Identification of the zinc-dependent endothelial cell binding protein for high molecular weight kininogen and factor XII: identity with the receptor that binds to the globular "heads" of C1q (gC1q-R).

High molecular weight kininogen (HK) and factor XII are known to bind to human umbilical vein endothelial cells (HUVEC) in a zinc-dependent and saturable manner indicating that HUVEC express specific binding site(s) for those proteins. However, identification and immunochemical characterization of the putative receptor site(s) has not been previously accomplished. In this report, we have identified a cell surface glycoprotein that is a likely candidate for the HK binding site on HUVECs. When solubilized HUVEC membranes were subjected to an HK-affinity column in the presence or absence of 50 microM ZnCl2 and the bound membrane proteins eluted, a single major protein peak was obtained only in the presence of zinc. SDS/PAGE analysis and silver staining of the protein peak revealed this protein to be 33 kDa and partial sequence analysis matched the NH2 terminus of gC1q-R, a membrane glycoprotein that binds to the globular "heads" of C1q. Two other minor proteins of approximately 70 kDa and 45 kDa were also obtained. Upon analysis by Western blotting, the 33-kDa band was found to react with several monoclonal antibodies (mAbs) recognizing different epitopes on gC1q-R. Ligand and dot blot analyses revealed zinc-dependent binding of biotinylated HK as well as biotinylated factor XII to the isolated 33-kDa HUVEC molecule as well as recombinant gC1q-R. In addition, binding of 125I-HK to HUVEC cells was inhibited by selected monoclonal anti-gC1q-R antibodies. C1q, however, did not inhibit 125I-HK binding to HUVEC nor did those monoclonals known to inhibit C1q binding to gC1q-R. Taken together, the data suggest that HK (and factor XII) bind to HUVECs via a 33-kDa cell surface glycoprotein that appears to be identical to gC1q-R but interact with a site on gC1q-R distinct from that which binds C1q.

Local densities orthogonal to beta-sheet amide planes: patterns of packing in globular proteins.

We have investigated the efficiency of packing by calculating intramolecular packing density above and below peptide planes of internal beta-pleated sheet residues in five globular proteins. The orientation of interest was chosen to allow study of regions that are approximately perpendicular to the faces of beta-pleated sheets. In these locations, nonbonded van der Waals packing interactions predominate over hydrogen bonding and solvent interactions. We observed considerable variability in packing densities within these regions, confirming that the interior packing of a protein does not result in uniform occupation of the available space. Patterns of fluctuation in packing density suggest that the regular backbone-to-backbone network of hydrogen bonds is not likely to be interrupted to maximize van der Waals interactions. However, high-density packing tends to occur toward the ends of beta-structure strands where hydrogen bonds are more likely to involve nonpolar side-chain groups or solvent molecules. These features result in internal protein folding with a central low-density core surrounded by a higher-density subsurface shell, consistent with our previous calculations regarding overall protein packing density.

Thermal unfolding of human high-density apolipoprotein A-1: implications for a lipid-free molten globular state.

Apolipoprotein A-1 (apoA-1) in complex with high-density lipoprotein is critically involved in the transport and metabolism of cholesterol and in the pathogenesis of atherosclerosis. We reexamined the thermal unfolding of lipid-free apoA-1 in low-salt solution at pH approximately 7, by using differential scanning calorimetry and circular dichroism. At protein concentrations <5 mg/ml, thermal unfolding of apoA-1 is resolved as an extended peak (25 degrees C-90 degrees C) that can be largely accounted for by a single reversible non-two-state transition with midpoint Tm 57 +/- 1 degree C, calorimetric enthalpy deltaH(Tm)= 200 +/- 20 kcal/mol (1 kcal = 4.18 kJ), van't Hoff enthalpy deltaHv(Tm) approximately 32.5 kcal/mol, and cooperativity deltaHv(Tm)/deltaH(Tm) approximately 0.16. The enthalpy deltaH(Tm) can be accounted for by melting of the alpha-helical structure that is inferred by CD to constitute approximately 60% of apoA-1 amino acids. Farand near-UV CD spectra reveal noncoincident melting of the secondary and tertiary structural elements and indicate a well-defined secondary structure but a largely melted tertiary structure for apoA-1 at approximately 37 degrees C and pH 7. This suggests a molten globular-like state for lipid-free apoA-1 under near-physiological conditions. Our results suggest that in vivo lipid binding by apoA-1 may be mediated via the molten globular apolipoprotein state in plasma.

