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Y Sugiura

Publications and source records attributed to Y Sugiura.

At least 19 recordsLinked to original sources
PubMed2001

Selected base sequence outside the target binding site of zinc finger protein Sp1.

Human transcription factor Sp1 contains three contiguous repeats of the C2H2-type zinc finger motif and binds to the decanucleotide sequence 5'-(G/T)GGGCGG(G/A)(G/A)(C/T)-3' (GC box). In order to determine whether the three-zinc finger peptide Sp1(530-623) has selectivity for sequence outside the GC box, we used a selection and amplification of binding experiment. The high affinity sequence generated from this selection is 5'-GGGTGGGCGTGGC-3' (s-GC box), which is flanked by a novel conserved guanine triplet on the 5'-side of the core decanucleotide. Gel mobility shift assays reveal that Sp1(530-623) binds to the s-GC box with 2.3-fold higher affinity than to the wild-type GC box, 5'-GGGGCGGGGC-3' (c-GC box). DNase I and hydroxyl radical footprinting analyses show that the area of the s-GC box protected by binding of Sp1(530-623) is wider by 1 nt than that of the c-GC box. On the other hand, alkylation interference analyses demonstrate that Sp1(530-623) forms only one special base contact at the guanine triplet. With respect to cleavage of the c-GC and s-GC boxes by the 1,10-phenanthroline-copper complex (OP-Cu), binding of Sp1(530-623) has no effect on the cleavage pattern of the s-GC box, whereas OP-Cu actually enhances cleavage of the c-GC box. Additionally, the extent of cleavage of the s-GC box by DNase I and OP-Cu is clearly different from that of the c-GC box under peptide-free conditions. The results strongly indicate that: (i) the conformation of the s-GC box is evidently distinct from that of the c-GC box; (ii) Sp1(530-623) binds to the s-GC box without induction of a conformational change in DNA detectable by cleavage with OP-Cu. The present study provides useful information for the design of multi-zinc finger proteins with various sequence specificities.

Amino Acid Sequence↗
PubMed2001

Alamethicin-leucine zipper hybrid peptide: a prototype for the design of artificial receptors and ion channels.

In this report, we describe a novel concept of extramembrane control of channel peptide assembly and the eventual channel current modulation. Alamethicin is a peptide antibiotic, which usually forms ion channels in various association states. By introducing an extramembrane leucine zipper segment (Alm-LeuZ), the association number of alamethicin was effectively controlled to produce a single predominant channel open state. The assembly was estimated to be a tetramer, by comparison of the channel conductance with that of the template-assembled Alm-LeuZ tetramer, which was prepared by the conjugation of a maleimide-functionalized peptide template with cysteine-derivatized Alm-LeuZ segments. Employment of an extramembrane segment of a random conformation provided higher levels of channel conductance. The result exemplified the possibility of channel current control by a conformational switch of the extramembrane segments.

Alamethicin↗
PubMed2001

Comparison of sensory and sympathetic innervation of the dura mater and posterior longitudinal ligament in the cervical spine after removal of the stellate ganglion.

Although cervical spinal tissues are rich in sensory and sympathetic fibers, which play a significant role in clinical phenomena, there is little information available regarding their anatomical characteristics. In this study, we compared the innervation of the cervical dura mater and the posterior longitudinal ligament (PLL) to that after removal of the stellate ganglion to determine whether the anatomical background plays a significant role in clinical manifestations. Immunoreactivities for calcitonin gene-related peptide (CGRP) and substance P (SP) were used as sensory markers, and immunoreactivity for neuropeptide Y (NPY) was used as a sympathetic marker. Sensory fibers in the cervical dura mater were distributed within each cervical segment, but those in the PLL extended beyond the segmental borders. A dense sensory fiber network forming a single layer was seen at the intervertebral disc region in the cervical PLL, whereas sympathetic fibers in this region were sparsely distributed. Sympathetic fibers were distributed not only around the vascular wall but also in the region independent from vessels, and some occasionally ran together with sensory fibers in both the dura mater and the PLL. Removal of the stellate ganglion had little effect on the distribution of sensory fibers but denervated the sympathetic fiber networks in the region independent from vessels of the upper ipsilateral cervical PLL. In conclusion, the cervical dura mater and the PLL have different sensory and sympathetic innervations. Sympathetic fibers pass through the stellate ganglion to project to the region independent from vessels in the upper cervical PLL. Clinical symptoms may be attributed to this characteristic innervation of the cervical spine.

