A study of the intracellular distribution of prion protein (PrP) in N2a neuroblastoma cells which had been infected with prions (ScN2a cells) revealed that most PrP is present in the cytoplasm. However, a significant amount of PrP is also present in the nucleus (predominantly in the nucleoli) of these cells, as analyzed by confocal laser scanning microscopy. By contrast, no PrP could be detected in the nucleus of uninfected N2a cells. The steady-state level of PrP mRNA did not markedly differ between the two cell strains. Likewise, no changes were found in the rate of transcription and in the half-life of PrP mRNA. A number of cellular proteins, among them the nuclear lectin CBP35, was identified that bound to the predicted RNA stem-loop structure of PrP RNA. CBP35 could also be detected in purified infections prions, suggesting a possible role in prion replication. Age-dependent studies revealed that the content of normal cellular PrP (PrPC) in brain extracts of rats did not change significantly during ageing, while the level of certain proteins that associate with PrPC mRNA decreases with age. In addition, we show that rat cortical cells when challenged with infectious PrP (PrPSc) undergo cell death (apoptosis) in vitro. This deleterious effect was prevented by memantine (1-amino-3,5-dimethyladamantane) and other blockers of N-methyl-d-aspartate (NMDA) receptor channels.

A cDNA clone encoding the sequence-specific DNA binding protein, serum response factor (SRF), has been isolated and analysed from the marine spongeGeodia cydonium. The 1443 bp long cDNA comprises an open reading frame of 1149 bp, from which an aa sequence with a Mr42,149 can be deduced. Sequence comparisons revealed that the aa sequence shares high homology to human andXenopus laevisSRFs. Like these vertebrate sequences also the sponge sequence displays the MADS-box motif present in the DNA-binding domain. The SRF, and also other transcription factors, such as ternary complex factors, recognizes the serum response element (CArG box) in promoter regions of a series of cellular immediate-early genes, whose expression is controlled by growth factors. A related CArG box sequence is present in sponge gene, encoding a receptor tyrosine kinase. Expression of SRF is strongly regulated by temperature stress; after incubation of the sponge at high temperature, the 1.6 kb transcript of SRF is downregulated. It is suggested that the SRF is involved in the transcriptional regulatory circuits, which control the response of the animal to altered environmental conditions.

Sponges (Porifera) are among the major phyla inhabiting the marine hard-substrate benthos, both in respect to the number of species and their biomass. Hence reliable biomarkers need to be developed to monitor the environmental load in those animals. Recently, the cDNA for the heat shock protein HSP70 has been isolated from the sponge Geodia cydonium and found to be a reliable indicator for temperature stress. In the present study, we have isolated the Rab GDP-dissociation inhibitor (GDI), which has previously been shown to be a key element in the intracellular traffic system. The 1521 bp long cDNA, encoding sponge GDI, has been isolated and analyzed. The deduced aa sequence (Mr = 50215) displays high homology to vertebrate, invertebrate and also yeast GDIs. Northern blot analysis revealed that the 1.6 kb sponge transcript that hybridized with the sponge GDI is down-regulated under temperature stress; in contrast, the transcription of the sponge HSP70 is strongly enhanced. Due to the high sequence conservation of the GDIs, one can assume that sponge GDI probes can be used as functional biomarkers for temperature stress, especially when applied simultaneously with the highly conserved HSP70 probes.

Proteasomes are large protein complexes that play a major role in selective degradation of intracellular proteins. Eukaryotes feature seven different α and β subunits. Two of the vertebrate housekeeping β-subunits have MHC-encoded homologues that can substitute for the housekeeping counterparts upon interferon-γ induction. In the present study we report the cloning of invertebrate β-subunit proteasome epsilon (PRCE), from the marine spongeGeodia cydoniumand from the colonial tunicateBotryllus schlosseri.Sequence comparisons revealed that the sponge and tunicate proteins are strikingly similar to vertebrate and yeast PRCEs and their MHC-linked counterparts the PRCCs (also termed LMP7), and to a lesser degree also to archaebacterial proteasome subunit β. Based on this comparison we suggest that all eukaryotic PRCEs and PRCCs feature a cleavable N-terminal propeptide, including the two mammalian PRCEs which appear to have been wrongly predicted from incomplete cDNAs. Our comparative analysis outlines 25 amino acid positions which appear to be unique for PRCCs, distinct from the corresponding residues in metazoan PRCEs.

The RNA stem-loop structure of the trans-activating region TAR sequence of human immunodeficiency virus-1 mRNA is the binding site for a number of host cell proteins. A virtually identical set of proteins from HeLa nuclear extracts was found to bind to the predicted RNA hairpin element of prion protein (PrP) mRNA, as demonstrated in UV cross-linking/RNase protection and Northwestern assays. We show that the cellular TAR loop-binding protein, p68, is among those proteins which associate with PrP RNA. Competition experiments with various TAR RNA mutants revealed that binding of partially purified p68 to PrP RNA stem-loop occurs sequence-specifically. The 100-kDa 2-5A synthetase which is involved in the cellular antiviral defense was able to bind to PrP mRNA stem-loop in Northwestern blots with cytosolic proteins from HeLa cells treated with interferon. However, the PrP RNA failed to activate this enzyme in vitro, in contrast to TAR RNA.

