Biomedical subjects

M Carmo-Fonseca

Publications and source records attributed to M Carmo-Fonseca.

At least 73 records · Page 4Linked to original sources

PubMed1991

In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells.

The in vivo distribution of snRNPs has been analysed by microinjecting fluorochrome-labelled antisense probes into the nuclei of live HeLa and 3T3 cells. Probes for U2 and U5 snRNAs specifically label the same discrete nuclear foci while a probe for U1 snRNA shows widespread nucleoplasmic labelling, excluding nucleoli, in addition to labelling foci. A probe for U3 snRNA specifically labels nucleoli. These in vivo data confirm that mammalian cells have nuclear foci which contain spliceosomal snRNPs. Co-localization studies, both in vivo and in situ, demonstrate that the spliceosomal snRNAs are present in the same nuclear foci. These foci are also stained by antibodies which recognize snRNP proteins, m3G-cap structures and the splicing factor U2AF but are not stained by anti-SC-35 or anti-La antibodies. U1 snRNP and the splicing factor U2AF closely co-localize in the nucleus, both before and after actinomycin D treatment, suggesting that they may both be part of the same complex in vivo.

Animals↗

PubMed1991

Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery.

The organization of the major snRNP particles in mammalian cell nuclei has been analysed by in situ labelling using snRNA-specific antisense probes made of 2'-OMe RNA. U3 snRNA is exclusively detected in the nucleolus while all the spliceosomal snRNAs are found in the nucleoplasm outside of nucleoli. Surprisingly, U2, U4, U5 and U6 snRNAs are predominantly observed in discrete nucleoplasmic foci. U1 snRNA is also present in foci but in addition is detected widely distributed throughout the nucleoplasm. An anti-peptide antibody specific for the non-snRNP splicing factor U2AF reveals it to have a similar distribution to U1 snRNA. Co-localization studies using confocal fluorescence microscopy prove that U2AF is present in the snRNA-containing foci. Antibody staining also shows the foci to contain snRNP-specific proteins and m3G-cap structures. The presence of major components of the nuclear splicing apparatus in foci suggests that these structures may play a role in pre-mRNA processing.

Antisense Elements (Genetics)↗

PubMed1991

The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre-rRNA processing in yeast.

The yeast snoRNP protein, NOP1, is structurally and functionally homologous to vertebrate fibrillarin and is essential for viability. A conditionally lethal allele was constructed by placing NOP1 expression under the control of a GAL promoter. Growth on glucose medium results in the depletion of NOP1 over several generations, during which cell growth is progressively impaired. Pulse labelling of proteins shows that NOP1 depleted strains are greatly impaired in the production of cytoplasmic ribosomes, and they have a reduced level of rRNA. Northern hybridization and pulse-chase labelling of pre-rRNA show a progressive impairment of all pre-rRNA processing steps. The pathway leading to 18S rRNA is particularly affected. Methylation of pre-rRNA is concomitantly impaired and unmethylated pre-rRNA accumulates and is not processed over long periods. NOP1 depletion does not prevent the accumulation of seven snoRNAs tested including U3; the levels of two species, U14 and snR190, decline. The snoRNAs synthesized in the absence of NOP1 retain TMG cap structures. Subnuclear fractionation and immunocytochemistry indicate that they continue to be localized in the nucleolus.

Base Sequence↗

PubMed1991

Across the nuclear pores with the help of nucleoporins.

Proteins targeted to specific intracellular organelles such as mitochondria or the endoplasmic reticulum are able to cross membranes. Yet, to enter or exit the nucleus, proteins and RNA must pass through nonmembranous "gates" of the nuclear envelope, the nuclear pore complexes. Recently, the primary amino acid sequence of a few nuclear pore proteins (the nucleoporins) became available. Nucleoporins from mammals, amphibians and yeast are structurally homologous indicating that nuclear pore structures are evolutionarily conserved in the eukaryotic cell. The role of nucleoporins in nucleocytoplasmic transport is still unclear: are nucleoporins involved in decoding nuclear targeting signals or are they mere transporters? Although definite answers are not yet available, data are rapidly accumulating from several laboratories using a variety of approaches.

