Biomedical subjects

R Bastos

Publications and source records attributed to R Bastos.

At least 19 recordsLinked to original sources

PubMed2000

Recombinant Nup153 incorporates in vivo into Xenopus oocyte nuclear pore complexes.

Nup153 is a molecular constituent of the nuclear basket of the nuclear pore complex (NPC) that plays a critical role in nuclear export of RNAs and proteins. In an effort to map this nucleoporin more precisely within the nuclear basket we have developed an experimental approach for localizing Nup153 expressed and incorporated in vivo into Xenopus oocyte NPCs. This approach involves the microinjection into the cytoplasm of Xenopus oocytes of in vitro synthesized mRNA from a vector encoding an epitope-tagged cDNA. Here we present results obtained by Western blots, fluorescence microscopy, and immuno-electron microscopy, which clearly document that the heterologous protein is properly expressed, targeted, and incorporated into preexisting Xenopus NPCs. This new approach for localizing nucleoporins within the structure of the NPC overcomes limitations of previous techniques and allows for greater specificity and resolution than have been possible with previous methods.

Animals↗

PubMed1999

The nucleolar antigen Nop52, the human homologue of the yeast ribosomal RNA processing RRP1, is recruited at late stages of nucleologenesis.

We report the molecular characterization of a novel nucleolar protein, Nop52, and its subcellular distribution during the cell cycle and nucleologenesis. This protein was originally identified with human autoantibodies which were subsequently used to clone its corresponding cDNA. Transfection experiments in mammalian cells have confirmed that this cDNA encodes a nucleolar protein that accumulates in the nucleoli and at the periphery of the chromosomes. Nop52 is the putative human homologue of the yeast ribosomal RNA processing protein RRP1 which is involved in pre-rRNA processing from 27S to 25S and 5.8S. In nucleoli, Nop52 is excluded from the ribosomal RNA transcription sites, accumulates in the granular external domain and mainly colocalizes with nucleolar proteins involved in the late processing step such as hPop1 and protein B23. During the building process of the nucleolus at the end of mitosis, a sequential order was observed in the assembly of nucleolar proteins of early and late processing mainly via the prenucleolar body pathway. The order is the following: fibrillarin, nucleolin, Nop52 together with protein B23 in the prenucleolar bodies, and simultaneously with hPop1, and finally Ki-67. The evolutionary conservation of Nop52 and the lethal effects observed in gene disruption experiments, predict a critical role for Nop52 in the generation of 28S rRNA.

Amino Acid Sequence↗

PubMed1999

Sequential recruitment of NPC proteins to the nuclear periphery at the end of mitosis.

Nuclear pore complexes (NPCs) are extremely elaborate structures that mediate the bidirectional movement of macromolecules between the nucleus and cytoplasm. With a mass of about 125 MDa, NPCs are thought to be composed of 50 or more distinct protein subunits, each present in multiple copies. During mitosis in higher cells the nuclear envelope is disassembled and its components, including NPC subunits, are dispersed throughout the mitotic cytoplasm. At the end of mitosis, all of these components are reutilized. Using both conventional and digital confocal immunofluorescence microscopy we have been able to define a time course of post-mitotic assembly for a group of NPC components (CAN/Nup214, Nup153, POM121, p62 and Tpr) relative to the integral nuclear membrane protein LAP2 and the NPC membrane glycoprotein gp210. Nup153, a component of the nuclear basket, associates with chromatin towards the end of anaphase, in parallel with the inner nuclear membrane protein, LAP2. However, immunogold labeling suggests that the initial Nup153 chromatin association is membrane-independent. Assembly of the remaining proteins follows that of the nuclear membranes and occurs in the sequence POM121, p62, CAN/Nup214 and gp210/Tpr. Since p62 remains as a complex with three other NPC proteins (p58, 54, 45) during mitosis and CAN/Nup214 maintains a similar interaction with its partner, Nup84, the relative timing of assembly of these additional four proteins may also be inferred. These observations suggest that there is a sequential association of NPC proteins with chromosomes during nuclear envelope reformation and the recruitment of at least eight of these precedes that of gp210. These findings support a model in which it is POM121 rather than gp210 that defines initial membrane-associated NPC assembly intermediates.

