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S Garzon

Publications and source records attributed to S Garzon.

At least 19 recordsLinked to original sources
PubMed1998

Characterization of the thermosensitive ts453 reovirus mutant: increased dsRNA binding of sigma 3 protein correlates with interferon resistance.

The mutation harbored by the reovirus ts453 thermosensitive mutant has been assigned to the S4 gene encoding the major outer capsid protein sigma 3. Previous gene sequencing has identified a nonconservative amino acid substitution located near the zinc finger of sigma 3 protein in the mutant. Coexpression in COS cells of the sigma 3 protein presenting this amino acid substitution (N16K), together with the other major capsid protein mu 1, has also revealed an altered interaction between the two proteins; this altered interaction prevents the sigma 3-dependent cleavage of mu 1 to mu 1C. This could explain the lack of outer capsid assembly observed during ts453 virus infection at nonpermissive temperature. In the present study, we pursued the characterization of this mutant sigma 3 protein. Although the N16K mutation is located close to the zinc finger region, it did not affect the ability of the protein to bind zinc. In contrast, this mutation, as well as mutations within the zinc finger motif itself, can increase the binding of the protein to double-stranded RNA (dsRNA). It also appears that the N16K mutant protein is more efficiently transported to the nucleus than the wild-type protein, an observation consistent with the postulated role of dsRNA binding in sigma 3 nuclear presence. The lack of association with mu 1, and/or the increased dsRNA-binding activity of sigma 3, could be responsible for a partial resistance of the ts453 virus to interferon treatment and this could have important consequences in the context of protein synthesis regulation during natural reovirus infection.

Animals↗
PubMed1995

The role of the major tegument protein VP8 of bovine herpesvirus-1 in infection and immunity.

The tegument of bovine herpesvirus-1 (BHV-1) carries an abundant protein of 96 kDa, termed VP8. Immunolabeling using VP8-specific antiserum and colloidal gold-labeled protein A as the electron-dense marker was used to identify VP8 in the virions and virus-infected cells. VP8 was confirmed to be a tegument protein that, like the herpes simplex virus-1 homologue VP13/14, contains O-linked carbohydrates. VP8 was found in the nucleus of virus-infected cells as early as 2 hr postinfection. Since VP8 is a gamma2 protein, this protein cannot be newly synthesized at this time and must be acquired from the inoculum. This supports the hypothesis that early during infection, VP8 has a function in modulation of alpha gene expression. Later during infection, VP8 was observed in the cytoplasm around nucleocapsids and in dense inclusions, which accumulated in the cisternae of the Golgi. In addition, de novo-synthesized VP8 continued to accumulate in the nucleus in dense areas and around nucleocapsids. In calves, VP8 stimulated T cell proliferation and antibody production, both after BHV-1 challenge and after immunization with purified VP8. These results suggest a role for VP8 in the induction of humoral and specifically cell-mediated immunity to BHV-1.

Animals↗
PubMed1994

Mutational analysis of the HIV-1 Vpu protein.

The human immunodeficiency virus type-1 (HIV-1) encoded Vpu protein facilitates the release of budding virions from the surface of infected cells and delays the rate of syncytium formation of the virus. Furthermore, Vpu induces rapid degradation of nascent CD4 molecules that are retained in the endoplasmic reticulum by the formation of gp160-CD4 complexes. Currently, little is known of the precise mechanism(s) by which Vpu function. Whether or not these different events are related remain unclear. In this report, we describe our recent structure/function studies on vpu suggesting that the protein may have independent biological activities during the HIV-1 infection.

Base Sequence↗
PubMed1993

Establishment of persistent reovirus infection in SC1 cells: absence of protein synthesis inhibition and increased level of double-stranded RNA-activated protein kinase.

