Clinical pharmacology of the new COMT inhibitor CGP 28 014

CGP 28 014 is a specific inhibitor of catechol-O-methyltransferase (COMT) in vivo. In humans, the inhibition was assessed by measuring urinary excretion of isoquinolines and with the levodopa test. Following administration of CGP 28 014, urinary excretion of isoquinolines was significantly increased. In rats, CGP 28 014 reduced plasma and striatal concentrations of 3-O-methyldopa (30MD) in a dose-dependent manner. Acute and subchronic administration of CGP 28 014 alone or in combination with the peripherally acting decarboxylase inhibitor benserazide decreased plasma 30MD as an index of COMT inhibition by about 50%. There seems to be a close relationship between the time-course of plasma concentrations of CGP 28 014 and the extent of COMT inhibition assessed by the 30MD/DOPA ratio in plasma.     

In vitro study of the proteolytic activity of rumen anaerobic fungi

Abstract To better define the antigenic structure of the outer cell membranes of Legionellae, a panel of 6 monoclonal antibodies was raised against partially purified outer membranes of Legionella pneumophila serogroup 1, Corby strain. This study describes the purification and characterization of one of these monoclonal antibodies reacting with a 135-kDa protein, which was shown to be common to all 14 serogroups of Legionella pneumophila . It shows no cross-reactivity with 20 other Legionella species, or 9 other Gram-negative species tested by SDS-PAGE and Western blotting procedures. The epitope would appear to be predominantly surface exposed and, from preliminary detergent extraction studies, not peptidoglycan-associated.     

Actin and spectrin-like (Mr= 260–240 000) proteins in gregarines

In Gregarina blaberae a M_r = 47 000 and a M_r = 260–240 000 doublet polypeptides reacted in immunoblotting: i) with a polyclonal monospecific rabbit antibody to frog muscular actin, a monoclonal anti-actin antibody against chicken gizzard; and ii) with polyclonal and monoclonal antibodies to human erythrocyte β-spectrin, respectively. The M_r = 47 000 actin-like protein is associated with the ghost and a contractille cytoplasmic extract. The presence of an actin-like protein in Gregarina and Lecudina and its cellular distribution in the cortex indicated that the gliding movement might involve an actin-myosin system in contrast to previous studies. Immunofluorescence showed clear differences between the anterior part of Gregarina and Lecudina which illustrated the high cell polarity of these protozoa. The M_r = 260–240 000 doublet was detected in SDS-PAGE from G. blaberae trophozoite ghosts but not in the cytoplasmic extracts or in extracts from sexual stages, indicating that the presence of these spectrin-like proteins is stage-dependent. Visualization of the M_r = 260–240 000 by immunofluorescence showed clear species differences, with rings arranged perpendicular to the longitudinal narrow folds of G. blaberae, with longitudinal lines underlying the folds of L. pellucida and with lines separating the large folds of Selenidium pendula. The cellular distribution is consistent with a stabilizer function of the spectrin-like proteins in the scaffolding of the cortex of gregarines according to the high diversity of the cell-shape and the cell motility systems in gregarines. The presence of spectrin-like proteins in protozoa and particularly in parasites from primitive arthropods indicated that ancestral spectrin genes could the M_r = 260–240 000 form.     

Characterization of the bioactive conformation of the C-terminal tripeptide Gly-Leu-Met-NH2 of substance P using [3-prolinoleucine10]SP analogues.

Residue Leu10 of substance P (SP) is critical for NK-1 receptor recognition and agonist activity. In order to probe the bioactive conformation of this residue, cis- and trans-3-substituted prolinoleucines were introduced in position 10 of SP. The substituted SP analogues were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in CHO cells transfected with the human NK-1 receptor. [trans-3-prolinoleucine10]SP retained affinity and potency similar to SP whereas [cis-3-prolinoleucine10]SP shows dramatic loss of affinity and potency. To analyze the structural implications of these biological results, the conformational preferences of the SP analogues were analyzed by NMR spectroscopy and minimum-energy conformers of Ac-cis-3-prolinoleucine-NHMe, Ac-trans-3-prolinoleucine-NHMe and model dipeptides were generated by molecular mechanics calculations. From NMR and modeling studies it can be proposed that residue Leu10 of SP adopts a gauche(+) conformation around the chi1 angle and a trans conformation around the chi2 angle in the bioactive conformation. Together with previously published results, our data indicate that the C-terminal SP tripeptide should preferentially adopt an extended conformation or a PPII helical structure when bound to the receptor.     

