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Biomedical subjects

L Carrasco

Publications and source records attributed to L Carrasco.

At least 145 records · Page 8Linked to original sources
PubMed1995

Cloning and inducible synthesis of poliovirus non-structural proteins in Saccharomyces cerevisiae.

Several coding regions of the poliovirus (PV) genome were cloned in the yeast Saccharomyces cerevisiae, and placed under the control of the inducible hybrid promoter pGAL/CYC, such that expression was triggered by incubating the cells in galactose (Gal)-containing medium. A number of PV genes encoding non-structural proteins, including 2Apro, 2B, 2C, 3A, 3AB and 3Cpro, were cloned and expressed in this eukaryotic system. The presence of these proteins after induction was detected by immunoblot analysis using specific antisera against each protein. The levels and the kinetics of protein synthesis after induction varied according to the PV protein analyzed. Thus, 2C was detected soon after Gal addition (3-5 h) and was one of the major polypeptides synthesized by yeast cells after 16 h of induction. In contrast, only low levels of synthesis were observed for 3A or 3AB, and then only after several hours of growth in Gal. The induction of the PV protease, 2Apro, was highly toxic for the cells such that growth was arrested after 5 h of induction and cell survival sharply declined.

3C Viral Proteases↗
PubMed1995

Induction of membrane proliferation by poliovirus proteins 2C and 2BC.

Poliovirus infection leads to the appearance of a number of cytoplasmic vacuoles involved in the replication of virus genomes. To characterize the viral proteins involved in membrane proliferation different poliovirus proteins have been expressed in HeLa cells. Two recombinant vaccinia viruses have been obtained that express poliovirus protein 2C, one under the 5' untranslated (UTR) sequence of poliovirus and another under the leader region of EMC virus. Expression of 2C was very efficient in both cases, although better results were obtained when poliovirus 2C was expressed under the 5'UTR sequence of EMC virus. Transient expression of poliovirus proteins 2B, 2C or 2BC placed under a T7 promoter was analyzed using a recombinant vaccinia virus that contains the bacteriophage T7 RNA polymerase. The expression of 2C, or 2BC, contrary to 2B, was able to induce the proliferation of vacuoles morphologically similar to those found during poliovirus infection. These findings indicate that poliovirus protein 2C, in addition to its NTPase and RNA binding activities, is also endowed with the capacity to induce the formation of cytoplasmic vacuoles.

Animals↗
PubMed1995

Ultrastructural study of the renal tubular system in acute experimental African swine fever: virus replication in glomerular mesangial cells and in the collecting ducts.

Despite the considerable attention given to kidney lesions in African swine fever (ASF), a number of questions remain to be answered. Structural and ultrastructural examination showed that a highly virulent isolate of ASF virus (Malawi 83) replicated in glomerular mesangial cells and renal collecting duct epithelial cells, with hyperplasia of the latter in infected pigs. Replication in mesangial cells may be due to their contact with the bloodstream, as well as to their phagocytic capacity and high metabolism rate. Virus replication in macrophages and endothelial cells of interstitial capillaries, and the necrosis of these infected cells gave rise to a large number of free virus in interstitial tissue. This, together with the lesser thickness of the basal membrane of collecting ducts in comparison to the rest of the tubular system, probably facilitates ASFV infection of tubular epithelial cells. Virus replication in these cells may account for the presence of virus in the urine of pigs with acute ASF where haematuria is not observed.

Acute Disease↗
PubMed1995

A pathological study of the perisinusoidal unit of the liver in acute African swine fever.

