Search PubMed⌕ Search

Biomedical subjects

P Todd

Publications and source records attributed to P Todd.

At least 19 recordsLinked to original sources
PubMed2001

Acoustic field assisted enhanced demixing of aqueous two-phase systems.

Aqueous two-phase extraction has been recognized as a versatile downstream processing technique for the recovery of biomolecules. A major deterrent to its industrial exploitation is the slow demixing of the two aqueous phases after extraction, due to their similar physical properties. A method to decrease the demixing times of these systems, employing a travelling acoustic wave field, is reported. The effects of phase composition and microbial cells on demixing in a polyethylene glycol/potassium phosphate two-phase system are studied in detail. As phase composition increased, demixing time decreased gradually. Phase volume ratio was found to have a significant effect on demixing time at low phase compositions. However, at intermediate and high phase compositions, only a small effect on demixing time was observed. The effect of phase composition and volume ratio on demixing behavior was explained based on the droplet size of the dispersed phase, which is the resultant effect of the physical properties of the phases. At all the phase compositions studied, the acoustically assisted process decreased the demixing time by 17-60% when compared to demixing under gravity alone. Increasing the cell concentration increased the demixing time markedly in case of yeast cells. However, it remained practically constant in the case of Lactobacillus casei cells. Application of an acoustic field reduced the demixing times up to 60% and 40% in the case of yeast and L. casei cells, respectively. Visual observations indicated that ultrasonication caused mild circulation currents in the phase dispersion enhancing droplet-droplet interaction, which in turn enhanced the rate of coalescence, eventually resulting in an enhanced demixing rate.

Countercurrent Distribution↗
PubMed2001

The NC1 domain of collagen IV encodes a novel network composed of the alpha 1, alpha 2, alpha 5, and alpha 6 chains in smooth muscle basement membranes.

Type IV collagen, the major component of basement membranes (BMs), is a family of six homologous chains (alpha1-alpha6) that have a tissue-specific distribution. The chains assemble into supramolecular networks that differ in the chain composition. In this study, a novel network was identified and characterized in the smooth muscle BMs of aorta and bladder. The noncollagenous (NC1) hexamers solubilized by collagenase digestion were fractionated by affinity chromatography using monoclonal antibodies against the alpha5 and alpha6 NC1 domains and then characterized by two-dimensional gel electrophoresis and Western blotting. Both BMs were found to contain a novel alpha1.alpha2.alpha5.alpha6 network besides the classical alpha1.alpha2 network. The alpha1.alpha2.alpha5.alpha6 network represents a new arrangement in which a protomer (triple-helical isoform) containing the alpha5 and alpha6 chains is linked through NC1-NC1 interactions to an adjoining protomer composed of the alpha1 and alpha2 chains. Re-association studies revealed that the NC1 domains contain recognition sequences sufficient to encode the assembly of both networks. These findings, together with previous ones, indicate that the six chains of type IV collagen are distributed in three major networks (alpha1.alpha2, alpha3.alpha4.alpha5, and alpha1.alpha2.alpha5.alpha6) whose chain composition is encoded by the NC1 domains. The existence of the alpha1.alpha2.alpha5.alpha6 network provides a molecular explanation for the concomitant loss of alpha5 and alpha6 chains from the BMs of patients with X-linked Alport's syndrome.

Animals↗
PubMed2001

Amyloplast sedimentation dynamics in maize columella cells support a new model for the gravity-sensing apparatus of roots.

Quantitative analysis of statolith sedimentation behavior was accomplished using videomicroscopy of living columella cells of corn (Zea mays) roots, which displayed no systematic cytoplasmic streaming. Following 90 degrees rotation of the root, the statoliths moved downward along the distal wall and then spread out along the bottom with an average velocity of 1.7 microm min(-1). When statolith trajectories traversed the complete width or length of the cell, they initially moved horizontally toward channel-initiation sites and then moved vertically through the channels to the lower side of the reoriented cell where they again dispersed. These statoliths exhibited a significantly lower average velocity than those sedimenting on distal-to-side trajectories. In addition, although statoliths undergoing distal-to-side sedimentation began at their highest velocity and slowed monotonically as they approached the lower cell membrane, statoliths crossing the cell's central region remained slow initially and accelerated to maximum speed once they reached a channel. The statoliths accelerated sooner, and the channeling effect was less pronounced in roots treated with cytochalasin D. Parallel ultrastructural studies of high-pressure frozen-freeze-substituted columella cells suggest that the low-resistance statolith pathway in the cell periphery corresponds to the sharp interface between the endoplasmic reticulum (ER)-rich cortical and the ER-devoid central region of these cells. The central region is also shown to contain an actin-based cytoskeletal network in which the individual, straight, actin-like filaments are randomly distributed. To explain these findings as well as the results of physical simulation experiments, we have formulated a new, tensegrity-based model of gravity sensing in columella cells. This model envisages the cytoplasm as pervaded by an actin-based cytoskeletal network that is denser in the ER-devoid central region than in the ER-rich cell cortex and is linked to stretch receptors in the plasma membrane. Sedimenting statoliths are postulated to produce a directional signal by locally disrupting the network and thereby altering the balance of forces acting on the receptors in different plasma membrane regions.

