Biomedical subjects

T Nagamune

Publications and source records attributed to T Nagamune.

At least 19 recordsLinked to original sources

PubMed2005

Single-stage autotrophic nitrogen-removal process using a composite matrix immobilizing nitrifying and sulfur-denitrifying bacteria.

We developed a novel single-stage autotrophic nitrogen-removal process comprised of two composite immobilized biomass layers-one of nitrifying bacteria and one of sulfur-denitrifying bacteria and elemental sulfur-in a Fe-Ni fibrous slag matrix. Nitrification and consumption of dissolved oxygen occurred in the outer part and sulfur denitrification in the anoxic inner part of the composite matrix, thus realizing autotrophic nitrogen removal in a single reactor. The complete conversion of ammonia into N2 in a single reactor was demonstrated in both batch-mode incubation and continuous-feed operation. The spatial profiles of the ammonia-oxidizing bacteria and denitrifying bacteria were evaluated by real-time PCR, targeting their functional genes, and stratification of these two types was observed in the matrix after several months of incubation. This process does not require any specific reactor type or conditions and thus has the potential to be applied to many different wastewater treatment processes due to its simplicity in both operation and construction.

Aerobiosis↗

PubMed2002

Real-time monitoring of ammonia-oxidizing activity in a nitrifying biofilm by amoA mRNA analysis.

Ammonia monooxygenase encoding mRNA (amoA mRNA) transcription in the wastewater treatment process was investigated using reverse transcription PCR (RT-PCR) as the model indicating specific function and activity in nitrifying processes. The dynamic response of amoA mRNA transcription and ammonia-oxidizing activity to the change of environmental conditions such as pH and concentration of ammonia was examined to determine the inductive factor and the inhibitor for amoA mRNA expression. Furthermore, we semiquantitatively investigated the response of amoA mRNA transcription to the pH fluctuation in a continuous fed nitrifying reactor. As a result, amoA mRNA oriented analysis enabled real-time assay of ammonia-oxidizing activity within 2 h as a response time. In contrast, rRNA and amoA encoding DNA were constantly detected at almost the same amount throughout the experiment. mRNA transcription was regulated by the many environmental conditions: ammonia seems to be one of the strong inducers for transcription of amoA mRNA, whereas low pH seems to be a strong inhibitor. These factors simultaneously affected the mRNA transcription and enzymatic activity leading to the complex phenomena of ammonia-oxidizing activity and amoA mRNA transcription in the continuous feeding reactors.

Ammonia↗

PubMed2001

Design of an artificial light-harvesting unit by protein engineering: cytochrome b(562)-green fluorescent Protein chimera.

We have generated a novel model protein for an artificial light-harvesting complex composed of two proteins, cytochrome b(562) (cytb(562)) and enhanced green fluorescent protein (EGFP), in which two chromophores are fixed in each protein matrix. Cytb(562) was appended to the N-terminus of EGFP via a Gly-Ser linker and the resultant fusion protein was successfully expressed in Escherichia coli as a mixture of the apo- and the holo-forms as to the cytb(562) moiety. The fluorescence of EGFP was substantially quenched when the apo-form was reconstituted with hemin. Based on the fluorescence lifetime measurements, it appeared that light energy entrapped by EGFP is transferred to the heme of cytb(562) by resonance energy transfer (energy transfer yield: 65%). Spatial organization of two chromophores using small and stable protein matrices will be promising toward the construction of an artificial light-harvesting complex by protein engineering.

Cytochrome b Group↗

PubMed2001

Replacing factor-dependency with that for lysozyme: affordable culture of IL-6-dependent hybridoma by transfecting artificial cell surface receptor.

Cytokines and growth factors are indispensable for the propagation and maintenance of factor-dependent mammalian cells. However, cytokines are often so expensive that the use of factor-dependent cells for industrial applications such as protein production is often not practical. Based on our previous design of a binary hen egg lysozyme (HEL)-specific receptor composed of portions of the anti-HEL antibody and the erythropoietin receptor, a new pair of chimeric receptors having the intracellular domain of gp130 were made and transfected to an interleukin-6 (IL-6)-dependent hybridoma, 7TD1. The clone expressing the two new receptors showed clear HEL dose-dependent cell growth and monoclonal antibody production in both serum-based and serum-free media without IL-6. These results establish the feasibility of applying receptor design to tailor cells for the inexpensive induction of cell growth for the purpose of producing therapeutic products.

