Efficient transfection of novel bovine papillomavirus 1 expression plasmids.

A series of novel bovine papillomavirus type 1 (BPV-1)-based expression plasmids was constructed and characterized in vitro as a starting point for the development of an in vivo gene therapeutic method. The order of transfection efficiency for different pBPVlacZ plasmids was pCGalBPV > pTKBPV > pSRalphaBPV in CV1-P cells. In the absence of selection pressure, the expression of pCGalBPVlacZ and pTKBPVlacZ was associated with long-term maintenance. In a comparison of pBPVlacZ with pSVlacZ, expression was maintained up to 12-17 and 8-12 days, respectively. The transfection of pBPVlacZ plasmids was efficient in secondary and primary, dividing and nondividing, neural and nonneural, and human cells and, furthermore, independent of the cell cycle as seen in growing as well as resting cells. All these characteristics are likely to be relevant for in vivo conditions, under which the percentage of proliferating cells could be quite low. In conclusion, the pBPV plasmids were efficiently delivered and expressed in different host cells, and therefore their performance in gene therapy is worth testing.

Replication of a chimeric origin containing elements from Epstein-Barr virus ori P and bovine papillomavirus minimal origin.

The bovine papillomavirus E2 protein is a multifunctional protein that activates viral transcription, co-operates in initiation of viral DNA replication, and is required for long-term episomal maintenance of viral genomes. The EBNA1 protein of Epstein-Barr virus is required for synthesis and maintenance of Epstein-Barr virus genomes. Both viral proteins act through direct interactions with their respective DNA sequences in their origins of replication. The chimeric protein E2:EBNA1, which consists of an transactivation domain of E2 and DNA binding domain of EBNA1 supported the replication of the chimeric origin that contained EBNA1 binding sites in place of the E2 binding sites principally as full-length E2 did in the case of papillomavirus minimal origin. This indicates that the chimeric protein E2:EBNA1 is competent to assemble a replication complex similar to the E2 protein. These data confirm the earlier observations that the only part of E2 specifically required for its activity in replication is the N-terminal activation domain and the function of the DNA binding domain of E2 in the initiation of replication is to tether the transactivation domain of E2 to the origin of replication.

Bovine papillomavirus oncoprotein E5 affects the arachidonic acid metabolism in cells.

The bovine papillomavirus type 1 (BPV-1) oncoprotein encoded by the E5 ORF is a small highly hydrophobic protein, which is capable of inducing oncogenic transformation of cells. We studied the effect of the BPV-1 E5 protein expression on the arachidonic acid metabolism in monkey (COS1) and human (C33A) cells. At relatively low protein concentrations the phospholipase A(2) (PLA(2)) activity and the arachidonic acid (AA) metabolism are activated. E5 mutant proteins, lacking cysteines responsible for the dimerisation of the protein (C37S, C37SC39S), and truncated E5, lacking the C-terminal region, are non-transforming and unable to stimulate the PLA(2) activity and AA metabolism. The transformation-defective mutant D33V, which does not activate the platelet-derived growth factor receptor (PDGFR), activates AA metabolism like wt E5. Our data suggest that the BPV-1 E5 protein could stimulate the AA metabolism independently of PDGF receptor.

Roles of the hinge region and the DNA binding domain of the bovine papillomavirus type 1 E2 protein in initiation of DNA replication.

The bovine papillomavirus (BPV-1) E2 protein is the regulator of extrachromosomal replication of papillomaviruses. The mutants with C-terminal truncations and in-frame internal deletions were constructed to study the role of structural domains of E2 in the initiation of DNA replication. We show that the replication initiation function of E2 is absolutely dependent on the ability of the protein to bind to DNA. Our study also confirms the borders of the functional domains of the E2 protein; residues 1-192 form the activation domain and residues 310-410 the DNA binding-dimerization domain. Some critical length and flexibility, but not the particular amino acid sequence between these two functional domains is required for the activity of the protein to support replication. The hinge region, including the major phosphorylation sites of E2, is also dispensable for the mediation of attachment of the BPV1 genome to the mitotic chromosomes.

DNA vaccination in mice using HIV-1 nef, rev and tat genes in self-replicating pBN-vector.