Animals↗
PubMed2001

A 'cassette' RNase: site-selective cleavage of RNA by RNase S equipped with RNA-recognition segment.

RNase S is a unique protein comprising the non-covalent association of two components, the S-peptide and the S-protein. An RNA-recognition segment derived from the human immunodeficiency virus (HIV)-1 Rev protein was conjugated with the S-peptide to form a complex with the S-protein. The resulting RNase S bearing the RNA-recognition segment preferentially hydrolyzed a single position of the RNA stem-loop derived from the specific binding site for the Rev protein.

Amino Acid Sequence↗
PubMed2001

Significant effect of linker sequence on DNA recognition by multi-zinc finger protein.

The unique linker sequence of the native nine zinc finger transcription factor IIIA (TFIIIA) appears to significantly affect its novel DNA recognition mode. An artificial new nine zinc finger peptide Sp1ZF9T has been created by connecting three units of the three zinc finger domains of Sp1 with the TFIIIA-type linker. The DNA-binding characteristics of Sp1ZF9T were evaluated by the gel mobility shift, DNase I footprinting, and methylation interference assays, and compared with those of the previous Sp1ZF9 with a Krüppel-type linker. The gel mobility shift assays revealed that Sp1ZF9T forms two complex species, a short-lived species (B-2) and a long-lived species (B-1), with GCIII DNA (5'-GGG GCG GGG GGG GCG GGG GGG GCG GGGCC-3'). The B-2 complex dissociated into the free peptide and DNA, whereas the B-1 complex was stable even after 72 h. The DNase I footprinting and methylation interference results indicated that 3'- and central portions of GCIII DNA are recognized by Sp1ZF9T in the B-1 complex. The present DNA binding mode of Sp1ZF9T is evidently different from that of Sp1ZF9. Namely, fingers 1-5 participate in the DNA contact of Sp1ZF9T, and fingers 1-9 in that of Sp1ZF9. Therefore, the linker sequence among the zinc finger domains has a significant effect on the specific DNA recognition by the multi-zinc finger proteins.

Animals↗
PubMed2001

Multiconnection of identical zinc finger: implication for DNA binding affinity and unit modulation of the three zinc finger domain.

Cys(2)-His(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules. They are expected to elevate the DNA binding affinity and specificity by increasing the number of finger modules. To investigate the relation between the number and the DNA binding affinity of the zinc finger, we have designed the two- to four-finger peptides by connecting the central zinc finger (finger 2) of Sp1 with the canonical linker sequence, Thr-Gly-Glu-Lys-Pro. Gel mobility shift assays reveal that the cognate three- and four-finger peptides, Sp1(zf222) and Sp1(zf2222), strongly bind to the predicted target sequences, but the two-finger peptide, Sp1(zf22), does not. Of special interest is the fact that the dissociation constant for Sp1(zf2222) binding to the target DNA is comparable to that for Sp1(zf222). The methylation interference, DNase I and hydroxyl radical footprintings, and circular permutation analyses demonstrate that Sp1(zf2222) binds to its target site with three successive zinc fingers and the binding of the fourth zinc finger is inhibited by DNA bending induced by the binding of the three-finger domain. The present results strongly indicate that the zinc finger protein binds to DNA by the three-finger domain as one binding unit. In addition, this information provides the basis for the design of a novel multifinger protein with high affinity and specificity for long DNA sequences, such as chromosomal DNAs.

Base Sequence↗
PubMed2001

Finger-positional change in three zinc finger protein Sp1: influence of terminal finger in DNA recognition.

