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Biomedical subjects

H Sakagami

Publications and source records attributed to H Sakagami.

At least 37 records · Page 2Linked to original sources
PubMed2000

Localization of mRNAs for phospholipase D (PLD) type 1 and 2 in the brain of developing and mature rat.

Phospholipase D (PLD) is known as one of the key enzymes in the lipid metabolism which produces several second messengers. We demonstrated the localization of the gene expression for PLD1 and PLD2 using in situ hybridization histochemistry in the brain of developing and mature rats. Whereas PLD1 mRNA expression was detected mainly in presumptive oligodendrocytes, PLD2 mRNA expression was detected mainly in presumptive astrocytes. In addition, the gene expression for PLDs were expressed in neuroepithelial cells of the ventricular/ependymal zones and the gene for PLD2 was expressed transiently in early postnatal gray matters, presumptive neurons.

Age Factors↗
PubMed2000

Aberration of chromosomes 8 and 11 in bladder cancer as detected by fluorescence in situ hybridization.

Although a bladder cancer-specific abnormality in chromosomes or genes has not been reported, chromosomal regions that tend to become abnormal have been recognized. In this study, we investigated abnormalities in chromosomes 8 and 11. There were 27 patients with bladder cancer, 16 males and 11 females, who participated in this study. Abnormalities in chromosomes 8 and 11 were investigated by the fluorescence in situ hybridization (FISH) method. Probes used in this study were chromosome 8 alpha-satellite and chromosome 11 alpha-satellite (Oncor Co.). Of 27 cases, 15 cases were positive for chromosome 8 (55.6%) and ten cases were positive for chromosome 11 (37.0%). Since the FISH method detects chromosomal abnormality by the number of signals generated in cancer cells, this method is objective and simple and thus may be applicable in clinical practice.

Adult↗
PubMed2000

Synthesis and biological activity of N-acylphenothiazines.

Previous studies have demonstrated the relationship between radical intensity and cytotoxic activity in water-soluble compounds. This relationship was investigated in lipophilic compounds. Several N-acylphenothiazines showed higher cytotoxic activity against human leukemic and squamous carcinoma cell lines than phenothiazine, the parent compound. Electron spin resonance (ESR) spectroscopy showed that these active compounds produced much lower amounts of radicals than phenothiazine. Several compounds failed to inhibit the cytopathic effects of human immunodeficiency virus (HIV) infection in MT-4 cells. It suggested that the radical-mediated-mechanisms has not involved in the induction of cytotoxic activity by lipophilic compounds, such as N-acylphenothiazines.

Anti-HIV Agents↗
PubMed2000

Cytotoxic activity of hydrolyzable tannins against human oral tumor cell lines--a possible mechanism.

Hydrolyzable tannins showed higher cytotoxic activity against human oral squamous cell carcinoma and salivary gland tumor cell lines than against normal human gingival fibroblasts, whereas gallic acid, a component unit of tannins, showed much weaker selective cytotoxicity. The cytotoxic activity of dimeric compounds was generally higher than that of monomeric compounds. Macrocyclic ellagitannin oligomers, such as oenothein B, woodfordin C and woodfordin D showed the greatest cytotoxic activity, and their activity (per given number of molecules) was one order higher than those of gallic acid and epigallocatechin gallate, a major component of green tea. These compounds induced apoptotic cell death characterized by DNA fragmentation (as demonstrated by the TUNEL method) and cleavage of cytokeratin 18 by activated caspase(s) (as demonstrated by M30 monoclonal antibody). ESR spectroscopy revealed that these macrocyclic compounds at higher concentrations produced their own radicals and significantly enhanced the radical intensity of sodium ascorbate, possibly by their prooxidant actions. Catalase failed to eliminate their apoptosis-inducing activity, reducing the possibility of the involvement of hydrogen peroxide production in the extracellular fraction. These observations suggested that the antitumor activity of macrocyclic ellagitannin oligomers reported previously might be explained by their apoptosis-inducing activity.

Antineoplastic Agents↗
PubMed2000

Induction of cytotoxicity by chlorogenic acid in human oral tumor cell lines.

