Isolation and characterization of a mouse protein C cDNA.

Protein C (PC) is a vitamin K-dependent serine protease, a deficiency of which results in thrombus. There is no spontaneously occurring mouse model of the disease. Attempts to create such a model in mice by using anti-sense gene technology requires isolation of a normal mouse PC cDNA. When a mouse liver (BALB/c) cDNA library was screened using a human PC cDNA as a probe, nine overlapping cDNA clones were isolated and sequenced. The cloned mouse PC cDNA comprised 1,512 nucleotides and the open reading frame of the cDNA encoded a polypeptide of 461 amino acids residues including a leader peptide composed of 41 amino acids. Mouse PC exhibited high homology to both human and bovine PCs. Mouse PC also had several structural features common in other PCs; locations of 23 Cys residues, location of putative beta-hydroxy Asp71, possible carbohydrate attachment sites involving Asp residues at amino acid positions 249, 314, and 330, and location of active sites such as His212, Asp258, and Ser361. Northern blot hybridization analysis identified a single species of mouse PC mRNA (2.0 kb in length) in mouse liver.

Photodynamic antisense regulation of human cervical carcinoma cell growth using psoralen-conjugated oligo(nucleoside phosphorothioate).

The antisense strategy has been applied to regulate gene expression in a sequence specific manner, which enables suppression of the proliferation of cancer cells and exploration of the functions of unknown genes. In order to generalize and to enhance the ability of the strategy, functionalization of antisense DNAs was done using a photo-crosslinking reagent, 4,5',8-trimethylpsoralen, and the possibility of photodynamic antisense regulation of gene expression was examined. Psoralen-conjugated oligo(nucleoside phosphorothioate)s (Ps-S-oligo) were prepared and used to inhibit the proliferation of human cervical carcinoma cells. Upon UVA irradiation of Ps-S-oligo treated cells, Ps-S-oligo complementary to the initiation codon region (Ps-P-As) of HPV18-E6*-mRNA of human cervical carcinoma cells inhibited drastically the cell growth (IC(50)=16 nM). In contrast, Ps-S-oligo with mismatched sequences and scrambled one showed lesser inhibitory effects than Ps-P-As. These results showed that the inhibition by Ps-S-oligo was dependent on (a) sequence, (b) UVA irradiation, (c) concentration and (d) cell line. The amount of intact HPV18-E6*-mRNA was decreased in a sequence dependent manner, indicating that the antiproliferative effect of Ps-P-As was an antisense manner. The psoralen-conjugated antisense DNA has significant potential to regulate gene expression, which may provide useful information to explore the novel gene regulating reagents.

[Recent development of nucleic acid drug].

Oligonucleotides and their analogs have been utilized for the regulation of gene expression since mid-1970. The so-called antisense strategy has recently acquired its reality of the application to clinical therapeutic uses. In the course of the development, it has been found that nucleic acids might have broad variety of abilities to regulate gene expression. The abilities are classified as ribozymes, antigenes, decoy DNAs (or RNAs) and aptamers, and they can be applied to clinical uses as nucleic acid drugs in the near future.

Effect of serine residue on the effectiveness of cationic polypeptide-based gene delivery.

Poly-L-lysine(pL) was chemically modified based on two essential features which we recently reported and subjected to the gene-transfer experiment in vitro. Introduction of 25 mol% serine residue to pL slightly enhanced the gene expression level, while trimethylation of epsilon-ammonium groups of lysine did not. Only when pL was modified in both way, giving N2-trimethyl poly(lysine-co-serine), markedly enhanced gene expression was observed. The cellular uptake and localization of DNA in the cells were similar for each cationic polypeptide. DNA forming complex with the polypeptides containing serine residue was found to be well transcribed in in vitro transcription/translation system, suggesting the hydrophilic nature may allow polypeptide/DNA complexes to be recognized by the transcriptional factors and lead the subsequent effective gene expression.

Gene introduction into mouse blastocysts via "pricking".

It is a well-known phenomenon that cultured mammalian cells that have been pricked in the presence of foreign DNA can be transformed. This micromanipulation 'pricking' technique was applied to mouse blastocysts to determine whether uptake of exogenous DNA would occur in the embryos. The middle region of the inner cell mass (ICM) was pricked three times in each blastocyst in a medium containing a linearized plasmid DNA. When the 60 treated blastocysts were transferred to the uterine horns of pseudopregnant females, 30 developing fetuses (50%) at the mid-gestation stage were obtained. Twenty-two of the 30 fetuses (73%) had less than 1 copy of the foreign DNA per diploid cell, as revealed by polymerase chain reaction (PCR)-Southern analysis, a sensitive technique combined with Southern blot processing of the PCR products. The 8 other fetuses were negative for the foreign DNA. When blastocysts were pricked in the presence of vector DNA coupling E. coli beta-galactosidase (beta-gal) gene to a mouse metallothionein-I (MT-I) promoter and assessed for beta-gal activity histochemically after 1 and 5 days of culture in the presence of 1 microM CdCl2, at least 65% of the embryos exhibited beta-gal activity mainly in the ICM region. These results indicate that mouse blastocysts can be transfected with a relatively high efficiency after pricking, and that the introduced gene expression occurs. This approach provides a means of mapping the regulatory elements of genes that are active in the mouse blastocyst ICM, and may be useful in investigating the fate of the ICM cells in an intact blastocyst by labeling them via pricking technique.

