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Biomedical subjects

N Misawa

Publications and source records attributed to N Misawa.

At least 55 records · Page 3Linked to original sources
PubMed1997

Primary human immunodeficiency virus type 1 viremia and central nervous system invasion in a novel hu-PBL-immunodeficient mouse strain.

We established four new mouse strains with defective T and B cells as well as defects in innate immunological reactions using an NK cell depletion antibody and showed that all mutant mouse strains efficiently received human peripheral blood leukocyte (PBL) engraftment (hu-PBL-scid mice). Higher levels of human immunodeficiency virus type 1 (HIV-1) replication were observed in these new hu-PBL-scid mice than in conventional hu-PBL-C.B-17-scid mice. In one particular strain, hu-PBL-NOD-scid mice, high levels of HIV-1 viremia (more than 10(6) 50% infectious doses per ml) were detected after infection with HIV-1. The plasma viral load was about 100 to 1,000 times higher than that observed in other hu-PBL-scid mice infected with HIV-1. Although high-level viremia did not correlate with the total amount of HIV-1 RNA in cells from infected mice, high levels of free virions were detected only in hu-PBL-NOD-scid mice. HIV-1 viremia induced systemic HIV-1 infection involving the liver, lungs, and brain. PCR in situ hybridization confirmed that HIV-1-infected cells invaded the brain tissue of the hu-PBL-NOD-scid mice. Our results suggest that the genetic background, including innate immunity, is critical in the development of primary HIV-1 viremia and subsequent central nervous system invasion with HIV-1. The hu-PBL-NOD-scid mouse represents a useful model for the study of the pathogenesis of HIV-1 in vivo, especially brain involvement, and therapy of primary HIV-1 viremia.

Animals↗
PubMed1997

High levels of viremia in hu-PBL-NOD-scid mice with HIV-1 infection.

We studied the compatibility of human lymphocyte engraftment and susceptibility to HIV-1 infection in 2 new immunodeficient mice. NOD/Shi-scid mice were generated by backcrossing of the scid mutation into NOD mice while C57BL/6-RAG2(0/0) were generated by knocking out the RAG-2 gene. Human T lymphocytes were reconstituted in new immunodeficient mouse strains. We found that the new immunodeficient mouse strains accepted human PBL engraftment and HIV-1 infection more efficiently than conventional C.B-17-scid mice. Especially in the hu-PBL-NOD/Shi-scid strain, we reproduced the high levels of HIV-1 viremia comparable to or at significantly higher levels than after HIV-1 primary infection. These results indicate that our hu-PBL-NOD-scid animal is useful for investigations of the activation mechanism in HIV-1 replication in vivo and after primary infection.

Acquired Immunodeficiency Syndrome↗
PubMed1997

[Antimicrobial activities of roxithromycin against recently obtained clinical isolates].

The purpose of our investigation was to monitor current trends in the susceptibility patterns of clinical bacterial isolates to roxithromycin (RXM). We measured the MICs of macrolide antibiotics, such as RXM, erythromycin (EM), clarithromycin (CAM), rokitamycin (RKM) and midecamycin (MDM), and other classes of antibacterial compounds against various clinical isolates at seven institutions between October and December in 1994 and 1995. RXM had excellent antibacterial activities for S. pyogenes, S. agalactiae, M. (B.) catarrhalis and methicillin sensitive S. aureus. Against methicillin sensitive S. epidermidis, RXM activity was fairly good but about 20% of the strains had MIC > or = 128 micrograms/ml. The activity against S. pneumoniae was not so potent and similar to activities of EM, CAM, MDM, and clindamycin. The vast majority of methicillin resistant S. aureus and S. epidermidis were also resistant to macrolide antibiotics and other classes of compounds tested. In conclusion, RXM is an unique macrolide antibiotic by retaining potent activity against S. pyogenes, S. agalactiae, S. aureus except MRSA, M. (B.) catarrhalis and M. pneumoniae.

