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Transactivation of inflammatory and immune mediators in asthma is tightly regulated by nuclear factor κB (NF-κB) and signal transducer and activator of transcription 6 (STAT6). Therefore, we investigated the efficacy of simultaneous inhibition of NF-κB and STAT6 using a chimeric decoy strategy to prevent asthma exacerbation. The effects of decoy oligodeoxynucleotides were evaluated using an ovalbumin-induced mouse asthma model. Ovalbumin-sensitized mice received intratracheal administration of decoy oligodeoxynucleotides 3 days before ovalbumin challenge. Fluorescent-dye-labeled decoy oligodeoxynucleotides could be detected in lymphocytes and macrophages in the lung, and activation of NF-κB and STAT6 was inhibited by chimeric decoy oligodeoxynucleotide transfer. Consequently, treatment with chimeric or NF-κB decoy oligodeoxynucleotides protected against methacholine-induced airway hyperresponsiveness, whereas the effect of chimeric decoy oligodeoxynucleotides was significantly greater than that of NF-κB decoy oligodeoxynucleotides. Treatment with chimeric decoy oligodeoxynucleotides suppressed airway inflammation through inhibition of overexpression of interleukin-4 (IL-4), IL-5, and IL-13 and inflammatory infiltrates. Histamine levels in the lung were reduced via suppression of mast cell accumulation. A significant reduction in mucin secretion was observed due to suppression of MUC5AC gene expression. Interestingly, the inhibitory effects on IL-5, IL-13, and histamine secretion were achieved by transfer of chimeric decoy oligodeoxynucleotides only. This novel therapeutic approach could be useful to treat patients with various types of asthma., Graphical Abstract

Angiotensin II is thought to participate in aneurysm formation, because of its ability to induce and perpetuate inflammation in the aortic wall. Because activation of renin is the first step of the renin–angiotensin system, renin inhibition could inhibit all components of this system effectively. Therefore, we examined the hypothesis that direct inhibition of renin activity could decrease the expansion of aortic aneurysm using a rabbit model. Aortic dilatation was induced by incubation with elastase around the rabbit abdominal aorta. Continuous administration of a direct renin inhibitor, aliskiren, was started at 1 week before incubation with elastase and continued for 5 weeks. Treatment with aliskiren markedly inhibited tissue renin activation and resulted in a significant reduction in angiotensin I and II production in the aneurysm wall. Consequently, the inhibition of renin activity prevented the expansion of experimental aortic aneurysm associated with preservation of the medial layer, independent of its blood pressure–lowering effect. Administration of aliskiren led to the inhibition of activation of NF-κB (nuclear factor-κB), AP-1 (activator protein-1), and CREB (cAMP response element–binding protein), which are thought to cooperatively regulate the inflammatory gene expression profile associated with aneurysm formation. As a result, treatment with aliskiren inhibited macrophage accumulation through suppression of MCP-1 (monocyte chemoattractant protein-1) and CCL4 (C-C motif chemokine ligand 4) expression, and TNF-α (tumor necrosis factor-α) production and activation of MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) were also suppressed in the aneurysm wall. In addition, inhibition of (pro)renin receptor elevation was also observed after treatment with aliskiren. Direct inhibition of renin activity using aliskiren prevented the progression of aortic aneurysm, suggesting it as a therapeutic option to treat abdominal aortic aneurysm.

