Changes in the reactivity of the immune system of children after the influenza A infection.
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Adolescent↗
Biomedical subjects
F Ciampor
Publications and source records attributed to F Ciampor.
Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17.
Carbonic anhydrase IX (CA IX) is a transmembrane protein whose expression is strongly induced by hypoxia in a broad spectrum of human tumours. It is a highly active enzyme functionally involved in both pH control and cell adhesion. Its presence in tumours usually indicates poor prognosis. Ectodomain of CA IX is detectable in the culture medium and body fluids of cancer patients, but the mechanism of its shedding has not been thoroughly investigated. Here, we analysed several cell lines with natural and ectopic expression of CA IX to show that its ectodomain release is sensitive to metalloprotease inhibitor batimastat (BB-94) and that hypoxia maintains the normal rate of basal shedding, thus leading to concomitant increase in cell-associated and extracellular CA IX levels. Using CHO-M2 cells defective in shedding, we demonstrated that the basal CA IX ectodomain release does not require a functional TNFalpha-converting enzyme (TACE/ADAM17), whereas the activation of CA IX shedding by both phorbol-12-myristate-13-acetate and pervanadate is TACE-dependent. Our results suggest that the cleavage of CA IX ectodomain is a regulated process that responds to physiological factors and signal transduction stimuli and may therefore contribute to adaptive changes in the protein composition of tumour cells and their microenvironment.
ADAM Proteins↗
Analysis of specificity of interaction between synthetic key saccharide components of lipopolysaccharides and monoclonal antibodies to Coxiella burnetii.
Two monoclonal antibodies (MAbs) against the lipopolysaccharides (LPSs) of Coxiella burnetii (C.b.) strains Priscilla and Nine Mile were prepared characterized by their interaction with synthetic glycoconjugates representing parts of LPSs of C.b. in virulent phase. Both MAbs were directed against immunodominant epitopes comprising core constituent of LPSs, Kdo (3-deoxy-alpha-D-manno-2-octulo-pyranosylonic acid). ELISA showed that the anti-Nine Mile MAb 4/11 bound preferably to disaccharides (alpha-Kdo (2 --> 4) alpha-Kdo and alpha-Kdo (2 --> 4) alpha-(5d) Kdo), while the anti-Priscilla MAb 1/4/H bound to all conjugates, though with various intensity. On the other hand, immunoelectron microscopy revealed a positive binding of only one glycoconjugate, namely the trisaccharide alpha-Kdo (2 --> 4) alpha-Kdo (2 --> 4) alpha-Kdo-BSA, to both MAbs. In competitive ELISA (cELISA), the anti-Priscilla MAb 1/4/H distinguished the strains Nine Mile and Priscilla, while the anti Nine Mile MAb 4/11 did not.
Animals↗
Vasodilatory activity in horsefly and deerfly salivary glands.
Salivary gland extract (SGE) of four horsefly species (Hybomitra bimaculata Macquart, Hybomitra ciureai Séguy, Tabanus bromius L., Tabanus glaucopis Meigen) and one deerfly species (Chrysops relictus Meigen) (Diptera: Tabanidae) were shown to contain vasodilatory activity. Aliquots equivalent to 1, 5 and 10 pairs of salivary glands (SG) relaxed rat femoral artery (with intact endothelium) pre-constricted with phenylephrine. Vasodilatory activity was dose-dependent. SGE of one horsefly species (Haematopota pluvialis L.) did not induce relaxation. The kinetics of vasodilation induced by SGE of four horsefly species differed from the deerfly. These results indicate that tabanid species may produce more than one type of vasodilator to aid blood feeding.
Animals↗
The ion channels coded by viruses.
Pathogenicity and virulence are multifactorial traits, depending on interaction of viruses with susceptible cells and organisms. The ion channels coded by viruses, viroporins, represent only one factor taking part in the cascade of interactions between virus and cell, leading to the entry of virus, replication and to profound changes in membrane permeability. The M2 protein from influenza A virus forms proton-selective, pH-regulated channel involved in regulating vesicular pH, a function important for the correct maturation of HA glycoprotein. The NB glycoprotein of influenza B viruses is an integral membrane protein with an ion channel activity. The CM2 protein of influenza C virus is an integral membrane glycoprotein structurally analogous to influenza A virus M2 and influenza B virus NB proteins. The picornavirus 3A protein is involved in cell lysis and shows homology with other lytic proteins. Vpu is an oligomeric integral membrane protein encoded by HIV-1, which forms ion channels. The togavirus 6K protein shows structural similarities with other viroporins.
