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Biomedical subjects

F Ishikawa

Publications and source records attributed to F Ishikawa.

At least 19 recordsLinked to original sources
PubMed2005

Efficient engraftment of primary adult T-cell leukemia cells in newborn NOD/SCID/beta2-microglobulin(null) mice.

Adult T-cell leukemia (ATL) develops via multiple oncogenic steps in human T-cell leukemia virus type I (HTLV-I) carriers. To better understand pathogenesis of ATL, we developed a novel xenogeneic engraftment model in which primary ATL cells are intravenously transplanted into neonatal nonobese diabetic (NOD)/severe-combined immunodeficiency (SCID)/beta2-microglobulin(null) (NOD/SCID/beta2m(null)) mice. Acute-type ATL cells engrafted in the peripheral blood and in the lymph nodes of recipients at a high efficiency. Engrafted ATL cells were dually positive for human CD4 and CD25, and displayed patterns of HTLV-I integration identical to those of donors by Southern blot analysis. These cells infiltrated into recipients' liver, and formed nodular lesions, recapitulating the clinical feature of each patient. In contrast, in smoldering-type ATL cases, multiple clones of ATL cells engrafted efficiently in NOD/SCID/beta2m(null) mice. When smoldering-type ATL cells were retransplanted into secondary NOD/SCID/beta2m(null) recipients, single HTLV-I-infected clones became predominant, suggesting that clones with dominant proliferative activity can be competitively selected in this xenogeneic system. Taken together, the NOD/SCID/beta2m(null) newborn system is useful to understand kinetics, metastasis, and disease progression of ATL in vivo.

Anemia, Refractory, with Excess of Blasts↗
PubMed2004

Expression of cholesteryl ester transfer protein (CETP) in germinal centre B cells and their neoplastic counterparts.

AIMS: Cholesteryl ester transfer protein (CETP) is known to facilitate the transfer of lipids between plasma lipoproteins. Previous studies on human tissues have determined that the spleen contains large amounts of CETP mRNA, while the exact location of CETP in such organs remains unknown. In the present study, our aim was to locate CETP protein expression at the cellular level in human normal and neoplastic lymphoid organs. METHODS AND RESULTS: In-situ hybridization (ISH) and immunohistochemistry were applied to pathology specimens. A specific rabbit anti-CETP antibody was used for immunohistochemical analysis, together with another CETP-specific monoclonal antibody. A riboprobe for ISH was derived from CETP cDNA. Immunohistochemically, CETP was localized in germinal centre B cells and a proportion of marginal zone B cells. ISH showed that CETP mRNA was located mostly in the same areas. When 141 malignant lymphomas of various subtypes were studied, high expression of CETP, equivalent to that found in normal germinal centre B cells, was demonstrated in lymphoma subtypes that are currently regarded as the neoplastic counterparts of primarily germinal centre B cells. CONCLUSION: CETP localizes B cells in germinal centres, a proportion of post-germinal centre B cells and their neoplastic counterparts.

Animals↗
PubMed2003

Evaluation of Bifidobacterium breve strain Yakult-fermented soymilk as a probiotic food.

We evaluated the effects of Bifidobacterium breve-fermented soymilk on probiotic function. An administered strain of B. breve strain Yakult was capable of growing in soymilk with no additives as high as 10(9) CFU/ml. During storage of the fermented soymilk at 10 degrees C for 20 days, viable counts of the strain did not change. The growth inhibition of the strain in a bile-containing medium was lessened by the addition of soy protein. In human feeding experiments, the administered B. breve was recovered at a level of over 10(9) CFU/g faeces, accompanied by an increase in the total number of bifidobacteria. These results indicate that fermented soymilk with B. breve strain Yakult could be a novel type of probiotic food.

Adult↗
PubMed2003

Composition and conservation of the telomeric complex.

The telomere is composed of telomeric DNA and telomere-associated proteins. Recently, many telomere-associated proteins have been identified, and various telomere functions have been uncovered. In budding yeast, scRap1 binds directly to telomeric DNA, and other telomere regulators (Sir proteins and Rif proteins) are recruited to the telomeres by interacting with scRap1. Cdc13 binds to the most distal end of the chromosome and recruits telomerase to the telomeres. In fission yeast and humans, TTAGGG repeat binding factor (TRF) family proteins bind directly to telomeric DNA, and Rap1 proteins and other telomere regulators are recruited to the telomeres by interacting with the TRF family proteins. Both organisms have Pot1 proteins at the most distal end of the telomere instead of a budding-yeast Cdc13-like protein. Therefore, fission yeast and humans have in part common telomeric compositions that differ from that of budding yeast, a result that suggests budding yeast has lost some telomere components during the course of evolution.

