Miniaturization of fluorescence polarization receptor-binding assays using CyDye-labeled ligands

Fluorescence polarization (FP) is an established technique for the study of biological interactions and is frequently used in the high-throughput screening (HTS) of potential new drug targets. This work describes the miniaturization of FP receptor assays to 1536-well formats for use in HTS. The FP assays were initially developed in 384-well microplates using CyDye-labeled nonpeptide and peptide ligands. Receptor expression levels varied from approximately 1 to 10 pmols receptor per mg protein, and ligand concentrations were in the 0.5- to 1.0-nM range. The FP assays were successfully miniaturized to 1536-well formats using Cy3B-labeled ligands, significantly reducing reagent consumption, particularly the receptor source, without compromising assay reliability. Z' factor values determined for the FP receptor assays in both 384- and 1536-well formats were found to be > 0.5, indicating the assays to be robust, reliable, and suitable for HTS purposes.     

A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function

Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.     

Recent ecological and biogeochemical changes in alpine lakes of Rocky Mountain National Park (Colorado, USA): a response to anthropogenic nitrogen deposition

Dated sediment cores from five alpine lakes (>3200 m asl) in Rocky Mountain National Park (Colorado Front Range, USA) record near‐synchronous stratigraphic changes that are believed to reflect ecological and biogeochemical responses to enhanced nitrogen deposition from anthropogenic sources. Changes in sediment proxies include progressive increases in the frequencies of mesotrophic planktonic diatom taxa and diatom concentrations, coupled with depletions of sediment δ¹⁵N and C : N values. These trends are especially pronounced since approximately 1950. The most conspicuous diatoms to expand in recent decades are Asterionella formosa and Fragilaria crotonensis. Down‐core species changes are corroborated by a year‐long sediment trap experiment from one of the lakes, which reveals high frequencies of these two taxa during autumn and winter months, the interval of peak annual limnetic []. Although all lakes record recent changes, the amplitude of stratigraphic shifts is greater in lakes east of the Continental Divide relative to those on the western slope, implying that most nitrogen enrichment originates from urban, industrial and agricultural sources east of the Rocky Mountains. Deviations from natural trajectories of lake ontogeny are illustrated by canonical correspondence analysis, which constrains the diatom record as a response to changes in nitrogen biogeochemistry. These results indicate that modest rates of anthropogenic nitrogen deposition are fully capable of inducing directional biological and biogeochemical shifts in relatively pristine ecosystems.     

Solid-state NMR structure determination

Biological applications of solid-state NMR (SS-NMR) have been predominantly in the area of model membrane systems. Increasingly the focus has been membrane peptides and proteins. SS-NMR is able to provide information about how the peptides or proteins interact with the lipids or other peptides/proteins in the membrane, their effect on the membrane and the location of the peptides or proteins relative to the membrane surface. Recent developments in biological SS-NMR have been made possible by improvements in labelling and NMR techniques. This review discusses aligned systems and magic angle spinning techniques, bilayers and bicelles, and measurement of chemical shift anisotropy and dipolar coupling. A number of specific experiments such as cross polarization, rotational resonance, REDOR, PISEMA, MAOSS and multidimensional experiments are described. In addition to 2H, 13C and 15N, recent solid-sate 1H, 19F and 17O NMR work is discussed. Several examples of the use of SS-NMR to determine the structure of membrane peptides and proteins are given.     

Influence of normal daytime fat deposition on laboratory measurements of torpor use in territorial versus nonterritorial hummingbirds

Fat deposition and torpor use in hummingbirds exhibiting distinct foraging styles should vary. We predicted that dominant territorial hummingbirds will use torpor less than subordinate nonterritorial species because unrestricted access to energy by territory owners allows for fat storage. Entry into torpor was monitored using open-flow respirometry on hummingbirds allowed to accumulate fat normally during the day. Fat accumulation was measured by solvent fat extraction. Territorial blue-throated hummingbirds (Lampornis clemenciae) had the highest fat accumulation and used torpor only 17% of the time. Fat storage by L. clemenciae averaged 26% of lean dry mass (LDM) in 1995 and 18% in 1996, similar to that measured for other nonmigratory birds. Fat storage by magnificent hummingbirds (Eugenes fulgens; trapliner) and black-chinned hummingbirds (Archilochus alexandri; nectar robber) averaged 19% and 16% of LDM, respectively, and they used torpor frequently (64% and 92% of the time, respectively). All species initiated torpor if total body fat dropped below 10% of LDM, indicating the existence of a torpor threshold. The ability of L. clemenciae to store enough fat to support nighttime metabolism is likely an important benefit of territoriality. Likewise, frequent torpor use by subordinates suggests that natural restrictions to energy intake can impact their energy budget, necessitating energy conservation by use of torpor.     