Animals↗
Characterization of RKZ isolate of ovine herpesvirus 1.
Cytopathic effect (CPE) characterized mainly by foci of rounded cells was observed in cultures of primary plexus choroideus cells from healthy lamb following cryopreservation. It was possible to transmit the infectious agent to other primary cells of ovine origin by co-cultivation with infected cells. By indirect immunofluorescence microscopy it was found that high percentage of sheep (65-80% in 3 different herds from Slovakia) are infected with this infectious agent. Electron microscopy of cells with CPE revealed the presence of herpesvirus particles. Viral DNA was isolated from infected cells using pulse-field gel electrophoresis and further used as probe in Southern blot analysis. The probe reacted specifically only with DNA from cells infected with Ovine herpesvirus 1 (OvHV-1) but not with DNA of other ruminant herpesviruses. Some of the HindIII restriction fragments of DNA of the obtained OvHV-1 isolate denominated RKZ were cloned. Part of the H9 clone was sequenced identifying a gene that encoded a polypeptide homologous to conserved herpesvirus VP23 structural protein. From comparison of the sequence of this clone with VP23 sequences of other herpesviruses it was deduced that OvHV-1 might be classified within the Rhadinovirus genus of the Gammaherpesvirinae subfamily. The sequencing of the H9 clone of DNA of RKZ isolate enabled establishment of sensitive and highly specific polymerase chain reaction (PCR) assay for detection of OvHV-1.
Amino Acid Sequence↗
Identification of MaTu-MX agent as a new strain of lymphocytic choriomeningitis virus (LCMV) and serological indication of horizontal spread of LCMV in human population.
In this study we elucidated the molecular character of MaTu-MX, previously described as an unusual transmissible agent. Amino acid sequencing of peptides generated from a 58-kDa MX-related protein purified from MaTu human carcinoma cells allowed us to identify it as a nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV). Northern blot analysis detected LCMV-specific RNAs in MaTu cells. Comparative immunoprecipitations showed cross-reactivity between NP of LCMV strain WE and MX NP. Using RT-PCR, we have cloned MX NP cDNA. According to sequence comparison, MX LCMV is as closely related to both LCMV strains WE and Armstrong as these strains are to one another. Based on this finding we propose that MX is a new strain of LCMV. We also showed that the stability of MX NP in MaTu cells is very high and that the virus is transmissible by cell-to-cell contact or by cell-free extract to human HeLa and monkey Vero cells, but not to human AGS, canine MDCK, mouse NIH 3T3, and hamster CHO cells. Finally, employing MX LCMV NP in immunoprecipitation and solid-phase radioimmunoassay, we found 37.5% prevalence of anti-LCMV antibodies in human sera, suggesting possible horizontal spread of the virus in the human population.
3T3 Cells↗
Sequence and characterisation of the Z gene encoding ring finger protein of the lymphocytic choriomeningitis virus MX strain.
We have cloned and characterised a cDNA encoding Z protein of recently identified MX strain of lymphocytic choriomeningitis virus (LCMV) persistently infecting human MaTu cells. Deduced amino acid sequence of LCMV MX Z protein showed 88.9% identity with that of the LCMV Armstrong (ARM) strain and 80.9% identity with that of the LCMV Traub (TRA) strain. It contained conserved zinc-binding RING finger domain and C-terminal proline-rich region. Northern blot analysis of total RNA from MaTu cells revealed presence of abundant truncated forms of L RNA. Z protein-specific rabbit antibodies were produced to glutathione S-transferase (GST)-Z fusion protein expressed in E. coli and used for the detection of Z protein in MaTu cells. Western blot and immunofluorescence analyses detected relatively high levels of Z protein indicating its role in maintenance of persistent LCMV.
Amino Acid Sequence↗
Effects of brefeldin A on the expression and transport of influenza A virus haemagglutinin, M1 and M2 proteins within the cell.
Brefeldin A (BFA) decreased the expression of influenza A virus haemagglutinin (HA) and M2 protein on the plasma membrane of virus-infected MDCK cells. It caused a retention of M1 protein in the cell nucleus and a decrease of its expression on the plasma membrane. On the other hand, an increased labelling of the cytoplasmic domain of M2 protein on the plasma membrane in BFA-treated cells was observed in contrast to the labelling in BFA-untreated cells. The effects of BFA on the microtubules and cellular motors are discussed.
Animals↗
Ultrastructural lectinocytochemistry of fowl plague virus-infected and uninfected MDCK cells.