Humans↗
PubMed2003

Human cord blood long-term engrafting cells are CD34+ CD38-.

There have been controversies about CD34 and CD38 expression by human cord blood (CB) stem cells. Using the newborn NOD/SCID/beta2-microglobulin-null mouse assay that we recently developed, we examined the in vivo engrafting capability of human CB cells. Almost all of the 4-5 months engrafting cells were found in CD34(+) population. The capability of secondary reconstitution was found only in the CD34(+) cells. When the CD34(+) CB cells were separated into CD38(-) and CD38(+) subpopulations and tested for engraftment, the majority of the engrafting cells were detected in the CD38(-) subpopulation. These findings are consistent with the results from studies of murine stem cells and strongly indicate that the phenotype of human CB stem cells is CD34(+) CD38(-).

ADP-ribosyl Cyclase↗
PubMed2002

Soymilk products affect ethanol absorption and metabolism in rats during acute and chronic ethanol intake.

In this study we evaluated the effects of soy products on ethanol metabolism during periods of acute and chronic consumption in rats. Gastric ethanol content and blood ethanol and acetaldehyde concentrations were investigated after the oral administration of ethanol (34 mmol/kg) plus soy products such as soymilk (SM) or fermented soymilk (FSM). The gastric ethanol concentration of the FSM group was greater than that of the control group, whereas portal and aortal blood ethanol concentrations of the FSM group were lower than in controls. The aortal acetaldehyde concentration in the FSM group was lower than that of the control group. The direct effect of isoflavones on liver function was investigated by using hepatocytes isolated from untreated rats. Genistein (5 micromol/L) decreased ethanol (P = 0.045) and tended to decrease acetaldehyde (P = 0.10) concentrations in the culture filtrate. Some variables of ethanol metabolism in the liver were investigated after chronic ethanol exposure for 25 d. Rats consumed a 5% ethanol fluid plus the SM diet, the FSM diet or a control diet. Microsomal ethanol oxidizing activity was significantly lower in the FSM group than the control group. Furthermore, cytosolic glutathione S-transferase activity was higher in the SM and FSM groups than in the control group. Acetaldehyde dehydrogenase activity (low K(m)) in the FSM group (P = 0.15), but not in the SM group (P = 0.31), tended to be greater than in the control group. The amount of thiobarbituric acid reacting substances in the liver of the SM and FSM groups tended to be less than that of the control group (P = 0.18 and 0.10, respectively). These results demonstrate that soymilk products inhibit ethanol absorption and enhance ethanol metabolism in rats.

Absorption↗
PubMed2001

Identification and analyses of the Xenopus TERT gene that encodes the catalytic subunit of telomerase.

The Xenopus telomerase catalytic component gene, xTERT (Xenopus telomerase reverse transcriptase), has been cloned. The production of xTERT recombinant protein together with the proposed Xenopus telomerase RNA (xTR) (Chen et al., 2000. Cell 100, 503-514) in a rabbit reticulocyte lysate system led to the reconstitution of active telomerase, indicating that both products are functional telomerase components. Both xTERT expression and telomerase activity are high from the early to the late blastula stage. However, they are decreased at the gastrula stage and thereafter, suggesting that the xTERT expression level is the primary mechanism for regulating telomerase activity in Xenopus development. This is the first report of a non-mammalian vertebrate TERT gene. Sequence comparison of xTERT with human and mouse TERTs has uncovered four regions conserved in the amino-terminal halves of vertebrate TERT proteins, the functions of which will be discussed herein.

Amino Acid Sequence↗
PubMed2001

spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast.

