Outline and discussion points of guidance for repeated dose tissue distribution studies.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
H Shindo
Publications and source records attributed to H Shindo.
Characterization of the binding of HU and IHF, homologous histone-like proteins of Escherichia coli, to curved and uncurved DNA.
The binding of E. coli histone-like protein HU to curved and uncurved DNA fragments containing adenine tracts was characterized by relative binding affinity assay, and compared with that of other homologous histone-like protein integration host factor (IHF). Both HU and IHF have about 3- to 5-fold higher affinity for overall curved DNA fragments such as (A6N4)11 and (A3T3N4)12 compared to a standard duplex fragment with mixed sequence. The binding manner of HU to the curved fragments was highly cooperative. However, loss of overall curvature for shorter fragments (< approximately 100 bp) reduced the preference of HU binding to curved (A3T3N4)n over uncurved (T3A3N4)n, indicating that the binding specificity of HU to curved DNA is length-dependent. Thus, the curved DNA configuration of the whole molecule facilitates the binding of several HU molecules to form the hierarchy of HU-DNA complex. Furthermore, it was shown that HU and IHF bind less well to (A6N9)n, which has a zig-zag straight structure, whereas they preferentially bind to uncurved (T3A3N4)14. These results suggested that not only intrinsically overall curvature but also the preferred orientations for DNA bending in the protein-DNA complex are important factors for affinities of HU and IHF.
Bacterial Proteins↗
Solution structure of the DNA binding domain of a nucleoid-associated protein, H-NS, from Escherichia coli.
The three-dimensional structure of the C-terminal domain (47 residues) obtained from the hydrolysis of H-NS protein with bovine trypsin was determined by NMR measurements and distance geometry calculations. It is composed of an antiparallel beta-sheet, an alpha-helix and a 3(10)-helix which form a hydrophobic core, stabilizing the whole structure. This domain has been found to bind to DNA. Possible DNA binding sites are discussed on the basis of the solution structure of the C-terminal domain of H-NS.
Amino Acid Sequence↗
The binding specificity and affinity of E. coli integration host factor (IHF) are influenced by the flexibility of flanking regions of its recognition sites.
To understand the roles of the 5'-flanking region of the recognition sites in binding specificity and the affinity of integration host factor (IHF), a variety of DNA fragments with a 13-bp consensus sequence, 5'-WATCAAN4TTR-3'[Friedman, Cell, 55, 545 (1988)], but with different 5'-flanking sequences were investigated by gel retardation and methylation interference assays. It has been well-established that the putative A/T rich element distal from the 5'-end of the consensus made a significant contributions to the binding of IHF. However, many of the DNA fragments used here revealed specific binding to IHF without such an A/T element. Several bases neighboring to the 5'-end of the consensus sequence had significant effects on the binding specificity as well as its affinity, and these results indicate that the sequence-directed bendability of the flanking region plays an important role in the specific recognition by IHF.
Autoradiography↗
[Anatomical study of the lumbar multifidus muscle and its innervation in human adults and fetuses].
The lumbar multifidus muscle was dissected with particular reference to its innervation in 10 Japanese adults and 10 fetuses from 5 to 10 months old. The results were as follows: 1) The multifidus muscle attached to the spinous process of a lumbar vertebra was segmentally innervated by the medial branch of the dorsal ramus of the lumbar nerve on a level with the spinous process. 2) The motor point of the nerve innervating the multifidus muscle in adults was slightly ventro-caudal to that portion of the lumbar spinous process with muscle attached. 3) The multifidus muscle in fetuses was thinner and flatter than that in adults. The motor point of the nerve innervating this muscle was slightly caudal at the lateral margin of the muscle to that portion of the lumbar spinous process with the muscle attached. These differences can be attributed to the fact that muscles used to oppose gravity are undeveloped in fetuses. 4) There was variation in 5 fascicles in total, in which the medial branch of the dorsal ramus of a lumbar nerve entered the multifidus muscle originating at the spinous process of a lower lumbar vertebra.
