The organization of the movement depends mainly on the anticipation of its sensory and emotional consequences.

Two sources of emotions influence directed actions, namely, those associated with the environment and those that are consequences of the action. The present study examines the impact of these emotions on movement preparation. It invokes theories from psychology, i.e., ideomotor theory and motor control's cognitive approach through movement analysis. In addition to their action readiness, emotions related to the environment can interfere with actions directed towards a goal. However, intentional action involves a goal that will cause satisfaction when achieved. While most studies consider each emotion's influence separately, few studies confront them to study their respective impact. In the current study, thirty-two right-handed young adults reach for a left target with a stylus that will reduce or enlarge an emotional picture that is initially present (nontarget stimulus). Kinematic analyses show that anticipating the pointing's emotional consequences impacts the final pointing position. All other results emphasize the impact of reducing or enlarging on the preparation and control of movement depending on the direction of movement. The emotional consequences of the action is a weighting factor that is relevant to the action goal and subject's intention, but it is less important than the action's visual consequences.

Molecular cloning and expression of two horse pancreatic cDNA encoding colipase A and B.

Pancreatic colipase plays an essential role in the intestinal fat digestion by anchoring lipase on lipid/water interfaces in the presence of bile salts. In contrast to other species, two molecular forms of colipase, A and B, have been found in horse. The two corresponding cDNAs were isolated from a horse pancreatic library and their nucleotide sequences were determined. Moreover, for the first time, active colipase has been obtained after transfection of COS cells by either colipase A or B cDNA.

Primary structure of the activation peptide from bovine pancreatic procarboxypeptidase A.

The complete sequence of the 94 residues composing the activation peptide of bovine procarboxypeptidase A has been determined by automated analysis of the intact activation segment and of three peptides resulting from enzymatic cleavages of the isolated peptide. The sequencing of a CNBr peptide isolated from procarboxypeptidase A allowed to connect the activation peptide with alpha-carboxypeptidase A (peptidylprolyl-L-amino-acid hydrolase, EC 3.4.17.1). The activation segment has a high content of acidic residues and a proline-rich region. Conformational prediction studies show that the bovine peptide, as the porcine and rat peptides, contains a high proportion of secondary structure and that the structural disposition of the regions in secondary structure is similar in the three peptides. The comparison of the sequence of the bovine, porcine and rat peptides, although exhibiting a striking homology, clearly shows that 40% of the substitutions have led to a charge change.

Amino acid sequence of horse colipase B.

The complete sequence of the 96 residues composing horse colipase B has been determined by automated analysis of the intact protein, of two CNBr peptides and two tryptic peptides arising, respectively, from the citraconylated chain and from the unreduced protein. The single histidine of the protein is located at position 29 as in horse colipase A. His86, present in the C-terminal region of the pig cofactor and supposed to play a role in the folding molecule, is not conserved in horse B. Large pieces of the pig and horse B chains were found to be identical or very similar, especially the N-terminal sequence and the central segment Ala49-Cys65 including the three tyrosines of the molecule. The four lysines and the ten half cystines are also conserved.

Crystallization and preliminary X-ray study of subunit III of the bovine pancreatic procarboxypeptidase A-S6 ternary complex.

Subunit III of the bovine pancreatic procarboxypeptidase A-S6 ternary complex was dissociated from the complex, purified and crystallized using the hanging- or sitting-drop method of vapour diffusion, with ammonium sulphate as the precipitant. The assays were carried out at pH 4.2 (20 mM-acetate buffer). An X-ray examination of the crystals shows that they are monoclinic, with a space group P21 and cell dimensions a = 47.9 A, b = 61.3 A, c = 39.0 A and beta = 95.0 degrees. The asymmetric unit contains one molecule of 25,800 Mr. The crystals are suitable for structure determination to at least 2.8 A resolution.

Crystallization and preliminary X-ray study of horse pancreatic lipase.

Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content). Lipase crystals strongly diffract to at least 1.8 A resolution. Some molecular properties of horse lipase compared to those of the better-known porcine enzyme are also presented.

Crystallographic study of a cleaved, non-activatable form of porcine zymogen E.

