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Biomedical subjects

T Shiba

Publications and source records attributed to T Shiba.

At least 19 recordsLinked to original sources

Decrease in culturability of Vibrio cholerae caused by glucose.

The culturability of Vibrio cholerae O1 serotype Inaba strain 569B was decreased by the addition of glucose to cell suspensions in starvation media. A similar effect was observed with sucrose, maltose, and fructose. We term this inhibitory effect glucose shock. It was not observed with arabinose or xylose or with carboxylates, such as acetate and pyruvate. No acidification of the medium occurred in the presence of these carbohydrates. Glucose shock was prevented by the addition of nitrogen or phosphorus sources. In the presence of phosphate, the bacterium produced formic acid from glucose. The phenomenon of glucose shock was also observed in V. cholerae O1 serotype Inaba strain RIMD 2203082 but not in strain RIMD 2203088 (O1 Inaba), IID 936 (O1 Ogawa), or RIMD 2214034 (non-O1). The culturability of Escherichia coli, Enterobacter aerogenes, and Listonella anguillarum did not decrease in starvation media with added glucose. Hence, the phenomenon should have ecological significance in determining the distribution of bacteria in marine ecosystems in situations where carbohydrates are abundant, but nitrogen and phosphorus are limiting.

Glucose

Purification and characterization of the Pseudomonas aeruginosa NfxB protein, the negative regulator of the nfxB gene.

The protein NfxB, involved in conferring resistance to quinolones in Pseudomonas aeruginosa, has a helix-turn-helix motif which is similar to that of other DNA-binding proteins. It appears to affect the membrane-associated energy-driven efflux of some antibiotics (H. Nikaido, Science 264:382-388, 1994). We constructed a plasmid that overproduced NfxB in Escherichia coli and purified the protein. Two species of NfxB (23 and 21 kDa), which are probably translated from different initiation codons, were isolated. Both proteins are also expressed in vivo in P. aeruginosa, with the 23-kDa NfxB being the major species. NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting. Expression of the phi (nfxB'-lacZ+) (Hyb) gene was repressed in the presence of the nfxB gene product provided by a second compatible plasmid in E. coli. In the P. aeruginosa wild-type strain (PAO2142), NfxB was undetectable by immunoblotting; however, it was detected in the nfxB missense mutant (PK1013E). These results suggested that NfxB negatively autoregulates the expression of nfxB itself. Since the 54-kDa outer membrane protein (OprJ) (N. Masuda, E. Sakagawa, and S. Ohya, Antimicrob. Agents Chemother. 39:645-649, 1995) was overproduced in nfxB mutants, NfxB may also regulate the expression of membrane proteins that are involved in the drug efflux machinery of P. aeruginosa.

Amino Acid Sequence

The gene for a major exopolyphosphatase of Saccharomyces cerevisiae.

The gene encoding a major exopolyphosphatase (scPPX1) in Saccharomyces cerevisiae (H. Wurst and A. Kornberg, J. Biol. Chem. 269:10996-11001, 1994) has been isolated from a genomic library. The gene, located at 57 kbp from the end of the right arm of chromosome VIII, encodes a protein of 396 amino acids. Overexpression in Escherichia coli allowed the ready purification of a recombinant form of the enzyme. Disruption of the gene did not affect the growth rate of S. cerevisiae. Lysates from the mutants displayed considerably lower exopolyphosphatase activity than the wild type. The enzyme is located in the cytosol, whereas the vast accumulation of polyphosphate (polyP) of the yeast is in the vacuole. Disruption of PPX1 in strains with and without deficiencies in vacuolar proteases allowed the identification of exopolyphosphatase activity in the vacuole. This residual activity was strongly reduced in the absence of vacuolar proteases, indicating a dependence on proteolytic activation. A 50-fold-lower protease-independent activity could be distinguished from this protease-dependent activity by different patterns of expression during growth and activation by arginine. With regard to the levels of polyP in various mutants, those deficient in vacuolar ATPase retain less than 1% of the cellular polyP, a loss that is not offset by additional mutations that eliminate the cytosolic exopolyphosphatase and the vacuolar polyphosphatases dependent on vacuolar protease processing.

Acid Anhydride Hydrolases

A gene that is related to SRY and is expressed in the testes encodes a leucine zipper-containing protein.

