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Biomedical subjects

K Fukada

Publications and source records attributed to K Fukada.

At least 19 recordsLinked to original sources

Time Dependent Anchoring of Adsorbed Cationic Surfactant Molecules at Mica/Solution Interface.

The nature of adsorbed cationic amphiphiles at the mica/solution interface was studied by XPS and contact angle measurements. The elemental analyses of freshly cleaved mica surfaces by XPS showed that the potassium atoms on the surface lattice of mica are not necessarily distributed equally to each surface on cleavage. The adsorbed cationic amphiphile molecules remaining on mica surfaces after rinsing with distilled water were found to be anchored to the surface by ion-exchange, replacing surface potassium and/or other cations. The ratio of adsorbed cationic amphiphile molecules with single alkyl chains to the maximum potassium ions on mica surface was estimated to be twice as large as that of amphiphiles having two alkyl chains. The contact angle of water drops placed on the adsorbed surface showed a gradual decrease with the elapse of time due to the dissolution of adsorbed surfactant into the water drop; however, the decrease was not observed for those mica surfaces when aged for more than 3 days in the adsorption bath. The anchoring of adsorbed molecules by ion-exchange was found to occur extremely slowly, however; the anchored molecules may not easily be desorbed when rinsed with deionized water. The time dependent anchoring of adsorbed molecules was studied in terms of adsorption time, alkyl chain length, and concentration of cationic surfactant. Copyright 1999 Academic Press.

Journal Article

Characterization of swollen lamellar phase of dimyristoylphosphatidylcholine-gramicidin A mixed membranes by DSC, SAXS, and densimetry.

For the fully hydrated multilamellar stack of dimyristoylphosphatidylcholine (DMPC) fluid membranes containing hydrophobic peptide gramicidin A (GrA), the membrane thickness and the bilayer-bilayer separation (i.e., water layer thickness) were determined by measuring small-angle X-ray scattering and the density of aqueous suspensions of DMPC-GrA mixtures. When the molar ratio of GrA to DMPC was 0.04, the membrane thickness decreased by 2-3 A by the incorporation of GrA molecules into DMPC bilayers, whereas the water layer thickness increased by 3-4 A. As the cause of the increment of water layer thickness, two possibilities were considered; (1) attractive van der Waals force acting between the bilayer membranes weakened by the decrease of membrane thickness, and (2) repulsive undulation force enhanced by the incorporation of GrA which may stabilize the gauche conformers of the lipid acyl chains.

Amino Acid Sequence

Microencapsulation of hepatitis B core antigen for vaccine preparation.

PURPOSE: To prepare poly(lactide-co-glycolide)(PLGA) microspheres containing recombinant hepatitis B core antigen (HBcAg; Mw = 3,600,000) by a w/o/w emulsion/solvent evaporation method and evaluate the possibility of this system as a potent long-acting carrier for hepatitis B core antigen in mice. METHODS: Various additives had been incorporated in the internal aqueous phase during the process of microencapsulating HBcAg, HBcAg antigenicity in the medium extracted from the prepared microspheres were measured by ELISA. Shape confirmation of the HBcAg antigen was performed by a sucrose gradient velocity centrifugal technique. For in vivo study, prepared microspheres were administered subcutaneously to Balb/C mice, and the serum IgG level was determined by ELISA. RESULTS: The inactivation of HBcAg by methylene chloride was dramatically reduced by the addition of gelatin (4-8% (w/v)) to the internal aqueous phase during the preparation. Further improvement of the loading efficiency to almost 61% resulted with cooling (4 degrees C). The prepared microspheres (4.27 microm+/-1.23 microm) containing 0.15% HBcAg displayed burst release (50-60% within 2 days). In subcutaneous inoculation, the adjuvant effect of PLGA microspheres was almost the same as that of the complete Freund's adjuvant. Whereas oral inoculation using the microspheres was not effective. CONCLUSIONS: The pH of the added gelatin seemed to be the key to the stabilization of HBcAg from various stability tests and CD spectrum study. Finally, the possibility of using this system as a potent long-acting hepatitis B vaccine was demonstrated.

Animals

Deprivation of leukemia inhibitory factor by its function-blocking antibodies augments T cell activation.

Leukemia inhibitory factor (LIF) is a cytokine that acts on a wide range of cell types in vitro, but knowledge of its physiological role is limited. High levels of LIF protein have been selectively detected in the thymus throughout postnatal development. LIF-deficient mice have shown impaired thymic T cell maturation, suggesting the possibility that T cells require LIF for maturation. We have used highly specific antibodies raised against native rat LIF to inhibit LIF function during a defined and restricted period of thymic T cell maturation (first postnatal week). Surprisingly, we observed increased T cell activation in the LIF-deprived wild-type rat. The increased T cell response is retained even 4 weeks after anti-LIF treatment when the level of LIF in the thymic microenvironment has returned to normal. Our results are in contrast to findings with LIF knockout mice, where decreased T cell activation was observed. These observations suggest that LIF may have alternative effects on various phases of T cell development and that LIF may be involved in the restriction of the T cell repertoire during maturation occurring in the first postnatal week.

Animals

Distribution of cholinergic neuronal differentiation factor/leukemia inhibitory factor binding sites in the developing and adult rat nervous system in vivo.

Cholinergic neuronal differentiation factor/leukemia inhibitory factor (CDF/LIF) is a multifunctional cytokine that affects neurons as well as many other cell types. Toward elucidating its neural functions in vivo, we previously investigated the distribution of CDF/LIF binding sites with iodinated native CDF/LIF in embryonic to postnatal day 0 (P0) rats. In the present study, we have extended our examination to postnatal ages and find that specific CDF/LIF binding sites are present at defined developmental stages in additional brain regions not previously exhibiting binding by P0. High levels of binding are detected in all P7 sensory and autonomic ganglia examined, but only in restricted postnatal central nervous system structures. Cranial motor and mesencephalic trigeminal neurons maintain high levels throughout, while binding to spinal motor neurons, which decreases to low levels at P0, reappears by P14 and increases with age. Most other structures, which show detectable binding by P0, exhibit higher levels at postnatal ages, including the red, deep, ventral cochlear, trapezoid, superior olivary, vestibular, ventral tegmental, and ventral posterior thalamic nuclei as well as the glomerular layer of the olfactory bulb. High levels are also detected in several structures for the first time after P0, including the cerebellar cortex (molecular and Purkinje cell layers), lateral reticular nucleus of the medulla and reticular formation, as well as the reticulotegmental, medial geniculate, solitary (rostral, dorsomedial, and commissural regions), medial septal, lateral mammillary, and lateral habenular nuclei. These results not only identify regions of potential CDF/LIF-responsive neurons and glia throughout development but suggest new CDF/LIF roles in the nervous system.

Animals

Immunohistochemistry of collagen types II and X, and enzyme-histochemistry of alkaline phosphatase in the developing condylar cartilage of the fetal mouse mandible.

We investigated the immunohistochemical localisation of types II and X collagen as well as the cytochemical localisation of alkaline phosphatase in the developing condylar cartilage of the fetal mouse mandible on d 14-16 of pregnancy. On d 14 of pregnancy, although no immunostaining for types II and X collagen was observed, alkaline phosphatase activity was detected in all cells in the anlage of the future condylar process. On d 15 of pregnancy, immunostaining for both collagen types was simultaneously detected in the primarily formed condylar cartilage. Alkaline phosphatase activity was also detected in chondrocytes at this stage. By d 16 of pregnancy, the hypertrophic cell zone rapidly increased in size. These findings strongly support a periosteal origin for the condylar cartilage of the fetal mouse mandible, and show that progenitor cells for condylar cartilage rapidly or directly differentiate into hypertrophic chondrocytes.

Alkaline Phosphatase

Tissue-specific and ontogenetic regulation of LIF protein levels determined by quantitative enzyme immunoassay.

To define the physiological role of leukemia inhibitory factor (LIF), it is essential to localize sites of LIF synthesis in vivo. We generated polyclonal antibodies specific for native rat LIF, and developed a two-site immunoassay to detect 10 pg LIF/ml. Using this immunoassay, we determined LIF content of 18 organs, CNS regions, and ganglia throughout postnatal development of rats. High levels of LIF protein (1.0-11.0 ng/g tissue) are present in relatively few tissues: the uterus at late proestrus to estrus and on day 5 of pregnancy, ovary at estrus to early metestrus-1, footpads during early postnatal development and thymus throughout. Intermediate levels (0.5-1.0 ng) are detected in the gut, skin, skeletal muscle, pancreas and lung at one or more postnatal ages. Low levels (0.1-0.5 ng) are observed in most other non-nervous and nervous tissues. LIF protein levels do not completely correspond to reported LIF mRNA levels.

Age Factors

Cholinergic neuronal differentiation factor (CDF)/leukemia inhibitory factor (LIF) binds to specific regions of the developing nervous system in vivo.

The cholinergic neuronal differentiation factor (CDF)/leukemia inhibitory factor (LIF) promotes survival and/or differentiation of specific neuronal populations in vitro. To investigate the spatial and temporal distribution of CDF/LIF binding sites in the developing rat nervous system, we localized binding of iodinated native rat CDF/LIF by radioautography. We find that specific CDF/LIF binding sites are present in distinct regions of the nervous system at defined developmental stages. CDF/LIF binding is detectable in all sensory ganglia examined, both neural crest- and placode-derived, and in all sympathetic and parasympathetic ganglia examined. In the central nervous system, in contrast, binding is restricted to specific regions in the motor, sensory, and limbic systems. These in vivo results not only support culture studies which suggest CDF/LIF involvement in development of specific populations of neurons, but also suggest new roles played by CDF/LIF.

Animals

Mutations in the transcriptional regulatory region of the precore and core/pregenome of a hepatitis B virus with defective HBeAg production.

Termination mutations in the precore open reading frame of hepatitis B virus (HBV) variants with defective hepatitis B e antigen (HBeAg) production have been demonstrated in both infected patients who have seroconverted to anti-HBe and those with fulminant hepatitis B. A donated plasma sample was found to be positive for the hepatitis B surface antigen, but negative for both HBeAg and anti-HBe. The HBV DNA titer in the plasma was estimated to be 32 pg/ml, and circulating virus-like particles were observed by electron microscopy. The entire nucleotide sequence of the virus was determined and at least 7 nucleotides were found to be unique when compared with previously reported sequences. These nucleotides created no termination codon in the precore/core, pol, preS/S and HBx open reading frames. The deduced amino acid substitutions were 28 Arg--Gln, 94 His--Tyr, 131 Val--Ile and 132 Phe--Tyr of HBx and 715 Met--Val and 789 Asp--Asn of pol. Furthermore, the precore and core/pregenome promoter contained altered 1764 A, 1766 T and 1768 A. Therefore, mutations in regions other than the precore open reading frame can cause defective HBeAg production.

Adult

Mother-to-child transmission of human T-lymphotropic virus type I (HTLV-I): an extended follow-up study on children between 18 and 22-24 years old in Okinawa, Japan.

The significant difference observed between the seroprevalence of HTLV-I in adults and in children is as yet unexplained. To evaluate a hypothetical explanation of the existence of seroconversion cases of "seronegative carriers" for this phenomenon, 21 of 55 children who had been born to seropositive mothers and who remained seronegative until the age of 18 years were further followed up at the ages of 22 and/or 24 years. None of the 21 seronegative children born to seropositive mothers seroconverted, either at 22 years or at 24 years. In addition, the polymerase-chain-reaction (PCR) technique could not prove the existence of the HTLV-I provirus genome in peripheral mononuclear cells (PBMC) of 10 of these children. Our results fail to prove the possibility of viral latency of HTLV-I in mother-to-child transmission. Therefore, the hypothetical seroconversion of "seronegative carriers" after adulthood cannot be an explanation.

Adolescent

Detection of antibodies to hepatitis C virus (HCV) structural proteins in anti-HCV-positive sera by an enzyme-linked immunosorbent assay using synthetic peptides as antigens.

We have defined 10 linear immunogenic regions encoded by the putative hepatitis C virus (HCV) structural proteins (core and envelope) by employing an enzyme-linked immunosorbent assay (ELISA) and by using 17 sequential synthetic peptides covering the N-terminal 330 amino acids of the structural polyproteins as antigens. These peptides correspond to amino acids 1 to 24, 21 to 44, 42 to 68, 64 to 91, and 100 to 120 of the putative core protein and amino acids 192 to 212, 223 to 238, 236 to 258, 250 to 266, and 307 to 330 of the putative envelope protein. In particular, the peptide covering amino acids 21 to 44 of the core protein was reactive with all but one (40 of 41) of the serum samples giving a positive signal in the passive hemagglutination assay (PHA) using the core and nonstructural proteins (NS 3/4) of the virus as antigens. We detected the HCV genome in 25 (61%) of 41 PHA-positive serum samples by the polymerase chain reaction (PCR) test. Of 25 PCR-positive serum samples, 17 serum samples had reactivity to the peptides derived from the envelope protein. On the other hand, only 1 of the 16 PCR-negative serum samples had reactivity to the peptides derived from the envelope protein. Interestingly, we often observed high serum alanine aminotransferase levels in PCR-positive individuals bearing antibodies to the envelope protein.

Alanine Transaminase

Development of a supersensitive polymerase chain reaction method for human T lymphotropic virus type II (HTLV-II) and detection of HTLV-II proviral DNA from blood donors in Japan.

A supersensitive polymerase chain reaction procedure was developed to detect human T-lymphotropic virus type II (HTLV-II) proviral genome. Six primer pairs covering the various regions of HTLV-II were compared and selected on the basis of specificity and sensitivity. Among them, one primer pair of the pol region of HTLV-II (II pol) was able to amplify and detect even 0.1 fg of the cloned plasmid HTLV-II DNA (seven copies) by regular ethidium bromide staining on polyacrylamide gel. By using this procedure, we screened 189 HTLV-I seropositive blood donors from Yamaguchi and Fukuoka Red Cross Blood Centers, Japan. There were four positive samples detectable with the HTLV-II-specific pol primer pair, as well as with the HTLV-I tax primer pair. The amplified DNAs of two specimens were cloned and sequenced. The sequences of the HTLV-I tax region from both specimens were identical to that of HTLV-I. On the other hand, those of the HTLV-II pol region were identical to that of HTLV-II, except for one base substitution in a clone from one subject. These results indicate that dual infection of HTLV-I and HTLV-II in the same persons occurs among Japanese blood donors.

