Synthesis and biological activity of anticoagulant heparan sulfate glycopolymers

Heparin has been used as an anticoagulant drug for more than 70 years. The global distribution of contaminated heparin in 2007, which resulted in adverse clinical effects and over 100 deaths, emphasizes the necessity for safer alternatives to animal-sourced heparin. The structural complexity and heterogeneity of animal-sourced heparin not only impedes safe access to these biologically active molecules, but also hinders investigations on the significance of structural constituents at a molecular level. Efficient methods for preparing new synthetic heparins with targeted biological activity are necessary not only to ensure clinical safety, but to optimize derivative design to minimize potential side effects. Low molecular weight heparins have become a reliable alternative to heparin, due to their predictable dosages, long half-lives, and reduced side effects. However, heparin oligosaccharide synthesis is a challenging endeavor due to the necessity for complex protecting group manipulation and stereoselective glycosidic linkage chemistry, which often result in lengthy synthetic routes and low yields. Recently, chemoenzymatic syntheses have produced targeted ultralow molecular weight heparins with high-efficiency, but continue to be restricted by the substrate specificities of enzymes.

To address the need for access to homogeneous, complex glycosaminoglycan structures, we have synthesized novel heparan sulfate glycopolymers with well-defined carbohydrate structures and tunable chain length through ring-opening metathesis polymerization chemistry. These polymers recapitulate the key features of anticoagulant heparan sulfate by displaying the sulfation pattern responsible for heparin’s anticoagulant activity. The use of polymerization chemistry greatly simplifies the synthesis of complex glycosaminoglycan structures, providing a facile method to generate homogeneous macromolecules with tunable biological and chemical properties. Through the use of in vitro chromogenic substrate assays and ex vivo clotting assays, we found that the HS glycopolymers exhibited anticoagulant activity in a sulfation pattern and length-dependent manner. Compared to heparin standards, our short polymers did not display any activity. However, our longer polymers were able to incorporate in vitro and ex vivo characteristics of both low-molecular-weight heparin derivatives and heparin, displaying hybrid anticoagulant properties. These studies emphasize the significance of sulfation pattern specificity in specific carbohydrate-protein interactions, and demonstrate the effectiveness of multivalent molecules in recapitulating the activity of natural polysaccharides.

A measurement of the proton elastic form factors for 1 ≤ Q^2 ≤ 3 (GeV/c)^2

We report measurements of the proton form factors, G^p_E and G^p_M, extracted from elastic electron scattering in the range 1 ≤ Q^2 ≤ 3 (GeV/c)^2 with uncertainties of <15% in G^p_E and <3% in G^p_M. The results for G^p_E are somewhat larger than indicated by most theoretical parameterizations. The ratio of Pauli and Dirac form factors, Q^2(F^p_2/F^p_1), is lower in value and demonstrates less Q^2 dependence than these parameterizations have indicated. Comparisons are made to theoretical models, including those based on perturbative QCD, vector-meson dominance, QCD sum rules, and diquark constituents to the proton. A global extraction of the form factors, including previous elastic scattering measurements, is also presented.

A unified strategy to ent-kauranoid natural products : total syntheses of (–)-maoecrystal Z, (–)-trichorabdal A, and (–)-longikaurin E

The diterpenoid constituents of the Isodon plants have attracted reasearchers interested in both their chemical structures and biological properties for more than a half-century. In recent years, the isolations of new members displaying previously unprecedented ring systems and highly selective biological properties have piqued interest from the synthetic community in this class of natural products.

Reported herein is the first total synthesis of such a recently isolated diterpenoid, (–)-maoecrystal Z. The principal transformations implemented in this synthesis include two highly diastereoselective radical cyclization reactions: a Sm^(II)-mediated reductive cascade cyclization, which forms two rings and establishes four new stereocenters in a single step, and a Ti^(III)-mediated reductive epoxide-acrylate coupling that yields a functionalized spirolactone product, which forms a core bicycle of maoecrystal Z.

