Effect Of Tissue Culture Irradiation On Orientation Of Cytoskeletal Fibers In Migrating Sheets Of Endothelial Cells

1981 ◽  
Author(s):  
A I Gotlieb
Keyword(s):  
Tissue Culture ◽  
Endothelial Cells ◽  
Immunofluorescence Microscopy ◽  
Fiber Bundles ◽  
Front Edge ◽  
Wound Edge ◽  
System A ◽  
Reduce Cell Proliferation ◽  
Made In

Irradiation of endothelial cells (EC) in tissue culture has been used to markedly reduce cell proliferation in order to study the effects of substances on the regulation of EC migration. Since irradiated EC (IRR-EC) migrate the same distance as do non-irradiated EC (NIRR-EC) over periods of up to six days, it is assumed that EC migration is not effected by irradiation. The purpose of this study was to examine the effect of irradiation on the cytoskeleton of migrating EC. The in-vitro experimental wound technique was used as a model system. A linear wound was made in confluent cultures of porcine thoracic aortic EC. EC were observed to migrate into the wound as a cohesive sheet of cells with only a few free unattached cells being present along the front edge of the wound. Cultures were irradiated one hour before wounding with 1500 rads. NIRR-EC migrated the same distance as did the IRR-EC although the latter were much flatter and each IRR-EC covered a larger surface than did the NIRR-EC. The orientation of cytoskeletal fiber bundles localized by immunofluorescence microscopy using antisera produced against electrophoretically purified porcine uterine myosin and chicken gizzard tropomyosin were different when comparing migrating IRR and NIRR-EC in the first row of the endothelial sheet. At 44 hours after wounding myosin localization showed that the main myosin stained fibers in 89% of the IRR-EC were roughly parallel to the wound edge while 11% were roughtly perpendicular to the wound edge. In NIRR-EC the figures were 54% and 46% respectively. Tropomyosin localization showed a similar diffence, 73% and 27% in IRR-EC and 45% and 55% in NIRR-EC. The data shows that there are differences in the extent of spreading of and in the orientation of cytoskeletal fiber bundles in migrating IRR-EC. These differences may reflect different cytoskeletal processes involved during migration of IRR and NIRR-EC.

scholarly journals Microtubule-organizing centers and cell migration: effect of inhibition of migration and microtubule disruption in endothelial cells.

1983 ◽  
Vol 96 (5) ◽  
pp. 1266-1272 ◽  
Author(s):  
A I Gotlieb ◽  
L Subrahmanyan ◽  
V I Kalnins
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Cytochalasin B ◽  
Leading Edge ◽  
Wound Edge ◽  
Microtubule Organizing Centers ◽  
Migration Effect ◽  
Microtubule Disruption ◽  
Made In ◽  
Experimentally Induced

We have previously shown that microtubule-organizing centers (MTOC's) become preferentially oriented towards the leading edge of migrating endothelial cells (EC's) at the margin of an experimentally induced wound made in a confluent EC monolayer. To learn more about the mechanism responsible for the reorientation of MTOC's and to determine whether a similar reorientation takes place when cell migration is inhibited, we incubated the wounded cultures with colcemid (C) and cytochalasin B (CB), which disrupt microtubules (MT's) and microfilaments (MF's), respectively. The results obtained showed that the MTOC reorientation can occur independent of cell migration since MTOC's reoriented preferentially toward the wound edge in the CB-treated cultures, even though forward migration of the EC was inhibited. In addition, the MTOC reorientation is inhibited by C, indicating that it requires an intact system of MT's and/or other intracellular structures whose distribution is dependent on that of MT's.

scholarly journals Translocation of protein tyrosine phosphatase Pez/PTPD2/PTP36 to the nucleus is associated with induction of cell proliferation

2000 ◽  
Vol 113 (17) ◽  
pp. 3117-3123 ◽  
Author(s):  
C. Wadham ◽  
J.R. Gamble ◽  
M.A. Vadas ◽  
Y. Khew-Goodall
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Nuclear Protein ◽  
Vascular Endothelial Cells ◽  
Tyrosine Phosphatase ◽  
Subcellular Localisation ◽  
Vascular Endothelial ◽  
Proliferating Cells ◽  
Wound Edge