Adult↗
Clinical efficacy of pravastatin for hyperlipidemia in patients with type 2 diabetes mellitus.
The efficacy of pravastatin (CAS 81131-70-6) on serum lipid levels in 91 type 2 diabetic patients with mean glycosylated hemoglobin of 8.5% was investigated up to 12 weeks. Oral administration of 10 to 20 mg/d of pravastatin significantly decreased total cholesterol by 18.4 +/- 1.5% after 4 weeks. When analyzed separately in type IIa and IIb hyperlipidemia, the reduction of total cholesterol by pravastatin was more prominent in the former. Low-density lipoprotein cholesterol were also significantly decreased 22.2 +/- 2.7% after 4 weeks. The effect of pravastatin in reducing triglyceride was more prominent in patients with higher triglyceride compared to those with lower triglyceride before the administration of the drug. High-density lipoprotein cholesterol showed a slight but significant increase by 4.2 +/- 1.9% after 4 weeks. Among the apolipoproteins examined, apolipoprotein B was significantly decreased after 4 weeks. Atherogenic index and apolipoprotein B/apolipoprotein A-I ratio were also significantly decreased after 4 weeks. The efficacy of pravastatin was also observed after 12 weeks to the same extent as after 4 weeks. No major side effects or abnormalities of laboratory parameters have been observed. These data lead to the conclusion that pravastatin is useful for the treatment of hyperlipidemia in type 2 diabetic patients with poor glycemic control without major adverse effects.
Adult↗
Mechanism of the formation of DNA triplex and effect of chemical modifications on its stability as studied by isothermal titration calorimetry.
The thermodynamic properties of DNA triplex formation with various single-stranded oligo-DNAs as well as mismatched sequences were investigated by isothermal titration calorimetry (ITC). For the triplex formation of perfectly matched sequences, enthalpy changes and dissociation constants were measured and their temperature-dependences suggest that conformational change of pyrimidine single-strand and the intermediate state(s) are involved in the triplex formation. Effects of mismatches and chemical modifications on stability and specificity of the triplex formation will also be discussed.
Base Sequence↗
Structural polymorphism and thermal stability of telomere DNAs (T2G4)n and (T4G4)n.
The ends of eukaryotic chromosomes, termed telomeres, contain a single-stranded 3' overhang composed of tandemly repeated guanine-rich sequences, such as (T2G4)n and (T4G4)n, along one strand. The sequences can form defined folded tetraplex structures. Here we have systematically examined structural polymorphism and thermal stability of a series of oligonucleotide sequences, Tet n: (T2G4)n and Oxy n: (T4G4)n (n = 1, 2, 3, 4), using circular dichroism (CD) spectroscopy. The CD spectra of Tet 1 and Oxy 1 are consistent with those observed in tetraplex structures consisting of four parallel strands (type I conformation); whereas the spectra of Tet 2, Oxy 2, Oxy 3, and Oxy 4 correspond with those observed for tetraplex conformations where the strands are antiparallel (type II conformation). The spectra of Tet 3 and Tet 4 suggests that Tet 3 and Tet 4 can adopt both type I and type II conformations and they are structurally polymorphic. The melting temperatures of Tet n and Oxy n (n = 1, 2, 3, 4) measured by CD melting are consistent with the previously reported values obtained from differential scanning calorimetry (DSC). Furthermore, the CD melting of Tet 4 suggests that the type II conformation of Tet 4 changes into type I conformation between 55 degrees C and 70 degrees C.
Base Sequence↗
Dynamics in isomerization of intramolecular DNA triplexes in supercoiled plasmids.
