ADNA-linked RNase H (Hybrid Enz-1) (Kanaya et al. (1992) J. Biol. Chem. 267, 8492-8498), in which dGTCATCTCC was attached to E. coli RNase H via a covalent linker of 21 A, was altered to improve the site-specific RNA cleavage by increasing the linker length. The sizes of the linkers on these hybrid enzymes (Hybrid Enz-2, -3, and -4) differed by 3 A, the axial rise of the DNA/RNA hybrid, to give 18-, 24-, and 27-A lengths. The conjugate with a size of A was able to cleave a synthetic 22mer RNA (5'-rAAGAUGUCUACGGAGAUGACCA-3'), containing the complementary 9mer RNA sequence (underlined), at one position, A16-U17. The kinetic parameters of Hybrid Enz-1, -2, -3, and -4 were examined using a 9mer RNA target. The results showed that longer linkers produced higher Km, kcat, and kcat/Km values, and the kcat/Km value of the conjugate with the 27-A linker reached 83% of that of the wild-type RNase H. Hybrid Enz-4 was found to be useful as an RNA restriction endonuclease.

Base Sequence↗

PubMedDate not supplied

Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the modified base and at adjacent residues.

It has been shown previously that deoxyguanosine residues in DNA are hydroxylated at the C-8 position both in vitro and in vivo to produce 8-hydroxydeoxyguanosine (8-OH-dG) by various agents that produce oxygen radicals such as reducing reagents-O2, metal ions-O2, polyphenol-H2O2-Fe3+, asbestos-H2O2 or ionizing radiation. These agents are mostly either mutagenic or carcinogenic; therefore, the formation of 8-OH-dG can also be considered a likely cause of mutation or carcinogenesis by oxygen radicals. It is of interest to know whether the 8-OH-dG residue in DNA is misread during DNA replication. To answer this question, we have examined the effect of the 8-OH-dG residue in DNA on the fidelity of DNA replication using a DNA synthesis system in vitro with Escherichia coli DNA polymerase I (Klenow fragment). The synthetic oligodeoxynucleotides, with or without an 8-OH-dG residue in a specified position, were chemically synthesized and used as templates for DNA synthesis under the conditions of the dideoxy chain termination sequencing method. Surprisingly, in addition to misreading of the 8-OH-dG residue itself, pyrimidines next to the 8-OH-dG residue (G has not yet been tested) were also misread.

8-Hydroxy-2'-Deoxyguanosine↗

PubMedDate not supplied

Design and anti-HIV-1 activity of hammerhead and hairpin ribozymes containing a stable loop.

Three ribozymes, a hairpin ribozyme (HR112) and two hammerhead ribozymes (RZ115 and RZ119) containing a 5'C(UUCG)G3' loop were designed to cleave the U5 region in the long terminal repeat (LTR) of HIV-1 RNA. The t1/2 values of chemically synthesized substrates mediated by three ribozymes were measured. The transformed CEM cells possessing these three ribozyme-encoding genes were challenged with a HIV-1IIIB strain, and two of these three ribozymes, HR112 and RZ119, were shown to possess strong anti-HIV-1 activity.

Anti-HIV Agents↗

PubMedDate not supplied

Rapid RNA cleavage using an antisense system with two terpyridine.Cu(II) complexes.

An effective approach to promote sequence-specific RNA cleavage by an antisense 2'-O-methyloligonucleotide with a terpyridine.Cu(II) complex attached at the 5'-end was developed. We have synthesized a Cu(II) complex 3'-conjugate, which when used in a tandem fashion, greatly enhanced the RNA cleavage efficiency.

Copper↗

PubMedDate not supplied

Expression of the amino terminal part of synthetic human growth hormone gene and somatomedin-like activity of expressed protein.

We have constructed three different plasmids containing parts of the human growth hormone gene using chemically synthesized oligomers and cloned them for the purpose of expressing them in Escherichia coli. AB, B and BC gene segments corresponding to ABhGH (residue 1-138), BhGH (residue 44-138) and BChGH (residue 44-192) were placed under the control of a tryptophan promoter in the expression vector. Upon induction with 3-indolylacrylic acid, ABhGH accumulated in cells but the BhGH and BChGH segments were not detected appreciably. Northern blotting analysis showed that the amount of mRNA transcribed from the AB gene segment was about ten-fold higher than that from the B or BC gene segment. ABhGH was found to have insulin-like growth factor I (IGF-I) activity, which could be explained by the hydrophilicity curves of these proteins.

Base Sequence↗

PubMedDate not supplied

A case of hepatocellular carcinoma with the sign of Leser-Trelat: a possible role of a cutaneous marker for internal malignancy.

A rare case of hepatocellular carcinoma who developed the complication of the sign of Leser-Trelat is reported. The patient, a 57-year-old male, visited our hospital with complaints of generalized malaise and anorexia. A diagnosis of hepatocellular carcinoma was made based on elevated alpha-fetoprotein measurement, ultrasonography, and hepatic arteriography findings. Chest x-ray film suggested pulmonary metastases of hepatocellular carcinoma. Thereafter, complications of the seborrheic keratosis developed in the trunk and the skin lesion was diagnosed as the sign of Leser-Trelat associated with hepatocellular carcinoma. The patient died of pneumonia 9 months after development of the sign of Leser-Trelat.

Biomarkers, Tumor↗

PubMedDate not supplied

Sjögren's syndrome and mixed connective tissue disease.

We investigated the clinical significance of the close association of Sjögren's syndrome (SS) with mixed connective tissue disease (MCTD) by analyzing the clinical manifestations, sialographic findings and immunological parameters of MCTD and primary SS. The prevalence of sialectasia or SS in MCTD was significantly higher than in any other connective tissue diseases. The prevalence of Raynaud's phenomenon, swollen fingers, arthralgias, lymphadenopathy, sclerodactyly, muscle weakness, fever and erythema was significantly higher in MCTD than in primary SS. There were no significant differences between these manifestations in MCTD patients with sialectasia or SS, and those in MCTD patients without sialectasia or SS. Although the levels or prevalence of the erythrocyte sedimentation rate, CRP, antinuclear factor, anti-DNA antibody and anti-RNP antibody were significantly greater in MCTD than in primary SS, there were no significant differences in the levels or the prevalence of laboratory abnormalities between MCTD with sialectasia or SS, and MCTD without sialectasia or SS. Moreover, there was a strict dissociation between the occurrence of anti-RNP antibody and anti-SS-B antibody both in MCTD and primary SS. These results suggest that the association of secondary SS or sialectasia in MCTD, although more common than in other connective tissue diseases, is merely a consequence of MCTD and does not influence the clinical course of MCTD.

Adult↗