Biomedical subjects

Michiko Kato

Publications and source records attributed to Michiko Kato.

13 recordsLinked to original sources

PubMed2006

A ubiquitin ligase complex assembles linear polyubiquitin chains.

The ubiquitin system plays important roles in the regulation of numerous cellular processes by conjugating ubiquitin to target proteins. In most cases, conjugation of polyubiquitin to target proteins regulates their function. In the polyubiquitin chains reported to date, ubiquitin monomers are linked via isopeptide bonds between an internal Lys and a C-terminal Gly. Here, we report that a protein complex consisting of two RING finger proteins, HOIL-1L and HOIP, exhibits ubiquitin polymerization activity by recognizing ubiquitin moieties of proteins. The polyubiquitin chain generated by the complex is not formed by Lys linkages, but by linkages between the C- and N-termini of ubiquitin, indicating that the ligase complex possesses a unique feature to assemble a novel head-to-tail linear polyubiquitin chain. Moreover, the complex regulates the stability of Ub-GFP (a GFP fusion protein with an N-terminal ubiquitin). The linear polyubiquitin chain generated post-translationally may function as a new modulator of proteins.

HeLa Cells↗

PubMed2006

Analysis of a processing system for proteases using yeast cell surface engineering: conversion of precursor of proteinase A to active proteinase A.

The display of a protease, carboxypeptidase Y (CPY) or procarboxypeptidase Y (proCPY), which is the vacuolar protease, on the yeast-cell surface was successfully performed using yeast-cell-surface engineering for the first time. Through that we could confirm the processing of vacuolar proteases containing proteinase A (PrA) and proteinase B (PrB) which are related to the maturation of proCPY, using a novel cell-surface engineering technique. Various protease-knockout strains of Saccharomyces cerevisiae with the CPY-displaying system were constructed to evaluate the operation of the activation process of CPY. The display of CPY (CPY-agg, which is a fusion protein of CPY with C-terminal half of alpha-agglutinin) on the cell surface was confirmed by immunofluorescence staining. The activity of the CPY-agg was determined after the conversion of proCPY to active CPY by treatment of whole cells with proteinase K. In the proCPY-displaying CPY-knockout strain and PrB-knockout strain, CPY was displayed as an active (mature) form, but in the proCPY-displaying PrA-knockout strain, CPY was present as an inactive form (proCPY). These facts indicate that PrA had been already activated before its transport to the vacuole and that active mature PrA might convert proCPY to CPY before the transport of proCPY to the vacuole. From these results, it was suggested that by using the yeast-cell-surface engineering at the location of the initial step, the autocatalytic activation from proPrA to PrA might occur before the vacuolar branch separates from the main secretory pathway.

Antigens, Surface↗

PubMed2006

Systems for the detection and analysis of protein-protein interactions.

The analysis of protein-protein interactions is important for developing a better understanding of the functional annotations of proteins that are involved in various biochemical reactions in vivo. The discovery that a protein with an unknown function binds to a protein with a known function could provide a significant clue to the cellular pathway concerning the unknown protein. Therefore, information on protein-protein interactions obtained by the comprehensive analysis of all gene products is available for the construction of interactive networks consisting of individual protein-protein interactions, which, in turn, permit elaborate biological phenomena to be understood. Systems for detecting protein-protein interactions in vitro and in vivo have been developed, and have been modified to compensate for limitations. Using these novel approaches, comprehensive and reliable information on protein-protein interactions can be determined. Systems that permit this to be achieved are described in this review.

Chromatography, Affinity↗

PubMed2006

Moderately roughened- and roughened-surface implants used as rigid orthodontic anchorage: a case series.

BACKGROUND: Osseointegrated implants, especially Brånemark turned-surface implants, have been shown to function as stable and efficient orthodontic anchors. While it is generally accepted that prostheses can be attached to implants that have been used as anchors, it has not been clarified if the same applies to moderately roughened- and roughened-surface implants. PURPOSE: The purpose of the present study was to assess the differences between moderately roughened- and roughened-surface implants that are used as orthodontic anchors and then bonded with prostheses and those that are bonded with prostheses without serving as orthodontic anchors. MATERIALS AND METHODS: A total of 43 moderately roughened- and roughened-surface implants (ITI titanium plasma spray TPS] Straumann AG, Waldenburg, Switzerland], ITI sandblasted large-grit acid-etched SLA] Straumann AG], Nobel TiUnite Nobel Biocare AB, Göteborg, Sweden]) were placed in 11 partially edentulous patients, aged 35-61 years (two men and nine women). After an appropriate healing period, orthodontic therapy was performed in 11 patients using 27 implants as orthodontic anchors. After completion of the orthodontic therapy, the prostheses were attached at the same time to both types of implants: the 27 implants that were used as anchors, and 16 implants that were not used as anchors. All 11 patients were followed up regularly. RESULTS: Regardless of use as orthodontic anchorage, all implants maintained osseointegration and continued to function properly. CONCLUSION: No differences existed in therapeutic results after prosthesis bonding whether or not moderately roughened- and roughened-surface implants were used as orthodontic anchors.

