VARIATION OF PLASTID AND MITOCHONDRIAL DNAS IN THE GENUS DAUCUS

Plastid and mitochondrial DNAs from suspension cultures of Daucus carota subsp. sativus cv. Danvers, D. carota subsp. gummifer, D. capillifolius, and D. pusillus were compared by restriction endonuclease fragment analysis. Organelles isolated from protoplasts of suspension cultures were purified using a one-step sucrose gradient. Plastid DNA fragment patterns for subsp. sativus, subsp. gummifer, and capillifolius were indistinguishable in Pstl, SalI and XhoI enzyme digests. No variation was detected in BamHI or HindIII digests between subsp. sativus and capillifolius. Pusillus plastid DNA varied significantly when compared to the other Daucus cultures. The size of the plastid genome of each species as determined by fragment addition, was estimated at 155 kb. Restriction digests of the mitochondrial DNAs generated a large number of fragments which when totaled established a size of 386–468 kb for the genomes. Densitometer scans of the fragment patterns indicate the bands were present in variable stoichiometry. Up to 26% of the fragments generated by PstI, SalI and HindIII digests were homologous in size among the four mitochondrial genomes. A further comparison of mitochondrial fragments indicated a closer homology of subsp. sativus to capillifolius than to subsp. gummifer or pusillus as was also found with the plastid genomes.     

Fourier transform IR spectroscopic appraisal of radiation damage in Micrococcus luteus

Fourier transform IR spectroscopy (FTIR) is used to analyze cells of Micrococcus luteus, the type species of the highly heterogeneous genus Micrococcus that belongs to the Micrococcaceae family. The cells of M. luteus, which is a Gram-positive and yellow-pigmented bacterium, are submitted to increasing doses of gamma radiation. Irradiation leads to the generation of reactive oxygen species that induce biochemical changes as shown in spectral profiles. Beyond a dose of 0.70 kGy, significant differences between samples are observed, particularly in the 1485-900 cm(-1) region, which contains information about membrane lipids, cell wall polysaccharides, and nucleic acids. After a dose of 16.50 kGy, M. luteus is reincubated for times ranging from 1 to 24 h. Postirradiation reincubated bacteria are found far from the control and irradiated cells (mainly in the 985-900 cm(-1) range), suggesting that a biomolecular rearrangement occurs as soon as reincubation begins in the growth medium. Thus, FTIR spectroscopy appears to be a very useful technique for the rapid visualization of the alterations induced by both the radiation and mutagenic response during reincubation. The use of mathematical methods gives good insight into the biomolecular compounds involved in these two mechanisms. In view of these preliminary results, we hypothesize that it can be successfully applied to any type of tissue and that it may be a future interesting tool for evaluating the effects of radiation in humans.     

Design and synthesis of benzoazepinone-derived cyclic malonamides and aminoamides as potent gamma-secretase inhibitors

We report the synthesis of benzoazepine-derived cyclic malonamides (2) and aminoamides (3) as gamma-secretase inhibitors for the potential treatment of Alzheimer's disease. The in vitro structure-activity relationships of 2 and 3 along with dog pharmacokinetic results are described.     

From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice

Therapeutic monoclonal antibodies have shown limited efficacy and safety owing to immunogenicity of mouse sequences in humans. Among the approaches developed to overcome these hurdles were transgenic mice genetically engineered with a 'humanized' humoral immune system. One such transgenic system, the XenoMouse, has succeeded in recapitulating the human antibody response in mice, by introducing nearly the entire human immunoglobulin loci into the germ line of mice with inactivated mouse antibody machinery. XenoMouse strains have been used to generate numerous high-affinity, fully human antibodies to targets in multiple disease indications, many of which are progressing in clinical development. However, validation of the technology has awaited the recent regulatory approval of panitumumab (Vectibix), a fully human antibody directed against epidermal growth factor receptor (EGFR), as treatment for people with advanced colorectal cancer. The successful development of panitumumab represents a milestone for mice engineered with a human humoral immune system and their future applications.     

