PubMed Health⌕ Search

Biomedical subjects

F Gaskin

Publications and source records attributed to F Gaskin.

At least 37 records · Page 2Linked to original sources
PubMed1986

Magnesium requirements for guanosine 5'-O-(3-thiotriphosphate) induced assembly of microtubule protein and tubulin.

Two conflicting interpretations on the role of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in microtubule protein and tubulin assembly have been previously reported. One study finds that GTP gamma S promotes assembly while another study reports that GTP gamma S is a potent inhibitor of microtubule assembly. We have examined the potential role of Mg2+ to learn if the conflicting interpretations are due to a metal effect. Turbidity, electron microscopy, and nucleotide binding and hydrolysis were used to analyze the effect of the Mg2+ concentration on GTP gamma S-induced assembly of microtubule protein (tubulin + microtubule-associated proteins) in the presence of buffer +/- 30% glycerol and in buffer with GTP added before or after GTP gamma S. GTP gamma S substantially lowers the Mg2+ concentration required to induce cross-linked or clustered rings of tubulin. These cross-linked rings do not assemble well into microtubules, and GTP only partially restores microtubule assembly. However, taxol will promote GTP gamma S-induced cross-linked rings of microtubule protein to assemble into microtubules. The effect of GTP gamma S on microtubule protein assembly in the presence of Zn2+ with and without added Mg2+ suggests that GTP gamma S also effects the formation of Zn2+-induced sheet aggregates. Purified tubulin was used in assembly experiments with Mg2+, Zn2+, and taxol to better understand GTP gamma S interactions with tubulin. The optimal Mg2+ concentration for assembly of tubulin is lower with GTP gamma S than with GTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Separation of assembly-competent tubulin from brain microtubule protein preparations using a fast-performance liquid chromatography procedure.

Fast-performance liquid chromatography was used to purify assembly-competent tubulin from porcine brain microtubule protein prepared by two cycles of assembly-disassembly. Microtubule protein (1-100 mg at 1.5-2.5 mg/ml) in buffer consisting of 0.1 M 2-(N-morpholino)ethanesulfonic acid, 0.5 mM MgCl2, 1 mM EGTA, 0.3 M KCl, and 0.02 mM GTP (pH 6.6) was applied to the Mono Q column (anion exchanger). The microtubule-associated proteins, GTP and GDP, eluted in the void volume. The tubulin fraction eluted at 0.45-0.50 M KCl with 65-80% recovery. The tubulin fraction contained trace enzymatic activities when compared with the starting microtubule protein, i.e., less than 1 versus 60 mU/mg/min of nucleoside diphosphate kinase, 0.2 versus 7.0 nmol/mg/min of Mg-ATPase at pH 6.6, and 0.2 versus 88 mU/mg/min of adenylate kinase. Both the Mono Q-purified tubulin and the pelleted microtubules that were assembled in 0.5 mM [3H]GTP contained 0.77 mol of labeled nucleotide/tubulin dimer. The Mono Q-purified tubulin fraction was competent to assemble, i.e., the critical concentration was 0.1 mg/ml in the presence of 0.03 mM taxol and 1 mM GTP at 37 degrees C. The Mono Q-purified tubulin fraction showed trace high-molecular-weight components, which were removed on Mono S (cation exchanger) columns. Alternatively, microtubule protein in buffer was applied to the Mono S column. Tubulin, trace nontubulin proteins, and several enzymatic activities came off in the void volume. A combination of Mono Q-Mono S or Mono S-Mono Q chromatography resulted in highly purified protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Neurofibrillary tangles in senile dementia of the Alzheimer type share an antigenic determinant with intermediate filaments of the vimentin class.