Allergic asthma is a common inflammatory disease of the airways characterized by reversible airflow obstruction and airway hyperresponsiveness (AHR) to bronchoconstrictor stimuli. The pathophysiology in asthma mainly occurs as a consequence of overexpression of the TH2 cytokines. These inflammatory factors are tightly regulated by a transcriptional network system. Nuclear factor kappaB (NFkB) and STAT6 are well known to regulate a set of gene associated with inflammatory and immune responses, and is thought to play an important role in the induction of allergic asthma. Therefore, we focused on the simultaneous inhibition of these important transcription factors using a decoy strategy to develop a novel therapeutic approach for treating asthma. For increased efficacy of decoy oligodeoxynucleotides (ODN) in vivo, we employed chimeric decoy ODN containing consensus sequences of both NFkB and STAT6 binding sites. In addition, two strands of decoy ODN were combined by the chemical spacer to increase its resistance to endonuclease for intratracheal administration. The therapeutic effect of chimeric decoy ODN was investigated using OVA-induced experimental asthma in mice. Mice were sensitized by intraperitoneal injections of 20 ug of ovalbumin (OVA) and 2 mg aluminum hydroxide constituted in 0.1 ml of saline on days 0 and 14. On days 21, 22 and 23, mice were challenged with 1% OVA aerosol for 20 min. At 24 hours after the last challenge, AHR was measured by methacholine-induced airflow obstruction. Intratracheal administration of decoy ODN was performed in OVA-sensitized mice at 3 days before the first challenge with aerosolized OVA. FITC-labeled chimeric decoy ODN could be detected in macrophages and monocytes migrating into the lung and airway, and NFkB and STAT6 activity were simultaneously inhibited by chimeric decoy ODN. Twenty-four hours after the last OVA challenge, treatment with chimeric or single transfection of NFkB decoy ODN was protected from methacholine-induced AHR, while mice treated with scrambled decoy ODN or saline developed a significantly increase in airway reactivity to methacholine. Importantly, this inhibitory effect of chimeric decoy ODN on airway hyperresponsiveness was significantly greater than that of NFkB decoy ODN. Treatment with chimeric decoy ODN markedly suppressed airway inflammation after OVA sensitization and challenge as compared with control and scrambled decoy ODN treatment. Inflammatory infiltrate, such as macrophage, was significantly inhibited by chimeric decoy ODN through suppression of ICAM-1 and eotaxin expression. In addition, secretion of Th2 cytokines including IL-4, IL-5 and IL-13 in BALF, and histamine in the whole lung were reduced by chimeric decoy ODN. Furthermore, a significant reduction of mucin secretion was observed by chimeric decoy ODN treatment accompanied by a suppression of MUC5AC gene expression. However, intratracheal administration of chimeric decoy ODN did not affected IgE synthesis. The present study provides a novel strategy for treating bronchial asthma by the simultaneous inhibition of both NFkB and STAT6 using chimeric decoy ODN. Further modification of chimeric decoy ODN would be useful to treat asthma as a decoy-based therapy.

A gene coding for human secretin elongated at the C-terminus by Gly-Lys-Arg was chemically synthesized and fused to the downstream of an artifical gene encoding [Leu 14 ]human growth hormone (hGH) which had a leucine residue instead of methionine at position 14 in the wild type hGH protein. The fusion gene was efficiently expressed under the control of the Escherichia coli trp promoter including the consensus Shine-Dalgarno sequence. After the fusion protein was cleaved in two parts with cyanogen bromide, the desired secretin-Gly-Lys-Arg (SecGKR) was finally purified to homogeneity using high-performance liquid chromatography, yielding 11 mg protein from 16 g of wet cells. The biological activities of secretin-Gly-Lys (SecGK) and secretin-Gly (SecG) obtained by digestion of SecGKR with carboxypeptidase B were compared with SecGKR and the mature human secretin. The activities of SecGKR and SecGK were 25–30% higher than that of the mature human secretin amidated at the carboxy-terminal amino acid, but that of SecG was 25% lower.

An artificial gene encoding [Leu 14 ]human growth hormone(hGH), which had a leucine residue at position 14 instead of methionine as in the wild type hGH protein, was constructed and expressed in Escherichia coli at a high level, as well as the [Met 14 ]hGH gene, under the control of the trp promoter including a consensus SD sequence. A human transforming growth factor α (TGFα) gene, which had a methionine codon attached to the 5′-terminal region, was chemically synthesized and fused to the [Leu 14 ]hGH gene. The fusion gene was efficiently expressed in E. coli , yielding 48 mg per gram of the wet cell weight. After the fusion protein was cleaved into two parts at the single methionine site by treatment with cyanogen bromide, the desired TGFα was made to form disulfide bonds by a refolding reaction, and then purified by HPLC. The structure of the TGHα was confirmed by amino acid sequence analysis. The biological activity of the purified TGFα was assessed.