Resistance to schistosome infection in Biomphalaria glabrata induced by gamma radiation

Two strains of Biomphalaria glabrata were studied with respect to the effects of ionizing radiation on their susceptibility to Schistosoma mansoni infection. Gamma radiation at levels of 3.5 and 5 krad did not induce susceptibility in the resistant S-3 strain, but was found to initiate resistance in the susceptible PR-1 strain. In an attempt to understand the induced resistance in irradiated snails, histopathologic examinations and analyses of snail hemolymph were performed. Results indicated that miracidia invading irradiated snails were quickly surrounded and encapsulated by amoebocytes. Similarly, alterations in the hemolymph of irradiated snails suggested that radiation induced aging. It is suggested that radiation-altered snails may be of value in studying the defense mechanisms of these organisms.     

Real-time kinetic modeling of voltage-gated ion channels using dynamic clamp

We propose what to our knowledge is a new technique for modeling the kinetics of voltage-gated ion channels in a functional context, in neurons or other excitable cells. The principle is to pharmacologically block the studied channel type, and to functionally replace it with dynamic clamp, on the basis of a computational model. Then, the parameters of the model are modified in real time (manually or automatically), with the objective of matching the dynamical behavior of the cell (e.g., action potential shape and spiking frequency), but also the transient and steady-state properties of the model (e.g., those derived from voltage-clamp recordings). Through this approach, one may find a model and parameter values that explain both the observed cellular dynamics and the biophysical properties of the channel. We extensively tested the method, focusing on Na_v models. Complex Markov models (10-12 states or more) could be accurately integrated in real time at >50 kHz using the transition probability matrix, but not the explicit Euler method. The practicality of the technique was tested with experiments in raphe pacemaker neurons. Through automated real-time fitting, a Hodgkin-Huxley model could be found that reproduced well the action potential shape and the spiking frequency. Adding a virtual axonal compartment with a high density of Na_v channels further improved the action potential shape. The computational procedure was implemented in the free QuB software, running under Microsoft Windows and featuring a friendly graphical user interface.     

Synthesis and SAR of hydroxyethylamine based phenylcarboxyamides as inhibitors of BACE

A series of N-((2S,3R)-1-(3,5-difluorophenyl)-3-hydroxy-4-(3-methoxybenzylamino)-butan-2-yl)benzamides has been synthesized as BACE inhibitors. A variety of P2 and P3 substituents has been explored, and these efforts have culminated in the identification of several 1,3,5-trisubstituted phenylcarboxyamides with potent BACE inhibitory activity.     

StAR-related lipid transfer domain protein 5 binds primary bile acids

Steroidogenic acute regulatory-related lipid transfer (START) domain proteins are involved in the nonvesicular intracellular transport of lipids and sterols. The STARD1 (STARD1 and STARD3) and STARD4 subfamilies (STARD4–6) have an internal cavity large enough to accommodate sterols. To provide a deeper understanding on the structural biology of this domain, the binding of sterols to STARD5, a member of the STARD4 subfamily, was monitored. The SAR by NMR [¹H-¹⁵N heteronuclear single-quantum coherence (HSQC)] approach, complemented by circular dichroism (CD) and isothermal titration calorimetry (ITC), was used. Titration of STARD5 with cholic (CA) and chenodeoxycholic acid (CDCA), ligands of the farnesoid X receptor (FXR), leads to drastic perturbation of the ¹H-¹⁵N HSQC spectra and the identification of the residues in contact with those ligands. The most perturbed residues in presence of ligands are lining the internal cavity of the protein. Ka values of 1.8·10−⁴ M⁻¹ and 6.3·10⁴ M⁻¹ were measured for CA and CDCA, respectively. This is the first report of a START domain protein in complex with a sterol ligand. Our original findings indicate that STARD5 may be involved in the transport of bile acids rather than cholesterol.