Amoxicillin↗
PubMed1996

The carotenoid 7,8-dihydro-psi end group can be cyclized by the lycopene cyclases from the bacterium Erwinia uredovora and the higher plant Capsicum annuum.

The genes for geranylgeranyl diphosphate synthase (crtE) and phytoene synthase (crtB) from the epiphytic bacterium Erwinia uredovora and the phytoene desaturase gene from the photosynthetic bacterium Rhodobacter capsulatus (Rc-crtI) were introduced into Escherichia coli, which resulted in the accumulation of the acyclic carotenoid, neurosporene. Further introduction of the lycopene cyclase gene from E. uredovora (crtY) or the higher plant Capsicum annuum (Icy) resulted in the production of a bicyclic carotenoid, 7,8-dihydro-beta-carotene, via monocyclic beta-zeacarotene. zeta-Carotene was also found to be cyclized to bicyclic 7,8,7',8'-tetrahydro-beta-carotene by the Erwinia cyclase. These results indicate that both lycopene cyclases can cyclize a 7,8-dihydro-psi end group to a 7,8-dihydro-beta end group, in addition to the usual cyclization of the psi end group to the beta end group. Furthermore, beta-carotene hydroxylase from Erwinia (CrtZ) was able to add a hydroxyl group to the 7,8-dihydro-beta end group and the beta end group.

Bacterial Proteins↗
PubMed1996

Expression in Escherichia coli and properties of the carotene ketolase from Haematococcus pluvialis.

High level expression of the functional beta-carotene ketolase gene bkt from Haematococcus pluvialis occurred in Escherichia coli transformants producing beta-carotene or zeaxanthin as a result of the presence of additional carotenoid genes from Erwinia uredovora. Requirement of molecular oxygen for the insertion of the keto group was demonstrated. The final product of this two-step ketolase reaction from beta-carotene is canthaxanthin (4,4'-diketo-beta-carotene) with the 4-monoketo derivative echinenone as an intermediate. A reaction sequence for the formation of astaxanthin from beta-carotene was established based on kinetic data on astaxanthin formation in E. coli transformants carrying the hydroxylase gene crtZ from Erwinia along with bkt. We conclude that the carotenoids zeaxanthin and adonixanthin which accumulate in addition to astaxanthin in this transformant are products of side reactions rather than direct precursors of astaxanthin. The possible mechanisms for the formation of the keto derivatives are discussed.

Bacterial Proteins↗
PubMed1996

Expression, purification and properties of lycopene cyclase from Erwinia uredovora.

Lycopene cyclase, an enzyme responsible for the formation of cyclic carotenoids from acyclic precursors has been purified to homogeneity in an active state. The Erwinia uredovora lycopene cyclase gene (crtY) was over-expressed in Escherichia coli. From this recombinant strain the enzyme was purified by immuno-affinity chromatography and its cyclization activity characterized as a two-step reaction in which both sides of the lycopene molecule are cyclized to beta-ionone rings with the monocyclic gamma-carotene as an intermediate. Furthermore, neurosporene as well as l-hydroxylycopene were cyclized to beta-zeacarotene and hydroxy-gamma-carotene respectively. In contrast, neither 1,1'- dihydroxylycopene nor the tetra-cis-prolycopene were accepted as substrates. The cofactors involved in the reaction were either NADH or NADPH. K(m) values were determined for lycopene and NADPH to be 1.8 microM and 2.5 mM respectively.

Amino Acid Sequence↗
PubMed1996

Stability and stabilization of Fusobacterium necrophorum hemolysin.

The stability and stabilization of the hemolytic activity of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were monitored over a period of four weeks using culture supernatants. The hemolytic activity was completely lost after one week at room temperature and 37 degrees C. After a two-week storage at 4 degrees C and -80 degrees C only trace activity was detected with -80 degrees C being the better of the two conditions. The addition of cysteine monohydrochloride, bovine serum albumin or Tween 80 as stabilizers, however, led to the detection of a considerable amount of the hemolytic activity in the sample stored at 4 degrees C and - 80 degrees C throughout the period investigated. The hemolytic activity appeared to be more stable in the presence of Tween 80 at -80 degrees C. Cysteine monohydrochloride was found to crystallize at - 80 degrees C and was therefore ineffective as a stabilizer at this temperature. Hemoglobin was also ineffective as a stabilizer.

