Tetrazolium Salt and Electron-Microscopic Studies of Cellular Degeneration and Necrosis in the Interdigital Areas of the Developing Chick Limb

1971 ◽  
Vol 8 (1) ◽  
pp. 229-251
Author(s):  
S. P. HAMMAR ◽  
N. K. MOTTET
Keyword(s):  
Cell Death ◽  
Enzyme Activity ◽  
Succinate Dehydrogenase ◽  
Hind Limb ◽  
Morphological Changes ◽  
Morphological Evidence ◽  
Tetrazolium Salt ◽  
Limb Buds ◽  
Cellular Degeneration ◽  
Intracellular Digestion

Cellular degeneration and necrosis were studied in the interdigital areas of developing hind limb buds of normal chick embryos by means of enzyme-specific tetrazolium salts and electron microscopy. Using succinic acid as a substrate and the tetrazolium salt, nitro blue tetrazolium, succinate dehydrogenase was specifically demonstrated via a colour reaction in which degenerating and necrotic cells--those with no enzyme activity--did not stain, while those with enzyme activity stained deeply blue-black. The interdigital cells exhibited near absent levels of succinate dehydrogenase as early as stage 26-27, one to two days prior to when morphological evidence of degeneration and necrosis was present. It was postulated that the mechanism of cell death resulted from decreased activity and/or loss of strategic cellular enzymes such as succinate dehydrogenase, with a subsequent fall in the cellular adenosine triphosphate (ATP) level and a resultant compromise in vital cellular processes, eventually leading to cell death. The evidence indicated that ‘biochemical degeneration’ occurred prior to morphological changes in cells. The ultrastructural events of degenerating and necrotic cells were also studied. Most ‘dying’ cells observed had already been phagocytosed and were observed in various stages of degeneration. The degenerating cells exhibited both nuclear and cytoplasmic changes. There was evidence of active intracellular digestion within the phagocytes. Numerous lysosomes were observed within these cells, and some appeared fused with the digestive vacuole's membranes. The origin of the phagocytes was not determined. No definite information was obtained concerning the utility of cellular degeneration and necrosis in the interdigital areas of the hind limb buds. It appeared to be at least partially causal in separation of the digits.

Ultrastructural Evidences of Caspase-Dependent Platelet Generation in ES Cell-Differentiation System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3621-3621
Author(s):  
Yumiko Matsubara ◽  
Mitsuru Murata ◽  
Hidenori Suzuki ◽  
Tamihiro Kamata ◽  
Aya Shimizu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Death ◽  
Morphological Changes ◽  
Es Cells ◽  
Chromatin Condensation ◽  
Embryonic Stem ◽  
Morphological Evidence ◽  
Hematopoietic Stem ◽  
Platelet Production ◽  
Sequential Experiments

Abstract Platelets are essential for thrombosis and hemostasis, and the elucidating the unique mechanism for platelet production from megakaryocyte is of major importance. However, extensive data on platelet generation have yet to be fully documented because it has been extremely difficult to obtain the sufficient amounts of hematopoietic stem cells. In this study, we therefore used murine embryonic stem (ES) cells that can proliferate and differentiate to megakaryocyte in the presence of thrombopoietin in vitro. ES-derived megakaryocytes and platelets were studied in detail by morphological analyses, and we especially focused on the relationship between cell death of megakaryocytes and platelet production in sequential experiments. A coculture system with ES/OP9 cells, stromal cells derived from M-CSF deficient mice, in the presence of thrombopoietin was used to differentiate mesodermal-like hematopoietic progenitors, immature megakaryocytes, mature megakaryocytes, and functional platelets from ES cells on days 5, 8, 12, and 15 of the culture, respectively, and the peak of cell count was observed on the day 12 for megakaryocytes and the day 15 for platelets. These were confirmed by morphology, Wright-Giemsa staining, CD41expression or fibrinogen binding assays. Interestingly, electron microscopy and immuno-electron microscopy during platelet production showed morphological changes supported by both “proplatelet theory” and “explosive fragmentation theory”, which have been controversial issues related to platelet biogenesis. On the days 8 and 12, megakaryocyte with tube-like (proplatelet-like) extensions of the periphery showed the expression of CD41, CD42c, or von Willebrand factor, in contrast, megakaryocyte-like large cells with smooth periphery had no expression of CD41 and CD42c. On the days 12 and 15, global fragmentation of megakaryocyte cytoplasm into individual platelets was frequently observed. For cell death of megakaryocytes, cells exhibit clear morphological evidence of nuclear change: chromatin condensation, typical of early apoptosis, on the day 8 and extensive condensation and apoptotic nucleus surrounded by a shortrim of cytoplasm in continuity with a portion of granulated cytoplasm on the days 12 and 15. These proceedings of cell death were confirmed by the TUNEL assay in each stage. Also, low production of platelet was observed by adding Z-DEVD-fmk, an inhibitor of caspases −3 and −7, on the day 8. Next, we examined the caspase activation in the different stage of the platelet production by western blotting, and anti-CD41 antibody was used to test the purity of the meg-lineage cells in each stage. Peak expressions of activated caspases −12, −9, and −7 or caspase 10 levels were observed on the day 8. Peak expression of activated caspase 3 was observed on the day 12. On the contrary, no different levels of caspase 6 were shown between the days 8 and 15. Together, present studies in the sequential experiments for megakaryocyte differentiation and platelet production develop previous theories for platelet generation and also suggest that the process for platelet production is focally associated with caspases −12, −10, −9, −7, and −3 dependent cell death of mrgakaryocytes.

