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Biomedical subjects

H Mizukami

Publications and source records attributed to H Mizukami.

At least 37 records · Page 2Linked to original sources
PubMed2001

Gamma-rays enhance rAAV-mediated transgene expression and cytocidal effect of AAV-HSVtk/ganciclovir on cancer cells.

Adeno-associated virus (AAV) vector has several unique properties suited for gene therapy applications. However, relatively low efficiency of transgene expression, which is mainly due to a limited second-strand synthesis from the single-stranded AAV genome, can be a problem in some applications that require potent gene expression such as antitumor applications. Recently, gamma-ray irradiation has been reported to enhance the second-strand synthesis of the AAV genome, and consequently transgene expression. We demonstrate here that an AAV vector harboring the herpes simplex virus type-1 thymidine kinase (HSVtk) is able to kill cancer cells more efficiently when used in combination with gamma-ray irradiation. A human maxillary sinus cancer cell line, NKO-1, was efficiently killed in combination with HSVtk transduction and ganciclovir (GCV), as expected. More importantly, gamma-ray irradiation of practical dosages augmented the cytocidal effect of the HSVtk/GCV system. Southern analysis indicated that gamma-rays enhanced the double-strand synthesis of the rAAV genome in NKO-1 cells. These findings suggest that the combination of rAAVtk/GCV suicide gene therapy with radiotherapy has synergistic effects in the treatment of cancers and may lead to a reduction of the potential toxicity of both rAAVtk/GCV and gamma-ray irradiation.

Antiviral Agents↗
PubMed2001

Expression of HGF/NK4 in ovarian cancer cells suppresses intraperitoneal dissemination and extends host survival.

Peritoneal dissemination is the most frequent progression pathway of ovarian cancer and is therefore a key step to improve the prognosis. NK4, a large part of the alpha-chain of hepatocyte growth factor, is known to inhibit cancer cell migration. To characterize the function of NK4 and investigate its potential role in gene therapy of ovarian cancer, we introduced NK4 cDNA to an ovarian cancer cell line HRA and investigated its effects both in vitro and in vivo. HRA cells were transfected with either NK4 or luciferase-expression plasmids. After selection, NK4-expressing HRA cells (HRA/NK4) and the control cells (HRA/LUC) were obtained. NK4 was detected in the culture supernatant of HRA/NK4 by Western analysis. Migration capabilities of the cells were evaluated in vitro by scratch wound healing assay. The number of migrated cells was significantly smaller in the HRA/NK4 cultures than that in the control cultures (HRA or HRA/LUC). Also, the culture supernatant of HRA/NK4 significantly suppressed migration of control cells. This suppressive effect was observed when NK4-expressing cells were mixed with control cells at the ratio of 25% or more. In the in vivo experiments, HRA transfectants were injected intraperitoneally. The number of intraperitoneal tumors of HRA/NK4 was much smaller than that of control. In mice injected with HRA/NK4, ascites formation was suppressed and the survival was significantly prolonged. These findings suggest that NK4-mediated gene therapy can improve the prognosis of ovarian cancer by suppressing peritoneal dissemination.

Animals↗
PubMed2001

Synthesis of 2-O-(4-coumaroyl)-3-(4-hydroxyphenyl)lactic acid, an important intermediate of rosmarinic acid biosynthesis.

A simple method to synthesize (+/-)-2-O-(4-coumaroyl)-3-(4-hydroxyphenyl)lactic acid (1), a key intermediate in rosmarinic acid biosynthesis in higher plant cells, was established by condensation of protected 4-coumaric acid and (+/-)-3-(4-hydroxyphenyl)lactic acid followed by deprotection. A stable supply of 1 thus attained will lead to biochemical and molecular biological characterization of later steps of rosmarinic acid biosynthesis.

Cinnamates↗
PubMed2001

Studies on chemical modification of monensin IX. Synthesis of 26-substituted monensins and their Na+ ion transport activity.

The C-26 modified monensin derivatives, 26-O-benzoylmonensin (3), 26-O-benzylmonensin (4) and 26-phenylaminomonensin (5) were prepared from monensin (1). Na+ ion transport activity through biological membrane and antibacterial activity of 3-5 were evaluated and compared with the activities reported for a 26-phenylurethane derivative (2). Among these compounds, 5 showed the largest Na+ ion transport and antibacterial activities. In these compounds, the formation of head-to-tail hydrogen bonds was suggested to be an important factor for Na+ ion transport and antibacterial activities.