The 1H-NMR spectra of native human epidermal growth factor (EGF) and a derivative lacking the final five residues have been assigned by two-dimensional methods, enabling their structures to be compared. The same structural features are observed for each protein, although the final five residues of native human EGF interact with residues earlier in the sequence. Comparison of the resonance shifts of human, rat and mouse EGF and human transforming growth factor alpha (TGF alpha) enables shifts characteristic of the EGF conformation to be identified, providing standards by which the structures of related proteins may be assessed.

For development of a novel method in protein engineering, we studied the use of nonprotein amino acids, namely, amino acids other than the 20 protein-constituting amino acids. We succeeded in in vivo production of human epidermal growth factor (hEGF) substituted with norleucine, an analog of methionine. In the present study we attempted production of hEGF substituted with p-fluorophenylalanine residue (pFPhe), although hEGF does not have Phe residues. We prepared two genes of mutant hEGF; Tyr22 or Tyr29 was replaced by Phe by oligonucleotide-directed mutagenesis. To enhance incorporation of p-fluorophenyl-alanine, these mutant hEGF genes were introduced into an Escherichia coli strain auxotrophic for phenylalanine. The optimum concentration of p-fluorophenylalanine for production of pFPhe-substituted hEGFs was 50 μg/ml. By gel filtration chromatography, and reverse-phase and ion-exchange high performance liquid chromatography, we succeeded in purification of pFPhe-substituted hEGFs. Fluorine substitution of the aromatic ring of each Phe residue probably induces a conformational change of hEGF.

We constructed a new vector containing the promoter and the signal sequence of E. coli phoA gene, the structural gene for the periplasmic alkaline phosphatase. One of the most useful characteristics of this vector is the unique HindIII restriction site located just at the end of the phoA signal sequence. This restriction site was generated by oligonucleotide-directed site-specific mutagenesis without changing the amino acid sequence of the signal peptide. Any kind of foreign structural gene can be easily inserted into the HindIII site by using synthetic oligonucleotides to construct a hybrid gene which has neither an extra sequence nor a deletion between the phoA signal sequence and the foreign structural gene. Human alpha-interferon gene was inserted into this HindIII site. When this hybrid gene was expressed under the control of the phoA promoter region, a low but significant activity was recovered in the cold water wash of the cells after an osmotic shock procedure.

Human insulin-like growth factor II (hIGF-II) was produced inEscherichia coli as a protein fused to human growth hormone. High level expression of the fusion protein was attained with pIBL-1 plasmid. The hIGF-II obtained byin vitro cleavage of the fusion protein with cyanogen bromide was highly purified and its biological activity was assessed.

Endeavoring to develop a method to biosynthesize proteins substituted with nonprotein amino acids, we attempted the incorporation of L-2-aminohexanoic acid (Ahx) into human epidermal growth factor (hEGF). Escherichia coli YK537 strain harboring plasmid pTA1522, which has the phoA promoter-phoA signal peptide-hEGF gene, was used. Cells were cultured first in high-phosphate medium and then, for induction of the hEGF-encoding gene, transferred to low-phosphate medium containing Ahx (0.25 mg/ml). hEGF and Ahx-substituted hEGF, [Ahx21]hEGF, secreted into the periplasm were recovered. After treatment with H2O2, [Ahx21]-hEGF was clearly separated from methionine-oxidized hEGF by one-step reverse-phase HPLC. Substitution of the methionine residue of hEGF with Ahx was confirmed by the amino acid analysis of [Ahx21]hEGF. The three biological activities of [Ahx21]hEGF were the same as those of hEGF. From the successful production of [Ahx21]hEGF, a basic strategy was established for preparing proteins substituted with nonprotein amino acid (alloprotein). Induction of the phoA promoter of pho regulon and secretion of the product to the periplasm may depress heat shock-like responses and subsequent hydrolysis of the product by cytoplasmic protease.

A hexanucleotide and an octanucleotide corresponding to tRNAMetf bases 41-46 and 47-54 were synthesized by stepwise addition of mononucleotides. The octanucleotide is the largest oligoribonucleotide synthesized by this method. A pentanucleotide (bases 61-65) and a hexanucleotide (bases 66-71) were synthesized via partially triesterified intermediates. The deblocked products were purified by ion-exchange chromatography and characterized by enzymatic hydrolysis. These oligonucleotides were used as substrates in joining reactions with RNA ligase.

