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Biomedical subjects

I Fujii

Publications and source records attributed to I Fujii.

At least 19 recordsLinked to original sources
PubMed2003

Display of a functional hetero-oligomeric catalytic antibody on the yeast cell surface.

A functional hetero-oligomeric protein was, for the first time, displayed on the yeast cell surface. A hetero-oligomeric Fab fragment of the catalytic antibody 6D9 can hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol. The gene encoding the light chain of the Fab fragment of 6D9 was expressed with the tandemly-linked C-terminal half of alpha-agglutinin. At the same time, the gene encoding the Fd fragment of the heavy chain of the Fab fragment was expressed as a secretion protein. The combined Fab fragment displayed and associated on the yeast cell surface had an intermolecular disulfide linkage between the light and heavy chains. This protein fragment catalyzed the hydrolysis of a chloramphenicol monoester derivative and exhibited high stability in binding with a transition-state analog (TSA). The catalytic reaction was also inhibited by the TSA. The successful display of a functional hetero-oligomeric catalytic antibody provides a useful model for the display of hetero-oligomeric proteins and enzymes.

Antibodies, Catalytic↗
PubMed2002

[Successful lingular segmentectomy using bronchoplasty in a patient with early hilar second primary lung carcinoma].

A 71-year-old man was admitted to our hospital with a small protrusive lesion at the lingular orifice of the left upper bronchus. He had undergone a right lower lobectomy and mediastinal dissection for lung carcinoma (large cell carcinoma, pT1N0M0) 14 months earlier. Early hilar squamous cell carcinoma was diagnosed by chest radiograph, CT and transbronchial biopsy. We performed a lingular segmentectomy with wedge resection of the left upper bronchus and N 1 lymph node dissection. The tumor was histopathologically diagnosed as early hilar second primary lung carcinoma. The patient's postoperative course was uncomplicated. At present, he is alive with good respiratory condition and without any evidence of recurrence. Segmentectomy is appropriate for a patient with contralateral second primary lung carcinoma as well as a patient with early hilar lung carcinoma. Bronchoplasty seems to increase the likelihood that such a patient will be a candidate for segmentectomy.

Aged↗
PubMed2001

Pentaketide melanin biosynthesis in Aspergillus fumigatus requires chain-length shortening of a heptaketide precursor.

Chain lengths and cyclization patterns of microbial polyketides are generally determined by polyketide synthases alone. Fungal polyketide melanins are often derived from a pentaketide 1,8-dihydroxynaphthalene, and pentaketide synthases are used for synthesis of the upstream pentaketide precursor, 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN). However, Aspergillus fumigatus, a human fungal pathogen, uses a heptaketide synthase (Alb1p) to synthesize its conidial pigment through a pentaketide pathway similar to that which produces 1,8-dihydroxynaphthalene-melanin. In this study we demonstrate that a novel protein, Ayg1p, is involved in the formation of 1,3,6,8-THN by chain-length shortening of a heptaketide precursor in A. fumigatus. Deletion of the ayg1 gene prevented the accumulation of 1,3,6,8-THN suggesting the involvement of ayg1 in 1,3,6,8-THN production. Genetic analyses of double-gene deletants suggested that Ayg1p catalyzes a novel biosynthetic step downstream of Alb1p and upstream of Arp2p (1,3,6,8-THN reductase). Further genetic and biochemical analyses of the reconstituted strains carrying alb1, ayg1, or alb1 + ayg1 indicated that Ayg1p is essential for synthesis of 1,3,6,8-THN in addition to Alb1p. Cell-free enzyme assays, using the crude Ayg1p protein extract, revealed that Ayg1p enzymatically shortened the heptaketide product of Alb1p to 1,3,6,8-THN. Thus, the protein Ayg1p facilitates the participation of a heptaketide synthase in a pentaketide pathway via a novel polyketide-shortening mechanism in A. fumigatus.

Alcohol Oxidoreductases↗
PubMed2001

Identification of Claisen cyclase domain in fungal polyketide synthase WA, a naphthopyrone synthase of Aspergillus nidulans.

