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Biomedical subjects

K Hirano

Publications and source records attributed to K Hirano.

At least 307 records · Page 17Linked to original sources
PubMed1997

Structural analysis of nucleic acids by using fluorescence resonance energy transfer (FRET).

We examined changes in the extent of fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In our system, FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. While, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. It should help us to estimate the suitability of designed functional molecules, such as antisense DNA and RNA and ribozymes, that target to specific sites.

DNA↗
PubMed1996

Interaction of protein phosphatase type 1 with a splicing factor.

A gizzard cDNA library was screened by the two-hybrid system using as bait the delta isoform of the catalytic subunit of protein phosphatase 1 (PP1delta). Among the proteins identified was a fragment of the polypyrimidine tract-binding protein-associated splicing factor (PSF) and for 242 residues was 97.1% identical to the human isoforms. Binding of PSF and PP1delta was confirmed by inhibition of phosphatase activity and by an overlay technique. The PP1delta binding site was contained in the N-terminal 82 residues of the PSF fragment. PSF may therefore act as a PP1 target molecule in the spliceosome.

Amino Acid Sequence↗
PubMed1996

Interactions and properties of smooth muscle myosin phosphatase.

Interactions of the type 1 phosphatase catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MBP) were compared using affinity columns. In the absence of ATP, MBP bound to dephosphorylated myosin, heavy meromyosin (HMM), and subfragment 1. In contrast, PP1c was not bound. In the presence of ATP, the binding of MBP occurred only with phosphorylated protein. The interaction of MBP with phosphorylated proteins also was demonstrated using thiophosphorylated proteins as competitive inhibitors. Kinetics parameters were determined. With phosphorylated light chains (P-LC20), the major difference between PP1c and MBP was a lower K(m) for the latter. With myosin, MBP showed a marked increase in kcat, compared to PP1c. ATP did not affect these parameters. To investigate the role of the large phosphatase subunit, two recombinant proteins representing the N-terminal two-thirds of the molecule were expressed. These activated PP1c, and activation was maximum at approximately an equimolar ratio. The equimolar mixture of recombinant fragment and PP1c exhibited K(m) values similar to MBP and increased kcat values, compared to PP1c alone. An affinity column was prepared using the recombinant fragment. Phosphorylated HMM and P-LC20 were bound in the presence and absence of ATP. The interaction of P-LC20 was not ATP-dependent. Dephosphorylated HMM did not bind in the presence of ATP. The N-terminal fragment of the large subunit also contained a binding site for PP1c. These results indicate that the N-terminal portion of the large subunit of MBP contained binding sites for P-LC20 and PP1c.

Adenosine Triphosphate↗
PubMed1996

Complete amino acid sequence and identification of the platelet glycoprotein Ib-binding site of jararaca GPIb-BP, a snake venom protein isolated from Bothrops jararaca.

Jararaca GPIb-BP, a snake venom protein composed of alpha and beta subunits purified from Bothrops jararaca, binds to platelet glycoprotein (GP)Ib and functions as a receptor blocker for von Willebrand factor binding to GPIb (Fujimura, Y., Ikeda, Y., Miura, S., Yoshida, E., Shima, H., Nishida, S., Suzuki, M., Titani, K., Taniuchi, Y., and Kawasaki, T. (1995) Thromb. Haemostasis 74, 743-750). We present here the entire 142- and 123-residue amino acid sequence of the respective alpha and beta subunits and also demonstrate that the platelet GPIb-binding site resides on the beta and not on the alpha subunit based on an enzyme-linked immunosorbent assay using biotin-labeled jararaca GPIb-BP and competing ligands. Sequences of the alpha and beta subunits were determined by analysis of the intact S-pyridylethylated proteins and their peptides generated by digestion with Achromobacter protease I, Staphyloccocus aureus V8 protease, pepsin, endoproteinase Asp-N, or L-1-tosylamino-2-phenylethyl chloromethyl ketone-trypsin. A 38-39% identity of amino acid sequence between the alpha and beta subunits of jararaca GPIb-BP was observed, as well as a high degree of sequence identities (38-64%) with the respective subunits of botrocetin (Usami, Y., Fujimura, Y., Suzuki, M., Ozeki, Y., Nishio, K., Fukui, H., and Titani, K (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 928-932) and the beta-chain of echicetin (Peng, M., Holt, J. C., and Niewiarowski, S. (1994) Biochem. Biophys. Res. Commun. 205, 68-72).

