Enzymatic characteristics of recombinant medium isozyme of 2'-5' oligoadenylate synthetase

P69 is an isozyme of the medium size class of human 2'-5' oligoadenylate synthetases. In this study, recombinant P69 was expressed and used for enzymological and structural investigations. Bacterially expressed P69 was inactive whereas the same protein expressed in insect cells was highly active. Whether this difference could be due to differential post-translational modifications of the protein was investigated. Mutations of appropriate residues showed that myristoylation of the protein was not necessary for enzyme activity. In contrast, inhibition of glycosylation of P69, by tunicamycin treatment of the insect cells, produced an enzymatically inactive protein. Recombinant P69 produced in insect cells was purified by affinity chromatography. It was a dimeric glycoprotein, very stable and completely dependent on double stranded (ds) RNA for activity. The enzyme catalyzed the non-processive synthesis of 2'-5'-linked oligoadenylate products containing up to 30 residues. 2'-O-Methylated dsRNA was incapable of activating P69 and a 25-base pair dsRNA was as effective as larger dsRNA. This expression system will be useful for large scale production of P69 and its mutants for structural studies.     

p59OASL, a 2'-5' oligoadenylate synthetase like protein: a novel human gene related to the 2'-5' oligoadenylate synthetase family

The 2'-5' oligoadenylate synthetases form a well conserved family of interferon induced proteins, presumably present throughout the mammalian class. Using the Expressed Sequence Tag databases, we have identified a novel member of this family. This protein, which we named p59 2'-5' oligoadenylate synthetase-like protein (p59OASL), shares a highly conserved N-terminal domain with the known forms of 2'-5' oligoadenylate synthetases, but differs completely in its C-terminal part. The C-terminus of p59OASL is formed of two domains of ubiquitin-like sequences. Here we present the characterisation of a full-length cDNA clone, the genomic sequence and the expression pattern of this gene. We have addressed the evolution of the 2'-5' oligoadenylate synthetase gene family, in the light of both this new member and new 2'-5' oligoadenylate synthetase sequence data from other species, which have recently appeared in the databases.     

Activation of 2'-5' oligoadenylate synthetase by single-stranded and double-stranded RNA aptamers

A number of small RNA molecules that are high affinity ligands for the 46-kDa form of human 2'-5' oligoadenylate synthetase have been identified by the SELEX method. Surface plasmon resonance analysis indicates that these RNAs bind to the enzyme with dissociation constants in the nanomolar range. Competition experiments indicate that the binding site for the small RNAs on the 2'-5' oligoadenylate synthetase molecule at least partially overlaps that for the synthetic double-stranded RNA, poly(I).poly(C). Several of the RNAs function as potent activators of 2'-5' oligoadenylate synthetase in vitro, although there is no correlation between binding affinity and ability to activate. The RNA aptamers having the strongest activation potential appear to have few base-paired regions. This suggests that 2'-5' oligoadenylate synthetase, which has previously been believed to be activated only by double-stranded RNA, can also be activated by RNA ligands with little secondary structure. Since 2'-5' oligoadenylate synthetase possesses no homology to other known RNA-binding proteins, the development of small specific ligands by SELEX should facilitate studies of RNA-protein interactions and may reveal novel features of the structure-function relationships involving this enzyme.     

Virus-induced cell death in plants expressing the mammalian 2',5' oligoadenylate system

The major components of the 2-5A system, responsible for the mammalian interferon-induced antiviral response, are the 2',5' oligoadenylate synthetase (2-5Aase) and 2',5' oligoadenylate (2-5A) dependent ribonuclease (RNase L). Transgenic tobacco plants expressing these two enzyme activities were produced by crossing the transgenic plants expressing RNase L with those expressing 2-5Aase. The double transgenic plants showed complete resistance against cucumber mosaic virus (CMV), infection with necrotic spots only forming on the virus-inoculated leaf. On the other hand, although plants inoculated with potato virus Y (PVY) formed necrotic spots on the inoculated leaf and virus amplification could not be detected, all plants died within 20 days of inoculation. The transgenic tobacco plants expressing either 2-5Aase or RNase L activity showed typical disease symptoms with CMV- or PVY-inoculation. These results suggest that the introduced 2-5A system is activated in tobacco cells by dsRNA, the replicating intermediates of RNA viruses, leading to death of the host cells, which has not been observed in mammalian cells.     