In the present study we report the establishment and characterization of an SC1 cell line persistently infected by reovirus. We observed that a significant percentage of SC1 cells was resistant to cell lysis upon infection with non-defective reovirus stocks. The apparent resistance of SC1 cells to the virus-induced inhibition of protein synthesis is probably an important factor favoring the establishment of such a persistence. The remaining cells, obtained following reovirus infection at a high multiplicity of infection, were kept as a continuous cell line and shown to have normal growth rate. They also released a high titer of virus that did not appear to differ from the original stock in neither infectivity nor genomic pattern. Electron microscopic examination further confirmed the presence of well-developed viral inclusions in the persistently infected cells. These cells were resistant to viral superinfection and exhibited a high constitutive level of the double-stranded RNA-activated protein kinase that might be involved in this resistance. We suggest that this cell line might be an interesting, and possibly more natural system than most previously used cell lines, for the continuing study of virus-host cell interactions during establishment of viral persistence using the much-studied model of reovirus infection.

Animals↗
PubMed1993

Structure on the capsular ligaments of the facet joints.

Microscopic studies showed that the capsular ligaments were mostly composed of collagenous tissue. The collagen fiber bundles were found to be crimped at right angles to their long axis. The collagen fibrils were connected transversely by proteoglycan filaments which were regularly associated with their periodicity. Among the collagen fiber bundles were varying quantities of elastic fibers and numerous fibroblasts. The highest concentration of elastic fibers was found in the posterior and inferior aspects of the capsular ligaments. These findings are discussed in relation to the mechanical behavior of the capsular ligaments.

Adolescent↗
PubMed1993

The effect of vpu on HIV-1-induced syncytia formation.

To investigate the role of vpu in the cytopathicity of human immunodeficiency type 1 (HIV-1), the MT4 CD4+ T-cell line was infected with viruses that were isogenic except for their ability to produce the vpu protein. The experiments described here demonstrate that expression of vpu reduces HIV-1 cytopathic effects by decreasing the rate of syncytia formation. By reducing the concentration of gp 120 at the cell surface, vpu limits cell killing by syncytia formation.

CD4-Positive T-Lymphocytes↗
PubMed1993

Muramidase-mediated damage to Candida yeast cells. Histochemical and immunochemical characterization of accumulating wall-like material.

Hen egg-white lysozyme is known to be fungicidal to blastoconidia of Candida albicans under defined in vitro conditions. This lethal action leads to changes in the layering of cell wall and to plasmolysis, caused by unremitting accumulation of wall-like material between the yeast cell wall and cytoplasmic membrane. Here, several methods were applied on ultrathin sections to define the nature of wall-like material: histochemical staining with periodic acid-thiocarbohydrazide-silver proteinate, periodic acid-alkaline bismuth, and phosphotungstic acid at low pH; the localization of the carbohydrate residues with lectin-gold complex; immunocytochemical staining with monospecific antibodies, factor 1 and 6, which recognized major cell wall antigens. The wall-like material was almost uniformly highlighted with periodic acid-thiocarbohydrazide-silver proteinate, factor 1 antibody, concanavalin A-gold and wheat germ agglutinin-ovomucoid-gold, indicating the presence of mannoproteins and chitin. The serotype A-specific epitope recognized by factor 6 antibody was not detected in the wall-like material, although it was demonstrated in the outer cell wall layers after 2 h of exposure to lysozyme.

Antibodies, Monoclonal↗
PubMed1992

Further characterization of the ts453 mutant of mammalian orthoreovirus serotype 3 and nucleotide sequence of the mutated S4 gene.

The sigma 3 protein of mammalian orthoreoviruses has multiple proven and postulated roles during viral multiplication. In this manuscript we took advantage of the availability of the ts453 thermosensitive mutant, already assigned to the S4 gene encoding sigma 3, to begin the elucidation of the relationship between the two main domains and the different roles of the sigma 3 viral protein. The alteration in the mutant appeared to affect the structural role of the protein. Nucleotide sequence determination indicated an especially significant change close to the zinc finger of the protein. These data suggest that the zinc-binding region might be especially important during the assembly of sigma 3 into the viral capsid.

Base Sequence↗
PubMed1991

Histochemical and immunochemical study of the fate of Candida albicans inside human neutrophil phagolysosomes.