Amino Acid Sequence↗
PubMed1996

Targeted disruption of the mouse apobec-1 gene abolishes apolipoprotein B mRNA editing and eliminates apolipoprotein B48.

A site-specific C to U editing reaction modifies nuclear apolipoprotein B100 (apoB100) mRNA, producing apolipoprotein B48 in the mammalian small intestine. This reaction is mediated by a multicomponent enzyme complex, which contains a catalytic subunit, Apobec-1. We have used gene targeting to disrupt mouse apobec-1 in order to establish its requisite importance in apoB mRNA editing and also, in view of its widespread tissue distribution in rodents, as a preliminary indication of other potential roles. Both heterozygous (apobec-1+/-) and homozygous (apobec-1-/-) gene-targeted mice appear healthy and fertile with no alterations in serum cholesterol or triglyceride concentrations. The apobec-1+/- mice demonstrated reduced levels of hepatic apoB mRNA editing. By contrast, levels of small intestinal apoB mRNA editing were indistinguishable in wild-type and apobec-1+/- animals, suggesting that Apobec-1 is expressed in limited quantities in the liver but not in the small intestine. The apobec-1-/- mice lacked detectable levels of Apobec-1 mRNA, expressed only unedited apoB mRNA in all tissues, and contained no apoB48 in their serum, demonstrating that there is no functional duplication of this gene.

APOBEC-1 Deaminase↗
PubMed1996

Primary structure of alboaggregin-B purified from the venom of Trimeresurus albolabris.

The complete amino acid sequences of alpha and beta subunits of alboaggregin-beta are presented. The alpha and beta subunits were separated by reversed-phase HPLC after reduction and S-pyridylethylation, and their sequences were determined by analysis of peptides generated by enzymatic or chemical digestion. The alpha and beta subunits consist of 133 and 123 amino acid residues, respectively. The sequences are highly homologous to each other (41.4% identity) and also to those of the alpha and beta subunits of botrocetin (a von Willebrand factor modulator) and the A and B chains of factor IX/X binding protein from other snake venoms. It is also homologous to C-type lectins with a homodimeric structure, but it shows no lectin-like activity.

Amino Acid Sequence↗
PubMed1996

Detection of undegraded oligonucleotides in vivo by fluorescence resonance energy transfer. Nuclease activities in living sea urchin eggs.

A method was investigated for monitoring the integrity of oligonucleotides in solution and in cells using fluorescence resonance energy transfer between two different fluorochromes attached to a single oligonucleotide. Ten-mer oligodeoxyribonucleotides labeled with fluorescein at one end and with rhodamine X at the other end were used. The oligomer had a specific absorption spectrum with peaks at 497 and 586 nm, which corresponded to fluorescein and rhodamine X, respectively. When excited at 494 nm, the oligomer had a specific fluorescence spectrum with peaks at 523 and 610 nm. The fluorescence intensity at 610 nm was 6-8 times higher than that at 523 nm. After digestion of the oligomer with an endonuclease, the fluorescence at 523 nm increased more than 12-15-fold but its fluorescence peak at 610 nm almost completely disappeared. To examine effects in vivo, sea urchin eggs were injected with a solution of the oligomer and excited with blue light at 470-490 nm. Two fluorescent images, a green image at 520-560 nm and a red image at above 580 nm, were obtained when a single egg was viewed under a fluorescence microscope. The ratio of the intensities of red to green fluorescence decreased in dependence on time after injection of the oligomer. These changes were not observed in eggs that had been injected with a solution of similarly double-labeled, phosphorothioate oligomer. These results indicated that unfertilized sea urchin eggs had nucleolytic activity. Analysis in vitro on supernatant of the egg homogenate indeed demonstrated the existence of nucleases. All together, our results indicate that the integrity of oligonucleotides can be estimated in living cells by monitoring the fluorescence resonance energy transfer of the double-labeled oligonucleotide.