SRY-related cDNA encoding a protein with a high-mobility-group (HMG) box and a leucine zipper motif, which was designated SOX-LZ, was isolated from a rainbow trout testis cDNA library. Comparison of this cDNA with the mouse homologous cDNA isolated from a testis cDNA library exhibits an overall amino acid sequence identity of 77%, which is in striking contrast to the abrupt loss of amino acid sequence homology outside the HMG box found among mammalian SRY genes. In both rainbow trout and mice, Northern (RNA) blot analyses have revealed the presence of a testis-specific 3-kb-long SOX-LZ mRNA, and this transcript appeared coincidentally with the protamine mRNA, suggesting its expression in the germ line. A recombinant HMG box region protein encoded by SOX-LZ could bind strongly with an oligonucleotide containing an AACAAT sequence, which is also recognized by mouse Sry and Sox-5. Upon cotransfection into CHO cells, SOX-LZ transactivated transcription through its binding motif when the region including the leucine zipper motif was deleted [SOX-LZ (D105-356)]; however, the intact SOX-LZ failed to transactivate. The intact SOX-LZ could form homodimers through the leucine zipper, which resulted in inhibition of DNA binding by the HMG box, while SOX-LZ (D105-356), which was incapable of dimerization, showed specific binding with the AACAAT sequence. Thus, the repressed transactivation of the intact SOX-LZ in CHO cells was primarily attributable to the low level of DNA binding of SOX-LZ homodimers.

Amino Acid Sequence

Cloning and analysis of the gene encoding lectin from the acorn barnacle Megabalanus rosa.

In the acorn barnacle Megabalanus rosa, two types of galactose-binding C-type lectins (BRA-2 and BRA-3) have been identified. Here, we report the isolation and characterization of BRA-2 cDNA and genomic clones. In contrast to the BRA-3 gene, which consists of four exons, BRA-2 is encoded by a single exon, implying differences in the physiological roles of the two lectins.

Amino Acid Sequence

Characterization of phorbol ester-stimulated serine phosphorylation of the human insulin receptor.

Phorbol 12-myristate 13-acetate (PMA)-stimulated phosphorylation of the human insulin receptor (IR) was characterized and compared in two cell types of different lineage: normal rat kidney epithelial (NRK) cells and Chinese hamster ovary (CHO) fibroblasts. PMA stimulation increased IR beta-subunit phosphorylation to 252 +/- 43 and 25- +/- 47% (+/- S.D.) of the unstimulated control in NRK and CHO cells respectively. Tryptic phosphopeptide analysis by Tricine/SDS/PAGE revealed significant differences in the PMA-stimulated phosphorylation of the IR in these two cell types. This phosphorylation of the IR was predominantly located in two tryptic phosphopeptides, and these phosphopeptides were absent in an IR mutant truncated by 43 C-terminal amino acids. The major PMA-stimulated tryptic phosphopeptide from in vivo-labelled CHO/IR was immunoprecipitated with an antibody against residues Ser1315 to Lys1329, and this precipitation was blocked with excess unlabelled peptide containing this sequence. Radiosequencing by manual Edman degradation revealed that this tryptic phosphopeptide was phosphorylated at Ser1315. This PMA-stimulated phosphorylation did not inhibit autophosphorylation of the IR in vivo. These results demonstrate that PMA-stimulated phosphorylation of the IR can exhibit significant differences when expressed in different cell types, and that Ser1315 is a major PMA-stimulated phosphorylation site on the human IR.

Amino Acid Sequence

Cellular and molecular abnormalities in the vascular endothelium of diabetes mellitus.

Diabetic vascular complications affect both micro- and macrovasculature, primarily in the retina, renal glomeruli, and multiple sites in the macrovessels. This review presents a summary of the abnormal function found in vivo and in cultured vascular cells exposed to elevated levels of glucose. We also discuss the various biochemical hypotheses that have been proposed to explain the adverse effects of hyperglycemia on vascular cells.

Diabetic Angiopathies

Normalization of retinal blood flow in diabetic rats with primary intervention using insulin pumps.