Chemically synthesized fragments corresponding to the 3' end of tRNAfMet from Escherichia coli were joined by T4-induced RNA ligase to yield a heptadecanucleotide (bases 61--77). The 3' terminus of C-C-A was modified by introduction of the ethoxymethylidene group to prevent intra- and intermolecular self-joining reactions at the 3' end. The terminal trimer was phosphorylated using polynucleotide kinase and joined to C-A-A with RNA ligase. The hexamer [C-A-A-C-C-A(ethoxymethylidene)] corresponding to bases 72--77 was obtained in a yield of 60%. An undecanucleotide (bases 61--71) which had been synthesized in a yield of 34% by similar enzymatic joining of U-C-C-G-G to pC-C-C-C-C-G was allowed to react with the 5'-phosphorylated hexamer (bases 72--77) using an excess of RNA ligase to yield the heptadecanucleotide U-C-C-G-G-C-C-C-C-C-G-C-A-A-C-C-A (bases 61--77). The product was identified by homochromatography and nearest neighbor analysis.

Various protecting groups were introduced to the phosphate of 2'-O-benzoyluridine 3'-phosphate. Stability and selective removal of these groups from the trisubstituted phosphate were investigated and nucleotides with trisubstituted phosphate were uesd for the synthesis of dinucleotides by condensation with a properly protected mononucleotide.

P1-Adenosine 5'-P2-o-nitrobenzyl pyrophosphate (nbzlppA) has been synthesized as a substrate for T4 RNA ligase catalyzed 3'-phosphorylation. Incubation of oligoribonucleotides and nbzlppA with RNA ligase yielded oligoribonucleotides having a 3'-o-(o-nitrobenzyl) phosphate. Photochemical removal of the o-nitrobenzyl group provided the free 3'-phosphate. Using [P2-32P] nbzlppA, 3'-termini of oligoribonucleotides could be labelled with 32P. This reaction was applied to modify the 3'-end of donor molecules in joining reaction with RNA ligase. A trinucleotide U-A-G was converted to U-A-Gpnbzl and phosphorylated with polynucleotide kinase. pU-A-Gpnbzl was then joined to an acceptor trinucleotide A-U-G to yield A-U-G-U-A-Gp.

Nascent short DNA chains could result from repair of incorporated uracil residues or be intermediates in discontinuous replication. We have characterized short DNA chains having apyrimidinic/apurinic-sites at 5' ends, the expected intermediates of repair, to distinguish them from RNA-linked replication intermediates. We have synthesized model substrates for the repair products; d(pRib[32P]poly(T)) and d(Rib[32P]poly(T)). Alkaline hydrolysis of both substrates has produced [5'-32P]poly(dT). Nascent short DNA was prepared from an Escherichia coli sof (dut) mutant, in this strain fragments from excision repair of uracil residues accumulate. The products of alkaline treatment are hardly digested by spleen exonuclease which selectively degrades 5'-hydroxyl-terminated DNA. These two results show that alkaline hydrolysis of the uracil repair fragments produces 5'-phosphoryl-terminated DNA, whereas it is known that 5'-hydroxyl-terminated DNA is generated from RNA-linked DNA molecules. The two types of nascent fragments thus can be distinguished by the 5'-terminal structure produced by an alkaline hydrolysis.

A tetranucleotide C-G-C-Gp (cytidylyl-(3'-5')-guanylyl-(3'-5')-cytidylyl-(3'-5')-guanosine 3'-phosphate) was synthesized by condensation of (Bz) bzC (Bz)-ibG (Bz) p (5) with C (Bz)-ibG (Bz) pNHC6H4S (O) CH3 (6) using 2, 4, 6-triisopropylbenzenesulfonyl chloride. This sequence corresponds to the E. coli tRNAMetf 5'-terminus. A base substituted analog U-G-C-Gp was also synthesized by a similar procedure to that used for C-G-C-Gp. These tetranucleotides were characterized by enzymic hydrolysis.