Compressibility as a means to detect and characterize globular protein states.

We report compressibility data on single-domain, globular proteins which suggest a general relationship between protein conformational transitions and delta kzeroS, the change in the partial specific adiabatic compressibility which accompanies the transition. Specifically, we find transitions between native and compact intermediate states to be accompanied by small increases in kzeroS of +(1-4) x 10(-6) cm3.g-1.bar-1 (1 bar = 100 kPa). By contrast, transitions between native and partially unfolded states are accompanied by small decreases in kzeroS of -(3-7) x 10(-6) cm3.g-1.bar-1, while native-to-fully unfolded transitions result in large decreases in kzeroS of -(18-20) x 10(-6) cm3.g-1.bar-1. Thus, for the single-domain, globular proteins studied here, changes in kzeroS correlate with the type of transition being monitored, independent of the specific protein. Consequently, kzeroS measurements may provide a convenient approach for detecting the existence of and for defining the nature of protein transitions, while also characterizing the hydration properties of individual protein states.

No. 1. of the New-milk cheese, or The comi-heroick theunderclap, a semi-globular publication, without beginning and without end. /

At head of title: (April 16, 1807)

EXCESSO DE PESO DE ADULTOS RESIDENTES EM UM AGLOMERADO URBANO SUBNORMAL

Objetivo: Avaliar a prevalência de excesso de peso e possíveis fatores de risco em adultos residentes em um aglomerado urbano subnormal. Métodos: Estudo observacional do tipo transversal, baseado em uma amostra de 582 adultos, na faixa etária de 20 a 59 anos, residentes no aglomerado urbano subnormal dos Coelhos (Recife). Definiu-se excesso de peso pelo índice de massa corporal (IMC) maior ou igual a 25kg/m2. Foram avaliadas possíveis associações com idade, sexo, raça/cor e fatores socioeconômicos (escolaridade e condição de trabalho). O efeito sobre a ocorrência de excesso de peso foi estimado pelo cálculo do Odds Ratio (OR), mediante o ajuste de modelos de regressão logística simples. A precisão e significância estatística desses ORs foram avaliadas através de intervalos de 95% de confiança e do teste de Wald. Adotou-se nível de significância de 5%. Resultados: A prevalência de excesso de peso foi de 62,5% (n=364), sendo maior nas mulheres (66,1%; n=251) do que nos homens (56,0%; n=113), com um aumento progressivo até a faixa etária de 40 a 49 anos, passando a declinar a partir de então. Nessa faixa, houve um risco de excesso de peso de 2,6 vezes. Além da idade, pertencer ao sexo feminino e não trabalhar também representaram condições de risco. Conclusão: A elevada prevalência do excesso de peso na população adulta residente no aglomerado urbano subnormal dos Coelhos se mostrou associada ao sexo, faixa etária e condição de trabalho, constituindo-se, assim, como possíveis fatores de risco do problema.

High porosity chained aggregates from the topsoil of the lunar regolith dust

The unusual behaviour of fine lunar regolith like stickiness and low heat conductivity is dominated by the structural arrangement of its finest fraction. Here, we show the previously unknown phenomenon of a globular 3D superstructure within the dust fraction of regolith. A study using the recently developed Transmission X-ray Microscopy (TXM) with tomographic reconstruction reveals a highly porous network of cellular voids in the finest dust fraction aggregates in lunar soil. Such porous chained aggregates are composed of sub-micron particles that form a network of cellular voids a few micrometers in diameter. Discovery of such a superstructure within the finest fraction of lunar topsoil enables a model of heat transfer to be constructed.

High porosity chained aggregates from the topsoil of the Lunar regolith Dust

The unusual behaviour of fine lunar regolith like stickiness and low heat conductivity is dominated by the structural arrangement of its finest fraction in the outer-most topsoil layer. Here, we show the previously unknown phenomenon of building a globular 3-D superstructure within the dust fraction of the regolith. New technology, Transmission X-ray Microscopy (TXM) with tomographic reconstruction, reveals a highly porous network of cellular void system in the lunar finest dust fraction aggregates. Such porous chained aggregates are composed of sub-micron in size particles that build cellular void networks. Voids are a few micrometers in diameter. Discovery of such a superstructure within the finest fraction of the lunar topsoil allow building a model of heat transfer which is discussed.