The immunogenicity of a self-replicating DNA-vector containing HIV-1 nef gene (pBN-Nef) was characterized using various DNA delivery methods. In addition, gene gun immunisation was used for assessing immunogenicity of two other HIV-1 genes (rev and tat) given in the same vector. The pBN-Nef was the most immunogenic raising both humoral and cell-mediated immune responses in mice; these responses lasted for up to six months. The pBN-Nef vector was immunogenic also when given intramuscularly or intradermally. The pBN-Rev construct did not elicit humoral responses but did elicit proliferative as well as CTL-response against the corresponding protein. The pBN-Tat was a poor immunogen in all respects. The antibodies elicited with various DNA delivery methods belonged to different antibody subclasses; however, two main epitopes in Nef were frequently recognized by all of them.

Diversity of Helicobacter pylori genotypes among Estonian and Russian patients with perforated peptic ulcer, living in Southern Estonia.

To compare the genomic variation of Helicobacter pylori in samples obtained from patients with perforated peptic ulcer, living in the same area of Estonia but belonging to different nationalities, 50 non-consecutive patients (32 Estonians and 18 Russians) admitted in the Tartu University Hospital in 1997-1999 were studied. Gastric samples of antral mucosa were obtained during operation and analysed histologically and with PCR for detection of different genotypes of H. pylori (cagA and vacA s and m subtypes). Among the 50 perforated peptic ulcer patients with histologically proven H. pylori colonisation no sample of gastric mucosa showed the s1b subtype of the vacA gene. The perforated peptic ulcer patients were mainly infected with cagA (82%) and s1 (98%) genotypes of H. pylori. The distribution of s1a/m1, s1a/m2 and s2/m2 subtypes of vacA genes was statistically different in Estonian and Russian patients (P<0.05). In conclusion differences in the distribution of vacA s and m subtypes of H. pylori were revealed between Estonian and Russian patients with perforated peptic ulcer from Southern Estonia.

The basis of prostaglandin synthesis in coral: molecular cloning and expression of a cyclooxygenase from the Arctic soft coral Gersemia fruticosa.

In vertebrates, the synthesis of prostaglandin hormones is catalyzed by cyclooxygenase (COX)-1, a constitutively expressed enzyme with physiological functions, and COX-2, induced in inflammation and cancer. Prostaglandins have been detected in high concentrations in certain corals, and previous evidence suggested their biosynthesis through a lipoxygenase-allene oxide pathway. Here we describe the discovery of an ancestor of cyclooxygenases that is responsible for prostaglandin biosynthesis in coral. Using a homology-based polymerase chain reaction cloning strategy, the cDNA encoding a polypeptide with approximately 50% amino acid identity to both mammalian COX-1 and COX-2 was cloned and sequenced from the Arctic soft coral Gersemia fruticosa. Nearly all the amino acids essential for substrate binding and catalysis as determined in the mammalian enzymes are represented in coral COX: the arachidonate-binding Arg(120) and Tyr(355) are present, as are the heme-coordinating His(207) and His(388); the catalytic Tyr(385); and the target of aspirin attack, Ser(530). A key amino acid that determines the sensitivity to selective COX-2 inhibitors (Ile(523) in COX-1 and Val(523) in COX-2) is present in coral COX as isoleucine. The conserved Glu(524), implicated in the binding of certain COX inhibitors, is represented as alanine. Expression of the G. fruticosa cDNA afforded a functional cyclooxygenase that converted exogenous arachidonic acid to prostaglandins. The biosynthesis was inhibited by indomethacin, whereas the selective COX-2 inhibitor nimesulide was ineffective. We conclude that the cyclooxygenase occurs widely in the animal kingdom and that vertebrate COX-1 and COX-2 are evolutionary derivatives of the invertebrate precursor.

Cell-specific modulation of papovavirus replication by tumor suppressor protein p53.

Small DNA tumor viruses like human papillomaviruses, simian virus 40, and adenoviruses modulate the activity of cellular tumor suppressor proteins p53 and/or pRB. These viruses replicate as nuclear multicopy extrachromosomal elements during the S phase of the cell cycle, and it has been suggested that inactivation of p53 and pRb is necessary for directing the cells to the S phase. Mouse polyomavirus (Py), however, modulates only the pRB protein activity without any obvious interference with the action of p53. We show here that Py replication was not suppressed by the p53 protein indeed in all tested different mouse cell lines. In addition, E1- and E2-dependent papillomavirus origin replication was insensitive to the action of p53 in mouse cells. We show that in hamster (Chinese hamster ovary) or human (osteosarcoma 143) cell lines the replication of both Py and papillomavirus origins was efficiently blocked by p53. The block of Py replication in human and hamster cells is not caused by the downregulation of large T-antigen expression. The deletion analysis of the p53 protein shows that the RPA binding, proline-rich regulatory, DNA-binding, and oligomerization domains are necessary for p53 action in both replication systems. These results indicate that in mouse cells the p53 protein could be inactive for the suppression of papovavirus replication.

