All-codon scanning identifies p53 cancer rescue mutations

In vitro scanning mutagenesis strategies are valuable tools to identify critical residues in proteins and to generate proteins with modified properties. We describe the fast and simple All-Codon Scanning (ACS) strategy that creates a defined gene library wherein each individual codon within a specific target region is changed into all possible codons with only a single codon change per mutagenesis product. ACS is based on a multiplexed overlapping mutagenesis primer design that saturates only the targeted gene region with single codon changes. We have used ACS to produce single amino-acid changes in small and large regions of the human tumor suppressor protein p53 to identify single amino-acid substitutions that can restore activity to inactive p53 found in human cancers. Single-tube reactions were used to saturate defined 30-nt regions with all possible codon changes. The same technique was used in 20 parallel reactions to scan the 600-bp fragment encoding the entire p53 core domain. Identification of several novel p53 cancer rescue mutations demonstrated the utility of the ACS approach. ACS is a fast, simple and versatile method, which is useful for protein structure–function analyses and protein design or evolution problems.     

Synthesis and evaluation of a new series of Neuropeptide S receptor antagonists

Administration of Neuropeptide S (NPS) has been shown to produce arousal, that is, independent of novelty and to induce wakefulness by suppressing all stages of sleep, as demonstrated by EEG recordings in rat. Medicinal chemistry efforts have identified a quinolinone class of potent NPSR antagonists that readily cross the blood–brain barrier. We detail here optimization efforts resulting in the identification of a potent NPSR antagonist which dose-dependently and specifically inhibited ¹²⁵I-NPS binding in the CNS when administered to rats.     

Characterization of photosystem I antenna proteins in the prasinophyte Ostreococcus tauri

Prasinophyceae are a broad class of early-branching eukaryotic green algae. These picophytoplankton are found ubiquitously throughout the ocean and contribute considerably to global carbon-fixation. Ostreococcus tauri, as the first sequenced prasinophyte, is a model species for studying the functional evolution of light-harvesting systems in photosynthetic eukaryotes. In this study we isolated and characterized O. tauri pigment-protein complexes. Two photosystem I (PSI) fractions were obtained by sucrose density gradient centrifugation in addition to free light-harvesting complex (LHC) fraction and photosystem II (PSII) core fractions. The smaller PSI fraction contains the PSI core proteins, LHCI, which are conserved in all green plants, Lhcp1, a prasinophyte-specific LHC protein, and the minor, monomeric LHCII proteins CP26 and CP29. The larger PSI fraction contained the same antenna proteins as the smaller, with the addition of Lhca6 and Lhcp2, and a 30% larger absorption cross-section. When O. tauri was grown under high-light conditions, only the smaller PSI fraction was present. The two PSI preparations were also found to be devoid of the far-red chlorophyll fluorescence (715-730 nm), a signature of PSI in oxygenic phototrophs. These unique features of O. tauri PSI may reflect primitive light-harvesting systems in green plants and their adaptation to marine ecosystems. Possible implications for the evolution of the LHC-superfamily in photosynthetic eukaryotes are discussed.     

Global warming and Bergmann’s rule: do central European passerines adjust their body size to rising temperatures?

Recent climate change has caused diverse ecological responses in plants and animals. However, relatively little is known about homeothermic animals’ ability to adapt to changing temperature regimes through changes in body size, in accordance with Bergmann’s rule. We used fluctuations in mean annual temperatures in south-west Germany since 1972 in order to look for direct links between temperature and two aspects of body size: body mass and flight feather length. Data from regionally born juveniles of 12 passerine bird species were analysed. Body mass and feather length varied significantly among years in eight and nine species, respectively. Typically the inter-annual changes in morphology were complexly non-linear, as was inter-annual variation in temperature. For six (body mass) and seven species (feather length), these inter-annual fluctuations were significantly correlated with temperature fluctuations. However, negative correlations consistent with Bergmann’s rule were only found for five species, either for body mass or feather length. In several of the species for which body mass and feather length was significantly associated with temperature, morphological responses were better predicted by temperature data that were smoothed across multiple years than by the actual mean breeding season temperatures of the year of birth. This was found in five species for body mass and three species for feather length. These results suggest that changes in body size may not merely be the result of phenotypic plasticity but may hint at genetically based microevolutionary adaptations.     

Structure of the antiviral assembly inhibitor CAP-1 complex with the HIV-1 CA protein

The CA domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein plays critical roles in both the early and late phases of viral replication and is therefore an attractive antiviral target. Compounds with antiviral activity were recently identified that bind to the N-terminal domain of CA (CA^N) and inhibit capsid assembly during viral maturation. We have determined the structure of the complex between CA^N and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)-N′-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) using a combination of NMR spectroscopy and X-ray crystallography. The protein undergoes a remarkable conformational change upon CAP-1 binding, in which Phe32 is displaced from its buried position in the protein core to open a deep hydrophobic cavity that serves as the ligand binding site. The aromatic ring of CAP-1 inserts into the cavity, with the urea NH groups forming hydrogen bonds with the backbone oxygen of Val59 and the dimethylamonium group interacting with the side-chains of Glu28 and Glu29. Elements that could be exploited to improve binding affinity are apparent in the structure. The displacement of Phe32 by CAP-1 appears to be facilitated by a strained main-chain conformation, which suggests a potential role for a Phe32 conformational switch during normal capsid assembly.     

Characterizing early events associated with the activation of target genes by 1,25-dihydroxyvitamin D3 in mouse kidney and intestine in vivo

In this report, we explore the interaction of the vitamin D receptor (VDR) at regulatory sites within both the Cyp24a1 and the Trpv6 genes using chromatin immunoprecipitation techniques in a mouse model in vivo. We show that exogenous 1,25(OH)(2)D(3) induces rapid VDR and RXR (retinoid X receptor) binding to the Cyp24a1 gene in both the kidney and the intestine and to the Trpv6 gene in the intestine. Separate studies of Trpv6 in vitro suggest that VDR binding occurs directly to VDR response elements located -2 and -4 kb upstream of the TSS. VDR binding is dose-dependent, demonstrating EC(50) values that are comparable with those for the induction of both Cyp24a1 and Trpv6 mRNA. Importantly, interaction of the VDR with these targets results in rapid changes in histone 4 acetylation as well as the recruitment of RNA polymerase II. The presence of both VDR and RNA polymerase II at these sites declines between 3-6 h, whereas the changes observed in acetylation decrease more slowly. Finally, we show that whereas mediator protein 1 is recruited to the Cyp24a1 promoter in the intestine, this coactivator is apparently not required for Trpv6 activation. These studies provide the first evidence for 1,25(OH)(2)D(3)-induced VDR interaction at key target genes in vivo, revealing the consequences of that interaction on the Cyp24a1 and Trpv6 genes.     

Effects of resource availability on the use of space by the mouse opossum<Emphasis Type="Italic">Marmosops paulensis</Emphasis> (Didelphidae) in a montane Atlantic forest area in southeastern Brazil

Food supply is an important determinant of animal movements. In the present study we tested the occurrence of an inverse relationship between daily movements ofMarmosops paulensis (Tate, 1931) and their food supply. This species is a member of the family Didelphidae, occurring in areas of high elevation in the Brazilian Atlantic Forest.M. paulensis moved mostly over the ground, although the understory was also used. Mean daily range ofM. paulensis estimated through spool-and-line device was about 0.40 ha; no differences were noted between body sizes and sexes. As expected, there was an inverse relationship between daily movements and food supply. This relationship resulted from the clumped distribution of fruits, particularlyPiper rather than the sparser distribution of arthropods.