Biomedical subjects

A Angulo

Publications and source records attributed to A Angulo.

At least 37 records · Page 2Linked to original sources

PubMed1996

A reliable method for Golgi staining of retina and brain slices.

Although the classical Golgi method is a powerful means for structural analysis of the brain, it is generally considered to be an unpredictable technique making anatomists wary of using it. Often, even when successful staining has occurred, deposits of silver chromate crystals on the surface of the tissue obscure examination. This paper describes a simple procedure for Golgi impregnation of retina and brain slices so that good, even staining is obtained and crystal formation is avoided. The most outstanding feature of the method is the consistency of results. This consistency is due to two factors: (1) the accurate determination of the optimal chromation by measuring the rise of pH in the solutions and (2) the uniform penetration of dichromate and silver nitrate to the specimen by using a freely hanging, sandwiching technique. We suggest that the method described here can be applied to other parts of the nervous system and will be a reliable way to identify and better classify new cell types.

Animals↗

PubMed1996

Characterization of the sequences of the human cytomegalovirus enhancer that mediate differential regulation by natural and synthetic retinoids.

Evidence exists to suggest that human cytomegalovirus (hCMV) may opportunistically use retinoic acid (RA) to advance its own replication, in which transcriptional activation of the viral major immediate-early promoter is a crucial control point. We demonstrate that the enhancer of the viral promoter contains three RA-response-elements that cooperate in mediating RA activation. These elements are direct repeats of two sequence motifs separated by 2 bp (DR2 site, REa) and 5 bp (DR5 sites, REb and c). DNA-binding experiments revealed that each of these elements bind RA receptor (RAR)-retinoid X receptor (RXR) heterodimers more efficiently than either homodimer. Apparent equilibrium dissociation constants of RAR-RXR heterodimers for sites REa, REb, and REc were estimated to be 5 nm, 10 nm, and 20 nm, respectively. The level of contribution of each of these elements to RA inducibility correlated with the strength of binding by RAR-RXR heterodimers to each site. These experiments demonstrate that RAR and RXR are necessary for RA responsiveness of the viral promoter. Using synthetic RA analogs, which selectively activate RARs and RXRs, the RAR partner within the heterodimeric complex appeared to be sufficient while the RXR partner was insufficient to independently activate transcription. However, joint activation of RARs and RXRs indicated that RXRs (in the presence of a transcriptionally active RAR) could contribute to transactivation. This restricted co-dependent ligand activation of RXR varied depending on the particular response element and the cell context. These studies further indicate that signaling of retinoid receptors (in particular RAR) by RA plays an important role in modulating hCMV infection.

Carcinoma↗

PubMed1995

Retinoid activation of retinoic acid receptors but not of retinoid X receptors promotes cellular differentiation and replication of human cytomegalovirus in embryonal cells.

The susceptibility of human embryonal cell line NT-2/D1 to replicate human cytomegalovirus (hCMV) is dependent on retinoic acid (RA) stimulation. Physiological responses to retinoic acid involve two distinct subfamilies of nuclear receptors, the RA receptors (RARs) and retinoid X receptors (RXRs), which function by activating transcription as heterodimeric or RXR homodimeric complexes from cis-acting DNA response elements. At present, it is not clear whether the association between these two classes of receptors can lead to multiple distinct induction pathways by signalling one or both receptor partners. Here we have determined, by selectively activating endogenous receptors with novel synthetic ligands specific for either RARs or RXRs, what ligand interaction is physiological in the retinoid receptor pathways necessary for inducing replication of hCMV in differentiated embryonal cells. We show that ligand binding to RAR alone is sufficient and that exclusive ligand activation of RXR is insufficient for inducing replication of hCMV. We also find that differentiation and inhibition of NT-2/D1 cell growth are promoted by compounds that signal the RAR pathway. These results provide direct evidence that RAR ligand-mediated physiological responses are separable and distinct from RXR ligand activation functions. Moreover, our results provide insight into a hormone response pathway for cellular differentiation that might be coopted by hCMV in the host.

Cell Differentiation↗

PubMed1995

Regulation of human cytomegalovirus by retinoic acid.

