Juan F. Santarén

African swine fever virus-induced polypeptides in vero cells

African swine fever virus induces in Vero cells 81 acid and 14 basic polypeptides which account for most of the information content of the virus DNA. The kinetics and the cytosine arabinoside sensitivity of the synthesis of the virus-induced polypeptides showed the existence of three classes of proteins, two early and one late. Most of the early proteins were synthesized along the whole replication cycle, but the synthesis of some of the early proteins was switched-off after virus DNA replication. Late proteins were defined as those whose synthesis did not take place in the presence of cytosine arabinoside. Cell iodination with chloramine T showed the presence in the membrane of the infected cells of two major virus-induced proteins of relative molecular weights 220K and 32K. Protein p220 was incorporated into the membrane very soon after its synthesis, before the appearance of intracellular infectious virus, whereas in the case of protein p32 there was a delay between synthesis and incorporation into the membrane.

Expression, cloning and cDNA sequence of a fibroblast serum-regulated gene encoding a putative actin-associated protein (p27)

A cDNA clone for a basic putative actin microfilament-associated protein, p27, highly induced in serum-stimulated NIH 3T3 cells, has been isolated by polyclonal antibodies and sequenced. p27 mRNA is a 1.2-kb molecule which is very low in resting NIH 3T3 cells but can be induced at least 100 times after 8 h of fetal calf serum stimulation. In contrast to other inducible mRNAs, p27 mRNA is stable, and its levels can be superinduced by cycloheximide mainly by prolonging transcription. The lack of expression of this messenger in mouse tissues, as well as in all cell lines so far tested, suggests that p27 may be an fibroblast-specific protein. One major open reading frame found in p27 cDNA codes for a 201 amino acid polypeptide not related to any previously described actin-binding protein. Interestingly, it shows alternative hydrophilic and hydrophobic domains of amino acids symmetrically arranged from the middle of the protein. The coordinate induction of p27 and actin mRNAs suggest that p27 may be involved in the cytoskeletal rearrangements induced early in cell growth and proliferation.

Transcriptional Inhibition of the Parvovirus Minute Virus of Mice by Constitutive Expression of an Antisense RNA Targeted against the NS-1 Transactivator Protein

We have assessed a genetic resistance approach based on antisense RNA to interfere with the prototype Minute Virus of Mice (MVMp), an autonomous parvovirus. MVMp is a cytolytic virus when infecting the permissive A9 mouse fibroblast cell line, and its gene expression is largely regulated at the level of transcription initiation by the nonstructural transactivator NS-1 protein, a multifunctional polypeptide also involved in viral DNA replication and cytotoxicity. An NS-1 specific antisense RNA constitutively expressed in transfected A9 clones increased several fold the proliferative viability of the cells upon high multiplicity virus infection, and cultures infected at low multiplicity reached confluence overcoming virus progression. All clones shared a common phenotype of resistance characterized by a lowered synthesis of viral DNA replicative intermediates and genomic forms, a significant reduction in the accumulation of the three viral messengers in the cytoplasmic and nuclear compartments, and a specific inhibition in viral protein synthesis. These results indicate that the constitutive antisense RNA mediates an overall repression of viral macromolecular synthesis by preventing the onset of NS-1 functions. Therefore, cytocidal parvoviruses may be hampered by engineered antisense RNA targeted against early regulators of virus growth.

Thermal and 13C-NMR study of the dynamic structure of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and 1-oleyl-2-palmitoyl-sn-glycero-3-phosphocholine in aqueous dispersions

Mixed-acid monounsaturated phosphatidylcholines containing palmitate and oleate chains have been synthesized by phospholipase A2 digestion of the appropriate single-acid phosphatidylcholine, followed by reacylation of the lysophosphatidylcholine with the desired fatty acid anhydride. The positional isomers 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and 1-oleyl-2-palmitoyl-sn-phosphocholine have been thus obtained. The thermotropic behavior of these lipids dispersed in excess water has been studied by differential scanning calorimetry. Positional isomers of mixed-acid monounsaturated phosphatidylcholines are found to have different gel to liquid-crystalline transition temperatures and enthalpies. It was found that mixtures of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine or 1,2-distearoyl-sn-glycero-3-phosphocholine exhibited inmiscibility in the phosphatidylcholine gel state. The dynamic structure of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and 1-oleyl-2-palmitoyl-sn-glycero-3-phosphocholine bilayers has been investigated by measuring the 13C nuclear spin-lattice relaxation times of sonicated aqueous dispersions. No difference was found between the two systems, suggesting that above the thermal transition the presence of the unsaturated acyl group in the 1 or 2 position does not affect significantly the dynamic structure of the bilayer.

African swine fever virus-induced polypeptides in porcine alveolar macrophages and in Vero cells: two-dimensional gel analysis.

High‐resolution two‐dimensional electrophoresis followed by computer analysis has been used to study quantitatively the patterns of protein synthesis produced in porcine alveolar macrophages and in Vero cells infected with African swine fever virus (ASFV). Initially, a protein database for each cell type was constructed. The porcine alveolar macrophage database includes 995 polypeptides (818 acidic, isoelectric focusing (IEF) and 177 basic, nonequilibrium pH gradient electrophoresis (NEPHGE)) whereas the Vero database contains 1398 polypeptides (1127 acidic, IEF and 271 basic, NEPHGE). Taking these databases as reference, ASFV highly virulent strain E70 induces 57 acid and 43 basic polypeptides in porcine alveolar macrophages, which account for most of the information content of the virus DNA. The kinetics of synthesis of the virus‐induced polypeptides showed the existence of three classes of proteins: one whose synthesis starts early after infection, continues for a period and then switches off; another whose synthesis also starts early but continues for prolonged periods; and a third which requires DNA replication. The attenuated, cell adapted, strain BA71V induces 92 acidic and 37 basic proteins in Vero cells. Significant differences were observed when comparing the patterns of polypeptides induced by the two viral strains. In both cell systems studied, ASFV infection produces a general shutoff of protein synthesis that affects up to 65% of the cellular proteins. Interestingly, 28 proteins of porcine alveolar macrophages and 48 proteins of Vero cells are stimulated at least two times by ASFV infection.