Animals↗

PubMed1998

Amino-terminal sequences that direct nucleoporin nup153 to the inner surface of the nuclear envelope.

Nup153 is a large O-linked glycoprotein that is a component of the basket-like structure that forms the nucleoplasmic face of nuclear pore complexes (NPCs). The Nup153 molecule has a tripartite structure consisting of N- and C-terminal domains flanking a central zinc finger domain. All of the targeting and assembly information contained within Nup153 is contributed by the N-domain. In fact this region of the molecule can target a cytosolic protein, pyruvate kinase, to the nucleoplasmic face of the NPC. The zinc finger and C-terminal domains appear to have no role in these targeting and assembly activities. Deletion analysis reveals that there are two distinct regions within the Nup153 N-domain that contain different targeting functions. One of these is directly involved in assembly into the NPC while a second overlapping region may target Nup153, as well as other reporter molecules, to the inner face of the nuclear envelope.

Amino Acid Substitution↗

PubMed1998

Urinary fluoride levels and prevalence of dental fluorosis in three Brazilian cities with different fluoride concentrations in the drinking water.

Urine samples from three daytime periods were collected from 545 5-50-year-old residents of three different Brazilian cities: Garça had fluoridated drinking water since 1973, Bauru since 1975 and Itápolis was not fluoridated. Dental fluorosis was examined in 985 5-24-year-olds using the Thylstrup-Fejerskov index (TF). The subjects were asked to estimate their daily intake of liquids and frequency of beverage consumption. The analysis of 94 water samples showed high variations in the fluoride content of the drinking water. The mean fluoride concentration of the water samples in Garça was 0.9 mg/L (range 0.75-1.2), in Bauru 0.64 mg/L (range 0.01-1.3), and in Itápolis 0.02 mg/L. Mean urinary fluoride concentration was 1.31 mg/L (s 0.61) in Garça, 0.88 mg/L (s 0.49) in Bauru, and 0.39 mg/L (s 0.21) in Itápolis. Self-reported daily liquid intake was not related to urinary fluoride concentration. The mean prevalence of fluorosis was 13.3% in Garça, 6.8% in Bauru, and 1.7% in Itápolis, with mainly categories TF 1 and TF 2 being recorded. Subjects with dental fluorosis tended to show a higher mean urinary fluoride concentration but the difference was not statistically significant. The study showed that fluoride exposure measured by urinary fluoride excretion was within the range expected for the level of fluoride concentration in the drinking water. However, enamel fluorosis tended to be markedly lower than expected. This study revealed that fluoride levels in the two cities with fluoridated drinking water were variable. To optimise anticaries benefits and minimise the risk of fluorosis greater control of the fluoride dosing of the drinking water is required.

Adolescent↗

PubMed1998

[Coronary intervention at a general hospital in the State of Piauí, Brazil].

PURPOSE: To evaluate retrospectively success and complication rates of coronary interventions performed in a general hospital with no digital equipment in the State of Piauí with a small annual number of procedures. METHODS: From August/91 to January/98, 122 patients underwent 146 procedures using a fluoroscope with camera and TV monitor of 525 lines. The variables analyzed included the initial success of procedures (residual stenosis less than fifty percent and TIMI III flow) and more serious complications (acute occlusion, infarcts, emergency surgeries and death). The clinical data and the morphological characteristics of the lesions (ACC/AHA Task Force) were also analyzed. RESULTS: Eighty five percent of the patients presented with an acute ischemic syndrome (acute myocardial infarction, post infarction angina, rest angina), 5% were in cardiogenic shock and 88% had type B lesions. The initial success rate was 93% while the more serious complications occurred in 3.5% of the cases. CONCLUSION: In spite of the small annual procedural volume and the use of conventional equipment, the initial success rate in our institution was excellent in a diverse and complex patient population while the complication rates were similar to that of the literature.