Production of recombinant goldfish growth hormone I in a baculovirus expression system

The authors studied the effect of a positioning orifice introduced in the anterior palatine region of occlusal splints for patients with craniomandibular disorders of swallowing and speech patterns. The patients were evaluated in four distinct situations. It was concluded that the splint orifice significantly favored swallowing and speech while the splint was being used, and is more comfortable for the patient.     

Production of recombinant porcine tumor necrosis factor alpha in a baculovirus expression system

PURPOSE: Stress is often noted by patients to be a precipitating factor in causing seizures. No precise data are, however, available. In 1995 for 250,000 inhabitants in The Netherlands, a serious life event occurred within a period of seven days. An extreme high water level in the province of Gelderland, with the possibility of a flood, made the government decide to evacuate people and their livestock. This retrospective study investigated the influence of this forced evacuation on the seizure frequency of patients with epilepsy, compared with patients of the same age and type of epilepsy living outside the evacuation area at the time of the threatening flood. METHODS: Information regarding epilepsy syndrome, seizure type, and frequency was derived from seizure diaries and medical histories of 30 evacuated patients and 30 matched control patients. RESULTS: Of the 30 evacuees, eight showed an increase and one a decrease in seizure frequency during or shortly after the evacuation period, compared with one and zero control patients, respectively. These results proved to be statistically significant (p < 0.05). CONCLUSIONS: Our data support the hypothesis that there is a relation, albeit small, between a stressful life event and seizure frequency.     

Identification of the reaction products of (2''-5'')oligoadenylate synthetase in the marine sponge

A new type of affinity cross-linking strategy has been developed in which His6-tagged proteins can be cross-linked to their binding partners in the presence of unmodified proteins (D. Fancy, K. Melcher, S. A. Johnston, and T. Kodadek, 1996, Chem. Biol. 3, 551-559). The chemistry involves the addition of Ni(II) to the His6 tag, followed by oxidation of the metal with a peracid. It is shown here that, in addition to the His6 tag, a tyrosine residue placed in close proximity to the metal-binding site can strongly stimulate the yield of cross-linked product. This finding has important practical implications in the use of the His6-Ni-based cross-linking reaction for the analysis of multiprotein complexes.     

The effect of (2'-5') and (3'-5') phosphodiester linkages on conformational and stacking properties of cytidylyl-cytidine in aqueous solution

Conformational properties of (2'-5') and (3'-5') CpC have been determined by proton magnetic resonance spectroscopy at 220 MHz. The ribose ring structures are predominantly 3E with the exception of the ring from the 2'-phosphate fragment of C(2'-5')pC which exhibits an 2E pucker. Bases are oriented anti with respect to the ribose and the conformations about C4'-C5', C5'-O5', C3'-O3' (C2'-O2') are gg, g'g', and g+ in equilibrium g-, respectively. The dimers exist as mixtures of stacked (g+g+ and g-g- about the P-O(C) bonds) and unstacked species at 20 degrees C. Stacking is estimated to be 35% in both dimers.     

