Biomedical subjects

C Risco

Publications and source records attributed to C Risco.

At least 37 records · Page 2Linked to original sources

PubMed1998

Optimization of phosphorus localization by EFTEM of nucleic acid containing structures.

Energy Filtered Transmission Electron Microscopy (EFTEM) has been used to study nucleic acids localization in unstained thin sections of virus-infected cells. For this purpose, phosphorus maps (P-maps) have been obtained by applying the N-windows Egerton model for background subtraction from data acquired by a non-dedicated TEM Jeol 1200EXII equipped with a post-column PEELS Gatan 666-9000 and a Gatan Image Filter (GIF-100). To prevent possible errors in the evaluation of elemental maps and thus incorrect nucleic acid localization, we have studied different regions of swine testis (ST) cells with similar local density containing either high concentration of nucleic acids (condensed chromatin and ribosomes) or a very low concentration (mitochondria). Special care was taken to optimize the sample preparation conditions to avoid as much as possible the traditional artifacts derived from this source. Selection of the best set of pre-edge images for background fitting was also considered in order to produce "true P-maps". A new software for interactive processing of images series has been applied to estimate this set. Multivariate Statistical Analysis was used as a filtering tool to separate the "useful information" present in the inelastic image series (characteristic signal) from the "non-useful information" (noise and acquisition artifacts). The reconstitution of the original image series preserving mainly the useful information allowed the computation of P-maps with improved signal-to-noise ratio (SNR). This methodology has been applied to study the RNA content of maturation intermediate coronavirus particles found inside infected cells.

Animals↗

PubMed1998

The fracture-flip technique reveals new structural features of the Escherichia coli cell wall.

With few exceptions, all bacteria possess a wall which protects them and controls their communication with the environment. In Gram-negative bacteria the cell wall exhibits a complex and unique multilayered organization. We have applied a modification of the freeze-fracture technique known as 'fracture-flip' to visualize the real surfaces of the different wall layers in a Gram-negative bacterium, Escherichia coli. In combination with treatments to weaken the interlayer connections, this technique has provided new insights into the structure of the bacterial wall. Large areas of an intermediate layer (most probably the peptidoglycan-containing matrix) have been visualized for the first time between the plasma membrane and the outer membrane of the wall. Extensive regions corresponding to the cytoplasmic face of the plasma membrane have also been obtained. These images provide new three-dimensional views of the bacterial cell wall and provide the structural framework for the analysis of the molecular relationships between the different cell wall components.

Cell Wall↗

PubMed1998

Expression of ORF A1 of infectious bursal disease virus results in the formation of virus-like particles.

A recombinant vaccinia virus inducibly expressing ORF A1 of infectious bursal disease virus (IBDV) has been constructed and characterized. Cells infected with this recombinant virus express the IBDV polyprotein, which is proteolytically processed to give mature VP2, VP3, and VP4 polypeptides. An electron microscopy study revealed that the cytoplasm of cells infected with the recombinant virus contains abundant IBDV-like particles (VLP). These VLP form close-packed paracrystalline arrays that are specifically recognized by anti-IBDV antibodies. The size and morphology of purified VLP were found to be akin to those of authentic IBDV particles.

Amino Acid Sequence↗

PubMed1998

Vaccinia virus 15-kilodalton (A14L) protein is essential for assembly and attachment of viral crescents to virosomes.

