Formation of a catalytically active dimer by tRNA(Val)-driven short ribozymes.

A minizyme is a hammerhead ribozyme with a short oligonucleotide linker instead of stem/loop II. Minizymes with low activity as monomers form active dimeric structures with a common stem. We explored the use of dimeric minizymes as gene-inactivating agents by placing minizymes under the control of a tRNA(Val) promoter. The tRNA(Val) portion of the transcript did not hinder dimerization as the tRNA-embedded minizyme formed an active dimeric structure. The cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNA(Val)-embedded conventional hammerhead ribozyme. The tRNA(Val)-driven minizyme inhibited reporter gene activity (95%) whereas the tRNA(Val)-driven hammerhead ribozyme resulted in approximately 55% inhibition.

Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3' end can be exported efficiently and specifically to the cytoplasm in mammalian cells.

Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNA(Val)-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3' end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5' and 3' ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells.

Control of the functional activity of an antisense RNA by a tetracycline-responsive derivative of the human U6 snRNA promoter.

In an effort to develop a regulatable derivative of the promoter of the human gene for U6 snRNA, we generated several constructs composed of the human U6 snRNA promoter and sequences derived from the gene for the tetracycline operator of a prokaryotic tetracycline resistance transposon. One of the constructs had strong transcriptional activity in the presence of tetracycline that was equivalent to 80% of the activity of the wild-type promoter. Furthermore, the transcriptional activity was almost completely repressed in the absence of tetracycline. Transcriptional activity became detectable within 4 hr after the addition of tetracycline to the culture medium. We used this system to control the functional activity of an antisense RNA for a chimeric gene derived from genes for the epidermal growth factor receptor (EGFR) and green fluorescent protein (GFP). A plasmid that expressed the chimeric gene and a plasmid that expressed the antisense RNA under the control of the inducible U6 promoter were used to cotransfect HeLa cells that were producing the tetracycline repressor protein (Tet R). Addition of tetracycline to the culture medium 12 hr after transfection resulted in the almost complete disappearance of the fluorescent signal due to the chimeric protein within 24 hr. Our results suggest that this expression system might be a useful tool for controlling the expression of functional RNAs, such as aptamers and antisense RNAs, both in basic research and in gene therapy.

Ribozymes: from mechanistic studies to applications in vivo.

The hammerhead ribozyme belongs to the class of molecules known as antisense RNAs. However, because of short extra sequences that form the so-called catalytic loop, it can act as an enzyme. Since the catalytic domain captures magnesium ions and magnesium ions can cleave phosphodiester bonds, hammerhead ribozymes are recognized as metalloenzymes. In general, the cleavage of phosphodiester bonds involves acid/base catalysis, with proton transfer occurring in the transition state. When the possibility of such a proton-transfer process was examined by measuring solvent isotope effects, it became apparent that no proton transfer occurs in the transition state during reactions catalyzed by a hammerhead ribozyme. It is likely, therefore, that hammerhead ribozymes exploit the general double-metal-ion mechanism of catalysis, with Mg2+ ions coordinating directly with the attacking and leaving oxygen moieties. Since the hammerhead ribozyme is one of the smallest RNA enzymes known and has potential as an antiviral agent, thus ribozyme has been extensively investigated for applications in vivo. Ribozymes are described that have possible utility as agents against HIV-1.

A simple assay system for examination of the inhibitory potential in vivo of decoy RNAs, ribozymes and other drugs by measuring the Tat-mediated transcription of a fusion gene composed of the long terminal repeat of HIV-1 and a gene for luciferase.

Nucleic acid-based drugs, including antisense RNA and DNA, ribozymes and decoys appear to have potential for the suppression of the expression of specific genes. To allow the examination of the potential of such agents in vivo as anti-HIV drugs in standard laboratories, where facilities for handling live virions are not available, we constructed a simple assay system (HIV-1 model) that allows measurement of the extent of inhibition of Tat-mediated transcription of HIV-1 by nucleic acid-based drugs and other agents. In cells that harbor a stable chimeric long terminal repeat (LTR)-Luc construct (a fusion gene consisting of the LTR of HIV-1 and the gene for luciferase), total luciferase activity in an aliquot of cell lysate is dose- and promoter-dependent on transfection with a Tat expression plasmid, reflecting the character of the LTR promoter of HIV. When HeLa cells were co-transfected with the Tat expression plasmid and another plasmid that encoded the U6 promoter or the promoter of the gene for tRNA(Val) linked to the trans-activating response (TAR) sequence, total luciferase activity was inhibited by 60 or 40%, respectively. The inhibition was also dependent on the dose of the TAR expression plasmid. These results demonstrate the usefulness of this simple assay system for detection of the efficacy of a decoy RNA or a ribozyme in vivo, without a requirement for HIV-infected cells, by measurement of luciferase activity in vitro.

Familial hypercholinesterasemia.

A case of familial hypercholinesterasemia was presented. Cholinesterase isoenzyme study revealed the extra C5 band nearer to the cathode than C4 on the gradient polyacrylamide gel electrophoresis in 6 out of 8 subjects in the family. No significant difference in the effect of inhibitors and activator on serum cholinesterase activity of the family with hypercholinesterasemia was found compared to that of the normal control. It was considered to be clinically useful for differential diagnosis of patients with elevated serum cholinesterase to find subjects with familial hypercholinesterasemia.

[Acute myocardial infarction without elevation of CK activity: analysis of CK-MM protein and gene of CK-MM].