Acid Etching, Dental↗

PubMed2006

Intrusion of molars with implants as anchorage: a report of two cases.

BACKGROUND: Osseointegrated implants do not move in response to orthodontic loads. PURPOSE: The aim of this study was to describe a method to intrude supra-erupted molars using prosthetic implants as anchorage. MATERIALS AND METHODS: A 51-year-old female and a 36-year-old male were treated with adjacent implants for orthodontic anchorage for supra-erupted teeth in the maxilla (16, 17) and in the mandible (46, 47), respectively. In both cases, the force of intrusion applied was 300 g. RESULTS: The therapy was completed without complications or abnormalities of the intruded teeth or anchorage implants. Intrusion required 13 months in the first case and 19 months in the latter. CONCLUSION: The present method makes it possible to secure enough space for prosthodontic therapy at edentulous segments and perform highly reliable implant therapy.

Adult↗

PubMed2005

Main-chain dominated amyloid structures demonstrated by the effect of high pressure.

It has been suggested that, while the globular native forms of proteins are a side-chain-dominated compact structure evolved by pursuing a unique fold with optimal packing of amino acid residues, amyloid fibrils are a main-chain-dominated structure with an extensive hydrogen bond network. To address this issue, the effects of hydrostatic pressure on amyloid fibrils of beta2-microglobulin (beta2-m), involved in dialysis-related amyloidosis, were studied. A systematic analysis at various pressures and concentrations of guanidine hydrochloride conducted by monitoring thioflavin T fluorescence, light-scattering, and tryptophan fluorescence revealed contrasting conformational changes occurring consecutively: first, a pressure-induced reorganization of fibrils and then a pressure-induced unfolding. The changes in volume as well as the observed structural changes indicate that the beta2-m amyloid fibrils under ambient pressure are less tightly packed with a larger number of cavities, consistent with the main-chain-dominated amyloid structure. Moreover, the amyloid structure without optimal packing will enable various isoforms to form, suggesting the structural basis of multiple forms of amyloid fibrils in contrast to the unique native-fold.

Amyloid↗

PubMed2005

Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2.

Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.

Amino Acid Motifs↗

PubMed2005

Construction of a selective cleavage system for a protein displayed on the cell surface of yeast.

We constructed a novel protein-purification system in which Saccharomyces cerevisiae with a protein displayed on the cell surface is harvested and the displayed protein is then cleaved from the cell surface. GFPuv was used as a model protein in this cell surface engineering experiment. In this system, the C-terminal 320 amino acids of alpha-agglutinin were bound to the C-terminal of GFPuv for display on the cell surface. In this novel system, the insertion of the recognition sequence-encoding gene of protease factor Xa between GFPuv and alpha-agglutinin was successfully carried out. The GFPuv, displayed by the insertion, was successfully cleaved from yeast cell surface by treatment with factor Xa, and could be easily recovered. By removing such a protease with well-known properties, the displayed protein could be isolated and purified with relative ease.

Base Sequence↗

PubMed2003

The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure.

The heat- and pressure-induced unfolding of the glycosylated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and/or by their intrinsic fluorescence in the temperature range of 20-75 degrees C or the pressure range of 0.1-700 MPa. Under all conditions, the precursor form showed a clear two-state transition from a folded to an unfolded state, regardless of the presence of the carbohydrate moiety. In contrast, the mature form, which lacks the propeptide composed of 91 amino acid residues, showed more complex behaviour: differential scanning calorimetry and pressure-induced changes in fluorescence were consistent with a three-step transition. These results show that carboxypeptidase Y is composed of two structural domains, which unfold independently but that procarboxypeptidase Y behaves as a single domain, thus ensuring cooperative unfolding. The carbohydrate moiety has a slightly protective role in heat-induced unfolding and a highly protective role in pressure-induced unfolding.

Calorimetry, Differential Scanning↗

PubMed2002

High pressure-induced changes of biological membrane. Study on the membrane-bound Na(+)/K(+)-ATPase as a model system.

