scholarly journals Distributions of vimentin and desmin in developing chick myotubes in vivo. II. Immunoelectron microscopic study.

1985 ◽  
Vol 100 (4) ◽  
pp. 1157-1166 ◽  
Author(s):  
K T Tokuyasu ◽  
P A Maher ◽  
S J Singer
Keyword(s):  
Intermediate Filaments ◽  
De Novo ◽  
Early Stage ◽  
Primary Role ◽  
Special Relationship ◽  
Frozen Sections ◽  
Intermediate Filament Proteins ◽  
Immunofluorescence Studies ◽  
Ultrathin Frozen Sections

The distribution of the intermediate filament proteins vimentin and desmin in developing and mature myotubes in vivo was studied by single and double immunoelectron microscopic labeling of ultrathin frozen sections of iliotibialis muscle in 7-21-d-old chick embryos, and neonatal and 1-d-old postnatal chicks. This work is an extension of our previous immunofluorescence studies of the same system (Tokuyasu, K. T., P. A. Maher and S. J. Singer, 1984, J. Cell Biol., 98:1961-1972). In immature myotubes of 7-11-d embryos, significant labeling for desmin and vimentin was found only in intermediate filaments, and these proteins coexisted in the same individual filaments. Each of the two proteins was present in irregular clusters along the entire length of a filament. No exclusively vimentin- or desmin-containing filaments were observed at this stage. In the early myotubes, the intermediate filaments were essentially all longitudinally oriented, even when they contained three times as much desmin as vimentin. No special relationship was recognized between the dispositions of the filaments and the organization of the myofibrils. Occasionally, several myofibrils were already aligned in lateral registry at this early stage, but labeling for desmin and vimentin was largely absent at the level of the Z bands. Instead, the Z bands appeared to be covered by elements of the sarcoplasmic reticulum. The confinement of intermediate filaments to the level of the Z bands occurred in the myotubes of later embryos after the extensive lateral registry of the Z bands. Thus, intermediate filaments are unlikely to play a primary role in producing the lateral registration of myofibrils during myogenesis, but may be important in determining the polarization of the early myotube and the alignment of its organelles. Throughout the development of myotubes, desmin and vimentin remained in the form of intermediate filaments, although the number of filaments per unit volume of myotube appeared to be reduced as myofibrils increased in number in maturing myotubes. This observation indicated that the transverse orientation of intermediate filaments in mature myotubes does not result from the de novo polymerization of subunits from Z band to Z band, but a continuous shifting of the positions and directions of intact filaments.

scholarly journals In vivo assembly of tight junctions in fetal rat liver.

1975 ◽  
Vol 67 (2) ◽  
pp. 310-319 ◽  
Author(s):  
R Montesano ◽  
D S Friend ◽  
A Perrelet ◽  
L Orci
Keyword(s):  
Tight Junctions ◽  
De Novo ◽  
Early Stage ◽  
Fetal Life ◽  
Bile Canaliculi ◽  
Linear Arrays ◽  
Fetal Rat ◽  
Particle Aggregates ◽  
Fetal Rat Liver

Examination of glutaraldehyde-fixed, freeze-fractured livers from 14-15-day rat fetuses provided the basis for the following observations. Membrane particles align in otherwise poorly particulated areas of the presumptive pericanalicular plasma membrane (A face), frequently forming a discontinuous "honey-comb" network joining small particle islands. Even at this early stage, contiguous B-fracture faces contain furrows, rather than rows of pits, distinguishing the linear particle aggregates on the A face as developing tight junctions rather than gap junctions. Short segments of these linear arrays merge with smooth ridges clearly identifiable as segments of discontinuous tight junctions. With the continuing confluence of particulate and smooth ridge segments, mature tight junctions become fully appreciable. We conclude that tight junctions form de novo by the alignment and fusion of separate particles into beaded ridges which, in turn, become confluent and are transformed into continuous smooth ones. At 21 days of fetal life, most of the images of assembly have disappeared, and the liver reveals well-formed bile canaliculi sealed by mature tight junctions.

scholarly journals Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments

1993 ◽  
Vol 122 (6) ◽  
pp. 1323-1335 ◽  
Author(s):  
GY Ching ◽  
RK Liem
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Neuronal Cell ◽  
Intermediate Filament Protein ◽  
Amino Acid Residues ◽  
Intermediate Filament Proteins ◽  
Transfected Cells ◽  
Type Iv

We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha-internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.