scholarly journals The Sensitizer 2,4-Dinitrofluorobenzene Activates Caspase-3 and Induces Cell Death in a Skin Dendritic Cell Line

2003 ◽  
Vol 22 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Maria Teresa Cruz ◽  
Carlos B. Duarte ◽  
Margarida Gonçalo ◽  
Américo Figueiredo ◽  
Arsélio P. Carvalho ◽  
...  
Keyword(s):  
Cell Death ◽  
Dendritic Cell ◽  
Cell Line ◽  
Caspase 3 ◽  
Morphological Changes ◽  
Mouse Skin ◽  
Annexin V ◽  
Tetrazolium Salt ◽  
Mtt Reduction

In this work, a dendritic cell line derived from mouse skin (FSDC) was used, as an in vitro experimental model, to evaluate the cytotoxic effect of two chemical sensitizers, a strong sensitizer (2,4-dinitrofluorobenzene, DNFB) and a weak sensitizer (2,4-dichloronitrobenzene, DCNB). The results indicated that DNFB reduces the cellular metabolism of FSDC, as evaluated by the reduction of the tetrazolium salt, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). All the DNFB concentrations tested, ranging from 5.2 μ M to 26 μM, significantly inhibited the MTT reduction after 1 hour of cell exposure to the sensitizer. In contrast, incubation of FSDC with the weak sensitizer DCNB had no significant effect on the MTT reduction assay. When the cells were incubated with DNFB (13 μ M), for 3 and 6 hours, morphological changes characteristics of cell death by apoptosis were observed, as assessed by propidium iodide (PI) DNA staining and annexin-V externalization analysis. These results correlate well with an increase of caspase-3-like activity after FSDC exposure to DNFB (13 μM) for 6 hours. Together, these results indicate that apoptotic death of skin dendritic cells occurs after exposure to the sensitizer DNFB, although necrotic cell death was also observed when the cells were incubated with high concentrations of DNFB (26 μM), or after long periods of cell exposure to the chemical DNFB (13 μM, for 6 hours).

Ultrastructural Study of a differentiating human colon carcinoma cell line

1990 ◽  
Vol 48 (3) ◽  
pp. 208-209
Author(s):  
Sylvie Polak-Charcon ◽  
Mehrdad Hekmati ◽  
Yehuda Ben Shaul
Keyword(s):  
Cell Line ◽  
Morphological Changes ◽  
Secretory Granules ◽  
Human Colon ◽  
Cell Types ◽  
Culture Conditions ◽  
Morphological Evidence ◽  
Human Colon Carcinoma Cell ◽  
Colon Cell

The epithelium of normal human colon mucosa “in vivo” exhibits a gradual pattern of differentiation as undifferentiated stem cells from the base of the crypt of “lieberkuhn” rapidly divide, differentiate and migrate toward the free surface. The major differentiated cell type of the intestine observed are: absorptive cells displaying brush border, goblet cells containing mucous granules, Paneth and endocrine cells containing dense secretory granules. These different cell types are also found in the intestine of the 13-14 week old embryo.We present here morphological evidence showing that HT29, an adenocarcinoma of the human colon cell line, can differentiate into various cell types by changing the growth and culture conditions and mimic morphological changes found during development of the intestine in the human embryo.HT29 cells grown in tissue-culture dishes in DMEM and 10% FCS form at late confluence a multilayer of morphologically undifferentiated cell culture covered with irregular microvilli, and devoid of tight junctions (Figs 1-3).

Oxidative Stress, Ocular Disease and Diabetes Retinopathy

2019 ◽  
Vol 24 (40) ◽  
pp. 4726-4741 ◽  
Author(s):  
Orathai Tangvarasittichai ◽  
Surapon Tangvarasittichai
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Cell Death ◽  
Protein Modification ◽  
Morphological Changes ◽  
Corneal Dystrophy ◽  
Age Related Macular Degeneration ◽  
Ocular Diseases ◽  
Retinal Light Damage ◽  
Age Related

Background: Oxidative stress is caused by free radicals or oxidant productions, including lipid peroxidation, protein modification, DNA damage and apoptosis or cell death and results in cellular degeneration and neurodegeneration from damage to macromolecules. Results: Accumulation of the DNA damage (8HOdG) products and the end products of LPO (including aldehyde, diene, triene conjugates and Schiff’s bases) were noted in the research studies. Significantly higher levels of these products in comparison with the controls were observed. Oxidative stress induced changes to ocular cells and tissues. Typical changes include ECM accumulation, cell dysfunction, cell death, advanced senescence, disarrangement or rearrangement of the cytoskeleton and released inflammatory cytokines. It is involved in ocular diseases, including keratoconus, Fuchs endothelial corneal dystrophy, and granular corneal dystrophy type 2, cataract, age-related macular degeneration, primary open-angle glaucoma, retinal light damage, and retinopathy of prematurity. These ocular diseases are the cause of irreversible blindness worldwide. Conclusions: Oxidative stress, inflammation and autophagy are implicated in biochemical and morphological changes in these ocular tissues. The development of therapy is a major target for the management care of these ocular diseases.