Kinetic and thermodynamic nature of two isomers, H-y3 and H-y5, for an intramolecular Pyr.Pur.Pyr triplex in supercoiled plasmid was studied by a specific chemical probe. H-y3 was found to be more stable in terms of enthalpy change by 6 to 10 kcal/mol than H-y5, and the time constant for the conversion from H-y5 to H-y3 was 0.6 hr in the absence of Mg2+, while it was 6 hr in the presence of 50mM Mg2+. The transition probability from the duplex form to either two isomers was also measured: the kinetic barrier was higher for the transition to H-y3 than that to H-y5. These results indicated that Mg2+ acts to increase kinetic barriers of interconversion between H-y3 and H-y5 isomers.
Base Sequence↗
The loop sequence plays crucial roles for isomerization of intramolecular DNA triplexes in supercoiled plasmids.
The effect of base composition in the central region of polypurine.polypyrimidine (Pur.Pyr) tracts on the formation of intramolecular DNA triplexes in plasmids was examined using chemical probes (diethyl pyrocarbonate and OsO4), and two-dimensional (2-D) agarose gel electrophoresis. Two isomers exist for an intramolecular triplex: one with the 3'-half of the Pyr strand as the third strand (H-y3) and the other with the 5'-half of the Pyr strand as the third strand (H-y5). It was shown that the content and position of G + C residues in the triplex loop region (the center of Pur.Pyr tracts) are primary determinants for the isomerization between the H-y3 and H-y5 triplexes. Divalent metal ions such as Mg2+ and negative supercoiling also modulate the isomerization: the H-y5 conformation is stabilized by the divalent metal ions and/or under relatively lower negative supercoiling. 2-D gel analyses revealed that two isomers, H-y3 and H-y5, are topologically non-equivalent: the H-y3 formation relaxes one more supercoil turn than H-y5. As the G + C content in the center of Pur.Pyr tracts increases, the triplex requires more supercoil energy for formation. Therefore, the base-pair opening in the center of Pur.Pyr tracts is the initial and critical step in the pathway for the formation of triplex as well as the isomerization. The role of the triplex loop sequence is explained by a model in which the nucleation process of H-y3 formation requires a wide range of base-pair opening compared to that of H-y5: such unwinding would not be favored for the central region of the duplex with high G + C content and so it would be in the presence of Mg2+, and thereby the H-y5 formation is promoted.
Base Composition↗
The effect of prostaglandin E1.alpha CD on vibratory threshold determined with the SMV-5 vibrometer in patients with diabetic neuropathy.
We studied the effect of prostaglandin E1.alpha CD (PGE1) on diabetic peripheral neuropathy by evaluating subjective symptoms and vibration sensation using a new vibrometer (SMV-5). Patients with diabetic neuropathy (n = 38) were divided into three groups; group A received no drugs (control), group B was treated with 1500 micrograms/day of oral methyl vitamin B12 (VB12) for four weeks, and group C received 1.2 micrograms/kg/day PGE1 intravenously for four weeks. There was a close relationship between symptom scores and vibratory threshold (VT). The effect of PGE1 on subjective symptoms and VT were compared with those in groups A and B. Patients who received PGE1 showed a significant improvement rate in pain and hypesthesia compared to patients in groups A and B, and in numbness compared to group A. During the study period, there was no significant change in VT in groups A and B, whereas VT was significantly improved at styloid process (P < 0.05) and at medial malleolus (P < 0.001) in group C. Our results confirmed that PGE1 significantly improved both subjective symptoms and VT, indicating that PGE1 therapy may be useful in diabetic neuropathy.
Administration, Oral↗
The linked roles of nitric oxide, aldose reductase and, (Na+,K+)-ATPase in the slowing of nerve conduction in the streptozotocin diabetic rat.