BACKGROUND: Based on the homology with fatty acid synthases and bacterial polyketide synthases (PKSs), thioesterase domains have been assigned at the C-terminus regions of fungal iterative type I PKSs. We previously overexpressed Aspergillus nidulans wA PKS gene in a heterologous fungal host and identified it to encode a heptaketide naphthopyrone synthase. In addition, expression of C-terminus-modified WA PKS gave heptaketide isocoumarins suggesting that the C-terminus region of WA PKS is involved in the cyclization of the second aromatic ring of naphthopyrone. To unravel the actual function of the C-terminus region, we carried out functional analysis of WA PKS mutants by C-terminus deletion and site-directed mutagenesis. RESULTS: Only the 32 amino acid deletion from the C-terminus of WA PKS caused product change to heptaketide isocoumarins from heptaketide naphthopyrone, YWA1 1, a product of intact WA PKS. Further C-terminus deletion mutant of WA PKS up to Ser(1967), an active site residue of so far called thioesterase, still produced isocoumarins. Site-directed mutagenesis of amino acid residues in this C-terminus region showed that even a single mutation of S1967A or H2129Q caused production of isocoumarin instead of naphthopyrone. Furthermore, the role of tandem acyl carrier proteins (ACPs), a typical feature of fungal aromatic PKSs, was examined by site-directed mutagenesis and the results indicated that both ACPs can function as ACP independently. CONCLUSIONS: Claisen-type cyclization is assumed to be involved in formation of aromatic compounds by some fungal type I PKSs. These PKSs have a quite identical architecture of active site domain organization, beta-ketoacyl synthase, acyltransferase, tandem ACPs and thioesterase (TE) domains. Since the C-terminus region of WA PKS of this type was determined to be involved in Claisen-type cyclization of the second ring of naphthopyrone, we propose that the so far called TE of these PKSs work not just as TE but as Claisen cyclase.

Amino Acid Motifs↗
PubMed2001

In vitro abzyme evolution to optimize antibody recognition for catalysis.

Enzymes have evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. To evolve abzymes toward higher catalytic activity, we have reconstructed an enzyme-evolutionary process in vitro. Thus, a phage-displayed combinatorial library from a hydrolytic abzyme, 6D9, generated by the conventional in vivo method with immunization of the transition-state analog (TSA), was screened against a newly devised TSA to optimize the differential affinity for the transition state relative to the ground state. The library format successfully afforded evolved variants with 6- to 20-fold increases in activity (kcat) as compared with 6D9. Structural analysis revealed an advantage of the in vitro evolution over the in vivo evolution: an induced catalytic residue in the evolved abzyme arises from double mutations in one codon, which rarely occur in somatic hypermutation in the immune response.

Amino Acid Sequence↗
PubMed2001

HIV inhibitor from Thai bitter gourd.

Thai bitter gourd protein (MRK29) was isolated from Momordica charantia ripe fruit and seed. The purification was performed by ammonium sulfate fractionation and gel filtration chromatography. MRK29 possessed one isoelectric point of (pI) > or = 9, and the time of flight mass spectrum (TOFMS) indicated its molecular weight at 28.6 kD. The twenty amino acid sequence from the N-terminus was in the following order: 1Asp Val Asn Phe Arg Leu Ser Gly Ala 10Asp Pro Arg X Tyr Gly Met Phe Ile Glu 20Asp. MRK29 inhibited the HIV-1 reverse transcriptase with 50% IR at the concentration of 18 micrograms/ml. MRK29 was concentrated in the 30-60% salt precipitated fraction, at which the concentration of 0.175 microgram/ml exerted 82% reduction of viral core protein p24 expression in HIV-infected cells. MRK29 might have modulatory role on immune cells, because it increased 3-fold TNF activity.

Amino Acid Sequence↗
PubMed2001

Characterization of anti-myocardial autoantibodies in Japanese patients with dilated cardiomyopathy.

Few previous reports have comprehensively screened all the anti-myocardial autoantibodies (AMCA) in relation to other clinical profiles in patients with idiopathic dilated cardiomyopathy (IDC), so the present study used both immunohistochemistry (FITC) and immunoblotting (IB) for screening patients with IDC in order to characterize the clinical significance of AMCA. Sera were collected from 100 patients with IDC and age-matched 100 healthy control subjects (CTL). For FITC, an unfixed frozen section of human myocardium was used for the standard indirect immunofluorescence; for IB, total cardiac homogenates of the same myocardium were blotted to serum at 2 sets of dilution (1:200 and 1:10,000). The positive rates of AMCA detection for each method were as follows (IDC vs CTL); 39% vs 6% for FITC, 38% vs 4% for IB (1:200), and 10% vs 0% for IB (1:10,000). Fifty-nine patients with IDC and 8 CTL were positive for AMCA by either method, and 18 patients with IDC and 2 CTL were positive for AMCA by both methods. IB-positivity at 1:200 was an independent predictor by multiple logistic regression analysis of non-sustained ventricular tachycardias as well as left ventricular end-diastolic diameter and plasma norepinephrine concentration.