Production and purification of active FGF2 via recombinant fusion protein

Basic fibroblast growth factor (FGF2) is involved in both cell proliferation and differentiation processes. Heparin may interfere in the stability and biological activities of FGFs. However, it is difficult to obtain FGF preparation without traces of heparin since heparin affinity chromatographies are routinely used to prepare this growth factor. We have therefore devised a means of production of active recombinant FGF2 devoid of heparin traces. The bovine FGF2 gene was inserted into the pMAL-c prokaryotic expression vector and the recombinant protein was synthesised as a fusion product between the maltose binding protein (MBP) and FGF2. Purification of the FGF2 fusion protein was performed by an amylose affinity chromatography. Yields were similar to those obtained by using a traditional heparin affinity column purification procedure. The fusion protein (MBP-FGF2) and the cleaved-off FGF2 were tested for some of their biological properties and compared to recombinant FGF2 purified by heparin affinity chromatography. Mitogenic activity on Chinese hamster lung fibroblasts (CCL39) and neurite outgrowth on pheochromocytoma culture cells (PC12) were used as biological assays. The cleaved-off FGF2 was as active as commercially available recombinant FGF2 (ED50 at 0.16 and 0.04 nM respectively). However MBP-FGF2 was less active (ED50 at 0.9 nM) in both tests.     

Effects of mutating specific residues present near the amino terminus of 2'-5'-oligoadenylate synthetase

In this study, we investigated the role of specific amino acid residues present near the amino terminus of the 9-2 isozyme of 2'-5'-oligoadenylate synthetase. In vitro expression of deletion mutants showed that residues 1-9 are required for enzyme activity. Within this region, residues 3, 7, and 8 were found to be conserved among all known isozymes of 2'-5'-oligoadenylate synthetase. Mutation of these residues singly or in combination resulted in partial or total loss of enzyme activity. Substitution of the proline residue at position 7 by different residues caused a partial or complete loss of activity. The properties of the inactive P7Q mutant were further explored by expressing the protein in bacteria. The bacterially expressed protein was also enzymatically inactive. The mutant protein could bind the substrate ATP and the activator double-stranded RNA normally. Oligomerization properties of the protein were examined by an affinity-based interaction assay and by glycerol gradient centrifugation; there was no detectable difference between the wild type and the P7Q mutant. These results demonstrated the importance of the proline residue at position 7 in conferring enzyme activity to the protein without affecting its other properties.     

Molecular cloning and expression of cynomolgus monkey interleukin-2 cDNA by the recombinant baculovirus system

Mice inoculated at 5, 21 and 28 days of age with 10(6) or 10(7) Cryptosporidium parvum oocysts became infected but did not exhibit any clinical signs of disease. Specific IgA antibodies were detected in faecal extracts from all infected mice by an indirect immunofluorescent assay. These antibodies first appeared between 11 and 37 days post-infection (dpi) and persisted until the end of the experiment at 55 dpl. They appeared earlier in older mice than in newborn mice. Reduction and resolution of oocyst shedding was not directly related, however, to IgA antibody levels in infected mice. Reactive C. parvum antigens were identified by immunoblotting techniques using faecal and serum samples from infected mice. IgA copro-antibodies reacted specifically with two antigens of 26 and 33 kDa, which were also identified by IgG antibodies in mouse serum. The role of these antibodies in the resolution of infections and the subsequent protection against challenge is unknown.     

Active recombinant rat dipeptidyl aminopeptidase I (cathepsin C) produced using the baculovirus expression system

An active form of rat dipeptidyl aminopeptidase I (DPPI, cathepsin C) was obtained by heterologous expression in insect cells. Baculoviruses carrying a cDNA sequence encoding the entire rat DPPI precursor was used to infect High Five cells in a serum-free medium. Recombinant DPPI (rDPPI) was secreted into the medium from which it was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography (HIC), and ion-exchange chromatography. A polyhistidine-tagged form of the enzyme (HT-rDPPI) was purified from the medium by immobilized metal affinity chromatography (IMAC). In vivo activation of native rat DPPI involves at least three chain cleavages per subunit and the ability of the expression system to imitate this processing was investigated. Both rDPPI and HT-rDPPI were secreted into the medium as unprocessed and inactive proenzymes and gradually converted into their active forms in the medium. This process was not completed at the time of harvest but mature enzyme processed similarly to native rat and human DPPI could be obtained by incubating the eluates from the HIC and IMAC columns at pH 4.5 and 5 degrees C for 18-40 h. The yield of purified and matured enzyme was approximately 50 mg/liter, and it was shown that rDPPI and HT-rDPPI were active against both a dipeptide-p-nitroanilide substrate and human growth hormone N-terminally extended with an Ala-Glu dipeptide.     