Animals↗

PubMed1991

Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast.

NOP1 is an essential nucleolar protein in yeast that is associated with small nucleolar RNA and required for ribosome biogenesis. We have cloned the human nucleolar protein, fibrillarin, from a HeLa cDNA library. Human fibrillarin is 70% identical to yeast NOP1 and is also the functional homologue since either human or Xenopus fibrillarin can complement a yeast nop1- mutant. Human fibrillarin is localized in the yeast nucleolus and associates with yeast small nucleolar RNAs. This shows that the signals within eucaryotic fibrillarin required for nucleolar association and nucleolar function are conserved from yeast to man. However, human fibrillarin only partially complements in yeast resulting in a temperature-sensitive growth, concomitantly altered rRNA processing and aberrant nuclear morphology. A suppressor of the human fibrillarin ts-mutant was isolated and found to map intragenically at a single amino acid position of the human nucleolar protein. The growth rate of yeast nop1- strains expressing Xenopus or human fibrillarin or the human fibrillarin suppressor correlates closely with their ability to efficiently and correctly process pre-rRNA. These findings demonstrate for the first time that vertebrate fibrillarin functions in ribosomal RNA processing in vivo.

Amino Acid Sequence↗

PubMed1991

Cytokeratin in early hamster embryogenesis and parthenogenesis: reorganization during mitosis and association with clusters of interchromatinlike granules.

In vivo obtained golden hamster embryos were used to study, by immunofluorescence and immunoelectron microscopy, the main cytokeratin pattern rearrangements during completion of meiosis and the first cleavage division. Our results point to three major re-organization steps: (1) diffuse immunofluorescent cytokeratin spots characteristic of recently ovulated oocytes rearrange into large cortical patches interconnected by fibrils in one-cell embryos; (2) during mitosis a homogeneous cytokeratin spotty pattern reappears; (3) in two-cell embryos cortical and perinuclear cytokeratin fibrillar networks become prominent. Parthenogenotic oocytes were able to mimic the major cytokeratin patterns observed until the first embryonic mitosis, supporting the concept of a maternally established common response to activation. Despite the lack of fibrillar immunofluorescent reactivity during mitosis, electron microscopy demonstrates persistence of 10 nm filament meshworks. These cytokeratin meshworks often associate with clusters of interchromatinlike granules, which persist in the cytoplasm for a short period after nuclear envelope reassembly.

Actin Cytoskeleton↗

PubMed1991

Human nucleoporin p62 and the essential yeast nuclear pore protein NSP1 show sequence homology and a similar domain organization.

NSP1 is an essential nuclear pore protein in yeast. We observed that anti-NSP1 antibodies label mammalian nuclear pore complexes and recognize nucleoporin p62. Also peptide antibodies raised against the NSP1 carboxy-terminal end cross-react with p62, a conserved component of the nuclear pore complex in higher eukaryotes. To further analyze the structural and functional similarity between NSP1 and mammalian nucleoporins, we cloned and sequenced nucleoporin p62 from a HeLa cDNA library. Human p62 consists of a carboxy-terminal domain homologous to the essential yeast NSP1 carboxy-terminal domain and an amino-terminal half resembling the repetitive middle domain of NSP1. The full-length p62 and a fusion protein consisting of cytosolic mouse dihydrofolate reductase (DHFR) and the p62 carboxy-terminal domain were expressed in transfected HeLa cells. Only overexpressed full-length p62, but not the DHFR-C-p62 fusion protein, binds wheat germ agglutinin (WGA). This suggests that modification by N-acetylglucosamine is mainly restricted to the repetitive amino-terminal half of p62 and implies a role of this type of repetitive sequences in nuclear transport. In the transfected HeLa cells, the DHFR-C-p62 fusion protein forms patchy aggregates that accumulate at the nuclear periphery but are also scattered through the cytoplasm. It is suggested that nucleoporin p62 may be targeted and anchored to the pore complex via its carboxy-terminal domain which reveals a hydrophobic heptad repeat organization similar to that found in lamins and other intermediate filament proteins.

