Rapid isolation of substrate-quality plasmid DNA without CsCl-dye density gradients

The isolation of plasmid DNA produced in transformed bacterial cells is essential for many molecular biology techniques. Two drawbacks to the widely used CsCl-ethidium bromide method of preparation are the need for ultracentrifuge time and the generation of ethidium bromide waste. In this article we describe a method for the quick isolation of plasmid DNA without the use of an ultracentrifuge or ethidium bromide.     

An automated method for determination of the sedimentation coefficient of macromolecules using a preparative centrifuge

Following centrifugation in a preparative ultracentrifuge at relatively high speeds for 1-3 h, quartz tubes containing approximately 150 microliter of macromolecular solution are optically scanned using an apparatus and method previously described (Attri, A. K., and Minton A. P. (1983) Anal. Biochem. 133, 142-152). The resulting data are processed by microcomputer immediately upon completion of scanning to yield the sedimentation coefficient of the solute. Calculated values agree to within a few percent with those found in the literature and with the results of control experiments carried out using an analytical ultracentrifuge.     

A rapid method for the isolation of intracytoplasmic membranes from Rhodopseudomonas sphaeroides using an air-driven ultracentrifuge

A method has been developed for the isolation intracytoplasmic (ICM) vesicles (chromatophores) from Rhodopseudomonas sphaeroides using an air-driven ultracentrifuge. Application of conventional techniques used for preparative scale equipment to the air-driven ultracentrifuge allows the rapid isolation of ICM vesicles from reduced quantities of starting material. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis profiles of ICM vesicles isolated in this fashion are essentially indistinguishable from those isolated by conventional means.     

A new CSLM-based method for determination of the phase behaviour of aqueous mixtures of biopolymers

On mixing different types of high molecular weight (bio)polymers in an aqueous solution, phase separation often occurs. In some cases, the occurrence of phase separation may be readily observed, because due to density differences the heavier of the two phases is accumulated at the bottom of the vessel in which the mixture is contained. By using classical techniques, the composition of the two phases may then be determined. In the case where the density differences are not so large, and the viscosity of the system is high, the two phases remain intimately mixed. An alternative route to determine the phase behaviour of these systems might be a microscopic technique (Confocal Scanning Laser Microscopy, CSLM), using the fluorescence intensity of labelled biopolymers to quantify their concentration and phase volume in the system. Experiments were performed with several mixtures of sodium alginate, labelled with fluorescein, and sodium caseinate, fluorescently labelled with Texas Red. The viscosity of the mixtures studied was low enough to allow bulk phase separation of the phases by using an ultracentrifuge. Results of the phase volumes, and the composition of the phases, obtained independently by applying the two different methods (CSLM, or analysis of the separate phases after centrifugation) were compared and found to be in reasonable agreement.     

An automated method for determining buoyant density of nucleic acids using a preparative ultracentrifuge

We present a technique for analytical buoyant density sedimentation of nucleic acids which is performed in a preparative ultracentrifuge, in contrast to an analytical ultracentrifuge. Following centrifugation in a preparative rotor, small cylindrical quartz tubes are optically scanned; upon completion of the scan the data are processed immediately by a microcomputer and the buoyant density of the nucleic acid is calculated. Experimental data are presented employing several different deoxyribonucleic acids banded in neutral and alkaline cesium sulfate. Results are independent of rotor speed, location of bands within the gradient, and loading density of the cesium sulfate solution. Derived buoyant density values agree within 0.5% of previously published values.     

A low-cost integrator for preparative ultracentrifuges

An integrator is described for the measurement of the time integral ∫₀^t rpm²dt in preparative ultracentrifuge where linearity exists either (a) between tachometer generator ac voltage amplitude and rpm (e.g., Sorvall RC2-B) or dc voltage and rpm, or (b) between the square-wave frequency from the tachometer generator and rpm (e.g., Beckman L2-65B). The construction and the precision levels of an integrator for Sorvall RC2-B preparative ultracentrifuge in the range 0–10,000 rpm and for Beckman L2-65B preparative ultracentrifuge in the range 0–40,000 rpm are described.     