A monoclonal antibody produced by a hybridoma between a plasmacytoma cell and a spleen cell from a mouse immunized with human brain microtubule fraction was demonstrated to stain neurofibrillary tangles of senile dementia of the Alzheimer type (SDAT). The antibody recognized at least 50% of the tangles in neuronal perikarya isolated from SDAT brains and stained a filamentous network in HeLa cells, fibroblasts, and astrocytes. It did not stain skin epithelial cells or neurons isolated from normal brains but reacted with Z bands in skeletal muscle. The monoclonal antibody stained coils in colchicine or colcemid-treated cultured cells in a pattern characteristic of 10-nm intermediate-sized filaments. Immunoblotting of Triton-insoluble cytoskeletal proteins of cultured cells electrophoresed in SDS polyacrylamide gels showed that the antigenic determinant is present in proteins of molecular weight 58,000 which comigrates with vimentin. Thus, it appears that the neurofibrillary tangles in SDAT share an antigenic determinant with vimentin.

Aged↗
PubMed1982

Further studies on the inflammatory response induced by zinc wire implants in the central nervous system of rats.

Implantation of zinc wires into the central nervous system of adult Lewis rats initiates an inflammatory reaction with a predominantly mononuclear cell profile. Extensive cuffs of small lymphocytes, mature plasma cells, macrophages, and histiocytes are found around the site of the wire implant and around adjacent blood vessels. The inflammatory response persists for at least 35 weeks but, with time , is gradually replaced by extensive fibrosis, collagen deposition, and proliferation of glial cells in the adjacent neuropil. The Lesion is not necrotic, nor is it characteristic of a foreign body granuloma, since neither giant cells nor organized epithelioid cells are found. No obvious clinical effect is observed. The fact that the implantation of zinc wire initiates an inflammatory response which resembles certain immune-mediated reactions is consistent with the reports of others that zinc affects immunological responses both in vivo and in vitro. Other metals (Be, Co, Mg, Pt, and Ni), implanted to control for the effect of zinc, initiated responses that were both distinct for the metal used and reproducible, and thus underscore the significance of studying the role of metals in both acute and chronic diseases.

Animals↗
PubMed1982

Nickel-induced changes and reappraisal of Rosenthal fibers in focal CNS lesions.

Nickel (Ni) wire implants into the CNS of Lewis rats induce the formation of structures morphologically similar to Rosenthal fibers (RF) seen in human conditions. We describe in detail the time courses of Ni-induced changes in rat astrocytes and compare the Ni-induced RF with RF occurring in focal lesions. Immunocytochemistry at the level of electron microscopy suggests that the Ni-induced RF are largely made up of plasma proteins. We suggest that the mechanism of RF formation and possibly the protein composition of RF depends on the condition in which they are found.

Animals↗
PubMed1981

Effects of zinc on the cytoskeletal proteins in the central nervous system of the rat.

To test for in vivo zinc neurotoxicity on the cytoskeleton of neurotubules and intermediate filaments, Zn wires were implanted into the brains of adult Lewis rats for periods of 1-35 weeks. After 16 weeks of implant, some neurons showed bundles of intermediate filaments which were often localized in the perinuclear area. At the same time, occasional 200 nm tubular-like structures were seen in swollen dendrites. These structures were morphologically similar to Zn ion-induced aggregates of pure tubulin and structures found in ZnSO4-treated dorsal root ganglion organotypic cultures. The 200 nm structures in dendrites and the intermediate filaments in neurons increased in frequency with time. After 35 weeks of Zn wire implant, few microtubules could be found in the lesion. All the animals showed an astrocyte and glial filament proliferation with axoglial membrane specialization. Other wires studied: Pt, Ni, Co, Mg, demonstrate that Zn wires have a specific effect on cytoskeletal proteins in the CNS of the rat and many of the effects can be explained by an interaction between Zn and tubulin.

Animals↗
PubMed1981

Nickel induction of Rosenthal fibers in rat brain.

Nickel wire implants into the central nervous system of Lewis rats induces the formation of structures morphologically similar to Rosenthal fibers. The nickel-induced lesions are similar to those seen in Alexander's disease and other human conditions in which Rosenthal fibers are found. This is the first report on the induction of Rosenthal fibers in animals. It might be used to design animal models to study Rosenthal fiber formation and possibly Alexander's disease.

Animals↗
PubMed1981

In vitro microtubule assembly regulation by divalent cations and nucleotides.