Metabolic and vascular factors have been invoked in the pathogenesis of diabetic neuropathy but their interrelationships are poorly understood. Both aldose reductase inhibitors and vasodilators improve nerve conduction velocity, blood flow, and (Na+,K+)-ATPase activity in the streptozotocin diabetic rat, implying a metabolic-vascular interaction. NADPH is an obligate cofactor for both aldose reductase and nitric oxide synthase such that activation of aldose reductase by hyperglycemia could limit nitric oxide synthesis by cofactor competition, producing vasoconstriction, ischemia, and slowing of nerve conduction. In accordance with this construct, N-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase reversed the increased nerve conduction velocity afforded by aldose reductase inhibitor treatment in the acutely diabetic rat without affecting the attendant correction of nerve sorbitol and myo-inositol. With prolonged administration, N-nitro-L-arginine methyl ester fully reproduced the nerve conduction slowing and (Na+,K+)-ATPase impairment characteristic of diabetes. Thus the aldose reductase-inhibitor-sensitive component of conduction slowing and the reduced (Na+,K+)-ATPase activity in the diabetic rat may reflect in part impaired nitric oxide activity, thus comprising a dual metabolic-ischemic pathogenesis.
Aldehyde Reductase↗
Effect of tyrphostin on cell growth and tyrosine kinase activity of epidermal growth factor receptor in human gliomas.
The effects of tyrphostin, a selective protein tyrosine kinase inhibitor, on epidermal growth factor (EGF)-stimulated cell growth and EGF-receptor tyrosine kinase activity were studied in four human glioma cell lines. Stimulation by EGF induced variable enhancements of cell growth as well as tyrosine phosphorylation of EGF receptor and intracellular target proteins in all glioma cell lines. The level of immunoreactive EGF receptor detected with antibodies against extra- and intracellular domains was moderate in all four glioma cell lines, but markedly decreased with the latter antibody in two glioma cell lines. This variation was associated with considerable reduction of the EGF-stimulated tyrosine autophosphorylation level. Tyrphostin inhibited dose-dependently the EGF-stimulated cell growth and tyrosine autophosphorylation in all glioma cell lines, and the optimum time for the maximum inhibitory effect on tyrosine autophosphorylation was 12 to 18 hours after treatment with tyrphostin. The antiproliferative activity of tyrphostin nearly correlated quantitatively with its potency as an inhibitor of the EGF-stimulated EGF receptor tyrosine kinase activity. Tyrphostin had no significant effect on the immunoreactive EGF receptor levels, on the affinity constants and numbers of EGF receptor, or on the down-regulation and specific internalization of EGF receptor in any glioma cell line, suggesting that the effects of tyrphostin are not likely to be the results of reduction in EGF receptor and EGF binding capacity. In addition, the serum-stimulated cell growth was also inhibited dose-dependently by higher concentrations of tyrphostin in all glioma cell lines. It might be suggested, therefore, that tyrphostin inhibits EGF-stimulated cell growth by a specific suppression of EGF receptor tyrosine kinase activity, and at higher concentrations there appears to be some degree of either nonspecific inhibition or inhibition of serum-stimulated protein tyrosine kinase activity to induce the cell growth inhibition of gliomas.
Catechols↗
[High resolution NMR for studying biomacromolecules].
Explore the source record for details and available documents.
Biocompatible Materials↗
Thermodynamic and kinetic studies of DNA triplex formation of an oligohomopyrimidine and a matched duplex by filter binding assay.
The filter binding method was found to be a powerful method for studying the formation of triplexes composed of a single-stranded homopyrimidine and a duplex with a matched purine-pyrimidine tract. With this technique, we were able to determine thermodynamic and kinetic parameters for triplex formation between a homopyrimidine 19-mer (5'-TCCTCTTCTTTTCTTTCTT-3') and a duplex with sequence 5'-GCAGGAGAAGAAAAGAAAGAACG-3' for the purine strand. The experiments were performed over a wide pH range (3.8-7.4) and a temperature range of 0-35 degrees C. pH and temperature dependencies of the thermodynamic parameters were best explained in terms of a three-state model for triplex formation at low temperatures relative to the melting point. The main results were as follows: (1) pH dependence of the dissociation constants of the triplex is a result of the rapid acid-base equilibrium of pyrimidine single strands; (2) the association rate for triplex formation decreases with increasing pH in accordance with the dissociation constants; (3) the dissociation constant is virtually temperature-independent at low pH, while it becomes strongly temperature-dependent with increasing pH (these results can be explained in terms of a negative, non-zero delta Cp for triplex formation at low pH); (4) the association rate decreases with increasing temperature, and the resulting negative activation energy indicates that the triplex formation process involves a quasi-stable intermediate; (5) the triplex formation is a second-order reaction at low pH, whereas it can be interpreted as a third-order reaction at neutral pH, suggesting that different triplex formation pathways are observed depending on the pH.