Adult↗
PubMed2000

Aspergillus fumigatus alb1 encodes naphthopyrone synthase when expressed in Aspergillus oryzae.

Conidial pigment biosynthesis is an important virulence factor in Aspergillus fumigatus, a human fungal pathogen. Involvement of DHN-melanin pathway in the biosynthesis of A. fumigatus conidial pigment implies that the Alb1p polyketide synthase (PKS) is a 1,3,6, 8-tetrahydroxynaphthalene (T4HN) synthase. The Alb1p, however, shows higher sequence similarity to a naphthopyrone synthase than to a T4HN synthase. To clarify the function of Alb1p, the alb1 gene was overexpressed in a heterologous host Aspergillus oryzae. The Alb1p PKS product in this heterologous expression system was identified as heptaketide naphthopyrone instead of pentaketide T4HN. The data suggest that Alb1p is a naphthopyrone synthase.

Aspergillus fumigatus↗
PubMed2000

Targeted and stable gene delivery into muscle cells by a two-step transfer system.

We developed a muscle-specific gene delivery system based on two-step gene transfer. The first step involved adenovirus-mediated transfer of the ecotropic retrovirus receptor (EcoRec) gene driven by the muscle-specific desmin promoter. Both human primary myoblasts and fibroblasts were efficiently transduced with this adenovirus vector. However, expression of EcoRec was detected only in myoblasts. In the second step, EcoRec-expressing myoblasts could be stably transduced with the ecotropic retroviral vector with the beta-galactosidase gene. Approximately 15% of myoblasts were transduced by this two-step strategy. When the transduced myoblasts were differentiated into myotubes, extensive cell-cell fusion occurred, and the apparent number of beta-galactosidase-positive cells increased to 28%. These results indicate that our two-step gene delivery system could be used for targeted and stable gene transfer into muscle cells.

Adenoviridae↗
PubMed2000

Enzymatic synthesis of 1,3,6,8-tetrahydroxynaphthalene solely from malonyl coenzyme A by a fungal iterative type I polyketide synthase PKS1.

The Colletotrichum lagenarium PKS1 gene encoding iterative type I polyketide synthase of 1,3,6,8-tetrahydroxynaphthalene (T4HN) was overexpressed in Aspergillus oryzae. SDS-PAGE analysis of the cell-free extract prepared from the transformant showed an intense band of 230000 which corresponded to the molecular weight of the deduced PKS1 protein. By using this cell-free extract, in vitro synthesis of T4HN was successfully confirmed as the first example of the fungal multi-aromatic ring polyketide synthase activity ever detected. To identify the starter unit for T4HN synthesis, (14)C-labeled acetyl CoA and/or (14)C-labeled malonyl CoA were used as substrates for T4HN synthase reaction. Observed was the incorporation of (14)C label into T4HN solely from malonyl CoA even in the absence of acetyl CoA and not from acetyl CoA. This in vitro result unambiguously identified that malonyl CoA serves as the starter as well as extender units in the formation of T4HN by fungal polyketide synthase PKS1.

Aspergillus oryzae↗
PubMed2000

Antibody-catalyzed removal of the p-nitrobenzyl ester protecting group: the molecular basis of broad substrate specificity.

Antibody catalysts for the removal of the p-nitrobenzyl ester protecting group have been generated to accommodate a broad range of substrates. Antibody 7B9, which was elicited against p-nitrobenzyl phosphonate 1, catalyzed the hydrolyses of p-nitrobenzyl monoesters of nonsubstituted, and beta- and gamma-substituted glutaric acids with almost identical Km and kcat values. In addition, 7B9 displayed substrate tolerance towards the a-substituents and accepted the p-nitrobenzyl esters of Leu, Norleu, and Phe. To define the molecular basis of the broad substrate tolerance, we have cloned and sequenced the antibody and constructed a model of the active-site-hapten complex. The model showed a relatively shallow pocket of the antigen-combining site that accommodates the p-nitrobenzyl moiety, and this is consistent with the observed substrate specificity. Thus, in the antibody-catalyzed reaction, the alpha-, beta-, and gamma-substituents of the substrates should be outside the combining site and ignored by the antibody recognition. A structural comparison of 7B9 with antibody D2.3, elicited against the structurally similar haptenic phosphonate, suggests the significance of the linker moiety in hapten design, which endows antibody catalysts with broad substrate specificity. These investigations provide new strategies for the generation of catalytic antibodies that accept a broad range of substrates for practical applications in organic synthetic chemistry.

