Biomedical subjects

T Shiba

Publications and source records attributed to T Shiba.

At least 19 recordsLinked to original sources

PubMed1992

Efficient amplification of Drosophila simulans copia directed by high-level reverse transcriptase activity associated with copia virus-like particles.

The number of retrotransposon copia per genome in Drosophila melanogaster cultured cells is two to three times higher than that in D. melanogaster embryo cells. Here, we have found that the genome of the related species, Drosophila simulans, contains in cultured cells more efficiently amplified copia DNA (approximately ten fold). Furthermore, we analyzed copia virus-like particles (VLPs) prepared from D. melanogaster and D. simulans cultured cells, which contain copia RNA and reverse transcriptase (RT) activity, and thus, play a major role in copia replication. The RT activity associated with the D. simulans VLPs was 25 times higher than that associated with the D. melanogaster VLPs. Taken together with the fact that copia is believed to transpose through an RNA intermediate, these results suggest that the amplification of copia DNA should relate to copia RNA-mediated transposition, and the higher RT activity associated with the D. simulans VLPs would lead to the efficient amplification of copia DNA. In a comparison between D. melanogaster and D. simulans copia nucleotide (nt) sequences, five nt substitutions, which cause the respective amino acid changes, were found in the copia RT-coding region. Polymerase chain reaction direct sequencing showed that these five substitutions are the vast majority in each Drosophila species. The substitutions, therefore, may be responsible for the high level of the RT activity associated with the D. simulans VLPs.

Amino Acid Sequence↗

PubMed1992

Metal-inducible activities of metallothionein promoters in fish cells and fry.

Activities of trout metallothionein-A promoter and mouse metallothionein-I promoter in fish cells and fry were examined using the chloramphenicol acetyltransferase (CAT) gene as a reporter. Transfection and transient CAT assays on a trout liver cell line indicated that both promoters were inducible by metals, including zinc. In vivo activities of both promoters were examined using microinjection system into medaka eggs. Some of the hatched fry were exposed to zinc and their CAT activities were assayed. Both promoters revealed considerable activities after exposure to zinc while only weak activities were detected in unexposed fry.

Animals↗

PubMed1992

Structure of Drosophila virilis glycerol-3-phosphate dehydrogenase gene and a comparison with the Drosophila melanogaster gene.

The complete glycerol-3-phosphate dehydrogenase gene of Drosophila virilis isolated by screening with alpha GPDHM cDNA of the adult fly was sequenced. The gene contains eight exons spread over a total of approximate 8 kb DNA. Its exon/intron organization is identical to that of D. melanogaster. A single transcription initiation site was determined by primer extension. The stop codons are located at the 3' end of each of the exons 6 to 8. TATA and CAAT boxes are present upstream of the transcriptional start site. Adult alpha GPDH protein is encoded by exons 1 to 6 and exon 8. Comparison of the sequence with that of D. melanogaster showed that the homology of the nucleotide sequence of the coding region is 85% and that the homology of the amino acid sequence is 98%. On the contrary, the non-coding region is quite different in length and nucleotide sequence.

Animals↗

PubMed1992

Production of a unique multi-lamella structure in the nuclei of yeast expressing Drosophila copia gag precursor.

Drosophila retrotransposon copia produces virus-like particles (VLPs) in the nuclei of cultured Drosophila cells. The VLPs contain copia RNA and reverse transcriptase activity, and thus, play a major role in copia replication. Here we have expressed the copia gag polyprotein precursor in yeast. The precursor, which includes copia protease itself, showed correct autoprocessing to produce a unique multi-lamella structure in the nuclei of the yeast cells. This expression system should be useful for the analysis of nuclear localization of the major copia VLP protein, and furthermore, would provide important information concerning the mechanism of copia VLPs formation.

Animals↗

PubMed1992

Evaluating retinal circulation using video fluorescein angiography in control and diabetic rats.