Base Sequence↗
Application of 13C-labeling and nuclear magnetic resonance spectroscopy to pharmacokinetic research: measurement of metabolic rate of benzoic acid to hippuric acid in the rat.
The use of 13C-labeling and nuclear magnetic resonance (NMR) spectroscopy to trace the biotransformation of benzoic acid (BA) to hippuric acid (HA) in the rat has been described. Novel [2,4,6,7-13C4]BA, which was labeled in the specific protonated carbons, was used in order to enhance the sensitivity of 13C NMR detection on the basis of the nuclear Overhauser enhancement and short spin-lattice relaxation time. The urinary excretion of [2,4,6,7-13C4]HA formed from intravenously administered [2,4,6,7-13C4]BA was followed by proton-decoupled 13C NMR spectroscopy (only 10 min accumulation time) without any separation procedures such as extraction and chromatography, using [2-13C]sodium acetate as an internal standard for quantitation. The heights of resonances for C2,6 of [2,4,6,7-13C4]HA and C2 of the internal standard were used to calculate [2,4,6,7-13C4]HA concentration. The lower limit of measurable amounts (ca. 40 nmol) was found to be improved about one order of magnitude over that of the method using commercially available [7-13C]BA. In general, this tracer technique has the potential for wide application to pharmacokinetic research since xenobiotic and endogenous metabolism can be followed by very simple and convenient procedures.
Animals↗
Stabilization of c-myc protein in human glioma cells.
The regulation of c-myc protein, product of c-myc/genes, was studied in four glioma cell lines by Northern blot, pulse-chase dot blot, immunoblot and immunoprecipitation analyses. Northern blot analysis revealed no overexpression of c-myc transcript, and pulse-chase dot blot analysis showed normal turnover rate of c-myc transcript, suggestive of no evidence of aberrant regulation of c-myc at post-transcriptional level. The synthesis levels of c-myc protein were shown by immunoprecipitation and closely associated with the c-myc transcript levels demonstrated by Northern blot, suggestive of no evidence of aberrant translational control of c-myc, whereas they were dissociated from the accumulation levels of c-myc protein shown by immunoblot, suggestive of an evidence of aberrant regulation of c-myc at post-translational level. The mean (+/- standard deviation) half-lives of c-myc protein in four glioma cell lines were calculated from the pulse-chase immunoprecipitation analysis, and being 98 +/- 8 to 143 +/- 11 min, were about four- to sixfold longer than normal. In surgical specimens, the immunostain of c-myc protein was not found in normal astrocytes but localized heterogenously in nuclei of reactive astrocytes and glioma cells, and increased in stained cell number in proportion to malignancy. Although this study was limited to four glioma cell lines, it suggests that the c-myc protein in glioma cells may be accumulated due to its prolonged half-life contributing to an uncontrolled proliferation.
Astrocytes↗
Thermodynamic and kinetic analyses of DNA triplex formation: application of filter-binding assay.
We have developed a simple and efficient method for studying equilibrium thermodynamics and kinetics of DNA triplex formation, which utilizes a filter-binding procedure. The application of this method to the triplex formation between a double-stranded homopurine-homopyrimidine and a single-stranded homopyrimidine oligonucleotides has demonstrated its ability in the quantitative estimation of equilibrium binding constants and rate constants under various conditions. Thus, this simple method can serve as a powerful tool for the systematic analysis of sequence and environmental effects on the equilibrium and kinetic quantities in the triplex formation.
Base Sequence↗