PURPOSE: Prior results have demonstrated a significant reduction in retinal blood flow in streptozotocin (STZ)-induced diabetic rats. These studies were extended to investigate whether retinal blood flow changes, in the diabetic rat model, could be prevented with strict glycemic control using insulin pumps. Retinal blood flow changes also were measured during hyperoxia and after intravitreal histamine infusion to validate the methodology. METHODS: Retinal blood flow changes were measured using video-based fluorescein angiography and computer-assisted image analysis. A total of 48 male Sprague-Dawley and 9 Brown Norway rats were used in these experiments. Retinal blood flow after primary insulin intervention was evaluated in diabetic rats implanted with mini-osmotic insulin pumps within 24 hours of STZ-induced diabetes. Diabetic rats, not treated with insulin, were used for comparison. RESULTS: Hyperoxia caused a significant (P = 0.001) reduction (54%) in retinal blood flow, whereas intravitreal infusion of 10(-3) M histamine caused a significant (P = 0.009) increase (108%) in retinal blood flow. Retinal blood flow in the primary insulin intervention group, after 1 week of diabetes, was not statistically different from retinal blood flow of nondiabetic controls as measured at baseline from the animals used in the hyperoxia and histamine infusion experiments. In contrast there was a significant (P = 0.0001) retinal blood flow reduction in the untreated diabetic group. CONCLUSIONS: The results showed that the local effect of histamine and hyperoxia on the retina produced the expected responses in retinal blood flow, further confirming the validity of the methodology. Primary insulin intervention demonstrated that strict glycemic control initiated immediately after induction of diabetes was sufficient to maintain normal retinal blood flow in STZ-induced diabetic rats.

Animals

[Late recurrence of renal cell carcinoma: report of three cases].

A 70-year-old woman underwent a left radical nephrectomy. The pathological examination revealed renal cell carcinoma (RCC), grade 1 and no lymph node metastasis. She suffered a recurrence in the renal fossa 11 years after the operation. The doubling time of the recurrent lesion was calculated to be very long for the cancer; about 1.3 years. Another case was in a 61-year-old woman, who underwent a right radical nephrectomy. The pathological examination revealed RCC, grade 1 with vascular invasion and no lymph node metastasis. Eleven years postoperatively she developed metastases in both lungs. The other case was in a 39-year-old woman, who underwent right radical nephrectomy. The pathological examination revealed RCC, grade 1 with vascular invasion and no lymph node metastasis. Eight years postoperatively she developed a recurrence in the left kidney. The doubling time of the recurrent lesion was about 0.9 year. All three patients responded to conservative therapy including interferon. Taking those doubling times into account, the recurrent lesions in the first and third cases seemed to have been growing very slowly and not rapidly immediately before clinically evident recurrence. Patients with a low grade RCC must have life-long followup.

Adult

X-Neu5Ac: a novel substrate for chromogenic assay of neuraminidase activity in bacterial expression systems.

A chromogenic substrate 1, 5-bromo-4-chloroindol-3-yl 5-acetamido-3,5-dideoxy-alpha-D-glycero-D-galacto-2-nonulopyranosidon ic acid (X-Neu5Ac), has been synthesized to facilitate the screening of bacterial colonies or plaques for the detection of either natural or mutant neuraminidase activity. Substrate 1 was hydrolyzed by neuraminidase isolated from Clostridium perfringens to release a halogenated indol-3-ol 2 that undergoes rapid aerobic oxidation to form the dark blue pigment, 5,5'-dibromo-4,-4'-dichloroindigo 3. Preliminary kinetic studies indicate that this compound is a good substrate (Km 0.89 x 10(-3) M) for neuraminidase and is quite stable under identical conditions in the absence of enzyme. These results suggest that X-Neu5Ac 1 can be useful to screen for bacterially-encoded enzyme production directly on agar plates.

Chromogenic Compounds

[Clinical study of allylestrenol (Perselin) on patients with prostatic hypertrophy].

The efficacy and safety of allylestrenol were studied in 22 patients with benign prostatic hypertrophy. A 25 mg allylestrenol tablet (Perselin tablet) was administered twice a day. A significant decrease in frequency of nocturnal urination was observed and improvement rates of subjective symptoms, such as sensation of residual urine, delay in start of urination, straining during urination and strength of urinary stream, were 59.1-68.2%. The rate of decrease of maximum area of transverse plane of prostate estimated by transrectal sonography was 13.2% and presumed circle area ratio (PCAR) improved significantly. The incidence of total side effects was 22.7%, whereas a decrease in sexual potency was observed in only 4.5% of the cases. The final global improvement rating of allylestrenol was 72.2% and the rate of usefulness was estimated in 63.6% of the patients. The present findings confirmed that allylestrenol is a useful and safe drug for the treatment of benign prostatic hypertrophy.

Administration, Oral

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

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

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

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

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

[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

[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