Millimolar concentrations of chlorogenic acid (CGA) showed higher cytotoxic activity against human oral squamous cell carcinoma (HSC-2) and salivary gland tumor (HSG) cell lines, as compared with that against human gingival fibroblast (HGF). The cytotoxic activity of CGA was significantly reduced by catalase or CoCl2, but not affected by FeCl3 or CuCl2. ESR spectroscopy showed that higher (millimolar) concentrations of CGA produced radicals under alkaline conditions, acting as a prooxidant, whereas lower concentrations of CGA scavenged superoxide and hydroxyl radical. CGA produced large DNA fragments (as identified by slightly faster migrating band of DNA on agarose gel electrophoresis) and nuclear condensation (as demonstrated by Hoechst (No. 33258) staining) in tumor cell lines. Activation of caspase was demonstrated by staining with M30 monoclonal antibody, which reacts with degradation products of cytokeratin 18. Contact with CGA for at least 6 h was necessary for irreversible cytotoxicity induction. Pretreatment of the cells with caspase 3 inhibitor partially inhibited the cytotoxic action of CGA. These date suggest that CGA induces cytotoxicity in oral tumor cell lines, possibly by hydrogen peroxide-mediated oxidation mechanism.

Anticarcinogenic Agents↗
PubMed2000

Classical protein kinase C and its hypoxic stimulus-induced translocation in the cat and rat carotid body.

The presence, subcellular distribution, species specificity and possible hypoxic stimulus-induced translocation of classical protein kinase C (cPKC) isozymes were examined in the carotid body. Carotid bodies were dissected from cats exposed in vivo to normoxic or acute hypoxic conditions and from normoxic rats. For immunohistochemistry isoform-specific monoclonal antisera to PKCalpha, PKCbetaI, PKCbetaII and PKCgamma were used. The immunoreactivity was visualized by fluorescein isothiocyanate (FITC) labelling. FITC/Texas red double-labelled specimens for the cPKC isozymes/tyrosine hydroxylase were used to demonstrate the chemoreceptor cell localization of cPKC isozymes. The immunofluorescence was detected using laser scanning confocal image technology. The results showed expression of the PKCalpha and PKCgamma but not PKCbeta isoforms in the cytoplasm of carotid body chemoreceptor cells. The double labelling provided evidence for the chemoreceptor cell localization of the cPKC isoforms detected. The immunostaining was most intense in the periphery of the perikarya, the nuclear envelope and, occasionally, the nucleoplasm. No major differences were found in the immunolocalization of PKCalpha and PKCgamma under normoxic and hypoxic conditions or between species. However, the immunoreactivity tended to accumulate more in the peripheral cytoplasm and away from the nucleus in the hypoxic chemoreceptor cell. This study demonstrates the presence of classical protein kinase C enzymes in chemoreceptor cells. The intensity of the immunoreactivity may suggest a role for the classical protein kinase C signalling pathway in shaping the hypoxic response at the carotid body. However, this study failed to provide firm evidence of this.

Animals↗
PubMed2000

Tetrahydrobiopterin impairs the action of endothelial nitric oxide via superoxide derived from platelets.

The mechanism by which exogenous tetrahydrobiopterin (BH(4)) impairs the action of endothelial nitric oxide (NO) in the presence of platelets was investigated. The endothelial NO generated by shear stress was determined by the anti-aggregating activity of indomethacin-treated endothelial cells and the cyclic GMP concentration in platelets. The inhibitory effect of exogenous BH(4) was suppressed by superoxide dismutase (SOD), or diclofenac sodium at concentrations inhibiting O(2)(-) generation, but not by allopurinol, a xanthine oxidase inhibitor. BH(4) similarly inhibited the anti-aggregatory effect of sodium nitroprusside (SNP), a NO donor. The inhibitory effect was suppressed by diphenyleneiodonium, a specific inhibitor of NADPH oxidase. Six(S)-BH(4), an inactive diastereoisomer of 6(R)-BH(4), and the 5,6,7,8-tetrahydropterin compounds inhibited the endothelial NO action, whereas sepiapterin and 7,8-dihydrobiopterin (BH(2)), 5,6-double bond pterins, were inactive. These tetrahydropterins, but not sepiapterin and BH(2), scavenged superoxide (O(2)(-)) generated by the hypoxanthine-xanthine oxidase reaction, possibly due to electron transfer during oxidation to its quinonoid-form. BH(4) markedly stimulated the O(2)(-) generation from platelets, in the presence of NADH, rather than that of NADPH. These findings suggest that BH(4) stimulates platelet NAD(P)H oxidase to generate O(2)(-), and inhibits the anti-aggregating effect of NO. SOD activity in the local environment may modify the effect of BH(4) on the endothelial NO activity.

