The use of NMR chemical shifts to analyse the MD trajectories: simulation of bovine pancreatic trypsin inhibitor dynamics in water as a test case for solvent influences.

In this paper the NMR secondary chemical shifts, that are estimated from a set of 3D-structures, are compared with the observed ones to appraise the behaviour of a known x-ray diffraction structure (of the bovine pancreatic trypsin inhibitor protein) when various molecular dynamics are applied. The results of a 200 ps molecular dynamics under various conditions are analysed and different ways to modify the molecular dynamics are considered. With the purpose of avoiding the time-consuming explicit representation of the solvent (water) molecules, an attempt was made to understand the role of the solvent and to develop an implicit representation, which may be refined. A simulation of hydrophobic effects in an aqueous environment is also proposed which seems to provide a better approximation of the observed solution structure of the protein.     

Replication of macrophage-tropic and T-cell-tropic strains of human immunodeficiency virus type 1 is augmented by macrophage-endothelial cell contact.

Macrophages perform a central role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection and have been implicated as the cell type most prominent in the development of central nervous system impairment. In this study, we evaluated the effect of interaction between macrophages and endothelial cells on HIV-1 replication. Upregulation of HIV-1 replication was consistently observed in monocyte-derived macrophages (hereafter called macrophages) cocultured with either umbilical vein endothelial cells or brain microvascular endothelial cells. HIV-1 p24 antigen production of laboratory-adapted strains and patient-derived isolates was increased 2- to 1,000-fold in macrophage-endothelial cocultures, with little or no detectable replication in cultures containing endothelial cells only. The upregulation of HIV-1 in macrophage-endothelial cocultures was observed not only for viruses with the non-syncytium-inducing, macrophage-tropic phenotype but also for viruses previously characterized as syncytium inducing and T-cell tropic. In contrast, cocultures of macrophages with glioblastoma, astrocytoma, cortical neuronal, fibroblast, and placental cells failed to increase HIV-1 replication. Enhancement of HIV-1 replication in macrophage-endothelial cocultures required cell-to-cell contact; conditioned media from endothelial cells or macrophage-endothelial cocultures failed to augment HIV-1 replication in macrophages. Additionally, antibody to leukocyte function-associated antigen (LFA-1), a macrophage-endothelial cell adhesion molecule, inhibited the enhanced HIV-1 replication in macrophage-endothelial cell cocultures. Thus, these data indicate that macrophage-endothelial cell contact enhances HIV-1 replication in macrophages for both macrophage-tropic and previously characterized T-cell-tropic strains and that antibody against LFA-1 can block the necessary cell-to-cell interaction required for the observed upregulation. These findings may have important implications for understanding the ability of HIV-1 to replicate efficiently in tissue macrophages, including those in the brain and at the blood-brain barrier.     

The palatability of Arctic willow for greater snow geese: the role of nutrients and deterring factors

Temporary feeding on willow buds and leaves by nesting greater snow geese provided us with an opportunity to test the relative importance of nutrients and deterrents in affecting the palatability for geese of a food plant with a high phenol content. Protein, total phenol and fiber (neutral and acid detergent fiber, and lignin) were analyzed in closed and open buds and in rolled and open leaves. Geese feed on willows at the open-buds and rolled-leaf stages but not at the closed-bud and open-leaf stages. Protein content was higher in open buds and rolled leaves (25–27%) than in closed buds and open leaves (19–21%). Phenol content increased during leaf emergence but was already high (14%) in rolled leaves. All plant fibers were very high in closed buds but declined rapidly during leaf emergence. The increase in phenol: protein ratio appeared to be more important than phenol concentration alone in explaining the cessation of feeding by geese on willow leaves whereas the high fiber content of closed buds may explain why they were not eaten. Our results illustrate the value of a multifactorial approach in the study of the food selection process in herbivores.     

Induction of apoptosis by protein kinase C pseudosubstrate lipopeptides in several human cells

Intracellular enzymes or receptors are interesting targets for thepharmacomodulation of cellular metabolism. We have previously shown thatmodification of relatively long peptides by a palmitoyl-lysine residue couldfacilitate their delivery into the cytoplasm of living cells. Severalpeptides containing pseudosubstrate sequences of protein kinase C (PKC) havebeen evaluated for their ability to modulate phosphorylation of modelsubstrate, neuronal morphology or tumor necrosis factor secretion. In thiswork we have evaluated the effect of palmitoyl-modified PKC-pseudosubstratepeptides on induction of apoptosis. We have established that these peptidesare able to induce apoptosis in different human cell types (primaryfibroblasts, T- and B-lymphocyte cell lines) as assessed by (terminal deoxynucleotidyl transferase dUTP nick-end labelling) and DNAfragmentation. In contrast, control peptides (non-lipidicPKC-pseudosubstrate peptides and irrelevant lipopeptides) had no or littleeffect on programmed cell death. This work highlights the pharmacologicalinterest of lipopeptides and argues in favor of the potential role of PKC(s)in the cell death machinery.     

