scholarly journals Are cross-bridging structures involved in the bundle formation of intermediate filaments and the decrease in locomotion that accompany cell aging?

1985 ◽  
Vol 100 (5) ◽  
pp. 1466-1473 ◽  
Author(s):  
E Wang
Keyword(s):  
Intermediate Filaments ◽  
Electron Microscopic Examination ◽  
Time Lapse ◽  
Cell Aging ◽  
Electron Microscopic ◽  
Cell Locomotion ◽  
Wide Range ◽  
Filament Bundles ◽  
Large Filament

Five different fibroblast strains derived from donors of a wide range of ages were used for investigation of senescence-associated changes in the organization of intermediate filaments (IFs) and the activity of cell locomotion. Results of immunofluorescence microscopy demonstrate that, in large and flat in vitro aged fibroblasts, vimentin-containing IFs are distributed as unusually organized large bundles. Electron microscopic examination shows that these large bundles are indeed composed of filaments of 8-10 nm. Such a profile of large bundles is rarely seen in young fibroblasts whose IFs are usually interdispersed among microtubules. Within the large filament bundles of senescent fibroblasts, cross-bridge-like extensions are frequently observed along the individual IFs. Immunogold labeling with antibody to one of the cross-bridging proteins, p50, further illustrates the abundance of interfilament links within the IF bundles. The senescence-related increase in interfilament association was also supported by the results of co-precipitation between vimentin and an associated protein of 50,000 D. Time-lapse cinematographic studies of cell locomotion reveal that accompanying aging, fibroblasts have a significantly reduced ability to translocate across a solid substratum. These results led me to suggest that the increased interfilament links via cross-bridges may in part contribute to the mechanism that orchestrates the formation of large filament bundles. The presence of enormous bundles in the cytoplasm may physically impede the efficiency of locomotion for these nondividing cells.

Tetrins: polypeptides that form bundled filaments in Tetrahymena

1990 ◽  
Vol 96 (2) ◽  
pp. 293-302
Author(s):  
J.E. Honts ◽  
N.E. Williams
Keyword(s):  
Intermediate Filaments ◽  
Tetrahymena Pyriformis ◽  
Ciliated Protozoan ◽  
Microtubule Associated Proteins ◽  
Fine Filament ◽  
Helical Content ◽  
In Vitro Assembly ◽  
Associated Proteins ◽  
Filament Bundles

The cortex of the ciliated protozoan Tetrahymena contains a number of fibrous elements, including a network of filaments that pervades the feeding organelle of this organism. The cluster of polypeptides (79–89K; K = 10(3) Mr) in Tetrahymena pyriformis GL-C that constitute these filaments has been purified by in vitro assembly after solubilization in 1.0 M KI. Four distinct sets of these polypeptides, designated ‘tetrins’, have been shown to be distinguishable from each other by immunochemical and biochemical criteria. The smallest filaments reassembled in vitro were 3–4 nm in diameter and these fine filaments were seen to be bundled together into thicker strands of varying diameters, similar to those within the cell. The thicker filament bundles were clearly distinguishable from intermediate filaments, but fine filaments in these bundles were superficially similar to the 2–5 nm filaments described as microtubule-associated proteins in other organisms. The ultrastructure of the tetrin filaments localized within the feeding organelle reveals a substantial presence of these filaments apart from microtubules. In addition, circular dichroism measurements indicate a relatively low alpha-helical content for these filaments and suggest that the tetrins may be substantially different from other fine filament proteins such as the tektins and giardins.

scholarly journals In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes.