Adult↗

PubMed1997

Nup84, a novel nucleoporin that is associated with CAN/Nup214 on the cytoplasmic face of the nuclear pore complex.

The short filaments extending from the cytoplasmic face of nuclear pore complexes are thought to contain docking sites for nuclear import substrates. One component of these filaments is the large O-linked glycoprotein CAN/Nup214. Immunoprecipitation studies carried out under nondenaturing conditions, and using a variety of antibodies, reveal a novel nonglycosylated nucleoporin, Nup84, that is tightly associated with CAN/Nup214. Consistent with such an association, Nup84 is found to be exposed on the cytoplasmic face of the nuclear pore complex. cDNA sequence analyses indicate that Nup84 contains neither the GLFG nor the XFXFG repeats that are a characteristic of a number of other nuclear pore complex proteins. Secondary structure predictions, however, suggest that Nup84 contains a coiled-coil COOH-terminal domain, a conclusion supported by the observation of significant sequence similarity between this region of the molecule and various members of the tropomyosin family. Mutagenesis and expression studies indicate that the putative coiled-coil domain is required for association with the cytoplasmic face of the nuclear pore complex, whereas it is the NH2-terminal region of Nup84 that contains the site of interaction with CAN/Nup214. These findings suggest a model in which Nup84 may function in the attachment of CAN/Nup214 to the central framework of the nuclear pore complex. In this way, Nup84 could play a central role in the organization of the interface between the pore complex and the cytoplasm.

Amino Acid Sequence↗

PubMed1997

Ventromedial hypothalamus lesions increase the dipsogenic responses and reduce the pressor responses to median preoptic area activation.

In this study, we investigated the participation of adrenergic receptors of the median preoptic area (MnPO) and the participation of ventromedial hypothalamus (VMH) in angiotensin II-(ANG II)-induced water intake and pressor responses. Male rats with sham or electrolytic VMH lesions and a stainless steel cannula implanted into the MnPO were used. Noradrenaline, clonidine (an alpha2-adrenergic receptor agonist), or phenylephrine (an alpha1-adrenergic receptor agonist) injected into the MnPO of sham-lesioned rats reduced water ingestion induced by ANG II injected into the same area. In VMH-lesioned rats ANG II-induced water intake increased with a previous injection of noradrenaline, phenylephrine, or isoproterenol. The pressor response induced by ANG II injected into the MnPO was reduced in VMH-lesioned rats, whereas the pressor response induced by clonidine was abolished. Previous treatment with noradrenaline and phenylephrine into the MnPO of sham-lesioned rats produced a pressor response, and a hypotensive response was obtained with the previous administration of noradrenaline, phenylephrine or isoproterenol into the MnPO of VMH-lesioned rats. These results show that VMH is essential for the dipsogenic and pressor responses induced by adrenergic and angiotensinergic activation of the MnPO in rats.

Adrenergic alpha-Agonists↗

PubMed1996

Targeting and function in mRNA export of nuclear pore complex protein Nup153.