African swine fever is an acute haemorrhagic disease of pigs which may serve as a model for the study of the pathogenesis of other viral haemorrhagic fevers. This paper describes an ultrastructural study of the sequence of lesions produced in the perisinusoidal functional unit of the liver of pigs inoculated with the Malawi '83 strain of African swine fever virus, which is classified as haemadsorbing and highly virulent. Virus replication was observed in Kupffer cells and monocytes from three days after inoculation, in hepatocytes and fat-storing cells at five and seven days after inoculation, and in sinusoidal endothelial cells at seven days after inoculation. Further observations included intravascular coagulation, which peaked at five days after inoculation, and fibroblast and myofibroblast transformation of fat-storing cells at seven days after inoculation. These results suggest that activated cells of the mononuclear phagocyte system may play a major role in this sequence of lesions and the possible role of the cytokines that may be released by these cells is discussed.

Acute Disease↗
PubMed1995

Pathological changes in the renal interstitial capillaries of pigs inoculated with two different strains of African swine fever virus.

African swine fever is a viral disease of pigs characterized predominantly by haemorrhagic lesions. This paper reports the lesions observed in the renal interstitial capillaries of pigs inoculated with African swine fever virus strains of differing virulence: the Malawi'83 strain (haemadsorbent and highly virulent) and the Dominican Republic'78 strain (haemadsorbent and moderately virulent). In pigs infected with the Malawi'83 strain, petechial haemorrhages and microhaemorrhages were observed 5 days after inoculation and lesions were evident in the renal capillaries. Signs of phagocyte activation were noticeable in endothelial cells, with enlarged fenestrations and even loss of endothelium, leaving the basement membrane of the vessels exposed. Platelet plugs and microthrombi were also observed in these vessels. At 7 days after inoculation these lesions had intensified, and were accompanied by virus replication in the endothelial cells. In pigs infected with the Dominican Republic'78 strain, haemorrhages were more abundant and more extensive, and although no endothelial cell lesions were observed, there was intense vasodilation with diapedesis of erythrocytes.

Africa↗
PubMed1995

Experimental African swine fever: apoptosis of lymphocytes and virus replication in other cells.

In order to determine the cause of cellular death of lymphocytes in pigs with acute African swine fever and the relationships between African swine fever virus (ASFV) and interstitial cells, ten pigs were inoculated with a highly virulent strain of ASFV (Malawi '83) and samples taken for ultrastructural study of hepatic and renal interstitial tissues. We demonstrated death by apoptosis of lymphocytes and virus replication in fibroblasts, smooth muscle cells and endothelial cells in the interstitial tissues of pigs inoculated with ASFV. From day 5 onwards, apoptotic lymphocyte and intense virus replication in hepatic interstitial macrophages and fibroblasts were observed. By day 7, apoptotic lymphocytes and virus replication in macrophages, interstitial capillary endothelial cells and fibroblasts in the kidney were observed. Virus replication was also seen in smooth muscle cells of hepatic and renal arterioles and venules. Our results suggest that mononuclear phagocyte system (MPS) cell activation, and the resulting release of cytokines, could induce apoptosis of lymphocytes and virus replication in non-MPS cells.

African Swine Fever↗
PubMed1995

A poliovirus 2A(pro) mutant unable to cleave 3CD shows inefficient viral protein synthesis and transactivation defects.