Actins↗
PubMed2000

The goodpasture autoantigen. Identification of multiple cryptic epitopes on the NC1 domain of the alpha3(IV) collagen chain.

Goodpasture (GP) disease is an autoimmune disorder in which autoantibodies against the alpha3(IV) chain of type IV collagen bind to the glomerular and alveolar basement membranes, causing progressive glomerulonephritis and pulmonary hemorrhage. Two major conformational epitope regions have been identified on the noncollagenous domain of type IV collagen (NC1 domain) of the alpha3(IV) chain as residues 17-31 (E(A)) and 127-141 (E(B)) (Netzer, K.-O. et al. (1999) J. Biol. Chem. 274, 11267-11274). To determine whether these regions are two distinct epitopes or form a single epitope, three GP sera were fractionated by affinity chromatography on immobilized NC1 chimeras containing the E(A) and/or the E(B) region. Four subpopulations of GP antibodies with distinct epitope specificity for the alpha3(IV)NC1 domain were thus separated and characterized. They were designated GP(A), GP(B), GP(AB), and GP(X), to reflect their reactivity with E(A) only, E(B) only, both regions, and neither, respectively. Hence, regions E(A) and E(B) encompass critical amino acids that constitute three distinct epitopes for GP(A), GP(B), and GP(AB) antibodies, respectively, whereas the epitope for GP(X) antibodies is located in a different unknown region. The GP(A) antibodies were consistently immunodominant, accounting for 60-65% of the total immunoreactivity to alpha3(IV)NC1; thus, they probably play a major role in pathogenesis. Regions E(A) and E(B) are held in close proximity because they jointly form the epitope for Mab3, a monoclonal antibody that competes for binding with GP autoantibodies. All GP epitopes are sequestered in the hexamer configuration of the NC1 domain found in tissues and are inaccessible for antibody binding unless dissociation of the hexamer occurs, suggesting a possible mechanism for etiology of GP disease. GP antibodies have the capacity to extract alpha3(IV)NC1 monomers, but not dimers, from native human glomerular basement membrane hexamers, a property that may be of fundamental importance for the pathogenesis of the disease.

Anti-Glomerular Basement Membrane Disease↗
PubMed2000

Engineering CHO cells to overexpress a secreted reporter protein upon induction from mouse mammary tumor virus promoter.

The mouse mammary tumor virus (MMTV) promoter is induced by the addition of a glucocorticoid hormone or analog such as dexamethasone. The hormone binds to its specific transcription factor, glucocorticoid receptor (GR), and the activated complex then binds to the glucocorticoid response elements (GREs) in the enhancer region of the MMTV promoter to induce the overexpression of downstream genes. We have constructed an expression vector for a reporter protein, secreted alkaline phosphatase (SEAP),controlled by the MMTV promoter and co-transfected this vector along with a GR expression cassette into Chinese hamster ovary (CHO) cells. High producers were cloned and grown in suspension cultures. A very high titer, over 0.4 mg/mL, of SEAP was obtained from this inducible overexpression system, about ten times that achievable with the same reporter protein using the strong constitutive SV40 promoter in CHO cells. A peak production rate of 187 pg SEAP per cell per day was observed within 3 days after induction, compared to the peak rate of 23 pg SEAP per cell per day expressed using the constitutive SV40 promoter. With the reduced or zero growth rate during the protein production phase, this novel MMTV overexpression system is highly suited for optimizing glycoprotein synthesis rates in high cell density fed-batch or perfusion bioreactors.

Alkaline Phosphatase↗
PubMed2000

Multistage electrophoresis system for the separation of cells, particles and solutes.