The crystal structure of a cleaved form of porcine zymogen E has been solved by molecular replacement using the bovine procarboxypeptidase A-S6 subunit III structure as search model. Crystallographic refinement using simulated annealing and energy minimization techniques resulted in a final R-factor of 0.189 for all data between 8 and 2.3 A resolution. The zymogen E three-dimensional model is very close to that of bovine subunit III and represents the second member of the zymogen E family for which the crystal structure is known. The two structures indicate that, in contrast to trypsinogen and chymotrypsinogen, zymogens of this family are highly organized molecules. The amino acid sequence of zymogen E has only been determined for the first 40 residues. Based on the electron density map, we have introduced six sequence changes relative to subunit III. Out of the 11 residues in the activation peptide, only the first six present well matching electron density; they are connected to the rest of the zymogen by an unexpected Cys1-Cys122 disulphide bridge (according to the bovine chymotrypsinogen A numbering system). Amino acid sequencing of protein solutions both from dissolved crystals and from the initial stock clearly indicated that the Val17-Asn18 bond had been cleaved during the crystallization process. This result adds weight to the assumption that the autolysis of the bovine zymogen E gives rise to subunit III and that this maybe a regulatory mechanism for protease E activity.

Horse pancreatic lipase. The crystal structure refined at 2.3 A resolution.

Pancreatic lipase (EC 3.1.1.3) plays a key role in dietary fat digestion by converting triacylglycerols into 2-monoacylglycerols and free fatty acids in the intestine. Although the crystallographic structures of the human pancreatic lipase and of a human lipase-porcine colipase complex have been solved, no refined structure of pancreatic lipase has yet been published. The crystal structure of the horse enzyme was solved by the molecular replacement method from the model of the human pancreatic lipase and subsequently refined to 2.3 A resolution. The final model contains two molecules of 449 amino acid residues each in the asymmetric unit, 705 well-defined water molecules and two calcium ions. The two molecules in the asymmetric unit of the orthorhombic crystals are related by a 2-fold non-crystallographic symmetry axis as in the case of the human lipase. However, the association between the two molecules in their respective crystal forms is different. The overall molecular structure of the horse lipase is very similar to that of the human enzyme. The horse lipase is made up of two well-defined domains. The N-terminal domain which bears the active centre has a typical alpha/beta hydrolase fold topology. The C-terminal domain which is devoted to colipase binding has a beta-sheet sandwich topology. Comparison of equivalent C alpha atom positions between the final model of the horse lipase and the human lipase structure shows only slight differences which are mainly located in the C-terminal domain. The horse enzyme possesses the common features of the known mammalian and microbial lipases, in particular the "flap" covering the catalytic triad. In addition to more precise information concerning these features, the elucidation of the horse lipase crystal structure allowed us to better understand the structural basis of the kinetic behaviour of pancreatic lipases towards a soluble substrate, p-nitrophenyl acetate, and the different sensitivity of these enzymes towards limited proteolysis.

Subunit III of ruminant procarboxypeptidase A-S6 complexes and pancreatic proteases E. A new family of pancreatic serine endopeptidases?

Subunit III (BSIII) of the bovine ternary complex of procarboxypeptidase A-S6 (PCPA-S6), a defective serine endopeptidase-like protein, actively synthesized by the pancreas of some ruminant species, is highly homologous to human protease E (HPE). Both proteins possess the same atypical disulfide bridge in position 98-99b. They are structurally related to porcine elastase 1 and human elastase 2 (about 56% identity). However, in contrast to those two enzymes which have an overall positive net charge, BSIII and HPE are negatively charged. Three-dimensional models of BSIII and HPE have been constructed from the crystallographic structure of porcine pancreatic elastase 1. The inhibitor-binding site for TFAI in these three proteins seems to be very similar; the atypical disulfide bridge does not seem to be involved in this binding site. The specific structural features of BSIII and HPE strongly support the assumption that BSIII is a truncated protease E and that both proteins belong to a separate serine endopeptidase family.

Further studies on the human pancreatic binary complexes involving procarboxypeptidase A.