Hierarchical multiscale model for biomechanics analysis of microfilament networks

The mechanisms of force generation and transference via microfilament networks are crucial to the understandings of mechanobiology of cellular processes in living cells. However, there exists an enormous challenge for all-atom physics simulation of real size microfilament networks due to scale limitation of molecular simulation techniques. Following biophysical investigations of constitutive relations between adjacent globular actin monomers on filamentous actin, a hierarchical multiscale model was developed to investigate the biomechanical properties of microfilament networks. This model was validated by previous experimental studies of axial tension and transverse vibration of single F-actin. The biomechanics of microfilament networks can be investigated at the scale of real eukaryotic cell size (10 μm). This multiscale approach provides a powerful modeling tool which can contribute to the understandings of actin-related cellular processes in living cells.

Hyperbranched polymer-gold nanoparticle assemblies : role of polymer architecture in hybrid assembly formation and SERS activity

Plasmonic gold nano-assemblies that self-assemble with the aid of linking molecules or polymers have the potential to yield controlled hierarchies of morphologies and consequently result in materials with tailored optical (e.g. localized surface plasmon resonances (LSPR)) and spectroscopic properties (e.g. surface enhanced Raman scattering (SERS)). Molecular linkers that are structurally well-defined are promising for forming hybrid nano-assemblies which are stable in aqueous solution and are increasingly finding application in nanomedicine. Despite much ongoing research in this field, the precise role of molecular linkers in governing the morphology and properties of the hybrid nano-assemblies remains unclear. Previously we have demonstrated that branched linkers, such as hyperbranched polymers, with specific anchoring end groups can be successfully employed to form assemblies of gold NPs demonstrating near-infrared SPRs and intense SERS scattering. We herein introduce a tailored polymer as a versatile molecular linker, capable of manipulating nano-assembly morphologies and hot-spot density. In addition, this report explores the role of the polymeric linker architecture, specifically the degree of branching of the tailored polymer in determining the formation, morphology and properties of the hybrid nano-assemblies. The degree of branching of the linker polymer, in addition to the concentration and number of anchoring groups, is observed to strongly influence the self-assembly process. The assembly morphology shifts primarily from 1D-like chains to 2D plates and finally to 3D-like globular structures, with increase in degree of branching. Insights have been gained into how the morphology influences the SERS performance of these nano-assemblies with respect to hot-spot density. These findings supplement the understanding of the morphology determining nano-assembly formation and pave the way for the possible application of these nano-assemblies as SERS bio-sensors for medical diagnostics.

Hybrid polymer/metal nanoparticles with surface enhanced raman tracking for potential use in nanomedicine

This project was a preliminary step towards the development of novel methods for early stage cancer diagnosis and treatment. Diagnostic imaging agents with high Raman signal enhancement were developed based on tailored assemblies of gold nanoparticles, which demonstrated potential for non-invasive detection from deep under the skin surface. Specifically designed polymers were employed to assemble gold nanoparticles into controlled morphologies including dimers, nanochains, nanoplates, globular and core-satellite nanostructures. Our findings suggest that the Raman enhancement is strongly dependent on assembly morphology and can be tuned to adapt to the requirements of the diagnostic agent.

A model for the interaction of trifluoroethanol with peptides and proteins

The structural stabilizing property of 2,2,2-trifluoroethanol (TFE) in peptides has been widely demonstrated, More recently, TFE has been shown to enhance secondary structure content in globular proteins, and to influence quaternary interactions in protein multimers. The molecular mechanisms by which TFE exerts its Influence on peptide and protein structures remain poorly understood. The present analysis integrates the known physical properties of TFE with a variety of experimental observations on the interaction of TFE with peptides and proteins and on the properties of fluorocarbons. Two features of TFE, namely the hydrophobicity of the trifluoromethyl group and the hydrogen bonding character (strong donor and poor acceptor), emerge as the most important factors for rationalising the observed effects of TFE. A model is proposed for TFE interaction with peptides which involves an initial replacement of the hydration shell by fluoroalcohol molecules, a process driven by apolar interactions and favourable entropy of dehydration. Subsequent bifurcated hydrogen-bond formation with peptide carbonyl groups, which leave intramolecular interactions unaffected, promotes secondary structure formation.