1982 ◽  
Vol 156 (6) ◽  
pp. 1604-1614 ◽  
Author(s):  
E H Burger ◽  
J W Van der Meer ◽  
J S van de Gevel ◽  
J C Gribnau ◽  
G W Thesingh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Electron Microscopic Examination ◽  
Osteoclast Formation ◽  
Mononuclear Phagocytes ◽  
Long Bone ◽  
Embryonic Liver ◽  
Blood Monocytes ◽  
Electron Microscopic ◽  
Embryonic Mouse

The origin of osteoclasts was studied in an in vitro model using organ cultures of periosteum-free embryonic mouse long-bone primordia, which were co-cultured with various cell populations. The bone rudiments were freed of their periosteum-perichondrium by collagenase treatment in a stage before cartilage erosion and osteoclast formation, and co-cultured for 7 d with either embryonic liver or mononuclear phagocytes from various sources. Light and electron microscopic examination of the cultures showed that mineralized matrix-resorbing osteoclasts developed only in bones co-cultured with embryonic liver or with cultured bone marrow mononuclear phagocytes but not when co-cultured with blood monocytes or resident or exudate peritoneal macrophages. Osteoclasts developed from the weakly adherent, but not from the strongly adherent cells of bone marrow cultures, whereas 1,000 rad irradiation destroyed the capacity of such cultures to form osteoclasts. In bone cultures to which no other cells were added, osteoclasts were virtually absent. Bone-resorbing activity of in vitro formed osteoclasts was demonstrated by 45Ca release studies. These studies demonstrate that osteoclasts develop from cells present in cultures of proliferating mononuclear phagocytes and that, at least in our system, monocytes and macrophages are unable to form osteoclasts. The most likely candidates for osteoclast precursor cells seem to be monoblasts and promonocytes.

scholarly journals Studies in vitro on the effects of 1H,2H,4H(5H)-octafluorocyclohexane and 1H,4H(2H)-nonafluorocyclohexane on enzymes and organelles

1969 ◽  
Vol 114 (4) ◽  
pp. 785-792 ◽  
Author(s):  
Jayasree Nath ◽  
H G Bray
Keyword(s):  
Adenosine Triphosphatase ◽  
Marked Decrease ◽  
Reductase Activity ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Toxic Compound ◽  
Marked Inhibition ◽  
Liver Homogenates ◽  
Nadh Cytochrome

A comparison has been made of the effect of 1H,2H,4H(5H)-octafluorocyclohexane, which is highly toxic (LD50 17mg./kg. in rats), and of 1H,4H(2H)-nonafluorocyclohexane, which is relatively non-toxic (LD50>440mg./kg. in rats), on the respiration of rat liver homogenates and mitochondria in vitro. 1H,2H,4H(5H)-Octafluorocyclohexane strongly inhibited the respiration of both homogenates and mitochondria, but neither compound had any significant effect on glycolysis or on glutamate dehydrogenase or NADH–cytochrome c reductase activity. 1H,2H,4H(5H)-Octafluorocyclohexane, however, caused a very marked inhibition of cytochrome oxidase activity, causing an almost complete lesion in this region of the respiratory chain. 1H,4H(2H)-Nonafluorocyclohexane was without effect in this respect. A marked decrease in turbidity of mitochondrial suspensions at 520nm. was caused by addition of both compounds, the effect being greater with 1H,2H,4H(5H)-octafluorocyclohexane. ATP, Mg2+ and bovine serum albumin did not reverse these changes. Mitochondrial adenosine triphosphatase activity was increased twofold by the toxic compound, but only slightly by the non-toxic compound. Electron-microscopic examination of mitochondria treated with 1H,2H,4H(5H)-octafluorocyclohexane revealed gross morphological damage, whereas the effect of 1H,4H(2H)-nonafluorocyclohexane appeared to be merely to cause swelling. The results obtained account, to some extent at any rate, for the toxic effects of 1H,2H,4H(5H)-octafluorocyclohexane.

scholarly journals Mice Deficient in the Axonemal Protein Tektin-t Exhibit Male Infertility and Immotile-Cilium Syndrome Due to Impaired Inner Arm Dynein Function

2004 ◽  
Vol 24 (18) ◽  
pp. 7958-7964 ◽  
Author(s):  
Hiromitsu Tanaka ◽  
Naoko Iguchi ◽  
Yoshiro Toyama ◽  
Kouichi Kitamura ◽  
Tohru Takahashi ◽  
...  
Keyword(s):  
Physiological Role ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Vitro Fertilization ◽  
Homozygous Mutant ◽  
Causal Genes ◽  
And Function ◽  
Ciliary Dyskinesia

ABSTRACT The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.

scholarly journals Phosphorylation of vimentin in mitotically selected cells. In vitro cyclic AMP-independent kinase and calcium-stimulated phosphatase activities.