Mg2+ and guanosine 5'-triphosphate (GTP) are usually used to assemble microtubule protein (tubulin + microtubule-associated proteins) or tubulin into microtubules in vitro. Recently, it has been shown that Mn2+ will substitute for Mg2+ in inducing pure tubulin + GTP to assemble into microtubules. We find that Mn2+ also substitute for Mg2+ in inducing two-cycle calf microtubule protein (MTP) + GTP to assemble into microtubules. Zn2+ or Co2+ induces MTP + GTP to form sheets with more than 13 protofilaments. We find that Co2+ also substitutes for Zn2+ in inducing tubulin to form 200-nm tubules similar to those reported with Zn2+. To learn whether metal-induced assembly is due to direct binding and/or a metal-GTP complex, metal-induced assembly of MTP and tubulin was studied in the presence of CrIIIGTP or taxol, two probes which promote assembly in the absence of added GTP. With CrGTP, Mg2+ and ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) were required for optimal assembly into microtubules, and Mn2+ could substitute for Mg2+. MTP incubated with Zn2+ and CrGTP assembled into sheets, shorter than but similar to those induced by Zn2+ + GTP. Mg2+-induced microtubules and Zn2+-induced sheets contained 0.45 mol of [8-3H]GTP/mol of tubulin if assembled from [8-3H]GTP and a mixture of 0.25 mol of [8-3H]CrGTP, 0.1 mol of [8-3H]GDP, and 0.05 mol of [8-3H]CrGDP/mol of tubulin if assembled from [8-3H]CrGTP. Zn2+ induced taxol-treated MTP to form sheets. Sheets were also induced from tubulin + Zn2+ and either CrGTP or taxol. These studies suggest that the Zn2+-induced structures are not due to a Zn-GTP complex and that Mg2+ does not promote assembly only through a Mg-GTP complex.

Animals↗
PubMed1981

Immunocytochemical studies of neurofibrillary tangles.

The molecular nature of neurofibrillary tangles of senile dementia of the Alzheimer type (SDAT) was studied by immunoperoxidase and immunofluorescence techniques. Five antiserums, including anti-humanbrain-2-cycle-purified-microtubule-fractions (2 x MT), anti-calf-brain-2 x MT, anti-sea-urchin-egg-tubulin, antibeef-brain-tubulin, and anti-human-brain-neurofilament(NF)-210-kilodalton(kd)-protein were tested for their binding to neurofibrillary tangles. The antihuman-2 x MT serum stained structures resembling neurofibrillary tangles, neurites of neuritic plaques, and microglialike cells in SDAT brains, but no such staining pattern was detected in normal brain sections. In neurons isolated from SDAT brains, about 40% of the tangles were labeled by the anti-human-2xMT serum with an identical pattern. Other antiserums tested did not preferentially bind tanglelike structures in tissue sections and bound to less than 5% of the tangles in isolated neurons. These results suggest that the antigenic sites of tubulin and NF proteins are not shared by neurofibrillary tangles. Different from the calf preparation, the human-2 x MT fractions contained a prominent protein band that was identical to ferritin in molecular weight and cross-reacted with anti-human-2 x MT and anti-human-ferritin serums. However, antiserums to this ferritinlike protein, or anti-ferritin, did not stain neurofibrillary tangles. Although neither the calf 2 x MT nor two other human MT fractions failed to elicit an antiserum that stained tangles, these fractions were able to remove the antihuman-2 x MT serum activity that binds to tangles. The data suggest that the protein (or proteins) that makes up neurofibrillary tangles of SDAT is present in various quantities in microtubule fractions of normal brain.

Alzheimer Disease↗
PubMed1978

Dynamic light scattering from solutions of microtubules.

Calf brain microtubule protein was assembled in vitro to form dilute solutions of microtubules (240 A diameter) having lengths greater than 1 micrometer. The microtubule solutions were examined by dynamic laser light scattering techniques. The angular dependence of the correlation function leads to the conclusion that the correlation function is measuring the translational diffusion constant of the particles. The length dependence of the correlation function, however, shows that the translational diffusion constant is not being measured and that the diffusion constant for the microtubules cannot be straightforwardly determined. These results suggest that a collective property of the rods is being measured by the laser light scattering. Although specific microtubule-microtubule interactions are a possible explanation for the observed results, we present arguments that suggest that the solution can be adequately modeled as a network of entangled polymers.

