Labeling with Nanogold and Undecagold: Techniques and Results

A significant new development in gold labeling for microscopy has been achieved through the use of gold cluster compounds that are covalently attached to antibodies or other probe molecules. These unique gold probes are smaller than most colloidal gold conjugates and exhibit improved penetration into tissues, higher labeling densities, and allow many new probes to be made with peptides, nucleic acids, lipids, drugs, and other molecules. A new fluorescent-gold conjugate is useful for examining localization by fluorescence microscopy, then visualizing the same label at the ultrastructural level in the electron microscope

Gold, Electron Microscopy, and Cancer Therapy

Radioactive gold has properties suitable for radiotherapy and can provide lethal irradiation to cells. If the gold is conjugated to a targeting molecule, such as an antibody, it may be possible to specifically deliver the dose to tumor cells. Various gold particles are possible candidates and include gold colloids with adsorption of antibodies or gold clusters with covalent attachment. Different sizes of gold particles are available and some may be preferred for certain situations. Problems with intravenous injection and in vivo delivery are numerous, and a more tractable application is the direct instillation into the urinary bladder of radiogold immunoconjugates to treat superficial bladder carcinoma. Preliminary studies indicate the feasibility of this approach

Evidence for a Second Type of Fibril Branch Point in Fibrin Polymer Networks, the Trimolecular Junction

Fibrin molecules polymerize to double-stranded fibrils by intermolecular end-to-middle domain pairing of complementary polymerization sites, accompanied by fibril branching to form a clot network. Mass/length measurements on scanning transmission electron microscopic images of fibrils comprising branch points showed two types of junctions. Tetramolecular junctions occur when two fibrils converge, creating a third branch with twice the mass/length of its constituents. Newly recognized trimolecular junctions have three fibril branches of equal mass/length, and occur when an extraneous fibrin molecule initiates branching in a propagating fibril by bridging across two unpaired complementary polymerization sites. When trimolecular junctions predominate, clots exhibit nearly perfect elasticity

The Location of the Carboxy-Terminal Region of γ Chains in Fibrinogen and Fibrin D Domains

Elongated fibrinogen molecules are comprised of two outer “D” domains, each connected through a “coiled-coil” region to the central “E” domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations that result in double-stranded fibrils. Factor XIIIa mediates crosslinking of the C-terminal regions of γ chains in each D domain (the γXL site) by incorporating intermolecular ɛ-(γ-glutamyl)lysine bonds between amine donor γ406 lysine of one γ chain and a glutamine acceptor at γ398 or γ399 of another. Several lines of evidence show that crosslinked γ chains extend “transversely” between the strands of each fibril, but other data suggest instead that crosslinked γ chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the γXL site in fibrinogen and assembled fibrin fibrils, we incorporated an amine donor, thioacetyl cadaverine, into glutamine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron dense gold cluster compound, undecagold monoaminopropyl maleimide (Au11). Fibrinogen was examined by scanning transmission electron microscopy to locate Au11-cadaverine-labeled γ398/399 D domain sites. Seventy-nine percent of D domain Au11 clusters were situated in middle to proximal positions relative to the end of the molecule, with the remaining Au11 clusters in a distal position. In fibrin fibrils, D domain Au11 clusters were located in middle to proximal positions. These findings show that most C-terminal γ chains in fibrinogen or fibrin are oriented toward the central domain and indicate that γXL sites in fibrils are situated predominantly between strands, suitably aligned for transverse crosslinking

The Relationship Between the Fibrinogen D Domain Self-Association/Cross-Linking Site (gammaXL) and the Fibrinogen Dusart Abnormality (Aalpha R554C-albumin): Clues to Thrombophilia in the Dusart Syndrome