Telomeres are essential for genome integrity. scRap1 (S. cerevisiae Rap1) directly binds to telomeric DNA and regulates telomere length and telomere position effect (TPE) by recruiting two different groups of proteins to its RCT (Rap1 C-terminal) domain. The first group, Rif1 and Rif2, regulates telomere length. The second group, Sir3 and Sir4, is involved in heterochromatin formation. On the other hand, human TRF1 and TRF2, as well as their fission yeast homolog, Taz1, directly bind to telomeric DNA and negatively regulate telomere length. Taz1 also plays important roles in TPE and meiosis. Human Rap1, the ortholog of scRap1, negatively regulates telomere length and appears to be recruited to telomeres by interacting with TRF2. Here, we describe two novel fission yeast proteins, spRap1 (S. pombe Rap1) and spRif1 (S. pombe Rif1), which are orthologous to scRap1 and scRif1, respectively. spRap1 and spRif1 are independently recruited to telomeres by interacting with Taz1. The rap1 mutant is severely defective in telomere length control, TPE, and telomere clustering toward the spindle pole body (SPB) at the premeiotic horsetail stage, indicating that spRap1 has critical roles in these telomere functions. The rif1 mutant also shows some defects in telomere length control and meiosis. Our results indicate that Taz1 provides binding sites for telomere regulators, spRap1 and spRif1, which perform the essential telomere functions. This study establishes the similarity of telomere organization in fission yeast and humans.

Amino Acid Sequence↗
PubMed2001

Structure of the C-terminal RNA-binding domain of hnRNP D0 (AUF1), its interactions with RNA and DNA, and change in backbone dynamics upon complex formation with DNA.

Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP) -type RNA-binding domains (RBDs), each of which can specifically bind to the UUAG-sequence. hnRNP D0 also binds specifically to single-stranded d(TTAGGG)(n), the human telomeric DNA repeat. We have already reported the structure and interactions with RNA of the N-terminal RBD (RBD1). Here, the structure of the C-terminal RBD (RBD2) determined by NMR is presented. It folds into a compact alpha beta structure comprising an antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. In addition to the four beta-strands commonly found in RNP-type RBDs, an extra beta-strand, termed beta 4(-), was found just before the fourth beta-strand, yielding a five-stranded beta-sheet. Candidate residues of RBD2 involved in the interactions with RNA were identified by chemical shift perturbation analysis. Perturbation was detected on the beta-sheet side, not on the opposite alpha-helix side, as observed for RBD1. It is notable that the beta 4(-) to beta 4 region of RBD2 is involved in the interactions in contrast to the case of RBD1. The chemical shift perturbation analysis also showed that RBD2 interacts with DNA in essentially the same way as with RNA. Changes in the backbone dynamics upon complex formation with DNA were examined by means of model free analysis of relaxation data. In free RBD2, the beta 4(-) to beta 4 region exhibits slow conformational exchange on the milli- to microsecond time scale. The exchange is quenched upon complex formation. The flexibility of free RBD2 may be utilized in the recognition process by allowing different conformational states to be accessed and facilitating induced fit. Additionally, faster flexibility on the nano- to picosecond time scale was observed for loop 3 located between beta 2 and beta 3 in free RBD2, which is retained by the complex as well.

Amino Acid Sequence↗
PubMed2001

Improved nasal bioavailability of elcatonin by insoluble powder formulation.

The bioavailability of elcatonin (ECT) via the nasal route was investigated with a powder dosage form utilizing water-insoluble calcium carbonate (CaCO(3)) in comparison with the liquid dosage form. Total radioactivity and the radioactivity of intact [3H]ECT were measured to evaluate the nasal absorption in vivo and the nasal mucosal transport in vitro. The systemic bioavailability of both total radioactivity and intact [3H]ECT following intranasal administration of the powder formulation in rats was significantly greater than in the case of the liquid formulation. In contrast, similar permeability of ECT across excised rabbit nasal mucosa was seen for both formulations, and was close to that of [14C]inulin, suggesting that the ECT transport is predominantly paracellular in each case. However, the powder formulation significantly prolonged the residence time of [3H]ECT in the rat nasal cavity, compared with the liquid formulation. We conclude that the powder formulation utilizing CaCO(3) improves the nasal bioavailability by increasing the residence time of ECT in the nasal cavity and is likely to be effective in increasing systemic drug delivery.

Administration, Intranasal↗
PubMed2001

Telomerase activation and p53 mutations in urethane-induced A/J mouse lung tumor development.