Nup153 is a large (153 kD) O-linked glyco-protein which is a component of the basket structure located on the nucleoplasmic face of nuclear pore complexes. This protein exhibits a tripartite structure consisting of a zinc finger domain flanked by large (60-70 kD) NH2- and COOH-terminal domains. When full-length human Nup153 is expressed in BHK cells, it accumulates appropriately at the nucleoplasmic face of the nuclear envelope. Targeting information for Nup153 resides in the NH2-terminal domain since this region of the molecule can direct an ordinarily cytoplasmic protein, pyruvate kinase, to the nuclear face of the nuclear pore complex. Overexpression of Nup153 results in the dramatic accumulation of nuclear poly (A)+ RNA, suggesting an inhibition of RNA export from the nucleus. This is not due to a general decline in nucleocytoplasmic transport or to occlusion or loss of nuclear pore complexes since nuclear protein import is unaffected. While overexpression of certain Nup153 constructs was found to result in the formation of unusual intranuclear membrane arrays, this structural phenotype could not be correlated with the effects on poly (A)+ RNA distribution. The RNA trafficking defect was, however, dependent upon the Nup153 COOH-terminal domain which contains most of the XFXFG repeats. It is proposed that this region of Nup153, lying within the distal ring of the nuclear basket, represents a docking site for mRNA molecules exiting the nucleus.

Animals↗

PubMed1995

Nuclear pore complex proteins.

The nuclear envelope forms the boundary between the nucleus and the cytoplasm and as such regulates the exchange of macromolecules between the two compartments. The channels through the nuclear envelop that actually mediate this macromolecular traffic are the nuclear pore complexes. These are extremely elaborate structures which in vertebrate cells exhibit a mass of approximately 120 MDa. They are thought to be composed of as many as 100 distinct polypeptide subunits. A major challenge in the field of nucleocytoplasmic transport is to identify these subunits and to determine their functions and interactions in the context of the three-dimensional structure of the nuclear pore complex. It is the aim of this review to summarize what is currently known of the 20 or so nuclear pore complex proteins that have been described in either vertebrate or yeast cells.

Amino Acid Sequence↗

PubMed1994

Sequence analysis of a cDNA encoding a human nuclear pore complex protein, hnup153.

Nuclear pore complexes represent the channels for the the bi-directional movement of macromolecules between the nucleus and cytoplasm, and are thought to contain upwards of 100 different polypeptide subunits. Many of these subunits belong to a growing family of polypeptides termed nucleoporins which are characterized by the presence of O-linked N-acetylglucosamine moieties and a distinctive pentapeptide repeat (XFXFG). This paper reports the primary structure of hnup153, the human homologue of the rat nucleoporin, nup153, with which it shares 82% amino acid identity. In addition to 33 copies of the XFXFG repeat, hnup153 exhibits four repeats of 37-38 amino acids each containing an apparent 'zinc finger motif'. These zinc fingers are most closely related to those found in the mouse oncoprotein mdm-2 and a product of Drosophila small optic lobes (sol) gene.

Amino Acid Sequence↗

PubMed1994

Role of adrenergic pathways of the medial preoptic area in ANGII-induced water intake and renal excretion in rats.

In this study, we investigated the participation of adrenergic neurotransmission in angiotensin II- (ANGII)-induced water intake and urinary electrolyte excretion by means of injection of the alpha 1-, alpha 2-, and beta-adrenoceptor antagonists and ANGII into the medial preoptic area (MPOA) in rats. Prazosin (an alpha 1-adrenergic antagonist) antagonized the water ingestion, Na+, K+ and urine excretion induced by ANGII, whereas yohimbine (an alpha 2-adrenergic antagonist) enhanced the Na+, K+ and urine excretion induced by ANGII. Propranolol (a nonselective beta-adrenoceptor blocker) antagonized the water ingestion and enhanced the Na+, and urine excretion induced by ANGII. Previous treatment with prazosin reduced the pressor responses to ANGII, whereas yohimbine had opposite effects. Previous injection of propranolol produced no effects in the pressor responses to ANGII. These results suggest that the adrenergic neurotransmission in the MPOA may actively participate in ANGII-induced dipsogenesis, natriuresis, kaliuresis and diuresis in a process that involves alpha 1-, alpha 2-, and beta-adrenoceptors.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed1994

Interactions and three-dimensional localization of a group of nuclear pore complex proteins.

