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K Nomura

Publications and source records attributed to K Nomura.

At least 19 recordsLinked to original sources
PubMed1997

Engineering of porcine pepsin. Alteration of S1 substrate specificity of pepsin to those of fungal aspartic proteinases by site-directed mutagenesis.

The S1 substrate specificity of porcine pepsin has been altered to resemble that of fungal aspartic proteinase with preference for a basic amino acid residue in P1 by site directed mutagenesis. On the basis of primary and tertiary structures of aspartic proteinases, the active site-flap mutants of porcine pepsin were constructed, which involved the replacement of Thr-77 by Asp (T77D), the insertion of Ser between Gly-78 and Ser-79 (G78(S)S79), and the double mutation (T77D/G78(S)S79). The specificities of the mutants were determined using p-nitrophenylalanine-based substrates containing a Phe or Lys residue at the P1 position. The double mutant cleaved the Lys-Phe(4-NO2) bonds, while wild-type enzyme digested other bonds. In addition, the pH dependence of hydrolysis of Lys-containing substrates by the double mutant indicates that the interactions between Asp-77 of the mutant and P1 Lys contribute to the transition state stabilization. The double mutant was also able to activate bovine trypsinogen to trypsin by the selective cleavage of the Lys6-Ile7 bond of trypsinogen. Results of this study suggest that the structure of the active site flap contributes to the S1 substrate specificity for basic amino acid residues in aspartic proteinases.

Amino Acid Sequence↗
PubMed1997

Sea urchin hatching enzyme (envelysin): cDNA cloning and deprivation of protein substrate specificity by autolytic degradation.

The hatching enzyme (envelysin) of the sea urchin Hemicentrotus pulcherrimus was purified from the medium of hatched blastulae. By cDNA cloning its deduced amino acid sequence and molecular architecture were revealed. The 591-residue precursor with calculated Mr of 66,123 consists of an 18-residue signal sequence, a 151-residue propeptide, and a 422-residue mature enzyme with N-terminal catalytic and C-terminal hemopexin-like domains. As compared with that of Paracentrotus lividus, its amino acid sequence is 69% identical and 10% similar. They share typical structural features with the mammalian MMP gene family members: cysteine switch, zinc-binding signature, methionine-turn, Cys residues near both ends of hemopexin-like domain, etc. However, its propeptide has a 70-residue extra sequence with an Asp- and Glu-rich stretch, supposedly involved in the proenzyme activation by binding Ca2+ ions in seawater. The hinge region is also longer than those of most MMPs, with an extra sequence rich in Thr and Arg residues. Mature 50K enzyme is highly susceptible to autolytic cleavage at Gln(503)-Leu(504), producing the 38K form retaining catalytic activity and substrate specificity against fertilization envelope. The 38K form and 15K fragment were coeluted from a gel-filtration column, suggesting that these two fragments are disulfide-bridged and that the tertiary structure is not much deviated. The 38K form further autolyzed to 32K form by cleaving Tyr(450)-Tyr(451) bond with the loss of protein-substrate specificity, retaining only nonspecific protease activity. Thus, the autolytic release of 2/3 of the C-terminal domain reduced the highly specific enzyme to a common nonspecific protease, implying that the size and structure of almost the entire hemopexin-like domain is essential for the protein substrate specificity. Moreover, autolytic degradation of envelysins from the two species follow quite different pathways despite their high homology in structure. The 38K and 32K forms were inhibited by bovine TIMP-1 with different IC50 values, indicating that its inhibitory activity depends on the extent of the interaction with the C-terminal domain of the enzyme.

Amino Acid Sequence↗
PubMed1997

Requirement of gamma-carboxyglutamic acid residues for the biological activity of Gas6: contribution of endogenous Gas6 to the proliferation of vascular smooth muscle cells.