Anti-Infective Agents↗
PubMed2001

Identity of Cypripedium calceolus (Orchidaceae) in Rebun Island: comparative DNA analysis of related species.

Cypripedium calceolus was found in 1980 in Rebun Island off the north coast of Hokkaido, Japan, but the origin of this plant has been a controversial issue. In this study, we have made a comparative study by chloroplast DNA sequencing analysis among C. calceolus which occurs in Rebun Island and populations of C. calceolus from western Europe, China and far eastern Russia (Nakhodka), and also as references, C. macranthos in Japan and other Cypripedium species in North America. A Cypripedium cf. "calceolus", found recently in eastern Hokkaido, was also included in this analysis. The C. calceolus samples analyzed were categorized into three groups, i.e., those from Western Europe, from China and far eastern Russia, and from Rebun Island. The C. calceolus in Rebun Island was clearly different from the others in terms of DNA sequence and morphological features. The C. cf. calceolus from eastern Hokkaido and one sample from Nadhodka, Russia, were also classified into the same group as those from Rebun Island, although some differences in their morphological features were observed. It is concluded that the C. calceolus found in Rebun Island is not identical with those growing in Europe and China. In addition, it was found that it may be possible to classify C. macranthos into two groups, namely groups which include or do not include var. rebunense. An unidentified Cypripedium species found in Rebun Island falls into the same group as var. rebunense.

Base Sequence↗
PubMed2000

Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson's disease.

Parkinson's disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.

Animals↗
PubMed2000

Inhibition of progressive reduction of islet beta-cell mass in spontaneously diabetic Goto-Kakizaki rats by alpha-glucosidase inhibitor.

The Goto-Kakizaki (GK) rat, an animal model of type 2 diabetes, exhibits mild hyperglycemia with a reduction of beta-cell mass. The mechanism for islet structural changes in this model and whether the changes are affected by metabolic control are not known. In the present study, we examined the process of islet changes in male GK rats aged 6, 8, 12, 24, and 36 weeks. Treatment effects with an alpha-glucosidase inhibitor (Voglibose; Takeda, Osaka, Japan) for 24 weeks (12 to 36 weeks of age) were also evaluated. The beta-cell mass increased until 8 weeks of age in both GK and control rats, but the increase was significantly (P < .01) smaller in GK rats versus at 8 weeks of age. Thereafter, the beta-cell mass decreased in GK rats, whereas it remained constant in controls. Voglibose treatment significantly (P < .01) inhibited the reduction of beta-cell mass in GK rats. Proliferative activity of beta cells as measured by bromodeoxyuridine (BrdU) uptake was significantly (P < .05) lower in GK rats versus control rats at 6 and 8 weeks, but the difference disappeared after 12 weeks of age, regardless of Voglibose treatment. The present study thus demonstrates a progressive loss of beta cells in GK rats that was mitigated by Voglibose treatment. We consider that the beta-cell loss in GK rats was due to an early impairment in proliferative activity and reduced survival. Voglibose did not appear to stimulate beta-cell proliferation, but exerted its effect via a reduction of hyperglycemia.

Aging↗
PubMed2000

The tissue distribution of lidocaine in acute death due to overdosing.

A 78-year-old woman committed double suicide with a bolus injection of 1 g of lidocaine hydrochloride. The tissue distribution of lidocaine in this fatal poisoning was investigated using an improved gas chromatographic method. The blood level was 42 microg/ml and the brain concentration was 70 miccro/g. Thus, the cause of death in this case was clearly diagnosed as lidocaine overdosing. The blood level of 42 microg/ml was higher than that calculated using the standard pharmacokinetic parameters. Lower blood/tissue ratios were also detected compared with the data described in the literature. These results indicated that the cardiac failure should have occurred immediately after the lidocaine injection. The present data show the distribution of lidocaine after acute death due to overdosing.

Journal Article↗
PubMed2000

Classification of standard alleles of the MN blood group system.