Using horseradish peroxidase (HRP)-conjugated lectins for pre-embedding labelling we have shown differences in ultrastructural localization of saccharides in cell compartments of fowl plague (FP) virus-infected and uninfected MDCK cells. Lectinochemical staining of the cell compartments in the case of FP virus-infected MDCK cells was less intensive as compared with uninfected cells. Also certain differences in the staining of subcompartments of cell organells were seen. Staining of uninfected cells with Pisum sativum agglutinin (PSA)-HRP revealed an extensive visualization of Golgi complex, mainly its cis-part, TGN vesicles and lysosomes. Staining of FP virus-infected cells with the same lectin marked very lightly rough endoplasmic reticulum and not at all the Golgi complex. Staining with Erythrina cristagalli agglutinin (ECA)-HRP revealed a picture very similar to PSA-HRP staining of uninfected and FP virus-infected cells. The differences in the lectinochemical staining of cell organelles of FP virus-infected and uninfected cells may be connected with the inhibition of cell protein synthesis during FP virus morphogenesis.
Animals↗
Influenza virus M2 protein and haemagglutinin conformation changes during intracellular transport.
The influenza virus M2 protein has an ion channel activity that permits ions to enter the virion during its uncoating and also modulates pH of intracellular compartments. M2 protein is a homotetramer consisting of either a pair of disulfide-linked dimers or a disulfide-linked tetramer. The M2 trans-membrane domain peptide adopts an alfa helical secondary structure. In polarized cells, M2 protein is expressed at the apical cell surface. The amantadine-induced, M2-mediated conversion of influenza A virus haemagglutinin (HA) to the low pH conformation occurs in an acidic trans-Golgi compartment. The M2 protein ion channel activity can affect the conformation of cleaved HA during intracellular transport. The equine influenza virus 1 HA expressed from cDNA does not require coexpression of a functional M2 protein to maintain HA in its native conformation.
Animals↗
Expression of MaTu-MN protein in human tumor cultures and in clinical specimens.
MaTu is a novel agent which may be of relevance in human oncogenesis, and has 2 components. One of them, the exogenous MX (coding for protein p58X), is transmissible to human fibroblasts, to HeLa and to HeLa x fibroblast (H/F) hybrids. The other component, MN, is a cellular gene. Its product, the protein p54/58N, is inducible by infecting HeLa cells with MX or by growing them in dense cultures. This p54/58N appears to be a tumor-associated antigen: it is expressed in HeLa and in tumorigenic cells (H/F-T), but not in fibroblasts or in nontumorigenic hybrid cells (H/F-N). Proteins related to p54/58N were also found on immunoblots prepared from human carcinomas of ovary, endometrium and uterine cervix, but not from normal tissues from corresponding organs or from placenta. Using genetically engineered MN protein, we developed a radioimmunoassay for MN-specific antibodies, and for quantitative determination of MN proteins in cell extracts. In HeLa cells infected with MX we observed conspicuous ultrastructural alterations: formation of abundant filaments on the cell surface and amplification of mitochondria. Using immunogold-staining, we visualized the p54/58N on the surface microvilli and in the nucleus, particularly in nucleoli.
Animals↗
Monoclonal antibodies demonstrate accessible HA2 epitopes in minor subpopulation of native influenza virus haemagglutinin molecules.
Haemagglutinin (HA) was detected on the surface of influenza virus infected cell with monoclonal antibodies (MAbs) against both HA glycopolypeptides, HA1 and HA2, however, the reactivity of HA2-specific MAbs was remarkably lower as compared with HA1-specific MAbs. Quantitative analysis with two MAbs, IB8 (anti-HA1) and IIF4 (anti-HA2) respectively, revealed that HA2 epitope was reachable for antibody only in minor subpopulation of the HA representing approximately 7% of all molecules (spikes). The basis of the HA heterogeneity is discussed.
Animals↗
Electron-microscopic study of the effect of various extractants on the morphology of Coxiella burnetii.
This paper presents the results of the effect of trichloroacetic acid (TCA) at two different temperatures and the effect of a mixture of detergents (D) on Coxiella burnetii. Both TCA and D caused a large destruction of C. burnetii cells. In both cases complexes of high-molar-mass components from the outer membrane were extracted. In the D case not only proteins but a mixture with other high-molar-mass structures were released from the destroyed cells. By extraction of TCA, the antigenic complex composed of lipopolysaccharides (LPS), proteins and phospholipids was released. The effect of the D mixture on C. burnetii causes their complete destruction. The TCA causes a surface destruction of cells but it does not cause complete disintegration. The small-cell variants (SCV) in both cases were shown to be more stable compared to the large-cell variants (LCV).