Early stages in vaccinia virus (VV) assembly involve the recruitment of cellular membranes from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to virus factories (or virosomes). The key viral factors involved in this process are not yet known. We have previously identified and characterized two viral proteins, of 21 kDa (A17L gene) and 15 kDa (A14L gene), that associate with tubulovesicular elements related to the ERGIC and are localized in viral membranes at all stages of virion assembly. We showed that the 21-kDa protein is not responsible for the recruitment of membranes from the ERGIC to viral factories. However, it appears to be essential for the organization of viral membranes. In this investigation we have generated a VV recombinant, VVindA14L, in which the expression of the A14L gene is inducibly regulated by the Escherichia coli lacI operator-repressor system. Repression of 15-kDa protein synthesis has a dramatic effect on virus yields and severely impairs plaque formation. Compared to wild-type VV, reduced amounts of 15-kDa protein are produced in VVindA14L-infected cells in the presence of IPTG (isopropyl-beta-D-thiogalactoside), and this correlates with a small-plaque phenotype and reduced VVindA14L yields under these conditions. In the absence of the 15-kDa protein, early and late viral protein syntheses proceed normally; however, proteolytic cleavage of the major core precursors is inhibited. Electron microscopic examination of cells infected with VVindA14L under nonpermissive conditions reveals the presence of numerous membranous elements that look like unfinished or disassembled crescents interspersed between electron-dense masses. These abnormal membrane elements are usually well separated from the surfaces of the dense structures. These findings show that the 15-kDa protein is essential for VV morphogenesis and indicate that this polypeptide is necessary both for the correct assembly of viral crescents and for their stable attachment to the surfaces of viral factories.

HeLa Cells↗

PubMed1998

Two types of virus-related particles are found during transmissible gastroenteritis virus morphogenesis.

The intracellular assembly of the transmissible gastroenteritis coronavirus (TGEV) was studied in infected swine testis (ST) cells at different postinfection times by using ultrathin sections of conventionally embedded infected cells, freeze-substitution, and methods for detecting viral proteins and RNA at the electron microscopy level. This ultrastructural analysis was focused on the identification of the different viral components that assemble in infected cells, in particular the spherical, potentially icosahedral internal core, a new structural element of the extracellular infectious coronavirus recently characterized by our group. Typical budding profiles and two types of virion-related particles were detected in TGEV-infected cells. While large virions with an electron-dense internal periphery and a clear central area are abundant at perinuclear regions, smaller viral particles, with the characteristic morphology of extracellular virions (exhibiting compact internal cores with polygonal contours) accumulate inside secretory vesicles that reach the plasma membrane. The two types of virions coexist in the Golgi complex of infected ST cells. In nocodazole-treated infected cells, the two types of virions coexist in altered Golgi stacks, while the large secretory vesicles filled with virions found in normal infections are not detected in this case. Treatment of infected cells with the Golgi complex-disrupting agent brefeldin A induced the accumulation of large virions in the cisternae that form by fusion of different membranous compartments. These data, together with the distribution of both types of virions in different cellular compartments, strongly suggest that the large virions are the precursors of the small viral particles and that their transport through a functional Golgi complex is necessary for viral maturation.

Animals↗

PubMed1998

Comparison of various antibiotic treatments for cows diagnosed with toxic puerperal metritis.

Holstein cows (n = 51) that had been diagnosed with toxic puerperal metritis were used to determine the treatment efficacy of various antibiotics. On the day of diagnosis, cows affected with toxic puerperal metritis were assigned randomly to three treatment groups. Cows in groups 1 and 2 received 22,000 IU/kg of procaine penicillin G i.m. for 5 d. In addition, cows in group 2 received an intrauterine infusion of 6 g of oxytetracycline on d 1, 3, and 5. Cows in group 3 received 2.2 mg/kg of ceftiofur sodium i.m. for 5 d. Dependent variables used to determine antibiotic efficacy included milk yield on d 1 through 12, rectal temperature on d 1 through 5, and serum haptoglobin concentration on d 1, 3, and 5. No difference was observed among groups for milk yield on d 1 and 12 or for temperature on d 1 and 5. Serum haptoglobin was elevated to > 10 mg/dl for cows in all groups; however, no difference was observed among groups on d 1 and 5. Because all groups showed a favorable response, this study suggests that there is no difference in treatment efficacy among antibiotics used to treat cows affected with toxic puerperal metritis.

Animals↗

PubMed1997

The coat and cylindrical inclusion proteins of a potyvirus are associated with connections between plant cells.