Human cytomegalovirus (HCMV) may opportunistically use retinoic acid (RA), a physiologically essential control molecule of cellular differentiation, to advance its own replication. At present, it is not clear whether RA stimulation of HCMV replication is exclusively a differentiation driven event. We report that exposure of permissively infected primary human foreskin fibroblast cells to RA transcriptionally activates the viral major immediate-early promoter (MIEP) and enhances the production of infectious particles. These experiments implicate a direct involvement of RA in modulating HCMV activation.

Cytomegalovirus↗

PubMed1994

Role of thyroid hormones in the maturation of interhemispheric connections in rats.

Hypothyroidism causes mental retardation secondary to changes in the organization of the CNS. These changes affect higher brain functions for which interhemispheric transfer of information is crucial. In present study, the anterior commissure (AC) and corpus callosum (CC) of normal (C) and hypothyroid (H) rats has been examined using quantitative electron microscopy. H rats received an antithyroid treatment with methimazole from embryonic day 14 (E14) and surgical thyroidectomy at postnatal day 6 (P6). In the AC, the number of axons (unmyelinated and myelinated) increased from 0.17 x 10(6) axons at E18 to 1.08 x 10(6) axons at P4 and it was almost the same at P180 (1.01 x 10(6) axons). In H rats the number of axons between P14 and P180 was similar to that of C rats. In contrast, there were only 0.11 x 10(6) myelinated axons at P180 resulting in a 66% reduction with respect to C rats (0.36 x 10(6) axons). In the CC of C rats, the number of myelinated axons increased from 1.76 x 10(3) axons at P12 to 3.34 x 10(6) axons at P184. In H rats, there were only 0.84 x 10(6) axons at P184 resulting in a 76% reduction with respect to C rats. This reduction was more important in the posterior sector of the CC (95%) than in the rest (on average 63%). Therefore these results show that thyroid hormones play an important role in the processes involved in the maturation of commissural axons.

Animals↗

PubMed1994

Golgi-Colonnier method: correlation of the degree of chromium reduction and pH change with quality of staining.

We examined the role of chromium reduction in the Golgi-Colonnier method, correlating the quality of neuronal impregnation with the levels of hexavalent (CrVI) and trivalent (CrIII) chromium in the tissue and in the chromation fluid (CF). The concentrations of both chromium species were assessed by measuring spectrophotometrically the CrVI before and after oxidizing the sample and by calculating the ratio of CrVI to total chromium (chromium ratio, CrR). The CrR was almost identical in the tissue and the CF, decreasing exponentially during chromation due to a progressive consumption of CrVI to form CrIII. Satisfactory cell impregnation was obtained only when the CrR was 0.45-0.7, regardless of other factors. The CrR values could be accurately predicted by the pH increase of the CF; this increase has proven to be a most reliable criterion to decide the endpoint of the chromation process. The dependence of cell staining on the [CrIII], together with the well-known ability of this species to bridge proteins, suggests that the key event for cell impregnation is the cross-linking of neuronal proteins by CrIII polymers.

Animals↗

PubMed1993

Virus-host interactions in African swine fever: the attachment to cellular receptors.

Biochemical and morphological techniques have shown that African swine fever virus (ASFV) enters susceptible cells by a mechanism of receptor-mediated endocytosis. The virus binds to a specific, saturable site in the cell and this interaction is required for a productive infection. A structural ASFV protein of 12kDa (p12) has been identified to be involved in the recognition of the cellular receptor, on the basis of the specific binding of the polypeptide to sensitive Vero cells. Protein p12 is externally located in the virus particle, forming disulfide-linked dimers with an apparent molecular mass of 17kDa. The gene has been mapped within the central region of the BA71V strain genome. Sequencing analysis has shown the existence of an open reading frame encoding a polypeptide of 61 amino acids characterized by the presence of a putative transmembrane domain, and a cysteine rich region in the C-terminal part which may be responsible for the dimerization of the protein. Transcripts of the p12 gene were only synthesized during the late phase of the infectious cycle. No posttranslational modifications of the polypeptide, such as glycosylation, phosphorylation or fatty acid acylation, have been found. The comparison of the amino acid sequence of protein p12 from 11 different virus strains has revealed a high degree of conservation of the polypeptide.

