scholarly journals Dynamics of microtubule depolymerization in monocytes.

1986 ◽  
Vol 102 (6) ◽  
pp. 2023-2032 ◽  
Author(s):  
L U Cassimeris ◽  
P Wadsworth ◽  
E D Salmon
Keyword(s):  
Average Length ◽  
Cold Treatment ◽  
Video Camera ◽  
Microtubule Assembly ◽  
Microtubule Depolymerization ◽  
Rate Limiting Step ◽  
Minor Population ◽  
High Concentrations ◽  
Tubulin Immunofluorescence ◽  
A Minor

Human monocytes, which contain few interphase microtubules (35.+/- 7.7), were used to study the dynamics of microtubule depolymerization. Steady-state microtubule assembly was abruptly blocked with either high concentrations of nocodazole (10 micrograms/ml) or exposure to cold temperature (3 degrees C). At various times after inhibition of assembly, cells were processed for anti-tubulin immunofluorescence microscopy. Stained cells were observed with an intensified video camera attached to the fluorescence microscope. A tracing of the entire length of each individual microtubule was made from the image on the television monitor by focusing up and down through the cell. The tracings were then digitized into a computer. All microtubules were seen to originate from the centrosome, with an average length in control cells of 7.1 +/- 2.7 microns (n = 957 microtubules). During depolymerization, the total microtubule polymer and the number of microtubules per cell decreased rapidly. In contrast, there was a slow decrease in the average length of the persisting microtubules. The half-time for both the loss of total microtubule polymer and microtubule number per cell was approximately 40 s for nocodazole-treated cells. The rate-limiting step in the depolymerization process was the rate of initiation of disassembly. Once initiated, depolymerization appeared catastrophic. Further kinetic analysis revealed two classes of microtubules: 70% of the microtubule population was very labile and initiated depolymerization at a rate approximately 23 times faster than a minor population of persistent microtubules. Cold treatment yielded qualitatively similar characteristics of depolymerization, but the initiation rates were slower. In both cases there was a significant asynchrony and heterogeneity in the initiation of depolymerization among the population of microtubules.

Polysaccharide Structures in the Outer Mucilage of Arabidopsis Seeds Visualized by AFM

2020 ◽  
Author(s):  
MAK Williams ◽  
V Cornuault ◽  
AH Irani ◽  
VV Symonds ◽  
J Malmström ◽  
...  
Keyword(s):  
Average Length ◽  
American Chemical Society ◽  
Md Simulations ◽  
Chemical Society ◽  
Side Chains ◽  
Rhamnogalacturonan I ◽  
Afm Imaging ◽  
Minor Population ◽  
The Stability ◽  
A Minor

© 2020 American Chemical Society. Evidence is presented that the polysaccharide rhamnogalacturonan I (RGI) can be biosynthesized in remarkably organized branched configurations and surprisingly long versions and can self-assemble into a plethora of structures. AFM imaging has been applied to study the outer mucilage obtained from wild-type (WT) and mutant (bxl1-3 and cesa5-1) Arabidopsis thaliana seeds. For WT mucilage, ordered, multichain structures of the polysaccharide RGI were observed, with a helical twist visible in favorable circumstances. Molecular dynamics (MD) simulations demonstrated the stability of several possible multichain complexes and the possibility of twisted fibril formation. For bxl1-3 seeds, the imaged polymers clearly showed the presence of side chains. These were surprisingly regular and well organized with an average length of ∼100 nm and a spacing of ∼50 nm. The heights of the side chains imaged were suggestive of single polysaccharide chains, while the backbone was on average 4 times this height and showed regular height variations along its length consistent with models of multichain fibrils examined in MD. Finally, in mucilage extracts from cesa5-1 seeds, a minor population of chains in excess of 30 μm long was observed.

scholarly journals Production of nitric oxide in the synovial membrane of rheumatoid and osteoarthritis patients.