Monitoring and purification of proteins using bovine papillomavirus E2 epitope tags.

We describe here the use of two newly mapped bovine papillomavirus type 1 (BPV-1) E2 protein epitopes as tags. We constructed several vector plasmids for overexpression as well as for moderate expression of single- or double-tagged proteins in either Escherichia coli or eukaryotic cells. The new tags were fused to several proteins, and the activity of the tagged proteins was tested in different assays. The tags were shown not to interfere with the function of these proteins in vivo and in vitro. Interaction of the monoclonal antibodies 3F12 and 1E2 with their respective epitopes was specific and had high affinity in a variety of conditions. We have demonstrated that the 3F12 antibody-epitope interaction tolerates high salt concentrations up to 2 M. This permits immunoprecipitation and immunopurification of the tagged proteins in high-salt buffers and reduction of the nonspecific binding of the contaminating proteins. We also provide a protocol for DNA binding and DNase I footprinting assays using the tagged, resin-bound DNA-binding proteins. The BPV-1 E2-derived tags can be recommended as useful tools for detection and purification of proteins.

Metronidazole and clarithromycin susceptibility and the subtypes of vacA of Helicobacter pylori isolates in Estonia.

The prevalence of metronidazole and clarithromycin resistance of Helicobacter pylori strains under different growth conditions (microaerophilic or anaerobic preincubation) was tested in 56 patients suffering from gastritis and peptic ulcer. vacA subtypes were detected in 46 H. pylori strains and were subsequently compared with the antibiotic resistance pattern. From 56 isolates, 26 proved resistant and 30 sensitive to metronidazole. The patients with peptic ulcer and gastritis were infected with both metronidazole-sensitive and metronidazole-resistant strains. In anaerobic preincubation all the strains were sensitive to metronidazole (MIC < 8 mg/l). All the strains were clarithromycin-sensitive (MIC < 2 mg/l). In the patients with gastritis and peptic ulcer s1 was the predominant vacA subtype. Comparison of vacA subtypes with the diagnoses revealed no correlation; different virulence factors such as vacA subtypes and antibiotic resistance to metronidazole in a microaerophilic milieu proved unrelated.

Inhibition of the bovine papillomavirus E2 protein activity by peptide nucleic acid.

The bovine papillomavirus type-1 E2 protein is the master regulator of the papillomavirus transcription and replication, the activity of which is regulated through sequence-specific DNA binding. Peptide nucleic acid (PNA) is a nucleic acid analogue, which associates with high affinity to complementary DNA, RNA or PNA, yielding in formation of stable complexes. The potential use of PNA as a sequence-specific inhibitor of the E2 protein activity is studied in this report. We demonstrate that replacement of one or both DNA strands with the complementary PNA reduced drastically the affinity of the BPV-1 E2 protein to its target site in the direct as well as in competitive binding as shown by in vitro gel-shift assays. We demonstrate that PNA could specifically bind to the double stranded E2 binding site by forming the complex with DNA oligonucleotide. In addition, PNA was able to bind specifically to the E2 binding site within the supercoiled plasmid DNA. Such binding of PNA to the E2 binding site within the origin of replication specifically abolished the activity of the E2 protein in the initiation of DNA replication in vivo.

Functional domains of the TOL plasmid transcription factor XylS.

The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.

Hepatitis C virus genotypes in Estonia.

Distribution of hepatitis C virus (HCV) geno(sub)types among 215 Estonian patients hospitalized with acute or chronic hepatitis and with HCV RNA-positive sera was investigated. For genotyping, both multiplex PCR with subtype-specific primers of the core region and RFLP analysis of cDNA of the 5' NCR region were used. These two methods permitted a correct characterization of genotypes, a more truthful characterization of mixed infections, and combined use of single-tube performances. They revealed, respectively, 200 and 202 (93.0% and 93.9%) HCV-positive samples of sera, subtype 1a- 0.9% and 0.9%, 1b- 56.3% and 64.2%, 3a- 13.9% and 22.3%, 2a- 6.5% and 5.6%, type 4 0.5% and 0%, mixed infections- 13.5% and 0%, and unidentified- 1.4% and 0.9%. In the majority of cases (84.7%) both methods gave completely or partially concordant results; in mixed infections, as determined by subtype-specific PCR, only one subtype was revealed by the RFLP method. In the remaining 15.3% of the cases (Ohno- 7.0%, RFLP- 8.3%) only one of the methods was positive. The epidemiological analysis of the dynamics of the subtypes' relative participation may indicate increasing 3a and decreasing 1b subtype infection during recent years.