We have used antibodies directed against a number of nuclear pore complex (NPC) proteins to determine their mutual interactions and location within the three-dimensional structure of the NPC. A monoclonal antibody, termed QE5, recognized three NPC polypeptides, p250, NUP153, and p62 on Western blots, and labeled the nuclear envelope of several cultured cell lines by immunofluorescence microscopy. These three polypeptides contained O-linked N-acetylglucosamine residues and were released from the NPC by detergent/high-salt treatment as discrete high molecular weight complexes. p250 was found in association with a novel 75 kD protein, NUP153 was released as a homo-oligomer of about 1 megadalton, and p62 was associated with polypeptides of 58 and 54 kD (previously reported by Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J. Forbes. 1991. J. Cell Biol. 114:169-183). p75, p58, and p54 were not galactosylated in vitro. Xenopus oocyte NEs were labeled with gold-conjugated QE5 and prepared for electron microscopy by quick freezing/freeze drying/rotary metal shadowing. This EM preparation method enabled us to more precisely localize the epitopes of this antibody to the cytoplasmic filaments and the nuclear basket of the NPC. Since QE5 recognizes three O-linked NPC glycoproteins, its labeling was compared with that of the lectin wheat germ agglutinin which recognizes O-linked N-acetylglucosamine moieties. The two probes were found to yield similar, although not identical, distributions of label. To identify the individual proteins with particular NPC components, we have used an anti-peptide antibody against NUP153 and a monospecific anti-p250 polyclonal antibody. Labeling with these two antibodies has documented that NUP153 is a constituent of the nuclear basket with at least one of its epitopes residing in its terminal ring, whereas p250 is a constituent of the cytoplasmic filaments.

Amino Acid Sequence↗

PubMed1993

High prevalence of sexually transmitted diseases in a rural area in Mozambique.

OBJECTIVE: To assess the extent of the sexually transmitted diseases (STDs) problem in a rural area of Mozambique. METHODS: A cross sectional study among pregnant women and patients presenting with genital complaints. Laboratory confirmation was done for gonorrhoea, chlamydial infection, active syphilis, trichomoniasis and HIV infection. SETTING: A primary health care setting in Vilanculos, Inhambane province, Mozambique. RESULTS: Evidence of one or more of the above STDs was found in 51% of 201 pregnant women, 56% of 85 women and 62% of 77 men with genital complaints. Neisseria gonorrhoea or Chlamydia trachomatis were found in 16% of pregnant women, 23% of female patients and 28% of male patients; genital ulcer disease was present in 6%, 28% and 36%, of respectively pregnant women, female and male patients. The prevalence of active syphilis was about the same in the three groups of study subjects, that is 15%. HIV infection was found in 4% of the male patients; no HIV infection could be detected in the female groups. CONCLUSION: STDs were a major health problem in this rural area in Mozambique. Though HIV infection was still low, the high prevalence of STDs indicates that the potential is there for an explosive spread of the HIV/AIDS epidemic.

Adolescent↗

PubMed1992

Increase of cytokeratin D during liver regeneration: association with the nuclear matrix.

An increase of a 45 kD protein (p45) in the nuclear matrix has been observed when rat liver cells were proliferatively activated in vivo by a partial hepatectomy. The maximal levels of the association of p45 with the nuclear matrix have been detected 24 hr after hepatectomy just at the time when DNA replication is also maximal. By amino acid sequence analysis, immunoblotting and immunocytochemical methods, it has been demonstrated that p45 is identical to rat cytokeratin D. Immunogold staining of nuclear matrix-intermediate filament preparations from cultured hepatocytes indicated that p45 is associated with cytoskeletal filaments that are strongly interconnected to the lamina, whereas no intranuclear localization of the protein has been detected. With an overlay assay a specific binding of labeled p45 to two nonidentified high-molecular weight proteins and also to lamin B has been observed. Northern blot analysis revealed a biphasic pattern of expression of the messenger RNA for cytokeratin D during liver regeneration. A sharp increase in the messenger RNA levels occurred in the prereplicative phase of liver regeneration a few hours before the accumulation of the protein in the nuclear matrix fraction, and a second peak occurred 48 hr after partial hepatectomy.

