Biomedical subjects

Hajime Mizukami

Publications and source records attributed to Hajime Mizukami.

At least 19 recordsLinked to original sources

PubMed2006

Semi-quantitative expression and knockdown of a target gene in single-cell mouse embryonic stem cells by high performance microinjection.

Interactions of multiple genes and associated factors are involved in the differentiation and de-differentiation of embryonic stem (ES) cells. Quantitative analysis of these genes and factors is essential for the elucidation of their mechanism. To meet this requirement, we have investigated various experimental conditions for high performance microinjection into mouse ES cells. A speedy and rhythmic operation was found to be important and was accomplished robotically by using a single-cell manipulation technique and XY-address registrable culture dishes. Among many experimental parameters, the tip size of an injection capillary, the pressure condition, and the DNA concentration in the injection capillary were of critical significance. Their optimum values were 0.5-0.8 microm, 0.7 kgf/cm(2) for 30 ms, and 1-100 ng/microl, respectively. Under these conditions, semi-quantitative control of the EGFP gene expression in mouse ES cells and its knockdown was successfully demonstrated.

Animals↗

PubMed2006

Sequence analysis of chloroplast chlB gene of medicinal Ephedra species and its application to authentication of Ephedra Herb.

Chloroplast chlB gene encoding subunit B of light-independent protochlorophyllide reductase was amplified from herbarium and crude drug specimens of Ephedra sinica, E. intermedia, E. equisetina, and E. przewalskii. Sequence comparison of the chlB gene indicated that all the E. sinica specimens have the same sequence type (Type S) distinctive from other species, while there are two sequence types (Type E1 and Type E2) in E. equisetina. E. intermedia and E. prezewalskii revealed an identical sequence type (Type IP). E. sinica was also identified by digesting the chlB fragment with Bcl I. A novel method for DNA authentication of Ephedra Herb based on the sequences of the chloroplast chlB gene and internal transcribed spacer of nuclear rRNA genes was developed and successfully applied for identification of the crude drugs obtained in the Chinese market.

Base Sequence↗

PubMed2006

Does a kampo medicine containing schisandra fruit affect pharmacokinetics of nifedipine like grapefruit juice?

Herb-drug interaction has attracted attention as medicinal topics recently. However, the drug information is sometimes confusing. Previous in vitro studies revealed that schisandra fruit had strong inhibitory effect on CYP3A4 and claimed the possibilities of its herb-drug interaction. In the present study, we evaluated the inhibitory effects of schisandra fruit and shoseiryuto, an herbal formula in Japanese traditional kampo medicine containing eight herbal medicines including schisandra fruit, on rat CYP3A activity in vitro, and the effect of shoseiryuto on pharmacokinetics of nifedipine in rats, in comparison with those of grapefruit juice, a well-characterized natural CYP3A inhibitor. Shoseiryuto and its herbal constituents, schisandra fruit, ephedra herb and cinnamon bark exhibited in vitro inhibitory effect of CYP3A. Although shoseiryuto inhibited rat CYP3A activity in vitro with a degree comparable to grapefruit juice, shoseiryuto did not significantly affect a plasma concentration profile of nifedipine in rats as grapefruit juice did. These results indicate that in vivo experiments using the extract of herbal medicine prepared with the same dosage form as patients take are necessary to provide proper information about herb-drug interaction.

Animals↗

PubMed2005

Systematic classification of alleles of the glycophorin A (MN blood group) gene.

Ten alleles (five M and five N alleles) of the MN blood group system with normal antigenicity were found by sequencing the glycophorin A (GPA) gene. This study demonstrates the systematic classification of these alleles to major or minor variations of the standard alleles. GPA-specific fragments ranging from 150 to 3.8 kb in length were amplified from the templates, and exons 1-7 and introns 1-6 were sequenced. The data were analyzed phylogenetically to classify these alleles into major groups or clusters. The ten alleles were grouped into four major clusters M10X (M101-M103), M20X (M201 and M202), N10X (N101-N104) and N20X (N201), where 'X' represents a digit indicating minor variations. This grouping was supported by phylogenetic analysis. The cluster system of GPA alleles is highly informative for genetic screening.