African Swine Fever↗

PubMed1993

Mapping and sequence of the gene encoding the African swine fever virion protein of M(r) 11500.

The gene encoding the African swine fever virus protein of M(r) 11,500, present in the virus particle, has been mapped and sequenced in the genome of the Vero cell-adapted virus strain BA71V. A serum raised against virion proteins of M(r) 12,000 to 13,000 isolated from polyacrylamide gels was used to screen a plasmid expression library, containing viral DNA random fragments, that expresses viral polypeptides fused to beta-galactosidase. Using this method, we have identified and sequenced the open reading frame (ORF) A137R, which initiates at the right end of the EcoRI A restriction fragment and extends into the EcoRI F fragment. Expression of the protein in Escherichia coli has confirmed that ORF A137R encodes a protein with an M(r) of about 12,000. A specific serum was raised against the E. coli-expressed protein, and has been used to identify the protein encoded by the ORF, which is translated at late times of infection and incorporated into the virus particle. Immunofluorescence experiments have shown that the protein localizes in virus factories.

African Swine Fever Virus↗

PubMed1993

Transcriptional mapping of a late gene coding for the p12 attachment protein of African swine fever virus.

The transcriptional characterization of the gene coding for the p12 attachment protein of the African swine fever virus is presented. The results obtained have been used to generate the first detailed transcriptional map of an African swine fever virus late gene. Novel experimental evidence indicating the existence of major differences between the mechanisms controlling the transcription of late genes in African swine fever virus and poxviruses is provided.

African Swine Fever Virus↗

PubMed1993

Inhibition of African swine fever virus binding and infectivity by purified recombinant virus attachment protein p12.

The African swine fever virus protein p12, involved in virus attachment to the host cell, has an apparent molecular mass of 17 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. We have also identified 12- and 10-kDa forms of the p12 protein in infected Vero cells and found that the mature 17-kDa protein is the only form present in virus particles. The p12 protein has been produced in large amounts in Spodoptera frugiperda insect cells infected with a recombinant baculovirus. A 17-kDa protein that possessed the biological properties of the viral protein was produced, since it bound to susceptible Vero cells and not to receptor-negative L cells, which do not support virus replication. The binding of the baculovirus-expressed protein p12 to Vero cells was specifically blocked by virus particles. In addition, the recombinant protein purified by immunoaffinity chromatography blocked the specific binding of virus particles to susceptible cells and prevented infection, demonstrating that the p12 protein mediates the attachment of virions to specific receptors and indicating that blocking the p12-mediated interaction between African swine fever virus and receptors in Vero cells can inhibit infection. However, although antibodies specific for protein p12 are induced in natural infections and in animals inoculated with inactivated virus or recombinant protein p12, these antisera did not inhibit virus binding to the host cell or neutralize virus infectivity.

African Swine Fever Virus↗

PubMed1993

Reproducibility of a commercial enzyme-linked immunosorbent assay for bovine paratuberculosis among eight laboratories.

Interlaboratory reproducibility of an absorbed enzyme-linked immunosorbent assay (ELISA) kit for detection of bovine serum antibodies to Mycobacterium paratuberculosis was evaluated. A panel of 30 bovine sera (15 positives and 15 negatives) was tested in triplicate microtiter wells on each of 2 days at 8 different laboratories. One laboratory had invalid results because of positive or negative serum control optical density (OD) readings beyond the acceptable range specified by the kit. The coefficient of variation (CV) for mean OD values was influenced by low ODs on test negative sera at 2 laboratories, thus the CVs on positive sera were considered a more representative measure of kit reproducibility. Between-well CVs averaged 6.7% +/- 2.8% (mean +/- standard deviation), and between-day CVs averaged 14.5% +/- 9.8% among the 7 laboratories with valid assays on the 15 positive sera. The OD values were converted to positive or negative classifications for each assay well, and the results were compared. Among 1,392 assays in 7 laboratories, 98.6% were in agreement. Eleven of 18 discrepant results were due to a sample that consistently gave OD values near the cutoff for a positive test. Exclusion of that serum from the analysis resulted in a 99.8% rate of agreement among laboratories. Results indicated that the absorbed ELISA kit provided reproducible results within and between laboratories.