1996 ◽  
Vol 184 (4) ◽  
pp. 1519-1524 ◽  
Author(s):  
I B McInnes ◽  
B P Leung ◽  
M Field ◽  
X Q Wei ◽  
F P Huang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Tnf Alpha ◽  
Nonspecific Esterase ◽  
No Production ◽  
No Donor ◽  
Minor Population ◽  
High Concentrations ◽  
A Minor ◽  
Necrosis Factor ◽  
High Output

We have demonstrated spontaneous nitric oxide (NO) production by primary synovial cultures from rheumatoid (RA) and osteoarthritis patients. Increased NO production followed addition of staphylococcal enterotoxin B. Immunochemical double staining with specific anti-human inducible NO synthase (iNOS) and nonspecific esterase (NSE), or anti-CD68 (markers for tissue macrophages) showed that although many lining layer cells in RA synovium expressed iNOS, most (approximately 90%) were NSE- and CD68-, with only a minor population (approximately 10%) which were iNOS+, CD68+/NSE+. These data demonstrate the capacity for high output of NO by human synovial tissue and show that, although human macrophages can express high levels of iNOS, the majority of cells expressing iNOS are fibroblasts. We also report that synoviocytes, and macrophage cell lines, cultured with the NO donor, S-nitroso-acetyl penicillamine, produced high concentrations of tumor necrosis factor (TNF)-alpha. These results suggest that NO may mediate pathology in RA through the induction of TNF-alpha production.

Interphase microtubule dynamics are cell type-specific

1990 ◽  
Vol 95 (1) ◽  
pp. 23-32
Author(s):  
P. Wadsworth ◽  
M. McGrail
Keyword(s):  
Epithelial Cell ◽  
Primary Cultures ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
Fibroblast Cells ◽  
Half Time ◽  
Quantitative Measurements ◽  
Cell Type Specific ◽  
High Concentrations ◽  
Tubulin Immunofluorescence

The rate and pattern of microtubule polymer loss in interphase cells have been examined using nocodazole to block microtubule assembly. Cells were incubated with high concentrations of nocodazole for various times and the pattern of microtubule disassembly was determined using tubulin immunofluorescence. Polymer loss was quantitated by measuring the decrease in percentage of cell area occupied by microtubules. The results demonstrate that microtubules in diverse cells disassemble individually and asynchronously. In addition, these quantitative measurements reveal that epithelial and fibroblast cells display strikingly different kinetics of polymer loss. In fibroblasts, polymer loss is rapid, with a half-time of 4 min at 37 degrees C. In epithelial cells, loss of 60% of the microtubules occurs with a half-time of 18 min; the remaining 40% of the microtubules disassemble much more slowly (average half-time of 72 min). To demonstrate that these differences were not due to species differences among various cells assayed in these experiments, epithelial and fibroblast cells derived from primary cultures of newt lung have been examined. Again, fibroblast and epithelial cell microtubule dynamics could be readily distinguished. To determine if modifications to epithelial cell microtubules contribute to their stability, microtubules were completely disassembled and allowed to regrow. The rate of polymer loss for recently regrown microtubules was more rapid than microtubules in control cells, indicating that stability increases with time after assembly.

scholarly journals An update on leucaena toxicity: Is inoculation with Synergistes jonesii necessary?

2019 ◽  
Vol 7 (2) ◽  
pp. 146-153
Author(s):  
H. Max Shelton ◽  
Graham Kerven ◽  
Scott A. Dalzell
Keyword(s):  
Rumen Fluid ◽  
Rumen Bacterium ◽  
Major Pathway ◽  
Bos Javanicus ◽  
Minor Population ◽  
High Concentrations ◽  
A Minor ◽  
Almost All ◽  
Short Time ◽  
Detoxification Pathway