Amino Acid Sequence↗

PubMed1990

Interactions of intermediate filaments with cell structures.

Intermediate filaments (IF) are unique components of the cytoskeleton of most eukaryotic cells. Also the nuclear lamins are now recognized to be IF-like proteins, providing the nucleus with a putative skeleton for chromatin attachment. Immunofluorescence and whole-mount electron microscopic studies reveal that IF form a cytoplasmic network that surrounds the nucleus and extends to cell surface, as 'mechanical integrators of cellular space'. It seems however unlikely that IF in the cell accomplish a merely structural role, considering the diversity of IF proteins and the complex regulation of their gene expression. In this work we primarily present electron microscopic data that points to the presence of interactions between IF and several cellular components, namely the nucleus, plasma membrane, other cytoskeletal elements, cytoplasmic organelles and ribonucleoproteins. Although the functional significance of such interactions remains to be demonstrated, assumptions like involvement of IF in information transfer or cytoskeleton-dependent control of gene expression represent attractive hypothesis for future research.

Animals↗

PubMed1990

Peptide containing nerves in human synovium: immunohistochemical evidence for decreased innervation in rheumatoid arthritis.

The innervation of normal and rheumatoid human synovium was studied by immunofluorescence microscopy. Antibodies against the general neuronal marker protein gene product (PGP) 9.5 and specific neuropeptides were used. We observed sensory nerves containing substance P (SP) and calcitonin gene related peptide (CGRP) as well as autonomic sympathetic fibers immunoreactive for neuropeptide tyrosine (NPY), its C terminal peptide (C-PON) and the catecholamine synthesizing enzyme tyrosine hydroxylase (TH). Three subpopulations of nerve fibers labelled with SP and CGRP were identified: some stained for SP or CGRP only and others contained both peptides. NPY/C-PON and TH labelled predominantly perivascular nerves. Quantification of immunostained nerves revealed a significantly decreased innervation of rheumatoid synovia. The densities of both PGP 9.5 and neuropeptide containing nerves were lower in all rheumatoid samples. Our results are compatible with a local release of neuropeptides into joint fluid and point to a disturbed neuronal control of rheumatoid synovial tissue.

Adolescent↗

PubMed1989

Identification of La ribonucleoproteins as a component of interchromatin granules.

Monoclonal antibodies raised against the La antigen were used to localize by preembedding immunoelectron microscopy, snRNPs containing this protein. The results demonstrate that La RNPs are localized in clusters of interchromatin granules, both in Triton X-100-extracted and DNase-digested nuclei. DNase-digested salt-extracted nuclei contained, in addition, labeled structures identified as perichromatin granules and fibers. A close association of labeled granules with the nucleoli was also observed. Digestion of nuclei with DNase yielded residual scaffolds of intermediate filaments and nuclear lamina devoid of interchromatin granules and La immunostaining. Release of the La antigen was tested in the presence of ATP and cytochalasin B. Only cytochalasin was effective suggesting a role for nuclear actin in anchorage of snRNPs.

Antibodies, Monoclonal↗

PubMed1989

Change of processing and nucleocytoplasmic transport of mRNA in HSV-1-infected cells.