Gas6 (encoded by growth-arrest-specific gene 6) is a gamma-carboxyglutamic acid (Gla)-containing protein which is released from growth-arrested vascular smooth muscle cells (VSMCs) and potentiates VSMC proliferation induced by Ca2+-mobilizing growth factors, but not that induced by receptor tyrosine kinases. In this study we examined the importance of Gla residues for the biological activities of Gas6 and tried to assess the importance of endogenous Gas6 in VSMC proliferation. We demonstrated that Gla-deficient Gas6 lacked receptor-binding and growth-potentiating activities. Therefore the vitamin K-dependent modification of Gas6 appeared to be essential for its biological activities. Next we used warfarin, an inhibitor of vitamin K-dependent gamma-carboxylation, to estimate the contribution of endogenous Gas6 to VSMC proliferation. Warfarin markedly inhibited the thrombin-induced proliferation of VSMC without affecting the mRNA or protein expression of Gas6. Therefore the inhibition seems to be due to prevention of the vitamin K-dependent modification of Gas6. However, warfarin did not affect epidermal growth factor-induced proliferation. A neutralizing antibody against Gas6 gave a similar result, i.e. it inhibited thrombin-induced VSMC proliferation but not that induced by epidermal growth factor. These results indicate that endogenously produced Gas6 is very important for VSMC proliferation induced by Ca2+-mobilizing growth factors.

1-Carboxyglutamic Acid↗
PubMed1997

Peripheral framework of carrot cell nucleus contains a novel protein predicted to exhibit a long alpha-helical domain.

A monoclonal antibody, CML-1, raised against carrot (Daucus carota L.) nuclear-matrix proteins selectively labeled the nuclear periphery of carrot protoplasts when visualized by confocal and electron microscopy. To identify the constituent proteins of higher plant cells structurally homologous to the vertebrate nuclear lamina, we cloned overlapping cDNAs partially encoding a CML-1-recognized protein and determined the entire sequence including the open reading frame. When the deduced amino acid sequence was compared with other known protein sequences contained in major databases, no protein was found to show high sequence identity across the whole region of the protein, while the partial sequence showed strong similarities with myosin, tropomyosin, and some intermediate filament proteins. The protein, designated NMCP1, had an estimated molecular mass of 133.6 kDa and showed three characteristic domains. The central domain contains long alpha-helices exhibiting heptad repeats of apolar residues, demonstrating structural similarity to that of filament-forming proteins. The terminal domains are predominantly nonhelical and contain potential sequence motifs for nuclear localization signals. NMCP1 has many recognition motifs for different types of protein kinases, including cdc2 kinase and PKC. These results suggest that NMCP1 protein forms coiled-coil filaments and is a constituent of the peripheral architecture of the higher plant cell nucleus.

Amino Acid Sequence↗
PubMed1997

Cyclic-AMP-dependent activation of an inter-phylum hybrid histone-kinase complex reconstituted from sea urchin sperm-regulatory subunits and bovine heart catalytic subunits.

A cAMP-dependent histone kinase was purified and characterized from spermatozoa of the sea urchin Hemicentrotus pulcherrimus. The molecular mass of the kinase was estimated to be 178 kDa by native PAGE and 400 kDa by gel chromatography on a Superose 6 HR 10/30 column. The enzyme, composed of two 39-kDa catalytic subunits and two 48-kDa regulatory subunits, phosphorylates the lysine-rich histone subspecies (H1 and H2B) isolated from H. pulcherrimus spermatozoa. We isolated cDNA clones encoding a 39-kDa catalytic subunit and a 48-kDa regulatory subunit of the enzyme. The cDNA clone for the 39-kDa subunit was 3881 bp, and the 352-residue deduced amino acid sequence showed 78% similarity with the catalytic subunit of/mammalian cAMP-dependent protein kinase (PKA). The cDNA for the 48-kDa subunit was 4589 bp and the 368-residue deduced amino acid sequence showed 57% similarity with the regulatory subunit of mammalian PKA, although the N-terminal 77 residues showed poor similarity. The mRNAs encoding both the catalytic subunit (7.5 kb) and the regulatory subunit (4.6 kb) were expressed in testis, ovary and egg. An inter-phylum hybrid enzyme, reconstituted from the regulatory subunit of cAMP-dependent histone kinase of sea urchin sperm and the catalytic subunit of bovine heart PKA, has a cAMP-dependent histone kinase activity. Thus, we suggest that the N-terminal 77-amino-acid residues of the regulatory subunit are not essential for inhibition by the regulatory subunit of the catalytic subunit, and that cAMP-dependent inhibitory activity of the regulatory subunit resides in the sequence between the inhibitory site and the C-terminus.

Amino Acid Sequence↗
PubMed1997

Selective homonuclear polarization transfer in the tilted rotating frame under magic angle spinning in solids.

