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H Inoue

Publications and source records attributed to H Inoue.

At least 1,369 records · Page 76Linked to original sources
PubMed1991

Proteolytic activity and cleavage specificity of cathepsin E at the physiological pH as examined towards the B chain of oxidized insulin.

Proteolytic activity and cleavage specificity of cathepsin E were investigated in a wide range of pHs from 3.0 to 10.5 using the B chain of oxidized insulin as substrate. Contrary to the previous notion that cathepsin E is virtually inactive above pH 6, significant proteolytic activity was observed at pH 7.4 and above. Further, cleavage specificity appeared to change significantly with pH and rather specific cleavage occurred at pH 7.4 and above as compared to pH 5.5 and 3.0. These results suggest that cathepsin E may function in vivo at the physiological pH with a rather restricted specificity.

Amino Acid Sequence↗
PubMed1991

The primary structure of Aspergillus niger acid proteinase A.

The complete amino acid sequence of the acid proteinase A, a non-pepsin type acid proteinase from the fungus Aspergillus niger var. macrosporus, was determined by protein sequencing. The enzyme was first dissociated at pH 8.5 into a light (L) chain and a heavy (H) chain, and the L chain was sequenced completely. Further sequencing was performed with the reduced and pyridylethylated or aminoethylated derivative of the whole protein, using peptides obtained by digestions with Staphylococcus aureus V8 protease, trypsin, chymotrypsin, and lysylendopeptidase. The location of the two disulfide bonds was determined by analysis of cystine-containing peptides obtained from a chymotryptic digest of the unmodified protein. These results established that the protein consists of a 39-residue L chain and a 173-residue H chain that associate noncovalently to form the native enzyme of 212 residues (Mr 22,265). This is, to our knowledge, the first time that such a protein with a rather short peptide chain associated noncovalently has been found. No sequence homology is found with other acid or aspartic proteinases, except for Scytalidium lignicolum acid proteinase B, an enzyme unrelated to pepsin by sequence, which has about 50% identity with the present enzyme. These two enzymes, however, are remarkably different from each other in some structural features.

Amino Acid Sequence↗
PubMed1991

The gene and deduced protein sequences of the zymogen of Aspergillus niger acid proteinase A.

Proteinase A obtained from the culture medium of Aspergillus niger var. macrosporus is a unique acid endopeptidase that is insensitive (or less sensitive) to specific inhibitors of ordinary acid or aspartic proteinases, such as pepstatin, diazoacetyl-DL-norleucine methyl ester, and 1,2-epoxy-3-(p-nitrophenoxy)-propane. In the preceding paper (Takahashi, K., Inoue, H., Sakai, K., Kohama, T., Kitahara, S., Takishima, K., Tanji, M., Athauda, S. B. P., Takahashi, T., Akanuma, H., Mamiya, G., Yamasaki, M. J. Biol. Chem. 266, 19480-19483), we reported the complete primary structure of the mature enzyme determined at the protein level. The enzyme has a unique two-chain structure with a 39-residue light (L) chain and a 173-residue heavy (H) chain linked noncovalently. As an extension of this study, we isolated genomic and cDNA clones encoding this proteinase and determined their nucleotide sequences. To isolate a genomic clone, the genomic DNA was selectively amplified by polymerase chain reaction using mixed oligonucleotide primers designed from the amino acid sequence of the H chain, and a specific probe thus generated was used for screening a lambda gt10 genomic library. A cDNA for the enzyme was also selectively amplified by polymerase chain reaction using primers synthesized based on the sequence of the genomic DNA. Sequencing of the cloned genomic DNA and cDNA revealed the nucleotide sequence of the structural gene for the enzyme of 846 base pairs without introns. It encodes the precursor form of proteinase A, including an NH2-terminal preprosequence of 59 residues, the L chain of 39 residues, an intervening sequence of 11 residues, and the H chain of 173 residues linked in that order. Thus, proteinase A is thought to be synthesized as a single peptide chain preproenzyme of 282 residues, which is processed to generate the mature two-chain form.