Animals↗
PubMed2000

Distinct immunohistochemical localization of two isoforms of Ca2+/calmodulin-dependent protein kinase kinases in the adult rat brain.

Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV) is thought to be involved in regulating gene expression by phosphorylating various transcriptional factors. CaM-KIV as well as CaM-KI are activated upon phosphorylation by two distinct isoforms of Ca2+/calmodulin-dependent protein kinase kinases, CaM-KKs alpha and beta. In this study, we raised isoform-specific monoclonal antibodies against CaM-KKs and examined the immunohistochemical localization of CaM-KKs in the rat brain, compared with that of CaM-KIV. CaM-KK alpha-immunoreactivity was rather widely distributed in neurons throughout the brain, except cerebellar cortex. The highest levels of CaM-KK alpha-immunoreactivity were observed in the cerebral cortex, facial nucleus and motor neurons of the spinal cord. Moderate CaM-KK alpha-immunoreactivity was observed in the hippocampal formation, pontine nuclei and various brain stem nuclei including trigeminal, vestibular, cochlear and hypoglossal nuclei. In contrast, CaM-KK beta-immunoreactivity was relatively restricted in some neuronal populations. The highest levels of CaM-KK beta-immunoreactivity were observed in the cerebellar granule cell layer, and moderate immunoreactivity was observed in the cerebral cortex, hippocampal formation, caudate putamen, pontine nuclei, cochlear nucleus and molecular layer of the cerebellum. In contrast to the prominent nuclear localization of CaM-KIV, both isoforms of CaM-KKs were localized in the perikaryal cytoplasm, dendrites and nerve terminals, but not in the cell nuclei. The distinct localization of two isoforms of CaM-KKs suggests that the complicated mechanisms for activation of CaM-KIV by CaM-KKs may be exerted in region-specific manners as well as intracellularly.

Animals↗
PubMed2000

Structure of the mouse glutamate decarboxylase 65 gene and its promoter: preferential expression of its promoter in the GABAergic neurons of transgenic mice.

GABA is synthesized by glutamate decarboxylase (GAD), which has two forms, GAD65 and GAD67. To elucidate the molecular mechanisms of mouse GAD65 (mGAD65) gene expression, we isolated and characterized the mGAD65 gene. The mGAD65 gene was found to be divided into 16 exons and spread over 75 kb. The sequence of the first exon and the 5'-flanking region indicated the presence of potential neuron-specific cis-regulatory elements. We used transgenic mice to examine the expression pattern conferred by a 9.2-kb promoter-proximal DNA fragment of the mGAD65 gene fused to the bacterial lacZ reporter gene. Transgenic mice showed high beta-galactosidase activity specifically in brain and testis. They also showed characteristic patterns of transgene expression in olfactory bulb, cerebellar cortex, and spinal cord, a similar expression pattern to that of endogenous mGAD65. However, no transgene expression was observed in the ventral thalamus or hypothalamus, in which high mGAD65 gene expression levels have been observed. These results suggest that the 9.2-kb DNA fragment of the mGAD65 gene is associated with its tissue-specific expression and its targeted expression in GABAergic neurons of specific brain regions but that additional regulatory elements are necessary to obtain fully correct expression.

Amino Acid Sequence↗
PubMed2000

Polyphenols from Eriobotrya japonica and their cytotoxicity against human oral tumor cell lines.

Three new flavonoid glycosides, together with 15 known flavonoids, have been isolated from the leaves of Eriobotrya japonica, and characterized as (2S)- and (2R)-naringenin 8-C-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosides, and cinchonain Id 7-O-beta-D-glucopyranoside, respectively, based on spectral analyses including two dimensional (2D) NMR techniques. Higher proanthocyanidin fraction in the water-soluble portion of the extract was characterized as a procyanidin oligomer mixture mainly composed of undecameric procyanidin. These polyphenols have also been assessed for cytotoxic activity against two human oral tumor (human squamous cell carcinoma and human salivary gland tumor) cell lines. Selective cytotoxicity of the procyanidin oligomer between tumor and normal gingival fibroblast cells, and its possible mechanism, were also described.

Antibodies, Monoclonal↗
PubMed2000

Antitumor potential and possible targets of phenothiazine-related compounds.