A monoclonal antibody (MAb) raised against the pore-complex lamina fraction from CV-1 cells was used to study alterations of gene expression in herpes simplex virus type 1 (HSV-1)-infected cells. This MAb, which recognized only one cellular polypeptide of 60,000 Da, selectively stained the nucleus in immunofluorescence microscopy, showing a punctuated pattern either at the nuclear surface or at the nuclear rim. By immunoelectron microscopy, the p60 antigen could be localized in the nuclear pore complex structure. Infection of CV-1 cells with HSV-1 resulted in a drastic change of the nuclear staining pattern. Four hours p.i., a clustering of the p60 antigen and, 12 h p.i., a formation of finger-like holes, penetrating the nucleus, occurred. Later in infection (22 h p.i.) the antigen was found to be almost absent. By RNA blot hybridization it was demonstrated that, after HSV-1 infection, the level of cellular mRNA (beta-tubulin) gradually decreased, while the level of HSV major DNA binding protein (DBP) mRNA increased, reaching maximal level 3-6 h p.i. Interestingly, the level of beta-tubulin gene transcripts changed differentially in the polysomal and in the nuclear fraction during the initial phase of infection, in contrast to the viral DBP transcripts, indicating that, after HSV infection, host cell transcripts accumulate in the nucleus. Evidence is presented indicating that this change is not due to altered nucleocytoplasmic mRNA transport but is due to an impaired splicing of host cell transcripts in HSV-infected cells. The MAb, directed against the nuclear pore p60 antigen, strongly inhibited the ATP-dependent efflux of both cellular and viral mRNA from isolated nuclei. The ATP-dependence of the efflux did not change during viral infection. However, the inhibitory potency of the MAb was found to be lost at the final stage of HSV infection, paralleling the loss of p60 antigen.

Adenosine Triphosphate↗

PubMed1989

Immunocytochemical localization and lectin-binding properties of the 22 kDa secretory protein from rat ventral prostate.

The distribution of the 22 kDa secretory protein from rat ventral prostate was studied by light and electron microscopic immunocytochemistry. An anti-22 kDa protein antiserum was raised in rabbits and its specificity was tested by Western blotting. With the immunofluorescence technique, the 22 kDa protein was detected in the luminal secretions and intracellular apical granules of the ventral prostate. No reaction was observed in the seminal vesicle or dorsolateral prostate. After castration, no intracellular immunoreactivity was detected in ventral prostate, although positively labeled secretory material was retained within the acinar lumen. Restoration of normal intracellular staining pattern was incomplete after 5 daily testosterone injections. At the ultrastructural level, labeling was confined to apical secretory granules and condensing vacuoles. The 22 kDa protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose was shown to bind intensely to wheat germ agglutinin (WGA) but only faintly to Concanavalin A. This protein was thus demonstrated to contain N-acetylglucosamine residues. Accordingly, on tissue sections, WGA reacted intensely with condensing vacuoles and secretory granules.

Animals↗

PubMed1989

Cytokeratin filaments are present in golden hamster oocytes and early embryos.

Light and electron microscope methods were used to study cytokeratin expression in the recently ovulated oocytes, fertilized eggs and early embryos from the golden hamster. Two cytokeratin polypeptides (Mr 51,000 and 58,000) were detected in oocyte lysates by immunoblotting using a polyclonal antiserum to prekeratin. In the oocyte, cytokeratin occurred as patchy aggregates consisting of short anastomosing 10-nm filaments that formed tight meshworks distributed throughout the cytoplasm. After fertilization the aggregates appeared to merge, becoming larger and concentrated at the cortical region. Prominent immunofluorescent fibrils were seen interconnecting the aggregates. In the 2-, 4- and 8-cell embryos, networks of cytokeratin filaments extended throughout the cortical and perinuclear regions, while aggregates progressively disappeared.

Animals↗

PubMed1988

Androgen-dependent nuclear proteins in rat ventral prostate are glycoproteins associated with the nuclear matrix.