Analysis of Variance↗

PubMed1992

Amino acid sequence and structural properties of protein p12, an African swine fever virus attachment protein.

The gene encoding the African swine fever virus protein p12, which is involved in virus attachment to the host cell, has been mapped and sequenced in the genome of the Vero-adapted virus strain BA71V. The determination of the N-terminal amino acid sequence and the hybridization of oligonucleotide probes derived from this sequence to cloned restriction fragments allowed the mapping of the gene in fragment EcoRI-O, located in the central region of the viral genome. The DNA sequence of an EcoRI-XbaI fragment showed an open reading frame which is predicted to encode a polypeptide of 61 amino acids. The expression of this open reading frame in rabbit reticulocyte lysates and in Escherichia coli gave rise to a 12-kDa polypeptide that was immunoprecipitated with a monoclonal antibody specific for protein p12. The hydrophilicity profile indicated the existence of a stretch of 22 hydrophobic residues in the central part that may anchor the protein in the virus envelope. Three forms of the protein with apparent molecular masses of 17, 12, and 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been observed, depending on the presence of 2-mercaptoethanol and alkylation with 4-vinylpyridine, indicating that disulfide bonds are responsible for the multimerization of the protein. This result was in agreement with the existence of a cysteine-rich domain in the C-terminal region of the predicted amino acid sequence. The protein was synthesized at late times of infection, and no posttranslational modifications such as glycosylation, phosphorylation, or fatty acid acylation were detected.

African Swine Fever Virus↗

PubMed1992

Comparison of the sequence of the gene encoding African swine fever virus attachment protein p12 from field virus isolates and viruses passaged in tissue culture.

Comparison of the amino acid sequence of the African swine fever virus attachment protein p12 from different field virus isolates, deduced from the nucleotide sequence of the gene, revealed a high degree of conservation. No mutations were found after adaptation to Vero cells, and a polypeptide with similar characteristics was present in an IBRS2-adapted virus. The sequence of the 5' flanking region was conserved among the isolates, whereas sequences downstream of the gene were highly variable in length and contained direct repeats in tandem that may account for the deletions found in different isolates. Protein p12 was synthesized in swine macrophages infected with all of the viruses tested.

African Swine Fever↗

PubMed1990

Morphology of the rat cochlear primary afferents during prenatal development: a Cajal's reduced silver and rapid Golgi study.

In this study, we analyse the process of spatial organisation of the cochlear root related to the morphological and topographical changes in the CN during the prenatal development of Wistar rats, placing special emphasis on aspects of the latero-medial distribution of the cochlear afferents. A total of 35 embryos from 8 Wistar rats was employed, corresponding to embryonic days 14, 16, 18 and 20. Twenty of these embryos were studied by the Cajal's reduced silver stain and 15 by the rapid Golgi method (osmium dichromate method). The otocyst, the vestibulo-cochlear ganglion and vestibulo-cochlear nerve were first observed at embryonic Day 14 (E14). At E16, a sharp separation between the cochlear and vestibular roots was distinguished. The final position of the primary afferents and their main branches (anterior and posterior) in the CN was observed at E18 and E20, when the total number of cochlear turns had been formed. The cochlear afferents coming from the apical coil, the last to be incorporated into the cochlear root, project their posterior branches at the bifurcation towards more medial portions of the PVCN and their anterior branches towards the more lateral regions of the AVCN.

Animals↗

PubMed1990

Prenatal development of the cochlear nerve in the albino rat.

Twenty-eight prenatal (14 to 20 days) Wistar albino rats were studied with Cajal-de Castro's silver reduced stain. The histological sections were oriented in a parasagittal oblique plane, parallel to the cochlear root, in order to observe the origin, course and destination of the cochlear fibres in as few consecutive sections as possible. In all the prenatal days studied, the fibres arising in the first half coil of the cochlea (hook's bundle), show a different orientation than those of the other cochlear nerve fibres, which constitute the spiralized bundle, originating in the basal, medial and apical portions of the cochlea. The hook-bundle does not participate in the curling of the cochlear nerve and it describes a particular course. At first, its fibres are situated in a lateral position in the nerve when it is inside the internal auditive duct. Then, the hook-fibres take a postero-inferior position just at the entry of the bulbar cochlear nuclei region. Furthermore, we have seen no hook's afferents branching in the ventral cochlear nucleus, and it could be suggested that these fibres project directly to the superficial dorso-medial area of the postero-ventral cochlear nucleus.

