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Amplification of DNA in vivo occurs in intracellular environments characterized by macromolecular crowding (MMC). In vitro Polymerase-chain-reaction (PCR), however, is non-crowded, requires thermal cycling for melting of DNA strands, primer-template hybridization and enzymatic primer-extension. The temperature-optima for primer-annealing and extension are strikingly disparate which predicts primers to dissociate from template during extension thereby compromising PCR efficiency. We hypothesized that MMC is not only important for the extension phase in vivo but also during PCR by stabilizing nucleotide hybrids. Novel atomistic Molecular Dynamics simulations elucidated that MMC stabilizes hydrogen-bonding between complementary nucleotides. Real-time PCR under MMC confirmed that melting-temperatures of complementary DNA–DNA and DNA–RNA hybrids increased by up to 8°C with high specificity and high duplex-preservation after extension (71% versus 37% non-crowded). MMC enhanced DNA hybrid-helicity, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA- single-strands.

Macromolecules crowd defined spaces, thereby excluding other like-sized molecules from the volume they occupy. These excluded-volume effect(s) (EVE) are well characterized for intracellular and partially for extracellular compartments such as blood plasma. We showed that EVE in fibroblast culture leads to faster enzymatic procollagen conversion and matrix deposition. Apparently, EVE can be applied to emulate in vivo conditions in an in vitro setting. Thus, we attempted to quantitatively capture the crowding potential of various macromolecules using dynamic light scattering under physiological conditions. We found that charged macromolecules like dextran sulfate (negative, 500 kDa) have a hydrodynamic radii of 46.4 ± 0.3 nm i.e. ~4 fold larger than that of neutral macromolecules like Ficoll (neutral, 400 kDa) and thus show greater EVE potential. At biologically effective concentrations viscosity was not increased. Unexpectedly, we observed a dramatic drop of hydrodynamic radii of all macromolecules tested above a threshold concentration. This suggested a hyper-crowding state in which the crowders compacted themselves mutually. We will use this hyper-crowding threshold to determine retrogradely rules that allow to predict the conditions for optimum crowding effects (such as the half-hyper-crowding concentration) in biological systems. We propose Dynamic Light Scattering (DLS) as a potential tool to estimate EVE in biotechnical applications.

Serglycin is a multifunctional molecule and one of the first proteoglycans to be cloned. In this manuscript, we examine the physiological roles of serglycin in immunity, hemostasis, cell growth, apoptosis, and reproduction. In addition, we review recent studies on the involvement of serglycin in various pathological conditions, including cancer, inflammatory diseases, and platelet disorders. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

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The actin cytoskeleton is intimately involved in most cellular functions, including cell motility, endo/exocytosis and intracellular trafficking. These processes are characterized by rapid oscillations of actin polymerization/depolymerization under tight temporal and spatial regulation. Hundreds of G-and F-actinbinding proteins, along with signaling and scaffolding proteins regulate the assembly of actin networks. Among these proteins, filament nucleators play a critical role by determining the time and location for actin polymerization, as well as the specific structures of the actin networks that they generate. Eukaryotic cells and certain pathogens use filament nucleators to stabilize actin nuclei (small oligomers of 2-4 actin subunits), whose formation is rate-limiting. Known filament nucleators include the Arp2/3 complex and its large family of Nucleation Promoting Factors (NPFs), Formins, Spire, Cobl, Lmod, VopL/VopF and TARP. Structural and functional studies are beginning to shed light on the diverse mechanisms used by these molecules to stabilize actin nuclei. Thus, with the exception of Formins known filament nucleators use the WASP-Homology 2 domain (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. A common architecture, found in Spire, Cobl and VopL/VopF, consists of tandem W domains that bind three to four actin subunits to form a nucleus. Structural considerations suggest that NPFs-Arp2/3 complex can also be viewed as a specialized form of tandem W-based nucleator. The nucleation activities of these proteins vary significantly, and the most effective nucleators are not necessarily those with the largest number of W domains. We show that the inter-W linkers play a critical role in determining the nucleation activities of filament nucleators and the structures of the actin nuclei that they generate. Furthermore, we present evidence that a previously neglected factor, oligomerization, is a major determinant of filament nucleation activity and nuclei structure.

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