Stabilization of Metals in Subsurface by Biopolymers: Laboratory Drainage Flow Studies

Environmental contamination with heavy metals and radionuclides remains a major problem worldwide. The current clean-up methodologies are based on energy-intensive engineering processes, which are disruptive and costly. A new universal technology targeted for the permanent enclosure and fixation of nuclear and other extreme hazardous metallic wastes in subsurface sites is needed. Such technology will be useful in treating contamination at many sites in the U.S., with specific applications to Department of Energy (DOE) sites. Biopolymers are potential tools for such an innovative technology. Biopolymers have repeated sequences, and therefore provide ample opportunity for chemical reactions with metals, soil particles, and other biopolymers. They also have the additional ability of creating cross-linking interpenetrating networks that can encapsulate the contaminants. Based on this concept, in the present work five biopolymers (xanthan, chitosan, polyhydroxy butyrate, guar gum, polyglutamic acid) were investigated for potential use in the stabilization of metals in the subsurface. The effects of these biopolymers (used alone and in combinations) on soil characteristics (permeability, shear strength) and their metal uptake ability have been studied using laboratory drainage flow systems. Biopolymer solutions were run through the experimental sandpack columns, followed by copper solution and leaching agents (distilled water and hydrochloric acid). The permeability and shear strength of sand were evaluated. Copper uptake capacity of each biopolymer and combination of biopolymers was also studied along with subsequent leaching. All biopolymers tested improved sand characteristics (by decreasing permeability and increasing shear strength) and had good metal uptake ability (60–90%) with relatively low leachability (10–22%). While biopolymers used alone were more efficient in metal uptake, the combination of two biopolymers (xanthan and chitosan) had an increasing plugging effect. These results show the potential of using biopolymers in subsurface metal stabilization.     

Production of renewable polymers from crop plants

Plants produce a range of biopolymers for purposes such as maintenance of structural integrity, carbon storage, and defense against pathogens and desiccation. Several of these natural polymers are used by humans as food and materials, and increasingly as an energy carrier. In this review, we focus on plant biopolymers that are used as materials in bulk applications, such as plastics and elastomers, in the context of depleting resources and climate change, and consider technical and scientific bottlenecks in the production of novel or improved materials in transgenic or alternative crop plants. The biopolymers discussed are natural rubber and several polymers that are not naturally produced in plants, such as polyhydroxyalkanoates, fibrous proteins and poly-amino acids. In addition, monomers or precursors for the chemical synthesis of biopolymers, such as 4-hydroxybenzoate, itaconic acid, fructose and sorbitol, are discussed briefly.     

Production and mass transfer characteristics of non-Newtonian biopolymers for biomedical applications

The market for microbial biopolymers is currently expanding to include several emerging biomedical applications. Specifically, these applications are drug delivery and wound healing. A fundamental understanding of the key fermentation parameters is necessary in order to optimize the production of these biopolymers. Considering that most microbial biopolymer systems exhibit non-Newtonian rheology, oxygen mass transfer can be an important parameter to optimize and control. In this article, we present a critical review of recent advances in rheological and mass transfer characteristics of selected biopolymers of commercial interest in biomedical applications.     

