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Biomedical subjects

L Carrasco

Publications and source records attributed to L Carrasco.

At least 109 records · Page 6Linked to original sources
PubMed1997

Poliovirus infection and expression of the poliovirus protein 2B provoke the disassembly of the Golgi complex, the organelle target for the antipoliovirus drug Ro-090179.

Infection of Vero cells with poliovirus results in complete disassembly of the Golgi complex. Milestones of the process of disassembly are the release to the cytosol of the beta-COP bound to Golgi membranes, the disruption of the cis-Golgi network into fragments scattered throughout the cytoplasm, and the disassembly of the stacked cisternae by a process mediated by long tubular structures. Transient expression of the viral protein 2B in COS-7 cells also causes the disassembly of the Golgi complex by a process preceded by the accumulation of the protein in the Golgi area. Vero cells infected for 3 h show no recognizable Golgi complexes at the ultrastructural level and display an enormously swollen endoplasmic reticulum (ER) with extensive areas of its surface heavily coated. Ro-090179 (Ro), a flavonoid isolated from the herb Agastache rugosa, provokes the specific swelling and disruption of the Golgi complex and strongly inhibits poliovirus infection. Ro provokes the swelling and the disruption of the stacked cisternae and trans-Golgi elements without affecting the cis-most Golgi cisternae much. Moreover, Ro inhibits the fusion of the Golgi complex with the ER in cells treated with brefeldin A and provokes the accumulation of the intermediate compartment membrane protein p58 into ERD2-positive Golgi elements but has no effect on the anterograde transport involved in protein secretion. Our results indicate that the secretory pathway and specifically the Golgi complex are preferential targets of poliovirus.

Animals↗
PubMed1997

Poliovirus protein 2BC increases cytosolic free calcium concentrations.

Poliovirus-infected cells undergo an increase in cytoplasmic calcium concentrations from the 4th h postinfection. The protein responsible for this effect was identified by the expression of different poliovirus nonstructural proteins in HeLa cells by using a recombinant vaccinia virus system. Synthesis of protein 2BC enhances cytoplasmic calcium concentrations in a manner similar to that observed in poliovirus-infected cells. To identify the regions in 2BC involved in modifying cytoplasmic calcium levels, several 2BC variants were generated. Regions present in both 2B and 2C are necessary to augment cellular free calcium levels. Therefore, in addition to inducing proliferation of membranous vesicles, poliovirus protein 2BC also alters cellular calcium homeostasis.

Calcium↗
PubMed1997

African swine fever virus infection of bone marrow: lesions and pathogenesis.

The effects of African swine fever (ASF) virus infection on bone marrow hematopoiesis and microenvironment were determined by studying the sequential development of ultrastructural lesions of bone marrow and blood cell changes. Eight pigs (two pigs/infected group) were inoculated by intramuscular route with 10(5) 50% hemadsorbing doses (HAD50) of the Malawi'83 ASF virus isolate. Two uninfected pigs were used as controls. Ultrastructural changes developed by day 3 postinoculation (PI), persisted through day 7 PI, and were characterized by activation of macrophages. From day 5 PI, viral replication was observed in monocytes/macrophages, reticular cells, immature neutrophils, and promonocytes. Also viral replication was detected in megakaryocytes, endothelial cells, and pericytes at day 7 PI. Vascular alterations consisted of activation of sinusoidal endothelial cells, intravascular coagulation, and fibrin strands interspersed among microenvironment and hematopoietic cells. No significant changes were observed in total white blood cells counts, percentage of monocytes, and platelet counts; however, severe lymphopenia and neutrophilia were detected from day 3 PI. Results of this experiment indicate that there is increased hematopoiesis in bone marrow during acute ASF, coinciding with macrophage activation. Neither vascular changes nor viral replication in different bone marrow cell populations gave rise to impaired bone marrow function. Increased hematopoiesis would exert a positive influence by preventing the early onset of thrombocytopenia and would exert a negative influence by stimulating the spread of the virus via neutrophils. Increased hematopoiesis would be unable to compensate for the lymphopenia.

Acute Disease↗
PubMed1997

Abnormal antler growth associated with testicular hypogonadism in red deer.

