A simple method for the large‐scale preparation of sucrose gradients

Baxter-Gabbard, K. L.

doi:10.1016/0014-5793(72)80031-0

Cited by 99 publications

(34 citation statements)

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“…Samples ‫002ف(‬ L) were loaded directly onto 4.8 mL of 5 to 25% sucrose gradient prepared by three freezing/thawing cycles (Baxter-Gabbard, 1972). Sucrose was dissolved in HEEM buffer containing 1 mM PMSF and 2 mM DTT.…”

Section: Velocity Sedimentationmentioning

confidence: 99%

Identification and Characterization of a Novel Microtubule-Based Motor Associated with Membranous Organelles in Tobacco Pollen Tubes

et al. 2000

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Pollen tube growth depends on the differential distribution of organelles and vesicles along the tube. The role of microtubules in organelle movement is uncertain, mainly because information at the molecular level is limited. In an effort to understand the molecular basis of microtubule-based movement, we isolated from tobacco pollen tubes polypeptides that cosediment with microtubules in an ATP-dependent manner. Major polypeptides released from microtubules by ATP (ATP-MAPs) had molecular masses of 90, 80, and 41 kD. Several findings indicate that the 90-kD ATP-MAP is a kinesin-related motor: binding of the polypeptide to microtubules was enhanced by the nonhydrolyzable ATP analog AMP-PNP; the 90-kD polypeptide reacted specifically with a peptide antibody directed against a highly conserved region in the motor domain of the kinesin superfamily; purified 90-kD ATP-MAP induced microtubules to glide in motility assays in vitro; and the 90-kD ATP-MAP cofractionated with microtubule-activated ATPase activity. Immunolocalization studies indicated that the 90-kD ATP-MAP binds to organelles associated with microtubules in the cortical region of the pollen tube. These findings suggest that the 90-kD ATP-MAP is a kinesin-related microtubule motor that moves organelles in the cortex of growing pollen tubes. INTRODUCTIONMicrotubule motor proteins are an important class of microtubule-associated proteins that transport specific cargo structures in a process driven by ATP hydrolysis. Microtubulebased motors are classified into two main superfamilies: kinesin, which comprises conventional kinesin and kinesinlike proteins (KLPs), and dynein; both superfamilies include several members that play important roles in such cellular mechanisms as organelle transport and mitosis. Motor proteins transport proteins, lipids, and other cell components to different parts of the cell at suitable velocities in membranous organelles. Intracellular transport is therefore essential for cellular morphogenesis and function . Microtubule-based motor proteins share some biochemical and functional features, such as nucleotide-dependent microtubule binding, microtubule-activated ATPase activity, and motor-driven microtubule translocation (Hirokawa, 1998). Although microtubule-based motor proteins are characterized primarily in animal cells, a large kinesin family has also been characterized in plant cells (Asada and Collings, 1997).The biochemical and structural properties of KLPs in plant cells are similar to those in animal cells. They have microtubule-stimulated ATPase activity (Mitsui et al., 1994), promote gliding of microtubules in motility assays in vitro (Song et al., 1997), and bind to microtubules in a nucleotidedependent manner (Mitsui et al., 1996). Although most KLPs identified in plants are probably involved in cell division (Asada and Shibaoka, 1994;Liu et al., 1996;Mitsui et al., 1996;, genetic analysis of Arabidopsis mutants having structural alterations of trichomes suggests that KLPs also take part in cell morphogenesis (Oppenheime...

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Section: Velocity Sedimentationmentioning

confidence: 99%

Identification and Characterization of a Novel Microtubule-Based Motor Associated with Membranous Organelles in Tobacco Pollen Tubes

et al. 2000

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“…Acridine-Posintro™ particles were ultracentrifuged (UC) in a continuous sucrose gradient prepared by the freezethaw method described previously (18). Briefly, 4.5 ml 25% (w/v) sucrose solution was placed in an ultraclear centrifuge tube (Beckman Coulter, CA, USA) and frozen at −20°C.…”

Section: Preparation and Characterization Of Posintro™ Nanoparticlesmentioning

confidence: 99%

In Vitro Cutaneous Application of ISCOMs on Human Skin Enhances Delivery of Hydrophobic Model Compounds Through the Stratum Corneum

Madsen

Ifversen

Madsen

et al. 2009

AAPS J

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Abstract. This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOMs) on human skin penetration of model compounds in vitro to evaluate their potential as a delivery system, ultimately for transcutaneous vaccination. Special focus was on elucidating the mechanisms of penetration. Preparation of ISCOMs was done by dialysis and subsequent purification in a sucrose density gradient. The penetration pathways of acridine-labeled ISCOMs were visualized using confocal laser scanning microscopy (CLSM). Transmission electron microscopy (TEM) was used to evaluate the ultrastructural changes in the skin after application of the ISCOMs with or without hydration. Transcutaneous permeation of the model compound, methyl nicotinate, was evaluated in diffusion cells. The prepared ISCOMs were 42-52 nm in diameter as evaluated by dynamic light scattering with zeta potentials of −33 to −26.1 mV. TEM investigations verified the presence of ISCOM structures. Penetration of acridine into skin was greatly increased by incorporation into ISCOMs as visualized by CLSM. Permeation of methyl nicotinate was enhanced in the presence of ISCOMs. Ultrastructural changes of the intercellular space in the stratum corneum after exposure of ISCOMs were observed on micrographs, especially for hydrated skin. In conclusion, cutaneous application of ISCOMs leads to increased penetration of hydrophobic model compounds through human stratum corneum and thus shows potential as a transcutaneous delivery system. The increased penetration seems to be reflected by a change in the intercellular space between the corneocytes, and the effect is most likely caused by the components of the ISCOMs rather than intact ISCOMs.

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“…Genomic DNA of F. necrophorum A25 was digested partially with restriction endonuclease Sau3AI, and the fragments were size-fractionated in a sucrose gradient (3). Ten-to 12-kb fragments were cloned into BamHI-digested and alkaline phosphatase-treated Lambda Zap Express vector (Stratagene Corp., La Jolla, Calif.) in accordance with the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Cloning, Sequencing, and Expression of the Leukotoxin Gene from Fusobacterium necrophorum

et al. 2001

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Fusobacterium necrophorum is a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF; lktA) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the whole lktA ORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin from F. necrophorum culture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed in Escherichia coli. Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against the F. necrophorum leukotoxin. Southern blot hybridization analysis revealed different patterns of lktA hybridizing bands between isolates of the two subspecies of F. necrophorum.

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A simple method for the large‐scale preparation of sucrose gradients

Cited by 99 publications

References 1 publication

Identification and Characterization of a Novel Microtubule-Based Motor Associated with Membranous Organelles in Tobacco Pollen Tubes

Identification and Characterization of a Novel Microtubule-Based Motor Associated with Membranous Organelles in Tobacco Pollen Tubes

In Vitro Cutaneous Application of ISCOMs on Human Skin Enhances Delivery of Hydrophobic Model Compounds Through the Stratum Corneum

Cloning, Sequencing, and Expression of the Leukotoxin Gene from Fusobacterium necrophorum

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