The nucleic acid fraction from cells of 6 species of bacterium and 2 kinds of vertebrate, calf and salmon, was extracted and purified by the same procedures as described previously. When the spleen cells from BALB/c mice were incubated with the nucleic acid fraction from either of the bacteria, natural killer (NK) activity of the cells was remarkably elevated and the cells produced factors to activate macrophages and to inhibit viral growth. It was shown that the factor to activate macrophages was interferon (IFN)-gamma and that to inhibit viral growth was IFNalpha/beta. On the other hand, the nucleic acid fraction from either of the vertebrate cells did not show such activities. Pretreatment of the bacterial nucleic acid fraction with DNase, but not with RNase, abrogated completely the biological activities. The activities of the bacterial nucleic acid were not influenced by the presence of polymyxin B, an inhibitor of lipopolysaccharide (LPS), and the spleen cells from not only BALB/c mice but also LPS-insensitive C3H/HeJ mice were activated, indicating that the activities of the fraction were not ascribed to LPS contaminated possibly into the fraction, but to DNA itself. Intralesional injection with the bacterial DNA fraction caused regression of mouse IMC tumors, but the injection with the vertebrate DNA fraction did not. These findings prompted us to examine the biological activities of DNA samples from a variety of animals and plants, which were provided from other laboratories or purchased from manufacturers. All of the DNA samples from cells of 5 kinds of bacterium, 2 of virus and. 4 of invertebrate augmented NK activity and induced IFN, more or less, in mouse spleen calls, while the DNA from 10 kinds of vertebrate, including 3 of fish and 5 of mammal, showed no such activities. The DNA from 2 species of plants, were also inactive. Possible mechanisms to explain the different biological activities of DNA from different cell sources were discussed based on our previous finding that the particular palindromic sequences with a G-C motif (s) are required for induction of IFNs and activation of NK cells with synthetic 30-mer oligonucleotides.We have demonstrated that a DNA-rich fraction, extracted fromMycobacterium bovis BCG and designated MY-1 (22), exhibited strong antitumor activities against 98 3
Specific palindromic sequences in synthetic oligonucleotides are required to induce IFN and augment IFN-mediated natural killer activity. To study the mechanism of IFN induction by oligonucleotides containing palindromic sequences, we investigated the possible target molecules of the oligonucleotides. Oligo-1, a 30mer single-stranded oligonucleotide with oligoG sequences next to the active palindromic sequence (AACGTT), had more activity than oligonucleotides with oligoA, oligoC, or oligoT sequences. The activity of oligo-1 was inhibited by a guanine homo-oligomer (G30), dextran sulfate, and polyvinyl sulfate. Oligo-1 bound to plastic-adherent mouse splenocytes, and the binding was inhibited by G30, dextran sulfate, and polyvinyl sulfate. Oligo-1 inhibited acetyl-LDL binding to the scavenger receptor on mouse splenocytes. These findings suggest that the binding of an extrapalindromic sequence to the scavenger receptor is required for the immunostimulatory activity of oligo-1.
Based on the previous finding that certain 30‐mer single‐stranded oligodeoxyribonucleotides (oligonucleotides) having particular 6‐mer palindromic sequences could induce interferon‐alpha and ‐gamma, and enhance natural killer activity, the present study was carried out to clarify the entire relationship between the activity and the sequence of 30‐mer oligonucleotides. The results indicated that the activity depended critically on the presence of particular palindromic sequences including the 5 ‐CG‐3 motif(s). The size and the number of palindromes as well as the extra‐palindromic sequences also influenced the activity. An oligonucleotide with a 10‐mer palindrome and extra‐palindromic oligoguanylate sequences showed the strongest activity among the oligonucleotides tested.
The DNAs of 20 strains of varicella-zoster virus (VZV) isolated from epidemiologically unrelated individuals, and of 15 strains isolated from vesicles of vaccinees with varicella or zoster after vaccination, were compared by restriction enzyme cleavage using HpaI. Differences were found in the sizes of the HpaI-F, -G and -K fragments of the wild strains. The gel migration patterns of the HpaI-F and -G fragments, but not of the HpaI-K fragment, were polymorphic in the different strains isolated from the vaccinees. The effects of serial passages in vitro and in humans on the genome stability of VZV were investigated by HpaI analysis. The DNA profiles of the HpaI-K fragments from six isolates recovered from room-mates infected in a single outbreak were identical, but the mobilities of their HpaI-F and -G fragments varied. The DNA profiles of the Oka vaccine virus after 10 and 85 passages in human embryo cells differed only in the HpaI-F fragment. The profiles of these fragments in DNA derived from two isolates obtained at different times from a vaccinee with varicella followed by zoster were compared with those of the Oka (parental) and Oka (vaccine) strains, and identical results were obtained for the two viruses. In addition, the same DNA profiles of HpaI fragments were obtained from three sequential isolates from one person and also from two isolates from another with varicella and zoster. Thus, it was concluded that: three variable fragments (HpaI-K, -F and -G) were not changed in the DNAs of isolates derived from the same patient; HpaI-K was stable both on passage in vitro and after human transmission in the case of the same outbreak, but was different among all wild-type strains isolated in epidemiologically unrelated outbreaks; HpaI-F was very unstable both on passage in vitro and in human infections by either vaccine or wild-type strains; HpaI-G was not influenced by passage in vitro but varied among wild-type strains. Using physical maps of VZV DNA established by others, three variable regions on the viral genome were identified. One was located near the 0.16 coordinate, which is covered by HpaI-K (variable region I, VRI). Another was represented by HpaI-F (VRII), the most unstable fragment, and mapped at about the 0.35 coordinate. The third was VRIII near the right terminus, covered by HpaI-G.
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