To further define the ultrastructural events associated with the killing of Candida albicans by human neutrophils, four methods were used: (1) the periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of vicinal-glycol-containing complex carbohydrates; (2) the localization of thermostable immunodeterminants of the yeast cell wall, mannans or mannoproteins, using monospecific antibodies and a protein A-gold complex (monAb-gold); (3) the localization of mannose residues with concanavalin A labeled with gold particles (Con A-gold); (4) the localization of chitin oligomers using wheat germ agglutinin and ovomucoid labeled with gold particles (WGA-gold). The mannan-rich cell wall layers were progressively lost as shown by altered PA-TCH-SP reactivity and a diffuse pattern of staining with Con A-gold and monAb-gold. The de novo appearance of conspicuous amounts of glycogen-like particles near the plasmalemma and in the cell wall was interpreted as evidence of a reparative process of the yeast cell wall. Chitin was seemingly unaltered and readily demonstrated by the WGA-gold in the wall remnants of ghost cells.

Candida albicans↗
PubMed1991

Cell walls of normal and lysozyme-damaged blastoconidia of Candida albicans: localization of surface factor 4 antigen and vicinal-glycol staining.

The fungicidal effect of lysozyme on Candida albicans involves ultrastructural modifications previously described (G. Marquis, S. Montplaisir, S. Garzon, H. Strykowski, and P. Auger, Lab. Invest. 46:627-636, 1982). To further define the action of lysozyme on the yeast cell wall, we used the following: (i) the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method to highlight vicinal-glycol-reactive sites of complex carbohydrates; (ii) a monospecific antiserum and a protein A-gold complex to study the expression of surface factor 4, a major Candida antigen; and (iii) the periodic acid-silver methenamine method to stain cell wall glycoproteins. All Candida cells were found to express surface factor 4 antigen. In normal blastoconidia, surface factor 4 was located in a glycoprotein-rich cell wall layer, underneath radially oriented bundles of filaments which form the outermost wall layer. In lysozyme-treated blastoconidia, this glycoprotein-rich layer was lost and the regular brushlike organization of the outer fibrillogranular layer was disrupted. PA-TCH-SP staining and localization of surface factor 4 antigen demonstrated an altered arrangement of bundles of filaments in the outer wall layers of blastoconidia which were morphologically intact but had abnormal cell wall appearance. Next, there was a reduction in thickness of the outer layer and the expression of surface factor 4 antigen was limited to the cytoplasmic membrane area. Later on, the cell wall was almost uniformly highlighted by PA-TCH-SP staining. These data evinced a highly plastic architecture of the cell wall in C. albicans.

Antigens, Fungal↗
PubMed1991

Identification of coronaviruses by the use of indirect protein A-gold immunoelectron microscopy.

Concentration by airfuge and protein A-colloidal gold immunoelectron microscopy (PAG-IEM) offered a rapid and sensitive method for detection and identification of coronaviruses from various species. The method was applied to partially purified tissue culture-adapted or egg-adapted mammalian and avian coronaviruses and to clarified fecal samples from diarrheic calves and turkey poults for detection of enteric coronaviruses. Aggregates of virus coated with specific antibody were seen in virus samples mixed with homologous antiserum but not in control samples containing preexposure serum. At least a 10-50-fold enhancement of the sensitivity of direct EM for virus detection was obtained using protein A-colloidal gold complex as an electron-dense marker. The PAG-IEM method demonstrated low nonspecific background labeling and permitted detection of soluble and particle-associated antigen. Reciprocal cross-reactivity was detected among the subgroup of mammalian hemagglutinating coronaviruses, and antisera to 4 members of other subgroups only recognized their homologous virus.

Animals↗
PubMed1990

Implication of mitochondria in the replication of Nodamura virus in larvae of the Lepidoptera, Galleria mellonella (L.) and in suckling mice.

The replication of Nodamura virus, type species of the family Nodaviridae, takes place principally into the interfibrillar spaces of the sarcoplasm in close association with mitochondria in the infected muscles of both G. mellonella and suckling mice. The most striking event in infection of G. mellonella muscle cells was the aggregation and shape modification of numerous mitochondria (elongation, interdigitation, and vesiculation) at an early stage of infection. These clusters of mitochondria were cemented by a thick electron-dense material at the periphery of which viral particles undergo maturation process. In a later stage, degenerated and dilated mitochondria showed a clear assembling of virus particles on their outer membrane and occasionally on some inner membranes. These facts and the localization of RNA by RNase-gold technique in the assembling sites of viruses around and inside the mitochondria allowed us to postulate a role for the mitochondria as supports and/or energy suppliers for viral RNA synthesis and translation.