Alleles↗

PubMed2005

Dermal mast cells play a central role in the incidence of scratching behavior in mice induced by multiple application of the hapten, 2,4,6-trinitrochlorobenzene.

Repeated application of 1% 2,4,6-trinitrochlorobenzene (TNCB) in acetone solution causes chronic skin inflammation in BALB/c mice. Associated scratching behavior gradually appeared, and chronic scratching behavior was established over 40 days after the initial application of TNCB. In order to explore the possible involvement of T cells and mast cells in the appearance of pruritus, we examined the response of athymic nude mice and genetically mast-cell-deficient mice. We could not detect either severe skin inflammation or immunoglobulin (Ig)E production in T-cell-deficient BALB/c nu/nu mice even after 80 days of TNCB treatment, whereas typical severe skin inflammation and IgE production were observed in mast-cell-deficient WBB6F1-W/Wv and WBB6F1-Sl/Sld mice. Furthermore, we observed persistent scratching behavior in WBB6F1-W/Wv mice, but not in BALB/c nu/nu and WBB6F1-Sl/Sld mice. Histological examination of TNCB-treated animals revealed the development of dermal mast cells in W/Wv mice but not in Sl/Sld mice. Degranulation of dermal mast cells was observed in the WBB6F1-W/Wv genotype, but most mast cells remained intact in TNCB-treated BALB/c nu/nu mice. These results suggest that mast cells play a pivotal role in the incidence of scratching behavior in this chronic pruritus model.

Acetone↗

PubMed2005

pX gene causes hypercholesterolemia in hypercholesterolemia-resistant BALB/c mice.

To investigate the high incidence of atherosclerosis in the patients affected with rheumatoid arthritis, we examined the effect of feeding a cholesterol-enriched diet on the development of hypercholesterolemia in pX transgenic mice, which spontaneously develop chronic inflammatory arthritis. Cholesterol feeding to pX transgenic mice induced a striking elevation in serum total cholesterol (ca. 500 mg/dl) compared with their littermates, BALB/c mice used as controls. The pX transgenic mice exhibited elevated mRNA levels of ACAT1, and ABCG5 in the small intestine compared with their littermates, and furthermore, apoA1, ABCA1, ABCG5, ACAT1, and ACAT2 mRNAs were induced more easily by a cholesterol-enriched diet in pX transgenic mice than their littermates. As ACAT1 mRNA in the small intestine is known not to be induced by feeding a cholesterol-enriched diet, a possibility was inferred that interferon-gamma induced by Tax, a pX gene product, might play an important role in the induction of ACAT1 mRNA and the following hypercholesterolemia. These findings suggest that pX gene plays an important role in inducing hypercholesterolemia in BALB/c mice, which are genetically less susceptible to hypercholesterolemia and atherosclerosis and that RA patients carrying HTLV-1 virus have a predilection for hypercholesterolemia, a main risk factor for cardiovascular diseases.

Animals↗

PubMed2005

[Possible influence of psychotropic drugs detected in blood when determining the cause of death in medicolegal autopsy cases in the Tokyo Medical Examiner's Office].

We reviewed the records of 118 medicolegal autopsy cases, in which psychotropic drugs were detected in blood, in the Tokyo Medical Examiner's Office in 1997, to explore how the drug levels were considered in determining the cause of death. Names and doses of the drugs were clear in 70 of 118 cases, and in most cases of the 70 cases, multiple drugs (up to 13 drugs) were prescribed to a person. It was also evident that 75 of the 118 cases had demonstrated psychosis for several months to 38 years prior to death. No information concerning prescriptions or history of psychosis could be obtained in the other cases. The causes of death in these 118 cases were as follows: deaths from specific diseases, 30 cases (25.4%); deaths from extrinsic factors excluding drug intoxication, 22 cases (18.6%); suicide related to drug intoxication, 31 cases (26.3%); deaths from extrinsic factors related to drug intoxication suggestive of suicide, but not confirmed, 19 cases (16.1%); non-suicide, including probable drug intoxication, 13 cases (11.0%); and deaths from malignant syndrome, 3 cases (2.5%). There were cases diagnosed as death from specific diseases based on morphological findings, though drug concentrations in blood were at a toxic or even lethal level. In some cases, drug intoxication was suspected, but drug levels in their blood were at a therapeutic level and there were no identifiable morphological changes directly associated with deaths, resulting in a cause of death other than drug intoxication being indicated. Thus, drug levels detected in the cadaver's blood are not always useful for determining the cause of death. This might be due to poor information on interactions between drugs (including alcohol), pathological changes or genetic variability of drug metabolism and excretion, and so on. Thus, further studies of these aspects are needed in order to make information on drugs detected in the cadaver more useful for determination of cause of death.