scholarly journals Sublethal Injury and Resuscitation of Candida albicans after Amphotericin B Treatment

2003 ◽  
Vol 47 (4) ◽  
pp. 1200-1206 ◽  
Author(s):  
Robert S. Liao ◽  
Robert P. Rennie ◽  
James A. Talbot
Keyword(s):  
Cell Death ◽  
Candida Albicans ◽  
Amphotericin B ◽  
Antifungal Agents ◽  
Sybr Green ◽  
Sequential Treatment ◽  
Tetrazolium Salt ◽  
Fungal Cell ◽  
Sublethal Injury

ABSTRACT Amphotericin B treatment was previously shown to inhibit Candida albicans reproduction and reduce the fluorescence of vitality-specific dyes without causing a corresponding increase in the fluorescence of the mortality-specific dyes bis-(1,3-dibutylbarbituric acid)trimethine oxonol and SYBR Green Ι. In the present study, we have confirmed these results and have shown that the numbers of CFU are reduced by 99.9% by treatment with 0.5 μg of amphotericin B per ml for 10 h at 35°C. This reduction was not due to fungal cell death. First, the level of reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide increased in the presence of concentrations of amphotericin B that caused greater than 90% reductions in the numbers of CFU. Second, fungal cells treated with amphotericin B at a concentration of 0.5 μg/ml were resuscitated by further incubation at 22°C for 15 h in the continued presence of amphotericin B. Third, recovery of the ability to replicate was prevented by sequential treatment with 20 μg of miconazole per ml, which also increased the fluorescence of mortality-specific dyes to near the maximal levels achieved with 0.9 μg of amphotericin B per ml. Sequential treatment with fluconazole and flucytosine did not increase the levels of staining with the mortality-specific dyes. Itraconazole was less effective than ketoconazole, which was less effective than miconazole. The practice of equating the loss of the capacity of C. albicans to form colonies with fungal cell death may give incorrect results in assays with amphotericin B, and the results of assays with caution with other antifungal agents that are lipophilic or that possess significant postantifungal effects may need to be interpreted.

scholarly journals Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

2021 ◽  
Vol 22 (15) ◽  
pp. 7906
Author(s):  
Alexey A. Komissarov ◽  
Maria A. Karaseva ◽  
Marina P. Roschina ◽  
Andrey V. Shubin ◽  
Nataliya A. Lunina ◽  
...  
Keyword(s):  
Cell Death ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Proteolytic Enzymes ◽  
Morphological Changes ◽  
Human Cells ◽  
Mitochondrial Potential ◽  
3C Protease ◽  
Wide Range

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

Motor projection patterns to the hind limb of normal and paralysed chick embryos

Development ◽  
1982 ◽  
Vol 72 (1) ◽  
pp. 269-286
Author(s):  
N. G. Laing
Keyword(s):  
Spinal Cord ◽  
Cell Death ◽  
Hind Limb ◽  
Muscle Volume ◽  
Chick Embryos ◽  
Relative Contribution ◽  
Hind Limbs ◽  
Lateral Motor Column ◽  
Before And After ◽  
Major Period

Counts were made of the number of motoneurons innervating the hind limbs of 10-day normal and paralysed chick embryos whose right hind limb buds had been subjected to varying degrees of amputation prior to innervation. The number of motoneurons on the intact sides of the paralysed embryos was found to be similar to the number present in normal embryos prior to the major period of motoneuron death. Since it has previously been shown that paralysis does not increase the number of motoneurons generated, this means that normal motoneuron death was largely prevented in the paralysed embryos. There were differences in the distributions of motoneurons in the rostrocaudal axis of the spinal cord between normal and paralysed embryos. Therefore, cell death does not eliminate a uniform fraction of motoneurons throughout the rostrocaudal extent of the chick embryo lumbar lateral motor column. It is also argued that there are differences in the relative contribution of the various lumbosacral levels to different parts of the limb, e.g. the shank, before and after the period of cell death. In both normal and paralysed embryos there was a linear relationship between the volume of limb muscle which developed after amputation and the number of motoneurons surviving in the spinal cord. There was no evidence of a ‘compression’ of motoneurons into the remaining muscle either after amputation alone or after amputation combined with paralysis. Motoneurons are therefore rigidly specified for certain parts of the limb. The relationship between motoneuron number and muscle volume on the amputated side differed from that of the intact side. For a similar increase in muscle volume there was a smaller increase in motoneuron number on the intact sides. This suggested a parallel to the paradoxically small increase in motoneuron number that occurs on the addition of a supernumerary limb.

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