Amino Acid Sequence↗

PubMed1992

[Meniscal injury in the plastic reconstruction of the anterior cruciate ligament. Meniscal suture or abstention].

The purpose of this study was to observe the change of the meniscal lesions, which are conservable by suture or simply by leaving-in-place within the frame of operated anterior instability. Forty-six knees which had been operated by the free transplant of bone-patellar tendon-bone, presented the associated lesions of conservable meniscus. These lesions were treated by suture of the meniscus in 15 cases and left in place in 31 cases. The programme of rehabilitation was the same in the two groups. All the patients have been followed-up for 26 months in average (12-40 months). In the group of meniscal suture, only one secondary meniscectomy was carried out. 8 patients presented moderate pain on the corresponding femorotibial joint line and 5/13 of the sportive patients recovered to the same level of activity. In the group of leaving-in-place, no meniscectomy has been carried out. Only 4 patients complained of pain and 23/27 recovered to the same activity level. The arthrographic and arthroscopic observations have been carried out in 23 cases. They confirmed the cicatrization of the sutured meniscus but also showed the possibility of total or partial cicatrization of the lesions left in place (12 on 13). The authors conclude that the peripheral meniscal lesions associated with the operation of the chronic anterior instability do not always require suture. The abstention on the meniscal lesion is possible if it is limited on the posterior segment: Then the functional results seem better.

Adolescent↗

PubMed1990

Decrease of calmodulin and actin in the plasma membrane of rat liver cells during proliferative activation.

After proliferative activation of rat liver cells in vivo by a partial hepatectomy a decrease of the calmodulin content in the three plasma membrane domains (blood sinusoidal, canalicular and lateral) was observed. At 24 hours after partial hepatectomy calmodulin was found to be 3 fold lower in the sinusoidal and lateral fractions whereas a 2 fold decrease was detected in the canalicular domain. Decreases on the actin levels have been also detected at 24 hours after a partial hepatectomy. Since at this time after surgery increases on nuclear actin and calmodulin have been reported, these results suggest the possibility that the actin and calmodulin dissociated from the plasma membrane after a partial hepatectomy could subsequently be translocated into the nuclei.

Actins↗

PubMed1990

Calmodulin-binding proteins in the nuclei of quiescent and proliferatively activated rat liver cells.

alpha-Spectrin, myosin light chain kinase (MLCK), and caldesmon have been detected in the nuclei of rat liver cells by 125I-calmodulin overlay, immunoblotting, and immunocytochemical methods. alpha-Spectrin is localized in the nuclear matrix, nuclear envelope, and nuclear pores. It has also been detected inside the nuclei in the form of small aggregates. MLCK is present in the nuclear matrix, envelope, nucleoli, and in a nuclease extract (S1 subfraction) but not in the nuclear pores. Caldesmon shows a diffuse distribution pattern inside the nuclei but it is not present in the nucleoli. Since all these proteins are components of the actin-myosin motility systems the presence of actin in the different nuclear subfractions has also been investigated: actin is present in the nuclear matrix, nuclear envelope, nucleoli, and nuclear pores. Proliferative activation of rat liver cells in vivo by partial hepatectomy induces the increase of alpha-spectrin, MLCK, and actin in different nuclear subfractions. This, together with the increase of nuclear calmodulin at the same time after hepatectomy (Pujol, M. J., Soriano, M., Aligúe, R., Carafoli, E., and Bachs, O. (1989) J. Biol. Chem. 264, 18863-18865), indicates that nuclear calmodulin could activate a nuclear contractile system during proliferative activation. A 62-kDa protein (p62) which binds to calmodulin columns and shows immunological similarities to caldesmon is specifically located in the region surrounding the nuclear envelope and is associated with the heterochromatin.

Animals↗