Oligo-2',5'-adenylate synthetase in pulmonary sarcoidosis and idiopathic pulmonary fibrosis

Oligo-2',5'-adenylate synthetase (2,5AS) is an enzyme induced by all types of interferon (IFN). We measured the levels of 2,5AS activity in peripheral blood mononuclear leukocytes (PBML) and bronchoalveolar lavage fluid (BALF) cells of patients with pulmonary sarcoidosis (SAR), idiopathic pulmonary fibrosis (IPF), and normal controls (NC). In NC, the levels of BALF cell 2,5AS activity were approximately seven times as high as the levels of PBML 2,5AS activity. The measurement 2,5AS activity from isolated cells showed that the levels of 2,5AS activity are independent of cell differential from PBML and BALF cells. The levels of PBML and BALF cell 2,5AS activity in SAR were both significantly high in comparison with those in NC. In patients with IPF, the levels of PBML 2,5AS activity were significantly increased as compared with those in NC, whereas there was no significant difference regarding the levels of BALF cell 2,5AS activity between patients with IPF and NC. These results suggest the following: (1) in patients with SAR, IFN production is enhanced both in the alveolar space and peripheral circulation; (2) in patients with IPF, IFN production is greatly enhanced in the circulation, whereas IFN production is not enhanced in the alveolar space; and (3) IFN may contribute to the pathogenesis of SAR and IPF.     

In vivo and in vitro analysis of baculovirus ie-2 mutants

Upon transient expression in cell culture, the ie-2 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) displays three functions: trans activation of viral promoters, direct or indirect stimulation of virus origin-specific DNA replication, and arrest of the cell cycle. The ability of IE2 to trans stimulate DNA replication and coupled late gene expression is observed in a cell line derived from Spodoptera frugiperda but not in a cell line derived from Trichoplusia ni. This finding suggested that IE-2 may exert cell line-specific or host-specific effects. To examine the role of ie-2 in the context of infection and its possible influence on the host range, we constructed recombinants of AcMNPV containing deletions of different functional regions within ie-2 and characterized them in cell lines and larvae of S. frugiperda and T. ni. The ie-2 mutant viruses exhibited delays in viral DNA synthesis, late gene expression, budded virus production, and occlusion body formation in SF-21 cells but not in TN-5B1-4 cells. In TN-5B1-4 cells, the ie-2 mutants produced more budded virus and fewer occlusion bodies but the infection proceeded without delay. Examination of the effects of ie-2 and the respective mutants on immediate-early viral promoters in transient expression assays revealed striking differences in the relative levels of expression and differences in responses to ie-2 and its mutant forms in different cell lines. In T. ni and S. frugiperda larvae, the infectivities of the occluded form of ie-2 mutant viruses by the normal oral route of infection was 100- and 1,000-fold lower, respectively, than that of wild-type AcMNPV. The reduction in oral infectivity was traced to the absence of virions within the occlusion bodies. The infectivity of the budded form of ie-2 mutants by hemocoelic injection was similar to that of wild-type virus in both species. Thus, ie-2 mutants are viable but exhibit cell line-specific effects on temporal regulation of the infection process. Due to its effect on virion occlusion, mutants of IE-2 were essentially noninfectious by the normal route of infection in both species tested. However, since budded viruses exhibited normal infectivity upon hemocoelic injection, we conclude that ie-2 does not affect host range per se. The possibility that IE-2 exerts tissue-specific effects has not been ruled out.     