Animals↗

PubMed2001

Observation of charge state and conformational change in immobilized protein using surface plasmon resonance sensor.

Behaviors of proteins immobilized on a solid surface were investigated using BIACORE, a biosensor utilizing surface plasmon resonance. This sensor is usually used for analyzing binding events during biomolecular interactions. Here we propose a novel use of this sensor to monitor two kinds of intramolecular changes in immobilized proteins. Several proteins were covalently attached to dextran chains on the sensor surface in the flow cell and were then exposed to a series of buffers with varying pH. Signal changes derived from changes of refractive index around the sensor surface were detected during and after the exposure to each of these buffers, which we denoted as in situ values and postvalues, respectively. The in situ value reflects the behavior of immobilized proteins in these buffers and was revealed to have a correlation with total charge state of the proteins, while the postvalue reflects how immobilized proteins react after the exposure and was suggested to represent the degree of conformational changes of the proteins. This method is expected to be applicable to various analyses and can provide us with new information about the behavior of proteins on solid phase.

Apoproteins↗

PubMed2001

Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer.

We describe a noncompetitive homogeneous bioluminescent immunoassay based on the antigen-dependent reassociation of antibody variable domains (open sandwich bioluminescent immunoassay, OS-BLIA). The reassociation of two chimeric proteins, an antibody heavy-chain fragment (V(H))-Renilla luciferase (Rluc) and an antibody light-chain fragment (V(L))-enhanced yellow fluorescent protein (EYFP), was monitored by a bioluminescence resonance energy transfer (BRET) between the two. Upon simple mixing of the reagents with the sample, an antigen-dependent increase in BRET was observed with a measurable concentration range of 0.1 to approximately 10 microg/ml antigen hen egg lysozyme. Compared with our comparable assays based on fluorescence resonance energy transfer (FRET), a 10-fold improvement in the sensitivity was attained, probably due to a reduction in reagent concentration.

Bacterial Proteins↗

PubMed2001

Monitoring of the refolding process for immobilized firefly luciferase with a biosensor based on surface plasmon resonance.

In order to examine the possibility of the use of a surface plasmon resonance (SPR) sensor for real-time monitoring of the process of refolding of immobilized proteins, the refolding of firefly luciferase immobilized on a carboxymethyldextran matrix layer was analyzed. The SPR signal of the immobilized luciferase decreased after unfolding induced by GdnCl and increased gradually in the refolding buffer, while there was no signal change in the reference surface lacking the immobilized protein. The decrease in the SPR signal on unfolding was consistent with the difference between the refractive indices of the native and unfolded protein solutions. The effects of blocking of the excess NHS-groups of the matrix layer on the refolding yield were examined by means of an SPR sensor. The results were consistent with those obtained with the enzymatic activity assay, indicating that the changes in the SPR signal reflected the real-time conformational changes of the immobilized protein. Hence, an SPR biosensor might be used for monitoring of the process of refolding of immobilized proteins and as a novel tool for optimization of the refolding conditions. This is the first demonstration that SPR signal changes reflect the conformational changes of an immobilized protein upon unfolding and refolding.

Animals↗

PubMed2001

A growth signal with an artificially induced erythropoietin receptor-gp130 cytoplasmic domain heterodimer.