Synthesis of two oligothymidylic acids, tridecamer and nonadecamer, is described by a rapid and simple solid-phase method on two kinds of polyacrylamide supports derivatized from commercially available Enzacryl Gel K-2. The syntheses were performed by the phosphotriester method using di- and tri-thymidylic acid blocks as the incoming 3'-phosphodiester component. High coupling yields were consistently obtained and the final product was isolated very easily by high performance liquid chromatography on Permaphase AAX.

An eicosanucleotide C--G--C--G--G--G--G--U--G--G--A--G--C--A--G--C--C--U--G--Gp corresponding to the bases 1--20 of the nascent sequence for the Escherichia coli tRNAfMet has been synthesized by the joining of the chemically synthesized oligonucleotides C--G--C--G, G--G--G--U--G--G and A--G--C--A--G--C--C--U--G--Gp using RNA ligase from T4-infected E. coli. The hexanucleotide and decanucleotide were phosphorylated with polynucleotide kinase and [gamma-32P]ATP prior to the joining reactions. The decanucleotide and eicosanucleotide were reconstituted respectively with the 3'-three-quarter molecule obtained by limited digestion with RNase T1 of the natural tRNAfMet from E. coli and the activity of the complex as a methionine acceptor was tested using purified methionyl-tRNA synthetase from E. coli. The amino acid acceptor activity of the reconstituted molecules was 11% and 84% with respect to that of the intact tRNAfMet.

N, N-Dimethyl-p-phenylenediamine, glycine amide and p-methylthioaniline were condensed with uridine 5'-phosphate and the phosphoramidates obtained were tested for their stability in anhydrous pyridine, 50% aqueous pyridine or 80% acetic acid. The p-methylthioanilidate (IIc) was oxidized to give p-methylsulfoxylanilidate of uridine 5'-phosphate (IId) which was found to be 5 times more stable than the p-methylthio compound. The p-methylsulfoxylanilidate of 2'-O-benzoyluridine 3'-phosphate was condensed with the mononucleotide to yield the dinucleotide, MMTrU(OBz)-p-U(OBz)-p in 28% yield.

Two oligonucleotides 14-bases long were synthesized, one complementary to rabbit beta-globin DNA (R beta G14A) and the other with the same sequence except for a single base change (T for C) (R beta G14B). Hybridization conditions were established such that R beta G14A would hybridize to globin DNA while R beta G14B would not. We also synthesized a mixture of 13-base long oligonucleotides (R beta G13Mix), representing eight of the possible coding sequences for amino acids 15-19 of rabbit beta-globin. One of the eight is complementary to globin DNA. R beta G13Mix was found to hybridize specifically to globin DNA under conditions where oligonucleotides forming single base pair mismatches do not. Furthermore, R beta G13Mix was shown to hybridize specifically to colonies containing a plasmid with a globin DNA insert. These results are discussed with respect to a general procedure for screening recombinant clones for those containing DNA coding for a protein of known amino acid sequence.

Synthesis of the milk proteins, casein and α-lactalbumin was not induced in cultured mammary explants from C3H/HeN castrated virgin mice in the presence of lactogenic hormones such as insulin, cortisol and PRL. Replacement therapy with 17-β-estradiol to the castrated mice completely restored the differentiative potentials of mammary explants, inducing synthesis of the two milk proteins. [3H] thymidine incorporation into DNA synthesized in cultured mammary explants was also decreased by castration to less than 50% (P< 0.001) of that obtained in intact mice, and 17-β-estradiol treatment to castrated animals restored DNA synthesis to 90% of the intact level. The addition of insulin to culture medium significantly (P< 0.001) enhanced [3H] thymidine incorporation into DNA in cultured mammary explants from both intact and 17-β-estradiol-treated castrated mice but not from castrated animals. Insulin binding sites (1710 ± 260 sites/cell) to mammary epithelial cells from castrated mice were significantly (P

A synthetic approach to the formylmethionine-tRNA from E. coli is described. The methods employed include 1) phosphodiester synthesis using block condensation or stepwise addition methods, 2) a mixed method combining di- and triester approaches, 3) the phosphotriester method using o-nitrobenzyl for 2′-OH, p-chlorophenyl and anilidate for phosphate and acyl groups for base protection, and 4) joining of the synthetic fragments with RNA ligase.