Amino Acid Sequence↗
PubMed1991

Cloning and functional characterization of a eucaryotic DNA photolyase gene from Neurospora crassa.

We cloned a genomic fragment of a photolyase gene from Neurospora crassa by polymerase chain reaction using synthesized oligonucleotide primers designed from the most conserved amino acid sequences among photolyases of various organisms. Using the cloned fragment as a hybridization probe we isolated a genomic fragment and cDNA clones encoding the complete photolyase gene of this organism. The amino acid sequence of the photolyase deduced from the determined nucleotide sequence indicates a protein consisting of 615 amino acid residues (Mr 69,971), which is most similar to that of Saccharomyces cerevisiae. Like yeast photolyase it contains a protruding amino terminus which is missing in photolyases of bacterial origin. Comparison of amino acids sequences among six photolyases suggests that the Neurospora crassa photolyase is more similar to photolyases of pterin type than those of deazaflavin type.

Amino Acid Sequence↗
PubMed1991

Leucine zipper motif in porcine renin-binding protein (RnBP) and its relationship to the formation of an RnBP-renin heterodimer and an RnBP homodimer.

An apparent leucine zipper motif was recognized in the predicted amino acid sequence of porcine kidney renin-binding protein (RnBP) by analysis of the nucleotide sequence of a cDNA encoding the protein (Inoue, H., Fukui, K., Takahashi, S., and Miyake, Y. (1990) J. Biol. Chem. 265, 6556-6561). To evaluate the role of this motif in the formation of an RnBP-renin heterodimer and an RnBP homodimer, a porcine mutant cDNA involving Leu185----Asp and Leu192----Asp substitutions was constructed and expressed in vitro and in Xenopus oocytes. The mutant protein neither binds to renin nor forms the homodimer. The results strongly suggest that the leucine zipper motif in the RnBP molecule mediates the formation of both the RnBP-renin heterodimer and the RnBP homodimer observed previously. The existence of the motif should facilitate elucidation of the role of RnBP in renin metabolism.

Amino Acid Sequence↗
PubMed1991

Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli.

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.

Base Sequence↗
PubMed1991

Ion-sensitive field effect transistor as a monovalent cation detector for ion chromatography and its application to the measurement of Na+ and K+ concentrations in serum.

An ion-sensitive field effect transistor, which is a small potentiometric ion-sensing probe, was applied as a detector in the ion chromatography of alkali-metal cations. The cation-sensing transistor was prepared by coating the gate part of the probe to form a poly(vinyl chloride) matrix membrane containing tetranactin, which enables detection of alkali-metal ions such as Na+, K+, Rb+, and Cs+. To be able to analyse amounts less than 1 microliter and make full use of the small ion-sensing probe, we constructed a miniature cation-exchange separation column (support 10 microns polystyrene) with a PTFE tube (20 mm x 1.5 mm I.D. x 2.1 mm O.D). As an application of the system, Na+ and K+ concentrations in serum were determined. The analytical results for these two cations were good agreement with those obtained by flame photometry.

Cations↗
PubMed1991

A new method for estimating preexcitation index without extrastimulus technique and its usefulness in determining the mechanism of supraventricular tachycardia.

The preexcitation index has been shown to be useful in determining the mechanism of paroxysmal supraventricular tachycardia (SVT) and the site of the accessory pathway in atrioventricular (AV) reentrant tachycardia. To test whether a preexcitation index could be computed analytically instead of by scanning the whole SVT cycle with extrastimuli, 19 patients with SVT were studied. The new index was computed using the following formula: (AV conduction time during SVT) + (ventriculoatrial conduction time during ventricular pacing at the SVT cycle length) - (SVT cycle length). There was a strong correlation between the preexcitation index determined by the extrastimulus technique and the new index in 15 patients in whom the preexcitation index could be determined (r = 0.99, p less than 0.01). The value on the new index was greater than 90 ms only in patients with dual AV nodal pathways. In the 4 patients in whom the preexcitation index could not be determined by the extrastimulus technique, the new index could differentiate AV reentrant tachycardia (index for 2 patients, 60 and 60 ms, respectively) from AV nodal reentrant tachycardia (index for 2 patients, 100 and 105 ms, respectively). In conclusion, the new index provided help in determining the mechanism of SVT, even when retrograde atrial preexcitation by a ventricular extrastimulus did not occur.