Four poliovirus mutants with modifications of tyrosine 88 in 2A(pro) were generated and introduced into the cloned poliovirus genome. Mutants Y88P and Y88L were nonviable, mutant Y88F showed a wild-type (WT) phenotype, and mutant Y88S showed a delayed cytopathic effect and formed small plaques in HeLa cells. Growth of Y88S in HeLa cells was restricted, giving rise to about 20% of the PFU production of the WT poliovirus. The 2A (Y88S) mutant synthesized significantly lower levels of viral proteins in HeLa cells than did the WT poliovirus, while the kinetics of p220 cleavage were identical for both viruses. Strikingly, the 2A (Y88S) mutant was unable to cleave 3CD, as shown by analysis of poliovirus proteins labeled with [35S]methionine or immunoblotted with a specific anti-3C serum. The ability of the Y88S mutant to form infectious virus and cleave 3CD can be complemented by the WT poliovirus. Synthesis of viral RNA was diminished in the Y88S mutant but less than the inhibition of translation of viral RNA. Experiments in which guanidine was used to inhibit poliovirus RNA synthesis suggest that the primary defect of the Y88S mutant virus is at the level of poliovirus RNA translation, while viral genome replication is much less affected. Transfection of HeLa cells infected with the WT poliovirus with a luciferase mRNA containing the poliovirus 5' untranslated sequence gives rise to a severalfold increase in luciferase activity. This enhanced translation of leader-luc mRNA was not observed when the transfected cells were infected with the 2A (Y88S) mutant. Moreover, cotransfection with mRNA encoding WT poliovirus 2A(pro) enhanced translation of leader-luc mRNA. This enhancement was much lower upon transfection with mRNA encoding 2A(Y88S), 2A(Y88L), or 2A(Y88P). These findings support the view that 2A(pro) itself, rather than the 3C' and/or 3D' products, is necessary for efficient translation of poliovirus RNA in HeLa cells.

Amino Acid Sequence↗
PubMed1995

Monensin and nigericin prevent the inhibition of host translation by poliovirus, without affecting p220 cleavage.

Addition of monensin or nigericin after poliovirus entry into HeLa cells prevents the inhibition of host protein synthesis by poliovirus. The infected cells continue to synthesize cellular proteins at control levels for at least 8 h after infection in the presence of the ionophore. Cleavage of p220 (gamma subunit of eukaryotic initiation factor 4 [eIF-4 gamma]), a component of the translation initiation factor eIF-4F, occurs to the same extent in poliovirus-infected cells whether or not they are treated with monensin. Two hours after infection there is no detectable intact p220, but the cells continue to translate cellular mRNAs for several hours at levels similar to those in uninfected cells. Nigericin or monensin prevented the arrest of host translation at all the multiplicities of poliovirus infection tested. At high multiplicities of infection, an unprecedented situation was found: cells synthesized poliovirus and cellular proteins simultaneously. Superinfection of vesicular stomatitis virus-infected HeLa cells with poliovirus led to a profound inhibition of vesicular stomatitis virus protein synthesis, while nigericin partially prevented this blockade. Drastic inhibition of translation also took place in influenza virus-infected Vero cells treated with nigericin and infected with poliovirus. These findings suggest that the translation of newly synthesized mRNAs is dependent on the integrity of p220, while ongoing cellular protein synthesis does not require an intact p220. The target of ionophore action during the poliovirus life cycle was also investigated. Addition of nigericin at any time postinfection profoundly blocked the synthesis of virus RNA, whereas viral protein synthesis was not affected if nigericin was added at 4 h postinfection. These results agree well with previous findings indicating that inhibitors of phospholipid synthesis or vesicular traffic interfere with poliovirus genome replication. Therefore, the action of nigericin on the vesicular system may affect poliovirus RNA synthesis. In conclusion, monensin and nigericin are potent inhibitors of poliovirus genome replication that prevent the shutoff of host translation by poliovirus while still permitting cleavage of p220.

Animals↗
PubMed1995

Requirement for vacuolar proton-ATPase activity during entry of influenza virus into cells.

The role that endosomal acidification plays during influenza virus entry into MDCK cells has been analyzed by using the macrolide antibiotics bafilomycin A1 and concanamycin A as selective inhibitors of vacuolar proton-ATPase (v-[H+]ATPase), the enzyme responsible for the acidification of endosomes. Bafilomycin A1 and concanamycin A, present at the low concentrations of 5 x 10(-7) and 5 x 10(-9) M, respectively, prevented the entry of influenza virus into cells when added during the first minutes of infection. Attachment of virion particles to the cell surface was not the target for the action of bafilomycin A1. N,N'-Dicyclohexylcarbodiimide, a nonspecific inhibitor of proton-ATPases, also blocked virus entry, whereas elaiophylin, an inhibitor of the plasma-proton ATPase, had no effect. The inhibitory actions of bafilomycin A1 and concanamycin A were tested in culture medium at different pHs. Both antibiotics powerfully prevented influenza virus infection when the virus was added under low-pH conditions. This inhibition was reduced if the virus was bound to cells at 4 degrees C prior to the addition of warm low-pH medium. Moreover, incubation of cells at acidic pH potently blocked influenza virus infection, even in the absence of antibiotics. These results indicate that a pH gradient, rather than low pH, is necessary for efficient entry of influenza virus into cells.

