Necrosis as Programmed Cell Death

The process of cell death is the mechanism through which organisms eliminate useless cells. Hence, it is a normal process that maintains homeostasis. Cell removal can be effectuated by several pathways that involve complex and regulated molecular events specific to each type of cell death. Diverse studies have evidenced different types of cell death: apoptosis, autophagy, and necrosis. This chapter presents a brief review of the apoptotic and autophagic cell death processes but focuses attention primarily on necrosis because it has previously been considered an accidental and uncontrolled form of cell death. More recent evidence, however, has shown that, under certain circumstances, necrosis is conducted by a controlled program called necroptosis, which is now included as a programmed cell death process

Endoplasmic Reticulum Stress during Mammalian Follicular Atresia

Follicles are ovarian structures that contain a single germ cell. During the mammalian reproductive lifetime, ovarian follicles mature through the process of follicular development, with the aim of selecting oocytes for ovulation. As part of this process, several follicles are eliminated by means of follicular atresia, a mechanism that mainly involves apoptosis. Nevertheless, it has been shown that there are other routes of programmed cell death in the ovary including autophagy, paraptosis, and necroptosis. Surprisingly, the endoplasmic reticulum is involved in these different programmed cell death pathways. Moreover, there are several evidences for the pathways triggered by intra- and extracellular signals in endoplasmic reticulum-induced cell death. Thus, it is important to analyze the participation of endoplasmic reticulum in follicular atresia

Fine structural organization of a non-reticulate plant cell nucleus. An ultracytochemical and immunocytochemical study

We studied the fine structural organization of the meristematic nucleus in roots of Lycopesicon esculentum (tomato) using ultracytochemical and immunocytochemical approaches. The nucleus has a non-reticulate (i.e. low DNA content) structure whose supramolecular organization differs in some respects from that in reticulate nuclei, principally in the organization of the chromocentres associated with the nuclear envelope, with which centromeric structures appear to be associated. The main difference at the nucleolar level is found in the fibrillar centres, which have a low amount of DNA labelling and in which inclusions of condensed chromatin are present only very rarely. The distribution of nucleolar DNA amongst the nucleolar compartments is similar to that in reticulate nucleoli as demonstrated using an anti-DNA monoclonal antibody. Tomato nuclei have nucleolus-associated bodies or karyosomes, like other plant species with a low DNA content and non-reticulate nuclear organization. The nuclear ribonucleoprotein structures in the inter- and perichromatin regions, namely inter- and perichromatin fibrils and granules, show similar ultrastructural and cytochemical characteristics in both types of nuclei.This work has been partially supported by CICYT (Spain) grant no. PB88-0037 and by funds of the agreement CSIC (Spain)/CONACYT (M6xiCO)

Firing of transcription and compartmentalization of splicing factors in tomato radicle nuclei during germination

[Background information]: Germination is a well-characterized process in which embryo cells of seeds experience a programmed transition from quiescence to proliferation. For this reason they constitute a very good system to analyse nuclear evolution from a dehydrated practically inactive state until the steady state of proliferation. We analysed the temporal and spatial organization of transcription and splicing factors in nuclei of tomato radicle cells during germination. To address this issue we performed in situ immunodetection of several markers of these processes: the Z-DNA stretches forming behind the active RNA polymerases, the splicing proteins U2B″ and Sm, and the trimethyl guanosin cap of small nuclear RNA. The concomitant structural changes of the different nuclear compartments were studied in meristematic nuclei by electron microscopy and high-resolution cytochemistry for DNA and ribonucleoproteins.[Results]: In quiescent cells practically no Z-DNA stretches were detected and splicing components localized mainly to one or two Cajal bodies associated to the nucleolus. In early germination, a massive de-condensation of chromatin and nucleolar Z-DNA conformation stretches were first detected, followed by the relocation of scarce splicing components to the small interchromatin spaces. Nucleoplasmic Z-DNA stretches were not detected until 4 h of imbibition and were accompanied by an important increase of splicing components in this nuclear domain. Soon after the post-germination stage, transcription and splicing topology and nuclear organization in meristematic nuclei resemble those in steady state growing tomato roots.[Conclusions]: Our results demonstrate that, in tomato, dormant nuclei splicing factors are stored in nucleolar Cajal bodies. In early germination, RNA polymerase I transcription is first activated, whereas mRNA transcription is fired later and is accompanied by a massive de-condensation of chromatin and accumulation of splicing factors in the interchromatin domains. Nucleoplasmic Cajal bodies appear later in germination.This work was supported by Spanish DGI project BFU 2006-00379 and by a CSIC-UNAM agreement for academic interchange

