scholarly journals Ca2+-dependent nuclease is involved in DNA degradation during the formation of the secretory cavity by programmed cell death in fruit of Citrus grandis ‘Tomentosa’

2020 ◽  
Vol 71 (16) ◽  
pp. 4812-4827 ◽  
Author(s):  
Mei Bai ◽  
Minjian Liang ◽  
Bin Huai ◽  
Han Gao ◽  
Panpan Tong ◽  
...  
Keyword(s):  
Cell Death ◽  
In Situ Hybridization ◽  
Programmed Cell Death ◽  
Nuclear Dna ◽  
Expression Patterns ◽  
Citrus Fruit ◽  
Dna Degradation ◽  
Secretory Cavity ◽  
Spatio Temporal

Abstract The secretory cavity is a typical structure in Citrus fruit and is formed by schizolysigeny. Previous reports have indicated that programmed cell death (PCD) is involved in the degradation of secretory cavity cells in the fruit, and that the spatio-temporal location of calcium is closely related to nuclear DNA degradation in this process; however, the molecular mechanisms underlying this Ca2+ regulation remain largely unknown. Here, we identified CgCaN that encodes a Ca2+-dependent DNase in the fruit of Citrus grandis ‘Tomentosa’, the function of which was studied using calcium ion localization, DNase activity assays, in situ hybridization, and protein immunolocalization. The results suggested that the full-length cDNA of CgCaN contains an ORF of 1011 bp that encodes a protein 336 amino acids in length with a SNase-like functional domain. CgCaN digests dsDNA at neutral pH in a Ca2+-dependent manner. In situ hybridization signals of CgCaN were particularly distributed in the secretory cavity cells. Ca2+ and Ca2+-dependent DNases were mainly observed in the condensed chromatin and in the nucleolus. In addition, spatio-temporal expression patterns of CgCaN and its protein coincided with the time-points that corresponded to chromatin degradation and nuclear rupture during the PCD in the development of the fruit secretory cavity. Taken together, our results suggest that Ca2+-dependent DNases play direct roles in nuclear DNA degradation during the PCD of secretory cavity cells during Citrus fruit development. Given the consistency of the expression patterns of genes regulated by calmodulin (CaM) and calcium-dependent protein kinases (CDPK) and the dynamics of calcium accumulation, we speculate that CaM and CDPK proteins might be involved in Ca2+ transport from the extracellular walls through the cytoplasm and into the nucleus to activate CgCaN for DNA degradation.

scholarly journals Zn2+-Dependent Nuclease Is Involved in Nuclear Degradation during the Programmed Cell Death of Secretory Cavity Formation in Citrus grandis ‘Tomentosa’ Fruits

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3222
Author(s):  
Minjian Liang ◽  
Mei Bai ◽  
Hong Wu
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Late Stage ◽  
Nuclear Dna ◽  
Dna Degradation ◽  
Secretory Cells ◽  
Cavity Formation ◽  
Secretory Cavity ◽  
Cytochemical Localization ◽  
Nuclear Degradation

Zn2+- and Ca2+-dependent nucleases exhibit activity toward dsDNA in the four classes of cation-dependent nucleases in plants. Programmed cell death (PCD) is involved in the degradation of cells during schizolysigenous secretory cavity formation in Citrus fruits. Recently, the Ca2+-dependent DNase CgCAN was proven to play a key role in nuclear DNA degradation during the PCD of secretory cavity formation in Citrus grandis ‘Tomentosa’ fruits. However, whether Zn2+-dependent nuclease plays a role in the PCD of secretory cells remains poorly understood. Here, we identified a Zn2+-dependent nuclease gene, CgENDO1, from Citrus grandis ‘Tomentosa’, the function of which was studied using Zn2+ ions cytochemical localization, DNase activity assays, in situ hybridization, and protein immunolocalization. The full-length cDNA of CgENDO1 contains an open reading frame of 906 bp that encodes a protein 301 amino acids in length with a S1/P1-like functional domain. CgENDO1 degrades linear double-stranded DNA at acidic and neutral pH. CgENDO1 is mainly expressed in the late stage of nuclear degradation of secretory cells. Further spatiotemporal expression patterns of CgENDO1 showed that CgENDO1 is initially located on the endoplasmic reticulum and then moves into intracellular vesicles and nuclei. During the late stage of nuclear degradation, it was concentrated in the area of nuclear degradation involved in nuclear DNA degradation. Our results suggest that the Zn2+-dependent nuclease CgENDO1 plays a direct role in the late degradation stage of the nuclear DNA in the PCD of secretory cavity cells of Citrus grandis ‘Tomentosa’ fruits.