Enzymatic activity of 2'-5'-oligoadenylate synthetase is impaired by specific mutations that affect oligomerization of the protein

Previous studies from our laboratory have shown that deletion of residues 321 to 344 of the 9-2 isozyme of 2'-5'-oligoadenylate (2-5(A)) synthetase causes a loss of its enzyme activity (Ghosh, S. K., Kusari, J., Bandyopadhyay, S. K., Samanta, H., Kumar, R., and Sen, G. C. (1991) J. Biol. Chem. 266, 15293-15299). Sequence comparison of this region among the different isozymes of 2-5(A) synthetases revealed that the residues at positions 330 to 333 are highly conserved. Alanine-scanning mutagenesis of these residues demonstrated that the residues present at 331, 332, and 333 are important for activity but the proline at position 330 was dispensable. The triple mutant containing Ala residues at 331, 332, and 333 was completely inactive. Different double mutants were slightly active, and the three single mutants were partially active. The triple mutant was further characterized for delineating the nature of its defect. The mutant protein was enzymatically inactive irrespective of whether it was synthesized in rabbit reticulocyte lysate, Escherichia coli or Trichoplusia ni insect cells. It could bind double-stranded RNA and ATP as efficiently as the wild type protein. It was, however, defective in oligomerization. Gel filtration and sedimentation velocity analyses of in vitro synthesized proteins revealed that the wild type protein, but not the triple mutant, formed tetramers. The tetrameric fraction, but not the monomeric fraction of the wild type protein was enzymatically active. The failure of the triple mutant to participate in homomeric protein-protein interaction was confirmed by in vivo assays in insect cells. These results indicate that tetramerization of the protein is required for the enzymatic activity of the small 2-5(A) synthetases.     

Activity of the interferon-induced 2',5'-oligoadenylate synthetase in patients with chronic hepatitis C

The interferon-induced 2',5'-oligoadenylate synthetase (2-5OAS) is responsible, at least in part, for the antiviral state established in cells in response to viral infections. The purpose of this work was to study the relationship between hepatitis C virus (HCV) infection and 2-5OAS in patients with chronic hepatitis C. Peripheral blood mononuclear cells (PBMC) of 27 patients with chronic hepatitis C were investigated, as well as PBMC of 10 control subjects. Then, the patients were treated with 3 mu interferon-alpha 2a three times per week. At month 3 of therapy, PBMC were sampled. Of the total PBMC samples obtained, half were used for determination of in vivo 2-5OAS activity. The remaining cells were cultured for 24 h in either the absence or presence of 500 U/ml of interferon-alpha 2a for the determination of in vitro 2-5OAS activity. The mean basal in vivo 2-5OAS activities were 3.6 +/- 2.8 nmol/10(6) cells in patients versus 1.6 +/- 1.1 nmol/10(6) cells in controls (p < 0.01). Basal in vivo 2-5OAS activity did not correlate with mean HCV viremia, quantified by a "branched DNA"-based assay. Before treatment, interferon-alpha was detected in the serum of 2 patients in 27. After a 24 h culture of PBMC in the presence of interferon, in vitro 2-5OAS activity was significantly induced in the PBMC of both the patients and the controls. However, in vitro induction of 2-5OAS activity was significantly lower in the PBMC of the patients than in the PBMC of the controls (p < 0.01). At month 3 therapy, in vivo 2-5OAS activity was significantly induced (20.5 +/- 17.9; p < 0.0001). In vitro IFN inductions of 2-5OAS activity in PBMC before treatment and at month 3 of therapy were not significantly different. In conclusion, in vivo 2-5OAS activity is significantly induced in patients with chronic hepatitis C, but endogenously produced interferon-alpha does not seem to be involved. Chronic induction of 2-5OAS activity results in a decreased sensitivity of PBMC to exogenous interferon induction. Whether this phenomenon plays a role in the resistance of chronic hepatitis C to interferon therapy remains uncertain.     