Adolescent↗
PubMed1991

NMR studies of a DNA containing 8-hydroxydeoxyguanosine.

The effects of hydroxylation at the C8 of a deoxyguanosine residue in DNA were studied by NMR analysis of a self-complementary dodecanucleotide, d(C1-G2-C3-oh8G4-A5-A6-T7-T8-C9-G10-C11-G12), which has an 8-hydroxy-2'-deoxyguanosine (oh8dG) residue at the 4th position. NMR data indicate that the 8-hydroxyguanine (oh8G) base takes a 6,8-diketo tautomeric form and is base-paired to C with Watson-Crick type hydrogen bonds in a B-form structure. The thermal stability of the duplex is reduced, but the overall structure is much the same as that of the unmodified d(CGCGAATTCGCG) duplex. The structural changes caused by 8-hydroxylation of the deoxyguanosine, if any, are localized near the modification site.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1991

Molecular cloning of a new class of cartilage-specific matrix, chondromodulin-I, which stimulates growth of cultured chondrocytes.

Here we report the structure and bioactivity of 25 kDa glycoprotein (chondromodulin-I) as a tissue-specific functional matrix component identified and cloned for the first time. Chondromodulin-I purified from fetal bovine cartilage markedly stimulated DNA synthesis of cultured growth-plate chondrocytes in the presence of basic fibroblast growth factor (FGF). Bovine chondromodulin-I cDNA revealed that the mature protein consists of 121 amino acids with three possible glycosylation sites and is coded as the C-terminal part of a larger precursor. On northern blot analysis, expression of chondromodulin-I mRNA was observed only in cartilage.

Amino Acid Sequence↗
PubMed1991

Prognostic implications of DNA histogram, DNA content, and histologic changes of regional lymph nodes in patients with lung cancer.

Forty-six cases of resected lung cancer, including 20 cases at Stages I and II and 26 cases at Stage III (N2), were subdivided into two groups: a good prognosis group with a longer survival period and a poor prognosis group in which the patients died earlier of the cancer. From paraffin-embedded lymph node tissues of these patients, the authors examined DNA histogram pattern and DNA content, using flow cytometry, and histologic hyperplasia of germinal center and paracortical area; they also evaluated their correlation with the prognosis. In the good prognosis group at Stages I and II, paracortical hyperplasia (PH) of the lymph nodes was observed significantly more frequently. In the good prognosis group at Stage III, the incidence of PH, G2M phase in the DNA histograms, and DNA content were all significantly higher. DNA content was positively correlated with the grade of PH.

Adenocarcinoma↗
PubMed1991

[Kinetic estimation of infused Lipiodol within a hepatoma using a CT dynamic study].

Distribution of Lipiodol within hepatic tumor was evaluated using serial computed tomography. Imagistic Lipiodol transition into the tumor via hepatic artery was slower than that of water soluble contrast medium, spreading to central part first and turn to peripheral part of the tumor. This method was thought to be useful to clarify Lipiodol distribution in hepatic tumor and to decide the method or necessity of additional trans-arterial therapy.

Aged↗
PubMed1991

Structures of regulatory regions in the human cytochrome P-450scc (desmolase) gene.