Phenothiazines and its related compounds have shown diverse biological activities including psychotropic, anticancer and other pharmacological activities. Recent studies have suggested the possible interactions between phenothiazines and their physiological targets or potential receptors. New types of phenothiazine, such as "half-mustard type" phenothiazines and benzo[a]phenothiazines, have been synthesized. These compounds stimulated T-cell blast formation, natural killer cell activity (possibly via activation of monocytes and macrophages) and antibody-dependent cellular cytotoxicity of human peripheral blood mononuclear cells, and showed cytotoxicity against several human cancer cell lines. Benzo[a]phenothiazines induced monocytic differentiation and apoptotic cell death (characterized by internucleosomal DNA fragmentation) in human myelogenous leukemic cell lines, but not in other cancer cell lines. These compounds also induced antimicrobial activity in vivo, possibly by host-mediated immunopotentiation. On the other hand, phenothiazines did not induce such immunopotentiation activity, but showed direct antibacterial activity in vitro. There was positive relation between their radical intensity and biological activities. These compounds did not show any apparent mutagenic activity, but rather be antimutagenic. These data suggest their possible applicability of "half-mustard type" phenothiazines and benzo[a]phenothiazines for cancer chemotherapy.

Animals↗
PubMed2000

Apoptosis-inducing activity of vitamin C and vitamin K.

Apoptosis-inducing activity of vitamins C and K and of their analogs are reviewed. Vitamin C shows both reducing and oxidizing activities, depending on the environment in which this vitamin is present. Higher concentrations of vitamin C induce apoptotic cell death in various tumor cell lines including oral squamous cell carcinoma and salivary gland tumor cell lines, possibly via its prooxidant action. The apoptosis-inducing activity of ascorbate is stimulated by Cu2+, lignin and ion chelator, and inhibited by catalase, Fe3+, Co2+ and saliva. On the other hand, at lower concentrations, ascorbic acid displays an antioxidant property, preventing the spontaneous and stress or antitumor agent-induced apoptosis. Sodium 5,6-benzylidene-L-ascorbate, intravenous administration of which induces degeneration of human inoperable tumors and rat hepatocellular carcinoma in vivo, induces apoptotic or non-apoptotic cell death, depending on the types of target cells. On the other hand, elevation of intracellular concentration of ascorbic acid by treatment with ascorbate 2-phosphate or dehydroascorbic acid makes the cells resistant to the oxidative stress-induced apoptosis. Vitamin K2, which has a geranylgeranyl group as a side chain,and vitamin K3 induces apoptosis of various cultured cells including osteoclasts and osteoblasts, by elevating peroxide and superoxide radicals. Synergistic apoptosis-inducing actions have been found between vitamins C and K, and between these vitamins and antiproliferative agents. The possible therapeutic application of these vitamins is discussed.

Animals↗
PubMed2000

Radical generation, radical-scavenging activity, and cytotoxicity of eugenol-related compounds.

To clarify the possible link between radicals and cytotoxicity of eugenol-related compounds, dimeric compounds were synthesized from eugenol (4-allyl-2-methoxy-phenol), butylated hydroxyanisole (BHA) (2-t-butyl-4-methoxyphenol) or MMP (2 methoxy-4-methylphenol); bis-EUG (3,3'-dimethoxy-5,5'-di-2-propenyl-1,1'-biphenyl-2,2'-diol), bis-BHA (3,3'-di-t-butyl-5,5'-dimethoxy-1,1'-biphenyl-2,2'-diol), and bis-MMP (3,3'-di-methoxy-5,5'-dimethyl-1,1'-biphenyl-2,2'-diol). The cytotoxic activity of these compounds was determined using a salivary gland tumor cell line (HSG), oral squamous cell carcinoma cell line (HSC-2) and human promyelocytic leukemia cell line (HL-60). A parabolic relationship between the cytotoxicity and log P (the octanol-water partition coefficient) was observed, showing that both BHA and bis-MMP, with a log P of 3-4, were the most cytotoxic. The cytotoxic activity of the 2-methoxy derivatives, eugenol, MMP and bis-MMP, against HSG cells was significantly enhanced by visible-light irradiation, possibly due to their high redox potential. Electron spin resonance (ESR) spectroscopy indicated that eugenol and BHA alone produced radicals under alkaline conditions (pH > 9.5), and eugenol most efficiently scavenges reactive oxygen species (O2-). Antioxidative reactivity of eugenol-related compounds was determined by measuring the inhibiting periods of the AIBN (2,2'-azobisisobutyronitrile)/MMA (methyl methacrylate) polymerization system, and the number of moles of peroxy radical trapped by moles of the relevant phenols (stoichiometric factor, n). It was found that the n values of eugenol and MMP were approximately 1, whereas those of BHA >2, suggesting that eugenol and MMP undergo dimerization through radical-radical couplings through quinone methides, whereas BHA undergoes the competitive interaction with poly-MMA radicals after oxidation by AIBN-peroxy radicals. BHA was an efficient peroxy radical-scavenger, but possibly reacted with polymer radicals of the lipid, thus mediating the cytotoxicity. The n value of bis-BHA was two, whereas those of bis-EUG and bis-MMP were 1.6-1.7, suggesting that the latter were further oxidized. The enthalpies of phenoxyl radical formation were determined using the semi-empirical PM3 quantum-mechanical method and the possible link to redox potential was discussed.

