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T Kuwabara

Publications and source records attributed to T Kuwabara.

At least 73 records · Page 4Linked to original sources
PubMed1997

Properties of hepatitis delta virus ribozyme, which consists of three RNA oligomer strands.

Properties of a hepatitis delta virus (HDV) RNA ribozyme system, which consists of three RNA oligomer strands (substrate 8-mer; enzyme 16-mer plus 35-mer) and contains a hybrid sequence of genomic and antigenomic RNA cores, are reported. Effects of Mg2+ concentration, divalent metal ion species, pH, and temperature on the cleavage activity were examined. The substrate cleavage activity increased with increasing Mg2+ concentration (0-100 mM). Ca2+ and Mn2+ ions were the most effective divalent cations and Mg2+ was less effective. The cleavage activity increased with increasing pH (5-7.5). The optimum temperature for the cleavage activity was 25-40 degrees C. The Mg2+ concentration, pH and temperature dependencies are different from those reported for the single-strand ribozymes (about 90-mer) although the divalent metal ion preference is very similar. Conformational change induced by Mg2+ ion titration was monitored by CD. The CD data and the activity-Mg2+ concentration data were analyzed by curve-fitting analysis using equations derived for multiple metal ion binding mechanisms. The data can be explained by a model in which three Mg2+ ions bind to one ribozyme unit.

Base Sequence↗
PubMed1997

Comparison of activities between hammerhead ribozymes and DNA enzymes targeted to L6 BCR-ABL chimeric (b2a2) mRNA.

For the treatment of chronic myelogenous leukemia (CML), attempts have been made to design hammerhead ribozymes that can specifically cleave BCR-ABL fusion mRNA. In the case of L6 BCR-ABL fusion mRNA (b2a2 type), which has no effective cleavage sites for conventional hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to cleave the chimeric mRNA specifically. Several hammerhead ribozymes with relatively long junction-recognition sequences have poor substrate-specificity. Therefore, we explored the possibility of using DNA enzymes, that was newly selected by Santoro & Joyce and that can cleave RNA molecules with high activity, to cleave of L6 BCR-ABL fusion (b2a2) mRNA. By contrast to the results with the conventional ribozymes, the newly designed DNA enzymes, having higher flexibility for selection of cleavage sites, were able to cleave this chimeric RNA molecule specifically at sites close to the junction, without any cleavage of the normal ABL or BCR mRNA.

Base Sequence↗
PubMed1997

Activities of tRNA-embedded dimeric minizymes.

Ribozymes have been shown to be potent inhibitors of gene expression and viral function. Effects of ribozyme-mediated repression to target gene in living cells are correlated with the amounts of expression and stabilities of ribozyme molecules. In our previous study, it was demonstrated that a minimized hammerhead ribozyme, minizyme, with high activity forms a dimeric structure with a common stem II. We constructed dimeric minizymes that could cleave the BCR-ABL chimeric (b2a2) mRNA which had been difficult target for conventional hammerhead ribozymes without damaging the normal ABL mRNA. In order to achieve high expression of these dimeric minizymes in vivo for the treatment of CML, we embedded the dimeric minizyme portion downstream of a tRNA(Val) promoter sequence which could be recognized by RNA polymerase III. We determined cleavage activities of tRNA-embedded dimeric minizymes and compared the activities between tRNA-embedded hammerhead ribozyme and tRNA-embedded dimeric minizymes. All tRNA-embedded dimeric minizymes tested were capable of cleavage the target substrate. The activity of the tRNA-embedded dimeric minizyme targeted at BCR-ABL mRNA was almost the same as that of the naked dimeric minizymes. Interestingly, the cleavage activity of tRNA-embedded dimeric minizymes was higher than that of tRNA-embedded conventional hammerhead ribozyme.

Base Sequence↗
PubMed1997

Structure-activity correlation for an HDV ribozyme composed of three RNA strands.

Hepatitis delta virus (HDV) RNA ribozyme system which consists of three RNA oligomer strands (substrate 8-mer; enzyme 16-mer plus 35-mer, Fig. 1) was designed. Effects of Mg2+ concentration on the pseudo first-order rate constant (kobs) of RNA cleavage reaction and on conformation of ribozyme complex were examined. The secondary structure of the complex was also analyzed by limited digestion with ribonucleases. The kobs and CD data were analyzed by curve-fitting analysis using equations derived for two-Mg2+ and three-Mg2+ ion binding models. The result revealed that a three-Mg2+ binding model can explain the Mg(2+)-concentration-dependent changes of both conformation and activity of the HDV ribozyme.

Base Sequence↗
PubMed1996

Characterization of several kinds of dimer minizyme: simultaneous cleavage at two sites in HIV-1 tat mRNA by dimer minizymes.

A minizyme is a hammerhead ribozyme with short oligonucleotide linkers instead of stem-loop II. In a previous study we demonstrated that a minizyme with high activity forms a dimeric structure with a common stem II. Because of their dimeric structure, minizymes are potentially capable of cleaving a substrate at two different sites simultaneously. In order to examine the properties of different kinds of minizyme, we constructed a number of minizymes with short oligonucleotide linkers (2-5 bases) instead of stem-loop II and examined their cleavage activities against HIV-1 tat mRNA. Analyses of melting curves, as well as Arrhenius plots, revealed that, in general, the longer the oligonucleotide linkers, the more stable and more active were the dimer minizymes. All minizymes examined cleaved the target substrate at two sites simultaneously. The activity of the dimer minizyme with a 5 nt linker was higher than that of the parental hammerhead ribozyme because the latter full-sized ribozyme was able to cleave at one site only.

Base Sequence↗
PubMed1996

Kinetic analysis of receptor-mediated endocytosis of G-CSF derivative, nartograstim, in rat bone marrow cells.

To elucidate the mechanism of the receptor-mediated clearance of granulocyte colony-stimulating factor (G-CSF), we performed kinetic analyses of the receptor-mediated endocytosis (RME) processes using a human G-CSF derivative, nartograstim (NTG), and isolated rat bone marrow cells. The first-order rate constants involved in RME processes were obtained by computerized model fitting of the time courses of the ligand-receptor complex on both the cell surface and in the cell interior and the degradation products in the medium in the pulse-chase experiment. They were also calculated based on a kinetic model involving the ligand concentration dependence of the initial binding rate, the steady-state degradation rate, and the steady-state amounts of ligand on both the cell surface and in the interior. The rate constants for the RME processes after receptor binding determined in the different experiments were similar, that is, the half-times for the dissociation, internalization, and degradation of the ligand-receptor complex were 770, 10-30, and 20 min, respectively. However, the association constant obtained by measuring the initial binding was fivefold greater than that calculated under steady-state conditions. These kinetic analyses support the hypothesis that the internalization of the receptor may be accelerated by ligand binding, causing downregulation of the receptor on the cell surface. These overall kinetic analyses based on steady-state and non-steady-state data of the RME processes clarify the dynamics of the interaction between NTG and its receptor.

Animals↗
PubMed1996

Elevation of cerebral lactate detected by localized 1H-magnetic resonance spectroscopy in migraine during the interictal period.

We used localized 1H-magnetic resonance spectroscopy (MRS) to study the metabolic changes in the brains of patients with migraine during the interictal period. Measurement of metabolite levels in the occipital visual cortex in six normal subjects and six patients disclosed high lactate levels in the five patients who had experienced a migraine attack within the previous 2 months. One patient who had not experienced a migraine attack in the previous 4 years did not show a lactate peak. We speculate that anaerobic glycolysis occurs in the brains of patients with migraine during the interictal period, and a long attack-free period may normalize the subclinical metabolic disturbance.

Adolescent↗
PubMed1996

Pharmacokinetics and pharmacodynamics of a recombinant human granulocyte colony-stimulating factor.

Granulocyte colony-stimulating factor (G-CSF), a hematopoietic growth factor, is a clinically effective drug used to promote neutrophil recovery in patients with chemo- or radiotherapy-induced neutropenia. We have reviewed the pharmacokinetic and pharmacodynamic properties of three kinds of G-CSFs: E. coli derived G-CSF, CHO-derived G-CSF, and mutein G-CSF. The clearances of G-CSFs are saturable and autoinducible in experimental animals and humans. That is, the systemic clearances of G-CSFs decrease as the dose injected increases and approaches a constant value. Both saturable and nonsaturable processes are involved in G-CSF elimination. Also, the systemic clearances of G-CSFs are increased by repeated administration of G-CSF. Although the relative bioavailability of G-CSFs after subcutaneous administration is approximately 60%, the increase in peripheral white blood cells or neutrophils is greater than that after intravenous administration at the same dose. The effects of G-CSFs seem to be time dependent rather than AUC dependent, considering that mean residence time of G-CSFs in the plasma is longer after subcutaneous administration than that after intravenous administration. There is a slight difference in the pharmacokinetics of E-coli- and CHO-G-CSF although they seem to be pharmacologically equivalent. The correlation between G-CSF clearance and peripheral neutrophil counts in the patients suggests that G-CSF receptors contribute to G-CSF clearance. Quantitative pharmacokinetic analysis using mutein G-CSF shows that the G-CSF receptor plays a major role in saturable G-CSF clearance, and that this saturable process accounts for approximately 80% of the total clearance at low doses. That is, the degradation following the receptor-mediated endocytosis in bone marrow might be a major clearance system of G-CSF at a physiological blood level. The G-CSF receptor in bone marrow might work not only as a signal transducer for differentiation and proliferation of granulopoietic precurcer cells but as a regulator of G-CSF levels in blood. In addition, at high doses, glomerular filtration in the kidneys is the major process for nonsaturable G-CSF clearance. At present, polyethylene glycol derivatives of G-CSF are being developed to reduce the frequency of G-CSF administration.

Animals↗
PubMed1995

The 60-kDa precursor to the dithiothreitol-sensitive tetrameric protease of spinach thylakoids: structural similarities between the protease and polyphenol oxidase.

The 60-kDa precursor to the 39-kDa dithiothreitol-sensitive protease was purified from photosystem II membranes of spinach. When partially purified 60-kDa protein was stored at 4 degrees C, the protein was degraded to fragments of 39 and 21 kDa. The 39-kDa fragment was suggested to be identical to the 39-kDa protease from effects of dithiothreitol on these polypeptides. The N-terminal amino acid sequences of the 60-kDa protein and the 39-kDa protease were the same, APILPDVEK-, suggesting that the latter was derived from the N-terminal portion of the former. Immunostaining with polyclonal antibodies against the 60-kDa protein indicated that the 60-kDa protein represents the species that occurs in the native thylakoids. These and other structural properties suggest that the protein might be identical to polyphenol oxidase.

Amino Acid Sequence↗
PubMed1995

Lactate rise in the basal ganglia accompanying finger movements: a localized 1H-MRS study.

To investigate whether motor activation can cause lactate elevation, we observed the metabolic changes in seven right-handed volunteers by localized 1H-magnetic resonance spectroscopy (MRS). The volume of interest (VOI) was centered on a region including portions of the putamen and globus pallidus. Finger opposition movements were applied as the motor tasks. On the side contralateral to the finger movements, lactate rose in all subjects. No lactate rise occurred in basal ganglia ipsilateral to the movements.

Basal Ganglia↗
PubMed1995

Toichiro Kuwabara on Dave Cogan. An interview. Interview by Sally Smith Hughes.

In this interview, Dr. Kuwabara recounts his first meeting with Dr. Cogan and describes his work at the Howe Laboratory, their pioneering research on diabetic retinopathy, histochemical and electronmicroscopical studies. This warm recollection exemplifies the mutual respect and deep friendship between Drs. Kuwabara and Cogan.

History, 20th Century↗
PubMed1995

Induction and intracellular localization of a 72-kDa heat shock protein in rat gastric mucosa after water-immersion stress.

We investigated the expression and changes in the intracellular localization of a 72-kDa heat shock protein (HSP72) in rat gastric pyloric and fundic mucosa before and after water-immersion stress. Severe mucosal damage was found in the fundic mucosal area of the stomach after this stress. However, no mucosal lesion developed in the pyloric mucosal area. HSP72 in both the soluble and insoluble fractions of the pyloric and the fundic mucosal areas was significantly increased after water-immersion stress, peaking 6h after the initiation of the stress. The increase in HSP72 was more significant in the pyloric mucosal area than in the fundic mucosal area under both normal and stress conditions. The increase of HSP72 in the pyloric mucosal cells occurred prior to the formation of the mucosal lesions, whereas the increase of HSP72 in the fundic mucosal cells was observed after ulcer formation. An immunohistochemical study showed that HSP72 was constitutively expressed in the cytoplasm of the gastric mucosal cells, and that the intranuclear induction of HSP72 was remarkably intense in the pyloric mucosal cells, especially in the proliferative zone, compared with the fundic mucosal cells. Our results may suggest that HSP72 has an important cytoprotective function in gastric mucosal cells and that there is a "biophysical" difference between pyloric and the fundic mucosal cells.