It is common to operate equilibrium-based separation methods, such as distillation and extraction, as multistage unit operations, in which equilibrium is presumably achieved within each stage. Two rate-based separation processes, free electrophoresis and magnetic particle separation, have now been operated in multistage mode. Preparative free electrophoresis of particles and solutes has resisted scale-up and is confined to a narrow range of ionic compositions. Natural convection induced in electrophoresis buffers by Ohmic heating has been a strong deterrent and has led to such measures as radial electrophoresis in Couette flow, free-flow electrophoresis, low-gravity electrophoresis, density gradient electrophoresis, and reorienting density gradient electrophoresis, to name a few. The short vertical electrophoresis path exploited in the last-mentioned forms the basis for multistage electrophoresis. A thin-layer countercurrent distribution apparatus was designed and constructed so that up to 20 fractions could be collected on the basis of electrophoretic mobility by applying an electric field. The mixture to be separated starts in a bottom cavity, and successive top cavities collect fractions as separand particles or molecules are electrophoresed upward out of the bottom cavity. Mathematical models of this process were developed, and experiments were performed to verify the predictions of the models by collecting and counting particles in each cavity after fractionation.

Animals↗
PubMed2000

Multistage magnetic and electrophoretic extraction of cells, particles and macromolecules.

Improved techniques for separating cells, particles, and macromolecules (proteins) are increasingly important to biotechnology because separation is frequently the limiting factor for many biological processes. Manufacturers of new enzymes and pharmaceutical products require improved methods for recovering intact cells and intracellular products. Similarly isolation, purification, and concentration of many biomolecules produced in fermentation processes is extremely important. Often such downstream processing contributes a large portion of the product cost. In conventional methods like centrifugation and even modern methods like chromatography, scale-up problems are enormous, making them uneconomical and prohibitively expensive unless the product is of very high value. Therefore there has been a need for efficient and economical alternative approaches to bioseparation processes to eliminate, reduce, or facilitate solids handling. Magnetic and electric field assisted separations may hold considerable potential for providing a future major improvement in bioseparation technology. In the present review the merits and demerits of the existing methods are discussed. We present mainly our own research on the development of unified multistage extraction processes that are versatile enough to handle cells and particles as well as macromolecules as described below. We describe multistage methods, namely ADSEP (Advanced Separator), MAGSEP (Magnetic Separator), and ELECSEP (Electrophoretic Separator), for quantitatively separating cells, particles, and solutes by using magnetically and electrophoretically assisted extraction processes. To the best of our knowledge, multistage magnetic and electrophoretic separations have not been reported in the earlier literature. The theoretical underpinnings of these separations are crucial to their success and to the identification of their advantages over other separation processes in particular applications. Hence mathematical modeling is stressed here, presenting our own models while also reviewing models reported in the literature. We also present suggestions for future work while analyzing the scale-up and economic aspects of these extraction processes. Commercial uses of the magnetic and electrophoretic processes, having both ground- and space-based research elements, also are presented in this review.

Biological Products↗
PubMed2000

Acoustic demixing of aqueous two-phase systems.

Aqueous two-phase systems demix slowly due to similar physical properties. This is one of the major drawbacks for their adaptation for industrial scale extraction of enzymes. In the present work, a method to accelerate the demixing rates of these systems, employing a traveling acoustic wave field is reported for the first time. Phase-demixing for three systems, viz. polyethylene glycol (PEG)/sodium sulfate, PEG/potassium phosphate and PEG/maltodextrin were studied. The acoustically assisted process decreased the demixing time significantly (about 2- to 3-fold in PEG/salt systems and about 2-fold in the PEG/maltodextrin system), compared to that in gravity alone. Ultrasonication apparently enhanced the coalescence of the dispersed phase droplets due to the mild circulation currents it caused in the dispersion. This in turn enhanced the rate of demixing due to the increased migration velocity of the larger droplets.

Acoustics↗
PubMed2000

Columella cells revisited: novel structures, novel properties, and a novel gravisensing model.

A hundred years of research has not produced a clear understanding of the mechanism that transduces the energy associated with the sedimentation of starch-filled amyloplast statoliths in root cap columella cells into a growth response. Most models postulate that the statoliths interact with microfilaments (MF) to transmit signals to the plasma membrane (or ER), or that sedimentation onto these organelles produces the signals. However, no direct evidence for statolith-MF links has been reported, and no asymmetric structures of columella cells have been identified that might explain how a root turned by 90 degrees knows which side is up. To address these and other questions, we have (1) quantitatively examined the effects of microgravity on the size, number, and spatial distribution of statoliths; (2) re-evaluated the ultrastructure of columella cells in high-pressure frozen/freeze-substituted roots; and (3) followed the sedimentation dynamics of statolith movements in reoriented root tips. The findings have led to the formulation of a new model for the gravity-sensing apparatus of roots, which envisages the cytoplasm pervaded by an actin-based cytoskeletal network. This network is denser in the ER-devoid central region of the cell than in the ER-rich cell cortex and is coupled to receptors in the plasma membrane. Statolith sedimentation is postulated to disrupt the network and its links to receptors in some regions of the cell cortex, while allowing them to reform in other regions and thereby produce a directional signal.