Begging, food provisioning, and nestling competition in great tit broods infested with ectoparasites

Ectoparasites are a ubiquitous environmental component of breedingbirds, and it has repeatedly been shown that hematoph-agousectoparasites such as fleas and mites reduce the quality andnumber of offspring of bird hosts, thereby lowering the valueof a current brood. Selection acting on the hosts will favorphysiological and behavioral responses that will reduce theparasites' impact. However, the results of the few bird studiesthat addressed the question of whether parasitism leads to ahigher rate of food provisioning are equivocal, and the beggingresponse to infestation has rarely been quantified. A changein begging activity and parental rate of food provisioning couldbe predicted in either direction: parents could reduce theirinvestment in the brood in order to invest more in future broods,or they could increase their investment in order to compensatefor the parasites' effect on the current brood. Since the nestlingsare weakened by the ectoparasites they may beg less, but onthe other hand they may beg more in order to obtain more food.In this study we show experimentally that (1) hen fleas (Ceratophyllusgallinae) reduce the body mass and size of great tit (Parusmajor) nestlings, (2) nestlings of parasitized broods more thandouble their begging rate, (3) the male parents increase thefrequency of feeding trips by over 50%, (4) the females do notadjust feeding rate to the lowered nutritional state of nestlings,and (5) food competition among siblings of parasitized broodsis increased. Ultimately the difference in the parental feedingresponse may be understood as the result of a sex-related differencein the trade-off of i0vesting in current versus future broods.     

Precise physical mapping of theEscherichia coli rnb gene, encoding ribonuclease II

Ribonuclease II (encoded byrnb) is one of the two main exonucleases involved in mRNA degradation inEscherichia coli. We report the precise physical mapping ofrnb to 29 min on the chromosomal map in the vicinity ofpyrF, and clarify the genetic and physical maps of thisE. coli chromosomal region. The results were confirmed by the construction of a strain partially deleted forrnb.     

Evolution of biological regulation networks under complex environmental constraints

This work investigates the influence of environmental inducers on the organization of cell regulation networks, using a connectionist approach. Protein interactions are modeled by an asymmetrical recurrent network, the units of which take continuous values. In contrast to classical models, we explicitly introduce a genome to encode the architecture of the system. This feature enables us to introduce an evolution model, in which a genetic algorithm that mimics the effects of evolution on proteins mutual interactions is used. We assume an efficient system to respond to persistent environmental stimuli, independently of their amplitude. Results are presented that show a structuration of the network with the emergence of specialized hierarchical structures. These structures seem to drive the system at the edge of chaos, so that it can present adapted responses to significant environmental changes.     

Long timestep dynamics of peptides by the dynamics driver approach

Previous experience with the Langevin/implicit-Euler scheme for dynamics (“LI”) on model systems (butane, water) has shown that LI is numerically stable for timesteps in the 5–20 fs range but quenches high-frequency modes. To explore applications to polypeptides, we apply LI to model systems (several dipeptides, a tetrapeptide, and a 13-residue oligoalanine) and also develop a new dynamics driver approach (“DA”). The DA scheme, based on LI, addresses the important issue of proper sampling, which is unlikely to be solved by small-time step integration methods or implicit methods with intrinsic damping at room temperature, such as LI. Equilibrium averages, time-dependent molecular properties, and sampling trends at room temperature are reported for both LI and DA dynamics simulations, which are then compared to those generated by a standard explicit discretization of the Langevin equation with a 1 fs timestep. We find that LI's quenching effects are severe on both the fast and slow (due to vibrational coupling) frequency modes of all-atom polypeptides and lead to more restricted dynamics at moderate timesteps (40 fs). The DA approach empirically counteracts these damping effects by adding random atomic perturbations to the coordinates at each step (before the minimization of a dynamics function). By restricting the energetic fluctuations and controlling the kinetic energy, we are able with a 60 fs timestep to generate continuous trajectories that sample more of the relevant conformational space and also reproduce reasonably Boltzmann statistics. Although the timescale for transition may be accelerated by the DA approach, the transitional. information obtained for the alanine dipeptide and the tetrapeptide is consistent with that obtained by several other theoretical approaches that focus specifically on the determination of pathways. While the trajectory for oligoalanine by the explicit scheme over the nanosecond timeframe remains in the vicinity of the full α_R-helix starting structure, and a high-temperature (6000°K) MD trajectory departs slowly from the a helical structure, the DA-generated trajectory for the same CPU time exhibits unfolding and refolding and reveals a range of conformations with an intermediate helix content. Significantly, this range of states is more consistent with spectroscopic experiments on small peptides, as well as the cooperative two-state model for helix–coil transition. The good, near-Boltzmann statistics reported for the smaller systems above, in combination with the interesting oligoalanine results, suggest that DA is a promising tool for efficiently exploring conformational spaces of biomolecules and exploring folding/unfolding processes of polypeptides. © 1995 Wiley-Liss, Inc.