Video fluorescein angiography has been used to evaluate retinal circulatory parameters in diabetic and non-diabetic Sprague-Dawley rats. Video fluorescein angiograms were recorded from the retina using a modified retinal fundus camera following a 5 ul bolus injection of sodium fluorescein dye into the jugular vein. Retinal circulatory parameters were measured using computer assisted image analysis. These analyses were performed on 25 diabetic rats with 1 week duration of diabetes and 26 matched, non-diabetic, rats. There was a significant (p = .0001) increase in retinal Mean Circulation Time (MCT) in the diabetic group (1.83 +/- 0.40 s) compared to the control group (1.09 +/- 0.27 s). There were no significant differences in arterial or venous diameters comparing diabetic and control groups. In a separate paired experiment, measurements were made from the same animals both before and after one week duration of diabetes. A paired t-test analysis demonstrated significantly increased MCT times in the 6 diabetic animals (p = .001) while there was no significant differences detected in the 4 corresponding control animals. These results indicate that significant increases in retinal circulation times can be measured as early as 1 week after streptozotocin induced diabetes in this animal model.

Animals↗

PubMed1992

[Anti-bacterial zeolite balloon catheter and its potential for urinary tract infection control].

We present here a production of anti-bacterial zeolite balloon catheter and investigated its potential for controlling urinary tract infection. This anti-bacterial balloon catheter showed a bactericidal effect against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli in vitro studies. The antibacterial effects were correlated with the concentration of anti-bacterial zeolite and size of catheter. We tried this catheter for 11 various urological patients who needed a long-term indwelling of a balloon catheter for lower urinary tract obstruction and neurogenic bladder. All patients were already indwelled silicon balloon catheter for 3 to 6 months and suffered with complicated urinary tract infection. Nine patients who had this anti-bacterial zeolite balloon catheter indwelled for 3 to 7 months and exchanged every 2 to 4 weeks, and no patient was taking antibiotics during this trial. Two patients (22.2%) showed good results by the urinary tract infection (UTI) criteria and 5 patients (55.4%) showed good effects by doctor's judgment. This anti-bacterial zeolite balloon catheter might be useful for patients who need long-term balloon catheter indwelling.

Aged↗

PubMed1992

[Prostatic cancer after transurethral resection for benign prostatic hypertrophy].

Five cases of prostatic cancer developed after transurethral resection of prostate for benign hypertrophy are reported. Duration of transurethral resection of prostate (TUR-P) to diagnosis of prostatic cancer ranged from one year and seven months to seven years and two months, on average four years and seven months and frequency of prostatic cancer after TUR-P was estimated at 1.2%. Four of five patients complained of macroscopic hematuria. The cystourethrogram showed the mass protruded in the dilated prostatic urethra or bladder-neck in four patients (80%), a remarkable finding, and four cases were at stage D. Risk of development of prostatic cancer is not decreased even after prostatectomy and prostatic carcinoma diagnosed after TUR-P often advances in stage. Therefore, periodical examinations of the patients who had a prior prostatectomy are very important.

Adenocarcinoma↗

PubMed1991

Stimulation of endothelin-1 gene expression by insulin in endothelial cells.

The present study characterized the regulation of the genetic expression of the vasoactive peptide endothelin-1 (ET-1) by insulin in bovine aortic endothelial cells. By RNA blot analysis, insulin (1.67 x 10(-8) M) increased ET-1 mRNA levels by 2.3-fold over the basal within 10 min and attained a maximum (5.3-fold increase) in 2 h. Dose-response studies showed that a maximum effect of insulin was reached at 1.67 x 10(-8) M although a significant increase can be observed at 1.66 x 10(-9) M. Radioligand receptor studies indicated that the affinity constant for insulin receptors on endothelial cells correlated closely with the dose response observed for ET-1 mRNA. The ET-1 mRNA half-life was estimated with actinomycin D studies to be 20 min in control cells and was not affected by insulin treatment. Moreover, the effects of phorbol 12-myristate 13-acetate (PMA) and insulin were additive in the induction of ET-1 gene expression. When protein kinase C in the bovine aortic endothelial cells was down-regulated by preincubation with 8 x 10(-7) M PMA for 24 or 48 h, insulin was still able to increase ET-1 mRNA levels whereas PMA was ineffective. Using a chloramphenicol acetyltransferase (CAT) fusion plasmid containing the CAT gene and the 5'-flanking region of the ET-1 gene (Lee, M. E., Bloch, K. D., Clifford, J. A., and Quertermous, T. (1990) J. Biol. Chem. 265, 10446-10450), we observed that 1.67 x 10(-8) M insulin increased CAT enzyme activity and mRNA levels. The insulin dose-response curve observed for CAT activity correlated with that observed for ET-1 mRNA levels. These results suggest that insulin stimulates expression of the ET-1 gene at the transcriptional level via its own receptors. This effect is mediated mostly through a protein kinase C-independent pathway, suggesting the existence of an insulin-responsive element in the ET-1 gene 5'-flanking sequence.