Animals↗
PubMed1996

Substitution of glycine for arginine-213 in extracellular-superoxide dismutase impairs affinity for heparin and endothelial cell surface.

Extracellular-superoxide dismutase (EC-SOD) levels in sera divide into two discontinuous groups: a low-level group below 400 ng/ml and a high-level group above 400 ng/ml [Adachi, Nakamura, Yamada, Futenma, Kato and Hirano (1994) Clin. Chim. Acta 229, 123-131]. Molecular genetic studies have shown that the donors in the high-level group have a single base substitution generating the exchange of glycine for arginine-213 (R213G) in the heparin-binding domain of EC-SOD [Sandström, Nilsson, Karlsson and Marklund (1994) J. Biol. Chem. 269, 19163-19166; Yamada, Yamada, Adachi, Goto, Ogasawara, Futenma, Kitano, Hirano and Kato (1995) Jpn. J. Hum. Genet. 40, 177-184]. The serum EC-SOD level in homozygote subjects was significantly higher than that in heterozygotes and in normal subjects. Serum EC-SOD from heterozygotes and homozygotes had equally decreased affinity for heparin, as judged by heparin-HPLC, as compared with that from normal donors. This result suggests that the serum EC-SOD in heterozygotes was mainly composed of the mutant form which has reduced heparin affinity. On the other hand, fibroblast cells derived from heterozygote subjects generated mRNA of both normal and mutant EC-SOD (m-EC-SOD), and expressed the corresponding proteins. EC-SOD is a tetrameric enzyme, and in heterozygote donors would be heterogeneous with regard to the constitution of normal and mutant subunits. The enzyme form consisting of only mutant subunits, the form with the weakest heparin affinity, can be preferentially driven out to the plasma phase, because EC-SOD in the vasculature exists in equilibrium between plasma and the endothelial cell surface. The binding of m-EC-SOD to bovine aortic endothelial cells was about 50-fold less than that of normal EC-SOD. This result suggests that the binding of m-EC-SOD to vascular endothelial cells is much decreased in vivo, which causes a high level of serum EC-SOD.

Animals↗
PubMed1996

Factors that influence bleeding in superficial-type colorectal tumors: study of endoscopic and histologic correlates.

To clarify the mechanisms responsible for superficial-type colorectal tumor (STT) bleeding, 74 STTs were studied to determine whether their size, shape, surface area features, colorectal site, and level of invasion were correlated with bleeding. The bleeding rate, the proportion of specimens from patients with positive fecal occult blood test (FOBT) results relative to the total number of specimens in each morphological group and probability of an STT being an invasive submucosal carcinoma were positively correlated with tumor size, and bleeding was more common in submucosal than in intramucosal lesions. Logistic regression analysis indicated that the level of submucosal invasion had a significant influence on bleeding. It was also found that the length of erosion on the surface of STTs was significantly greater in bleeding tumors, and that the potential for invasion into the submucosa increased with the area of erosion.

Colon↗
PubMed1996

Dural caroticocavernous fistula with both ophthalmic arteries arising from middle meningeal arteries.

A 60-year-old woman presented with a dural caroticocavernous fistula (CCF) in whom both by ophthalmic arteries arose from the middle meningeal arteries instead of from the internal carotid arteries. Transarterial embolisation of the CCF was contraindicated because of the risk of blindness. Transvenous coil embolisation was therefore performed. Bilateral origin of the ophthalmic artery from the middle meningeal artery is extremely rare.

Brain Diseases↗
PubMed1996

Least microtransfusion from mother to fetus in elective cesarean delivery.