Molecular chaperones and the biogenesis of mitochondria and peroxisomes

Summary— A review of the proteinaceous machinery involved in protein sorting pathways and protein folding and assembly in mitochondria and peroxisomes is presented. After considering the various sorting pathways and targeting signals of mitochondrial and peroxisomal proteins, we make a comparative dissection of the protein factors involved in: i) the stabilization of cytosolic precursor proteins in a translocation competent conformation; ii) the membrane import apparatus of mitochondria and peroxisomes; iii) the processing of mitochondrial precursor proteins, and the eventual processing of certain peroxisomal precursor, in the interior of the organelles; and iv) the requirement of molecular chaperones for appropriate folding and assembly of imported proteins in the matrix of both organelles. Those aspects of mitochondrial biogenesis that have developed rapidly during the last few years, such as the requirement of molecular chaperones, are stressed in order to stimulate further parallel investigations aimed to understand the origin, biochemistry, molecular biology and pathology of peroxisomes. In this regard, a brief review of findings from our group and others is presented in which the role of the F1‐ATPase α‐subunit is pointed out as a molecular chaperone of mitochondria and chloroplasts. In addition, data are presented that could question our previous indication that the immunoreactive protein found in the rat liver peroxisomes is due to the presence of the F1‐ATPase α‐subunit.

The expression of heat shock protein HSP60A reveals a dynamic mitochondrial pattern in Drosophila melanogaster embryos.

The evolutionarily conserved hsp60 ( heat-shock protein 60) family of molecular chaperones ensures the correct folding of nuclear-encoded proteins after their translocation across the mitochondrial membrane during development as well as after heat-shock treatment. Although the overexpression of HSP60 proteins and their localization in the cytoplasm have been linked with many humans pathologies, the detailed pattern of their expression in different animal models and their subcellular localization during normal development and in stress conditions are little-known. In this report, we have used two-dimensional gel electrophoresis followed by MALDI-TOF to identify and purify heat shock protein HSP60A of Drosophila melanoagaster. We demonstrate that it is heat-shock inducible and describe two novel antisera, specifically designed to recognize the denatured and native polypeptide, respectively, in Drosophila. Immunoelectron microscopy and immunostaining of Drosophila cells with these antibodies reveals that HSP60A is always localized to the inner membrane of mitochondria. Expression of HSP60A is post-transcriptionally regulated in a highly dynamic pattern during embryogenesis, even under heat-shock conditions. In contrast, in very stressful situations, its expression is upregulated transcriptionally over the entire embryo. These findings suggest novel roles for HSP60 family proteins during normal Drosophila development.

Specific antibody against a protein (p27) present in nonestablished fibroblasts. A putative microfilament-associated protein

A specific polyclonal antibody has been raised against a basic cytoplasmic protein (p27) which is induced by serum in growth-arrested NIH 3T3 cells but is constitutively expressed in nonestablished fibroblasts. Immunoblotting analysis and [35S]methionine labeling show that p27 is absent in tissues and established cell lines of different types. However, it is present in fibroblasts from human, rat, mouse, and chicken origin and is highly conserved as determined by two-dimensional gel electrophoresis. Double immunofluorescence shows that p27 colocalizes with actin filaments. These observations would suggest that p27 is an actin-associated protein expressed in nonestablished fibroblasts.

2D gene expression parameters of wing imaginal disc of Drosophila for developmental analysis.

Abstract By using high resolution two-dimensional (2D) gel electrophoresis coupled with computer-analysis we have established a quantitative Drosophila wing imaginal disc protein database of third instar larvae as a reference to be used for comparative purposes in genetic studies. A general catalogue integrated by 1,184 35S-methionine-labelled polypeptides from wing imaginal disc has been obtained. The level of expression for all the proteins has been quantitatively determined. The quantitative reproducibility of the analysis system has been estimated and all the controls studied as database reference to interpret the results of experiments with mutant discs. One example, corresponding to iro1 mutation, has been used to show how some of the changes observed with mutant discs clearly extend out of the limits defined by the controls. This enables us to generate comparative parameters for the study of proliferation, morphogenesis and differentiation of Drosophila and opens the possibility of rapidly defining the nature and quantity of changes in patterns of gene expression in developmental genetic studies.

Thermal and NMR studies of chick embryo lecithins

Abstract Proton magnetic resonance and differential scanning calorimetry have been used to investigate model membranes composed of known molecular species of lecithins from liver, lung and brain of 19-day chick embryos. Above the chain-melting temperature the three systems studied show a similar behaviour, with the T1 increasing with temperature and giving a similar activation energy in each case. Throughout the temperature range analyzed only a single T1 is observed. These T1 values can be interpreted in terms of the chemical composition of molecular species of lecithins. Calorimetric techniques show different behaviour in the three models. Thermodynamic parameters of the gel-to-liquid transition are calculated and interpreted in terms of the molecular species of lecithins.