Adenocarcinoma↗
PubMed1999

Alpha-trifluoromethylated acyloins induce apoptosis in human oral tumor cell lines.

Cytotoxic activity of newly synthesized trifluoromethyl ketones and related compounds was studied using two human oral tumor cell lines (HSG and HSC-2). Among them, alpha-trifluoromethylacyloins (1 and 2) were found to induce apoptotic cell death, as judged by the terminal deoxynucleotidyl transferase (TdT) dUTP nick end-labeling (TUNEL) method which detects DNA nick or fragments. Furthermore, the cytoplasm of 1 or 2 treated HSG cells was stained by M30 monoclonal antibody, which detects the product resulting from the cleavage of cytokeratin 18 by activated caspase.

Antineoplastic Agents↗
PubMed1999

Novel related cDNAs (C184L, C184M, and C184S) from developing mouse brain encoding two apparently unrelated proteins.

Three related cDNAs (C184L, C184M, and C184S) were isolated from a developing mouse brain cDNA library. C184S is the 5'-end portion and C184M is the 3'-end portion, respectively, of C184L. C184S and C184M have open reading frames of 199 amino acids (ORF1) and 189 amino acids (ORF2), respectively; C184L has both ORF1 and ORF2 (dicistronic structure), but seems to translate only ORF1. Southern blot analysis suggests that all of the three related mRNAs are transcribed from the same single gene. The intervening region of C184L cDNA between ORF1 and ORF2 contained a promoter sequence for C184M mRNA, which is transcribed from the corresponding genomic sequence. Very recently, a cDNA encoding human homologue of ORF1 (human autoantigen p27) and a cDNA encoding a different mouse isoform of ORF2 (mammary tumor virus receptor) were reported. Our results indicate that the mRNAs encoding these apparently unrelated proteins are transcribed within an adjacent or overlapping area on the genome, suggesting the same origin of the two transcription units.

Aging↗
PubMed1999

Localization of mRNA for Dri 42, subtype 2b of phosphatidic acid phosphatase, in the rat brain during development.

The expression of a gene termed Dri 42, a differentiation-related gene originally identified from intestine and a gene encoding phosphatidic acid phosphatase 2b isoform, was localized in developing and matured rat brains by in situ hybridization histochemical analysis. The gene expression was dominant in the ventricular germinal zone without significant expression in the intermediate, mantle and marginal zones throughout embryonic brain and spinal cord. The dominant expression in the ventricular germinal zone was maintained at P0 and P7, but it markedly decreased at later postnatal stages, while persistently high expression was detected in Bergmann glial cells of the cerebellar Purkinje cell layer throughout the postnatal development.

Animals↗
PubMed1999

Immunohistochemical distribution and functional characterization of an organic anion transporting polypeptide 2 (oatp2).

The rabbit polyclonal antibody against rat organic anion transporting polypeptide 2 (oatp2) was raised and immunoaffinity-purified. Western blot analysis for oatp2 detected two bands ( 74 and 76 kDa) in rat brain and a single band (76 kDa) in the liver. By immunohistochemical analysis, the oatp2 immunoreactivity was specifically high at the basolateral membrane of rat hepatocytes. Functionally, the oatp2-expressing oocytes were found to transport dehydroepiandrosterone sulfate, delta1 opioid receptor agonist [D-Pen2,D-Pen5]enkephalin, Leuenkephalin, and biotin significantly, as well as the substrates previously reported. These data reveal the exact distribution of the rat oatp2 at the protein level in the liver, and that oatp2 appears to be involved in the multispecificity of the uptaking substrates in the liver and brain.

Animals↗