Supramolecular organization of a chromocentric plant nucleus

Cytochemical staining (EDTA procedure for ribonucleoprotein (RNP) and osmium ammine staining), immunocytochemistry (mAbs anti DNA and trimethyl guanosine cap), hypotonic-formaldehyde fixation and in situ hybridization have been used to study the macromolecular architecture of the interphase nucleus of meristematic cells of a dicotyledon plant with chromocentric nuclei, Chirantodendron pentadactylon. The perichromatin region contains extended chromatin loops, perichromatin fibrils as well as typical and immature perichromatin granules. Hypotonic fixation combined with immunolocalization of trimethyl guanosine cap showed that perichromatin fibrils are associated with snURNPs. These results indicate that in the perichromatin region the splicing of newly synthesized pre-mRNA is taking place. Thus, the same functions are localized in the perichromatin region of plant and animal cell nuclei. RNA-containing granules morphologically similar to perichromatin granules, but significantly smaller in size, were found in the interchromatin region. These granules are frequently grouped in clusters. Filaments of extended chromatin pervade the interchromatin region. These observations show that these structures correspond to Lacandonia granules, previously found in some monocotyledons with reticulate nucleus, but not observed in dicotyledons with a chromocentric nucleus. The presence of these granules in both monocotyledons and dicotyledons suggests that they probably constitute an ancestral character of angiosperms. Perichromatin and Lacandonia granules pediculate and irregular in shape corresponding to immature forms are described for the first time in the nucleus of plants. Immunolocalization indicates that the snURNPs are associated with fibrils, interchromatin granules, coiled bodies and nucleolus associated bodies (NABs). Taken as whole these features demonstrate that intense transcription and splicing occur in the ample interchromatin region of meristematic nuclei of Chirantodendron pentadactylon.This work was partially supported by a grant CONACYT (México) CSIC (Spain) and by a grant of Intercambio Académico UNAM.Peer reviewe

High resolution detection of rRNA and rDNA in plant nucleoli with different activities by in situ hybridization

In the present work we perform in situ hybridization with probes to different stretches of rDNA and electron microscopy of nucleoli with different activities, to gain insight into the ultrastructural organization of transcription and processing in the plant nucleolus. The main ultrastructural nucleolar components: fibrillar centers (FC), dense fibrillar component (DFC), and granular component (GC), are arranged in different ways depending on nucleolar activity. Heterogeneous FCs containing RNP fibrils and nucleolar perichromatin granules are frequently seen in nucleoli in the process of activation. DNA-RNA in situ hybridization with biotinylated probes spanning different sequences of the rDNA unit followed by immunogold detection of biotin, demonstrated the localization of the ribosomal transcripts in DFC., mainly in the zones around the FCs, in GC, and in the periphery of pale FC. The internal region of the heterogeneous FCs is labeled only in cells in the process of activation of transcription after dormancy. The distribution of the U3 probe indicates that the processing of the rRNA takes place in the DFC and inside the heterogeneous FCs, in which transcription occurs. DNA-DNA hybridization demonstrates the presence of rDNA in the compact and extended chromatin located in the interior and at the periphery of FCs and in nucleolar associated chromatin. Our results support the view that the plant nucleolus has a highly dynamic morphological and functional organization composed of a bipartite domain formed by FCs surrounded by DFC., which is associated with rRNA transcription and processing, and the GC representing a store of preribosomal particles.This work has been supported by grants of the ClCYT (Spain) projects PB95-0117 and PB960909, and of the agreements CSIC (Spain)/CONACYT (Mexico) and CSIC (Spain)/UNAM (Mexico). Pre-t-RNA detection in plant nucleolus Bassy e

Evidencia ultraestructural del nucléolo de Entamoeba histolytica

Although cell structure of Entamoeba histolytica is well known, the presence of nucleolar material has not been described with the electron microscope. Here we use light and electron microscopy cytochemical techniques to search for evidence1 of nucleolar material in this amoeba trophozoyte. Toluidine blue for RNA and silver staining for nucleolar organizer stain peripheral intranuclear material2. With the electron microscope, a3 similar material is fibro-granular and is contrasted with techniques for ribonucleoproteins and nucleolar organizer, but it is negative for DNA. These results show ultrastructural evidence for the presence of a peripheral and ring-shaped nucleolus4 in the nucleus of E. histolytica. It is suggested that this intranuclear organelle is a general feature in eukaryotes.Aunque la estructura celular de Entamoeba histolytica se conoce con detalle, no se ha descrito la presencia de material nucleolar con el microscopio electrónico. En este trabajo utilizamos técnicas citoquímicas para microscopía de luz y electrónica para evidenciar la presencia del nucléolo en trofozoitos de esta amiba. Con la técnica de azul de toluidina para RNA y la técnica de impregnación argéntica para organizador nucleolar, se observa contraste de material intranuclear periférico. Con el microscopio electrónico, un  material similar de naturaleza fibro-granular se contrasta también con las técnicas para ribonucleoproteínas y para organizador nucleolar. Una zona similar es negativa cuando se aplican técnicas para DNA. Estos resultados muestran evidencia ultraestructural de la presencia de un  nucléolo periférico y anular en el núcleo de E. histolytica. Se sugiere que este organelo intranuclear es un caracter general de los eucariontes

EVIDENCIA ULTRAESTRUCTURAL DEL NUCLÉOLO DE Entamoeba histolytica

Aunque la estructura celular de Entamoeba histolytica se conoce con detalle, no se ha descrito la presencia de material nucleolar con el microscopio electrónico. En este trabajo utilizamos técnicas citoquímicas para microscopía de luz y electrónica para evidenciar la presencia del nucléolo en trofozoitos de esta amiba. Con la técnica de azul de toluidina para RNA y la técnica de impregnación argéntica para organizador nucleolar, se observa contraste de material intranuclear periférico. Con el microscopio electrónico, un material similar de naturaleza fibro-granular se contrasta también con las técnicas para ribonucleoproteínas y para organizador nucleolar. Una zona similar es negativa cuando se aplican técnicas para DNA. Estos resultados muestran evidencia ultraestructural de la presencia de un nucléolo periférico y anular en el núcleo de E. histolytica. Se sugiere que este organelo intranuclear es un caracter general de los eucariontes

Differential distribution and association of repeat DNA sequences in the lateral element of the synaptonemal complex in rat spermatocytes

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC