Biomedical subjects

L Carrasco

Publications and source records attributed to L Carrasco.

At least 73 records · Page 4Linked to original sources

PubMed1999

Pathological and immunohistochemical study of the liver and hepatic lymph nodes in goats infected with one or more doses of Fasciola hepatica.

Lesions produced by Fasciola hepatica in the liver, gall-bladder and hepatic lymph nodes (HLNs) of four groups of five goats are described; in addition, the distribution of CD3+ T lymphocytes and IgG-lambda light chain-bearing cells was analysed in the hepatic lesions and HLNs. One group of goats received a single oral dose of metacercariae, but the other four groups received four or five doses at different intervals over a period of 11 weeks. Animals that survived were killed 53-55 weeks after the first infective dose. Goats were more susceptible to multiple doses than to a single dose, even when the total number of metacercariae was the same. This greater susceptibility was manifested by the occurrence of deaths and the severity of hepatic lesions. CD3+ lymphocytes were sparse in the infiltrate surrounding the acute migratory tracts, suggesting inhibition of the local cell-mediated immune response. Goats with numerous hepatic calcareous granulomas showed the most severe hepatic damage, including marked cirrhosis, with a striking infiltrate of CD3+ T lymphocytes and lambda IgG- plasma cells, replacing extensive areas of hepatic parenchyma, in which hypertrophy of the smooth endoplasmic reticulum of hepatocytes was evident. These findings were observed mainly in the goats given more than one infective dose.

Animals↗

PubMed1999

The role of pulmonary intravascular macrophages in the pathogenesis of African horse sickness.

African horse sickness (AHS) is a disease of equids, characterized by severe pulmonary oedema and caused by an orbivirus. To determine the role of pulmonary intravascular macrophages (PIMs) in the development of pulmonary microvascular changes in this disease, five horses were given an intravenous inoculation of 10(6)TCID50of serotype 4 of AHS virus. Viral replication was detected in endothelial cells, PIMs, interstitial macrophages and fibroblasts. Alveolar and interstitial oedema, and changes in pulmonary microvasculature, consisting mainly of the sequestration of neutrophils and the formation of platelet aggregates and fibrinous microthrombi, were related to endothelial changes and to a high degree of PIM activation. This suggested that the PIMs, once activated, contributed to these vascular changes by releasing chemical inflammatory mediators.

African Horse Sickness↗

PubMed1999

Pathogenesis of African horse sickness: ultrastructural study of the capillaries in experimental infection.

African horse sickness (AHS) was induced in five horses by inoculation, to determine the ultrastructural changes in endothelial cells of capillaries in the myocardium, lung, spleen and liver. The animals developed cardiac and mixed forms of the disease. Alterations detected in the endothelial cells of the vessels of infected animals included: the presence of structures associated with viral infection, hypertrophy, degenerative changes, appearance of cytoplasmic projections, changes in permeability, alteration of intercellular junctions, loss of endothelium, subendothelial deposition of cell debris and fibrin, and vascular repair. In association with these changes, oedema, haemorrhages and microthromboses were detected, particularly in the myocardium and lung. This study showed that infection of, and changes to, the capillary endothelial cells of the organs under study was independent of the form in which the disease manifested itself but was dependent on the organ and blood vessel type. Thus, different levels of viral tropism were observed for the endothelial cells of the vessels in different organs. Viral infection was commonest in the endothelial cells of myocardial vessels, followed by those in the lung, whereas in the spleen and liver, endothelial cell infection was rare and, in the case of the liver, limited to the interstitial capillaries.

African Horse Sickness↗

PubMed1999

Genetic selection of poliovirus 2Apro-binding peptides.

The yeast two-hybrid system has been used to identify mammalian clones that interact with poliovirus 2A proteinase (2Apro). Eight clones which encode previously unidentified human proteins were selected from a HeLa cell cDNA expression library. In addition, five clones encoding short peptides that interact with poliovirus 2Apro were also identified. The lengths of these peptides range from 6 to 30 amino acids, but all of them contain the Leu-X-Thr-Z motif (X represents any amino acid; Z represents a hydrophobic residue). This sequence is invariably located just at the carboxy terminus of each peptide. This approach raises the possibility of designing substrate analogue inhibitors of 2Apro. Thus, two nonhydrolyzable peptides containing the Leu-X-Thr-Z motif prevented cleavage of eukaryotic initiation factor 4G by poliovirus 2Apro in vitro. A more general method for identifying peptides with antiproteinase activity is discussed.

Amino Acid Sequence↗

PubMed1999

Cleavage of eukaryotic translation initiation factor 4G by exogenously added hybrid proteins containing poliovirus 2Apro in HeLa cells: effects on gene expression.

Efficient cleavage of both forms of eukaryotic initiation factor 4G (eIF4G-1 and eIF4G-2) has been achieved in HeLa cells by incubation with hybrid proteins containing poliovirus 2Apro. Entry of these proteins into cells is promoted by adenovirus particles. Substantial levels of ongoing translation on preexisting cellular mRNAs still continue for several hours after eIF4G degradation. Treatment of control HeLa cells with hypertonic medium causes an inhibition of translation that is reversed upon restoration of cells to normal medium. Protein synthesis is not restored in cells lacking intact eIF4G after hypertonic treatment. Notably, induction of synthesis of heat shock proteins still occurs in cells pretreated with poliovirus 2Apro, suggesting that transcription and translation of these mRNAs takes place even in the presence of cleaved eIF4G. Finally, the synthesis of luciferase was examined in a HeLa cell line bearing the luciferase gene under control of a tetracycline-regulated promoter. Transcription of the luciferase gene and transport of the mRNA to the cytoplasm occurs at control levels in eIF4G-deficient cells. However, luciferase synthesis is strongly inhibited in these cells. These findings indicate that intact eIF4G is necessary for the translation of mRNAs not engaged in translation with the exception of heat shock mRNAs but is not necessary for the translation of mRNAs that are being translated.

Cysteine Endopeptidases↗

PubMed1999

Nasal zygomycosis and pulmonary aspergillosis in an American bison.

Concomitant nasal zygomycosis and pulmonary aspergillosis was diagnosed in a 3-mo-old female American bison calf (Bison bison) in Pennsylvania (USA). Etiologic diagnosis was made by immunohistochemistry using a panel of monoclonal antibodies and heterologously absorbed polyclonal antibodies. In the lungs fungal infection was accompanied by hemorrhage, fibrin exudation, and infiltration with neutrophils. Fungi were observed to penetrate apparently normal epithelial lining of the nasal turbinates, and there was hemorrhage, edema, and invasion of blood vessels in the submucosa. In vessels fungi were typically associated with thrombosis. The calf may have been infected due to a high level of exposure to mouldy feed and litter in the environment in combination with a collapse of it's natural defence mechanisms.

Animals↗

PubMed1998

Interaction of adenosine nucleotide analogs with Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.

The substrate characteristics and interactions of different adenosine nucleotide analogs with Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase were investigated by steady-state kinetic analysis and calculations of interaction energies. Comparison of Vmax/Km values showed that analogs substituted at C8 in the adenine ring (8-Br-ATP, 8-N3-ATP, 8-N3-ADP) gave almost the same kinetic values as ATP and ADP, whereas those substituted in the ribose hydroxyls (3'(2')-O-(N-methylanthraniloyl)-ATP (MANT-ATP), 3'(2')-O-(N-methylanthraniloyl)-ADP (MANT-ADP), 2'(3')-O-(2,4,6-trinitrophenyl)-ADP (TNP-ADP), 2'(3')-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP)) showed 1-8% the value for the corresponding physiological substrate. A comparison between the experimental results and molecular mechanics calculations was performed, employing a model for the S. cerevisiae PEP carboxykinase-ATP-Mn2+ complex. The calculated interaction energies of S. cerevisiae PEP carboxykinase with ATP, MANT-ATP, TNP-ATP, 8-Br-ATP, and 8-N3-ATP were linearly related (correlation coefficient 0.92) with -ln(Vmax/Km). This good correlation supports the proposal that the interaction of the substituent with the enzyme affects the interaction of the common region of ATP with the active site, thus leading to effects in Vmax.

Adenine Nucleotides↗

PubMed1998

Malignant schwannoma in a red deer (Cervus elaphus).

A five-year-old female red deer (Cervus elaphus) was in poor condition and severely lame on the left hindleg owing to a 19.4 cm x 15.9 cm mass involving and destroying the distal end (head) of metatarsal bones III and IV, the proximal sesamoid bones and the first phalanges (III and IV). The histopathological analysis revealed a spindle cell tumour with frequent palisade arrangement (Antoni type A pattern), and with highly anaplastic tumour cells in some areas. Structures resembling peripheral nerves were identified within the tumour. The neoplastic cells reacted with vimentin in a cytoplasmic pattern, and almost all of them reacted with S-100 protein in a nuclear and cytoplasmic pattern and did not express neurofilament, glial fibrillary acidic protein or keratins. This immunophenotype and the histopathological features were consistent with a diagnostic of malignant schwannoma. It was atypical because of the species affected, the location and the local malignancy.

Animals↗

PubMed1998

Mutational analysis of poliovirus 2Apro. Distinct inhibitory functions of 2apro on translation and transcription.

Transient expression of poliovirus 2Apro in mammalian cells by means of the recombinant vaccinia virus vT7 expression system leads to drastic inhibition of both cellular and vaccinia virus gene expression (Aldabe, R., Feduchi, E., Novoa, I., and Carrasco, L. (1995) FEBS Lett. 377, 1-5; Aldabe, R., Feduchi, E., Novoa, I., and Carrasco, L. (1995) Biochem. Biophys. Res. Commun. 215, 928-936). To obtain further insights into the molecular basis of this inhibition, a number of 2Apro variants were generated and expressed in COS-1 cells. The effect of these variants on cellular translation, on vaccinia virus-specific translation, and on transcription of the reporter gene luciferase was analyzed. The ability of the different 2Apro variants to block cellular translation depends on their capacities to cleave eIF-4G. The blockade exerted by 2Apro on transcription of the luciferase gene reinforces the notion that this protease is a potent inhibitor of RNA polymerase II-mediated transcription. Some of the 2Apro variants tested failed to block luciferase transcription, despite the fact that eIF-4G cleavage and inhibition of translation were observed. Two reconstituted polioviruses mutated in 2Apro were defective in inhibiting luciferase transcription, yet were still able to cleave eIF-4G and block translation. These findings indicate that 2Apro interferes with cellular gene expression at both the transcriptional and translational levels. Moreover, these two effects probably reflect the inactivation of different host proteins by poliovirus 2Apro.

Amino Acid Sequence↗

PubMed1998

The human immunodeficiency virus type 1 Vpu protein enhances membrane permeability.

Infection of T lymphocytes by the human immunodeficiency virus causes drastic alterations in the intracellular cation content of the infected cells. The human immunodeficiency virus type 1 genome encodes several accessory proteins, including Vpu, an integral membrane protein that forms ion channels in planar lipid bilayers. The effect of Vpu on the permeability of the plasma membrane to several molecules has been analyzed. Expression of vpu in Escherichia coli cells increases membrane permeability to a number of molecules such as 2-nitrophenyl beta-D-galactopyranoside, uridine, the impermeable translation inhibitor hygromycin B, and lysozyme. In addition, transient expression of Vpu in eukaryotic COS cells enhances entry of charged molecules such as hygromycin B and neurobiotin into these cells. The effect of Vpu on cell membrane permeability resembles that reported for other membrane-active proteins from different animal viruses, including influenza M2, Semliki Forest virus 6K, and poliovirus 2B and 3A proteins.

Animals↗

PubMed1998

Poliovirus 2A proteinase cleaves directly the eIF-4G subunit of eIF-4F complex.

The initiation of translation on eukaryotic mRNA is governed by the concerted action of polypeptides of the eIF-4F complex. One of these polypeptides, eIF-4G, is proteolytically inactivated upon infection with several members of the Picornaviridae family. This cleavage occurs by the action of virus-encoded proteinases: 2Apro (entero- and rhinovirus) or Lpro (aphthovirus). An indirect mode of eIF-4G cleavage through the activation of a second cellular proteinase has been proposed in the case of poliovirus. Although cleavage of eIF4G by rhino- and coxsackievirus 2Apro has been achieved directly in vitro, a similar activity has not been documented to date for poliovirus 2Apro. We report here that a recombinant form of poliovirus 2Apro fused to maltose binding protein (MBP) directly cleaves human eIF-4G from a highly purified eIF-4F complex. Efficient cleavage of eIF-4G requires magnesium ions. The presence of other initiation factors such as eIF-3, eIF-4A or eIF-4B mimics in part the stimulatory effect of magnesium ions and probably stabilizes the cleavage products of eIF-4G generated by 2Apro. These results suggest that efficient cleavage of eIF4G by MBP-2Apro requires a proper conformation of this factor. Finally, MBP-2Apro protein cleaves an eIF-4G-derived synthetic peptide at the same site as rhino- and coxsackievirus 2Apro (R485-G486).

Carrier Proteins↗

PubMed1998

Immunohistochemical characterization of hepatic lesions associated with Elaeophora elaphi parasitism in red deer (Cervus elaphus).