Pez is a non-transmembrane tyrosine phosphatase with homology to the FERM (4.1, ezrin, radixin, moesin) family of proteins. The subcellular localisation of Pez in endothelial cells was found to be regulated by cell density and serum concentration. In confluent monolayers Pez was cytoplasmic, but in cells cultured at low density Pez was nuclear, suggesting that it is a nuclear protein in proliferating cells. This notion is supported by the loss of nuclear Pez when cells are serum-starved to induce quiescence, and the rapid return of Pez to the nucleus upon refeeding with serum to induce proliferation. Vascular endothelial cells normally exist as a quiescent confluent monolayer but become proliferative during angiogenesis or upon vascular injury. Using a ‘wound’ assay to mimic these events in vitro, Pez was found to be nuclear in the cells that had migrated and were proliferative at the ‘wound’ edge. TGFbeta, which inhibits cell proliferation but not migration, inhibited the translocation of Pez to the nucleus in the cells at the ‘wound’ edge, further strengthening the argument that Pez plays a role in the nucleus during cell proliferation. Together, the data presented indicate that Pez is a nuclear tyrosine phosphatase that may play a role in cell proliferation.

scholarly journals Restoring the natural tropism of AAV2 vectors for human liver

2020 ◽  
Vol 12 (560) ◽  
pp. eaba3312
Author(s):  
Marti Cabanes-Creus ◽  
Claus V. Hallwirth ◽  
Adrian Westhaus ◽  
Boaz H. Ng ◽  
Sophia H.Y. Liao ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Tissue Culture ◽  
Human Liver ◽  
Xenograft Model ◽  
Human Hepatocytes ◽  
Targeted Gene Therapy ◽  
Tissue Culture Propagation ◽  
Made In

Recent clinical successes in gene therapy applications have intensified interest in using adeno-associated viruses (AAVs) as vectors for therapeutic gene delivery. Although prototypical AAV2 shows robust in vitro transduction of human hepatocyte–derived cell lines, it has not translated into an effective vector for liver-directed gene therapy in vivo. This is consistent with observations made in Fah−/−/Rag2−/−/Il2rg−/− (FRG) mice with humanized livers, showing that AAV2 functions poorly in this xenograft model. Here, we derived naturally hepatotropic AAV capsid sequences from primary human liver samples. We demonstrated that capsid mutations, likely acquired as an unintentional consequence of tissue culture propagation, attenuated the intrinsic human hepatic tropism of natural AAV2 and related human liver AAV isolates. These mutations resulted in amino acid changes that increased binding to heparan sulfate proteoglycan (HSPG), which has been regarded as the primary cellular receptor mediating AAV2 infection of human hepatocytes. Propagation of natural AAV variants in vitro showed tissue culture adaptation with resulting loss of tropism for human hepatocytes. In vivo readaptation of the prototypical AAV2 in FRG mice with a humanized liver resulted in restoration of the intrinsic hepatic tropism of AAV2 through decreased binding to HSPG. Our results challenge the notion that high affinity for HSPG is essential for AAV2 entry into human hepatocytes and suggest that natural AAV capsids of human liver origin are likely to be more effective for liver-targeted gene therapy applications than culture-adapted AAV2.

An Automatic Suturing Machine for Intestinal Anastomosis: Advantages Compared With Hand-Suturing Technique

2018 ◽  
Vol 26 (2) ◽  
pp. 209-218 ◽  
Author(s):  
Sanaz Mosafer Khoorjestan ◽  
Gholamreza Rouhi
Keyword(s):  
Mechanical Strength ◽  
Intestinal Anastomosis ◽  
Tensile Tests ◽  
Point Of View ◽  
In Vitro Tests ◽  
System A ◽  
Expert Surgeon ◽  
Suturing Methods ◽  
Made In

One of the main procedures in intestinal surgery is anastomosis, which is mostly performed by stapling or hand suturing. Due to limitations of these methods, a novel automatic suturing machine was designed and fabricated in this study, equipped with a needle-driving system; a thread control mechanism, and a linear mechanism, which is applicable in intestinal anastomosis by making continuous sutures. The main advantages of the fabricated machine are employing biocompatible suture, from the tissue’s adaptation point of view, and making a uniform suturing pattern, independent of surgeon’s skill, and thus offering a greater strength than the hand-sutured specimen. In order to evaluate the capability of the fabricated machine and investigate the validity of the hypothesis made in this study, that is, a more uniform suture will result in a greater mechanical strength of the sutured tissue, in vitro tests were performed on human intestine specimens, which were manually sutured by an expert surgeon and by the automatic suturing machine. The tensile tests with an elongation rate of 5 mm/min were done for 90 specimens, in 9 groups with various suturing configurations. The optimum pattern, from the mechanical strength point of view, was found to be the same in both manual and automatic suturing methods, that is, h7 d6 ( h = distance of suture from the edge of the tissue = 7 mm, and d = distance between stitches = 6 mm). It was also shown that the maximum breaking strength, for the best suturing pattern, h7 d6, is significantly greater when the automatic suturing machine was employed, compared with the hand-sutured tissue ( P < .001).