Autopsy↗

PubMed2004

Different response to exogenous L-arginine in nitric oxide production between hippocampus and striatum of conscious rats: a microdialysis study.

We previously showed that systemic administration of a nitric oxide (NO) precursor, L-arginine (L-Arg), failed to reverse suppression by NO synthase (NOS) inhibitors of chemically induced shaking behavior in rats, leading to the hypothesis that exogenous L-Arg might be non-uniformly supplied to brain regions susceptible to NOS inhibitors. In the present study, therefore, we examined the effect of exogenous L-Arg on the extracellular levels of the oxidative nitric oxide (NO) products, nitrite (NO2-) and nitrate (NO3-), in two different brain regions, the hippocampus and the striatum, of conscious rats by means of in vivo brain microdialysis. The basal NO2- levels in the two brain regions were comparable, while the NO3- level was significantly lower in the hippocampus than the striatum. The addition of 10 mM L-Arg, but not D-Arg, to the perfusing solution significantly increased NO2- and NO3- in the hippocampus and NO2- alone in the striatum. These increases were abolished by 1 mM N(omega)-nitro-L-arginine, an NOS inhibitor. L-Arg at 1mM was able to significantly increase NO2-, but not NO3-, in the hippocampus to a level comparable with that at 10 mM L-Arg, while it had no effect in the striatum. L-Arg (500 mg/kg, i.p.) induced a significant increase in NO2- and NO3- in the hippocampus, but not in the striatum. These results suggest that the striatum may have a lower ability to enhance NO production by utilising exogenous L-Arg than the hippocampus, despite higher basal NO production.

Analysis of Variance↗

PubMed2004

Molecular cloning and characterization of a glucosyltransferase catalyzing glucosylation of curcumin in cultured Catharanthus roseus cells.

Catharanthus roseus cell suspension cultures are capable of converting exogenously supplied curcumin to various glucosides. The glucosylation efficiency is enhanced by addition of methyl jasmonate (MJ) to the cultures prior to curcumin administration. Two cDNAs encoding UDP-glucosyltransferases (CaUGT1 and CaUGT2) were isolated from a cDNA library of cultured C. roseus cells, using a PCR method directed at the conserved UDP-binding domain of plant glycosyltransferases. The sequence identity between their deduced amino acid sequences was 27%. The expression of both genes was up-regulated by addition of MJ to the cell cultures although the mRNA level of CaUGT1 was much lower than that of CaUGT2. The corresponding cDNAs were expressed in Escherichia coli as fusion proteins with maltose-binding protein. The recombinant CaUGT1 exhibited no glucosylation activity with either curcumin or curcumin monoglucoside as substrate, whereas the recombinant CaUGT2 catalyzed the formation of curcumin monoglucoside from curcumin and also conversion of curcumin monoglucoside to curcumin diglucoside. The use of the recombinant CaUGT2 may provide a useful new route for the production of curcumin glucosides.

Catalysis↗

PubMed2004

Effect of N- and C-terminal deletions on the RNA N-glycosidase activity and the antigenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica.

Karasurin-A, from root tubers of Trichosanthes kirilowii var. japonica, is a type I ribosome-inactivating protein (RIP) that displays activity of RNA N-glycosidase to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in the ribosome. We expressed recombinant proteins of karasurin-A and its various mutants with N- or C-terminal deletions in Escherichia coli as fusion proteins with maltose-binding protein (MBP), and compared their enzymatic activities and antigenicities. Muteins of karasurin-A generated by deleting either the first 100 N-terminal or the last 30 C-terminal amino acid residues lost activity of RNA N-glycosidase. The mutant proteins whose 80 N-terminal or 20 C-terminal amino acids were deleted could depurinate rRNA although the activities were decreased drastically. The antigenicities of the recombinant proteins were considerably reduced by deleting 20 amino acid residues from either N- or C-terminal regions.