Animals↗
PubMed1990

Ultrastructure of the human interspinous ligament and ligamentum flavum. A preliminary study.

The ultrastructure of ligamenta flava (LF) and interspinous ligaments (ISL) obtained from four patients who underwent surgery for vertebral fracture (control group) and five patients operated for disc herniation was studied. The fine structure of LF was composed of elastic and elaunin fibers. Small diameter collagen fibrils were found between the elastic system fibers. The ISL was constituted predominantly of collagen fibrils. Elastic fibers were seen in the most ventral part of the ligament. In ISL and LF of the control group, the cells were fibroblastic-like cells. Chondrocytes were present only near their attachment sites. The proteoglycans were demonstrated between the collagen fibrils, and they appeared to form a regular interfibrillar linking. In ligaments obtained from patients with disc herniation, several modifications were found. The fibroblasts transformed into chondrocytic cells, which were surrounded by a pericellular matrix rich in proteoglycan filaments. A few cells that had suffered necrosis were found. Alterations in the collagen-proteoglycans arrangement also were evidenced. The proteoglycan filaments were randomly oriented to the collagen fibrils.

Adult↗
PubMed1990

Production and characterization of monoclonal antibodies to cell wall antigens of Aspergillus fumigatus.

Two murine monoclonal antibodies (MAbs) against Aspergillus fumigatus were produced and characterized. Splenocytes from cell wall-immunized BALB/c mice were fused with SP2/0 myeloma cells. The hybridomas were screened with a cold alkali (CA) extract of mycelium containing protein, mannose, and galactose, and two MAbs of the immunoglobulin M class were purified from ascites fluid. MAbs 1 and 40 were characterized by double immunodiffusion against CA antigen, indirect enzyme immunoassay with mannans of Candida albicans serotypes A or B or Candida tropicalis, indirect immunofluorescence with C. albicans- or A. fumigatus-infected tissues, indirect immunofluorescence with smears of other pathogenic fungi, Western blotting (immunoblotting) with the lectin concanavalin A or BS-1 from the seeds of Bandeirea simplicifolia, and immunoelectron microscopy. MAb 1 did not cross-react with Candida mannan and recognized a periodate-sensitive, pronase- and heat-resistant epitope in CA antigen and three mannose- and galactose-containing components (80, 62, and 49 kilodaltons) of a mycelial homogenate. Immunoelectron microscopy demonstrated binding of MAb 1 to the inner cell wall and intracellular membranes of hyphae and conidia of A. fumigatus. Circulating antigen was detected in experimental invasive aspergillosis by inhibition enzyme immunoassay with MAb 1 and CA antigen. MAb 40 was a nonprecipitating antibody cross-reactive with Candida species, and competition for an epitope located diffusely in the cell wall of A. fumigatus hyphae was demonstrated by incubating MAb 40 with mannan of C. albicans serotype A. These results suggest that MAb 1 recognizes immunodominant oligogalactoside side chains of A. fumigatus galactomannan, while MAb 40 binds to mannopyranosyl side chains common to A. fumigatus galactomannan and C. albicans mannan.

Animals↗
PubMed1989

Identification and location of the structural glycoproteins of a tissue culture-adapted turkey enteric coronavirus.