The subcellular locations of two potyviral proteins, the coat (CP) and nonstructural cylindrical inclusion (CI) proteins of tobacco vein mottling virus (TVMV), during early stages in the development of systemic infections in plants, have been investigated. Ultrathin sections of newly emerged leaves in infected plants were treated with antibodies specific to these proteins and then with gold-labeled secondary antibodies and examined by electron microscopy. CI was detected near plasmodesmatal connections between mesophyll cells prior to the appearance of CP or any virus-induced features or effects. Further accumulation of CI was evident in the form of conical structures, many of which appeared to penetrate the cell wall and to be connected to cones in neighboring cells. Prior to its appearance in other parts of the cells, the viral CP was detected, often in linear arrays, near the vertices or inside the cones and in plasmodesmata. In situ hybridization analysis of similar tissue sections with a TVMV RNA-specific oligoribonucleotide probe revealed the presence of the viral RNA in plasmodesmata. These results lend support to the notion that the formation of specific structures by potyviral CI proteins is required for and plays a direct role in the intercellular passage of viral genetic material, in the form of virus particles or complexes containing viral CP and RNA, in infected plants.

Capsid↗

PubMed1997

Characterization of early stages in vaccinia virus membrane biogenesis: implications of the 21-kilodalton protein and a newly identified 15-kilodalton envelope protein.

Vaccinia virus (VV) membrane biogenesis is a poorly understood process. It has been proposed that cellular membranes derived from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) are incorporated in the early stages of virion assembly. We have recently shown that the VV 21-kDa (A17L gene) envelope protein is essential for the formation of viral membranes. In the present work, we identify a 15-kDa VV membrane protein encoded by the A14L gene. This protein is phosphorylated and myristylated during infection and is incorporated into the virion envelope. Both the 21- and 15-kDa proteins are found associated with cellular tubulovesicular elements related to the ERGIC, suggesting that these proteins are transported in these membranes to the nascent viral factories. When synthesis of the 21-kDa protein is repressed, organized membranes are not formed but numerous ERGIC-derived tubulovesicular structures containing the 15-kDa protein accumulate in the boundaries of the precursors of the viral factories. These data suggest that the 21-kDa protein is involved in organizing the recruited viral membranes, while the 15-kDa protein appears to be one of the viral elements participating in the membrane recruitment process from the ERGIC, to initiate virus formation.

Amino Acid Sequence↗

PubMed1996

Cooperation between transmissible gastroenteritis coronavirus (TGEV) structural proteins in the in vitro induction of virus-specific antibodies.

Following infection of haplotype defined NIH-miniswine with virulent transmissible gastroenteritis coronavirus (TGEV), isolated mesenteric lymph node CD4+ T-cells mounted a specific proliferative response against infectious or inactivated purified virus in secondary in vitro stimulation. A specific, dose-dependent response to the three major recombinant viral proteins: spike (S), membrane (M), and nucleoprotein (N), purified by affinity chromatography, was characterized. Induction of in vitro antibody synthesis was analyzed. The purified recombinant viral proteins induced the in vitro synthesis of neutralizing TGEV-specific antibodies when porcine TGEV-immune cells were stimulated with each of the combinations made with two of the major structural proteins: S + N, S + M, and to a minor extent with M + N, but not by the individual proteins. S-protein was dissociated from purified virus using NP-40 detergent and then micellar S-protein oligomers (S-rosettes) were formed by removing the detergent. These occurred preferentially by the association of more than 10 S-protein trimmers. These S-rosettes in collaboration with either N or M-proteins elicited TGEV-specific antibodies with titers up to 84 and 60%, respectively, of those induced by the whole virus. N-protein could be partially substituted by a 15-mer peptide that represents a T helper epitope previously identified in N-protein (Antón et al. (1995)). These results indicate that the induction of high levels of TGEV-specific antibodies requires stimulation by at least two viral proteins, and that optimum responses are induced by a combination of S-rosettes and the nucleoprotein.

Animals↗

PubMed1996

Inducible expression of the vaccinia virus A17L gene provides a synchronized system to monitor sorting of viral proteins during morphogenesis.