The connection of functional modules is effective for the design of DNA binding molecules with the desired sequence specificity. C(2)H(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules and are expected to recognize any DNA sequences by permutation, multi-connection, and the substitution of various sets of zinc fingers. To investigate the effects of the replacement of the terminal finger on the DNA recognition by other fingers, we have constructed the three zinc finger peptides with finger substitution at the N- or C-terminus, Sp1(zf223), Sp1(zf323), and Sp1(zf321). From the results of gel mobility shift assays, each mutant peptide binds preferentially to the target sequence that is predicted if the fingers act in a modular fashion. The methylation interference analyses demonstrate that in the cases of the N-terminal finger substitution mutants, Sp1(zf223) and Sp1(zf323), the N-terminal finger recognizes bases to different extents from that of the wild-type peptide, Sp1(zf123). Of special interest is the fact that the N-terminal finger of the C-terminal finger substitution mutant, Sp1(zf321), shows a distinct base recognition from those of Sp1(zf123) and Sp1(zf323). DNase I footprinting analyses indicate that the C-terminal finger (active finger) induces a conformational change in the DNA in the region for the binding of the N-terminal finger (passive finger). The present results strongly suggest that the extent of base recognition of the N-terminal finger is dominated by the binding of the C-terminal finger. This information provides an important clue for the creation of a zinc finger peptide with the desired specificity, which is applicable to the design of novel drugs and biological tools.

Base Composition↗
PubMed2001

Synthetic inhibitors of the processing of pretransfer RNA by the ribonuclease P ribozyme: enzyme inhibitors which act by binding to substrate.

2,2'-p-Phenylene bis[6-(4-methyl-1-piperazinyl)]benzimidazole, 2,2'-bis(3,5-dihydroxyphenyl)-6,6'-bis benzimidazole, and 2,2'-bis(4-hydroxyphenyl)-6,6'-bis benzimidazole are shown by UV-visible and fluorescence spectrophotometry to be strong ligands for tRNA, giving simple, hyperbolic binding isotherms with apparent dissociation constants in the micromolar range. Hydroxyl radical footprinting indicates that they may bind in the D and T loops. On the basis of this tRNA recognition as a rationale, they were tested as inhibitors of the processing of precursor tRNAs by the RNA subunit of Escherichia coli RNase P (M1 RNA). Preliminary studies show that inhibition of the processing of Drosophila tRNA precursor molecules by phosphodiester bond cleavage, releasing the extraneous 5'-portion of RNA and the mature tRNA molecule, was dependent on both the structure of the inhibitor and the structure of the particular tRNA precursor substrate for tRNA(Ala), tRNA(Val), and tRNA(His). In more detailed followup using the tRNA(His) precursor as the substrate, experiments to determine the concentration dependence of the reaction showed that inhibition took time to reach its maximum extent. I(50) values (concentrations for 50% inhibition) were between 5.3 and 20.8 microM, making these compounds among the strongest known inhibitors of this ribozyme, and the first inhibitors of it not based on natural products. These compounds effect their inhibition by binding to the substrate of the enzyme reaction, making them examples of an unusual class of enzyme inhibitors. They provide novel, small-molecule, inhibitor frameworks for this endoribonuclease ribozyme.

Binding Sites↗
PubMed2001

Beneficial effects of immediate enteral nutrition after esophageal cancer surgery.

This study was conducted to determine the effects of immediate enteral nutrition (EN) on nutritional status, immunological competence, and the suppression of excessive inflammatory responses in patients following esophageal cancer surgery. Twenty-four patients who underwent the same elective operation for thoracic esophageal carcinoma were randomized into an immediate enteral nutrition (IEN) group who received EN from postoperative day (POD) 1 and a parenteral nutrition (PAN) group. Both groups received comparable volumes and calories on the same POD. Laboratory studies were carried out preoperatively and on PODs 1-7. Other nutritional and immunological assessments were repeated on PODs 1 and 7. Plasma concentrations of nitrate and nitrite were also measured. All of the patients in the IEN group tolerated enteral feeding well. There were no significant differences in the results of nutritional assessments, lymphocyte function, or plasma nitrate and nitrite levels between the two groups. The IEN group showed a significantly earlier recovery of the total lymphocyte count. The serum levels of total bilirubin and C-reactive protein were significantly attenuated in the IEN group. These results indicate that immediate EN may have beneficial effects on immunological competence and the suppression of excessive inflammatory responses in patients following esophagectomy. Patients undergoing radical esophageal surgery who are subjected to severe surgical stress might benefit the most from early EN.