X-ray structure of recombinant horse L-chain apoferritin at 2.0 Å resolution: implications for stability and function

The X-ray structure of recombinant horse L-chain (rL) apoferritin, solved at 2.0?Å resolution with a final R factor of 17.9%, gives evidence that the residue at position 93 in the sequence is a proline and not a leucine, as found in earlier sequencing studies. The structure is isomorphous with other apoferritin structures, and we thus draw particular attention to those structural features which can be related to the stability and function of the protein. Analysis of hydrogen bonding and salt bridge interactions shows that dimers and tetramers are the most stable molecular entities within the protein shell: a result confirming earlier biophysical experiments. The stability of horse rL apoferritin to both dissociation into subunits at acidic pH values and to complete unfolding in guanidine chloride solutions is compared with that of other apoferritins. This emphasizes the role played by the salt bridge in the stability of this protein family. The horse rL apoferritin is significantly more resistant to denaturation than horse spleen ferritin, which in turn is more resistant than any human rH apoferritins, even those for which a salt bridge is restored. Finally, this structure determination not only establishes that a preformed pocket exists in L-chain apoferritin, at a site known to be able to bind porphyrin, but also underlines the particular function of a cluster of glutamic acids (E53, E56, E57 and E60) located at the entrance of this porphyrin-binding pocket.     

Selection and Methionine Accumulation in the Fat Body Protein 2 Gene (FBP2), a Duplicate of the Drosophila Alcohol Dehydrogenase (ADH) Gene

The Drosophila fat body protein 2 gene (Fbp2) is an ancient duplication of the alcohol dehydrogenase gene (Adh) which encodes a protein that differs substantially from ADH in its methionine content. In D. melanogaster, there is one methionine in ADH, while there are 51 (20% of all amino acids) in FBP2. Methionine is involved in 46% of amino acid replacements when Fbp2 DNA sequences are compared between D. melanogaster and D. pseudoobscura. Methionine accumulation does not affect conserved residues of the ADH-ADH^r-FBP2 multigene family. The multigene family has evolved by replacement of mildly hydrophobic amino acids by methionine with no apparent reversion. Its short-term evolution was compared between two Drosophila species, while its long-term evolution was compared between two genera belonging respectively to acalyptrate and calyptrate Diptera, Drosophila and Sarcophaga. The pattern of nucleotide substitution was consistent with an independent accumulation of methionines at the Fbp2 locus in each lineage. Under a steady-state model, the rate of methionine accumulation was constant in the lineage leading to Drosophila, and was twice as fast as that in the calyptrate lineage. Substitution rates were consistent with a slight positive selective advantage for each methionine change in about one-half of amino acid sites in Drosophila. This shows that selection can potentially account for a large proportion of amino acid replacements in the molecular evolution of proteins. Received: 12 December 1994 / Accepted: 15 April 1996     

Mapping of a Gene for Long QT Syndrome to Chromosome 4q25-27

Long QT syndrome (LQTS) is a heterogeneous inherited disorder causing syncope and sudden death from ventricular arrhythmias. A first locus for this disorder was mapped to chromosome 11p15.5. However, locus heterogeneity has been demonstrated in several families, and two other loci have recently been located on chromosomes 7q35-36 and 3p21-24. We used linkage analysis to map the locus in a 65-member family in which LQTS was associated with more marked sinus bradycardia than usual, leading to sinus node dysfunction. Linkage to chromosome 11p15.5, 7q35-36, or 3p21-24 was excluded. Positive linkage was obtained for markers located on chromosome 4q25-27. A maximal LOD score of 7.05 was found for marker D4S402. The identification of a fourth locus for LQTS confirms its genetic heterogeneity. Locus 4q25-27 is associated with a peculiar phenotype within the LQTS entity.     

Excess of deletions of maternal origin in the DiGeorge/Velo-cardio-facial syndromes. A study of 22 new patients and review of the literature

We have determined the parental origin of the deleted chromosome 22 in 29 cases of DiGeorge syndrome (DGS) using a CA-repeat mapping within the commonly deleted region, and in one other case by using a chromosome 22 short arm heteromorphism. The CA-repeat was informative in 21 out of 29 families studied and the deleted chromosome was of maternal origin in 16 cases (72%). When these data are pooled with recent results from the literature, 24 de novo DGS, velo-cardio-facial syndrome (VCFS) and isolated conotruncal cardiac disease deletions are found to be of maternal origin and 8 of paternal origin, yielding a ² of 8 with a probability level lower than 0.01. These data, and review of the literature on familial DGS/VCFS and isolated conotruncal cardiopathies suggest that there is a strong tendency for the 22q11.2 deletions to be of maternal origin.     

Physico-chemical properties and evidence for electrophoretic variants of rat transcortin

Isolation of rat plasma transcortin was carried out by affinity chromatography, as previously described for human. The protein was shown to be pure by PAGE and one single N-terminal amino acid was identified (Ser), which suggested that the protein molecule has a single polypeptide chain. This assumption is supported by SDS-PAGE. The amino acid composition was reported and compared with the one of human transcortin. The purified protein always migrated in PAGE (with or without SDS) as a double band; the faster component being more intense than the slower one. Whether transcortin was free or bound to corticosterone, the same aspect was observed. Molecular weight of these two variants were determined by SDS-PAGE as 65,900 and 75,800. Polymers only appeared after irreversible denaturation of the protein, as previously described for human transcortin. Various other physical parameters were determined: a sedimentation coefficient of 3.71 S +/- 0.18 was calculated by ultracentrifugation in sucrose gradient, association constants at 4 degrees C for corticosterone and cortisol (2.7 X 10(9) M-1 and 4.2 X 10(8) M-1, respectively).