BACKGROUND AND OBJECTIVES: We had classified the M alleles of the MN blood group system into two subtypes, M(G) (standard M) and M(T), based on a G/T substitution in intron 1 of the glycophorin A (GPA) gene. This study provides further study on nucleotide sequences of M(G), M(T) and N alleles to profile the new allele, M(T). MATERIALS AND METHODS: M(G), M(T) and N alleles of the GPA gene were amplified using GPA gene-specific primers to avoid co-amplification of the genes of glycophorins B and E. Then the 5'-flanking region, exons 1-7 and introns 1-4 of the alleles were analyzed by DNA sequencing. RESULTS: There were 17 nucleotide substitutions and deletions between M(G) (standard M) and N alleles. Ten of the M(T) nucleotides were M(G)-type but the other 7 were N-type. M(T) allele also showed one base change and one deletion that were observed in neither the M(G) nor the N allele. Moreover, we found nucleotide substitutions within each allele, allowing further classification of the alleles. CONCLUSION: By the sequence data of M(G), M(T) and N alleles, the three alleles could be further classified into M101 and M102, M201 and M202, and N101 and N102, respectively.

Alleles↗
PubMed2000

Identification of the crude drug atractylodes rhizome (Byaku-jutsu) and atractylodes lancea rhizome (So-jutsu) using chloroplast TrnK sequence as a molecular marker.

Novel methods for molecular authentication of Atractylodes-derived crude drugs (Jutsu) were established based on PCR-restriction fragment length polymorphism (RFLP) and direct sequencing of chloroplast trnK. Two regions inside the chloroplast trnK were selected as molecular markers for identification and discrimination of Atractylodes Rhizome (Byaku-jutsu) and Atractylodes Lancea Rhizome (So-jutsu). The Region 1 fragment (260 bp) amplified from So-jutsu and Wa-byaku-jutsu (Atractylodes Rhizome derived from A. japonica) gave 2 bands of 180 bp and 80 bp on agarose gel electrophoresis after digestion with a restriction endonuclease HinfI, whereas the fragment amplified from Kara-byaku-jutsu (Atractylodes Rhizome derived from A. ovata) remained undigested, which allowed unambiguous identification of Kara-byaku-jutsu. By direct sequencing of Region 2 (436 bp) and comparison of the nucleotide sequence data sets we could not only discriminate Byaku-jutsu and So-jutsu but also identify the original plant species of each crude drug specimen. A simple and reliable protocol for rapid preparation of DNA suitable for PCR from as little as 1 mg of Atractylodes-derived crude drugs was also described.

Asteraceae↗
PubMed2000

Sesquiterpenoid derivatives from Ferula ferulaeoides [correction of ferulioides]. IV.

Four novel prenyl-furocoumarin type sesquiterpenoid derivatives, 2,3-dihydro-7-hydroxy-2S*,3R*-dimethyl-3-[4,8-dimethyl-3(E),7-nonadie nyl]-furo[3,2-c]coumarin, 2,3-dihydro-7-hydroxy-2R*,3R*-dimethyl-3-[4,8-dimethyl-3(E),7-nonadie nyl]-furo[3,2-c]coumarin, 2,3-dihydro-7-hydroxy-2S*,3R*-dimethyl-3-[4-methyl-5-(4-methyl-2-furyl)- 3(E)-pentenyl]-furo[3,2-c]coumarin, and 2,3-dihydro-7-methoxy-2S*,3R*-dimethyl-3-[4,8-dimethyl-3(E),7-nonadie nyl]-furo]3,2-c]coumarin were isolated from the roots of Ferula ferulaeoides [corrected]. Their structures were established by detailed spectral analysis and the biosynthetic pathway leading to these prenyl-furocoumarin type sesquiterpenoids is proposed based on these structures.

Ferula↗
PubMed1999

Forensic diagnosis of death from cold.

To clarify the forensic diagnosis of hypothermia, the postmortem changes in the bodies of 211 cases of hypothermia (partly including death from diseases) encountered between January 1990 and January 1999 were studied as follows:i) The rectal temperature had been measured in 83 cases in which the time after death was known relatively clearly, and the rate of decrease in the rectal temperature per hour was evaluated. The rate of decrease in the rectal temperature was 2.3 +/- 1.2 degrees C, 2.7 +/- 1.5 degrees C and 3.6 +/- 1.9 degrees C per hour (mean +/- SD) when the ambient temperature was > or = 0 degrees C, 0 degrees C > to >-10 degrees C and -10 degrees C > or =, respectively. ii) The color of blood collected from the right and left hearts was different in 43 (33.6%) of the 128 cases of death from cold that underwent inquest and in 20 (87.0%) of the 23 cases that underwent forensic autopsy. This color difference was useful for the diagnosis of fatal hypothermia. iii) Gastric or duodenal submucosal bleeding (Wishnewski's spots) was noted in 10 (43.5%) of the 23 cases that underwent forensic autopsy. iv) Paradoxical undressing was observed in 35 (21.3%) of the 164 cases that underwent inquest.