1989 ◽  
Vol 108 (1) ◽  
pp. 67-78 ◽  
Author(s):  
R M Evans
Keyword(s):  
Phosphatase Activity ◽  
Kinase Activity ◽  
Electron Microscopic Examination ◽  
Intermediate Filament Protein ◽  
Electron Microscopic ◽  
Intact Cells ◽  
Activity Peak ◽  
Casein Kinases ◽  
Phosphopeptide Mapping

The phosphorylation of the intermediate filament protein vimentin was examined under in vitro conditions. Cell cytosol and Triton-insoluble cytoskeleton preparations from nonmitotic and mitotically selected mouse L-929 cells exhibited vimentin kinase activity that is apparently cAMP and Ca2+ independent. The level of vimentin kinase activity was greater in preparations from mitotically selected cells than nonmitotic cells. Addition of Ca2+ to mitotic cytosol decreased net vimentin phosphorylation. Dephosphorylation experiments indicated that there is phosphatase activity in these preparations which is stimulated by addition of Ca2+. Fractionation of cytosol from nonmitotic cells on DEAE-Sephacel and phosphocellulose revealed a single major vimentin kinase activity (peak I). Fractionation of cytosol from mitotically selected cells yielded a similar activity (peak I) and an additional vimentin kinase activity (peak II) that was not found in nonmitotic preparations. Based on substrate specificity and lack of inhibition to characteristic inhibitors, the semipurified peak I and II vimentin kinase activities appear to be cAMP-independent enzymes that are distinct from casein kinases I and II. Phosphopeptide mapping studies indicated that both peak I and peak II vimentin kinases phosphorylate tryptic peptides in the NH2-terminal region of vimentin that are phosphorylated in intact cells. Electron microscopic examination of reconstituted vimentin filaments phosphorylated with both semipurified kinases indicated that phosphorylation induced filament disassembly. These experiments indicate that the increased phosphorylation of vimentin during mitosis may be catalyzed by a discrete cAMP-independent protein kinase. In addition, preparations from mitotic cells exhibited a Ca2+-stimulated phosphatase activity, suggesting that Ca2+ may play a regulatory role in vimentin dephosphorylation during mitosis.

A light and electron microscope study of the interaction of soil bacteria with Phytophthora cinnamomi Rands

1977 ◽  
Vol 23 (11) ◽  
pp. 1518-1525 ◽  
Author(s):  
N. Malajczuk ◽  
H. J. Nesbitt ◽  
A. R. Glenn
Keyword(s):  
Soil Bacteria ◽  
Phytophthora Cinnamomi ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Microbial Association ◽  
Reproductive Structures ◽  
Streptomyces Spp ◽  
Lysis Inhibition ◽  
Zoospore Production

Light- and electron-microscopic examination showed that bacteria became associated with the hyphae and asexual reproductive structures of P. cinnamomi in soil. In suppressive soils this association appears to be correlated with hyphal lysis, inhibition of zoospore production, and sporangial breakdown. One notable feature of the microbial association between P. cinnamomi and soil bacteria is the formation of extensive slime material. Many of the bacteria isolated from the fungal hyphosphere display antagonism to the growth of P. cinnamomi in vitro. The bacteria are morphologically varied and include Pseudomonas, Bacillus, and Streptomyces spp. These observations suggest that the appropriate manipulation of the antagonistic bacteria may provide a means of biological control of P. cinnamomi.