The vaccinia virus (VV) A17L gene encodes a 21- to 23-kDa virion component that forms a stable complex with the 14-kDa envelope protein (A27L gene). In a previous report, we described the construction of a VV recombinant, VVindA17L, in which the expression of the A17L gene is inducibly regulated by isopropyl-beta-D-thiogalactoside (IPTG). We demonstrated that shutoff of the A17L gene results in a blockade of virion morphogenesis at a very early stage (D. Rodríguez, M. Esteban, and J. R. Rodríguez, J. Virol. 69:4640-4648, 1995). In the present study, we show that virus growth is restored if the inducer is provided not later than 6 h postinfection. Immunofluorescence and immunoelectron microscopy analysis of VVindA17L-infected cells revealed that in the absence of the 21- to 23-kDa protein, the 14-kDa protein is distributed throughout the cytoplasm. After IPTG addition, the 14-kDa protein can be detected around viral factories and immature virions; at later times, it localizes in the external membranes of intracellular mature virions. Immunoelectron microscopy with anti-21- to 23-kDa antibodies showed that soon after induction, the protein accumulates in membranes of the rough endoplasmic reticulum and in the nuclear envelope. With time, the protein localizes in viral crescents and subsequently associates to the membranes of immature and intracellular mature virions. These results are consistent with a model in which the 21- to 23-kDa protein would be synthesized at the endoplasmic reticulum, from where the protein could be translocated to the membranes of the intermediate compartment to generate the precursors of the viral membranes. Also, these results argue that 14-kDa envelope protein becomes posttranslationally associated to viral membranes through its interaction with the 21-kDa protein.

Animals↗

PubMed1996

The transmissible gastroenteritis coronavirus contains a spherical core shell consisting of M and N proteins.

Coronaviruses are enveloped RNA viruses involved in a variety of pathologies that affect animals and humans. Existing structural models of these viruses propose a helical nucleocapsid under the virion envelope as the unique internal structure. In the present work, we have analyzed the structure of the transmissible gastroenteritis coronavirus. The definition of its organization supports a new structural model for coronaviruses, since a spherical, probably icosahedral, internal core has been characterized. Disruption of these cores induces the release of N-protein-containing helical nucleocapsids. Immunogold mapping and protein analysis of purified cores showed that they consist of M and N proteins, M being the main core shell component. This surprising finding, together with the fact that M protein molecules are also located in the virion envelope, indicates that a reconsideration of the assembly and maturation of coronaviruses, as well as a study of potential M-protein subclasses, is needed.

Animals↗

PubMed1995

Cellular functions during activation and damage by pathogens: immunogold studies of the interaction of bacterial endotoxins with target cells.

Bacterial endotoxins (lipopolysaccharides or LPS) are active components of Gram-negative bacteria that act on numerous cellular functions through the processes of cell activation and damage. The molecular mechanisms involved in the "endotoxic phenomenon" are not defined yet, although extensive studies have been carried out. Immunogold and electron microscopy (EM) have contributed to identify the primary target cells of endotoxins and the subcellular systems that receive the direct action of these bacterial agents. Here, we review our studies on immunogold detection of endotoxins in cellular and subcellular systems. The analysis of the interaction between endotoxins and cells was focussed on the following aspects: (1) morphological characteristics of the LPS aqueous suspensions used in experimental work; (2) binding of endotoxins to the plasma membrane of type II pneumocytes and alveolar macrophages (two of their cellular targets), and influence of the state of aggregation of the LPS; (3) movement and distribution of endotoxins inside the cell, from the plasma membrane to the nucleoplasm; and (4) interaction of LPS with microtubules and its effects on the integrity of the microtubular network. These approaches provide information at the molecular level as well as data for the establishment of physiological models of endotoxicity.

Animals↗

PubMed1995

Ultrastructure of human astrovirus serotype 2.