A wild 5-yr-old red deer (Cervus elaphus) was eulled from a privately owned herd because of deformed antlers, retained velvet and bilateral symmetrical testicular hypogonadism. The clinical and pathological changes seen in this deer were most consistent with congenital hypoplasia, but testicular atrophy was an alternative possibility for the etiology of their condition.

Animals↗
PubMed1997

Subcellular changes in the tonsils of pigs infected with acute African swine fever virus.

A study of the pathogenesis of acute African swine fever (ASF) was carried out in pigs inoculated with a highly virulent strain of ASF virus to determine the sequential development of the subcellular changes in a particular lymphoepithelial organ, the tonsil. The apoptosis of the lymphocytes and the inhibition of lymphocyte proliferation were the main changes that occurred in the tonsillar lymphoid structures. This may explain the early lymphopenia observed in acute ASF. Moreover, vascular changes, consisting of increased vascular permeability, activation of endothelial cells and loss of these cells, might have been the cause of the characteristic haemorrhages found in the lymphoid organs during this disease. Virus replication has been observed in the epithelial cells, fibroblasts and reticular cell beginning on day 5 post-infection. The activation of the endothelial cells, apoptosis of lymphocytes, decreased lymphocyte mitosis and virus replication in non-mononuclear phagocyte system (MPS) cells all occurred after an intense proliferation and activation of the tonsillar macrophages and coincide with virus replication, which occurs in the macrophages 5 days post infection.

African Swine Fever↗
PubMed1997

Development of microscopic lesions in splenic cords of pigs infected with African swine fever virus.

Acute forms of African swine fever are characterized by hemorrhagic lesions in the lymphoid organs. This paper reports the evolution of lesions in the splenic cords of pigs inoculated with African swine fever (ASF) virus (strain Malawi'83). Ultrastructural examination of the splenic cords of the infected pigs revealed numerous macrophages attached to the muscle cells harboring virus replication center and cytopathic effects at 3 dpi (days post-infection). From 5 dpi, the splenic cords contained a large number of erythrocytes associated with abundant fibrin deposits, mainly arranged around the muscle cells, from which macrophages had disappeared. It is likely that the ASF virus replication, and consequent cytopathic effects, observed in the fixed macrophages of splenic cords, may be responsible for the fibrin deposition.

African Swine Fever↗
PubMed1996

Human immunodeficiency virus (HIV) Nef is an RNA binding protein in cell-free systems.

The function of human immunodeficiency virus nef gene product has been much debated but the precise activity of this protein in the HIV replication cycle remains unknown. HIV-1 Nef was obtained as a fusion protein with maltose binding protein (MBF), purified by amylose column chromatography and separated from MBP by cleavage with factor Xa. Purified HIV-1 Nef protein, but not the fusion protein MBP-Nef, binds to RNA in vitro as tested by three different assays, radioactive or non-radioactive. North-western analysis, UV cross-linking or band-shift analysis. This activity was lost in a deletion mutant lacking 22 amino acids from the amino terminus of HIV-1 Nef, while a deletion of 44 residues from the carboxy terminus of the protein does not impair the RNA binding activity. Moreover, a single amino acid replacement, Arg to Gly at position 22 produces a Nef variant deficient in its ability to interact with RNA. Different Nef proteins from HIV-1, HIV-2 or SIV were fused to MBP and cleaved with factor Xa. The different Nef proteins were all endowed with RNA-binding capacity. Sequence similarities between several RNA binding proteins, including picornavirus 2C and different Nef proteins are observed. The function of Nef during the HIV replication cycle is discussed on the basis of the present findings.

Amino Acid Sequence↗
PubMed1996

Membrane permeabilization by poliovirus proteins 2B and 2BC.

Poliovirus infection leads to drastic alterations in membrane permeability late during infection. Transient expression of each nonstructural protein of poliovirus by means of recombinant vaccinia virus encoding the T7 RNA polymerase indicates that proteins 2B and 2BC strongly enhance membrane permeability to hygromycin B in HeLa cells. Almost no effect on expression of proteins 2C, 3A, 3AB, and 3C was found. Deletions and point mutations in 2B and 2BC have identified sequences in 2B involved in membrane permeabilization. Regions located at both ends of 2B are necessary to bring about these permeability alterations. A deletion of 11 amino acids of 2BC at the junction between 2B and 2C, as well as long deletions in 2C encompassing the GTPase motifs of this protein, do not impair the capacity of 2BC to modify the permeability of the membrane. The release of compounds such as choline or uridine from preloaded cells is also augmented by 2B and 2BC expression.