Amino Acid Sequence↗

PubMed1991

Attenuation of ischemia-induced regional myocardial acidosis by LP-805, a newly developed vasodilator, in dogs.

The effect of 8-tert-butyl-6,7-dihydropyrrolo[3,2-e]-5-methyl- pyrazolo[1,5-a]pyrimidine-3-carbonitrile (LP-805), a newly developed vasodilator, on myocardial acidosis induced by ischemia was studied in anesthetized open-chest dogs. Ischemia was induced by partially occluding the left anterior descending coronary artery. The coronary flow was artificially reduced to about 1/3 of the original flow. Myocardial pH was measured with a glass micro pH electrode inserted into the left ventricular wall perfused by the occluded artery. Myocardial pH decreased from about 7.5 to about 6.9 after the onset of ischemia and remained at this low level until the occluded coronary artery was released. After 30 min of ischemia, either saline containing 0.1 N HCl or 10, 30 or 100 micrograms/kg of LP-805 was injected intravenously. LP-805 attenuated the decrease in myocardial pH induced by ischemia in a dose-dependent manner. In conclusion, LP-805 may reduce the influence of ischemia on the myocardium.

Acidosis↗

PubMed1991

SOS-inducible DNA repair proteins, RuvA and RuvB, of Escherichia coli: functional interactions between RuvA and RuvB for ATP hydrolysis and renaturation of the cruciform structure in supercoiled DNA.

The ruv operon is induced by treatments that damage DNA and is regulated by the LexA repressor. It encodes two proteins, RuvA and RuvB, that are involved in DNA repair, recombination in RecE and RecF pathways, and mutagenesis. RuvB protein was previously purified and has ATP-binding activity and weak ATPase activity. To study the biochemical properties of RuvA and its interaction with RuvB, we purified RuvA protein to near homogeneity from an over-producing strain. RuvA bound more efficiently to single-stranded DNA than to double-stranded DNA. RuvA bound to DNA greatly enhanced the ATPase activity of RuvB; the enhancing effect of various forms of DNA was in the order of supercoiled DNA greater than single-stranded DNA greater than linear double-stranded DNA. UV irradiation further enhanced the ATPase stimulatory effect of supercoiled DNA dose dependently. The RuvA-RuvB complex has an activity that renatures the cruciform structure in supercoiled DNA. From these experiments and previous work, we infer that the RuvA-RuvB complex may promote branch migration in recombination and may correct irregular structures in DNA, such as cruciforms and hairpins, to facilitate DNA repair using ATP as the energy source.

Adenosine Triphosphatases↗

PubMed1991

Identification of an unusual structure in the Drosophila melanogaster transposable element copia: evidence for copia transposition through an RNA intermediate.

The Drosophila melanogaster transposable element copia is usually 5 kb long with long terminal repeats (LTRs), and its major transcripts are a full-length 5-kb RNA and a 2-kb RNA. We have previously shown that the 2-kb RNA is generated through splicing. Here, we have cloned a genomic intronless copia using an oligodeoxyribonucleotide probe which is specific for the junction of the two exons. The unusual copia is bounded by two LTRs and lacks precisely the intron of the 2-kb copia RNA. Identification of genomic intronless copia strongly suggests that copia transposes through an RNA intermediate. Moreover, we have found that copia virus-like particles (VLPs), in which reverse transcription of copia RNA seems likely to occur, packages the spliced copia RNA much less efficiently than the full-length copia RNA. This result leads to the suggestion that much lower copy number of genomic intronless copia, as compared with that of 'normal' copia, may be responsible for the inefficient packaging of the spliced copia RNA into the VLP.