The ultrastructure of human astrovirus serotype 2 (H-Ast2) grown in cell culture was analysed by electron microscopy of thin sections and negatively stained preparation. Infected LLCMK2 cells, as visualized in thin sections, contained cytoplasmic aggregates of dense or hollow-cored particles that aggregated in quasicrystalline arrays and were specifically labelled using a rabbit polyclonal anti-Ast2 antiserum. H-Ast2 particles from the supernatant of infected LLCMK2 cells in thin sections after flat- embedding were similar in size to intracellular virions. In negatively stained preparations, these virus particles had an external diameter of 41 nm and exhibited a well defined layer of surface spikes. Pentagonal and hexagonal contours were occasionally visible, and probably correspond to the projections of icosahedral structures. Star-like morphologies and particles with surface triangular hollows were seen in dark areas of the preparations only after a short treatment of the viruses of pH 10. Incubation of the viruses at pH 10.5 induced a rapid disassembly of the virus particles. The finding that the particles with icosahedral geometry and surface spikes are fully infective allows an alternative morphological model to the traditional one for astroviruses to be proposed.

Cell Line↗

PubMed1995

Membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion.

The binding domains of four monoclonal antibodies (MAbs) specific for the M protein of the PUR46-MAD strain of transmissible gastroenteritis coronavirus (TGEV) have been located in the 46 carboxy-terminal amino acids of the protein by studying the binding of MAbs to recombinant M protein fragments. Immunoelectron microscopy using these MAbs demonstrated that in a significant proportion of the M protein molecules, the carboxy terminus is exposed on the external surface both in purified viruses and in nascent TGEV virions that recently exited infected swine testis cells. The same MAbs specifically neutralized the infectivity of the PUR46-MAD strain, indicating that the C-terminal domain of M protein is exposed on infectious viruses. This topology of TGEV M protein probably coexists with the structure currently described for the M protein of coronaviruses, which consists of an exposed amino terminus and an intravirion carboxy-terminal domain. The presence of a detectable number of M protein molecules with their carboxy termini exposed on the surface of the virion has relevance for viral function, since it has been shown that the carboxy terminus of M protein is immunodominant and that antibodies specific for this domain both neutralize TGEV and mediate the complement-dependent lysis of TGEV-infected cells.

Animals↗

PubMed1995

Intracellular transport of the murine leukemia virus during acute infection of NIH 3T3 cells: nuclear import of nucleocapsid protein and integrase.

The entry and intracellular transport of Moloney-murine leukemia virions inside mouse NIH 3T3 cells have been followed by electron microscopy techniques. Five viral proteins--matrix (MA, p15), capsid (CA, p30), nucleocapsid (NC, p10), integrase (IN), and the envelope glycoprotein (SU, gp70)--were located by immunolabeling using gold probes. After entering the cells, viral particles were frequently detected inside cytoplasmic vesicles of variable size. Their viral envelope was apparently lost during intracytoplasmic transport. When the unenveloped viral cores reached the nuclear membrane or its vicinity, they were disrupted. Two of the immunolabeled proteins, NC and IN, were detected entering the nucleus of non-dividing cells, where both were targeted to the nucleolus. However, MA and CA were found only in the cytoplasm. NC is a nucleic acid-binding protein which contains potential nuclear localization signals. We suggest that NC could enter the nucleus as part of a nucleoprotein complex, associated with IN, and possibly, also with viral DNA.

3T3 Cells↗

PubMed1994

Effect of sequential treatment with prostaglandin F2 alpha and/or oxytocin on estrus and pregnancy rate of lactating dairy cows.