Biological Transport↗
PubMed1996

Effects of cold ischemia time on the graft after orthotopic liver transplantation. A bile cytological study.

We conducted a daily analysis of bile cellularity in 16 orthotopic liver transplant patients fitted with a T-tube, and correlated the cytological parameters (number of cells per slide, and percentage of difference cell types) with the duration of cold ischemia time (CIT). Two groups were established: one comprised patients whose CIT was less than 7 hr (CIT averaged 345 min) and the other comprised patients with a CIT of more than 7 hr (CIT averaged 505 min). The control group consisted of 15 patients who had received cholecystectomy for biliary lithiasis and were fitted with a T-tube. All 3 groups showed the highest cell density on the 1st postoperative day (control: 53.3 +/- 15.5 cells/slide; short ischemia: 70 +/0 21.4 cells/slide; long ischemia: 158.8 +/- 53.2 cells/slide), which steadily ischemia group showed a higher cell density than did the control group for the first 2 days, although this was not significant. The long ischemia group showed the highest cell density, although only significantly for the first 2 days when compared with the controls, and basically at the expense of a increase in ductal epithelial cells. Our results show that prolonged cold ischemia causes an increase in bile cell density at the expense of ductal epithelial cells: the longer the preservation time, the greater the increase.

Adolescent↗
PubMed1996

The lesional changes and pathogenesis in the kidney in African swine fever.

African swine fever is a viral haemorrhagic disease of pigs which has been used as a model for the study of viral haemorrhagic diseases in man. The acute course of the disease is characterized by acute proliferative glomerulonephritis, with viral replication in mesangial cells and occasional focal necrosis of the renal tubular system; hyperplasia of the collecting ducts is associated with evident virus replication. Haemorrhages have been attributed to endothelial dysfunction, aggravated by virus replication in endothelial cells in the final stages of the disease. The renal interstitium displays intense oedema and an infiltrate largely composed of macrophages. Virus replication has also been observed in fibroblasts and in the smooth-muscle cells of arterioles and venules. In subacute-chronic forms of the disease, various types of glomerulonephritis are observed, ranging from mesangial proliferative glomerulonephritis to focal and segmental hyalinosis associated with immune-mediated phenomena. No striking changes are reported in the renal tubular system. Interstitial haemorrhages are associated with diapedesis due to immunologically mediated events. The interstitium has also been found to contain a lymphohistiocytic infiltrate with abundant plasma cells. No evidence has been reported of viral replication in any cell population.

African Swine Fever↗
PubMed1996

Caprine mastitis due to aspergillosis and zygomycosis: a pathological and immunohistochemical study.

Of 73 goats on a dairy farm, 27 developed mycotic mastitis in the post-partum period. Purulent mammary secretion, progressive induration of the affected glands, slight fever and weight loss were observed. As treatment produced no improvement within 2-3 weeks, all diseased animals were slaughtered. At post-mortem examination, widespread acute and chronic mycotic lesions were seen throughout the affected glands. Infection was thought to have spread through the milk ducts as an ascending infection resulting from prophylactic intramammary treatment with antibiotic before parturition. In all lesions, aspergillus hyphae were identified by indirect immunofluorescent labelling, which gave a strong and uniform reaction with polyclonal and monoclonal antibodies to Aspergillus fumigatus. In a single granulomatous lesion, zygomycotic hyphae were also identified immunohistochemically. At terminal swellings of aspergillus hyphae, "yeast-like bodies" were produced, a phenomenon which seems to be associated with special, but unknown, circumstances.

Animals↗
PubMed1996

Structural and ultrastructural study of glomerular changes in African swine fever.

The pathological effect of haemorrhagic fever viruses on the kidney have not been clearly documented. This study reports glomerular lesions in African swine fever. In the acute form of the disease there was an acute diffuse proliferative glomerulonephritis, which was believed to be related to virus replication in circulating monocytes and glomerular mesangial cells, and to the presence of abundant circulating cell debris resulting from viral replication at other sites. In the subacute form, the proliferative mesangial glomerulonephritis observed may have been associated with systemic immune-mediated phenomena, and with subendothelial and mesangial deposits of immunoglobulins and complement components.