Cyclic diacyl groups for protection of the N6-amino group of deoxyadenosine in oligodeoxynucleotide synthesis.

Three kinds of substituted phthaloyl groups and a succinyl group were introduced onto the N6-amino function of deoxyadenosine derivatives. Among them, the succinyl group was found to be the most effective for prevention of depurination upon detritylation in acidic media and the most stable in basic media. Protection of the N6-amino function of 5'-O-dimethoxytrityldeoxyadenosine and introduction of a succinate linker into the 3'-hydroxyl were achieved simultaneously by a one-step reaction with succinic anhydride. A tetradeoxyribonucleotide, dTpTpTpA containing a 3'-terminal deoxyadenosine was successfully synthesized on a polystyrene support via the phosphotriester method.

Structure/function relationship in the polyplexes containing cationic polypeptides for gene delivery.

Various cationic polypeptides of linear or highly branched structures were synthesized by introducing tertiary or quaternary ammonium groups and hydroxyl groups to poly(L-lysine) (PL) or polyamideamine (PAMAM) dendrimers. These polycations were mixed with plasmid DNA to form polyplexes and subjected to in vitro gene introduction experiment. The transient gene expression was greatly affected by the side groups of PL derivatives or the surface cation charge density of PAMAM dendrimers. This difference in gene expression was found to result from two independent factors as follows: one is the cellular uptake of the polyplexes and the other is the compaction of the polyplexes. Lower charge density of PAMAM dendrimers suppressed the polyplex formation and cellular uptake, resulting the lower gene expression. Only the polycations that form polyplexes compacted at an adequate extent lead an effective gene expression, suggesting that the physicochemical properties of the polyplexes defined by the chemical structures of the polycations play an important role in the effective gene transfer.

Gene regulation by decoy approach (I): synthesis and properties of photo-crosslinked oligonucleotides.

To explore gene regulation by double stranded oligonucleotides as decoy molecules for transcriptional factors, oligodeoxyribonucleotides (ODN) and the complementary oligo(deoxyribonucleoside phosphorothioate)s (OPT) were photo-crosslinked by use of psoralen. UV melting curves showed that the thermal stability of crosslinked duplexes increased compared with that of non-crosslinked ones. CD spectra showed that the photo-crosslinked duplexes formed B-DNA structure. In addition, cross-linked duplexes were resistant against digestion by snake venom phosphodiesterase. These results suggest that the photo-crosslinked duplexes have desirable characteristics as decoy molecules.

Characterization of RNA structure by bis-pyrene-labeled 2'-O-methyloligonucleotides.

Properties of 2'-O-methyloligoribonucleotides containing two consecutive 2'-O-(1-pyrenylmethyl)uridine were investigated as a fluorescent probe to search the single strand regions of RNA. The bis-pyrene-labeled 2'-O-methyloligoribonucleotide (OMUpy2) induced the formation of pyrene dimer upon hybridization with the complementary oligoribonucleotides and showed remarkable appearance of broad structureless fluorescence at 480 nm. Contrarily, when OMUpy2 was hybridized with the complementary oligodeoxyribonucleotides, such enhancement of fluorescence was scarcely observed. When various OMUpy2 were applied to E. coli 5S-rRNA, the fluorescence intensity at 480 nm was varied in a sequence specific manner.

Molecular design of a eukaryotic messenger RNA and its chemical synthesis.

A designed mRNA consisting of 42 ribonucleotides having the cap structure was synthesized. The capped leader sequence of the brome mosaic virus (BMV) mRNA 4, m7G5'pppGUAUUAAUA (F-1), was synthesized by the phosphotriester method and followed by the capping reaction. A 32-mer consisting of an initiation codon (AUG), the coding region corresponding to a bacterial pheromone cAD1 and two stop codons, was constructed by the 18-mer (F-2) and 14-mer (F-3), which were synthesized by the phosphoramidite method. 2'-,3'-O-Methoxymethylene-guanosine 5'-phosphate was condensed with F-3 using P1-2',3'-O-methoxymethyleneguanosine-5'-yl P2-adenosine-5'-yl pyrophosphate (9) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated successively with T4 RNa ligase to afford the whole RNA molecule.