scholarly journals In Vitro Tissue Culture in Brachypodium: Applications and Challenges

2020 ◽  
Vol 21 (3) ◽  
pp. 1037 ◽  
Author(s):  
Alexander Betekhtin ◽  
Karolina Hus ◽  
Magdalena Rojek-Jelonek ◽  
Ewa Kurczynska ◽  
Candida Nibau ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Transformation Efficiency ◽  
Brachypodium Distachyon ◽  
Low Frequency ◽  
Transformation Techniques ◽  
Bioenergy Grasses ◽  
Embryogenic Potential ◽  
Gradual Loss ◽  
Made In

Brachypodium distachyon has become an excellent model for plant breeding and bioenergy grasses that permits many fundamental questions in grass biology to be addressed. One of the constraints to performing research in many grasses has been the difficulty with which they can be genetically transformed and the generally low frequency of such transformations. In this review, we discuss the contribution that transformation techniques have made in Brachypodium biology as well as how Brachypodium could be used to determine the factors that might contribute to transformation efficiency. In particular, we highlight the latest research on the mechanisms that govern the gradual loss of embryogenic potential in a tissue culture and propose using B. distachyon as a model for other recalcitrant monocots.

scholarly journals Microvascular pericytes contain muscle and nonmuscle actins.

1985 ◽  
Vol 101 (1) ◽  
pp. 43-52 ◽  
Author(s):  
I M Herman ◽  
P A D'Amore
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Stress Fibers ◽  
Fiber Bundles

We have affinity-fractionated rabbit antiactin immunoglobulins (IgG) into classes that bind preferentially to either muscle or nonmuscle actins. The pools of muscle- and nonmuscle-specific actin antibodies were used in conjunction with fluorescence microscopy to characterize the actin in vascular pericytes, endothelial cells (EC), and smooth muscle cells (SMC) in vitro and in situ. Nonmuscle-specific antiactin IgG stained the stress fibers of cultured EC and pericytes but did not stain the stress fibers of cultured SMC, although the cortical cytoplasm associated with the plasma membrane of SMC did react with nonmuscle-specific antiactin. Whereas the muscle-specific antiactin IgG failed to stain EC stress fibers and only faintly stained their cortical cytoplasm, these antibodies reacted strongly with the fiber bundles of cultured SMC and pericytes. Similar results were obtained in situ. The muscle-specific antiactin reacted strongly with the vascular SMC of arteries and arterioles as well as with the perivascular cells (pericytes) associated with capillaries and post-capillary venules. The non-muscle-specific antiactin stained the endothelium and the pericytes but did not react with SMC. These findings indicate that pericytes in culture and in situ possess both muscle and nonmuscle isoactins and support the hypothesis that the pericyte may represent the capillary and venular correlate of the SMC.

scholarly journals Photoautotrophic System: A Review and Potential Application for Plant Propagation In Vitro

2018 ◽  
Vol 5 (2) ◽  
pp. 73-78
Author(s):  
Krisantini Krisantini ◽  
Ni Made Armini Wiendi
Keyword(s):  
Tissue Culture ◽  
Culture Media ◽  
Co2 Enrichment ◽  
Advantages And Disadvantages ◽  
System A ◽  
Leaf Chlorophyll ◽  
Micro Propagation ◽  
Agar Media ◽  
Propagation Methods

AbstractThe standard method of in vitro plant micro propagation uses of tightly closed culture bottles using agar media containing macro and micro nutrients and sucrose as a source of carbon for the explants. The closed bottle culture is usually kept in a temperature and light controlled environment which is lower and of different quality from the natural sunlight, resulting in high relative humidity and no air exchange inside the bottles.  Explants produced in vitro have malfunctioned stomata, undeveloped cuticles and lower leaf chlorophyll levels, and hyper hydration of the plantlets. Photoautotrophic tissue culture is micro propagation without or with a reduced sugar level in the culture media, so the growth or accumulation of carbohydrates of the explants is dependent fully upon photosynthesis and inorganic nutrient uptake. This method is usually combined with ventilation or CO2 enrichment, and recently, with incorporating porous materials such as vermiculite, gum or paper pulp to the agar media to promote better root system of the explants. This article discuss the advantages and disadvantages of the photoautotrophic micro propagation compared to the standard micro propagation methods, and provided the results of the photo autotrophic micro propagation studies conducted at Laboratory of Tissue Culture II of the Department of Agronomy and Horticulture, Bogor Agricultural University, Indonesia.