Albumins↗
PubMed1996

Experimental hepatitis induced by Campylobacter jejuni infection in Japanese quail (Coturnix coturnix japonica).

To establish an experimental model for vibrionic hepatitis caused by Campylobacter jejuni, Japanese quails (Coturnix coturnix japonica) were inoculated with C. jejuni strains isolated from chicken hepatitis (BL107) and human diarrhea (HP5113). Necrotic liver lesions were formed by intra-pancreaticoduodenal vein injection by which the bacteria reached the liver directly via the portal vein, but not by intra-gastric infection. These liver lesions were observed from day 1 to 7 after the infection. The pathological changes were weak and no clinical signs were observed throughout the experimental period. By immunohistochemistry, the bacterial antigens were detected in the hepatocytes, and intercellular spaces between the hepatocytes, and in the macrophages during the early stage of the infection. When focal hepatocyte necrosis was formed, the antigen was detected more frequently in the intact hepatocytes at the periphery of the lesions than within necrotic foci. The bacteria were not detected from the liver, spleen or blood according to raising the serum agglutination titer. In contrast, the bacteria immediately invaded the bile in 5 min post-infection and were retained in the gallbladder for long periods. The present study showed that necrotizing hepatitis was formed by intra-pancreaticoduodenal vein infection of the quail with C. jejuni.

Animals↗
PubMed1996

Detection of serum-dependent cytotoxic activity of Campylobacter jejuni and its characteristics.

We previously set forth appropriate three assay systems using Chinese hamster ovary cells to detect the Campylobacter jejuni cytotoxin. Although we could not reach a conclusion because the cytotoxins shown in this study were not purified, at least three different cytotoxins were detected in these assay systems. The first cytotoxin in the presence of fetal calf serum (FCS) was heat-labile and the molecular weight (Mw) was estimated at 50-100 k by ultrafiltration. The second cytotoxin detected in the presence of newborn calf serum (NCS) was heat-stable and Mw was estimated at 0.5-3.0 k. The third cytotoxin detected in serum-free culture (SFC) assay was heat-labile and non dialyzable. However, Mw was not estimated since the low Mw and heat-stable cytotoxin was also detected in this assay. The cytotoxic activity detected in FCS and NCS assays, but not that detected in SFC assay, was completely abolished by treatment with a reducing agent. In contrast, the cytotoxicity detected in both FCS and NCS assays was not inactivated by such an enzyme as trypsin, lipase, neuraminidase, and beta-galactosidase. When the filtrate was heated at 100 degrees C to inactivate the heat-labile cytotoxin, the cytotoxic activity was detected in the NCS assay but not in FCS assay. However, when NCS was added to this heated filtrate, the cytotoxicity was restored in FCS assay. Furthermore, when normal rabbit serum (NRS) was added, no cytotoxicity was restored. The cytotoxic activity in SFC assay was completely inactivated with FCS or NRS. These findings suggest that the cytotoxic activity is dependent on serum added to tissue culture medium and that the substance amplifying and/or inhibiting the cytotoxic activity may be present in serum.

Animals↗
PubMed1996

Comparison of haemolytic activity between Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme in vitro and in vivo.

Haemolytic activity of two subspecies of Fusobacterium necrophorum was compared in vitro and in vivo. F. necrophorum subsp. necrophorum (Fnn) showed a stronger activity than F. necrophorum subsp. funduliforme (Fnf) in vitro. Haemolytic activity of Fnn and Fnf was 57.97%+/-1.90 and 17.33%+/-1.44, respectively, compared to complete haemolysis by distilled water. In the mice injected with Fnn, haemolysin was detected in the liver at a titre of from 1 : 16 to 1 : 128, and Fnn was recovered from all mice at a viable bacterial count of 10(5) to 10(6) cells per gram liver tissue. In the mice injected with Fnf, haemolysin titre was <1 : 2 to 1 : 32. No liver abscess was formed. The viable count of recovered bacteria was 10(3) to 10(5) cells per gram, except for two mice in which no Fnf was detected. The results suggest that haemolysin might be a virulence factor in this species.