Keynote paper presented at the International Leucaena Conference, 1‒3 November 2018, Brisbane, Queensland, Australia.Concern about mimosine toxicity and its management has contributed to the restricted adoption of leucaena as a forage for ruminants. The toxicity is a function of the antimitotic effects of mimosine, which is rapidly converted to also toxic compounds, isomers of hydroxypyridone (DHP), by plant and microbial enzymes. Work by R.J. Jones and colleagues (1960‒1994) identified a rumen bacterium (Synergistes jonesii) capable of degrading DHP, and rumen fluid containing this bacterium was subsequently made available in Australia as a commercial inoculum for cattle producers.Research by University of Queensland and CSIRO over 15 years, commencing in 2003, found evidence for another pathway of toxin management in Indonesia, where hundreds of Balinese farmers had fed uninoculated Bali bulls (Bos javanicus) up to 100% leucaena without experiencing toxicity symptoms, apart from an initial 1‒2 week period while their cattle became adapted to the new diet. Tests showed that the Indonesian cattle were not degrading all DHP, as it appeared in high concentrations in urine samples, predominantly as 2,3-DHP and almost all (>97%) in a conjugated form. The conjugating compounds (glucuronic acid and sulfate compounds), produced in the liver, appeared to be the major pathway for neutralizing the toxicity of DHP. Other work revealed that S. jonesii was a ubiquitous organism detectable in the rumen fluid of animals in all countries but always as a minor population, often below the level of detection.Since the Indonesian cattle fed leucaena suffered mimosine toxicity symptoms for only a short time before quickly recovering, we hypothesize that conjugation of DHP by the liver was the major detoxification pathway for these animals. This detoxification pathway is also operative in Australia and other countries but further studies are needed to determine its significance.

Polysaccharide Structures in the Outer Mucilage of Arabidopsis Seeds Visualized by AFM

2020 ◽  
Author(s):  
MAK Williams ◽  
V Cornuault ◽  
AH Irani ◽  
VV Symonds ◽  
J Malmström ◽  
...  
Keyword(s):  
Average Length ◽  
American Chemical Society ◽  
Md Simulations ◽  
Chemical Society ◽  
Side Chains ◽  
Rhamnogalacturonan I ◽  
Afm Imaging ◽  
Minor Population ◽  
The Stability ◽  
A Minor

© 2020 American Chemical Society. Evidence is presented that the polysaccharide rhamnogalacturonan I (RGI) can be biosynthesized in remarkably organized branched configurations and surprisingly long versions and can self-assemble into a plethora of structures. AFM imaging has been applied to study the outer mucilage obtained from wild-type (WT) and mutant (bxl1-3 and cesa5-1) Arabidopsis thaliana seeds. For WT mucilage, ordered, multichain structures of the polysaccharide RGI were observed, with a helical twist visible in favorable circumstances. Molecular dynamics (MD) simulations demonstrated the stability of several possible multichain complexes and the possibility of twisted fibril formation. For bxl1-3 seeds, the imaged polymers clearly showed the presence of side chains. These were surprisingly regular and well organized with an average length of ∼100 nm and a spacing of ∼50 nm. The heights of the side chains imaged were suggestive of single polysaccharide chains, while the backbone was on average 4 times this height and showed regular height variations along its length consistent with models of multichain fibrils examined in MD. Finally, in mucilage extracts from cesa5-1 seeds, a minor population of chains in excess of 30 μm long was observed.

scholarly journals Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine.

1984 ◽  
Vol 99 (3) ◽  
pp. 1066-1075 ◽  
Author(s):  
E D Salmon ◽  
M McKeel ◽  
T Hays
Keyword(s):  
Rate Constant ◽  
Electron Micrographs ◽  
Average Length ◽  
Intrinsic Rate ◽  
Microtubule Assembly ◽  
Spindle Microtubules ◽  
Intracellular Concentrations ◽  
High Concentrations