An application of the R2TR method (1995, Chem. Phys. Lett. 232, 424) to selective homonuclear polarization transfer under magic angle spinning is proposed. It is shown that, for a spinning speed fast enough to remove the maximum homonuclear dipolar coupling constant omegaD involved, the flip-flop and flop-flop mechanisms are suitable for recoupling the spins with a chemical shift difference larger than omegaD and a difference comparable to or smaller than omegaD, respectively. It is also shown that, for fast polarization transfer, the off-resonance frequencies should be much higher than the RF intensity in the flip-flop condition, while for the flop-flop condition, the off-resonance frequencies should be much lower than the RF intensity. Some one- and two-dimensional experiments are proposed by utilizing the capability of the R2TR method to abruptly switch on and off the recoupling condition, and are demonstrated for triply 13C-enriched l-alanine. The mixing time required for population transfer was found to be ca. 0.5 ms for the methine and methyl 13C spins separated by 1.5 A and ca. 5 ms for the methyl and the carboxyl carbons separated by 2.5 A. The experimental results and theoretical simulations show that selective polarization transfer is achieved when the difference in the isotropic chemical shifts between the relevant pair of spins and a neighboring spin is more than 1000 Hz.

Alanine↗
PubMed1997

A keratin K10 gene mutation in a Japanese patient with epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis (EHK), or bullous congenital ichthyosiform erythroderma, is characterized by generalized erythroderma, ichthyosiform skin and blistering, and is caused by an aberration of the keratin intermediate filaments. In this study, we examined keratin K10 and 1 gene mutations in a Japanese EHK patient who had severe ichthyosiform erythroderma at birth and developed subsequent blistering. The patient had a G to A transition at codon 156 of the keratin K10 gene, which resulted in an arginine (Arg)-->histidine (His) substitution in the helix initiation peptide of the highly-conserved 1A domain in keratin K10. This is the first mutation report of a Japanese patient with EHK, although the position and mode of the mutation identified here did not differ from those in reported Western cases.

Amino Acid Sequence↗
PubMed1997

Plasma concentration of endothelin-1 does not reflect renal vasoconstriction as estimated by duplex ultrasonography in cirrhosis.

Endothelin, a potent vasoconstrictor, is thought to play a role in liver cirrhosis-related functional kidney failure. Our aim was to investigate the correlation between renal vasoconstriction, as detected by a Doppler ultrasound technique, and plasma concentrations of endothelin in cirrhotic patients. Fifty cirrhotic patients underwent Doppler examinations to detect renal vasoconstriction. The plasma concentration of endothelin was measured in 10 patients who exhibited vasoconstriction of the renal microvessels diagnosed by Doppler waveform analysis and was compared to that of patients in whom there was no sign of such vasoconstriction. No difference was observed in the plasma concentration of endothelin between patients in whom renal vasoconstriction was diagnosed and those in whom it was not. Our results suggested that the circulating endothelin does not reflect renal vasoconstriction, at least in the early phase of the functional renal failure associated with cirrhosis of the liver.

Endothelin-1↗
PubMed1997

Ovarian atypical endometriosis: its close association with malignant epithelial tumours.

The incidence of ovarian atypical endometriosis and its association with malignant epithelial tumours in a consecutive series of cases during the period 1987 to 1995 were studied. Atypical glandular changes were observed in four (1.7%) of 255 ovarian endometriosis cases and one patient with ovarian atypical endometriosis developed subsequent endometrioid carcinoma in the abdominal wall. Fifty-four (24.1%) of the 224 ovarian cancers were associated with ovarian endometriosis; 21 with typical and 33 with atypical endometriosis. Clear cell carcinomas and endometrioid carcinomas were most frequently associated with endometriosis, with 54% (27 of 50 cases) and 41.9% (13 of 31), respectively. Atypical endometriosis was found in 18 clear cell carcinomas, in seven endometrioid carcinomas, in four serous carcinomas, in three mucinous borderline tumours, and in one serous borderline tumour. In 13 cases, the atypical endometriosis was in contiguity with malignant epithelial tumours. We consider that atypical endometriosis possesses a precancerous potential or is most frequently associated with clear cell and endometrioid carcinomas. Close screening of cellular atypia or hyperplasia in ovarian endometriosis and careful long-term follow-up of patients with atypical endometriosis is required.

Adult↗
PubMed1997

Vulval angiomyofibroblastoma. Clinicopathologic analysis of six cases.