Carrier Proteins↗

PubMed2004

Antioxidants from rape (Brassica campestris vir. Japonica Hara) oil cake.

A simple but novel compound, S-1-methoxy-1-(3,5-dimethoxy-4-hydroxyphenyl)ethane, was isolated as a moderately antioxidative compound from rape (Brassica campestris L. subsp. napus) oil cake together with 5 known compounds. Three of these compounds, indolacetonitrile, 4-hydroxyindolacetonitrile, and 4-hydroxyphenylacetonitrile, showed strong antioxidative activity evaluated by the ferric thiocyanate method.

Antioxidants↗

PubMed2004

Suppressive effect of Kanzo-bushi-to, a Kampo medicine, on collagen-induced arthritis.

Kanzo-bushi-to (KBT) is a traditional Japanese herbal medicine (Kampo medicine), which is used in Japan to treat rheumatoid arthritis. In the present study, we investigated the suppressive effect of KBT on collagen-induced arthritis (CIA) and further studied the underlying mechanism. CIA was induced in male DBA/1J mice by immunization with bovine type II collagen, followed by a booster injection 21 d later. KBT was given at a dose of 430 mg/kg/d from three days before the first immunization to the end of the experiment. KBT suppressed CIA development effectively and further protected focal bone erosion and bone destruction as evidenced by the reduced histological score. Histochemical examination revealed that KBT decreased TRAP-positive cells at the synovium-bone interface and at the sites of focal bone erosion, coincident with the findings that RANKL/OPG mRNA ratio was significantly reduced by KBT treatment. KBT also decreased mRNA levels of M-CSF and iNOS in joints and of iNOS in peritoneal macrophages. In conclusion, KBT prevented osteoclast generation by decreasing RANKL/OPG ratio and M-CSF mRNA levels, resulting in reduction in bone erosion and destruction. In addition, KBT has anti-inflammatory effect such as the suppression of iNOS expression in peritoneal macrophages and joints of CIA mice. These finding suggests that KBT is a potential new therapeutic agent for the treatment of RA.

Animals↗

PubMed2004

Suppressive effect of Dai-bofu-to on collagen-induced arthritis.

Dai-bofu-to (DBT) is a traditional Japanese herbal medicine (Kampo medicine) used for the treatment of rheumatoid arthritis (RA). In the present study, to establish the usefulness of DBT, we examined the effect of DBT on collagen-induced arthritis (CIA). DBT (1.72 g/kg/d) significantly reduced the severity of arthritis throughout the experiment and significantly delayed the onset of arthritis. The induction of CIA decreased T cells and increased B cells in popliteal lymph nodes close to the affected joints, while the treatment of CIA with DBT counteracted the changes in T and B cells. In pX transgenic mice as a spontaneously developed arthritis model, a decrease in T cells and increase in B cells in popliteal lymph nodes were observed, as compared to BALB/c mice, the littermates of pX transgenic mice. In contrast, DBT returned the cell number of T and B cells to the level of BALB/c mice. As osteoclastogenesis is regulated by some T cell cytokines and osteotropic factors, we examined the effect of DBT on the receptor activator of NF-kappa B (RANK), RANK ligand (RANKL), osteoprotegerin (OPG) and M-CSF mRNAs, which were induced by arthritis induction. Although DBT had no effect on RNAK or RANKL mRNA levels, DBT stimulated an increase in OPG mRNA levels and suppressed an increase in M-CSF mRNA level. These results suggest that DBT may possess an anti-osteoclastogenetic effect, which is brought by reducing the ratio of RANKL/OPG and by decreasing M-CSF mRNA levels. In conclusion, immunomodulatory and anti-osteoclastogenetic effects might be involved in the suppression of arthritis by DBT.

Animals↗

PubMed2003

Production of unnatural glucosides of curcumin with drastically enhanced water solubility by cell suspension cultures of Catharanthus roseus.