Animals↗

PubMed1990

Tectorial membrane changes in hypothyroid rats during postnatal development.

The morphological changes of the tectorial membrane (TM) during the postnatal development (0, 3, 6, 12 and 25 day old) of the organ of Corti were studied by light microscopy in 20 control and hypothyroid rats. Hypothyroidism was induced by daily administration of propylthioruracil (PTU) until the end of lactation. The auditive receptor in the cochlea of the hypothyroid animals shows serious structural alterations compared with those of normal ones: abnormal persistence of Kölliker's organ, immaturity of sensory cells and supporting cells and a specific distortion of the TM. Differences with controls were first observed on the sixth postnatal day of the hypothyroid rats. The inner spiral sulcus was not shaped and the TM was attached to the Kölliker's organ. In older stages (12 and 25 days), Kölliker's organ was still present. The TM acquired a shap hump with an abnormal fibrillar arrangement in its middle part. It was still attached to the outer supporting cells by a remnant of the marginal net. It was suggested that the TM is secreted by the inner spiral limbus and Kölliker's organ. An abnormal persistence of these structures in the hypothyroidism results in a retardation of Corti's organ development. However, this conclusion does not explain the absence of the outer portion of the TM. Our study confirms the hypothesis that the secretion of any components of the marginal zone of TM is made by outer supporting cells which in PTU-treated animals appear very immature and with hypoplasia.

Animals↗

PubMed1989

Retroperitoneal sacrocolpopexy for correction of prolapse of vaginal vault.

The problem of prolapse of vaginal vault has a clearly defined anatomic basis and so does it surgical restoration. Herein, we present our experience with a technique that is a modification of the abdominal sacrocolpopexy of Randall and Nichols. Our procedure is characterized by the use of a segment of Teflon (polytetrafluoroethylene) vascular prosthesis placed in the retroperitoneum at the sacral concavity to fasten the vaginal vault to the promontory. We have obtained excellent results with its application in 18 patients, who had a return to normal sexual activity and no recurrences during a follow-up period of 36 months.

Abdominal Muscles↗

PubMed1988

The occurrence of salmonellae in kitchens of care institutions.

Sporadic cases of salmonellosis always by the same serotypes occurred in two different care institutions. In the first institution the cases had been caused by S. binza and S. eimsbuettel, in the second by S. mons and S. braenderup. The cases of salmonellosis prompted studies into the occurrence of these Salmonella serotypes in the kitchens of the relevant institutions, as well as into the occurrence of salmonellas in kitchens of care institutions in general. Using Moore's swabs the relevant Salmonella serotypes could be demonstrated in the kitchens of the institutions with sporadic cases of salmonellosis, while in kitchens of 15 other arbitrary chosen institutions Salmonella could not be detected at all. This demonstrated that the occurrence of sporadic cases of salmonellosis was related to the occurrence of salmonellas in the kitchens. Then investigations were started to establish the origin of the salmonellas. There was a mice plague in one institution. Mouse-dung was found positive for the relevant Salmonella serotype(s). Cases of salmonellas did not occur any more when the mice had been eradicated. It was concluded that the sporadic cases of salmonellosis in that institution were strongly associated with the presence of mice. In the other institution it appeared that disinfection was inadequate and that salmonellae were spread all over the kitchen environment during cleaning, leading to recontamination. After thorough disinfection and adaptation of cleaning and disinfection procedures sporadic cases of salmonellosis did not occur anymore. Results of these investigations demonstrated the important role of vermin and proper cleaning and disinfection in food hygiene. Moreover, the sporadic cases of salmonellosis came to light because gastro-enteritis was a reason for examination of faecal specimens of patients. This is not usual in many care institutions. However, in this way problems in hygiene can be traced, especially in the kitchen. For this reason it is recommended strongly to examine faecal specimens in cases of gastro-enteritis, even in sporadic cases.

Accident Prevention↗