Glucose-dependent insulinotropic polypeptide receptor null mice exhibit compensatory changes in the enteroinsular axis

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut hormones that act via the enteroinsular axis to potentiate insulin secretion from the pancreas in a glucose-dependent manner. Both GLP-1 receptor and GIP receptor knockout mice (GLP-1R(-/-) and GIPR(-/-), respectively) have been generated to investigate the physiological importance of this axis. Although reduced GIP action is a component of type 2 diabetes, GIPR-deficient mice exhibit only moderately impaired glucose tolerance. The present study was directed at investigating possible compensatory mechanisms that take place within the enteroinsular axis in the absence of GIP action. Although serum total GLP-1 levels in GIPR knockout mice were unaltered, insulin responses to GLP-1 from pancreas perfusions and static islet incubations were significantly greater (40-60%) in GIPR(-/-) than in wild-type (GIPR(+/+)) mice. Furthermore, GLP-1-induced cAMP production was also elevated twofold in the islets of the knockout animals. Pancreatic insulin content and gene expression were reduced in GIPR(-/-) mice compared with GIPR(+/+) mice. Paradoxically, immunocytochemical studies showed a significant increase in beta-cell area in the GIPR-null mice but with less intense staining for insulin. In conclusion, GIPR(-/-) mice exhibit altered islet structure and topography and increased islet sensitivity to GLP-1 despite a decrease in pancreatic insulin content and gene expression.     

Mitochondrial biogenesis and remodeling during adipogenesis and in response to the insulin sensitizer rosiglitazone

White adipose tissue is an important endocrine organ involved in the control of whole-body metabolism, insulin sensitivity, and food intake. To better understand these functions, 3T3-L1 cell differentiation was studied by using combined proteomic and genomic strategies. The proteomics approach developed here exploits velocity gradient centrifugation as an alternative to isoelectric focusing for protein separation in the first dimension. A 20- to 30-fold increase in the concentration of numerous mitochondrial proteins was observed during adipogenesis, as determined by mass spectrometry and database correlation analysis. Light and electron microscopy confirmed a large increase in the number of mitochondrion profiles with differentiation. Furthermore, mRNA profiles obtained by using Affymetrix GeneChips revealed statistically significant increases in the expression of many nucleus-encoded mitochondrial genes during adipogenesis. Qualitative changes in mitochondrial composition also occur during adipose differentiation, as exemplified by increases in expression of proteins involved in fatty acid metabolism and of mitochondrial chaperones. Furthermore, the insulin sensitizer rosiglitazone caused striking changes in mitochondrial shape and expression of selective mitochondrial proteins. Thus, although mitochondrial biogenesis has classically been associated with brown adipocyte differentiation and thermogenesis, our results reveal that mitochondrial biogenesis and remodeling are inherent to adipose differentiation per se and are influenced by the actions of insulin sensitizers.     

The Ku heterodimer performs separable activities at double-strand breaks and chromosome termini

The Ku heterodimer functions at two kinds of DNA ends: telomeres and double-strand breaks. The role that Ku plays at these two classes of termini must be distinct, because Ku is required for accurate and efficient joining of double-strand breaks while similar DNA repair events are normally prohibited at chromosome ends. Toward defining these functional differences, we have identified eight mutations in the large subunit of the Saccharomyces cerevisiae Ku heterodimer (YKU80) which retain the ability to repair double-strand breaks but are severely impaired for chromosome end protection. Detailed characterization of these mutations, referred to as yku80(tel) alleles, has revealed that Ku performs functionally distinct activities at subtelomeric chromatin versus the end of the chromosome, and these activities are separable from Ku's role in telomere length regulation. While at the chromosome terminus, we propose that Ku participates in two different activities: it facilitates telomerase-mediated G-strand synthesis, thereby contributing to telomere length regulation, and it separately protects against resection of the C-strand, thereby contributing to protection of chromosome termini. Furthermore, we propose that the Ku heterodimer performs discrete sets of functions at chromosome termini and at duplex subtelomeric chromatin, via separate interactions with these two locations. Based on homology modeling with the human Ku structure, five of the yku80(tel) alleles mutate residues that are conserved between the yeast and human Ku80 proteins, suggesting that these mutations probe activities that are shared between yeast and humans.