Animals↗

PubMed1991

Autoprocessing of Drosophila copia gag precursor to generate a unique laminate structure in Escherichia coli.

Drosophila copia protease is likely to be encoded in the gag gene. We have expressed copia gag polyprotein precursor in E. coli. The gag precursor was correctly processed to generate a unique laminate structure in E. coli. The processing was almost completely blocked by a mutation at the putative active site of copia protease, and resulted in accumulation of the precursor. Furthermore, the laminate structure was not found in E. coli expressing the mutant precursor. These results indicate that the protease is involved in cleaving the gag precursor itself. Also, the assembly of copia gag protein should correlate to the autoprocessing of copia gag polyprotein precursor.

Animals↗

PubMed1991

Structure-activity relationships of sarafotoxins: chemical syntheses of chimera peptides of sarafotoxins S6b and S6c.

The three chimera peptides of sarafotoxins S6b (SRTb) and S6c (SRTc), [Thr2]SRTb, [Asn4]SRTb and [Glu9]SRTb, were synthesized chemically. From the comparisons of lethality, vasoconstrictor activity and receptor binding activity of SRTb, SRTa [( Asn13]SRTb), SRTc [( Thr2,Asn4,Glu9,Asn13]SRTb), [Thr2]SRTb, [Asn4]SRTb and [Glu9]SRTb, it appears that the Lys9 to Glu9 substitution greatly diminishes these activities while the Lys4 to Asn4 substitution does not affect them, and the Ser2 to Thr2 substitution or the Tyr13 to Asn13 substitution slightly diminishes these activities. These results suggest that the very low activities of SRTc are caused mainly by the Lys9 to Glu9 substitution, but not by the Ser2 to Thr2 substitution, which was suggested to be responsible for the weak bioactivities of SRTd [( Thr2,Ile19]SRTb).

Amino Acid Sequence↗

PubMed1991

Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure.

Genetic evidence suggests that the Escherichia coli ruvC gene is involved in DNA repair and in the late step of RecE and RecF pathway recombination. To study the biochemical properties of RuvC protein, we overproduced and highly purified the protein. By employing model substrates, we examined the possibility that RuvC protein is an endonuclease that resolves the Holliday structure, an intermediate in genetic recombination in which two double-stranded DNA molecules are linked by single-stranded crossover. RuvC protein cleaves cruciform junctions, which are formed by the extrusion of inverted repeat sequences from a supercoiled plasmid and which are structurally analogous to Holliday junctions, by introducing nicks into strands with the same polarity. The nicked ends are ligated by E.coli or T4 DNA ligases. Analysis of the cleavage sites suggests that DNA topology rather than a particular sequence determines the cleavage site. RuvC protein also cleaves Holliday junctions which are formed between gapped circular and linear duplex DNA by the function of RecA protein. However, it does not cleave a synthetic four-way junction that does not possess homology between arms. The active form of RuvC protein, as studied by gel filtration, is a dimer. This is mechanistically suited for an endonuclease involved in swapping DNA strands at the crossover junctions. From these properties of RuvC protein and the phenotypes of the ruvC mutants, we infer that RuvC protein is an endonuclease that resolves Holliday structures in vivo.

Amino Acid Sequence↗

PubMed1991

Thoracoabdominal aortic aneurysm associated with Marfan's syndrome--report of a case.

A case of Marfan's syndrome associated with thoracoabdominal aortic aneurysm and mitral regurgitation in a 29 year old male is reported herein. The aneurysm was replaced with a Y-shaped graft using Crawford's technique, while the major branches of the abdominal aorta were separately cannulated from inside the aneurysm and perfused via partial extracorporeal circulation using a left femoro-femoral bypass. We found this technique useful in the prevention of tissue ischemia during the operation. The patient's postoperative course was uneventful and he has encountered no problems in the year and half since his operation.