The major rat ventral prostate androgen-dependent nuclear proteins were studied using isolated nuclei, nuclear matrix and nuclear envelope fractions. Nuclear and subnuclear fractions obtained were characterized by electron microscopy and SDS-polyacrylamide gel electrophoresis. A group of approximately 20 kDa peptides is demonstrated to be present in nuclei, nuclear matrices and nuclear envelopes from normal prostate. Time course experiments indicate that the 20 kDa peptides become drastically reduced after 7 or 10 days following castration and are incompletely restored after 3 daily testosterone injections. Lectin binding studies demonstrate that the 20 kDa peptides bind both to Concanavalin A and Wheat Germ Agglutinin. These peptides represent the major nuclear Concanavalin A binding glycoproteins from normal prostate nuclei and nuclear matrices.

Androgens↗

PubMed1988

Nuclear compactness as assessed by ferritin permeation: a critical evaluation of the method.

The fracture-permeation method was applied in order to assess changes in nuclear compactness associated with different metabolic conditions. Rat ventral prostate was used as a model because transcription in the secretory cells of this organ is highly dependent on androgens. Two major problems that raise important questions concerning the validity of the method were encountered: (1) poorly preserved cells are massively permeated by ferritin and (2) within the same experimental group the permeation pattern was quite variable. In order to objectify such variability, the percentage of permeated nuclear cross-fractures was quantified. Different permeation patterns could be detected in nuclei from normal, castrated, and testosterone-treated prostatic cells, possibly reflecting changes in chromatin compactness.

Animals↗

PubMed1988

Filamentous cross-bridges link intermediate filaments to the nuclear pore complexes.

Intermediate filament-nuclear matrix interactions were studied in cultured rat ventral prostate cells and isolated rat uterine epithelial cells. Cytokeratin filaments were identified by immunoelectron microscopy. In addition to conventional thin section of Triton X-100 treated cells, subcellular residues composed of intermediate filaments and nuclear matrix were critical-point dried and platinum-carbon replicated. The results demonstrate the presence of a previously unrecognized type of filamentous cross-bridges that link intermediate filaments to the nuclear pore complexes.

Animals↗

PubMed1986

Quantitative ultrastructural autoradiographic study of RNA transport in rat ventral prostate.

The nucleocytoplasmic RNA transport in rat ventral prostate was studied by electron microscope autoradiography. Isolated prostate acini from normal, castrated, and DHT-treated animals were labeled in vitro with [3H]uridine for 5 min and chased for 0, 15, 30 min and 4 hr. The results show that DHT induces a significant nucleolar enlargement but intranuclear migration of rRNA is not apparently affected by androgens; migration of RNA through euchromatin is delayed by castration and stimulated by DHT; migration through the nuclear envelope is androgen-dependent. In addition prostate acini were maintained for 24 hr in suspension culture in order to study the in vitro effects of DHT. The result show that DHT stimulates uridine uptake and/or incorporation but induces no nucleolar enlargement; DHT has no clear effects on RNA migration kinetics; cytoplasmic transport of RNA in cells cultured in medium with or without DHT is severely impaired but is restored after supplementation of medium with insulin and dexamethasone.

Animals↗

PubMed1985

Different patterns of filipin-sterol labeling in prostate nuclear membranes from normal and castrated rats.

Filipin was used as cytochemical probe for sterol detection in freeze-fractured prostate nuclear membranes from rats under different hormonal conditions. Isolated prostate acini and nuclei were fixed in glutaraldehyde and post-treated with filipin, according to Robinson and Karnovsky (1980). In general, most plasma and intracellular cytoplasmic membranes displayed a marked response to filipin in either epithelial and stromal cells from normal and castrated animals. Nuclear membranes from epithelial secretory cells were systematically negative to filipin labeling in normal animals, although after castration a positive response was detected. Stromal nuclear membranes were labeled both in normal and castrated animals. Filipin-treated isolated nuclei displayed the same overall labeling pattern but there was a different distribution of induced deformations relative to intact cell nuclei. These observations indicate that: a) nuclear membranes from different cell types have different responses to filipin; b) a change in the molecular organization of nuclear membranes from prostate secretory cells follow castration; c) nuclei isolation affects the distribution of filipin induced deformations on the membranes.

Animals↗