African Swine Fever↗
PubMed1996

Subcellular changes in platelets in acute and subacute African swine fever.

The morphological changes in platelets in acute and subacute African swine fever (ASF) and their relationship to pathogenesis were studied. Eight pigs were inoculated with a highly virulent strain of African swine fever (Malawi '83) and 14 with a moderately virulent strain (Dominican Republic '78) for ultrastructural study of platelets, monocyte/macrophages and vascular structures in the liver, spleen, lymph node, bone marrow, lung and kidney. Both viruses produced activation and degranulation of platelets from day 3 after inoculation onwards, coinciding with activation of the mononuclear phagocyte system and virus replication in monocyte/macrophages. Platelet aggregation and viscous metamorphosis of platelets were observed at 5 and 7 days after inoculation with the highly virulent strain, coinciding with endothelial alterations, but platelet aggregation was less prevalent and there was no sign of viscous metamorphosis in animals inoculated with the moderately virulent strain. Virions within platelets were observed at the final stage of acute ASF and at 5-7 days after inoculation in subacute ASF. This suggests that platelets assist in disseminating ASF virus within the body, especially in subacute infections.

Acute Disease↗
PubMed1996

Apoptosis in lymph nodes in acute African swine fever.

This paper reports apoptosis of lymph-node lymphocytes in swine experimentally inoculated with a virulent African swine fever (ASF) virus isolate (Malawi '83). Apoptosis was observed in both compartments of cortical tissue, but was more intense in diffuse lymphoid tissue (T area). Lymphopenia detected in peripheral blood was associated with T-lymphocyte depletion. No evidence of ASF virus replication was observed in lymphocytes in the lymph nodes studied. This finding, together with the high rate of virus replication recorded in macrophages in diffuse lymphoid tissue as compared with the low rate recorded for lymphoid follicles, suggests a mechanism for the induction of apoptosis related to virus replication in cells of the mononuclear phagocyte system.

African Swine Fever↗
PubMed1996

The pathogenic role of pulmonary intravascular macrophages in acute African swine fever.

Recent studies of pulmonary intravascular macrophages have led to the re-examination of the mechanisms giving rise to alveolar oedema. A highly virulent isolate of African swine fever virus was replicated in pulmonary intravascular macrophages, interstitial and alveolar macrophages, fibroblasts and neutrophils. The alveolar oedema-characteristic of acute forms of African swine fever-and the vascular changes observed, which consisted of the formation of fibrin microthrombi in septal capillaries and the vacuolisation of endothelial cells, may have been due, however, to the activation of pulmonary intravascular macrophages, and not to the cytopathic effect subsequent to the replication of the African swine fever virus. Furthermore, it was observed that virus replication in cells not belonging to the mononuclear phagocyte system-such as fibroblasts and neutrophils-occurred earlier than in cells belonging to that system.

African Swine Fever↗
PubMed1996

Screening for membrane-permeabilizing mutants of the poliovirus protein 3AB.

Synthesis of the poliovirus polypeptide 3AB in bacterial cells results in an increase in membrane permeability. The alterations observed resemble those elicited by bacteriophage lytic proteins, which are presumed to cause pore formation in biological membranes. This property has been exploited in the development of an in vivo screening system that allows morphological differentiation of Escherichia coli clones expressing either wild-type 3AB or variant 3AB proteins lacking the ability to permeabilize bacteria. Expression of the wild-type 3AB gene in the presence of a chromogenic beta-galactosidase substrate causes E. coli clones to stain dark blue. In contrast, bacterial mutants that synthesize 3AB proteins with alterations in the hydrophobic domain lack pore-forming activity and stain to a light blue colour, allowing differentiation from wild-type clones. This phenotypic property correlates with the rate of entry of the beta-galactosidase substrate into the bacteria. The method developed here was used to screen more than 8000 E. coli clones after random PCR mutagenesis of the poliovirus 3AB gene. Our results show the existence of three different domains involved in the permeabilizing activity of 3AB protein. Twenty individual amino acid substitutions were identified in clones that showed the mutant phenotype and such bacteria displayed different reduced levels of permeability towards ONPG, hygromycin B, lysozyme and uridine. The procedure reported here may be of general interest to understand structure-function relationships in other eukaryotic proteins known to form pores.

Binding Sites↗