The preparation of two additional ent-kauranoid natural products, (–)-trichorabdal A and (–)-longikaurin E, is also described from a derivative of this key spirolactone. These syntheses are additionally enabled by the palladium-mediated oxidative cyclization reaction of a silyl ketene acetal precursor that is used to install the bridgehead all-carbon quaternary stereocenter and bicyclo[3.2.1]octane present in each natural product. These studies have established a synthetic relationship among three architecturally distinct ent-kaurane diterpenoids and have forged a path for the preparation of interesting unnatural ent-kauranoid structural analogs for more thorough biological study.

Digested sludge: delineation and modeling for ocean disposal

Experimental work was performed to delineate the system of digested sludge particles and associated trace metals and also to measure the interactions of sludge with seawater. Particle-size and particle number distributions were measured with a Coulter Counter. Number counts in excess of 10¹² particles per liter were found in both the City of Los Angeles Hyperion mesophilic digested sludge and the Los Angeles County Sanitation Districts (LACSD) digested primary sludge. More than 90 percent of the particles had diameters less than 10 microns.

Total and dissolved trace metals (Ag, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) were measured in LACSD sludge. Manganese was the only metal whose dissolved fraction exceeded one percent of the total metal. Sedimentation experiments for several dilutions of LACSD sludge in seawater showed that the sedimentation velocities of the sludge particles decreased as the dilution factor increased. A tenfold increase in dilution shifted the sedimentation velocity distribution by an order of magnitude. Chromium, Cu, Fe, Ni, Pb, and Zn were also followed during sedimentation. To a first approximation these metals behaved like the particles.

Solids and selected trace metals (Cr, Cu, Fe, Ni, Pb, and Zn) were monitored in oxic mixtures of both Hyperion and LACSD sludges for periods of 10 to 28 days. Less than 10 percent of the filterable solids dissolved or were oxidized. Only Ni was mobilized away from the particles. The majority of the mobilization was complete in less than one day.

The experimental data of this work were combined with oceanographic, biological, and geochemical information to propose and model the discharge of digested sludge to the San Pedro and Santa Monica Basins. A hydraulic computer simulation for a round buoyant jet in a density stratified medium showed that discharges of sludge effluent mixture at depths of 730 m would rise no more than 120 m. Initial jet mixing provided dilution estimates of 450 to 2600. Sedimentation analyses indicated that the solids would reach the sediments within 10 km of the point discharge.

Mass balances on the oxidizable chemical constituents in sludge indicated that the nearly anoxic waters of the basins would become wholly anoxic as a result of proposed discharges. From chemical-equilibrium computer modeling of the sludge digester and dilutions of sludge in anoxic seawater, it was predicted that the chemistry of all trace metals except Cr and Mn will be controlled by the precipitation of metal sulfide solids. This metal speciation held for dilutions up to 3000.

The net environmental impacts of this scheme should be salutary. The trace metals in the sludge should be immobilized in the anaerobic bottom sediments of the basins. Apparently no lifeforms higher than bacteria are there to be disrupted. The proposed deep-water discharges would remove the need for potentially expensive and energy-intensive land disposal alternatives and would end the discharge to the highly productive water near the ocean surface.

Geology of the Pacoima Area, Los Angeles County, California

The Pacoima area is located on an isolated hill in the northeast section of the San Fernando, the northeast portion of the Pacoima Quadrangle, Los Angeles County, California. Within it are exposed more than 2300 feet of Tertiary rocks, which comprise three units of Middle Miocene (?) age, and approximately 950 feet of Jurassic (?) granite basement. The formations are characterized by their mode of occurrence, marine and terrestial origin, diverse lithology, and structural features.

The basement complex is composed of intrusive granite, small masses of granodiorite and a granodiorite gneiss with the development of schistosity in sections. During the long period of erosion of the metamorphics, the granitic rocks were exposed and may have provided clastic constituents for the overlying formations.