Humoral and cellular immune responses to HIV-1 nef in mice DNA-immunised with non-replicating or self-replicating expression vectors.

OBJECTIVE: HIV accessory protein Nef is expressed early in the infectious cycle of the virus and has been shown to be an effective immunogen in humoral and cellular immune responses. We have used two different self-replicating pBN vectors and one non-replicating pCGal2 derived (pCG) vector expressing HIV-1 Nef in DNA immunisation of mice in order to determine their efficiency in raising humoral and cellular immune responses. DESIGN AND METHODS: The expression of Nef by the three plasmids was tested by transfections into COS-1 cells. Balb/c mice were immunised with the pBN-NEF and pCGE2-NEF constructs using gold particle bombardment. Immunoblotting and immunocytochemistry were used to detect in vitro expression of Nef. 51Cr release assay, ELISA and immunoblotting were used to detect cellular and humoral immune responses in immunised mice. RESULTS: Efficient in vitro expression of Nef was detected in pBN and pCGE2-NEF transfected cells, in pBN-NEF transfected cells the expression lasting up to three weeks. Anti-Nef antibodies in sera of 13 of 16 pBN-NEF immunised mice were detected within four weeks after the last immunisation, whereas only 2 of 12 pCGE2-NEF immunised mice had very weak anti-Nef antibodies. Twelve of the pBN-NEF immunised mice (75%) and 6 the pCGE2-NEF immunised mice (50%) showed Nef-specific cytotoxic T lymphocyte (CTL) responses within four weeks. CONCLUSIONS: We conclude that the three eukaryotic expression vectors tested are capable of inducing a cell mediated immune response towards HIV-1 Nef and should be considered as part of a genetic HIV vaccine.

Distribution of Acinetobacter baumannii in a neurointensive care unit.

In a 1-month prospective case-matched study we found Acinetobacter baumannii was a prevailing microbe simultaneously colonizing respiratory tract and skin of neurointensive care unit patients who stayed in our neurointensive care unit for more than 3 d. A. baumannii was not isolated from healthy case-matched controls. Based on their phenotypic properties and the results of amplified ribosomal DNA restriction analysis the 12 strains of Acinetobacter spp. isolated were identified as belonging to DNA group 2 (A. baumannii). For epidemiological typing, Biolog system results of 95-carbon source oxidation, antibiograms and restriction endonuclease analysis were used. One predominant A. baumannii strain was found in all colonized patients, skin and respiratory tract were found mainly to be colonized with the same strain. The starting point of A. baumannii colonization seemed to vary with the individual patient. Environmental strains were different from patients' strains: they were metabolically more active, more resistant and had a different restriction endonuclease analysis profile.

Effect of bovine papillomavirus E2 protein-specific monoclonal antibodies on papillomavirus DNA replication.

The bovine papillomavirus type 1 (BPV-1) E2 protein is the master regulator of papillomavirus replication and transcription. We have raised a panel of monoclonal antibodies (MAbs) against the BPV-1 E2 protein and used them to probe the structure and function of the protein. Five MAbs reacted with linear epitopes, and four MAbs recognized conformation-dependent epitopes which mapped within the C-terminal DNA-binding and dimerization domain. MAb 1E2 was able to recognize the replication- and transactivation-defective but not the competent conformation of the transactivation domain of the E2 protein. MAb 5H4 prevented efficiently the formation of E2-DNA as well as E2-dependent E1-E2-origin complexes and also dissociated preformed complexes in a concentration-dependent manner. Cotransfection of several MAbs with the BPV-1 minimal origin plasmid pUCAlu into CHO4.15 cells resulted in a dose-dependent inhibition of replication. Inhibition of replication by MAb 5H4 and the Fab' fragment of 5H4 correlated with their ability to dissociate the E2 protein from the DNA. MAb 3F12 and MAbs 1H10 and 1E4, directed against the hinge region, were also capable of inhibiting BPV-1 origin replication in CHO4.15 cells. However, the Fab' fragments of 1H10 and 3F12 had no effect in the transient replication assay. These data suggest that MAbs directed against the hinge region sterically hinder the inter- or intramolecular interactions required for the replication activity of the E2 protein.