Helix-coil stability constants for amino acids in nonaqueous solvents. Studies of random poly(-benzyl-L-glutamate-co-L-alanine) and poly(-benzyl-L-glutamate-co-L-leucine) in a mixed solvent

The host-guest technique has been applied to the determination of the helix-coil stability constants of two naturally occurring amino acids, L-alanine and L-leucine, in a nonaqueous solvent system. Random copolymers containing L-alanine and L-leucine, respectively, as guest residues and -benzyl-L-glutamate as the host residue were synthesized. The polymers were fractionated and characterized for their amino acid content, molecular weight, and helix-coil transition behavior in a dichloroacetic acid (DCA)-1,2-dichloroethane (DCE) mixture. Two types of helix-coil transitions were carried out on the copolymers: solvent-induced transitions in DCA-DCE mixtures at 25°C and thermally induced transitions in a 82:18 (wt %) DCA-DCE mixture. The thermally induced transitions were analyzed by statistical mechanical methods to determine the Zimm-Bragg parameters, and s, of the guest residues. The experimental data indicate that, in the nonaqueous solvent, the L-alanine residue stabilizes the -helical conformation more than the L-leucine residue does. This is in contrast to their behavior in aqueous solution, where the reverse is true. The implications of this finding for the analysis of helical structures in globular proteins are discussed.

Folding and Selective Exit of Reporter Proteins from the Yeast Endoplasmic Reticulum

The present study analyses the traffic of Hsp150 fusion proteins through the endoplasmic reticulum (ER) of yeast cells, from their post-translational translocation and folding to their exit from the ER via a selective COPI-independent pathway. The reporter proteins used in the present work are: Hsp150p, an O-glycosylated natural secretory protein of Saccharomyces cerevisiae, as well as fusion proteins consisting of a fragment of Hsp150 that facilitates in the yeast ER proper folding of heterologous proteins fused to it. It is thought that newly synthesized polypeptides are kept in an unfolded form by cytosolic chaperones to facilitate the post-translational translocation across the ER membrane. However, beta-lactamase, fused to the Hsp150 fragment, folds in the cytosol into bioactive conformation. Irreversible binding of benzylpenicillin locked beta-lactamase into a globular conformation, and prevented the translocation of the fusion protein. This indicates that under normal conditions the beta-lactamase portion unfolds for translocation. Cytosolic machinery must be responsible for the unfolding. The unfolding is a prerequisite for translocation through the Sec61 channel into the lumen of the ER, where the polypeptide is again folded into a bioactive and secretion-competent conformation. Lhs1p is a member of the Hsp70 family, which functions in the conformational repair of misfolded proteins in the yeast ER. It contains Hsp70 motifs, thus it has been thought to be an ATPase, like other Hsp70 members. In order to understand its activity, authentic Lhs1p and its recombinant forms expressed in E. coli, were purified. However, no ATPase activity of Lhs1p could be detected. Nor could physical interaction between Lhs1p and activators of the ER Hsp70 chaperone Kar2p, such as the J-domain proteins Sec63p, Scj1p, and Jem1p and the nucleotide exchange factor Sil1p, be demonstrated. The domain structure of Lhs1p was modelled, and found to consist of an ATPase-like domain, a domain resembling the peptide-binding domain (PBD) of Hsp70 proteins, and a C-terminal extension. Crosslinking experiments showed that Lhs1p and Kar2p interact. The interacting domains were the C-terminal extension of Lhs1p and the ATPase domain of Kar2p, and this interaction was independent of ATPase activity of Kar2p. A model is presented where the C-terminal part of Lhs1p forms a Bag-like 3 helices bundle that might serve in the nucleotide exchange function for Kar2p in translocation and folding of secretory proteins in the ER. Exit of secretory proteins in COPII-coated vesicles is believed to be dependent of retrograde transport from the Golgi to the ER in COPI-coated vesicles. It is thought that receptors escaping to the Golgi must be recycled back to the ER exit sites to recruit cargo proteins. We found that Hsp150 leaves the ER even in the absence of functional COPI-traffic from the Golgi to the ER. Thus, an alternative, COPI-independent ER exit pathway must exists, and Hsp150 is recruited to this route. The region containing the signature guiding Hsp150 to this alternative pathway was mapped.