OBJECTIVE: To examine the variability of maternal-fetal microtransfusion in different modes of delivery, as measured by hepatitis B surface antigen (HBsAg) and placental alkaline phosphatase. METHODS: We recruited 97 HBsAg-positive pregnant women. The mode of delivery included elective cesarean in 16, normal spontaneous vaginal delivery in 56, vacuum or forceps delivery in 12, and emergency cesarean after labor in 13. We measured HBsAg and placental alkaline phosphatase levels in 97 pairs of maternal and fetal blood samples collected at delivery. RESULTS: The mean maternal placental alkaline phosphatase levels did not differ among these four groups. The mean cord placental alkaline phosphatase level of the elective cesarean group was the lowest (P < .05). All samples of cord sera for this group were negative for HBsAg, compared with 38 of 56, eight of 12, and seven of 13 in the spontaneous vaginal, vacuum or forceps, and emergency cesarean groups, respectively (P < .05). CONCLUSION: The level of mother-to-fetus microtransfusion least in the elective cesarean group, as revealed by both the lowest cord placental alkaline phosphatase and HBsAg levels. These observations may have implications for reducing perinatal transmission of blood-borne viruses.

Alkaline Phosphatase↗
PubMed1996

The localization of tissue factor and apolipoprotein(a) in atherosclerotic lesions of the human aorta and their relation to fibrinogen-fibrin transition.

We examined the immunohistochemical distribution of tissue factor (TF), apolipoprotein (a) (apo(a)) in atherosclerotic intimas of human thoracic aortas obtained from 51 autopsies in order to analyze the mechanism of fibrinogen-fibrin transition as a part of thrombogenic properties of atherosclerotic intimas. TF was overexpressed mainly by macrophages in both fatty streaks and more advanced lesions, while it was also scatteringly deposited in the matrix of advanced lesions, especially in the atheromatous gruel. TF-positive macrophages were frequently intermingled at the base of fibrin thrombi formed on the eroded intimas. On the other hand, apo(a) was localized in the stroma and within some macrophages, and also in the mural thrombi. Fibrinogen and fibrin were more frequently detected in the matrix of advanced lesions than in that of early lesions. Fibrin was occasionally co-located with cell- and matrix-associated TF and apo(a) deposited in matrix. These findings suggest that the overexpressed TF in the atherosclerotic intima plays a critical role in the initiation of fibrin formation. This could result from either fibrinogen permeating into the intima or from rupture of the fibrous cap overlying atheromas. Apo(a) deposited in the atherosclerotic intima may also participate in the persistent deposition of fibrin.

Adult↗
PubMed1996

Resistance to antituberculosis drugs in Japan.

SETTING: Five years after the last survey of drug-resistant tuberculosis in Japan, a serious new phenomenon has gradually begun to appear. A nationwide survey was conducted by the Tuberculosis Research Committee. OBJECTIVE: To determine resistance patterns to five anti-tuberculosis drugs and risk factors. DESIGN: Cultures were obtained from patients hospitalized at 38 hospitals in various districts of Japan throughout 6 months, from 1 June through 30 November in 1992. Drug susceptibility testing was carried out in the national reference laboratory. RESULTS AND CONCLUSIONS: Resistance to one or more drugs was found in 5.6% of new cases and 27.8% of recurrent cases (P < 0.001). About 88% of drug resistant isolates from the new cases were resistant to one drug, while 50.8% of the drug resistant isolates from the recurrent cases had resistance to two or more drugs (P < 0.001). Resistance rates to both isoniazid and rifampin in new cases was very low (only 0.14%). Primary drug resistance rates were higher in age groups less than 60 years old, compared to those of 60 years and over (P = 0.05). Compared with the rate in Japanese patients, foreign-born individuals had a higher resistance rate in the recurrent cases (P = 0.034). This survey indicated a similar trend in resistance rates to five antituberculosis drugs to those of the last survey in 1987.

Adolescent↗
PubMed1996

Purification and partial characterization of an indomethacin hydrolyzing enzyme from pig liver.

PURPOSE: Indomethacin is well known to be metabolized via O-demethylation and N-deacylation. In this paper we found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. METHODS: An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsomes using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of rho-chlorobenzoic acid liberated from indomethacin by HPLC. RESULTS: The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in alpha-naphthylacetate and rho-nitrophenylacetate, which are typical substates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Michaelis constant (K(m)) and maximum velocity (Vmax) values for indomethacin were 67.8 microM and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage showed high homology with a mouse carboxylesterase isozyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand, purified human liver carboxylesterases pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic activity for indomethacin. CONCLUSIONS: These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associated with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.