scholarly journals Intermediate filaments exchange subunits along their length and elongate by end-to-end annealing

2009 ◽  
Vol 185 (5) ◽  
pp. 769-777 ◽  
Author(s):  
Gülsen Çolakoğlu ◽  
Anthony Brown
Keyword(s):  
Intermediate Filaments ◽  
Actin Filaments ◽  
Fusion Proteins ◽  
Cultured Cells ◽  
Intermediate Filament Proteins ◽  
Subunit Exchange ◽  
End To End ◽  
Vimentin Intermediate Filament ◽  
Vimentin Filaments

Actin filaments and microtubules lengthen and shorten by addition and loss of subunits at their ends, but it is not known whether this is also true for intermediate filaments. In fact, several studies suggest that in vivo, intermediate filaments may lengthen by end-to-end annealing and that addition and loss of subunits is not confined to the filament ends. To test these hypotheses, we investigated the assembly dynamics of neurofilament and vimentin intermediate filament proteins in cultured cells using cell fusion, photobleaching, and photoactivation strategies in combination with conventional and photoactivatable fluorescent fusion proteins. We show that neurofilaments and vimentin filaments lengthen by end-to-end annealing of assembled filaments. We also show that neurofilaments and vimentin filaments incorporate subunits along their length by intercalation into the filament wall with no preferential addition of subunits to the filament ends, a process which we term intercalary subunit exchange.

scholarly journals Visualization of longitudinally-oriented intermediate filaments in frozen sections of chicken cardiac muscle by a new staining method.

1983 ◽  
Vol 97 (2) ◽  
pp. 562-565 ◽  
Author(s):  
K T Tokuyasu
Keyword(s):  
Skeletal Muscle ◽  
Cardiac Muscle ◽  
Intermediate Filaments ◽  
Ethyl Cellulose ◽  
Staining Method ◽  
Uranyl Acetate ◽  
Frozen Sections ◽  
Cell Biol ◽  
Ultrathin Frozen Sections

When ultrathin frozen sections of chicken cardiac muscle were osmicated, dehydrated in ethanol, embedded in ethyl cellulose, and stained with acidic uranyl acetate, filaments of 10-12 nm width were visualized in wide interfibrillar spaces. Immunostaining of the frozen sections for desmin resulted in exclusive labeling of such filaments. These observations indicated that longitudinally oriented networks of intermediate filaments were present in the interfibrillar spaces, in addition to the transversely oriented networks that surround myofibrils at the level of Z band. As in skeletal muscle (Tokuyasu, K. T., A. H. Dutton, and S. J. Singer, 1983, J. Cell Biol. 97:1727-1735), desmin in chicken cardiac muscle is believed to be largely, if not entirely, in the form of intermediate filaments.

scholarly journals Reorganization of cytoplasmic structures during cell fusion

1991 ◽  
Vol 100 (3) ◽  
pp. 431-442 ◽  
Author(s):  
Q.A. Zheng ◽  
D.C. Chang
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Fusion ◽  
Intermediate Filaments ◽  
Early Stage ◽  
Cytoskeletal Proteins ◽  
The Other ◽  
Primary Role ◽  
Cytoplasmic Bridges ◽  
Cytoskeletal Networks ◽  
Cytoplasmic Structures

In order to provide a better understanding of the dynamic process of cell fusion, we studied the reorganization of cytoplasmic structures in electro-fused CV-1 cells. Using fluorescence microscopy and double staining methods, we examined correlations between the structural patterns of the major cytoskeletal proteins (microtubules, actin and vimentin intermediate filaments) and the distribution of various organelles (endoplasmic reticulum, mitochondria and nuclei) at different stages of cell fusion. Our results suggest that microtubules appear to play a primary role in the process of cytoplasmic reorganization. At the early stage of cell fusion, microtubules were observed to infiltrate rapidly into the newly formed cytoplasmic bridges and establish a connection between the cytoskeletal networks of fusing cells. The reorganization of microtubules was found to be correlated with the redistribution of endoplasmic reticulum (ER), vimentin intermediate filaments, mitochondria, and the aggregation of nuclei. The F-actin system, on the other hand, appeared to be independent of the reorganization of the other cytoplasmic structures. The principal function of F-actin during cell fusion is probably to widen the cytoplasmic bridges by lamellipodial extension.

scholarly journals SITES OF GLYCOGEN SYNTHESIS IN RAT LIVER CELLS AS SHOWN BY ELECTRON MICROSCOPE RADIOAUTOGRAPHY AFTER ADMINISTRATION OF GLUCOSE-H3