Animals↗
PubMed1977

Characterization of heterogeneous solutions using laser light scattering: study of the tubulin system.

Laser light scattering has been applied to a systematic study of a heterogeneous solution of tubulin at low temperature--conditions under which tubulin assembly into microtubules does not take place. Methods of analyzing laser light scattering results obtained from solutions containing multiple components are discussed. Data analysis techniques are described and their application to the determination of diffusion constants from experimental data is extensively illustrated. Multiple components were found under the conditions that the tubulin was studied. We have identified one component having D20,w = 4.41 X 10(-7) cm2/s (sigma = 0.54 X 10(-7) cm2/s) which has the expected value for tubulin dimer. In addition, we have found two components which are significantly larger than tubulin. One large component has D 20,w approximately 0.55 X 10(-7) cm2/s and is present in all samples at 4 degrees C even after centrifugation to remove components greater than 10 S. Another large component having 3.2 X 10(-7) cm2/s greater than or equal to D20,w greater than or equal to 1.5 X 10(-7) cm2/s has been found to sediment with 10 S less than or equal to s less than 20 S.

Animals↗
PubMed1977

Equilibrium components of tubulin preparations.

Laser light scattering techniques are used to examine the existence of a tubulin-ring or tubulin-aggregate equilibrium. Three principal components in addition to tubulin dimer have been observed. One large component having D20,w = 0.65 +/- 0.25 X 10(-7) cm2/s is in equilibrium with tubulin (dimer) at 4 degrees C. A second large component having D20,w = 0.74 +/- 0.05 X 10(-7) cm2/s is metastable at 4 degrees C and is formed when tubulin solutions are warmed to 36 degrees C and cooled back to 4 degrees C. The properties of the component reported in the previous paper (Gethner, J.S., Flynn, G.W., Berne, B.J., and Gaskin, F. (1977), Biochemistry 16 (preceding paper in this issue)) to sediment at 10 S to 20 S are shown to be consistent with a small, metastable aggregate of tubulin.

Animals↗
PubMed1977

Zinc ion-induced assembly of tubulin.

Zinc ion-induced assembly of tubulin was followed using electron microscopy and turbidimetric measurements. A scheme utilizing repeated cycles of assembly and disassembly was used to prepare tubulin and microtubule-associated proteins (MAPs) (Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 765-768). Tubulin was further purified by phosphocellulose chromatography to remove the MAPs (Weingarten, M., Lockwood, A. H., Hwo, S-Y, and Kirschner, M. W. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1858-1862). In tubulin preparations containing MAPs and added GTP, Zn2+-induced sheets of 15 to 60 protofilaments oriented in parallel. In the absence of MAPs and/or added GTP, Zn2+ induced the formation of sheets which wrapped quite specifically and serial sections were often consistent with a tubular structure of approximately 220 nm. The assembly of recycled tubulin + GTP and 0 to 1 mM Zn2+ was analyzed by A350 as a function of time at 30 degrees. The greater the concentration of Zn2+, the shorter the lag time, the faster the rate after the lag, and the greater the plateau value of A350. Although turbidimetric measurements can be used to quantitate microtubules, they are not quantitative for Zn2+-induced sheets.

Animals↗
PubMed1976

Uterine tubulin production during early pregnancy in the rabbit.

Rabbit uterine tubulin levels rise rapidly in the preimplantation period to peak values of about 10-fold in the endometrium and about 4-5 fold in the myometrium on days 3-5 of pregnancy. Endometrial tubulin falls to non-pregnancy levels by mid-pregnancy while myometrial tubulin declines much more slowly. The rise in tubulin exceeds the early rate of increase in total uterine protein and DNA. Following implantation, tubulin content of the decidual basal plate area increases rapidly to peak about day 12 then declines. Production of uterine tubulin appears capable of a major, short term response to gonadal hormone stimulation.

Animals↗