We report a strategy for generating efficient signal transduction with unnatural heterologous receptor combinations. As previously described [Ueda, H., Kawahara, M. et al. (2000) J. Immunol. Methods 241, 159-170], chimeric receptors composed of the V(H)/V(L) domains of anti-hen egg lysozyme antibody HyHEL-10 and N-terminally truncated erythropoietin receptor (EpoR) can be activated by lysozyme. When the cytoplasmic domains of these receptors were substituted with one derived from gp130, IL-3 dependent Ba/F3 cells expressing both V(H)-gp130 and V(L)-gp130 grew dose-dependently when given lysozyme without IL-3. However, cells expressing the heterologous pair of V(H)-gp130 and V(L)-EpoR also showed more efficient and stricter lysozyme-dependent proliferation in the absence of IL-3, indicating this combination is as an efficient and strict signal transducer as wild-type EpoR. The immunoprecipitation data indicated the existence of a preformed V(H)-gp130 and V(L)-EpoR heterodimer in the absence of lysozyme, suggesting the crucial role of a receptor conformational change in signal triggering as well as wild-type EpoR and gp130. Phosphorylation of JAK2, STAT3, and STAT5 was observed upon the addition of lysozyme, suggesting the activation of both EpoR- and gp130-derived signals.

Animals↗

PubMed2001

How does heme axial ligand deletion affect the structure and the function of cytochrome b(562)?

We have recently generated a new mutant of cytochrome b(562) (cytb(562)) in which Met7, one of the axial heme ligands, is replaced by Ala (M7A cytb(562)). The M7A cytb(562) can bind heme and the UV-visible absorption spectrum is of a typical high-spin ferric heme. To investigate the effect of the lack of Met7 ligation on the structural integrity of cytb(562), thermal transition analyses of M7A cytb(562) were conducted. From the thermodynamic parameters obtained, it is concluded that the folding of M7A cytb(562) is comparable to the apoprotein despite the presence of heme. On the other hand, exogenous ligands such as cyanide and azide ions are readily bound to the heme iron, indicating that the axial coordination site is available for substrate binding. The peroxidase activity of this mutant is thus examined to evaluate new enzymatic function at this site and M7A cytb(562) was found to catalyze an oxidation reaction of aromatic substrates with hydrogen peroxide. These observations demonstrate that the Met7/His102 bis-ligation to the heme iron is crucial for the stable folding of cytb(562), whereas the functional conversion of cytb(562) is successfully achieved by the loose folding together with the open coordination site.

Amino Acid Substitution↗

PubMed2001

Design of the linkers which effectively separate domains of a bifunctional fusion protein.

With the aim of separating the domains of a bifunctional fusion protein, the ability of several lengths of helix-forming peptides to separate two weakly interacting beta-can domains was compared with that of flexible linkers or of a three alpha-helices bundle domain. We introduced helix-forming peptide linkers A(EAAAK)nA (n = 2-5) between two green fluorescent protein variants, EBFP and EGFP, and investigated their spectral properties. The fluorescence resonance energy transfer from EBFP to EGFP decreased as the length of the linkers increased. The circular dichroism spectra analysis suggested that the linkers form an alpha-helix and the alpha-helical contents increased as the length of the linkers increased. The results clearly suggested the ability of the helical linkers to control the distance and reduce the interference between the domains. This 'linker engineering' may open a way to the rational design of linkers which maximize the multiple functions of fusion proteins or de novo multi-domain proteins.

Circular Dichroism↗

PubMed2001

Expanded-bed protein refolding using a solid-phase artificial chaperone.

An efficient solid-phase protein refolding method based on artificial chaperone-assisted refolding is proposed. The method employs insoluble cyclodextrin polymer beads and the expanded-bed technique. Alpha-glucosidase, whose spontaneous refolding yield from a urea-denatured state is up to 30% at a protein concentration of up to 10 microg/ml, could be refolded with a yield that was improved more than two-fold at a protein concentration more than five-fold higher when protein solution was circulated through an expanded bed under optimized conditions. Unlike the conventional liquid-phase artificial system, further steps to purify the refolded product, which are generally needed to remove detergent-cyclodextrin complex and excess cyclodextrin, were unnecessary. In addition, the polymer beads were reusable after simple washing with water, and the continuous system is suitable for easy-scale-up using commercially available devices. This new method is considered to be a powerful means of achieving large-scale protein refolding for industrial protein production.

Journal Article↗

PubMed2001

Construction of green fluorescent protein reporter genes for genotoxicity test (SOS/umu-test) and improvement of mutagen-sensitivity.