The mouse telomerase holoenzyme, which synthesizes telomeric DNA de novo, is a ribonucleoprotein complex that includes the mouse telomerase RNA component (mTERC), mouse telomerase-associated protein (mTEP1) and mouse telomerase reverse transcriptase (mTERT). To determine the role of telomerase in urethane-induced lung tumorigenesis in A/J mice we examined telomerase activity and the expression of each telomerase subunit in 20 tumor samples, harvested at 16, 28, 40 and 50 weeks after urethane treatment. The telomeric repeat amplification protocol assay showed that statistically significant telomerase activation occurred both early and late in tumorigenesis. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that mRNA expression levels of mTEP1 and mTERT were up-regulated during tumor progression, while mTERC expression was not significantly different between tumors and normal lung. We further examined mTEP1 protein expression in normal lung tissue and lung tumors; western blot analysis showed preferential expression of mTEP1 protein in lung tumors compared with normal lung and immunohistochemistry revealed that a majority of the adenoma cells were positively stained in the nucleus, whereas only a few of the adjacent normal alveolar cells were immunoreactive. In addition, we investigated DNAs of the 20 tumor samples by single strand conformation polymorphism and sequencing analyses to examine whether alterations of the p53 gene in exons 5-8 were associated with telomerase activity. Although we found one nonsense, two missense, two silent and one simultaneous double mutation at different codons in six late stage tumors, there was no apparent correlation between telomerase activity and p53 mutations. Collectively, these results suggest that mTEP1 as well as mTERT may be involved in the regulation of telomerase activity and that telomerase activation may contribute to lung tumorigenesis in A/J mice independently of p53 gene alterations.

Animals↗
PubMed2001

Telomerase-inhibitory effects of the triphosphate derivatives of some biologically active nucleosides.

Telomerase is a ribonucleoprotein reverse transcriptase that uses its internal RNA moiety as a template for synthesis of telomere repeats. To clarify the susceptibility of telomerase to HIV-1 reverse transcriptase inhibitors (RT), we investigated the inhibitory effects of 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP), which is known to be a potent HIV-1 RT inhibitor, and acyclovir triphosphate (ACVTP). Lineweaver-Burk plot analyses showed that the inhibition mode of these compounds was competitive with the substrate dNTP counterpart. However, inhibition by AZTTP was weak (Ki = 15 microM, Km of dTTP = 7.1 microM). Interestingly, ACVTP showed considerable inhibition. The Ki value of ACVTP was 5.0 microM, being smaller than the Km of dGTP (12 microM).

Acyclovir↗
PubMed2001

In vitro reconstitution of the end replication problem.

The end replication problem hypothesis proposes that the ends of linear DNA cannot be replicated completely during lagging strand DNA synthesis. Although the idea has been widely accepted for explaining telomere attrition during cell proliferation, it has never been directly demonstrated. In order to take a biochemical approach to understand how linear DNA ends are replicated, we have established a novel in vitro linear simian virus 40 DNA replication system. In this system, terminally biotin-labeled linear DNAs are conjugated to avidin-coated beads and subjected to replication reactions. Linear DNA was efficiently replicated under optimized conditions, and replication products that had replicated using the original DNA templates were specifically analyzed by purifying bead-bound replication products. By exploiting this system, we showed that while the leading strand is completely synthesized to the end, lagging strand synthesis is gradually halted in the terminal approximately 500-bp region, leaving 3' overhangs. This result is consistent with observations in telomerase-negative mammalian cells and formally demonstrates the end replication problem. This study provides a basis for studying the details of telomere replication.

Animals↗
PubMed2001

[Identification system for Sildenafil in health foods].

A substantially available identification system for Sildenafil in health foods was established using 3 different analytical methods; i.e. TLC, preparative TLC/MS and HPLC/photo-diode array. Sildenafil in health foods was extracted with ethyl acetate under alkaline conditions as sample solutions for TLC and preparative TLC, and also extracted with 50% methanol and then diluted with solution of HPLC mobile phase for HPLC. The sample solution for TLC was applied to Silica gel 60 F254 plates with chloroform/methanol/28% ammonia (90:1:5, under layer) as mobile phase. Spots were located under UV radiation at 254 nm and 366 nm, and spraying dragendorff reagent. The conditions for preparative TLC were the same as these of TLC method, and samples abtained from preparative TLC were determined by MS with APCI interface, under both positive and negative modes. The HPLC analysis was carried out on a column of Cosmosil 5C18-AR (4.6 mm x 150 mm, 5 microns) with 0.05 mol/l phosphate buffer pH 3.0/acetonitrile(73:27) as mobile phase and the eluate was monitored by a photo-diode array detector. The quantitative analysis was available, when the peak of this sample on HPLC was detected at 290 nm. When this system was applied to commercial health foods, Sildenafil was identified and their contents were 25 mg-45 mg/tablet or bottle. These contents nearly correspond to that in Viagra, 25 mg, 50 mg/tablet. Therefore, there is a fear of side effects for Sildenafil, when it is taken as health foods.