Journal Article↗
PubMed1999

DNA profiling of Acorus calamus chemotypes differing in essential oil composition.

The phylogenetic relationship of Acorus gramineus and three types of Acorus calamus was analyzed by comparing the 700 bp sequence of a 5S-rRNA gene spacer region. Although there was no intra-specific variation in the essential oil profile of A. gramineus which contained a phenylpropanoid (Z-asarone) as a predominant constituent, A. calamus was classified into two chemotypes: chemotype A in which Z-asarone is a major essential oil constituent and chemotype B which contained sesquiterpenoids predominantly. An intermediate type (M) of these two chemotypes in various ratios was also observable. The NJ tree constructed based on the sequences revealed that A. gramineus was clearly distinguished from any of the chemotypes of A. calamus and that the phylogenetic relationship predicted by the spacer region data correlated well with the essential oil chemotype pattern of A. calamus.

Base Sequence↗
PubMed1999

A simple and rapid protocol for preparation of crude drug DNA suitable for PCR.

A protocol for rapid purification of DNA from a variety of herbal crude drugs, starting from samples as small as 1 mg, has been developed. Neither phenol extraction nor alcohol precipitation is involved in the procedure. Total nucleic acids are first extracted with the lysis buffer containing SDS. After CHCl3 extraction, DNA is bound to glass fiber fleece in the presence of guanidine thiocyanate, while unbound impurities are washed away. The entire procedure can be carried out in 1.5-ml microtubes. The purified DNA obtained serves as a template for PCR.

DNA, Plant↗
PubMed1999

Hematopoietic stem cell gene therapy: a current overview.

Retrovirus-mediated gene transfer into murine hematopoietic stem cells and reconstitution of syngeneic mice have demonstrated persistence and functioning of the transgenes over extended periods of time. In contrast, clinically relevant levels of gene transfer into large animal and human stem cells have not been widely achieved. Results of current clinical gene transfer studies have raised fundamental questions about the physiology of primitive human hematopoietic cells and gene therapy vectors. Efforts are being undertaken to answer these problems and to develop more efficient gene therapy strategies.

Animals↗
PubMed1998

Accelerated loss of islet beta cells in sucrose-fed Goto-Kakizaki rats, a genetic model of non-insulin-dependent diabetes mellitus.

The Goto-Kakizaki (GK) rat is a spontaneously diabetic animal model of non-insulin-dependent diabetes mellitus, which is characterized by progressive loss of beta cells in the pancreatic islets with fibrosis. In the present study, we examined the effects of sucrose feeding on the islet pathology in this model. Six-week-old GK rats were fed with 30% sucrose for 6 weeks to induce severe hyperglycemia, and their condition was compared with that of nontreated rats. Age-matched normal Wistar rats were also given sucrose for the same periods and used for comparison. The sucrose-treated GK rats showed elevated blood glucose levels on oral glucose tolerance tests at 60 minutes and 120 minutes, representing 123% and 127% of values in untreated GK rats, respectively. At the end of the study, the mean beta-cell volume density in GK rats was 50% less than that in untreated Wistar rats. Sucrose feeding further reduced the volume densities of beta cells to only 50% of the levels of age-matched GK rats. Apoptotic cells were found in islet beta cells only in GK rats fed sucrose (mean 0.067%). There appeared to be more islets that immunohistochemically stained strongly positive with 8-hydroxy-deoxyguanosine as a marker of oxidative damage of DNA in GK rats fed sucrose compared with those not given sucrose. GK rats not fed sucrose showed significantly lower proliferative activity of beta cells measured by 5-bromo-2'-deoxyuridine uptake and intensified expression of Bcl-2 immunoreactivities at 6 weeks of age compared with those in age-matched Wistar rats. These two indices were reduced in both GK and Wistar rats with increasing age and were not affected by sucrose feeding in either group. The present study thus indicated that sucrose feeding promoted the apoptosis of beta cells in GK rats through increased oxidative stress without altering their proliferative activity.

8-Hydroxy-2'-Deoxyguanosine↗