Green fluorescent protein (GFP) reporter genes for the bacterial umu-test were constructed. Utilization of tandem, lacUV5 and chimeric trp/umu promoters, and coexpression of the Escherichia coli recA5327 mutant enhanced the GFP expression level fourteen-fold over that of the system with only the umu promoter, thereby improving the sensitivity of the umu-test.

Journal Article↗

PubMed2001

Single cell reporter assay using cell surface displayed Vargula luciferase.

Reporter genes such as firefly luciferase are common tools to monitor gene expression in various systems. As reporter gene represents the expression level of the gene of interest with its enzyme activity, firefly luciferase is most frequently used because its luminescent activity is highly sensitive and less time consumable for assay. However, since firefly luciferase is expressed internally in the cell, lysis of the cell is a critical step, and thus it is difficult to monitor the gene expression level continuously. In this report, we utilized secretive Vargula hilgendorfii luciferase modified to cell surface displayed one by fusing with human EGFR transmembrane sequence. This modified Vargula luciferase was expressed on cell surface without losing its bioluminescent activity. Co-transfection with secretive alkaline phosphatase showed that the behaviors of cell surface displayed Vargula luciferase and secretive alkaline phosphatase are comparable to each other. Furthermore, the luminescence of a single cell expressing cell surface displayed Vargula luciferase can be monitored by using photon counting CCD camera, which indicates that this reporter gene can monitor gene expression in a single cell without cell lysis.

Journal Article↗

PubMed2000

Open sandwich enzyme-linked immunosorbent assay for the quantitation of small haptens.

The quantitation of low-molecular-weight haptens has been difficult with conventional sandwich immunoassays due to their small size. Many researchers have attempted to develop sandwich assays for haptens due to the significant advantages of the sandwich format over competitive assays including greater dynamic range, ease of automation, and sensitivity. Here we apply the open-sandwich ELISA (OS-ELISA), an immunoassay based on antigen-dependent stabilization of antibody variable regions (V(H) and V(L) domains), to hapten quantitation. Two fusion proteins, the high-affinity mutant V(H) domain from anti-4-hydroxy-3-nitrophenacetyl (NP) antibody B1-8 tethered with Escherichia coli alkaline phosphatase (V(H)(W33L)-PhoA) and the V(L) domain from the same antibody tethered with Streptococcus sp. protein G, were made. These fusion proteins when added together achieved Fv reassociation consequent to the addition of NP. Signal was generated in a direct relationship to the NP concentration with better sensitivity compared with competitive immunoassay, demonstrating this assay to be a quick noncompetitive alternative to the conventional assays for small compounds, such as environmental pollutants, drugs of abuse, and therapeutic drugs. With our previous demonstration that the OS-ELISA works well with large proteins, the OS-ELISA becomes the first practical immunoassay approach capable of quantifying any molecule regardless of their size.

Animals↗

PubMed2000

Cell-growth control by monomeric antigen: the cell surface expression of lysozyme-specific Ig V-domains fused to truncated Epo receptor.

Previously we have shown that the V(H) and V(L) fragments of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 are weakly associated but can be driven together by antigen. By joining these antibody variable domains to the cytoplasmic portion of the murine erythropoietin receptor, we created a chimeric growth factor receptor that could be activated by HEL. After co-transfection with two plasmids encoding the respective chimeric receptors in IL-3 dependent murine pro-B Ba/F3 cells, a portion of the cells survived under antigen dependent stimulation without IL-3. These surviving cells all showed coexpression of the two chimeric receptor chains and demonstrated HEL dose-dependent growth stimulation without IL-3. When another IL-3 dependent cell line 32D was transfected with a variant of such chimeric receptor with a linker peptide (Gly-Ser-Gly) inserted between V(H)/V(L) and EpoR domains, an improved growth response was attained. These observations suggest the utility of heterodimeric Fv chimeric receptors in creating cells that respond to monomeric antigen.

Cell Membrane↗

PubMed2000

Direct comparison of electron transfer properties of two distinct semisynthetic triads with non-protein based triad: unambiguous experimental evidences on protein matrix effects.