The 2',5'-oligoadenylate (2-5A) system is involved in the defense of mammalian cells against virus infection. In a previous study [25], we demonstrated that the activities of the enzymes which synthesize and degrade 2-5A [2-5A synthetase (2-5OAS) and 2',3'-exoribonuclease] and of the enzyme that is activated by 2-5A (ribonuclease L) change during aging and development in different tissues of rat. The age-dependent decrease in 2-5OAS activity and increase in 2-5A nuclease activity results in a decrease in the cellular 2-5A content, suggesting that the efficiency of the antiviral 2-5A system is impaired in aged rats. Here we determined the age-dependent changes in the level of mRNA coding for the class I isoenzyme of 2-5OAS (M(r) 40-46 kDa) in rat liver and brain using a cDNA which was recently cloned from rat hippocampus. We found that the decrease in 2-5OAS activity is accompanied by a decrease in the level of 2-5OAS mRNA; in old animals (32-33 months old), the amount of 2-5OAS mRNA was reduced to 20-30% compared to young adult (2-3 months old) (100%) and middle-aged adult animals (12 months old) (110-120%). In addition, Western-blotting experiments revealed that the amount of class I 2-5OAS capable of binding to its activator, poly(I).poly(C), is also diminished in the livers and brains of old rats compared to those of young adult and middle-aged adult animals.

A number of cellular proteins were identified that bind to the predicted RNA stem-loop structure of prion protein (PrP) RNA; a virtually identical set of RNA-binding proteins was found to associate with the trans-activating region TAR of the human immunodeficiency virus-1. The predicted hairpin elements of the PrP mRNA contain, like TAR RNA, a CUGGG sequence in the loop and a uridine and adenine bulge in the stem; these features are unique among cellular RNAs. UV cross-linking of RNA.protein complexes formed between PrP RNA and HeLa nuclear protein yielded four prominent RNase-resistant complexes, in addition to some minor bands, which migrated at approximate to 90, 68, 42, and 37kDa under denaturing conditions. The presence of multiple PrP RNA-binding, as well as TAR RNA-binding polypeptides was also demonstrated in Northwestern assays with nuclear extracts from mouse ascites, liver, and spleen, whereas only one PrP RNA-binding protein (a doublet with an approximate molecular mass of 35kDa) was found in brain extract from rat. The nuclear beta-galactoside-specific lectin, CBP35 (carbohydrate-binding protein with a molecular mass of 35 kDa), which has been identified in nuclear ribonucleoprotein (RNP) complexes from a variety of mammalian tissues and cells, was among those proteins which bind to PrP RNA. The cellular prion protein, PrPC, was found to be unable to bind PrP RNA directly; however, this protein could be detected in the RNP/CBP35 complex formed between PrP RNA and rat brain extracts. Association of PrPC with RNP/CBP35 complex was abolished by RNase treatment. CBP35 could be also detected in purified infectious scrapie prions, suggesting a possible role in prion replication.

The mismatched double-stranded RNA (dsRNA), poly(I).poly(C12U), also termed Ampligen, exhibits a strong antiviral and cytoprotective effect on cells (human T-lymphoblastoid CEM cells and human T-cell line H9) infected with the human immunodeficiency virus type 1 (HIV-1). Untreated H9 cells infected with HIV-1 start to release the virus 3 days post-infection, while in the presence of 40 micrograms/ml (80 micrograms/ml) of poly(I).poly(C12U) the onset of virus production and release is retarded and does not occur before day 5 (day 6). We demonstrate that poly(I).poly(C12U) markedly extends the duration of the transient increase of 2',5'-oligoadenylate (2-5A) synthetase mRNA level and activity preceding virus production after infection of cells with HIV-1. Treatment of HeLa cells with poly(I).poly(C12U) was found to cause a significant increase in total (activated plus latent) 2-5A synthetase activity; no evidence was obtained that the level of latent (nonactivated) 2-5A synthetase is changed in cells treated with dsRNA plus interferon (IFN). Poly(I).poly(C12U) is able to bind and to activate 2-5A synthetase(s) from HeLa cell extracts. Addition of poly(I).poly(C12U) to HeLa cell extracts results in production of longer 2-5A oligomers (> or = 3 adenylate residues), which are better activators of RNase L. Both free and immobilized poly(I).poly(C12U) also bind to the dsRNA-dependent protein kinase (p68 kinase), resulting in autophosphorylation of the enzyme. Activation of the kinase by the free RNA occurs within a limited concentration range (10(-7) to 10(-6) grams/ml). Addition of HIV-1 Tat protein does not affect binding and activation of p68 kinase to poly(I).poly(C12U)-cellulose but strongly reduces the binding of the kinase to immobilized TAR RNA of HIV-1. We conclude that poly(I).poly(C12U) may antagonize Tat-mediated down-regulation of dsRNA-dependent enzymes.