Antigens, Bacterial↗
Evidence that the amantadine-induced, M2-mediated conversion of influenza A virus hemagglutinin to the low pH conformation occurs in an acidic trans Golgi compartment.
Amantadine treatment of cells infected with H7 strains of influenza A viruses causes an M2 protein-mediated conversion of hemagglutinin (HA) from its native to its low pH conformation. Immunofluorescence and electron microscopic observations showed that the structural alteration and hence drug action occur shortly after HA exits from the Golgi complex during its passage through the strans Golgi region. Using the DAMP/anti-DNP pH probe it is evident that virus infection causes increased acidity of the trans Golgi region and that vesicles containing low pH HA in amantadine-treated virus-infected cells are particularly acidic. These results indicate therefore that the alteration in HA is the direct consequence of exposure to an adverse low pH and provide further support for the conclusion that the M2 protein, the target of amantadine action, is involved in regulating vesicular pH, a function important for the correct maturation of the HA glycoprotein.
Amantadine↗
Regulation of pH by the M2 protein of influenza A viruses.
Inhibition of the function of the M2 protein by amantadine can cause a conformational change in the haemagglutinin (HA) of H7 influenza A viruses and the consequent expression of the low pH form of the glycoprotein on the surface of virus-infected cells. Immunofluorescence studies showed that this conversion occurs shortly after HA exists from the Golgi complex apparently during its transport through the trans Golgi network and using the pH probe, DAMP/anti-DNP, that it is the direct result of reduced vesicular pH. The lowest pHs encountered were estimated using mutant HAs differing in pH stability to be approximately 5.2 and 5.6 in virus-infected CEF or MDCK cells, respectively, in the absence of functional M2. Depending on the particular M2, this protein was responsible for increases in vesicular pH of up to 0.8 units. The influence of mutations in both HA and M2 on the maturation of native HA illustrates the important relationship between the structural and functional properties of these two proteins. Using the fluorescent probe SNARF-1 the M2 protein was also shown to be largely responsible for the 0.3-0.4 unit reduction in intracellular pH of virus-infected cells. The data thus provide further evidence for the pH regulatory function of M2 and its importance for the maturation of the HA glycoprotein.
Amantadine↗
Common and different antigenic properties of the rabies virus glycoprotein of strains SAD-Vnukovo and Pitman-Moore.
Two fixed rabies virus strains, SAD-Vnukovo and Pitman-Moore (PM) were used as combined immunogens for the generation of hybridomas secreting specific monoclonal antibodies (MoAbs). The obtained hybridomas were primarily screened by an ELISA for production of MoAbs to antigen of SAD-Vnukovo strain. Six positive clones were established. A panel of MoAbs has been characterized according to reactivity in immunofluorescence, immunoblot, ELISA and neutralization tests. All MoAbs were positive in immunofluorescence when cells infected with the SAD-Vnukovo strain were used. By immunoblot, four MoAbs showed specificity for the viral glycoprotein of both SAD-Vnukovo and PM rabies strains. This pattern of reactivity indicated the existence of shared conformation-independent epitopes located on the related antigens. However, in ELISA, the tested MoAbs did not recognize viral glycoproteins of the PM strain. This indicates, that the different strain-specific conformations of the native glycoprotein determine the accessibility of the common linear determinants for respective antibodies. Only one antibody, with conformation-dependent glycoprotein specificity, was capable to neutralize the CVS strain of rabies virus.
Animals↗
Localization of nucleocapsid associated polypeptides in measles virus-infected cells by immunogold labelling after resin embedding.
The nucleo-, phospho- and matrix protein of measles virus were localized at high resolution within infected cells by use of post-embedding immunogold labelling techniques. In general, labelling with monospecific antibodies as well as with a polyvalent rabbit anti-measles hyperimmune antiserum revealed measles virus polypeptides to be distributed non-randomly within infected cells with the label largely confined to specific sites, namely inclusions of nucleocapsids and assembled virus structures at the plasma membrane. Immunogold double labelling indicated that the phosphoprotein strictly co-localized with the nucleoprotein in cytoplasmic inclusions of nucleocapsids and in budding virions, whereas intranuclear inclusions of nucleocapsids were devoid of phosphoprotein labelling. Antibodies to the matrix protein clearly labelled assembled virus structures at the plasma membrane but exhibited no significant cytoplasmic or intranuclear reaction. The data indicate that the composition of nucleocapsids varies with the cellular compartment with which they are associated, supporting the view of a rapid assembly of paramyxovirus nucleocapsid polypeptides, and emphasize the proposed selective role of the matrix protein in virus assembly and budding at the plasma membrane.
Capsid↗