Biomedical subjects
Publications and source records attributed to J Imai.
Allyl group serves as a useful protecting group for an protection of sugar hydroxyls and amino and imide moieties of by brief treatment with a palladium catalyst and a variety of nucleophiles at room temperature.
Explore the source record for details and available documents.
In vitro studies have shown that the translational inhibitory activity of 2-5 A can be blocked by the oligoribonucleotide 2',5'-(pA)3. We have examined the effect of simultaneous introduction of inhibitor and antagonist into intact mouse cells using calcium phosphate coprecipitation. Upon introduction of 10(-4) M 2',5'-(pA)3 and 10(-6) M 2-5 A, inhibition of protein synthesis was prevented. Efficiency of calcium phosphate precipitation of 2-5 A in the presence or absence of 2',5'-(pA)3 was comparable. Introduction of 2',5'-(pA)3 analogs showed that nucleotides which do not bind well to the 2-5 A dependent endonuclease do not prevent 2-5 A inhibitory activity. Thus, 2',5'-(pA)3 functions as an antagonist of 2-5 A in vivo.
To define the epitopes involved in binding anti-oligonucleotide antibodies, several hybridomas producing monoclonal antibodies directed against 2',5'-oligoadenylate were established. A solid-phase enzyme-linked immunoassay that employed microtiter wells coated with Ficoll-2',5'-oligoadenylate conjugates proved useful in screening and characterizing hybridoma supernatants. Control experiments demonstrated that the conjugates were irreversibly adsorbed to polystyrene wells under the conditions employed in the assay. Reactivity of monoclonal antibodies with numerous analogues of 2',5'-oligoadenylate was measured by using a competition assay. Several monoclonal antibodies originating from different mice immunized with the same or different immunogens possessed distinctive fine specificities. At least one 2',5'-phosphodiester bond was important in forming each epitope, suggesting that the ribose phosphate backbone is a critical element in defining an antigenic domain of an oligonucleotide. The purine bases were also important, and modification of the bases had varied effects on the extent of antibody recognition. The length of the oligonucleotide and the nature of the termini were also of some importance. In several instances the modification created by linkage of 2',5'-oligoadenylate to carrier protein also contributed to the determinant. The monoclonal antibody most specific for 2',5'-oligoadenylates was relatively insensitive to ionic strength. In contrast, a monoclonal antibody with a 2',5'-oligopurine specificity appeared to bind 2',5'-oligoadenylate through one ion pair, whereas the binding of a monoclonal antibody with a low degree of base specificity appeared to bind through two ion pairs. The results demonstrated that 2',5'-linked oligoadenylate-protein complexes possess at least three distinct oligonucleotide-related antigenic surfaces that can be recognized with high apparent affinity by monoclonal antibodies. A model for the three epitopes is presented.
Analogs of the triphosphate 2'-5'-linked adenylate trimer (ppp5'A2'p5'A2'p5'A, called 2-5A) which contain 3'-deoxyadenosine (cordycepin) instead of adenosine either in positions one and two, or in all three positions, are 10-100-fold less potent than is parent 2-5A in inhibition of protein synthesis in intact cells, when utilizing calcium co-precipitation techniques to introduce the 5'-triphosphate oligonucleotides into the cells. That the inhibition of protein synthesis was a consequence of activation of the 2-5A-dependent endonuclease by the 3'-deoxyadenosine analogs of 2-5A was demonstrated in obtaining the ribosomal RNA cleavage pattern that is characteristic of endonuclease activation by parent 2-5A. Additional results (i.e. lack of activity by the dimer species ppp5'(3'dA)2'p5'-(3'dA) or the monomer 3'dA) as well as kinetic analysis both in intact cells and in cell-free extracts provided further evidence that the inhibition of protein synthesis observed with these 3'-deoxyadenosine 2-5A analogs was not due to their degradation to the antimetabolite monomer unit 3'-deoxyadenosine.
We have synthesized a series of 2-5A (ppp5'-A2'p5'A2'p5'A) analogs in which each adenosine residue has been sequentially replaced by inosine: viz., ppp5'I2'p5'A2'p5'A, ppp5'A2'p5'I2'p5'A, and ppp5'A2'p5'A2'p5'I. These transformations enabled us to delineate the role of each of the three purine N-6 amino groups of 2-5A in determining oligonucleotide binding to and activation of the 2-5A-dependent endoribonuclease, RNase L. With the RNase L activity of both mouse L cells and human Daudi lymphoblastoid cells, we found that the N-6 amino group of the first adenosine nucleotide residue (from the 5'-terminus) is of crucial importance in determining binding to the endonuclease; however, removal of the N-6 amino moieties of the second or third adenosine nucleotide residues resulted in only a minimal decrease in binding to the endonuclease. On the other hand, conversion of the third adenosine residue to inosine effected a dramatic (10,000-fold compared to 2-5A) loss in ability to activate the nuclease; however, execution of the same N-6 amino group conversion at either the first or second adenosine residue did not cause a major change in nuclease activation ability when the accompanying decreased endonuclease binding was considered. These results clearly demonstrate that the N-6 amino group of the first adenosine residue of 2-5A is critical in RNase L binding whereas the N-6 amino function of the third adenosine residue of 2-5A is crucial for the activation of RNase L.