Natural course of joint destruction and fluctuation of serum C1q levels in patients with rheumatoid arthritis.

Using the number of joints with erosion in a total of 68 joints throughout the body, we studied a population of patients with rheumatoid arthritis whose disease duration was 10-15 years. Three groups, each showing a Poisson distribution, were found: the subset with least erosive disease (LES), the subset with more erosive disease (MES), and the subset with mutilating disease (MUD). The mean number of joints with erosion was 10.9 in LES, 32.2 in MES, and 53.5 in MUD. In LES, erosive articular changes were primarily limited to the peripheral smaller joints. In MES, the larger axial joints were also involved. Almost all joints were extensively damaged in MUD. During the early period of disease, differences between the 3 groups were highly significant in the rapidity of carpal bone destruction, as assessed by the yearly reduction of carpal height ratio (P less than 0.001), and in the serum C1q level (P less than 0.001).

Synthesis of N2, N2, 7-trimethylguanosine cap derivatives.

Several derivatives of N2,N2-7-trimethylguanosine (m3(2,2,7G)-cap, which was found at the 5' ends of small nuclear RNAs, were synthesized by use of S-phenyl N2,N2,7-trimethylguanosine 5'-phosphorothioate (PhSpm3(2,2,7)G) as a key intermediate. This compound was activated by iodine in the presence of phosphoric acid and diphosphoric acid to give N2,N2,7-trimethylguanosine-5'-diphosphate (ppm3(2,2,7)G) and 5'-triphosphate (ppm3(2,2,7)G), respectively. Similar reactions of PhSpm3(2,2,7)G with ADP and GDP gave capped dinucleoside triphosphates, m3(2,2,7)G5'pppA and m3(2,2,7)G5'pppG, respectively.

The pathology of the involved tendons in patients with familial arthropathy and congenital camptodactyly.

In 2 sisters with congenital camptodactyly and joint effusions, abnormalities in tendons were restricted to the portion within synovial sheaths. This implied a disease of the tenosynovium, rather than one of the tendon itself. In areas of chronic involvement, some tendons were replaced by fibrous tissue. Significant portions of the tendons in fingers with camptodactyly were replaced by hard scars. Congenital camptodactyly is probably the result of an intrauterine tenosynovitis, rather than an isolated congenital anomaly.

Serum C1q levels as a prognostic guide to articular erosions in patients with rheumatoid arthritis.

C1q was measured serially by single radial immunodiffusion in 54 rheumatoid arthritis (RA) patients over a period of more than 5 years, and values were correlated with laboratory, radiographic, and clinical findings. The number of joints with erosion (NJE) was determined retrospectively from radiographs of patients who had RA of greater than 7 years duration. In patients with clinically "burned out" RA, C1q levels were not statistically different from those of healthy adults. During the period of active disease, each patient's C1q level remained very constant, irrespective of erythrocyte sedimentation rate, C-reactive protein (CRP) level, or whether the RA was active or in remission. No sustained correlation was found between the C1q level and the other 2 acute phase reactants, but patients with C1q levels of at least 250 micrograms/ml showed a positive CRP over a period of years, in contrast to those with C1q levels below 250 micrograms/ml. Patients with an initial C1q above 250 micrograms/ml had more erosive RA when compared with those having C1q levels below 250 micrograms/ml. These data suggest that active RA can be classified into two subsets by C1q levels, one with persistent inflammation and a high NJE and another without persistent inflammation and with a low NJE.

2'-Pyrene modified oligonucleotide provides a highly sensitive fluorescent probe of RNA.

Oligonucleotide 9mers containing 2'-O-(1-pyrenylmethyl)uridine [U(pyr)] at the center position were synthesized by using a protected U(pyr) phosphoramidite. The UV melting behaviors indicate that the pyrene-modified oligonucleotides can bind to both their complementary DNA and RNA in aqueous solution. When compared with the unmodified oligonucleotides, the pyrene-modified oligonucleotides showed higher affinity for DNA while exhibiting lower affinity for RNA. The pyrene-modified oligonucleotides in diluted solution exhibited fluorescence typical of pyrene monomer emission [lambdamax 378 (band I) and 391 nm (band III)]. When these oligomers bound to DNA, the fluorescence intensity ratio of band III/band I was increased. With this fluorescence change, a new broad emission (lambdamax 450 nm) due to exciplex between the pyrene and an adjacent nucleobase appeared. In contrast, addition of RNA to the pyrene oligonucleotides resulted in enhancement of the pyrene monomer emission with decrease in the fluorescence band ratio. The extent of the emission enhancement was found to be highly dependent on the nucleobase adjacent to the U(pyr) in the pyrene oligomers. The pyrene oligonucleotide containing dC at the 3'-site of the modification showed remarkable increase (approximately 250 times) in fluorescence (375 nm) upon binding to complementary RNA. The present findings would open the way to the design of a highly sensitive fluorescent probe of RNA.

