Use of restrained molecular dynamics in water to determine three-dimensional protein structure: prediction of the three-dimensional structure of Ecballium elaterium trypsin inhibitor II

Refinement of distance geometry (DG) structures of EETI-II (Heitz et al.: Biochemistry 28:2392-2398, 1989), a member of the squash family trypsin inhibitor, have been carried out by restrained molecular dynamics (RMD) in water. The resulting models show better side chain apolar/polar surface ratio and estimated solvation free energy than structures refined "in vacuo." The consistent lower values of residual NMR constraint violations, apolar/polar surface ratio, and solvation free energy for one of these refined structures allowed prediction of the 3D folding and disulfide connectivity of EETI-II. Except for the few first residues for which no NMR constraints were available, this computer model fully agreed with X-ray structures of CMTI-I (Bode et al.: FEBS Lett. 242:285-292, 1989) and EETI-II complexed with trypsin that appeared after the RMD simulation was completed. Restrained molecular dynamics in water is thus proved to be highly valuable for refinement of DG structures. Also, the successful use of apolar/polar surface ratio and of solvation free energy reinforce the analysis of Novotny et al. (Proteins 4:19-30, 1988) and shows that these criteria are useful indicators of correct versus misfolded models.     

The productive gene for alpha-H chain disease protein MAL is highly modified by insertion-deletion processes

alpha-H chain diseases (HCD) is a human lymphoproliferative disorder, characterized by the production of truncated alpha-Ig H chains, without associated L chains. In this study, we have analysed the serum protein, the alpha-HCD mRNA and the rearranged alpha-HCD gene from the leukemic cells of a patient (MAL) with alpha-HCD. The abnormal MAL serum Ig consisted of short alpha 1-chains, lacking VH and CH1 domains (only CH2 and CH3 domains were present). The alpha-HCD mRNA (1.2 kb) was shorter than a normal alpha-mRNA (2 kb); the corresponding cDNA had sequences for the leader, a 84-bp sequence of unknown origin and the CH2 and CH3 exons. The establishment of the sequence of the productive alpha-HCD MAL allele revealed two major deletions; that of the VH region as well as that of the CH1 region. The JH region is altered by multiple mutations, small insertions and a duplication of the psi JH3 region. A large insert (INS1), of 360 bp (containing the 84 bp exon found in the cDNA), replaces the deleted VH region. INS1 is non-Ig related and apparently of nongenomic origin. A large second insert (509 bp), is located between the enhancer and the switch region. Insert2 contains repetitive non-Ig-related sequences and a small Ig-related sequence. All these alterations resulted in an abnormal mRNA, which comprises the leader, a 84-bp alien exon derived from INS1 and the CH2 and CH3 exons of the alpha 1-gene.     

Nitrification and nitrate uptake: Leaching balance in a declined forest ecosystem in eastern France

Nitrate uptake and leaching were measured during one year in a declined fir forest on the Vosges highlands (eastern France), in order to investigate whether excess nitrification could be responsible for a deleterious acidification of the ecosystem. Nitrate uptake by the vegetation was active mainly from spring to early fall, and then reached about 66 kg N ha^-1. No significant leaching loss occurred during the growth period of the vegetation. Significant nitrate leaching occurred in winter (about 17 kg N ha^-1). During fall and winter the nitrification rate was of the same magnitude as values reported for other ecosystems, and, thus, was not considered to be abnormaly strong. No abnormal temporal discoupling of nitrate production and nitrate uptake occurred in the ecosystem, and forest decline must therefore have some other cause.     

Gramicidin channels that have no tryptophan residues.

In order to understand how aromatic residues modulate the function of membrane-spanning proteins, we examined the role of the four tryptophans in gramicidin A (gA) in determining the average duration and permeability characteristics of membrane-spanning gramicidin channels; the tryptophan residues were replaced by tyrosine (gramicidin T, gT), tyrosine O-benzyl ether [gramicidin T(Bzl), gT(Bzl)], naphthylalanine (gramicidin N, gN), and phenylalanine (gramicidin M enantiomer, gM-). These analogues form channels with durations and conductances that differ some 10- and 16-fold, respectively. The single-channel conductance was invariably decreased by the Trp----Yyy replacement, and the relative conductance alterations were similar in phosphatidylcholine (DPhPC) and monoglyceride (GMO) bilayers. The duration variations exhibited a more complex pattern, which was quite different in the two membrane environments: in DPhPC bilayers, gN channels have an average duration that is approximately 2-fold longer than that of gA channels; in GMO bilayers, the average duration of gN channels is about one-tenth that of gA channels. The sequence-dependent alterations in channel function do not result from alterations in the channels' peptide backbone structure, because heterodimers can form between the different analogues and gramicidine A, and there is no energetic cost associated with heterodimer formation [cf. Durkin, J. T., Koeppe, R. E., II, & Andersen, O. S. (1990) J. Mol. Biol. 211, 221]. The alterations in permeability properties are consistent with the notion that Trp residues alter the energy profile for ion permeation through long-range electrostatic interactions.     

Lactate removal ability and graded exercise in humans

Venous lactate concentrations of nine athletes were recorded every 5 s before, during, and after graded exercise beginning at a work rate of 0 W with an increase of 50 W every 4th min. The continuous model proposed by Hughson et al. (J. Appl. Physiol. 62: 1975-1981, 1987) was well fitted with the individual blood lactate concentration vs. work rate curves obtained during exercise. Time courses of lactate concentrations during recovery were accurately described by a sum of two exponential functions. Significant direct linear relationships were found between the velocity constant (gamma 2 nu) of the slowly decreasing exponential term of the recovery curves and the times into the exercise when a lactate concentration of 2.5 mmol/l was reached. There was a significant inverse correlation between gamma 2 nu and the rate of lactate increase during the last step of the exercise. In terms of the functional meaning given to gamma 2 nu, these relationships indicate that the shift to higher work rates of the increase of the blood lactate concentration during graded exercise in fit or trained athletes, when compared with less fit or untrained ones, is associated with a higher ability to remove lactate during the recovery. The results suggest that the lactate removal ability plays an important role in the evolution pattern of blood lactate concentrations during graded exercise.     

Fatal attack on an infant by an adult female tonkean macaque

We describe a fatal attack on a 10-month-old female infant by an adult female Tonkean macaque (Macaca tonkeana)in a semi-free-ranging group. During an intense conflict involving many group members, an adult female, the second most dominant in the group, attacked the infant of a lower-ranking female. The attacker remained near the victim for approximately 20 min, grabbing and biting it repeatedly, until the mother retrieved the infant. She died 2 days later. An autopsy revealed no lethal injury;the main cause of death was starvation or dehydration or both. We discuss social conditions under which a fatal attack by an adult female might occur.