Synthesis, X-ray Analysis, and Biological Evaluation of a New Class of Stereopure Lactam-Based HIV-1 Protease Inhibitors

In an effort to identify a new class of druglike HIV-1 protease inhibitors, four different stereopure beta-hydroxy gamma-lactam-containing inhibitors have been synthesized, biologically evaluated, and cocrystallized. The impact of the tether length of the central spacer (two or three carbons) was also investigated. A compound with a shorter tether and (3R,4S) absolute configuration exhibited high activity with a K-i of 2.1 nM and an EC50 of 0.64 mu M. Further optimization by decoration of the P1' side chain furnished an even more potent HIV-1 protease inhibitor (K-i = 0.8 nM, EC50 = 0.04 mu M). According to X-ray analysis, the new class of inhibitors did not fully succeed in forming two symmetric hydrogen bonds to the catalytic aspartates. The crystal structures of the complexes further explain the difference in potency between the shorter inhibitors (two-carbon spacer) and the longer inhibitors (three-carbon spacer)

Molecular analysis of the gut microbiota of identical twins with Crohn's disease

Increasing evidence suggests that a combination of host genetics and the composition of the gut microbiota are important for development of Crohn's disease (CD). Our aim was to study identical twins with CD to determine microbial factors independently of host genetics. Fecal samples were studied from 10 monozygotic twin pairs with CD (discordant n=6, concordant n=4) and 8 healthy twin pairs. DNA was extracted, 16S rRNA genes were PCR amplified and T-RFLP fingerprints generated using general bacterial and Bacteroides group specific primers. The microbial communities were also profiled based on their % G+C contents. Bacteroides 16S rRNA genes were cloned and sequenced from a subset of the samples. The bacterial diversity in each sample and similarity indices between samples were estimated based on the T-RFLP data using a combination of statistical approaches. Healthy individuals had a significantly higher bacterial diversity compared to individuals with CD. The fecal microbial communities were more similar between healthy twins than between twins with CD, especially when these were discordant for the disease. The microbial community profiles of individuals with ileal CD were significantly different from healthy individuals and those with colonic CD. Also, CD individuals had a lower relative abundance of B. uniformis and higher relative abundances of B. ovatus and B. vulgatus. Our results suggest that genetics and/or environmental exposure during childhood in part determine the gut microbial composition. However, CD is associated with dramatic changes in the gut microbiota and this was particularly evident for individuals with ileal CD

The 14-3-3 protein family - Conserved structure, diverse functions

While once abstruse, 14-3-3 proteins are now well known as players in several cellular processes. These processes range from apoptosis and mitosis in mammals and yeast, to regulation of primary metabolism in plants. To date, 104 proteins have been shown to interact with 14-3-3s of which 85 are found in mammals and yeast, and 21 are found in plants. Their consensus action is to bind to phosphorylated motifs within target proteins resulting in activation, inhibition, stabilization, or translocation. Although 14-3-3s have been crystallized, are ubiquitous in all Eukaryotes, and extremely well studied, several questions regarding their mechanism of interaction with other proteins remain. In plants, 14-3-3s inhibit enzymes in primary metabolism such as nitrate reductase and sucrose phosphate synthase, leading to accumulation of substrates, whilst the plasma membrane (PM) H+ATPase is activated. At present, the PM H+ATPase 14-3-3 interaction is the best characterized in plants. The identification of the deviating 14-3-3 binding motif in the PM H+ATPase allowed examination of the possibility of isoform specificity. Indeed, differences were observed in binding specificity between nine different Arabidopsis thaliana14-3-3 isoforms and a phosphopeptide corresponding to the binding motif in the A. thaliana PM H+ATPase isoform AHA2. Examinations of the three-dimensional structure of 14-3-3s have been made in an attempt to identify residues responsible for functional specificity. With the presumption that evolutionary insight may allow more exact extrapolation of observations made in unicellular organisms to multicellular organisms, we data-mined public databases, identifying all 14-3-3 isoforms present. Phylogenetic analysis showed kingdom-wise clustering. Within Metazoa, isoforms grouped between species, whilst isoform groupings observed within Viridiplantae occurred within species. When examining only full-length plant 14-3-3s approximately 42% of all residues are conserved, at a threshold of 98%, with an apparently higher percentage within each cluster. This large divergence, resulting from numerous gene duplications, negates the possibility of functional extrapolation outside of families of species in plants. The inability of all nine tested A. thaliana 14-3-3 isoforms to bind to a phosphopeptide corresponding to the motif in the A. thaliana PM H+ATPase isoform AHA9 suggested the existence of additional 14-3-3 isoforms in A. thaliana. The completion of the Arabidopsis genome sequencing allowed identification of all 14-3-3 genes, authentic and putative. In addition to the 10 previously cloned 14-3-3s in A. thaliana, five novel genes were identified. Subsequent expression analysis showed that two out of the five were transcribed, bringing the total number of expressed isoforms to twelve

Redox signalling in chloroplasts and mitochondria: genomic and biochemical evidence for two-component regulatory systems in bioenergetic organelles

Redox chemistry is central to the primary functions of chloroplasts and mitochondria, that is, to energy conversion in photosynthesis and respiration. However, these bioenergetic organelles always contain very small, specialized genetic systems, relics of their bacterial origin. At huge cost, organellar genomes contain, typically, a mere 0.1% of the genetic information in a eukaryotic cell. There is evidence that chloroplast and mitochondrial genomes encode proteins whose function and biogenesis are particularly tightly governed by electron transfer. We have identified nuclear genes for 'bacterial' histidine sensor kinases and aspartate response regulators that seem to be targeted to chloroplast and mitochondrial membranes. Sequence similarities to cyanobacterial redox signalling components indicate homology and suggest conserved sensory and signalling functions. Two-component redox signalling pathways might be ancient, conserved mechanisms that permit endogenous control over the biogenesis, in situ, of bioenergetic complexes of chloroplasts and mitochondria

Postprandial effects on plasma lipids and satiety hormones from intake of liposomes made from fractionated oat oil: two randomized crossover studies.

The composition and surface structure of dietary lipids influence their intestinal degradation. Intake of liposomes made of fractionated oat oil (LOO) is suggested to affect the digestion process and postprandial lipemia and also induce satiety

Evolution and isoform specificity of plant 14-3-3 proteins

The 14-3-3 proteins, once thought of as obscure mammalian brain proteins, are fast becoming recognized as major regulators of plant primary metabolism and of other cellular processes. Their presence as large gene families in plants underscores their essential role in plant physiology. We have examined the Arabidopsis thaliana 14-3-3 gene family, which currently is the largest and most complete 14-3-3 family with at least 12 expressed members and 15 genes from the now completed Arabidopsis thaliana genome project. The phylogenetic branching of this family serves as the prototypical model for comparison with other large plant 14-3-3 families and as such may serve to rationalize clustering in a biological context. Equally important for ascribing common functions for the various 14-3-3 isoforms is determining an isoform-specific correlation with localization and target partnering. A summary of localization information available in the literature is presented. In an effort to identify specific 14-3-3 isoform location and participation in cellular processes, we have produced a panel of isoform-specific antibodies to Arabidopsis thaliana 14-3-3s and present initial immunolocalization studies that suggest biologically relevant, discriminative partnering of 14-3-3 isoforms