Behavioural properties of chick somitic mesoderm and lateral plate when explanted in vitro

Development ◽  
1980 ◽  
Vol 56 (1) ◽  
pp. 41-58
Author(s):  
Ruth Bellairs ◽  
P. A. Portch ◽  
E. J. Sanders
Keyword(s):  
Time Lapse ◽  
Lateral Plate Mesoderm ◽  
General Shape ◽  
Electron Microscopic ◽  
Lateral Plate ◽  
Plastic Substrates ◽  
Different Types ◽  
Somitic Mesoderm ◽  
Microscopic Techniques

Tissue culture, time-lapse cinematographic and electron microscopic techniques have been used to study the properties of chick mesoderm at several stages of differentiation. Lateral plate, unsegmented mesoderm (segmental plate), and newly formed somites were dissected from stage-12 embryos, whilst dermo-myotomes and sclerotomes were dissected from stage-18 embryos. Each type of mesoderm was found to exhibit a characteristic pattern of behaviour. The explants from the unsegmented mesoderm, from the newly formed somites and from the older embryos could be placed in a developmental sequence; with increasing differentiation they settled and spread on the substrate more readily, whether explanted as pieces of tissue or as individual cells, and it was concluded that this implied an increased adhesion to the substrate. Similarly, with increasing differentiation, the cells segmented at a faster rate. No significant differences could be discerned in the internal structure of the different types of cells, although differences in the general shape were apparent. The lateral plate mesoderm cells, which bear some resemblances to the unsegmented mesoderm cells in the embryo, also show some morphological resemblances to them in vitro. However, the lateral plate cells had a much greater success in attaching to glass or plastic substrates. They were also found to have the highest speed of locomotion of all the tissues studied, whereas the unsegmented had the lowest. It is concluded therefore, that although cells may look similar to one another morphologically, their behaviour may differ greatly, probably because they are already partially determined.

scholarly journals Synergy, Pharmacodynamics, and Time-Sequenced Ultrastructural Changes of the Interaction between Nikkomycin Z and the Echinocandin FK463 against Aspergillus fumigatus

2001 ◽  
Vol 45 (12) ◽  
pp. 3310-3321 ◽  
Author(s):  
Christine C. Chiou ◽  
Nikolaos Mavrogiorgos ◽  
Elizabeth Tillem ◽  
Richard Hector ◽  
Thomas J. Walsh
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Rhizopus Oryzae ◽  
Ultrastructural Changes ◽  
Synergistic Activity ◽  
Partial Recovery ◽  
Electron Microscopic ◽  
Wide Range ◽  
Nikkomycin Z

ABSTRACT We investigated the potential synergy between two cell wall-active agents, the echinocandin FK463 (FK) and the chitin synthase inhibitor nikkomycin Z (NZ), against 16 isolates of filamentous fungi. Susceptibility testing was performed with a broth macrodilution procedure by NCCLS methods. The median minimal effective concentration (MEC) of FK against all Aspergillus species was 0.25 μg/ml (range, 0.05 to 0.5 μg/ml). For Fusarium solaniand Rhizopus oryzae, MECs of FK were >512 μg/ml. The median MEC of NZ against Aspergillus fumigatus was 32 μg/ml (range, 8 to 64 μg/ml), and that against R. oryzae was 0.5 μg/ml (range, 0.06 to 2 μg/ml); however, for the other Aspergillus species, as well as F. solani, MECs were >512 μg/ml. A checkerboard inhibitory assay demonstrated synergy against A. fumigatus (median fractional inhibitory concentration index = 0.312 [range, 0.15 to 0.475]). The effect was additive to indifferent against R. oryzae and indifferent against other Aspergillus spp. and F. solani. We further investigated the pharmacodynamics of hyphal damage by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and examined the time-sequenced changes in hyphal ultrastructure. Significant synergistic hyphal damage was demonstrated with the combination of NZ (2 to 32 μg/ml) and FK (0.03 to 0.5 μg/ml) over a wide range of concentrations (P < 0.001). The synergistic effect was most pronounced after 12 h of incubation and was sustained through 24 h. Time-sequenced light and electron microscopic studies demonstrated that structural alterations of hyphae were profound, with marked transformation of hyphae to blastospore-like structures, in the presence of FK plus NZ, while fungi treated with a single drug showed partial recovery at 24 h. The methods used in this study may be applicable to elucidating the activity and interaction of other cell wall-active agents. In summary, these two cell wall-targeted antifungal agents, FK and NZ, showed marked time-dependent in vitro synergistic activity against A. fumigatus.

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