Assignment1 of the programmed cell death 4 gene (PDCD4) to human chromosome band 10q24 by in situ hybridization

1999 ◽  
Vol 87 (1-2) ◽  
pp. 113-114 ◽  
Author(s):  
H. Soejima ◽  
O. Miyoshi ◽  
H. Yoshinaga ◽  
Z. Masaki ◽  
I. Ozaki ◽  
...  
Keyword(s):  
Cell Death ◽  
In Situ Hybridization ◽  
Programmed Cell Death ◽  
Human Chromosome ◽  
Chromosome Band ◽  
Programmed Cell Death 4 ◽  
Human Chromosome Band

Genetic Loci Controlling Lethal Cell Death in Tomato Caused by Viral Satellite RNA Infection

2012 ◽  
Vol 25 (8) ◽  
pp. 1034-1044 ◽  
Author(s):  
Ping Xu ◽  
Hua Wang ◽  
Frank Coker ◽  
Jun-ying Ma ◽  
Yuhong Tang ◽  
...  
Keyword(s):  
Cell Death ◽  
In Situ Hybridization ◽  
Programmed Cell Death ◽  
Minus Strand ◽  
Satellite Rna ◽  
Dominant Trait ◽  
Systemic Necrosis ◽  
Backcross Population ◽  
Major Qtl

Cucumber mosaic virus (CMV) associated with D satellite RNA (satRNA) causes lethal systemic necrosis (LSN) in tomato (Solanum lycopersicum), which involves programmed cell death. No resistance to this disease has been found in tomato. We obtained a line of wild tomato, S. habrochaitis, with a homogeneous non-lethal response (NLR) to the infection. This line of S. habrochaitis was crossed with tomato to generate F1 plants that survived the infection with NLR, indicating that NLR is a dominant trait. The NLR trait was successfully passed on to the next generation. The phenotype and genotype segregation was analyzed in the first backcross population. The analyses indicate that the NLR trait is determined by quantitative trait loci (QTL). Major QTL associated with the NLR trait were mapped to chromosomes 5 and 12. Results from Northern blot and in situ hybridization analyses revealed that the F1 and S. habrochaitis plants accumulated minus-strand satRNA more slowly than tomato, and fewer vascular cells were infected. In addition, D satRNA-induced LSN in tomato is correlated with higher accumulation of the minus-strand satRNA compared with the accumulation of the minus strand of a non-necrogenic mutant D satRNA.

scholarly journals Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.

1992 ◽  
Vol 119 (3) ◽  
pp. 493-501 ◽  
Author(s):  
Y Gavrieli ◽  
Y Sherman ◽  
S A Ben-Sasson
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Dna Fragmentation ◽  
Nuclear Dna ◽  
Nuclear Periphery ◽  
Lymphoid Tissues ◽  
Pooled Dna ◽  
Nuclear Dna Fragmentation ◽  
Small And Large Intestine

Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy-transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.

scholarly journals Expression of epiregulin and amphiregulin in the rat ovary

2004 ◽  
Vol 33 (1) ◽  
pp. 281-291 ◽  
Author(s):  
T Sekiguchi ◽  
T Mizutani ◽  
K Yamada ◽  
T Kajitani ◽  
T Yazawa ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Expression Patterns ◽  
Mrna Levels ◽  
Rat Ovary ◽  
Cox 2 ◽  
Spatio Temporal ◽  
Egf Family

We have previously reported that the epidermal growth factor (EGF) family growth factor, epiregulin, is expressed in rat ovarian granulosa cells by induction with pregnant mare serum gonadotropin (PMSG). In this study, we report that amphiregulin, another member of the EGF family, was also induced in the rat ovary by gonadotropin treatment. Northern blot analysis revealed that PMSG treatment induced the expression of both epiregulin and amphiregulin mRNA after 24 h, but the expression then decreased 48 h after treatment. Further treatment with human chorionic gonadotropin (hCG) rapidly induced the expression of both epiregulin and amphiregulin genes and maximal levels were reached 4 h after hCG treatment. A marginal increase in amphiregulin mRNA levels was also observed 6 h after PMSG treatment. In situ hybridization revealed that epiregulin and amphiregulin mRNAs were localized in the granulosa cells of large antral follicles. These spatio-temporal expression patterns were similar to those of cyclo-oxygenase-2 (COX-2) and progesterone receptor (PR). In adult cycling rats, epiregulin and amphiregulin were strongly induced at 1800 and 2000 h on proestrus coinciding with the preovulatory LH surge. An in situ hybridization study also showed that epiregulin and amphiregulin mRNAs were detectable in the granulosa cells of preovulatory ovarian follicles at 2000 h on proestrus, where transcripts of COX-2 and PR were co-localized with those of epiregulin and amphiregulin. These observations suggested that the EGF family members, epiregulin and amphiregulin, may play a role in the ovulatory process of cycling rats as well as in the induction of ovulation in immature rats.