The ribofuranose oxygens of the three adenosine residues of the 5'-monophosphate of the 2-5A core [adenylyl(2'----5')adenylyl(2'----5')adenosine] were replaced by methylenes through the synthesis of an aristeromycin [9-[(1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl]adenosine] analogue. In the synthetic approach, the chlorophosphite triester procedure was employed together with the use of dimethoxytrityl and tert-butyldimethylsilyl protecting groups. The final product 14 was bound to the 2-5A-dependent endonuclease of mouse, rabbit, or human cells 100-300 times less effectively than parent p5'A2'p5'A2'p5'A. In extracts of human Daudi cells where the monophosphate p5'A2'p5'A2'p5'A was able to effect ribosomal RNA cleavage at 2 X 10(-7) M, 14 required a concentration of 2 X 10(-5) M to bring about discernible rRNA cleavage.
Monoclonal antibodies directed against 2-5A were developed and characterized. 2',5'-Oligoadenylate-protein complexes possess at least three distinct antigenic surfaces defined primarily by the ribose-phosphate backbone. A schematic model for the three epitopes is presented. Antibodies directed against 2-5A, in conjunction with other techniques, were employed to quantify 2-5A in various tissues of pathogen-free mice. Levels of 2-5A were in the range of 400-800 fmole/gm. Mice injected with poly(I).poly(c) or encephalomyocarditis virus (EMCV) showed elevated levels of 2-5A. Administration of poly(I).poly(C) or EMCV increased the level of 2-5A in different tissues to different extents. Raman spectroscopy was employed to explore the conformation of 2-5A. Distinct differences were observed in bands arising from the backbone portion of 2-5A relative to those of 3-5A. The most striking finding was the appearance of a strong, sharp band at 1460 cm-1 in spectra of 5'-monophosphorylated 2-5A's. This band was barely detectable in core and triphosphorylated 2-5A. Its presence suggests that 5'-monophosphorylated 2-5A's possess a unique conformational feature that distinguish them from cores and 5'-triphosphorylated forms.
A survey of carriers of adult T-cell leukemia virus (ATLV) detected as anti-ATLV associated antigens was made in Taiwan. Among 2545 adults aged 30 years or more examined, seropositive donors amounted to 0.9% in the Han Chinese but none in the aborigines.
Explore the source record for details and available documents.
Recently developed chemical synthetic methodology for facile preparation of sequence-specific 2'5'-oligonucleotides has allowed more exacting questions to be asked regarding the biological role of each of the nucleotide residues of 2-5A. We have found, employing analogs in which each adenosine residue has been sequentially replaced by adenosine (viz, ppp5'I2'p5'A2'p5'I, ppp5'A2'p5'I2'p5'A, ppp5'A2'p5'A2'p5'I) that the N6 amino group of the first (or 5'-terminal) adenosine residue of 2-5A trimer is critical in RNase L binding whereas the N6 amino moiety of the third (or 2'-terminal) adenosine residue of 2-5A is crucial for the activation of RNase L. The second or middle adenosine unit of 2-5A does not seem to be critical for either binding or activation. Similarly, in studies on sequence-specific tubercidin analogs of 2-5A, activation of but not binding to mouse RNase L was dependent on the presence of the purine N7 atoms of the first and third adenosine residues of 2-5A, but, as with the N6 amino group, the N7 moiety of the second adenosine residue of 2-5A was not essential for either binding to or activation of the 2-5A-dependent endonuclease. Finally, sequence-specific purine 8-bromination provided analogs of dramatically varying biological properties, and provided a 5'-monophosphate, p5'A2'p5'(br8A)2'p5'(br8A), which possessed 8% of the translational inhibitory action of 2-5A itself. This latter result may represent an important impetus toward obtaining a 2-5A derivative with biological activity in an intact cell.