The mechanism of regulatory expression of human cytochrome P-450scc gene by cAMP was investigated in a transient expression system using Y-1 cells (mouse adrenal tumor cell line) and a chimeric DNA composed of the structural gene for bacterial chloramphenicol acetyltransferase and the 5' flanking upstream sequence of the cytochrome P-450scc (cholesterol desmolase) gene which was revealed to contain a DNA element(s) responsive to cAMP [Inoue, H. et al. (1988) Eur. J. Biochem. 171, 435-440]. Introduction of deletions and point mutations in the upstream regulatory sequence demonstrated that three regions were mainly required for response to cAMP. These regions contained a short similar sequence. All of them have a 5-bp motif GTCAT (or ATGAC) in common, and have at least two motifs which conserve four out of five base pairs of the consensus sequence of the cAMP-responsive element (CRE), CGTCA (or TGACG). They are all apparently necessary for regulation by cAMP. Gel mobility shift assays suggested that a binding factor(s) to these regions was present in the nuclear extracts of Y-1 cells and adrenal cortex tissues and appeared to be different from the somatostatin CRE-binding protein. Deletion analysis also suggested that the region around -44 was essential to the basal transcriptional activity. This region shows some similarity to the CTF NF-1 binding site [Johnson and McKnight (1989) Annu. Rev. Biochem. 58, 799-839].

Animals↗
PubMed1991

Bone morphogenetic proteins (BMP-2 and BMP-3) promote growth and expression of the differentiated phenotype of rabbit chondrocytes and osteoblastic MC3T3-E1 cells in vitro.

We studied the effects of highly purified bone morphogenetic protein 2 and 3 (BMP-2 and -3) on growth plate chondrocytes and osteoblastic cells in vitro and compared to TGF-beta. A mixture of BMP-2 and 3 (BMPs) strongly stimulated DNA synthesis of chondrocytes in the presence of fibroblast growth factor (FGF). BMPs induced rapid maturation of chondrocytes at a growing stage: BMPs transformed the cells into rounded cells and induced marked accumulation of cartilage matrix; TGF-beta slightly reduced matrix accumulation and changed cell morphology into spindle-like in the presence of FGF. Moreover, exposure of chondrocytes to BMPs resulted in a dramatic increase of the putative approximately 80 kD PTH receptors expressed on the cell surface. In multilayered chondrocytes at the calcifying stage, BMPs stimulated alkaline phosphatase (ALPase) activity but TGF-beta inhibited it. In osteoblastic MC3T3-E1 cells, BMPs were found to be the most potent stimulator of ALPase activity thus far described: ALPase in the cells treated with approximately 100 ng/ml of BMPs reached 5- to 20-fold over the basal, whereas TGF-beta inhibited expression of ALPase activity in these cells. The stimulatory action of BMPs overrode the inhibition of ALPase activity by TGF-beta when the cells were incubated with TGF-beta and BMPs. BMPs also upregulated expression of the approximately 80 kD PTH receptor on the cells. These results suggest that BMPs have unique biologic activities in vitro that lead to growth and phenotypic expression of cells playing a critical role in endochondral bone formation.

Alkaline Phosphatase↗
PubMed1991

Simple system for isolation of cellular and viral mutants for transformation by retrovirus.

To investigate the cellular mechanism of transformation by retroviruses, we established a system for isolation of cellular and viral mutants for transformation of a rat cell line. Mutagenized untransformed cells of this line were infected with recombinant murine retrovirus containing the src gene of Rous sarcoma virus and the selective marker gene, neo. After reaching confluence, cells transformed by the src gene tend to overgrow and die. Utilizing this property of src transformed rat cells and the selective marker gene, we could easily select untransformed cell clones containing the retrovirus genome. Expression of the src gene product in the flat clones selected was examined by in vitro assay of src kinase activity. To determine whether the mutations of these flat clones were viral or cellular, the susceptibilities of the clones to transformation were examined after superinfection with the wild-type virus and also characterized the retroviruses recovered from these clones. With this system, two novel clones were isolated. One had a defect in viral information affecting the transformed phenotype, but still retained src kinase activity like fully transformed cells. The other showed low src kinase activity but retained wild-type transforming virus, suggesting that a cellular gene involved in viral gene expression was mutated.

Animals↗