Aged↗
PubMed2001

Long-term survival after gastric cancer and liver and paraaortic lymph node metastases: report of a case.

We report the case of a 46-year-old man in whom successful resection of carcinoma of the stomach with liver and paraaortic lymph node metastases was carried out. The carcinoma was removed completely with combined resection of the lower esophagus, total stomach, distal pancreas, spleen, two metastatic liver nodes, and groups 1 and 2 and abdominal paraaortic lymph nodes. Adjuvant chemotherapy was administered postoperatively. The patient is currently well with a grade 1 performance status and no signs of recurrence 12 years after his operation. This experience suggests that even the presence of metastatic paraaortic lymph nodes and liver metastases is not necessarily a contraindication to surgery when the carcinoma can be resected curatively and safely.

Carcinoma↗
PubMed2001

Design of novel zinc finger proteins: towards artificial control of specific gene expression.

In this review, we summarize design strategies for generating proteins with desired sequences such as long contiguous base pairs and diverse sequence specificities based on the nature of Cys(2)-His(2) zinc finger proteins. Recent progress towards artificial DNA binding proteins has been achieved by structure-based design processes and by selection strategies. Indeed, a multi-zinc finger protein with an 18 (or 27)-base pair address, and new zinc finger proteins for diverse DNA target sites (TATA-box and p53 binding site) have been created successfully. Such novel zinc finger proteins will probably be useful tools in molecular biology and potentially in human medicine.

Animals↗
PubMed2001

Coupling between substrate binding and allosteric regulation in ribozyme catalysis.

The contribution of substrate binding to allosteric regulation in the ribozyme catalysis has been investigated using allosteric ribozymes consisting of the hammerhead ribozyme and a flavin mononucleotide (FMN) aptamer. Kinetic parameters were measured for various lengths of the substrates with a wide range of binding energy. The maximum cleavage rate of each ribozyme was retained with the long substrates. However, the cleavage rates largely decreased by the truncation of the substrates according to loss in the free energy of substrate binding. The high sensitivity to the substrate lengths is attributed to the increase in the energetic requirement for the catalytic core folding, which is caused by the incorporation of the aptamer region. One role of FMN binding is assisting the promotion of the core folding through the stabilization of the aptamer domain. The allosteric effect is significantly expressed only when the substrate binding energy is insufficient for the core folding of the ribozyme-substrate complex. This type of allosteric interaction dominates the substrate dependency of another type of regulation. These results demonstrate that an adequate correlation between the type of regulation and the substrate binding is responsible for the effective allosteric interaction in the kinetic process.

Allosteric Regulation↗
PubMed2001

Effect of arginine mutation of alanine-556 on DNA recognition of zinc finger protein Sp1.

Human transcription factor Sp1, which contains three Cys(2)His(2)-class zinc finger motives, specifically binds to the so-called GC box DNA. It has been indicated that finger 1 has a unique DNA-binding mode compared with fingers 2 and 3, or the Zif268 model. Therefore, we investigate the role of Ala at position 6 on the recognition helix, which is not responsible for guanine recognition and highly conserved among Sp1 family. Several Ala-556 mutations of Sp1 bind to DNA with different DNA-binding features. In particular, the Ala-->Arg substitution alters the DNA-binding contribution of the three zinc fingers in Sp1. In this case, the DNA-binding specificity of each finger decreases in the order 2>1>3. This result reveals that one amino acid in position 6 plays an important role not only for the selectivity to the putative finger 1 subsite, but also for the binding mode of the three fingers to each finger subsite. Probably, Ala-556 is indispensable to characterize the binding mode of the Sp1 zinc fingers, namely the diverse binding contribution of finger 1 and the rigid binding one of finger 3. In Sp1, the N-terminal finger 1 serves as a 'hinge finger'.