Animals↗
PubMed1995

Canthaxanthin biosynthesis by the conversion of methylene to keto groups in a hydrocarbon beta-carotene by a single gene.

Compounds that include (a) keto group(s) in a molecule are ubiquitous natural components. A novel gene involved in ketocompound biosynthesis, designated crtW, was isolated from the marine bacteria Agrobacterium aurantiacum and Alcaligenes PC-1 that produce ketocarotenoids such as astaxanthin. When this gene was introduced into Escherichia coli that accumulated beta-carotene due to the Erwinia carotenogenic genes, the E. coli transformants synthesized canthaxanthin, one of ketocarotenoids, which was identified after purification by its visible, FD-MS and 1H-NMR spectral analysis. It has been demonstrated for the first time that one gene encodes an enzyme "ketolase" that catalyzes the conversion of methylene groups of a hydrocarbon beta-carotene to keto groups for synthesizing canthaxanthin via echinenone.

Alcaligenes↗
PubMed1995

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli.

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, beta-carotene ketolase (beta-carotene oxygenase), which converted beta-carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3'S), which was identified after purification by a variety of spectroscopic methods.

Amino Acid Sequence↗
PubMed1995

Production of monoclonal antibodies directed to Hanganutziu-Deicher active gangliosides, N-glycolylneuraminic acid-containing gangliosides.

We have established three kinds of monoclonal antibodies against gangliosides containing N-glycolylneuraminic acid (NeuGc) by immunization of BALB/c mice with the purified gangliosides inserted into liposomes comprising Salmonella minnesota R595 lipopolysaccharides, and fusion of spleen cells with a mouse myeloma cell line. One monoclonal antibody, SHS-1, which was generated by immunizing mice with purified i-active ganglioside(NeuGc), reacted specifically with the i-active ganglioside(NeuGc) used as an immunogen. Structurally related gangliosides, such as GM3(NeuGc), sialosylparagloboside (SPG) (NeuGc), or I-active ganglioside(NeuGc), corresponding gangliosides [GM3 containing N-acetylneuraminic acid (NeuAc), SPG(NeuAc), i-active ganglioside(NeuAc), and I-active ganglioside(NeuAc)], other gangliosides, or neutral glycosphingolipid (GSL) were not recognized by the monoclonal antibody. These findings indicate that the SHS-1 monoclonal antibody may be specific for NeuGc-containing i-active ganglioside. On the other hand, the other two monoclonal antibodies, MSG-1 and SPS-20, which were generated by immunizing mice with purified ganglioside GM3(NeuGc) and SPG(NeuGc), respectively, showed cross-reactivity to structurally related gangliosides. The MSG-1 monoclonal antibody exhibited reactivity to ganglioside GM3(NeuAc). The SPS-20 monoclonal antibody also cross-reacted with SPG(NeuAc), i-active ganglioside(NeuGc), and i-active ganglioside(NeuAc). Neither MSG-1 nor SPS-20 reacted with corresponding gangliosides, other gangliosides, or neutral GSLs tested. Using the SHS-1 antibody specific for i-active ganglioside(NeuGc), we studied the expression of NeuGc-containing antigen in human colon cancer tissue. An NeuGc-containing glycoconjugate was detected in the colon cancer tissue.

Animals↗
PubMed1995

Cytotoxin detection in Campylobacter jejuni strains of human and animal origin with three tissue culture assay systems.