The survey on soil-transmitted helminthiasis was carried out in the three villages of Asahan Regency, North Sumatra, Indonesia, between October 1981 and December 1982. The prevalence rates of geohelminthiasis proved to be extremely high (average 97%) in the three villages and more than 70% harboured three or more helminths, especially Ascaris lumbricoides, Trichuris trichiura and hookworm. Necator americanus and Ancylostoma duodenale were detected in 61.1% and 52.2% of 835 fecal specimens, respectively. The effects of two anthelmintics, Combantrin and Trivexan, were essentially the same against A. lumbricoides and hookworm, although Trivexan was better than Combantrin against T. trichiura. The kinetic changes of parasitic infection in community after mass treatment were examined. Ten months after the first drug treatment, the incidence of Ascariasis or trichuriasis reverted nearly to pre-treatment level, while hookworm infection rate remained significantly low. Four months after the second mass treatment, the reinfection rate of A. lumbricoides was most prevalent followed by trichuriasis and hookworm infection. The reinfection rate (63.6%) of A. lumbricoides in children was about six times higher than that (10.4%) in adults at four month after the mass treatment. This study indicates that mass treatment with Trivexan at two month intervals for children and four month intervals for adults is necessary for the effective helminth control scheme in highly endemic areas of North Sumatra.
A series of tubercidin (7-deazaadenosine) analogues of 2-5A of the general formula p5'(c7A)2'p[5'(c7A)-2'p]n5'(c7A) (n = 0-5) were prepared by lead ion catalyzed polymerization of the 5'-phosphoroimidazolidate of tubercidin. Through the corresponding imidazolidates, these oligonucleotide 5'-monophosphates were converted to the 5'-triphosphates. All reported structures were corroborated by enzyme digestion and 1H or 31P nuclear magnetic resonance. When evaluated for its ability to bind to the 2-5 A-dependent endonuclease of mouse L cells, the tubercidin analogue of trimeric 2-5A, namely, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), and the corresponding tetramer were bound as effectively as 2-5A itself; nonetheless, it and the corresponding tetramer, ppp5'-(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), failed to stimulate the 2-5A-dependent endonuclease as judged by its inability to inhibit translation in extracts of mouse L cells programmed with encephalomyocarditis virus RNA and to give rise to ribosomal RNA cleavage in the same cell system under conditions where 2-5A showed activity at 10(-9) M. The trimer, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), was an antagonist of 2-5A action in the L cell extract. In the lysed rabbit reticulocyte system, both the trimeric and tetrameric tubercidin 2-5A analogues were bound to the 2-5A-dependent endonuclease as well as 2-5A, but in this case, the tetramer triphosphate, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), was just as potent an inhibitor of translation as 2-5A tetramer triphosphate. Moreover, this inhibition was prevented by the established 2-5A antagonist p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)
The oligonucleotides A5'pp5'A2'p5'A2'p5'A and A5'ppp5'A2'p5'A2'p5'A were prepared by reaction of AMP or ADP, respectively, with the 5'-(phosphoimidazolidate) of A2'p5'A2'p5'A. A5'pppp5'A2'(p5'A)n (n = 1-3) were synthesized by reaction of p5'A2'(p5'A)n (n = 1-3) with adenosine 5'-trimetaphosphate. All structures were confirmed by enzyme digestion and 1H and 31P nuclear magnetic resonance (NMR). The products A5'pppp5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were found to be identical with two of the products of the 2-5A synthetase catalyzed reaction of Ap4A with ATP, thus confirming the structural assignments made by earlier investigators. In extracts of mouse L cells programmed with encephalomyocarditis virus RNA, A5'pppp5'A2'p5'A2'p5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were equipotent with 2-5A itself as inhibitors of translation. The oligomers A5'ppp5'A2'p5'A2'p5'A and A2'pppp5'A2'p5'A were about 100 times less active than 2-5A, and A5'pp5'A2'p5'A2'p5'A was without translational inhibitory activity. When affinity for the 2-5A-dependent endonuclease was determined (by displacement of 2-5A[32P]pCp from endonuclease), all of the analogues, as well as 2-5A itself, had similar affinities for the endonuclease except for A5'pppp5'A2'p5'A, which was bound approximately 100 times less effectively. Under conditions of the radiobinding assay, A5'pppp5'A2'p5'A2'p5'A was degraded (t1/2 = 2 h) to ATP, ADP, AMP, ppp5'A2'p5'A2'p5'A, and p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)
It is thought that nonkeratinizing or undifferentiated squamous cell carcinoma in the nasopharynx (NPC) is intimately correlated with Epstein-Barr Virus (EBV). Twenty-one patients with NPC were followed in Kyoto University Hospital and 4 in Osaka Red Cross Hospital during the past 2 years from 1980 to 1981. These patients were classified histopathologically according to the WHO classification in 1978 and staged with the TNM classification in Union Internationale Contre le Cancer (UICC) in 1978. The incidence rate of NPC among the head and neck tumors was 5.6% in the authors' university from 1980 to 1981. The sex ratio of male to female was nearly equal. The mean age of NPC patients was 56.7 years. Sera from these 25 patients with nasopharyngeal carcinoma were collected at intervals of 3 to 8 months over a 2-year period, and were examined for their spectra and titers of antibodies of EBV-related antigens. They were titrated for IgG, IgA and IgM antibodies to EB viral capsid antigen (VCA), for IgG and IgA antibodies to early antigen-DR component (EA) and for antibodies to EBV-associated nuclear antigen (EBNA). All of these patients were primarily treated with radiation, while a few who did not respond to this therapy were subsequently treated with surgery or chemotherapy. EBV antibodies of VCA-IgG, -IgA, EA(DR)-IgG, and -IgA and EBNA were elevated in 73% and 90% of the nonkeratinizing and undifferentiated NPC patients, respectively. The VCA-IgM was elevated in almost none of the cases. In contrast to this, these values were all in a normal range in the NPC patients with keratinizing squamous cell carcinoma and malignant lymphoma. Also 9% and 10% of nonkeratinizing and undifferentiated carcinomas showed the normal ranges of EBV antibodies, possibly indicating a nonassociation with EBV. When NPC disappeared with radiation therapy, EBV antibodies became normal for 6-18 months. However, those whose NPC did not respond to the combined therapy with radiation, surgery and chemotherapy maintained high titers of EBV antibodies. The prognosis was the poorest in the patients with undifferentiated carcinoma, 40% of whom died within 4 years after diagnosis.