Alanine↗
PubMed2001

Altered expression of heme oxygenase-1 in the livers of patients with portal hypertensive diseases.

This study was designed to determine changes in expression of heme oxygenase (HO)-1, the stress-inducible and carbon monoxide-producing enzyme, in normotensive and portal hypertensive human livers. GTS-1, a monoclonal antibody against rat HO-1 cross-reacted with the human HO-1 and blocked its enzyme activity, allowing us to examine the activity and localization of HO-1. In controls, approximately 50% of the total HO activity was from HO-1 as judged by the sensitivity to GTS-1, while the rest of activity was from other isozymes such as HO-2. HO-1 was expressed mainly in a subpopulation of Kupffer cells, and the expression in hepatic stellate cells, sinusoidal endothelial cells, and hepatocytes was little, if any. The HO-1 expression exhibited quite different pictures in the livers of portal hypertensive diseases. In cirrhotic livers, which undergo portal hypertension through increases in intrasinusoidal resistance and regenerative changes in the parenchyma, HO-1 occurred in a majority of Kupffer cells and was also observed in hepatocytes. Consequently, the total HO-1 activities became significantly greater in these tissues than those from normal individuals. By contrast, livers of idiopathic portal hypertension that are characterized by an increase in presinusoidal resistance displayed a significant decrease in the HO-1 expression in Kupffer cells, and its hepatocellular expression was not detectable. Although factors involved in altered HO-1 expression in these cells remain unknown, the results suggest that Kupffer cells could alter their expression of HO-1 in response to local hemodynamic changes associated with chronic portal hypertension in humans.

Antibodies, Monoclonal↗
PubMed2001

Incidence of genetic subgroups of hereditary spinocerebellar ataxia in Fukushima Prefecture.

The prevalence of each type of hereditary spinocerebellar ataxias (SCAs) was genetically determined in Fukushima Prefecture, and the results were compared to those in other areas of Japan. The genetic analyses were done in 29 patients with dominant SCA and 5 patients with SCA with negative family history. Machado-Joseph disease was identified in 41.3% of the cases, SCA6 17.2%, dentatorubral-pallidoluysian atrophy (DRPLA) 6.9% and unknown 34.5%. The incidence is clearly different from those of Miyagi and Yamagata Prefectures as SCA1 has not been identified in our region, and is in fact similar to that of Hokuriku or Kanto Provinces. An apparent difference in the incidence of each SCA may be attributed to the historical and geographic regional difference in the distribution of inhabitants and also to the small size of the SCA population we have so far investigated. In addition, 2 of the 3 genetically identified DRPLA in this study were not clinically diagnosed, and one of them was thought to be sporadic. Late onset DRPLA may thus be misdiagnosed to other disease categories, when dementia was not apparent at the time of onset.

Genetic Diseases, Inborn↗
PubMed2001

Arrangement of D-periodic collagen fibrils and association of proteoglycans with fibrils in the synovium of the mouse temporomandibular joint.

The present study was performed to examine changes in the arrangement of D-periodic collagen fibrils in the synovium of the growing temporomandibular joint in mice. At 1 week of age, the mandibular condyle was undeveloped, and only a few collagen fibrils were recognizable in the subintimal layer of the synovium. At 8 weeks, the mandibular condyle was structurally developed with an increase of collagen fibrils in the synovium; a fully mature condyle was observed at 6 months of age. The close association of proteoglycans with collagen fibrils in the synovium of the growing joint was examined by both conventional and energy-filtering transmission electron microscopy of cupromeronic blue-stained specimens. Proteoglycans were associated with D-periodic collagen fibrils in the short filamentous form in random fashion at 1 week of age, but in a regular pattern with D-periodicity at 6 months. These associations in the synovium could be correlated with the mechanical character of the temporomandibular joint.

Age Factors↗