Cytotoxin (CTX) production in 34 human and 22 animal strains of Campylobacter jejuni isolated in Japan and other countries was studied by three assay systems described previously. Furthermore, cholera-like enterotoxin production by these strains was tested by reversed passive latex agglutination (RPLA). CTX titres in the fetal calf serum (FCS) and newborn calf serum (NCS) assays were relatively lower, with a maximum of 4 and 8, respectively, than the maximum of 128 for the serum-free culture (SFC) assay. CTX detection rates were 62, 85 and 100% in human isolates and 64, 77 and 100% in animal isolates for the FCS, NCS and SFC assay systems, respectively. There was no significant difference in the detection rate of CTX between human and animal isolates, or between human isolates from Japan and other countries. With the three assay systems, the strains were divided into four groups from the pattern of CTX detection; 54% of strains gave positive results in all three assay systems, and 9% of them were positive in the SFC assay only. Morphological changes on CHO cells showed distended instead of rounded cells with eight of 21 strains negative in the FCS assay. Cholera-like enterotoxin was not detected in the culture filtrate of any of the strains when tested by RPLA. These results indicate that cytotoxin production by C. jejuni is complex as compared with that of other enteric pathogens.

Animals↗
PubMed1995

Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level.

A carotenoid biosynthesis gene cluster for the production of astaxanthin was isolated from the marine bacterium Agrobacterium aurantiacum. This cluster contained five carotenogenic genes with the same orientation, which were designated crtW, crtZ, crtY, crtI, and crtB. The stop codons of individual crt genes except for crtB overlapped the start codons of the following crt genes. Escherichia coli transformants carrying the Erwinia uredovora carotenoid biosynthesis genes provide suitable substrates for carotenoid biosynthesis. The functions of the five crt genes of A. aurantiacum were determined through chromatographic and spectroscopic analyses of the pigments accumulated in some E. coli transformants carrying various combinations of the E. uredovora and A. aurantiacum carotenogenic genes. As a result, the astaxanthin biosynthetic pathway is proposed for the first time at the level of the biosynthesis genes. The crtW and crtZ gene products, which mediated the oxygenation reactions from beta-carotene to astaxanthin, were found to have low substrate specificity. This allowed the production of many presumed intermediates of astaxanthin, i.e., adonixanthin, phoenicoxanthin (adonirubin), canthaxanthin, 3'-hydroxyechinenone, and 3-hydroxyechinenone.

Amino Acid Sequence↗
PubMed1995

A transformation system for the yeast Candida utilis: use of a modified endogenous ribosomal protein gene as a drug-resistant marker and ribosomal DNA as an integration target for vector DNA.

We have developed a transformation system for the yeast Candida utilis. A novel strategy was applied to construct the transformation system, since auxotrophic mutants which could be used as hosts for transformation are not available. A gene encoding the ribosomal protein L41 was cloned from C. utilis, which is sensitive to cycloheximide, and used as a marker gene conferring cycloheximide resistance after modification of its amino acid sequence. The marker gene was constructed by substitution of the proline codon at position 56 with the glutamine codon by in vitro mutagenesis, as it had been reported previously that the 56th amino acid residue of L41 is responsible for the cycloheximide sensitivity of various organisms (S. Kawai, S. Murao, M. Mochizuki, I. Shibuya, K. Yano, and M. Takagi, J. Bacteriol. 174:254-262 1992). The ribosomal DNA (i.e., DNA coding for rRNA) of C. utilis was also cloned and used as a multiple-copy target for the integration of vector DNA into the genome, which resulted in a high transformation efficiency. Transformants were obtained by electroporation with a maximum efficiency of approximately 1,400 transformants per 1 microgram of linearized DNA carrying the gene for cycloheximide resistance and part of the ribosomal DNA. No transformants were obtained with intact plasmids. Multiple copies of the linearized plasmid were integrated into the host chromosome by homologous recombination. Southern analysis of the transformants in which vector DNA was integrated at the L41 gene locus indicated that there are two copies of gene for the L41 protein per cell, suggesting that C. utilis is diploid. Transformants were obtained from a variety of C. utilis strains, indicating that this method is applicable to the transformation of other C. utilis strains, even though there is significant heterogeneity in chromosomal karyotypes among these strains.

Amino Acid Sequence↗