The Minnesota strain of turkey enteric coronavirus (TCV) was grown on a human rectal tumor (HRT-18) cell line in the presence of radiolabeled amino acids and glucosamine to analyse virion structural proteins. In addition to the 52,000 unglycosylated nucleocapsid protein, three major glycoprotein species were found to be associated with the viral envelope. A predominant glycosylated protein with a molecular weight of 22-24,000 represented the transmembrane matrix protein. Larger glycoproteins with apparent molecular weights of 180-200,000 (gp 200), 120-125,000 (gp 120) and 95-100,000 (gp 100) were associated to the characteristic large bulbous projections (peplomers) located at the surface of the virion. The gp 100 and gp 120 species apparently arose from a proteolytic cleavage of gp 200, as suggested by digestion studies with trypsin and chymotrypsin. An additional large glycoprotein with mol. wt. of 140,000 (gp 140), that behaved as a disulfide-linked dimer of a 66,000 molecule, was found to be associated to granular projections located near the base of the large peplomers. Digestion studies with trypsin, bromelain and pronase demonstrated that gp 140 was related to the hemagglutinating activity of the virus. An inner membranous sac or tongue-shaped structure could be visualized in the interior of the viral particles following treatment with pronase. In contrast, trypsin or chymotrypsin treatments resulted in evaginations ("budding") on the virus surface. Progeny viral particles produced in TCV-infected cell cultures in the presence of tunicamycin lacked both types of surface projections, as demonstrated by electron microscopy and electrophoresis. The matrix protein also appeared to be reduced to its unglycosylated form, concomitant with a considerable loss of its antigenicity. Thus, with respect to its morphological and biochemical characteristics, TCV resembles viruses belonging to the group of mammalian hemagglutinating coronaviruses, but differs in that both types of envelope glycoproteins are N-glycosylated as in case of the avian infectious bronchitis virus.

Coronaviridae↗
PubMed1989

Intracellular synthesis and processing of the structural glycoproteins of turkey enteric coronavirus.

Pulse labeling of cells with [35S]methionine or [3H]glucosamine at different times after infection, followed by SDS-PAGE and Western immunoblotting analysis using rabbit anti-TCV hyperimmune serum, was used to resolve and identify TCV-induced intracellular proteins. The viral structural proteins (gp 200, gp 140/gp 66, gp 100/gp 120, p 52, and gp 24/p 20) were detected in radiolabeled cell extracts by 9 to 12 hours post-infection, as well as two possible non-structural proteins with apparent mol.wts. of 36,000 and 32,000. The predominant 52,000 nucleocapsid protein could be detected in cell lysates as soon as 6 to 8 hours after infection; it was initially resolved as a complex of 3 closely migrating species with mol.wts. ranging from 46,000 to 52,000. Pulse-chase and immunoprecipitation experiments indicated that gp 200 arose from a putative precursor with mol.wt. of 150,000 to 170,000, that underwent glycosylation. Proteolytic cleavage of gp 200, in turn, probably yielded the gp 100 and gp 120 species. The unique TCV hemagglutinin protein originated from a primary precursor with mol.wt. of 60,000, which underwent rapid dimerization by disulfide bond formation and glycosylation to yield gp 140. The peplomeric and matrix proteins were both shown to be N-glycosylated, as indicated by their sensitivity to tunicamycin (TM) and their resistance to sodium monensin (SM). In the presence of TM, proteins with mol.wts. of 90,000, 120-130,000, and 150,000 accumulated in TCV-infected cells rather than peplomeric glycoproteins, and the matrix protein E1 was only detected in its unglycosylated form. The addition of TM to the culture medium interfered with the maturation of progeny viral particles, as suggested by the absence of peplomers at the surface of the intravacuolar and extracellular virions, and the accumulation of amorphous material not found in the absence of the glycosylation inhibitor. High yields of virus replication were obtained, in the presence of SM, even at concentrations which greatly affected the cellular functions.

Blotting, Western↗
PubMed1989

Ultrastructure and protein A-gold immunolabelling of HRT-18 cells infected with turkey enteric coronavirus.

The Minnesota strain of turkey enteric coronavirus (TCV) was propagated in HRT-18 cells, a cell line derived from human rectum adenocarcinoma. A productive non-cytopathic infection was established, without a previous adaptation, in these cells as shown by the specific hemagglutinating activity in cell culture supernatants. A post-embedding immunochemical technique, using specific antiserum directed against the original egg-adapted virus and colloidal-gold-labelled protein A as the electron-dense marker, was used for the identification of the virus and related antigens in the cells by electron microscopy. Budding of typical coronavirus particles, through intracytoplasmic membranes and accumulation of complete virus within cytoplasmic vesicles or the lumen of rough endoplasmic reticulum, were the main features of the viral morphogenesis. Late in infection, numerous progeny viral particles were shown at the outer surface of infected cells, but budding could not be demonstrated at this level. Two different types of surface projections were observed on the extracellular particles of this avian coronavirus. These morphological characteristics have been thus far described only for mammalian hemagglutinating coronaviruses.

Animals↗