In order to understand the roles of protein matrix in electron transfer processes (ET) within biological systems, a heme-based donor (Zn-heme: ZnPP)-sensitizer (Ru2+(bpy)3)-acceptor (cyclic viologen: BXV4+) triad 1 was used as a probe molecule. Two semi-synthetic systems, Cyt-b562(1) and Mb(1), in which the triad is incorporated into cytochrome b562 (Cyt-b562) or into myoglobin (Mb), were constructed by cofactor reconstitution. These two semi-synthetic proteins were compared with the triad itself (i.e., without the protein matrix) using absorption spectroscopy, steady state emission and excitation studies, laser flash photolysis experiments, and molecular modeling. Photoexcitation of the ZnPP moiety of Cyt-b562(1) or Mb(1) leads to a direct ET from the triplet state of ZnPP state (3ZnPP) to BXV4+ to generate a final charge-separated (CS) state, Cyt-b562(Zn+)-Ru2+-BXV3+* or Mb(Zn+)-Ru2+-BXV3+*. On the other hand, direct ET from the excited ZnPP moiety to the BXV4+ moiety is also involved in 1 in the absence of the protein matrix, but the excited state of ZnPP involved is not 3ZnPP, but the singlet excited state (1ZnPP) in this pathway. When the Ru2+(bpy)3 moiety of Cyt-b562(1) or Mb(1) is excited, a stepwise ET relay occurs with the ion-pair, Cyt-b562(Zn)-Ru3+-BXV3+* or Mb(Zn)-Ru3*-BXV3+*, as an intermediate, leading to the same final CS state as that generated in the direct ET pathway. The lifetimes of the corresponding final CS states were determined to be 300 ns for 1 in the absence of the protein matrix, 600-900 ns for Cyt-b562(1) and 1.1-18 micros for Mb(1), the values of which are greatly affected by the protein matrix. Molecular modeling study of the three systems consistently explained the differences of their photophysical behavior.

Cytochrome b Group↗

PubMed2000

Ribosomal RNA supplementation highly reinforced cell-free translation activity of wheat germ.

We have constructed an inexpensive, highly efficient eukaryotic cell-free translation system. Wheat germ rRNA (WG rRNA) was prepared by phenol/chloroform (P/C) extraction, a simple and quick method, from wheat germ, an inexpensive and commercially available by-product of flour production. Addition of a small amount of WG rRNA into a wheat germ cell-free translation system increased the protein productivity of the system 6- to 8-fold. Isolated 18S or 28S rRNA alone enhanced the protein production only 2-fold or 3.9-fold, respectively, at maximum. On the other hand, their equimolar mixture enhanced the production as much as the whole WG rRNA, indicating 18S and 28S rRNA synergistically functioned to enhance protein synthesis. Addition of WG rRNA slightly improved the stability of mRNA in the cell-free translation system, which explained only partly the enhancement of protein production. Addition of WGE or ribosome containing approximately the same amount of rRNA in the form of protein-rRNA complex as WG rRNA added to the system did not increase the protein production in the translation system. When ribosome in the cell-free translation system was replaced with WG rRNA, the system did not exhibit any detectable translation activity, indicating that the translation activity of WG rRNA is negligible in comparison with that of ribosome. These results indicated that WG rRNA affected some mechanisms regulating the translation rate in wheat germ cell-free system, resulting in increased protein production.

Journal Article↗

PubMed2000

Refolding of firefly luciferase immobilized on agarose beads.

The renaturation yield of the denatured firefly luciferase decreased strongly with increasing protein concentration in a renaturation buffer, because of aggregation. In this study, firefly luciferase was immobilized on agarose beads at a high concentration. Although the protein concentration was extremely high (about 100-fold) compared to that of soluble luciferase, the renaturation yield was comparable with that for the soluble one. Thus, immobilization was shown to be effective for avoiding aggregation of firefly luciferase. It was also shown that the optimum buffer conditions for renaturation of the immobilized luciferase were the same as those for the renaturation in solution. Also, it was indicated that electrostatic interactions between a protein and the matrix have a negative effect on renaturation of the immobilized luciferase since the renaturation yield decreased at acidic pH only for the immobilized luciferase. These novel observations are described in detail in this paper.

Animals↗