Animals↗
PubMed1996

Phase-contrast X-ray computed tomography for observing biological soft tissues.

Biological soft tissues are almost transparent to hard X rays and therefore cannot be investigated without enhancement with a contrast medium, such as iodine. On the other hand, phase-contrast X-ray imaging is sensitive to light elements (1-8). This is because the X-ray phase shift cross section is almost thousand times larger than the X-ray absorption cross section for light elements such as hydrogen, carbon, nitrogen and oxygen (4,5). Hence, phase-contrast X-ray imaging is a promising technique for observing the structure inside biological soft tissues without the need for staining and without serious radiation exposure. We have devised a means of observing biological tissues in three dimensions using a novel X-ray computed tomography (CT) by modifying the phase-contrast technique. To generate appropriate CT input data, we used phase-mapping images obtained using an X-ray interferometer (6) and computer analysis of interference patterns (9). Now, we present a three-dimensional observation result of a nonstained sample of a cancerous rabbit liver, using a synchrotron X-ray source. Phase-contrast X-ray CT was able to clearly differentiate the cancer lesion from the normal tissue. Moreover, fine structures corresponding to cancerous degeneration and fibrous tissues were clearly depicted.

Animals↗
PubMed1996

Macrophage-colony stimulating factor (M-CSF) enhances proteinuria and recruitment of macrophages into the glomerulus in experimental murine nephritis.

In this study, we examined the effects of macrophage-colony stimulating factor (M-CSF) on glomerular macrophages in lipopolysaccharide (LPS)-induced murine nephritis. Mice injected intraperitoneally with either M-CSF plus LPS, LPS alone, M-CSF alone or saline every day for 8 days were examined for the degree of urine albumin excretion and lymphocyte-function associated antigen-1-positive (LFA-1+) cells in peripheral blood as well as renal pathology. From our results, LPS or M-CSF combined with LPS emphasized the degree of proteinuria, glomerular deposition of immunoglobulins and mesangial proliferation, associated with accumulation of macrophages in the glomeruli. However, in immunohistological examination of kidneys from these nephritic mice, neither intercellular adhesion molecule-1 (ICAM-1), which may play an important role in the recruitment of macrophages into glomeruli, M-CSF receptor nor the number of LFA-1+ cells in peripheral blood was enhanced by M-CSF. On the other hand, M-CSF alone induced neither proteinuria nor any pathological changes and did not increase the number of glomerular Mac-1+ cells above that in saline-treated controls. These results indicate that M-CSF does not directly cause glomerulonephritis but might participate in accelerating the glomerular inflammatory process by stimulating a potent chemoattractant to recruit monocytes-macrophages into the glomeruli.

Animals↗
PubMed1996

Significance of placental alkaline phosphatase (PLAP) in the monitoring of patients with seminoma.

OBJECTIVE: To investigate the significance of placental alkaline phosphatase (PLAP) as a tumour marker for seminoma. PATIENTS AND METHODS: A total of 673 serum samples from 116 patients with testicular germ cell tumours (78 seminomas and 38 non-seminomas) were analysed for several markers including PLAP, total alkaline phosphatase (AL-P), alpha fetoprotein (AFP), beta human chorionic gonadotrophin (HCG-B), and lactic dehydrogenase (LDH). RESULTS: Serum PLAP was increased initially in about 50% of patients with seminoma, and the mean magnitude of elevation was about five times the normal value. There was no significant correlation between PLAP and LDH or between PLAP and HCG-B. Therefore, a combination of these three markers was of value, and resulted in a positive identification rate of 82% of patients with seminoma. False-positive results for PLAP appeared in 1.6% of 673 samples investigated. CONCLUSION: The monitoring of serum PLAP might be of value, as fluctuations in this marker provide information about disease status and prognosis.

Adult↗