Study on RNA structure by pyrene-labeled 2'-O-methyloligoribonucleotides.

Properties of 2'-O-methyloligoribonucleotides containing 2'-O-(1-pyrenylmethyl)uridine were investigated as the fluorescent probe to search the single strand regions on RNA secondary and tertiary structure. The pyrene-labeled 2'-O-methyloligoribonucleotide (OMUpy) showed remarkable increase of fluorescence intensity to 333-fold at 375 nm when hybridized with the complementary oligoribonucleotide. When OMUpy, complementary to loop or stem regions, was applied to E. coli 5S-rRNA, the fluorescence intensities were increased in a sequence specific manner. The difference of the fluorescence intensities corresponds to the higher-order structure of 5S-rRNA, suggesting that pyrene-labled 2'-O-methyloligoribonucleotide can be applicable to search single strand regions of RNA.

Photodynamic antisense regulation using psoralen-conjugated oligo(nucleoside phosphorothioate)s (I). Growth regulation of cervical carcinoma cells.

To increase the antisense regulatory effect of oligo(nucleoside phosphorothioate)s (S-Oligo), a photo-crosslinking reagent, 4, 5', 8-trimethylpsoralen, was used in this study. Psoralen-conjugated oligo(nucleoside phosphorothioate) (Ps-S-Oligo) complementary to the human papillomavirus type 18 (HPV18) mRNA drastically inhibited the cellular proliferation of cervical cancer cells only upon UVA-irradiation. In contrast, Ps-S-Oligos with mismatched sequences and scrambled one showed lesser inhibitory effects than that with matched one. These results suggest that psoralen-conjugated antisense S-Oligo has significant potential to regulate gene expression upon UVA-irradiation.

Pre-mRNA with a trimethylguanosine cap structure can be spliced efficiently in vitro.

A chemically synthesized 2,2,7-trimethylguanosine cap (TMG-cap) analogue was added to an in vitro pre-mRNA splicing system. A slight inhibition of the splicing was observed, but it was much less remarkable than that by a 7-methylguanosine cap analogue. This result does not favor a direct role of the TMG-cap structure of small nuclear RNAs in pre-mRNA splicing, and suggests that the TMG-cap does not effectively interact with the 7-methylguanosine cap binding factors. Using the SP6 in vitro transcription system and a TMG-cap analogue, we prepared a beta-globin transcript which has a TMG-cap at the 5' end. This TMG-capped transcript was spliced accurately and efficiently in vitro.

Synthesis of RNA having cap structure.

RNA consisting 43 nucleotides bearing cap structure was synthesized (Figure). In the first place, 9 mer of a leader sequence with the cap structure (F-1) was synthesized by the phosphotriester method and followed by the capping reaction. Next, 32 mer of a cistron was divided into two fragments and each was synthesized by the phosphoramidite method. The 3'-end nucleotide of the RNA, a modified guanosine 5'-phosphate, was introduced to F-3 by use of P1-2',3'-O-methoxymethylene guanosine-5'-yl P2-adenosine-5'-yl diphosphate (A5' ppGmM) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated with T4 RNA ligase to afford the desired RNA.

Photo-cross-linked oligonucleotide duplex as a decoy-DNA for inhibition of restriction endonuclease activity.

As a novel type of regulator molecule for DNA-recognizing proteins, a photo-cross-linked oligonucleotide duplex was designed and synthesized. The molecule regulated the activity of a restriction endonuclease by being recognized as a substrate. This type of regulating molecule is regarded as a decoy-DNA. 4,5',8-[4-Aminoethylaminomethyl]-trioxalen (aeAMT) was conjugated with an oligodeoxyribonucleotide (ODN) at the 5'-end and the aeAMT was cross-linked with the thymine residue of the complementary oligonucleotide upon UVA irradiation. The terminally cross-linked oligonucleotides, singly clipped (SC) decoy-DNA, acquired thermal stability. An oligonucleoside phosphorothioate (OPT) was also introduced as one or both components, yielding three types of decoy-DNAs, SC-ODN-ODN (SC.DD), SC-OPT-ODN (SC.SD), and SC-OPT-OPT (SC.SS). The SC decoy-DNAs inhibited the function of the restriction endonuclease, AatII, in a sequence-specific and concentration-dependent manner with an appreciable IC50 value (1.3 microM for SC.DD, 0.016 microM for SC.SD, 0.002 microM for SC.SS). The SC decoy-DNAs were found to be effective for regulating the DNA recognizing proteins.