At metaphase, the amount of tubulin assembled into spindle microtubules is relatively constant; the rate of tubulin association equals the rate of dissociation. To measure the intrinsic rate of dissociation, we microinjected high concentrations of colchicine, or its derivative colcemid, into sea urchin embryos at metaphase to bind the free tubulin, thereby rapidly blocking polymerization. The rate of microtubule disassembly was measured from a calibrated video signal by the change in birefringent retardation (BR). After an initial delay after injection of colchicine or colcemid at final intracellular concentrations of 0.1-3.0 mM, BR decreased rapidly and simultaneously throughout the central spindle and aster. Measured BR in the central half-spindle decreased exponentially to 10% of its initial value within a characteristic period of approximately 20 s; the rate constant, k = 0.11 +/- 0.023 s-1, and the corresponding half-time, t 1/2, of BR decay was approximately 6.5 +/- 1.1 s in this concentration range. Below 0.1 mM colchicine or colcemid, the rate at which BR decreased was concentration dependent. Electron micrographs showed that the rapid decrease in BR corresponded to the disappearance of nonkinetochore microtubules; kinetochore fiber microtubules were differentially stable. As a control, lumicolchicine, which does not bind to tubulin with high affinity, was shown to have no effect on spindle BR at intracellular concentrations of 0.5 mM. If colchicine and colcemid block only polymerization, then the initial rate of tubulin dissociation from nonkinetochore spindle microtubules is in the range of 180-992 dimers per second. This range of rates is based on k = 11% of the initial polymer per second and an estimate from electron micrographs that the average length of a half-spindle microtubule is 1-5.5 micron. Much slower rates of tubulin association are predicted from the characteristics of end-dependent microtubule assembly measured previously in vitro when the association rate constant is corrected for the lower rate of tubulin diffusion in the embryo cytoplasm. Various possibilities for this discrepancy are discussed.

The treatment of iron oxalate leach liquors in a UASB with sulfate reduction

1997 ◽  
Vol 36 (6-7) ◽  
pp. 383-390 ◽  
Author(s):  
J. E. Teer ◽  
D. J. Leak ◽  
A. W. L. Dudeney ◽  
A. Narayanan ◽  
D. C. Stuckey
Keyword(s):  
Bacterial Species ◽  
Upflow Anaerobic Sludge Blanket ◽  
Anaerobic Sludge ◽  
Desulfovibrio Vulgaris ◽  
Hydrogenotrophic Methanogenesis ◽  
Irreversible Effects ◽  
High Concentrations ◽  
A Minor ◽  
Anaerobic Sludge Blanket ◽  
Reducing Bacteria

The presence of small amounts of iron (>0.013% Fe) in sand creates problems in the manufacture of high quality glass. Removal by hot sulphuric acid is possible, but creates environmental problems, and is costly. Hence organic acids such as oxalic have been investigated since they are effective in removing iron, and can be degraded anaerobically. The aim of this work was to identify key intermediates in the anaerobic degradation of oxalate in an upflow anaerobic sludge blanket reactor (UASB) which was removing iron from solution in the sulphide form, and to determine the bacterial species involved. 2-bromoethanesulfonic acid (BES) and molybdenum were selected as suitable inhibitors for methanogenic and sulphate reducing bacteria (SRB) respectively. 40mM molybdenum was used to inhibit the SRB in a reactor with a 12hr HRT. Total SRB inhibition took place in 20 hrs, with a complete breakthrough of influent sulphate. The lack of an immediate oxalate breakthrough confirmed Desulfovibrio vulgaris subspecies oxamicus was not the predominant oxalate utilising species. Nevertheless, high concentrations of molybdenum were found to inhibit oxalate utilising bacteria in granular reactors but not in suspended population reactors; this observation was puzzling, and at present cannot be explained. Based on the intermediates identified, it was postulated that oxalate was degraded to formate by an oxalate utilising bacteria such as Oxalobacter formigenes, and the formate used by the SRBs to reduce sulphate. Acetate, as a minor intermediate, existed primarily as a source of cell carbon for oxalate utilising bacteria. Methanogenic inhibition identified that 62% of the CH4 in the reactor operated at 37°C originated from hydrogenotrophic methanogenesis, whilst this figure was 80% at 20°C. Possible irreversible effects were recorded with hydrogenotrophic methanogens.

scholarly journals Developmental-Dependent Action of Microtubule Depolymerization on the Function and Structure of Synaptic Glycine Receptor Clusters in Spinal Neurons

2004 ◽  
Vol 91 (2) ◽  
pp. 1036-1049 ◽  
Author(s):  
Brigitte van Zundert ◽  
Francisco J. Alvarez ◽  
Juan Carlos Tapia ◽  
Hermes H. Yeh ◽  
Emilio Diaz ◽  
...  
Keyword(s):  
Average Length ◽  
Glycine Receptor ◽  
Colchicine Treatment ◽  
Morphological Properties ◽  
Microtubule Depolymerization ◽  
Spinal Neurons ◽  
Functional Changes ◽  
Microtubule Disruption ◽  
Immature Neurons ◽  
Mature Neurons