Precipitation of protein by ultracentrifuge with angle rotor

Precipitation of a protein by ultracentrifuge with an angle rotor was investigated experimentally. Experimental data for Lysozyme and Bovine serum albumin were compared with the calculated distribution of concentration, and an agreement between calculated curves and experimental data during the course of ultracentrifugation was obtained. From the first approximated model presented in our previous paper, it is observed that the precipitation of a protein by ultracentrifuge with an angle rotor proceeds more rapidly than that with a swing rotor. Part 1 of this article was published in Vol. 5. No. 2     

Analytical ultracentrifugation and the characterization of chromatin structure

This mini review consists of two parts. The first part will provide a brief overview of the theoretical aspects involved in the two kinds of experiments that can be conducted with the analytical ultracentrifuge (sedimentation velocity and sedimentation equilibrium) as they pertain to the study of chromatin. In the following sections, I describe the analytical ultracentrifuge experiments which, in my opinion, have contributed the most to our understanding of chromatin. Few other biophysical techniques, with the exception of X-ray scattering and diffraction, have contributed as extensively as the analytical ultracentrifuge to the characterization of so many different aspects of chromatin structure. In the course of his scientific career, Professor Henryk Eisenberg has made many important contributions to the theoretical aspects underlying ultracentrifuge analysis, especially in the analysis of solutions of polyelectrolytes and biological macromolecules [H. Eisenberg, Biological macromolecules and polyelectrolytes in solution, Clarendon Press, Oxford, 1976]. As an example he has devoted some of his research effort to the characterization of chromatin in solution. This review includes these important contributions.     

Challenges of the utilization of wood polymers: how can they be overcome?

Diminishing fossil fuel resources as well as growing environmental and energy security concerns, in parallel with growing demands on raw materials and energy, have intensified global efforts to utilize wood biopolymers as a renewable resource to produce biofuels and biomaterials. Wood is one of the most abundant biopolymer composites on earth that can be converted into biofuels as well as used as a platform to produce bio-based materials. The major biopolymers in wood are cellulose, hemicelluloses, and lignin which account for >90% of dry weight. These polymers are generally associated with each other in wood cell walls resulting in an intricate and dynamic cell wall structure. This mini-review provides an overview of major wood biopolymers, their structure, and recent developments in their utilization to develop biofuels. Advances in genetic modifications to overcome the recalcitrance of woody biomass for biofuels are discussed and point to a promising future.     

Production and Mass Transfer Characteristics of Non-Newtonian Biopolymers for Biomedical Applications

ABSTRACT:?

The market for microbial biopolymers is currently expanding to include several emerging biomedical applications. Specifically, these applications are drug delivery and wound healing. A fundamental understanding of the key fermentation parameters is necessary in order to optimize the production of these biopolymers. Considering that most microbial biopolymer systems exhibit non-Newtonian rheology, oxygen mass transfer can be an important parameter to optimize and control. In this article, we present a critical review of recent advances in rheological and mass transfer characteristics of selected biopolymers of commercial interest in biomedical applications.     

A practical approach to optimization and validation of a HPLC assay for analysis of polyribosyl-ribitol phosphate in complex combination vaccines

The use of multi-factor statistical experimental design methodology minimized the vaccine material and laboratory resources required for optimization and validation of an HPLC assay for quantitation of depolymerized and total PRP. Components of the assay selected for optimization were adjuvant dissolution, ultracentrifuge conditions including ultracentrifuge model, sample diluent, mobile phase and column oven temperature. Previous experience has shown these components of the assay to be most troublesome and therefore required optimization prior to validation. Specificity, linearity, precision, accuracy and ruggedness were confirmed through a validation of the optimized assay. The validation also established the assay to be stability indicating, by showing that changes to the integrity of the PRP-OMPC conjugate could be detected.     

Studies on polydisperse systems using an air-driven ultracentrifuge: application to phosphatidylcholine vesicles.

A high-speed air-driven ultracentrifuge (Airfuge) has been used to determine the molecular weight and effective specific volume of phosphatidylcholine vesicles. The method used to determine the effective specific volume involved varying the solution density until zero sedimentation of the vesicles occurred. The value obtained for the effective specific volume of 0.9885 ml/g agrees well with previously reported values. The determination of the molecular weight of the vesicles is based on a method in which the fraction of vesicles remaining in an upper fraction of the solution column is compared with the values obtained using standard proteins. The values obtained for the molecular weight of the vesicles range from 1.7 X 10(6) to 2.3 X 10(6) and are in good agreement with results obtained using the analytical ultracentrifuge and with previously reported results. Possible effects due to the polydispersity of the solute are assessed using theoretical calculations and the possibility of using the Airfuge for the study of other polydisperse systems is discussed.     