Long-term episomal maintenance of bovine papillomavirus type 1 plasmids is determined by attachment to host chromosomes, which Is mediated by the viral E2 protein and its binding sites.

Papillomavirus genomes are stably maintained as extrachromosomal nuclear plasmids in dividing host cells. To address the mechanisms responsible for stable maintenance of virus, we examined nuclear compartmentalization of plasmids containing the full-length upstream regulatory region (URR) from the bovine papillomavirus type 1 (BPV1) genome. We found that these plasmids are tightly associated with the nuclear chromatin both in the stable cell lines that maintain episomal copies of the plasmids and in transiently transfected cells expressing the viral E1 and E2 proteins. Further analysis of viral factors revealed that the E2 protein in trans and its multiple binding sites in cis are both necessary and sufficient for the chromatin attachment of the plasmids. On the other hand, the BPV1 URR-dependent plasmid replication and chromatin attachment processes are clearly independent of each other. The ability of the plasmids to stably maintain episomes correlates clearly with their chromatin association function. These data suggest that viral E2 protein-mediated attachment of BPV1 genomes to the host cell chromatin could provide a mechanism for the coupling of viral genome multiplication and partitioning to the host cell cycle during viral latent infection.

Human papillomavirus type 18 E1 protein is translated from polycistronic mRNA by a discontinuous scanning mechanism.

Papillomaviruses are small double-stranded DNA viruses that replicate episomally in the nuclei of infected cells. The full-length E1 protein of papillomaviruses is required for the replication of viral DNA. The viral mRNA from which the human papillomavirus type 18 E1 protein is expressed is not known. We demonstrate that in eukaryotic cells, the E1 protein is expressed from polycistronic mRNA containing E6, E7, and E1 open reading frames (ORFs). The translation of adjacent E7 and E1 ORFs is not associated; it is performed by separate populations of ribosomes. The translation of the downstream E1 gene is preceded by ribosome scanning. Scanning happens at least at the 5' end of the polycistronic mRNA and also approximately 100 bp in front of the E1 gene. Long areas in middle of the mRNA are bypassed by ribosomes, possibly by ribosomal "shunting." Inactivation of short minicistrons in the upstream area of the E1 gene did not change the expression level of the E1 gene.

p53 protein is a suppressor of papillomavirus DNA amplificational replication.

p53 protein was able to block human and bovine papillomavirus DNA amplificational replication while not interfering with Epstein-Barr virus oriP once-per-cell cycle replication. Oligomerization, intact DNA-binding, replication protein A-binding, and proline-rich domains of the p53 protein were essential for efficient inhibition, while the N-terminal transcriptional activation and C-terminal regulatory domains were dispensable for the suppressor activity of the p53 protein. The inhibition of replication was caused neither by the downregulation of expression of the E1 and E2 proteins nor by cell cycle block or apoptosis. Our data suggest that the intrinsic activity of p53 to suppress amplificational replication of the papillomavirus origin may have an important role in the virus life cycle and in virus-cell interactions.

Opioid antagonist naloxone potentiates anxiogenic-like action of cholecystokinin agonists in elevated plus-maze.

This study investigated the interplay of cholecystokinin (CCK) and endogenous opioid peptides in the regulation of anxiety. The acute administration of non-selective CCK agonist caerulein (1 and 5 microg/kg) and a selective CCK(B) receptor agonist BOC-CCK-4 (1, 10 and 50 microg/kg) induced a dose-dependent anxiogenic-like action in the plus-maze model of anxiety. BOC-CCK-4 displayed a similar efficacy with caerulein, indicating that the described effect was mediated via CCK(B) receptor subtype. The opioid antagonist naloxone itself (0.5 mg/kg) did not change the exploratory activity of rats in the plus-maze. However, the combination of naloxone with the sub-effective doses of caerulein (1 microg/kg) and BOC-CCK-4 (1 microg/kg) induced a significant inhibition of exploratory behaviour in rats. Accordingly, CCK and endogenous opioid peptides have an antagonistic role in the exploratory model of anxiety in rats.