Collagen targeting using multivalent protein-functionalized dendrimers

Collagen is an attractive marker for tissue remodeling in a variety of common disease processes. Here we report the preparation of protein dendrimers as multivalent collagen targeting ligands by native chemical ligation of the collagen binding protein CNA35 to cysteine-functionalized dendritic divalent (AB₂) and tetravalent (AB₄) wedges. The binding of these multivalent protein constructs was studied on collagen-immobilized chip surfaces as well as to native collagen in rat intestinal tissues. To understand the importance of target density we also created collagen-mimicking surfaces by immobilizing synthetic collagen triple helical peptides at various densities on a chip surface. Multivalent display of a weak-binding variant (CNA35-Y175K) resulted in a large increase in collagen affinity, effectively restoring the collagen imaging capacities for the AB₄ system. In addition, dissociation of these multivalent CNA35 dendrimers from collagen surfaces was found to be strongly attenuated.     

Transposition of the gene cluster CYC1-OSM1-RAD7 in yeast

A mutant of the yeast Saccharomyces cerevisiae contains an increased amount of iso-1-cytochrome c because two copies of a segment, denoted COR, were transposed to a new position on chromosome VII, while the original COR region was retained at the normal position on chromosome X; this COR segment encompasses the CYC1, OSM1 and RAD7 loci which determine, respectively, iso-1-cytochrome c, osmotic sensitivity and ultraviolet light sensitivity. The analysis of genomic DNA with cloned probes indicates that the length of the COR segment is approximately 12,000 base-pairs. We suggest that certain normal strains of yeast, which possibly may contain reiterated sequences, can produce extended transpositions similar to prokaryotes.     

Anaerobic utilization of phosphite and hypophosphite by Bacillus sp.

A Bacillus sp. capable of utilizing phosphite and hypophosphite under anaerobic conditions was isolated from Cape Canerval soil samples. The organism was isolated on a glucose-mineral salts medium with phosphate deleted. Anaerobic cultivation of this isolate resulted in decreases in the hypophosphite or phosphite concentration, increases in turbidity, cell count, and dry-cell weight, and decreases in pH and glucose concentration. The optimum hypophosphite concentration for this isolate was 60 microgram/ml, whereas the optimum phosphate concentration was greater than 1,000 microgram/ml, suggesting that higher concentrations of hypophosphite may be toxic to this isolate. Hypophosphite or phosphite utilization was accompanied by little or no detectable accumulation of phosphate in the medium, and 32P-labeled hypophosphite was incorporated into the cell as organic phosphate. When phosphate was present in the medium, the isolate failed to metabolize phosphite. In the presence of phosphite and hypophosphite, the isolate first utilized phosphite and then hypophosphite.     

How does Sec63 affect the conformation of Sec61 in yeast?

The Sec complex catalyzes the translocation of proteins of the secretory pathway into the endoplasmic reticulum and the integration of membrane proteins into the endoplasmic reticulum membrane. Some substrate peptides require the presence and involvement of accessory proteins such as Sec63. Recently, a structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins was determined by cryo-electron microscopy (cryo-EM). Here, we show by co-precipitation that the Sec61 channel subunit Sbh1 is not required for formation of stable Sec63-Sec61 contacts. Molecular dynamics simulations started from the cryo-EM conformation of Sec61 bound to Sec63 and of unbound Sec61 revealed how Sec63 affects the conformation of Sec61 lateral gate, plug, pore region and pore ring diameter via three intermolecular contact regions. Molecular docking of SRP-dependent vs. SRP-independent signal peptide chains into the Sec61 channel showed that the pore regions affected by presence/absence of Sec63 play a crucial role in positioning the signal anchors of SRP-dependent substrates nearby the lateral gate.     

Energetics of liposomes encapsulating silica nanoparticles

Nanoparticles may be taken up into cells via endocytotic processes whereby the foreign particles are encapsulated in vesicles formed by lipid bilayers. After uptake into these endocytic vesicles, intracellular targeting processes and vesicle fusion might cause transfer of the vesicle cargo into other vesicle types, e.g., early or late endosomes, lysosomes, or others. In addition, nanoparticles might be taken up as single particles or larger agglomerates and the agglomeration state of the particles might change during vesicle processing. In this study, liposomes are regarded as simple models for intracellular vesicles. We compared the energetic balance between two liposomes encapsulating each a single silica nanoparticle and a large liposome containing two silica nanoparticles. Analytical expressions were derived that show how the energy of the system depends on the particle size and the distance between the particles. We found that the electrostatic contributions to the total energy of the system are negligibly small. In contrast, the van der Waals term strongly favors arrangements where the liposome snugly fits around the nanoparticle(s). Thus the two separated small liposomes have a more favorable energy than a larger liposome encapsulating two nanoparticles.     

Augmenting Endogenous Wnt Signaling Improves Skin Wound Healing

Wnt signaling is required for both the development and homeostasis of the skin, yet its contribution to skin wound repair remains controversial. By employing Axin2^LacZ/+ reporter mice we evaluated the spatial and temporal distribution patterns of Wnt responsive cells, and found that the pattern of Wnt responsiveness varies with the hair cycle, and correlates with wound healing potential. Using Axin2^LacZ/LacZ mice and an ear wound model, we demonstrate that amplified Wnt signaling leads to improved healing. Utilizing a biochemical approach that mimics the amplified Wnt response of Axin2^LacZ/LacZ mice, we show that topical application of liposomal Wnt3a to a non-healing wound enhances endogenous Wnt signaling, and results in better skin wound healing. Given the importance of Wnt signaling in the maintenance and repair of skin, liposomal Wnt3a may have widespread application in clinical practice.     

Polycyclic Aromatic Hydrocarbon-Induced Signaling Events Relevant to Inflammation and Tumorigenesis in Lung Cells Are Dependent on Molecular Structure

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental and occupational toxicants, which are a major human health concern in the U.S. and abroad. Previous research has focused on the genotoxic events caused by high molecular weight PAHs, but not on non-genotoxic events elicited by low molecular weight PAHs. We used an isomeric pair of low molecular weight PAHs, namely 1-Methylanthracene (1-MeA) and 2-Methylanthracene (2-MeA), in which only 1-MeA possessed a bay-like region, and hypothesized that 1-MeA, but not 2-MeA, would affect non-genotoxic endpoints relevant to tumor promotion in murine C10 lung cells, a non-tumorigenic type II alveolar pneumocyte and progenitor cell type of lung adenocarcinoma. The non-genotoxic endpoints assessed were dysregulation of gap junction intercellular communication function and changes in the major pulmonary connexin protein, connexin 43, using fluorescent redistribution and immunoblots, activation of mitogen activated protein kinases (MAPK) using phosphospecific MAPK antibodies for immunoblots, and induction of inflammatory genes using quantitative RT-PCR. 2-MeA had no effect on any of the endpoints, but 1-MeA dysregulated gap junctional communication in a dose and time dependent manner, reduced connexin 43 protein expression, and altered membrane localization. 1-MeA also activated ERK1/2 and p38 MAP kinases. Inflammatory genes, such as cyclooxygenase 2, and chemokine ligand 2 (macrophage chemoattractant 2), were also upregulated in response to 1-MeA only. These results indicate a possible structure-activity relationship of these low molecular weight PAHs relevant to non-genotoxic endpoints of the promoting aspects of cancer. Therefore, our novel findings may improve the ability to predict outcomes for future studies with additional toxicants and mixtures, identify novel targets for biomarkers and chemotherapeutics, and have possible implications for future risk assessment for these PAHs.