Cross-linking of fibrinogen at its COOH-terminal gamma chain cross-linking site occurs in the presence of factor XIIIa due to self-association at a constitutive D domain site ( gammaXL ). We investigated the contribution of COOH-terminal regions of fibrinogen Aalpha chains to the gammaXL site by comparing the gamma chain cross-linking rate of intact fibrinogen (fraction I-2) with that of plasma fraction I-9, plasmic fraction I-9D, and plasmic fragment D1, which lack COOH-terminal Aalpha chain regions comprising approximately 100, approximately 390, and 413 residues, respectively. The cross-linking rates were I-2 \u3e I-9 \u3e 1-9D = D1, and indicated that the terminal 100 or more Aalpha chain residues enhance gammaXL site association. Fibrinogen Dusart, whose structural abnormality is in the COOH-terminal alphaC region of its Aalpha chain (Aalpha R554C-albumin), is associated with thrombophilia ( Dusart Syndrome ), and is characterized functionally by defective fibrin polymerization and clot structure, and reduced plasminogen binding and tPA-induced fibrinolysis. In the presence of XIIIa, the Dusart fibrinogen gamma chain cross-linking rate was about twice that of normal, but was normalized in proteolytic fibrinogen derivatives lacking the Aalpha chain abnormality, as was reduced plasminogen binding. Electron microscopy showed that albumin-bound Dusart fibrinogen alphaC regions were located in the vicinity of D domains, rather than at their expected tethered location near the fibrinogen E domain. In addition, there was considerable fibrinogen aggregation that was attributable to increased intermolecular COOH-terminal Aalpha chain associations promoted by untethered Dusart fibrinogen aC domains. We conclude that enhanced Dusart fibrinogen self-assembly is mediated through its abnormal alphaC domains, leads to increased gammaXL self-association and gamma chain cross-linking potential, and contributes to the thrombophilia that characterizes the Dusart Syndrome

Conformational analysis of 16 S ribosomal RNA from Escherichia coli by scanning transmission electron microscopy

AbstractDigitized images of molecules of 16 S rRNA from Escherichia coli, obtained by scanning transmission electron microscopy (STEM), provide quantitative structural information that is lacking in conventional electron micrographs. We have determined the morphology, total molecular mass, mass distribution within individual rRNA molecules and apparent radii of gyration. From the linear density (M/L) we have assessed the number of strands in the structural backbone of rRNA and studied the pattern of branching and folding related to the secondary and tertiary structure of rRNAs under various buffer conditions. Even in reconstitution buffer 16 S RNA did not show any resemblance to the native 30 S subunit

The Covalent Structure of Factor XIIIa Crosslinked Fibrinogen Fibrils

When factor XIIIa-mediated crosslinking of fibrin or fibrinogen occurs, reciprocal intermolecular isopeptide bonds form first between paired carboxy terminal γ chain donor-acceptor sites in outer molecular D domains, resulting in γ chain dimers. Their location in the fibrin polymer is not certain, but some evidence suggests they are situated at the outermost ends of the D domains of linearly aligned molecules comprising each strand of double-stranded fibrils ( DD-long ). Other experiments indicate that γ chain bonds are located between D domains in opposing fibril strands ( transverse ). To distinguish between these possible arrangements, we evaluated the ultrastructure of fibrils and fibers found in factor XIIIa-fibrinogen crosslinking mixtures, based on this reasoning: if DD-long bonding occurs, single-stranded fibrils should result, whereas transverse positioning will result in double-stranded fibrils. Fibrils formed in partially crosslinked fibrinogen solutions consisted of two parallel strands, as discerned visually from scanning transmission electron microscopic images and confirmed by mass per unit length fibril measurements. Neighboring fibrinogen D domains in each fibril strand were aligned end-to-end and were in register with a fibrinogen E domain in the opposite strand, creating a half-staggered molecular arrangement with -22.5-nm periodicity corresponding to half the length of fibrinogen. Ribbon-like fibrinogen fibers, like fibrils, displayed 22.5-nm periodicity, as expected from laterally associated double-stranded fibrils with D domains in register. Taken together, these results indicate that carboxy terminal γ chain bonds are positioned transversely between strands and are represented by thin filamentous structures bridging the D domains of opposing fibril strand— it follows that the same γ chain crosslink arrangement occurs in fibrin

Small, Long Blood Half-Life Iodine Nanoparticle for Vascular and Tumor Imaging

Abstract Standard clinical X-ray contrast agents are small iodine-containing molecules that are rapidly cleared by the kidneys and provide robust imaging for only a few seconds, thereby limiting more extensive vascular and tissue biodistribution imaging as well as optimal tumor uptake. They are also not generally useful for preclinical microCT imaging where longer scan times are required for high resolution image acquisition. We here describe a new iodine nanoparticle contrast agent that has a unique combination of properties: 20 nm hydrodynamic diameter, covalent PEG coating, 40 hour blood half-life, 50% liver clearance after six months, accumulation in tumors, and well-tolerated to at least 4 g iodine/kg body weight after intravenous administration in mice. These characteristics are unique among the other iodine nanoparticles that have been previously reported and provide extended-time high contrast vascular imaging and tumor loading. As such, it is useful for preclinical MicroCT animal studies. Potential human applications might include X-ray radiation dose enhancement for cancer therapy and vascular imaging for life-threatening situations where high levels of contrast are needed for extended periods of time