Animals↗
PubMed1995

Membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41.

The transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1) has been implicated in the cytopathology observed during HIV infection. The first amino acids located at the amino terminus are involved in membrane fusion and syncytium formation, while sequences located at the carboxy terminus have been predicted to interact with membranes and modify membrane permeability. The HIV-1 gp41 gene has been cloned and expressed in Escherichia coli cells by using pET vectors to analyze changes in membrane permeability produced by this protein. This system is well suited for expressing toxic genes in an inducible manner and for analyzing the function of proteins that modify membrane permeability. gp41 enhances the permeability of the bacterial membrane to hygromycin B despite the low level of expression of this protein. To localize the regions of gp41 responsible for these effects, a number of fragments spanning different portions of gp41 were inducibly expressed in E. coli. Two regions of gp41 were shown to increase membrane permeability: one located at the carboxy terminus, where two highly amphipathic helices have been predicted, and another one corresponding to the membrane-spanning domain. Expression of the central region of gp41 comprising this domain was highly lytic for E. coli cells and increased membrane permeability to a number of compounds. These findings are discussed in the light of HIV-induced cytopathology and gp41 structure.

Base Sequence↗
PubMed1995

Enhanced intracellular calcium concentration during poliovirus infection.

The infection of human fibroblasts by poliovirus leads to a notable increase in the intracellular calcium concentration, [Ca2+]i, measured by microfluorimetry or by flow cytometry. [Ca2+]i increases from 2 to 3 h postinfection, and by the fifth hour there is a 5- to 10-fold increase in [Ca2+]i. At this time postinfection there is active viral protein synthesis. The modifications in [Ca2+]i are not observed in the presence of cycloheximide, guanidine, or Ro 09-0179, indicating that virus gene expression is required for the increase in [Ca2+]i. Attempts to identify the source of the intracellular Ca2+ by using different inhibitors of calcium fluxes suggest that calcium enters from the culture medium through voltage-sensitive calcium channels.

Calcium↗
PubMed1995

Elaeophorosis in red deer caused by Elaeophora elaphi: lesions of natural disease.

This is the first study of the lesions produced by Elaeophora elaphi, a filarial worm first reported in 1986. Only the morphologic characteristics of this species have been described previously. Samples for the study were taken from 74 red deer (Cervus elaphus), 1-10 years old, slaughtered in central Spain. Grossly, filariae were observed in the transverse, left, or right branches of the portal vein of 18 animals. In addition to filariae, whitish thrombi were found in the right and left branches of the portal vein in nine animals. Thrombi of similar characteristics were also observed in 12 deer in which no adult filariae were found. Microscopic changes were observed only in the right and left branches of the portal vein and consisted of thickening of the intima with surface formation of villi. Stroma of villi contained a moderate to intense infiltrate composed mainly of eosinophils and mononuclear cells. The tunica media of affected vessels was uniformly hypertrophic and in some cases presented a moderate inflammatory infiltrate similar to that observed in the stroma of villi. Thrombi consisted of a central area containing calcified filariae or cuticle debris, surrounded by giant cells and numerous lymphoid cells forming clearly defined nodules resembling lymphoid follicles. Thrombi were externally surrounded by a strip of connective tissue and beyond by endothelial cells that were occasionally continuous with the tunica intima. Other lesions probably associated with the infestation were multifocal granulomas in the periportal areas, lymphoid tissue formations in the hepatic parenchyma, and an interstitial nephritis.