1966 ◽  
Vol 30 (1) ◽  
pp. 151-175 ◽  
Author(s):  
Antonio Coimbra ◽  
C. P. Leblond
Keyword(s):  
De Novo ◽  
Early Stage ◽  
Glycogen Synthesis ◽  
Light And Electron Microscopy ◽  
Rat Liver Cells ◽  
Silver Grains ◽  
Fasted Rats ◽  
Partly Smooth

Glycogen synthesis was investigated by giving tritium (H3)-labeled glucose with carrier to fasted rats in vivo or incubating liver slices from fasted rats in vitro using a glucose-H3-containing medium. After 15 min or 1 hr, pieces of liver were fixed and radioautographed for light and electron microscopy. In vivo and in vitro, radioautographic reactions appeared over "glycogen areas" and over zones transitional between these areas and ergastoplasm. Treatment of sections by alpha amylase removed all but about 5% of the radioactivity, so that about 95% of it consisted of glycogen (synthesized during the 15 min or 1 hr elapsing after administration of glucose-H3). Within glycogen areas and transitional zones, most silver grains were over or very close to glycogen granules and smooth (or partly smooth) vesicles. Presumably, much of the label was added onto growing glycogen granules, in accord with the biochemical view that glycogen may serve as substrate for further glycogen synthesis. The few silver grains located far from glycogen granules—15% at the 15 min interval in vivo—approximated smooth (or partly smooth) vesicles of endoplasmic reticulum. This observation raised the possibility that smooth membranes play a role in glucose uptake at an early stage in de novo formation of glycogen granules.

scholarly journals Mutation of Ki-ras and N-ras oncogenes in myelodysplastic syndromes

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1707-1712 ◽  
Author(s):  
J Lyons ◽  
JW Janssen ◽  
C Bartram ◽  
M Layton ◽  
GJ Mufti
Keyword(s):  
Myelodysplastic Syndromes ◽  
De Novo ◽  
Early Stage ◽  
Refractory Anemia ◽  
In Vivo Model ◽  
Ras Mutation ◽  
Mutational Status ◽  
Oligonucleotide Hybridization

Abstract Somatic mutation of the N-ras oncogene occurs frequently in de novo acute myeloid leukemia (AML). By virtue of their relation to AML, myelodysplastic syndromes (MDS) provide an in vivo model of human leukemogenesis. By using a strategy for analysis of gene mutation based on in vitro amplification of target sequences by the polymerase chain reaction (PCR) and selective oligonucleotide hybridization we analyzed the mutational status of codons 12, 13, and 61 of Ha-ras, K-ras, and N- ras in peripheral blood (PB) and/or bone marrow (BM) in 34 cases of primary MDS. Mutations at codon 12 of Ki-ras or N-ras were detected in three cases (9%): one of six cases of refractory anemia with excess blasts (RAEB) and two of nine cases of chronic myelomonocytic leukemia (CMML). The nucleotide substitution differed in each. In all cases the mutant allele was detectable in PB cells. A sustained hematologic remission was achieved after low-dose cytarabine therapy in the case of RAEB. Neither case of CMML exhibited signs of disease progression during follow-up at 7 and 12 months. In contrast, four of 31 patients without the ras mutation underwent transformation to AML within 12 months of genetic analysis. We conclude that ras mutations in MDS are heterogeneous and may develop at an early stage during the evolution of MDS. Their detection in PB cells illustrates the potential utility of ras mutation as a clonal marker in myeloid malignancy.

scholarly journals Immunocytochemical analysis of intermediate filaments in embryonic heart cells with monoclonal antibodies to desmin.

1984 ◽  
Vol 98 (6) ◽  
pp. 2179-2191 ◽  
Author(s):  
S I Danto ◽  
D A Fischman
Keyword(s):  
Monoclonal Antibodies ◽  
Cardiac Myocytes ◽  
Intermediate Filaments ◽  
Embryonic Heart ◽  
Immunofluorescent Staining ◽  
Heart Cells ◽  
Frozen Sections ◽  
Adult Chicken ◽  
Intermediate Filament Proteins ◽  
Filament Bundles