Adult↗

PubMed1991

Properties of the Escherichia coli RuvA and RuvB proteins involved in DNA repair, recombination and mutagenesis.

The ruvA and ruvB genes constitute an operon, which is regulated by the SOS system and involved in DNA repair, recombination and mutagenesis. RuvA protein binds to both single-stranded and double-stranded DNA. RuvB protein has weak ATPase activity. RuvA bound to DNA greatly enhances ATPase activity of RuvB. UV-irradiation to supercoiled DNA further enhances the stimulatory effect of RuvA on the RuvB ATPase activity. In the presence of ATP the RuvA-RuvB complex has an activity that renatures cruciform structures formed by heating and gradually cooling supercoiled DNA with an inverted repeat. These findings suggest that the RuvA-RuvB complex interacts with an irregular conformation in damaged DNA and induces conformational changes in DNA using energy provided by ATP hydrolysis, so that it facilitates DNA repair, recombination and error prone replication.

Bacterial Proteins↗

PubMed1991

Synthetic low-toxicity muramyl dipeptide and monophosphoryl lipid A replace Freund complete adjuvant in inducing growth-inhibitory antibodies to the Plasmodium falciparum major merozoite surface protein, gp195.

The Plasmodium falciparum major merozoite surface protein (gp195) is a protective antigen against lethal malaria. However, increasing evidence indicates that the efficacy of a malaria vaccine will require a strong adjuvant that is safe for human use. We compared the efficacies of two low-toxicity synthetic immunomodulators, B30-MDP (a lipophilic muramyl dipeptide derivative) and LA-15-PH (a synthetic equivalent of monophosphoryl lipid A), with that of Freund complete adjuvant (FCA) in eliciting an antibody response to gp195. Rabbits were immunized with native gp195 and B30-MDP, LA-15-PH, or the two in combination, with liposomes as the vehicle. Aluminum hydroxide and FCA were used as reference adjuvants. Results showed that adjuvant formulations based on B30-MDP alone or in combination with LA-15-PH induced high antibody titers to gp195, as compared with FCA. LA-15-PH alone was less effective. Aluminum hydroxide induced significantly lower antibody titers. The functional activity of the rabbit anti-gp195 antibodies induced by different adjuvants was evaluated in an in vitro parasite growth inhibition assay previously shown to correlate with anti-gp195 immunity in the Aotus monkey model. All rabbits immunized with B30-MDP-LA-15-PH and two of three rabbits immunized with B30-MDP alone produced sera that strongly inhibited parasite growth. The degree of growth inhibition was similar to that with FCA. The antibody titers of the rabbits receiving B30-MDP-LA-15-PH strongly correlated with the degree of in vitro growth inhibition. Our findings provided strong evidence that adjuvant formulations based on synthetic B30-MDP and LA-15-PH can replace FCA as adjuvants in stimulating protective immunity specific for gp195.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1991

Deletion analysis of the 5' untranslated leader sequence of tobacco mosaic virus RNA.

To determine the sequences essential for viral multiplication in the 5' untranslated leader sequence of tobacco mosaic virus RNA, mutant TMV-L (a tomato strain) RNAs which carry several deletions in this 71-nucleotide sequence were constructed by an in vitro transcription system and their multiplication was analyzed by introducing mutant RNA into tobacco protoplasts by electroporation. Large deletions of the sequence from nucleotides 9 to 47 or 25 to 71 abolished viral multiplication; when about 10-nucleotide deletions were introduced throughout this 5' leader sequence, only deletion of the sequence from nucleotides 2 to 8 abolished detectable viral multiplication. This mutant RNA, however, directed the synthesis of the 130,000-molecular-weight protein in a rabbit reticulocyte lysate in vitro translation system, and consequently this 5'-proximal portion appears likely to be essential for replication.

Base Sequence↗