In order to study the pressure-induced changes of biological membrane, hydrostatic pressures of from 0.1 to 400 MPa were applied to membrane-bound Na(+)/K(+)-ATPase from pig kidney as a model system of protein and lipid membrane. The activity showed at least a three-step change induced by pressures of 0.1-100 MPa, 100-220 MPa, and 220 MPa or higher. At pressures of 100 MPa or lower a decrease in the fluidity of lipid bilayer and a reversible conformational change in transmembrane protein is induced, leading to the functional disorder of membrane-associated ATPase activity. A pressure of 100-220 MPa causes a reversible phase transition in parts of the lipid bilayer from the liquid crystalline to the gel phase and the dissociation of and/or conformational changes in the protein subunits. These changes could cause a separation of the interface between alpha and beta subunits and between protein and the lipid bilayer to create transmembrane tunnels at the interface. Tunnels would be filled with water from the aqueous environment and take up tritiated water. A pressure of 220 MPa or higher irreversibly destroys and fragments the gross membrane structure, due to protein unfolding and interface separation, which is amplified by the increased pressure. These findings provide an explanation for the high pressure-induced membrane-damage to subcellular organelles.

Adenosine Triphosphatases↗

PubMed2002

Increased monoamine concentration in the brain and blood of fetal thalidomide- and valproic acid-exposed rat: putative animal models for autism.

Autism is defined as a congenital neurodevelopmental disorder in which serotonergic dysfunction may be involved in its pathogenesis. One of the characteristic laboratory findings in autistic patients is hyperserotonemia, although its mechanism has not been elucidated to date because of difficulties in studying human patients. Recent reports have demonstrated that thalidomide or valproic acid exposure during early embryonic days (first trimester) in humans causes higher incidence of autism. Morphologic abnormalities found in autism (e.g. cerebellar anomalies, reduced motor neuron numbers) have been reported in animals administered with these teratogens prenatally, suggesting the possibility of the use of these animals as an experimental autistic model. In this study, we evaluated monoamine levels in the brain and blood of rats exposed to teratogens prenatally. Of the groups exposed to thalidomide on embryonic day (E)2, E4, E7, E9, and E11, a significant increase of hippocampal serotonin was only observed in the group exposed on E9. Furthermore, E9 thalidomide and valproic acid exposure both resulted in an increase of hippocampal serotonin, frontal cortex dopamine, and hyperserotonemia. These results thus indicate that two potentially autism-inducing teratogens, thalidomide and valproic acid, have the same effect on early monoamine system development in the brain and the blood, which may explain the pathogenesis of autism.

Animals↗

PubMedDate not supplied

Construction of a cultivation system of a yeast single cell in a cell chip microchamber.

A novel single cell screening system was constructed using a yeast cell chip in combination with the yeast cell surface engineering [NanoBiotechnology 2005, 1, 105-111]. Enzymes or functional proteins displayed on a yeast cell surface can be used as a protein cluster. To achieve high-throughput screening of protein libraries on the cell surface, a catalytic reaction by a single cell-surface-engineered yeast cell was successfully carried out in the microchamber on the yeast cell chip. After screening, to replicate a target cell for use in measuring of activity, DNA sequencing, and preservation, a novel single cell cultivation system in the yeast cell chip was constructed. To avoid damage of the rapid dry up of medium in the microchamber array, the yeast cell chip was modified with a protection sheet, so that the modified chip was like a micro-culture tank constructed on the yeast cell chip microchamber. As a result, single yeast cell cultivation in the yeast cell chip microchamber was observed, and the modified yeast cell chip was evaluated to be good for a single cell selection. The improvement showed that the single cell screening system coupled with the single cell cultivation using the modified yeast cell chip may be superior to that by a cell sorter for the isolation of a target cell and its practical use.

Cell Count↗

PubMedDate not supplied

Enhancement of cellulase activity by clones selected from the combinatorial library of the cellulose-binding domain by cell surface engineering.

To improve the cellulolytic activity of a yeast strain displaying endoglucanase IotaIota (EG II) from Trichoderma reesei, a combinatorial library of the cellulose-binding domain (CBD) of EG II was constructed by using cell surface engineering. When EG II degrades celluloses, CBD binds to cellulose, and its catalytic domain cleaves the glycosidic bonds of cellulose. CBD had a flat face, composed of five amino acids for binding. It was supposed that the three hydrophobic amino acid residues of the five amino acid residues were essential for binding to cellulose. Therefore, by improving the two remaining amino acid residues, construction of mutants with a combinatorial library of the two amino acids in CBD was carried out and binding ability and hydrolysis activity were measured. In the first screening by halo assay using the Congo Red staining method, about 200 of the 2000 colonies formed clear halos, and then five colonies with the clearest halos were finally selected. In the second screening, the binding ability of the five mutants to phosphoric acid-swollen Avicel was measured. In addition, the measurement of hydrolysis activity toward carboxymethylcellulose (CMC) using the screened mutants was carried out. As a result, the mutated EG II exhibiting higher binding ability (1.5-fold) had higher hydrolysis activity (1.3-fold) compared to the parent EG II-displaying yeast cell, demonstrating that CBD has confirmatively some effect on the cellulase activity through its binding ability of the enzyme to cellulose.

Binding Sites↗