A total of 335 lactating dairy cows was used to determine the effect of oxytocin or PGF2a given 8 h after treatment with a luteolytic dosage of PGF2a on the percentage of cows exhibiting estrus within 7 d after treatment, and the pregnancy rate to a single insemination at this time. On the initial day of treatment (Day 0), cows with a palpable corpus luteum on the ovary were treated with 25 mg, im of PGF2a. At 8 h later, the cows were divided into 3 groups. Cows in Group 1 (n = 112) were treated with oxytocin (0.33 IU/kg bwt im); cows in Group 2 (n = 112) were treated with 25 mg, im of PGF2a; and cows in Group 3 (n = 111) served as the untreated controls. Cows in all 3 groups were continuously observed for estrus visually or by way of an activated heatmount detector within 7 d after treatment, and were inseminated within 12 h of the observed estrus.Plasma progesterone (P4) concentration was determined using radioimmunoassay on Day 0 and Day 2. Of the cows with P4 greater than 1 ng/ml on Day 0, the percentage of cows observed in estrus within 7 d after treatment was 75, 89 and 72% for cows in Group 1, Group 2 and Group 3, respectively. When all cows were evaluated, the percentage of cows observed in estrus within 7 d after treatment was 60, 70 and 55% for cows in Group 1, Group 2 and Group 3, respectively. In both instances, the value for cows in Group 2 was significantly higher than that for either cows in Group 1 or Group 3. The pregnancy rate for cows inseminated within 7 d was similar for cows in all 3 groups. The results of this study demonstrated that treatment of dairy cows with 2 luteolytic dosages of PGF2a at an 8-h interval resulted in more cows being observed in estrus within 7 d than with 1 treatment with PGF2a, or with oxytocin given at an 8-h interval after a luteolytic dosage of PGF2a.

Journal Article↗

PubMed1994

Type II pneumocytes revisited: intracellular membranous systems, surface characteristics, and lamellar body secretion.

BACKGROUND: Type II pneumocytes, the producers of pulmonary surfactant, have been extensively studied during the last 20 years because of the importance of their metabolism in lung function and integrity. The ultrastructural studies of the 1970s and 1980s have shown that these cells present unique elements. EXPERIMENTAL DESIGN: In this work, we used thin-section, freeze-fracture, and fracture-flip electron microscopy techniques to obtain new information on the ultrastructural peculiarities of isolated rat type II pneumocytes, focusing our study on the intracellular membranous systems and their interrelationships and the microanatomy of their plasma membrane during secretory process. RESULTS: In thin-sections of pneumocytes postfixed with osmium tetroxide and potassium ferricyanide, we observed that lamellar bodies (LBs) are usually connected to membranes of the endoplasmic reticulum, and seem to emerge and grow from them. Unusual connections between the endoplasmic reticulum and mitochondria were detected, as well as numerous "bar-like structures" (BLSs), most of them in the early stages of development and often generating from the nuclear membrane. Membranes of the smooth endoplasmic reticulum that closely follow the outlines of mitochondria also appear to be the origin of some BLSs. Possible transition forms, BLS--LB, were also detected, although they were rare. New images of the surface of the pneumocytes and its changes during LB secretion showed a segregation and clearing of membrane particles at the areas of LB extrusion. CONCLUSIONS: We propose that LBs can originate directly from membranes of the endoplasmic reticulum or from BLSs. An indirect participation of mitochondria appears possible. The plasma membrane of pneumocytes displays structural changes associated with the secretion of LBs as visualized by a redistribution of intramembrane and surface particles.

Animals↗

PubMed1993

Biochemical and electron microscopy analysis of the endotoxin binding to microtubules in vitro.

The mechanisms involved in cellular activation and damage by bacterial endotoxins are not completely defined. In particular, there is little information about possible intracellular targets of endotoxins. Recently, the participation of a microtubule associated protein in endotoxin actions on macrophages has been suggested. In the present work, we have studied the effect of E. coli lipopolysaccharide on the polymerization of microtubular protein in vitro. Electrophoretic analysis of the polymerization mixtures showed that the endotoxin inhibited the polymerization when present at high concentrations. At lower concentrations, LPS selectively displaced the microtubule associated protein MAP-2 from the polymerized microtubules. Electron microscopy showed that LPS binds to microtubules of tubulin + MAPs and to microtubules of purified tubulin (without MAPs) polymerized with taxol. Gel filtration experiments confirmed the binding of LPS to tubulin, and by ligand blot assays an interaction LPS-MAP-2 was detected. The ability of LPS to interact with microtubular proteins suggests a possible participation of microtubules on the cellular effects of endotoxins.

Animals↗