scholarly journals Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis.

1994 ◽  
Vol 127 (6) ◽  
pp. 1717-1727 ◽  
Author(s):  
S Estus ◽  
W J Zaks ◽  
R S Freeman ◽  
M Gruda ◽  
R Bravo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
In Situ Hybridization ◽  
Programmed Cell Death ◽  
Sympathetic Neurons ◽  
Genetic Program ◽  
Nuclear Staining ◽  
Initial Stimulus ◽  
Induced Genes

We have examined the hypothesis that neuronal programmed cell death requires a genetic program; we used a model wherein rat sympathetic neurons maintained in vitro are deprived of NGF and subsequently undergo apoptosis. To evaluate gene expression potentially necessary for this process, we used a PCR-based technique and in situ hybridization; patterns of general gene repression and selective gene induction were identified in NGF-deprived neurons. A temporal cascade of induced genes included "immediate early genes," which were remarkable in that their induction occurred hours after the initial stimulus of NGF removal and the synthesis of some required ongoing protein synthesis. The cascade also included the cell cycle gene c-myb and the genes encoding the extracellular matrix proteases transin and collagenase. Concurrent in situ hybridization and nuclear staining revealed that while c-jun was induced in most neurons, c-fos induction was restricted to neurons undergoing chromatin condensation, a hallmark of apoptosis. To evaluate the functional role of the proteins encoded by these genes, neutralizing antibodies were injected into neurons. Antibodies specific for either c-Jun or the Fos family (c-Fos, Fos B, Fra-1, and Fra-2) protected NGF-deprived neurons from apoptosis, whereas antibodies specific for Jun B, Jun D, or three nonimmune antibody preparations had no protective effect. Because these induced genes encode proteins ranging from a transcription factor necessary for death to proteases likely involved in tissue remodeling concurrent with death, these data may outline a genetic program responsible for neuronal programmed cell death.

scholarly journals Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).

1993 ◽  
Vol 12 (1) ◽  
pp. 371-377 ◽  
Author(s):  
M.C. Peitsch ◽  
B. Polzar ◽  
H. Stephan ◽  
T. Crompton ◽  
H.R. MacDonald ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Nuclear Dna ◽  
Dna Degradation

scholarly journals RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

2021 ◽  
Vol 4 (1) ◽  
pp. 20
Author(s):  
Mujeeb Shittu ◽  
Tessa Steenwinkel ◽  
William Dion ◽  
Nathan Ostlund ◽  
Komal Raja ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Expression Patterns ◽  
Drosophila Species ◽  
Gene Expression Patterns ◽  
Probe Design ◽  
Tissue Preparation ◽  
Design And Synthesis ◽  
Rna In Situ Hybridization

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

scholarly journals Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lenka Ulrychová ◽  
Pavel Ostašov ◽  
Marta Chanová ◽  
Michael Mareš ◽  
Martin Horn ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Schistosoma Mansoni ◽  
Rna Sequencing ◽  
Serine Proteases ◽  
Expression Patterns ◽  
High Sensitivity ◽  
Host Parasite Interactions ◽  
Highly Sensitive ◽  
Host Parasite

Abstract Background The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host–parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. Methodology Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. Results FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. Conclusions The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host–parasite interactions. Graphic abstract

Expression of programmed cell death ligand 1 (PD‐L1) at in situ and invasive extramammary Paget’s disease and literature review

2021 ◽  
Author(s):  
Junji Kato ◽  
Shintaro Sugita ◽  
Kohei Horimoto ◽  
Sayuri. Sato ◽  
Daisuke Yoneta ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Literature Review ◽  
Paget’S Disease ◽  
Paget's Disease ◽  
Extramammary Paget’S Disease ◽  
Extramammary Paget's Disease
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