Base Sequence

Some chemical properties of the HCl-methanol extract from the puparial cuticle of Drosophila melanogaster.

1. The HCl-methanol (HCl-MeOH) soluble fraction from the puparial cuticle of yellow, black and ebony of D. melanogaster was hydrolyzed in hydrochloric acid and examined for beta-alanine, ketocatechol, and acetic acid. 2. Between beta-alanine and ketocatechol and between beta-alanine and acetic acid, a quantitatively inverse relationship was found, respectively. The former relationship was further confirmed by the feeding experiment of beta-alanine to black. 3. Of total beta-alanine in the HCl-MeOH extract, the proportion of those having free amino group was 74.8 per cent. 4. All these results indicate that the HCl-MeOH soluble fraction of the puparial cuticle may be useful for investigating the cross-link structure of the cuticle.

Acetates

Function-blocking antibodies against cholinergic neuronal differentiation factor.

Cholinergic neuronal differentiation factor, CDF, causes a transition from noradrenergic to cholinergic phenotype in cultured sympathetic neurons. Moreover, its identification with leukemia inhibitory factor has shown that CDF is a multifunctional cytokine. To examine the physiological role of CDF and to further elucidate the as yet unknown effects of CDF on the nervous system, two kinds of function-blocking antibodies were generated. One type, raised against whole native CDF, completely blocks CDF activity, whereas the other type, raised against a synthetic peptide corresponding to the N-terminal amino acid region of CDF, blocks activity partially. All three anti-CDF and two antipeptide polyclonal antibodies tested in this study significantly inhibit CDF function.

Animals

Immunoaffinity purification and dose-response of cholinergic neuronal differentiation factor.

A glycoprotein from heart cell-conditioned medium, cholinergic neuronal differentiation factor (CDF), causes a transition from noradrenergic to cholinergic phenotype in cultured rat sympathetic neurons. Although the transition has been known to occur in a dose-dependent manner and CDF has been purified, the examination of a complete dose-response of neurons to CDF has not been possible because sufficient quantities of pure CDF have not been available. A complete dose-response curve is essential for evaluating the biological response of the neurons, for assessing the physiological role of CDF and for understanding the mechanism of action of CDF. We report here an immunoaffinity-purification procedure for CDF with a 73.1% recovery using antibodies raised against a synthetic peptide homologous with the N-terminal region of CDF. This method produced pure CDF in quantities sufficient for examination of the full dose-response range of the neurons. Our main findings are the following. The dose-responses of acetylcholine and catecholamine metabolisms to CDF are different, although the same molecule affects both transmitters. While the half-maximal concentrations for acetylcholine induction (0.20 nM) and for catecholamine suppression (0.28 nM) are similar, the response of catecholamine metabolism begins slowly and saturates at a CDF concentration (5-20 nM) considerably higher than that of acetylcholine (0.6 nM). This may indicate that CDF affects multiple processes in catecholamine metabolism.

Amino Acid Sequence

Follow-up of asymptomatic HTLV-I carriers among blood donors in Kyushu, Japan.

We examined mortality from adult T-cell leukemia/lymphoma (ATL/ATLL) and other diseases alleged to be associated with human T-lymphotropic virus type I (HTLV-I) among anti-HTLV-I antibody-positive blood donors in Kyushu, Japan. During 1984-87, a total of 3,991 blood donors aged 40 years or over were followed from the date of donation to the date of death or the end of the study. Crude mortality rates from ATL (with 95 percent confidence intervals) were 68 per 100,000 (13-202) for males and 36 per 100,000 (3-132) for females. The rates were underestimated by approximately 50 percent because of self-selection and short observation periods. Neither death rates from other cancers nor death rates from all cancers were elevated.

Adult

The cholinergic neuronal differentiation factor from heart cells is identical to leukemia inhibitory factor.

A protein secreted by cultured rat heart cells can direct the choice of neurotransmitter phenotype made by cultured rat sympathetic neurons. Structural analysis and biological assays demonstrated that this protein is identical to a protein that regulates the growth and differentiation of embryonic stem cells and myeloid cells, and that stimulates bone remodeling and acute-phase protein synthesis in hepatocytes. This protein has been termed D factor, DIA, DIF, DRF, HSFIII, and LIF. Thus, this cytokine, like IL-6 and TGF beta, regulates growth and differentiation in the embryo and in the adult in many tissues, now including the nervous system.

Amino Acid Sequence