Monoclonal antibodies ( McAbs ) have been generated against a preparation of intermediate filament proteins (IFP) from adult chicken gizzard. Two antibodies, D3 and D76 , have been characterized in detail. They bind specifically to desmin but recognize different epitopes. In the adult chicken, both McAbs produced equivalent immunofluorescent staining patterns, reacting in frozen sections with all forms of muscle tissue, including vascular smooth muscle, but with no other tissue types. In isolated skeletal myofibrils and in longitudinal frozen sections of cardiac and skeletal muscle, desmin was detected with both McAbs at the Z-band and in longitudinally-oriented filament bundles between myofibrils. In contrast to these results in the adult, the intermediate filaments (IF) of embryonic cardiac myocytes in primary cultures were decorated only with McAb D3, whereas McAb D76 was completely unreactive with these cells. Similarly, frozen sections through the heart at early stages of embryonic chick development (Hamburger-Hamilton stages 17-18) revealed regions of myocytes, identified by double immunofluorescence with myosin-specific McAbs , that were unstained with McAb D76 even though similar regions were stained by McAb D3. That McAb D76 reacted with desmin in all adult cardiac myocytes but not with all embryonic heart cells indicates that embryonic and adult cardiac IF are immunologically distinct and implies a conversion in IF immunoreactivity during cardiac development.

scholarly journals Mutation of Ki-ras and N-ras oncogenes in myelodysplastic syndromes

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1707-1712 ◽  
Author(s):  
J Lyons ◽  
JW Janssen ◽  
C Bartram ◽  
M Layton ◽  
GJ Mufti
Keyword(s):  
Myelodysplastic Syndromes ◽  
De Novo ◽  
Early Stage ◽  
Refractory Anemia ◽  
In Vivo Model ◽  
Ras Mutation ◽  
Mutational Status ◽  
Oligonucleotide Hybridization

Somatic mutation of the N-ras oncogene occurs frequently in de novo acute myeloid leukemia (AML). By virtue of their relation to AML, myelodysplastic syndromes (MDS) provide an in vivo model of human leukemogenesis. By using a strategy for analysis of gene mutation based on in vitro amplification of target sequences by the polymerase chain reaction (PCR) and selective oligonucleotide hybridization we analyzed the mutational status of codons 12, 13, and 61 of Ha-ras, K-ras, and N- ras in peripheral blood (PB) and/or bone marrow (BM) in 34 cases of primary MDS. Mutations at codon 12 of Ki-ras or N-ras were detected in three cases (9%): one of six cases of refractory anemia with excess blasts (RAEB) and two of nine cases of chronic myelomonocytic leukemia (CMML). The nucleotide substitution differed in each. In all cases the mutant allele was detectable in PB cells. A sustained hematologic remission was achieved after low-dose cytarabine therapy in the case of RAEB. Neither case of CMML exhibited signs of disease progression during follow-up at 7 and 12 months. In contrast, four of 31 patients without the ras mutation underwent transformation to AML within 12 months of genetic analysis. We conclude that ras mutations in MDS are heterogeneous and may develop at an early stage during the evolution of MDS. Their detection in PB cells illustrates the potential utility of ras mutation as a clonal marker in myeloid malignancy.

A Histological Assessment of the Mechanism of Early-Stage Healing of a Biphasic Calcium Phosphate in an In Vivo Rabbit Model

2018 ◽  
Vol 782 ◽  
pp. 275-282
Author(s):  
Ian R. Dunkley ◽  
Scott M. Vickers ◽  
Jeffrey Badura ◽  
Jeffrey Toth
Keyword(s):  
Calcium Phosphate ◽  
Biphasic Calcium Phosphate ◽  
De Novo ◽  
Early Stage ◽  
Femoral Condyle ◽  
Rabbit Model ◽  
Complete Healing ◽  
Histological Assessment ◽  
Cortical Shell

The healing mechanism of osteoconductive biphasic calcium phosphate granules was investigated by a histological assessment of early-stage bone deposition and remodeling. The deposition of de novo bone on the scaffold granules was observed to initiate at the defect periphery by week one and in the bulk of the defect incorporating the granules by week four. New bone tissue was deposited in the space provided by the macroporosity and was observed in direct apposition to the implanted material confirming the bioactivity of the biphasic calcium phosphate. The granules were removed through a cell-mediated resorption process that was observed to begin as early as week two following surgery. Mature lamellar bone, fatty bone marrow, and vascularization was observed throughout the bulk of the defect with the cortical shell healed by week twelve. This healing mechanism was found to balance bone formation and implant resorption resulting in complete healing of the corticocancellous defect in the rabbit femoral condyle.

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