In contrast to procarboxypeptidase B which has always been reported to be secreted by the pancreas as a monomer, procarboxypeptidase A occurs as a monomer and/or associated to one or two functionally different proteins, depending on the species. Recent studies showed that, in the human pancreatic secretion, procarboxypeptidase A is mainly secreted as a 44 kDa protein involved in at least three different binary complexes. As previously reported, two of these complexes associated procarboxypeptidase A to either a glycosylated truncated protease E or zymogen E. In this paper, we identified proelastase 2 as the partner of procarboxypeptidase A in the third complex, thus reporting for the first time the occurrence of a proelastase 2/procarboxypeptidase A binary complex in vertebrates. Moreover, from N-terminal sequence analyses, the 44 kDa procarboxypeptidase A involved in these complexes was identified as being of the A1 type. Only one type of procarboxypeptidase B, the B1 type, has been detected in the analyzed pancreatic juices, thus emphasizing the previously observed genetic differences between individuals.

Identification of a procarboxypeptidase A-truncated protease E binary complex in human pancreatic juice.

The characterization, in human pancreatic juice, of a binary complex associating procarboxypeptidase A with a 32 kDa inactive glycoprotein (G32) is reported in this paper. Free G32 was isolated after dissociation of the binary complex. N-terminal sequence analysis revealed a complete homology between this protein and human protease E (HPE 1), except for the two strongly hydrophobic N-terminal residues (Val-Val) which are missing in G32. This protein might be a truncated protease E highly analogous to the subunit III of the ruminant procarboxypeptidase A-S6 ternary complex. The analogy with bovine subunit III is further supported by interspecies reassociation experiments showing that bovine procarboxypeptidase A can specifically bind human G32.

A cross-linked complex between horse pancreatic lipase and colipase.

The water soluble carbodiimide N-cyclohexyl-N'-2-morpholinoethyl-carbodiimide-methyl-p-toluolsulfona te was found to effectively covalently cross-link pancreatic colipase to lipase as evidenced by Western blotting experiments using antibodies directed either against lipase or colipase. Moreover the resulting covalent complex has a Mr consistent with a stoichiometry of 1 mol colipase per mol lipase. Cross-linked lipase and colipase retain their activity implying a correct covalent binding between the two proteins. The specificity of the lipase-colipase binding was further supported by the very low amount of cross-linked products when lipase or colipase alone were incubated in the presence of carbodiimide. The formation of a covalent lipase-colipase complex in the presence of carbodiimide clearly demonstrates that the binding between both proteins involves ion pairing. Furthermore, the formation of an active covalent complex strongly suggests that the lipase-colipase binding site is distinct from the colipase interfacial recognition site as well as from the lipase catalytic site.

Interactions of bile salt micelles and colipase studied through intermolecular nOes.

Colipase is a small protein (10 kDa), which acts as a protein cofactor for the pancreatic lipase. Various models of the activated ternary complex (lipase-colipase-bile salt micelles) have been proposed using detergent micelles, but no structural information has been established with bile salt micelles. We have investigated the organization of sodium taurodeoxycholate (NaTDC) micelles and their interactions with pig and horse colipases by homonuclear nuclear magnetic resonance (NMR) spectroscopy. The NMR data supply evidence that the folding of horse colipase is similar to that already described for pig colipase. Intermolecular nuclear Overhauser effects have shown that two conserved aromatic residues interact with NaTDC micelles.

The bovine pro-carboxypeptidase A-S6 ternary complex: a rare case of a secreted protein complex.

Up to now, a non-covalent ternary complex in which the pro-carboxypeptidase A (subunit I) is associated to two functionally different proteins (subunits II and III) has only been found in the pancreas of ruminant species. In the other species studied so far, the pro-carboxypeptidase A is secreted either as a monomer or as a binary association with a functionally different protein. Subunit I is the immediate precursor of carboxypeptidase A. Subunit II is a chymotrypsinogen of the C-type, involved, like subunit I, in the degradation of proteins and peptides. Although closely related to the pancreatic serine endopeptidases, subunit III appears to be devoid of any specific enzymatic activity. Information about the spatial organization of the subunits in the ternary complex has been deduced from the sequential dissociation of the complex. In contrast to the mechanism of activation of subunits I and II, which is independent of their aggregation state, the catalytic properties of the resulting enzymes are sensitive to their aggregation state. Moreover, the structural basis of inactivity of subunit III as well as the physiological role of the ternary complex are also discussed in this review.