This study was based on the immunohistochemical characterization of hepatic lesions caused by Elaeophora elaphi in 15 red deer. In eight animals filariae were found in branches of the portal vein. "Web-like" endophlebitis (15 cases), lymph nodule-like thrombi associated with filarial debris (12 cases) and fibrinous thrombi (three cases) were also observed in the portal vein. Multifocal granulomatous hepatitis (six cases) and lymph nodule-like tissue (three cases) were found in the liver parenchyma. The distribution of CD3 antigen, IgG-lambda light chains, and S-100 protein in lymphoid tissue in the portal vein thrombi and hepatic parenchyma was similar to that found in the cortex of the lymph nodes. In these lesions, lymphoid follicles with large germinal centres (mainly composed of IgG+ B lymphocytes, with occasional S-100+ follicular dendritic cells) predominated over interfollicular lymphoid tissue (mainly composed of CD3+ T lymphocytes and S-100+ interdigitating cells). This suggested that the local humoral immune response was more important than the cellular response in this parasitic disease. The presence of interdigitating and follicular dendritic cells confirmed the high degree of organization of the abnormal lymphoid tissue and suggested that antigen presentation to T and B lymphocytes and the subsequent proliferation of the latter was particularly efficient in such tissue. The latter may therefore be superior to diffuse granulomatous infiltrate in terms of the production of a local immune response.

Age Factors↗

PubMed1998

Thrombocytopenia associated with apoptotic megakaryocytes in a viral haemorrhagic syndrome induced by a moderately virulent strain of African swine fever virus.

A viral haemorrhagic syndrome was induced in 14 pigs by inoculation with an African swine fever (ASF) virus strain of moderate virulence, to determine changes in megakaryocyte (MK) numbers and morphology and thus to assess the role of these cells in the thrombocytopenia characteristic of subacute ASF. The strain tested induced changes in the proportion of different types of MK (typical nucleated MKs, apoptotic MKs and immature MKs); it also caused subcellular lesions over the first 7 days post-inoculation (dpi). At 7 dpi, severe thrombocytopenia was observed. There was a statistically significant increase in apoptotic MK numbers. The MKs showed three stages in the course of the disease: a compensatory stage, represented by cytoplasmic projections, a hypermaturity stage, represented by apoptotic MKs, and a regenerative stage, represented by clusters of immature MKs. These changes, especially the presence of numerous apoptotic MKs, may explain the early and transitory thrombocytopenia detected in subacute ASF. The large number of apoptotic MKs observed may be associated with the accelerated maturation of these cells, resulting from the action of cytokines, or peripheral platelet consumption, or both.

African Swine Fever↗

PubMed1998

Immunohistochemical and ultrastructural evidence of hog cholera virus infection of megakaryocytes in bone marrow and spleen.

Twelve pigs were inoculated with a highly virulent strain of hog cholera virus (HCV) to study viral infection of megakaryocytes in the bone marrow and spleen. Immunohistochemical and ultrastructural examination revealed HCV infection in a small proportion (2.5-9.0%) of these cells from the 2nd to the 9th day after inoculation, at which time the experiment was terminated. Megakaryocyte infection accounts for the presence of viral antigens in platelets. The latter may represent a passive vehicle for spreading the virus in the animal.

Animals↗

PubMed1998

Use of bile cytology for early diagnosis of complications in orthotopic liver transplantation.

We conducted a daily analysis of bile cellularity in 25 patients undergoing 29 orthotopic liver transplants (OLT) and correlated the cytological parameters with the clinical outcome of each patient. The 16 patients without complications only showed slides with cells during the first 4-5 postoperative days. The four patients with primary non-function (PNF) of the graft had a high cell density up to the time of the retransplant, with a preponderance of polymorphonuclear (PMN) leucocytes (59.2%) and epithelial cells (29.2%). During the episodes of sepsis (n = 3) and rejection (n = 7) we noted the sudden appearance of high cellularity, almost exclusively PMN leucocytes (96.5%), and a preponderance of PMN leucocytes (84.2%) with appreciable percentages of mononuclear cells and macrophages as well as the early appearance of lymphoblasts in the rejection episodes. Our results show that bile cytology can be a useful method for diagnosing graft complications in liver transplantation.

Adult↗

PubMed1998

Prognostic factors in primary gastrointestinal non-Hodgkin's lymphoma: a multivariate analysis of 76 cases.