As a result of rapid sedimentation in a transitional environment, the Middle Miocene Twin Peaks formation was laid down unconformably on the granite. This formation is essentially a large thinning bed of gray to buff pebble and cobble conglomerate grading to coarse yellow sandstone. The contact of conglomerate and granite is characterized by its faulted and depositional nature.

Beds of extrusive andesite, basalt porphyry, compact vesicular amygdaloidal basalts, andesite breccia, interbedded feldspathic sands and clays of terrestial origin, and mudflow breccia comprise the Pacoima formation which overlies the Twin Peaks formation unconformably. A transgressing shallow sea accompanied settling of the region and initiated deposition of fine clastic sediments.

The marine Topanga (?) formation is composed of brown to gray coarse sandstone grading into interbedded buff sandstones and gray shales. Intrusions of rhyolitedacite and ash beds mark continued but sporatic volcanism during this period.

The area mapped represents an arch in the Tertiary sediments. Forces that produced the uplift of the granite structural high created stresses that were relieved by jointing and faulting. Vertical and horizontal movement along these faults has displaced beds, offset contacts and complicated their structure. Uplift and erosion have exposed the present sequence of beds which dip gently to the northeast. The isolated hill is believed to be in an early stage of maturity.

On the transport properties of fluid-particle flow

The hydrodynamic forces acting on a solid particle in a viscous, incompressible fluid medium at low Reynolds number flow is investigated mathematically as a prerequisite to the understanding of transport processes in two-phase flow involving solid particles and fluid. Viscous interaction between a small number of spherical particles and continuous solid boundaries as well as fluid interface are analyzed under a “point-force” approximation. Non-spherical and elastic spherical particles in a simple shear flow area are then considered. Non-steady motion of a spherical particle is briefly touched upon to illustrate the transient effect of particle motion.

A macroscopic continuum description of particle-fluid flow is formulated in terms of spatial averages yielding a set of particle continuum and bulk fluid equations. Phenomenological formulas describing the transport processes in a fluid medium are extended to cases where the volume concentration of solid particles is sufficiently high to exert an important influence. Hydrodynamic forces acting on a spherical solid particle in such a system, e.g. drag, torque, rotational coupling force, and viscous collision force between streams of different sized particles moving relative to each other are obtained. Phenomenological constants, such as the shear viscosity coefficient, and the diffusion coefficient of the bulk fluid, are found as a function of the material properties of the constituents of the two-phase system and the volume concentration of solid. For transient heat conduction phenomena, it is found that the introduction of a complex conductivity for the bulk fluid permits a simple mathematical description of this otherwise complicated process. The rate of heat transfer between particle continuum and bulk fluid is also investigated by means of an Oseen-type approximation to the energy equation.

I. Studies on the activation of Drosophila phenol oxidase. II. Studies on serum insulin in normal subjects and diabetics

Part I

Phenol oxidase is the enzyme responsible for hardening and pigmentation of the insect cuticle. In Drosophila, phenol oxidase is a latent enzyme. Enzyme activity is produced by the interaction of a number of protein components. A minimal activation scheme consisting of six protein components, designated Pre S, S activator, S, P. P' and Ʌ₁ is described. Quantitative assays have been developed for the S activator, S, P and P' proteins and these components have been partially purified. Experiments describing the interactions of the six components have been conducted and a model for the activation of phenol oxidase in a minimal system is proposed. Possible mechanisms of the reactions between the constituents of the activating system and potential regulatory mechanisms involved in phenol oxidase production and function are discussed.

Part II

A method has been developed for the partial purification of insulin from human serum. A procedure for the determination of the electrophoretic mobility of serum insulin on polyacrylamide gels is described. An electrophoretic analysis of insulin isolated from a normal subject is reported and in addition to a major band, the existence of a number of minor bands of immunoreactive insulin is described. A comparison of the electrophoretic patterns of insulin isolated from normal and diabetic subjects was carried out and indications that differences between them may occur are reported.