Microtubules have been proposed to interact with gephyrin/glycine receptors (GlyRs) in synaptic aggregates. However, the consequence of microtubule disruption on the structure of postsynaptic GlyR/gephyrin clusters is controversial and possible alterations in function are largely unknown. In this study, we have examined the physiological and morphological properties of GlyR/gephyrin clusters after colchicine treatment in cultured spinal neurons during development. In immature neurons (5-7 DIV), disruption of microtubules resulted in a 33 ± 4% decrease in the peak amplitude and a 72 ± 15% reduction in the frequency of spontaneous glycinergic miniature postsynaptic currents (mIPSCs) recorded in whole cell mode. However, similar colchicine treatments resulted in smaller effects on 10-12 DIV neurons and no effect on mature neurons (15-17 DIV). The decrease in glycinergic mIPSC amplitude and frequency reflects postsynaptic actions of colchicine, since postsynaptic stabilization of microtubules with GTP prevented both actions and similar reductions in mIPSC frequency were obtained by modifying the Cl- driving force to obtain parallel reductions in mIPSC amplitude. Confocal microscopy revealed that colchicine reduced the average length and immunofluorescence intensity of synaptic gephyrin/GlyR clusters in immature (approximately 30%) and intermediate (approximately 15%) neurons, but not in mature clusters. Thus the structural and functional changes of postsynaptic gephyrin/GlyR clusters after colchicine treatment were tightly correlated. Finally, RT-PCR, kinetic analysis and picrotoxin blockade of glycinergic mIPSCs indicated a reorganization of the postsynaptic region from containing both α2β and α1β GlyRs in immature neurons to only α1β GlyRs in mature neurons. Microtubule disruption preferentially affected postsynaptic sites containing α2β-containing synaptic receptors.

Neurofilament proteins are co-expressed with desmin in heart conduction system myocytes

1990 ◽  
Vol 97 (1) ◽  
pp. 11-21
Author(s):  
M. Vitadello ◽  
M. Matteoli ◽  
L. Gorza
Keyword(s):  
Subcellular Distribution ◽  
Ultrastructural Level ◽  
Rabbit Heart ◽  
Cardiac Cells ◽  
Structural Components ◽  
Minor Population ◽  
Neuronal Proteins ◽  
Tissue Homogenates ◽  
A Minor

We have recently shown that specialized myocytes of the rabbit heart express a cytoskeletal protein similar to the M subunit of neurofilaments (NF). Since this result was obtained using a single anti-NF-M monoclonal antibody, we tested on conduction myocytes a panel of five anti-NF antibodies, specific for each of the three NF subunits and for phosphorylated and non-phosphorylated epitopes. Two antibodies, one specific for the L subunit and one for phosphorylated M subunit of NF, reacted with specialized myocytes in immunohistochemistry. In immunoblots on conduction tissue homogenates the two antibodies recognized two polypeptides with electrophoretic mobility and solubility properties identical to those of NF-L and NF-M in the sciatic nerve. The subcellular distribution of NF immunoreactivity in specialized myocytes was very similar to desmin localization; namely, it was distributed on large filamentous bundles and on fine filaments localized transversely at the level of the Z line. At the ultrastructural level, immunoreactive filaments were localized in the intermyofibrillar space and connected myofibrils with mitochondria. Co-expression of NF proteins and desmin was also observed in vitro in a minor population of cardiac myocytes cultured from embryonic rabbit heart. In most cases NF immunoreactivity co-localized with desmin, especially where filaments were well organized, but in some cells anti-NF and anti-desmin antibodies labelled different filamentous structures. These results indicate that NF proteins are structural components of the cytoskeleton of specialized myocytes and show a subcellular distribution very similar to desmin. Such a composition of intermediate filaments indicates that in these cardiac cells muscle differentiation is compatible with the expression of neuronal proteins.

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