Clinicopathologic, immunohistochemical, ultrastructural, and flow cytometric analyses were performed on vulvar angiomyofibroblastomas in six women (ages 32 to 46 years; average age, 41.3 years). The tumors were all well circumscribed, and measured 2 to 9 cm in greatest dimension. Five tumors were clinically diagnosed as Bartholin's gland cysts. At histologic analysis, the tumors were characterized by alternating hypercellular and hypocellular edematous areas with abundant blood vessels. The hypercellular areas showed a perivascular proliferation of spindle-shaped and round cells, which often formed small nests or epithelioid arrangements, and a short fascicular pattern of spindle cells. The proliferating vessels were thin-walled and capillary-sized. Cellular atypia and mitotic figures were absent. Two tumors contained an adipose element, and one showed a prominent vascular proliferation reminiscent of a capillary hemangioma. At immunohistochemical analysis, the tumor cells were strongly positive for vimentin and desmin. Some cells were weakly positive for HHF35 and alpha-smooth muscle actin. Ultrastructurally, one neoplasm showed fibroblastic differentiation. Flow cytometry revealed three tumors were DNA diploid and two were aneuploid. All tumors were excised, and none have recurred (average follow-up, 28.3 months). These results indicate that angiomyofibroblastomas contain heterogeneous components.

Adult↗
PubMed1997

Application of antisense ribonucleic acid complementary to O6-methylguanine-deoxyribonucleic acid methyltransferase messenger ribonucleic acid for therapy of malignant gliomas.

OBJECTIVE: A derivative of chloroethylnitrosoureas, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), is a drug of choice for the chemotherapy of human malignant brain tumors. However, the cytocidal effect of ACNU is effectively repressed through repair of ACNU-mediated deoxyribonucleic acid lesions by O6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT). Because a variety of human tumors, including brain tumors, contain high levels of MGMT activity, we investigated the effect of antisense ribonucleic acid (RNA) complementary to MGMT messenger RNA on ACNU resistance in tumor cells. METHODS: We established a stable ACNU-resistant clone, C6AR, from the rat glioma cell line C6 exposed to a stepwise increasing concentration of ACNU. We transfected a plasmid deoxyribonucleic acid-encoding antisense MGMT RNA under the control of the human metallothionein promoter into C6AR cells and determined the effect of the antisense RNA on ACNU resistance of tumor cells by a colony-forming efficiency assay. RESULTS: C6AR cells expressed abundant MGMT messenger RNA, although the transcription level of the MGMT gene in parental C6 cells was below the lower limits of detection under the same assay conditions. ACNU resistance of C6AR cells was significantly repressed by transfected gene-dependent antisense MGMT RNA expression that resulted in decreased survival of the tumor cells. CONCLUSION: ACNU resistance resulting from the expression of MGMT in rat glioma cells is significantly overcome by the expression of antisense MGMT RNA. This result suggests that the antisense MGMT RNA system might be a useful strategy for overcoming ACNU resistance in the treatment of intractable malignant gliomas.

Animals↗
PubMed1997

In vivo transfer of a foreign gene to keratinocytes using the hemagglutinating virus of Japan-liposome method.

The hemagglutinating virus of Japan (HVJ)-liposome method involves the entrapment of DNA and nuclear protein within liposomes and the use of HVJ to enhance liposome fusion with cell membranes. This method has been used successfully for in vivo gene transfer to various types of tissue. In this study, we investigated whether this method transfers genes effectively to normal and malignantly transformed keratinocytes in vivo. We applied HVJ-liposome complex (HLC) containing the beta-galactosidase gene to the tape-stripped skin of hairless rats and detected the enzyme activity in the keratinocytes of the treated skin. Comparison of this method with the naked DNA injection method, which was shown recently to be useful for in vivo gene transfer to keratinocytes, demonstrated that the transfer efficiency of the latter was about 5 times higher than that of the former. We assessed the efficacy of the HVJ-liposome method for gene transfer to transformed keratinocytes by examining the effect of HLC containing the herpes simplex virus thymidine kinase gene on the growth of mouse squamous cell carcinomas. Local injection of HLC into the tumors followed by administration of ganciclovir to mice resulted in tumor growth inhibition. These results indicate that the HVJ-liposome method is suitable for in vivo gene transfer to keratinocytes; also that this method may prove a good tool for basic research into keratinocyte biology and future keratinocyte gene therapy.

Animals↗
PubMed1997

Glycine substitution mutations in the type VII collagen gene (COL7A1) in dystrophic epidermolysis bullosa: implications for genetic counseling.