OBJECTIVE: To present our experience with the treatment of primary gastrointestinal (GI) non-Hodgkin's lymphoma, evaluate prognostic factors, and give our recommendations for treatment. DESIGN: Retrospective study. SETTING: Teaching hospital, Spain. SUBJECTS: 76 patients (47 men and 29 women, mean age 51 years) treated over the 15 years 1980-1994. INTERVENTIONS: 52 patients had radical resections, 19 palliative resections, and 5 biopsy alone. 42 (55%) also had adjuvant chemotherapy and 20 (26%) radiotherapy. RESULTS: Patients with primary intestinal lymphoma were slightly but not significantly younger than those with gastric lymphoma (43 compared with 56 years). 43 Patients (57%) had tumours in the stomach, 26 (34%) in the small bowel, and 7 (9%) in the colon. At presentation 34 had stage I disease, 25 stage IIE1 disease, and the remaining 17 stage IIE2; 14 were classified as low grade, 41 as intermediate, and 21 as high grade. 60 (79%) had a B-cell phenotype. Overall 5-year survival was 53%. Of the 11 variables tested by univariate analysis for their prognostic effect only abdominal mass (p < 0.001), clinical stage (p < 0.001), type of operation (p < 0.001), tumour size (p < 0.05), and histological grade (p < 0.05) achieved significance, but when Cox's multivariate analysis was applied only clinical stage was significant (p < 0.01). CONCLUSION: Operation is the treatment of choice, but chemotherapy and radiotherapy may have a role though as yet there are no standard guidelines for their use.

Chemotherapy, Adjuvant↗

PubMed1998

Co-expression of human eIF-4G and poliovirus 2Apro in Saccharomyces cerevisiae: effects on gene expression.

The poliovirus 5' untranslated region (5' UTR) confers on mRNAs the capacity to be translated by internal initiation. The functionality of this RNA motif has been tested in yeast cells (Saccharomyces cerevisiae) using luciferase (luc) as a reporter gene. Although some luciferase is synthesized from luc mRNA containing the poliovirus 5' UTR (Leader-luc mRNA), much more luciferase is synthesized in cells that express luc mRNA devoid of the poliovirus 5' UTR. Since poliovirus 2Apro enhances the translation of Leader-luc mRNAs after eIF-4G cleavage in mammalian cells, yeast cells were produced that synthesize three heterologous proteins, luciferase, poliovirus 2Apro and human eIF-4G. Initially, S. cerevisiae cells constitutively expressing human eIF-4G were isolated. The human eIF-4G gene does not complement yeast cells defective in the initiation factor counterpart, p150, indicating that the human and yeast eIF-4G are not interchangeable. Expression of poliovirus 2Apro in an inducible manner does not affect p150, but led to the efficient cleavage of human eIF-4G in yeast cells. Induction of 2Apro was detrimental to luciferase synthesis either from luc mRNA or Leader-luc mRNA irrespective of the presence or absence of human eIF-4G. 2Apro blocked luciferase expression at the transcriptional level. Finally, the effects of 16 point mutations of poliovirus 2Apro on luciferase expression and human eIF-4G cleavage were analysed. Only those 2Apro variants that generate viable polioviruses actively cleave eIF-4G in yeast.

Amino Acid Sequence↗

PubMed1998

Genetic analysis of poliovirus protein 3A: characterization of a non-cytopathic mutant virus defective in killing Vero cells.

A mutational and genetic analysis of the poliovirus protein 3A has led to the identification of a single amino acid mutant virus with a restrictive phenotype to form plaques in Vero cells. This mutant (I46T 3A) can be grown and amplified in HeLa cells, where virus replication takes place at wild-type levels. However, Vero cells infected with this virus cannot complete the growth cycle. I46T 3A virus has a defect in the ability to kill Vero cells, as estimated by FACS analysis of propidium iodide uptake by dead cells. Since these defects are observed under conditions where no abnormalities in the rate of synthesis and processing of the mutant polyprotein occur, the inability to induce the cytopathic effect in infected Vero cells denotes the existence of a defect in the activity of 3A, but not the level of expression of the viral genome. As a consequence of this impaired capability to generate the cytopathic effect, I46T 3A mutant viruses cannot be titrated by plaque assay in Vero cells. Only revertant viruses with the wild-type sequence arise and form lysis plaques in Vero cells. Our results suggest a role for the 3A protein (or a precursor thereof) in the virus-induced cytopathic effect. The mutant virus characterized in this work may be a useful tool to understand how poliovirus kills infected cells and carries out the final step of its life-cycle, the release of virus progeny.

Amino Acid Substitution↗