A number of 2',5'-linked oligoadenylates and their analogues were prepared and evaluated for their ability to interact with the 5'-O- triphosphoadenylyl -(2'----5')-adenylyl-(2'----5')-adenosine (2-5A) dependent endoribonuclease of mouse L cells. The oligonucleotides were assayed for their ability to antagonize the action of 2-5A, to displace a radiolabeled probe from the 2-5A-dependent nuclease, or to inhibit translation in a cell-free system. These experiments demonstrated the following: (1) Three AMP residues in a 5'-phosphorylated oligonucleotide were needed for maximum interaction with the endonuclease, and higher oligomers (greater than or equal to 4 AMP residues) did not show significantly higher binding. (2) The third (2'-terminal) adenosine residue was required for optimal binding activity. (3) 5'-Phosphorylation of the oligonucleotide was necessary for maximum binding to the endonuclease, but the first (from the 5' terminus) internucleotide phosphate of higher unphosphorylated or core oligomers, such as A2'p5'A2'p5'A2'p5'A, may partly replace the requirement for a 5'-monophosphate moiety; in agreement with this, the 5'-methyl ester of 5'pA2'p5'A2'p5'A, i.e., Me-p5'A2'p5'A2'p5'A, was bound to the endonuclease as well as or better than the higher core oligomers but approximately 100 times more effectively than the trimer core, A2'p5'A2'p5'A. (4) Base-modified analogues, such as p5'C2'p5'C2'p5'C, p5'U2'p5'U2'p5'U, or p5'I2'p5'I2'p5'I, were at least 2000 times less effectively bound to the endonuclease than p5'A2'p5'A2'p5'A. (5) The triphosphate ppp5 'I2'p5'I2'p5'I was 10 000 times less active than 2-5A as an inhibitor of translation. These latter two points implied the critical role of the adenine N1-nitrogen and/or exocyclic amino group in the binding of 2-5A to the endonuclease.
We inoculated Coxsackie virus B1 (CVB1) intraperitoneally into 36 2-week-old Syrian golden hamsters. Serial ECGs were recorded from 10 hamsters which survived the acute and electrocardiographically manifest myocarditis. In a mean 17.8 week (range 9 to 31 weeks) follow-up period, chronic ventricular premature contractions (VPC) followed the acute ECG changes in three of the animals. VPC were recorded in a uniform contour in a hamsters, which had a ventricular aneurysm and were of two kinds of contour in the other two hamsters, each of which had two ventricular aneurysms. No VPC was recorded from the other seven hamsters, which had no aneurysms in the heart. None of the inoculated animals died during the follow-up period, irrespective of the existence of ventricular aneurysm, after surviving the first 2 weeks post-infection. The results demonstrate a close relationship between chronic VPC of uniform contours following CVB1 myocarditis and the formation of ventricular aneurysms in Syrian golden hamsters. The hamsters were able to live for a considerably long period with the aneurysms. Ventricular aneurysms following CVB1 myocarditis in Syrian golden hamsters may be an experimental model of human ventricular aneurysms of unknown etiology.
A new gelatin particle agglutination test was developed for assay of natural antibodies to adult T-cell leukemia virus (ATLV/HTLV-I). Partially purified viral antigen from culture fluid of a virus-producer cell line was coated on artificial gelatin particle carriers. A high correlation was observed between the titers of antibodies determined by the agglutination test and by indirect immunofluorescence assay. The agglutination test is simple, sensitive and specific, and should be useful for mass screening of human sera for viral antibodies.