Dystrophic epidermolysis bullosa (DEB) is an inherited mechanobullous disorder characterized by fragility of the skin and mucous membranes. The anchoring fibril protein, type VII collagen, is encoded by COL7A1, which harbors mutations in this group of diseases. In this study, we report novel glycine substitution mutations in COL7A1 in two Japanese families with DEB. The mutation detection strategy consisted of PCR amplification of genomic DNA, followed by heteroduplex analysis and nucleotide sequencing of the PCR products demonstrating altered mobility. The first case is a patient with clinically severe recessive DEB. The proband was shown to have a homozygous glycine-to-valine substitution (G2671V) in exon 108. The clinically unaffected parents were heterozygous carriers of this mutation, indicating that this glycine substitution in one allele is "silent" when combined with a normal COL7A1 allele. Thus, this patient appeared to be affected with DEB inherited in an autosomal recessive pattern. The second case was a DEB patient with a heterozygous glycine-to-glutamic acid substitution (G2079E) in exon 75. The parents were clinically unaffected and neither had this mutation in their peripheral blood leukocyte DNA. Haplotype analyses suggested that this case arose as a de novo occurrence of autosomal dominant DEB. These cases illustrate the consequences of COL7A1 glycine substitution mutations underlying DEB in terms of the mode of inheritance and the phenotype, with profound implications for genetic counseling of individuals at risk for recurrence of DEB in subsequent offspring or future generations.

Adult↗
PubMed1997

Small cell carcinoma of the ovary: a case report of large cell variant.

An extremely rare case of large cell variant of ovarian small cell carcinoma is described. A 34-year-old woman (gravida 1, para 1) had a unilateral ovarian mass measuring 17cm in greatest diameter and a metastatic lesion in the omentum. Microscopically, the tumor showed a diffuse arrangement of large, closely packed epithelial cells with abundant eosinophilic cytoplasm and large nuclei with prominent nucleoli. The tumor cells also were arranged in follicle-like and trabecular structures and cords. Immunohistochemically, many tumor cells were diffusely positive for epithelial membrane antigen and some cells contained cytokeratin CAM5.2, vimentin, neurofilament, neuron-specific enolase, or alpha-1 antitrypsin. However, no specific lineage was detected. The tumor was aneuploid by flow cytometry. The patient received chemotherapy postoperatively. However, the patient showed metastases in the inguinal and retroperitoneal lymph nodes. The serum calcium level, which was not measured preoperatively, was mildly elevated postoperatively. The patient was well with no evidence of disease 17 months after diagnosis. This tumor must be distinguished from other primary or metastatic 'undifferentiated' neoplasms, especially ovarian small cell carcinoma of pulmonary type and granulosa cell tumor.

Adult↗
PubMed1997

Chronic urticaria associated with aseptic meningitis: an atypical urticarial vasculitis?

The patient was a 7-year-old girl with early onset urticarial cutaneous lesions and was later complicated with aseptic meningitis. Her skin lesions occurred in the infantile period and were diagnosed as urticaria, but did not disappear with antihistamines and were recurrent and persistent. In addition, she had experienced an episode of headache about once a month since 1991, when she was 4 years old, and was diagnosed as aseptic meningitis. All studies including skin biopsy for urticarial vasculitis (UV) and systemic lupus erythematosus (SLE) were negative except for the data from non-specific inflammations. A systemic corticosteroid therapy dramatically reduced her symptoms. An unusual clinical course for this patient is described. It might suggest that this case is a presentation of the disease entity of UV, chronic urticaria and possibly SLE. To our knowledge, a similar case has not been previously reported.

Child↗
PubMed1997

Quantitative evaluation of inflammatory cells in seasonal allergic conjunctivitis.

This study aimed to investigate the inflammatory changes occurring in the allergic conjunctiva due to different allergens. Ninety-two allergic conjunctivitis patients were divided into two groups: group I showed cedar pollen sensitivity only; group II showed sensitivities to cedar pollen and household dust or mites. Using brush cytology procedures, quantitative evaluation of inflammatory cells in the conjunctiva was made. The mean proportion of the neutrophils and lymphocytes in group II was significantly higher than the percentage of neutrophils in group I. The counts of eosinophils showed no significant difference between groups I and II. Our results suggest that the eosinophil numbers are not different between the allergic conjunctivitis groups. The neutrophilic and lymphocytic increase in allergic conjunctivitis may be due to two distinct groups of antigens.

Adolescent↗