Catharanthus roseus cell suspension cultures converted exogenously supplied curcumin to a series of glucosides, none of which has been found in nature so far. The efficiency of glucosylation was dependent on culture stage of the cells and medium sucrose concentration. Methyl jasmonate and salicylic acid enhanced the glucoside formation only when they were added to the cultures prior to the addition of curcumin. The glucoside yield was 2.5 micromol/g fresh weight of the cells at an optimal culture condition. The water solubility of curcumin-4',4"-O-beta-D-digentiobioside was 0.65 mmol/ml, which was 20 million-fold higher than that of curcumin.

Acetates↗

PubMed2003

RNA interference: potentials for the prevention of HIV infections and the challenges ahead.

Presently, Phase III efficacy trials with subunit immune antigens against the human immunodeficiency virus (HIV) infection are occurring in both the USA and some Southeast Asian countries. However, there is no feeling of dèja vu within the scientific community that these studies will demonstrate meaningful protection against HIV infection, perhaps because the immune responses crucial for containment of the virus might not be elicited by this approach. Under this prevailing gloomy setting for international acquired immune deficiency syndrome (AIDS) therapy, RNA interference has been identified as a likely strategy in the understanding that inhibition of HIV replication can be mediated by post-transcriptional gene silencing through the production of double-stranded RNA (dsRNA) nucleotide intermediates. This review briefly explores the role of the nef gene during HIV-1 infection and highlights the expectation that RNA interference might serve as a new sequence-specific therapeutic arsenal in AIDS prevention, and possibly treatment, in the years ahead.

Acquired Immunodeficiency Syndrome↗

PubMed2003

Molecular cloning and characterization of a cDNA encoding a novel apoplastic protein preferentially expressed in a shikonin-producing callus strain of Lithospermum erythrorhizon.

A cDNA (LEPS-2) encoding a novel cell wall protein was cloned from shikonin-producing callus tissues of Lithospermum erythrorhizon by differential display between a shikonin-producing culture strain and a non-producing strain. The LEPS-2 cDNA encoded a polypeptide of 184 amino acids. The deduced amino acid sequence exhibited no significant homology with known proteins. Expression of LEPS-2 gene as well as accumulation of LEPS-2 protein was highly correlated with shikonin production in L. erythrorhizon cells in culture. In the intact plant, expression of LEPS-2 was detected only in the roots where shikonin pigments accumulated. Cell fractionation experiments and immunocytochemical analysis showed that the protein was localized in the apoplast fraction of the cell walls. The shikonin pigments were also stored on the cell walls as oil droplets. These results indicate that expression of the LEPS-2 is closely linked with shikonin biosynthesis and the LEPS-2 protein may be involved in the intra-cell wall trapping of shikonin pigments.

Amino Acid Sequence↗

PubMed2003

Sho-saiko-to and Saiko-keisi-to, the traditional Chinese and Japanese herbal medicines, altered hepatic drug-metabolizing enzymes in mice and rats when administered orally for a long time.

As the consumption of herbal remedies has increased, the opportunity that such herbal medicines are co-administered with other drugs has also risen gradually and we are, therefore, very much concerned about herb-drug interactions. We examined the effects of pre-administration of Kampo medicines (Sho-saiko-to, Saiko-keishi-to, Shigyaku-san and Dai-saiko-to) on the pentobarbital-induced sleeping time in mice and rats, to clarify the possibility that they could affect the drug-metabolizing enzymes. The administration of Sho-saiko-to and Saiko-keishi-to for 4 weeks significantly shortened the pentobarbital-induced sleeping time in mice and the administration of Sho-saiko-to for 2 weeks significantly reduced the sleeping time in rats. Furthermore, we tried to identify the molecular species of rat cytochrome P450s (CYPs) affected by Sho-saiko-to and Saiko-keishi-to by competitive RT-PCR. The oral administration of Sho-saiko-to for 2 weeks upregulated the mRNA expression of CYP2B, CYP3A1, CYP2E1 and CYP4A1 in rats. The treatment with Saiko-keishi-to for 2 weeks also up-regulated the mRNA expression of CYP2B, CYP3A1 and CYP4A1. Sho-saiko-to and Saiko-keishi-to may potentially influence the drug-metabolizing enzymes in man, and would thus require much attention when used in the clinical situation.

Animals↗

PubMed2002

CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4'-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis.

Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4'-hydroxyphenyllactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid biosynthesis.

Acetates↗