Characteristics of human optic gliomas in tissue culture

1977 ◽  
Vol 46 (1) ◽  
pp. 78-84 ◽  
Author(s):  
Robert L. Martuza ◽  
Paul L. Kornblith ◽  
Theodore M. Liszczak
Keyword(s):  
Tissue Culture ◽  
Bipolar Cells ◽  
Fiber Bundles ◽  
Rosenthal Fibers ◽  
Content Type ◽  
Cellular Processes ◽  
Dense Deposits ◽  
Well Differentiated

✓ Nine human optic gliomas were examined in tissue culture. Typically, growth from the explants revealed well differentiated bipolar cells with abundant 9 to 10 nm fibers similar to those observed in the surgical specimens. Multinucleation was rare except for one culture, which had as many as 20 nuclei arranged in a palisading fashion along the periphery of some of the cells. Degenerative changes of the 9 to 10 nm fiber bundles with the production of amorphous electron-dense deposits were observed both in vivo and in vitro, and were thought to represent the formation of Rosenthal fibers. A distinctive feature of some of the optic gliomas was the ability of their long, thin cellular processes to form fibrous tangles in tissue culture. The correlation of these fibrous tangles in culture with Rosenthal fibers in vivo is still uncertain.

The distribution of microfilament bundles in rabbit endothelial cells in the intact aorta and during wound healing in situ

1989 ◽  
Vol 67 (9) ◽  
pp. 553-562 ◽  
Author(s):  
Kem A. Rogers ◽  
Martin Sandig ◽  
Nancy H. McKee ◽  
Vitauts I. Kalnins
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Blood Flow ◽  
Vessel Wall ◽  
Balloon Catheter ◽  
Wound Edge ◽  
Microfilament Bundles

The distribution of microfilament (MF) bundles in rabbit thoracic aortic endothelial cells (EC) fixed in situ was examined using en face preparations and the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In the normal aorta, prominent peripheral MF bundles are seen near the cell borders running the full length of each cell, parallel to the direction of blood flow, while shorter less prominent bundles are seen in the more central regions. In EC covering the flow dividers at intercostal ostia, the central MF bundles are more prominent, longer, and more numerous than in the other regions of the aorta examined. This increase in the number, size, and length of central MF bundles may result from the response of the cells to the higher shear forces present in this region of the vessel wall. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, there is an initial reduction in the size of all of the MF bundles in cells near the wound edge. This is followed by an increase in the number and size of the central MF bundles. At 48 h after wounding, strongly stained central MF bundles could be detected in EC up to 0.75 mm from the wound edge. Adjacent to the wounds that had failed to reendothelialize 10 months after denudation, some regions had EC with prominent peripheral MF bundles and others, EC with prominent central MF bundles. At the very edge of the wound, the EC and their MF bundles were oriented with their long axes parallel to the wound edge and perpendicular to the direction of blood flow. The failure of the wounded vessel wall to become fully reendothelialized may be related to the orientation of EC at the wound edge. These results show that EC migration in situ is accompanied by a dramatic change in the organization of MF in which different stages can be identified. Microfilament bundles in rapidly migrating cells in vivo, 24 and 48 h after wounding, resemble stress fibers seen in EC migrating in vitro and in slowly migrating fibroblasts and epithelial cells.Key words: microfilaments, in situ, endothelium, wound healing, aorta.

scholarly journals Cytochalasin B inhibition of endothelial proliferation at wound edges in vitro.

1979 ◽  
Vol 81 (2) ◽  
pp. 348-354 ◽  
Author(s):  
S C Selden ◽  
S M Schwartz
Keyword(s):  
Tissue Culture ◽  
Endothelial Cells ◽  
Cytochalasin B ◽  
Background Rate ◽  
Low Background ◽  
Endothelial Proliferation

Cytochalasin B prevents both migration and wound-associated replication when applied to wounded monolayers of bovine endothelium in tissue culture. The normal low background rate of replication in undisturbed areas of the cultures is not inhibited by cytochalasin B. These results suggest that some form of movement may be required for initiation of wound-associated replication by endothelial cells.

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