Chromatography, High Pressure Liquid↗
PubMed2001

Hypocholesterolemic effect of indigestible fraction of Chlorella regularis in cholesterol-fed rats.

The effects of Chlorella regularis powder (CP) and Chlorella regularis indigestible fraction (CIF) on serum and liver lipid concentrations and on fecal steroid excretion were estimated in rats fed diets containing 5 g/kg cholesterol and 2.5 g/kg sodium cholate. The ingestion of 12.7% CP or 5.3% CIF did not influence food intake or growth. CP and CIF decreased the levels of serum cholesterol, but had no effect on the levels of serum triacylglycerol and phospholipid. Liver cholesterol contents were lower in the CP and CIF groups than in the control group, but CP and CIF did not affect liver triacylglycerol content. CP and CIF increased the total amount of fecal neutral steroids excreted, but did not modify the total bile acid excretion. However, the soluble bile acid concentrations of reconstituted fecal water in the rats fed CP and CIF diets were lower than the control value. Moreover, CP and CIF had a high bile acid binding capacity in vitro. These results indicated that CIF had a hypocholesterolemic effect and enhanced fecal neutral steroid excretion while decreasing the soluble fecal bile acid concentration.

Animals↗
PubMed2000

Recognition of 2'-deoxy-l-ribonucleoside 5'-triphosphates by human telomerase.

Telomerase is classified as one of the reverse transcriptases (RTs). To clarify whether l-enantiomers of natural 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) are recognized by human telomerase, a quantitative telomerase assay based on the "stretch PCR" method was developed and used for kinetic analysis of the inhibitory effects of these compounds on the enzyme. Among the four l-enantiomers of dNTPs, l-dTTP and l-dGTP inhibited telomerase activity and the others showed slight or no inhibitory effect. Lineweaver-Burk plot analysis showed that the inhibition modes of l-dTTP and l-dGTP were partially competitive (mixed type) and competitive with the corresponding substrate dNTP, respectively. However, the K(i) values of l-dTTP and l-dGTP (21 and 15 microM) were several times larger than the K(m) values (3-6 microM). These results suggest that the active site of telomerase is not able to discriminate strictly the chirality of dNTPs, although it is more discriminatory than HIV-1 RT.

Deoxyadenine Nucleotides↗
PubMed2000

Human chromatid cohesin component hRad21 is phosphorylated in M phase and associated with metaphase centromeres.

Sister chromatids duplicated in S phase are connected with each other during G(2) and M phase until the onset of anaphase. This chromatid cohesion is essential for correct segregation of genetic material to daughter cells. Recently, understanding of the molecular mechanisms governing chromatid cohesion in yeast has been greatly advanced, whereas these processes in mammalian cells remain unclear. We report here biochemical and cytological analyses of human Rad21, a homologue of the yeast cohesin subunit, Scc1p/Mcd1p. hRad21 is a nuclear phosphorylated protein. Its abundance does not change during the cell cycle, and it becomes hyperyphosphorylated in M phase. Most hRad21 is not associated with chromatin when the nuclear envelope breakdown takes place in prophase. However, a detailed analysis of the spread chromosomes indicated that hRad21 remains associated with prometaphase-like chromosomes along their entire lengths. The mitotic chromatin-bound hRad21 becomes dissociated in a highly regulated manner because hRad21 remains specifically at the centromeres but disappears from the arm regions on metaphase-like chromosomes. Interestingly, hRad21 at the metaphase centromeres appears to be present at the inner pairing domain where the two sister chromatids are supposed to be in intimate contact. These results suggest that hRad21 has a critical role in chromatid cohesion in human mitotic cells.

Antibodies↗