Neurotrophic effects of PACAP in the cerebellar cortex

In the rodent cerebellum, PACAP is expressed by Purkinje neurons and PAC1 receptors are present on granule cells during both the development period and in adulthood. Treatment of granule neurons with PACAP inhibits proliferation, slows migration, promotes survival and induces differentiation. PACAP also protects cerebellar granule cells against the deleterious effects of neurotoxic agents. Most of the neurotrophic effects of PACAP are mediated through the cAMP/PKA signaling pathway and often involve the ERK MAPkinase. Caspase-3 is one of the key enzymes implicated in the neuroprotective action of PACAP but PACAP also inhibits caspase-9 activity and increases Bcl-2 expression. PACAP and functional PAC1 receptors are expressed in the monkey and human cerebellar cortex with a pattern of expression very similar to that described in rodents, suggesting that PACAP could also exert neurodevelopmental and neuroprotective functions in the cerebellum of primates including human.     

Solution structure of the region 51-160 of human KIN17 reveals an atypical winged helix domain

Human KIN17 is a 45-kDa eukaryotic DNA- and RNA-binding protein that plays an important role in nuclear metabolism and in particular in the general response to genotoxics. Its amino acids sequence contains a zinc finger motif (residues 28-50) within a 30-kDa N-terminal region conserved from yeast to human, and a 15-kDa C-terminal tandem of SH3-like subdomains (residues 268-393) only found in higher eukaryotes. Here we report the solution structure of the region 51-160 of human KIN17. We show that this fragment folds into a three-alpha-helix bundle packed against a three-stranded beta-sheet. It belongs to the winged helix (WH) family. Structural comparison with analogous WH domains reveals that KIN17 WH module presents an additional and highly conserved 3(10)-helix. Moreover, KIN17 WH helix H3 is not positively charged as in classical DNA-binding WH domains. Thus, human KIN17 region 51-160 might rather be involved in protein-protein interaction through its conserved surface centered on the 3(10)-helix.     

Development of potent, orally active 1-substituted-3,4-dihydro-2-quinolone glycogen phosphorylase inhibitors

A series of substituted 3,4-dihydro-2-quinolone glycogen phosphorylase inhibitors, which have potential as antidiabetic agents, is described. Initial members of the series showed good enzyme inhibitory potency but poor physical properties. Optimisation of the 1-substituent led to 2,3-dihydroxypropyl compounds which showed good in vitro potency and improved physical properties, together with good DMPK profiles and acute in vivo efficacy in a rat model. X-ray crystallographic data are presented, showing an unexpected variety of binding orientations at the dimer interface site.     

Embryonic stem cell therapy for diabetes mellitus

There is a compelling need to develop novel therapies for diabetes mellitus. Recent successes in the transplantation of islets of Langerhans are seen as a major breakthrough. However, there is huge disparity between potential recipients and the availability of donor tissue. Human embryonic stem cells induced to form pancreatic beta cells could provide a replenishable supply of tissue. Early studies on the spontaneous differentiation of mouse embryonic stem cells have laid the foundation for a more directed approach based on recapitulating the events that occur during the development of the pancreas in the mouse. A high yield of definitive endoderm has been achieved, and although beta-like cells can be generated in a step-wise manner, the efficiency is still low and the final product is not fully differentiated. Future challenges include generating fully functional islet cells under Xeno-free and chemically defined conditions and circumventing the need for immunosuppression.     

Efficient surface patterning of oligonucleotides inside a glass capillary through oxime bond formation

The efficient surface patterning of oligonucleotides was accomplished onto the inner wall of fused-silica capillary tubes as well as on the surface of glass slides through oxime bond formation. The robustness of the method was demonstrated by achieving the surface immobilization of up to three different oligonucleotide sequences inside the same capillary tube. The method involves the preparation of surfaces grafted with reactive aminooxy functionalities masked with the photocleavable protecting group, 2-(2-nitrophenyl) propyloxycarbonyl group (NPPOC). Briefly, NPPOC-aminooxy silane 1 was prepared and used to silanize the glass surfaces. The NPPOC group was cleaved under brief irradiation to unmask the reactive aminooxy group on surfaces. These reactive aminooxy groups were allowed to react with aldehyde-containing oligonucleotides to achieve an efficient surface immobilization. The advantage associated with the present approach is that it combines the high-coupling efficiency of oxime bond formation with the convenience associated with the use of photolabile groups. The present strategy thus offers an alternative approach for the immobilization of biomolecules in the microchannels of "labs on a chip" devices.     