Endoplasmic Reticulum↗
PubMed1999

Combined effects of space flight factors and radiation on humans.

The probability that a dose of ionizing radiation kills a cell is about 10,000 times the probability that the cell will be transformed to malignancy. On the other hand, the number of cells killed required to significantly impact health is about 10,000 times the number that must be transformed to cause a late malignancy. If these two risks, cell killing and malignant transformation, are about equal, then the risk that occurs during a mission is more significant than the risk that occurs after a mission. The latent period for acute irradiation effects (cell killing) is about 2-4 weeks; the latent period for malignancy is 10-20 years. If these statements are approximately true, then the impact of cell killing on health in the low-gravity environment of space flight should be examined to establish an estimate of risk. The objective of this study is to synthesize data and conclusions from three areas of space biology and environmental health to arrive at rational risk assessment for radiations received by spacecraft crews: (1) the increased physiological demands of the space flight environment; (2) the effects of the space flight environment on physiological systems; and (3) the effects of radiation on physiological systems. One physiological system has been chosen: the immune response and its components, consisting of myeloid and lymphoid proliferative cell compartments. Best-case and worst-case scenarios are considered. In the worst case, a doubling of immune-function demand, accompanied by a halving of immune capacity, would reduce the endangering dose to a crew member to around 1 Gy.

Animals↗
PubMed1999

Migration of Polystyrene Latex Particle Bands in Density-Gradient Electrophoresis.

Electrophoretic migration of sulfated polystyrene latex particle bands was investigated in a density-gradient electrophoresis apparatus. The bands spread with time, and their velocity was found to depend on particle concentration and to decrease during the initial stages of electrophoresis. A change in pH during electrophoresis occurred, and this change may have caused variations in band velocity. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

The goodpasture autoantigen. Mapping the major conformational epitope(s) of alpha3(IV) collagen to residues 17-31 and 127-141 of the NC1 domain.

The Goodpasture (GP) autoantigen has been identified as the alpha3(IV) collagen chain, one of six homologous chains designated alpha1-alpha6 that comprise type IV collagen (Hudson, B. G., Reeders, S. T., and Tryggvason, K. (1993) J. Biol. Chem. 268, 26033-26036). In this study, chimeric proteins were used to map the location of the major conformational, disulfide bond-dependent GP autoepitope(s) that has been previously localized to the noncollagenous (NC1) domain of alpha3(IV) chain. Fourteen alpha1/alpha3 NC1 chimeras were constructed by substituting one or more short sequences of alpha3(IV)NC1 at the corresponding positions in the non-immunoreactive alpha1(IV)NC1 domain and expressed in mammalian cells for proper folding. The interaction between the chimeras and eight GP sera was assessed by both direct and inhibition enzyme-linked immunosorbent assay. Two chimeras, C2 containing residues 17-31 of alpha3(IV)NC1 and C6 containing residues 127-141 of alpha3(IV)NC1, bound autoantibodies, as did combination chimeras containing these regions. The epitope(s) that encompasses these sequences is immunodominant, showing strong reactivity with all GP sera and accounting for 50-90% of the autoantibody reactivity toward alpha3(IV)NC1. The conformational nature of the epitope(s) in the C2 and C6 chimeras was established by reduction of the disulfide bonds and by PEPSCAN analysis of overlapping 12-mer peptides derived from alpha1- and alpha3(IV)NC1 sequences. The amino acid sequences 17-31 and 127-141 in alpha3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.

Amino Acid Sequence↗
PubMed1999

Bacterial growth in space flight: logistic growth curve parameters for Escherichia coli and Bacillus subtilis.

Previous investigations have reported that bacterial suspension cultures grow to higher stationary concentrations in space flight than on Earth; however, none of these investigations included extensive ground controls under varied inertial conditions. This study includes extensive controls and cell-growth data taken at several times during lag phase, log phase, and stationary phase of Escherichia coli and Bacillus subtilis. The Marquardt-Levenberg, least-squares fitting algorithm was used to calculate kinetic growth parameters from the logistic bacterial growth equations for space-flight and control growth curves. Space-flight cultures grew to higher stationary-phase concentrations and had shorter lag-phase durations. Also, evidence was found for increased exponential growth rate in space.

Bacillus subtilis↗
PubMed1999

Autolysis of Escherichia coli and Bacillus subtilis cells in low gravity.

