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A recombinant bait region mutant of human α2-macroglobulin exhibiting an altered proteinase-inhibiting spectrum (1999)

Ikai, Atsushi ; Ookata, Kayoko ; Shimizu, Masaru ; [et al.]

Springer

Cytotechnology 31 (1999), S. 53-60

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ISSN: 1573-0778

Keywords: alpha-2-macroglobulin ; α2M ; bait region ; CHO cells ; human ; lysyl endopeptidase

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract Alpha 2-macroglobulin (α2M), a plasma glycoprotein produced in the liver, inhibits a variety of proteinases and thus considered to play important homeostatic roles in the body. This broad inhibitory spectrum has been explained by the trapping theory by which a proteinase recognizes a region of 25–30 amino acid peptide in α2M called bait region and cleaves it, leading to the conformational change of α2M, and to the subsequent entrapment and inhibition of the proteinase. We constructed α2M cDNAs with mutated DNA sequences in the bait region, and obtained recombinant CHO cell lines producing either wild type α2M, or mutant α2Ms, i.e., α2M/K692 and α2M/K696, each with substitution of Arg with Lys at codons 692 and 696, respectively. We tested if lysyl endopeptidase is not inhibited by wild type α2M, but could be inhibited by these engineered mutant α2Ms. Thus, recombinant α2M/K696 protein successfully inhibited lysyl endopeptidase activity, while recombinant α2M/K692 protein was not sensitive to lysyl endopeptidase, suggesting that not all bait region peptide bonds can equally be accessible and susceptible to proteinases. The present results not only provided the trapping theory with additional supportive evidence, but the first experimental evidence for the value of engineered α2M-derived proteinase inhibitor with an artificial proteinase inhibitory spectrum of potential industrial and/or therapeutic usefulness.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1008011919876

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Detection and cDNA cloning of H-strand mitochondrial regulatory region RNAs in cultured human cells and human tissues (2000)

Nakamichi, Noboru ; Ito, Mamiko ; Maeda, Takuji ; [et al.]

Springer

Cytotechnology 33 (2000), S. 175-188

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ISSN: 1573-0778

Keywords: cDNA ; differentiation ; gene expression ; growth ; humancells ; human tissues ; mitochondrial DNA ; mitochondrial RNA ; polyadenylated RNA

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract In the mitochondrion, essential genetic elements for replication and transcription are mostly housed within a shortsegment of its DNA located between tRNAPhe and tRNAPro genes, which is called mitochondrial regulatoryregion (mrr). RNAs are known to be transcribed from mrr, thestructures and the functions of which are yet to be fullycharacterized.We detected ca. 1.3 kb H-strand transcripts of mrr (mrrH-RNAs),and 0.2 kb L-strand transcripts of mrr (mrrL-RNAs) in varioushuman cultured cells and tissues using double stranded mrrDNAprobes. The steady state levels of mrrL-RNAs were generally highin cultured cells, while they varied among tissues. On the otherhand, the levels of mrrH-RNAs varied among tissues and amongcultured cells. A tendency was observed in these cells andtissues that a high level of mrrL-RNA is associated with cellproliferation, and a high level of mrrH-RNA withdifferentiation. Several cDNA clones to 1.3 kb mrrH-RNA were obtained from humanskeletal muscle polyadenylated RNAs. The 5′ terminus of the 1.3 kb RNA was determined to be at nucleotide position 15953 whichis immediately downstream of tRNAThr sequence.Polyadenylation site for most of the clones was demonstrated tobe at nucleotide position 576 which is immediately upstream oftRNAPhe sequence. The longest cDNA insert obtained was 1177 bps long spanning from nucleotide positions 15969 to 576 which could code for a peptide of 76 amino acids. The cDNAs isolatedhere are the first cDNA clones reported to human mrrH-RNAs.These results, together with previous results, furthersubstantiate that polyadenylated mrrH- and mrrL-RNAs are commonly present at varying levels among human tissues andcells. The 3′ end sequences of the cloned mrrH-cDNA provideswith insights into the mechanisms of transcription termination.The cDNA clones will provide tools to further the study of thefunction of mrr RNAs.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1008154027997

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Simultaneous fabrication of nanogap electrodes using field-emission-induced electromigration (2015)

Mitsuki Ito, Mamiko Yagi, Kohei Morihara and Jun-ichi Shirakashi

American Institute of Physics (AIP)

In: Journal of Applied Physics

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Publication Date: 2015-07-02

Description: We present a simple technique for simultaneous control of the electrical properties of multiple Ni nanogaps. This technique is based on electromigration induced by a field emission current and is called “activation.” Simultaneous tuning of the tunnel resistance of multiple nanogaps was achieved by passing a Fowler–Nordheim (F-N) field emission current through an initial group of three Ni nanogaps connected in series. The Ni nanogaps, which had asymmetrical shapes with initial gap separations in the 80–110-nm range, were fabricated by electron-beam lithography and a lift-off process. By performing the activation procedure, the current–voltage properties of the series-connected nanogaps were varied simultaneously from “insulating” to “metallic” via “tunneling” properties by increasing the preset current of the activation procedure. We can also simultaneously control the tunnel resistances of the series-connected nanogaps, which range from a resistance of the order of 100 TΩ–100 kΩ, by increasing the preset current from 1 nA to 30 μ A. This tendency is quite similar to that of individually activated nanogaps, and the tunnel resistance values of the simultaneously activated nanogaps were almost the same at each preset current. These results clearly imply that the electrical properties of the series-connected nanogaps can be controlled simultaneously via the activation procedure.

Print ISSN: 0021-8979

Electronic ISSN: 1089-7550

Topics: Physics