taxonomic determination and study of phyletic relations. A new subgenus, Meyeriscus, is created for Tapirus pannonicus, the most evolved European tapirid.

Résumé

Une nouvelle espèce de tapir est décrite, Tapirus balkanicus du Miocène supérieur de Bulgarie. La comparaison avec les espèces européennes du Miocène moyen (Tapirus teilen) au Villafranchien (Tapirus arvernensis) amènent à privilégier la denture supérieure sur l'inférieure pour la détermination et les rapports phylétiques éventuels entre les espèces. Pour l'espèce la plus évoluée de tous les Tapiridés européens, Tapirus pannonicus est proposé un nom de sous-genre particulier, Meyeriscus nov.     

Test of dynamic closed-loop baroreflex and autoregulatory control of total peripheral resistance in intact and conscious sheep

This is the first study able to examine and delineate the actual actions of the physiological mechanisms responsible for the dynamic couplings between cardiac output (CO), arterial pressure (Pa), right atrial pressure (PRA), and total peripheral resistance (TPR) in an individual subject without altering the underlying regulatory mechanisms. Eight conscious male sheep were used, where both types of baroreceptors were independently exposed to simultaneous beat-to-beat pressure perturbations under intact closed-loop conditions while CO, Pa, PRA, and TPR were measured. We applied the cardiovascular system identification method proposed in a companion paper (4) to quantitatively characterize the dynamic closed-loop transfer relations CO-->Pa, PRA-->Pa, Pa-->TPR, and PRA-->TPR from the measured signals. To validate the dynamic properties of the estimated transfer relations, the essential parts of the linear dynamics of the model were independently and comprehensively evaluated via error model cross-validation, and the overall model's steady-state behavior was compared with a separate random effects regression approach. In addition to numerous physiological findings, we found that the cardiovascular system identification results were exceptionally consistent with the analytically derived solutions previously discussed in Ref. 4. In conclusion, this study presents the first time validation of a cardiovascular system identification method by means of experimentally acquired animal data in the intact and conscious animal and offers a set of powerful quantitative tools essential to advancing our knowledge of cardiovascular regulatory physiology.     

Enhanced rates of P700<Superscript>+</Superscript> dark-reduction in leaves of<Emphasis Type="Italic"> Cucumis sativus</Emphasis> L. photoinhibited at chilling temperature

The changes in electron transport within photosystem I (PSI) were studied in detached leaves of Cucumis sativus L. during the course of irradiation with moderate white light (300 mol photons m^–2 s^–1) at 4°C. When intact leaves were exposed to the combination of moderate light and low temperature, the amplitude of far-red light-induced P700 absorbance changes at 820 nm (A₈₂₀), a relative measure of PSI, progressively decreased as the light treatment time increased. Almost no oxidation of P700 was noticeable after 5 h. Methyl viologen accelerated the oxidation of P700 to a steady-state level and also increased the magnitudes of A₈₂₀ changes in photoinhibited leaves, reflecting the rapid removal of electrons from native carriers. Photoinhibition under moderate light and chilling temperature also accelerated the rate of P700⁺ reduction after far-red light excitation as the half-times of the two exponential components of P700⁺ decay curves decreased relative to the control ones. A detailed analysis of the kinetics of P700⁺ reduction using diuron alone or the combination of diuron and methyl viologen strongly favours an increased rate of electron donation from stromal reductants to PSI through the plastoquinone pool following photoinhibitory treatment. Importantly, the marked acceleration of P700⁺ re-reduction is the consequence of the irradiation of leaf segments at low temperature and not caused by chilling stress alone.Abbreviations A ₀ and A ₁ Primary acceptor chlorophyll and secondary electron acceptor phylloquinone - FR Far-red light - F _X , F _A , and F _B Iron–sulfur centers - MT Multiple-turnover flash - MV Methyl viologen - Ndh NAD(P)H-dehydrogenase - PQ Plastoquinone - PS Photosystem - P700 Reaction-center chlorophyll of PSI - ST Single-turnover flash     

Methoxyoxalamido chemistry in the synthesis of novel amino linker and spacer phosphoramidites: a robust means for stability, structural versatility, and optimal tether length

We have developed a general route to the synthesis of novel amino linker and spacer phosphoramidites utilizing methoxyoxalamido (MOX) chemistry. The synthesis makes use of readily available and inexpensive primary aliphatic amino alcohols and diamines to produce a rich and diverse variety of phosphoramidites. Among these are monomers with exceptionally long (up to 56 atoms in length) amphipathic tethering arms. The chemistry bestows exceptional control over the physical characteristics within the tethers through the selection of appropriate building blocks. Furthermore, MOX chemistry enables fairly rapid assembly of these discrete-length tethers in a modular fashion. All novel phosphoramidites were successfully used in automated syntheses of 5'-modified oligonucleotides.     

Bidirectional replication from an internal ori site of the linear N15 plasmid prophage

The prophage of coliphage N15 is not integrated into the chromosome but exists as a linear plasmid molecule with covalently closed hairpin ends (telomeres). Upon infection the injected phage DNA circularizes via its cohesive ends. Then, a phage-encoded enzyme, protelomerase, cuts the circle and forms the hairpin telomeres. N15 protelomerase acts as a telomere-resolving enzyme during prophage DNA replication. We characterized the N15 replicon and found that replication of circular N15 miniplasmids requires only the repA gene, which encodes a multidomain protein homologous to replication proteins of bacterial plasmids replicated by a theta-mechanism. Replication of a linear N15 miniplasmid also requires the protelomerase gene and telomere regions. N15 prophage replication is initiated at an internal ori site located within repA and proceeds bidirectionally. Electron microscopy data suggest that after duplication of the left telomere, protelomerase cuts this site generating Y-shaped molecules. Full replication of the molecule and subsequent resolution of the right telomere then results in two linear plasmid molecules. N15 prophage replication thus appears to follow a mechanism that is distinct from that employed by eukaryotic replicons with this type of telomere and suggests the possibility of evolutionarily independent appearances of prokaryotic and eukaryotic replicons with covalently closed telomeres.