Effects of non-linearity on cell–ECM interactions

Filamentous biopolymers such as F-actin, vimentin, fibrin and collagen that form networks within the cytoskeleton or the extracellular matrix have unusual rheological properties not present in most synthetic soft materials that are used as cell substrates or scaffolds for tissue engineering. Gels formed by purified filamentous biopolymers are often strain stiffening, with an elastic modulus that can increase an order of magnitude at moderate strains that are relevant to cell and tissue deformation in vivo. This review summarizes some experimental studies of non-linear rheology in biopolymer gels, discusses possible molecular mechanisms that account for strain stiffening, and explores the possible relevance of non-linear rheology to the interactions between cell and extracellular matrices.     

An optical pulse modulation system for laser interference studies in the analytical ultracentrifuge

The mode of operation and construction of a high-speed laser light modulator is presented that is designed for use with the analytical ultracentrifuge. The system may be used with continuous wave lasers having optical powers not exceeding 100 W and has an optical bandwidth from 200 nm to 800 nm. The electronic modulation circuitry described is capable of producing optical pulses of 0.8 μsec duration at a firing frequency in excess of 100,000 pps and is designed to permit the laser source to operate in either a pulsed or nonpulsed mode. The unit is extremely versatile, and its characteristics permit full advantage to be taken of pulsed laser interferometry in the ultracentrifuge.     

Can Nanotechnology Improve the Sustainability of Biobased Products?

Recent developments in nanotechnology, especially in the area of nanoclay composites, are improving the technical performance of biobased polymers and moving them toward technical and economic competitiveness with petroleum‐based polymers and conventional composites. We assess whether these developments also improve the environmental sustainability of biopolymers, by using a life cycle approach. We estimate energy use and emissions from the nanoclay production process and compare these with prior life cycle data for biopolymers as well as other fibers, and we find that nanoclay production results in lower energy use and greenhouse gas emissions than production of many common biopolymers and glass fibers. Nanoclay composites hence can improve the life cycle environmental performance of several common biopolymers. However, for some biopolymers the relative performance depends on the functional unit.     

Protein Diffusion on Charged Biopolymers: DNA versus Microtubule

Protein diffusion in lower-dimensional spaces is used for various cellular functions. For example, sliding on DNA is essential for proteins searching for their target sites, and protein diffusion on microtubules is important for proper cell division and neuronal development. On the one hand, these linear diffusion processes are mediated by long-range electrostatic interactions between positively charged proteins and negatively charged biopolymers and have similar characteristic diffusion coefficients. On the other hand, DNA and microtubules have different structural properties. Here, using computational approaches, we studied the mechanism of protein diffusion along DNA and microtubules by exploring the diffusion of both protein types on both biopolymers. We found that DNA-binding and microtubule-binding proteins can diffuse on each other’s substrates; however, the adopted diffusion mechanism depends on the molecular properties of the diffusing proteins and the biopolymers. On the protein side, only DNA-binding proteins can perform rotation-coupled diffusion along DNA, with this being due to their higher net charge and its spatial organization at the DNA recognition helix. By contrast, the lower net charge on microtubule-binding proteins enables them to diffuse more quickly than DNA-binding proteins on both biopolymers. On the biopolymer side, microtubules possess intrinsically disordered, negatively charged C-terminal tails that interact with microtubule-binding proteins, thus supporting their diffusion. Thus, although both DNA-binding and microtubule-binding proteins can diffuse on the negatively charged biopolymers, the unique molecular features of the biopolymers and of their natural substrates are essential for function.