Head-to-head bis-hairpin polyamide minor groove binders and their conjugates with triplex-forming oligonucleotides: studies of interaction with target double-stranded DNA

Two hairpin hexa(N-methylpyrrole)carboxamide DNA minor groove binders (MGB) were linked together via their N-termini in head-to-head orientation. Complex formation between these bis-MGB conjugates and target DNA has been studied using DNase I footprinting, circular dichroism, thermal dissociation, and molecular modeling. DNase I footprint revealed binding of these conjugates to all the sites of 492 b.p. DNA fragment containing (A/T)(n)X(m)(A/T)(p) sequences, where n>3, p>3; m=1,2; X = A,T,G, or C. Binding affinity depended on the sequence context of the target. CD experiments and molecular modeling showed that oligo(N-methylpyrrole)carboxamide moieties in the complex form two short antiparallel hairpins rather than a long parallel head-to-head hairpin. Binding of bis-MGB also stabilized a target duplex thermodynamically. Sequence specificity of bis-MGB/DNA binding was validated using bis-conjugates of sequence-specific hairpin (N-methylpyrrole)/(N-methylimidazole) carboxamides. In order to increase the size of recognition sequence, the conjugates of bis-MGB with triplex-forming oligonucleotides (TFO) were synthesized and compared to TFO conjugated with single MGB hairpin unit. Bis-MGB-oligonucleotide conjugates also bind to two blocks of three and more A.T/T.A pairs similarly to bis-MGB alone, independently of the oligonucleotide moiety, but with lower affinity. However, the role of TFO in DNA recognition was demonstrated for mono-MGB-TFO conjugate where the binding was detected mainly in the area of the target sequence consisting of both MGB and TFO recognition sites. Basing on the molecular modeling, three-dimensional models of both target DNA/bis-MGB and target DNA/TFO-bis-MGB complexes were built, where bis-MGB forms two antiparallel hairpins. According to the second model, one MGB hairpin is in the minor groove of 5'-adjacent A/T sequence next to the triplex-forming region, whereas the other one occupies the minor groove of the TFO binding polypurine tract. All these data together give a key information for the construction of MGB-MGB and MGB-oligonucleotide conjugates possessing high specificity and affinity for the target double-stranded DNA.     

Conceptual bases for quantifying the role of the environment on gene evolution: the participation of positive selection and neutral evolution

To demonstrate that a given change in the environment has contributed to the emergence of a given genotypic and phenotypic shift during the course of evolution, one should ask to what extent such shifts would have occurred without environmental change. Of course, such tests are rarely practical but phenotypic novelties can still be correlated to genomic shifts in response to environmental changes if enough information is available. We surveyed and re-evaluated the published data in order to estimate the role of environmental changes on the course of species and genomic evolution. Only a few published examples clearly demonstrate a causal link between a given environmental change and the fixation of a genomic variant resulting in functional modification (gain, loss or alteration of function). Many others suggested a link between a given phenotypic shift and a given environmental change but failed to identify the underlying genomic determinant(s) and/or the associated functional consequence(s). The proportion of genotypic and phenotypic variation that is fixed concomitantly with environmental changes is often considered adaptive and hence, the result of positive selection, even though alternative causes, such as genetic drift, are rarely investigated. Therefore, the second aim herein is to review evidence for the mechanisms leading to fixation.     

On the use of ultrasounds to quantify the longitudinal threshold force to detach osteoblastic cells from a conditioned glass substrate

Cell adhesion on a biomaterial is an important phase of the cell-material interactions and the quality of this phase governs the success of the biomaterial integration. Understanding of the phenomena of cell adhesion and in particular understanding of cell adhesion on biomaterials is of crucial importance for the development of new biomaterials with excellent biocompatibility. One of the physical quantitative indexes to evaluate the quality of cell-material adhesion is its strength. Determining the strength of adhesive bonds requires applying external forces to the cells. Thus, a few methods have been developed to evaluate the strength of cell-material adhesion (micropipette, microplates, microcantilever, ...). These methods apply shear forces on adherent cells. The aim of our work is the development of a new ultrasonic characterization method of cellular adhesion on substrates. With our method, longitudinal acoustic waves are applied on cell culture to impose a longitudinal strain on cells. Only the cells subjected to a sufficient level of strain will be detached from the substrate. The idea is to correlate cell detachment rate to the longitudinal strain threshold supported by cells. From this result, we can deduce the critical force just sufficient to detach the cell. This global method can be adapted for different cell types and for different substrates. This method can provide an evaluation of the effect of functionalization on substrates. The technique is investigated for the 200 kHz ultrasound frequency. An insonificator adapted to the use of cell culture boxes was developed and calibrated. Tests were carried out on a glass substrate with or without biological conditioning. We used the MC3T3-E1 osteoblastic cell line. Our results to date provide the value of the necessary force to detach with reproducibility osteoblastic cells from glass.     

Top-down mass spectrometry of intact membrane protein complexes reveals oligomeric state and sequence information in a single experiment

Here we study the intact stoichiometry and top-down fragmentation behavior of three integral membrane proteins which were natively reconstituted into detergent micelles: the mechano-sensitive ion channel of large conductance (MscL), the Kirbac potassium channel and the p7 viroporin from the hepatitis C virus. By releasing the proteins under nondenaturing conditions inside the mass spectrometer, we obtained their oligomeric sizes. Increasing the ion activation (collision energy) causes unfolding and subsequent ejection of a highly charged monomer from the membrane protein complexes. Further increase of the ion activation then causes collision-induced dissociation (CID) of the ejected monomers, with fragments observed which were predominantly found to stem from membrane-embedded regions. These experiments show how in a single experiment, we can probe the relation between higher-order structure and protein sequence, by combining the native MS data with fragmentation obtained from top-down MS.