Amino Acid Sequence↗
PubMed2000

Neuroaxonal leukodystrophy associated with congenital cutis laxa: report of an autopsy case.

A male patient, who was born with congenital cutis laxa characterized by cutaneous laxity due to the degeneration of elastic fibers, presented with an arrest of mental and motor development at the age of 3 years. The progressive decline of the psychomotor abilities led to the patient's death at the age of 4 years and 9 months. An autopsy revealed extensive white matter degeneration, characterized by the formation of numerous neuroaxonal spheroids and a diffuse loss of axons and myelin sheaths. The centrum semiovale and the cerebellar white matter were the most severely affected. The ultrastructure of the spheroids was consistent with a dystrophic type of axonal swelling. Neurons of the cerebral cortex, cerebellar cortex, and some brain stem nuclei were lost in moderate to severe degrees, and there were relatively few neuroaxonal spheroids in the gray matter. The pallidum and substantia nigra were well preserved. Neuroaxonal leukodystrophy, in which the spheroid formation predominantly affects the white matter, is the rarest variant of primary neuroaxonal dystrophies, and there are very few reports of autopsied cases. Among the reported cases, two Japanese siblings had congenital skin lesions similar to those of our case. The unique association of neuroaxonal leukodystrophy and congenital cutis laxa may form a distinct variant in this disease category.

Brain↗
PubMed1999

Diverse structural solutions to catalysis in a family of antibodies.

BACKGROUND: Small organic molecules coupled to a carrier protein elicit an antibody response on immunisation. The diversity of this response has been found to be very narrow in several cases. Some antibodies also catalyse chemical reactions. Such catalytic antibodies are usually identified among those that bind tightly to an analogue of the transition state (TSA) of the relevant reaction; therefore, catalytic antibodies are also thought to have restricted diversity. To further characterise this diversity, we investigated the structure and biochemistry of the catalytic antibody 7C8, one of the most efficient of those which enhance the hydrolysis of chloramphenicol esters, and compared it to the other catalytic antibodies elicited in the same immunisation. RESULTS: The structure of a complex of the 7C8 antibody Fab fragment with the hapten TSA used to elicit it was determined at 2.2 A resolution. Structural comparison with another catalytic antibody (6D9) raised against the same hapten revealed that the two antibodies use different binding modes. Furthermore, whereas 6D9 catalyses hydrolysis solely by transition-state stabilisation, data on 7C8 show that the two antibodies use mechanisms where the catalytic residue, substrate specificity and rate-limiting step differ. CONCLUSIONS: Our results demonstrate that substantial diversity may be present among antibodies catalysing the same reaction. Therefore, some of these antibodies represent different starting points for mutagenesis aimed at boosting their activity. This increases the chance of obtaining more proficient catalysts and provides opportunities for tailoring catalysts with different specificities.

Antibodies, Catalytic↗
PubMed1999

A new pathway for polyketide synthesis in microorganisms.

Chalcone synthases, which biosynthesize chalcones (the starting materials for many flavonoids), have been believed to be specific to plants. However, the rppA gene from the Gram-positive, soil-living filamentous bacterium Streptomyces griseus encodes a 372-amino-acid protein that shows significant similarity to chalcone synthases. Several rppA-like genes are known, but their functions and catalytic properties have not been described. Here we show that a homodimer of RppA catalyses polyketide synthesis: it selects malonyl-coenzyme-A as the starter, carries out four successive extensions and releases the resulting pentaketide to cyclize to 1,3,6,8-tetrahydroxynaphthalene (THN). Site-directed mutagenesis revealed that, as in other chalcone synthases, a cysteine residue is essential for enzyme activity. Disruption of the chromosomal rppA gene in S. griseus abolished melanin production in hyphae, resulting in 'albino' mycelium. THN was readily oxidized to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin), which then randomly polymerized to form various coloured compounds. THN formed by RppA appears to be an intermediate in the biosynthetic pathways for not only melanins but also various secondary metabolites containing a naphthoquinone ring. Therefore, RppA is a chalcone-synthase-related synthase that synthesizes polyketides and is found in the Streptomyces and other bacteria.

Acyltransferases↗