Minireview on pancreatic lipase and colipase.

By hydrolyzing the dietary triacylglycerols, pancreatic lipase causes catalysis in heterogeneous medium. In vivo, lipase action cannot take place without colipase due to the presence of bile salts. The cofactor enables lipase anchoring to the water-lipid interface. The lipase-colipase system furnishes an excellent example of specific interactions (protein-protein and protein-lipid). The studies of lipase catalytic properties brought to light the importance of certain parameters related to the 'quality of the interface'. The structure-function relationship analyses revealed a certain number of functional amino acid residues in lipase and colipase involved either in the catalytic site of the enzyme or in the recognition sites (lipase-colipase and protein-interface). Comparisons of the sequences of lipases derived from different sources display interesting similarities in certain cases.

The lipase/colipase complex is activated by a micelle: neutron crystallographic evidence.

The catalytic activity of most lipases depends on the aggregation state of their substrates. It is supposed that lipase activation requires the unmasking and structuring of the enzyme's active site through conformational changes involving the presence of oil-in-water droplets. This phenomenon has been called interfacial activation. Here, we report the crystal structure of the pancreatic activated lipase/colipase/micelle complex as determined using the D2O/H2O contrast variation low resolution neutron diffraction method. We find that a disk-shaped micelle interacts extensively with the concave face of colipase (CL) and the distal tip of the C-terminal domain of lipase away from the active site of the enzyme. Such interaction appears to help stabilizing the lipase-CL interaction. Consequently, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and it is mediated by CL and a micelle.

Adsorption and activation of pancreatic lipase at interfaces.

The first step of the lipase-catalyzed hydrolysis of insoluble long chain triglycerides is the adsorption of the enzyme to the interface. This adsorption, which is spontaneous when the interface is hydrophobic, is hindered by bile salts. Under these conditions, a small protein cofactor designated colipase adsorbs first and then anchors lipase at the interface. Interfacial adsorption enhances lipase activity, due, at least in part, to an acceleration of the rate-limiting deacylation step of the reaction. In this respect, lipase appears to be a most interesting model of an enzyme being activated by the presence of a lipid. The 3 steps of the heterogeneous catalysis induced by lipase, interfacial adsorption, interfacial activation and catalysis proper are under the control, respectively, of a serine hydroxyl group, a carboxyl and a histidine imidazole.

Reptiles and mammals have differentially retained long conserved noncoding sequences from the amniote ancestor.

Many noncoding regions of genomes appear to be essential to genome function. Conservation of large numbers of noncoding sequences has been reported repeatedly among mammals but not thus far among birds and reptiles. By searching genomes of chicken (Gallus gallus), zebra finch (Taeniopygia guttata), and green anole (Anolis carolinensis), we quantified the conservation among birds and reptiles and across amniotes of long, conserved noncoding sequences (LCNS), which we define as sequences ≥500 bp in length and exhibiting ≥95% similarity between species. We found 4,294 LCNS shared between chicken and zebra finch and 574 LCNS shared by the two birds and Anolis. The percent of genomes comprised by LCNS in the two birds (0.0024%) is notably higher than the percent in mammals (<0.0003% to <0.001%), differences that we show may be explained in part by differences in genome-wide substitution rates. We reconstruct a large number of LCNS for the amniote ancestor (ca. 8,630) and hypothesize differential loss and substantial turnover of these sites in descendent lineages. By contrast, we estimated a small role for recruitment of LCNS via acquisition of novel functions over time. Across amniotes, LCNS are significantly enriched with transcription factor binding sites for many developmental genes, and 2.9% of LCNS shared between the two birds show evidence of expression in brain expressed sequence tag databases. These results show that the rate of retention of LCNS from the amniote ancestor differs between mammals and Reptilia (including birds) and that this may reflect differing roles and constraints in gene regulation.