The role of gravity in the autolysis of Bacillus subtilis and Escherichia coli was studied by growing cells on Earth and in microgravity on Space Station Mir. Autolysis analysis was completed by examining the death phase or exponential decay of cells for approximately 4 months following the stationary phase. Consistent with published findings, the stationary-phase cell population was 170% and 90% higher in flight B. subtilis and E. coli cultures, respectively, than in ground cultures. Although both flight autolysis curves began at higher cell densities than control curves, the rate of autolysis in flight cultures was identical to that of their respective ground control rates.

Bacillus subtilis↗
PubMed1999

Early root cap development and graviresponse in white clover (Trifolium repens) grown in space and on a two-axis clinostat.

White clover (Trifolium repens) was germinated and grown in microgravity aboard the Space Shuttle (STS-60, 1994; STS-63, 1995), on Earth in stationary racks and in a slow-rotating two-axis clinostat. The objective of this study was to determine if normal root cap development and early plant gravity responses were dependent on gravitational cues. Seedlings were germinated in space and chemically fixed in orbit after 21, 40, and 72 h. Seedlings 96 h old were returned viable to earth. Germination and total seedling length were not dependent on gravity treatment. In space-flown seedlings, the number of cell stories in the root cap and the geometry of central columella cells did not differ from those of the Earth-grown seedlings. The root cap structure of clinorotated plants appeared similar to that of seedlings from microgravity, with the exception of three-day rotated plants, which displayed significant cellular damage in the columella region. Nuclear polarity did not depend on gravity; however, the positions of amyloplasts in the central columella cells were dependent on both the gravity treatment and the age of the seedlings. Seedlings from space, returned viable to earth, responded to horizontal stimulation as did 1 g controls, but seedlings rotated on the clinostat for the same duration had a reduced curvature response. This study demonstrates that initial root cap development is insensitive to either chronic clinorotation or microgravity. Soon after differentiation, however, clinorotation leads to loss of normal root cap structure and plant graviresponse while microgravity does not.

Fabaceae↗
PubMed1999

Particulates from PTFE degradation in terrestrial and microgravity.

It has recently been discovered that the ultrafine particles generated during polymer thermodegradation are a major health hazard, owing to their unique pathway of processing in the lung. This hazard in manned spacecraft is poorly understood because the particulate products of polymer thermodegradation are generated under low gravity conditions. Particulates generated from the degradation of polytetrafluoroethylene (PTFE), insulation coating for 20 AWG copper wire (representative of spacecraft application) under intense current overload, were studied in terrestrial gravity and microgravity. Microgravity tests were done in a 1.2-s drop tower at the Colorado School of Mines. Thermophoretic sampling was used for particulate collection. Transmission electron microscopy (TEM) was used to examine the smoke particulates. The pigmentation of PTFE insulation seems to have an overwhelming effect on size, shape and morphology of the particulate. Nanometer-sized particles were found in all cases, but their extent of aggregation and size distribution were dependent on both PTFE pigmentation and gravity; higher aggregation occurred in low gravity. Four different color insulations (viz. white, black, red and yellow) were studied.

Air Pollution, Indoor↗
PubMed1999

Effects of space flight and mixing on bacterial growth in low volume cultures.

Previous investigations have shown that liquid suspension bacterial cultures grow to higher cell concentrations in spaceflight than on Earth. None of these studies included ground-control experiments designed to evaluate the fluid effects potentially responsible for the reported increases. Therefore, the emphasis of this research was to both confirm differences in final cell concentration between 1g and microgravity cultures, and to examine the effects of mixing as a partial explanation for this difference. Flight experiments were performed in the Fluid Processing Apparatus (FPA), aboard Space Shuttle Missions STS-63 and STS-69, with simultaneous 1g static and agitated controls. Additional static 1g, agitated, and clino-rotated controls were performed in 9-ml culture tubes. This research revealed that both E. coli and B. subtilis samples cultured in space flight grew to higher final cell densities (120-345% increase) than simultaneous static 1g controls. The final cell concentration of E. coli cells cultured under agitation was 43% higher than in static 1g cultures and was 102% higher with clino-rotation. However, for B. subtilis cultures grown while being agitated on a shaker or clino-rotated, the final cell concentrations were nearly identical to those of the simultaneous static 1g controls. Therefore, these data suggest that the unique fluid quiescence in the microgravity environment (lack of sedimentation, creating unique transfer of nutrients and waste products), was responsible for the enhanced bacterial proliferation reported in this and other studies.

Bacillus subtilis↗