Three-compartment model: critical evaluation based on neutron activation analysis

There is renewed interest in Siri's classic three-compartment (3C) body composition model, requiring body volume (BV) and total body water (TBW) estimates, because dual-energy X-ray absorptiometry (DEXA) and in vivo neutron activation (IVNA) systems cannot accommodate subjects with severe obesity. However, the 3C model assumption of a constant ratio (alpha) of mineral (M) to total body protein (TBPro) and related residual mass density (D(RES)) based on cadaver analyses might not be valid across groups differing in sex, race, age, and weight. The aim of this study was to derive new 3C model coefficients in vivo and to compare these estimates to those derived by Siri. Healthy adults (n = 323) were evaluated with IVNA and DEXA and the measured components used to derive alpha and D(RES). For all subjects combined, values of alpha and D(RES) (means +/- SD, 0.351 +/- 0.043; 1.565 +/- 0.023 kg/l) were similar to Siri's proposed values of 0.35 and 1.565 kg/l, respectively. However, alpha and D(RES) varied significantly as a function of sex, race, weight, and age. Expected errors in percent body fat arising by application of Siri's model were illustrated in a second group of 264 adults, including some whose size exceeded DEXA limits but whose BV and TBW had been measured by hydrodensitometry and (2)H(2)O dilution, respectively. Extrapolation of predictions by newly developed models to very high weights allows percent fat error estimation when Siri's model is applied in morbidly obese subjects. The present study results provide a critical evaluation of potential errors in the classic 3C model and present new formulas for use in selected populations.     

Structural basis of HP1/PXVXL motif peptide interactions and HP1 localisation to heterochromatin

HP1 family proteins are adaptor molecules, containing two related chromo domains that are required for chromatin packaging and gene silencing. Here we present the structure of the chromo shadow domain from mouse HP1beta bound to a peptide containing a consensus PXVXL motif found in many HP1 binding partners. The shadow domain exhibits a novel mode of peptide recognition, where the peptide binds across the dimer interface, sandwiched in a beta-sheet between strands from each monomer. The structure allows us to predict which other shadow domains bind similar PXVXL motif-containing peptides and provides a framework for predicting the sequence specificity of the others. We show that targeting of HP1beta to heterochromatin requires shadow domain interactions with PXVXL-containing proteins in addition to chromo domain recognition of Lys-9-methylated histone H3. Interestingly, it also appears to require the simultaneous recognition of two Lys-9-methylated histone H3 molecules. This finding implies a further complexity to the histone code for regulation of chromatin structure and suggests how binding of HP1 family proteins may lead to its condensation.     

Aminoimidazo[1,2-a]pyridines as a new structural class of cyclin-dependent kinase inhibitors. Part 1: Design, synthesis, and biological evaluation

We have identified a novel structural class of protein serine/threonine kinase inhibitors comprised of an aminoimidazo[1,2-a]pyridine nucleus. Compounds from this family are shown to potently inhibit cyclin-dependent kinases by competing with ATP for binding to a catalytic subunit of the protein. Structure-based design approach was used to direct this chemical scaffold toward generating potent and selective CDK2 inhibitors. The discovery of this new class of ATP-site directed protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, provides the basis of new medicinal chemistry tool in search for an effective treatment of cancer and other diseases that involve protein kinase signaling pathways.     

Primer design and marker clustering for multiplex SNP-IT primer extension genotyping assay using statistical modeling

MOTIVATION: The optimization of the primer design is critical for the development of high-throughput SNP genotyping methods. Recently developed statistical models of the SNP-IT primer extension genotyping reaction allow further improvement of primer quality for the assay. RESULTS: Here we describe how the statistical models can be used to improve primer design for the assay. We also show how to optimize clustering of the SNP markers into multiplex panels using statistical model for multiplex SNP-IT. The primer set failure probability calculated by a model is used as a minimization function for both primer selection and primers clustering. Three clustering algorithms for the multiplex genotyping SNP-IT assay are described and their relative performance is evaluated. We also describe the approaches to improve the speed of primer design and clustering calculations when using the statistical models. Our clustering decreases the average failure probability of the marker set by 7-25%. The experimental marker failure rate in the multiplex reaction was reduced dramatically and success rate can be achieved as high as 96%. AVAILABILITY: The primer design using statistical models is freely available from www.autoprimer.com.     

Subcellular distribution of the V-ATPase complex in plant cells,and <Emphasis Type="Italic">in vivo</Emphasis> localisation of the 100 kDa subunit VHA-a within the complex

Background  

Vacuolar H⁺-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed.     

AFM study of the elastin-like biopolymer poly(ValGlyGlyValGly)

In this paper, we report an AFM study on the supramolecular structures adopted by the synthetic polypentapeptide poly(ValGlyGlyValGly), whose monomeric sequence is an abundant, simple building block of elastin. The polypeptide was analyzed by deposition from both methanolic and aqueous suspensions, showing different behaviors. In methanol, the polypeptide is able to evolve, in a time-dependent way, from layers to ribbons to beaded filaments. When the equilibrium is reached, the formation of well-defined dendritic structures is also observed. This restructuring of the polypentapeptide seems to be reminiscent of a sort of Rayleigh instability. When deposited from aqueous suspensions, the polypeptide self-assembles either in fibrillar networks or in amyloid-like patterns, both of them being found in elastin or elastin-related polypeptides. As a general finding, poly(ValGlyGlyValGly) seems to constitute an excellent mimetic of the supramolecular properties of native elastin.     

Floxed allele for conditional inactivation of the voltage-gated sodium channel Scn8a (NaV1.6)

The sodium channel gene Scn8a encodes the channel NaV1.6, which is widely distributed in the central and peripheral nervous system. NaV1.6 is the major channel at the nodes of Ranvier in myelinated axons. Mutant alleles of mouse Scn8a result in neurological disorders including ataxia, tremor, paralysis, and dystonia. We generated a floxed allele of Scn8a by inserting loxP sites around the first coding exon. The initial targeted allele containing the neo-cassette was a severe hypomorph. In vivo deletion of the neo-cassette by Flp recombinase produced a floxed allele that generates normal expression of NaV1.6 protein. Ubiquitous deletion of the floxed exon by Cre recombinase in ZP3-Cre transgenic mice produced the Scn8a(del) allele. The null phenotype of Scn8a(del) homozygotes confirms the in vivo inactivation of Scn8a. Conditional inactivation of the floxed allele will make it possible to circumvent the lethality that results from complete loss of Scn8a in order to investigate the physiologic role of NaV1.6 in subpopulations of neurons.