Animals↗
PubMed1995

Immunohistochemical distribution pattern of intermediate filament proteins in 50 feline neoplasms.

Twenty-eight epithelial and 22 nonepithelial feline tumors were studied immunohistochemically. Epithelial tumors were 10 squamous cell carcinomas, two basal cell tumors, two sebaceous gland carcinomas, three apocrine gland carcinomas, three thyroid papillary carcinomas, one thyroid solid carcinoma, one renal clear cell carcinoma, one renal papillary carcinoma, one endometrial carcinoma, and four lung bronchioloalveolar carcinomas. Nonepithelial tumors were 10 fibrosarcomas, one liposarcoma, one leiomyosarcoma, one rhabdomyosarcoma, one hemangiosarcoma, two mast cell tumors, one osteosarcoma, three melanomas, and two lymphomas. Commercially available antibodies directed against high- and low-molecular-weight keratins (keratin, RCK-102, NCL-5D3), vimentin, desmin, glial fibrillary acidic protein (GFAP), and neurofilament intermediate filament (IF) proteins were used in the avidin-biotin-peroxidase complex technique on formalin-fixed, paraffin-embedded tumor tissue samples. All epithelial tumors except the endometrial carcinoma expressed some type of keratin protein. Squamous cell carcinomas expressed high-molecular-weight keratins exclusively. Coexpression of high- and low-molecular-weight keratins was observed in one basal cell tumor, sebaceous and apocrine adenocarcinomas, and thyroid, renal, and lung carcinomas. In addition to keratins, vimentin immunoreactivity was found in all basal cell tumors, all sebaceous gland, thyroid papillary, renal, and lung adenocarcinomas, and one of the apocrine gland adenocarcinomas. Immunoreactivity with GFAP antibody was found in one basal cell tumor and one sebaceous gland adenocarcinoma. The endometrial carcinoma did not react with any of the antibodies applied. Nonepithelial tumors analyzed expressed either vimentin (fibrosarcomas, liposarcoma, haemangiosarcoma, mast cell tumors, osteosarcomas, melanomas) or vimentin and desmin (leiomyosarcoma, rhabdomyosarcoma, one fibrosarcoma) IF proteins exclusively. Lymphomas did not react with any of the antibodies employed. These findings indicate that IF proteins antibodies can be included in diagnostic panels of antibodies for immunocharacterization of feline tumors. In addition, they can be used as a basis for the diagnoses of poorly differentiated or undifferentiated feline neoplasms.

Animals↗
PubMed1995

Description of a new population of fixed macrophages in the splenic cords of pigs.

The ultrastructure of porcine splenic cords was analysed by light and electron microscopy after both perfusion and immersion fixation. The external aspect of the splenic cords was found to be composed of a network of smooth muscle cells arranged between the trabeculae and the venous sinus walls. Macrophages frequently attached to smooth muscle cells surrounded the whole of their surfaces and were attached by slender cytoplasmic projections. Intercellular junctions in the form of electron-dense areas 200-350 nm in length and 45-55 nm in thickness were observed in the macrophage membrane close to the muscle cells. In the splenic cords of nonperfused animals, in addition to the macrophage population adhering to smooth muscle cells, numerous macrophages without cell junctions were also seen and identified as free macrophages.

Actins↗
PubMed1995

Improved factor Xa cleavage of fusion proteins containing maltose binding protein.

The addition of five glycine residues at a position adjacent to the factor Xa cleavage site of a MBP-2C fusion protein, comprising maltose binding protein (MBP) and poliovirus 2C, allowed factor Xa to generate both of the component proteins. If, however, MBP-2C was without the above modification, it was cleaved only at a site located internally within poliovirus 2C, and this protein was not, therefore, generated by factor Xa cleavage. A simple procedure is described that uses PCR for the introduction of five glycines adjacent to the factor Xa recognition site.

Amino Acid Sequence↗