The morphology of the polyhedra of a host range-expanded recombinant baculovirus and its parents

The host range-expanded recombinant baculovirus, RecB-8 was isolated from BmN-4 cells coinfected with Autographa californica and Bombyx mori nuclear polyhedrosis viruses. Its genome was compared with those of its parents by restriction endonuclease digestion and their polyhedra compared in an electron microscope. Interestingly, the polyhedra of RecB-8 were tetrahedral although the polyhedrin gene was the same as that of the BmNPV parent which has icosahedral polyhedra. Thus the morphology of the RecB-8 polyhedra resulted from host cell factors and/or another viral genome in the host cells.     

Molecular cloning and characterization of two related and interferon-induced 56-kDa and 30-kDa proteins highly similar to 2''-5'' oligoadenylate synthetase

The capability of subjects to categorize (as short or long) visually marked empty time intervals was investigated in three experiments. Two visual signals, located 18 degrees to the left (L) and to the right (R) of a fixation point in the visual field, established four marking conditions, two unilaterally presented (L-L and R-R) and two bilaterally presented (L-R and R-L). In Experiments 1 and 2, the results show that discrimination is better with unilateral sequences than with bilateral sequences and that the perceived duration is longer with an L-R than with an R-L sequence. In addition, Experiment 2 shows that, in comparison with a condition in which Markers 1 and 2 remain identical for a complete session, varying the markers from trial to trial does not decrease discrimination. Also, Experiment 2 shows that discrimination is better when both visual markers are presented at fovea than it is in the unilateral conditions. Experiment 3 shows that bilateral intervals are perceived as being longer and are better discriminated than are intervals marked by an intermodal sequence (auditory-visual or visual-auditory). The general discussion reports the implications of having different perceived duration and sensitivity levels, in various marker-type conditions, for an internal-clock hypothesis. Some implications of these results for a lateralized-timer hypothesis are also discussed.     

Non-enzymatic, template-directed ligation of 2'-5' oligoribonucleotides. Joining of a template and a ligator strand

Decauridylate containing exclusively a 2'-5' phospho-diester bond ([2'-5']U10) served as a template for the synthesis of oligoadenylates [oligo(A)s] from the 5'-phosphorimidazolide of 2'-5' diadenylate (ImpA-2'p5'A). Joining of [2'-5']U10and ImpA2'p5'A also took place in substantial amounts to yield long-chain oligoribonucleotides in the template-directed reaction. An unusual CD spectrum ascribed to helix formation between [2'-5']U10and [2'-5'](pA)2was observed under the same conditions as that of the template-directed reaction. The 3'-5' linked decauridylate ([3'-5']U10) also promoted the template-directed synthesis of oligo(A)s from ImpA2'p5'A, but more slowly compared with [2'-5']U10. The results indicate that short-chain RNA oligomers with a 2'-5' phosphodiester bond could lead to longer oligoribonucleotides by template-directed chain elongation.     

A 5'-3' exonuclease activity involved in forming the 3' products of histone pre-mRNA processing in vitro

Histone RNA 3' processing in vitro produces one or more 5' cleavage products corresponding to the mature histone mRNA 3' end, and a group of 3' cleavage products whose 5' ends are mostly located several nucleotides downstream of the mRNA 3' end. The formation of these 3' products is coupled to the formation of 5' products and dependent on the U7 snRNP and a heat-labile processing factor. These short 3' products therefore are a true and general feature of the processing reaction. Identical 3' products are also formed from a model RNA containing all spacer nucleotides downstream of the mature mRNA 3' end, but no sequences from the mature mRNA. Again, this reaction is dependent on both the U7 snRNP and a heat-labile factor. Unlike the processing with a full-length histone pre-mRNA, this reaction produces only 3' but no 5' fragments. In addition, product formation is inhibited by addition of cap structures at the model RNA 5' end, indicating that product formation occurs by 5'-3' exonucleolytic degradation. This degradation of a model 3' product by a 5'-3' exonuclease suggests a mechanism for the release of the U7 snRNP after processing by shortening the cut-off histone spacer sequences base paired to U7 RNA.