Animals↗
PubMed1995

Renal clearance of a recombinant granulocyte colony-stimulating factor, nartograstim, in rats.

PURPOSE: To clarify the role of the kidney in the elimination of a recombinant human granulocyte colony-stimulating factor, nartograstim, we have investigated its pharmacokinetics in rats with renal failure. METHODS: The steady-state clearance (CLss) were determined by the intravenous infusion for 4 hr to unilateral renally-ligated and cisplatin-treated rats, whose renal functions were about 50 and 10% of controls, respectively. RESULTS: CLss of nartograstim (27 ml/hr/kg) in the renally-ligated rats at a high infusion rate was significantly lower (25%) than in control rats (p < 0.05). CLss in these rats, at a low infusion rate was 95 ml/hr/kg, 14% lower than in control rats. The saturable CLss in these rats, 68 ml/hr/kg, was not significantly different from control rats (75 ml/hr/kg, p > 0.05). Also, CLss in cisplatin-treated rats with extensive renal failure, at a high infusion rate, decreased to 57% of controls. Furthermore, the total body clearances (CLtot) of nartograstim after bolus intravenous administration to renally-ligated and cisplatin-treated rats were reduced to 33-49% of controls. CONCLUSIONS: These results suggest that the kidney may be responsible for 40-50% of the nonsaturable clearance of nartograstim. Thus, the kidney should make a major contribution to the elimination of nartograstim when rats are given a high dose of nartograstim, which saturates the receptor-mediated clearance.

Animals↗
PubMed1995

Expression in Escherichia coli of the extrinsic 18-kDa protein of photosystem II of spinach.

A cDNA encoding the precursor for the 18-kDa protein of PSII of spinach was expressed in Escherichia coli. When the cell lysate was incubated at 7 degrees C, the precursor was degraded by proteases of E. coli to a polypeptide of 18 kDa (P18) that consisted of the mature protein moiety plus the last four residues of the transit peptide. P18 was able to reconstitute the water-oxidizing complex of NaCl-treated PSII membranes supplemented with the 23-kDa protein. Moreover, P18 was cleaved by the prolyl endoproteinase of spinach specifically at the Pro-12-Leu-13 bond, as was the authentic 18-kDa protein. These properties of P18 indicate that the present expression system is potentially useful for studies of the substrate specificity of the endoproteinase, as well as of the structure-function relationships of the 18-kDa protein.

Amino Acid Sequence↗
PubMed1995

Reversible changes in conformation of the 23-kDa protein of photosystem II and their relationship to the susceptibility of the protein to a proteinase from photosystem II membranes.

An aspartic proteinase was partially purified from PSII membranes of spinach. The 23-kDa protein of PSII was resistant to the proteinase when in its native form but became susceptible upon modification by p-chloromercuriphenylsulfonic acid (pCMPS), upon incubation at pH 3, and upon incubation in dilute SDS. Conformational changes caused by pCMPS seemed to be restricted to a localized region since the modified protein reconstituted the water-oxidizing complex with slightly less efficiency as compared with the untreated protein. Under the three proteolytic conditions, cleavage occurred at the Asn-58-Leu-59 bond, suggesting that the protein underwent similar conformational changes in each case. Dithiothreitol, dialysis at pH 7, and neutral detergents suppressed the facilitative effects of pCMPS, pH 3, and SDS on proteolysis, respectively. With the reversible changes in susceptibility, reversible changes in circular dichroism of the protein also occurred at 200-208 nm. These observations indicate that the conformational changes are reversible and the renaturation of the substrate was associated with the suppression of the susceptibility. These results suggest that the scissile bond becomes reversibly exposed and susceptible to the proteinase in response to environmental changes.

Amino Acid Sequence↗