A 56 year old female patient had acute myocardial infarction which was confirmed by autopsy. However the patient showed no elevation of CK activity in the serum. We proved the deficiency of CK-MM protein in her serum as well as in the myocardial tissues. To further elucidate the molecular mechanism we isolated and characterized genomic DNA and cDNA of CK-MM from the myocardial tissue of the patient and demonstrated the depression of mRNA of CK-MM which might be related with the deficiency of CK-MM protein. Point mutation at codon 54 [Exon 2, GAC (Asp)-->GGC(Gly)] was also demonstrated on the beta-sheet of CK-MM. Association of point mutation with depressed CK-MM is to be clarified.

[Measurement of serum PSA values by DELFIA PSA and its clinical significance on diagnosis and follow-up of prostate cancer patients].

Serum prostate specific antigen (PSA) values detected by DELFIA PSA were evaluated for usefulness in the diagnosis and follow-up of patients with prostate cancer. The system is time-resolved fluoroimmunoassay using europium as a tracer, which has a detectable range of 0.10-500 ng/ml with a small sample volume (25 microliters) and reliable quality control data. Furthermore, serum PSA values detected by the assay were equivocal to those detected by Tandem-R PSA. From the mean +3 S.D. of serum PSA values obtained on 227 normal males, 1.98 ng/ml was decided as an upper normal level. Serum PSA values in benign prostatic hyperplasia (BPH) (n = 69) and prostate cancer (n = 86) patients were statistically higher than those in normal males. However, when 1.98 ng/ml was used as a cut-off value, the false positive rate in BPH cases elevated up to 80%. Therefore, in the differential diagnosis of prostate cancer and BPH, we recommend 11.7 ng/ml (mean + S.D. in BPH cases) as a cut-off value, in which sensitivity is 72.1%, 88.4% are true negative in BPH, and efficacy is 79.4%. Serially determined serum PSA values in following up the patients with prostate cancer were confirmed to be highly effective for diagnosing recurrence and evaluating treatment responses. These findings suggest that DELFIA PSA is a useful tool for determination of serum PSA values.

[Measurement of serum tissue polypeptide antigen (TPA) in patients with malignant tumor using prolifigen TPA-M "Daiichi" kit].

Serum tissue polypeptide antigen (TPA) was measured using a newly developed Prolifigen TPA-M "Daiichi" kit in 1,236 healthy subjects, 2,867 patients with malignant tumors, and 901 with benign diseases. Because 94.0% of healthy subjects had serum TPA under 70 U/l, the cut-off value was set at 70 U/l. Serum TPA was elevated in more than 50% of patients with head and neck cancer, lung cancer, liver cancer, gallbladder or bile duct cancer, pancreatic cancer, colorectal cancer, ovarian cancer, and prostate cancer. The overall positive rate in malignant tumors was 55.5%. Serum TPA was higher in advanced cancer than in earlier stage cancer, and decreased after the resection of the tumor. The false positive rate in benign diseases was 31.3%. ROC analysis revealed the usefulness of TPA as a tumor marker in many cancers. The correlation coefficient between TPA and CYFRA 21-1, and between TPA and TPSA, was 0.747 and 0.694, respectively. In conclusion, measurement of serum TPA using the new kit is useful in the management of patients with various malignant tumors.

[Gamma Glutamyltransferase].

Gamma-Glutamyltransferase (GGT), formerly called gamma-glutamyltranspeptidase, is predominantly a membrane-bound enzyme. The estimation of enzyme activity in serum is useful in monitoring hepatobiliary complaints. The electrophoresis with surfactant (Triton X-100) developed by the authors demonstrates five distinct bands of enzyme activity in the serum from patients with hepatitis. These bands are called isoenzyme GGT1 to 5 from anode to cathode, respectively. Four isoenzymes GGT2 to 5, except GGT1 are demonstrated in normal adult serum. The affinity electrophoresis is more variable to identify the hepatoma associated isoenzyme, namely HA-GGT. Concanavalin A used in the method has no affinity with HA-GGT and this isoenzyme is separated from GGT2. The diagnostic sensitivity and specificity for the measurement of HA-GGT were 58% and 83%, respectively.

Activities of HIV-RNA targeted ribozymes transcribed from a 'shot-gun' type ribozyme-trimming plasmid.

We have constructed a "shot-gun" type ribozyme-trimming system. By concatenating several units, each consisting of a trans-acting ribozyme (targeted to HIV-RNA) and cis-acting ribozymes (trimming 5'- and 3'-ends of the trans-acting ribozyme), several kinds of trans-acting ribozymes can be liberated upon transcription and self-cleavage. Since each liberated HIV-RNA-targeted ribozymes can work independently, they can simultaneously cleave HIV-RNA at several different sites. Ribozymes were targeted at relatively conserved GUC-containing sites at LTR, gag and tat regions.

A familial study of C5+cholinesterase and its frequency in the normal population.

A case of familial hypercholinesterasemia was presented. A cholinesterase isoenzyme study revealed the extra C5 band nearer to the cathode than C4 on the gradient polyacrylamide gel electrophoresis in 6 out of 8 subjects in the family. No significant difference in the effect of inhibitors and activator was found when compared with the normal control. We investigated the frequency of variant with C5+cholinesterase in normal healthy subjects. The frequency of the extra C5 band found in the normal healthy population in Japan was 1.2%. It was considered to be clinically useful for differential diagnosis of patients with elevated serum cholinesterase to find subjects with familial hypercholinesterasemia.